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Immunomodulatory Activity of Methanolic Extracts of Ficus Glomerata Roxb. Leaf, Fruit and Bark in Cyclophosphamide Induced Mice

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  • HKES college of pharmacy, Kalaburagi (Gulbarga), Karnataka, India

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To evaluate the immunomodulatory activity of methanolic extracts of Ficus glomerata Roxb. leaves, fruits and bark on cyclophosphamide induced immunosuppression in mice. Methanolic extracts of leaves, fruits and bark of Ficus glomerata Roxb. (500mg/kg p.o.) were administered 13 days to albino mice and cyclophosphamide (30mg/kg i.p.) was administered on 11th,12th and 13th days 1 hour after the administration of the respective treatment. On the 14th day blood was collected by retro orbital puncture and the activity was evaluated by determining the RBC, Hb%, Platelet, total WBC and differential counts. Methanolic extracts of leaves, fruits & bark of Ficus glomerata Roxb. showed very significant (p<0.001) counteracting effect to cyclophosphamide induced reduction in total WBC, DLC and platelet counts & significant (P<0.01) effect to that of reduction in RBC counts and Hb %. The significant inmmunostimulant effect of the methanolic extracts of Ficus glomerata Roxb. leaf, fruit & barks on cyclophosphamide induced myelosuppression may be attributed towards the collective presence of saponins, sterols and tannins in the extracts, which suggest the immunomodulatory activities of the methanolic extracts of Ficus glomerata Roxb. leaves, fruits & bark.
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International Journal of Modern Botany 2011; 1(1): 4-7
DOI: 10.5923/j.ijmb.20110101.02
Immunomodulatory Activity of Methanolic Extracts of
Ficus Glomerata Roxb. Leaf, Fruit and Bark in
Cyclophosphamide Induced Mice
Sanjeev Heroor1,*, Arunkumar Beknal1, Nitin Mahurkar2
1Dept. of Pharmacogonsy and Phytochemistry, HKES’s MTR institute of Pharmaceutical Sciences, Gulbarga, Karnataka, India
2Dept. of Pharmacology, HKES’s MTR institute of Pharmaceutical Sciences, Gulbarga, Karnataka, India
Abstract To evaluate the immunomodulatory activity of methanolic extracts of Ficus glomerata Roxb. leaves, fruits and
bark on cyclophosphamide induced immunosuppression in mice. Methanolic extracts of leaves, fruits and bark of Ficus
glomerata Roxb. (500mg/kg p.o.) were administered 13 days to albino mice and cyclophosphamide (30mg/kg i.p.) was
administered on 11th,12th and 13th days 1 hour after the administration of the respective treatment. On the 14th day blood
was collected by retro orbital puncture and the activity was evaluated by determining the RBC, Hb%, Platelet, total WBC and
differential counts. Methanolic extracts of leaves, fruits & bark of Ficus glomerata Roxb. showed very significant (p<0.001)
counteracting effect to cyclophosphamide induced reduction in total WBC, DLC and platelet counts & significant (P<0.01)
effect to that of reduction in RBC counts and Hb %. The significant inmmunostimulant effect of the methanolic extracts of
Ficus glomerata Roxb. leaf, fruit & barks on cyclophosphamide induced myelosuppression may be attributed towards the
collective presence of saponins, sterols and tannins in the extracts, which suggest the immunomodulatory activities of the
methanolic extracts of Ficus glomerata Roxb. leaves, fruits & bark.
Keywords Ficus Glomerata Roxb, Cyclophosphamide, Immunomodulatory, Myelosuppression
1. Introduction
Indian traditional systems of medicine like Siddha and
Ayurveda have suggested to increase the body’s natural
resistance to disease under the concept of ‘Rasayana’. A
number of medicinal plants as Rasayanas such as Tinospora
codifolia, Mangifera indica, etc. have been claimed to pos-
sess immunomodulatory activity[1]. Immunomodulation is
any procedure which can alter the immune system of an
organism by suppression and stimulation of the cells and
organs of the immune system[2].
Immunostimulation in a drug induced immunosuppres-
sion model and immuno suppression in an experimental
hyper-reactivity model by the same preparation can be said
to be true immunomodulation[3]. Certain agents have been
shown to possess acitivity to normalize or modulate patho-
physiological processes and hence are called immuno-
modulatory agents[4]. A number of medicinal plants have
been screened systematically for their immunomodulatory
activity such as Tinospara cordifolia, Mangifera indica[5].
