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Effect of some Indian herbs on macrophage functions in ochratoxin A treated mice
Journal of Ethnopharmacology 58 (1997) 15 – 20
Effect of some Indian herbs on macrophage functions in ochratoxin A treated mice J.N. Dhuley Department of Pharmacology and Toxicology, Research Centre, Hindustan Antibiotics Limited, Pimpri, Pune-411018, India Received 27 January 1997; received in revised form 23 June 1997; accepted 25 June 1997
Abstract The effect of Indian herbs namely, Asparagus racemosus, Tinospora cordifolia, Withania somnifera and Picrorhiza kurrooa on the functions of macrophages obtained from mice treated with the carcinogen ochratoxin A (OTA) was investigated. The chemotactic activity of murine macrophages was significantly decreased by 17 weeks of treatment with OTA compared with controls. Production of interleukin-1 (IL-1) and tumor necrosis factor (TNF) was also markedly reduced. Treatment with Asparagus racemosus, Tinospora cordifolia, Withania somnifera and Picrorhiza kurrooa significantly inhibited OTA-induced suppression of chemotactic activity and production of IL-1 and TNF-a by macropahges. Moreover, we found that Withania somnifera treated macrophage chemotaxis and that Asparagus racemosus induced excess production of TNF-a when compared with controls. © 1997 Elsevier Science Ireland Ltd. Keywords: Indian herbs; Carcinogen (ochratoxin A); Macrophage; Cytokine production
1. Introduction From antiquity to modern times, man has been obsessed with a quest for rejuvenation and an innate desire to extend his lifespan. Ayurveda is an original holistic system of diagnosis and treatment involving nutrition, hygiene and rejuvenation, developed and perfected in India over 5000 years ago. Ayurveda (Ayur, life; Veda, knowledge), is the knowledge of healthy living and is not merely confined to the treatment of illness. The modulation of immune response by using
various agents in order to alleviate the disease has been of interest over many years and the concept of ‘Rasayan’ in Ayurveda has similarity with it (Samhita, 1949). Some Indian herbs have been shown to exert anti-inflammatory (Singh and Atal, 1986), anti-stress and anti-cancer (Thatte, 1996) effects by modulating the immune system. Asparagus racemosus (AR), Withania somnifera (WS), Tinospora cordifolia (TC) and Picrorhiza kurrooa (PK) have been found to enhance host resistance and reduce the side effects of other toxic agents.
0378-8741/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 3 7 8 - 8 7 4 1 ( 9 7 ) 0 0 0 7 2 - X
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J.N. Dhuley / Journal of Ethnopharmacology 58 (1997) 15–20
Ochratoxin A (OTA) is a mycotoxin consisting of a dihydroisocoumarin moiety linked through its carboxyl group to L-b-phenylalanine. It has been found as a frequent contaminant in various food and feed commodities including cereals, grains and to a lesser extent coffee, milk and foods from animal origin (IPCS, 1990). It is a well-known tumor promoter that contributes to cancer of the kidney, liver and mammary gland (Kane et al., 1986; Castegnaro et al., 1987; Huff, 1991; Marquardt and Frohlich, 1992; PfohlLeskowicz et al., 1993). In the present study, an attempt has been made to investigate the effects of Asparagus racemosus Roxb. (Liliaceae, root), Withania somnifera L. (Solanaceae, root and leaves), Tinospora cordifolia Willd. (Menispermaceae, whole plant) and Picrorhiza kurrooa Royle (Scrophulariaceae, rhizome) on OTA-induced suppression of chemotaxis and cytokine production in murine macrophages.
ground. The material was then extracted with 80% ethanol (3 g/20 ml) at room temperature. After 3 h, the macerate was filtered on Whatman no. 1 paper and the alcoholic extracts dried at 40°C for 1 week. The dried material was maintained in the dark in a cool and dry place. Before using, it was dissolved in water (10% w/v final concentration), filter-sterilized and maintained at 5°C.
2.3. Treatment Six week-old mice received a daily 1.5 mg/kg oral dose of OTA dissolved in an isotonic solution of sodium bicarbonate for 17 weeks. Crude alcoholic extracts of AR (100 mg/kg), WS (100 mg/ kg), PK (100 mg/kg) and TC (100 mg/kg) prepared in distilled water were administered orally to mice, once daily for 17 weeks. Mice were sacrificed 17 weeks after the completion of treatment and their peritoneal macrophages were collected. Control group received distilled water (l0 ml/kg) orally for 17 weeks.
2. Materials and methods
2.4. Phytochemical screening 2.1. Animals Inbred male albino mice (4 weeks old) of Hindustan Antibiotics (HA) strain were used. Mice were housed at 249 1°C and 10 h light: 14 h dark cycle was maintained throughout the experimental period. All the animals were given HA diet and water ad libitum. Each group consisted of six animals.
The protocol followed for phytochemical screening was that described by Chhabra et al. (1984). Each powdered plant material (15 g) was extracted by heating on a water bath for 15 min with (3× 50 ml) methanol. The extract was concentrated under reduced pressure and chemical reactions were performed with each extract for the detection of different constituents using thin layer chromatography (Wagner et al., 1984).
