HOODIA GORDONII: QUALITY CONTROL AND

Transcription

HOODIA GORDONII: QUALITY CONTROL AND
BIOPHARMACEUTICAL ASPECTS
by
ILZE VERMAAK
Submitted in partial fulfillment of the requirements for the degree
DOCTOR TECHNOLOGIAE
in the
Department of Pharmaceutical Sciences
FACULTY OF SCIENCE
TSHWANE UNIVERSITY OF TECHNOLOGY
Supervisor: Prof AM VILJOEN
Co-Supervisor: Prof JH HAMMAN
April 2011
DECLARATION BY CANDIDATE
“I hereby declare that the thesis submitted for the degree of DTech:
Pharmaceutical Sciences, at Tshwane University of Technology, is my own
original work and has not previously been submitted to any other institution of
higher education. I further declare that all sources cited or quoted are indicated
and acknowledged by means of a comprehensive list of references”.
I Vermaak
Copyright© Tshwane University of Technology 2011
ii
ACKNOWLEDGEMENTS
I would like to acknowledge the following people:
§
First and foremost my supervisor Prof Alvaro Viljoen for his excellent
supervision and mentorship and for sharing with me his expertise and
research insight. He is my role model of a successful researcher and the
one who inspires me to become a researcher of note.
§
My co-supervisor Prof Sias Hamman for his thoughtful advice, endless
support and encouragement as well as attention to detail.
§
Dr Gill Enslin (Head of Department) and my colleagues at the Department
of Pharmaceutical Sciences for their support and encouragement.
§
My fellow students at the Department of Pharmaceutical Sciences: Thank
you for always being willing to assist me when needed and for your support
and encouragement. It certainly has been fun.
§
The people who provided Hoodia gordonii samples:
§
The Kalahari Hoodia Growers in Namibia. A special thank you for
your kind hospitality during our visits.
§
Conrad Geldenhuys and Nico Laubscher from the Northern Cape
Department of Environmental Affairs and Nature Conservation.
§
Dr Marietjie Stander, Kobus Engelbrecht and Adolf Joubert.
§
Dr Weiyang Chen (HPLC) for developing the quantification method for P57.
§
Dr Malgorzata Baranska (Raman spectroscopy) for teaching us about
Raman spectroscopy and the productive collaboration that followed. Your
hospitality during our visit to Poland was much appreciated.
§
Dr Jan Masthoff (CAMAG) for his assistance with HPTLC-densitometry
training and hospitality during our visit to Switzerland.
§
Tshwane University of Technology
(TUT) and National Research
Foundation (NRF) for financial assistance. A special note of thanks to TUT
for granting me sabbatical leave enabling me to complete the write-up of my
thesis.
§
My special friends: your support and encouragement and especially your
patience with me is greatly appreciated.
§
My family for their support and interest in my studies. Lastly and most
importantly, I wish to thank my parents and sister for their encouragement,
love and support, making it possible to complete this degree.
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ABSTRACT
Hoodia gordonii is a popular weight loss product highly susceptible to adulteration.
This highlights the need for rapid and simple quality control methods for
authentication of raw material and products.
High performance thin layer
chromatography analysis was used to authenticate raw material. Quantitative high
performance thin layer chromatography analysis as well as near infrared, midinfrared and Raman spectroscopy combined with chemometric techniques were
used to develop alternative quantification methods for P57 (the perceived active
ingredient of H. gordonii) in raw material and/or products. Liquid chromatography
coupled to mass spectrometry was used to determine the concentration of P57
and used as reference data to develop calibration models based on the partial
least squares projections to latent structures regression algorithm for the infrared
spectra. The performance of each calibration model was evaluated according to
the correlation coefficient (R2) and root mean square error of prediction.
The high performance thin layer chromatography system produced good
separation of compounds including that of the P57 band.
Quantitative
determination for P57 resulted in linear calibration curves with good correlation
coefficient (R2) values of 0.9706-0.9993. For the near infrared spectroscopy data,
the partial least squares projections to latent structures model with 2nd derivative
pre-processing predicted P57 content with an R2 value of 0.9629 and a root mean
square error of prediction of 0.03%. Pre-processing of the Raman data with
orthogonal signal correction yielded a partial least squares projections to latent
structures model with an R2 value of 0.9986 and a root mean square error of
prediction of 0.004%. The qualitative and quantitative high performance thin layer
chromatography
analyses
provided
a
chemical
fingerprint
technique
for
authentication and confirmation of the presence of P57 in H. gordonii raw material
and products as well as for quantification of P57.
The parameters of the
calibration models demonstrated that both near infrared and Raman spectroscopy
shows potential to rapidly quantify P57 in H. gordonii raw material.
As H. gordonii is one of the most widely consumed anti-obesity products of natural
origin, it is disturbing that key aspects such as pharmacokinetic and
biopharmaceutical aspects have not been sufficiently investigated. The in vitro
iv
transport of pure P57 as well as P57 from crude plant extracts across excised
porcine intestinal and buccal tissue was investigated using a Sweetana-Grass
diffusion apparatus.
In addition to using buffer as transport medium, in vivo
conditions were mimicked with the use of saliva and simulated intestinal fluid. The
apparent permeability coefficient and flux values were calculated and statistically
analysed. Pure P57 was transported at a much lower rate and extent than P57
from the crude extract across porcine intestinal tissue.
P57 was transported
across the buccal mucosa when applied in the form of a crude plant extract but no
transport could be detected when P57 was applied in the pure form. The transport
of P57 across intestinal and buccal mucosal tissues is significantly affected by
exposure to conditions simulating an in vivo environment.
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TABLE OF CONTENTS
PAGE
ACKNOWLEDGEMENTS ....................................................................................... iii
ABSTRACT ............................................................................................................ iv
LIST OF FIGURES ................................................................................................. xi
LIST OF TABLES ................................................................................................. xvi
GLOSSARY ......................................................................................................... xvii
CHAPTER 1 – Introduction
1.1
INTRODUCTION....................................................................................... 1
1.2
AIMS ......................................................................................................... 4
1.3
OBJECTIVES ............................................................................................ 4
1.4
THESIS LAYOUT ...................................................................................... 5
1.5
REFERENCES.......................................................................................... 6
CHAPTER 2 – Hoodia gordonii and other plants used for their anti-obesity
property
2.1
INTRODUCTION....................................................................................... 7
2.2
HOODIA GORDONII ................................................................................. 7
2.2.1
Classification and botanical aspects ......................................................... 7
2.2.2
Ethnopharmacology ................................................................................ 10
2.2.3
Commercialisation................................................................................... 10
2.2.4
Intellectual property rights and benefit sharing issues ............................ 14
2.2.5
Phytochemistry........................................................................................ 15
2.2.6
Biological activity ..................................................................................... 16
2.2.6.1 Anti-obesity activity ................................................................................. 16
2.2.6.2 Other biological activities ........................................................................ 19
2.2.7
Toxicity .................................................................................................... 20
2.3
PLANTS USED FOR THEIR ANTI-OBESITY PROPERTY .................... 21
2.3.1
Introduction ............................................................................................. 21
2.3.2
Mechanisms of action of anti-obesity preparations ................................. 22
2.3.3
Other plants popularly used as anti-obesity agents ................................ 25
2.4
CONCLUSIONS ...................................................................................... 42
2.5
REFERENCES........................................................................................ 44
vi
SECTION 1 – Quality control ............................................................................. 68
REFERENCES ..................................................................................................... 69
CHAPTER 3 – Chromatographic techniques
3.1
LIQUID CHROMATOGRAPHY ............................................................... 70
3.1.1
INTRODUCTION..................................................................................... 70
3.1.2
BACKGROUND AND LITERATURE REVIEW ....................................... 71
3.1.3
MATERIALS AND METHODS ................................................................ 73
3.1.3.1 Plant material, reagents and sample preparation ................................... 73
3.1.3.2 Liquid chromatography coupled to mass spectrometry (LC-MS) ............ 74
3.1.3.3 High performance liquid chromatography (HPLC) .................................. 74
3.1.4
RESULTS................................................................................................ 75
3.1.4.2 High performance liquid chromatography (HPLC) .................................. 77
3.1.5
CONCLUSIONS ...................................................................................... 77
3.2
QUALITATIVE HIGH PERFORMANCE THIN LAYER
CHROMATOGRAPHY (HPTLC) ............................................................. 79
3.2.1
INTRODUCTION..................................................................................... 79
3.2.2
3.2.2.1 Analysis of plant material ........................................................................ 80
3.2.3
3.2.3.1 Plant material and reagents .................................................................... 81
3.2.3.2 High performance thin layer chromatography (HPTLC) analysis ............ 81
3.2.3.3 Liquid chromatography coupled to mass spectrometry (LC-MS)
analysis .................................................................................................... 83
3.2.4
RESULTS AND DISCUSSION................................................................ 84
3.2.4.1 High performance thin layer chromatography (HPTLC) .......................... 84
3.2.5
CONCLUSIONS ...................................................................................... 92
3.3
QUANTITATIVE HIGH PERFORMANCE THIN LAYER
CHROMATOGRAPHY (HPTLC) ............................................................. 92
3.3.1
INTRODUCTION..................................................................................... 92
3.3.2
vii
3.3.3
3.3.3.1 Plant material and reagents .................................................................... 94
3.3.3.2 Quantitative high performance thin layer chromatography analysis ....... 94
3.3.4
RESULTS AND DISCUSSION................................................................. 96
3.3.4.1 Quantitative high performance thin layer chromatography analysis ....... 96
3.3.5
CONCLUSIONS .................................................................................... 100
3.4
REFERENCES...................................................................................... 102
CHAPTER 4 – Vibrational spectroscopy
4.1
INTRODUCTION................................................................................... 104
4.2
BACKGROUND AND LITERATURE REVIEW ..................................... 105
4.2.1
Infrared spectroscopy (IR)..................................................................... 105
4.2.2
Chemometric data analysis ................................................................... 107
4.3
MATERIALS AND METHODS .............................................................. 108
4.3.1
Plant material ........................................................................................ 108
4.3.2
Near infrared spectroscopy (NIR) ......................................................... 109
4.3.3
Mid-infrared spectroscopy (MIR) ........................................................... 109
4.3.4
Raman spectroscopy ............................................................................ 110
4.3.5
Raman mapping .................................................................................... 111
4.3.6
Data analysis......................................................................................... 111
4.3.7
P57 quantification with LC-MS analysis ................................................ 112
4.4
RESULTS AND DISCUSSION.............................................................. 112
4.4.1
Chemometric data analysis methods and parameters .......................... 112
4.4.2
NIR spectroscopy .................................................................................. 113
4.4.3
MIR spectroscopy ................................................................................. 116
4.4.4
Raman spectroscopy ............................................................................ 117
4.4.5
Raman mapping .................................................................................... 118
4.5
CONCLUSIONS .................................................................................... 120
4.6
REFERENCES...................................................................................... 122
viii
SECTION 2 – Biopharmaceutical aspects ...................................................... 125
CHAPTER 5 – Buccal and intestinal transport of P57 from Hoodia gordonii
5.1
INTRODUCTION................................................................................... 126
5.2
BACKGROUND AND LITERATURE REVIEW ..................................... 127
5.2.1
Introduction ........................................................................................... 127
5.2.2
In vitro models for intestinal permeation studies .................................. 130
5.2.2.1 Membrane-based models ..................................................................... 130
5.2.2.2 Cell culture models................................................................................ 131
5.2.2.3 Isolated tissue models........................................................................... 131
5.2.3
In vitro models for buccal permeation studies ....................................... 133
5.2.3.1 Isolated tissue models........................................................................... 133
5.2.3.2 Cell culture models................................................................................ 133
5.3
METHODS AND MATERIALS .............................................................. 134
5.3.1
In vitro intestinal and buccal permeation studies .................................. 134
5.3.1.1 Plant material and reagents .................................................................. 134
5.3.1.2 Tissue preparation ................................................................................ 134
5.3.1.3 Preparation of simulated intestinal fluid and artificial saliva .................. 136
5.3.1.4 Transport studies .................................................................................. 137
5.3.1.5 Quantification of P57 ............................................................................. 138
5.3.1.6 Data analysis and statistical evaluation ................................................ 138
5.4
RESULTS AND DISCUSSION.............................................................. 139
5.4.1
In vitro intestinal transport studies......................................................... 139
5.4.2
In vitro buccal transport studies ............................................................ 141
5.5
CONCLUSIONS .................................................................................... 143
5.6
REFERENCES...................................................................................... 144
CHAPTER 6 – General conclucions and recommendations
6.1
GENERAL CONCLUSIONS.................................................................. 147
6.2
RECOMMENDATIONS ......................................................................... 148
ANNEXURE 1: Chemical structures
1.1.
Chemical structures of the steroidal glycosides isolated from H. gordonii
with their corresponding aglycones indicated (*,#,$,@,^) ............................. 150
ix
ANNEXURE 2: Publications emanating from this thesis
2.1.
Publications in accredited journals ........................................................ 158
2.2.
Publication in a non-accredited journal ................................................. 158
2.3.
Publications submitted to accredited journals ....................................... 158
ANNEXURE 3: Conference contributions
3.1.
Oral papers ........................................................................................... 159
3.2.
Poster .................................................................................................... 159
x
LIST OF FIGURES
PAGE
FIGURE 1.1:
Examples of H. gordonii products available for purchase on
2
the internet
FIGURE 1.2:
Examples of H. gordonii products that are available in
3
various forms such as patches, oral strips, capsules, topical
gels, fruit bars and chewing gum
FIGURE 2.1:
Hoodia gordonii in flower (Photograph by AM Viljoen)
9
FIGURE 2.2:
Hoodia gordonii cross-sections of dried and freshly-cut
9
stems (Photographs by AM Viljoen)
FIGURE 2.3:
The natural distribution of H. gordonii in South Africa and
9
Namibia indicated in orange
FIGURE 2.4:
Timeline of the most important developments in the research
11
and commercial development of H. gordonii
FIGURE 2.5:
An article on H. gordonii published in the Sunday Times
13
newspaper on 20 September 2009
FIGURE 2.6:
Photographs of commercially cultivated H. gordonii in
13
greenhouse tunnels in Namibia and South Africa and the
processing of the raw material by slicing (Photographs by
AM Viljoen)
FIGURE 3.1:
Schematic illustration of an HPLC apparatus coupled to an
73
ultraviolet detector and a mass spectrometer (LC-UV-MS)
FIGURE 3.2:
LC-MS chromatogram of the plant material used as the
75
H. gordonii control standard for the LC-MS analytical method
FIGURE 3.3:
An extracted mass (861.5 m/z) chromatogram of P57 (A)
76
and the H. gordonii control standard plant material (B)
FIGURE 3.4:
Calibration curve for the P57 reference standard as obtained
76
by LC-MS
FIGURE 3.5:
The percentage of P57 determined with LC-MS in
77
H. gordonii stem samples (n = 145)
FIGURE 3.6:
An HPLC chromatogram of the H. gordonii plant material
from a commercial capsule
xi
78
FIGURE 3.7:
The calibration curve for the P57 reference standard with the
78
HPLC method
FIGURE 3.8:
A diagram of the CAMAG semi-automated HPTLC system
including
the
developing
automatic
chamber
TLC
ADC2,
Sampler
4,
automatic
chromatogram
immersion
82
device, TLC plate heater III and documentation device
Reprostar 3
FIGURE 3.9:
The HPTLC chromatogram used to determine the limit of
84
detection for P57. P57 was spotted at 5 µg (track 1), 3 µg
(track 2), 2 µg (track 3), 1.5 µg (track 4), 1 µg (track 5), 0.5
µg (track 6), 0.25 µg (track 7), 0.15 µg (track 8) and 0.1 µg
(track 9)
FIGURE 3.10: An HPTLC plate viewed under UV at 365 nm.
Track 1:
85
H. gordonii standard raw material, track 2-8: H. gordonii
commercial products (1-7) from different manufacturers and
track 9: the P57 reference standard
Track 1:
86
commercial products (8-14) from different manufacturers
and track 9: the P57 reference standard
Track 1:
86
commercial products (15-20) from different manufacturers
and track 9: the P57 reference standard
FIGURE 3.13: An HPTLC plate viewed under UV at 365 nm. Track 1: wild
population sample of H. gordonii from South Africa, track 2:
wild population sample of H. gordonii from Namibia, track 3:
cultivated sample of H. gordonii from South Africa, track 4:
cultivated sample of H. gordonii from Namibia, track 5: 2
year old actively growing plant sample, track 6: 2 year old
dormant plant sample, track 7: a 3 year old actively growing
cultivated plant, track 8: a 3 year old dormant cultivated
plant and track 9: the P57 reference standard
xii
88
Track 1:
89
H. gordonii standard raw material, track 2-7: the same
sample of H. gordonii standard raw material as for track 1
adulterated with O. ficus-indica in concentrations of 10%,
20%, 40%, 50 %, 60%, 80%, respectively, track 8: O. ficusindica raw material, track 9: the P57 reference standard
Track 1:
90
H. gordonii standard raw material, Tracks 2 and 3: Opuntia
ficus-indica samples, Tracks 4 and 5: Aloe ferox samples,
Track 6: Cereus jamacaru sample, Track 7: Agave
americana sample and Track 8: the P57 reference standard
FIGURE 3.16: LC-MS chromatograms of the P57 standard (A) and the
91
preparative TLC sample (B)
FIGURE 3.17: Mass spectra of the P57 reference standard (A) and the
91
preparative TLC sample (B)
FIGURE 3.18: A photograph of the CAMAG TLC scanner 3 used in the
94
quantification of P57 from commercial samples (Photograph
by I Vermaak)
FIGURE 3.19: An example of an HPTLC chromatogram that was used for
96
the quantification of P57 from H. gordonii commercial
products.
Track 1: 65.50 ng P57, Track 2-3: Product 1,
Track 4: 75.00 ng P57, Track 5-6: Product 2, Track 7:
100.00 ng P57, Track 8-9: Product 3, Track 10: 125.00 ng
P57, Track 11-12: Product 4
FIGURE 3.20: An example of the baseline chromatogram for one track (A)
97
and the corresponding integration of the P57 peak as
indicated (B)
FIGURE 3.21: The linear calibration curve for four standard levels of P57
97
(black) and the four H. gordonii products (green) in duplicate
in terms of peak height
FIGURE 3.22: The linear calibration curve for four standard levels of P57
(black) and the four H. gordonii products (green) in duplicate
in terms of peak area
xiii
98
FIGURE 4.1:
Photograph of the Buchi NIRFlex N500 Fourier transform
near
infrared
spectrophotometer
and
sample
109
tray
(Photograph by I Vermaak)
FIGURE 4.2:
Photographs of the Bruker Alpha-P Fourier transform
110
infrared spectrophotometer (Photographs by I Vermaak)
FIGURE 4.3:
Photographs of the Bruker Multiram Fourier transform
110
Raman spectrometer (Photographs by I Vermaak)
FIGURE 4.4:
Typical FT-NIR spectra for H. gordonii powdered raw
114
material (A) and the spectrum for the P57 reference
standard (B)
FIGURE 4.5:
Loadings line plot from the PLS calibration model using FT-
115
NIR data before (A) and after (B) removal of X variables with
low loading weights (10000 – 7800 cm-1)
FIGURE 4.6
PLS calibration model using the 2nd derivative pre-
116
processing technique calculated from FT-NIR data
FIGURE 4.7
PLS calibration model with OSC pre-processing calculated
118
from FT-NIR-Raman data
FIGURE 4.8
A microscopic image of a fresh H. gordonii slice with the
119
marked area where the Raman mapping measurement was
performed (A), and chemical maps of the clusters (B) and
the borders of the clusters (C) showing the distribution of
various plant components (blue – cellulose; violet –
carotenoids; green – lignin)
FIGURE 4.9
A single Raman spectrum of H. gordonii extracted from the
119
three clusters coloured accordingly. At the bottom a Raman
spectrum of pure P57 standard is presented
FIGURE 5.1:
Schematic illustration of the drug absorption pathways
across intestinal epithelial cells. (A) paracellular diffusion,
(B) paracellular diffusion through opened tight junctions, (C)
transcellular
passive
diffusion
with
(C*)
intracellular
metabolism, (D) carrier-mediated transcellular transport, (E)
efflux and (F) vesicular transcytosis (Hunter and Hirst,
1997:131)
xiv
128
FIGURE 5.2:
The preparation and mounting of porcine intestinal mucosa
135
in Sweetana-Grass diffusions chambers (Photographs by JH
Hamman)
FIGURE 5.3:
A photograph of a diffusion chamber cell as well as a
136
schematic diagram of an assembled cell indicating the
excised mucosal tissue mounted between two half-cells
FIGURE 5.4:
Transport of pure P57 as well as P57 from a crude plant
139
extract across porcine intestinal tissue in the absorptive
direction ( ) and secretory direction ( ) expressed in terms
of apparent permeability coefficient (Papp) values
FIGURE 5.5:
Transport of pure P57 as well as P57 from a crude plant
extract across porcine buccal tissue in the absorptive
direction expressed in terms of apparent permeability
coefficient (Papp) values
xv
142
LIST OF TABLES
PAGE
TABLE 2.1:
Summary of studies on the in vivo anti-obesity activity of
17
H. gordonii and P57
TABLE 2.2:
Summary of the plants investigated for their anti-obesity
26
properties in alphabetical order by species name
TABLE 3.1:
Comparison of the P57% LC-MS results and HPTLC results
87
TABLE 3.2:
Peak height and area comparison for the calculation of intra-
99
day precision
TABLE 3.3:
Peak height and area comparison for the calculation of inter-
100
day precision
TABLE 3.4:
Accuracy of the P57 standards determined by recovery
101
analysis
TABLE 4.1:
Number of PLS factors, R2, Q2, RMSEP and RMSEE values
113
for determining P57 content with NIR, MIR and Raman
spectroscopy utilising a PLS model with different preprocessing methods
TABLE 4.2:
P57 content of the outer layers and cortex samples of
120
H. gordonii determined by LC-MS analysis
TABLE 5.1:
Experimental groups used for the intestinal and buccal
138
transport studies
TABLE 5.2:
Flux (J) values (µg/cm2/h) for P57 across porcine intestinal
140
mucosa
TABLE 5.3:
Flux (J) values (µg/cm2/h) for P57 across porcine buccal
mucosa
xvi
142
GLOSSARY
ACC
Acetyl coenzyme A carboxylase
ADC
Automatic developing chamber
AMPK
5ʹ Adenosine monophosphate-activated protein kinase
ANOVA
Repeated one-way analysis of variance
APCI
Atmospheric pressure chemical ionisation
ATP
Adenosine triphosphate
cAMP
3ʹ-5ʹ-Cyclic adenosine monophospate
CBD
Convention on Biological Diversity
CCAAT
Cytidine-cytidine-adenosine-adenosine-thymidine
CCK
Cholesystokinin
C/EBPs
CCAAT-enhancer-binding proteins
CF-FAB
Continuous flow-fast atom bombardment
CITES
Convention on International Trade in Endangered Species
CNS
Central nervous system
CoA
Coenzyme A
CSIR
Council for Scientific and Industrial Research
CYP
Cytochrome P
ESI
Electrospray ionisation
FAS
Fatty acid synthase
FDA
Food and Drug Administration
FT
Fourier transform
GDPH
Glycerol-3-phosphate dehydrogenase
HPLC
High performance liquid chromatography
HPTLC
High performance thin layer chromatography
5-HTP
5-Hydroxytryptophan
IAM
Immobilised artificial membrane
i.c.v.
Intracerebroventricular
IR
Infrared
LB
Liebermann-Burchard
LC
Liquid chromatography
LC-MS
Liquid chromatography coupled to mass spectrometry
LC-UV
Liquid chromatography coupled to ultraviolet detection
xvii
LC-UV-MS
Liquid chromatography coupled to ultraviolet detection and
mass spectrometry
LLC-PK1
Lewis lung carcinoma-porcine kidney 1
MDCK
Madin-Darby canine kidney
MIR
Mid-infrared
MS
Mass spectrometry
MSC
Multiplicative signal correction
NIR
Near infrared
OPLS
Orthogonal
partial
least
squares
projections
structures
OSC
Orthogonal signal correction
PAMPA
Parallel membrane permeability assays
PCA
Principal component analysis
P-gp
P-glycoprotein
PLS
Partial least squares projections to latent structures
PPAR
Peroxisome proliferator-activated receptors
Q
2
Cumulative overall cross-validated R2X
R2
Correlation coefficient
RBP
Retinoid binding protein
Rf
Retention front
RMSEP
Root mean square error of prediction
ROS
Reactive oxygen species
SIF
Simulated intestinal fluid
SNV
Standard normal variate
SREBP
Sterol-regulatory-element-binding protein
TG
Triglyceride
TLC
Thin layer chromatography
TSP
Thermospray
UCP
Uncoupling proteins
UPLC
Ultra performance liquid chromatography
UV
Ultraviolet
xviii
to
latent
CHAPTER 1
Introduction
1.1
INTRODUCTION
Hoodia gordonii (Masson) Sweet ex. Decne. is a spiny succulent plant with fleshy,
thorny stems, belonging to the Apocynaceae family of flowering plants.
It is
frequently erroneously referred to as the desert cactus (Cactaceae) in the media
due to its appearance.
According to traditional knowledge passed down from
generation to generation, the San bushmen of Southern Africa consumed the flesh
of this plant to suppress hunger and thirst while on long hunting trips or in times of
low food supply (Van Heerden, 2008:435; Glasl, 2009:300). Not surprisingly, in a
world intensely focused on obesity and its associated illnesses, the discovery of
this plants’ promise as an appetite suppressant initiated substantial research and
commercial interest. According to the World Health Organisation, 1.6 billion adults
worldwide are overweight, a number expected to increase by 40% within the next
decade (WHO, 2006:S.a.). This global fat-epidemic gives rise to a multimillion
dollar market for weight loss products. Hoodia-containing products exploded onto
the market being sold as the new miracle anti-obesity preparation. It is extremely
popular, as evidenced by the more than 5 million links that appear when the word
Hoodia is typed into the GoogleTM search engine, and has been widely advertised
in magazines as well as on several television shows (Figure 1.1).
The credible history attached to the use of H. gordonii, the aggressive advertising,
the worldwide obesity problem and the general misconception by the public that all
plant medicinal products are of natural origin and therefore safe makes H. gordonii
the ‘perfect’ candidate to seize a large portion of the anti-obesity products market.
Unfortunately, the shortage of plant material due to the slow growth rate of H.
gordonii, the need for a collecting permit and the high market demand leads to the
distribution of low quality products produced by unscrupulous companies. Many
commercially sold “Hoodia” weight-loss preparations have been proven to be fake
(Holt and Taylor, 2006:111).
1
FIGURE 1.1: Examples of H. gordonii products available for purchase on the
internet
The quality and authenticity of H. gordonii raw plant material purchased by
manufacturers and H. gordonii products is determined by the quantity of P57AS3
(commonly referred to as P57) it contains. P57 is one of the steroidal glycosides
isolated by the pioneering group of scientists who investigated Hoodia species for
appetite suppression (Van Heerden et al., 2007:2546) and is the perceived active
anti-obesity compound in raw plant material. Since then several other researchers
have identified steroidal glycosides from H. gordonii which may have potential as
appetite-suppressants but according to the herbal industry, P57 content is
currently the only benchmark of quality. The preferred analytical method currently
used to quantify P57 is high performance liquid chromatography (HPLC) coupled
to mass spectrometry (LC-MS) although more recently Janssen et al. (2008:200)
quantified steroidal glycosides by using HPLC coupled to an ultraviolet detector
(LC-UV).
These analytical methods are expensive, require extensive sample
preparation, and needs to be performed by a well-trained technician.
This
highlights the need for simpler, faster, and less expensive quality control analytical
methods such as vibrational spectroscopy including near infrared (NIR), midinfrared (MIR) and Raman spectroscopy.
2
High performance thin layer
chromatography (HPTLC) can provide a simpler means of authentication of raw
material, confirm the presence of P57, and in addition quantify its content by
densitometry.
In addition to the need for quality control, the most basic biopharmaceutical
properties to validate the pharmacological effect of H. gordonii were not
determined before it went into commercial production. It is only recently that some
of these aspects such as the in vitro pharmacokinetic profile of P57 have been
investigated. To complicate matters, H. gordonii products are available in many
different forms including tablets, capsules, patches, oral strips, topical gels, fruit
bars and even chewing gum (Figure 1.2).
FIGURE 1.2: Examples of H. gordonii products that are available in various forms
such as patches, oral strips, capsules, topical gels, fruit bars and chewing gum
Many of these commercial products such as the Hoodia patches are sold
regardless of the fact that there is no precedent proving benefit (Holt and Taylor,
2006:111). Currently, it has not even been concluded whether oral H. gordonii has
the desired anti-obesity effect, let alone whether P57 and other H. gordonii
phytoconstituents permeate through barriers such as the skin to the extent of
eliciting a systemic effect. It is evident that much research is needed before the
3
extensive worldwide use of H. gordonii can be validated. In addition, the more
immediate problem of a faster quality control method for H. gordonii products
could increase the safety and reputation of these products.
1.2
AIMS
The first aim of the research project was to develop and optimise rapid quality
control methods for H. gordonii raw material and products. The second aim was to
determine whether the perceived active component of H. gordonii (P57) is
transported across porcine intestinal and buccal mucosa.
1.3
OBJECTIVES
§
To determine the quantity of P57 in various plant material samples using
liquid chromatography coupled to mass spectrometry (LC-MS).
§
To develop a chemical fingerprint technique for H. gordonii plant material
and products using high performance thin layer chromatography (HPTLC).
§
To develop a method to detect adulteration of H. gordonii raw material and
products and to confirm the presence or absence of P57 in these samples
using qualitative HPTLC.
§
To use quantitative HPTLC to quantify P57 in H. gordonii products.
§
To acquire vibrational spectroscopy data for powdered H. gordonii raw
material using near infrared (NIR), mid infrared (MIR) and Raman
spectroscopy and to determine the location of P57 within the plant stem
using Raman mapping.
§
To develop calibration models using the LC-MS quantification data in
conjunction with the NIR, MIR and Raman spectroscopy data using
chemometric analysis.
§
To determine whether pure P57 and P57 in crude extract form is
transported across porcine intestinal mucosa in Sweetana-Grass diffusion
chambers and to quantify the amount with high performance liquid
chromatography (HPLC).
§
To determine whether pure P57 and P57 in crude extract form is
transported through porcine buccal mucosa in Sweetana-Grass diffusion
chambers and to quantify the amount with HPLC.
4
1.4
THESIS LAYOUT
The thesis is divided into the following chapters: Introduction (Chapter 1), Hoodia
gordonii and other plants used for their anti-obesity property (Chapter 2),
chromatographic techniques (Section 1, Chapter 3), Vibrational spectroscopy
(Section 1, Chapter 4), Buccal and intestinal transport of P57 from H. gordonii
(Section 2, Chapter 5) and General conclusions and recommendations (Chapter
6). The experimental part was divided into two sections dealing with the two main
topics, i.e. quality control and biopharmaceutical aspects.
5
1.5
REFERENCES
GLASL, S. 2009. Hoodia: A herb used in South African traditional medicine – A
potential cure for overweight? Pharmacognostic review of history, composition,
health-related claims, scientific evidence and intellectual property rights.
Schweizerische Zeitschrift für Ganzheitsmedizin, 21(6):300-306.
HOLT, S., TAYLOR, T.V. 2006. Hoodia gordonii: An overview of biological and
botanical characteristics: Part 1. Townsend Letter for Doctors and Patients,
280:104-113.
JANSSEN, H.-G., SWINDELLS, C., GUNNING, P., WANG, W., GRÜN, C.,
MAHABIR, K., MAHARAJ, V.J., APPS, P.J. 2008. Quantification of appetite
suppressing steroid glycosides from Hoodia gordonii in dried plant material,
purified extracts and food products using HPLC-UV and HPLC-MS methods.
Analytica Chimica Acta, 617(1-2):200-207.
VAN HEERDEN, F.R. 2008. Hoodia gordonii: A natural appetite suppressant.
Journal of Ethnopharmacology, 119:434-437.
VAN HEERDEN, F.R., HORAK, R.M., MAHARAJ, V.J., VLEGGAAR, R., SENABE,
J.V., GUNNING, P.J. 2007. An appetite suppressant from Hoodia species.
Phytochemistry, 68:2545-2553.
WHO see WORLD HEALTH ORGANISATION. 2006. Fact sheet no 311.
Available on: http://www.who.int/mediacentre/factsheets/fs311/en/print.html
[Accessed: 2009/09/29].
Figure references (www)
FIGURE 1.1
http://webforhealth.com/wp-content/uploads/2009/01/hoodiastar.gif [Accessed:
2010/12/13].
http://hoodiadietpill.cn/images/hoodia-Gordonii_header.jpg [Accessed:
2010/12/13].
http://free99.net/images/Hoodia%20diet%20patch.gif [Accessed: 2010/12/13].
http://www.dietsinreview.com/images/cache/75x35_hoodia-stack-logo.gif
[Accessed: 2010/12/13].
http://www.fortmaywood.com/news/i/User%2520buy%2520hoodia.jpg [Accessed:
2010/12/13].
http://www.tvtopten.com/images/hoodia2.jpg[Accessed:2010/12/13].
http://i.inteletrack.com/images/purehoodia/update2/PureHoodia_Email.jpg
[Accessed: 2010/12/13].
http://www.hoodiaworx.com/images/hoodiaplus430x600.gif [Accessed:
2010/12/13].
6
CHAPTER 2
Hoodia gordonii and other plants used for their antiobesity property
2.1
INTRODUCTION
Hoodia gordonii has enjoyed a great deal of popularity as the new miracle weightloss product and has established itself, from 2005 onwards, as one of the bestselling weight-loss supplements in the USA, Canada and Western Europe. The
commercialisation of this plant has been highly controversial due to intellectual
property rights and benefit sharing issues as well as the fact that several
prominent pharmaceutical companies involved in its development have withdrawn
from further commercial ventures. Despite the lack of convincing scientific studies
on the quality and efficacy of H. gordonii products, it is widely consumed raising
concerns about safety. The long history of use of fresh raw plant material sets a
precedent for safety but it must be kept in mind that this cannot be extrapolated to
the use of extracts or synthetic chemical analogues developed (Holt and Taylor,
2006a:111). The investigation into H. gordonii as an anti-obesity agent in this
study led to further exploration of the numerous other plants that are used for the
treatment of obesity. In most cases the scientific basis for worldwide consumption
is clearly absent and information on toxicity is limited. This emphasises the need
for natural products to be regulated more rigorously similar to the regulation of
conventional pharmaceuticals.
2.2
HOODIA GORDONII
2.2.1 Classification and botanical aspects
Hoodia gordonii is part of the genus classified as stapeliads within the tribe
Ceropegieae of the subfamily Asclepiadoideae belonging to the Apocynaceae
family.
The previous classification of Trichocaulon is not recognised as all
Trichocaulon species were reclassified as Hoodia and Hoodia is no longer
considered part of the Ascepliadaceae. The complete botanical name Hoodia
gordonii (Masson) Sweet ex. Decne. was derived based on the discoveries of
7
several people. Hoodia pilifera was the first Hoodia species to be discovered by
Carl P. Thunberg and Francis Masson in 1774. Robert Gordon observed a Hoodia
species in 1779 and made a drawing of the plant which was published by Masson
as Stapelia gordonii Masson. Robert Sweet of England placed Stapelia gordonii
into a new genus in 1830. The genus was Hoodia, named after Mr Hood, a well
known succulent grower in Britain.
Finally, Hoodia gordonii was first validly
published by Joseph Decaisne in 1844 after the previous generic names were
declared invalid (Bruyns, 2005:92; Van Heerden, 2008:434-435).
H. gordonii is a spiny succulent that has rows of small thorns present along the
fleshy, grey-green to grey-brown stems. Only one stem is produced in the early
stages but as the plant matures as many as 50 individual branches are formed
from the common base. It is usually about as broad as it is tall and can attain a
height of up to 1 metre. The large flowers, borne on or near the terminal apex, are
usually flesh-coloured but more intense purple-red flowers are found in some
Namibian populations.
Variation in flower size is common (50-100 mm) with
flowers decreasing in size as the flowering period advances. The plant usually
flowers in August/September and the large number of flowers almost completely
obscure the stems. Pollination occurs as a result of the attraction of flies and
blowflies to the flowers due to their unpleasant carrion-like smell.
The seed
capsules, produced in October and November, resemble antelope or goat horns
and contain numerous flat, light brown seeds with silky seed hairs attached to one
end (Van Wyk and Wink, 2004:171; Bruyns, 2005:115,117, Olivier, 2005:S.a.).
Figure 2.1 shows H. gordonii in flower and Figure 2.2 the intact stem as well as
cross-sections of dried and freshly cut stems.
H. gordonii is widely distributed throughout South Africa and Namibia (Figure 2.3).
It grows in parts of the Western Cape, the north and northwestern regions of the
Northern Cape as far as Kimberley and parts of the southern Free State as well as
in southwestern Namibia. It has a wide tolerance of growing habitats and can
survive extreme heat (> 40 °C) as well as relatively low temperatures (-3 °C), but
is susceptible to frost and mainly grows in summer rainfall areas. It is found in a
variety of habitats such as dry sand, stony slopes or barren, flat areas (Bruyns,
2005:115; Olivier, 2005:S.a.).
8
FIGURE 2.1: Hoodia gordonii in flower (Photograph by AM Viljoen)
FIGURE 2.2: Hoodia gordonii cross-sections of dried and freshly-cut stems
(Photographs by AM Viljoen)
FIGURE 2.3: The natural distribution of H. gordonii in South Africa and Namibia
indicated in orange
9
2.2.2 Ethnopharmacology
Hoodia species are reported to have been consumed by the San people of South
Africa and Namibia as appetite and thirst suppressants during long hunting trips
and when food supplies were low (Glasl, 2009:300). Usually, a small piece of the
fresh stem is consumed after peeling to remove the thorns. Hoodia currorii, H.
flava, H. gordonii and H. pilifera have all been consumed fresh as appetite and
thirst suppressants and H. currori has been used to treat indigestion, hypertension,
diabetes and stomach ache while Hoodia officinalis subsp. officinalis was used to
treat pulmonary tuberculosis.
According to records H. pilifera (‘ghaap’) was
consumed more often as it has a cool and watery taste, while H. gordonii has a
persistent bitter taste and was considered inferior, emphasised by the disparaging
vernacular names such as ‘muishondghaap’ and ‘jakkalsghaap’ referring to it only
being fit for consumption by animals. However, it is said that the bitterness is less
prominent after good rains and the juicy stems would then be eaten raw or
cooked.
Adding to confusion, several species of stapeliads are referred to
vernacularly as ‘ghaap’ (Van Wyk and Gericke, 2000:70; Van Wyk and Wink,
2004:171; Van Heerden, 2008:345; Glasl, 2009:302).
In many papers on H. gordonii it is wrongly stated that it was also used as a cure
for abdominal cramps, diabetes, indigestion, hypertension and so forth, but these
applications are in fact attributed to other Hoodia species as mentioned previously.
The source quoted refers to Hoodia species in general and these uses as well as
the uses for H. gordonii are described in a paper by Van Heerden (2008:345).
From this detailed ethnopharmacological account, H. gordonii was also used for
the treatment of tuberculosis and the honey from the flowers could be used to treat
cancer (Foden, 2005:S.a; Van Heerden, 2008:345).
2.2.3 Commercialisation
The most important developments in the research and commercial development of
H. gordonii is depicted in Figure 2.4. The use of Hoodia species, especially H.
pilifera, by the San as a source of food and as a water substitute was recorded in
1932 and 1937. As a result of this the National Food Research Institute which is
part of the Council for Scientific and Industrial Research (CSIR) in South Africa
included Hoodia species in an investigation into edible wild plants in 1963. The
10
CSIR further investigated the appetite-suppressant activity of extracts of Hoodia
species making a breakthrough with the structure elucidation of the compound
called P57AS3 (commonly known as P57). The process to extract and synthesise
P57 was patented in South Africa in 1995. In 1998, this was followed by a patent
granted by the World Intellectual Property Organisation on pharmaceutical
compositions with appetite suppressant activity.
In the same year a licensing
agreement to further develop P57 was signed between the CSIR and Phytopharm.
Phytopharm, a British pharmaceutical company specialising in phytomedicines, in
turn sub-licensed the pharmaceutical company Pfizer for further development and
commercialisation. Due to a merger between Pfizer and Pharmacia in 2003, the
natureceuticals branch responsible for the development of P57 was closed (Van
Heerden, 2008:434; Glasl, 2009:301; Wynberg, Schroeder and Chennels,
2009:95). There were also unconfirmed reports that additional reasons for the
discontinuation included difficulty in synthesising the P57 molecule and that the
synthetic molecules were not as effective as the natural molecules.
