Picrorhiza scrophulariiflora, from traditional use to immunomodulatory activity

Abstract

Natural products have been an important resource for the maintenance of life for ages. Evaluation of traditionally used medicines, keeping into account the traditional principles that are applied in drug therapy, may supply leads towards effective drug discovery. This thesis deals with the pre-clinical evaluation of the immunomodulatory activity of Picrorhiza scrophulariiflora and the activity-guided isolation of active constituents, in order to assess its usefulness in rheumatoid arthritis or related conditions. Rhizomes of two Picrorhiza species happen to be available on South-Asian markets, e.g. P. kurrooa Royle and P. scrophulariiflora Pennell (Scrophulariaceae). Both species are known under the same local name, and used for similar purposes. However, an inventory of the geographical distribution of both Picrorhiza species provided sufficient information to establish the proper identity of the material under investigation as P. scrophulariiflora. Picrorhiza species are commonly used in Ayurveda, the traditional medicine of South Asia. The applicability of a plant for the treatment of a particular disease can be derived from generalized traditional principles, or obtained directly from traditional uses. Both approaches suggest that Picrorhiza might be useful in the prevention of amavata (rheumatoid arthritis). For the treatment of established amavata, however, it is not recommended, because it might enhance the joint damage. Review of the experimental literature on Picrorhiza species revealed the accumulation of cucurbitacin glucosides, iridoid glucosides, phenylethanoid glucosides, and acetophenone glucosides in the rhizomes of Picrorhiza species. These compounds have a wide range of biological activities, among which anti-inflammatory effects seem to prevail. The effects described relate to a protection of the liver against experimentally induced damage, the inhibition of pro-inflammatory responses, the interference with the formation of reactive oxygen species, and the enhancement of the physiological antioxidant system. The second part of the thesis describes experimental work on Soxhlet extracts of P. scrophulariiflora. The diethyl-ether Soxhlet extract was the most anti-inflammatory of all extracts prepared, concerning both nonspecific (complement and respiratory burst) and specific (T-cell proliferation) immune responses. Furthermore, prolonged administration of this extract showed inhibition of carrageenan-induced paw edema in mice. This effect is in line with the effects observed in vitro for this extract. Moderate inhibitory activity on the release of reactive oxygen species by activated neutrophils in vitro, was mainly attributed to apocynin. However, this compound did not show any anti-inflammatory effect in the acute inflammation model, suggesting that compounds other than apocynin are responsible for the anti-inflammatory effects observed. Collagen-induced arthritis in mice was used as a model to determine the effects of P. scrophulariiflora extracts on chronic inflammation. Treatment with the diethyl-ether extract or with apocynin reduced the incidence of arthritis, while other extracts tested were not active and none of the extracts affected the severity of arthritis. These results suggest that the inhibitory activity of the diethyl-ether extract can be attributed to apocynin. The in vivo effects were not associated with significant toxicity, as tested in mice by a semi-chronic assay involving body weight and weights of critical lymphoid organs. Activity-guided purification of the diethyl-ether extract revealed the presence of several compounds in the light petroleum and the diethyl ether extracts, which potently inhibit the classical pathway of complement. Part of these compounds might be phytosterols, while the nature of other active compounds has not yet been demonstrated. Guided by inhibitory activity in a mitogen-induced human T-lymphocyte proliferation assay, two cucurbitacins were isolated from the diethyl-ether extract: picracin and deacetylpicracin. Both compounds exhibited a potent inhibition of PHA-induced T-cell proliferation, which could not be ascribed to direct cytotoxic effects. Furthermore, incubation of activated T cells with picracin or deacetylpicracin for 24 h showed a marked inhibition of IL-2 release. However, flow-cytometric analysis showed a significant induction of apoptosis by picracin, while deacetylpicracin was not different from untreated cells. In addition, incubation of human monocytes with picracin for 24 h inhibited the release of IL-1b and TNFa, while deacetylpicracin was not active. Delayed-type hypersensitivity, a valuable model to demonstrate cell-mediated immune responses, was used to determine the effects of picracin and deacetylpicracin in vivo. Both picracin and deacetylpicracin significantly inhibited paw edema, at doses of 100 mg/kg and 30 mg/kg, respectively, after intraperitoneal administration. These effects, however, could not be enhanced by local, subcutaneous administration. Instead, an increased swelling was observed, and control experiments revealed a concentration-dependent direct inflammatory response. These effects might be related to the induction of T-cell apoptosis in vivo. The intraperitoneal administration of high doses of picracin or deacetylpicracin might induce a strong influx of leukocytes secondary to T-cell apoptosis. Leukocytes might as well be withheld from the paw, leaving behind a reduced number of inflammatory cells to elicit a local immune response after subcutaneous injection of antigen. In conclusion, several compounds play a role in immunomodulatory effects induced by Picrorhiza extracts. Picracin and deacetylpicracin induce a local immune response upon local administration, while they tend to decrease a local inflammatory response after systemic (i.p.) administration. Although the latter results are promising with regard to the anti-inflammatory activity of Picrorhiza, it cannot be excluded that these compounds enhance local inflammation and for example aggravate rheumatoid arthritis. Testing of picracin and deacetylpicracin in experimentally induced arthritis should be performed in order to evaluate such effects. Both compounds are present in the diethyl-ether extract, which decreased the onset of arthritis. However, apocynin was shown to exhibit similar effects, and is present in this extract as well, suggesting that it might be responsible for the effects observed for the diethyl-ether extract in collagen-induced arthritis. Neither the diethyl-ether extract, nor apocynin decreased the severity of joint inflammation in the dosages used. These findings are in agreement with the contraindication of Picrorhiza for the treatment of rheumatoid arthritis, according to both contemporary use and to Ayurvedic principles. However, the experimental results as well as the conclusions drawn from the Ayurvedic properties of Picrorhiza suggest that it can play an important role in the prevention of rheumatoid arthritis.