2.2. Chemicals and plant material Ochratoxin A, RPMI 1640 medium, fetal calf serum, guinea pig serum, lipopolysaccharide (LPS) were purchased from Sigma (St. Louis, MO). Authenticated plant material was supplied by Ayurvedic Rasashala, Pune. Voucher specimens of samples were retained (A. racemosus, ARS 1:8 97; W. somnifera, ARS 23: 97; T. cordifolia, ARS 20: 97 and P. kurrooa, ARS 16: 97) and have been deposited at Ayurvedic Rasashala, Pune. Plant materials (as mentioned above) were finely
2.5. Macrophage culture and determination of macrophage chemotaxis Macrophages were obtained from the peritoneal cavities of mice treated with Indian herbs and/or OTA according to the method of Tasaka et al. (1985) and washed with RPMI 1649 medium supplemented with 10% fetal calf serum, 50 U/ml of penicillin G and 50 mg/ml of streptomycin (HA Ltd, Pimpri, Pune). Macrophages were centrifuged at 500×g for 5 min at 4°C and the resulting cell pellet was resuspended in the culture
J.N. Dhuley / Journal of Ethnopharmacology 58 (1997) 15–20
medium. Chemotactic activity was determined according to the Boyden chamber method (Dejana et al., 1985). In brief, peritoneal macrophages (200 m1, 2 ×105 cells/ml) were loaded into the upper chamber and 200 ml of zymosan-activated guinea pig serum used as the chemotactic factor, was loaded into the lower chamber by a millipore filter 0.5 mm (Sigma, St. Louis, MO). After 90 min incubation, the millipore filter was stained with Giemsa reagent and the number of macrophages that had migrated onto the filter was counted with a light microscope. The number of macrophages was counted in five fields per mouse.
2.6. Preparation of the macrophage culture supernatant Macrophages obtained from mice treated with OTA and/or Indian herbs were cultured in 3 ml of the culture medium in a concentration of 2 × 105 cells/ml and allowed to adhere to the plastic surface of culture dishes for 6 h. The adherent macrophages were stimulated with an appropriate concentration (25 mg/ml) of LPS from Escherichia coli. The culture supernatant of the stimulated macrophages was collected with a sterilized filter (pore size 0.45 mm) after 48 h and stored at 70°C for further use. The IL-1 concentration in the sample was measured by an indirect enzyme-linked immunosorbent assay using a mouse interleukin-la ELISA kit (Intertest-la XTM, Genzyme, Cambridge, MA). The TNF-a concentration in the samples was measured by an enzyme-linked immunosorbent assay using a mouse tumor necrosis factor-a ELISA kit. (Factor-Test mTNF-a, Genzyme).
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3. Results
3.1. Chemotactic acti6ity of peritoneal macrophages The chemotactic activity of macrophages estimated by the number of migrated macrophages per field, was significantly decreased after 17 weeks treatment with OTA when compared with normal controls. Treatment with AR, WS, TC and PK significantly inhibited OTA-induced suppression of chemotactic activity (Fig. 1).
3.2. IL-a production by macrophages IL-a production in OTA-treated macrophages was significantly suppressed at week 17 when compared with normal macrophages. Treatment with AR, WS, TC and PK significantly inhibited OTA-induced suppression of IL-la production (Fig. 2).
3.3. TNF-a production by macrophages TNF-a production in OTA-treated macrophages was significantly lowered as compared to normal control mice. Treatment with AR, WS, TC and PK significantly enhanced the TNF-a
2.7. Statistical analysis Control and treatment groups consisted of six mice. Results are presented as the mean9 S.E. Data were analysed by the use of Student’s twotailed t-test. A P value of less than 0.05 was considered to be statistically significant.
Fig. 1. Chemotactic activity of peritoneal macrophage. AR, Asparagus racemosus; WS, Withania somnifera; TC, Tinospora cordifolia; PK, Picrorhiza kurrooa; OTA, Ochratoxin A. Each value represents mean 9S.E. * PB0.01 when compared with control group; ** PB 0.005 and *** PB 0.02 when compared with OTA-treated group.
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J.N. Dhuley / Journal of Ethnopharmacology 58 (1997) 15–20 Table 1 Results of phytochemical screening
Fig. 2. IL-1a production by macrophages. AR, Asparagus racemosus; WS, Withania somnifera; TC, Tinospora cordifolia; PK, Picrorhiza kurrooa; OTA, Ochratoxin A. Each value represents mean 9 S.E. * PB0.001 when compared with control group; ** P B0.005 and *** PB 0.002 when compared with OTA-treated group.
levels compared to noraml control macrophages. Moreover, the production of TNF-a was much higher in OTA + AR and OTA + WS treated animals than in normal control macrophages (Fig. 3).
