Research Article

In Vivo Approaches to Investigate the Immune Response of Plant-Based Anti Tumour Drug, Elephantopus scaber L

Balakrishnan Sreedevi Geetha1*, Panickamparambil Gopalakrishnan Latha2, Sivasankaran Nair Mangalam3 and Prathapan Remani4
1Kerala State Council for Science, Technology and Environment, India
2Jawaharlal Nehru Tropical Botanic Garden and Research Institute, India
3National Institute for Interdisciplinary Science and Technology (CSIR), India
4Regional Cancer Centre, India


*Corresponding author: Balakrishnan Sreedevi Geetha, Department of Pharmacology, Kerala State Council for Science, Technology and Environment Thiruvananthapuram, 695004, India


Published: 17 Jan, 2017
Cite this article as: Geetha BS, Latha PG, Mangalam SN, Remani P. In Vivo Approaches to Investigate the Immune Response of Plant-Based Anti Tumour Drug, Elephantopus scaber L. Ann Pharmacol Pharm. 2017; 2(1): 1011.

Abstract

Inspired by our earlier research reports towards the ability of E. scaber and its phytochemical constituents in regimens for inhibiting tumours with extensive proliferative potencies, the active fraction of E. scaber (ES) was chosen in the present study for investigating its immuno modulatory activity, anti tumour activity and toxicity, in in vivo animal models. The influence of the administration of ES (100 mg/kg, i.p) on the humoral and cellular immune response was investigated in mice. The immuno potentiating effect of ES were evaluated by determining the immune profile of treated mice such as circulating antibodies, anti-SRBC antibody producing cells (PFC), antibody dependent complement mediated cytotoxicity (ACC) of tumour and normal mice, delayed type hypersensitivity reaction (DTH), and the number of peritoneal exudates cells and macrophages. ES significantly increased the production of specific antibody to sheep red blood cell (SRBC) antigen in immunized mice. ES enhanced splenocyte proliferation and showed maximum peak value of spleen cells on day 5. After EAC tumour transplantation, the antibody complement mediated cytotoxicity (ACC) showed a significant cytotoxic activity in the ES treated group on day 10 for an antibody dilution of 1:4. The effects of ES for 7 days elicited a significant dose related increase in 4h and delayed 24h DTH response in mice Elevated delayed type hypersensitivity reaction (DTH), observed in this study suggested the activation of the cellular immune response. Treatment with ES for 7 consecutive days resulted in a two fold increase in number of peritoneal exudate cells and macrophage count. The results of immuno modulatory studies using in vivo models demonstrated the effect of ES to stimulate both the humoral and cell-mediated components of the immune response in the experimental mice. Growth inhibitory effect of ES on EAC tumour cells implanted into the peritoneal cavity of mice were evaluated by determination of animal survival, recorded and expressed as mean survival time (MST) in days and the percentage increase in life span (% ILS).ES (100 mg/kg, i.p) was found effective in prolonging the lifespan of (Ehrlich Ascites Carcinoma) EAC tumour bearing mice and exhibited significant in vivo anti tumour efficacy against EAC tumour cells. The influence of the administration of ES for the short term toxicity studies was investigated in Wistar rats. The short term toxicity studies in the animals treated with ES were judged by monitoring behavioral changes, hematological parameters, serum enzymes activities and other biochemical parameters. The effect of ES on serum enzyme activities, alkaline phosphatase (SAKP), alanine transaminase (SALT), aspartate transaminase (SAST), and other biochemical parameters like serum urea, serum creatinine, serum calcium and serum glutamyl transferase (γ-GT) levels of rats indicated that there was no toxicity even at higher concentrations upto1000 mg/kg. Also haematological profiles such as red blood cell count (RBC), white blood cell count (WBC) and differential count of WBC such as neutrophil, lymphocytes, monocytes and basophils, haemoglobin content, hematocrit (Hct), platelet count and erythrocyte indices such as mean cell volume (MCV), mean cell haemoglobin (MCH) and mean cell haemoglobin concentration (MCHC), also did not show any treatment related effects. The results of the short term toxicity studies indicated that high dose upto 1000 mg/kg of ES was tolerated by rats, without producing any toxic symptoms thus indicating a wide margin of safety of the drug used. In conclusion, our results indicated that ES with its nontoxic nature, is as a good candidate for further investigations in order to develop a natural compound as an immuno modulator and anticancer agent. Our earlier research work reported the presence of sesquiterpene lactones, deoxyelephantopin and isodeoxyelephantopin as the major phytochemical constituents of ES. Hence its constituents can be used as potential therapeutic tools to regulate inflammatory responses/immunological disorders and prevent carcinogenesis.
Keywords: Elephantopus scaber L; Immune response; Toxicity; Tumour growth