FIGURE 2.4: Timeline of the most important developments in the research and
commercial development of H. gordonii
11
Despite wide media speculation on the safety of H. gordonii, no cases of adverse
effects have been confirmed except for one study reporting two cases, one of
acute hepatitis and another of anticholinergic syndrome for preparations
containing Hoodia and concomitant drugs. In 2004, a patent was licensed to the
company Unilever for the incorporation of H. gordonii extracts into food products to
produce functional foods. Despite having invested vast sums of money in this
project, including the development of an extraction facility, these plans were
abandoned in December 2008 due to safety and efficacy concerns. Unilever also
terminated all activities related to Hoodia in South Africa on 31 March 2009.
Phytopharm remains optimistic about the Hoodia programme and insists that it will
find other partners for further development of what should be a very lucrative
industry (Wynberg, Schroeder and Chennels, 2009:96-97).
Various pharmaceutical companies invested vast sums of money before
withdrawing from this project. In addition, many farmers invested in H. gordonii
crops during the “Hoodia boom” or uprise in the commercial interest in this plant.
Cultivation of the H. gordonii plant is not easy and it takes a long time to mature
(Van Heerden, 2008:435). Plants were initially grown from seed, very expensive
at the time, greenhouses had to be built, and watering systems were needed.
After all this the distribution of fake products led to a substantial decrease in the
price of H. gordonii raw material as described in the newspaper article depicted in
Figure 2.5. H. gordonii is cultivated for commercial purposes on large scale in
South Africa and Namibia (Figure 2.6) as well as other parts of the world, though
interest in continued cultivation has waned.
12
FIGURE 2.5: An article on H. gordonii published in the Sunday Times newspaper
on 20 September 2009
FIGURE 2.6: Photographs of commercially cultivated H. gordonii in greenhouse
tunnels in Namibia and South Africa and the processing of the raw material by
slicing (Photographs by AM Viljoen)
13
2.2.4 Intellectual property rights and benefit sharing issues
The San, more popularly known as ‘Bushmen’, are the oldest inhabitants of Africa
and possess a wealth of knowledge about local biodiversity in Botswana, Namibia,
South Africa and Angola. These hunter-gatherers used to live in small nomadic
groups covering a vast expanse of land but oppression, genocide and
dispossession has led to loss of land, culture and identity. Only a minority of the
San people have secured rights to live on their own land, while others work as
labourers on commercial game/cattle ranches or reside in government
resettlement villages. The common factor among all San is the hopeless poverty
they live in. The use of Hoodia by the San as a thirst and appetite suppressant is
undisputed.
Records of this knowledge resulted in subsequent research and
patents on Hoodia and licensing agreements for its commercialisation were
granted without the knowledge of the San who are the originators of this
knowledge (Wynberg, Schroeder and Chennels, 2009:89-94).
Until 2001,
development and commercialisation continued without acknowledgement of the
San leading them to launch a painstakingly long and hard challenge against the
CSIR on their Hoodia patent. This only occurred after the foreign media were
informed of this potential exploitation of the San people, depicted as poor and
emaciated, in stark contrast with images of rich obese Westerners.
This act
portrayed as biopiracy by large pharmaceutical companies resulted in an eagerly
followed high-profile law suit and forced negotiations concerning benefit-sharing
(Wynberg, Schroeder and Chennels, 2009:100-102). This case raised issues on
ownership of knowledge and resources.
Previously, renewable resources such as plants, animals and microorganisms
were regarded as common heritage of humankind. Any person could remove
these resources from nature and use them to develop commercial products.
However, the establishment of the Convention on Biological Diversity (CBD) in
1992 declared that biological resources fall under the sovereignty of the states.
The convention encompasses the conservation of biological diversity and the
sustainable use of its components as well as fair and equitable sharing of benefits.
Traditional knowledge is considered to be removed from the common heritage of
humankind and therefore is subject to formal prior informed consent and benefits
must be shared equitably (Wynberg, Schroeder and Chennels, 2009:11). The San
14
opted to share in the royalties rather than continue with an expensive law suit to
prevent use of their knowledge culminating in a mutual benefit agreement which
was finally signed in 2003. It was important to ensure that the agreement was
inclusive of all of the San people and would benefit all equally as the traditional
knowledge is extended into several countries (Wynberg, Schroeder and Chennels,
2009:102). According to the agreement between the CSIR, its licensees and the
South African San Council, the parties will share royalties from potential sales of
drugs or other products derived from Hoodia. The money, 8% of all payments that
the CSIR receives from licensees and 6% of all royalties after commercialisation,
will be paid into a Trust controlled by the San to be used to the benefit of the San
people (Glasl, 2009:302). However, the payment of these royalties is currently not
applicable since no products have yet been developed out of this agreement. The
Sunday Times newspaper of 20 September 2009 (Figure 2.6) describes how the
San are still waiting for their royalties.
It is clear that the commodification of
traditional knowledge is complex and the laws concerning intellectual property
rights are continuously evolving to adapt to issues relevant to each natural
resource.
2.2.5 Phytochemistry
The pioneering group of scientists who investigated Hoodia species, Van Heerden
et al. (2007:2545), isolated two steroidal glycosides named Compound 1 and 2
from H. gordonii and H. pilifera extracts. Compound 1 is referred to as P57AS3,
more commonly known as P57. Since then many new structural analogues have
been isolated from Hoodia gordonii: eleven oxypregnane glycosides – hoodigoside
A-K (Pawar et al., 2007:524); ten pregnane glycosides – hoodigoside L-U (Pawar,
Shukla and Khan, 2007:882-884); ten steroidal glycosides – gordonoside A-I, L
(Dall’Acqua and Innocenti, 2007:559); seven pregnane glycosides – hoodigoside
V-Z and hoodistanaloside A-B (Shukla et al., 2009:675); and Formula 7-8 and 12
(Abrahamse, Povey and Rees, 2007:4-5). The backbone chemical structures of
these compounds are the aglycones hoodigogenin A, calogenin, hoodistanal,
dehydrohoodistanal and isoramanone formed as a result of acid and/or enzymatic
hydrolysis. The structures of the compounds isolated from H. gordonii and the
respective aglycone of each analogue is depicted in Annexure 1.1. The discovery
of numerous new compounds provides the opportunity for research into their
15
biological activity and has also been used to develop new quality control methods
for H. gordonii products (Avula et al., 2008:722).
2.2.6 Biological activity
2.2.6.1
Anti-obesity activity
Scientific studies on the effect of H. gordonii and/or P57 on weight loss is scarce
because much of the necessary research has not been done but also partly due
the fact that some of the information is proprietary.
The first study that
investigated the mechanism of action of P57 was that of MacLean and Luo
(2004:1-11) who determined the effect of P57 (P57AS3) injected directly into the
third ventricle of the brain in rats. They found that intracerebroventricular (i.c.v)
injection of P57 led to a reduced food intake of 50-60% over the 24-h period
following injection. The duration of effect was about 24-48 h and dose-dependent.
In addition, pair-fed rats showed increased adenosine triphosphate (ATP) in
hypothalamic neurons compared to animals treated with the solvent only. As it
has been reported that intrahypothalamic 5ʹ adenosine monophosphate-activated
protein kinase (AMPK) may regulate food intake and that AMPK is regulated in
part by ATP, this central nervous system mechanism of action of P57 is plausible.
However, recently a paper was published which seems to refute these findings.
Pharmacokinetic studies after oral administration of P57 revealed that P57 did not
reach the brain indicating that its mechanism of action may not be due to
increased ATP in the hypothalamus (Madgula et al., 2010a:1585).
The first scientific reports of in vivo anti-obesity activity were by Tulp and Harbi
(2001:A404; 2002:A654) where food intake in both lean (moderate reduction) and
obese (marked reduction) rats were voluntarily reduced with a concomitant
reduction in body weight. The effective dose of Hoodia species was determined to
be 1.8-2.7 kg/kg of bodyweight (Tulp and Harbi, 2001:A404). In the follow-up
study, 3% (w/v) aqueous homogenate of dehydrated Hoodia plant material was
administered to lean and obese mice resulting in a decreased food intake within
48 h. The body weight of the obese rats, initially twice the weight of the lean rats,
decreased to near normal weights after two to three weeks of Hoodia feeding and
the body fat mass in lean and obese rats were decreased compared to rats fed a
normal diet (Tulp and Harbi, 2002:A654).
16
However, these studies were only
published as abstracts which severely limits the value in terms of interpretation of
the results. In addition, the administration of Hoodia species is indicated and not
H. gordonii specifically. In another study, P57 isolated from H. gordonii led to a
decreased food consumption and body mass over an 8-day period after
administration by oral gavage to rats in doses of 6.25-50 mg/kg. In comparison
with the known appetite-suppressant compound fenfluramine at 15 mg/kg/day,
P57 (30 mg/kg/day) caused a suppression in body weight while fenfluramine
administration resulted in a small decrease in food intake and an increase in body
weight, though less than in the control group (Van Heerden et al., 2007:2545;
2549-2553). H. gordonii has also been fed to broiler chickens to determine the
effect on productivity and carcass characteristics. Daily supplementation with an
H. gordonii meal (300 mg) had no effect of feed intake, digestibility and growth but
fat pad weights were reduced by 40%. Therefore H. gordonii supplementation
may have a beneficial effect on carcass quality (Mohlapo et al., 2009:1591), but
the mechanisms need to be further investigated.
Clinical studies in humans have apparently been done but remain unpublished
partly due to the proprietary nature of drug development research.
In human
studies undertaken by Phytopharm, 20 obese free-feeding volunteers were treated
with an extract of H. gordonii. Exercise was not required and it was not expected
of volunteers to follow any eating plan. The subjects who received the capsule
containing the extract reduced their daily calorie intake of their own accord, by
approximately 1000 calories per day.
The decreased food intake resulted in
weight loss of about 2 kg and a reduction in blood glucose and triglycerides (Holt
and Taylor, 2006a:108; Glasl, 2009:305).
The Phytopharm press release
reporting on this study has since been removed from their website. In another
paper on H. gordonii, the authors report an open-label observational study
performed by one of the authors. A specific product called Hoodia Supreme® said
to contain 400 mg of pure H. gordonii per capsule was supplied to eight obese
participants twice daily for a 4-week period.
Data was gathered by direct
questioning on eating habits as well as weight measurement. Weight loss (± 1-7
kg), reduction in appetite, a voluntary reduction in calorie intake (estimated 5001000 calories/day), a reduction in craving for carbohydrate-containing foods and a
mild energising effect was reported. In the same paper there was reference to a
17
study by Goldfarb, but the original source as mentioned in the references could not
be located on the internet.
It stated that 7 participants receiving DEX-L10®
containing 500 mg of H. gordonii for a 28-day period reported a median weight
loss of ± 5 kg with no alteration in dietary or daily activity habits (Holt and Taylor
2006b:103). A summary of the in vivo studies performed on H. gordonii and P57
is shown in Table 2.1.
TABLE 2.1: Summary of studies on the in vivo anti-obesity activity of H. gordonii
and P57
In vitro/In
vivo
Tested in:
Hoodia gordonii or P57
used and
administration
Effect(s)
In vivo
(animals)
Rats
Hoodia species crude
aqueous
homogenate/semipurified extracts (oral)
Reduced food intake, body
weight, gonadal fat pad weight
and blood glucose
Rats
P57 (oral gavage)
Reduction in food intake and
body weight
Rats
P57 (i.c.v injection)
Reduction in food intake
Chickens
H. gordonii meal (oral)
Reduction of fat pad weights
Obese
males (20)
Hoodia extract (oral)
Reduction in food intake, body
weight, blood glucose and
triglycerides*
Obese
subjects
(2 male, 6
female)
Hoodia Supreme®
capsules containing
Hoodia gordonii raw
material (oral)
Reduction in food intake,
appetite, body weight, craving
for carbohydrates and a mild
energising effect reported
Overweight
subjects (7)
DEX-L10® capsules
containing Hoodia
gordonii raw material
(oral)
Reduction in body weight and
calorie intake*
In vivo
(humans)
*
Could not be verified as the original source as cited could not be located
The information related to clinical studies is scarce and for most of these studies
the data are not currently accessible, raising even more questions about the use of
H. gordonii as an anti-obesity preparation. It is evident that proper clinical studies
in humans are needed to validate claimed anti-obesity effects. These intriguing
and promising results may have triggered the entry of several drug development
partners into agreements with Phytopharm, but despite the positive results the
18
question remains why two renowned pharmaceutical companies were unable to
publish data and abandoned what seems to be a potential goldmine.
2.2.6.2
Other biological activities
Hoodigosides A-K and P57 was evaluated for cytotoxic and antioxidant properties
in vitro. These compounds were found not to be cytotoxic and did not show any
inhibition of growth against a panel of cell lines including SK-MEL, KB, BT-549,
SKOV-3, VERO and LLC-PK1 at the highest concentration of 25 µg/ml.
No
antioxidant activity was recorded in HL-60 cells and intracellular reactive oxygen
species (ROS) generation was not inhibited (Pawar et al., 2007:533). In a US
patent application the gastroprotective effect of P57 and the effects on gastric acid
secretion and gastric motility were described.
Gastric damage due to oral
administration of aspirin was reduced by 91.5% for P57 and 93.2% for cimetidine
(a pharmaceutically registered gastric acid secretion inhibitor). Oral administration
of a spray dried extract at a dose of 50 mg/kg inhibited acid output by 50% and
gastric emptying by 26%. Intraduodenal administration of purified P57 at a dose of
10 mg/kg decreased acid output by 88% while subcutaneous administration
decreased acid output by 43% (Hakkinen, Horak and Maharaj, 2005:5-6). These
effects were promising but Madgula et al. (2010b:65) found 100% degradation of
P57 in simulated gastric fluid, which raised more questions about the actual active
molecule.
Antidiabetic activity has also been claimed, but ethnopharmacalogically it is H.
currori that has been used as an antidiabetic (Van Heerden, 2008:435). In 2006, a
patent was filed for antidiabetic properties from extracts of Hoodia. An acute (20
to 160 mg/kg) and chronic study (60 and 120 mg/kg) was conducted with P57
(Compound 1) and food and water intake, bodyweight, glucose, insulin and leptin
levels in male diabetic ZDF rats were assessed. In the chronic study the doses
were reduced to 60 and 30 mg/kg on day 7 due to concerns about the 50%
reduction in food intake. The results showed that P57 had positive effects on
bodyweight, normal glycaemia was maintained and water intake of diabetic rats
were reduced within 4 days to that of the control lean rats (Rubin, Bindra and
Cawthorne, 2006:11-14).
19
2.2.7 Toxicity
Data pertaining to the toxicity/safety of H. gordonii is scarce. In a five year study in
Italy, 233 spontaneous reports on adverse reactions to natural health products
were collected and analysed.
Anti-cholinergic syndrome was noted with one
preparation containing 100 mg H. gordonii raw material (Menniti-Ippolito et al.,
2008:629). However, it also contained 200 mg Ma huang (Ephedra sinica) raw
material which most likely caused this effect (Homoud, 2009:658).
Another
product containing Hoodia was associated with a case of acute hepatitis, but six
pharmaceutical preparations were taken concomitantly (Menniti-Ippolito et al.,
2008:269). Acute toxicity studies were described in a patent application by Van
Heerden et al. (2002:52-53). A plant extract administered orally to mice in doses
of 100 mg/kg up to 3028.5 mg/kg, revealed no clinical signs of toxicity. Doserelated reversible histopathological liver changes in the form of moderate cloudy
swelling and hydropic degeneration of hepatocytes was recorded for a dose of 200
mg/kg. The number of animals used in this study were small (n = 2 per dosage
group) but the fact that all of the groups except one showed liver changes on
histopathological examination warrants further investigation. During anti-obesity
activity experiments in congenic lean and obese rats, high quantities of H. gordonii
were orally administered and no adverse effects were noted (Tulp and Harbi,
2002:A654). No adverse effects were reported for the clinical study performed in
humans but it should be kept in mind that not much was reported at all due to the
proprietary nature and the occurrence of side effects is certainly not the type of
information a company developing a product would attract attention to.
Its
traditional use by the San for decades bodes well for safety, but is only applicable
to fresh H. gordonii and not extracts or molecules isolated from the plant as these
would be in concentrated form.
The scarcity of information in terms of scientific studies on the efficacy and safety
of H. gordonii, which is such a widely-consumed anti-obesity product, stimulated
an investigation into other plants used for obesity.
These plants are also
consumed by the general public despite the fact that safety and efficacy studies
are in most cases lacking. In addition, the safety of some of these products has
been called into question as side-effects and even deaths were reported
highlighting the need for proper clinical studies on all natural products.
20
2.3
PLANTS USED FOR THEIR ANTI-OBESITY PROPERTY
2.3.1 Introduction
Obesity is a serious health problem that has become one of the most common
health concerns of modern times and should be considered a global epidemic. In
2005 it was estimated that 1.6 billion adults were overweight globally with at least
400 million classified as obese. These numbers are projected to increase to 2.3
billion overweight and 700 million obese adults by 2015. The number of obese
teenagers and children in particular has increased dramatically in recent years
leading to heightened awareness of this global problem.
At least 20 million
children under the age of five years were overweight in 2005.
Obesity is
associated with and can lead to many disease conditions including type-2
diabetes,
cardiovascular
disease,
hypertension,
sleep
apnoea
and
cerebrovascular accidents amongst many others (WHO, 2006:S.a.).
Obesity occurs as a result of a higher energy intake than energy expenditure and
weight loss can only be achieved by a negative energy balance (Finer, 2002:718)
such as through a combination of exercise and dieting. The body mass index
(BMI = [weight in kg]/[height in m]2) is a clinical calculation based on the weight
and height of the patient that can be used for diagnosis of obesity. The normal
range for BMI is 19.8 – 26.0, while a range between 26.1 – 29.0 indicates an
overweight state and a value above 30 would diagnose a patient as obese (Mycek,
Harvey and Champe, 2000:474).
The commercial market for anti-obesity preparations is considerable due to public
awareness of the ill-effects of obesity and the general perception that being obese
is less than attractive. This multibillion dollar industry expands on a daily basis as
new ‘miracle cure’ products become available. Anti-obesity preparations of natural
origin are particularly appealing to consumers as the general perception is that if it
is natural it must be effective and safe.
Many plant products are currently
commercially available but unfortunately most of them have been poorly
researched, if at all. Not surprisingly, associations between consumption of herbal
products and instances of toxicity have been and are still being made.
For
example, severe neurologic and cardiovascular complications have been
associated with the consumption of ephedra alkaloids (Ephedra sinica) in
21
hundreds of cases. Ephedra-containing products were banned by the Food and
Drug Administration (FDA) in 2004 compelling manufacturers to develop “ephedrafree” products. Since then Citrus aurantium (bitter orange), which contains the
sympathomimetic agent synephrine, has been frequently used as an ephedra
replacement in supplements. Synephrine consumption has been associated with
tachycardia, exercise-induced syncope and myocardial infarction (Nykamp, Fackih
and Compton, 2004:812; Bouchard et al., 2005:541; Firenzuoli, Gori and Galapai,
2005:247). Conventional or allopathic medicines are subjected to rigorous testing
procedures before they are allowed to enter the commercial market. In the natural
products industry, the most basic scientific studies which should precede
commercial development are in general lacking. If nothing else, toxicity studies
should at least be conducted. In addition, medical practitioners should recognise
the extensive use of natural products and include this information in patient history
files as this would ensure that more information especially in terms of drug
interactions is obtained. It is imperative that these plants and herbal products be
extensively investigated in terms of quality, efficacy and especially safety in order
to validate their widespread consumption.
2.3.2 Mechanisms of action of anti-obesity preparations
Weight loss can only occur if energy consumption is less than energy expenditure,
i.e. if a negative energy balance exists.
The obvious approach of exercise
combined with dieting has a relatively low success rate and therefore alternative
forms of obesity treatment including surgery and medications have been
developed (Goodman et al., 1992:217). A substance causing a reduction in food
intake through satiety mechanisms increases energy expenditure and efficiency,
resulting in dietary compensation.
Sympathetic nervous system stimulation
causes β 1-, β 2-, β 3- and α -adrenoreceptor activation resulting in decreased
appetite.
Compounds that selectively stimulate β
3-receptors,
cause the
thermogenic effects of sympathomimetic agents but do not exhibit the side effects
associated with general stimulation such as increased blood pressure, increased
heart rate (β1), increased smooth muscle contraction and tremors (β2) (Finer,
2002:734; Ohkoshi et al., 2007:1257; Arbo et al., 2008:2770; Arbo et al.,
2009:114). Activation of 3ʹ-5ʹ-cyclic adenosine monophospate (cAMP) promotes
the breakdown of stored fat, increases the basal metabolic rate, increases
22
utilisation of body fat and regulates the thermogenic response to food (WesterterpPlantenga, Lejeune and Kovacs, 2004:1196). Acetyl coenzyme A carboxylase
(ACC) inhibitors moderately retard fat accumulation as it affects fatty acid
biosynthesis (Watanebe, Kawabata and Kasai, 1999:489). Activation of the 5ʹ
adenosine monophosphate-activated protein kinase (AMPK) pathway results in
increased fatty acid oxidation, inhibition of cholesterol synthesis, lipogenesis and
inhibition of adipocyte lipolysis and lipogenesis.
Increased expression of
uncoupling proteins UCP1, UCP2, and UCP3 results in increased thermogenesis
and this process is induced by substances like caffeine which results in the
dissipation of energy in the form of heat (Matsuda et al., 2003:2408; WesterterpPlantenga, Lejeune and Kovacs, 2004:1196-1197; Chen et al., 2009:785;792;
Murase et al., 2009:78).
Conventional pharmaceutical anti-obesity drugs are few and are classified either
as centrally acting or peripherally acting.
Centrally-acting products include
sympathomimetics with central nervous system (CNS) stimulant properties such
as
phentermine
(Duromine®),
phendimetrazine
(Obex-LA®,
Obesan
X®),
amfepramone (Tenuate Dospan®), norpseudoephedrine (Nobese No.1®) and
agents that specifically inhibit re-uptake of noradrenaline and serotonin in the CNS
such as sibutramine (Reductil®).
Peripherally acting products such as orlistat
(Xenical®) inhibits lipase in the gastrointestinal tract leading to decreased dietary
fat absorption (Gibbon, 2008:69). Not all of these preparations are registered in all
countries, as the final decision lies with the regulatory authorities in each
respective
country.
Amfepramone,
phentermine,
phendimetrazine
and
norpseudoephedrine (less potent) exhibits amphetamine-like effects including
increased energy and wakefulness, the main reasons for its abuse potential, and
decreased appetite.
Due to extensive abuse, norpseudoephedrine containing
preparations were recently converted from over-the-counter to prescription-only
medicines in South Africa.
Sibutramine inhibits the re-uptake of released
serotonin and noradrenaline from hypothalamic neurons causing a predominant
effect on satiety and causes thermogenesis. It has no effects on dopaminergic
neurons.
Adverse effects include nausea, insomnia, dry mouth, rhinitis and
constipation and in some individuals increases heart rate and blood pressure.
Patient deaths have been attributed to sibutramine (Finer, 2002:727) and
23
ephedrine use. Orlistat inhibits pancreatic and gastric lipases which functions to
catalyse the hydrolysis of ingested fats, resulting in a reduction of dietary fat
absorption and consequently high faecal fat excretion. This is bound to have a
considerable effect as between 35 and 45% of energy intake in western diets is
attributed to dietary fat.
Unfortunately, malabsorption of fat leads to rather
uncomfortable side effects such as flatus with discharge, oil spotting from the
rectum, faecal incontinence, faecal urgency, loose or liquid stools and
malabsorption of fat-soluble vitamins (Finer, 2002:730; Gibbon, 2008:69).
However, only 1% of patients experience side-effects severe enough to withdraw
from treatment, while 25% of patients may report such side-effects. It has also
been suggested that these side-effects may aid in modification of the patient’s
lifestyle by avoiding high-fat meals to avoid the side-effects and thereby it may
contribute to healthier choices in food intake (Finer, 2002:730).
Many new mechanisms to induce weight loss have been proposed as a result of
ongoing research and new molecules that utilise ‘old’ pathways are still
discovered. The focus of many research groups has been on discovering new
effective pancreatic lipase inhibitors from plants with fewer side effects. This may
be due to the fact that it is not a complicated study and large numbers of plants
can easily be screened. In these studies, effects are compared to mainly Orlistat®
(positive control) with few studies reporting on associated side-effects.
What
makes Orlistat® and other pancreatic lipase inhibitors desirable is that they are
acting peripherally thereby decreasing chances of systemic side effects. Similar to
pancreatic lipase, α-amylases function to digest carbohydrates into easily
absorbable sugars. Inhibition of these enzymes therefore results in decreased
absorption of sugars which have been shown to have a beneficial effect in
diabetes and most probably obesity.
Leptin is a protein hormone that is encoded by an obesity gene and regulates
appetite and energy expenditure.
Circulating levels of leptin are generally
proportional to the amount of body fat (decreased body fat = decreased leptin).
However, in leptin gene deficient individuals injections of leptin decreases food
intake and increases thermogenesis and physical activity.
Peroxisome
proliferator-activated receptors (PPARs) and CCAAT (cytidine-cytidine-adenosine24
adenosine-thymidine) -enhancer-binding proteins (or C/EBPs) are important
adipogenic transcription factors involved in the inhibition of differentiation into
adipocytes. Inhibition of differentiation of preadipocytes into adipocytes is another
common mechanism exhibited by many of the plants used as anti-obesity
preparations. Hormone-sensitive lipase is a rate-limiting enzyme catalysing the
lipolysis of triacylglycerol and diacylglycerol following the phosphorylations of the
enzyme by protein kinase A. Peptide YY is released predominantly from the lower
intestine following a meal and decreases appetite in both lean and obese subjects.
Ghrelin is predominantly released from the stomach and its level increases in
times of hunger and decrease immediately post-prandially.
Administration of
ghrelin to rats and humans increases appetite (Murray et al., 2008:747; Pang,
Choi and Park, 2008:183-184; Shi et al., 2009:72;75; Smeets and WesterterpPlantenga, 2009:230).
Adiponectin levels are inversely correlated to body fat
percentage in adults (Kang et al., 2007:4389;4395). Cholesystokinin A (CCK A)
acts as a physiological satiety factor and activation of its receptors peripherally,
slows gastric emptying and induces satiety.
Fatty acid synthase (FAS), an
important enzyme in the fatty acid synthesis pathway, catalyses the reductive
synthesis of long-chain fatty acid from acetyl-coenzyme A (CoA) and malonyl-CoA.
FAS inhibition leads to inhibition of feeding and weight loss. Down-regulation of
sterol-regulatory-element-binding protein (SREBP) decreases sterol biosynthesis
(Watanabe, Kawabata and Kasai, 1999:489; Chen et al., 2009:790-792).
It is clear that many targets for anti-obesity agents exist but the only effect that
should qualify a product as an anti-obesity preparation is its positive effect on
weight loss.
The treatment can only be considered successful if it induces
sufficient weight loss from the initial body weight (5-10%), prevents further weight
gain and allows long term maintenance of the weight loss once it is achieved
(Alarcon-Aguilar, 2007:69).
2.3.3 Other plants popularly used as anti-obesity agents
Many plants are being consumed for their anti-obesity properties. Table 2.2 lists
the botanical names, family, vernacular names, plant parts used, mechanisms of
action, in vitro or in vivo studies, toxicity and anti-obesity compounds isolated for
63 plants that have been investigated scientifically for their anti-obesity properties.
25
TABLE 2.2: Summary of the plants investigated for their anti-obesity properties in alphabetical order by species name
Species name
Common
name
Plant parts
used
Proposed mechanism of
action
In
vitro
In
vivo
anim*
In
vivo
hum#
Toxicity
Active anti-obesity
compounds isolated
References
Acanthopanax
senticosus (Rupr. et
Maxim.) Harms.
Siberian
ginseng
Fruit, cell
cultured
herb extract
Pancreatic lipase inhibitor
Yes
Yes
No
Unknown,
expect similar
to A.
sessiliflorus
Silphioside F,
copteroside B,
hederagenin 3-O-β-Dglucuronopyranoside 6ʹO-methyl ester,
gypsogenin 3-O-β-Dglucuronide
Cha, Rhee and Heo,
2004:422-428; Li et al.,
2007a:1087-1089
Wu jia
Leaves
Yes
Yes
No
Not significant
Chiisanoside, 11deoxyisochiisanoside,
isochiisanoside,
sessiloside
Yoshizumi et al., 2006:335341; Yoshizumi et al.,
2008:1126-1128
Japanese
horsechestnut
Seeds
Pancreatic lipase inhibitor,
suppresses blood
triacylglycerol level,
suppresses increase in body
weight, increases
triacylglycerol level in feaces
Yes
Yes
No
Unknown
Escin Ia, Escin Ib, Escin
IIa, Escin IIb
Van Wyk and Wink,
2004:34; Kimura et al.,
2006:1657-1665; Hu et al.,
2008:12-15; Kimura et al.,
2008:4783-4788
Lesser
galangal
Rhizomes
Yes
Yes
No
Unknown
3-methylethergalangin,
5-hydroxy-7-(4ʹ-hydroxy3ʹ-methoxyphenyl)-1phenyl-3-heptanone
Shin, Han and Kim,
2003:854-856; Shin et al.,
2004:138-140; Van Wyk and
Wink, 2004:43
Peanut
Nuts, shells
(peanut shell), lipolysis of 3T3L1 adipocytes (peanut shell),
increases satiety, increase in
energy efficiency and
expenditure, dietary
compensation
Yes
Yes
Yes
None
observed
(peanut shell)
and none
known
(peanuts)
No
Coelho et al., 2006:585591; Moreno et al.,
2006a:2797-2802; Higgs,
2007:353-356; Mattes, KrisEtherton and Foster,
2008:1741S-1744S
Araliaceae
Acanthopanax
sessiflorus (Rupr. et
Maxim) Seem.
Araliaceae
26
Aesculus turbinata
Blume
Hippocastanaceae
Alpinia officinarum
Hance
Zingiberaceae
Arachis hypogaea L.
Fabaceae
26
Species name
Common
name
Plant parts
used
action
In
vitro
In
vivo
anim*
In
vivo
hum#
Toxicity
Active anti-obesity
compounds isolated
References
Camellia sinensis
(L.) Kuntze
Tea
Leaves
Stimulates sympathetic
nervous system,
thermogenesis, inhibits gastric
and pancreatic lipase,
increase fat oxidation,
suppresses leptin, increase
triacylglycerol excretion,
decrease adipocyte cell size,
suppresses food intake,
suppress adipocyte
differentation, increase
expression of genes involved
in fatty acid synthesis and
oxidation, decrease lipoprotein
lipase, decrease hormonesensitive lipase, decrease
UCP-2, activation of AMPK,
increase adiponectin, increase
ghrelin
Yes
Yes
Yes
Hepatotoxicity
Epigallocatechin gallate
Van Wyk and Wink,
2004:75; Han et al.,
1999:98-104; Sayama et al.,
2000:481-484; Han et al.,
2001:1459-1464; Chantre
and Lairon, 2002:3-8; Lin,
Della-Fera and Baile,
2005:982-989; WesterterpPlantenga, Lejeune and
Kovacs, 2004:1195-1203;
Auvichayapat et al.,
2008:486-491; Hsu et al.,
2008:363-369; Sarma et al.,
2008:469-482; Chen et al.,
2009:784-792; Kapetanovic
et al., 2009:28-36; Lambert
et al., 2010:409-415
Caper
Fruits
Unknown
No
Yes
No
Unknown
No
Inocencio et al., 2000:70-73;
Eddouks, Lemhadri and
Michel, 2005:345-349;
Lemhadri et al., 2007:106109
Red pepper
Fruits
Thermogenesis, increase
energy expenditure, increase
secretion of catecholamines,
enhance expression of the
adiponectin gene, activation of
AMPK, reduce food intake,
increase satiety, decrease
ghrelin levels, down-regulates
expression of UCP1 and
UCP2, suppress fatty acid
Yes
Yes
Yes
Not significant
Capsaicin, capsiate,
dihydrocapsiate, 9oxooctadeca-10,12dienoic acids
Watanabe, Kawabata and
Kasai, 1999:489-492; Kanki
et al., 2003:1337-1342;
Masuda et al., 2003:24082415; Chanda et al., 2005:
66-75; Hwang et al.,
2005:694-698; WesterterpPlantenga, Smeets and
Lejeune, 2005:682-688;
Kang et al., 2007:4389-
Theaceae
27
Capparis spinosa L.
Capparaceae
Capsicum annuum
L.
Solanaceae
27
Species name
Common
name
Plant parts
used
action
In
vitro
In
vivo
anim*
In
vivo
hum#
Toxicity
Active anti-obesity
compounds isolated
biosynthesis (ACC inhibitor)
Caralluma fimbriata
Wall.
References
4395; Smeets and
Westerterp-Plantenga,
2009:229-234
Caralluma
Stems
Unknown
No
No
Yes
Unknown
No
Kuriyan et al., 2007:338-343
Nomame
herba
Aerial parts,
fruits
Yes
Yes
No
Unknown
Proanthocyanidin, flavan
dimers ((2S)-3ʹ,4ʹ,7trihydroxyflavan-(4αà8)catechin)
Hatano et al., 1997:893-899;
Yamamoto et al., 2000:758763
Khat
Leaves
Sympathomimetic effect,
decrease leptin levels,
decrease blood triacylglycerol
level
Yes
Yes
Yes
Liver, urinary,
reproductive
Cathinone, cathine
Al-Habori et al., 2002:209216; Van Wyk and Wink,
2004:84; Al-Hebshi and
Skaug, 2005:299-305; AlDubai, Al-Habori and AlGeiry, 2006:632-635; AlZubairi et al., 2008:298-302;
Gibbon, 2008:401;
Mahmood and Lindequist,
2008: 271-277; Murray et
al., 2008:747-750
Roman
chamomile
Aerial parts
Unknown
No
Yes
No
Unknown
No
Lemhadri et al., 2007:106109
Bitter orange
Fruits
Increased sympathomimetic
activity, stimulates lipolysis,
increased thermogenesis
Yes
Yes
Yes
Sympathomimetic effects
Synephrine, octopamine
Calapai et al., 1999:586592; Colker et al., 1999:145152; Fugh-Berman and
Apocynaceae
Cassia mimosoides
L. var. nomame
Makino
Fabaceae
28
Catha edulis (Vahl)
Endl.
Celastraceae
Chamaemelum
nobile (L.) All.
Asteraceae
Citrus aurantium L.
Rutaceae
28
Species name
Common
name
Plant parts
used
action
In
vitro
In
vivo
anim*
In
vivo
hum#
Toxicity
Active anti-obesity
compounds isolated
References
Meyers, 2004:698-703;
Marchei et al., 2006:14681472; Arbo et al.,
2008:2770-2774; Arbo et al.,
2009:114-117
Citrus unshiu
Marcow.
Satsuma
mandarin
orange
Fruit (peel,
segment
wall extract,
membranes)
Inhibits lipase activity,
stimulates lipolysis
Yes
Yes
No
Unknown
(synephrine
content
suggest
similar to C.
aurantium)
Hesperidin, pectin
Kawaguchi et al., 1997:102103; Tsujita et al., 2003:340345; Tsujita and Takaku,
2007:547-551; Edashige,
Murakami and Tsujita,
2008:409-414; Tsujita and
Takaku, 2008:102
Coleus
Roots
Activates cAMP
Yes
Yes
Yes
Not significant
Forskolin
Lieberman, 2004:330-332;
Godard, Johnson and
Richmond, 2005:1335-1342;
Han et al., 2005a:449;
Henderson et al., 2005:5462; Canová et al., 2006:200209
Turmeric
Rhizomes
Inhibits adipokine-induced
angiogenesis, suppresses
3T3-L1 differentiation,
increases oxidation and fatty
acid esterification, downregulates the expression of
key transcription factors
Yes
Yes
No
High dose
CNS
stimulation,
haematological changes
Curcumin,
demethoxycurcumin,
bisdemethoxycurcu-min
Qureshi, Shah and Ageel,
1992:124-126; Asai and
Miyazawa, 2001:2932-2934;
Van Wyk and Wink,
2004:118; Ejaz et al.,
2009:919-924
Sweet tea
tree
Leaves
Yes
Yes
No
Unknown
No
Kurihara et al., 2003:383385; Zhang et al.,
2010:1491-1496
Rutaceae
Coleus forskohlii
Briq.
29
Lamiaceae
Curcuma longa L.
Zingiberaceae
Cyclocarya paliurus
(Batal.) Iljinsk.
Juglandaceae
29
Species name
Common
name
Plant parts
used
action
In
vitro
In
vivo
anim*
In
vivo
hum#
Toxicity
Active anti-obesity
compounds isolated
References
Dioscorea nipponica
Makino
Wild yam
Roots/
rhizomes
suppresses blood
triacylglycerol level
Yes
Yes
No
Not significant
except at high
doses
(anorexia,
asthenia,
weight loss,
piloerection,
ataxia,
syncope)
Dioscin, diosgenin,
gracillin, prosapogening
A, prosapogenin C
Kwon et al., 2003:14511456; Son et al., 2007:30633069; Qin et al., 2009:543550;
Ma Huang
Aerial parts
Increase release of
norepinephrine and inhibition
of re-uptake (stimulates α- and
β-adrenergic receptors )
Yes
Yes
Yes
Toxic effects
known,
sympathomimetic
stimulation
Ephedrine
Fontanarosa, Rennie and
DeAngelis, 2003:1568-1570;
Shekelle et al., 2003:15371545; Van Wyk and Wink,
2004:134; Morton,
2005:242-247; Kim et al.,
2008b:128-137
Bee bee
tree, Evodia
Fruits
Increase lipolysis in BAT,
enhances heat dissipation,
reduced food intake, decrease
expression of orexigenic
peptides (NPY, AgRP),
increase leptin levels, inhibit
adipogenesis in 3T3-L1 cells,
inhibit differentiation in human
white preadipocytes
Yes
Yes
Yes
None reported
Evodiamine, rutecarpine
Kobayashi et al., 2001:628632; Kim et al., 2008b:128137; Yang, 2008:1317-1321;
Hu et al., 2010:259-267; Kim
et al., 2009b:437-442; Shi et
al., 2009:71-77; Wang,
Wang and Yamashita,
2009:3655-3659
Goat's rue
Aerial parts
Activates AMPK in H4IIE rat
hepatoma, HEK293 human
kidney cells, 3T3-L1
adipocytes and L6 myotubes,
down-regulation of fatty acid
synthase and sterolregulatory-element-binding
protein (SREBP)
Yes
Yes
No
Liver, lung
effects
Galegine, 1-(4
chlorobenzyl) guanidine
hemisulfate
Palit, Furman and Gray,
1999:1313-1318; Van Wyk
and Wink, 2004:150;
Mooney et al., 2008:16691676; Rasekh et al.,
2008:21-26; Coxon et al.,
2009:3457-3463
Dioscoreaceae
Ephedra sinica
Stapf.
Ephedraceae
30
Evodia rutaecarpa
(Juss.) Benth.
Rutaceae
Galega officinalis L.
Fabaceae
30
Species name
Common
name
Plant parts
used
action
In
vitro
In
vivo
anim*
In
vivo
hum#
Toxicity
Active anti-obesity
compounds isolated
References
Garcinia cambogia
Desr.
Brindal berry
Fruit rind
Inhibit ATP-citrate lyase
(suppress fatty acid
synthesis), suppresses
appetite (decreased food
intake), decrease leptin levels
Yes
Yes
Yes
Testicular
toxicity in rats
(disputed)
(-)-Hydroxycitric acid
Mattes and Bormann,
2000:87-93; Van Loon et al.,
2000:1445-1449; Preuss et
al., 2004:171-179; Soni et
al., 2004:1513-1527;
Burdock, 2005:1683-1684;
Saito et al., 2005:411-418
Gardenia
Fruit
suppresses serum triglyceride
and cholesterol level
Yes
Yes
No
Genotoxic
(genipin,
gardenia
yellow),
hepatotoxic
(genipin)
Crocin, crocetin
Ozaki et al., 2002:16031609; Lee et al., 2005:21062110; Sheng et al.,
2006:116-122
Liquorice
Roots
Blocks 11β-hydroxysteroid
dehydrogenase type 1
(topical), down-regulation of
genes for fatty acid synthesis
(mice)
Yes
Yes
Yes
Hypermineralo
corticoidism,
pseudoaldosteronisms (due
to glycyrrhizic
acid), not
relevant to
LFO, blood
abnormalities
and
anticoagulation (rats LFO)
Glycyrrhetinic acid
(topical)
Armanini et al., 2005:538542; Isbrukcer and Burdock,
2006:167-188; Tominaga et
al., 2006:672-682;
Kamisoyama et al.,
2008:3225-3231; Nakagawa
et al., 2008:2349-2357;
Tominaga et al., 2009:169177
Chinese
liquorice
Roots
Inhibits pancreatic lipase
Yes
No
No
Unknown
Licochalcone A
Won et al., 2007:1046-1049
Clusiaceae
Gardenia
jasminoides Ellis
Rubiaceae
Glycyrrhiza glabra L.
