Immunomodulatory activity of Alcoholic extracts of Tinospora cordifolia Stem

 

Sanjiv Kumar Biradar, Chandra Kishore Tyagi

Faculty of Pharmacy, Sri Satya Sai University of Technology and Medical Sciences,

Village- Pachama, District- Sehore - 466001, MP.

*Corresponding Author E-mail:

 

ABSTRACT:

Tinospora cordifolia is a plant well known for its medicinal value in Indian ayurveda and Indian traditional medicine system. However, to prove its efficiency for the clinical utilization, more experimental data will be beneficial. In the present investigation, evaluated the immunomodulatory activity of the alcoholic extracts of Tinospora cordifolia stem on various in-vivo experimental models such as neutrophil adhesion test, phagocytic index by carbon clearance test, Hemagglutinating antibody (HA) titre and delayed type hypersensitivity (DTH) responses. The evaluation of immunomodulatory potential by oral administration of alcoholic stem extracts (50, 100, 200 and 300mg/kg b.w, p.o) evoked a significant increase in percent neutrophil adhesion to nylon fibers as well as a dose dependent increased in antibody titre values, and potentiated delayed type hypersensitivity reaction induced by sheep red blood cells and significant response towards phagocytosis in carbon clearance assay (*p<0.05, *p<0.01, ***p<0.001). This may be due to concentrations of active phytochemicals present in particular plant extract. Hence, it was concluded that the plant extracts increased humoral as well as cell mediated immunity.

 

KEYWORDS: Tinospora cordifolia, Alcoholic, Immunomodulatory Phytochemicals and Sheep Red Blood.

 

 


INTRODUCTION:

The immune system is involved in the etiology as well as pathophysiologic mechanisms of many diseases. Ayurveda, the Indian traditional system of medicine, lays emphasis on promotion of health concept of strengthening host defenses against different diseases1. Indian medicinal plants are a rich source of substances which are claimed to induce both specific and nonspecific immunity2. Plants are the essential and integral part in complementary and alternative medicine system because they develop the ability for the formation of secondary metabolites like proteins, flavonoids, alkaloids, steroids and phenolic substances which are in turn used to restore health and heal many diseases3.

 

 

Tinospora cordifolia, commonly known as Gulvel/ Guduchi/ Gulancha belongs to plant family Menispermaceae, a climbing shrub widely distributed throughout Indian subcontinent and China4. In Hindi, the plant is commonly known as Giloya, which is a Hindu mythological term that refers to the heavenly elixir that have saved celestial beings from old age and kept them eternally young. Guduchi is widely used in veterinary folk medicine/ayurvedic system of medicine for its general tonic, antiperiodic, anti-spasmodic, anti-inflammatory, antiarthritic, anti-allergic and anti-diabetic properties5-9. The plant is used in ayurvedic, “Rasayanas” to improve the immune system and the body resistance against infections. In view of the above information and folklore use of the root and fruits of this plant, the present study was undertaken to evaluate the immunomodulatory activity of the alcoholic extracts of Tinospora cordifolia stem in various in vivo experimental models.

 

MATERIALS AND METHODS:

Plant Material:

Fresh Tinospora cordifolia stem were obtained and identified from authentic sources. A voucher specimen has been identified and deposited in the Departmental herbarium, Faculty of Pharmacy, Sri Satya Sai University of Technology and Medical Sciences, Sehore, M. P. The collected stem were dried in shade, to prevent the loss off active constituents, crushed to coarse powder using mechanical grinder and used for further studies.

 

Preparation of Extract:

Powdered plant materials (35gm) were soaked in 75% ethanol and methanol (500ml) separately for 72h in a conical flask. Then the extracts were filtered, concentrated and the solvent was removed by rotary evaporator. The extract was dried over desiccators and to give amorphous mass with a percentage yield of (2.54% w/w and 2.65% w/w). Then the extracts were stored in 4ºC for further studies.

 

Drugs:

Accurately weighted quantities of the ethanol and methanol extracts were suspended in 5% dimethyl sulfoxide (DMSO) for preparation of desired dosages in the experiment.

 

Preliminary Phytochemical Screening:

Phytochemical analysis was done by using standard procedure10.

 

Experimental animals:

Wistar albino rats (age 6-8 weeks, body weight 150-180gm) of either sex were used for the study of the crude extracts. Institutional Animal Ethics Committee has approved the protocol (Reg. No. -379/01/ab/CPCSEA) and was in accordance with International Standard on the care and use of experimental animals (CCAC, 1993). Animals were housed under standard conditions of temperature (25ºC±2ºC), 12 h/12 h light/dark cycles, relative humidity 44–56% respectively, for 1 week before and during the experiments and fed with standard diet pellet and water ad libitum (Hindustan lever Ltd. Kolkata, India). After performing the experiments all the waste material disposed in a safe and sanitary manner.