Cyclophosphamide is an alkylating agent widely used in
* Corresponding author:
ssheroor@gmail.com (Sanjeev Heroor)
Published online at http://journal.sapub.org/ijmb
Copyright © 2011 Scientific & Academic Publishing. All Rights Reserved
anti-neoplastic therapy. It is effective against a variety of
cancers such as lymphoma, myeloma and chronic lympho-
cytic leukemia[6]. Cyclosphamide acts on both cyclic and
intemitotic cells, resulting in general depletion of immune
competant cells[7]. Cyclophosphamide induced immuno-
suppression is reported to prompt various types of infec-
tions[8].
Figure 1. Image of the plant of Ficus glomerata Roxb
(Figure 1) Ficus glomerata Roxb. (Syn. Ficus racemosa)
family: moraceae, an evergreen tree of 15-18m height.
Young shoots are glabrous, pubescent with ovate and ta-
pering leaves. The tree bears a few aerial roots, figs pubes-
cent, reddish when ripened[9]. Survey of literature shows
International Journal of Modern Botany 2011; 1(1): 4-7 5
the presence of glycosides, tannins and wax[10]. Tradition-
ally leaves are used in bilious affection and diarrhea. Fruit
is edible and is given in menorrhagia, haemoptysis and in
diabetes. Bark is used in the form of fine powder in dysen-
tery, diabetes and in combination with gingelly oil it is ap-
plied to cancerous affections[11]. The present study was
aimed at screening of methanolic extracts of leaves, fruits
and bark Ficus glomerata Roxb. for immunomodulatory
activity on cyclophosphamide induced immune suppressed
albino mice.
2. Materials & Methods
2.1. Plant Material
Leaves, fruits and bark of Ficus glomerata Roxb. were
collected from local areas of North Karnataka & a voucher
specimen has been deposited at the departmental herbarium.
The mentioned parts of the plant were dried and pulverized
to particle size (#) 40 and then were first defatted with pe-
troleum ether (40-600C) and extracted with methanol using
Soxhlet apparatus for 48h to obtain methanolic extracts of
leaves, fruits and barks of the plant respectively. The filtrates
of the extracts were concentrated to dryness at 400C under
reduced pressure in a rota evaporator.
The yields of the methanolic extracts of leaves, fruits and
barks were 23.97gm (14.47%w/w), 9.95gm (9.25%w/w) and
52.85gm (33.02%w/w) respectively.
2.2. Preliminary Phytochemical Studies
The phytochemical screening and TLC studies[12,13] of
the methanolic extracts of leaves, fruits and barks indicated
the presence of carbohydrates, glycosides, wax, steroids,
saponins and tannins but only the fruit extract showed the
presence of proteins instead of wax.
2.3. Animals
Swiss albino mice of either sex, weighing 25-30g house-
hold in standard conditions of temperature, humidity and
light were used. They were fed with standard rodent diet and
water ad libitum.
2.4. Acute Toxicity Studies
Acute toxicity studies were conducted as per OECD
guideline by 425 method to assess LD50[14]. The animals
did not show mortality at the dose of 5000mg/kg and hence
its 1/10th dose i.e., 500mg/kg was used as the therapeutic
dose for the methanolic extracts of the study.
2.5. Test Samples
Weighed quantities of test extracts were suspended in 1%
sodium carboxy methyl cellulose to prepare a suitable dos-
age form[15]. The control animals were given an equivalent
volume of sodium CMC vehicle.
2.6. Drugs
Cyclophosphamide was used as a standard immunosup-
pressant, Cycloxan® (Biochem pharmaceutical industries
Ltd. Mumbai) containing 200mg cyclosphosphamide, was
procured from the market and dilutions were made using
sterile water for injection as mentioned on the label of the
marketed product.
2.7. Cyclophosphamide Induced Myelosuppression[16]
Animals were divided into four groups of six animals each.
Group I served as Control group, received the vehicle(1%
sodium CMC) for a period of 13 days. Group II (Cyclo-
phosphamide group) received the vehicle (1% sodium CMC)
for a period of 13 days and on 11th, 12th and 13th days was
injected with cyclophosphamide (30mg/kg i.p). Group III,
IV and V were administered methanolic extracts of leaves,
fruits and bark of the plant (500mg/kg p.o.) daily for 13 days.
The animals of groups III, IV and V were injected with
Cyclophosphamide (30mg/kg i.p) on the 11th, 12th & 13th
days, 1 hour after the administration of the respective
treatment. Blood samples were collected on the 14th day of
experiment by retro orbital puncture and hematological pa-
rameters were studied for RBC, Hb %, Platelets, total WBC
counts and differential leucocytes counts (DLC).
Statistical analysis:Data were expressed as mean ± SEM
and differences between the groups were statistically de-
termined by analysis of variance followed by Dunnet’s test.
3. Results and Discussions
Cyclophosphamide at the dose of 30mg/kg. i.p. caused a
significant reduction in total WBC count, differential leu-
cocyte counts and platetes and to some extent reduction in
RBC & HB % as compared to control group (Group I).