31
Fabaceae
Glycyrrhiza uralensis
Fisch.
Fabaceae
31
Species name
Common
name
Plant parts
used
action
In
vitro
In
vivo
anim*
In
vivo
hum#
Toxicity
Active anti-obesity
compounds isolated
References
Griffonia simplicifolia
(Vahl ex DC)
Griffonia
Seeds
Increases serotonin and
circulating leptin levels
Yes
Yes
Yes
Serotonin
syndrome in
rats at high
doses
5-HTP
Yamada, Sugimoto and
Ujikawa, 1999:49-51; Amer
et al., 2004:137-142; Turner,
Loftis and Blackwell,
2006:325-335
Gurmar
Leaves
Decrease fat digestibility,
decreased food and water
intake (1 study)
Yes
Yes
Yes
(in
combi
nation)
None reported
Gymnemic acids,
gymnemate
Shigematsu et al., 2001:713716; Ogawa et al., 2004:8;
Preuss et al., 2004:171-179;
Kanetkar, Singhal and
Kamat, 2007:77-81; Luo et
al., 2007:93-97
Jiaogulan
immortality
herb
Aerial parts
Yes
No
No
None
observed, mild
adverse
effects
Dammarane-type
triterpenoids (20S, 23R)3β-,20βdihydroxydamma-24dien-21-oic acid 21,23lactone, (20S, 24S)20,24epoxydammarane3β,12β,25-triol
Attawish et al., 2004:539550; Razmovski-Naumovski
et al., 2005:197-214; Bai et
al., 2010:306-309
Roselle
Calyces,
dried flowers
Inhibition of expression of
adipogenic transcription
factors C/EBPα and PPARγ
through PI3K and MAPK
pathway - inhibition of
differentiation into adipocytes
Yes
Yes
No
Testicular
toxicity reduced
sperm count
and
spermatogenesis
No
Akindahunsi and Olaleye,
2003:161-164; Kim et al.,
2003:499-504; Orisakwe,
Husaini and Afonne,
2004:295-297; Van Wyk and
Wink, 2004:170; AlarconAguilar et al., 2007:66-70;
Kim et al., 2007:260-266;
Sowemimo et al., 2007:427432; Carvajal-Zarrabal et al.,
2009:in press
Fabaceae
Gymnema sylvestre
R.Br.
Asclepiadaceae
Gynostemma
pentaphyllum
(Thunb.) Makino
32
Cucurbitaceae
Hibiscus sabdariffa
L.
Malvaceae
32
Species name
Common
name
Plant parts
used
action
In
vitro
In
vivo
anim*
In
vivo
hum#
Toxicity
Active anti-obesity
compounds isolated
References
Hoodia gordonii
(Masson) Sweet ex.
Decne.
Hoodia
Stems
Increase hypothalamic ATP
Yes
Yes
Yes
Not significant
P57
MacLean and Luo, 2004: 111; Holt and Taylor
2006a:104-113; Van
Heerden et al., 2007: 25452553; Glasl, 2009:300-305;
Mohlapo et al., 2009:15911595
Yerba mate
Leaves
Thermogenic, decreases food
intake, reduces size of
adipocytes, reduces plasma
leptin, up-regulate PPARγ2,
SREBP1, FAS and HMR,
increases UCP2 and UCP3
expression, increases AMPK
phosporylation, inhibits
pancreatic lipase
Yes
Yes
Yes
Genotoxic,
mutagenic
Caffeine
Andersen and Fogh,
2001:243-249; Ruxton,
2004:25-28; Van Wyk and
Wink, 2004:179; Heck and
De Meija, 2007:138-139;
Pang, Choi and Park,
2008:178-184; Martins et al.,
2010:42-47
Manchurian
walnut
Fruits
Yes
Yes
No
Unknown
1,4,8trihydroxynaphthalene1-O-β-d-[6ʹ-O-(3ʹ,4ʹ,5ʹtrihydroxybenzoyl)]gluco
pyranoside, αhydrojuglone-4glucoside
Han et al., 2007:184-186; Li
et al., 2007b:846-851
Chinese
juniper
Heartwood,
berries
Enhanced gene transcription,
elevated AMPK protein
expression, phosphorylation in
visecral adipose tissue
Yes
Yes
No
Unknown
(-)-cedrol
Sánchez de Medina et al.,
1994:197-200; Van Wyk and
Wink 2004:184; Ju et al.,
2008:110-114; Kim et al.,
2008c:1415-1420;
Sciencelab 2008:S.a.; Park,
Jung and Kim, 2009:513
Banaba
Leaves
Inhibits differentiation in 3T3L1 adipocytes, inhibits
Yes
Yes
No
Kidney and
liver effects,
Tannic acid
Glick and Joslyn, 1970:509514; Suzuki et al., 1999:791-
Apocynaceae
Ilex paraguariensis
A. St.-Hil.
Aquifoliaceae
33
Juglans
mandshurica Maxim.
Juglandaceae
Juniperus chinensis
L.
Cuppressaceae
Lagerstroemia
33
Species name
Common
name
Plant parts
used
speciosa (L.) Pers.
action
In
vitro
In
vivo
anim*
In
vivo
hum#
expression of PPAR-γ2 and
PPAR- γ
795; Liu et al., 2001:22422246; Liu et al., 2005:165171; Sigma-Aldrich,
2006:S.a.; Klein et al.,
2007:401-406
Bark, leaves
Inhibits pancreatic and
lipoprotein lipase, inhibits
lipolysis of 3T3-L1
preadipocytes, increases
feacal fat excretion, downregulates obesity-related
genes in the liver and
epididymal fat, inhibits αglucosidase, inhibits αamylase
Yes
Yes
No
Not significant
Mangiferin
Prasanth, Padmaja and
Samiulla, 2001:179-181;
Prasanth et al., 2001:686688; González et al.,
2007:2526-2532;
Yoshikawa, 2002:18191824; Moreno et al.,
2006b:21-26; Birari and
Bhutani, 2007:885; Rodeiro
et al., 2007:2506-2511;
Wauthoz et al., 2007:112117
Bitter melon
Fruits (juice)
Increases sympathomimetic
activity (increase plasma
epinephrine and
norepinephrine), increases
lipolysis, decreases blood and
white adipose tissue leptin,
decreases resistin mRNA
expression, activates AMPK
pathway
Yes
Yes
Yes
Abortefacient,
reduced
fertility, high
dose toxicity
Momordicoside S,
karaviloside XI
Chen, Chan and Li,
2003:1088-1093; Virdi et al.,
2003:107-111; Grover and
Yadav, 2004:123-129; Chen
and Li, 2005:747-753; Shih,
Lin and Lin, 2008:134-143;
Tan et al., 2008:263-273;
Kasbia, Arnason and
Imbeault, 2009:127-132
Chinese
bayberry
Bark
Inhibits lipase, suppresses
increase in blood triglyceride
levels
Yes
Yes
No
None reported
Myricitrin, myricetin,
gallic acid
Kobayashi et al., 2008b:289292
34
Cucurbitaceae
Myrica rubra Sieb. et
Zucc.
References
Mango
Anacardiaceae
Momordica
charantia L.
Active anti-obesity
compounds isolated
mutagenic,
carcinogenic
(tannic acid)
Lythraceae
Mangifera indica L.
Toxicity
Myricaceae
34
Species name
Common
name
Plant parts
used
action
In
vitro
In
vivo
anim*
In
vivo
hum#
Toxicity
Active anti-obesity
compounds isolated
References
Nelumbo nucifera
Gaertn.
Lotus
Leaves
Inhibit α-amylase and
pancreatic lipase, increase
lipolytic activity 3T3-L1
adipocytes, increase UCP3
(thermogenesis)
Yes
Yes
Yes
Yes (brine
shrimp)
Flavanoids (quercetin 3O-α-arabinopyranosyl(1α2)-βgalactopyranoside, (+)catechin, hyperoside,
isoquercitrin, astragalin)
Van Wyk and Wink,
2004:213; Huralikuppi,
Christopher and Stephen,
2006:217-223; Ono et al.,
2006:238-243; Rai et al.,
2006; Ohkoshi et al.,
2007:1255-1258
Black cumin
Seeds
Unknown
No
No
Yes
High dose
thymoquinone
No
Zaoui et al., 2002:69-73;
Gilani, Jabeen and Khan,
2004:441-447; Le et al.,
2004:251-258; Van Wyk and
Wink, 2004:216; Najmi et al.,
2008:11-13; Khader,
Bresgen and Eckl,
2009:129-132; Qidwai et al.,
2009:639-644; Sultan, Butt
and Anjum, 2009:2768-2775
Asian
ginseng
Roots,
berries
Inhibit pancreatic lipase,
decreased hypothalamic
expression of orexigenic
peptide Y, anorexigenic
cholecystokinin increased,
reduced leptin levels, inhibit
lipid accumulation in 3T3-L1
adipocytes, increased PPARγ, and lipoprotein lipase,
activates AMPK
Yes
Yes
No
Safe
Ginsenosides (Rg3,
Rh2), protopanaxadiol
saponins,
protopanaxatriol
saponins
Carabin, Burdock and
Chatzidakis, 2000:293-301;
Attele et al., 2002:18511857; Coon and Ernst,
2002:323-344; Xie et al.,
2002:254-258; Dey et al.,
600-605; Van Wyk and
Wink, 2004:224; Hwang et
al., 2007:1002-1007; Karu,
Reifen and Kerem,
2007:2824-2827; Hwang et
al., 2008:262-266; Kim et al.,
2008a:78-85; Mollah et al.,
2008:220-225; Kim et al.,
2009c:596-601
Nelumbonaceae
Nigella sativa L.
Ranunculaceae
35
Panax ginseng C.A.
Mey.
Araliaceae
35
Species name
Common
name
Plant parts
used
action
In
vitro
In
vivo
anim*
In
vivo
hum#
Toxicity
Active anti-obesity
compounds isolated
References
Panax japonicus
C.A. Mey.
Japanese
ginseng
Rhizomes
decrease plasma
triacylglycerol
Yes
Yes
No
Unknown
Chikusetsusaponins II,
IV, and V and 28deglucosylchikuketsusa
ponins IV and V
Han et al., 2005b:9-17;
Gibbon, 2008:69
American
ginseng
Stems,
leaves
decrease plasma
triacylglycerol
Yes
Yes
No
Unknown
Ginsenosides
R[b.sub.1], R[b.sub.2],
Rc, Rd
Van Wyk and Wink,
2004:224; Liu et al.,
2008:S.a.
Guarana
Seeds
Unknown (thermogenesis)
No
Yes
No
Considered
safe, mild
adverse
effects
Methylxanthines
Espinola et al., 1997:223229; Mattei et al., 1998:111116; Van Wyk and Wink,
2004:228; Lima et al.,
2005:1019-1027; Ruxton
and Gardner, 2005:111-125
Yohimbe
Bark
α2-adrenergic antagonist
(enhance lipid mobilisation)
No
No
Yes
CNS
overstimulation,
irritability and
anxiety attacks
Yohimbine
Tam, Worcel and Wyllie,
2001:215-239; Van Wyk and
Wink, 2004:229; Ostojic,
2006:289-299
White kidney
bean
Beans
Inhibits α-amylase,
phytohemoagglutinin-mediated
(lectin) release of
cholecystokinin and glucagonlike peptides
Yes
Yes
Yes
Toxic in high
doses
Phytohemagglutinin
Pusztai et al., 1998:213-220;
Pittler and Ernst, 2004:529536; Van Wyk and Wink,
2004:237; Carai et al.,
2009:145-153
Indian
psyllium
Seeds,
husks
Lower food intake, increased
fullness, reduces ghrelin gene
expression, lowers plasma
leptin levels
Yes
Yes
Yes
Minor adverse
effects
No
Turnbull and Thomas,
1995:338-342; Van Wyk and
Wink, 2004:245; Galisteo et
al., 2005:2399-2403; Singh,
2007:1-10; Wang et al.,
Araliaceae
Panax
quinquefolium L.
Araliaceae
Paullinia cupana
Mart. var. sorbilis
Mart.
36
Sapindaceae
Pausinystalia
yohimbe (K.
Schum.) Beille
Rubiaceae
Phaseolus vulgaris
L.
Fabaceae
Plantago ovata
Forssk.
Plantaginaceae
36
Species name
Common
name
Plant parts
used
action
In
vitro
In
vivo
anim*
In
vivo
hum#
Toxicity
Active anti-obesity
compounds isolated
References
2007:1635-1641; Gibbon,
2008:56; Rodríguez-Morán,
Guerrero-Romero and
Lazcano-Burciaga,
1998:273-277
Platycodon
grandiflorum (Jacq.)
A. DC
Platycodon
root
Roots
Inhibits pancreatic lipase,
reduce plasma and liver
triacylglycerol, inhibits 3T3-L1
differentiation, suppressed
FABP expression, inhibit
hepatic FAS, reduced food
intake (saponin fractions)
Yes
Yes
No
Unknown
Platycodin A, platycodin
C, deapioplatycodin D,
platycodin D (34.8%),
deapioplatycodin D3,
platycodin V, platycodin
D2, SK1
Han et al., 2000:2760-2764;
Han et al., 2002:2241-2244;
Van Wyk and Wink,
2004:247; Xu et al.,
2005:180-184; Zhao et al.,
2005:983-990; Park, Yoon
and Ahn, 2007:3493-3498;
Jeong et al., 2008:245-251;
Kim et al., 2009a:629-635;
Shin et al., 2009:47-53
Japanese
bitter orange
Fruits
Increases intestinal transit
time
Yes
Yes
No
Not significant
No
Shim et al., 2009:294-299
Roseroot,
Golden root
Rhizomes
Inhibits lipase, decreases lipid
absorption
Yes
Yes
No
None reported
Rhodionin, rhodiosin
Kobayashi et al.,
2008a:1716-1719;
Blomkvist, Taube and
Larhammar, 2009:11871189
Rosemary
Leaves
Inhibit adipocyte
differentiation, increase
intracellular GSH, induce
phase2 enzymes (involved in
GSH metabolism)
Yes
Yes
No
None known
Carnosic acid, carnosol
Van Wyk and Wink,
2004:276; Takahashi et al.,
2009:549-554
Campanulaceae
37
Poncirus trifoliata
(L.) Raf.
Rutaceae
Rhodiola rosea L.
Crassulaceae
Rosmarinus
officinalis L.
Lamiaceae
37
Species name
Common
name
Plant parts
used
action
In
vitro
In
vivo
anim*
In
vivo
hum#
Toxicity
Active anti-obesity
compounds isolated
References
Salacia reticulata
Wight
Kotahla
himbutu
Roots,
stems
Inhibit pancreatic lipase,
lipoprotein lipase, lipolytic
effects, suppresses white
adipose tissue accumulation,
reduce size of the adipocytes,
decrease triacylglycerol levels,
decrease plasma adiponectin,
leptin and free fatty acids
Yes
Yes
No
Low birth
weight, greater
postimplantation
losses, low
survival ratio
and viability
index
(-)-Epigallocatechin, (-)epicatechin-(4βà8)-(-)4ʹ-Omethylepigallocatechin,
(-)-epicatechin, 3β,22βdihydroxyolean-12-en29-oic acid, mangiferin,
(-)-4ʹ-Omethylepigallocatechin,
maytenfolic acid
Shimoda et al., 2001:527;
Yoshikawa et al.,
2002:1819-1824;
Ratnasooriya, Jayakody and
Premakumara, 2003:931935; Jayawardena et al.,
2005:215-217; Kishino et al.,
2006:433-438; Im et al.,
2008:645-651
Chinese
willow
Leaves
Inhibit α-amylase, enhances
norepinephrine-induced
lipolysis in fat cells, inhibits
palmitic acid uptake into small
intestinal brush border
membranes
Yes
Yes
No
Unknown
Apigenin-7-O-dglucoside, luteolin-7-Od-glucoside, chrysoeriol7-O-d-glucoside
Han et al., 2003a:11881194; Han et al.,
2003b:1195-1198; Li et al.,
2008:1530-1536
Sage
Leaves
Inhibit pancreatic lipase, inhibit
triglyceride increase
Yes
Yes
No
Neurotoxicity
of essential oil
due to thujone
Carnosic acid, carnosol,
royleanonic acid, 7methoxyrosmanol,
oleanolic acid
Ninomiya et al., 2004:19431946; Van Wyk and Wink,
2004:283
Scabiosa
Whole plant
Yes
No
No
Unknown
Scabiosaponins E, F, G,
I, hookerosides A, B,
and prosapogenin 1b
Zheng et al., 2004:604-613;
Birari and Bhutani,
2007:881; Guo et al.,
2009:1167-1174;
Mahabala,
country
mallow
Leaves
Unknown
No
Yes
Yes
Unknown
No
Patel et al., 2009:233237;Thounaojam et al.,
2009:413-429
Clove
Dried
unopened
flower buds
decrease plasma
triacylglycerol
Yes
Yes
No
Organ
damage in rats
Not characterized
Van Wyk and Wink,
2004:315; Kim et al.,
2005:84-87; Yu, Shirai and
Celestraceae
Salix matsudana
Koidzumi
Salicaceae
38
Salvia officinalis L.
Lamiaceae
Scabiosa tschiliensis
Grun.
Dipsacaceae
Sida rhomboidea.
Roxb
Malvaceae
Syzygium
aromaticum (L.)
Merr. et Perry
38
Species name
Common
name
Plant parts
used
action
In
vitro
In
vivo
anim*
In
vivo
hum#
Toxicity
Active anti-obesity
compounds isolated
Myrtaceae
Taraxacum officinale
Weber ex Wigg.
Suzuki, 2007:155-161;
Agbaje, Adeneye and
Daramola, 2009:241-254
Dandelion
Roots,
leaves
Yes
Yes
No
Unknown
No
Rácz-kotilla, Rácz and
Solomon, 1974:212-217;
Van Wyk and Wink,
2004:318; Zhang et al.,
2008:200-203; RodriguezFragoso et al., 2008:130
Triumphetta
Leaves
Unknown
No
Yes
No
Unknown
No
Ngondi et al., 2006:200
Fenugreek
Seeds, fiber
Inhibit lipid absorption,
decreased expression of
adipogenic factors, increased
satiety (reduced energy
intake)
Yes
Yes
Yes
CNS effects at
high doses in
mice, none
observed
during human
studies
4-hydroxyisoleucine,
galactomannan
Petit et al., 1995:674-680;
Muralidhara et al., 1999;
831-837; Abdel-Barry and
Al-Hakiem, 2000:65-68; Van
Wyk and Wink, 2004:325;
Handa et al., 2005:11861187; Mathern et al.,
2009:1543-1548;
Vijayakumar et al.,
2010:667-674
Grape vine
Seeds
Inhibits pancreatic lipase,
inhibits lipoprotein lipase,
inhibits lipolysis, increase
adiponectin, reduce
leptinaemia, reduce energy
intake, reduce PPARγ and
C/EBPα, inhibits adipocyte
differentiation through cell
Yes
Yes
Yes
None
observed
Resveratrol, stilbesterol,
ampelopsin A, vitisin B,
3,4ʹ,5-trimethoxy
stilbene, vitisin A
Bentivegna and Whitney,
2002:1731-1743; Yamakoshi
et al., 2002:599-607;
Moreno et al., 2003: 876879; Van Wyk and Wink,
2004:344;Vogels, Nijs and
Westerterp-Plantenga,
2004:667-673; Décordé et
Asteraceae
Triumphetta
cordifolia A. Rich
References
Tiliaceae
39
Trigonella foenumgraecum L.
Fabaceae
Vitis vinifera L.
Vitaceae
39
Species name
Common
name
Plant parts
used
action
In
vitro
In
vivo
anim*
In
vivo
hum#
Toxicity
Active anti-obesity
compounds isolated
cycle arrest
Wasabia japonica
(Miq.) Matsum.
Leaves,
rhizomes
and stalks
Suppress differentiation of
preadipocytes, suppress
increase in glycerol-3phosphate dehydrogenase
(GDPH) activity and
triglyceride (TG) accumulation
Yes
No
No
Unknown
No
Hou et al., 2000:195-199;
Morimitsu et al., 2000:125133; Ogawa et al.,
2009:239-244
Japanese
ginger
Rhizomes
Suppress differentiation of
preadipocytes, suppress
increase in glycerol-3phosphate dehydrogenase
(GDPH) activity and
triglyceride (TG) accumulation
Yes
Yes
No
Unknown
No
Iwashita, Yamaki and
Tshushida, 2001:164-169;
Miyoshi et al., 2003:113-118
Ginger
Rhizomes
Reduced expression of
retinoid binding protein (RBP)
and mRNA in the liver and
visceral fat
Yes
Yes
No
None known
No
Weidner and Sigwart,
2000:513-519; Iwashita,
Yamaki and Tshushida,
2001:164-169; Van Wyk and
Wink, 2004:349; WesterterpPlantenga et al., 2006:8591; Matsuda et al.,
2009:903-907
Zingiberaceae
40
Zingiber officinale
Roscoe
al., 2008:659-666; Kim et al.,
2008d:108-113
Wasabi
Brassicaceae
Zingiber mioga
Roscoe
Zingeberaceae
* in vivo animal studies
#
References
in vivo human studies
40
This investigation showed that several plants including Cassia mimosoides,
Dioscorea nipponica, Glycyrrhiza glabra and Panax ginseng possess promising
anti-obesity activity with the potential to be developed into anti-obesity agents or
blueprints for anti-obesity compounds, but only after additional research and
clinical trials have been conducted. It was also revealed that the toxicity of an
astonishing 35% of the plants included in the table has not been investigated while
the toxicity of most of the other plants has not been investigated comprehensively.
Consider the following example: Tea is the most consumed beverage worldwide
and hence it is not surprising that tea products would automatically be considered
safe. However, instances of hepatotoxicity have been linked to consumption of
green tea extracts (Sarma et al., 2008:477). These extracts are prepared
differently from ordinary tea and are therefore higher in tea catechins which may
cause adverse effects. Marked mortality was observed when purified green tea
extract was administered to Beagle dogs. The highest dose administered had to
be decreased from 1000 mg/kg/day to 800 mg/kg/day whereafter unscheduled
mortality occurred in 16 out of 24 dogs. The cause could not be determined due to
extensive organ and system involvement and the severity of the toxicity. In a
follow-up study at a lower dosage (200 mg/kg) the dogs presented with digestive
disturbances, mild liver damage, moderate to progressive anaemia, mild
disturbances in haematopoiesis and transient ocular symptoms (Kapetanovic et
al., 2009:30-35).
After a safety review by the United States Pharmacopoeia
Dietary Supplement Information Expert Committee it was concluded that 27 out of
34 reports pertaining to liver damage were probably caused by the consumption of
green tea products (Sarma et al., 2008:469-470). In addition to safety, the quality
and efficacy of these plants were also neglected. Another concern is that much of
the research conducted has been of an in vitro nature. Clinical trials in humans
have not been done for an alarming 62% of these plants, yet they are freely sold
and consumed daily.
For most of the plants the molecule(s) responsible for the anti-obesity activity has
not been isolated. Isolation of single compounds is important in basic science to
determine the mechanism by which plants exert their effect. Effects may then be
attributed to a single compound and efforts can then be made to substitute certain
functional groups on a molecule either to increase its activity or to ameliorate side
41
effects. On the other hand, ease of interpretation should not detract from the way
in which these plants or plant combinations were used traditionally as it is wellknown that different compounds in one plant extract act on different target sites
resulting in synergistic interactions.
One such example is a study on the
antimicrobial activity of T. camphoratus, where the combination of the volatile and
non-volatile fractions exhibited increased efficacy compared to the individual
fractions (Van Vuuren and Viljoen, 2009:505). An example of a synergistic plant
combination is the increased antimicrobial activity of extracts of Salvia
chameleaegnea and Leonotis leonurus in combination against Gram-positive
organisms compared to the extracts in isolation (Kamatou et al., 2006:634). A
combination strategy seems particularly suited to obesity as body weight is
controlled by many factors. However, this does not imply that many plants should
be randomly combined into one anti-obesity formulation with the hope that this will
have a synergistic anti-obesity effect as the combination may exhibited more side
effects and interactions.
2.4
CONCLUSIONS
The statistics on obesity prove that it should be considered a threat to the general
health of the world population. This threat leads to exploitation of the public by
manufacturing companies focused on profit with little consideration towards the
overall effect that these products may have on the consumer.
This is further
compounded by the fact that there is a general misconception by the public that
‘green is good’. These products are deemed natural or derived from nature and
are therefore considered harmless despite the fact that more and more evidence
to the contrary is emerging. Manufacturers of herbal supplements often claim that
their products are potent and effective with no or few adverse effects. If these
supplements possess such potent biological activity, they should be considered to
be conventional drugs and treated as such in terms of quality control and proof of
effectivity (Fontanarosa, Rennie and DeAngelis, 2003:1569).
At this stage,
research into side effects and toxicity seems to take place only once serious harm
or even death of patients has occurred. For the treatment of obesity and the
treatment of any disease the risk-benefit ratio must be considered. Future studies
should therefore focus on pursuing promising anti-obesity activity displayed by
42
many plants on in vivo models other than rats or mice to eventually progress to in
vivo human studies.
It should also be kept in mind that caution should be exercised when clinical
results are interpreted.
In some studies, dietary modification and exercise
regimens were introduced at the same time as the administration of the plant or
product to be tested. This means that weight loss cannot be ascribed to the action
of the plant or product only as dietary modification and exercise will have a
beneficial effect on weight loss. It is evident that the investigation into plants used
for their anti-obesity activity raised numerous concerns and questions which by
nature can only be answered through more research.
H. gordonii is a prime example of most of the abovementioned statements. It is
unscrupulously marketed as a miracle drug to cure obesity and is one of the bestsellers, but it is also frequently adulterated with other plant material and
substances which may have deleterious effects on the consumer. In contrast to
other plants, the correct route of research and development of a drug was followed
to a large extent for H. gordonii. However, the aspiration of developing P57 into a
pharmaceutical anti-obesity preparation has not been realised due to numerous
factors and H. gordonii products are still freely sold. The in vivo studies that have
been performed to prove anti-obesity activity and numerous other aspects that
should be investigated prior to consumption are insufficient.
Ideally, all plant
products should be regulated like conventional or allopathic medicines as they
should conform to the cardinal rule of ‘safety, efficacy and quality’. In addition,
basic research to substantiate the worldwide use of medicinal plants must
underpin and precede commercial development.
43
2.5
REFERENCES
ABDEL-BARRY, J.A., AL-HAKIEM, M.H.H. 2000. Acute intraperitoneal and oral
toxicity of the leaf glycosidic extract of Trigonella foenum-gracum in mice. Journal
of Ethnopharmacology, 70:65-68.
ABRAHAMSE, S.L., POVEY, K.J., REES, D.D. 2007. Appetite suppressant
compositions. US Patent 2007/0207227.
AGBAJE, E.O., ADENEYE, A.A., DARAMOLA, A.O. 2009. Biochemical and
toxicological studies of aqueous extract of Syzigium aromaticum (L.) MERR. &
PERRY (Myrtaceae) in rodents. The African Journal of Traditional Complementary
and Alternative Medicines, 6(3):241-254.
AKINDAHUNSI, A.A., OLALEYE, M.T. 2003. Toxicological investigation of
aqueous-methanolic extract of the calyces of Hibiscus sabdariffa L. Journal of
Ethnopharmacology, 89:161-164.
ALARCON-AGUILAR, F.J., ZAMILPA, A., PEREZ-GARCIA, M.D., ALMANZAPEREZ, J.C., ROMERO-NUÑEZ, CAMPOS-SEPULVEDA, E.A., VAZQUEZCARRILLO, L.I., ROMAN-RAMOS, R. 2007. Effect of Hibiscus sabdariffa on
obesity in MSG mice. Journal of Ethnopharmacology, 114:66-71.
AL-DUBAI, W., AL-HABORI, M., AL-GEIRY, A. 2006. Human khat (Catha edulis)
chewers have elevated plasma leptin and nonesterified fatty acids. Nutrition
Research, 26:632-636.
AL-HABORI, M., AL-AGHBARI, A., AL-MAMARY, M., BAKER, M. 2002.
Toxicological evaluation of Catha edulis leaves: a long term feeding experiment in
animals. Journal of Ethnopharmacology, 83:209-217.
AL-HEBSHI, N.N., SKAUG, N. 2005. Khat (Catha edulis) – an updated review.
Addiction Biology, 10:299-307.
AL-ZUBAIRI, A., ISMAIL, P., PEI PEI, C., RAHMAT, A. 2008. Genotoxic effect of
Catha edulis (khat) crude extract after sub-chronic administration in rats.
Environmental Toxicology and Pharmacology, 25:298-303.
AMER, A., BREU, J., MCDERMOTT, J., WURTMAN, R.J., MAHER, T.J. 2004. 5Hydroxy-L-tryptophan suppresses food intake in food-deprived and stressed rats.
Pharmacology Biochemistry and Behaviour, 77:137-143.
ANDERSEN, T., FOGH, J. 2001. Weight loss and delayed gastric emptying
following a South American herbal preparation in overweight patients. Journal of
Human Nutrition and Dietetics, 14(3):243-250.
ARBO, M.D., LARTENTIS, E.R., LINCK, V.M., ABOY, A.L., PIMENTEL, A.L.,
HENRIQUES, A.T., DALLEGRAVE, E., GARCIA, S.C., LEAL, M.B., LIMBERGER,
R.P. 2008. Concentrations of p-synephrine in fruits and leaves of Citrus species
(Rutaceae) and the acute toxicity testing of Citrus aurantium extract and psynephrine. Food and Chemical Toxicology, 46:2770-2775.
44
ARBO, M.D., SCHMITT, G.C., LIMBERGER, M.F., CHARAO M.F., MORO, A.M.,
RIBEIRO, G.L., DALLEGRAVE, E., GARCIA, S.C., LEAL, M.B., LIMBERGER,
R.P. 2009. Subchronic toxicity of Citrus aurantium L. (Rutaceae) extract and psynephrine in mice. Regulatory Toxicology and Pharmacology, 54:114-117.
ARMANINI, D., NACAMULLI, D., FRANCINI-PESENTI, F., BATTAGIN, G.,
RAGAZZI, E., FIORE, C. 2005. Glycyrrhetinic acid, the active principle of licorice,
can reduce the thickness of subcutaneous fat through topical application. Steroids,
70:538-542.
ASAI, A., MIYAZAWA, T. 2001. Dietary curcuminoids prevent high-fat diet-induced
lipid accumulation in rat liver and epididymal adipose tissue. The Journal of
Nutrition, 131:2932-2935.
ATTAWISH, A., CHIVAPAT, S., PHADUNGPAT, S., BANSIDDHI, J.,
TECHADAMRONGSIN, Y., MITRIJIT, O., CHAORAI, B., CHAVALITTUMRONG,
P. 2004. Chronic toxicity of Gynostemma pentaphyllum. Fitoterapia, 75:539-551.
ATTELE, A.S., ZHOU, Y.-P., XIE, J.-T., WU, J.A., ZHANG, L., DEY, L. PUGH, W.,
RUE,, P.A., POLONSKY, K.S., YUAN, C.-S. 2002. Antidiabetic effects of Panax
ginseng berry extract and the identification of an effective component. Diabetes,
51:1851-1858.
AUVICHAYAPAT, P., PRAPOCHANUNG, M., TUNKAMNERDTHAI, O.,
SRIPANIDKULCHAI, B.-O., AUVICHAYAPAT, N., THINKHAMROP, B.,
KUNHASARA, S., WONGPRATOOM, S., SINAWAT, S., HOGPRAPAS, P. 2008.
Effectiveness of green tea on weight reduction in obese Thais: A randomized,
controlled trial. Physiology and Behaviour, 93:486-491.
AVULA, B., WANG, Y.-H., PAWAR, R.S., SHUKLA, Y.J., SMILLIE, T.J KHAN, I.A.
2008. A rapid method for chemical fingerprint analysis of Hoodia species, related
genera, and dietary supplements using UPLC-UV-MS. Journal of Pharmaceutical
and Biomedical Analysis, 48:722-731.
BAI, M.-S., GAO, J.-M, FAN, C., YANG, S.-X., ZHANG, G., ZHENG, C.-D. 2010.
Bioactive dammarane-type triterpenoids derived from the acid hydrosylate of
Gynostemma pentaphyllum saponins. Food Chemistry, 119:306-310.
BENTIVEGNA, S.S., WHITNEY, K.M. 2002. Subchronic 3-month oral toxicity
study of grape seed and grape skin extracts. Food and Chemical Toxicology,
40:1731-1743.
BIRARI R.B., BHUTANI, K.K. 2007. Pancreatic lipase inhibitors from natural
sources: unexplored potential. Drug Discovery Today, 12(19/20):879-889.
BLOMKVIST, J., TAUBE, A., LARHAMMAR, D. 2009. Perspective on roseroot
(Rhodiola rosea) studies. Planta Medica, 75:1187-1190.
BOUCHARD, N.C., HOWLAND, M.A., GRELLER, H.A., HOFFMAN, R.S.,
NELSON, L.S. 2005. Ischemic stroke associated with use of an ephedra-free
45
dietary supplement containing synephrine. Mayo Clinic Proceedings, 80(4):541545.
BRUYNS, P.V. 2005. Stapeliads of Southern Africa and Madagascar volume 1.
Pretoria: Umdaus Press.
BURDOCK, G. 2005. Letter to the editor: Garcinia cambogia toxicity is misleading.
Food and Chemical Toxicology, 43:1683-1684.
CALAPAI, G., FIRENZUOLI, F., SAITTA, A., SQUADRITO F., ARLOTTA, M.R.,
COSTANTINO, G., INFERRERA, G. 1999. Antiobesity and cardiovascular toxic
effects of Citrus aurantium extracts in the rat: a preliminary report. Fitoterapia,
70:586-592.
CANOVÁ, N.K., LINCOVÁ, D., KMONÍČKOVÁ, E., KAMENÍKOVÁ, L, FARGHALI,
H. 2006. Nitric oxide production from rat adipocytes is modulated by β 3-adrenergic
receptor agonists and is involved in a cyclic AMP-dependent lipolysis in
adipocytes. Nitric Oxide, 14:200-211.
CARABIN, I.G., BURDOCK, G.A., CHATZIDAKIS, C. 2000. Safety assessment of
Panax ginseng. International Journal of Toxicology, 19(4):293-301.
CARAI, M.A.M., FANTINI, N., LOI, B., COLOMBO, G., RIVA, A., MORAZZONI, P.
2009. Potential efficacy of preparations derived from Phaseolus vulgaris in the
control of appetite, energy intake, and carbohydrate metabolism. Diabetes
Metabolic Syndrome and Obesity: Targets and Therapy, 2:145-153.
CARVAJAL-ZARRABAL, O., HAYWARD-JONES, P.M., ORTA-FLORES, Z.,
NOLASCO-HIPÓLITO, C., BARRADAS-DERMITZ, D.M., AGUILAR-USCANGA,
M.G., PEDROZA-HERNÁNDEZ, M.F. 2009. Effect of Hibiscus sabdariffa L., dried
calyx ethanol extract on fat absorption-excretion, and body weight implication in
rats.
Journal
of
Biomedicine
and
Biotechnology,
in
press:doi:
10.1155/2009/394592.
CHA, Y.-S., RHEE, S.-J., HEO, Y.-R. 2004. Acanthopanax senticosus extract
prepared from cultured cells decreases adiposity and obesity indices in C57BL/6J
mice fed a high fat diet. Journal of Medicinal Food, 7(4):422-429.
CHANDA, S., MOULD, A., ESMAIL, A., BLEY, K. 2005. Toxicity studies with pure
trans-capsaicin delivered to dogs via intravenous administration. Regulatory
Toxicology and Pharmacology, 43:66-75.
CHANTRE, P., LAIRON, D. 2002. Recent findings of green tea extract AR25
(Exolise) and its activity for the treatment of obesity. Phytomedicine, 9:3-8.
CHEN, N., BEZZINA, R., HINCH, E., LEWANDOWSKI, P.A., CAMERON-SMITH,
D., MATHAI, M.L., JOIS, M., SINCLAIR, A.J., BEGG, D.P., WARK, J.D.,
WEISINGER, H.S., WEISINGER, R.S. 2009. Green tea, black tea, and
epigallocatechin modify body composition, improve glucose tolerance, and
46
differentially alter metabolic gene expression in rats fed a high-fat diet. Nutrition
Research, 29:784-793.
CHEN, Q., CHAN, L.L.Y., LI, E.T.S. 2003. Bitter melon (Momordica charantia)
reduces adiposity, lowers serum insulin and normalizes glucose tolerance in rats
fed a high fat diet. The Journal of Nutrition, 133:1088-1093.
CHEN, Q., LI, E.T.S. 2005. Reduced adiposity in bitter melon (Momordica
charantia) fed rats is associated with lower tissue triglyceride and higher plasma
catecholamines. British Journal of Nutrition, 93:747-754.
COELHO, S.B., LOPES DE SALES, R., IYER, S.S., BRESSAN, J., COSTA,
N.M.B, LOKKO, P., MATTES, R. 2006. Effects of peanut oil load on energy
expenditure, body composition, lipid profile, and appetite in lean and overweight
adults. Nutrition, 22:585-592.
COLKER, C.M., KALMAN, D.S., TORINA G.C., PERLIS, T., STREET, C. 1999.
Effects of Citrus aurantium extract, caffeine, and St. John’s Wort on body fat loss,
lipid levels, and mood states in overweight healthy adults. Current Therapeutic
Research, 60(3):145-153.
COON, J.T., ERNST, E. 2002. Panax ginseng: a systematic review of adverse
effects and drug interactions. Drug Safety, 25(5):323-344.
COXON, G.D., FURMAN, B.L., HARVEY, A.L., MCTAVISH, J., MOONEY, M.H.,
ARASTOO, M., KENNEDY, A.R., TETTEY, J.M., WAIGH, R.D. 2009.
Benzylguanidines and other galegine analogues inducing weight loss in mice.
Journal of Medicinal Chemistry, 52(11):3457-3463.
DÉCORDÉ, K., TEISSÉDRE, P.-L., SUTRA, T., VENTURA, E., CRISTOL, J.-P.,
ROUANET, J.-M. 2008. Chardonnay grape seed procyanidin extract
supplementation prevents high-fat diet-induced obesity in hamsters by improving
adipokine imbalance and oxidative stress markers. Molecular Nutrition and Food
Research, 53(5):659-666.
DALL’ACQUA, S., INNOCENTI, G. 2007. Steroidal glycosides from Hoodia
gordonii. Steroids, 72:559-568.
DEY, L., XIE, J.T., WANG, A., WU, J., MALECKAR, S.A., YAN, C.-S. 2003. Antihyperglycemic effects of ginseng: comparison between root and berry.
Phytomedicine, 10:600-605.
EDASHIGE, Y., MURAKAMI, N., TSUJITA, T. 2008. Inhibitory effects of pectin
from the segment membrane of citrus fruits on lipase activity. Journal of Nutritional
Science and Vitaminology, 54:409-415.
EDDOUKS, M., LEMHADRI, A., MICHEL, J.-B. Hypolipidemic activity of aqueous
extract of Capparis spinosa L. in normal and diabetic rats. Journal of
47
EJAZ, A., WU, D., KWAN, P., MEYDANI, M. 2009. Curcumin inhibits adipogenesis
in 3T3-L1 adipocytes and angiogenesis and obesity in C57/BL mice. The Journal
of Nutrition, 139(5):919-925.
ESPINOLA, E.B., DIAS, R.F., MATTEI, R., CARLINI, E.A. 1997. Pharmacological
activity of guarana (Paullinia cupana Mart.) in laboratory animals. Journal of
FINER, N. 2002. Pharmacotherapy of obesity. Best Practice and Research Clinical
Endocrinology and Metabolism, 16(4):717-742.