Background

The treatment of many diseases owes much to plants-derived drugs, and the treatment of cancer is no exception. In addition to cytotoxic drugs, the potentiation of host defense mechanism has been recognized as a possible means of inhibiting tumour growth. Therefore searching for immuno modulatory materials from natural herbs and characterising the immune enhancement effects may have great potential in cancer treatment, based on combination of time honored traditional usage and ongoing scientific research.
The plant proposed for the present study was Elephantopus scaber L. (Astereacea), which is genus of herb, collected from Kerala region of South India. This plant species have been used since ancient times as folk remedies to treat wound healing, liver disease, hepatitis, leucoderma, sexually transmitted diseases, to prevent inflammation after birth rheumatism, fever, cold, goitre and bone fracture including cancer [1].
Our previous research findings have shown the tumour inhibitory activity of the active fraction of E. scaber (ES) against chemically induced tumours and its ability to inhibit the development of solid tumours [2]. Further our research work have shown that sesquiterpene lactones, deoxyelephantopin and isodeoxyelephantopin isolated from ES inhibited the Phyto-heamagglutinin stimulated proliferating human lymphocyte and growth of tumour cell lines and induces apoptosis in vitro [3]. Inspired by the ability of E. scaber and its phytochemical constituents in regimens for inhibiting tumours with extensive proliferative potencies, the active fraction of E. scaber (ES) was chosen in the present study for investigating its immuno modulatory activity, anti tumour activity and its toxicity, using in vivo animal models.


