a study on the retention of pyrroloquinazoline alkaloid

WORLetDal.JOURNAL OF
PHAJournal
RMAC
AND PH
ARPharmaceutical
MACEUTICSciences
AL SCIENCES
Srinivasan
World
ofYPharmacy
and
SJIF Impact Factor 5.210
Volume 4, Issue 05, 948-957.
Research Article
ISSN 2278 – 4357
A STUDY ON THE RETENTION OF
PYRROLOQUINAZOLINE ALKALOID VASICINE IN
NUTRACEUTICAL FORMULATIONS OF ADHATODA VASICA
K. Srinivasan1* and C. Sivasubramanian2
1
Research Scholar, Department of Environment & Herbal Science, Tamil University,
Thanjavur, Tamil Nadu, India.
2
Head of the Department, Department of Environment & Herbal Science, Tamil University,
Thanjavur, Tamil Nadu, India.
Article Received on
24 Feb 2015,
Revised on 18 March 2015,
Accepted on 08 April 2015
ABSTRACT
Vasicine, a quinazoline alkaloid is the major active compound of
Adhatoda vasica, an Indian traditional herb which gives its antiasthma
property in the treatment of asthma disorder. The purpose of this
research
*Correspondence for
work
is
to
evaluate
the
retention
of
pyrroloquinazoline alkaloid vasicine in Adhatoda vasica raw herb and
Author
K. Srinivasan
its formulations through microencapsulation and extrusion process
Research Scholar,
using high performance liquid chromatography technique. The analysis
Department of
result indicated that the higher retention i.e. 73.18% of vasicine in gum
Environment & Herbal
acacia encapsulated Adhatoda vasica leaf powder compared to initial
Science, Tamil University,
vasicine content of hot air died powder (378 ppm). The retention
Thanjavur, Tamil Nadu,
percentage of vasicine was found to be 69.57%, 64.72%, 49.50% and
India.
44.24% in maltodextrin encapsulated, gum acacia encapsulated and
extruded, raw herb spray dried and raw herb extruded respectively. This study will help
the functional food product developers to exploit the microencapsulated Adhatoda vasica leaf
powder to resist high temperature and pressure in their formulations.
KEYWORDS: Adhatoda vasica, vasicine, alkaloids, microencapsulation, extrusion.
1. INTRODUCTION
Adhatoda vasica Nees, a medicinal shrub in South Asian traditional medicine is well known
for its use in respiratory ailments. This plant is considered like mother to doctors in
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traditional Indian medicine system. The plant leaf is valued for containing bronchodilator
alkaloids, mainly vasicine, quinazoline, vasicinone, deoxyvasicine (Gulfraz, 2006). This
quinazoline alkaloid vasicine was first isolated from the leaves of Adhatoda vasica Nees by
Hooper and subsequently in Peganum harmala in the name of Peganine. The salts are
obtained as crystals. The crude drug from Adhatoda vasica is used in India as a remedy for
asthma and the pure alkaloid acts as a bronchodilator (Glasby, 1978).
Microencapsulation is a modern technology to stabilize compounds of interest, such as
bioactive principles. Encapsulation of food ingredients such as flavors, lipids, and carotenoids
through spray drying process has been in use for many decades. Preparation of stable
emulsions, atomization of the emulsions into fine droplets, and dehydration of the atomized
particles with a heated gas stream are the major basic steps involved in producing
microencapsulated compounds. The spray drying process is one of the microencapsulation
techniques which rely on achieving high retention of the core material especially volatile
compounds during process and storage (Jafari et.al., 2008). Maltodextrins are widely used for
flavors and polyphenol encapsulation. Maltodextrins are reported to improve shelf life of
orange oil (Reineccius et. al., 1986) and carrot carotene. Kanakdande, Bhosale and Singhal
(2007) reported that gum arabic is a better wall material for encapsulation of cumin oleoresin
by spray-drying as compared to other wall materials. Spray drying produces powders with
particle sizes in the micrometre scale, which would have a smoother mouth feel than microbeads and should allow the addition of microencapsulated powder to a wider range of foods
(Lina Yonekura et.al, 2013).
Even though an increasing demand for the herbal drug treatment of respiratory ailments have
aroused, Adhatoda vasica, an indigenous plant medicine having its beneficial effects,
particularly in asthma is poorly utilized because of its bitterness. Further, the formulations of
this plant like kada (Iyengar et.al, 1994) and Fermiforte (Shete, 1993) having its drawback by
losing the alkaloids during the preparation process. Because of the hard cell wall of the
Adhatoda vasica leaves, simply crushing or squeezing will not yield juice extract. Further,
there is an increasing demand for nutritive and healthy foods in the market and this fact has
led the food industry to focus their research in products of this nature (Helena C.F.et al.,
2013). Hence, this study focus on the gaps in nutraceuticals developments from Adhatoda
vasica and its antiasthma alkaloids especially vasicine and to overcome the bitter taste. Thus,
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the objective of the study is to evaluate the retention of alkaloids especially vasicine in
different techniques like microencapsulation & extrusion with microencapsulation.
