Download Immunomodulatory activity of MunroniapumilaWight. (Meliaceae

Volume 3, Issue 1, January-February 2014
ISSN No.: 2319-7536
Available Online at www.gpublication.com/crbps
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RESEARCH PAPER
Immunomodulatory
extraction in rats
activity
of
MunroniapumilaWight.
(Meliaceae)
leaf
Ediriweera P.S. Chandana *1, Shashindra K.K. Hansini 2 and Sameera T.D.K.C. Gunasekara3,
*1Department of Zoology, Faculty of Science, University of Ruhuna, Matara, Sri Lanka.
[email protected]
2
Department of Zoology, Faculty of Science, University of Ruhuna, Matara, Sri Lanka.
[email protected]
3
Department of Zoology, Faculty of Science, University of Ruhuna, Matara, Sri Lanka.
[email protected] 2
Abstract
MunroniapumilaWight. (Local name: “Bimkohomba”) is a medicinal plant endemic to Sri Lanka. It has long been used in Sri
Lankan traditional medicine to treat a variety of ailments on the belief that it can enhance immune system. However, scientific
evidence supporting this notion is lacking. Present study was undertaken to study immune responses of M. pumila leaf
extractions in rats. The crude product of the dried leaves M. pumila was prepared by ethanol extraction. The crude product was
dissolved in saline at a concentration of 40 mg/ml to prepare the test solutions. The test solution was administered orally at a
dose of 200 mg/kg/day in each rat for 14 days in Wistar albino rats (n=12). Control group rats (n=12) was received normal
saline for 14 days. Paw edema reaction (acute inflammatory reaction), total & differential WBC counts and blood IL-4 levels
were measured. In M. pumila treated group acute inflammatory reaction was significantly high (paw edema recovery). Total
WBC count and lymphocyte count were significantly high in M. pumila treated group. These results indicate a possible
enhancement of cell mediated immunity in M. pumila treated rats. However blood IL-4 level did not differ significantly between
control & M. pumila treated rats indicating a possible non-involvement of adaptive immune response. Taken together Ethanolic
leaf extract of M. pumila enhanced the cell mediated immunity in the present experimental system.
Keywords
Munroniapumila, inflammatory response, IL4, WBC count, Cell mediated immunity.
INTRODUCTION
Cause of various diseases can be attributed to
immunosuppression or weak immunomodulatory
effects [1]. Our immune system whether it is cell based
or humoral, need of activation at proper times to combat
with various ailments. In western medicine there are
many antibiotics designed to combat with various
infections. There are also several drugs designed to
activate immune system (immunomodulators). These
medications cause serious side effects which give rise to
various medical conditions. Western medicines are
basically not designed to activate the body’s own
immune system. In contrast traditional plant based
medicines are believed to cause fewer side effects and
strengthen the body’s natural defense mechanisms.
Many western scientists are now looking to develop
traditional (indigenous) medicines in order to combat
various diseases [2].
Traditional medicines were developed based on
knowledge systems which evolved over generations
within various societies. It includes Ayurveda, Siddha
medicine, Unani medicine, DeshiyaChikithsawa
(traditional Sri Lankan medicine) etc. In most cases
plant based immunomodulators are used to treat
diseases although their mechanistic basis of healing is
not known. Recently, scientists are paying much
attention to unravel the mechanistic basis of
immunomodulatory effects of plant based medicines.
One emerging field in the medical science is
Aurgenomics where scientists are trying to understand
the mechanistic basis of the traditional medicine at
genomic level [3].
The traditional medical practice in Sri Lanka (mainly
based on herbal extractions) boasts glorious history with
its roots tracing back to pre-aryan civilization [4]. Sri
Lanka possesses a very diverse flora in which medicinal
plants are a significant component. There are many
endemic plants which are used by traditional medical
practitioners to enhance the immune system. In Sri
Lanka, however, a very few research is undertaken to
characterize immunomodulatory effects of these plant
based medicines.
Acute inflammatory reaction is a direct
measurement of cell mediated innate immunity, while
blood IL-4 level is a measurement of adaptive &
humoral immunity. Total WBC count and differential
WBC count are also measurements of cell mediated
immunity [5]. Paw edema method, total WBC count &
the differential WBC count were used as direct
measurements of cell mediated innate immunity.
Adaptive immune response was studied by measuring
blood IL-4 which is a key regulator in the adaptive
immunity [6].