Methods

Animals
Inbred strains of Swiss albino mice (males) in the age group of 8–10 weeks, weighing 20–30 g and Wistar rats (males) weighing 160-200 g were used for the experiments. The animals were housed under conventional laboratory conditions and fed with standard pellet diet (Lipton India Ltd., Mumbai, India) and boiled water ad libitum. All experiments involving animals were done, strictly adhering to the guidelines for animal experimentation and handling, issued by the Government of India.
Tumour Cells
Ehrlichs Ascitic Carcinoma (EAC) tumours were maintained as ascites by serial transplantation in mice, by intra peritoneal (i.p.) injection of 1x106  cells/mouse. The tumor cells were aspirated from the tumour-bearing mice aseptically and washed thrice in phosphate-buffered saline before transplantation [4-6].
Sheep Red Blood Cells (SRBC)
SRBC was obtained from sheep maintained at the animal house of the institute. Sheep venous blood was aseptically collected and mixed with an equal volume of sterile Alsevier’s solution. At the time of use it was then washed thrice with normal mice and adjusted to a concentration of 5×106 cell/ml.
Chemicals
RPMI-1640 medium was obtained from Gibco BRL, USA, and the antibiotics streptomycin, penicillin, and gentamycin sulphate from Hi-Media Laboratories, Mumbai. Foetal calf serum, Trypsin, Histopaque, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and trypan blue were obtained from Sigma Chemical Company, USA. Heparin was obtained from DIFCO, USA. The solvents used for the phytochemical analyses were LR grade, but except for final purification and spectroscopic studies, AR or spectroscopic grade solvents were used. Silica gel was obtained from Sisco Research Laboratories and Merck Laboratories, Mumbai. All the biochemicals required for the experiment were purchased from Hi-Media Laboratories, Mumbai. The chemicals used were obtained from BDH Laboratories, Mumbai. The reagents used were of analytical grade.
Experimental Protocol
Preparation of active fraction of E. scaber(ES): Fresh E. scaber plants were collected from JNTBGRI campus, India, and after authentication by the plant taxonomist of the Institute with a voucher specimen of the plant (TBGT 25419) were deposited in the Herbarium of the Institute. The preparation of active fraction of E. scaber referred to as (ES) was done by partially purifying the chloroform extract of plant by column chromatography as previously reported by [2]. It was suspended in 10% DMSO, to required concentrations and used for the experiments.
Evaluation of Immuno modulatory effect of E: The immuno potentiating effect of ES were evaluated by determining the immune profile of treated mice such as circulating antibodies, anti-SRBC antibody producing cells (PFC), antibody dependent complement mediated cytotoxicity (ACC) of tumour and normal mice, delayed type hypersensitivity reaction (DTH), the number of peritoneal exudates cells and macrophages.
Quantitation of circulating antibodies and determination of anti-SRBC antibody producing cells: The Swiss albino mice were divided into two groups of 24 animals per group and were immunized by an i.p. injection of 0.25 ml of 2 % SRBC (2.5x106 /ml) in normal saline. After 30 minutes, the group I animals received 0.5 ml of DMSO, i.p. and group II animals received ES, at a dose of 100 mg/kg body weight i.p. in 0.5 ml of DMSO. On days 3, 4, 5 and 6 after immunization, 6 mice from each group were sacrificed, blood was collected by heart puncture for evaluating haemagglutination antibody titre as described by [7] and spleen was removed aseptically from the immunized groups of mice for performing Jerne’s plaque-forming assay for determining the T lymphocyte-dependent antibody responses by the method of [8]. The number of plaque forming cells in the spleen was quantified and expressed as PFC/106 spleen cells.
Assay of Haemagglutination Antibody Titre: Briefly, two fold dilutions of sera were prepared in 0.15 M phosphate buffered saline (pH 7.2) and 50 µl of each dilution was dispensed into 96-well microtitre plates. 25µl of fresh 1% SRBC suspension was dispersed into each well and mixed thoroughly. The plates were incubated at 28ºC for 2h and agglutination was evaluated macroscopically. The value of the reciprocal of the highest serum dilution causing visible haemagglutination was taken as the antibody titre and the mean value of the titre was calculated.
Jerne’s Plaque Forming Cell (PFC) Assay: Briefly, the spleen was removed and gently teased in cold RPMI-1640 medium, using the plastic plunger of a disposable syringe and passed over a nylon gauze to get a single cell suspension in RPMI-1640 medium. Cells were washed twice with the same medium and suspended in RPMI-1640 medium to a density of 1x106/ml. Petri dishes were layered with 1.2% agarose in 0.15 M NaCl to form a bottom layer. A mixture consisting of 2 ml of 0.6% agarose in RPMI-1640 medium, 0.1 ml of the suspension of 20% SRBC and 1x106 spleen cells was poured over the bottom layer of agarose. The Petri dishes were then incubated at 37ºC for 90 min. 2 ml of 1:10 diluted fresh rabbit serum with PBS was added as complement to each Petri dish and incubation was further continued for 45 min. Plaques formed on the agarose surface were then counted and the average number of PFC were determined.
Effect of ES on Antibody Dependent Complement Mediated Cytotoxicity (ACC): The Swiss albino mice were divided into four groups of 24 animals per group. Group I animals received 0.5 ml of DMSO, group II animals received ES 100 mg/kg body weight i.p., group III animals were 0.5x106 EAC cells/mouse i.p. of single dose and received 0.5 ml of DMSO, till the end of the experiment and group IV animals were challenged with 0.5x106/mouse EAC cells i.p. of single dose and received ES (100 mg/kg body weight i.p.) till the end of the experiment. Blood was collected from six animals from each group at definite intervals of time after the treatments and the separated serum was used for determination of ACC activity according to [9]. The cytotoxicity was assessed by trypan blue exclusion method [10].
