|Year : 2014 | Volume
| Issue : 1 | Page : 49-53
Experimental studies on immunomodulatory potential of Cissus quadrangularis Linn
Priyanka Yadav1, Aditya Ganeshpurkar1, Nisha Sonkar1, Divya Bansal2, Nazneen Dubey1
1 Drug Discovery Laboratory, Shri Ram Institute of Technology-Pharmacy, Jabalpur, Madhya Pradesh, India
2 Pharmaceutics Research Laboratory, Shri Ram Institute of Technology-Pharmacy, Jabalpur, Madhya Pradesh, India
|Date of Web Publication||1-Jul-2014|
Drug Discovery Laboratory, Shri Ram Institute of Technology-Pharmacy, Jabalpur - 482 002, Madhya Pradesh
Source of Support: None, Conflict of Interest: None
Introduction: In ancient times Cissus quadrangularis Linn. was used as a general chow item in China. The stout fleshy stem of C. quadrangularis is an edible plant found throughout the hotter parts of India, Malaya, West Africa, and Ceylon. The plant part contains a number of phytoconstituents like alkaloids, glycosides, polyphenols, flavonoids, sterols, etc. It has demonstrated antiulcer and cytoprotective properties. Aim: The present study was designed to evaluate immune-modulatory potential of C. quadrangularis. Materials and Methods: Plant material was powdered and extraction was done with ethanol phytochemical screening and estimations of phenolics and flavonoids were carried out. Acute toxicity studies were performed as per Organization for Economic Co-operation and Development (OECD) guidelines. The extract was further evaluated for immunomodulatory activity through carbon clearance test, effect on serum immunoglobulins, determination of delayed-type hypersensitivity (DTH) and phagocytosis of killed Candida albicans tests were done to determine immunoprophylactic potential. Result: Results of phytochemical and phytoanalytical tests demonstrated that C. quadrangularis is rich in polyphenolic and flavonoid content. C. quadrangularis also showed promising immunomodulatory potential on various models like DTH, carbon clearance test and effect on serum immunoglobulins. Discussion and Conclusion: These studies conclude that C. quadrangularis possess immunomodulatory activity which might be due to presence of phytochemicals like flavonoids and phenolic compounds.
Keywords: Cissus quadrangularis , flavonoids, immunomodulatory, polyphenols
|How to cite this article:|
Yadav P, Ganeshpurkar A, Sonkar N, Bansal D, Dubey N. Experimental studies on immunomodulatory potential of Cissus quadrangularis Linn. Niger J Exp Clin Biosci 2014;2:49-53
|How to cite this URL:|
Yadav P, Ganeshpurkar A, Sonkar N, Bansal D, Dubey N. Experimental studies on immunomodulatory potential of Cissus quadrangularis Linn. Niger J Exp Clin Biosci [serial online] 2014 [cited 2019 Jul 19];2:49-53. Available from: http://www.njecbonline.org/text.asp?2014/2/1/49/135730
| Introduction|| |
Alteration in activity of the immune system is caused by various diseases like arthritis, ulcerative colitis, asthma, allergy, parasitic diseases, cancer and infectious diseases which reduce the number and phagocytic function of the neutrophils and macrophages, as well as an impairment of the intracellular bactericidal capacity of these cells. Due to this, the body becomes abnormally susceptible to infections by this microbial environment which depends on the extent of dysfunctioning of immune system.
Rasayana in Ayurveda has been interested for many years in the modulation of the immune response to alleviate the disease. The function and efficacy of the immune system may be influenced by many exogenous factors like food and pharmaceuticals, physical and psychological stress hormones, etc., resulting in either immunostimulation or immunosuppression. ,,
C. quadrangularis Linn. (Vitaceae) known as bone setter, is often used as a general chow item in India.  The stout fleshy stem of C. quadrangularis is an edible plant part found throughout the hotter parts of India, Malaya, West Africa and Ceylon.  The stem is used for the treatment of eye and ear diseases, irregular menstruation, asthma, piles, tumors, fractures of bones, wounds, and scurvy. 
