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Immunostimulant effects of Capparis zeylanica Linn. leaves
Journal of Ethnopharmacology 108 (2006) 311–315
Short communication
Immunostimulant effects of Capparis zeylanica Linn. leaves B.V. Ghule a,∗ , G. Murugananthan b,1 , P.D. Nakhat a , P.G. Yeole a a
Institute of Pharmaceutical Education and Research, Borgaon, Meghe, Wardha 442001 (M.S.), India b J.S.S. College of Pharmacy, S.S. Nagar, Mysore 570 015, Karnataka, India Received 31 March 2005; received in revised form 16 March 2006; accepted 30 March 2006 Available online 7 May 2006
Abstract The present study was undertaken to explore the immunomodulatory activity of ethanolic and water extracts of Capparis zeylanica Linn. (family: Capparidaceae) leaves on neutrophil adhesion test, humoral response to sheep red blood cells, delayed-type hypersensitivity, phagocytic activity and cyclophosphamide-induced myelosuppression. Pre-treatment of water extract (300 mg/kg, oral) of Capparis zeylanica evoked a significant increase in neutrophil adhesion to nylon fibres. The augmentation of humoral immune response to sheep red blood cells by ethanolic and water extracts (150–300 mg/kg) is evidenced by increase in antibody titres in mice. A dose-related increase in both primary and secondary antibody titre was observed. Oral administration of ethanolic and water extracts of Capparis zeylanica leaves, at doses of 150 and 300 mg/kg in mice, dose dependently potentiated the delayed-type hypersensitivity reaction induced by sheep red blood cells. Immunomodulatory activity was also assessed by serological and haematological tests. Capparis zeylanica extracts prevented myelosuppression in mice treated with cyclophosphamide drug. The study comprised the acute toxicity and preliminary phytochemical screening of the ethanol and water extracts. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Capparis zeylanica leaves; Immunostimulation; Neutrophil adhesion test; HA titre; DTH response; Phagocytic response
1. Introduction The immune system is known to be involved in the etiology as well as pathophysiological mechanisms of many diseases (Sharma, 1983). Ayurveda gives emphasis on promotion of health—a concept of strengthening host defenses against different diseases (Thatte and Dahanukar, 1986). ‘Rasayana’ plants are particularly recommended for the treatment of immune disorders (Wagner, 1999). Ayurveda (with particular reference to plants) may play an important role in modern health care, particularly where satisfactory treatment is not available. There is need to evaluate the potential of Ayurvedic remedies as adjuvants to counteract side effects of modern therapy and compare the cost effectiveness of certain therapies vis-a-vis modern therapeutic schedules (Dhanukar and Thatte, 1997). Development of agents capable of moving ‘patients’ immune system from a state of immune deficiency to one of more normal function would ∗
Corresponding author. Tel.: +91 7152 240284; fax: +91 7152 241684. E-mail addresses: [email protected] (B.V. Ghule), [email protected] (G. Murugananthan). 1 Fax: +91 821 2495900. 0378-8741/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2006.03.041
likely to have a significant impact on disease and the patient it affects. Such agent would not be a cure, but would control the manifestation and course of disease (Gottlieb et al., 1987). These plants have also been evaluated for their anabolic, antistress/adaptogenic, nootropic, antioxidant and antiageing effects (Upadhyaya, 1997a,b). The isolation, purification and chemical characterization of the immunoactive phytochemicals have been carried out in some of these plants (Wagner, 1984). These phytochemicals and Indian medicinal plants are claimed to induce paraimmunity, the non-specific immunomodulation of essentially macrophages, granulocytes, NK cells and lymphocytes and complement functions (Upadhyaya, 1997a,b). Capparis zeylanica, Linn. (family: Capparidaceae) is commonly known as Indian caper, is a climbing shrub found throughout India and has been used as a ‘Rasayana’ drug in the traditional Ayurvedic system of medicine. ‘Rasayana’ plants are particularly recommended for the treatment of immune disorders (Wagner, 1999). In Northern India, the leaves are widely used as counter-irritant, febrifuge and as a cataplasm in swellings, boils and piles (Chopra, 1969). The various species of genus Capparis are useful in the treatment of cough, asthma, inflammation, fevers, cholera and also useful as poultice in gout and
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B.V. Ghule et al. / Journal of Ethnopharmacology 108 (2006) 311–315
rheumatism (Kirtikar and Basu, 1987; Chopra, 1969). Modern phytochemical screening of the plant has shown the presence of fatty acids (Haque et al., 2004) and flavonoids (Sharaf, 1997) in the leaves. Flavonoids have been known to possess antioxidant, antineoplastic, antiulcer, anti-inflammatory and antimicrobial activities (Narayana et al., 2001). The present study was therefore undertaken to explore the preliminary phytochemical screening, acute toxicity studies and immunomodulatory activity of ethanolic and water extracts of Capparis zeylanica leaves on cellular and humoral immune responses to the antigenic challenge by sheep RBCs and by neutrophil adhesion test, phagocytic activity and cyclophosphamide-induced myelosuppression. 2. Experimental 2.1. Plant material The fresh leaves of the Capparis zeylanica plant were collected at the flowering stage (month: December) in Nagpur District, Maharashtra State, India and authenticated by the authority of Botany Department, Nagpur University, Nagpur, India. A voucher specimen (no.: CSV/B-1 (12), 2004) is deposited in the Institute of pharmaceutical Education and Research, Wardha for future reference. 2.2. Extraction The air-dried, coarsely powdered leaves (500 g) were successively extracted with petroleum ether (60–80◦ ) and 95% ethanol by soxhletion. The dried marc was cold macerated to obtain water extract. The extracts were evaporated to dryness in vacuo (40 ◦ C). The yield of petroleum ether, ethanolic and water extracts was obtained 6.8, 12.3 and 15.5%, respectively. 2.3. Preliminary phytochemical screening The petroleum ether, ethanolic and water extracts of Capparis zeylanica were subjected to preliminary phytochemical screening (Trease and Evans, 1983) for the detection of various plant constituents. 2.4. Animals Swiss mice (18–20 g) of either sex kept at the Laboratory Animal Center of the Institute of Pharmaceutical Education and Research, Wardha, India were used. The animals were housed under standard environmental conditions had free access to standard pellet diet (Goldmohar brand, Lipton India Ltd.) and water ad libitum. 2.5. Acute toxicity studies The method described by Lorke (1983) was employed in the determination of the LD50 . Fifty mice were separated into two sets of five groups of animals, each consisting of five male and female mice (n = 5). They were fasted overnight and then the
