Biomedical & Pharmacology Journal, March 2019. Vol. 12(1), p.

251-258

Amelioration of Cyclophosphamide Induced

Immunosupression by the hydro-Alcoholic Extract of
Gymnema Sylvestre Leaves in Albino Rats

Padmaja Priyadarshini Kar1, Bandana Rath1,

Y. Roja Ramani2 and C.S. Maharana1
1
Department of Pharmacology, MKCG Medical College, Berhampur, Odisha, India.
2
Department of Pharmacology, SLN Medical College, Koraput, Odisha, India.

http://dx.doi.org/10.13005/bpj/1635

(Received: 12 October 2018; accepted: 02 February 2019)

Cyclophosphamide (CP), the most commonly used anti-neoplastic agent causes

immunosuppression and toxic effects on various organs that are the limiting factors of cancer
treatment. It can be hypothesized that addition of new immunopotentiating agents with
detoxification properties would have beneficial role in cancer therapy. Many researchers
have proved that, if certain plant products are combined with cancer chemotherapeutic agents,
reduce toxicities and improve tumour response. In Ayurveda, Gymnema sylvestre is commonly
used for diabetes, obesity and asthma. Also it possesses anti-inflammatory, astringent and
digestive properties. Reports on the immunostimulatory activity of Gymnema sylvestre leaves
are available from some in vitro and in vivo experiments. With this background the present
study was undertaken to evaluate the potential beneficial role of hydro-alcoholic extract of
Gymnema sylvestre leaves (GSE) on cyclophosphamide induced immunnosupression in rats.
In this experiment, five groups (n=6 in each) of wistar albino rats were randomly divided to
receive drugs and vehicle orally for 21 days. Gr I and II received vehicle. Gr III, IV and V were
administered with Levamisole 50 mg/kg, GSE 25mg/kg and GSE 50 mg/kg respectively. Except Gr
I rats, all rats were injected intraperitoneally with Cyclophosphamide (100mg/kg) on day 9th and
16th of drug treatment. The effects on various organ weights, rise in Haemagglutination titre to
Sheep RBC Antigen, delayed type of hypersensitivity (DTH) response to Sheep RBC, percentage
of neutrophil adhesion to nylon fibre and phagocytic index from carbon clearance test were
evaluated. Humoral and cellular immunity were measured from HA titre and DTH response
respectively. It has been observed that, GSE 50 mg/kg significantly increased the antibody titre,
percentage neutrophil adhesion and phagocytic index in CP induced immunosuppressed rats.
It also restored the CP induced changes in organ weights and the DTH response at 24 and 48
hours of antigen challenge. But these effects were not comparable to that of Levamisole. Our
study shows that Gymnema sylvestre reduced the CP induced immunotoxicities and therefore,
it could be a safe supplement to cyclophosphamide chemotherapy.

Keywords: Gymnem sylvestre, Cyclophosphamide, Immunomodulation, Albino rats.

Several cytotoxic drugs are being used lupus erythematosus, rheumatoid arthritis and
as cancer chemotherapeutics as well as for multiple sclerosis but has toxic effects on normal
long term immunosuppressive therapy in organ cells too.2-4 Apart from its therapeutic efficacy, its
transplant subjects.1 Cyclophosphamide is most immunosuppressive and cytotoxic effects bring an
commonly used chemotherapeutic drug against impairment of host defence mechanism leading
a variety of cancers and disorders like systemic to significant morbidity and mortality which is

This is an Open Access article licensed under a Creative Commons license: Attribution 4.0 International (CC-BY).
Published by Oriental Scientific Publishing Company © 2019
252 Kar et al., Biomed. & Pharmacol. J, Vol. 12(1), 251-258 (2019)