Assay of antibody complement mediated cytotoxicity (ACC): The serum for ACC activity was heat inactivated at 56ºC for 30 min to eliminate intrinsic complement activity. The specificity of tumour specific antibodies in the tumour serum was confirmed by testing normal mouse serum which did not show any cytotoxicity against EAC cells in the presence of complement. EAC cells were washed thrice in RPMI-1640 medium, viability checked and finally made up to a concentration of 1x106 cells/ml in the medium. Antiserum was serially diluted with culture medium so as to get 1:1, 1:2, 1:4 and 1:8 dilutions of antibody. Fresh rabbit serum, diluted with phosphate buffer saline (PBS) in the ratio of 1:10 was used as a source of complement. The heat inactivated serum (100 µl) and diluted rabbit serum (50 µl) were added to 1 ml RPMI-1640 medium containing 1x106 EAC cells. The total volume was made up to 2 ml and incubated at 37ºC for 3 h. The cytotoxicity was assessed by trypan blue exclusion method [11-13].
Effect of ES on delayed hypersensitivity reaction (DTH) in mice: Delayed type hypersensitivity response to sheep red blood cell (SRBC) was induced in mice following the method of [14]. Swiss albino mice were divided into three groups of 6 animals per group. All the three groups of animals were immunized by injecting 20µl of 5x109 SRBC/ml subcutaneously (s.c) into the right hind foot pad on day 0 and challenged 7 days later by injecting intradermally (i.d.) the same amount of SRBC into the left hind foot pad. The thickness of foot pad was measured at 24 h after challenge using a caliper. The difference in the thickness of right hind paw and left hind paw expressed in mm was taken as a measure of DTH reaction. ES in doses of 100 and 200 mg/kg was administered intra peritoneally (i.p.) respectively on each of 3 days prior to immunization, on the day of immunization and each of 3 days after immunization (days -3 -2, -1, 0, +1, +2, +3) to group II and group III animals, simultaneously and group I animals (control) received DMSO.
Effect of ES on mouse peritoneal macrophages: Swiss albino mice were divided into three groups of mice of 6 animals per group. Group I animals were given 10% DMSO (0. 5 ml) i.p., and served as the control, group II animals were treated once with ES (100 mg/kg) i.p., and group III animals received the ES (100 mg/kg) i.p., for seven consecutive days. Peritoneal cells from control and treated groups were collected as described by [15]. Cold normal saline (2 ml) was injected into the peritoneal cavity of all three groups of mice, under mild ether anaesthesia. After a gentle massage, the peritoneal fluid was collected and transferred into siliconized test tubes containing 3 ml of RPMI-1640 medium (pH 7.2-7.4) containing heparin (5 units/ml). The cells were washed twice by low speed centrifugation (400-500 rpm) and suspended in RPMI-1640 medium. Macrophages were separated by adhering on glass surface at 37ºC in heparin-free medium. The adhered cells were collected in cold RPMI-1640 medium and counted using haemocytometer. Total peritoneal exudate cells and the number of macrophages (stained with 1% neutral red) were counted after 24 h of treatment and compared with the control.
Evaluation of antitumour activity of ES on the EAC ascitictumour reduction in mice: The animals were divided into four groups of 9 animals per group. All the four groups of animals were transplanted intra peritoneally (i.p.) with 1x106 EAC cells. After 24 h, animals of group I serving as control were given 0.5 ml of 10 % DMSO, group II and group III animals were given ES 100 mg/kg, i.p and200 mg/kg, i.p., respectively and group IV animals were given cisplatin 3 mg/kg, i.p. for 5 alternate days. The administration of ES was continued once daily for 10 days, 24h after tumour transplantation. Growth inhibitory effect of ES on EAC tumour cells implanted into the peritoneal cavity of mice were evaluated by determination of animal survival, recorded and expressed as mean survival time (MST) in days and the percentage increase in life span (% ILS) as described by [16].
Evaluation of short toxicity of ES in normal rats: The short term toxicity in the animals treated with ES were judged by monitoring behavioral changes, hematological parameters, serum enzymes activities and other biochemical parameters. To determine short term (15 days) toxicity, male rats were divided into 5 groups of 6 animals in each group. The group I control animal was given 1 ml of 10% DMSO (i.p) daily and groups II, III, IV and V were administered intra peritoneally (i.p) with 100 m/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg respectively of ES in 10% DMSO for 15 days. Body weight, food and water intake and general behavioral changes were monitored. 24 h after the last administration of the drug, the rats were killed and the blood samples were collected for biochemical estimations and haematological examination.
Serum were separated by centrifugation and the enzymes, alkaline phosphatase (SAKP), alanine transaminase (SALT), aspartate transaminase (SAST), serum urea, serum creatinine, serum calcium and serum glutamyl transferase (γ-GT) were assayed by standard methods [17-21]. Haematological parameters such as red blood cell count (RBC), white blood cell count (WBC) and differential counts of WBC such as neutrophil, lymphocytes, monocytes and basophils, haemoglobin content, hematocrit (Hct), platelet count and erythrocyte indices such as mean cell volume (MCV), mean cell haemoglobin (MCH) and mean cell haemoglobin concentration (MCHC) were determined as per recommended methods [22].Gross pathological examination was done of major organs viz., liver, spleen, heart, lung and kidney (data not shown).