first set was administered the ethanolic and water extracts at the following doses: 1, 10, 100, 1000 and 2000 mg/kg, p.o. while the second set was similarly treated i.p. Animals were observed for 24 h after treatment and the final LD50 value was calculated as the square root of the product of the lowest lethal dose and highest non-lethal dose, i.e. the geometric mean of the consecutive doses for which 0 and 100% survival rates were recorded. 2.6. Investigations into the immunomodulatory activity of ethanolic and water extracts of Capparis zeylanica 2.6.1. Animals Swiss mice (18–20 g) of either sex kept at the Laboratory Animal Center of the Institute of Pharmaceutical Education and Research, Wardha, India were used. 2.6.2. Antigenic material The sheep red blood cells (SRBCs) were used as an antigenic material. The sheep blood was obtained from Sheep farm, Veterinary College, Nagpur and collected in Alsevers solution. During the experimentation, adequate amount of SRBCs were washed three times with pyrogen-free normal saline (0.9%, w/v NaCl). The settled SRBCs were then suspended in normal saline. 2.6.3. Suspension of test samples Aqueous suspension of (1%, w/v) ethanolic and water extracts of Capparis zeylanica were prepared in (10%, v/v) propylene glycol solution. 2.6.4. Neutrophil adhesion test The method originally described by Wilkinson (1978) was employed. Mice of Group I was served as control and received 10% propylene glycol solution, whereas Groups II, III, IV and V were pretreated with ethanolic (150–300 mg/kg, oral) and water (150–300 mg/kg, oral) extracts, respectively. On 14th day of drug treatment, blood samples were collected (before challenge) by puncturing retro-orbital plexus into heparinized vials and analyzed for total leukocyte cell (TLC) and differential leukocyte cell (DLC) counts. After initial counts, blood samples were incubated with 80 mg/ml of nylon fibres for 15 min at 37 ◦ C. The incubated blood samples were again analyzed for TLC and DLC. The product of TLC and percent neutrophil gives neutrophil index of blood sample. The percent neutrophil adhesion was calculated as shown below: NIu − NIt Neutrophil adhesion (%) × 100 NIu where NIu is the Neutrophil index of untreated blood samples and NIt is the Neutrophil index of treated blood samples. 2.6.5. Haemagglutinating antibody (HA) titre The method used was similar to that described previously by Puri et al. (1993). On 14th and 21st day of drug treatment, each mouse was immunized with 0.5 × 109 SRBCs/ml/mice by i.p. route, including mice of control group. On 21st and 27th day of the treatment, primary and secondary antibody
B.V. Ghule et al. / Journal of Ethnopharmacology 108 (2006) 311–315
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individual animals and analyzed for haematological and serological parameters.
titres were determined by titrating serum dilutions with SRBCs (0.025 × 109 cells). The microtitre plates were incubated at 37 ◦ C for 2 h and examined visually for agglutination.The highest number dilution of serum showing haemagglutination has been expressed as HA titre.
2.6.9. Statistical analysis All data were expressed as mean ± S.D. and analyzed statistically by using Unpaired t-test and Dunnett’s t-test. A difference was considered significant at P < 0.05.
2.6.6. Delayed-type hypersensitivity (DTH) response Six animals per group (control and treated) were immunized (Nelson and Mildenhall, 1967) by i.p. administration of 0.5 × 109 SRBCs/mice and challenged by s.c. administration of 0.025 × 109 SRBCS/ml into right hind foot pad on day +14.The ethanolic and water extracts of Capparis zeylanica were administered orally from day – 14 until day +13.DTH response was measured at 24 and 48 h after SRBCs challenge on days +14 and +15 and expressed as mean percent increase in paw volume by using Plethysmometer.
3. Results 3.1. Phytochemical screening Preliminary phytochemical screening revealed that petroleum ether extract contains fats whereas ethanolic and water extracts showed the presence of alkaloids, saponins, terpenoids, steroids and flavonoids, tannins, carbohydrates and proteins, respectively.
2.6.7. Phagocytic response The method described by Hudson and Hay (1980) was followed. Groups of six mice each were injected intravenously with 0.2 ml of (1.6%, w/v) suspension of carbon particles (size, 20–25 um) stabilized in gelatin 30 min after the last dose of ethanol and water extracts (150–300 mg/kg, p.o.) of Capparis zeylanica Blood samples were collected from the tip of the tail at different intervals after injection and percent transparency was determined spectrophotometrically at 675 nm (UV–vis 2401 PC spectrophotometer, Schimadzu Ltd., Japan) until the transparency equivalent to the standard (original preinjection blood sample) was obtained. The rate of carbon clearance (phagocytic index, K) was calculated from the slope of each timeconcentration curve drawn by plotting 100-mean transmittance values as ordinate on semilogarithmic paper against time as abscissa.
3.2. Acute toxicity studies The LD50 was estimated to be 1624.5 ± 42.6, 1428 ± 62.8 mg/kg (p.o.) and 324.6 ± 12.4, 310 ± .4 ± 11.8 mg/kg (i.p.) in mice. During observation the animals exhibited decreased mobility, respiratory distress (gasping) with eventual immobility but no convulsions or loss of righting reflex prior to death. 3.3. Neutrophil adhesion test Pretreatment of water extract (at a dose of 300 mg/kg, oral) evoked a significant (P < 0.05) increase in the in vitro neutrophil adhesion to nylon fibres, which correlates the increase in percent neutrophils. However, ethanolic extract (150–300 mg/kg) did not show any significant increase in neutrophil adhesion when compared with respective control (Table 1).
