Moringa peregrina - The University of Jordan

Transcription

All Rights Reserved - Library of University of Jordan - Center of Thesis Deposit
II
III
I dedicate this work to my father & mother
and my wife and my daughter Mariam.
Special dedication to all my family, specially my brother Abdullah Majali
(Abu- Salameh) and his wife and their babies (Salameh and Fatimah) and
all my friends.
AL-Majali I.S.
DEDICATION
IV
ACKNOWLEDGEMENT
appreciation goes to my supervisor, Prof. Dr Sawsan Oran, for her patience, guidance,
assistance and technical advice. Her astute comments and suggestions have added
considerably to the clarity and organization of this work. May God richly bless her and her
family.
My appreciation also goes to my Co-Supervisors Prof. Khaled Khleifat and Dr. Mona
Hassuneh for their diverse and valuable contributions towards the success of this project
and special thanks goes to Prof. Khalid Matalka from Petra University for his helpful
comments and guidance in the preparation of this dissertation.
Great thanks to Miss Sara AL-Qaise from Dr. Hassuneh’s laboratory for her technical
assistance.
My sincere thanks also go to all my friends in Mutah school and at the
University of Jordan Dr. Jameel Al-Bzour, Dr. Wamidh Talib, Mona Abbas and Soha
Abudoleh who helped me in various ways and special thanks to Mr. Mashour AL-Assaf for
helping me in various ways.
Finally, the spiritual and moral support given by my sisters and their husbands are highly
appreciated.
AL-Majali I.S.
I thank the Almighty God for blessing me to complete this project. Special thanks and
LIST OF CONTENTS
Subject
Page
Committee Decision
II
Dedication
III
Acknowledgement
IV
List of Contents
V
List of Tables
IX
List of Figures
X
List of Abbreviations
XII
Abstract
XIII
1. Introduction, Specific Aims and Significance
1.1. Introduction
1
1
1.1.1.
Medicinal plants
1
1.1.2.
Moringaceae
2
1.1.3.
Antibacterial medicinal plants
3
1.1.4.
The immunomodulatory medicinal plants
4
1.1.5.
Median Lethal dose (LD50)
6
1.1.6.
Antioxidant
8
1.2. Specific Aims
9
1.3. Significance
11
2. Literature Review
2.1 Antibacterial effect of Moringa peregrina ethanolic extract
12
13
V
VI
2.2 Imunomodulatory and Antioxidant effects of Moringa peregrina
13
ethanolic extracts.
3.1 Plant material
18
20
3.1.1 Preparation of ethanolic extract
20
3.2 Determination of Moringa peregrina extracts median lethal dose
20
3.3 Bacterial strains and antibacterial activity
21
3.3.1 Determination of Minimum Inhibitory Concentration (MIC)
3.4 Immunomodulatory Activity
21
22
3.4.1 Animals
22
3.4.2 Treatment
22
3.4.3 Body and Relative Organ Weight
23
3.4.4 Measuring hematological parameters
23
3.4.5 Single cell suspension (SCS) of mononuclear cells
24
3.4.6 Lymphocyte proliferations (Blastogenesis) in response to
24
mitogen
3.4.7 Mixed Lymphocytes Response (MLR) Assay
25
3.4.8 IgM plaque-forming cell (PFC) assay
26
3.5 Ferric reducing antioxidant power (FRAP) assay
27
3.6 Statistical Analysis.
28
4. Results
30
4.1 Dry weight and (%) yield of plant extracts
31
4.2 Antibacterial effect of Moringa peregrina ethanolic extracts
31
4.3 Moringa peregrina extracts median lethal dose (LD50)
34
4.4 Imunomodulatory effect of Moringa peregrina ethanolic extracts on
36
3. Materials and Methods
VII
4.4.1 Effect of Moringa peregrina extracts on mice relative body weight
36
4.4.2 Effect of Moringa peregrina extracts on relative weights of lymphoid
38
organs (Spleen, Thymus and Lymph nodes)
42
4.4.3 Effect of Moringa peregrina extracts on Spleen Cellularity
44
4.4.4 Effect of Moringa peregrina extracts on hematological parameters
51
4.4.5 Effect of Moringa peregrina extracts on peripheral blood total white
cells WBCs counts
53
4.4.6 Effect of Moringa peregrina extracts on Mitogen Blastogenesis
Response
55
4.4.7 Effect of Moringa peregrina extraction Mixed Lymphocyte Reaction
4.4.8 Effect of Moringa peregrina extracts on Immunoglobulin-M (IgM)
57
Plaque Forming Cell (PFC) Assay
4.5 Antioxidant effect of Moringa peregrina ethanolic extracts
59
5. Discussion
65
6.Conclusions and Recommendations
67
7.References
79
8.Abstract (in Arabic)
Balb/c Mice
VIII
NUMBER
TABLE CAPTION
PAGE
4.1
Zone of inhibition for ethanolic extract of Moringa peregrina
plant at different concentrations on the bacterial growth of E.