3.4. Phytochemical screening Results of the phytochemical screening are shown in Table 1. Of the compounds mentioned
Fig. 3. TNF-a production by macrophages. AR, Asparagus racemosus; WS, Withania somnifera; TC, Tinospora cordifolia; PK, Picrorhiza kurrooa;. OTA, Ochratoxin A. Each value represents mean 9 S.E. * P B0.001 when compared with control group; ** PB 0.005 and *** PB 0.002 when compared with OTA-treated group.
Class of compounds
AR
WS
TC
PK
Alkaloids Anthocyanins Fatty acids Coumarins Glycosides Saponins Steroids Tannins Volalite oils Flavonoids
− − − − ++ +++ − − − −
+++ − − − − − ++ − − −
+++ − − − − − ++ − − −
− − − − +++ + − − − −
AR, Asparagus racemosus; WS, Withania somnifera; TC, Tinospora cordifolia; PK, Picrorhiza kurrooa.
in the table, alkaloids, saponins, glycosides, steroidal lactones frequently have pharmacological activities.
4. Discussion Macrophages play an important role in nonspecific and specific immune responses. In innate immunity, the phagocytosis of foreign materials by macrophages and other phagocytes facilitates the effector function of these cells in homeostasis, host defence and inflammation. In acquired immunity, macrophages and other phagocytes contribute to regulation of both humoral and cellular immune responses. Macrophages recognise and process foreign materials and then present them to B- and T-lymphocytes. Together with cytotoxic T-lymphocytes and natural killer cells, macrophages served as effector cells to provide immune surveillance against tumor cells. In addition to these cell-to-cell interactions, macrophages also influence the immune system by secretion of cytokines in both an autocrine and paracrine manner to protect the host against infectious agents, tumor cells and toxic agents as well as to modulate the behaviour of cells in the environment of triggered cells. The alcoholic extract of AR contains four saponins, viz. shatavarin I–IV, which are known to possess anti-oxytocic, lactogenic and anti-
J.N. Dhuley / Journal of Ethnopharmacology 58 (1997) 15–20
diarrheal activity (Wealth of India, 1985) and have an inhibitory effect on the growth of Entamoeba histolytica in vitro (Agarwaal, 1996). In the present study, AR increased significantly, not only chemotaxis, but also IL-1 and TNF-a production in macrophages. TNF production, in particular, was higher in AR-treated macrophages than in controls, though not significantly. WS has been used as relaxant, anti-spasmodic, anti-ulcerative and in arthopathies. A number of withanolides have so far been isolated from this plant and reported to possess both immunosuppression and immunostimulatory properties in different sets of studies (Buddhiraja and Sudhir, 1987; Ziauddin et al., 1996) Withaferin-A has been found to inhibit the growth of Ehrlich ascites carcinoma in mice within 24 h followed by complete disappearance of tumor cells after 2 – 4 days of treatment by activating immune responses (Shohat and Joshua, 1971; Funger, 1973; Shohat et al., 1978). In the present study, WS increased significanly, not only chemotakis but also IL-1 and TNF-a production in macrophage. TNF-a and chemotactic activity in particular was higher in WS-treated macrophage than in controls, though not significantly. TC has been used as a tonic and also as hepatoprotective agent (Rege et al., 1984; Singh et al., 1984). It contains the glucosides and gilosterol which are claimed to possess potent immunomodulatory activity (Bhattacharya et al., 1991) and have been shown to produce significant leucocytosis and predominant neutrophilia in animal models (Thatte et al., 1988). In the present study TC significantly increased not only chemotaxis but also IL-1 and TNF-a production in macrophages. PK has been used as a tonic and also as laxative, anti-periodic and hepatoprotective agent (Dwivedi et al., 1991; Visen et al., 1993). It contains the glucoside picrorhizin which has been shown to possess immunostimulant activity (Patwardhan et al., 1991). In the present study, PK significantly increased not only chemotaxis but also IL-1 and TNF-a production in macrophages. The basic principle in traditional Indian medicine, Ayurveda, is to give greater priority to the life and health of the patient rather than to make selective attack on target region involved in
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an ailment (Singh, 1996). It uses non-pharmacological methods of cure profusely and utilises remedies essentially drawn from nature and hence is considered to be both eco-and biofriendly. Using an in 6i6o macrophage model, it is shown that AR, WS, TC and PK stimulate chemotaxis and modulate cytokine production in macrophages obtained from mice with OTA-induced immunosuppression.