FIRENZUOLI, F., GORI, L., GALAPAI, C. 2005. Adverse reaction to an adrenergic
herbal extract (Citrus aurantium). Phytomedicine, 12:247-248.
FODEN, W. 2005. Information document on trade in Hoodia gordonii and other
Hoodia species. Available from: http://www.plantzafrica.com/planthij/hoodia.htm
[Accessed: 29/26/2010].
FONTANAROSA, P.B., RENNIE, D., DEANGELIS, C.D. 2003. The need for
regulation of dietary supplements – lessons from ephedra. Journal of the American
Medical Association, 289(12):1568-1570.
FUGH-BERMAN, A., MYERS, A. 2004. Citrus aurantium, an ingredient of dietary
supplements marketed for weight loss: current status of clinical and basic
research. Experimental Biology and Medicine, 299:698-704.
GALISTEO, M., SÁNCHEZ, M., VERA, R., GONZÁLEZ, M., ANGUERA, A.,
DUARTE, J., ZARZUELO, A. 2005. A diet supplemented with husks of Plantago
ovata reduces the development of endothelial dysfunction, hypertension and
obesity by affecting adiponectin and TNF-α in obese Zucker rats. The Journal of
Nutrition, 135:2399-2404.
GIBBON, C.J. (ed.). 2008. South African Medicines Formulary 8th ed. Cape Town:
Health and Medical Publishing group.
GILANI, A.H., JABEEN, Q. KHAN, M.A.U. 2004. A review of medicinal uses and
pharmacological activities of Nigella sativa. Pakistan Journal of Biological
Sciences, 7(4):441-451.
Schweizerische Zeitschrift für GanzheitsMedizin, 21(6):300-306.
GLICK, Z., JOSLYN, M.A. 1970. Food intake depression and other metabolic
effects of tannic acid in the rat. The Journal of Nutrition, 100:509-515.
GODARD, M.P., JOHNSON, B.A., RICHMOND, S.R. 2005. Body composition and
hormonal adaptations associated with forskolin consumption in overweight and
obese men. Obesity Research, 13(8):1335-1343.
48
GOODMAN, A.G., RALL, T.W., NIES, A.S., TAYLOR, P. (eds). 1992. Goodman
and Gilman’s: The pharmacological basis of therapeutics 8th ed vol 1. New York:
McGraw-Hill.
GONZÁLEZ, J.E., RODRÍGUEZ, M.D., RODEIRO, I., MORFFI, J., GUERRA, E.,
LEAL, F., GARCÍA, H., GOICOCHEA, E., GUERRERO, S., GARRIDO, G.,
DELGADO, R., NUÑEZ-SELLES, A.J. 2007. Lack of in vivo embryotoxic and
genotoxic activities of orally administered stem bark aqueous extract of Mangifera
indica L. (Vimang®). Food and Chemical Toxicology, 45:2526-2532.
GROVER, J.K., YADAV, S.P. 2004. Pharmacological actions and potential uses of
Momordica charantia: a review. Journal of Ethnopharmacology, 93:123-132.
GUO, T., LIU, Q., WANG, P., ZHANG, L., ZHANG, W., LI, Y. 2009. Facile
synthesis of three bidesmosidic oleanolic acid saponins with strong inhibitory
activity on pancreatic lipase. Carbohydrate Research, 344:1167-1174.
HAKKINEN, J., HORAK, R.M., MAHARAJ, V. 2005. Gastric acid secretion. US
Patent 2005/0079233 A1.
HAN, L.-K., TAKAKU, T., LI, J., KIMURA, Y., OKUDA, H. 1999. Anti-obesity action
of oolong tea. International Journal of Obesity, 23:98-105.
HAN, L-K., XU, B.-J., KIMURA, Y., ZHENG, Y.-N., OKUDA, H. 2000. Platycodi
radix affects lipid metabolism in mice with high fat diet-induced obesity. The
Journal of Nutrition, 130:2760-2764.
HAN, L.-K., KIMURA, Y., KAWASHIMA, M., TAKAKU, T., TANIYAMA, T.,
HAYASHI, T., ZHENG, Y.N., OKUDA, H. 2001. Anti-obesity effects in rodents of
dietary teasaponin, a lipase inhibitor. International Journal of Obesity,
25(10):1459-1464.
HAN, L.-K., ZHENG, Y.-N., XU, B.-J., OKUDA, H., KIMURA, Y. 2002. Saponins
from Platycodi radix ameliorate high fat diet-induced obesity in mice. The Journal
of Nutrition, 132:2241-2245.
HAN, L.-K., SUMIYOSHI, M., ZHANG, J., LIU, M.-X., ZHANG, X.-F., ZHENG, Y.N., OKUDA, H., KIMURA, Y. 2003a. Anti-obesity action of Salix matsudana leaves
(Part 1). Anti-obesity action by polyphenols of Salix matsudana in high fat-diet
treated rodent animals. Phytotherapy Research, 17(10):1188-1194.
HAN, L.-K., SUMIYOSHI, M., ZHENG, Y.-N., OKUDA, H., KIMURA, Y. 2003b.
Anti-obesity action of Salix matsudana leaves (Part 2). Isolation of anti-obesity
effectors from polyphenol fractions of Salix matsudana. Phytotherapy Research,
17(10):1195-1198.
HAN, L.-K., MORIMOTO, C., YU, R.H., OKUDA, H. 2005a. Effects of Coleus
forskohlii on fat storage in ovariectomized rats. Yakugaku-Zasshi, 125(5):449-453.
49
HAN, L.-K., ZHENG, Y.-N, YOSHIKAWA, M., OKUDA, H., KIMURA, Y. 2005b.
Anti-obesity effects of chikusetsusaponins isolated from Panax japonicus
rhizomes. BMC complementary and Alternative Medicine, 5:9-18.
HAN, L., LI, W., NARIMATSU, S., LIU, L., FU, H., OKUDA, H., KOIKE, K. 2007.
Inhibitory effects of compounds isolated from fruit of Juglans mandshurica on
pancreatic lipase. Journal of Natural Medicines, 61:184-186.
HANDA, T., YAMAGUCHI, K., SONO, Y., YAZAWA, K. 2005. Effects of fenugreek
seed extract in obese mice fed a high-fat diet. Bioscience Biotechnology and
Biochemistry, 69(6):1186-1188.
HATANO, T., YAMASHITA, A., HASHIMOTO, T., ITO, H., KUBO, N.,
YOSHIYAMA, M., SHIMURA, S., ITOH, Y., OKUDA, T., YOSHIDA, T. 1997.
Flavan dimers with lipase inhibitory activity from Cassia nomame. Phytochemistry,
46(5):893-900.
HECK, C.I., DE MEJIA, E.G. 2007. Yerba Mate Tea (Ilex paraguariensis): A
comprehensive review on chemistry, health implications, and technological
considerations. Journal of Food Science, 72(9):138-151.
HENDERSON, S., MAGU, B., RASMUSSEN, C., LANCASTER, S., KERKSICK,
C., SMITH, P., MELTON, C., COWAN, P., GREENWOOD, M., EARNEST, C.,
ALMADA, A., MILNOR, P., MAGRANS, T., BOWDEN, R., OUNPRASEUTH, S.,
THOMAS, A., KREIDER, R.B. 2005. Effects of Coleus forskohlii supplementation
on body composition and hematological profiles in mildly overweight women.
Journal of the International Society of Sports Nutrition, 2(2):54-62.
HIGGS, J. 2007. The potential role of peanuts in the prevention of obesity.
Nutrition and Food Science, 35(5):353-358.
HOLT, S., TAYLOR, T.V. 2006a. Hoodia gordonii. An overview of biological and
botanical characteristics: Part 1. Townsend Letter for Doctors and Patients,
280:104-113.
HOLT, S., TAYLOR, T.V. 2006b. Hoodia gordonii. Separating science from
speculation: Part 2. Townsend Letter for Doctors and Patients, 281:99-104.
HOMOUD, M.K. 2009. The sale of weight-loss supplements on the Internet: A
lurking health crisis waiting to strike. Heart Rhythm, 6(5):658-662.
HOU, D., FUKUDA, M, FUJII M., FUKE, Y. 2000. Transcriptional regulation of
nicotinamide adenine dinucleotide phosphate: quinine oxidoreductase in murine
hepatoma cells by 6-(methylsufinyl)hexyl isothiocyanate, an active principle of
wasabi (Eutrema wasabi Maxim). Cancer Letters, 161:195-200.
HSU, C.-H., TSAI, T.-H., KAO, Y.-H., HWAN, K.-C., TSEN, T.-Y., CHOU, P. 2008.
Effect of green tea extract on obese women: A randomized, double-blind, placebocontrolled clinical trial. Clinical Nutrition, 27:363-370.
50
HU, J.-N, ZHU, X.-M., HAN, L.-K., SAITO, M., SUN, Y.-S., YOSHIKAWA, M.,
KIMURA, Y., ZHENG, Y.-N. 2008. Anti-obesity effects of escins extracted from the
seeds of Aesculus turbinata Blume (Hippocastanaceae). Chemical and
Pharmaceutical Bulletin, 56(1):12-16.
HU, Y., FAHMY, H., ZJAWIONY, J.K. DAVIES, G.E. 2010. Inhibitory effect and
transcriptional impact of berberine and evodiamine on human white preadipocyte
differentiation. Fitoterapia, 81(4):259-268.
HURALIKUPPI, J.C., CHRISTOPHER, A.B., STEPHEN, P.M. 2006. Antidiabetic
effect of Nelumbo nucifera Gaertn. extract: Part II. Phytotherapy Research,
5(5):217-223.
HWANG, J.-T., PARK, I.-J., SHIN, J.-I., LEE, Y.K., LEE, S.K., BAIK, H.W., HA, J.,
PARK, O.J. 2005. Genistein, EGCG, and capsaicin inhibit adipocyte differentiation
process via activating AMP-activated protein kinase. Biochemical and Biophysical
Research Communications, 338:694-699.
HWANG, J.-T., KIM, S.-H., LEE, M.-S., KIM, S.H., YANG, H.-J., KIM, M.-J., KIM,
H.-S., HA, J., KIM, M.S., KWON, D.Y. 2007. Anti-obesity effects of ginsenoside
Rh2 are associated with the activation of AMPK signaling pathway in 3T3-L1
adipocyte. Biochemical and Biophysical Research Communications, 364:10021008.
HWANG, J.-T., LEE, M.-S., KIM, H.-J., SUNG, M.-J., KIM, H.Y., KIM, M.S.,
KWON, D.Y. 2008. Antiobesity effect of ginsenoside Rg3 involves the AMPK and
PPAR-γ signal pathways. Phytotherapy Research, 23(2):262-266.
IM, R., MANO, H., NAKATUNI, S., SHIMIZU, J., WADA, M. 2008. Aqueous extract
of Kotahla himbutu (Salacia reticulata) stems promotes oxygen consumption and
suppresses body fat accumulation in mice. Journal of Health Science, 54(6):645653.
INOCENCIO C, RIVERA D, ALCARAZ F, TOMÁS-BARBERÁN FA. 2000.
Flavonoid content of commercial capers (Capparis spinosa, C. sicula and C.
orientalis) produced in Mediterranean countries. European Food Research and
Technology, 212:70-74.
ISBRUCKER, R.A., BURDOCK, G.A. 2006. Risk and safety assessment on the
consumption of licorice root (Glycyrrhiza sp.), its extract and powder as a food
ingredient, with emphasis on the pharmacology and toxicology of glycyrrhizin.
Regulatory Toxicology and Pharmacology, 46:167-192.
IWASHITA, K., YAMAKI, K., TSHUSHIDA, T. 2001. Mioga (Zingiber mioga Rosc.)
extract prevents 3T3-L1 differentiation into adipocytes and obesity in mice. Food
Science and Technology Research, 7(2):164-170.
JAYAWARDENA, M.H.S., DE ALWIS, N.M.W., HETTIGODA, V., FERNANDO,
D.J.S. 2005. A double-blind randomized placebo controlled cross over study of a
51
herbal preparation containing Salacia reticulata in the treatment of type 2 diabetes.
JEONG, S., CHAE, K., JUNG, Y.S., RHO, Y.H., LEE, J., HA, J., YOON, K.H., KIM,
G.C., OH, K.S., SHIN, S.S., YOON, M. 2008. The Korean traditional medicine
Gyeongshingangjeehwan inhibits obesity through regulation of leptin and PPARα
action in OLETF rats. Journal of Ethnopharmacology, 119:245-251.
JU, J.B., KIM, J.S., CHOI, C.W., LEE, H.K., OH, T.-K., KIM, S.C. 2008.
Comparison between ethanolic and aqueous extracts from Chinese juniper berries
for hypoglycaemic and hypolipidemic effects in alloxan-induced diabetic rats.
KAMATOU, G.P.P., VILJOEN, A.M., VAN VUUREN, S.F., VAN ZYL, R. 2006. In
vitro evidence of antimicrobial synergy between Salvia chamelaeagnea and
Leonotis leonurus. South African Journal of Botany, 72:634-636.
KAMISOYAMA, H., HONDA, K., TOMINAGA, Y., YOKOT, S., HASEGAWA, S.
2008. Investigation of the anti-obesity action of licorice flavonoid oil in diet-induced
obese rats. Bioscience Biotechnology and Biochemistry, 72(12):3225-3231.
KANETKAR, P., SINGHAL, R., KAMAT, M. 2007. Gymnema sylvestre: A Memoir.
Journal of Clinical Biochemistry and Nutrition, 41:77-81.
KANG, J.-H., KIM, C.-S., HAN, I.-S., KAWADA, T., YU, R. 2007. Capsaicin, a
spicy component of hot peppers, modulates adipokine gene expression and
protein release from obese-mouse adipose tissues and isolated adipocytes, and
suppresses the inflammatory responses of adipose tissue macrophages. FEBS
Letters, 581:4389-4396.
KANKI, K., NISHIKAWA, A., FURUKAWA, F., KITAMURA, Y., IMAZAWA, T.,
UMEMURA, T., HIROSE, M. 2003. A 13-week subchronic toxicity study of paprika
color in F344 rats. Food and Chemical Toxicology 41:337-1343.
KAPETANOVIC, I.M., CROWELL, J.A., KRISHNARAJ, R., ZAKHAROV, A.,
LINDEBLAD, M., LYUBIMOV, A. 2009. Exposure and toxicity of green tea
polyphenols in fasted and non-fasted dogs. Toxicology, 260:28-36.
KARU, N., REIFEN, R., KEREM, Z. 2007. Weight gain reduction in mice fed
Panax ginseng saponin, a pancreatic lipase inhibitor. Journal of Agricultural and
Food Chemistry, 55:2824-2828.
KASBIA, G.S., ARNASON, J.R., IMBEAULT, P. 2009. No effect of acute, single
dose oral administration of Momordica charantia L., on glycemia, energy
expenditure and appetite: A pilot study in non-diabetic overweight men. Journal of
KAWAGUCHI, K., MIZUNO, T., AIDA, K., UCHINO, K. 1997. Hesperidin as an
inhibitor of lipases from porcine pancreas and Pseudomonas. Bioscience
Biotechnology and Biochemistry, 61(1):102-104.
52
KHADER, M., BRESGEN, N., ECKL, P.M. 2009. In vitro toxicological properties of
thymoquinone. Food and Chemical Toxicology, 47:129-133.
KIM, J.H., KANG, S.A., HAN, S.-M., SHIM, I. 2008a. Comparison of the antiobesity
effects of protopanaxadiol-and protopanaxatriol-type saponins of red ginseng.
Phytotherapy Research, 23(1):78-85.
KIM, J.-Y., SO, H., YOUN, M.-J, KIM, H.-J., KIM, Y., PARK, C., KIM, S.-J., HA, Y.A, CHAI, K.-Y., KIM, S.-M., KIM, K.-Y., PARK, R. 2007. Hibiscus sabdariffa L.
water extract inhibits the adipocyte differentiation through the PI3-K and MAPK
pathway. Journal of Ethnopharmacology, 114:260-267.
KIM, M.S., KIM, J.K, KIM, H.J, MOON, S.R., SHIN, B.C., PARK, K.W., YANG,
H.O., KIM, S.M., PARK, R. 2003. Hibiscus extract inhibits the lipid droplet
accumulation and adipogenic transcription factors expression of 3T3-L1
preadipocytes. Journal of Alternative and Complementary Medicine, 9(4):499-504.
KIM, S.-K., KIM, Y.-M., HONG, M.-J., RHEE, H.-I. 2005. Studies on the inhibitory
effects of Eugenia aromaticum extract on pancreatic lipase. Agricultural Chemistry
and Biotechnology, 48(2):84-88.
KIM, H.-J., PARK, J.-M., KIM, J.-A., KO, B.-P. 2008b. Effect of herbal Ephedra
sinica and Evodia ruteacarpa on body composition and resting metabolic rate: A
randomized, double-blind clinical trial in Korean premenopausal women. Journal of
Acupuncture and Meridian Studies, 1(2):128-138.
KIM, S.-J., JUNG, J.Y., KIM, H.W., PARK, T. 2008c. Anti-obesity effects of
Juniperus chinensis extract are associated with increased AMP-activated protein
kinase expression and phosphorylation in the visceral adipose tissue of rats.
Biological and Pharmaceutical Bulletin, 31(7):1415-1421.
KIM, S.-H., PARK, H.-S., LEE, M.-S., CHO, Y.-J., KIM, Y.-S., HWANG, J.-T.,
SUNG, M.J., KIM, M.S., KWON, D.Y. 2008d. Vitisin A inhibits adipocyte
differentiation through cell cycle arrest in 3T3-L1 cells. Biochemical and
Biophysical Research Communications, 372:108-113.
KIM, J.-Y., MOON, K.-D., SEO, K.-I., PARK, K.-W., CHOI, M.-S., DO, G.-M.,
JEON, Y.-K., CHO, Y.-S., LEE, M.-K. 2009a. Supplementation of SK1 from
Platycodi radix ameliorates obesity and glucose intolerance in mice fed a high-fat
diet. Journal of Medicinal Food, 12(3):629-636.
KIM, S.-J., LEE, S.-J., LEE, S., CHAE, S., HAN, M.-D., MAR, W. NAM, K.-W.
2009b. Rutecarpine ameliorates bodyweight gain through the inhibition of
orexigenic neuropeptides NPY and AgRP in mice. Biochemical and Biophysical
Research Communications, 389:437-442.
KIM, S.N., LEE, J.H., SHIN, H., SON, S.H.,KIM, Y.S. 2009c. Effects on in vitrodigested ginsenosides on lipid accumulation in 3T3-L1 adipocytes. Planta Medica,
75:596-601.
53
KIMURA, H., OGAWA, S., JISAKA, M., KIMURA, Y., KATSUBE, T., YOKOTA, K.
2006. Identification of novel saponins from edible seeds of Japanese horse
chestnut (Aesculus turbinata Blume) after treatment with wooden ashes and their
nutraceutical activity. Journal of Pharmaceutical and Biomedical Analysis,
41:1657-1665.
KIMURA, H., OGAWA, S., KATSUBE, T., JISAKA, M., YOKOTA, K. 2008.
Antiobese effects of novel saponins from edible seeds of Japanese horse chestnut
(Aesculus turbinata Blume) after treatment with wood ashes. Journal of
Agricultural and Food Chemistry, 56(12):4783-4788.
KISHINO, E., ITO, T., FUJITA, K., KIUCHI, Y. 2006. A mixture of the Salacia
reticulata (Kotahala himbutu) aqueous extract and cyclodextrin reduces the
accumulation of visceral fat mass in mice and rats with high-fat diet-induced
obesity. The Journal of Nutrition, 136:433-439.
KLEIN, G., KIM, J., HIMMELDIRK, K., CAO, Y., CHEN, X. 2007. Antidiabetes and
anti-obesity activity of Lagerstroemia speciosa. Evidence-based Complementary
and Alternative Medicine, 4(4):401–407.
KOBAYASHI, Y., NAKANO, Y., KIZAKI, M., HOSHIKUMA, K., YOKOO, Y.,
KAMIYA, T. 2001. Capsaicin-like anti-obese activities of evodiamine from fruits of
Evodia rutaecarpa, a vanilloid receptor agonist. Planta Medica, 67:628-633.
KOBAYASHI, K., YAMADA, K., MURATA, T., HASEGAWA, T., TAKANO, F,
KOGA, K., FUSHIYA, S., BATKHUU, J., YOSHIZAKI, F. 2008a. Constituents of
Rhodiola rosea showing inhibitory effect on lipase activity in mouse plasma and
alimentary canal. Planta Medica, 74:1716-1719.
KOBAYASHI, K., IHARA, S., KOBATA, A., ITOH, K., KUSUNOLI, N., YOSHIZAKI,
F. 2008b. Inhibitory effect of Myrica bark on lipase activity in mouse plasma and
gastrointestinal tract. Journal of Medicinal Food, 11(2):289-293.
KURIHARA, H., ASAMI, S., SHIBATA, H., FUKAMI, H., TANAKA, T. 2003.
Hypolipemic effect of Cyclocarya paliurus (Batal) Iljinskaja in lipid-loaded mice.
Biological and Pharmaceutical Bulletin, 26(3):383-385.
KURIYAN, R., RAJ, T., SRINIVAS, S.K., VAZ, M., RAJENDRAN, R., KURPAD,
A.V. 2007. Effect of Caralluma fimbriata extract on appetite, food intake and
anthropometry in adult Indian men and women. Appetite, 48:38-344.
KWON, C.-S., SOHN, H.Y., KIM, S.H., KIM, J.H., SON, K.H., LEE, J.S., LIM, J.K.,
KIM, J.-S. 2003. Anti-obesity effect of Dioscorea nipponica Makino with lipaseinhibitory activity in rodents. Bioscience Biotechnology and Biochemistry,
67(7):1451-1456.
LAMBERT, J.D., KENNET, M.J., SANG, S., REUHL, K.R., JU, J., YANG, C.S.
2010. Hepatotoxicity of high oral dose (-)-epigallocatechi-3-gallate in mice. Food
and Chemical Toxicology, 48:409-416.
54
LE, P.M., BENHADDOU-ANDALOUSSI, A., ELIMADI, A., SETTAF, A.,
CHERRAH, Y., HADDAD, P.S. 2004. The petroleum ether extract of Nigella sativa
exerts lipid-lowering and insulin-sensitizing actions in the rat. Journal of
LEE, I.-A., LEE, J.H., BAEK, N.-I., KIM, D.-H. 2005. Antihyperlipidaemic effect of
crocin isolated from the fructus of Gardenia jasminoides and its metabolite
crocetin. Biological and Pharmaceutical Bulletin, 28(11):2106-2110.
LEMHADRI, A., EDDOUKS, M., SULPICE, T., BURCELIN, R. 2007. Antihyperglycaemic and anti-obesity effects of Capparis spinosa and Chamaemelum
nobile aqueous extracts in HFD mice. American Journal of Pharmacology and
Toxicology, 2(3):106-110.
LI, F., LI, W., FU, H., ZHANG, Q., KOIKE, K. 2007a. Pancreatic lipase-inhibiting
triterpenoid saponins from fruits of Acanthopanax senticosus. Chemical and
LI, X., LIU, Z., ZHANG, X., WANG, L., ZHENG, Y., YUAN, C., SUN, G. 2008.
Isolation and characterization of phenolic compounds from the leaves of Salix
matsudana. Molecules, 13:1530-1537.
LI, Z.-B., WAND, J.-Y., JIANG, B., ZHANG, X.-L., AN, L.-J., BAO, Y.-M. 2007b.
Benzobijuglone, a novel cytotoxic compound from Juglans mandshurica, induced
apoptosis in HeLa cervical cancer cells. Phytomedicine, 14:846-852.
LIEBERMAN, S. 2004. A new potential weapon for fighting obesity. Forskolin – the
active diterpene in Coleus. Alternative and Complementary Therapies, 10(6):330333.
LIMA, W.P, CARNEVALI JR, L.C., EDER, R., COSTA ROSA, L.F.B.P., BACCHI,
E.M., SEELAENDER, M.C.L. 2005. Lipid metabolism in trained rats: Effect of
guarana (Paullinia cupana Mart.) supplementation. Clinical Nutrition, 24:10191028.
LIN, J., DELLA-FERA, M.A., BAILE, C.A. 2005. Green tea polyphenol
epigallocatechin gallate inhibits adipogenesis and induces apoptosis in 3T3-L1
adipocytes. Obesity Research, 13(6):982-990.
LIU F., KIM, J,-K., LI, Y., LIU, X.-Q., LI, J., CHEN, X. 2001. An extract of
Lagerstroemia speciosa L. has insulin-like glucose uptake-stimulatory and
adipocyte differentiation-inhibitory activities in 3T3-L1 cells. The Journal of
Nutrition, 131:2242-2247.
LIU, W., ZHENG, Y., HAN, L., WANG, H., SAITO, M., LING, M., KIMURA, Y.
2008. Saponins (ginsenosides) from stems and leaves of Panax quinquefolium
prevented high-fat diet-induced obesity in mice. International Journal of
Phytotherapy
and
Phytopharmacology,
available
online
at:
http://www.thefreelibrary.com/Saponins+(ginsenosides)+from+stems+and+
leaves+of+Panax+quinquefolium...-a0191955988 [Accessed: 27/11/2009].
55
LIU, X., KIM, J.-K., LI, Y., LI, J., LIU, F., CHEN, X. 2005. Tannic acid stimulates
glucose transport and inhibits adipocyte differentiation in 3T3-L1 cells. The Journal
of Nutrition, 135:165-171.
LUO, H., KASHIWAGI, A., SHIBAHARA, T., YAMADA, K. 2007. Decreased
bodyweight without rebound and regulated lipoprotein metabolism by gymnemate
in genetic multifactor syndrome animal. Molecular and Cellular Biochemistry,
299(1-2):93-98.
MACLEAN, D.B., LUO, L.-G. 2004. Increased ATP content/production in the
hypothalamus may be a signal for energy-sensing of satiety: studies of the
anorectic mechanism of a plant steroidal glycoside. Brain Research, 1020:1-11.
MADGULA, V.L.M., ASHFAQ, M.K., WANG, Y.-H., AVULA, B., KHAN, I.A.,
WALKER, L.A., KHAN, S.I. 2010a. Bioavailability, pharmacokinetics, and tissue
distribution of the oxypregnane steroidal glycoside P57AS3 (P57) from Hoodia
gordonii in mouse model. Planta Medica, 76(14):1582-1586.
MADGULA, V.L.M., AVULA, B., PAWAR, R.S., SHUKLA, Y.J., KHAN, I.A.,
WALKER, L.A., KHAN, S.I. 2010b. Characterization of in vitro pharmacokinetic
properties of hoodigenin A from Hoodia gordonii. Planta Medica, 76(1):62-69.
MAHMOOD, S.A., LINDEQUIST, U. 2008. A pilot study on the effect of Catha
edulis Frosk., (Celastraceae) on metabolic syndrome in Wokw rats. African
Journal of Traditional Complementary and Alternative Medicines, 5(3):271-277.
MARCHEI, E., PICHINI, S., PACIFICI, R., PELLEGRINI, M., ZUCCARO, P. 2006.
A rapid and simple procedure for the determination of synephrine in dietary
supplements by gas chromatography-mass spectrometry. Journal of
Pharmaceutical and Biomedical Analysis, 41:1468-1472.
MARTINS, F., NOSO, T.M, PORTO, V.B., CURIEL, A., GAMBERO, A., BASTOS,
D.H.M., RIBEIRO, M.L., CARVALHO, P., 2010. Maté tea inhibits in vitro pancreatic
lipase activity and has hypolipidaemic effect on high-fat diet-induced obese mice.
Obesity, 18(1):42-47.
MASUDA, Y., HARAMIZU, S., OKI, K., OHNUKI, K., WATANABE, T., YAZAWA,
S., KAWADA, T., HASHIZUME, S.-I., FUSHIKI, T. 2003. Upregulation of
uncoupling proteins by oral administration of capsiate, a non-pungent capsaicin
analog. Journal of Applied Physiology, 95:2408-2415.
MATHERN, J.R., RAATZ, S.K., THOMAS, W., SLAVIN, J.L. 2009. Effect of
fenugreek fiber on satiety, blood glucose and insulin response and energy intake
in obese subjects. Phytotherapy Research, 23(11):1543-1548.
MATSUDA, A., WANG, Z., TAKAHASHI,S., TOKUDA, T., MIURA, N.,
HASEGAWA, J. 2009. Upregulation of mRNA of retinoid binding protein and fatty
acid binding protein by cholesterol enriched-diet and effect of ginger on lipid
metabolism. Life Sciences, 84:903-907.
56
MATTEI, R., DIAS, R.F., ESPÍNOLA, E.B., CARLINI, E.A., BARROS, S.B.M.
1998. Guarana (Paullinia cupana): toxic behavioural effects in laboratory animals
and antioxidant activity in vitro. Journal of Ethnopharmacology, 60:111-116.
MATTES, R.D., BORMANN, L. 2000. Effects of (-)-hydroxycitric acid on appetitive
variables. Physiology and Behaviour, 71:87-94.
MATTES, R.D., KRIS-ETHERTON, P.M., FOSTER, G.D. 2008. Impact of peanuts
and tree nuts on body weight and healthy weight loss in adults. The Journal of
Nutrition, 138(9):1741S-1745S.
MENNITI-IPPOLITO F., MAZZANTI, G., SANTUCCIO, C., MORO, PA., CALAPAI,
G., FIRENZUOLI F., VALERI, A., RASCHETTI, R. 2008. Surveillance of suspected
adverse reactions to natural health products in Italy. Pharmacoepidemiology and
Drug Safety, 17:626-635.
MIYOSHI, N., NAKAMURA, Y., UEDA, Y., ABE, M., OZAWA, Y., UCHIDA, K.,
OSAWA,T. 2003. Dietary ginger constituents, galanals A and B, are potent
apoptosis inducers in Human T lymphoma Jurkat cells. Cancer Letters, 199:113119.
MOHLAPO, T.D., NG’AMBI, J.W., NORRIS, D., MALATJE, M.M. 2009. Effect of
Hoodia gordonii meal supplementation at finisher stage on productivity and
carcass characteristics of Ross 308 broiler chickens. Tropical Animal Health and
Production, 41:1591-1596.
MOLLAH, M.L., KIM, G.-S., MOON, H.-K., CHUNG, S.-K., CHEON, Y.-P., KIM, J.K., KIM, K.-S. 2008 Antiobesity effects of wild ginseng (Panax ginseng C.A.
Meyer) mediated by PPAR-γ, GLUT4 and LPL in ob/ob mice. Phytotherapy
Research, 23(2):220-225.
MOONEY, M.H., FOGARTY, S., STEVENSON, C., GALLAGHER, A.M., PALIT,
P., HAWLEY, S.A., HARDIE, DG., COXON, G.D., WAIGH, R.D., TATE, R.J.,
HARVEY, A.L., FURMAN, B.L. 2008. Mechanisms underlying the metabolic
actions of galegine that contribute to weight loss in mice. British Journal of
Pharmacology, 153:1669-1677.
MORENO, D.A., ILIC, N., POULEV, A., BRASAEMLE, D.L., FRIED, S.K.,
RASKIN, I. 2003. Inhibitory effects of grape seed extract on lipases. Nutrition,
19:876-879.
MORENO, D.A., ILIC, N., POULEV, A., RASKIN, I. 2006a. Effects of Arachis
hypogaea nutshell extract on lipid metabolic enzymes and obesity parameters. Life
Sciences, 78:2797-2803.
MORENO, D.A., RIPOLL, C., ILIC, N., POULEV, A., AUBIN, C., RASKIN, I.
2006b. Inhibition of lipid metabolic enzymes using Mangifera indica extracts.
International Journal of Food Agriculture and Environment, 4(1):21-26.
57
MORIMITSU Y., HAYASHI, K., NAKAGAWA, Y., FUJII, H., HORIO, F., UCHIDA,
K., OSAWA, T. 2000. Antiplatelet and anticancer isothiocyanates in Japanese
domestic horseradish, Wasabi. Mechanisms of Ageing and Development,
116:125-134.
MORTON, S.C. 2005. Ephedra. Statistical Science, 20(3):242-248.
MURALIDHARA, NARASIMHAMURTHY, K., VISWANATHA, S., RAMESH, B.S.
1999. Acute and subchronic toxicity assessment of debitterized fenugreek powder
in the mouse and rat. Food and Chemical Toxicology, 37:831-838.
MURASE, T., MISAWA, K., HARAMIZU, S., HASE, T. 2009. Catechin-induced
activation of the LKB1/AMP-activated protein kinase pathway. Biochemical
MURRAY, C.D.R., LE ROUX, C.W., EMMANUEL, A.V., HALKET, J.M.,
PRZYBOROWSKA, A.M., KAMM, M.A., MURRAY-LYON, I.M. 2008. The effect of
Khat (Catha edulis) as an appetite suppressant is independent of ghrelin and PYY
secretion. Appetite, 51:747-750.
MYCEK, M.J., HARVEY, R.A., CHAMPE, P.C. 2000. Lippincott’s illustrated
reviews: Pharmacology 2nd ed. Philadelphia: Lippincott-Raven publishers.
NAJMI, A., NASIRUDDIN, M., KHAN, R.A., HAQUE, S.F. 2008. Effect of Nigella
sativa oil on various clinical and biochemical parameters of insulin resistance
syndrome. International Journal of Diabetes in Developing Countries, 28(1):11-14.
NAKAGAWA, K., KITANO, M., KISHIDA, H., HIDAKA, T., NABAE, K., KAWABE,
M., HOSOE, K. 2008. 90-Day repeated-dose toxicity study of licorice flavonoid oil
(LFO) in rats. Food and Chemical Toxicology, 46:2349-2357.
NGONDI, J.L., MAKAMTO, S.C., ETAME, S.L., OBEN, J. 2006. Effect of
Triumphetta cordifolia on body weight and blood lipids in normolipidemic guinea
pigs. Drug Development Research, 66(3):200-203.
NINOMIYA, K., MATSUDA, H., SHIMODA, H., NISHIDA, N., KASAJIMA, N.,
YOSHINO, T., MORIKAWA, T., YOSHIKAWA, M. 2004. Carnosic acid, a new
class of lipid absorption inhibitor from sage. Bioorganic and Medicinal Chemistry
Letters, 14:1943-1946.
NYKAMP, D.L., FACKIH, M.N, COMPTON, A.L. 2004. Possible association of
acute lateral-wall myocardial infarction and bitter orange supplement. The Annals
of Pharmacotherapy, 38(5):812-816.
OGAWA, T., TABATA, H., KATSUBE, T., OHTA, Y., YAMASAKI, Y., YAMASAKI,
M., SHIWAKU, K. 2009. Suppresive effect of hot water extract of wasabi (Wasabia
japonica Matsum.) leaves on the differentiation of 3T3-L1 preadipocytes. Food
Chemistry, 118:239-244.
58
OGAWA, Y., SEKITA, K., UMEMURA, T., SAITO, M., ONO, A., KAWASAKI, Y.,
UCHIDA, O., MATSUSHIMA, Y., INOUE, T., KANNO, J. 2004. Gymnema
sylvestre leaf extract: A 52-week dietary toxicity study in wistar rats. Shokuhin
Eisegaku Zasshi, 45(1):8-18.
OHKOSHI, E., MIYAZAKI, H., SHINDO, K., WATANABE, H., YOSHIDA, A.,
YAJIMA, H. 2007. Constituents from the leaves of Nelumbo nucifera stimulate
lipolysis in the white adipose tissue of mice. Planta Medica, 73:1255-1259.
OLIVIER, I. 2005. Hoodia gordonii (Masson) Sweet ex Decne. Available from
http://www.plantzafrica.com/planthij/hoodgord.htm [Accessed: 05/02/2008].
ONO, Y., HATTORI, E., FUKAYA, Y., IMAI, S., OHIZUMI, Y. 2006. Anti-obesity
effect of Nelumbo nucifera leaves extract in mice and rats. Journal of
ORISAKWE, O.E., HUSAINI, D.C., AFONNE, O.J. 2004. Testicular effects of subchronic administration of Hibiscus sabdariffa calyx aqueous extract in rats.
Reproductive Toxicology, 18:295-298.
OSTOJIC, S.M. 2006. Yohimbine: The effects on body composition and exercise
performance in soccer players. Research in Sports Medicine, 14(4):289-299.
OZAKI, A., KITANO, M., FURUSAWA, N., YAMAGUCHI, H., KURODA, K., ENDO,
G. 2002. Genotoxicity of gardenia yellow and its components. Food and Chemical
Toxicology, 40:1603-1610.
PALIT, P., FURMAN, B.L., GRAY, A.I. 1999. Novel weight-reducing activity of
Galega officinalis in mice. Journal of Pharmacy and Pharmacology, 51(11):13131319.
PANG, J., CHOI, Y., PARK, T. 2008. Ilex paraguariensis extract ameliorates
obesity induced by high-fat diet: Potential role of AMPK in the visceral adipose
tissue. Archives of Biochemistry and Biophysics, 476:178-185.
PARK, T., JUNG, H.J., KIM, H.W. 2009. (-)-Cedrol isolated from Juniperus
chinensis reduces diet-induced obesity through increased uncoupling protein
expression in the visceral adipose tissue of mice. New Biotechnology, 255:513.
PARK, Y.S., YOON, Y., AHN, H.S. 2007. Platycodon grandiflorum extract
represses up-regulated adipocyte fatty acid binding protein triggered by a high fat
feeding in obese rats. World Journal of Gastroenterology, 13(25):3493-3499.
PATEL, D.K., PATEL, K.A., PATEL, U.K., THOUNAOJAM, M.C., JADEJA, R.N.,
ANSARULLAH,
PADATE,
G.S.,
SALUNKE,
S.P.,
DEVKAR,
R.V.,
RAMACHANDRAN, A.V. 2009. Assessment of lipid lowering effect of Sida
rhomboidea.Roxb methanolic extract in experimentally induced hyperlipidemia.
Journal of Young Pharmacists, 1(3):233-238.
59
PAWAR, R.S., SHUKLA, Y.J., KHAN, S.I., AVULA, B., KHAN, I.A. 2007. New
oxypregnane glycosides from appetite suppressant herbal supplement Hoodia
gordonii. Steroids, 72:524-534,
PAWAR, R.S., SHUKLA, Y.J., KHAN, I.A. 2007. New calogenin glycosides from
Hoodia gordonii. Steroids, 72:881-891.
PETIT, P.R., SAUVAIRE, Y.D., HILLAIRE-BUYS, D.M., LECONTE, O.M.,
BAISSAC, Y.G., PONSIN, G.R., RIBES, G.R. 1995. Steroid saponins from
fenugreek seeds: Extraction, purification, and pharmacological investigation on
feeding behavior and plasma cholesterol. Steroids, 60:674-680.
PITTLER, M.H., ERNST, E. 2004. Dietary supplements for body-weight reduction:
a systematic review. American Journal of Clinical Nutrition, 79:529-536.
PRASANTH, D., PADMAJA, R., SAMIULLA, D.S. 2001. Effect of certain plant
extracts on α-amylase activity. Fitoterapia, 72:179-181.
PRASANTH D., AMIT, A., SAMIULLA, D.S., ASHA, M.K., PADMAJA, R. 2001. αglucosidase inhibitory activity of Mangifera indica bark. Fitoterapia, 72:686-688.
PREUSS, H.G., BAGCHI, D., BAGCHI, M., RAO, C.V.S., DEY, D.K.,
SATYANARAYANA, S. 2004. Effects of a natural extract of (-)-hydroxycitric acid
(HAS-SX) and a combination of HCA-SX plus niacin-bound chromium and
Gymnema sylvestre extract on weight loss. Diabetes Obesity and Metabolism,
6:171-180.
PUSZTAI, A., GRANT, G., BUCHAN, W.C., BARDOCZ, S., DE CARVALHO,
A.F.F.U., EWEN, S.W.B 1998. Lipid accumulation in obese Zucker rats is reduced
by inclusion of raw kidney bean (Phaseolus vulgaris) in the diet. British Journal of
Nutrition, 79:213-221.
QIDWAI, W., BIN HAMZA, H., QURESHI, R., GILANI, A. 2009. Effectiveness,
safety and tolerability of powdered Nigella sativa (Kalonji) seed in capsules on
serum lipid levels, blood sugar, blood pressure, and body weight in adults: results
of a randomized, double-blind controlled trial. The Journal of Alternative and
Complementary Medicine, 15(6):639-644.