2.6.8. Cyclophosphamide-induced immunosuppression This method used was as described by Ziauddin et al. (1996). Albino mice were divided into six groups designated as I–VI, each group containing six mice. The control Group I received 10% propylene glycol solution. Group II was administered with only cyclophosphamide at the dose of 30 mg/kg, i.p. while Groups III, IV, V and VI mice received cyclophosphamide alongwith test extracts, i.e. ethanolic and water extracts (150–300 mg/kg, p.o.) of Capparis zeylanica for 10 days. On day 11, blood sample was collected from the retro-orbital plexus of
3.4. Haemagglutinating antibody (HA) titre The HA titre was used to assess humoral immune response.A dose-related increase in both primary and secondary antibody titre was observed in mice treated with ethanolic and water extracts of Capparis zeylanica. The augmentation of the humoral immune response to SRBCS by ethanolic and water extracts is evidenced by increase in the antibody titres in the blood of mice (Table 2).
Table 1 Effects of ethanolic and water extracts of Capparis zeylanica on neutrophil adhesion test Groups
Treatment (mg/kg, p.o.)
Neutrophil index UB
Control Ethanolic extract Ethanolic extract Water extract Water extract
– 150 300 150 300
Tabulated values are expressed as mean ± S.D. a P < 0.05, significant; blood.
275.00 277.45 281.70 302.46 315.46
Neutrophil adhesion (%) FTB
± ± ± ± ±
ns P > 0.05,
70.21 41.12 50.12 45.12 42.45
226.20 225.17 224.15 239.12 241.70
± ± ± ± ±
50.12 46.15 60.15 56.12 46.21
17.98 18.84 20.42 20.93 23.98
± ± ± ± ±
6.50 7.21ns 7.82ns 5.61ns 6.45a
non-significant; Dunnett’s t-test (n = 6); UB, untreated blood; FTB, fibre treated
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Table 2 Effects of ethanolic and water extracts of Capparis zeylanica leaves on antibody titres to antigenically challenged mice Group
Treatment (mg/kg, p.o.)
Mean haemagglutinating antibody (HA) titre 1◦ HA titre
Control Ethanolic extract Ethanolic extract Water extract Water extract
– 150 300 150 300
lanica for 5 days and 30 min prior to carbon injection exhibited a dose-related increase in the clearance rate of carbon by the cells of the RES (Table 3).
07.33 08.16 09.58 08.50 10.13
± ± ± ± ±
3.7. Cyclophosphamide-induced immunosuppression
2◦ HA titre
0.51 0.75b 0.37a 0.54a 0.45a
08.83 09.16 12.17 10.37 13.78
± ± ± ± ±
0.40 0.75a 0.81a 0.81a 0.89a
Cyclphosphamide at the dose of 30 mg/kg, i.p. caused a significant reduction in the haemoglobin, RBCs, WBCs and platlets count. Combined treatment of cyclophosphamide and ethanol and water extracts (150–300 mg/kg, p.o.) of Capparis zeylanica resulted in a restoration of bone marrow activity as compared with cyclophosphamide treatment alone (Table 4).
Tabulated values are mean ± S.D. a P < 0.01, highly significant; b P < 0.05, significant; Dunnett’s t-test (n = 6); 1◦ HA, primary haemagglutinating; 2◦ HA, secondary haemagglutinating.
4. Discussion and conclusions 3.5. Delayed-type hypersensitivity (DTH) reactions Modulation of the immune response through stimulation or suppression may help in maintaining a disease-free state. Agents that activate host defense mechanisms in the presence of an impaired immune responsiveness can provide supportive therapy to conventional chemotherapy (Wagner, 1984). The results obtained in the present study indicate that Capparis zeylanica is a potent immunostimulant, stimulating both the specific and non-specific immune mechanisms. The neutrophil, an end cell unable to divide and with limited capacity for protein synthesis is, nevertheless, capable of a wide range of responses, in particular chemotaxis, phagocytosis, exocytosis and both intracellular and extracellular killing (Dale and Foreman, 1984). In the present study, water extract (300 mg/kg, p.o.) of Capparis zeylanica evoked a significant increase in per-
The cell-mediated immune response was assessed by DTH reaction, i.e. foot pad reaction. Both ethanolic and water extracts produced a significant, dose-related increase in DTH reactivity in mice.Increase in DTH reaction in mice in response to T cell dependent antigen revealed the stimulatory effect of ethanolic and water extracts on T cells (Table 3). 3.6. Phagocytic response The phagocytic activity of the reticulo-endothelium system was measured by the rate of removal of gelatin-stabilized carbon particles from the blood circulation. Oral administration of ethanolic and water extracts (150–300 mg/kg) of Capparis zey-
Table 3 Effects of ethanolic and water extracts of Capparis zeylanica on foot pad reaction of antigenically challenged mice and phagocytic response Group(s)
Treatment (mg/kg, p.o.)
Foot pad thickness, i.e. mean (%) oedema 24 h
Control Ethanolic extract Ethanolic extract Water extract Water extract
– 150 300 150 300
29.12 31.19 34.95 32.20 37.57
Phagocytic index
48 h ± ± ± ± ±
1.29 1.96ns 2.45a 1.24b 1.80a
23.12 24.71 27.85 25.50 31.24
± ± ± ± ±
0.98 2.00ns 1.85a 1.45ns 2.27a
0.22 0.26 0.31 0.27 0.30
± ± ± ± ±
0.02 0.03ns 0.02a 0.02b 0.04a
Values are mean ± S.D. a P < 0.01, highly significant; b P < 0.05, significant; ns P > 0.05, non-significant (compared to respective control); Dunnett’s t-test (n = 6).