Phytoconstituents like α-amyrin, α-amyrone, β-sitosterol, ketosteroid, oxosteroid, onocer-7-ene-3α, 21β-diol and onocer-7-ene3β, 21α-diol, stilbene derivatives and quercetin are found in it,  herb is also rich in β-carotene.  Extract of C. quadrangularis was reported to show antiulcer and cytoprotective property in various experimental ulcer models. , The aim of current work was to explore immunomodulatory potential of C. quadrangularis.
| Materials and methods|| |
The stem of the plant C. quadrangularis, purchased from a local market in Jabalpur, was identified and authenticated by Dr. AB Tiwari, Professor and Head, Department of Crop and Herbal Physiology, Jawahar Lal Nehru Krishi Vishwaviyalaya, Jabalpur, Madhya Pradesh, India. C. quadrangularis were collected in the month of November, it was dried in shade, and then extracted.
Sabouraud broth, Hank's balanced salt solution (HBSS), Giemsa stain, sodium carbonate solution, and zinc sulphate were purchased from Central Drug House, Mumbai, India. All the chemicals used were of analytical grade.
Preparation of Plant Extracts
The powder of C. quadrangularis was subjected to solvent extraction using soxhlet apparatus. The plant part was extracted with ethanol and was filtered through No. 4 Whatman filter paper. Extract was then evaporated at 40°C to dryness, and stored at 4°C for further use.
Phytochemical and Phytoanalytical Studies
Phytochemical screening of ethanolic extract was done by standard method to test the presence of alkaloids, glycosides, phenolics, tannins, carbohydrates, lipids, etc. 
Assay for Total Flavonoids Content
Total flavonoid content was determined using the method given elsewhere.  The concentrations of flavonoid compounds were calculated according to the following equation that was obtained from the standard quercetin graph:
Absorbance = 0.0338 quercetin (μg) - 0.0002; R 2 = 0.9998
Determination of Total Phenolic Compounds
Total soluble phenolic compounds in the extract were determined with Folin-Ciocalteu reagent according to the reported method  using pyrocatechol as a standard phenolic compound. The total concentration of phenolic compounds in the extract determined as microgram of pyrocatechol equivalent by using an equation that was obtained from standard pyrocatechol graph:
Absorbance = 0.0054 × total phenols (pyrocatechol equivalent (μg)) - 0.0058.
Healthy adult Wistar albino rats between 8 and 12 months of age and weighing about 150-200 g were used for the study. The animals were housed in polypropylene cages, maintained under standard conditions (12 h light:12 h dark cycle; 25 ± 3°C and 35-60% humidity). They were fed with standard rat pellet diet (Hindustan Lever Ltd., Mumbai, India) and water ad libitum. All the experimental protocols were approved by the Institutional Animal Ethical Committee, Shri Ram Institute of Technology-Pharmacy, Jabalpur, India (Institutional Ethical Committee Number SRIT/MPY/008).
For experimental procedure, Wistar albino rats were divided in the following four groups containing five rats in each group. 
Group I (n = 5): Rats treated with distilled water 10 ml/kg (control).
Group II (n = 5): Rats treated with ethanolic extract of Cissus quadrangularis (200 mg/kg).
Group III (n = 5): Rats treated with ethanolic extract of C. quadrangularis (400 mg/kg).
Group IV (n = 5): Positive control: Rats treated with levamisole 50 mg/kg.
Serum red blood cell (SRBC) collected in Alsever's solution, were washed three times in large volumes of pyrogen free 0.9% normal saline and adjusted to a concentration of 5 Χ 10 9 cells/ml for immunization and challenge.
Acute Toxicity Studies
These studies were performed according to Organization for Economic Co-operation and Development (OECD) guidelines, received draft guidelines No. 423  adopted from Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Social Justice and Empowerment, Government of India. Rats weighing between 150 and 200 g in groups of five were used (n = 5). The animals were fasted for 4 h with free access to water only. The C. quadrangularis extracts was administered orally in dose of 2,000 mg/kg to different groups of mice and observed over 14 days for mortality and physical/behavioral changes. The experiments were performed after the experimental protocols had been approved by the Institutional Animal Ethical committee.
Carbon clearance test
The four groups of rats were administered with drug/vehicle/extract for 5 days orally. After 48 h of the last dose of the drug, mice were injected with 0.1 ml of Indian ink via the tail vein. Blood samples were withdrawn at 0 and 15 min. A 50 μl blood sample was mixed with 4 ml of 0.1% sodium carbonate solution and the absorbance of this solution was determined at 660 nm. The phagocytic index (PI) K was calculated using the following equation. ,
K = (log e OD 1 - log e OD 2 )/15
Where OD 1 and OD 2 are the optical densities at 0 and 15 min, respectively.