coli
32
4.2
plant at different concentrations on the bacterial growth of S.
32
aureus
4.3
plant at different concentrations on the bacterial growth of K.
33
pneumonia
4.4
Minimum Inhibitory Concentration (MIC) of Moringa
peregrina ethanolic extracts of leaves, roots and seeds
against E. coli, S. aureus and K. pneumonia
33
LIST OF TABLES
IX
NUMBER
4.1
4.2
4.3
4.4
4.5
FIGURE CAPTION
% Mortality of Moringa peregrina seed extract
(mg/Kg) following a single oral dose
% Mortality of Moringa peregrina leaves extract
% Mortality of Moringa peregrina roots extract
Effect of Moringa peregrina leaves extract on the
average body weight of mice.
Effect of Moringa peregrina roots extract on the
PAGE
34
34
35
36
37
4.6
Effect of Moringa peregrina seeds extract on the
4.7
relative lymphoid organs' weights.
A : Spleen relative mass.
B : Lymph Nodes relative mass.
C : Thymus relative mass.
38
38
39
4.8
relative lymphoid organs' weights
39
40
40
relative lymphoid organs' weights
41
42
42
4.9
37
4.10
Effect of M. peregrina leaves extract on the cellularity
of the spleen as compared with the control group.
43
4.11
Effect of M. peregrina roots extract on the cellularity of
the spleen as compared with the control group.
43
4.12
Effect of M. peregrina seeds extract on the cellularity
of the spleen as compared with the control group.
44
LIST OF FIGURES
4.13
4.14
4.15
4.16
4.17
4.18
Effect of M. peregrina leaves extract on hematological
parameters.
A :RBCx106/µl
B : Hemoglobin g/dl
C : Platelets x103 /µl
D : PCV %.
45
45
46
46
Effect of M. peregrina roots extract on hematological
parameters.
A :RBCx106/µl
B : Hemoglobin g/dl
D : PCV %.
47
47
48
48
Effect of M. peregrina seeds extract on hematological
parameters.
A :RBCx106/µl
B : Hemoglobin g/dl
D : PCV %.
49
49
50
50
Effect of Moringa peregrina leaves extract on total
WBC number.
Effect of Moringa peregrina roots extract on total WBC
number.
Effect of Moringa peregrina seeds extract on total
WBC number.
51
52
52
4.19
The proliferative response of lymphocyte obtained from
the spleen of mice treated groups with different dose of
Moringa peregrina leaves extract in the absence or the
presence of different mitogen ( LPS, Con-A and PHA).
53
4.20
Moringa peregrina roots extract in the absence or the
54
4.21
Moringa peregrina seeds extract in the absence or the
54
X
4.22
Effect of Moringa peregrina leaves extract on the T
lymphocyte proliferation. R: Respond cells, S:
Stimulators cells, M: cRPMI-1640 complex media.
55
4.23
Effect of Moringa peregrina roots extract on the T
56
4.24
Effect of Moringa peregrina seeds extract on the T
56
4.25
humoral response as assessed by the IgM- Plaque
Forming Cell Assay (PFCs).
57
4.26
58
4.27
58
4.28
Standard curve of ascorbic acid.