2. MATERIAL AND METHODS
2.1. Collection of Plant Material
The leaves of plant Adhatoda vasica Nees (Family- Acanthaceae) were collected from the
Herbal garden, Tamil University, Thanjavur and was identified and authenticated. The
voucher specimen has been deposited in their repository herbarium for future reference. The
leaves were shade dried and powdered.
2.2. Chemicals & Reagents
Reference standard vasicine (purity 98%, w/w), kindly provided as a gift sample provided by
Sami Lab Pvt Ltd., Mumbai. Methanol (HPLC grade) and water (HPLC grade) were obtained
from Merck Limited, India and Nice Chemicals Pvt. Ltd., respectively, while all other
chemicals used in the experiment were of analytical grade.
2.3. Microencapsulation
Microcapsules were produced using Adhatoda vasica leaf powder as a core and gum acacia &
maltodextrin as wall materials. To increase the recovery percentage, the coating material was
prepared with Adhatoda vasica leaf powder in different ratios of 5:1, 5:2, 5:3, 5:4 and 5:5
which gives concentrations of 10, 20, 30, 40 and 50% of leaf powder. The inlet temperature
and feed flow rate of the spray dryer were optimized for Adhatoda vasica extract. The
experiment is conducted in Randomized Block Design (RBD) with different temperature (ie.
80, 90, 100,120, 140 & 160˚ C) and feed rate (180 & 300 rpm). Wall materials were
evaluated in terms of total phenolic and alkaloid content retention, color of the powder and
recovery percentage. Gum acacia with finer emulsion characteristics (Viscosity - 12.82
centipoises) at 5:5 ratio shows higher retention after the encapsulation.
2.4. Extrusion technique
Extrusion was performed in a corotating twin screw extruder (Hermann Berstorff Laboratory
Co-rotating Twin Screw Extruder ZE25(33D) which consisted of 7 parts of barrel ended with
a 24.5 mm thick die plate and one circular die hole (diameter 3.0 mm). The length to diameter
(L/D) ratio for the extruder was 870/25. Extruded products were prepared from a mixture of
rice flour, salt (1%), pepper (1%) and jeera (1%) with 5% Adhatoda vasica leaf powder with
and without microencapsulation. A central composite 32 factorial design in Randomized
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Block Design (RBD) was employed to investigate the interaction of operating conditions at
varying temperature (120, 140 and 160° C) and feed moisture (19, 22 and 25%). There are 13
runs (treatment combinations) in the design. (Hashimoto and Grossmann, 2003).
2.5. High Performance Liquid Chromatography Study
High Performance Liquid chromatography was performed with a Shimadzu (Kyoto, Japan)
system consisting of a column oven (model CTO-10ASVP), a UV–visible diode-array
detector (model SPD-M10 Avp), a degasser (model DGU14 A), and a liquid chromatography
pump (model LC-10AT-VP); The sample (10 µL) was injected into the HPLC with a syringe
(Hamilton, Reno, NV, USA). The HPLC column used was a reversed-phase C18 (150 mm ×
4.6 mm, 5 µm; #504955) from Supelco (Bellefonte, PA, USA). Methanol and water in the
ratio of 40:60 was used as a mobile phase. Flow rate was maintained at 0.7 ml/min and
detection carried out at λmax at 298 nm was used (Ram et al., 2007). The standard stock
solution of vasicine was prepared by dissolving in methanol to obtain standard solution of
concentration 100 µg/ml. Further dilutions were made with methanol to get aliquot
concentrations 20, 40 60, 80 and 100 µg/ml. Twenty microlitres of this standard solutions
were injected into HPLC system and the peak area value and retention time were recorded.
The raw Adhatoda vasica raw herb leaf powder and its formulations through
microencapsulation and extrusion were extracted with three 5 ml volumes of methanol. The
pooled methanol extract, dried over anhydrous sodium sulphate was evaporated to dryness in
vacuo. The dried methanol extract, reconstituted with 1 ml of methanol was injected into
HPLC system. Peak area was noted for all the concentrations tested and the standard plot was
plotted with concentration (µg/ml) on the abscissa and peak area on the ordinate.
3. RESULTS & DISCUSSION
The retention of major alkaloid vasicine in techniques like microencapsulation and extrusion
of Adhatoda vasica was analyzed through High Performance Liquid Chromatography with
pure vasicine as standard. The pure vasicine standard plot was found to be linear for the
concentrations studied with a linear regression coefficient R2 of 0.999. The retention time of
standard vasicine was around 9.167 minutes (Fig.1). In hot air dried Adhatoda vasica leaf
powder, under the same conditions, the retention time of vasicine was around 9.508 minutes
(Fig. 2). The vasicine content was calculated as 377.982 ppm.
