Title of Invention	A NOVAL ANTIFUNGAL/ANTICANCER COMPOSITION
Abstract	The present invention relates to a novel antifungal/anticancer composition. More particularly, the present invention relates to methyl-ß-orcinol carboxylate of formula 1 isolated from a lichen (Everniastrum cirrhatum), for treating pathogenic fungal infections of humans that are resistant to polyene and azole antibiotics such as amphotericin B, nystatin, clotrimazole etc.

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Field of the invention The present invention relates to a novel antifungal/anticancer composition. More particularly, the present invention relates to methyl-ß-orcinol carboxylate of formula 1 isolated from a lichen (Everniastrum cirrhatum), for treating pathogenic fungal infections of humans that are resistant to polyene and azote antibiotics such as amphotericin B, nystatin, clotrimazole etc. (Figure Removed) Lichens are symbiotic associations between fungi, green algae and/or cyanobacteria. They have a varied chemistry and produce many polyketide-derived compounds, including some, such as depsides and depsidones that are rarely reported elsewhere. Depsides are a class of compounds, which appear to be unique to the lichens. These compounds are dimeric esters of variously substituted orsellinic acids and are the major source of the so-called lichen acids. Although lichens have been appreciated in traditional medicines, their value has largely been ignored by the modern pharmaceutical industry because difficulties in establishing axenic cultures and conditions for rapid growth preclude their routine use in most conventional screening processes. The association between fungi and algae is specific and selective. The name of the fungal component is given to the whole lichen and there are >13500 described species, including almost one-fifth of all known fungi (Hawcksworth and Hill, 1984; The Lichen Forming Fungi, Mecorquodale Ltd). Although the individual mycobionts and photobionts (the fungi and the photosynthetic algae or cyanobacteria, respectively) are small and nondescript if cultured in a laboratory dish, the symbiotic components together in nature present a full range of varied and beautiful forms, and some such as Ramalina menziesii (the 'fishnet' lichen) can drape entire trees, creating a prominent display (Arvis,W.O.,20QO; Lichens, Smithsonian Situation Press). They perform a variety of ecological roles such as colonizing marginal habitat in Antarctica, stabilizing soil in the semi-arid desert of Australia and contributing to nitrogen turnover in the northern pacific forests of North America. They produce characteristic secondary metabolites that are unique with respect to those of higher plants. Lichens produce a wide range of chemical compounds, among which approximately 350 secondary metabolites have been identified. These mycobiont derived products usually accumulate as extra cellular crystals on the cell walls of the symbionts, and account for up to 10% (in exceptional cases, up to 40%) of thallus dry mass (Galun, M. and Shomer-llan, A. (1988) in CRC Handbook of Lichenology, Vol. Ill; Galun, M., ed.), pp,3- 8.CRC Press); many are unique to lichens. Most lichen secondary compounds are formed by the polyketide pathway, while others derive from the shikimic acid and mevalonic acid pathways these are key routes for secondary metabolism in all organisms. Several lichen extracts have been used for various remedies in folk medicine, and screening test with lichens have indicated the frequent occurrence of metabolites with antibiotic, antimycobacterial, antiviral, analgesic, and antipyretic properties. Furthermore, a distinct class of lichen metabolites is the depsides. These types of compounds are formed by condensation of two or more hydroxybenzoic acids whereby the carboxyl group of one molecule is esterified with a phenolichydroxyl group of a second molecule. Owing to the phenolic nature of their chemical structures, these molecules are interesting candidates for evaluating their effects on leukotriene biosynthesis, as a major class of inhibitors often contains a hydroxylated aromatic ring (Fitzsimmons et al 1989). Moreover, two small-molecule lichen-derived metabolites, protolichesterinic acid and lobaric acid, have been reported to inhibit 5-LO from porcine leukocytes (Ogmundsdottir et al 1998). The latter has also been shown to inhibit peptide leukotriene formation (Gissurarson