 

Antigen:

Fresh blood was collected from sheep sacrificed in the local slaughter house. Sheep red blood cells (SRBCs) were washed three times in large volumes of Alsever’s solution and adjusted to a concentration of 0.5×109 cells/ml for immunization and challenge.

 

Toxicity assay:

Dried alcoholic stem extractive were dissolved in 5% DMSO and administered orally to different groups of rats in dosages ranging from 100 to 1000mg/kg for the LD50 study using the modified method11.

 

Neutrophil Adhesion test:

Neutrophil adhesion test was performed according to the method suggested by12. The rats were treated orally with vehicle (5% DMSO) and extracts (50, 100, 200 and 300mg/kg body weight) for 14 days. On day 14, blood samples were collected from the retro-orbital plexus into heparinized vials and analyzed for total leukocyte count (TLC), differential leukocyte count (DLC). After the initial counts, blood samples were incubated with 80mg/ml of nylon fibres for 15 min at 37°C. The incubated blood samples were again analyzed for TLC and DLC. The percentage of neutrophils in the treated and untreated blood was determined and the difference was taken as index of neutrophil adhesion.

 

Carbon Clearance Test:

Carbon clearance test was performed by method suggested by13 with slide modification. Wistar albino rats were treated with the drug and vehicle orally for 7 days. On 7th day, all the animals of the entire groups received the treatment of an intravenous injection of Indian ink dispersion (10ml/body weight) in tail vein. Blood samples were withdrawn at 0 and 10 min after injection. A 50μl blood sample was mixed with 4ml of 0.1% sodium carbonate solution to lyses the erythrocyte and the absorbance of this solution was determined at 675nm. After 10min of blood collection animals were sacrificed and the livers and spleens were collected and weighed. Rate of carbon clearance (K) and phagocytic index (a) were calculated by using following formula Where OD2 is the log absorbance of blood at 2min; OD10 is log absorbance of blood at 10 min; T2 is the last time point of blood collection; T1 is the first time point of blood collection.

 

Rate of carbon clearance and phagocytic index of treated group animals were compared with the control group animals.

 

Where OD2 is the log absorbance of blood at 2min; OD10 is log absorbance of blood at 10 min; T2 is the last time point of blood collection; T1 is the first time point of blood collection.

 

Rate of carbon clearance and phagocytic index of treated group animals were compared with the control group animals.

Haemagglutinating antibody (HA) titre:

Haemagglutinating antibody titre was done according to14 The animals were immunized by injecting 0.1ml of SRBCs suspension containing 0.5×109 cells intraperitoneally on 0 day. Blood samples were collected in microcentrifuge tubes from individual animal by retro-orbital puncture on 7th day. The blood samples were centrifuged at 2500 rpm for 10 minutes and serum was obtained15. Antibody levels were determined by the haemagglutination technique. Equal volumes of individual serum samples of each group were pooled. Two fold serial dilutions of pooled serum samples made in 25μl volume of normal saline in micro-titer plates was added to 25μl of 1% suspension of SRBCs in saline. After mixing, the plates were incubated at 37ºC for 1h and examined for hemagglutination under microscope. The reciprocal of the highest dilution of the test serum agglutination was taken as the antibody titre.

 

SRBC-Induced Delayed-type hypersensitivity (DTH) response:

SRBC-Induced Delayed-type hypersensitivity (DTH) response was carried out by [14]. On 7th day the thickness of right hind foot pad was measured using vernier caliper. The animals were then challenged by injecting 0.5x109 SRBCs in right hind foot pad. A foot pad thickness was measured again 24 h after the challenge. The difference between pre and post challenge foot pad thickness expressed in mm was taken as a measure of (DTH) and the mean value obtained for treatment groups were compared with that of control group. The data obtained was subjected to statistical analysis.

 

Statistical analysis:

Data were expressed as the mean standard deviation (S.D.) of the means and statistical analysis was done by one-way ANOVA. Differences between the data were considered significant at *p<0.05, **p<0.01, ***p<0.001.

 

RESULTS:

Phytochemical analysis:

Preliminary phytochemical study showed that, ethanolic extract contains alkaloids, tannins, flavonoids, saponins, glycosides and phenolic compounds. While methanolic extract contain alkaloids, tannins, flavonoids, saponins and glycosides compounds as its active phytoconstituents.