QIN, Y., WU, X., HUANG, W., GONG, G., LI, D., HE, Y., ZHAO, Y. 2009. Acute
toxicity and sub-chronic toxicity of steroidal saponins from Dioscorea zingiberensis
C.H. Wright in rodents. Journal of Ethnopharmacology, 126:543-550.
QURESHI, S., SHAH, A.H., AGEEL, A.M. 1992. Toxicity studies on Alpinia
galanga and Curcuma longa. Planta Medica, 58:124-127.
RÁCZ-KOTILLA, E., RÁCZ, G., SOLOMON, A. 1974. The action of Taraxacum
officinale extracts on the body weight and diuresis of laboratory animals. Planta
Medica, 26:212-217.
60
RAI, S., WAHILE, A., MUKHERJEE, K., SAHA, B.P., MUKHERJEE, P.K. 2006.
Antioxidant activity of Nelumbo nucifera (sacred lotus) seeds. Journal of
RASEKH, H.R., NAZARI, P., KAMLI-NEJAD, M.., HOSSEINZADEH, L. 2008.
Acute and subchronic oral toxicity of Galega officinalis in rats. Journal of
RATNASOORIYA, W.D., JAYAKODY, J.R.A.C., PREMAKUMARA, G.A.S. 2003.
Adverse pregnancy outcome in rats following exposure to a Salacia reticulata
(Celestraceae) root extract. Brazilian Journal of Medical and Biological Research,
36(7):931-935.
RAZMOVSKI-NAUMOVSKI, V., HUANG, T.H.-W., TRAN, V.H., LI, G.Q., DUKE,
C.C., ROUFOGALIS, B.D. 2005. Chemistry and pharmacology of Gynostemma
pentaphyllum. Phytochemistry Reviews, 4:197-219.
RODEIRO, I., DONATO, M.T., JIMÉNEZ, N., GARRIDO, G., DELGADO, R.,
GÓMEZ-LECHÓN, M.J. 2007. Effects of Mangifera indica L. aqueous extract
(Vimang) on primary culture of rat hepatocytes. Food and Chemical Toxicology,
45:2506-2512.
RODRIGUEZ-FRAGOSO, L., REYES-ESPARZA, J., BURCHIEL, S.W.,
HERRERA-RUIZ, D., TORRES, E. 2008. Risks and benefits of commonly used
herbal medicines in Mexico. Toxicology and Applied Pharmacology, 227:125-135.
RODRÍGUEZ-MORÁN, M., GUERRERO-ROMERO, F., LAZCANO-BURCIAGA,
G. 1998. Lipid-and glucose-lowering efficacy of Plantago psyllium in Type II
diabetes. Journal of Diabetes and its Complications, 12(5):273-278.
RUBIN, I.D., BINDRA, J.S., CAWTHORNE, M.A. 2006. Extracts, compounds and
pharmaceutical compositions having anti-diabetic activity and their use. US Patent
2006/0159779 A1.
RUXTON, C.H.S. 2004. Efficacy of Zotrim: a herbal weight loss preparation.
Nutrition and Food Science, 34(1):25-28.
RUXTON, C.H.S., GARDNER, E.J. 2005. A review of the efficacy and safety of
key ingredients of over-the-counter products for weight management. British Food
Journal, 107(2):111-125.
SAITO, M., UENO, M., OGINO, S., KUBO, K., NAGATA. J., TAKEUCHI, M. 2005.
High dose of Garcinia cambogia is effective in suppressing fat accumulation in
developing male Zucker obese rats, but highly toxic to testis. Food and Chemical
Toxicology, 43:411-419.
SÁNCHEZ DE MEDINA, F., GÁMEZ M.J., JIMÉNEZ, I., JIMÉNEZ, J., OSUNA,
J.I., ZARZUELO, A. 1994. Hypoglycemic activity of juniper “berries”. Planta
Medica, 60:197-200.
61
SARMA, D.N., BARRETT, M.L., CHAVEZ, M.L., GARDINER, P., KO, R.,
MAHADY, G.B. MARLES, R.J., PELLICORE, L.S., GIANCASPRO, G.I., LOW
DOG, T. 2008. Safety of green tea extracts: A systematic review by the US
Pharmacopoeia. Drug Safety, 31(6):469-484.
SAYAMA, K., LIN, S., ZHENG, G., OGUNI, I. 2000. Effects of green tea on growth,
food utilization and lipid metabolism in mice. In Vivo, 14(4):481-484.
SCIENCELAB. 2008. Material safety data sheet: Juniper oil. Available at:
http://www.sciencelab.com/xMSDS-Juniper_oil-9924428 [Accessed: 07/12/2009].
SHEKELLE, P.G., HARDY, M.L., MORTON, S.C., MAGLIONE, M., MOJICA,
W.A., SUTTORP, M.J., RHODES, S.L., JUNGVIG, L., GAGNÉ, J. 2003. Efficacy
and safety of ephedra and ephedrine for weight loss and athletic performance: a
meta-analysis. Journal of the American Medical Association, 289(12):1537-1545.
SHENG, L., QIAN, Z., ZHENG, S., XI, L. 2006. Mechanisms of hypolipidaemic
effect of crocin in rats: crocin inhibits pancreatic lipase. European Journal of
SHI, J., YAN, J., LEI, Q., ZHAO, J., CHEN, K., YANG. D., ZHAO, X., ZHANG, Y.
2009. Intragastric administration of evodiamine suppresses NPY and AgRP gene
expression in the hypothalamus and decreases food intake in rats. Brain
Research, 1247:71-78.
SHIH, C.-C., LIN, C.-H., LIN, W.-L. 2008. Effects of Momordica charantia on
insulin resistance and visceral obesity in mice on high-fat diet. Diabetes Research
and Clinical Practice 81:134-143.
SHIGEMATSU, N., ASANO, R., SHIMOSAKA, M., OKAZAKI, M. 2001. Effect of
administration with the extract of Gymnema sylvestre R. Br leaves on lipid
metabolism in rats. Biological and Pharmaceutical Bulletin, 24(6):713-717.
SHIM, W.-S., BACK, H., SEO, E.-K., LEE, H.-T., SHIM, C.-K. 2009. Long-term
administration of an aqueous extract of dried, immature fruit of Poncirus trifoliata
(L.) Raf. suppresses body weight gain in rats. Journal of Ethnopharmacology,
126:294-299.
SHIMODA, H., FURUHASHI, T., KAZUYOSHI, N., TAKAHIKO, N., MASAAKI, O.
2001. Thirteen-week repeat dose oral toxicity study of Salacia reticulata extract in
rats. Japanese Journal of medicine and Pharmaceutical Science, 46(4):527-540.
SHIN, J.-E., HAN, M.J., KIM, D.-H. 2003. 3-Methylethergalangin isolated from
Alpinia officinarum inhibits pancreatic lipase. Biological and Pharmaceutical
Bulletin, 26(6):854-857.
SHIN, J.-E., HAN, M.J., SONG, M.-C., SONG, M.-S., BAEK, N.-I., KIM, D.-H.
2004.
5-Hydroxy-7-(4ʹ-hydroxy-3ʹ-methoxyphenyl)-1-phenyl-3-heptanone:
a
pancreatic lipase inhibitor isolated from Alpinia officinarum. Biological and
62
SHIN, S.S., JUNG, Y.S., YOON, K.H., CHOI, S., HONG, Y., PARK, D., LEE, H.,
SEO, B.I., LEE, H.Y., YOON, M. 2009. The Korean traditional medicine
Gyeongshingangjeehwan inhibits adipocyte hypertrophy and visceral adipose
tissue accumulation by activating PPARα action in rat white adipose tissue.
SHUKLA, Y.J., PAWAR, R.S., DING, Y., LI, X.-C., FERREIRA, D., KHAN, I.A.
2009. Pregnane glycosides from Hoodia gordonii. Phyochemistry, 70:675-683.
SIGMA-ALDRICH. 2006. Material Safety Data Sheet: Tannic acid. Available
from: http://people.ccmr.cornell.edu/~ralph/MSDS/tannic_acid.pdf [Accessed:
07/12/2009]
SINGH, B. 2007. Psyllium as therapeutic and drug delivery agent. International
Journal of Pharmaceutics, 334:1-14.
SMEETS, A.J., WESTERTERP-PLANTENGA, M.S. 2009. The acute effects of a
lunch containing capsaicin on energy and substrate utilisation, hormones and
satiety. European Journal of Nutrition, 48:229-234.
SON, I.S., KIM, J.H., SOHN, H.Y., SON, K.H., KIM, J.-S., KWON, C.-S. 2007.
Antioxidative and hypolipidemic effects of diosgenin, a steroidal saponin of yam
(Dioscorea spp.), on high-cholesterol fed rats. Bioscience Biotechnology and
Biochemistry, 71(12):3063-3071.
SONI, M.G., BURDOCK, G.A., PREUSS, H.G., STOHS, S.J. OHIA, S.E. BAGCHI,
D. 2004. Safety assessment of (-)-hydroxycitric acid and Super CitriMax®, a novel
calcium/potassium salt. Food and Chemical Toxicology, 42:1513-1529.
SOWEMIMO, A.A., FAKOYA, F.A., AWOPETU, I., OMOBUWAJO, O.R.,
ADESANYA, S.A. 2007. Toxicity and mutagenic activity of some selected Nigeria
plants. Journal of Ethnopharmacology, 113:427-432.
SULTAN, M.T., BUTT, M.S., ANJUM, F.M. 2009. Safety assessment of black
cumin fixed and essential oil in normal Sprague dawley rats: Serological and
hematological indices. Food and Chemical Toxicology, 47:2768-2775.
SUZUKI, Y., UNNO, T., USHITANI, M., HAYASHI, K., KAKUDA, T. 1999.
Antiobesity activity of extracts from Lagerstroemia speciosa L. leaves on female
KK-Ay mice. Journal of Nutritional Science and Vitaminology, 45(6):791-795.
TAKAHASHI, T., TABUCHI, T., TAMAKI, Y., KOSAKA, K., TAKIKAWA, Y.,
SATOH, T. 2009. Carnosic acid and carnosol inhibit adipocyte differentiation in
mouse 3T3-L1 cells through induction of phase2 enzymes and activation of
glutathione metabolism. Biochemical and Biophysical Research Communications,
382:549-554.
TAM, S.W., WORCEL, M., WYLLIE, M. 2001. Yohimbine: a clinical review.
Pharmacology and Therapeutics, 91:215-243.
63
TAN, M.-J., YE, J.-M., TURNER, N., HOHNEN-BEHRENS, C., KE, C.-Q., TANG,
C.-P., CHEN, T., WEISS, H.-C., GESING, E.-R., ROWLAND, A., JAMES, D.E.,
YE, Y. 2008. Antidiabetic activities of triterpenoids isolated from bitter melon
associated with activation of the AMPK pathway. Chemistry and Biology, 15:263273.
THOUNAOJAM,
M.,
JADEJA,
R.,
ANSARULLA,
DEVKAR,
R.,
RAMACHANDRAN, A.V. 2009. Dysregulation of lipid and cholesterol metabolism
in high fat diet fed hyperlipidemic rats: protective effect of Sida rhomboidea. roxb
leaf extract. Journal of Health Science, 55(3):413-420.
TOMINAGA, Y., MAE, T., KITANO, M., SAKAMOTO, Y., IKEMATSU, H.,
NAKAGAWA, K. 2006. Licorice flavonoid oil effects body weight loss by reduction
of body fat mass in overweight subjects. Journal of Health Science, 52(6):672-683.
TOMINAGA, Y., NAKAGAWA, K., MAE, T., KITANO, M., YOKOTA, S., ARAI, T.,
IKEMATSU, H., INOUE, S. 2009. Licorice flavonoid oil reduces total body fat and
visceral fat in overweight subjects: a randomized, double-blind, placebo-controlled
study. Obesity Research and Clinical Practice, 3:169-178.
TSUJITA, T., SUMIYOSHI, M., HAN, L.-K., FUJIWARA, T., TSUJITA, J., OKUDA,
H. 2003. Inhibition of lipase activities by citrus pectin. Journal of Nutritional
Science and Vitaminology, 49(5):340-345.
TSUJITA, T., TAKAKU, T. 2007. Lipolysis induced by segment wall extract from
Satsuma mandarin orange (Citrus unshu Mark). Journal of Nutritional Science and
Vitaminology, 53:547-551.
TSUJITA, T., TAKAKU, T. 2008. Lipolysis induced by various citrus fruits. Nippon
Shokuhin Kagaku Kogaku Kaishi, 55(3):102-108.
TULP, O.L., HARBI, N. 2001. Effect of Hoodia plant on food intake and body
weight in lean and obese LA/Ntul//cp-rats. FASEB Journal, 15(4):A404.
TULP, O.L., HARBI, N. 2002. Effect of Hoodia plant on weight loss in congenic
obese LA/Ntul//-cp rats. FASEB Journal, 16(4):A654.
TURNBULL, W.H., THOMAS, H.G. 1995. The effect of a Plantago ovata seed
containing preparation on appetite variables, nutrient and energy intake.
International Journal of Obesity, 19(5):338-342.
TURNER, E.H., LOFTIS, J.M., BLACKWELL, A.D. 2006. Serotonin a la carte:
supplementation
with
the
serotonin
precursor
5-hydroxytryptophan.
Pharmacolopgy and Therapeutics, 109:325-338.
VAN HEERDEN, F.R., VLEGGAAR, R., HORAK, R.M., LEARMONTH, R.A.,
MAHARAJ, V., WHITTAL, R.D. 2002. Pharmaceutical compositions having
appetite suppressant activity. US Patent 6,376,657 B1.
64
VAN HEERDEN, F.R. 2008. Hoodia gordonii: A natural appetite suppressant.
VAN HEERDEN, F.R., HORAK, R.M., MAHARAJ, V.J., VLEGGAAR, R., SENABE,
J.V., GUNNING, P.J. 2007. An appetite suppressant from Hoodia species.
Phytochemistry, 68:2545-2553.
VAN LOON, L.J.C., VAN ROOIJEN, J.J.M., NIESEN, B., VERHAGEN, H., SARIS,
W.H.M., WAGENMAKERS, A.J.M. 2000. Effects of acute (-)-hydroxycitrate
supplementation on substrate metabolism at rest and during exercise in humans.
American Journal of Clinical Nutrition, 72:1445-1449.
VAN VUUREN, S.F., VILJOEN, A.M. 2009. Interaction between the non-volatile
and volatile fractions on the antimicrobial activity of Tarchonanthus camphoratus.
South African Journal of Botany, 75:505-509.
VAN WYK, B.-E, GERICKE, N. 2000. People’s plants. Pretoria: Briza publications.
VAN WYK, B.-E., WINK, M. 2004. Medicinal plants of the world. Pretoria: Briza
publications.
VIJAYAKUMAR, M.V., PANDEY, V., MISHRA, G.C., BHAT, M.K. 2010.
Hypolipidemic effect of fenugreek seeds is mediated through inhibition of fat
accumulation and upregulation of LDL receptor. Obesity, 18(4):667-674.
VIRDI, J., SIVAKAMI, S., SHAHANI, S., SUTHAR, A.C., BANAVALIKAR, M.M.,
BIYANI, M.K. 2003. Antihyperglycemic effects of three extracts from Momordica
charantia. Journal of Ethnopharmacology, 88:107-111.
VOGELS, N., NIJS, I.M.T., WESTERTERP-PLANTENGA, M.S. 2004. The effect
of grape-seed extract on 24h energy intake in humans. European Journal of
Clinical Nutrition, 58:667-673.
WANG, Z.Q., ZUBERI, A.R., ZHANG, X.H., MACGOWAN, J., QIN, J., YE, X.,
SON, L., WU, Q., LIAN, K., CEFALU, W.T. 2007. Effects of dietary fibers on
weight gain, carbohydrate metabolism, and gastric ghrelin gene expression in
mice fed a high-fat diet. Metabolism, 56:1635-1642.
WANG, T., WANG, Y., YAMASHITA, H. 2009. Evodiamine inhibits adipogenesis
via the EGFR-PKCα-ERK signaling pathway. FEBS Letters, 583:3655-3659.
WATANABE, J., KAWABATA, J., KASAI, T. 1999. 9-Oxooctadeca-10,12-dienoic
acids as acetyl-CoA carboxylase inhibitors from red pepper (Capsicum annuum
L.). Bioscience Biotechnology and Biochemistry, 63(3):489-493.
WAUTHOZ, N., BALDE, A., BALDE, E.S., VAN DAMME, DUEZ, P. 2007.
Ethnopharmacology of Mangifera indica L. bark and pharmacological studies of its
main C-glucosylxanthone mangiferin. International Journal of Biomedical and
Pharmaceutical Sciences, 1(2):112-119.
65
WEIDNER, M.S., SIGWART, K. 2000. The safety of a ginger extract in the rat.
WESTERTERP-PLANTENGA, M., DIEPVENS, K., JOOSEN, A.M.C.P, BÉRUBÉPARENT, S, TREMBLAY, A. 2006. Metabolic effects of spices, teas, and caffeine.
Physiology and Behaviour, 89:85-91
WESTERTERP-PLANTENGA, M.S., LEJEUNE, M.P.G.M., KOVACS, E.M.R.
2004. Body weight loss and weight maintenance in relation to habitual caffeine
intake and green tea supplementation. Obesity Research, 13(7):1195-1204.
WESTERTERP-PLANTENGA, M.S., SMEETS, A., LEJEUNE, M.P.G. 2005.
Sensory and gastrointestinal satiety effects of capsaicin on food intake.
International Journal of Obesity, 29:682-688.
WON S.-R., KIM, S.-K., KIM, Y.-M, LEE, P.-H., RYU, J.-H., KIM, J.-W., RHEE, H.I. 2007. Licochalcone A: A lipase inhibitor from the roots of Glycyrrhiza uralensis.
Food Research International, 40:1046-1050.
WHO see WORLD HEALTH ORGANISATION. 2006. Fact sheet no 311.
Available on: http://www.who.int/mediacentre/factsheets/fs311/en/print.html
[Accessed: 2009/09/29].
WYNBERG, R., SCHROEDER, D., CHENNELS, R. (eds.). 2009. Indigenous
peoples, consent and benefit sharing. Lessons from the San-Hoodia case.
London: Springer.
XIE, J.T., ZHOU, Y.-P., DEY, L., ATTELE, A.S., WU, J.A., GU, M., POLONSKY,
K.S., YUAN, C.-S. 2002. Ginseng berry reduced blood glucose and body weight in
db/db mice. Phytomedicine, 9:254-258.
XU, B.J., HAN, L.K., ZHENG, Y.N., LEE, J.H., SUNG, C.K. 2005. In vitro inhibitory
effect of triterpenoidal saponins from Platycodi radix on pancreatic lipase. Archives
of Pharmacal Research, 28(2):180-185.
YAMADA, J., SUGIMOTO, Y., UJIKAWA, M. 1999. The serotonin precursor 5hydroxytryptophan elevates serum leptin levels in mice. European Journal of
YAMAKOSHI, J., SAITO, M., KATAOKA, S., KIKUCHI, M. 2002. Safety evaluation
of proanthocyanidin-rich extract from grape seeds. Food and Chemical Toxicology,
40:599-607.
YAMAMOTO, M., SHIMURA, S., ITOH, Y., OHSAKA, T., EGAWA, E., INOUE, S.
2000. Anti-obesity effects of lipase inhibitor CT-II, an extract from edible herbs,
Nomame Herba, on rats fed a high-fat diet. International Journal of Obesity,
24:758-764.
66
YANG, X.W. 2008. Toxicological assessment on safety of water and 70%
ethanolic extracts of nearly ripe fruit of Evodia rutaecarpa. Zhongguo Zhong Yao
Za Zhi, 33(11):1317-1321.
YOSHIKAWA, M., SHIMODA, H., NISHIDA, N., TAKADA, M., MATSUDA, H.
2002. Salacia reticulata and its polyphenolic constituents with lipase inhibitory and
lipolytic activities have mild antiobesity effects in rats. The Journal of Nutrition,
132:1819-1824.
YOSHIZUMI, K., HIRANO, K., ANDO, H., HIRAI, Y., IDA, Y., TSUJI, T., TANAKA,
T., SATOUCHI, K., TERAO, J. 2006. Lupane-type saponins from leaves of
Acanthopanax sessiliflorus and their inhibitory activity on pancreatic lipase.
Journal of Agricultural and Food Chemistry, 54:335-341.
YOSHIZUMI, K., MUROTA, K., WATANABE, S., TOMI, H., TSUJI, T., TERAO, J.
2008. Chiisanoside is not absorbed but inhibits oil absorption in the small intestine
of rodents. Bioscience Biotechnology and Biochemistry, 72(4):1126-1129.
YU, L., SHIRAI, N., SUZUKI, H. 2007. Effects of some Chinese spices on body
weights, plasma lipids, lipid peroxides, and glucose, and liver lipids in mice. Food
Science and Technology Research, 13(2):155-161.
ZAOUI, A., CHERRAH, Y., MAHASSINI, N., ALAOUI, K., AMAROUCH, H.,
HASSAR, M. 2002. Acute and chronic toxicity of Nigella sativa fixed oil.
Phytomedicine, 9:69-74.
ZHAO, H.L., SIM, J.-S., SHIM, S.H., HA, Y.W., KANG, S.S., KIM, Y.S. 2005.
Antiobese and hypolipidemic effects of platycodin saponins in diet-induced obese
rats: evidence for lipase inhibition and calorie intake restriction. International
Journal of Obesity, 29:983-990.
ZHANG, J., KANG, M.-J., KIM, M.-J., KIM, M.-E., SONG, J.-H., LEE, Y.-M., KIM,
J.-I. 2008. Pancreatic lipase inhibitory activity of Taraxacum officinale in vitro and
in vivo. Nutrition Research and Practice, 2(4):200-203.
ZHANG, J., SHEN, Q., LU, J.-C., LI, J.-Y., LIU, W.-Y., YANG, J.-J., LI, J., XIAO, K.
2010. Phenolic compounds from the leaves of Cyclocarya paliurus (Batal.)
Ijinskaja and their inhibitory activity against PTP1B. Food Chemistry, 119:14911496.
ZHENG, Q., KOIKE, K., HAN, L.-K., OKUDA, H., NIKAIDO, T. 2004. New
biologically active triterpenoid saponins from Scabiosa tschiliensis. Journal of
Natural Products, 67(4):604-613.
67
SECTION 1
Quality control
In this section of the study, the development of rapid and cost-effective quality
control analytical techniques for H. gordonii raw material and products were
investigated.
Quality control of products from botanical origin is of great
importance as they are widely consumed by the general public and it is well-known
that these products often vary in quality and chemical constituent profiles due to
environmental and other factors (Smillie and Khan, 2010:175). In a recent paper
Smillie and Khan (2010:175-185) eloquently described a comprehensive approach
towards identifying and authenticating botanical products.
The available tools
suggested for authentication include macroscopic and microscopic techniques,
genetic fingerprinting techniques, analytical chemical fingerprinting techniques
such as ultra or high performance thin layer chromatography (UPLC/HPLC), liquid
chromatography coupled to mass spectrometry (LC/MS), and high performance
thin layer chromatography (HPTLC), as well as chemometric/metabolomic
techniques such as proton nuclear magnetic resonance 1H-NMR and infrared (IR)
spectroscopy coupled with chemometric analysis (Smillie and Khan 2010:175185).
If an objective would be to satisfy this suggested approach using H. gordonii as an
example, the scope of this study fills some of the current voids. Several analytical
techniques have been developed for H. gordonii and its constituents using
UPLC/LC-MS and HPTLC.
However, this is the first time quantitative
determination of P57 was attempted using other suggested tools such as IR
spectroscopy in combination with chemometric analysis and quantitative HPTLC
analysis.
The traditional as well as new methods are all comprehensively
discussed in the following two chapters.
Microscopic and macroscopic and
genetic fingerprint techniques such as polymerase chain reaction (PCR) have
been attempted by Joshi et al. (2009:253-264) to distinguish between different
species of Hoodia, the common adulterant O. ficus-indica as well as other related
species with limited success.
Macroscopically and microscopically, Hoodia
68
species could be distinguished from each other as well from all the other species.
However, in powder form, differentiation between the species of Hoodia was not
possible due to the similar physical characteristics of the small particles. Genomic
deoxyribonucleic acid (DNA) analysis by PCR was useful to distinguish between
different species of Hoodia and O. ficus-indica. In addition, the PCR method could
distinguish between H. gordonii, H. parviflora and H. ruschii from dried plant
tissue. However, when tablets/capsules were analysed, this method was rendered
useless possibly due to degradation of DNA during manufacturing processes
(Joshi et al., 2009:253-264).
REFERENCES
JOSHI, V., TECHEN, N., SCHEFFLER, B.E., KHAN, I.A. 2009. Identification and
differentiation between Hoodia gordonii (Masson) Sweet ex. Decne., Opuntia ficus
indica (L.) P. Miller, and related Hoodia species using microscopy and PCR.
Journal of Herbs Spices and Medicinal Plants, 15:253-264.
SMILLIE, T.J., KHAN, I.A. 2010. A comprehensive approach to identifying and
authenticating botanical products. Clinical Pharmacology and Therapeutics,
87(2):175-185.
69
CHAPTER 3
Chromatographic techniques
3.1
LIQUID CHROMATOGRAPHY
3.1.1 INTRODUCTION
The most commonly used method to analyse raw plant material for P57 content is
liquid chromatography (LC). As P57 is the only recognised anti-obesity compound
in H. gordonii, the first analytical methods were focused on its quantification only.
When liquid chromatography coupled to ultraviolet detection (LC-UV) is used,
detection of P57 is performed at 220 nm. This absorbance is due to the presence
of a tigloyl group, present in all H. gordonii steroidal glycosides (Jansen et al.,
2008:202). Avula et al. (2006:606) developed a method for the quantification of
P57 using liquid chromatography electrospray ionisation mass spectrometry (LCESI-MS) and liquid chromatography with ultraviolet detection (LC-UV). The LCMS method had a total run time of 45 min including a 5 min wash and 15 min
calibration period while the total run time for the LC-UV method was 55 min.
Product testing revealed that 4 of the 6 products tested did not contain P57 or was
below the detection limit and no P57 was detected in the common adulterant
Opuntia ficus-indica (prickly pear). The P57 results for LC-UV and LC-MS were
very similar but the limit of detection of LC-MS (10 ng/ml) was lower than that of
LC-UV (100 ng/ml) (Avula et al., 2006:607-611).
The same research group
discovered many novel compounds from H. gordonii and subsequently followed up
these discoveries with a chemical fingerprinting technique for Hoodia species and
11 oxypregnane glycosides with LC-UV. Due to the complexity of these plant
extracts and abundance of similar compounds the run time was increased to 80
minutes and various other adjustments were made to the chromatographic
conditions. This method could distinguish between Hoodia species and 23 other
closely related genera, but could not authenticate H. gordonii as all 11 compounds
were present in all Hoodia species (Avula et al., 2007:1527-1530). In a follow-up
study ultra performance liquid chromatography (UPLC) in combination with UV
and MS was used for chemical fingerprinting of Hoodia species (12 hoodigosides)
and related genera and the quantification of P57 in several different dosage forms
70
of dietary supplements claiming to contain Hoodia. The use of UPLC enabled a
considerable reduction in analysis time from 80 min for HPLC to 15 min. The
UPLC-UV method was used for quantification of P57 and all the plant extracts and
products were analysed with LC-MS.
P57 was not detected in 26 of the 35
commercially available products tested. The nine other products had low amounts
of P57 and with UPLC-UV one product tested negative for P57 while its presence
was confirmed with the more sensitive UPLC-MS method.
This paper also
provided extraction methods for the many different dosage forms in which
products claiming to contain Hoodia is sold (Avula et al., 2008:722-730). Another
HPLC-UV quantification method was developed by Janssen et al. (2008:200).
This method can be used to quantify several steroidal glycosides from plant
material and even complex food products such as yoghurt and maize oil.
However, this paper is more focused on method development and quantification
results as such are not shown. The development of these methods contributes
greatly towards the quality control of H. gordonii plant material and products.
However, liquid chromatography methods are expensive, time-consuming, and
laborious and require skilled personnel.
Thus, simpler and faster methods of
quality control of H. gordonii raw materials and products are desirable and were
therefore investigated in this study. For the purposes of this study, plant samples
were sent to an independent analytical laboratory for P57 quantification by LC-MS
analysis. These results were used as reference values in the study to develop
alternative analytical methods for quantification of P57. Further on in the study, an
in-house HPLC method with photodiode array (PDA) detection was developed for
the quantification of P57 in the samples generated from the in vitro transport
studies. The LC-MS and HPLC quantification methods are briefly described in this
chapter.
3.1.2 BACKGROUND AND LITERATURE REVIEW
In the field of phytochemistry, chromatography is the key to obtaining pure
compounds for structure elucidation, pharmacological testing or for development
into therapeutic entities. In addition, it is the main analytical technique used in the
quality control and standardisation of phytochemical products.
Crude plant
extracts are very complex, contain many different molecules and metabolites and
the chemical nature of these constituents varies considerably within a given
71
extract (Wolfender, Rodriguez and Hostettmann, 1998:299-300; Marston,
2007:2785). High performance liquid chromatography (HPLC) has the ability to
separate, tentatively identify and quantify the compounds present in any sample
that can be dissolved in a liquid. Coupled to ultraviolet (UV) photodiode array
detection (PDA) it allows the running of a chromatographic separation with
simultaneous detection at different wavelengths.
The resultant UV spectra of
natural products provide information on the type or class of constituents contained
within a sample.
However, the variability of the physicochemical and spectroscopic parameters of
these compounds causes numerous detection problems. For example, a product
that does not contain a chromophore cannot be detected by an UV PDA detector.
HPLC chromatograms can be used as fingerprints to authenticate plant material
for quality control purposes and different species can be detected by comparison
of their chemical composition (Wolfender, Rodriguez and Hostettmann, 1998: 300;
Marston, 2007:2787-2789).
Since every molecule possesses a specific molecular weight, detection of
compounds using mass spectrometry (MS) can be ideally considered as a
universal detection method.
LC-MS is one of the most sensitive analytical
methods and can be used to obtain information on the molecular weight and
structure of compounds. Different LC/MS interfaces have been developed due to
basic incompatibilities between HPLC and MS.
The compounds within plant
extracts have to be ionised to allow detection with MS, and the complexity of these
crude plant extracts leads to difficulties as not one interface can detect all of the
compounds contained within such an extract. For the analysis of plant chemical
constituents and their secondary metabolites, the main interfaces used include
electrospray ionisation (ESI), atmospheric pressure chemical ionisation (APCI),
thermospray (TSP) and continuous flow-fast atom bombardment (CF-FAB). Each
of these interfaces possesses unique characteristics and ESI is used for detection
of larger polar molecules such as saponins while TSP allows for detection of
smaller moderately polar molecules (Wolfender, Rodriguez and Hostettmann,
1998:299-300; Marston, 2007:2789-2788). The use of LC-hyphenated techniques
plays a significant role as a strategic tool in phytochemical investigations as it
72
provides a great deal of preliminary information about the nature and composition
of crude plant extracts with only a small amount of plant material. Unfortunately
the availability of general LC-MS libraries of spectra of crude plant extract
constituents still presents challenges (Wolfender, Ndjoko and Hostettmann,
2003:453). A schematic diagram of an LC-UV-MS setup is shown in Figure 3.1.
HPLC pump
HPLC pump
Injector
8-port valve
Waste
Cation exchange column
RPLC column
Mass spectrometer
UV detector
Waste
or
fraction
collector
FIGURE 3.1: Schematic illustration of an HPLC apparatus coupled to an ultraviolet
detector and a mass spectrometer (LC-UV-MS)
3.1.3 MATERIALS AND METHODS
3.1.3.1
Plant material, reagents and sample preparation
For the LC-MS method, the pure P57 reference standard (ChromaDexTM Inc
California, USA), was dissolved in 50% acetonitrile, and diluted to concentrations
of 0.005, 0.01, 0.025, 0.05 and 0.1 mg/ml.
The in-house control sample of
authentic unadulterated H. gordonii plant material and the samples (n=145) to be
tested were prepared in the same way for analysis. Ground dry plant material (0.5
g) was weighed off on an analytical balance and extracted in 10 ml of 50%
acetonitrile and 0.1% formic acid for 2 h in an ultrasonic bath (Sonorex digital 10p).
The filtered extract was transferred to an HPLC vial for analysis.
73
For the HPLC method a crude plant extract was prepared by extraction of the
powder (2.4 g) from a commercial H. gordonii capsule product with acetonitrile in
an ultrasonic bath (Sonorex digital 10p) for 10 min at 25 °C. The process was
repeated three times to ensure maximal extraction, the filtered extracts combined
and air-dried. The dried extract was redissolved in methanol for analysis. The
P57 content of the commercial H. gordonii product was determined by LC-MS
analysis in an independent laboratory. Pure P57 (ChromaDexTM Inc, California,
USA) as reference standard was dissolved in methanol in concentrations of 1, 2,
3, 5, 7.5, 10 and 20 µg/ml to develop a calibration curve.
3.1.3.2
Liquid chromatography coupled to mass spectrometry (LC-MS)
P57 was quantified using a Waters (MA, USA) API Q-TOF Ultima system
equipped with a BEH C18 column (1.7 µm, 2.1 x 50 mm). The MS detector was
operated in electron impact mode with the capillary voltage at 3.5 kV and the cone
voltage at 35 kV (positive switching - ES+). The flow rate of the HPLC was 0.4
ml/min, the cone gas flow rate was 50 l/h and the desolvation gas flow rate was
350 l/h. The source temperature was 100 °C and the desolvation temperature
350 °C. The accurate mass (861.5 m/z) was extracted in ES+ mode within a mass
range of 200 to 1800 m/z. The injection volume was 2 µl. Gradient elution was
used with 0.1% formic acid in water (Solvent A) and acetonitrile (Solvent B) as
mobile phases.
The gradient started with 100% solvent A, changed to 20%
solvent B over 0.5 min, to 100% solvent B over 11.5 min, followed by a 2 min
isocratic step and a return to initial conditions for 1 min for a total run time of 15
min. Masslynx® (version 4.2) software was used for data analysis. The extracted
mass chromatogram of m/z 861.5 which corresponds to the [M-H2O+H]+ was used
for quantification.
3.1.3.3
High performance liquid chromatography (HPLC)
An HPLC system consisting of a Waters (MA, USA) 2690 separation module and a
Waters 996 photodiode array detector was used and separation was achieved
using a Gemini C18 column (250 x 4.6 mm; 5 µm particle size; Phenomenex)
equipped with a guard column (4 mm x 3.0 mm, Phenomenex). The mobile phase
consisted of water containing 0.05% acetic acid (A) and acetonitrile (B). Gradient
elution was used as follows: 100% A at 0 min, adjusted over 40 min to 20% A and
74
80% B. Each run was followed by a 5 min wash with 100% B and an equilibration
period of 8 min.
The flow rate was 1.0 ml/min, the column operated at a
temperature of 30 °C and the detection wavelength was 220 nm.
The
chromatographic data was collected and analysed with Empower® software.
3.1.4 RESULTS
3.1.4.1
H. gordonii is a complex plant containing an abundance of chemical constituents
as illustrated in Figure 3.2, which is an example of the LC-MS chromatogram for
the plant material used as the in-house control standard in this method.
FIGURE 3.2: LC-MS chromatogram of the plant material used as the H. gordonii
control standard for the LC-MS analytical method
An example of an extracted mass (861.5 m/z) chromatogram of both P57 (A) and
the H. gordonii control standard material (B) is shown in Figure 3.3. An example
of a calibration curve calculated from the area under the peaks for the respective
reference standards is shown in Figure 3.4. A good correlation coefficient (R2) of
0.9991 was obtained for the calibration curve.
75
FIGURE 3.3: An extracted mass (861.5 m/z) chromatogram of P57 (A) and the H.
gordonii control standard plant material (B)
FIGURE 3.4: Calibration curve for the P57 reference standard as obtained by LCMS
The percentage content of P57 in several (n=145) H. gordonii plant material
samples as determined by LC-MS is shown in Figure 3.5. It can be seen that
76
there is a high variation in percentage P57 content in stem samples of H. gordonii
plant materials collected from different sites, which range from 0.000-0.430%.
FIGURE 3.5: The percentage of P57 determined with LC-MS in H. gordonii stem
samples (n = 145)
3.1.4.2
High performance liquid chromatography (HPLC)
An example of the HPLC chromatogram for H. gordonii plant material from a
commercial capsule product that was used in the transport study is shown in
Figure 3.6. Figure 3.7 shows the calibration curve for the P57 reference standard
for dilutions of 1, 2, 3, 5, 7.5, 10 and 20 µg/ml. A good correlation coefficient of
0.9967 was obtained for this calibration curve.
3.1.5 CONCLUSIONS
The correlation coefficients for the calibration curves for the LC-MS method
(R2=0.9991) as well as the HPLC method (R2 = 0.9967) indicated that good
linearity was obtained for both these analytical methods over concentration ranges
of P57 applicable to the samples that were analysed in this study. Thus, the LCMS method was acceptable to quantify P57 in H. gordonii raw materials and
products while the HPLC method was an acceptable analytical method to quantify
P57 concentrations in the transport studies.
77
0.40
0.35
0.30
AU
0.25
0.20
0.15
0.10
0.05
0.00
-0.05
-0.10
5.00
10.0
0
15.0
0
20.0
0
25.0
0
Time (min)
30.0
0
35.0
0
40.0
0
45.0
0
FIGURE 3.6: An HPLC chromatogram of the H. gordonii plant material from a
commercial capsule
FIGURE 3.7: The calibration curve for the P57 reference standard with the HPLC
method
78
3.2
QUALITATIVE HIGH PERFORMANCE THIN LAYER
CHROMATOGRAPHY (HPTLC)
3.2.1 INTRODUCTION
The high consumer demand and limited geographical distribution of H. gordonii
leads to gross adulteration of up to 75% of H. gordonii products. Allegedly some
of these products contain small quantities of real H. gordonii material blended with
adulterant plant materials like Opuntia ficus-indica (prickly pear), Agave
americana, Aloe ferox and Cereus jamacaru (Adams, 2006:S.a.; Avula et al.,
2006:606; Widmer, Reich and DeBatt, 2008:21).
Most companies selling H.
gordonii products are acutely aware of the high rate of adulteration of these
products and tend to highlight positive analytical test results accompanied by an
example of a Convention on International Trade in Endangered Species (CITES)
certificate to demonstrate authenticity. Some companies also indicate that their
products contain high percentages of P57.
The triterpene glycoside (named P57) was first isolated by Van Heerden et al.
(1998) who pioneered the investigation into Hoodia species, and is currently the
only perceived active compound in H. gordonii.
Therefore the quality of H.
gordonii raw material and products is determined in part by the presence of P57.
The preferred analytical method currently used for determination of P57 content is
high performance liquid chromatography (HPLC) coupled to a mass spectrometer
(LC-MS) or HPLC coupled to a photodiode array detector (LC-UV) (Widmer, Reich
and DeBatt, 2008:21). However, LC-MS requires skilled personnel and in addition
it is both laborious and costly. Thin layer chromatography (TLC) is an option that
has distinct advantages over several other chromatographic methods such as its
simplicity, low cost, high sample capacity, and rapid availability of results. It is
unique due to the fact that it presents the results as an easy-to-interpret image or
chemical fingerprint (Marston, 2007:2787).
Several papers discussing the authentication of H. gordonii and to detect the
presence of P57 have been published. Widmer, Reich and DeBatt (2008:21-25)
first investigated the HPTLC method used by Alkemists Pharmaceuticals, wellknown for the authentication of H. gordonii raw material before embarking on the
development of their own method. Extraction from raw material and development
79
conditions were optimised and enabled H. gordonii to be distinguished from
H. currorii, H. parviflora and O. ficus-indica. They also concluded that β-sitosterol
can be used to check for the presence of P57 as it has the same Rf value
(Widmer, Reich and DeBatt, 2008:23-24).
This was criticised in a paper by
Rumalla et al. (2008: 3959) as they mention that using only P57 or a non-specific
compound like β -sitosterol may not establish the complete chromatographic
identity of H. gordonii which comprises of at least two chemically distinct steroidal
aglycones. The elegant method developed by Rumalla et al. (2008:3961-3964)
provided a characteristic fingerprint for Hoodia species, but not H. gordonii
specifically, with Rf values for 11 pregnane glycosides used as standards. The
chemical fingerprint of H. gordonii was compared with 24 closely related plant
species and none was comparable to H. gordonii.