Table 4 Effects of ethanolic and water extracts of Capparis zeylanica on blood cells of mice treated with cyclophosphamide for 10 days Group
Haemoglobin concentration in g
I II III IV V VI
14.20 11.82 14.50 14.76 12.89 13.01
± ± ± ± ± ±
0.18 0.22a 0.20c 0.24b 0.17b 0.21b
RBC count in (%) million (cmm) 4.40 4.15 4.61 4.97 4.21 4.32
± ± ± ± ± ±
0.19 0.14c 0.12ns 0.16b 0.20ns 0.16ns
WBC count in thousands (cmm) 11.02 09.32 11.55 12.21 10.63 11.32
± ± ± ± ± ±
0.32 0.31a 0.36c 0.30b 0.28b 0.31b
Platelet count in thousands (cmm) 502.40 436.20 541.20 547.09 558.30 498.30
± ± ± ± ± ±
30 21a 21ns 25b 28c 31c
Group I: control (without any drug treatment); Group II: cyclophosphamide-treated mice group; Group III: treatment with ethanolic extract (300 mg/kg, p.o.) of Capparis zeylanica; Group IV: treatment with water extract (300 mg/kg, p.o.) of Capparis zeylanica; Group V: cyclophosphamide (30 mg/kg, i.p.) and ethanolic extract (300 mg/kg, p.o.) treated group; Group VI: cyclophosphamide (30 mg/kg, i.p.) and water extract (300 mg/kg, p.o.) treated group. a P < 0.0001, extremely significant; b P < 0.05, significant; c P < 0.01, very significant; ns P > 0.05, non-significant. Comparison of Group II with Group I, (Unpaired t-test, n = 6). Comparison of Group I with Groups III and IV, (Dunnett’s t-test, n = 6). Comparison of Group II with Goups V and VI, (Dunnett’s t-test, n = 6).
B.V. Ghule et al. / Journal of Ethnopharmacology 108 (2006) 311–315
cent neutrophils. This may help in increasing immunity of body against microbial infections (Benacerraf, 1978). Antibody molecules, a 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, neutralization of toxins, etc. (Miller, 1991). The augmentation of the humoral immune response to SRBCs by Capparis zeylanica, as evidenced by increase in the antibody titre in mice (Table 2) indicated the enhanced responsiveness of T and B lymphocyte subsets, involved in the antibody synthesis (Benacerraf, 1978). Cell-mediated immunity (CMI) involves effector mechanisms carried out by T lymphocytes and their products (lymphokines). CMI responses are critical to defense against infectious organisms, infection of foreign grafts, tumour immunity and delayed-type hypersensitivity reactions (Miller, 1991). Therefore, increase in DTH reaction in mice in response to T cell dependent antigen revealed the stimulatory effect of ethanolic and water extracts of Capparis zeylanica on T cells (Table 3). Phagocytosis is the process by which certain body cells, collectively known as phagocytes, ingest and removes microorganisms, effete or malignant cells, inorganic particles and tissue debris (Miller, 1991). Capparis zeylanica appeared to enhance the phagocytic function by exhibiting a dose related increase in clearance rate of carbon by the cells of the reticulo-endothelium system (Table 3). Since Capparis zeylanica augmented the circulating antibody titre, it was thought worthwhile to evaluate its effect on peripheral blood count and cyclophosphamide-induced immunosuppression. The administration of Capparis zeylanica significantly ameliorated the total WBCs count, RBCs count, haemoglobin and platelets count and also restored the myelosuppressive effects induced by cyclophosphamide (Table 4). The present investigation suggests that Capparis zeylanica may stimulate both cellular and humoral immune responses. Further studies to elucidate the exact immunostimulatory mechanisms of Capparis zeylanica are in progress. Acknowledgements We owe our thanks to the authority of Botany Department, Nagpur University, Nagpur for the authentication of plant specimen. The facilities provided by the Institute of Pharmaceutical Education and Research, Wardha during this course of study are gratefully acknowledged. References Benacerraf, B.A., 1809. Hypothesis to relate the specificity of T lymphocytes and the activity of I region specific Ir genes in macrophages and B lymphocytes. Journal of Immunology 120.
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Chopra, R.N., 1969. Glossary of Indian Medicinal Plants. Council of Scientific and Industrial Research, New Delhi, p. 49. Dale, M.M., Foreman, J.C., 1984. Textbook of Immunopharmacology. Blackwell, London, pp. 52–54. Dhanukar, S.A., Thatte, U.M., 1997. Current status of Ayurveda in phytomedicine. Phytomedicine 4, 359. Gottlieb, A.A., Gottlieb, M.S., Scholes, V.E., 1987. Reconstitution of immune functions in AIDS/ARC. Concepts in Immunopathology 4, 261– 274. Haque, M.E., Rahman, M.M., Mossadik, M.A., Sarker, S.D., 2004. E-octadec-7en-ynoic acid from the roots of Capparis zeylanica. Fitoterapia 74, 130–133. Hudson, L., Hay, F.C., 1980. Practical Immunology, second ed. Blackwell, London, pp.73–92. Kirtikar, K.R., Basu, B.D., 1987. Indian Medicinal Plants, vol. 1., second ed. Oriental Enterprises, Dehradun, India, pp. 200–201. Lorke, D.A., 1983. New approach to acute toxicity testing. Archives of Toxicology 54, 275–287. Miller, L.E., 1991. In: Ludke, H.R., Peacock, J.E., Tomar, R.H. (Eds.), Manual of Laboratory Immunology. Lea and Febiger, London, pp. 1– 18. Narayana, K.R., Reddy, M.S., Chaluvadi, M.R., Krishna, D.R., 2001. Bioflavonoids classification, pharmacological, biochemical effects and therapeutic potential. Indian Journal of Pharmacology 33, 2– 16. Nelson, D.A., Mildenhall, P., 1967. Studies on cytophilic antibodies: The production by mice of macrophage cytophilic antibodies to sheep erythrocytes: relationship to the production of other antibodies and development of delayed type hypersensitivity. Australian Journal of Experimental Biology and Medical Sciences 45, 113. Puri, A., Saxena, R.P., Saxena, K.C., 1993. Immunostimulant agents from Andrographis peniculata. Journal of Natural Product 56, 995–999. Sharaf, M.A., 1997. Flavonoids of four Cleome and three