Munroniapumila, locally known as “Bimkohomba” is
an endemic medicinal plant in Sri Lanka which is
believed to be a strong immune-stimulant. As indicated
by various traditional medical practitioners in Sri
Lanka, all parts of M.pumila are highly effective in
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Ediriweera P.S. Chandana et al, Current Research in Biological and Pharmaceutical Sciences, 3 (1) Jan-Feb 2014, 01-04
boosting immune system. However, there is no
scientific evidence to support this notion. Therefore the
present study has been undertaken to investigate the
immunomodulatory activity of leaves of M. pumila in a
rat model.
MATERIALS AND METHODS
Collection of plant material
Munroniapumila (“Bimkohomba”) was obtained from
Haldummulla Medicinal Plant Research Garden and
was authenticated by Dr.PiyalMarasinghe, the Chief
Botanist, Haldummulla Medicinal Plant Research
Garden, Haldummulla, Sri Lanka.
Preparation of the extract
The M. pumila leaf extractions were prepared
as described elsewhere [7]. Leaves were washed &
dried at 450C under shady condition. Dried plant
material was placed in 100% ethanol for 6 days & on
6th day plant material was sieved. The extraction was
evaporated at 700C & the crude product was obtained.
Test solution was prepared by dissolving crude extract
in normal saline at a concentration of 40mg/ml.
IL-4 measuring kit was purchased from
Cusabio Biotech Company Ltd. Blood IL-4 level was
measured using specific antibody coated micro-wells
according to the protocol developed by the
manufacturer. Plasma of blood samples of control &
treated rats were filled to the wells according to the
plate layout & several wells was kept for the control.
Micro plate was sealed & incubated for two hours at
370C. After incubation micro plate was tapped on a
blotting paper & 100μl of Biotin solution was added to
each well. It was incubated for an hour at 370C. After
three washes using 200μl of washing buffer for each
wash, the micro plate was tapped on a blotting paper &
100μl of Horse Radish Peroxidase was added to each
well. The micro plate was incubated for one hour at
370C. After five washes, 90μl of Tetra Methyl
Benzediene substrate was added to each well in dark
environment & it was incubated for 15 minutes. Then
the stop solution was added to stop the reaction. Finally
the absorbance of the each well was recorded using
ELISA reader at 450nm. Blood IL-4 levels were
calculated using the standard curve.
RESULTS
Experimental animals
Acute inflammation
Female Wistar Albino rats weighing 150 –
200g were used in the present study. The experimental
animals were obtained from Medical Research Institute
in Colombo, Sri Lanka & were maintained under
standard laboratory conditions in an animal house under
12/12 light/dark cycle. They were fed with commercial
pellet diet & water ad libitum. The animals were
allowed to acclimatize to laboratory conditions prior to
experiment.
Paw edema inflammation was totally recovered after
120 minutes in M. pumila treated group. In the control
group, however, it was recovered after 270 minutes.
This time gap between the recoveries of treated &
control rats was significantly different
(p=0.012).
Therefore the total inflammatory reaction (the time
taken for the development of the highest paw edema &
the time taken for the recovery) was faster in M .pumila
treated group than the control group (Figure.1).
Dose
Animals were randomly separated into
treatment and control groups. Treatment group (n=10)
was orally fed with 1 ml of test solution for 14 days
where each rat received 200 mg of crude extract per
1000g b.w. (body weight) per day [8]. The control
group (n=10) was received the 1ml of normal saline.
Acute inflammation
After 1 hour of the last treatment on 14th day,
paw edema (acute inflammation) was induced in each
rat by injecting 0.1ml of chick red blood cells into the
right hind paw as described elsewhere [9, 10]. Size of
the paw thickness was measured in each rat in every 30
minutes using a manual Vernier Caliper. Recovering
time of treated groups was compared with control group
using SPSS repeated measurements One Way ANOVA.
WBC differential count
Blood smears were prepared as described by
Jagzap, et al., 2012 [10]. Differential WBC count was
recorded using a light microscope.
Total WBC count
Total WBC was counted for each blood sample
using a hemocytometer.
Measuring blood IL-4 level (Using ELISA)
Differential WBC count
Numbers of all the types of WBCs were higher in M.
pumila treated group than that of control group
(Figure.2).
Total WBC count
Total WBC count of Bimkohomba treated
group was significantly higher* than that of control
group (p = 0.019) (Figure.3).
Measuring blood IL-4 level (Using ELISA)
Mean blood IL-4 level in Bimkohomba treated
rats and control group rats were 2.572 ± 1.042 pg/ml
2.317 ± 0.549 pg/ml respectively. There was no
significance difference of blood IL-4 levels between
control & M. pumila treated rats (t = 0.43, p = 0.687).
