Two new lanostane-type triterpenes, 1 and 2, were isolated from Astraeus hygrometricus. The structures were established by IR, (1)H- and (13)C-NMR, MS, and X-ray crystallographic experiments. The triterpenes exhibited excellent in vitro toxicities against Candida albicans, comparable to standard antifungal antibiotics. The triterpene 2 significantly inhibited the growth of Leishmania donovani promastigotes in vitro. The triterpene skeleton may be considered a template structure in search for new compounds with anticandidal and leishmanicidal activity.

We claim the present invention as substantial in depth evidences that mushroom derived active molecules can be exploited as target specific, comparatively nontoxic leads for antileishmanial therapy.

eIn our previous report, we showed that astrakurkurone, a triterpene isolated from the Indian mushroom Astraeus hygrometricus (Pers.) Morgan, induced reactive oxygen species, leading to apoptosis in Leishmania donovani promastigotes, and also was effective in inhibiting intracellular amastigotes at the 50% inhibitory concentration of 2.5 g/ml. The aim of the present study is to characterize the associated immunomodulatory potentials and cellular activation provided by astrakurkurone, leading to effective antileishmanial activity in vitro and in vivo. Astrakurkurone-mediated antileishmanial activity was evaluated by real-time PCR and flow cytometry. The involvement of Toll-like receptor 9 (TLR9) was studied by in vitro assay in the presence of a TLR9 agonist and antagonist and by in silico modeling of a three-dimensional structure of the ectodomain of TLR9 and its interaction with astrakurkurone. Astrakurkurone caused a significant increase in TLR9 expression of L. donovani-infected macrophages along with the activation of proinflammatory responses. The involvement of TLR9 in astrakurkurone-mediated amastigote killing has been evidenced from the fact that a TLR9 agonist (CpG, ODN 1826) in combination with astrakurkurone enhanced the amastigote killing, while a TLR9 antagonist (bafilomycin A1) alone or in combination with astrakurkurone curbed the amastigote killing, which could be further justified by in silico evidence of docking between mouse TLR9 and astrakurkurone. Astrakurkurone was found to reduce the parasite burden in vivo by inducing protective cytokines, gamma interferon and interleukin 17. Moreover, astrakurkurone was nontoxic toward peripheral blood mononuclear cells of immunocompromised patients with visceral leishmaniasis. Astrakurkurone, a nontoxic antileishmanial, enhances the immune efficiency of host cells, leading to parasite clearance in vitro and in vivo.

This study explored the efficacy of Fa fraction of Tricholoma giganteum against Ehrlich's ascites carcinoma (EAC). Mechanisms of apoptogenic effect of the fraction were delineated. The flow cytometric analysis of EAC cells, showed an increase in number of cells in sub-G(0)/G(1) population and reduction in the G(2)/M phase due to the treatment thus suggesting apoptosis. The induction of apoptosis has also been confirmed by nuclear staining that demonstrated distinctive morphological features of apoptosis. Our data also revealed an increase in the expression of pro-apoptotic protein p53 in EAC and induced factors contributing to apoptosis. Pro-apoptotic gene Bax was up-regulated during p53-mediated apoptosis. No significant change in the expression of anti-apoptotic protein Bcl-2 was observed ensuing in decrease of the Bcl-2/Bax ratio. p53-mediated growth arrest involves p21 as a major effecter, which interestingly showed moderate elevation. All these observations indicate that Fa fraction of T. giganteum induces apoptogenic signal in EAC.

In various forms of purified collagen (powder of insoluble collagen from bovine skin, fibers from rat tail tendons, membrane from bovine gut), carboxyl groups were activated by carbodiimide to allow covalent binding of heparin. Collagen powder and collagen fibers from rat tail tendons were also incubated in a heparin solution under the same reaction conditions but without carbodiimide present to account for other forms of collagen-heparin interaction. It was found that the linkage of heparin to collagen formed in the presence of carbodiimide is stable, as heparin was minimally extractable by 0.2M buffers with a pH ranging from 5 to 9. Collagen powder incubated with heparin in the absence of carbodiimide released heparin almost completely into Tris buffer of pH 9.0. As a consequence of covalent binding of heparin to collagen, the collagen fibers became more stable as shown by their significantly reduced swelling capacity and significantly increased shrinkage temperature. Collagen fibers interacted with heparin in the absence of carbodiimide also showed some stabilization of their structure, which was, however, significantly less than with carbodiimide reaction. By two independent methods it was shown that heparin linked to collagen by a stable bond retains its anticoagulant activity. It is concluded that, in the presence of carbodiimide, heparin covalently binds to collagen thus forming an antithrombogenic surface. At the same time, collagen is crosslinked. Incubation of collagen in the solution of heparin without carbodiimide also stabilizes collagen structure, but to a significantly lesser degree. Such a linkage is unstable as heparin dissociates and is readily extractable into 0.2M Tris buffers with pH 7-9.

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