This thesis provides an alternative rapid and simple HPTLC method to
authenticate H. gordonii raw material and products with good band separation
confirmed by LC-MS as an accurate and reliable method for the confirmation of
the presence of P57 in raw material and product samples. The new mobile phase,
the use of Liebermann-Burchard reagent and the specificity for P57 which is still
the main measure of quality as determined by industry standards distinguishes this
method from previously published methods.
3.2.2.1
Analysis of plant material
The quality of plant material is traditionally divided into two aspects. Qualitative
analysis deals with the authentication of material while quantitative analysis is
focused on the measurement of certain biomarkers or active substances (Reich
and Schibli, 2006:1).
The complexity of plant material presents difficulty in
analysis as plants contain numerous constituents including active and inactive
compounds as well as unknown compounds.
In addition, there is an innate
variability in the composition of plant material due to differences in maturity,
geographical origin and environmental factors such as mineral nutrition and
exposure to stress. Chemical composition differences from grower to grower as
well as from crop to crop are common and postharvest handling and storage also
has an effect (Lazarowych and Pekos, 1998:497; Reich and Schibli, 2006:3).
80
For
herbal
extracts,
regulatory
authorities
often
recommend
fingerprint
chromatography for proper identification purposes and the British Herbal
Pharmacopoeia for example emphasises the use of TLC profiles for this purpose
(Lazarowych and Pekos, 1998:497; Marston, 2007:2787). An authentic botanical
sample
which
is
extracted
and
chromatographed
provides
a
unique
chromatographic fingerprint and primary reference for comparison against test
samples. TLC is the ideal screening method as it not only provides authentication
or identification with determination of adulteration in some cases but also
quantitative or semi-quantitative determination of biomarkers (Reich and Schibli,
2006:4; Marston, 2007:2787).
HPTLC is an improvement on TLC in that the
stationary phase particle size on the plate is much smaller, 5 µm as opposed to 12
µm, providing greater separation efficiency, reproducibility, improved detection,
and faster separation over shorter developing distances (Marston, 2007:2787).
3.2.3.1
Plant material and reagents
A number of H. gordonii stem samples were collected at various locations
throughout South Africa and Namibia from natural populations as well as from
cultivated sites.
The aerial parts of Opuntia ficus-indica, Aloe ferox, Cereus
jamacaru and Agave americana from wild populations were collected in South
Africa.
The samples were hand-picked, sliced and air-dried.
The dried plant
material was ground with a Retsch® 400 MM ball mill (Haan, Germany) at a
frequency of 30 Hz for 90 s. H. gordonii containing products (capsules, tablets,
chips, spray, topical gel, patch, fruit bar, compote, instant drink mix, oral strips)
were purchased from various sources including retail stores in South Africa as well
as internet purchases from international companies. The P57 reference standard
was purchased from ChromaDexTM Inc (California, USA).
3.2.3.2
High performance thin layer chromatography (HPTLC) analysis
HPTLC analysis was performed on a CAMAG semi-automated HPTLC system
(Muttenz, Switzerland) including the automatic TLC Sampler 4 (connected to a
nitrogen tank), automatic developing chamber ADC2, chromatogram immersion
device, TLC plate heater III, and documentation device Reprostar 3 (Figure 3.8)
with winCATS version 1.4.4.6337 planar chromatography manager software.
81
FIGURE 3.8: A diagram of the CAMAG semi-automated HPTLC system including
the
automatic
TLC
Sampler
4,
automatic
developing
chamber
ADC2,
chromatogram immersion device, TLC plate heater III and documentation device
Reprostar 3
The powdered raw plant material and most products were extracted with
acetonitrile in an ultrasonic bath (Sonorex digital 10p) at 100% power and 25 °C
for 20 min and filtered.
This process was repeated three times with fresh
acetonitrile to maximise extraction efficiency and the filtrates were combined and
air-dried. In a preliminary study, the combination of acetonitrile and sonication
was determined to be a good extraction method for compounds from H. gordonii.
The buccal spray product was placed in a vacuum oven (Vismara) at 40 °C to dry
by evaporation.
The compote, fruit bar and oral strips were extracted with a
mixture of acetonitrile and water (50:50) to aid in dissolution and release of P57
from poorly soluble excipients used in these products. The dried extracts were
made up to a concentration of 50 mg/ml and 8 µl were spray-applied with a 25 µl
syringe together with 2 µl of a 1 mg/ml solution of the reference standard P57 as
10 mm bands on 20 x 10 cm silica gel plates (Silica gel 60 F254, Merck, Germany).
No chamber saturation was performed prior to development. Temperature and
humidity were not controlled via the HPTLC system but the experiments were
carried out in a temperature controlled laboratory.
The bands were sprayed
10 mm from the bottom of the plate, with the first position at 30 mm from the side
82
edge. After a pre-drying phase of 5 min, ascending development was carried out
over a path of 85 mm with the mobile phase (15 ml) consisting of toluene,
chloroform and ethanol (40:40:12.5 v/v/v).
This mobile phase and chamber
conditions resulted in plates with good resolution and well-defined spots. A 5 min
drying
phase
followed
development.
To
visualise
the
chromatogram,
derivatisation was performed by immersion of the plate in Liebermann-Burchard
(LB) reagent and heating at 100 °C for 5-10 min. This reagent is traditionally used
for detection of triterpenes and steroids (saponins, bitter principles) and therefore it
was expected that P57 (a triterpene glycoside) would be clearly visualised. This is
the first time LB reagent has been used as derivatising agent in an HPTLC quality
control method for H. gordonii raw material and products.
LB reagent was
prepared by adding 10 ml of acetic anhydride and 10 ml of concentrated sulphuric
acid to 100 ml of absolute ethanol while cooling on ice (Wagner and Bladt,
1996:362). The images were captured under ultraviolet light at 366 nm.
The limit of detection for P57 was determined by spotting dilutions containing 5 µg,
3 µg, 2 µg, 1.5 µg, 1 µg, 0.5 µg, 0.25 µg 0.15 µg and 0.1 µg per 5 µl (spot volume),
respectively. The smallest amount of the analyte that provided a visual response
was considered the limit of detection for P57. To establish that the co-elution of
compounds (especially P57 analogues) does not occur in the area of the reference
standard and to confirm that the bands perceived to be P57 were indeed P57,
preparative TLC was done. Several plates were spotted and developed using a
sample with a known high quantity of P57 as determined by LC-MS. The bands
corresponding to the reference compound (P57) were scraped off from the plates,
washed with methanol and analysed by LC-MS.
3.2.3.3
analysis
P57 was quantified using a Waters (MA, USA) API Q-TOF Ultima system
equipped with a BEH C18 column (1.7µm, 2.1 x 50 mm). The MS detector was
operated in electron impact mode with the capillary voltage at 3.5 kV and the cone
voltage at 35 kV (positive switching - ES+).
The flow rate of the HPLC was
0.4 ml/min, the cone gas flow rate was 50 l/h and the desolvation gas flow rate
was 350 l/h. The source temperature was 100 °C and the desolvation temperature
83
350 °C. The accurate mass (861.5 m/z) was extracted in ES+ mode within a mass
range of 200 to 1800 m/z. The injection volume was 2 µl. Gradient elution was
used with 0.1% formic acid in water (Solvent A) and acetonitrile (Solvent B) as
mobile phases.
The gradient started with 100% solvent A, changed to 20%
solvent B over 0.5 min, to 100% solvent B over 11.5 min, followed by a 2 min
isocratic step and a return to initial conditions for 1 min for a total run time of
15 min. The LC-MS data was analysed using Masslynx® (version 4.2) software.
The specific mass of 861.5 (m/z) was used for quantification of P57.
3.2.4 RESULTS AND DISCUSSION
3.2.4.1
High performance thin layer chromatography (HPTLC)
Figure 3.9 displays the HPTLC chromatogram for determining the limit of detection
for P57.
The smallest amount spotted that provided a visual response was
considered the limit of detection. Although there is a band visible in track 9, it is so
unclear that the limit of detection should be considered 0.15 µg (track 8) or even
0.25 µg (track 7). This indicated that the HPTLC method will be able to confirm
the presence of P57 even in highly diluted samples.
The P57 band eluted
consistently at a retention front (Rf) value of 0.42 on consecutive days, indicating
acceptable precision and reproducibility of the method.
FIGURE 3.9: The HPTLC chromatogram used to determine the limit of detection
for P57. P57 was spotted at 5 µg (track 1), 3 µg (track 2), 2 µg (track 3), 1.5 µg
(track 4), 1 µg (track 5), 0.5 µg (track 6), 0.25 µg (track 7), 0.15 µg (track 8) and
0.1 µg (track 9)
84
Figures 3.10-3.12 display HPTLC chromatograms for several weight loss
consumer products claiming to contain H. gordonii viewed under UV light at 365
nm with the first track being raw H. gordonii standard material and the last track
being the P57 reference standard for each figure.
FIGURE 3.10: An HPTLC plate viewed under UV at 365 nm. Track 1: H. gordonii
standard raw material, track 2-8: H. gordonii commercial products (1-7) from
different manufacturers and track 9: the P57 reference standard
On comparison with H. gordonii raw material (track 1), it is evident that all the
products presented in Figure 3.10 contain H. gordonii plant material and P57
where the lowest concentrations were obtained for Product 3 (track 4), Product 4
(track 5), Product 6 (track 7) and Product 7 (track 8) and the highest concentration
for product 5 (track 6) as detected qualitatively through visual inspection of band
intensity.
In Figure 3.11 it is shown that products 13 and 14 (tracks 7 and 8) contain the
lowest concentration of P57 and that all the products presented in this figure
contain H. gordonii.
85
It is evident that none of the products presented in Figure 3.12 contain H. gordonii
nor P57 (or negligible amounts below the limit of detection) based on comparison
with the standard H. gordonii raw material chromatogram (chemical fingerprint) in
track 1, and these include Product 15 topical gel (0.006%), Product 16 transdermal
patch (0.000%), Product 17 fruit bar (0.001%), Product 18 compote (0.003%),
Product 19 instant drink mix (0.000%) and Product 20 oral strips (0.000%). These
results were confirmed by the LC-MS results (Table 3.1)
86
TABLE 3.1: Comparison of the P57% LC-MS results and HPTLC results
Figure
Track
3.10
1
3.11
3.12
3.13
3.14
Sample description
LC-MS result
%P57
HPTLC result
+ or – for P57
Namibian cultivated raw material
0.186
+
2
Product 1 (Capsules)
0.248
+
3
0.162
+
4
0.131
+
5
0.080
+
6
0.304
+
7
0.191
+
8
0.102
+
1
0.186
+
2
0.134
+
3
0.134
+
4
Product 10 (Tablets)
0.064
+
5
0.180
+
6
0.169
+
7
Product 13 (Chips – sliced H.
gordonii)
0.058
+
8
Product 14 (Buccal spray)
0.048
+
1
0.186
+
2
Product 15 (Topical gel)
0.006
-
3
Product 16 (Transdermal patch)
0.000
-
4
Product 17 (Fruit bar)
0.001
-
5
Product 18 (Compote)
0.003
-
6
Product 19 (Instant drink mix)
0.000
-
7
Product 20 (Oral strips)
0.000
-
1
South African wild
0.127
+
2
Namibian wild
0.161
+
3
South African cultivated
0.115
+
4
Namibian cultivated
0.186
+
5
2 yr old actively growing cultivated
0.131
+
6
2 yr old dormant cultivated
0.288
+
7
3 yr old actively growing cultivated
0.048
+
8
3 yr old dormant cultivated
0.112
+
1
0.186
+
3.15
1
Namibian wild raw material
0.161
+
+ = visually detectable on the HPTLC plate; - = not visually detectable on the HPTLC plate
87
Figure 3.13 is an HPTLC plate of two wild population samples from different
localities and samples of differing maturity and growing stages. There are some
qualitative differences in terms of band intensity between the samples for example
on comparison of track 2, a wild population sample and track 4, a cultivated site
sample.
This can be explained by the fact that certain phytoconstituents in
cultivated plants can sometime be much lower compared to those in wild
populations of the same plant species.
Plants manufacture secondary
metabolites, which are mainly responsible for the medicinal properties of the plant,
under certain conditions of stress in their natural environment which may not be
expressed when cultivated under more controlled conditions (Schippmann,
Leaman and Cunningham, 2006:81). In addition, the two wild samples (tracks 1
and 2) showed a red band situated right below the P57 band which was absent in
all the cultivated samples (tracks 3 to 8). It was visually evident from the intensity
of the bands that both the 3 yr old plants (tracks 7 and 8) contained less P57
compared to the 2 yr old plants (tracks 5 and 6).
This difference in the
concentration of P57 was confirmed with LC-MS (Table 3.1).
Track 1: wild
population sample of H. gordonii from South Africa, track 2: wild population sample
of H. gordonii from Namibia, track 3: cultivated sample of H. gordonii from South
Africa, track 4: cultivated sample of H. gordonii from Namibia, track 5: 2 year old
actively growing plant sample, track 6: 2 year old dormant plant sample, track 7: a
3 year old actively growing cultivated plant, track 8: a 3 year old dormant cultivated
plant and track 9: the P57 reference standard
88
Figure 3.14 is an HPTLC plate spotted with H. gordonii raw plant material and H.
gordonii raw plant material with gradually increasing adulteration concentrations of
O. ficus-indica plant material.
standard raw material, track 2-7: the same sample of H. gordonii standard raw
material as for track 1 adulterated with O. ficus-indica in concentrations of 10%,
20%, 40%, 50 %, 60%, 80%, respectively, track 8: O. ficus-indica raw material,
track 9: the P57 reference standard
It is evident that P57 was present in the samples spotted from track 1-7, but the
intensity decreased as the percentage of adulteration increased up to a barely
visible band with low intensity in track 7. Furthermore, O. ficus-indica (track 8)
clearly does not contain all the compounds found in H. gordonii (including P57)
and displays only eight bands for this mobile phase. Another visible difference
between the composition of H. gordonii plant material and O. ficus-indica plant
material is at an Rf value of 0.4 where H. gordonii shows a black band and O.
ficus-indica yielded a red band.
Figure 3.15 shows HPTLC chromatograms of plant materials allegedly used to
adulterate H. gordonii products, including O. ficus-indica, A. ferox, C. jamacaru
and A. americana.
It is evident from these plates that it would be easy to
distinguish A. ferox, C. jamacaru and A. americana from H. gordonii raw material
89
with HPTLC but that adulteration with O. ficus-indica would be more difficult to
detect as shown in Figure 3.14.
standard raw material, Tracks 2 and 3: Opuntia ficus-indica samples, Tracks 4 and
5: Aloe ferox samples, Track 6: Cereus jamacaru sample, Track 7: Agave
americana sample and Track 8: the P57 reference standard
3.2.4.2
Figure 3.16 displays the LC-MS chromatograms used to establish that co-elution
of compounds at the specific Rf value (0.42) of P57 does not occur.
Chromatogram A in this figure shows the P57 reference standard and
chromatogram B shows the band isolated with preparative TLC. It is evident from
LC-MS analysis that the major peak obtained for the extracted compounds was
P57 with an accurate mass of 861.5 m/z.
90
6.76
100
%
A
6.94
0
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
13.00
14.00
15.00
6.77
100
%
B
6.95
6.66
0
1.00
2.00
3.00
4.00
5.00
6.00
7.24
7.00
14.57
8.00
9.00
10.00
11.00
12.00
13.00
14.00
Time
15.00
FIGURE 3.16: LC-MS chromatograms of the P57 standard (A) and the preparative
TLC sample (B)
The mass spectra for each of the chromatograms in Figure 3.16 are displayed in
Figure 3.17.
313.190
100
A
%
313.209
861.435
457.256
314.201
761.388
314.216
458.260
315.195
381.209
413.240
557.316
466.233
862.440
761.417
762.392
601.327
763.408
862.503
901.423
829.408
981.390
0
313.190
100
%
B
861.435
457.256
314.201
761.388
458.260
314.216
315.195
0
300
350
381.209 413.240
400
557.316
466.228
450
500
550
862.440
762.392
601.327
600
862.503
763.408
650
700
750
800
829.402
850
901.423
900
950
m/z
1000
FIGURE 3.17: Mass spectra of the P57 reference standard (A) and the preparative
TLC sample (B)
91
Co-elution of bands is a common problem with HPTLC analysis of complex
samples such as plant material but clearly this method establishes that P57 elutes
as a single compound at Rf 0.42. This was confirmed with preparative TLC and
LC-MS analysis indicating specificity of this method. Thus, the HPTLC method
has the ability to clearly confirm the presence of P57 with the specified mobile
phase and derivatising agent used. To validate that P57 was indeed present in all
the H. gordonii raw material and product samples that were spotted on the TLC
plates, the concentration of P57 was determined with LC-MS and the results are
collectively presented in Table 3.1.
3.2.5 CONCLUSIONS
The quality of commercial H. gordonii-containing products is a cause for concern
especially because these products are consumed on a daily basis by many people
worldwide. The lack of quality in terms of P57 content in several commercially
available products is highlighted in this study. Some of the products investigated,
especially the topical products and some oral dosage forms, contain very low
concentrations or no P57 at all. Since P57 is currently considered to be the only
active ingredient in H. gordonii for weight-loss, this simple HPTLC technique
provides a fast and cost-effective method for screening products for the presence
of P57.
Furthermore, developing new rapid and accurate methods for basic
quality control could contribute to the distribution of only high quality raw material,
and consequently, more safe high quality consumer products.
This technique
could be developed further to perform the quantitative determination of P57 in raw
materials and products.
3.3
QUANTITATIVE HIGH PERFORMANCE THIN LAYER
CHROMATOGRAPHY (HPTLC)
3.3.1 INTRODUCTION
Several HPTLC-based quality control methods for H. gordonii raw material and
products have been developed as discussed in the previous section.
To the
author’s knowledge quantitative HPTLC has not been used before to quantify the
amount of P57 within H. gordonii products. It has been used however to quantify
marker molecules found in several plant species. Eugenol and gallic acid from
Syzygium aromaticum (L.) Merr. et Perry (Myrtaceae) (Pathak et al., 2004:241),
92
harpagoside from Harpagophytum procumbens DC (Pedaliaceae) (Günther and
Schmidt 2005:817), luteolin from Thymus vulgaris L. (Lamiaceae) (Bazylko and
Strzelecka,
2007:931)
and
steviol
glycosides
(steviolbioside,
stevioside,
rebaudioside-A) from Stevia rebaudiana Bertoni (Asteraceae) (Jaitak et al.,
2008:790) amongst others have all been successfully quantified from powdered
samples using HPTLC coupled to densitometry. The techniques traditionally used
to quantify P57 (LC-MS, LC-UV) are laborious, time-consuming and expensive.
HPLTC-densitometric quantification is a method of choice due to high sample
throughput at lower operating costs and shorter analysis times. Quantification of
the important biomarker, P57, from H. gordonii samples will contribute greatly to
quality control of raw material and products.
Densitometry involves quantifying the bands on a chromatogram by measuring the
amount of light reflected from the plate to a detector. It is particularly useful and
accurate for the quantification of marker compounds and is sensitive because of
its spectral selectivity.
Light in the range of 190-800 nm from different light
sources can be used to measure absorption or fluorescence with various light
sources such as a deuterium lamp (190-450 nm), tungsten lamp (370-800 nm) and
a mercury vapour lamp with high-energy output at several individual wavelengths
(including 254 and 366 nm). A narrow beam of light of a specified wavelength and
geometrical dimension is allowed to reach the plate and after detection the
chromatogram data is integrated and mathematically evaluated to produce peak
height and area for each band or substance zone.
A calibration function can be calculated when a set of standards are
chromatographed together with the samples to be quantified. The measurements
of the unknown compound will then be relative to the standard. The functional
relationship between the signal and the amount of substance is more complicated
in TLC since light is reflected from both the surface of the plate as well as the
surrounding area. With fluorescence measurement the signal is proportional to
the intensity of the light used for excitation and a simple linear equation usually
applies. For most absorption measurements the data is best fitted by polynomial
functions (Reich and Schibli, 2006:61, 63, 116).
93
3.3.3.1
H. gordonii containing products (capsules) were purchased from various sources
including retail stores in South Africa as well as internet purchases from
international companies.
The P57 reference standard was purchased from
ChromaDexTM Inc (California, USA).
3.3.3.2
Quantitative high performance thin layer chromatography analysis
This quantitative method was based upon the previously discussed qualitative
HPTLC method (3.2.3.2) used to detect the presence of P57 with minor
modifications.
HPTLC analysis was performed on a CAMAG semi-automated
HPTLC system (CAMAG, Muttenz, Switzerland) including the automatic TLC
Sampler 4 (connected to a nitrogen tank), automatic developing chamber ADC2,
chromatogram immersion device, TLC plate heater III, documentation device
Reprostar 3 and TLC Scanner 3 (Figure 3.20) with winCATS version 1.4.4.6337
planar chromatography manager software.
FIGURE 3.18: A photograph of the CAMAG TLC scanner 3 used in the
quantification of P57 from commercial samples (Photograph by I Vermaak)
94
The powdered raw plant material (1 g) from the commercial capsules were
extracted with acetonitrile in an ultrasonic bath (Sonorex digital 10p) at 100%
power and 25 °C for 20 min and filtered. This process was repeated three times
with fresh acetonitrile to maximise extraction efficiency, the filtrates combined and
air-dried. The dried extracts of four products were made up to a concentration of
10 mg/ml, sonicated for 1 min to facilitate dissolution and 3000 nl were sprayapplied in duplicate under a flow of nitrogen gas with a 25 µl syringe together with
four concentrations (65.50, 75.00, 100.00, 125.00 ng) of the P57 reference
standard (0.125 mg/ml). The 6 mm bands were applied 25 mm from the sides and
10 mm from the bottom on 20 x 10 cm silica gel plates (Silica gel 60 F254, Merck,
Germany). Fresh samples were prepared daily and the plates were washed with
methanol prior to use.
The developing chamber was saturated with 25 ml of
mobile phase and a saturation pad for 20 min ±30°C and conditioned to 47%
relative humidity (RH) using potassium thiocyanate (KSCN). After a plate predrying phase of 5 min, ascending development was carried out over a path of 85
mm with the mobile phase (15 ml) consisting of toluene, chloroform and ethanol
(40:40:12.5 v/v/v).
The bands were visualised by immersion of the plate in
Liebermann-Burchard (LB) reagent and heating at 100 °C for 5-10 min.
LB
reagent was prepared daily by adding 10 ml of acetic anhydride and 10 ml of
concentrated sulphuric acid to 100 ml of absolute ethanol while cooling on ice
(Wagner and Bladt, 1996:362). Image acquisition and archiving was done with the
Reprostar 3 documentation device at a wavelength of 366 nm.
Quantitative evaluation of the plate was performed with a mercury lamp (Hg) in
remission-fluorescence mode with a K400 optical filter at a wavelength of 366 nm.
The slit dimensions were 4.00 x 0.30 mm, scanning speed 20 mm/s and data
resolution 100 µm/step. The results were compared in terms of height as well as
area and the two values were averaged to evaluate the method. The method was
validated with respect to intra-day precision (repeatability), inter-day precision
(intermediate precision) and accuracy. Intra-day precision is the precision of the
method over a short interval of time (day) under the same operating conditions
while intermediate precision is the variation on different days. The analyses were
done three times on one day to determine intra-day precision and on two
consecutive days to determine inter-day precision. Accuracy of the standards was
95
expressed as a percentage by dividing the observed concentration by the
theoretical concentration spotted multiplied by 100 (Jaitak et al., 2008:790).
3.3.4 RESULTS AND DISCUSSION
3.3.4.1
Quantitative high performance thin layer chromatography analysis
The chamber conditions and mobile phase consisting of toluene, chloroform and
ethanol (40:40:12.5 v/v/v) gave good resolution of P57. In addition, the Rf value of
0.49 was consistent on the same day as well as on consecutive days indicating
precision and reproducibility of the method.
Figure 3.19 is an example of a
chromatogram that was used for the densitometric quantification of P57 from H.
gordonii products. The test samples were spotted in duplicate next to each other
and the four reference standard levels of P57 (65.50, 75.00, 100.00, 125.00 ng)
were spread over the plate.
FIGURE 3.19: An example of an HPTLC chromatogram that was used for the
quantification of P57 from H. gordonii commercial products. Track 1: 65.50 ng
P57, Track 2-3: Product 1, Track 4: 75.00 ng P57, Track 5-6: Product 2, Track 7:
100.00 ng P57, Track 8-9: Product 3, Track 10: 125.00 ng P57, Track 11-12:
Product 4
Figure 3.20 is an example of the baseline graph for one track and the
corresponding integrated peak where P57 is identified.
96
FIGURE 3.20: An example of the baseline chromatogram for one track (A) and the
corresponding integration of the P57 peak as indicated (B)
Figures 3.21 and 3.22 show the calibration curves for four standard levels of P57
and the four H. gordonii products in duplicate in terms of graph height and area,
respectively.
FIGURE 3.21: The linear calibration curve for four standard levels of P57 (black)
and the four H. gordonii products (green) in duplicate in terms of peak height.
97
FIGURE 3.22: The linear calibration curve for four standard levels of P57 (black)
and the four H. gordonii products (green) in duplicate in terms of peak area
The calibration curve of the peak area at 366 nm (y) versus concentration (x) was
found to be linear in the concentration range of 65.50-125.00 ng for P57 with good
correlation coefficients of 0.9706-0.9992 based on peak height and 0.9720-0.9993
based on peak area.
From Table 3.2 it can be seen that the amount of P57 detected for the same
sample varied throughout the day (n=3) for product 1 from 57.19-67.33 ng, product
2 from 77.47-92.10 ng, product 3 from 62.86-74.72 ng and product 4 from 60.1470.94 ng. The results for the first test of the day were consistently higher than for
the other two testing times.
Table 3.3 displays the inter-day precision results for
the duplicates of all the test samples. The amount of P57 detected for the same
sample varied across the two days for product 1 from 63.19-67.37 ng, product 2
from 65.64-74.72 ng, product 3 from 79.27-92.10 ng and product 4 from 70.4493.56 ng. There was a consistent correlation between height and area for all the
test samples on same day as well as on consecutive day testing.
98
TABLE 3.2: Peak height and area comparison for the calculation of intra-day
precision
Product
Duplicate
Height
(ng)
Area
(ng)
Average of height
and area
(ng)
Average
(total)
(ng)
Time 1
1
2
3
4
1
70.14
72.05
71.01
2
62.96
64.32
63.64
1
93.11
95.02
94.07
2
89.50
90.76
90.13
1
75.43
76.87
76.15
2
72.71
73.86
73.29
1
72.04
74.45
73.25
2
67.04
70.19
68.62
67.33
92.10
74.72
70.94
Time 2
1
2
3
4
1
57.17
57.66
57.42
2
56.98
56.93
56.96
1
80.14
78.52
79.33
2
76.87
74.94
75.91
1
64.77
64.01
64.39
2
61.36
61.30
61.33
1
62.47
64.34
63.41
2
55.84
57.87
56.86
57.19
77.62
62.86
60.14
Time 3
1
2
3
4
1
60.55
62.03
61.29
2
54.89
56.26
55.58
1
79.06
80.61
79.84
2
74.55
75.63
75.09
1
68.02
69.89
68.96
2
67.53
69.83
68.68
1
67.96
69.28
68.62
2
61.51
62.86
62.19
99
58.44
77.47
68.82
65.41
TABLE 3.3: Peak height and area comparison for the calculation of inter-day
precision
Product
Duplicate
Height
(ng)
Area
(ng)
Average of height
and area
(ng)
Average
(total)
(ng)
Day 1
1
2
3
4
1
67.17
70.72
68.95
2
56.32
58.51
57.42
1
64.55
66.86
65.71
2
64.59
66.52
65.56
1
76.45
82.11
79.28
2
74.14
78.38
76.26
1
95.56
104.95
100.26
2
83.68
90.03
86.86
63.19
65.64
79.27
93.56
Day 2
1
2
3
4
1
70.14
72.05
71.10
2
62.96
64.32
63.64
1
75.43
76.87
76.15
2
72.71
73.86
73.29
1
93.11
95.02
94.07
2
89.50
90.76
90.13
1
72.04
74.45
72.25
2
67.04
70.19
68.62
67.37
74.72
92.10
70.44
The average percentage recovery at four different levels of P57 was good at
95.32-107.52 % on day one and 98.29-101.99% on day two (Table 3.4).
3.3.5 CONCLUSIONS
The proposed quantitative HPTLC method for the estimation of P57 from
commercially available H. gordonii products was found to be accurate, precise,
specific and reproducible with good correlation coefficients and a good correlation
between height and area for the P57 estimations.
A major advantage of
quantification by HPTLC as opposed to LC-MS/HPLC is the ability to analyse
several samples at one time with a small quantity of mobile phase thereby
reducing the time and cost of analysis.
100
TABLE 3.4: Accuracy of the P57 standards determined by recovery analysis
Standard
Amount spotted
(ng)
Amount
detected
(ng)
Average of height
and area
(ng)
Recovery
(%)
Day 1
1
65.50
65.85 (height)
67.54 (area)
66.70
101.82
2
75.00
76.92 (height)
76.47 (area)
76.70
102.26
3
100.00
96.16 (height)
94.48 (area)
95.32
95.32
4
125.00
132.12 (height)
136.67 (area)
134.40
107.52
Day 2
1
65.50
62.73 (height)
66.02 (area)
64.38
98.29
2
75.00
77.13 (height)
75.84 (area)
76.49
101.99
3
100.00
100.12 (height)
97.78 (area)
98.95
98.95
4
125.00
123.83 (height)
129.04 (area)
126.44
101.15
101
3.4
REFERENCES
ADAMS, M. 2006. Hoodia fraud: Counterfeit Hoodia gordonii weight loss pills
dominate
the
market.
Natural
news.com.
Available
from:
http://www.naturalnews.com/018677.html [Accessed: 03/06/2009].
AVULA, B., WANG, Y.H., PAWAR, R.S., SHUKLA, Y.J., SCHANEBERG, B.,
KHAN, I.A. 2006. Determination of the appetite suppressant P57 in Hoodia
gordonii plant extracts and dietary supplements by liquid chromatography
/electrospray ionization mass spectrometry (LC-MSD-TOF) and LC-UV methods.
Journal of AOAC International, 89(3):606-611.
AVULA, B., WANG, Y.H., PAWAR, R.S., SHUKLA, Y.J., KHAN, I.A. 2007.
Chemical fingerprinting of Hoodia species and related genera: Chemical analysis
of oxypregnane glycosides using High-performance liquid chromatography with
UV detection in Hoodia gordonii. Journal of AOAC International, 90(6):1526-1531.
AVULA, B., WANG, Y.-H., PAWAR, R.S., SHUKLA, Y.J., SMILLIE, T.J., KHAN,
I.A. 2008. A rapid method for chemical fingerprint analysis of Hoodia species,
related genera, and dietary supplements using UPLC-UV-MS. Journal of
Pharmaceutical and Biomedical Analysis, 48:722-731.
BAZYLKO, A., STRZELECKA, H. 2007. A HPTLC densitometric determination of
luteolin in Thymus vulgaris and its extracts. Fitoterapia, 78:931-395.
GÜNTHER, M., SCHMIDT, P.C. 2005. Comparison between HPLC and HPTLCdensitometry for the determination of harpagoside from Harpagophytum
procumbens CO2-extracts. Journal of Pharmaceutical and Biomedical Analysis,
37:817-821.
JAITAK, V., GUPTA, A.P., KAUL, V.K., AHUJA, P.S. 2008. Validated highperformance thin-layer chromatography method for steviol glycosides in Stevia
rebaudiana. Journal of Pharmaceutical and Biomedical Analysis, 47:790-794.
JANSSEN, H.-G., SWINDELLS, C., GUNNING, P., WANG, W., GRÜN, C.,
MAHABIR, K., MAHARAJ, V.J., APPS, P. J. 2008. Quantification of appetite
suppressing steroid glycosides from Hoodia gordonii in dried plant material,
purified extracts and food products using HPLC-UV and HPLC-MS methods.
Analytica Chimica Acta, 617(1-2):200-207.
LAZAROWYCH, N.J., PEKOS, P. 1998. Use of fingerprinting and marker
compounds for identification and standardisation of botanical drugs: strategies for
applying pharmaceutical HPLC analysis to herbal products. Drug Information
Journal, 32:497-512.
MARSTON, A. 2007. Role of advances in chromatographic techniques in
phytochemistry. Phytochemistry, 68:2785-2797.
PATHAK, S.B., NIRANJAN, K., PADH, H., RAJANI, M. 2004. TLC densitometric
method for the quantification of eugenol and gallic acid in clove. Chromatographia,
60(3/4):241
102
SCHIPPMANN, U., LEAMAN, D., CUNNINGHAM, A.B. 2006. A comparison of
cultivation and wild collection of medicinal and aromatic plants under sustainability
aspects. In: BOGERS, R.J., CRAKER, L.E., LANGE, D. (eds.). Medicinal and
aromatic plants. The Netherlands: Springer:75-79.
REICH, E., SCHIBLI, A. 2006. High-performance thin-layer chromatography for
the analysis of medicinal plants. New York: Thieme.
RUMALLA, C.S., AVULA, B., SHUKLA, Y.J., WANG, Y.H., PAWAR, R.S.,
SMILLIE, R.J., KHAN, I.A. 2008. Chemical fingerprint of Hoodia species, dietary
supplements, and related genera by using HPTLC. Journal of Separation Science,
31(22):3959-3964.
VAN HEERDEN, F.R., VLEGGAAR, R., HORAK, R.M., LEARMONTH, R.A.,
MAHARAJ, V., WHITTAL, R.D. 1998. Pharmaceutical compositions having
appetite suppressant activity. PCT/GB98/01100.
WAGNER, H., BLADT, S. 1996. Plant Drug Analysis: A thin layer chromatography
atlas 2nd ed. Heidelberg: Springer-Verlag.
WIDMER, V., REICH, E., DEBATT, A. 2008. Validated HPTLC method for
identification of Hoodia gordonii. JPC – Journal of Planar Chromatography –
Modern TLC, 21:21-26.
WOLFENDER, J.-L., RODRIGUEZ, S., HOSTETTMANN, K. 1998. Liquid
chromatography coupled to mass spectrometry and nuclear magnetic resonance
spectroscopy for the screening of plant constituents. Journal of Chromatography
A, 794:299-316.
WOLFENDER, J.-L., NDJOKO, K., HOSTETTMANN, K. 2003. Liquid
chromatography with ultraviolet absorbance-mass spectrometric detection and
with nuclear magnetic resonance spectroscopy: a powerful combination for the online structural investigation of plant metabolites. Journal of Chromatography A,
1000:437-455.
103
CHAPTER 4
Vibrational spectroscopy
4.1
INTRODUCTION
Despite the discovery of new potentially appetite-suppressing compounds from H.
gordonii, the quality and therefore the purchase of raw material by manufacturers
from H. gordonii growers is still determined by quantifying P57.
Traditionally,
liquid-chromatography coupled to mass spectrometry (LC-MS) is used to
determine the P57 content of raw material.
LC-MS is cumbersome, requires
considerable expertise, is expensive and requires extensive sample preparation.
Vibrational spectroscopy techniques, including mid-infrared (MIR), near-infrared
(NIR) and Raman, offer an attractive alternative to traditional analytical methods
due to rapid analysis time, little or no pretreatment of samples, reduction in the use
of chemicals and the possibility to evaluate several components simultaneously in
different physical states (solids, liquids, gases) and perhaps most importantly, it is
non-destructive in nature. The most commonly used technique in terms of number
of publications has been NIR, despite its lower level of spectral information,
followed by MIR and Raman spectroscopy (Moros, Garrigues and De La Guardia,
2010:578-579). NIR and MIR spectroscopy has been used with success in the
food industry for quantifying various components such as peptides in cheeses
(González-Martín et al., 2009:1564) adulterants in Mexican honeys (GallardoVelázquez et al., 2009:313), caffeine in green tea powder and granules (Sinija and
Mishra, 2009:998), the acid value of peanut oil (Rao et al., 2009:249) and aflatoxin
B1 in chilli powder (Tripathi and Mishra, 2009:840). It has also found application
as an analytical technique in the agricultural (Camps and Christen, 2009:1125;
Xie, Ying and Ying, 2009:34), petrochemical (Balabin and Safieva, 2008:2745;
Zhan et al., 2008:165) and pharmaceutical (Qu et al., 2008:1146; Mantanus et al.,
2009:186) industries amongst others. In combination with chemometric analysis,
Raman spectroscopy has found application in various diverse industries such as;
the medical industry to identify liver cirrhosis, differences between benign and
malignant tumours and to detect teeth caries; the pharmaceutical industry for
quantification of polymorphs in powder mixtures; in botany as a tool in taxonomy,
104
to optimise breeding and cultivation and for quality control of plant products like
essential oils; in the food and textile industries for quality control; and to analyse
works of art and gemstones for characterisation, restoration, conservation, dating
and authentication (Schrader et al., 2000:35; Kachrimanis, Braun and Griesser,
2007:407; Braun et al., 2010:29; Moros, Garrigues and De La Guardia, 2010:580).
Raman spectroscopy combined with micro-equipment can provide detailed
molecular information with high spatial resolution at the cellular level.
For
example, mapping has been used to show the spatial distribution of certain
components or active ingredients in plants and pharmaceutical products
(Baranska et al., 2005:1; Zhang, Henson and Sekulick, 2005:262; Baranska and
Proniewicz, 2008:153).
Thus, it can be used to determine where a specific
component is localised in a plant. Raman mapping as well has the advantages
that it is non-destructive, does not require laborious sample preparation and
provides extensive data about the sample. The large amount of information in the
form of spectral data (multivariate data matrices) that is generated with vibrational
spectroscopy techniques requires the use of chemometric analysis for
interpretation. Regression methods like the partial least squares projections to
latent structures (PLS) algorithm are used to link the spectra to quantifiable
properties of samples, in this instance P57.
Mathematical pre-processing
techniques such as orthogonal signal correction (OSC) and multiplicative signal
correction (MSC) that are applied to data decrease interferences thereby
enhancing the interpretation of the data (Moros, Garrigues and De La Guardia,
2010:580). The aim of this study was to develop calibration models using NIR,
MIR and Raman spectroscopy in combination with chemometric data processing
which may potentially be used for simpler, less expensive and faster quantification
of P57 in raw material. In addition, Raman mapping was investigated to determine
where P57 is localised within the stem.
4.2
BACKGROUND AND LITERATURE REVIEW
4.2.1 Infrared spectroscopy (IR)
Infrared spectroscopy techniques are advantageous because the cover a wide
wavenumber interval and is customarily divided into three wavelength regions
depending on the type of instrumentation used to acquire the spectral information.
NIR covers wavelengths 750-2500 nm or 13333-400 cm-1, MIR 2500-25000 nm or
105
4000-400cm-1 and far infrared 25-1000 µm or 400-10 cm-1. However, as some
overlap exists, a new spectral region known as short-wavelength near-infrared
(SW-NIR) (600-1000 nm) has been introduced which includes part of the visible
light region (700-750 nm) (Moros, Garrigues and De La Gaurdia, 2010:578; Lin et
al., 2009:119-120).
A typical NIR instrument consists of a radiation source, a wavelength selection
device, a sample holder, a photoelectric detector that measures the intensity of
detected light and converts it to electrical signals and a computer system for
acquisition and processing of the spectral data (Lin et al., 2009:122). Radiation
sources used in analytical spectroscopy are divided into continuum sources and
line sources. Continuum sources emit light with a relatively stable and continuous
intensity over a wide range of wavelengths and include tungsten filament lamps,
deuterium tungsten light, high pressure mercury or xenon arc lamps and heated
solid ceramics or heated wires.
The tungsten filament lamp and deuterium
tungsten light are used for the NIR region while heated solid ceramics or heater
wires are used for the MIR region. NIR light provided by the radiation source can
penetrate much further into samples than MIR radiation, 13 mm versus a couple of
micrometers, therefore samples may be scanned within containers that are
transparent to NIR light. MIR light on the other hand, does not penetrate through
these materials but it provides better specificity (Lin et al., 2009:139-141). Line
sources emit light at only a few discrete wavelengths and the intensity varies at
different wavelengths.
Examples include hollow cathode lamps, electrodeless
discharge lamps, light-emitting diodes (LED), sodium or mercury vapour lamps
and lasers. Lasers emit monochromatic radiation with high intensity and are used
in Raman spectrometers (Lin et al., 2009:123-124). Several different radiation
sources of NIR and MIR instruments are classified according to the mechanism of
light separation into filter-based, monochromator-based, and Fourier transform
(FT) instruments.