 

Toxicity study:

Acute toxicity studies with extract revealed that LD50 is above 1000mg/kg body weight. There was no lethality in any of the groups after 7 days of treatment.

 

Neutrophil Adhesion test:

Effect of ethanolic and methanolic extracts on neutrophil activation by the neutrophil adhesion test is shown in (Table 1 and 4). The percentage of neutrophil adhesion was significantly (*p<0.05, **p <0.01, ***p<0.001) increased by both the extracts at concentration dependent manner when compared with the control group. Ethanolic extract of Tinospora cordifolia stem showed most potent neutrophil adhesion (18.07±4.21, 22.87±3.63, 28.69±0.22** and 30.42±1.56** %) to nylon fibers as compared to control group (12.42±1.35%) and methanolic extract (14.89±3.99, 18.91±0.47, 22.45±0.6* and 25.58±1.68**%) at the respective concentrations of 50, 100, 200 and 300mg/kg body weight. Cytokines are secreted by activated immune cells for margination and extravasations of the phagocytes mainly polymorphonuclear neutrophil. Alcoholic extract significantly evoked increased in the adhesion of neutrophil to nylon fibers which correlates to the process of margination of cells in blood vessels.


 

Table 1: Effect of ethanolic extract of Tinospora cordifolia stem on neutrophil adhesion test

Animal

TLC (103mm-3) [X]

Neutrophil [Y]

Neutrophil index [XY]

% Of neutrophil group Badhesion (mg/kg)

 

UB

FTB

UB

FTB

UB

FT

 

Control

7.70±0.14

7.42±0.17

46.32±1.25

42.1±1.53

356.66±0.22

312.38±1.22

12.42±1.35

50

8.24±0.10

7.6±0.22

49.76±2.45

44.2±2.88

410.02±3.25

335.92±0.33

18.07±4.21

100

8.78±0 .33

7.7±0.14

53.1±2.78

46.7±3.44

466.22±0.15

359.59±0.22

22.87±3.63

200

9.82±0.45

8.1±0.17

59.8±3.33

51.7±1.67

587.24±2.92

418.77±3.12

28.69±0.22**

300

11.72±0.27

9.32±0.42

62.63±.1.34

54.8±2.10

734.02±4.2

510.74±3.10

30.42±1.56***

 

Table 2. Effect of ethanolic extract of Tinospora cordifolia stem on phagocytic index

Animal Group

Treatment dose (mg/kg) b.wt.

Phagocytic index

1.

Control (10ml/kg vehicle)

2.22 ±0.57

2.

50

2.52±0.54

3.

100

2.76 ±0.19

4.

200

4.38 ± 0.34*

5.

300

5.88 ± 0.43***

Values are mean± S.D. (n=6) * P< 0.05, ***p<0.001 significant.

 


Carbon Clearance Test:

Effect of alcoholic extract on the phagocytic activity by the carbon clearance test is shown in (Table 2 and 5). The phagocytic activity of the reticuloendothelial system is generally measured by the rate of removal of carbon particles from the blood stream. In carbon clearance test, all the extract treated groups exhibited significantly high phagocytic index. The phagocytic index of the plant extracts showed significant (*p<0.05, **p<0.01, ***p<0.001) increased in phagocytic index when compared to control group. This indicated stimulation of the reticuloendothelial system.

 

Haemagglutinating antibody (HA) titre:

In the present study Tinospora cordifolia stem significantly improved humoral antibody response to SRBCs challenge. The mean values of haemagglutination antibody titre of both the extracts were compared to vehicle control (3 and 6). In haemagglutination test, SRBCs that act as specific antigens were injected intraperitoneally which triggered production of specific antibodies that were estimated in serum samples of different treated groups. The mean antibody titer values of extracts treated were significantly increased as compared to control group (*p<0.05, ***p<0.001) confirming their effect on humoral immunity

 

SRBC-Induced Delayed-type hypersensitivity (DTH) response:

Delayed-type hypersensitivity (DTH) response to SRBC was calculated as a measure of paw oedema thickness (mm) for 50,100, 200 and 300mg/kg body weight of each animal after the treatment with both the extracts and compared with control. As shown in (Table 3 and 6) the ethanolic and methanolic extract produced a significant, dose-related increase in DTH activity in rats while reduction in paw oedema in later hours may be due to quick action of various enzymes, hormones that increased the phagocytosis, which activate macrophages on the invader. This may be due to saponins and similar type of compounds in the extracts which might increase the metabolic activity of the neighboring cells to release serine protease and immunohormones (Cytokines). These metabolites and activated macrophages eliminate the causative agents hence the oedema gradually reduces.