Capparis species. Biochemical Systematics and Ecology 25, 161–166. Sharma, P., 1983. Charaka Samhita, Chikitasasthana. Chaukhamba Orientalia, Varanasi, India, p. 54. Thatte, U.M., Dahanukar, S.A., 1986. Ayurveda and contemporary scientific thought. Trends in Pharmacological Sciences 7, 247. Trease, G.E., Evans, M.C., 1983. Textbook of Pharmacognosy, 12th ed. Balliere, Tindall, London, pp. 343–383. Upadhyaya, S.N., 1997a. Immunomodulation. In: Sainis, K.B., Sumariwalla, P.F., Goel, A., Chintalwar, G.J., Sipahimalani, A.T., Banerji, A. (Eds.), Immunomodulatory Properties of Stem Extracts of Tinospora cordifolia: Cell Targets and Active Principles. Narosa Publishing House, New Delhi, India, p. 95. Upadhyaya, S.N., 1997b. In: Katiyar, C.K., Brindavanam, N.B., Tiwari, P., Narayana, D.B.A. (Eds.), Immunomodulation. Narosa Publishing House, New Delhi, pp. 163–187. Wagner, H., 1984. In: Hiroshi Hikino, N.R., Farnsworth (Eds.), Economic and Medicinal Plant Research, vol. I. Academic Press, London, pp. 113– 153. Wagner, H., 1999. In: Dhanukar, S.A., Thatte, U.M., Rege, N.M., Birkhauser (Eds.), Immunomodulatory agents from plants. Verlag, Basel, Switzerland, pp. 289–323. Wilkinson, P.C., 1978. Neutrophil adhesion test. In: Vane, J.K., Ferreria, S.H. (Eds.), Handbook of Experimental Pharmacology, I, first ed. Springer Verlag, Berlin, p. 109. Ziauddin, M., Phansalkar, N., Patki, P., Diwanay, S., Patwardhan, B., 1996. Studies on the immunomodulatory effects of Ashwagandha. Journal of Ethnopharmacology 50, 69–76.
Short communication
Immunostimulant effects of Capparis zeylanica Linn. leaves B.V. Ghule a,∗ , G. Murugananthan b,1 , P.D. Nakhat a , P.G. Yeole a a
Institute of Pharmaceutical Education and Research, Borgaon, Meghe, Wardha 442001 (M.S.), India b J.S.S. College of Pharmacy, S.S. Nagar, Mysore 570 015, Karnataka, India Received 31 March 2005; received in revised form 16 March 2006; accepted 30 March 2006 Available online 7 May 2006
Abstract The present study was undertaken to explore the immunomodulatory activity of ethanolic and water extracts of Capparis zeylanica Linn. (family: Capparidaceae) leaves on neutrophil adhesion test, humoral response to sheep red blood cells, delayed-type hypersensitivity, phagocytic activity and cyclophosphamide-induced myelosuppression. Pre-treatment of water extract (300 mg/kg, oral) of Capparis zeylanica evoked a significant increase in neutrophil adhesion to nylon fibres. The augmentation of humoral immune response to sheep red blood cells by ethanolic and water extracts (150–300 mg/kg) is evidenced by increase in antibody titres in mice. A dose-related increase in both primary and secondary antibody titre was observed. Oral administration of ethanolic and water extracts of Capparis zeylanica leaves, at doses of 150 and 300 mg/kg in mice, dose dependently potentiated the delayed-type hypersensitivity reaction induced by sheep red blood cells. Immunomodulatory activity was also assessed by serological and haematological tests. Capparis zeylanica extracts prevented myelosuppression in mice treated with cyclophosphamide drug. The study comprised the acute toxicity and preliminary phytochemical screening of the ethanol and water extracts. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Capparis zeylanica leaves; Immunostimulation; Neutrophil adhesion test; HA titre; DTH response; Phagocytic response
1. Introduction The immune system is known to be involved in the etiology as well as pathophysiological mechanisms of many diseases (Sharma, 1983). Ayurveda gives emphasis on promotion of health—a concept of strengthening host defenses against different diseases (Thatte and Dahanukar, 1986). ‘Rasayana’ plants are particularly recommended for the treatment of immune disorders (Wagner, 1999). Ayurveda (with particular reference to plants) may play an important role in modern health care, particularly where satisfactory treatment is not available. There is need to evaluate the potential of Ayurvedic remedies as adjuvants to counteract side effects of modern therapy and compare the cost effectiveness of certain therapies vis-a-vis modern therapeutic schedules (Dhanukar and Thatte, 1997). Development of agents capable of moving ‘patients’ immune system from a state of immune deficiency to one of more normal function would ∗
Corresponding author. Tel.: +91 7152 240284; fax: +91 7152 241684. E-mail addresses: [email protected] (B.V. Ghule), [email protected] (G. Murugananthan). 1 Fax: +91 821 2495900. 0378-8741/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2006.03.041
likely to have a significant impact on disease and the patient it affects. Such agent would not be a cure, but would control the manifestation and course of disease (Gottlieb et al., 1987). These plants have also been evaluated for their anabolic, antistress/adaptogenic, nootropic, antioxidant and antiageing effects (Upadhyaya, 1997a,b). The isolation, purification and chemical characterization of the immunoactive phytochemicals have been carried out in some of these plants (Wagner, 1984). These phytochemicals and Indian medicinal plants are claimed to induce paraimmunity, the non-specific immunomodulation of essentially macrophages, granulocytes, NK cells and lymphocytes and complement functions (Upadhyaya, 1997a,b). Capparis zeylanica, Linn. (family: Capparidaceae) is commonly known as Indian caper, is a climbing shrub found throughout India and has been used as a ‘Rasayana’ drug in the traditional Ayurvedic system of medicine. ‘Rasayana’ plants are particularly recommended for the treatment of immune disorders (Wagner, 1999). In Northern India, the leaves are widely used as counter-irritant, febrifuge and as a cataplasm in swellings, boils and piles (Chopra, 1969). The various species of genus Capparis are useful in the treatment of cough, asthma, inflammation, fevers, cholera and also useful as poultice in gout and