a major limiting factor in cancer treatment.5 The plant Gymnema sylvestre were procured from
toxic effects of cyclophosphamide (CP) are mainly Indian Herbs Saharanpur (UP)
due to generation of its active metabolites namely Animals
phosphoramide mustard and acrolein, the most Thirty wistar albino rats of either sex
toxic agent6. The molecular mechanism of CP of 2-3 months old (weighing between 120-
inducing immunosuppression has been explained 150gms) were procured from a registered breeder.
by Huang and Li in 20137 As IL-10 and IFN (no.526/02/bc/CPCSEA) The animals were
–ã play an important role in cyclophosphamide housed in central animal house of MKCG Medical
induced immunosuppression, the model of CP College Berhampur (Regd No. 472/CPCSEA/)
induced immunosupression is employed to and maintained at 22±1º C with 55±10% relative
evaluate the potential immunomodulatory effects humidity and 12/12 hour light/ dark cycle. All rats
of test substances.8-10 Hence, discovery of new were fed with standard pellet diet and water ad
immunopotentiating agents with detoxification libitum. The animals were allowed to acclimatize
properties would have potential beneficial effects to laboratory conditions one week prior to the day
in cancer treatment. of experimentation. Prior to the experimentation,
A large number of plants such as the experimental protocol was approved by the
Tinospora cordifolia, Moringa indica, Ocimum Institutional Animal Ethics Committee (IAEC)
sanctum, Azadirachta indica, Abulitum indicum, and care of animals was taken as per CPCSEA
Curcuma longa, Moringa Olifera, Centilla aciatica guidelines.
have been shown to possess a wide array of Drugs and chemicals
immunomodulatory effects in rat and mice.11 Many All the drugs and chemicals were
studies have reported that combining certain plant of analytical grade. Levamisole (Khandewal
products with chemotherapy may improve quality pharmaceutical Ltd. Mumbai), cyclophasphamide
of life, tumour response and reduce toxicities of (Biochem Pharmaceutical, Mumbai), colloidal
chemotherapy.eg Ganoderma lucidum,5 Kigelia carbon (Indian Ink, camel India pvt. Limited),
africana12, Onion lectin1 hemicellulose of bamboo Alsevers solution and Phosphate buffer saline were
shavings, 8 Decalepis hamiltonii 13,14 Roscorea procured from Hi-Media Laboratories Pvt. Limited.
procera rhizomes15 etc. Test compound preparation
Gymnema sylvestre R Br. (Family The dilution of hydro-alcoholic extract
Asclipedeceae) leaves, commonly known as of Gymnema sylvestre leaves was prepared in 1%
Gudmar has been widely used in Indian Ayurveda gum acacia prior to oral administration. Freshly
traditional medicine to treat diabetes, obesity and prepared solution was used for experiment.
asthma.16 The leaves of this plant has also been Acute toxicity study
used as anti-inflammatory, astringent, anodyne, Acute toxicity study was done as per
digestive and liver tonic.17 The effect of Gymnema OECD guidelines 423 [limit test]19. The extract
leaf extract on fat and glucose metabolism is also at doses 5, 50, 1000 and 2000 mg/kg were given
reported.18 In some in - vitro experimental models orally to different group of overnight fasted rats.
the immunostimulatory effect of Gymnema has The animals had free access to water only. The
been observed11 and hence this activity in in-vivo animals were observed for 24 hours. There was no
models needs to be explored. With this background, mortality or behavioural changes observed during
the present study was aimed to investigate the the study period.
immunomodulatory activity of hydro-alcoholic Phytochemical screening of plant material
extract of Gymnema sylvestre leaves using Preleminary phytochemical screening
experimental models of cyclophosphamide- of hydro-alcoholic extract of this plant revealed
induced immunosuppression in Wistar albino rats. presence of alkaloids, triterpenoids, flavonoids,
steroids, tannins and phenolic compounds.
Materials and Methods Experimental Design
The rats were randomly divided into
Plant Material five different groups for each experimental
The hydroalcoholic extract of leaves of model. All the drugs and vehicles were given
 Kar et al., Biomed. & Pharmacol. J, Vol. 12(1), 251-258 (2019) 253

orally for 21 days. On 9th and 16thday of study, Body and Organ weight
cylophosphamide was injected intraperitoneally The initial body weight of each animal
(i.p) at a dose of 100mg/kg to all the rats except was weighed before starting the experiment and
that of Gr-I (control)20 weighed at weekly interval after administration
Gr.I- (Control) Received vehicle, 1% gum acacia of drug or vehicle. At the end of experiment, the
Gr.II- (Disease control) received Cyclophosphamide animals were sacrificed. The liver, spleen and
-100mg/kg ip and1% gum acacia orally kidney were removed and weighed immediately.
Gr.III- (standard) Cyclophosphamide + Levamisole- Haemagglutination Ab (HA) titre
50mg/kg On 14th day of drug treatment, rats of all
Gr.IV -Cyclophosphamide+Gymnema sylvestre the groups were immunized with 0.5X 109 sheep
-25mg/kg RBC intraperitoneally. The day of immunization
Gr.V - Cyclophosphamide+Gymnema sylvestre was referred as day 0. The drug treatment was
-50mg/kg continued for another 7 more days. On 21st day, 1
Experimental procedure hour after the last test dose, blood samples were
Preparation of sheep RBC antigen collected from retro orbital plexus and serum was
For this purpose, 5 ml of sheep blood separated.
was collected in sterile Alsevere’s solution in 1:1 The Ab titre was determined by
preparation and centrifuged at 2000rpm for 10 challenging 25 µL of two fold diluted sera with
minutes. Then it was washed with buffer saline 0.025X109 Sheep RBC suspensions in 96 well
(PBS) for 4-5 times and kept in refrigerator for microtitre plates. PBS was used as diluents in all
further use. On the day of experiment, the RBC samples. The plates were incubated at 37oC for
suspension was adjusted to a concentration of 0.5 1hr and then observed for haemagglutination. The
X 109 cells after RBC count in Neubers chamber. highest dilution showing haemagglutination was
This suspension was used for immunisation and taken as Ab titre.23
antigen challenge.21, 22