et al 1997). Lichen depsides have also been described to inhibit prostaglandin biosynthesis (Sankawa et al 1982). Lichens and lichen products have been used in traditional medicines for centuries and still hold considerable interest as alternative treatments in various parts of the world. Indeed, today a variety of lichen-based tonics, lotions and lozenges can be purchased in Iceland, where they're medicinal. However, lichens have been essentially ignored by the modern pharmaceutical industry, despite the fact that lichens produce a large number of low molecular weight molecules with diverse structures and that studies have provided evidence of biological activity in extracts from whole lichens (Table-1). There are two contributing reasons for this; (1) lichens are slow growing in nature and (2) they are difficult to propagate and resynthesize in culture (Ahmadjian , 1993; The Lichen Symbiosis, Blaisdell Publishing Company). Industrial scale harvests are neither ecologically sensible nor sustainable and for many species are not feasible. Even if the lichen cultures are established in-vitro they do not produce the typical lichen substances and the techniques to encourage this are still unknown. Table-1: Previously described bioactive constituents from different Lichens. (Table Removed) It is thought that most secondary metabolites of lichens are made by the mycobiont (Huneck, and Yoshimura, (1996) Identification of Lichen Substances, Springer-Verlag). This is not surprising because fungal compounds are well known in medicine (e.g. penicillin and cyclosporin). It is possible, however, that the photobionts also contribute to the repertoire of lichen metabolites. Cyanobacteria produce many bio-active secondary metabolites (Namikoshi, M. and Rinehart, K.L. (1996) Bioactive compounds produced by cyanobacteria. J. Ind. Microbiol. 17, 373-143) and there is an example of a patented anti fungal compound produced by a strain of Nostoc isolated from a lichen (US Pat.No. 4, 946, 835, Merck & Co). There are compelling reasons for expanding the search for natural-product drugs because previously reliable standard antibiotics are becoming less and less effective against new strains of multi drug-resistant pathogens. It has even been suggested that the end of the antibiotic era is fast approaching. In the past, search for pharmaceutically active molecules concentrated on the products of microbes that can be cultivated in the laboratory. More recently synthetic chemical methodologies have attracted a great deal of attention and combinatorial chemistry has been promoted as a source of molecules for automated highthroughput screening methods. Although these approaches have provided some lead molecules there is still a great need to discover novel chemical entities for therapeutic use. Systemic and superficial fungal infections affect millions of people throughout the world. Most of these diseases are caused by Candida albicans, Cryptococcus neoformans, Aspergillus sp., Trichophyton sp., Microsporum gypseum, Epidermophyton floccossum that are infectious in nature. In India, large number of people are involved in agriculture with majority of them living in villages where due to the prevailing unhygienic conditions the incidence of mycotic infections are severe. Fungal infections are also assuming increasing importance on account of decrease in immune systems mainly because of organ transplant operations, cancer chemotherapy and acquired immune deficiency syndrome (AIDS). Moreover the skin infections spread rapidly due to poor hygienic conditions and over population as well as increasing level of environmental pollution. To counter these infections only a handful of antifungal agents such as greseofulvine, amphotericin and nystatin are available in the market, although the available antibacterials are replete. Most of these antifungals are synthetic derivatives with known side effects to human and animals. Compounding this problem is the development of resistance towards commonly used drugs thus rendering the chemotherapy less useful. Therefore new antifungal substances from natural sources have to be generated to counter the resistance phenomenon. During 1990-96 the world market for antifungals was over US $ 1500 millions representing 1.5% of the total global anti-infective market. Currently anti-fungals (both topical and systemic) represent more than 6% of the total anti-infective agents. The world