References Agarwaal, K.K., 1996. Herbal insight: Asparagus racemosus. Indian Journal of Clinical Practice: Medinews 10:28. Bhattacharya, S.K., Maity, P.C., Mediratta, P., Sen, P., 1991. Modulation of humoral immume responses by Tinospora malabarica in experimental animals. Indian Journal of Experimental Biology 29, 971 – 972. Buddhiraja, R.D., Sudhir, S., 1987. Review of biological activity of withanolides. Journal of Scientific and Industrial Research 46, 488 – 491. Castegnaro, M., Bartsch, H., Chernozemsky, I., 1987. Endemic nephropathy and urinary tract tumors in the Balkans. Cancer Research 47, 3608 – 3609. Chhabra, S.C., Uiso, F.C., Mshiu, E.N., 1984. Phytochemical screening of Tanzanian plants. Journal of Ethnopharmacology 11, 159 – 179. Dejana, E., Languino, L.R., Polentarutti, N., Balconi, G., Ryckewaert, J.J., Larrieu, M.J., Donati, M.B., Mantovoni, A., Margurie, G., 1985. Interaction between fibrinogen and cultured endothelical cells. Induction of migrationand specific binding. Journal of Clinical Investigations 75, 11 – 18. Dwivedi, Y., Rastogi, R., Sharma, S.K., Garg, N.K., Dhawan, E.N., 1991. Picroliv affords protection against thioacetamide induced hepatic damage in rats. Planta Medica 23, 143 – 145. Funger, V.A., 1973. Hemmung immunologisch bedingter Entziindungen durch das pflanzensteroid Withaferin A. Arzeimittel Forschung 23, 932 – 935. Huff, J.E., 1991. Carcinogenicity of ochratoxin A in experimental animals. In: Castegnaro, M., Plestina, R., Dirheimer, G., Chernozemsky, I.N., Bartsch, H. (Eds.), Mycotoxins, Endemic Nephropathy and Urinary Tract Tumors. International Agency for Research in Cancer, Lyon, France, pp. 229 – 244. IPCS, 1990. Selected mycotoxins: Ochratoxins, trichothecens, ergot. Environmental Health Criteria 105, 27 – 70. Kane, A., Creppy, E.E., Koth, R., Roschenthaler, R., Dirheimer, G., 1986. Distribution of (3H)-label from low doses of radioactive ochratoxin A ingested in rats, and evidence for single-strand breaks caused in liver and kidneys. Archives of Toxicology 58, 219 – 224. Marquardt, R.R., Frohlich, A.A., 1992. A review of recent advances in understanding ochratoxicosis. Journal of Animal Science 70, 2968 – 3988.
J.N. Dhuley / Journal of Ethnopharmacology 58 (1997) 15–20
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Patwardhan, B., Kalbag, D., Patki, P.S., Nagsampagi, B.A., 1991. Search of immunomodulatory agents—a review. Indian Drugs 28, 249 – 254. Pfohl-Leskowicz, A., Grosse, Y., Kane, A., Creppy, E.E., Dirheimer, G., 1993. Differential DNA adduct formation and disappearance in three mouse tissues after treatment with the mycotoxin ochratoxin A. Mutation Research 289, 265 – 273. Rege, N., Dahanukar, S., Karandikar, S.M., 1984. Hepatoprotective effects of Tinospora cordifolia against carbon tetrachloride induced liver damage. Indian Drugs 21, 1–12. Samhita, C., 1949. Ayurvedic Society, (translated by Shree Gulabkunverba), Jamnagar, India. Shohat, B., Joshua, H., 1971. Effect of withaferin A on Ehrlich ascites tumor cells. II. Target tumor cell distruction in vivo by immune activation. International Journal of Cancer 8, 487 – 496. Shohat, B., Kirson, I., Lavie, D., 1978. Immunosuppression activity of two plant steroidal lactones, withaferin A and withanolide E. Biomedicine 28, 18–24. Singh, B., Sharma, M.L., Grupta, D.K., Atal, C.K., Arya, R.K., 1984. Protective effect of Tinospara cordifolia on carbon tetrachloride induced hepatotoxicity. Indian Journal of Pharmacology 16, 139–142. Singh, G.B., Atal, C.K., 1986. Pharmacology of an extract of salai guggal ex-Boswellia serrata, new non-steroidal antiinflammotory agent. Agents and Actions 8, 407–412.
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Singh, R.H., 1996. Nutritional dynamics of drug action in Ayurveda: A fundamentally new approach. Indian Journal of Clinical Practice: Medinews 7, 18. Tasaka, K., Nakayama, Y., Hasegawa, M., Kurokawa, K., 1985. A new method of eliciting delayed hypersensitivity in the mouse abdominal cavity. Journal of Immunological Methods 77, 331 – 341. Thatte, U.M., Chabria, S., Karandikar, S.M., Dahanukar, S., 1988. Immunotherapentic modification by Indian medical plants. Indian Drugs 25, 95 – 97. Thatte, U.M., 1996. Ayurveda: An important source of medicine. Indian Journal of Clinical Practice: Medinews 5, 18. Visen, P.K.S., Shukla, B., Patnaik, G.K., Kaul, A., Kapoor, N.K., Dhawan, B.N., 1993. Hepatoprotective activity of picroliv, the active principle of Picrorhiza kurrooa, on rat hepatocytes against paracetamol foxicity. Planta Medica 59, 37 – 39. Wagner, H., Bladt, S., Zgainski, E.M., 1984. Plant Analysis — A Thin Layer Chromatography Atlas, 2nd edn. SpringerVerlag, New York, pp. 291 – 295. Wealth of India, 1985. Raw Materials, vol. 470 – 472. Ziauddin, M., Phansalkar, N., Patki, P., Diwanay, S., Patwardhan, B., 1996. Studies on the immunomodulatory effects of Ashwagandha. Journal of Ethnopharmacology 50, 69 – 76.