Table 1. Effect of different drugs and vehicle on relative organ weights of rats

Group Drug and dose Mean relative organ weight (mg/ 100gm B.W.) ±
SEM
Spleen Liver Kidney

I (Control) 1% Gum Acacia- 0.5ml/ Rat orally 0.52±0.029 4.77±0.293 0.87±0.027

II- CP control 100mg/kg (i.p) +GA 0.5 ml orally 0.21±0.029a 2.46±0.233a 0.56±0.024a
III CP+LEV -50 mg/kg 0.41±0.0149c 4.16±0.197c 0.76±0.202c
IV CP+GSE-25mg/kg 0.30±0.009b 3.40±0.178b 0.65±0.021
V CP+GSE-50mg/kg 0.39±0.013c 3.81±0.152b 0.72±0.021c

n=6 in each group. One way ANOVA test reveals - a: p<0.05 vs normal control, b- p<0.05, c- p<0.001 vs CP control group

Table 2. Effect of different drugs and vehicle on haemagglutination titer,

percentage neutrophil adhesion and phagocytic index in rats

Group HA titer % NA Phagocytic Index

I(control) 138.7±25.69 28.47±2.06 0.068±0.0633

II(CP) 24.0±3.58a 13.25±1.18a 0.026±0.0031a
III(CP + LEV) 106.7±13.4c 24.52±1.27c 0.061±0.0041c
IV(CP+ GSE 25mg/kg) 53.33±6.75 16.92±1.45 0.037±0.0026
V(CP+GSE-50 mg/kg) 85.33±13.49b 21.83±1.34b 0.047±0.0035b

n=6 in each group. One way ANOVA test reveals - a : p<0.05 vs normal control, b- p<0.05,
c- p<0.001 vs CP control group.
254 Kar et al., Biomed. & Pharmacol. J, Vol. 12(1), 251-258 (2019)