 

Table 3: Effect of ethanolic extract of Tinospora cordifolia stem on HA titre and DTH response using SRBCs as an antigen in rats.

Group

Treatment group

(mg/kg b.w p.o)

DTH response (h)

24

48

72

HA titre

1.

Control

0.19±0.08

0.14 ± 0.06

0.08±0.01

24.12±1.22

2.

50

0.22±0.07

0.17 ± 0.02

0.07±0.04

27.11±1.57

3.

100

0.31±0.09

0.25 ± 0.09

0.12±0.05

38.44±3.78

4.

200

0.54±0.08*

0.39± 0.10

0.28±0.07

54.03±5.10***

5.

300 0.

72±0.04***

0.50±0.10*

0.36±0.06**

73.23±2.23***

 


DISCUSSION:

The present study was carried out to evaluate the immunomodulatory activity of ethanolic and methanolic extracts of Tinospora cordifolia stem at various dose levels and results showed that both the extracts significantly increased immunomodulatory activity at concentration dependent manner. The results indicated that the plant increased cell mediated as well as humoral immunity. Cell adherence property of neutrophil is one of the earliest responses of both immunological and physical injury12. In neutrophil adhesion test, cell adherence property of neutrophil was assessed in blood sample from different groups, by treating with nylon fibers to which the neutrophil adhere. Adherence to nylon fibers was increased in the alcoholic extracts of Tinospora cordifolia stem immunized group respectively. This may be due to the up regulation of ß 2 integrins and also by decreased corticosterone level. Phagocytosis by neutrophil constituents an essential arm of the host to defense against foreign antigen. Neutrophil normally have receptors for fragments crystallizable (Fc) complement components (C3b) which are involved in the uptake of foreign antigens. Ethanolic extract of Tinospora cordifolia stem on neutrophil adhesion test showed much better activity as compared to methanolic extract of T. cordifolia stem. The carbon clearance assay was used to evaluate the effect on reticuloendothelial cell mediated phagocytosis13-16. When ink containing colloidal carbon is injected intravenously, the macrophages engulf the carbon particles of the ink. Rate of clearance of (carbon particles) ink from blood is known as phagocytic index. The extract produced an increased in phagocytic index suggesting its effect on reticuloendothelial system. Antibodies, product of B- lymphocytes and plasma cells, are central to humoral immune responses. IgG and IgM are the major immunoglobulins which are involved in the complement activation, opsonization and neutralization of toxins17. Immunoglobulin like IgM can overcome electric barrier and get cross-linking with red blood cells, which lead to subsequent agglutination. The augmentation of the humoral immune response to SRBCs by plant extracts as evidenced by an increased in the antibody titre in rats indicated that enhanced responsiveness of T and B lymphocyte subsets, which is involved in the antibody synthesis18. The humoral immunity involves interaction of B cells with the antigen and their subsequent proliferation and differentiation to antibody secreting plasma cells19. Delayed type Hypersensitivity required the specific recognition of given antigen by activated T lymphocytes, which subsequently proliferate and release cytokines. DTH is a part of the process of graft rejection, tumor immunity and most important, immunity to many intracellular microorganisms. It can also be due to activation of complement, release of reactive oxygen or nitrogen species by activated phagocytes and pro-inflammatory cytokines20. Delayed type hypersensitivity (DTH) is antigen specific and cause erythema induction at the site of antigen infection in immunized animals. The histology of DTH can be different for different species but the general characteristics are influx of immune cells at the site of injection, macrophages and basophills in rat’s induction become apparent within 24-72h21. Increased in paw oedema after the treatment of extracts suggested that they have boosted cellular immunity against SRBC. According to22 DTH is involved in host defense against organisms that replicate intracellularly where macrophages act as a principle effectors cells. Treatment with both the extracts resulted in immunostimulation, which can be attributed to effective macrophage function or to the enhanced synthesis of lymphokines. Alcoholic extracts of Tinospora cordifolia stem showed significant immunomodulatory activity, which differs in each extract. This may be due to concentration of active phytochemical present in particular plant extract.

 

CONCLUSSION:

The present investigation suggested that alcoholic extracts derived from Tinospora cordifolia stem may stimulate both cellular and humoral immune responses. The extracts not only potentiate nonspecific immune response, but also improve humoral as well as cell-mediated immunity effectively. Thus, from the results obtained, it can be concluded that Tinospora cordifolia has therapeutic potential and could be served as an effective immunomodulatory candidate.