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Effect of some Indian herbs on macrophage functions in ochratoxin A treated mice J.N. Dhuley Department of Pharmacology and Toxicology, Research Centre, Hindustan Antibiotics Limited, Pimpri, Pune-411018, India Received 27 January 1997; received in revised form 23 June 1997; accepted 25 June 1997
Abstract The effect of Indian herbs namely, Asparagus racemosus, Tinospora cordifolia, Withania somnifera and Picrorhiza kurrooa on the functions of macrophages obtained from mice treated with the carcinogen ochratoxin A (OTA) was investigated. The chemotactic activity of murine macrophages was significantly decreased by 17 weeks of treatment with OTA compared with controls. Production of interleukin-1 (IL-1) and tumor necrosis factor (TNF) was also markedly reduced. Treatment with Asparagus racemosus, Tinospora cordifolia, Withania somnifera and Picrorhiza kurrooa significantly inhibited OTA-induced suppression of chemotactic activity and production of IL-1 and TNF-a by macropahges. Moreover, we found that Withania somnifera treated macrophage chemotaxis and that Asparagus racemosus induced excess production of TNF-a when compared with controls. © 1997 Elsevier Science Ireland Ltd. Keywords: Indian herbs; Carcinogen (ochratoxin A); Macrophage; Cytokine production
1. Introduction From antiquity to modern times, man has been obsessed with a quest for rejuvenation and an innate desire to extend his lifespan. Ayurveda is an original holistic system of diagnosis and treatment involving nutrition, hygiene and rejuvenation, developed and perfected in India over 5000 years ago. Ayurveda (Ayur, life; Veda, knowledge), is the knowledge of healthy living and is not merely confined to the treatment of illness. The modulation of immune response by using
various agents in order to alleviate the disease has been of interest over many years and the concept of ‘Rasayan’ in Ayurveda has similarity with it (Samhita, 1949). Some Indian herbs have been shown to exert anti-inflammatory (Singh and Atal, 1986), anti-stress and anti-cancer (Thatte, 1996) effects by modulating the immune system. Asparagus racemosus (AR), Withania somnifera (WS), Tinospora cordifolia (TC) and Picrorhiza kurrooa (PK) have been found to enhance host resistance and reduce the side effects of other toxic agents.
0378-8741/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 3 7 8 - 8 7 4 1 ( 9 7 ) 0 0 0 7 2 - X
16
J.N. Dhuley / Journal of Ethnopharmacology 58 (1997) 15–20
Ochratoxin A (OTA) is a mycotoxin consisting of a dihydroisocoumarin moiety linked through its carboxyl group to L-b-phenylalanine. It has been found as a frequent contaminant in various food and feed commodities including cereals, grains and to a lesser extent coffee, milk and foods from animal origin (IPCS, 1990). It is a well-known tumor promoter that contributes to cancer of the kidney, liver and mammary gland (Kane et al., 1986; Castegnaro et al., 1987; Huff, 1991; Marquardt and Frohlich, 1992; PfohlLeskowicz et al., 1993). In the present study, an attempt has been made to investigate the effects of Asparagus racemosus Roxb. (Liliaceae, root), Withania somnifera L. (Solanaceae, root and leaves), Tinospora cordifolia Willd. (Menispermaceae, whole plant) and Picrorhiza kurrooa Royle (Scrophulariaceae, rhizome) on OTA-induced suppression of chemotaxis and cytokine production in murine macrophages.
ground. The material was then extracted with 80% ethanol (3 g/20 ml) at room temperature. After 3 h, the macerate was filtered on Whatman no. 1 paper and the alcoholic extracts dried at 40°C for 1 week. The dried material was maintained in the dark in a cool and dry place. Before using, it was dissolved in water (10% w/v final concentration), filter-sterilized and maintained at 5°C.
2.3. Treatment Six week-old mice received a daily 1.5 mg/kg oral dose of OTA dissolved in an isotonic solution of sodium bicarbonate for 17 weeks. Crude alcoholic extracts of AR (100 mg/kg), WS (100 mg/ kg), PK (100 mg/kg) and TC (100 mg/kg) prepared in distilled water were administered orally to mice, once daily for 17 weeks. Mice were sacrificed 17 weeks after the completion of treatment and their peritoneal macrophages were collected. Control group received distilled water (l0 ml/kg) orally for 17 weeks.
2. Materials and methods
2.4. Phytochemical screening 2.1. Animals Inbred male albino mice (4 weeks old) of Hindustan Antibiotics (HA) strain were used. Mice were housed at 249 1°C and 10 h light: 14 h dark cycle was maintained throughout the experimental period. All the animals were given HA diet and water ad libitum. Each group consisted of six animals.
The protocol followed for phytochemical screening was that described by Chhabra et al. (1984). Each powdered plant material (15 g) was extracted by heating on a water bath for 15 min with (3× 50 ml) methanol. The extract was concentrated under reduced pressure and chemical reactions were performed with each extract for the detection of different constituents using thin layer chromatography (Wagner et al., 1984).