market for antifungals is expanding at the rate of 20% per annum and is estimated to reach over US $ 600 million/annum. However, many of the synthetic drugs produce side effects in immune stressed individuals. On the other hand natural products and their formulations made out of herbal sources will have more acceptances than the synthetic antifungals. Objects of the invention The main object of the present invention is to provide a novel antifungal/anticancer composition. The main object of the present invention to identify Lichen extract, which can specifically kill the polyene drug resistant fungal infections of humans. It is also the object of the invention to isolate, characterize and establish the nature of the bioactive molecule from the active lichen extract by bioactivity-guided fractionation. Still another object of the invention is to test the ergosterol binding ability of the bioactive molecule using in-vitro assays. Summary of the invention Accordingly the present invention provides a novel antifungal/anticancer composition comprising a pharmaceutically effective amount of methyl-p-orcinol carboxylate of formula I and a pharmaceutically acceptable carrier (Figure Removed) In one embodiment of the invention, the composition is anti-fungal and the methyl-ßorcinol carboxylate of formula I is present in a concentration in the range of 10-400 μg/ml. In another embodiment of the invention, the composition is anticancer and the methyl-p-orcinol carboxylate of formula I is present in concentration in range of 1-10 μg/ml. In another embodiment of the invention, the fungus is from the group of yeasts comprising of Candida sp, exemplified by Candida albicans. In another embodiment of the invention, the cancer is liver, colon, ovarian or mouth (oral) cancer of humans. The invention also relates to a method of treatment of fungal infections in a subject comprising administering to the subject an anti-fungal composition comprising a pharmaceutically effective amount of methyl-p-orcinol carboxylate of formula I and a pharmaceutically acceptable carrier. (Figure Removed) In one embodiment of the invention, the methyl-p-orcinol carboxylate of formula I is isolated from lichen Everniastrum cirrhatum. In another embodiment of the invention, the fungus comprises a multiple or single drug resistant strain. In another embodiment of the invention, the methyl-p-orcinol carboxylate of formula I is present in a concentration in the range of 10-400 jig/ml. In a further embodiment of the invention, the fungus is from the group of yeasts comprising of Candida sp, exemplified by Candida albicans. In a further embodiment of the invention, the fungus is a polyene drug resistant strain, the polyene drug being exemplified by nystatin and anphotericin In yet another embodiment of the invention, the fungus comprises an azole resistant strain, the azole drug being exemplified by clotrimazole, flucanoazole, itracanoazole and micanazole. In yet another embodiment of the invention, the fungus is simultaneously resistant to both polyene and azole classes of antibiotics. The subject is preferably human. The present invention also provides a method for the treatment of cancer in a subject such as a human being, the cancer being either of liver, colon, ovarian and mouth (oral) cancer comprising administering to the subject a pharmaceutically effective amount of methyl-p-orcinol carboxylate of formula I and a pharmaceutically acceptable carrier. In one embodiment of the invention, the concentration of methyl-p-orcinol carboxylate of formula I is in the range of 1-10 μg/ml. The present invention also relates to the use of methyl-p-orcinol carboxylate of formula I for the treatment of fungal infection or cancer in a subject. Detailed description of the invention The present invention relates to a novel biomolecule methyl-p-orcinolcarboxylate of formula I isolated from a lichen (Everniastrum cirrhatum), for treating pathogenic fungal infections of humans that are resistant to polyene antibiotics such as amphotericin B, nystatin etc. However, the biomolecule does not possess ergosterolbinding property. Infections due to Candida sp account for about 80% of all major systemic fungal infections. Candida is now the fourth most prevalent organism found in bloodstream infections and is the most common cause of fungal infections in immuno-compromised people. Vaginal candidiasis commonly affects women, including those