Figure 1

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Figure 1
Effect of ES on anti-SRBC antibody titre. Values are mean± SD of three separate assays, ***p<0.00.

Figure 2

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Figure 2
Effect of ES on the number of PFC in mice. Values are mean ± SD of three separate assays, ***p<0.001.

Figure 3

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Figure 3
Effect of ES on ACC activity of normal & EAC tumour cells. Values are mean of 3 separate assays, ***p<0.001.

Figure 4

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Figure 4
Effect of ES on ACC activity of EAC tumour cells. Maximum ACC activity noted with antibody dilution of 1:4.

Table 1

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Table 1
Effect of ES on SRBC-induced DTH activity in mice % change in DTH reaction is shown in parentheses, n=6/group, **p<0.01, *p<0.05.

Table 2

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Table 2
Effect of ES on enhancement of peritoneal cells in normal mice.

Table 3

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Table 3
Effect of i.p administration of ES on ILS of EAC-tumour bearing mice.

Table 4

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Table 4
Effect of ES on serum enzyme activities and biochemical parameters in rats after 15 days treatment.

Table 5

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Table 5
Effect of ES on serum enzyme activities and biochemical parameters in rats after 15 days treatment.

Results

The administration of ES induced a statistically significant enhancement in haemagglutination antibody titre value of 469.33 on day 6 after SRBC immunization, compared to the control animals which showed maximum titre value on day 5 (Figure 1). The administration of ES enhanced the number of PFCs in the spleen of treated animals after SRBC immunization from day 3 and showed maximum peak value of 1038/106 spleen cells on day 5. The PFCs formed in the control group of immunized animals increased upto day 5 and the activity declined there after (Figure 2). ES enhanced the humoral immune response demonstrated by antibody complement mediated cytotoxicity (ACC). In EAC tumour bearing mice, the ACC activity was observed on day 6 and peaked on day 10 with 38.83 % of cell death, whereas in tumour animals treated with ES, the ACC activity peaked on day 10 with 68.92 % of cell death for an antibody dilution of 1:4 (Figure 3,4). The effect of ES for 7 days elicited a significant dose related increase in 4h and delayed 24h delayed DTH response in mice (Table 1). Treatment of ES for 7 consecutive days resulted in a two fold increase in number of peritoneal exudate cells (Table 2).
Palpable ascetic tumours appeared in the peritoneal cavity of the mouse, within 7-12 days after i.p, injection of EAC tumour cells. ES (100 mg/kg,i.p) treated EAC tumour bearing mice survived about 28.8±1.34 days, whereas control animals died within 17.3±0.87 days (Table 3).
In the short term toxicity study after 15 days, even though there a very slight increase in serum activities of SAKP, SAST and SALT levels, these variations are were not very significant. There was no significant change in other biochemical parameters studied (Table 4). There was also a slight increase in Hb (2.04%) (p<0.05) and RBC (3.06%), whereas other hematological parameters did not show significant change in the treated group, after i.p. administration of ES for 15 days (Table 5). There was no notable change in the food intake, and behaviour compared to the control group. The control and the treated group showed normal increase in the body weight which was not significantly different from each other. Pathological examination of major organs viz. liver, spleen, heart, lung and kidney did not exhibit any gross morphological changes (data not shown).
Statistical Analysis
All experimental data were expressed as the mean ± standard deviation (S.D.) and statistical analysis was performed by using the Student’s t-test [23]. A ‘p’ value lesser than 0.05 was taken as significant.
Determination of PFC
Plaques formed on the agarose surface were counted and the average number of PFC were determined and the values have been expressed as plaque forming cells per million spleen cells using the formula: No. of plaques/million spleen cells= No. of plaques formed/No. of spleen cells added x 106
Determination of animal survival
Animal survival were determined using the formula % ILS = T-C/C x 100, where T is the mean survival in days of treated group and C is the mean survival time in days of control group [4,11,12,16].
Determination of Haematological parameters
Total white blood cells (WBC) x 103/mm3; red blood cells count (RBC) x 106/mm3
Hematocrit (Hct) expressed in percentage, Hct(%)=(RBC x MCV) /10.
Mean Cell volume (MCV) expressed in cubic micro meters, MCV (µm3) =hematocritx10/erythrocyte count (106/µl).
Mean Cell Haemoglobin (MCH) expressed in pictogram, MCH (pg)=Hb concentration (g/dl) x 10/erythrocyte count (106/µl).
Mean Cell Haemoglobin Concentration (MCHC) expressed in gram per deciliter of haemoglobin, (MCHC) (g/dl)=haemoglobin (g/dl) x 100/hematocrit (%).