2.2. Chemicals and plant material Ochratoxin A, RPMI 1640 medium, fetal calf serum, guinea pig serum, lipopolysaccharide (LPS) were purchased from Sigma (St. Louis, MO). Authenticated plant material was supplied by Ayurvedic Rasashala, Pune. Voucher specimens of samples were retained (A. racemosus, ARS 1:8 97; W. somnifera, ARS 23: 97; T. cordifolia, ARS 20: 97 and P. kurrooa, ARS 16: 97) and have been deposited at Ayurvedic Rasashala, Pune. Plant materials (as mentioned above) were finely
2.5. Macrophage culture and determination of macrophage chemotaxis Macrophages were obtained from the peritoneal cavities of mice treated with Indian herbs and/or OTA according to the method of Tasaka et al. (1985) and washed with RPMI 1649 medium supplemented with 10% fetal calf serum, 50 U/ml of penicillin G and 50 mg/ml of streptomycin (HA Ltd, Pimpri, Pune). Macrophages were centrifuged at 500×g for 5 min at 4°C and the resulting cell pellet was resuspended in the culture
J.N. Dhuley / Journal of Ethnopharmacology 58 (1997) 15–20
medium. Chemotactic activity was determined according to the Boyden chamber method (Dejana et al., 1985). In brief, peritoneal macrophages (200 m1, 2 ×105 cells/ml) were loaded into the upper chamber and 200 ml of zymosan-activated guinea pig serum used as the chemotactic factor, was loaded into the lower chamber by a millipore filter 0.5 mm (Sigma, St. Louis, MO). After 90 min incubation, the millipore filter was stained with Giemsa reagent and the number of macrophages that had migrated onto the filter was counted with a light microscope. The number of macrophages was counted in five fields per mouse.
2.6. Preparation of the macrophage culture supernatant Macrophages obtained from mice treated with OTA and/or Indian herbs were cultured in 3 ml of the culture medium in a concentration of 2 × 105 cells/ml and allowed to adhere to the plastic surface of culture dishes for 6 h. The adherent macrophages were stimulated with an appropriate concentration (25 mg/ml) of LPS from Escherichia coli. The culture supernatant of the stimulated macrophages was collected with a sterilized filter (pore size 0.45 mm) after 48 h and stored at 70°C for further use. The IL-1 concentration in the sample was measured by an indirect enzyme-linked immunosorbent assay using a mouse interleukin-la ELISA kit (Intertest-la XTM, Genzyme, Cambridge, MA). The TNF-a concentration in the samples was measured by an enzyme-linked immunosorbent assay using a mouse tumor necrosis factor-a ELISA kit. (Factor-Test mTNF-a, Genzyme).
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3. Results
3.1. Chemotactic acti6ity of peritoneal macrophages The chemotactic activity of macrophages estimated by the number of migrated macrophages per field, was significantly decreased after 17 weeks treatment with OTA when compared with normal controls. Treatment with AR, WS, TC and PK significantly inhibited OTA-induced suppression of chemotactic activity (Fig. 1).
3.2. IL-a production by macrophages IL-a production in OTA-treated macrophages was significantly suppressed at week 17 when compared with normal macrophages. Treatment with AR, WS, TC and PK significantly inhibited OTA-induced suppression of IL-la production (Fig. 2).
3.3. TNF-a production by macrophages TNF-a production in OTA-treated macrophages was significantly lowered as compared to normal control mice. Treatment with AR, WS, TC and PK significantly enhanced the TNF-a
2.7. Statistical analysis Control and treatment groups consisted of six mice. Results are presented as the mean9 S.E. Data were analysed by the use of Student’s twotailed t-test. A P value of less than 0.05 was considered to be statistically significant.
Fig. 1. Chemotactic activity of peritoneal macrophage. AR, Asparagus racemosus; WS, Withania somnifera; TC, Tinospora cordifolia; PK, Picrorhiza kurrooa; OTA, Ochratoxin A. Each value represents mean 9S.E. * PB0.01 when compared with control group; ** PB 0.005 and *** PB 0.02 when compared with OTA-treated group.
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J.N. Dhuley / Journal of Ethnopharmacology 58 (1997) 15–20 Table 1 Results of phytochemical screening
Fig. 2. IL-1a production by macrophages. AR, Asparagus racemosus; WS, Withania somnifera; TC, Tinospora cordifolia; PK, Picrorhiza kurrooa; OTA, Ochratoxin A. Each value represents mean 9 S.E. * PB0.001 when compared with control group; ** P B0.005 and *** PB 0.002 when compared with OTA-treated group.
levels compared to noraml control macrophages. Moreover, the production of TNF-a was much higher in OTA + AR and OTA + WS treated animals than in normal control macrophages (Fig. 3).
3.4. Phytochemical screening Results of the phytochemical screening are shown in Table 1. Of the compounds mentioned
Fig. 3. TNF-a production by macrophages. AR, Asparagus racemosus; WS, Withania somnifera; TC, Tinospora cordifolia; PK, Picrorhiza kurrooa;. OTA, Ochratoxin A. Each value represents mean 9 S.E. * P B0.001 when compared with control group; ** PB 0.005 and *** PB 0.002 when compared with OTA-treated group.