with normal immunity, especially after antibiotic use. Lichens were collected from Narayan Ashram, Pithoragarh; Uttaranchal, India in the month of April 2002. Subsequently the lichens were identified taxonomically as Everniastrum cirrhatum. The collected lichen was air dried in shade and ground to fine powder. The powdered lichen material was used further for chemical analysis. Ethanol extract was prepared and tested against Candida albicans MTCC 1637 (equivalent to ATCC 18804) the fungi that cause different forms of candidiasis in humans and drug resistant mutants of the fungi. Amphotericin and nystatin are standard polyene antifungal drugs used in chemotherapy. Candida albicans isolates resistant to these polyene antibiotics are already reported. High-level resistance to amphotericin B, seen in all the major Candida species, is most common in neutropenic patients who have received prolonged courses of amphotericin B. Such drug resistant infections are clinically difficult to treat and are physician's nightmare. Hence, we developed such polyene resistant strains of Candida albicans in-vitro and evaluated the anti-candidial effect of lichen extracts / compounds against them. The extract and subsequent solvent (hexane and ethyl acetate) fractions were found to be active against amphotericin and nystatin resistant Candida. Bioactivity guided fractionation of the active fractions resulted in the isolate of active compounds by column chromatography. The active compound could be crystallized from 96% hexane: 4% ethyl acetate fraction. The purified compound was analyzed by spectroscopic techniques using 'H & 13C NMR, LC-MS etc to decipher the chemical structure. Ccompound was identified as methyl-(3-orcinolcarboxylate, of formula I. The compound is a colorless crystal with melting temperature of 137°C. Caccamese et al (1985) have already found that the methyl-(3-orcinolcarboxylate inhibit the growth of yeast strains such as Saccharomyces cerevisiae. However, in this study we shown a unique property of methyl-p-orcinolcarboxylate wherein the compound specifically inhibits the growth of polyene and azole drug resistant strains of Candida albicans and Saccharomyces cerevisiae. The principal sterol in the fungal cytoplasmic membrane, is the target site of action of amphotericin B and the azoles. Amphotericin B, a polyene, binds irreversibly to ergosterol, resulting in disruption of membrane integrity and ultimately cell death. Therefore, the ability of lichen compounds to bind to ergosterol was also investigated using in-vitro ergosterol binding assay (Antonio & Molinski 1993; J.Natl.Prod.56:54-6\). The results indicated that the compounds do not possess any specificity to ergosterol in the wild type and drug resistant strains of Candida sp. The present invention therefore provides an antifungal/anticancer composition comprising a pharmaceutically effective amount of methyl-p-orcinol carboxylate of formula I and a pharmaceutically acceptable carrier. A concentration of methyl-p-orcinol carboxylate of formula I in the range of 10-400 μg/ml provides antifungal activity against the group of yeasts comprising of Candida sp, exemplified by Candida albicam. A concentration of methyl-p-orcinol carboxylate of formula I in the range of 1-10 μg/ml provides anticancer activity against liver, colon, ovarian or mouth (oral) cancer of humans. The methyl-p-orcinol carboxylate of formula I is isolated from lichen Everniastrum cirrhatum. The fungus can be either a multiple drug resistant or single drug resistant strain. For example, the fungus can be from the group of yeasts comprising of Candida sp, exemplified by Candida albicans. The drugs in question can be a polyene drug exemplified by nystatin and anphotericin or a azole drug exemplified by clotrimazole, flucanoazole, itracanoazole and micanazole. The following examples are illustrative and should not be construed as limiting the scope of the invention in any manner. Examples 1.Isolation of polyene drug resistant mutant strains of Candida albicans MTCC 1637 (equivalent to ATCC 18804) C. albicans was grown to log phase in Sabouraud's dextrose broth (5 ml) for 48 hrs at 37°C in a shaker at 250rpm. The cells were pelleted by centrifugation at SOOOrpm at 4°C and the pellet was dissolved in 5 ml phosphate buffered saline PBS (6.8pH). The culture was divided in to five groups of 1 ml each in eppendrof tubes. Ethyl methane sulfonate (EMS) was added to each