59
XI
XII
AFC
Antibody Forming Cell
APC
Antibody Presenting Cell
BSA
Bovine Serum Albumin
b.wt
Body weight
Con A
Concanavalin A
cRPMI 1640
Complete RPMI medium
cRPMI 1640-10percentage
Complete RPMI medium with 10 percentage
cRPMI 1640-5percentage
Complete RPMI medium with 5 percentage
Exp
Experimental (treatment ) groups
FBS
Fetal Bovine Serum
Gm
Gram
Hb
Hemoglobin
HEPES
N-2-hydroxyethylpiperazin -N-2-ethanesulfonic
acid
IgM
Immunoglobulin M
IL
Interleukin
Kg
Kilogram
L
Litter
LPS
Lipopolysaccharide
MBA
Mitogen Blastogenesis Assay
Mg
milligram
MHC
Major Histocompatability Complex
µg
Microgram
µl
Microlitter
MIC
Minimum Inhibitory Concentrations
MLR
Mixed Lymphocyte Reaction
MTT
3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide
NK cell
Natural Killer cell
LIST OF ABBREVIATIONS OR SYMBOLS
PABA
Para-amino benzoic acids
PBS
Phosphate Buffer Solution
PCV
Packed Cell Volume
PFC
Plaque Forming Cell
PHA
Phytohemagglutinin
RBCs
Red Blood Cell
S.D.
Standard Deviation
S.E.
Standard Error
SCS
Single Cell Suspensions
SRBCs
Sheep Red Blood Cell
TNF-α
Tumor Necrosis Factors alpha
TNF-β
Tumor Necrosis Factors beta
TPTZ
2,4,6, Tripyridyl-s-Triazine
WBCs
White Blood Cell
WHO
World Health Organizations
XIII
XIV
By
Ibrahim Salameh AL-Majali
Supervisor
Prof. Sawsan Oran
Co- Supervisor
Prof. Khalid Khleifat
Co- Supervisor
Dr. Mona R. Hassuneh
ABSTRACT
The antibacterial, antioxidant and immunomodulatory effects of Moringa peregrina
(leaves, roots and seeds) ethanolic extracts were investigated. The effect of plant
extracts were tested against three bacterial species: Escherichia coli (E. coli ATCC
25922), Staphylococcus aureus (S. aureus ATCC 43300) and Klebsiella pneumoniae
(K. pneumoniae ATCC 13883). Moringa peregrina ethanolic extracts showed
significant antibacterial effect on the three tested bacterial strains using the disc
diffusion methods. The inhibition zones caused by leaf ethanolic extracts were 14-30,
8-19 and 9-22 mm in diameter against E. coli, K. pneumonia, and S. aureus
respectively. Root ethanolic extracts showed inhibition zones as 18-42, 44-59 and 3445 mm in diameter against E. coli, K. pneumonia, and S. aureus respectively. Seed
extract caused inhibitory zones of 16-38, 6-32 and 6-18mm in diameter against E. coli,
K. pneumonia, and S. aureus respectively. The results showed that the zones of
inhibition for the three bacterial species increased in a dose dependant manner and that
the Moringa peregrina root ethanolic extract exhibited more potent inhibition. The
other test done to assess the antibacterial effect of Moringa peregrina ethanolic extracts
was the minimum inhibitory concentrations (MIC). Such test was conducted on the
same three bacterial species, where the MIC for the Moringa peregrina leaf extract
against E. coli, K. pneumoniae and S. aureus were 12.0 mg/ml, 15.0 mg/ml and
18mg/ml respectively. The MIC for seed ethanolic extract were 13.0 mg/ml, and 7.0
mg/ml against E. coli, K. pneumoniae and S. aureus respectively. Also, the MIC for
root ethanolic extract of Moringa peregrina against E. coli, K. pneumoniae and S.
aureus were, 9.0 mg/ml and 3 mg /ml, 2 mg/ml and 5 mg/ml respectively. Such low
MIC values especially for the root extract represent strong potential for Moringa
peregrina as an antibacterial agent.
The antioxidant activity was obtained by ferric reducing antioxidant power (FRAP)
assay using ethanolic extracts of Moringa peregrina. The leaves demonstrated the
highest activity comparing to root and seed extract. Moringa peregrina extracts at 0.3
mg/ml exhibited antioxidant activity equivalent to 4.1 mM (0.72 mg/ml) ascorbic acid
for leaves, whereas roots’ and seeds’ extracts were equivalent to 2.46 (0.433 mg/ml)
and 2.61 mM (0.45 mg/ml) ascorbic acid, respectively. Such results, suggests that
ASSESSMENT OF THE ANTIBACTERIAL, ANTIOXIDANT AND
IMMUNOMODULATORY EFFECTS OF Moringa peregrina
EXTRACTS.