Delayed Type Hypersensitivity (DTH) response 7.0 Values of p < 0.05 were considered to be the
On 7th day of treatment the paw volume of minimum level of significance.
right hind foot pad of all rats were measured using
plethysmometer by mercury displacement method. Results
The animals were then immunized by injecting
20µl of 0.5X109 SRBC’s intra-peritoneally. On 21st Effect on lymphoid organ weight
day of drug treatment, all rats were challenge with The relative weights of liver spleen
0.025X109 SRBC S.C into right hind paw. After 24 and kidney with respect to their body weights
and 48 hr of challenge, the right hind paw volumes in CP control rats were significantly reduced in
were measured again. The difference between pre comparison to normal control rats. (p<0.001) With
and post challenge paw volumes was expressed as treatment of levamisole, and GSE 25 as well as 50
DTH reaction.23, 24 mg/kg the relative organ weights were significantly
Neutrophil adhesion test increased in comparison to that of CP treated rats
On day 14, from all rats the blood samples as shown in Table-1.
from retro orbital puncture were collected in EDTA Effect on Humoral immunity
containing vials and analyzed for TLC, Differential Administration of Cyclophosphamide
count(DLC). After initial count, blood samples decreased HA titre to a highly significant extent as
were incubated with 80mg/ml of nylon fiber at compared to that of control rats. (p <0.001) The
37°C for 15mins. The incubated blood samples mean Ab titre of control rats was 138.7±25.69
were again analyzed for TLC and DLC whereas the CP treated rats had 24.0±3.58.
The product of TLC and % Neutrophil Levamisole increased HA titre (106.7±13.4)
gives Neutrophil index of the sample.25 to a highly significant extent in CP induced
immunosuppressed rat when compared to that of
Neutrophil adhesion % = [Neutrophil index CP treated control rats. (p<0.001) But this effect
of untreated blood sample - Neutrophil is not comparable to that of normal rats. GSE with
index of treated blood sample/ Neutrophil 50 mg/kg , significantly increased the HA titre to
index of untreated blood sample]X 100 85.33±13.49 in comparison to CP control group
of rats (p<0.01) revealing stimulation of humoral
Carbon clearance test immune response to sheep RBC. (Table-2)
The animals were divided into five groups Effect of Gymnema sylvestre on cell mediated
and drug or vehicle treatment was given for 21 days immunity
exactly as per the above experimental design. On Administration of Cyclophosphamide
21st day, 3 hours after the last dose all the animals of (100mg/kg) i.p. produced a significant decrease
each group were injected intravenously (tail vein) in paw edema at 24 and 48 hours of sheep antigen
with carbon ink suspension (1:50 dilution of Indian challenge to a highly significant extent(p<0.01)
ink, Camel) in a dose of 0.5 ml/ 100gm body weight. in comparison to control animals. The mean paw
Blood was withdrawn from retro-orbital venous edema volume of control rats at 24 and 48 hours
plexus (25µl) at 0 and 15 minutes after injection were 0.47±0.018ml and 0.42±0.02 ml respectively.
of colloidal carbon ink and was lysed with 0.1% The Standard drug Levamisole with CP elicited a
of sodium carbonate solution (3ml). The optical highly significant increase in paw edema volume
density was measured spectrophotometrically to 0.41 ± 0.022 ml and 0.35± 0.013 ml at 24
at 650 nm.24, 26 The Phagocytic index (K) was and 48 hours respectively.(p<0.001) Gymnema
calculated using the formula: sylvestre 25 and 50mg/kg also elicited a significant
K = (ln OD1-ln OD2) / t1-t2 increase in Delayed hypersensitivity response in
Where OD1 and OD2 are the optical densities at cyclophosphamide treated immunosuppressed
time t1 and t2 respectively. rats suggesting activation of cellular immunity.
Statistical analysis (p<0.05). (Figure-1)
The data were analyzed by One- Effect on %Neutrophil adhesion (NA)
way ANOVA with post hoc Tukey’s multiple In this test, the percentage of neutrophil
comparison‘t’ test using Graph pad Prism version adhesion in normal control and CP control rats
 Kar et al., Biomed. & Pharmacol. J, Vol. 12(1), 251-258 (2019) 255

n=6 in each group. One way ANOVA test reveals - a : p<0.05 vs normal control, b- p<0.05, c- p<0.001 vs CP
control group.
Fig. 1. Paw oedema of various treatment groups at different time intervals

were 28.47 ±2.06 and 13.25±1.18 respectively Immunosuppression may occur due to diseases
which revealed a highly significant decrease or certain chemotherapeutic substances which
in neutrophil adhesion with administration of reduce resistance against infection and stress.
Cyclophosphamide. (p<0.001) Levamisole and Immunostimulation implies the stimulation
Gymnema sylvestre (50mg/kg) increased % of some non specific system ie. Macrophage,
neutrophil adhesion to a significant extent in CP complement, granulocytes and T-lymphocytes. In
induced immunosuppressed rats when compared recent years, research in ethnopharmacology has
with that of CP control group but in comparison to focused on the role of various plant constituents in
Control rats, this effect was not significant.(Table- boosting immunological response.
2) With Levamisole, GSE-25mg/kg and GSE-50 Cyclophosphamide (CP) has been
mg/kg, the percentage NA were24.52±1.27, 16.92 widely used in various cancer chemotherapeutic
± 1.45 and 21.83 ± 1.34 respectively. Increase in regimens and prevention of Graft rejection
% neutrophil adhesion shown by GSE correlates w h i c h a l s o s u p p r e s s e s i m m u n i t y. T h e
with margination of cells in blood vessels. immunosuppressive models have been used to
Effect on Phagocytic index evaluate the immunoregulatory properties of
The phagocytic index with CP treatment various drugs and medicinal plant products. 7
was significantly reduced to 0.026± 0.0031 in Hence in the present study, cyclophosphamide
comparison to normal control group of rats (0.068± (CP) induced immunosupression in wistar rats has
0.0633). Levamisole treated rats had phagocytic been used as a suitable model for investigating the
index of 0.061± 0.0041 which was a highly immunomodulatory potential of hydroalcoholic
significant increase as compared to CP controlled extract of Gymnema sylvestre leaves.
rats. Administration of GSE 50 mg/kg b.w. In the present study, Levamisole and
significantly enhanced phagocytic activity showing GSE restored the changes in relative weights of
phagocytic index of 0.047±0.0035 .(p<0.05) The lymphoid organs like spleen, liver, kidney in CP
results therefore indicate an improvement in induced immunosuppressed rats. (Table 1)
immunity by treatment with GSE (Table 2). In the present study Cyclophosphamide
significantly suppressed the antibody titer in
Discussion response to sheep RBC. Levamisole and Gymnema
sylvestre leaf extract (GSE) at 50 mg/kg augmented
Immune response is a complex system that the humoral immune response by increasing
involves a network of biochemical mechanisms. antibody titer in CP injected rats. (Table-2.) The
256 Kar et al., Biomed. & Pharmacol. J, Vol. 12(1), 251-258 (2019)