of the culture tube @ 0.1% (v/v) and allowed to grow for 40 min. Then the mutagen was completely washed off thrice by repeatedly pelleting the cells and re-dissolving in PBS. The mutagenized stocks was then diluted in Sabouraud's dextrose broth two folds and allowed to grow for 6 hrs at 37°C in a shaker at 250rpm. Titre of the cells before treatment with EMS and immediately after treatment with EMS was calculated to obtain the killing percentage in each of the five tubes. The mutagenized and fixed cultures were then plated in Sabouraud's dextrose agar containing different concentration of amphotericin, nystatin and clotrimazole. The colonies found growing after 5thday from each of the five mutagenized stocks were then purified thrice separately by streaking in the same medium containing the antibiotics. 2. Drug resistance of mutant strains against polyenes and azoles The drug resistance property of the mutants was studied by standardized disc diffusion assay (Bauer at al 1966, American Journal of Clinical Pathology 45: 493-496) with slight modifications. The discs were prepared (5 mm diameter made of Whatman #3 filter paper) by impregnating 8 μl of test compound and placing them on pre-inoculated agar surface. A disc containing only the solvent was used as the control. A zone of growth inhibition surrounding the disc is indicative of the resistant nature of the strains to antibiotics. As is evident from this example the results indicate that all the mutant strains were highly resistant to amphotericin and nystatin as the zone of growth inhibition was far less in mutants than that of the wild type parent strain. However only Amph C7R, Amph C6R, Clo 31R and Clo 28R were only resistant to clotrimazole indicating of less zone of growth inhibition. Table 2: (Table Removed) 3.Collection and extraction of lichen materials: Two kg of the lichen (Everniastrum cirrhatum) material were collected from Narayan Ashram, Pithoragargh, Uttaranchal, during the month of April 2002. They were separated and air-dried at room temperature (35°C-40°C) in shade. After air drying they were ground and sieved to fine powder in a mixer grinder. 1.5 kg of the powdered materials were dipped in absolute ethanol in a percolator for 72 hrs at room temperature (35°C-40°C). Ethanol extract was filtered using Whatman filter paper No.l and concentrated at the 60°C under reduced pressure. The ethanolic extract was then lyophilized to obtain 15.5 g of crude extract. Stock of 100 mg/ml was made in DMSO and tested for bio-activity. 4. Bioactivity guided fractionation of the lichen materials Solvent fractionation of the active crude extracts was undertaken to isolate the active principle. Ethanolic extract was dissolved in 500ml of hexane. Then it was filtered using Whatman No. 1 filter paper. The insoluble portion was dissolved in 500ml of ethyl acetate. All the solvent fractions were concentrated at 40°C under reduced pressure to obtain 3g of hexane and 1.5g of ethyl acetate extract and tested. The results indicate that both ethyl acetate and hexane fraction obtained from the crude extract possessed the bioactivity against drug resistant strains of C.albicans. The hexane fraction was considerably more active than the ethyl acetate extract. Table3 (Table Removed) 5. Purification and characterization of the active molecule The hexane and ethyl acetate fractions thus obtained are mixed together and further fractionated in a glass column having an internal diameter of 3.0 cm and length of 72.0 cm. Hexane was used as the initial mobile phase and silica gel (particle size 60-120 mesh) as the stationary phase. Different fractions of approximately 100 ml were collected and dried under vacuum. Concentrated fractions were then run on TLC plates and fractions of similar TLC pattern were pooled together. After about 3 liter of hexane fraction collected the polarity of the mobile phase was slightly increased from fraction No. 36 by adding ethyl acetate to hexane (4% of ethyl acetate in final volume). Similarly fractions No.64 to 78 were combined together based on identical 7-spot bands as appeared in TLC. Above fractions were dissolved in 50 ml of acetone and kept at room temperature (25-30°C) for crystallization of compounds. Crystals thus obtained were again properly washed with acetone and TLC of crystals was carried out by using a mobile phase of benzene 98% plus acetone 2%. TLC plates showed a single spot on exposing to iodine