Effect on Serum Immunoglobulins
The four groups of rats were treated with drug/vehicle/extract orally for 21 days. Six hours after the last dose, blood samples were collected and the serum was separated by centrifugation, the collected serum was used for estimation of immunoglobulin levels. Briefly, for each serum sample to be analyzed, a control tube containing 6 ml of distilled water and a test tube containing 6 ml of zinc sulphate solution were prepared. To each, 0.1 ml of serum was added from a pipette. They were inverted to enable complete mixing of the reagents and left to stand for 1 h at room temperature in plugged tubes. The pH of the solution was monitored throughout the experimental period using pH meter. The first tube served as blank and the second tube was taken as sample the turbidity developed was measured spectrophotometrically at 580 nm. 
Determination of Delayed-Type Hypersensitivity (DTH)
The effect of the C. quadrangularis ethanol extract on the antigen specific cellular immune response in experimental animals was measured by determining the degree of DTH response using the foot paw swelling test. The rats were divided into four groups. Seven days later (day +7), the same animals were injected subcutaneously with 0.2 ml of SRBC suspended in 50 μl of phosphate buffered saline (PBS) pH 7.2 into the right hind foot pad for elicitation of the DTH reaction. The left hind foot pad was injected with 50 μl of PBS as control. The difference between the means of right and left hind footpad thickness gave a degree of foot pad swelling which was used for group comparisons. The control group was administered with 0.1 ml of PBS. The footpad thicknesses were measured after 24 h of sensitization by using vernier caliper. 
Blood samples were collected by the retro-orbital plexus puncture method from overnight fasted rats under light ether anesthesia by one drop system.
Phagocytosis of Killed Candida Albicans Preparation of C. Albicans Suspension
C. albicans culture was incubated in Sabouraud broth overnight and then centrifuged to form a cell bottom and supernatant was discarded. The cell button washed with sterile HBSS and centrifuged again. This was done three to four times. The final cell button was mixed with a mixture of sterile HBSS and human serum in proportion of 4:1. The final cell suspension of concentration 1 × 10 8 was used for the experiment.
Rat blood (0.2 ml) was obtained by tail prick method on sterile glass slide and incubated at 37°C for 25 min to allow clotting. The blood clot was removed very gently and slide was drained slowly with sterile normal saline, taking care not to wash the adhered neutrophils (invisible). The slide consisting of polymorphonuclear neutrophils (PMNs) was flooded with extract and incubated at 37°C for 15 min. The PMNs were covered with C. albicans suspension and incubated at 37°C for 1 h. The slide was drained, fixed with ethanol, and stained with Giemsa stain.
The mean number of Candida cells phagocytosed by PMNs on the slide was determined microscopically for 100 granulocytes using morphological criteria. This number was taken as PI and was compared with basal PI of control. This procedure was repeated for different concentrations (10, 20, 40, 100, and 1000 μg/ml) of test sample. The mean particle number (MPN) associated with each cell was calculated. 
The statistical significance was assessed using one-way analysis of variance (ANOVA) followed by Dennett's comparison test. The values are expressed as mean ± standard error of the mean (SEM) and P < 0. 01 was considered significant.
| Results|| |
The results of preliminary phytochemical analysis extract of C. quadrangularis Linn. showed abundant presence of alkaloids, terpenoids, saponins, tannins, and polyphenols. Screening of phenolic compounds with NaOH and FeCl 3 revealed their presence and quantification was done. The total amount of phenolic content present in extract was found to be 715.2 ± 2.57 mg PE (Pyrocatechol Equivalent)/100 g. By using the standard curve of quercetin (R 2 = 0.9998), the total flavonoid content of extract was found to be 169.2 ± 1.97 mg QE (Quercetin Equivalent)/100 g. Total alkaloidal content in the extract was found to be 17.2 mg/kg dry basis.
Acute Toxicity Studies
Acute oral toxicity was carried out by OECD Guidelines. It is found that C. quadrangularis extract was safe at limit dose of 2,000 mg/kg, with no mortality in studied subjects. Thus 1/10 th of this dose, that is, 400 and 200 mg/kg for C. quadrangularis were used in the subsequent study.