 

REFERENCES:

1.      Thatte UM, Dhanukar SA, Ayurveda and contemporary scientific thought, Trends Pharmacol Sci, 7, 1986, 247.

2.      Sainis KB, Sumariwalla PF, Goel A, Chintalwar GJ, Sipahimalani AT, Banerji A, Immunomodulatory properties of stem extracts of Tinospora cordifolia: cell targets and active principles. (Eds.) Narosa Publishing House, New Delhi, India, 1997, 95.

3.      Perianayagam JB, Sharma SK, Joseph A, Christiana AJM, Evaluation of Antidiarrheal potential of Emblica officinalis, J. Ethnopharmacol, 95(1), 2004, 85-87.

4.      Singh SS, Pandey SC, Shrivastava S, Gupta VS, Palio B, Chemistry and medicinal properties of Tinospora cordifolia, Indian. J. Pharmacol, 35, 2003, 83–9.

5.      Nadkarni KM, Nadkarni AK, editors. Indian Materia Medica, 3rd ed, Vol 1, Mumbai, M/S Popular Prakasan Pvt. Ltd; 1976.

6.      Kirtikar KR, Basu BD, Indian Medicinal Plants, Vol 1. 2nd ed. New Connaught Place, Dehra Dun: M/S Bishen Singh, Mahendra Pal Singh, 1975, 858.

7.      Chopra RN, Nayar SL, Chopra IC, editors. Glossary of Indian Medicinal plants. New Delhi: CSIR; 1956.

8.      Chopra RN, Chopra LC, Handa KD, Kapur LD, editors. Indigenous Drugs of India. 2nd ed. Kolkata: M/S Dhar VN and Sons; 1982.

9.      Zhao TF, Wang X, Rimando AM, Che C, Folkloric medicinal plants: Tinospora sagittata var. cravaniana and Mahonia bealei, Planta Med, 1991, 57: 505.

10.   Trease GE, Evans MC, Textbook of Pharmacognosy, 12th ed. Balliere, Tindall, London, 1983, 343–383.

11.   Ghosh MN, Fundamentals of Experimental Pharmacology. Scientific Book Agencies, Calcutta, India, 1971, 84–88.

12.   Shinde UA, Phadke AS, Nair AM, Mungantiwar AA, Dikshit VJ, Saraf MN, Preliminary studies on the immunomodulatory activity of Cedrus deodara wood oil. Fitoterapia, 70, 1999, 333–339.

13.   Gokhale AB, Damre AS, Saraf MN, Investigations into the immunomodulatory activity of Argyreia speciosa, J Ethnopharmacol, 84, 2003, 109–114.

14.   Puri A, Saxena R, Saxena RP, Saxena KC, Immunomodulant agents from Andrographis paniculata. J Nat Prod 56(56), 1993, 995-999.

15.   Kumar SVS, Mishra SH, Hepatoprotective activity of extracts from Pergularia daemia Forsk against carbon tetrachloride-induced toxicity in rats, Phcog Mag, 3(11), 2007, 187-191.

16.   Jayathirtha, MG, Mishra SH, Preliminary immunomodulatory activities of methanol extracts of Eclipta alba and Centella asiatica, Phytomedicine, 11, 2004, 361-65.

17.   Miller LE, In: Ludke, H.R., Peacock, J.E., Tomar, R.H. (Eds.), Manual of Laboratory Immunology. Lea and Febiger, London, 1991 1–18.

18.   Benacerraf B, A hypothesis to relate the specificity of T lymphocytes and the activity of I region specific Ir genes in macrophages and borrower lymphocytes. J of Immunol, 120, 1978, 1809–1832.

19.   Ose EE, Muenster OA, A method for evaluation of vaccines containing Pasteurella multocida. Am J Vet Res, 29, 1968, 1863- 66.

20.   Smith HF, Kroes BH, Immunomodulatory and antiinflamatory activity of Picrorhiza scrophulariflora, J Ethnopharmacol, 73, 2000, 101-109.

21.   Poulter LW, Seymour GJ, Duke O, Janossy G, Panayi G, Immunohistological analysis of delayed-type hypersensitivity in man. Cell Immunol, 74, 1982, 358–369.

22.   Dikshit V, Damre AS, Kulkarni KR, Gokhale A, Saraf MN, Preliminary screening of imunocin for immunomodulatory activity, Indian J Pharm Sci, 62(4), 2000, 257-260.

 

 

 

Received on 23.01.2021         Modified on 19.02.2021

Accepted on 10.03.2021  ©AandV Publications All right reserved

Res. J. Pharmacognosy and Phytochem. 2021; 13(2):73-77.

DOI: 10.52711/0975-4385.2021.00012