fume. These TLC plates exhibited a single dark red colored spot when dipped in bacopa reagent (vaniline 3.5 g, H2SO4 17.8 g, absolute alcohol 332.5 ml) and heated at 120 ° C for 5 minutes. About 40 mg of the crystal could be collected from the above run. The melting temperature of crystal thus obtained was found to be 137 ° C. The active spot obtained by TLC was further purified by repetitive column chromatography, which can be performed by a person skilled in the art and then analyzed by H & 13C NMR, LC-MS to determine the structure of the active pure compound. On the basis of spectroscopic data the compound isolated was identified as Methyl-p-orcinolcarboxylate. 6. Specific anticandidial activity of methyl-p-orcinolcarboxylate acid against polyene and azole resistant strains The pure compound isolated was then tested against polyene and azole resistant strains of Candida albicans. The data described below indicates that the compound methyl-ßorsellinic acid was able to inhibit the growth of drug resistant strains in a dose dependant manner whereas it was inactive against the wild type strain. In another experiment welldefined amphotericin and nystatin resistant strains of Saccharomyces cerevisiae were used in the assay. These strains designated as erg 1 and erg 6 carry mutations in the ergosterol biosynthetic pathway and therefore are unable to synthesize ergosterol which are the binding site of polyene drugs. Therefore absence of ergostreol results in polyene resistance. The results suggests that methyl-p-orcinolcarboxylate was able to specifically inhibit the growth of polyene drug resistant Saccharomyces cerevisiae. Table 4 (Table Removed) 7. Anticancer property of methyl p-orsellinic acid against human cancer cell lines Cytotoxicity testing in vitro was done by the method of Woerdenbag et al.,1993; J.Nat.Prod. 56 (6): 849-856). 2x103 cells/well were incubated in the 5% CO2 incubator for 24h to enable them to adhere properly to the 96 well polysterene microplate (Grenier, Germany). Test compounds dissolved in 100% DMSO (Merck,Germany) in atleast five doses were added and left for 6h after which the compound plus media was replaced with fresh media and the cells were incubated for another 48h in the CO? incubator at 37°C. The concentration of DMSO used in our experiments never exceeded 1.25%, which was found to be non-toxic to cells. Then, 10 μl MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma M 2128] was added, and plates were incubated at 37°C for 4 h. 100 μl dimethyl sulfoxide (DMSO, Merck, Germany) were added to all wells and mixed thoroughly to dissolve the dark blue crystals. After a few minutes at room temperature to ensure that all crystals were dissolved, the plates were read on a SpectraMax 190 Microplate Elisa reader (Molecular Devices Inc., USA), at 570 nm. Plates were normally read within 1 h of adding the DMSO. The experiment was done in triplicate and the inhibitory concentration (1C) values were calculated as follows: % inhibition =[1-OD (570 nm) of sample well / OD (570 nm) of control well] x 100. IC9o is the concentration μg/mL required for 90% inhibition of cell growth as compared to that of untreated control. The results described indicate that the ethanolic crude extract of the lichen and the isolated pure compound methyl-Pore inolcarboxylate was active against liver (WRL-68); colon (Caco-2); ovarian (MCF-7 & PA-1) and oral (KB 403) human cancer cell lines. Table 6 (Table Removed) We Claim: 1. A novel antifungal/anticancer composition comprising a pharmaceutically effective amount of methyl-ß-orcinol carboxylate of formula I and a pharmaceutically acceptable carrier. (Figure Removed) 2. A composition as claimed in claim 1, wherein the composition is anti-fungal and the methyl-p-orcinol carboxylate of formula I is present in a concentration in the range of 10- 400 µg/ml. 3. A composition as claimed in claim 1 wherein the composition is anticancer and the methyl-p-orcinol carboxylate of formula I is present in a concentration in the range of 1- l0 µg/ml. 4. A composition as claimed in claim 2 wherein the fungus is from the group of yeasts comprising of Candida sp. 5. A composition as claimed in claim 3, wherein the cancer is liver, colon, ovarian or mouth (oral) cancer of humans. 6. An antifungal/anticancer composition substantially as herein described withreference to the foregoing examples.

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