DISCUSSION
M. pumila is an endemic plant which is used as a
medicinal plant in Sri Lankan traditional medicine. It is
believed
that
M.
pumila is a
powerful
immunomodulator and in the present study
immunomodulatory effect of ethanolic extract of M.
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Ediriweera P.S. Chandana et al, Current Research in Biological and Pharmaceutical Sciences, 3 (1) Jan-Feb 2014, 01-04
Acute Inflammation
9
8
7
Paw Thickness/ mm
pumila leaves was studied in Wistar albino rats with a
special reference to cell mediated immunity.
M. pumila showed marked increase in cell
mediated immune response in rats. The acute
inflammatory response, which is a direct correlate of
cell mediated immunity [11], was found to be
significantly increased in M. pumila treated rats than
control group. Increased acute inflammatory response
indicates that ethanolic extract of M. pumila leaves have
a stimulatory effect on lymphocytes and accessory cell
types required for the expression of the reaction.
Various studies have indicated that the
increased differential WBC count is an indication of the
enhancement of the immune function [12]. Therefore,
increased differential WBC counts in M. pumila treated
rats indicates immune function enhancement.
Total WBC count of M. pumila treated rats
was significantly higher than control group, which
indicates the enhanced production of WBC. In fact,
bone marrow is the most affected organ during any
immunomodulation therapy [13]. Treatment of M.
pumila was found to increase the total WBC indicating
that this medicinal plant might have stimulated the bone
marrow activity in experimental rats. These data further
highlight the increased cell mediated immunity in the
M. pumila treated rats.
IL-4 levels did not show any significant
difference in the M. pumila treated group when
compared to the control group. Hence IL-4 might not be
the cytokine mediator of M. pumilaimmunomodulatory
action. These data might support the notion that M.
pumila might have the ability to stimulate innate
immunity function via powerful acute inflammatory
reaction & the significant elevation of total WBC
counts. Since IL-4 level has not been increased in the
M. pumila treated group it can be deduced that M.
pumila might not enhance adaptive immunity in the
present experimental set up. IL-4 is a key regulator in
humoral and adaptive immunity [6]. According to the
present results the use of M. pumila leaves as an
immunomodulator may be possible in future.
Further studies are required to test the toxicity
& the dose dependent activity. Similarly isolation of
active ingredients is also essential.
6
5
4
3
2
1
0
0
30
60
control
90
120
150
180
Time/min
Bimkohomba
Figure 2: Differential WBC counts in M. pumila treated
group & the control group.
CONCLUSION
Within the scope of findings of the present study, the
ethanolic extract of Munroniapumila Wight. leaves can
exert its bioactivity on immune system as a potent
enhancer of cell mediated immunity.
Figure 3: Total WBC count of M. pumila treated group
& the control group
FIGURES AND ILLUSTRATIONS
Figure 1: Acute inflammatory reaction of M. pumila
treated group & the control group.
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Ediriweera P.S. Chandana et al, Current Research in Biological and Pharmaceutical Sciences, 3 (1) Jan-Feb 2014, 01-04
Total WBC count in Bimkohomba treated
group and control group
14
5. Shailasree S, Ruma K, Kini KR, NiranjanaSR,Prakash
HS.(2012) Potential anti-inflammatory bioactives from
medicinal plants of Western Ghats, India- Review. Phcog.
Commn.2(2): 2-9.
12
WBC count * 10^3/µl
4. Abeysekara SAC. (2007)Traditional medicine in Sri Lanka
and neighbouringcountries,S.A.C. Abeysekara: Colombo, Sri
Lanka.p292
10
6. Hershey GK, Friedrich MF, Esswein LA, Thomas ML,
Chatila TA.(1997)The association of atopy with a gain-offunction mutation in the alpha subunit of the interleukin-4
receptor. N. Engl. J. Med. 337(24):1720–1725.
8
6
4
7. Zulfiker AHM, Rahman MM, Hossain MK, Hamid K,
Mazumder, Rana MS. (2010) In vivo analgesic activity
ofethanolic extracts of two medicinal plants –
ScopariadulcisL. andFicusracemosa Linn. Biol.Med. 2:42-48.
2
0
Control
Binkohomba
ACKNOWLEDGEMENT
Authors are grateful for the research grant
(DVC/Pro/19/2) offered by UOR under TURIS
(Transforming University of Ruhuna into International
Standards).
Authors
also
wish
to
thank
Dr.PiyalMarasinghe, Chief Botanist, Haldummulla
Medicinal Plant Research Garden, Sri Lanka for his
valuable support.
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