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rheumatism (Kirtikar and Basu, 1987; Chopra, 1969). Modern phytochemical screening of the plant has shown the presence of fatty acids (Haque et al., 2004) and flavonoids (Sharaf, 1997) in the leaves. Flavonoids have been known to possess antioxidant, antineoplastic, antiulcer, anti-inflammatory and antimicrobial activities (Narayana et al., 2001). The present study was therefore undertaken to explore the preliminary phytochemical screening, acute toxicity studies and immunomodulatory activity of ethanolic and water extracts of Capparis zeylanica leaves on cellular and humoral immune responses to the antigenic challenge by sheep RBCs and by neutrophil adhesion test, phagocytic activity and cyclophosphamide-induced myelosuppression. 2. Experimental 2.1. Plant material The fresh leaves of the Capparis zeylanica plant were collected at the flowering stage (month: December) in Nagpur District, Maharashtra State, India and authenticated by the authority of Botany Department, Nagpur University, Nagpur, India. A voucher specimen (no.: CSV/B-1 (12), 2004) is deposited in the Institute of pharmaceutical Education and Research, Wardha for future reference. 2.2. Extraction The air-dried, coarsely powdered leaves (500 g) were successively extracted with petroleum ether (60–80◦ ) and 95% ethanol by soxhletion. The dried marc was cold macerated to obtain water extract. The extracts were evaporated to dryness in vacuo (40 ◦ C). The yield of petroleum ether, ethanolic and water extracts was obtained 6.8, 12.3 and 15.5%, respectively. 2.3. Preliminary phytochemical screening The petroleum ether, ethanolic and water extracts of Capparis zeylanica were subjected to preliminary phytochemical screening (Trease and Evans, 1983) for the detection of various plant constituents. 2.4. Animals Swiss mice (18–20 g) of either sex kept at the Laboratory Animal Center of the Institute of Pharmaceutical Education and Research, Wardha, India were used. The animals were housed under standard environmental conditions had free access to standard pellet diet (Goldmohar brand, Lipton India Ltd.) and water ad libitum. 2.5. Acute toxicity studies The method described by Lorke (1983) was employed in the determination of the LD50 . Fifty mice were separated into two sets of five groups of animals, each consisting of five male and female mice (n = 5). They were fasted overnight and then the
first set was administered the ethanolic and water extracts at the following doses: 1, 10, 100, 1000 and 2000 mg/kg, p.o. while the second set was similarly treated i.p. Animals were observed for 24 h after treatment and the final LD50 value was calculated as the square root of the product of the lowest lethal dose and highest non-lethal dose, i.e. the geometric mean of the consecutive doses for which 0 and 100% survival rates were recorded. 2.6. Investigations into the immunomodulatory activity of ethanolic and water extracts of Capparis zeylanica 2.6.1. Animals Swiss mice (18–20 g) of either sex kept at the Laboratory Animal Center of the Institute of Pharmaceutical Education and Research, Wardha, India were used. 2.6.2. Antigenic material The sheep red blood cells (SRBCs) were used as an antigenic material. The sheep blood was obtained from Sheep farm, Veterinary College, Nagpur and collected in Alsevers solution. During the experimentation, adequate amount of SRBCs were washed three times with pyrogen-free normal saline (0.9%, w/v NaCl). The settled SRBCs were then suspended in normal saline. 2.6.3. Suspension of test samples Aqueous suspension of (1%, w/v) ethanolic and water extracts of Capparis zeylanica were prepared in (10%, v/v) propylene glycol solution. 2.6.4. Neutrophil adhesion test The method originally described by Wilkinson (1978) was employed. Mice of Group I was served as control and received 10% propylene glycol solution, whereas Groups II, III, IV and V were pretreated with ethanolic (150–300 mg/kg, oral) and water (150–300 mg/kg, oral) extracts, respectively. On 14th day of drug treatment, blood samples were collected (before challenge) by puncturing retro-orbital plexus into heparinized vials and analyzed for total leukocyte cell (TLC) and differential leukocyte cell (DLC) counts. After initial counts, blood samples were incubated with 80 mg/ml of nylon fibres for 15 min at 37 ◦ C. The incubated blood samples were again analyzed for TLC and DLC. The product of TLC and percent neutrophil gives neutrophil index of blood sample. The percent neutrophil adhesion was calculated as shown below: NIu − NIt Neutrophil adhesion (%) × 100 NIu where NIu is the Neutrophil index of untreated blood samples and NIt is the Neutrophil index of treated blood samples. 2.6.5. Haemagglutinating antibody (HA) titre The method used was similar to that described previously by Puri et al. (1993). On 14th and 21st day of drug treatment, each mouse was immunized with 0.5 × 109 SRBCs/ml/mice by i.p. route, including mice of control group. On 21st and 27th day of the treatment, primary and secondary antibody
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individual animals and analyzed for haematological and serological parameters.
titres were determined by titrating serum dilutions with SRBCs (0.025 × 109 cells). The microtitre plates were incubated at 37 ◦ C for 2 h and examined visually for agglutination.The highest number dilution of serum showing haemagglutination has been expressed as HA titre.
2.6.9. Statistical analysis All data were expressed as mean ± S.D. and analyzed statistically by using Unpaired t-test and Dunnett’s t-test. A difference was considered significant at P < 0.05.