Class of compounds
AR
WS
TC
PK
Alkaloids Anthocyanins Fatty acids Coumarins Glycosides Saponins Steroids Tannins Volalite oils Flavonoids
− − − − ++ +++ − − − −
+++ − − − − − ++ − − −
+++ − − − − − ++ − − −
− − − − +++ + − − − −
AR, Asparagus racemosus; WS, Withania somnifera; TC, Tinospora cordifolia; PK, Picrorhiza kurrooa.
in the table, alkaloids, saponins, glycosides, steroidal lactones frequently have pharmacological activities.
4. Discussion Macrophages play an important role in nonspecific and specific immune responses. In innate immunity, the phagocytosis of foreign materials by macrophages and other phagocytes facilitates the effector function of these cells in homeostasis, host defence and inflammation. In acquired immunity, macrophages and other phagocytes contribute to regulation of both humoral and cellular immune responses. Macrophages recognise and process foreign materials and then present them to B- and T-lymphocytes. Together with cytotoxic T-lymphocytes and natural killer cells, macrophages served as effector cells to provide immune surveillance against tumor cells. In addition to these cell-to-cell interactions, macrophages also influence the immune system by secretion of cytokines in both an autocrine and paracrine manner to protect the host against infectious agents, tumor cells and toxic agents as well as to modulate the behaviour of cells in the environment of triggered cells. The alcoholic extract of AR contains four saponins, viz. shatavarin I–IV, which are known to possess anti-oxytocic, lactogenic and anti-
J.N. Dhuley / Journal of Ethnopharmacology 58 (1997) 15–20
diarrheal activity (Wealth of India, 1985) and have an inhibitory effect on the growth of Entamoeba histolytica in vitro (Agarwaal, 1996). In the present study, AR increased significantly, not only chemotaxis, but also IL-1 and TNF-a production in macrophages. TNF production, in particular, was higher in AR-treated macrophages than in controls, though not significantly. WS has been used as relaxant, anti-spasmodic, anti-ulcerative and in arthopathies. A number of withanolides have so far been isolated from this plant and reported to possess both immunosuppression and immunostimulatory properties in different sets of studies (Buddhiraja and Sudhir, 1987; Ziauddin et al., 1996) Withaferin-A has been found to inhibit the growth of Ehrlich ascites carcinoma in mice within 24 h followed by complete disappearance of tumor cells after 2 – 4 days of treatment by activating immune responses (Shohat and Joshua, 1971; Funger, 1973; Shohat et al., 1978). In the present study, WS increased significanly, not only chemotakis but also IL-1 and TNF-a production in macrophage. TNF-a and chemotactic activity in particular was higher in WS-treated macrophage than in controls, though not significantly. TC has been used as a tonic and also as hepatoprotective agent (Rege et al., 1984; Singh et al., 1984). It contains the glucosides and gilosterol which are claimed to possess potent immunomodulatory activity (Bhattacharya et al., 1991) and have been shown to produce significant leucocytosis and predominant neutrophilia in animal models (Thatte et al., 1988). In the present study TC significantly increased not only chemotaxis but also IL-1 and TNF-a production in macrophages. PK has been used as a tonic and also as laxative, anti-periodic and hepatoprotective agent (Dwivedi et al., 1991; Visen et al., 1993). It contains the glucoside picrorhizin which has been shown to possess immunostimulant activity (Patwardhan et al., 1991). In the present study, PK significantly increased not only chemotaxis but also IL-1 and TNF-a production in macrophages. The basic principle in traditional Indian medicine, Ayurveda, is to give greater priority to the life and health of the patient rather than to make selective attack on target region involved in
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an ailment (Singh, 1996). It uses non-pharmacological methods of cure profusely and utilises remedies essentially drawn from nature and hence is considered to be both eco-and biofriendly. Using an in 6i6o macrophage model, it is shown that AR, WS, TC and PK stimulate chemotaxis and modulate cytokine production in macrophages obtained from mice with OTA-induced immunosuppression.