FTIR has established itself as one of the most powerful
techniques for chemical analysis. Most modern FT spectrometers have an optical
device called a Michelson interferometer that allows the controlled generation of
interference
patters
or
interferograms.
Fourier
transformation
(FT),
a
mathematical procedure, is applied to interferograms to obtain spectra of the
106
scanned samples (Lin et al., 2009:125-126; Subramanian and Rodriguez-Saona,
2009:146, 155).
The interpretation of spectra obtained from IR spectrometers is based upon the
overtones and vibrations of the atoms of a molecule when passing IR radiation
through a sample.
The energy at which any peak in an absorption spectrum
appears corresponds to the frequency of a vibration of a part of the sample
molecule. Therefore a range of functional groups can be identified according to
absorption peaks at specific wavelengths or matched with characteristic vibrations
(Lin et al., 2009:139). Overtone peaks in NIR spectra are mainly due to O-H, C-H,
S-H, and N-H stretching modes and combination bands which are formed when
two or more vibrations combine through addition and subtractions of energies to
form a single band. NIR spectra are complex and overlapping peaks and large
baseline variations may present difficulties in the interpretation highlighting the
need for processing methods such as derivatisation to improve spectral
characteristics (Subramanian and Rodriguez-Saona, 2009:161).
With MIR
spectroscopy, a chemical profile of the sample is obtained by monitoring the
fundamental vibrational and rotational stretching of molecules.
It provides
information useful to analyse the composition of a sample as well as the structure
of chemical molecules. Although MIR spectra are easier to interpret than NIR
spectra, chemometric analysis may still be required to draw meaningful
information from the spectral data (Subramanian and Rodriguez-Saona, 2009:164165).
4.2.2 Chemometric data analysis
Chemometrics is used to extract useful information efficiently from spectra and is
defined by the International Chemometrics Society (IC) as the science of relating
measurements made on a chemical system to the state of the system via the
application of mathematical or statistical methods (Moros, Garrigues and De La
Guardia, 2010:580). The power of chemometrics is that it can be used to model
systems that are both largely unknown and complex such as spectral data.
Chemometrics has a holistic, exploratory approach to data and process modelling.
No assumptions are made about the data prior to analysis because in
chemometrics the process or data structures reveal the relations themselves
107
(Swanepoel and Esbensen, 2007:S.a.).
Data from vibrational spectrometers
combined with chemometrics provides calibration models and classification tools.
Classification methods, also called discriminant analysis, places samples into
different classes through pattern recognition while regression methods are used to
construct calibration models as it links the spectra to quantifiable properties of the
samples. Various pre-processing methods (mathematical pretreatments), such as
derivatives and multiplicative corrections, are used to remove spectral variation
unrelated to sample components from measured spectra to enhance spectral
information.
This is considered a critical step before calibration models are
constructed as this can significantly enhance the prediction accuracy of the model.
In addition, selection of spectral regions may enhance predictive ability as many
wavenumbers in a spectrum are not relevant to the sample
(Moros, Garrigues
and De La Guardia, 2010:580-582). The combination of NIR, MIR and Raman
with chemometric analysis provides fast tools for multi-component analysis of
various samples as an attractive alternative to the tedious, time-consuming
traditional analytical methods (Moros, Garrigues and De La Guardia, 2010:590).
4.3
MATERIALS AND METHODS
4.3.1 Plant material
Natural population (wild) and cultivated site samples of H. gordonii stems (n = 145)
were hand-picked throughout South Africa and Namibia, sliced and air-dried.
Random sampling was performed and all possible aspects of variation were
considered (e.g. young vs mature plants).
To construct a good predictive
calibration model, samples must be selected for their variability rather than for
quantity (Moros, Garrigues and De La Guardia, 2010:582). H. gordonii plants
were harvested at different levels of maturity according to market need, thus it was
important to include this variation. The dried plant material was powdered with a
Retsch® 400 MM ball mill at a frequency of 30.0 Hz for 90 s and sieved (500 µm)
to control the particle size distribution. For the Raman mapping experiment an
intact, fresh H. gordonii stem was sliced transversely to a thickness of 6 mm. For
LC-MS confirmation, the outer layers and cortex of 5 stems from different plants
were separated with a knife and processed as above.
The P57 reference
standard was purchased from ChromaDexTM Inc (California, USA).
108
4.3.2 Near infrared spectroscopy (NIR)
The powdered H. gordonii samples were placed in Chromacol® glass vials. Near
infrared spectra of the powdered samples were collected from 10 000-4000 cm-1
on a Büchi® (Flawil, Switzerland) NIRFlex N500 Fourier transform near infrared
(FT-NIR) spectrophotometer (Figure 4.1) with NIRWare® (version 1.2.3000
advanced edition) software in reflectance mode.
Spectra were collected at a
spectral resolution of 4 cm-1 with 32 scans per sample.
FIGURE 4.1: Photograph of the Buchi NIRFlex N500 Fourier transform near
infrared spectrophotometer and sample tray (Photograph by I Vermaak)
4.3.3 Mid-infrared spectroscopy (MIR)
FT-MIR spectra were generated with a Bruker® (Ettlingen, Germany) Alpha-P
Fourier transform infrared spectrophotometer (Figure 4.2) in absorbance mode
and recorded with OPUS® (version 6.5) software. Spectra were collected from
4000-375 cm-1 at intervals of 4 cm-1 and the data for each spectrum was the
average spectrum of 32 scans. The spectra for each sample were collected three
times and the mean of the three spectra was used in further analysis steps. A
blank background measurement was recorded after every three samples scanned.
109
FIGURE 4.2: Photographs of the Bruker Alpha-P Fourier transform infrared
spectrophotometer (Photographs by I Vermaak)
4.3.4 Raman spectroscopy
A Bruker® (Ettlinger, Germany) Multiram Fourier transform Raman spectrometer
(Figure 4.3) was used to generate Raman spectra of the powders in the range of
4000-100 cm-1, with a spectral resolution of 4 cm-1 set to 128 scans per sample.
The spectrometer was equipped with a diode-pumped Nd:YAG laser set to 30
mW, emitting at 1064 nm, and a germanium detector cooled with liquid nitrogen.
The Raman spectra were recorded and the instrument controlled with OPUS®
(version 6.5) software. Spectral measurements performed on the powder were
collected in duplicate and averaged.
FIGURE 4.3: Photographs of the Bruker Multiram Fourier transform Raman
spectrometer (Photographs by I Vermaak)
110
4.3.5 Raman mapping
Two-dimensional mapping of fresh H. gordonii plant material was done with the
same spectrometer as above and settings as in 4.3.4 using a 6 mm cross-section
of the stem. The stem sample was mapped at an area of 18.5 mm x 12.5 mm with
a spatial resolution of 570 µm. A focused laser beam at a power of 50 mW with a
diameter of ca. 50 µm was used and 128 scans were collected at each point.
4.3.6 Data analysis
The spectral data was exported to Microsoft® Excel (2007) to enable analysis
using Simca-P® 11.0 (NIR, MIR) and Simca-P+® 12.0 (Raman) chemometric
analysis software. Criteria such as the correlation coefficient (R2), the number of
partial least squares projections to latent structures (PLS) factors and the root
mean square error of prediction (RMSEP) were used to assess the ability of these
models to accurately predict the chosen quality parameter (Mantanus et al.,
2009:1750).
The spectral data were analysed using partial least squares
projections to latent structures (PLS) and/or orthogonal projections to latent
structures (O-PLS) regression methods and various mathematical pre-processing
methods were investigated to develop a mathematical calibration model (Thermo
Scientific, 2009).
extremely
Partial least squares regression (PLS-R) represents an
successful
new
extension
of
traditional
statistical
regression
methodology, developed over the last 35 years within the discipline of
chemometrics. In PLS the independent data (X) is related to the dependent data
(Y) and PLS attempts to capture variance and achieve correlation between X and
Y-data. O-PLS is based on PLS but includes an orthogonalisation step. Strong
systematic variance present in X may be unrelated to Y and is therefore irrelevant
for the prediction of Y. Removing the information in X which is orthogonal to Y,
termed structured noise, will not influence the variation between X and Y.
Therefore the interpretation of the model is improved while the correlation remains
the same (Swanepoel and Esbensen, 2007:S.a.). A work set and training was
randomly chosen by the software. The workset consisted of 30% of the Y-values
(P57 concentration determined by LC-MS) while the other 70% was used as the
training set. Due to this randomised approach there will be slight differences in the
results but this gives an indication of the robustness of the model if the results are
fairly consistent. Several repetitions were done for each pre-processing method to
111
determine whether results remained consistent and differences were not
significant.
The training and work sets remained constant for all the pre-
processing methods investigated.
4.3.7 P57 quantification with LC-MS analysis
Extracts of the H. gordonii stem samples (0.2 g) were prepared in an ultrasonic
bath (Sonorex digital 10p) for 2 h with 4 ml of 50% acetonitrile and 0.1% formic
acid. The detection and quantification of P57 in was performed using a Waters
(MA, USA) API Q-TOF Ultima system equipped with a BEH C18 column (1.7µm,
2.1 x 50 mm). The MS detector was operated in electron impact mode with the
capillary voltage at 3.5 kV and the cone voltage at 35 kV (positive switching - ES+).
The flow rate of the HPLC was 0.4 ml/min, the cone gas flow rate was 50 l/h and
the desolvation gas flow rate was 350 l/h. The source temperature was 100 °C
and the desolvation temperature 350 °C. Accurate mass (861.5 m/z) was used to
quantify P57 in ES+ mode within a mass range of 200 to 1800 m/z. The injection
volume was 2 µl.
Gradient elution was used with 0.1% formic acid in water
(Solvent A) and acetonitrile (Solvent B) as mobile phases. The gradient started
with 100% solvent A, changed to 20% solvent B over 0.5 min, to 100% solvent B
over 11.5 min, followed by a 2 min isocratic step and a return to initial conditions
for 1 min for a total run time of 15 min. The LC-MS data was analysed using
Masslynx® (version 4.2) software.
4.4
RESULTS AND DISCUSSION
4.4.1 Chemometric data analysis methods and parameters
Many different calibration models were generated with the sample set (n=145)
according to the method of pre-processing used and are shown in Table 4.1
together with the most important parameters used to evaluate these models. It is
important to avoid overfitting by choosing a model with a small number of PLS
factors, which is an indication of the fit of the model. The correlation coefficient
(R2) is a measure of how well the model fits the data. An R2 value close to one is
a necessary condition for a good model, but even models with a good R2 can
suffer from poor prediction ability.
A poor R2 is found when there is poor
reproducibility (a lot of noise) in the training data set, or (with PLS) when for any
other reason X does not explain Y. RMSEP is the root mean square error of
112
prediction for observations in the prediction set. The Q2 value is an indication of
the predictive ability of the model and is considered good when greater than 0.5.
There are various causes for a poor Q2 such as noisy data, when the relationship
of X to Y is poor (with PLS), or when the model is dominated by a few scattered
outliers in the training set (SIMCA-P+ analysis advisor, 2008:S.a.).
TABLE 4.1:
Number of PLS factors, R2, Q2, RMSEP and RMSEE values for
determining P57 content with NIR, MIR and Raman spectroscopy utilising a PLS
model with different pre-processing methods
Pretreatment
method
None
1st
derivative
2nd
derivative
MSC
SNV
OSC
NIR spectroscopy
PLS factors
1
3
6
3
4
2
R2
0.2021
0.5716
0.9629
0.2497
0.3424
0.7074
RMSEP
0.10
0.09
0.03
0.13
0.11
0.06
MIR spectroscopy
PLS factors
3
3
1
2
2
1
R2
0.2158
0.5918
0.2285
0.4997
0.4522
0.6630
RMSEP
0.07
0.06
0.10
0.07
0.09
0.04
Raman spectroscopy
PLS factors
4
3
2
4
5
2
R2
0.3486
0.6851
0.7253
0.3856
0.5324
0.9986
RMSEP
0.054
0.052
0.056
0.062
0.062
0.004
MSC – mulitiplicative signal correction; SNV – standard normal variate; OSC – orthogonal
signal correction; R2 – correlation coefficient; RMSEP – root mean square error of
prediction.
4.4.2 NIR spectroscopy
The general profile of the FT-NIR spectra of H. gordonii powdered raw material (A)
and the P57 reference standard (B) is depicted in Figure 4.4.
From visual
inspection the wavenumbers 10000-7800 cm-1 does not contribute to the model as
evident from the low absorbance values. Between 5000-4000 cm-1 strong peaks
from combination bands are present in the P57 reference standard spectrum (B).
These peaks were also present in the H. gordonii powdered raw material spectra
(A) but overlapping of peaks occur due to “masking” by other components. CH2
113
and CH3 absorption peaks were observed between 7000-5000 cm-1.
Many
different calibration models can be generated according to the method of preprocessing used.
The best calibration model was constructed using the 2nd
derivative pre-processing method and this model was further developed.
FIGURE 4.4: Typical FT-NIR spectra for H. gordonii powdered raw material (A)
and the spectrum for the P57 reference standard (B)
The T vs U plot, describing the intercorrelation between the X and Y illustrates the
potential of a correlation that exists between these two variables. About 42% of
the variance of X is explained by the PLS model after the exclusion of X-variables
with low loading weights (wavenumbers 10000-7800 cm-1) as indicated on the
loadings line plot before and after exclusion (Figure 4.5). This is a considerable
improvement as before exclusion only 24% of the variance of X was explained.
After construction of the PLS model six outliers were removed which improved the
accuracy of prediction or the R2 value.
114
FIGURE 4.5: Loadings line plot from the PLS calibration model using FT-NIR data
before (A) and after (B) removal of X variables with low loading weights (100007800 cm-1)
The PLS model had an R2 value of 0.9629, RMSEP of 0.03% and 6 PLS factors
(Figure 4.6). The cumulative overall cross-validated R2X (Q2) for this model was
75.6%, indicating a good prediction ability of this model.
An OPLS model
separates the PLS components into two groups, those related to Y (predictive) and
those orthogonal to Y. The Y-orthogonal component may be part of the model, but
is not useful for the prediction of Y.
The main benefit of OPLS is model
interpretation, while maintaining the predictive ability of the PLS method. As for
the PLS model, the OPLS model had an R2 value of 0.9629, RMSEP of 0.03% and
6 PLS factors and in addition the T vs U plot indicated good correlation of 0.9848
between the observed values (obtained from LC-MS measurements) and
predicted values for P57 (obtained from the spectra by FT-NIR measurements).
The cumulative overall cross-validated R2X (Q2) for the O-PLS model was 56.5%.
115
This indicated that the prediction ability of the OPLS model is not as good as the
prediction ability of the PLS model as some of the X-variables removed are in fact
not orthogonal to the first component. However, the O-PLS model was only used
for interpretation and the PLS model was used as the final calibration model.
These results indicate that FT-NIR may be used for rapid determination of the
concentration of P57 in H. gordonii raw plant materials. Considering the high cost
of other analytical methods, the small range of error of prediction (RMSEP) using
the FT-NIR method is negligible.
FIGURE 4.6: PLS calibration model using the 2nd derivative pre-processing
technique calculated from FT-NIR data
4.4.3 MIR spectroscopy
Chemometric analysis of the FT-MIR data did not reveal promising results and
even with the removal of several outliers the predictive ability of the model did not
improve appreciably. Several pre-processing methods were applied to the FT-MIR
data with little success as can be seen in Table 4.1 where the highest R2 value
(i.e. the best correlation between LC-MS values and predicted FT-MIR values)
was 0.6630. This implies that the accuracy of predicting P57 in H. gordonii plant
material will be very low and this method was consequently not further
investigated.
116
4.4.4 Raman spectroscopy
The results for the model assessment criteria for several models after the
application of various pre-processing techniques (Table 4.1) show that the best
model was constructed after applying the OSC pre-processing method to the data.
OSC is used to correct the X data matrix (spectral data) by removing information
that is linearly unrelated or orthogonal to the response matrix Y of the calibration
model. It is widely applied for the correction of excessive background noise of
spectral data collected with several analytical techniques such as NIR and nuclear
magnetic resonance (NMR) (Blanco et al., 2001:126; Qu, Ou and Cheng,
2005:838). It has been used successfully to develop calibration models for plant
extracts and whole soybean protein with NIR spectra, and in a pharmaceutical
setting for D-mannitol and paracetamol polymorphs with FT-Raman spectroscopy
(Qu, Ou and Cheng., 2005:838; Kachrimanis, Braun and Griesser, 2007:407; Igne
et al., 2009:57; Braun et al., 2010:29). Two principal components were used to
describe the OSC calibration model with 66.2% of the variation in X explained by
PLS factor 1 and 32.3% by PLS factor 2 (R2 X (cum) = 0.985). The predictive
ability of the model is 99.9 % as indicated by the overall cross-validated R2X (Q2)
value of 0.999.
The calibration model shows good correlation between the
observed values (obtained from LC-MS measurements) and predicted values for
P57 (obtained from the spectra by FT-Raman measurements) with a regression of
0.9986 indicating high accuracy of this model (Figure 4.7).
The difference between R2 and Q2 was less than 0.2 and the RMSEP value for this
model was 0.004, indicators of a good model. Internal validation by response
permutation (100) confirmed the validity of the model as Q2 intercepts the Y-axis
below zero and R2 and Q2 are closely correlated. In addition, this indicated that
noise is not being modeled and the model is not overfitted. No outliers were
removed for any of the models and internal validation and overfitting of the model
was assessed with 100 permutations.
These results clearly demonstrate the
feasibility of FT-Raman spectroscopy for rapidly determining the concentration of
P57 in H. gordonii raw plant materials with a negligible error of prediction.
117
FIGURE 4.7: PLS calibration model with OSC pre-processing calculated from FTNIR-Raman data
4.4.5 Raman mapping
Raman mapping was performed to determine where the perceived active molecule
P57 is localised in the plant. Spectra collected from the plant show some diversity
corresponding to the tissue and its various chemical compositions. Generally it is
possible to obtain maps, showing the distribution of individual components, based
on the integration of characteristic Raman bands. However, because of the low
concentration of P57 in the plant material this procedure could not be applied in
this case since in the measured spectra signals characteristic of P57 were hardly
visible. For this reason chemometrics was used to analyse Raman mapping data.
Application of cluster analysis in the spectral range from 1500 to 1800 cm-1
together with pre-processing of vector normalisation allowed grouping the spectra
into three clusters (Figure 4.8 B,C). This wavenumber range was chosen since it
covers the most specific bands of P57.
A single spectrum extracted from the three clusters shown in Figure 4.9
demonstrates several bands which can be assigned to individual plant
components. An outer part of the slice contains mainly carotenoids (violet ring)
and additionally lignin (green spots), whereas the cortex is composed of cellulose
(blue center). Spectra extracted from the inner part of the slice also shows bands
with weak intensity which can be assigned to P57.
118
FIGURE 4.8: A microscopic image of a fresh H. gordonii slice with the marked
area where the Raman mapping measurement was performed (A), and chemical
maps of the clusters (B) and the borders of the clusters (C) showing the
distribution of various plant components (blue – cellulose; violet – carotenoids;
green – lignin)
lignin
Raman Intensity
carotenoids
cellulose
H.gordonii
P57
H.gordonii
H.gordonii
P57 standard
3500
3000
2500
2000
1500
Wavenumber / cm-1
1000
500
FIGURE 4.9: A single Raman spectrum of H. gordonii extracted from the three
clusters coloured accordingly.
At the bottom a Raman spectrum of pure P57
standard is presented
The distribution is not homogenous and P57 is mainly localised in the inner fleshy
circle of the plant. This was confirmed with LC-MS of several samples where the
119
outer layers and cortex of the stem were analysed separately (Table 4.2). In effect
the inclusion of the outer layers with the thorns of the plant containing very small
amounts of P57 causes dilution of the final P57 content of the raw material.
TABLE 4.2. P57 content of the outer layers and cortex samples of H. gordonii
determined by LC-MS analysis
Sample
Outer layers
(% P57)
Cortex
(% P57)
1
0.000
0.031
2
0.017
0.074
3
0.023
0.165
4
0.051
0.173
5
0.021
0.084
4.5
CONCLUSIONS
The quality of raw plant material is determined to a large extent by the content of
P57.
The results show that there was a high variation of P57 concentration
ranging from 0.000-0.430% P57. The unwritten industry standard of good quality
H. gordonii raw plant material is considered to be more than 0.300% P57 content.
A mere 4% of the 145 plant samples collected and analysed contain more than
this limit of P57 content, all from natural populations.
If a 20% deviation is
allowed, as per the industry standard of 0.300% P57 content, raw plant material
containing more than 0.240% P57 would be acceptable and only 12% of the plant
samples collected attained this standard. In addition, it is evident that this quality
standard may be unrealistic in cultivated H. gordonii raw material as 9% of the
12% samples that comply with this standard were collected from natural
populations in contrast to only 3% that was collected from cultivated site samples.
In general the concentration of P57 is much lower in cultivated site samples
compared to natural population samples. It is known that active ingredient levels
can be higher in wild plant populations compared to cultivated stock, usually due
to differences in growth rates. In addition, the conditions under which plants are
cultivated are different compared to natural populations which may also account
for these differences (Schippmann, Leaman and Cunningham, 2006:81). Raman
mapping was used to observe the spatial distribution of P57 within the plant. This
120
information may be used to only include the part of the plant containing the highest
concentration of P57 in the final raw material. By preventing dilution, the final P57
content of cultivated raw material may be increased. However, removing these
parts may not be viable in the industry as this would considerably lengthen the
processing procedures. Both FT-NIR and FT-Raman spectroscopy demonstrates
potential as alternative methods to LC-MS to rapidly quantify P57 in H. gordonii
raw material with high accuracy and a small error of prediction.
121
4.6
REFERENCES
BALABIN, R.M., SAFIEVA, R.Z. 2008. Motor oil classification by base stock and
viscosity based on near infrared (NIR) spectroscopy data. Fuel, 87(12):2745-2752.
BARANSKA, M., PRONIEWICZ, L.M. 2008. Raman mapping of caffeine alkaloid.
Vibrational Spectroscopy, 48:153-157.
BARANSKA, M., SCHULZ, H., SIUDA, R., STREHLE, M.A., RÖSCH, P., POPP,
J., JOUBERT, E., MANLEY, M. 2005. Quality control of Harpagophytum
procumbens and its related phytopharmaceutical products by means of NIR-FTRaman spectroscopy. Biopolymers, 77:1-8.
BLANCO, M., COELLO, J., MONTOLIU, I., ROMERO, M.A. 2001. Orthogonal
signal correction in near infrared calibration. Analytica Chimica Acta, 434:125-132.
BRAUN, D.E., MAAS, S.G., ZENCIRCI, N., LANGES, C., URBANETZ, N.A.,
GRIESSER, U.J. 2010. Simultaneous quantitative analysis of ternary mixtures of
D-mannitol polymorphs by FT-Raman spectroscopy and multivariate calibration
models. International Journal of Pharmaceutics, 385:29-36.
CAMPS, C., CHRISTEN, D. 2009. Non-destructive assessment of apricot fruit
quality by portable visible-near infrared spectroscopy. LWT – Food Science and
Technology, 42(6):1125-1131.
GALLARDO-VELÁZQUEZ, T., OSORIO-REVILLA, G., ZUÑIGA-DE LOA, M.,
RIVERA-ESPINOZA. 2009. Application of FTIR-HATR spectroscopy and
multivariate analysis to the quantification of adulterants in Mexican honeys. Food
Research International, 42:313-318.
GONZÁLEZ-MARTÍN, I., HERNÁNDEZ-HIERRO, J.M., VIVAR-QUINTANA, A.,
REVILLA, I., GONZÁLEZ-PÉREZ, C. 2009. The application of near infrared
spectroscopy technology and a remote reflectance fibre-optic probe for the
determination of peptides in cheeses (cow’s, ewe’s and goat’s) with different
ripening times. Food Chemistry, 114:1564-1569.
IGNE, B., ROGER, J.-M., ROUSSEL, S., BELLON-MAUREL, V., HURBURGH,
C.R. 2009. Improving the transfer of near infrared prediction models by orthogonal
methods. Chemometrics and Intelligent Laboratory Systems, 99:57-65.
KACHRIMANIS, K., BRAUN, D.E., GRIESSER, U.J. 2007. Quantitative analysis of
paracetamol polymorphs in powder mixtures by FT-Raman spectroscopy and PLS
regression. Journal of Pharmaceutical and Biomedical Analysis, 43:407-412.
LIN, M., RASCO, B.A., CAVINATO, A.G, AL-HOLY, M. 2009. Infrared (IR)
spectroscopy – near-infrared spectroscopy and mid-infrared spectroscopy. In:
SUN, D. (ed.). Infrared spectroscopy for food quality analysis and control.
Massachusetts: Academic Press:119-143.
122
MANTANUS, J., ZIÉMONS, LEBRUN, P., ROZET, E., KLINKENBERG, R.,
STREEL, B., EVRARD R., HUBERT, PH. 2009. Moisture content determination of
pharmaceutical pellets by near infrared spectroscopy: Method development and
validation. Analytica Chimica Acta, 642(1-2):186-192.
MOROS, J., GARRIGUES, S., DE LA GUARDIA, M. 2010. Vibrational
spectroscopy provides a green tool for multi-component analysis. Trends in
Analytical Chemistry, 29(7):578-591.
QU, H.-B., OU, D.-L., CHENG, Y.-Y. 2005. Background correction in near-infrared
spectra of plant extracts by orthogonal signal correction. Journal of Zhejiang
University SCIENCE B, 6(8):838-843.
QU, N., MINGCHAO, Z., MI, H., DOU, Y., REN, Y. 2008. Nondestructive
determination of compound amoxicillin powder by NIR spectroscopy with the aid of
chemometrics. Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy, 70(5):1146-1151.
RAO, Y., XIANG, B., ZHOU, X., WANG, Z., XIE, S., XU, J. 2009. Quantitative and
qualitative determination of acid value of peanut oil using near-infrared
spectrometry. Journal of Food Engineering, 93:249-252.
SCHIPPMANN, U., LEAMAN, D., CUNNINGHAM, A.B. 2006. A comparison of
cultivation and wild collection of medicinal and aromatic plants under sustainability
aspects. In: BOGERS, R.J., CRAKER, L.E., LANGE, D. (eds.). Medicinal and
aromatic plants. The Netherlands: Springer:75-95.
SCHRADER, B., SCHULZ, H., ANDREEV, G.N., KLUMP, H.H., SAWATZKI, J.
2000. Non-destructive NIR-FT-Raman spectroscopy of plant and animal tissues, of
food and works of art. Talanta, 53:35-45.
SIMCA-P+ analysis advisor. 2008. SIMCA-P+ version 12.0.0. Sweden: Umetrics
AB.
SINIJA, V.R., MISHRA, H.N. 2009. FT-NIR spectroscopy for caffeine estimation in
instant green tea powder and granules. LWT – Food Science and Technology,
42:998-1002.
SUBRAMANIAN, A., RODRIGUEZ-SAONA, L. 2009. Fourier transform infrared
(FTIR) spectroscopy. In: SUN, D. (ed.). Infrared spectroscopy for food quality
analysis and control. Massachusetts: Academic Press:145-178.
SWANEPOEL, M., ESBENSEN, K H. 2007. Representative sampling at the grape
intake: A critical issue - often forgotten. 35th Technical Issue - Australian
Grapegrower and Winemaker.
THERMO SCIENTIFIC. 2009. http://www.thermo.com/com/cda/resources/
resources_detail/1,2166,13465,00.html [Accessed: 13/05/2009].
123
TRIPATHI, S., MISHRA, H.N. 2009. A rapid FT-NIR method for estimation of
aflatoxin B1 in red chilli powder. Food Control, 20:840-846.
XIE, L., YING, Y., YING, T. 2009. Classification of tomatoes with different
genotypes by visible and short-wave near-infrared spectroscopy with leastsquares support vector machines and other chemometrics. Journal of Food
Engineering, 94(1):34-39.
ZHAN, J., YIN, W., SHANG, H., LIU, C. 2008. In situ FT-IR spectroscopy
investigations of carbon nanotubes supported Co-Mo catalysts for selective
hydrodesulfurization of FCC gasoline. Journal of Natural Gas Chemistry,
17(2):165-170.
ZHANG, L., HENSON, M.J., SEKULICK, S.S. 2005. Multivariate data analysis for
raman imaging of a model pharmaceutical tablet. Analytica Chimica Acta,
545:262-278.
124
SECTION 2
Biopharmaceutical aspects
In Part A, the issue of quality of H. gordonii materials and products was
comprehensively discussed. If the authenticity of H. gordonii raw materials and
products has been established then it is necessary to move towards meeting
efficacy requirements. One of the first steps in the development of an effective
product is to determine biopharmaceutical aspects related to the active
constituents of H. gordonii such as P57. The most basic scientific research would
be to establish that the purported active ingredient (P57) of H. gordonii is in fact
absorbed to provide acceptable systemic bioavailability. Information in this area
has been lacking for H. gordonii though several recent studies have addressed
some of these aspects as discussed in the following chapter. This project aimed
to contribute to and build upon current pharmacokinetic and biopharmaceutical
knowledge of P57.
125
CHAPTER 5
Buccal and intestinal transport of P57 from Hoodia
gordonii
5.1
INTRODUCTION
As mentioned before, Hoodia gordonii was traditionally used by the San people of
South Africa and Namibia to suppress hunger and thirst while on long hunting trips
or in times of famine. The fresh stems were peeled to remove the thorns and the
flesh was chewed and/or retained in the buccal cavity before swallowing (Van Wyk
and Gericke, 2000:70; Lee and Balick, 2007:404-405; Glasl, 2009:300). Research
on
biopharmaceutical
aspects
that
should
be
investigated
prior
to
commercialisation of products claiming biological activity such as the bioavailability
of the active compound has been lacking, though recently some of the
biopharmaceutical aspects have been the focus of in vitro and in vivo
investigations in animals.
A mouse model was used to determine the
bioavailability, pharmacokinetic profile and tissue distribution of pure P57
(Madgula et al., 2010b:1582-1586).
In addition, the in vitro metabolic stability of P57 and interactions with drug
metabolising enzymes (Madgula et al., 2008:1269-1275), the pharmacokinetic
profile of hoodigogenin A, the aglycone of P57, and the stability of P57 in
simulated gastrointestinal fluids (Madgula et al., 2010a:62-69) have been studied.
The intestinal transport of pure P57 using the Caco-2 cell model has been
investigated before (Madgula et al., 2010a:65-67), but this study is the first to
compare the intestinal transport of pure P57 with that of P57 in a crude extract
across intestinal mucosa. In addition, no transport studies across buccal tissue
have been conducted yet for P57, which may contribute to the overall
bioavailability of P57 specifically when the traditional use of the plant is
considered. It was therefore the aim of this study to contribute to the current
biopharmaceutical knowledge of the active ingredient of H. gordonii. This was
done by investigating the in vitro transport of P57 across porcine intestinal and
126
buccal tissues in a Sweetana-Grass diffusion apparatus. In addition, the transport
obtained for pure P57 with the transport of P57 when applied in the form of a
crude plant extract was compared.
5.2
BACKGROUND AND LITERATURE REVIEW
5.2.1
Introduction
The oral route of medicine administration is the most frequently used and
preferred administration route when a systemic effect is required. The oral route is
preferred over other routes of administration due to several reasons that include
the following aspects: it is safe, simple, convenient, patients find it acceptable,
good patient compliance, the administration of medicines can be controlled by the
patients themselves and does not require skilled medical intervention, a large
surface area for systemic absorption is available, many drugs are well-absorbed
from the gastrointestinal tract and it enables the use of both immediate release
and sustained release dosage forms (Chan, Lowes and Hirst, 2004:26; Daugherty
and Mrsny, 1999:144; Hamman, 2007:64). Disadvantages associated with the
oral route include a relatively slow onset of action, the possibility of irregular
absorption, exposure to the first-pass metabolism effect and destruction of certain
medicines by the enzymes and secretions of the gastrointestinal tract (York,
2007:7).
The gastrointestinal tract is a hollow muscular tube that is divided into the
oesophagus, the stomach, the small intestine and the colon. The small intestine
can be further sub-divided into the duodenum, jejunum and ileum. The wall of the
gastrointestinal tract consists of four main layers namely the mucosa, submucosa,
muscularis externa and the serosa (Hamman, 2007:90-91).
The intestinal
epithelium separates the lumen of the gastrointestinal tract from the systemic
circulation and is the main physical barrier to the absorption of orally administered
compounds from the gastrointestinal tract. This membrane is semi-permeable and
allows rapid transport of some molecules while impeding the transport of others.
Lipophilic molecules are transported across the bilayer of apical membrane, while
water and small hydrophilic molecules are transported through its numerous
aqueous pores. The two main mechanisms of transport by which molecules can
traverse the intestinal epithelium in both directions is transcellular (across the
127
cells) and paracellular (between the cells). The transcellular pathway is further
divided into passive diffusion, carrier-mediated transport (active and facilitated)
and endocytosis (fluid-phase endocytosis or pinocytosis, receptor-mediated
endocytosis, phagocytosis and transcytosis). Several absorption pathways are
schematically illustrated in Figure 5.1 (Hunter and Hirst, 1997:131; Ashford,
2007a:279-283).
FIGURE 5.1:
Schematic illustration of the drug absorption pathways across
intestinal epithelial cells.
(A) paracellular diffusion, (B) paracellular diffusion
through opened tight junctions, (C) transcellular passive diffusion with (C*)
intracellular metabolism, (D) carrier-mediated transcellular transport, (E) efflux and
(F) vesicular transcytosis (Hunter and Hirst, 1997:131)
The rate of transport of a substance is determined by its physicochemical
properties, the nature of the membrane and the concentration gradient of the
substance across the membrane. A balance between the aqueous solubility of the
molecule, which dictates the amount of drug available for absorption, and its
lipophilicity, which is important for membrane permeability, will provide the best
oral bioavailability (Ashford, 2007a:279).
The apical intestinal epithelial cell
membrane also contains countertransport efflux proteins that actively expels
molecules back into the lumen of the gastrointestinal tract after absorption. One of
the key countertransport proteins is P-glycoprotein (P-gp), which is expressed at
high levels on the apical surface of columnar cells in the jejunum. Efflux via P-gp
is recognised as a mechanism which may significantly decrease bioavailability of
128
orally administered substances, especially at low doses. The P-gp transporters
may also be blocked when two substances are co-administered allowing increased
transport of one substance while the other substance blocks the P-gp transporter.
The P-gp transporters are also saturable meaning that administration of high
doses of a substance may cause saturation of the transporters (Ashford,
2007a:283; Hamman, 2007:108). Several in vitro methods have been investigated
to determine intestinal and buccal permeability as an indicator of drug absorption
in vivo.
Drug delivery through the buccal mucosa (the cheek lining of the oral cavity) has
several advantages as an alternative route of administration compared to oral
administration.
These include: easy access to the oral cavity and patient
acceptance, avoidance of first-pass metabolism in the liver and intestine,
avoidance of pre-systemic elimination within the gastrointestinal tract, bypassing
peroral absorption variability as a result of gastric emptying rate or the presence of
food, its relatively high permeability and good vascular supply (Shojaei, 1998:15,
27; Nicolazzo and Finnin, 2008:90; Obradovic and Hidalgo, 2008:167).
The
buccal route of administration is more suited to sustained release formulations and
delivery of less permeable molecules than the sublingual mucosa. The sublingual
mucosa is more permeable and is used when a rapid onset of action is required.
A major disadvantage of the buccal mucosa is low flux resulting in low
bioavailability, therefore penetration enhancers are often included in buccal
formulations (Shojaei, 1998:19). The structure of the buccal mucosa consists of
the stratified squamous epithelium (outermost layer) and lamina propria separated
by the basement membrane and the submucosa. The buccal mucosa thickness is
about 500-800 µm and the epithelium consists of about 40-50 cell layers. The
buccal and sublingual areas within the oral cavity are non-keratinised while the
gingivae and hard palate are keratinised (Shojaei, 1998:16; Nicolazzo and Finnin,
2008:91).
The major barrier to the transport of molecules across the buccal mucosa is onethird to one-quarter from the apical surface of the buccal epithelium.
Other
barriers include the salivary film and mucous layer, the basement membrane and
a metabolic barrier. Permeation of molecules through the buccal mucosa typically
129
occurs via passive diffusion but carrier-mediated transport has also been
demonstrated for certain nutrients such as D-glucose (Nicolazzo and Finnin,
2008:91, 94). Passive diffusion involves transcellular (through the cells) as well as
paracellular (between the cells) routes and is influenced by the physicochemical
properties of the molecule (lipophilicity or hydrophilicity) as well as the pH partition
theory.
Lipophilic molecules and also the unionised form of a substance,
predominantly permeates via the transcellular route while hydrophilic molecules
permeate mainly via the paracellular route (Nicolazzo and Finnin, 2008:94;
Obradovic and Hidalgo, 2008:167).
5.2.2
In vitro models for intestinal permeation studies
5.2.2.1
Membrane-based models
One of the first steps to assess oral absorption is to determine the partition
coefficient of a molecule indicating its lipophilicity which can then be used to
predict the ability of a molecule to permeate through a biological membrane
(Ashford, 2007b:307). This in itself is not sufficient and membrane-based models
can be used to screen the transport of substances across lipophilic membranes.
Parallel artificial membrane permeability assays (PAMPA) involve the use of
organic solutions of artificial lipids to form bilayer structures simulating the cell
membrane. The bilayer separates the aqueous donor and acceptor chambers and
passive transport of small molecules is mimicked.
It provides complementary
information to other models in that passive transport permeability is assessed but
it lacks paracellular and active transport mechanisms. It is a popular screening
method due to its rapid results, low cost, versatility and easy adaptability in terms
of increasing permeability of the membrane to investigate specific molecules
(Bermejo et al., 2004:430; Hamman, 2007:17). In immobilised artificial membrane
(IAM) chromatography, artificial monolayers of synthetic phospholipid analogues
are bound to the silica of a high performance liquid chromatography (HPLC)
column which then simulates a biological membrane.
It therefore provides a
measure of how well the solute in the aqueous phase will partition into biological
membranes through determination of its retention on the column.
When
partitioning dominates the interaction of the molecule with the membranes and
when it is the rate limiting step of drug movement, it may be used to predict the
permeation of molecules (Ashford, 2007b:308; Hamman, 2007:18).
130
5.2.2.2
Cell culture models
Cell culture techniques are well-accepted models to predict absorption of
molecules. The most widely used cell line, Caco-2, is a human epithelial colon
adenocarcinoma cell line that was first proposed and characterised as a model for
oral drug absorption in 1989.
The Caco-2 cell model has many advantages
including determining the mechanisms of absorption of a substance, it can be
used as a rapid screening tool, requires only a small amount of the substance for
transport studies and it can be used as an indicator of cellular toxicity. In culture,
Caco-2 cells spontaneously differentiate to form a monolayer of polarised
enterocytes with distinct apical and basolateral sides that are separated by tight
junctions.
These
enterocytes
resemble
those
in
the
small
intestine
morphologically and biochemically, in that they contain microvilli and many of the
transport systems present in the small intestine, for example the P-gp efflux
transporter.
Disadvantages of the Caco-2 cells are that the paracellular
permeability is more comparable to the colon than the small intestine, they lack a
mucus layer and lipophilic compounds may be retained within the cell layer
(Ashford, 2007b:308-309; Hamman, 2007:18-23). To determine absorption, the
substance in question is added to the apical chamber which is separated from the
basolateral chamber by the Caco-2 monolayer. The appearance of the substance
in the basolateral chamber is determined over time. If the disappearance of the
substance from the apical chamber does not match its appearance in the
basolateral chamber, the substance may be susceptible to enzyme degradation as
it is transported through the cells. The substance may also be susceptible to
metabolism within the epithelial cells due to the presence of cytochrome P450
(CYP) enzymes (Ashford, 2007b:310).
Other cell models used in drug
permeability studies include the Madin-Darby canine kidney (MDCK), Lewis lung
carcinoma-porcine kidney 1 (LLC-PK1) and HT-29-A18C1 (subclone of human
intestinal adenocarcinoma) cell lines as well as a TC-7 clone of Caco-2 cells.
However, these cell models are used infrequently due to various disadvantages
(Ashford, 2007b:310; Hamman, 2007:24).
5.2.2.3
Isolated tissue models
Isolated tissue models for use in absorption studies include mounting excised
sheets of intestinal mucosa in a diffusion chamber, the use of everted intestinal
131
sacs or solutions containing brush border membrane vesicles (Ashford,
2007b:310, Hamman, 2007:24-26).