In gum acacia encapsulated Adhatoda vasica leaf powder under the same conditions
described above the retention time of vasicine was around 9.467 minutes (Fig.3). The
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vasicine content was calculated as 276.607 ppm. The percentage retention of vasicine in gum
acacia encapsulated Adhatoda vasica leaf powder compared to initial hot air dried powder
was 73.18%.
The retention time of vasicine was around 9.592 minutes (Fig.4) in maltodextrin encapsulated
Adhatoda vasica leaf powder. The vasicine content was calculated as 262.955 ppm. The
percentage retention of vasicine in maltodextrin encapsulated Adhatoda vasica leaf powder
compared to initial hot air dried powder was 69.57%.
In the spray dried Adhatoda vasica leaf powder without wall material, the retention time of
vasicine was around 9.558 minutes (Fig.5). The vasicine content was calculated as
187.112 ppm. The percentage retention of vasicine in spray dried Adhatoda vasica leaf
powder compared to initial hot air dried powder was 49.50%.
The gum acacia found to be suitable carrier material for encapsulating Adhatoda vasica leaf
powder based on the retention of total phenol and alkaloid content. The extruded product
with microencapsulation using gum acacia was studied under the same chromatographic
conditions described above. The retention time of vasicine was around 9.550 minutes (Fig.6).
The vasicine content was calculated as 244.634 ppm. The percentage retention of vasicine in
encapsulated and extruded Adhatoda vasica leaf powder compared to initial hot air dried
powder was 64.72 %. Further the extruded Adhatoda vasica leaf powder without
microencapsulation was studied. The retention time of vasicine was around 9.542 minutes
(Fig.7). The vasicine content was calculated as 167.214 ppm. The percentage retention of
vasicine in extruded Adhatoda vasica leaf powder compared to initial hot air dried powder
was 44.24 %.
It is in agreement with published data showing that the encapsulation of maltodextrins in
volatiles retention is not significant compared to gum acacia. It was also reported that gum
acacia retains the active phytochemicals than maltodextrins and the poor retention of
compounds by maltodextrins is related to the lack of emulsification properties (Reineccius et
al., 2003).
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Table. 1 – Retention of vasicine alkaloid in different preparations of Adhatoda vasica
leaf powder through HPLC Study
Treatments of Adhatoda vasica
Retention Time
leaf powder
(min)
Hot air dried
9.508
Gum acacia microencapsulated
9.467
Maltodextrin microencapsulated
9.592
Raw Adhatoda vasica spray dried
9.558
Encapsulated and Extruded
9.550
Raw Adhatoda vasica Extruded
9.542
Note: The retention percentage is calculated by comparing
Vasicine
Retention
(mg/Kg)
percentage
377.982
276.607
73.18
262.955
69.57
187.112
49.50
204.634
64.72
167.214
44.24
with the initial hot air dried
Adhatoda vasica leaf powder.
Fig.1 - HPLC Chromatogram of pure vasicine standard
Fig.2 - HPLC Chromatogram of hot air dried Adhatoda vasica leaf powder
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Fig.3 - HPLC Chromatogram of gum acacia encapsulated Adhatoda vasica leaf powder
Fig.4 - HPLC Chromatogram of maltodextrin encapsulated Adhatoda vasica leaf
powder
Fig.5 - HPLC Chromatogram of spray dried Adhatoda vasica leaf powder without wall
material
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Fig.6 - HPLC Chromatogram of of encapsulated and extruded Adhatoda vasica leaf
powder
Fig.7 - HPLC Chromatogram of extruded Adhatoda vasica leaf powder without
microencapsulation
4. CONCLUSIONS
The retention percentage of vasicine quantitatively analyzed through HPLC in all the
products through hot air drying, microencapsulation and extrusion experiments. The results
clearly indicate that the gum acacia micro encapsulated Adhatoda vasica leaf powder showed
a better retention of vasicine comparing to the other treatments and hence protects the
vasicine content under high pressure and temperature during extrusion process. The results of
this study will help the functional food product developers to exploit the microencapsulated
Adhatoda vasica leaf powder in processing techniques involving extreme temperature and
pressure in their formulations. This promising research work should be tested on multiple
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product development in both the pharmaceutical and nutraceuticals field to deliver the active
ingredients through food and other matrices.
ACKNOWLEDGMENT
The authors thank the support from Dr. K. Singaravadivel and Dr. S. Kumaravel, Indian
Institute of Crop Processing Technology, Thanjavur.
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