References Agarwaal, K.K., 1996. Herbal insight: Asparagus racemosus. Indian Journal of Clinical Practice: Medinews 10:28. Bhattacharya, S.K., Maity, P.C., Mediratta, P., Sen, P., 1991. Modulation of humoral immume responses by Tinospora malabarica in experimental animals. Indian Journal of Experimental Biology 29, 971 – 972. Buddhiraja, R.D., Sudhir, S., 1987. Review of biological activity of withanolides. Journal of Scientific and Industrial Research 46, 488 – 491. Castegnaro, M., Bartsch, H., Chernozemsky, I., 1987. Endemic nephropathy and urinary tract tumors in the Balkans. Cancer Research 47, 3608 – 3609. Chhabra, S.C., Uiso, F.C., Mshiu, E.N., 1984. Phytochemical screening of Tanzanian plants. Journal of Ethnopharmacology 11, 159 – 179. Dejana, E., Languino, L.R., Polentarutti, N., Balconi, G., Ryckewaert, J.J., Larrieu, M.J., Donati, M.B., Mantovoni, A., Margurie, G., 1985. Interaction between fibrinogen and cultured endothelical cells. Induction of migrationand specific binding. Journal of Clinical Investigations 75, 11 – 18. Dwivedi, Y., Rastogi, R., Sharma, S.K., Garg, N.K., Dhawan, E.N., 1991. Picroliv affords protection against thioacetamide induced hepatic damage in rats. Planta Medica 23, 143 – 145. Funger, V.A., 1973. Hemmung immunologisch bedingter Entziindungen durch das pflanzensteroid Withaferin A. Arzeimittel Forschung 23, 932 – 935. Huff, J.E., 1991. Carcinogenicity of ochratoxin A in experimental animals. In: Castegnaro, M., Plestina, R., Dirheimer, G., Chernozemsky, I.N., Bartsch, H. (Eds.), Mycotoxins, Endemic Nephropathy and Urinary Tract Tumors. International Agency for Research in Cancer, Lyon, France, pp. 229 – 244. IPCS, 1990. Selected mycotoxins: Ochratoxins, trichothecens, ergot. Environmental Health Criteria 105, 27 – 70. Kane, A., Creppy, E.E., Koth, R., Roschenthaler, R., Dirheimer, G., 1986. Distribution of (3H)-label from low doses of radioactive ochratoxin A ingested in rats, and evidence for single-strand breaks caused in liver and kidneys. Archives of Toxicology 58, 219 – 224. Marquardt, R.R., Frohlich, A.A., 1992. A review of recent advances in understanding ochratoxicosis. Journal of Animal Science 70, 2968 – 3988.
J.N. Dhuley / Journal of Ethnopharmacology 58 (1997) 15–20
20
Patwardhan, B., Kalbag, D., Patki, P.S., Nagsampagi, B.A., 1991. Search of immunomodulatory agents—a review. Indian Drugs 28, 249 – 254. Pfohl-Leskowicz, A., Grosse, Y., Kane, A., Creppy, E.E., Dirheimer, G., 1993. Differential DNA adduct formation and disappearance in three mouse tissues after treatment with the mycotoxin ochratoxin A. Mutation Research 289, 265 – 273. Rege, N., Dahanukar, S., Karandikar, S.M., 1984. Hepatoprotective effects of Tinospora cordifolia against carbon tetrachloride induced liver damage. Indian Drugs 21, 1–12. Samhita, C., 1949. Ayurvedic Society, (translated by Shree Gulabkunverba), Jamnagar, India. Shohat, B., Joshua, H., 1971. Effect of withaferin A on Ehrlich ascites tumor cells. II. Target tumor cell distruction in vivo by immune activation. International Journal of Cancer 8, 487 – 496. Shohat, B., Kirson, I., Lavie, D., 1978. Immunosuppression activity of two plant steroidal lactones, withaferin A and withanolide E. Biomedicine 28, 18–24. Singh, B., Sharma, M.L., Grupta, D.K., Atal, C.K., Arya, R.K., 1984. Protective effect of Tinospara cordifolia on carbon tetrachloride induced hepatotoxicity. Indian Journal of Pharmacology 16, 139–142. Singh, G.B., Atal, C.K., 1986. Pharmacology of an extract of salai guggal ex-Boswellia serrata, new non-steroidal antiinflammotory agent. Agents and Actions 8, 407–412.
.
Singh, R.H., 1996. Nutritional dynamics of drug action in Ayurveda: A fundamentally new approach. Indian Journal of Clinical Practice: Medinews 7, 18. Tasaka, K., Nakayama, Y., Hasegawa, M., Kurokawa, K., 1985. A new method of eliciting delayed hypersensitivity in the mouse abdominal cavity. Journal of Immunological Methods 77, 331 – 341. Thatte, U.M., Chabria, S., Karandikar, S.M., Dahanukar, S., 1988. Immunotherapentic modification by Indian medical plants. Indian Drugs 25, 95 – 97. Thatte, U.M., 1996. Ayurveda: An important source of medicine. Indian Journal of Clinical Practice: Medinews 5, 18. Visen, P.K.S., Shukla, B., Patnaik, G.K., Kaul, A., Kapoor, N.K., Dhawan, B.N., 1993. Hepatoprotective activity of picroliv, the active principle of Picrorhiza kurrooa, on rat hepatocytes against paracetamol foxicity. Planta Medica 59, 37 – 39. Wagner, H., Bladt, S., Zgainski, E.M., 1984. Plant Analysis — A Thin Layer Chromatography Atlas, 2nd edn. SpringerVerlag, New York, pp. 291 – 295. Wealth of India, 1985. Raw Materials, vol. 470 – 472. Ziauddin, M., Phansalkar, N., Patki, P., Diwanay, S., Patwardhan, B., 1996. Studies on the immunomodulatory effects of Ashwagandha. Journal of Ethnopharmacology 50, 69 – 76.
.