humoral immunity involves interaction of B cells integrins through which neutrophils firmly adhere
with the antigen and their subsequent proliferation to nylon fibres. Thus it can be explained that
and differentiation into antibody secreting cells. GSE causes stimulates margination of neutrophils
Thus rise in antibody titre to sheep RBC antigen towards the site of inflammation. Such effect with
shows the index of humoral immunity.27 Our plant extacts of Trichopus zeylanicus was also
observations corroborate with that of P.K.M. observed by R.S Bachhav.29
Nagarathna et al (2014) who have reported the In Carbon clearance test, the phagocytic
immunostimulant properties of Kigelia africana.12 activity was enhanced by Levamisole and GSE
DTH is a part of process of graft rejection, 50mg/kg b.w. It indicates an improvement in non
tumor immunity and mainly responsible for specific immunity involving R.E. system. Such a
development of immunity to intracellular micro- correlation was explained in the study of V. Sharma
organisms. It is an immuno-inflammatory reaction et al 2010 who have experimented with the plant
in which TH1 and macrophages take an important Anacyclus pyrethrum.31
role. DTH is mediated by a specific antigen which In an in vitro study, the findings of V.K.
activate T-Lymphocytes and subsequently release Singh et al 2015 explains that presence of active
cytokines. In the present study, Sheep RBC was compounds, Gymnemic acid in methanolic extact
used as an antigenic substance which elicits of Gymnema sylvestre leaf stimulates both myeloid
hypersensitivity reaction. In comparison to CP and lymphoid components of immune system and
control group, rats treated with Levamisole ,GSE therefore can restore the innate immune function.
(25 and 50 mg/kg) significantly enhanced DTH Thus our in vivo experiment is also in accordance
reaction as measured from increase in hind foot to V K Singh et al.11
paw edema volume (Fig.1) suggesting infiltration This is important to note that
of macrophages to inflammatory site. Macrophages enhancing immune response is one of the best
are one of the important phagocytic cells and play strategies to reverse the host defence system in
a vital role in innate immunity response, also immunosuppressed individuals. In the present
activate adaptive immune response against any study, the restoration of immunity is characterized
foreign object invading host cells including tumor by rise in DTH response, HA titer and percentage
cells28 The index of macrophage activation and neutrophil adhesion, phagocytic index and lymphoid
pro-inflammatory response is reflected from rise in organ weights in CP induced immunosuppressed
level of NO, TNF-á and COX-2. It has been shown rats. In this experiment, the preliminary screening
in the study of V.P. Kumar et al that Allium cepa for identifying the phytoconstituents of hydro-
agglutinin increased the level of NO, TNF-á and alcoholic extract of Gymnema sylvestre leaves
COX-2 in CP treated immunosuppressed rats.1 revealed the presence of alkaloids, triterpenoids,
In many other studies also the effect of various flavonoids, steroids, tannins and phenolic
plant extracts have been shown to increase DTH compounds. In many researches, scientifically it
response reflecting their immunostimulant effect.29 has been proved that, the alkaloid, terpenoid and
In the neutrophil adhesion test, the flavonoid components of plant extracts possess
adhesion of neutrophils to nylon fibres describes immunostimulatory property.12,29,32 In an in vitro
the margination of cells to blood vessels and that test, the immunostimulatory property of Gymnema
is mediated through the interaction of â2 integrins sylvestre plant has been explained due to presence
present on the surface of the neutrophils. 30 of gymnemic acid.11 In the present study, probably
Neutrophils are also capable of wide range of this immunostimulatory activity possessed by
responses in particular chemotaxis, phagocytosis, Gymnema could be due to the presence of the above
exocytosis also both intacellular and extracellular phytoconstituents.
killing. In this test Levamisole and GSE (50mg/kg)
significantly increased % of neutrophil adhesion Conclusion
to nylon fibers as compared to CP treated control
group but this effect was not comparable to that The present investigation suggests that,
of normal control rats. (Table-2) This effect of orally administered Gymnema sylvestre may
Gymnema might be due to the upregulation of â2 accelerate the recovery of cyclophosphamide-
 Kar et al., Biomed. & Pharmacol. J, Vol. 12(1), 251-258 (2019) 257

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