2.6.6. Delayed-type hypersensitivity (DTH) response Six animals per group (control and treated) were immunized (Nelson and Mildenhall, 1967) by i.p. administration of 0.5 × 109 SRBCs/mice and challenged by s.c. administration of 0.025 × 109 SRBCS/ml into right hind foot pad on day +14.The ethanolic and water extracts of Capparis zeylanica were administered orally from day – 14 until day +13.DTH response was measured at 24 and 48 h after SRBCs challenge on days +14 and +15 and expressed as mean percent increase in paw volume by using Plethysmometer.
3. Results 3.1. Phytochemical screening Preliminary phytochemical screening revealed that petroleum ether extract contains fats whereas ethanolic and water extracts showed the presence of alkaloids, saponins, terpenoids, steroids and flavonoids, tannins, carbohydrates and proteins, respectively.
2.6.7. Phagocytic response The method described by Hudson and Hay (1980) was followed. Groups of six mice each were injected intravenously with 0.2 ml of (1.6%, w/v) suspension of carbon particles (size, 20–25 um) stabilized in gelatin 30 min after the last dose of ethanol and water extracts (150–300 mg/kg, p.o.) of Capparis zeylanica Blood samples were collected from the tip of the tail at different intervals after injection and percent transparency was determined spectrophotometrically at 675 nm (UV–vis 2401 PC spectrophotometer, Schimadzu Ltd., Japan) until the transparency equivalent to the standard (original preinjection blood sample) was obtained. The rate of carbon clearance (phagocytic index, K) was calculated from the slope of each timeconcentration curve drawn by plotting 100-mean transmittance values as ordinate on semilogarithmic paper against time as abscissa.
3.2. Acute toxicity studies The LD50 was estimated to be 1624.5 ± 42.6, 1428 ± 62.8 mg/kg (p.o.) and 324.6 ± 12.4, 310 ± .4 ± 11.8 mg/kg (i.p.) in mice. During observation the animals exhibited decreased mobility, respiratory distress (gasping) with eventual immobility but no convulsions or loss of righting reflex prior to death. 3.3. Neutrophil adhesion test Pretreatment of water extract (at a dose of 300 mg/kg, oral) evoked a significant (P < 0.05) increase in the in vitro neutrophil adhesion to nylon fibres, which correlates the increase in percent neutrophils. However, ethanolic extract (150–300 mg/kg) did not show any significant increase in neutrophil adhesion when compared with respective control (Table 1).
2.6.8. Cyclophosphamide-induced immunosuppression This method used was as described by Ziauddin et al. (1996). Albino mice were divided into six groups designated as I–VI, each group containing six mice. The control Group I received 10% propylene glycol solution. Group II was administered with only cyclophosphamide at the dose of 30 mg/kg, i.p. while Groups III, IV, V and VI mice received cyclophosphamide alongwith test extracts, i.e. ethanolic and water extracts (150–300 mg/kg, p.o.) of Capparis zeylanica for 10 days. On day 11, blood sample was collected from the retro-orbital plexus of
3.4. Haemagglutinating antibody (HA) titre The HA titre was used to assess humoral immune response.A dose-related increase in both primary and secondary antibody titre was observed in mice treated with ethanolic and water extracts of Capparis zeylanica. The augmentation of the humoral immune response to SRBCS by ethanolic and water extracts is evidenced by increase in the antibody titres in the blood of mice (Table 2).
Table 1 Effects of ethanolic and water extracts of Capparis zeylanica on neutrophil adhesion test Groups
Treatment (mg/kg, p.o.)
Neutrophil index UB
Control Ethanolic extract Ethanolic extract Water extract Water extract
– 150 300 150 300
Tabulated values are expressed as mean ± S.D. a P < 0.05, significant; blood.
275.00 277.45 281.70 302.46 315.46
Neutrophil adhesion (%) FTB
± ± ± ± ±
ns P > 0.05,
70.21 41.12 50.12 45.12 42.45
226.20 225.17 224.15 239.12 241.70
± ± ± ± ±
50.12 46.15 60.15 56.12 46.21
17.98 18.84 20.42 20.93 23.98
± ± ± ± ±
6.50 7.21ns 7.82ns 5.61ns 6.45a
non-significant; Dunnett’s t-test (n = 6); UB, untreated blood; FTB, fibre treated
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Table 2 Effects of ethanolic and water extracts of Capparis zeylanica leaves on antibody titres to antigenically challenged mice Group
Treatment (mg/kg, p.o.)
Mean haemagglutinating antibody (HA) titre 1◦ HA titre
Control Ethanolic extract Ethanolic extract Water extract Water extract
– 150 300 150 300
lanica for 5 days and 30 min prior to carbon injection exhibited a dose-related increase in the clearance rate of carbon by the cells of the RES (Table 3).
07.33 08.16 09.58 08.50 10.13
± ± ± ± ±
3.7. Cyclophosphamide-induced immunosuppression
2◦ HA titre
0.51 0.75b 0.37a 0.54a 0.45a
08.83 09.16 12.17 10.37 13.78
± ± ± ± ±
0.40 0.75a 0.81a 0.81a 0.89a
Cyclphosphamide at the dose of 30 mg/kg, i.p. caused a significant reduction in the haemoglobin, RBCs, WBCs and platlets count. Combined treatment of cyclophosphamide and ethanol and water extracts (150–300 mg/kg, p.o.) of Capparis zeylanica resulted in a restoration of bone marrow activity as compared with cyclophosphamide treatment alone (Table 4).
Tabulated values are mean ± S.D. a P < 0.01, highly significant; b P < 0.05, significant; Dunnett’s t-test (n = 6); 1◦ HA, primary haemagglutinating; 2◦ HA, secondary haemagglutinating.