Carbon Clearance Assay
Administration of C. quadrangularis 200 and 400 mg/ kg, p.o. produced increase in clearance of carbon particles from blood as indicated by a significant increase in PI (P < 0.01). The levamisole also produced a significant increase (P < 0.001) in the PI [Figure 1].
|Figure 1: Effect of Cissus quadrangularis on phagocytic index in rats. Results are given as mean ± standard error of the mean (SEM) of five animals in each group. Control group compared with rest of the treated groups. Significance at *P < 0.05; **P < 0.01; ***P < 0.001|
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Serum Immunoglobulin Levels
C. quadrangularis showed a significant (P < 0.01) increase in the serum immunoglobulin levels with dose of 200 and 400 mg/kg/day, response was 19.481 ± 0.3231 and 22.191 ± 0.1080, respectively in comparison to control group; whereas, levamisole also showed significant increase in the serum immunoglobulin levels (P < 0.001) when compared to control [Figure 2].
|Figure 2: Effect of Cissus quadrangularis on serum immunoglobulins levels in rats. Results are given as mean ± SEM of five animals in each group. Control group compared with rest of the treated groups. Significance at *P < 0.05; **P < 0.01; ***P < 0.001|
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The DTH response to SRBC which corresponds to cell mediated immunity showed a dose dependent increase due to treatment with C. quadrangularis. With dose of 200 and 400 mg/kg/day the DTH response was 0.756 ± 0.003 and 0.823 ± 0.002, respectively in comparison to corresponding value of 0.293 ± 0.008 for untreated control group. The differences in DTH response were statistically significant (P < 0.01) [Figure 3].
|Figure 3: Effect of Cissus quadrangularis on mean foot pad thickness in rats. Results are given as mean ± SEM of five animals in each group. Control group compared with rest of the treated groups. Significance at *P < 0.05; **P < 0.01; ***P < 0.001|
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Phagocytosis of Killed C. albicans
The ethanolic extracts of C. quadrangularis stimulated the phagocytosis of killed C. albicans. The MPNs were found to be 5, 4, and 3 for ethanol extract; 2, 1, and 0 for HBSS (control). At concentration of 1,000, 100, and 40 μg/ml, respectively, when compared to positive control (standard), that is, pooled serum (6, 4-5, and 4, respectively) at the same concentrations [Figure 4]. However, at low concentration of 20 and 10 μg/ml, the stimulation of phagocytic activity was comparatively lower.
|Figure 4: Effect of Cissus quadrangularis on mean percentage of killed Candida. Results are given as mean ± SEM of five animals in each group. Control group compared with rest of the treated groups. Significance at *P < 0.05; **P < 0.01; ***P < 0.001|
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| Discussion|| |
Immunomodulatory agents from plants and animals augment the immune responsiveness of the body against pathogens by stimulating the nonspecific immune system.  Pharmaceuticals which turn on host defense mechanisms in the presence of an immune responsiveness could endow with supportive therapy to conventional chemotherapy.
Carbon clearance test was carried out to evaluate the effect of drugs on the reticuloendothelial system (RES) and mononuclear phagocyte system (MPS), both are diffuse system of phagocytic cells. C. quadrangularis at experimental doses showed significant increase in the PI, by increasing the production of phagocytic cells. 
Ethanolic extract of C. quadrangularis increased WBC counts and bone marrow cells that stimulate hemopoetic system resulting in the activation of immunomodulatory activity in DTH reaction. C. quadrangularis showed a significant increase in the serum immunoglobulin levels as evident by zinc sulphate turbidity test. This test provides a rough estimation of the amount of immunoglobulins present in the serum. Zinc sulphate causes precipitation of the immunoglobulins making the solution cloudy. The estimation of serum immunoglobulin levels is used to evaluate the increase in serum immunoglobulin production after the administration of the drugs. ,
The present study was carried out to estimate the immunomodulatory activity of ethanolic extract of C. quadrangularis. By enhancing PI, serum immunoglobulin level WBC count, extract dictated its beneficial effect on immune system. C. quadrangularis was found to have a significant immunostimulatory activity on both the specific and nonspecific immune mechanisms.
The present study demonstrates the immunostimulant potential of the ethanolic extract of C. quadrangularis which is revealed by a significant increase in the phagocytic function of human neutrophils, when compared with the control indicating the possible immunostimulating effect. Neutrophils stimulation increased in number of cells acting as chemoattractant.
In conclusion, it is revealed that the ethanolic extracts of C. quadrangularis stimulate immune system by acting through cellular and humoral immunity in both in vitro and in vivo experimental models. This could be due to the presence of various chemical constituents that increases the PI and serum immunoglobulin levels in human blood. However, a more detailed systemic study is required to extrapolate results in human.
| Acknowledgements|| |
Authors are thankful for Rewa Shiksha Samiti for providing support for research purpose and also thankful to Dr. A.B. Tiwari for authenticating the sample of Ciccus quadrangularis.
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