Moringa peregrina ethanolic extracts of leaves, roots and seeds may have strong
potential as an antioxidant agent.
The median lethal dose (LD50) was determined for each Moringa peregrina ethanolic
extract; seeds, leaves, and roots. Twelve groups, each group consists of 10 Balb/c mice,
each group was treated orally with either 750, 1000, 1250 or 1500 mg/Kg of each
extract. The obtained LD50 values of Moringa peregrina seeds, leaves and roots extracts
were 1230, 1178, and 1030 mg/Kg, respectively.
Furthermore, the immunomodulatory effects of Moringa peregrina leaves, seeds and
roots ethanolic extracts was assessed in Balb/c mice. The assessment was done two
levels: levels I (non-functional) and II (functional). To achieve this, groups of 5 Balb/c
mice were given a single acute subtoxic oral doses of the extracts at 1.13, 11.30, 23.40
and 113.4mg/Kg and immunomodulation was assessed on the 6th day following
ingestion of the different doses. In level I assessment, the following were assessed:
body weight, relative lymphoid organ weighs, splenic cellularity and peripheral blood
hematologic parameters. The ingestion of Moringa peregrina plant extracts acute doses
slightly increased the weight of the treated mice, especially in mice that were given seed
extracts. The relative lymphoid organs weight were also slightly affected by the acute
doses of Moringa peregrina extracts where statically significant increase in spleen and
lymph nodes weights was noticed in treated mice with leaves and roots extracts. While
the seeds' extracts did not affect their relative weights. As for the thymus, only the roots
extract signifcanlty increased its weight. Spleen cells significantly increased at low
doses of leaves and roots extracts, and seeds extract increased mice spleen numbers in
dose dependent manner. Furthermore, increasing concentration of leaves extract
significantly increased RBCs, hemoglobin, platelets and PCV %, roots extracts
increased hemoglobin and PCV %. Seeds extracts did not affect any of the
hematological parameters. In addition, WBCs in peripheral blood increased in dose
dependent manner. For level II immunomodulation assessment, the effect of Moringa
peregrina leaves, seeds and roots ethanolic extracts on the following functional criteria
was conducted: 1) effect on the proliferative responses of T and B lymphocytes in
mitogen blastogenesis and mixed allogeneic MLR assays; 2) IgM-Plaque forming cells
assay to T-dependant Ag sRBCs. , In the in vitro splenic lymphocytes mitogen induced
blastogenesis, the response to both T cell mitogens (ConA and PHA) and B cell mitogen
(LPS) was elevated in all groups that ingested leaves, roots and seeds extracts. The only
exception is the ConA response in higher doses of root extract where no significant
increase from vehicle control was detected. Similar results from mixed lymphocyte
reaction MLR assay were observed where T cell dependant proliferation increase was
noted for responder cells from all Moringa peregrine extracts fed mice. Furthermore,
the oral administration of an acute dose of Moringa peregrina leaves and seeds extracts
significantly increased PFCs/106 splenocytes in a dose dependent manner. Interestingly,
low doses of roots extract significantly increased PFCs/106 splenocytes more than
higher doses. These findings indicated that certain constituents of Moringa peregrina
leaves, roots and seeds ethanolic extracts possess potent effects on the cellular and
humoral immunity. In conclusion, Moringa peregrina ethanolic extracts from leaves,
seeds, or roots are safe when used at doses around or below 1:10th of LD50, because all
acute oral doses from 113.4 – 1.13 mg/kg given showed no toxicity at all hematologic
and immunological parameters tested. Interestingly, such subtoxic acute doses of
Moringa peregrina extracts demonstrated significant potential as an immunomodulatory
agent even at the lowest dose of 1.13 mg/kg. Thus, the current study stresses the
importance of preserving such an important endemic plant and warrants for further
studies to assess the specific mechanism by which it exerts its beneficial effects.
XV
74
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76
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Moringa peregrina - The University of Jordan

Transcription

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