4. Discussion and conclusions 3.5. Delayed-type hypersensitivity (DTH) reactions Modulation of the immune response through stimulation or suppression may help in maintaining a disease-free state. Agents that activate host defense mechanisms in the presence of an impaired immune responsiveness can provide supportive therapy to conventional chemotherapy (Wagner, 1984). The results obtained in the present study indicate that Capparis zeylanica is a potent immunostimulant, stimulating both the specific and non-specific immune mechanisms. The neutrophil, an end cell unable to divide and with limited capacity for protein synthesis is, nevertheless, capable of a wide range of responses, in particular chemotaxis, phagocytosis, exocytosis and both intracellular and extracellular killing (Dale and Foreman, 1984). In the present study, water extract (300 mg/kg, p.o.) of Capparis zeylanica evoked a significant increase in per-
The cell-mediated immune response was assessed by DTH reaction, i.e. foot pad reaction. Both ethanolic and water extracts produced a significant, dose-related increase in DTH reactivity in mice.Increase in DTH reaction in mice in response to T cell dependent antigen revealed the stimulatory effect of ethanolic and water extracts on T cells (Table 3). 3.6. Phagocytic response The phagocytic activity of the reticulo-endothelium system was measured by the rate of removal of gelatin-stabilized carbon particles from the blood circulation. Oral administration of ethanolic and water extracts (150–300 mg/kg) of Capparis zey-
Table 3 Effects of ethanolic and water extracts of Capparis zeylanica on foot pad reaction of antigenically challenged mice and phagocytic response Group(s)
Treatment (mg/kg, p.o.)
Foot pad thickness, i.e. mean (%) oedema 24 h
Control Ethanolic extract Ethanolic extract Water extract Water extract
– 150 300 150 300
29.12 31.19 34.95 32.20 37.57
Phagocytic index
48 h ± ± ± ± ±
1.29 1.96ns 2.45a 1.24b 1.80a
23.12 24.71 27.85 25.50 31.24
± ± ± ± ±
0.98 2.00ns 1.85a 1.45ns 2.27a
0.22 0.26 0.31 0.27 0.30
± ± ± ± ±
0.02 0.03ns 0.02a 0.02b 0.04a
Values are mean ± S.D. a P < 0.01, highly significant; b P < 0.05, significant; ns P > 0.05, non-significant (compared to respective control); Dunnett’s t-test (n = 6).
Table 4 Effects of ethanolic and water extracts of Capparis zeylanica on blood cells of mice treated with cyclophosphamide for 10 days Group
Haemoglobin concentration in g
I II III IV V VI
14.20 11.82 14.50 14.76 12.89 13.01
± ± ± ± ± ±
0.18 0.22a 0.20c 0.24b 0.17b 0.21b
RBC count in (%) million (cmm) 4.40 4.15 4.61 4.97 4.21 4.32
± ± ± ± ± ±
0.19 0.14c 0.12ns 0.16b 0.20ns 0.16ns
WBC count in thousands (cmm) 11.02 09.32 11.55 12.21 10.63 11.32
± ± ± ± ± ±
0.32 0.31a 0.36c 0.30b 0.28b 0.31b
Platelet count in thousands (cmm) 502.40 436.20 541.20 547.09 558.30 498.30
± ± ± ± ± ±
30 21a 21ns 25b 28c 31c
Group I: control (without any drug treatment); Group II: cyclophosphamide-treated mice group; Group III: treatment with ethanolic extract (300 mg/kg, p.o.) of Capparis zeylanica; Group IV: treatment with water extract (300 mg/kg, p.o.) of Capparis zeylanica; Group V: cyclophosphamide (30 mg/kg, i.p.) and ethanolic extract (300 mg/kg, p.o.) treated group; Group VI: cyclophosphamide (30 mg/kg, i.p.) and water extract (300 mg/kg, p.o.) treated group. a P < 0.0001, extremely significant; b P < 0.05, significant; c P < 0.01, very significant; ns P > 0.05, non-significant. Comparison of Group II with Group I, (Unpaired t-test, n = 6). Comparison of Group I with Groups III and IV, (Dunnett’s t-test, n = 6). Comparison of Group II with Goups V and VI, (Dunnett’s t-test, n = 6).
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cent neutrophils. This may help in increasing immunity of body against microbial infections (Benacerraf, 1978). Antibody molecules, a 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, neutralization of toxins, etc. (Miller, 1991). The augmentation of the humoral immune response to SRBCs by Capparis zeylanica, as evidenced by increase in the antibody titre in mice (Table 2) indicated the enhanced responsiveness of T and B lymphocyte subsets, involved in the antibody synthesis (Benacerraf, 1978). Cell-mediated immunity (CMI) involves effector mechanisms carried out by T lymphocytes and their products (lymphokines). CMI responses are critical to defense against infectious organisms, infection of foreign grafts, tumour immunity and delayed-type hypersensitivity reactions (Miller, 1991). Therefore, increase in DTH reaction in mice in response to T cell dependent antigen revealed the stimulatory effect of ethanolic and water extracts of Capparis zeylanica on T cells (Table 3). Phagocytosis is the process by which certain body cells, collectively known as phagocytes, ingest and removes microorganisms, effete or malignant cells, inorganic particles and tissue debris (Miller, 1991). Capparis zeylanica appeared to enhance the phagocytic function by exhibiting a dose related increase in clearance rate of carbon by the cells of the reticulo-endothelium system (Table 3). Since Capparis zeylanica augmented the circulating antibody titre, it was thought worthwhile to evaluate its effect on peripheral blood count and cyclophosphamide-induced immunosuppression. The administration of Capparis zeylanica significantly ameliorated the total WBCs count, RBCs count, haemoglobin and platelets count and also restored the myelosuppressive effects induced by cyclophosphamide (Table 4). The present investigation suggests that Capparis zeylanica may stimulate both cellular and humoral immune responses. Further studies to elucidate the exact immunostimulatory mechanisms of Capparis zeylanica are in progress. Acknowledgements We owe our thanks to the authority of Botany Department, Nagpur University, Nagpur for the authentication of plant specimen. The facilities provided by the Institute of Pharmaceutical Education and Research, Wardha during this course of study are gratefully acknowledged. References Benacerraf, B.A., 1809. Hypothesis to relate the specificity of T lymphocytes and the activity of I region specific Ir genes in macrophages and B lymphocytes. Journal of Immunology 120.
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