The diffusion chamber method involves
sourcing intestine and removing the musculature to isolate the intestinal mucosa.
The intestinal tissue from various animal species including pigs and rats has been
used to investigate drug permeation and it has been shown that permeability data
obtained using porcine intestinal tissue correlated well with human data (Ashford,
2007b:310; Nejdfors et al., 2000:501). The intestinal mucosa is cut into small
pieces and mounted between the donor and acceptor chambers of a diffusion cell.
The substance is added to the donor chamber and samples are removed from the
acceptor chamber at specific time intervals and analysed.
In addition, the
chambers can be analysed independently allowing fluxes from the apical to
basolateral and basolateral to apical sides to be measured. Advantages of this
method include careful control of the experimental conditions by maintenance of
the system at 37 °C and oxygen is provided to the tissue mimicking the in vivo
condition. In addition, the pH of the buffer solutions can easily be altered and
permeability across different sections of the intestine can be assessed (Ashford,
2007b:310, Hamman, 2007:26).
In the everted intestinal sac method segments of the whole intestine are used as
opposed to excised sheets of intestinal mucosa. The intestines are excised from
rats, everted, and cut into segments which are tied up at both ends to form sacs.
These sacs are placed into a solution containing the test substance for a period of
time to allow substance uptake and samples are withdrawn at various time
intervals and analysed. The method can distinguish between passive and active
transport and can also show the influence of P-gp.
The disadvantage of this
method is the rapid loss of viability of the tissue (Ashford, 2007b:311; Hamman,
2007:25). The brush border membrane vesicle method involves the formation of
vesicles, produced from intestinal scrapings, which contain luminal surface
proteins and phospholipids with most of the brush border enzymatic and carrier
activity. After incubation of the substance in a buffer solution with the vesicles, the
vesicles are removed and the amount of substance absorbed by the vesicles is
calculated (Hamman, 2007:24).
132
5.2.3
In vitro models for buccal permeation studies
5.2.3.1
Isolated tissue models
The most widely used in vitro method for buccal permeation assessment is via
excised buccal tissue from various animal sources mounted in diffusion chambers
(Franz-type, flow-through, modified Ussing chamber).
Many animal tissues
including rabbit, monkey, dog, hamster, rat and pig buccal mucosa have been
investigated as permeation models due to the shortage of sufficient quantities of
human buccal mucosa. It has been found that the use of most of these tissues are
limited due to the small area of non-keratinised tissue in the case of rabbits, the
epithelium of dogs and monkeys is much thinner and therefore more permeable,
and the surfaces of rat and hamster tissues are keratinised. The most commonly
utilised tissue is porcine buccal mucosa as it is similiar to human mucosa in terms
of structure, morphology, composition, thickness and shows good correlation in
terms of permeability. In addition, porcine buccal mucosa is easily acquired at low
cost from slaughterhouses (Shojaei, 1998:21; Nicolazzo and Finnin, 2008:97-99;
Obradovic and Hidalgo, 2008:168). However, the use of isolated tissue models
suffers several disadvantages including the limited surface area, large variability in
permeation among replicates, the presence of tissue damage from mastication,
minimal potential for automisation of the assay and the laborious and timeconsuming process of tissue excision (Obradovic and Hidalgo, 2008:172).
5.2.3.2
Cell culture models
In addition to isolated tissue models, cell cultures have been developed as an in
vitro technique to assess the permeability of compounds across buccal epithelia,
particularly to overcome some of the disadvantages of isolated tissue models
(Obradovic and Hidalgo, 2008:172).
Cultures must be harvested from an
appropriate source and grown under controlled conditions (temperature and
humidity) using suitable growth medium. It was found that cultures from hamster
cheek pouch do not differentiate into keratinised cells and therefore may be an
appropriate model to predict permeability as the human buccal mucosa is nonkeratinised.
Human buccal mucosa has been cultured from human buccal
carcinoma cells (TR 146 cell line) but it is more permeable rendering it a less
suitable model (Nicolazzo and Finnin, 2008:102). The most suitable cell culture
model derived from healthy human buccal mucosa showed good correspondence
133
to intact human buccal mucosa in terms of morphology, protein expression and
lipid composition with good reproducibility. In addition, good correlation was found
on comparison between the human buccal cell culture and isolated human buccal
mucosa in terms of permeability but only a limited amount of substances have
been investigated. These cultures may also be used to evaluate drug metabolism,
previously difficult to investigate due to the lack of fresh human buccal mucosa
(Nicolazzo and Finnin, 2008:102; Obradovic and Hidalgo, 2008:172-173).
5.3.
METHODS AND MATERIALS
5.3.1
In vitro intestinal and buccal permeation studies
5.3.1.1
A crude plant extract was prepared by extraction of the powder from a commercial
H. gordonii capsule product with acetonitrile in an ultrasonic bath (Sonorex digital
10p) for 10 min at 25 °C.
The process was repeated three times to ensure
maximal extraction, the filtered extracts combined and air-dried. The P57 content
of the commercial H. gordonii product was determined by LC-MS analysis. The
pure P57 was purchased from ChromaDexTM Inc (California, USA). Solvents used
in high performance liquid chromatograpy (HPLC) for quantification of P57 were of
analytical grade.
5.3.1.2
Tissue preparation
The numbers in the brackets in this section refers to Figure 5.2, which depicts
photographs of the different step during tissue preparation for transport studies in
the Sweetana-Grass diffusion apparatus. Porcine buccal and intestinal tissues
from the oral and peritoneal cavities respectively were obtained from freshlyslaughtered pigs at a local abattoir (R&R Abattoir, Pretoria, South Africa) on each
study day and washed with as well as transported in cold Krebs-Ringer
bicarbonate buffer (referred to henceforth as buffer) (1). The intestinal tissue was
prepared by sliding the intestine onto a glass tube and removing the serosal layer
from the intestinal mucosa with blunt dissection (2). Thereafter the mucosal layer
was cut along the mesenteric border (3) (Legen, Salobir, and Kerč, 2005:185),
rinsed with buffer, placed on filter paper and cut into smaller pieces (4). The
intestinal mucosa was placed on one half of the clamp, the filter paper removed (5)
134
FIGURE 5.2: The preparation and mounting of porcine intestinal mucosa in
Sweetana-Grass diffusions chambers (Photographs by JH Hamman)
135
and the clamp was closed (6). The clamps containing the intestinal mucosa with
an exposed surface area of 1.13 cm2 (7-8) were mounted between the two halfcells or chambers of the Sweetana-Grass diffusion apparatus with the mucosal
side facing the apical chamber (Grass and Sweetana, 1988:374). The buccal
mucosa was prepared by carefully removing excess underlying connective tissue
using fine-point forceps and a scalpel, rinsed with buffer, clamped and mounted in
the Sweetana-Grass diffusion half-cells (Ceschel et al., 2002:S46; Van Eyk and
Van der Bijl, 2004:388). The assembled cells were placed in the six-cell aluminum
block heater of the Sweetana-Grass apparatus, which is connected to a water bath
maintaining the temperature at 37 °C. The apical and basolateral chambers were
filled with 5 ml of fresh warmed buffer (37 °C). The tissues were continuously
oxygenated with medical oxygen and an equilibration period of 30 min was
allowed before commencement of the transport studies (Figures 5.2 (7-8) and 5.3).
FIGURE 5.3: A photograph of a diffusion chamber cell as well as a schematic
diagram of an assembled cell indicating the excised mucosal tissue mounted
between two half-cells
5.3.1.3
Preparation of simulated intestinal fluid and artificial saliva
Simulated intestinal fluid (SIF) was prepared by dissolving 6.8 g monobasic
potassium phosphate (KH2PO4) in 250 ml water.
After mixing, 190 ml 0.2 M
sodium hydroxide (NaOH), 400 ml water and 10.0 g pancreatin (Sigma Aldrich)
were added. The pH was adjusted to 7.5 ± 0.1 with 0.2 M sodium hydroxide and
distilled water was added to provide a volume of 1000 ml (USP, 1990:1789). The
artificial saliva was prepared by dissolving 5.208 g NaHCO3, 1.369 g
K2HPO4.3H2O, 0.877 g NaCl, 0.447 g KCl, 0.441 g CaCl2.2H2O, 2.160 g mucin
136
and 200,000 U of hog pancreas α -amylase (Sigma Aldrich) in distilled water and
making up the resulting solution to one litre. The resulting artificial saliva solution
was adjusted to a pH of 7.0 ± 0.1 (Boland et al., 2004:402).
5.3.1.4
Transport studies
Bi-directional
intestinal
transport
experiments
(apical-to-basolateral
and
basolateral-to-apical) were conducted for pure P57 and for P57 in H. gordonii
crude extract across porcine intestinal tissue. Buccal transport experiments in the
apical-to-basolateral direction were conducted for pure P57 and P57 in H. gordonii
crude extract across porcine buccal tissue. In each experiment, 5% methanol was
used to dissolve the pure P57 or the crude extract to increase solubility before it
was made up to volume with the specific transport medium. Solutions made up to
volume with buffer was adjusted to pH 7.4 ± 0.1, while that of SIF was adjusted to
pH 7.5 ± 0.1 (USP, 1990:1789) and for artificial saliva to pH 7.0 ± 0.1 (Boland et
al., 2004:402).
A concentration of 200 µM of P57 was added to the chambers as previously
reported by Madgula et al. (2008:1272) in an in vitro transport study using a Caco2 cell model. Therefore pure P57 was added to each chamber in a concentration
of 1.2 mg/5 ml. It was calculated that 2.4 g of plant material had to be extracted to
provide approximately 1.2 mg of P57 per chamber based on the LC-MS result of
0.304% P57 determined for the commercial H. gordonii product. Samples (200 µl)
drawn from each of the original solutions before they were applied to the tissues in
the transport studies were analysed for P57 content. The transport studies were
initiated by adding 5 ml of each solution as indicated in Table 5.1 to the donor
chamber (the apical or basolateral chamber, respectively). Samples (200 µl) were
drawn from the receiver chamber over a period of 2 h at 20 min intervals and the
volume of each sample was replaced with warm (37 °C) transport medium.
137
TABLE 5.1: Experimental groups used for the intestinal and buccal transport
studies
Intestinal transport studies
Absorptive direction
Secretory direction
Apical chamber
Basolateral
chamber
Apical chamber
Basolateral chamber
Pure P57 + buffer
Buffer
Buffer
Pure P57 + buffer
Extract + buffer
Buffer
Buffer
Extract + buffer
Extract + SIF
Buffer
SIF
Extract + buffer
Buccal transport studies
Absorptive direction
Apical chamber
Basolateral chamber
Pure P57 + buffer
Buffer
Pure P57 + artificial saliva
Buffer
Extract + buffer
Buffer
Extract + artificial saliva
Buffer
5.3.1.5
Quantification of P57
Quantification of P57 was performed with an HPLC system consisting of a Waters
(MA, USA) 2690 separation module and a Waters 996 photodiode array detector
and separation achieved using a Gemini C18 column (250 x 4.6 mm; 5 µm particle
size; Phenomenex) equipped with a guard column (4 mm x 3.0 mm,
Phenomenex). The mobile phase consisted of water containing 0.05% acetic acid
(A) and acetonitrile (B). Gradient elution was used as follows: 100% A at 0 min,
adjusted in the following 40 min to 20% A and 80% B. Each run was followed by a
5 min wash with 100% B and an equilibration period of 8 min. The flow rate was
1.0 ml/min, the column was temperature controlled at 30 °C and the detection
wavelength was 220 nm. The chromatographic data was collected and analysed
with Empower® software.
5.3.1.6
Data analysis and statistical evaluation
The transport results were corrected for dilution and the cumulative P57 transport
was plotted as a function of time. The apparent permeability coefficient (Papp)
values (cm/s) were calculated according to the following equation (Hansen and
Nilsen, 2009:88):
138
Papp = (dC/dt) (1/A.60.C0) (cm/s)
where dC/dt is the transport rate (slope), A is the surface area of the tissue (1.13
cm2) and C0 is the initial concentration of P57 applied. Statistical analysis of the
Papp values for both the buccal and intestinal transport experiments was done by
means of a repeated one-way analysis of variance (ANOVA) to indicate whether
differences are significant (p ≤ 0.05) or not.
The P57 flux (J) values were calculated according to the following equation
(Hansen and Nilsen, 2009:88):
J = V(dC/dt)/A (µg/cm2/h)
where V is the receiver chamber volume, dC is the receiver chamber P57
concentration (µg/ml), dt is the transport time (h) and A is the tissue surface area
(cm2). The net flux of P57 (JNET) was calculated as follows (Hansen and Nilsen,
2009:88):
JNET = JB-A – JA-B (µg/cm2/h)
where JB-A is the flux of P57 in the B-A direction and JA-B is the flux of P57 in the AB direction.
5.4
RESULTS AND DISCUSSION
5.4.1 In vitro intestinal transport studies
The Papp values for P57 transport across intestinal mucosa are shown in Figure
5.4, while the flux (J) values for P57 are shown in Table 5.2.
FIGURE 5.4: Transport of pure P57 as well as P57 from a crude plant extract
across porcine intestinal tissue in the absorptive direction (
direction (
) and secretory
) expressed in terms of apparent permeability coefficient (Papp) values
139
In general, the in vitro transport of pure P57 in buffer across porcine intestinal
tissue was very low, but it was significantly higher in the secretory direction (Papp =
0.059 x 10-6 cm/s) than in the absorptive direction (Papp = 0.022 x 10-6 cm/s)
indicating that P57 is actively effluxed by intestinal membrane transporters. This is
also shown by the positive JNET flux value (Table 5.2).
TABLE 5.2: Flux (J) values (µg/cm2/h) for P57 across porcine intestinal mucosa
Transport
direction
A-B
(µg/cm2/h)
B-A
(µg/cm2/h)
JNET flux
(µg/cm2/h)
Pure P57 +
buffer
0.174
0.501
0.3265
Extract +
buffer
10.523a
10.476a
-0.0469
Extract + SIF
1.357b
1.356b
-0.0009
A-B: Apical to basolateral; B-A: Basolateral to apical. Values with the same superscript
letter (a,b) are not significantly different from each other.
These results are congruent with a previous study by Madgula et al. (2008:1272)
where the intestinal transport of pure P57 across Caco-2 cell monolayers revealed
lower transport in the absorptive direction than in the secretory direction. In this
previous Caco-2 cell study it was also shown that in the presence of inhibitors of
two membrane transporters namely verapamil which inhibits P-gp and MK-571
which inhibits multidrug resistance-associated protein, the permeability of P57
increased in the absorptive direction indicating it is a substrate of these two active
membrane transporters. In the absence of these efflux pump inhibitors, the P57
permeability was concentration-dependent indicating saturation of transporters
(Madgula et al. 2008:1273).
The intestinal transport of P57 applied as a H.
gordonii crude extract in buffer had a significantly higher Papp value of 74.479 x 106
cm/s compared to the Papp value of pure P57 of 0.022 x 10-6 cm/s in the
absorptive direction as well as in the secretory direction Papp of 73.466 x 10-6 for
the crude extract and 0.059 x 10-6 cm/s for pure P57. Furthermore, P57 transport
from the crude extract in buffer was slightly but not significantly higher in the
absorptive direction (Papp = 74.479 x 10-6 cm/s) than in the secretory direction (Papp
73.466 x 10-6 cm/s) with a negative flux value of -0.0469 µg/cm2/h indicating that
phytochemicals in the crude extract influences P57 transport by means of efflux
140
inhibition but other mechanisms such as changes to P57 solubility and membrane
permeability cannot be excluded. The phenomenon that crude extracts are in
some cases more biologically active than the pure compounds has been
established before.
For example, pure aspalathin was transported to a lower
extent across Caco-2 cell monolayers compared to aspalathin in a crude Rooibos
tea extract (Huang et al., 2008).
The transport of P57 in the H. gordonii crude extract dissolved in SIF was
significantly decreased in both the absorptive and secretory directions (Papp =
10.617 x 10-6; 14.608 x 10-6 cm/s) compared to the P57 transport from the crude
extract dissolved in buffer (Papp = 74.479 x 10-6; 73.466 x 10-6 cm/s). The transport
in SIF was significantly higher in the secretory direction compared to the
absorptive direction. These lower transport results can be explained by the fact
that the stability of P57 was significantly affected by SIF. This corresponds to a
stability study by Madgula et al. (2010a:65) where it was determined that 8.6% of
the initial concentration of pure P57 applied was degraded within 180 min when
exposed to SIF. In addition, 100% of pure P57 was degraded in simulated gastric
fluid (SGF) after 60 min. As the simulated intestinal fluid is used to mimic in vivo
conditions, these results indicate that the bioavailability of orally administered P57
may be significantly affected by enzymatic degradation and/or hydrolysis. An in
vivo study by Madgula et al. (2010b:1582) in CD1 female mice revealed a peak
plasma level of P57 after 0.6 h and moderate bioavailability of 47.5% after oral
administration of a methanolic extract of H. gordonii (equivalent to a dose of 25
mg/kg P57).
The moderate oral bioavailability is likely due to degradation in
gastric and intestinal fluids. Acid hydrolysis in the stomach may be circumvented
by the use of enteric-coating which enables release of a substance from a dosage
form only upon reaching the small intestines. In addition, the buccal route of
administration may be considered as an alternative route of administration to avoid
the harsh conditions in the gastrointestinal tract that degrades P57.
5.4.2 In vitro buccal transport studies
The Papp values of the buccal transport of P57 from H. gordonii crude extract
across porcine buccal mucosa are shown in Figure 5.5 while the flux values (J) are
shown in Table 5.3.
141
FIGURE 5.5: Transport of pure P57 as well as P57 from a crude plant extract
across porcine buccal tissue in the absorptive direction expressed in terms of
apparent permeability coefficient (Papp) values
TABLE 5.3: Flux (J) values (µg/cm2/h) for P57 across porcine buccal mucosa
Transport direction
A-B
(µg/cm2/h)
Pure P57 + buffer
0
Pure P57 + saliva
0
Extract + buffer
2.223a
Extract + saliva
2.204a
Values with the same superscript letter (a) are not significantly different from each other.
In this study, no buccal transport of pure P57 in either buffer or artificial saliva was
obtained at the concentration tested. However, when crude extract was dissolved
in buffer, the Papp value was 19.149 x 10-6 cm/s (Figure 5.5), indicating that
phytoconstituents present in the crude extract significantly enhanced the transport
of P57 across the buccal tissue. Interestingly, the transport was significantly
higher when the crude extract was dissolved in artificial saliva (Papp = 22.081 x 10-6
cm/s) as compared to the buffer indicating that α-amylase does not degrade P57.
These results further indicate that significantly more P57 is transported across the
buccal membrane when in vivo conditions are simulated probably by effects of the
saliva such as solubility improvement.
142
5.5
CONCLUSIONS
It was determined with in vitro transport experiments that pure P57 is not
transported across excised porcine buccal mucosa, while it is transported across
excised porcine intestinal mucosa. The transport of P57 from a crude extract was
significantly higher than the transport obtained for pure P57 across both porcine
intestinal and buccal mucosal tissues.
This indicates that the crude extract
contains phytoconstituents that influence the transport of P57 across these
mucosal tissues. The results obtained reinforce the credibility of the traditional
method of chewing and oral consumption of the plant itself as opposed to oral
administration of the pure isolated compound, P57. It can further be concluded
that buccal transport of P57 from plant material may contribute to the overall
bioavailability and therefore the pharmacological effects of P57, the perceived
active ingredient of H. gordonii.
143
5.6
REFERENCES
ASHFORD, M. 2007a. Gastrointestinal tract – physiology and drug absorption.
In: AULTON, M.E. (ed.). Aulton’s Pharmaceutics: the design and manufacture of
medicines. Hungary: Elsevier Limited:270-285.
ASHFORD, M. 2007b. Assessment of biopharmaceutical properties. In:
AULTON, M.E. (ed.). Aulton’s Pharmaceutics: the design and manufacture of
medicines. Hungary: Elsevier Limited:304-323.
BERMEJO, M., AVDEEF, A., RUIZ, A., NALDA, R., RUELL, J.A., TSINMAN, O.,
GONZÁLEZ, I., FERNÁNDEZ, C., SÁNCHEZ, G., GARRIGUES, T.M., MERINO,
V. 2004. PAMPA – a drug absorption in vitro model. 7. Comparing rat in situ,
Caco-2, and PAMPA permeability of fluoroquinolones. European Journal of
Pharmaceutical Sciences, 21:429-441.
BOLAND, A.B., BUHR, K., GIANNOULI, P., VAN RUTH, S.M. 2004. Influence of
gelatin, starch, pectin and artificial saliva on the release of 11 flavour compounds
from model gel systems. Food Chemistry, 86:401-411.
CESCHEL, G.C., MAFFEI, P., SFORZINI, A., LOMBARDI BORGIA, S., YASIN, A.,
RONCHI, C. 2002. In vitro permeation through porcine buccal mucosa of caffeic
acid phenetyl ester (CAPE) from a topical mucoadhesive gel containing propolis.
Fitoterapia, 73 (Suppl.1):S44-S52.
CHAN, L.M.S., LOWES, S., HIRST, B.H. 2004. The ABCs of drug transport in
intestine and liver: efflux proteins limiting drug absorption and bioavailability.
European Journal of Pharmaceutical Sciences, 21:25-51.
DAUGHERTY, A.L., MRSNY, R.J. 1999. Transcellular uptake mechanisms of the
intestinal epithelial barrier Part one. Pharmaceutical Science and Technology
Today, 2(4):144-151.
Schweizerische Zeitschrift für GanzheitsMedizin, 21(6):300-306.
GRASS, G.M., SWEETANA, S.A. 1988. In vitro measurement of gastrointestinal
tissue permeability using a new diffusion cell. Pharmaceutical Research, 5(6):372376.
HAMMAN, J.H. 2007. Oral drug delivery: biopharmaceutical principles, evaluation
and optimisation 2nd ed. Pretoria: Content solutions.
HANSEN, T.S., NILSEN, O.G. 2009. Echinacea purpurea and P-glycoprotein drug
transport in Caco-2 cells. Phytotherapy Research, 23:86-91.
HUANG, M.J., DU PLESSIS, J., DU PREEZ, J., HAMMAN, J.H., VILJOEN, A.M.
2008. Transport of aspalathin, a Rooibos tea flavonoid, across the skin and
intestinal epithelium. Phytotherapy Research, 22:699-704.
144
HUNTER, J. HIRST, B.H. 1997. Intestinal secretion of drugs. The role of Pglycoprotein and related drug efflux systems in limiting oral drug absorption.
Advanced Drug Delivery Reviews, 25:129-157.
LEE, R.A., BALICK, M.J. 2007. Indigenous use of Hoodia gordonii and appetite
suppression. Explore, 3(4):404-406.
LEGEN, I., SALOBIR, M., KERČ, J. 2005. Comparison of different intestinal
epithelia as models for absorption enhancement studies. International Journal of
Pharmaceutics, 291:183-188.
WALKER, L.A., KHAN, S.I. 2008. In vitro metabolic stability and intestinal transport
of P57AS3 (P57) from Hoodia gordonii and its interaction with drug metabolizing
enzymes. Planta Medica, 74:1269-1275.
WALKER, L.A., KHAN, S.I. 2010a. Characterization of in vitro pharmacokinetic
properties of hoodigonenin A from Hoodia gordonii. Planta Medica, 75(1):62-69.
MADGULA, V.L.M., ASHFAQ, M.K., WANG, Y.-H., AVULA, B., KHAN, I.A.,
WALKER, L.A., KHAN, S.I. 2010b. Bioavailability, pharmacokinetics, and tissue
distribution of the oxypregnane steroidal glycoside P57AS3 (P57) from Hoodia
gordonii in mouse model. Planta Medica, 76(14):1582-1586.
NEJDFORS, P., EKELUND, M., JEPPSSON, B., WESTRÖM, B.R. 2000. Mucosal
in vitro permeability in the intestinal tract of the pig, the rat, and man: species- and
region-related differences. Scandinavian Journal of Gastroenterology, 35(5):501507.
NICOLAZZO, J.A., FINNIN, B.C. 2008. In vivo and in vitro models for assessing
drug absorption across the buccal mucosa. In: EHRHARDT, C., KIM, K.-J. (eds.).
Drug Absorption Studies - In Situ, In Vitro, and In Silico Models. New York:
Springer:89-111.
OBRADOVIC, T., HIDALGO, I.J. 2008. In vitro models for investigations of buccal
drug permeation and metabolism. In: EHRHARDT, C., KIM, K.-J. (eds.). Drug
Absorption Studies - In Situ, In Vitro, and In Silico Models. New York: Springer:67181.
SHOJAEI, A.H. 1998. Buccal mucosa as a route for systemic drug delivery: a
review. Journal of Pharmacy and Pharmaceutical Sciences, 1(1):15-30.
USP. 1990. United States Pharmacopoeia 22nd ed. Rockville: The United States
Pharmacopoeial convention, Inc:1789.
VAN EYK, A.D., VAN DER BIJL, P. 2004. Comparative permeability of various
chemical markers through human vaginal and buccal mucosa as well as porcine
buccal and mouth floor mucosa. Archives of Oral Biology, 49:387-392.
145
VAN WYK, B.-E., GERICKE, N. 2000. People’s plants. Pretoria: Briza publications.
YORK, P. 2007. Design of dosage forms. In: Aulton, M.E. (ed.). Aulton’s
Pharmaceutics: the design and manufacture of medicines. Hungary: Elsevier
Limited:1-14.
Figure references (www)
FIGURE 5.3 (left):
http://warneronline.com/img_lg/690993_p2300_gr.jpg [Accessed: 2010/12/12].
146
CHAPTER 6
General conclusions and recommendations
6.1
GENERAL CONCLUSIONS
The occasional use of H. gordonii and other Hoodia species as appetite and thirst
suppressants by people indigenous to South Africa has been well-documented
leading to the popular consumption of H. gordonii preparations as anti-obesity
preparations.
Use of products of natural origin is particularly appealing to
consumers due to the general perception that if it is natural it must be effective and
safe. All of the quality control studies performed to date showed evidence of fake
products claiming to contain H. gordonii therefore quality control has been the
main focus of scientific studies.
Furthermore, the supply of H. gordonii plant
material is limited due to its sparse geographical distribution, slow maturation rate,
need for a permit to cultivate or export material as well as high public demand,
contributing to adulteration of a large amount of products. Despite the isolation of
numerous steroidal glycosides from H. gordonii, the main focus has been on the
pregnane glycoside P57, considered to be the active ingredient and marker
molecule to determine quality of H. gordonii raw material and products.
The first aim of this thesis was to develop and optimise rapid quality control
methods for H. gordonii raw material and products. In terms of quality control a
chemical fingerprint for H. gordonii raw material was developed and the presence
or absence of P57 in raw material and products was successfully detected using
high
performance
thin
layer
chromatography
(HPTLC).
Several
novel
quantification methods for P57 were developed using near infrared (NIR) and
Raman spectroscopy coupled with chemometric data analysis as well has
quantitative HPTLC. These methods are advantageous in that they allow simpler
and faster quantification methods as opposed to the traditional analytical methods
such as liquid chromatography coupled to a photo diode-array detector (LC-UV)
and liquid chromatography coupled to a mass spectrometer (LC-MS). The wellestablished traditional methods were used to develop reference sets needed to
expand the current quality control methods. Adulteration of raw material could be
147
detected to a certain extent with HPTLC and the location of P57 within a stem
sample was determined using Raman mapping.
Publications based on scientific studies of key aspects such as in vivo
biopharmaceutics, the biological activity of all chemical constituents, clinical
efficacy and especially safety are insufficient or completely absent for H. gordonii
This causes great concern as H. gordonii is one of the most widely consumed antiobesity products of natural origin. Therefore, the second aim was to contribute
towards increasing the biopharmaceutical knowledge of P57. It was shown in this
study for the first time that P57 is transported across porcine intestinal mucosa
which is closely related to human mucosa to a significantly higher extent when
applied in the form of a crude extract as compared to pure P57. Furthermore, P57
in its pure form was not transported across porcine buccal mucosa but it was
transported in the crude extract form. The results therefore indicate the possibility
that some of the more than 40 other compounds in H. gordonii crude extract may
beneficially affect P57 transport across mucosal epithelia. The use of media such
as artificial saliva and simulated intestinal fluid which closely represents in vivo
conditions revealed that saliva may have a beneficial effect in the transport of P57
while exposure to intestinal fluid has a deleterious effect on P57.
In addition to meeting the aims and objectives of the study, comprehensive
reviews on the literature pertaining to H. gordonii as well as anti-obesity
preparations of plant origin were conducted. The H. gordonii case encompasses
all issues related to the development of commercial products from natural origin
including intellectual property rights, benefit sharing and biodiversity issues,
protection of natural resources, the complexity of plant extracts, the paramount
importance of quality control methods and the need for proper research including
clinical studies prior to commercialisation.
6.2
RECOMMENDATIONS
As a result of this study, new questions have arisen which should be investigated.
Many of the Hoodia species have been reported to be used for weight loss and
contain P57 and other steroidal glycosides, but only H. gordonii has been
scientifically investigated to some extent. All pharmacokinetic studies completed
148
up to this point indicated that the oral bioavailability of P57 is low suggesting that
other molecules or metabolites may be responsible for the anti-obesity effect. The
aglycone of P57, hoodigogenin A, is a prime candidate for further research. In
addition, the investigation of other Hoodia species for anti-obesity activity should
be considered.
Many aspects that should be investigated prior to commercialisation of products
claiming biological activity such as bioavailability of the active ingredient has been
lacking. For example, H. gordonii is also marketed in the form of a topical gel, yet
there is no evidence to support sufficient transdermal permeation to produce
pharmacological effects.
A future avenue of research should be to determine
whether P57 and other compounds from H. gordonii are in fact transported across
the skin.
A void still exists in terms of quality control of commercial H. gordonii products as
compounds unique to H. gordonii has not been found and none of the methods
developed showed that it could distinguish between different species of Hoodia in
powder form used to manufacture products. In addition, the HPTLC quantification
method developed during this study should be optimised for possible
implementation as a standard quality control method in practice.
149
ANNEXURE 1: Chemical structures
1.1
Chemical structures of the steroidal glycosides isolated from H. gordonii
with their corresponding aglycones indicated (*,#,$,@,^)
O
O
O
HO
O
O
H3CO
OH
O
O
OH
O
O
OCH3
OCH3
P57/Compound 1/Formula 6*
O
O
O
HO
H3CO
O
O
O
OH
O
OH
OCH3
Hoodigoside A*
O
O
O
HO
H3CO
O
O
O
OH H CO
3
O
O
OH
O
OH
OCH3
Hoodigoside B*
O
O
O
HO
O
H3CO
OH
O
O
O
O
OCH3
O
O
OCH3
OCH3
Hoodigoside C/Gordonoside C*
150
OH
O
O
O
O
O
HO
H3CO
O
O
O
O
OH H3CO
O
O
OH
OH
O
OCH3
Hoodigoside D
*
OCH3
O
O
O
OH
O
HO
HO
OH
O
O
H3CO
OH
O
O
O
O
OH
O
OCH3
OCH3
Hoodigoside E/Gordonoside H*
O
OH
O
HO
HO
O
O
O
O
O
O
OH H3CO
O
O
H3CO
O
O
OH
OH
O
OCH3
Hoodigoside F
OCH3
*
O
OH
O
HO
HO
O
O
O
O
O
O
OH
O
O
H3CO
OCH3
O
O
OH
OH
O
OCH3
Hoodigoside G
OCH3
*
O
OH
HO
HO
O
O
O
O
O
O
O
OH
OCH3
O
O
OCH3
O
O
OCH3
Hoodigoside H
151
*
OH
O
OCH3
O
OH
O
HO
O
O
HO
O
O
O
O
OH H3CO
O
O
OCH3
OH
O
O
O
OCH3
Hoodigoside I
OCH3
*
O
OH
O
HO
O
O
HO
O
O
O
O
OH H3CO
O
O
O
O
OH
OH
O
OCH3
Hoodigoside J
OCH3
*
O
OH
O
O
O
HO
HO
O
OH HO
O
O
O
HO
OH
O
OH
OCH3
Hoodigoside K*
H
HO
O
O
OH
OH HO
O
O
O
O
H3CO
O
O
OH H3CO
OH
OH
OH HO
O
O
O
OH
OH
O
HO
Hoodigoside L#
H
HO
O
OH
OH HO
O
HO
H3CO
O
O
OH H3CO
O
O
OH
O
OH
OH HO
O
O
OH
OH
O
Hoodigoside M#
152
HO
H
OH
O
HO
H3CO
O
O
H
OH 3CO
OH
O
Hoodigoside N#
H
HO
O
OH
OH HO
O
O
O
O
O
H3CO
O
O
H3CO
OH
OH
O
HO
Hoodigoside O
#
H
HO
O
O
OH
OH HO
O
O
O
O
H3CO
O
O
OH
OH
OH HO
O
O
O
OCH3
HO
#
H
HO
O
O
OH
OH HO
O
O
O
O
O
O
O
OCH3
OH
O
OCH3
HO
#
H
HO
O
O
OH
OH HO
O
O
O
O
O
O
OCH3
OH
OH
OH HO
O
O
O
OCH3
HO
H
HO
O
O
OH
OH HO
O
O
O
O
OCH3
O
O
O
O
O
OCH3
OH
OH
O
Hoodigoside R#
O
H3CO
OH
OH
O
Hoodigoside Q
O
OH
OH HO
O
O
OH
O
H3CO
OH
OH
O
Hoodigoside P
O
H3CO
OH
OH
O
OH
O
OCH3
OH
OH HO
O
O
OH
OH
O
Hoodigoside S#
153
HO
H
HO
O
O
OH
OH HO
O
O
O
H3CO
O
O
O
O
O
O
O
OCH3
OH
O
OCH3
OH
OH
O
HO
OCH3
Hoodigoside T#
H
HO
O
O
OH
OH HO
O
O
O
O
O
OCH3
OCH3
O
O
O
O
O
OCH3
OH
OH
OH HO
O
O
O
OCH3
OH
OH
O
HO
Hoodigoside U#
HO
O
O
H
O
OH
OH
OH
O
O
H3CO
O
O
H
OH 3CO
OH
O
Hoodigoside V/Compound 2#
O
O
O
O
HO
H3CO
O
O
H3CO
O
O
O
O
OH
OH
O
OCH3
Hoodigoside W
OCH3
*
O
OH
O
HO
H3CO
O
O
O
O
OH
OH
O
OCH3
OCH3
Hoodigoside X$
HO
O
H
O
OH
OH
OH
HO
H3CO
O
O
OH H3CO
O
OH
O
Hoodigoside Y#
154
HO
O
H
OH
OH
O
O
OH
O
O
H3CO
O
O
O
O
OH
O
OH
OCH3
OCH3
Hoodigoside Z#
O
O
O
HO
O
H3CO
OH
O
O
O
O
O
O
H3CO
OH
O
OCH3
Gordonoside B
*
OCH3
O
O
O
O
HO
O
O
O
O
O
O
H3CO
HO
OH
O
OCH3
Gordonoside D
*
OCH3
O
O
O
HO
O
O
O
H3CO
O
O
OH
O
O
H3CO
O
OCH3
Gordonoside E
*
OCH3
O
O
O
HO
O
O
O
O
O
H3CO
OCH3
O
O
OCH3
OCH3
Gordonoside F/Formula 9*
155
OH
O
O
O
O
O
HO
O
O
O
O
H3CO
O
O
OCH3
OH
O
OCH3
OCH3
Gordonoside G/Formula 10*
O
O
O
O
HO
O
O
H3CO
HO
O
O
O
O
O
O
H3CO
OH
O
OCH3
Gordonoside I
OCH3
*
O
O
O
O
OH
O
O
H3CO
O
O
H3CO
O
O
OCH3
O
O
OH
O
OCH3
Gordonoside L/Formula 11
*
OCH3
HO
O
H
O
O
H3CO
O
OH
H
O
H
O
O
O
OH
OH
OH
OH
OH
CHO
OCH3
Hoodistanaloside A@
HO
O
H
O
O
H3CO
H
O
H
O
O
O
OH
OCH3
Hoodistanaloside B^
156
OH
CHO
O
OH
OH
OH
O
O
O
O
HO
O
O
O
O
H3CO
OCH3
O
O
OH
OH
O
OCH3
Formula 7
OCH3
*
O
O
O
O
HO
O
O
O
O
H3CO
O
O
OH
OH
O
OCH3
Formula 8
OCH3
*
O
O
O
O
HO
O
O
O
O
H3CO
O
O
OCH3 OH
OH
O
OCH3
Formula 12
*
OCH3
Chemical structures of aglycones
O
O
O
OH
OH
H
OH
OH
HO
OH
HO
Hoodigogenin A*
/Gordonoside A
HO
Calogenin#
Isoramanone$
OH
OH
H
H
H
H
HO
OH
H
H
OH
CHO
Hoodistanal
O
HO
@
OH
CHO
Dehydrohoodistanal^
157
ANNEXURE 2: Publications emanating from this thesis
2.1.
Publications in accredited journals
§
Vermaak, I., Hamman, J.H., Viljoen A.M. 2010. High performance thin layer
chromatography as a method to authenticate Hoodia gordonii raw material
and products. South African Journal of Botany, 76:119-124.
§
Vermaak, I., Hamman, J.H., Viljoen A.M. 2010. A rapid spectroscopic
method for quantification of P57 in Hoodia gordonii raw material. Food
Chemistry, 120:940-944.
§
Vermaak, I., Viljoen A.M., Hamman, J.H., Baranska, M. 2010. The potential
application of FT-Raman spectroscopy for the quantification and mapping of
the steroidal glycoside P57 in Hoodia gordonii. Phytochemistry Letters,
3:156-160.
§
Vermaak, I., Hamman, J.H., Viljoen, A.M. 2010. Hoodia gordonii: An up-todate review of a commercially important anti-obesity plant. Planta Medica,
in press.
§
Vermaak, I., Viljoen, A.M., Chen, W., Hamman, J.H. In vitro transport of
the steroidal glycoside P57 from Hoodia gordonii across excised porcine
intestinal and buccal tissue. Phytomedicine, in press.
2.2.
Publication in a non-accredited journal
§
Vermaak, I., Viljoen, A.M. 2008. Indigenous South African Medicinal Plants.
Part 9: Hoodia gordonii. South African Pharmaceutical Journal, 75(4):37.
2.3.
Publication in preparation for submission to an accredited journal
§
Vermaak, I., Hamman, J.H., Viljoen, A.M. The quantification of P57 from H.
gordonii using HPTLC coupled to densitometry.
158
ANNEXURE 3: Conference contributions
3.1.
Oral papers
§
Vermaak, I., Viljoen, A.M., Manley, M. Towards a rapid quality control
method for Hoodia gordonii. Indigenous Plant Use Forum (IPUF) 2008, 7-11
July, Graaff-Reinet, South Africa.
§
Vermaak, I., Viljoen, A.M., Manley, M. Towards a rapid quality control
method for Hoodia gordonii raw materials and products. World Conference
on Medicinal and Aromatic Plants IV 2008, 9 – 14 November, Cape Town,
South Africa.
§
Vermaak, I., Viljoen, A.M., Hamman, J.H. Quality control of Hoodia gordonii
raw material and products with HPTLC and FT-NIR spectroscopy. 5th
International Conference on Pharmaceutical and Pharmacological Sciences
(ICPPS) 2009, 23 – 26 September, Potchefstroom, South Africa.
§
raw material and products. 5th University of Johannesburg Botany
Symposium 2009, 27 October, Johannesburg, South Africa.
§
raw material and products. TUT Faculty of Science Research Day 2010, 4
August, Pretoria, South Africa.
§
Vermaak, I., Viljoen, A.M., Hamman, J.H. In vitro permeability of the
steroidal glycoside P57 from Hoodia gordonii through porcine buccal and
intestinal
mucosa.
31st
Annual
Conference
of
the
Academy
of
Pharmaceutical Sciences South Africa (APSSA) 2010, 14 – 17 September,
Polokwane, South Africa.
§
Vermaak, I., Viljoen A.M., Hamman, J.H. In vitro permeability of the
steroidal glycoside P57 from Hoodia gordonii through porcine buccal and
intestinal mucosa. Department of Pharmaceutical Sciences (TUT) Research
showcase 2010, 22 October, Pretoria, South Africa.
3.2.
Poster
§
raw material and products. 58th International Congress and Annual Meeting
of the Society for Medicinal Plant and Natural Product Research (GA) 2010,
29 August – 2 September, Berlin, Germany.
159

HOODIA GORDONII: QUALITY CONTROL AND

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