Methanolic extracts of leaves, fruits and bark showed very
significant (P<0.001) increase in total WBC, DLC and
Platelets and significant (P<0.01) increase in RBC and Hb %
when compared with cyclophosphamide group (Group II).
However the significant effects of fruits and bark extracts of
methanol were more pronounced than that of the methanolic
extract of leaves.
Table 1. Effect of methanolic extracts of Ficus glomerata Roxb. leaf, fruit and barks on cyclophosphamide induced myelosuppression
Animal group n=6
RBC (106/mm3)
Hb(g%)
Platelets (105/mm3)
WBC (103/mm3)
I
6.332±0.060
9.667±0.076
6.467±0.071
4.550±0.133
II
5.047±0.090
8.100±0.118
4.783±0.047
2.300±0.208
III
5.728*±0.148
9.133*±0.105
5.700**±0.129
3.250**±0.076
IV
5.777*±0.127
9.150*±0.056
5.717*±0.130
3.150*±0.160
V
5.748*±0.127
9.217*±0.090
5.750**±0.128
3.700**±0.077
6 Sanjeev Heroor et al.: Immunomodulatory Activity of Methanolic Extracts of Ficus Glomerata Roxb. Leaf,
Fruit and Bark in Cyclophosphamide Induced Mice
Tab le 2. Effect of methanolic extracts of Ficus glomerata Roxb. leaf, fruit and Barks on cyclophosphamide induced myelosuppression
Animal Gr. n=6
Lymphocytes (%)
Eosinophils (%)
Basophils (%)
Monocytes (%)
I
73.50±0.428
2.667±0.210
1.667±0.210
1.667±0.210
II
64.00±0.730
0.666±0.210
0.333±0.210
0.166±0.167
III
69.83*±0.477
1.500*±0.223
0.500*±0.223
0.666*± 0.210
IV
69.17**± 0.749
1.333**± 0.210
0.333**± 0.210
0.500*± 0.223
V
69.33**± 0.802
1.500*± 0.223
0.500**± 0.223
0.666*± 0.210
Immunomodulatory activity of methanolic extract of
leaves, fruits and bark of Ficus glomerata Roxb. was ex-
plored by evaluating their effects on cyclophosphamide
induced myelosuppression in mice. Results of the study
revealed the counteracting effect of the extracts to the
cyclophosphamide induced bone marrow activity suppres-
sion i.e. myelosuppression, as indicated by increase in RBC,
total WBC platelet counts, Hb % and DLC in the extract
treated groups (Group III & IV), when compared to cyclo-
phosphamide treated group (Group II).
Bone marrow is a site of continued proliferation and
turnover of blood cells and is also a source of cells involved
in immune activity. A high degree of cell proliferation ren-
ders bone marrow a sensitive agent, particularly to cytotoxic
drugs. In fact bone marrow is the organ most affected during
any immunosuppression therapy with this class of drugs.
Loss of stem cells and inability of bone marrow to regenerate
new blood cells will result in thrombocytopenia and leuco-
penia[17].
The results indicate modulation of bone marrow activity,
namely suppression when used cyclophosphamide alone
and stimulation to counteract the cyclophosphamide induced
myelosuppression in pretreated methanolic extract groups of
leaves, fruits and bark of Ficus glomerata Roxb.
4. Conclusions
From the phytochemical investigation, it was found that
the major chemical constituents of the methanolic extracts of
leaves, fruits and bark were steroids, saponins, tannins,
proteins and carbohydrates. Saponins are either triterpenoid
or steroidal glycosides proven as important phytoconstituent
with different pharmacological activities such as antiallergic,
cytotoxic, antitumour, antiviral, immunomodulating, anti-
hepatotoxic, and antifungal activities. Recently three dios-
genyl saponins isolated from Paris polyphylla reported for
immunostimulating activity[18]. Tannins are also known to
possess immunostimulating activites. The well known ay-
urvedic formulation, Triphala Churna contains Terminalia
chebula, Terminalia belenica and Emblica officialis, which
are rich in tannins have been reported for immunostimulating
activity[19]. Hence the collective presence of steroids,
saponins and tannins in the methanolic extracts would be
attributed for immunostimulating activity. However metha-
nolic extracts of fruits and bark of Ficus glomerata Roxb.
exhibited more immune-potentiating activity than that of
methanolic extract of leaves. Further studies are underway to
find out the exact mechanism of immunostimulating effect of
Ficus glomerata Roxb.
ACKNOWLEDGEMENTS
Authors are thankful to authorities of HKESociety and
MTR Institute of Pharmaceutical sciences, Gulbarga, Kar-
nataka India, for providing necessary facilities to carry out
the study.
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