Conclusion

From the data presented here, the effect of ES on both cellular and humoral immune response is well demonstrated. The augmentation of the humoral response to SRBC by ES as evidenced by increase in haemagglutination titre and number of plaque forming cells to SRBC indicated enhanced responsiveness of macrophages and B-lymphocytes subsets, involved in antibody synthesis [24]. In view of the pivotal role played by macrophages in coordinating the processing and presentation of antigen to T and B cells, the augmentation of the humoral response to SRBC reveals that ES may enhance the effect by facilitating these processes. The immunological status in normal and tumour bearing animals reflected in ACC activity, the one of the mechanism by which the host immune system can kill tumour cells, were also assessed in the present study. From the study, a significant cytotoxic activity has been observed in the drug treated group on day 10 for an antibody dilution of 1:4 compared to other days. These results are strengthened by the significant increase in anti- SRBC antibody producing cells in the spleen of animals treated with ES indicating the augmentation of humoral immune response elicited by tumour antigen expressed during malignant transformation of cells. The effect of ES on the antigen specific cellular immune response in experimental animals was measured by determining the degree of DTH reaction using the foot pad swelling test where a significant increase in paw edema was observed compared to control. During the cell mediated immune response the sensitized T-lymphocytes challenged by the antigen are converted to lymphoblasts and secrete lymphokines which is an important event/ marker of cell mediated immune response, attracting more scavenger cells to the site of reaction and the infiltrating cells are thus immobilized to promote inflammatory (defensive) reaction The increase in paw edema with ES treatment for 7 days around SRBC immunization thus suggested the enhancement of cell mediated immune response. Increase in delayed type hypersensitivity (DTH) reaction in response to thymus dependent antigen also revealed the stimulatory effect of ES on T lymphocytes and accessory cell types required for the expression of the reaction which is also noted in the case of Picrorhiza kurroa [24,25]. The increased hypersensitivity noted, may be due to the simultaneous presence of high antibody titre, promoting the elimination of antigen by non-presenting phagocytes. This also indicates that treatment with ES shifts the immune response towardsTH1 type of immune response. The fact that ES enhanced peritoneal exudate cells and macrophage count suggest that ES treatment indirectly inhibited tumour cell growth, probably mediated through activation of macrophage inside the peritoneal cavity.
Increase in life span is a reliable criterion for judging the value of any anticancer drug. The increase of life span of EAC tumour bearing mice by treatment with ES is a positive result and supports the anti tumour effect of the drug. The results of toxicity studies indicate that showed that high doses of ES are tolerated by the rats without producing any toxic symptoms, thus indicating a wide margin of safety of the drug used.
The results of immuno modulatory studies using in vivo models demonstrated the effect of ES to stimulate both the humoral and cell-mediated components of the immune response in the experimental mice, suggesting the therapeutic usefulness of E. scaber in a variety of ailments. The anti tumour efficacy of ES is also well demonstrated in the present study. From our previous research reports, the phytochemical studies indicated the presence of sesquiterpene lactones, deoxyelephantopin and isodeoxyelephantopin in ES [2]. These may be the compounds responsible for the stimulation of immune response by ES. These findings are in agreement with studies of diterpenes from Andrographispaniculata that play an important role in stimulating both the humoral and cell-mediated response [26]. In conclusion, our results indicated that ES with its non-toxic nature serve as a good candidate for further investigations in order to develop a natural compound as an immuno modulator and anticancer agent. Also its phytochemical constituents particularly the sesquiterpene lactones can be used as potential therapeutic tools to regulate inflammatory responses and prevent carcinogenesis.


Acknowledgment

The authors thank Kerala Forest and Wildlife Department, Government of Kerala, India for the financial support. The lead author, Dr. Geetha B.S thank Science and Engineering Research Board, Department of Science and Technology, Government of India and The Centre For International
Co-operation in Science, Chennai, for the travel grants provided to participate the Global Conference on Biological Engineering and Natural Science held at Singapore in 18-20 February 2016.


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