JP4498930B2 - Methyl-β-orcinol carboxylate from lichen plant (Everniastrum cirrhatum) for use in the treatment of fungal infections and cancer - Google Patents

Description

The present invention relates to a formula isolated from a lichen (Everniastrum cirrhatum) for treating human pathogenic fungal infections resistant to polyenes and azole antibiotics such as amphotericin B, nystatin, clotrimazole and the like. It relates to a novel use of the already known biomolecule I of methyl-β-orcinol carboxylate.

  Lichen plants have a symbiotic relationship between fungi, green algae, and / or cyanobacteria. They have a variety of chemicals and produce many polyketide-derived compounds, including those that have not been well reported, such as depsides and depsidons. Depsides are a class of compounds and appear to be unique to lichens. These compounds are dimeric esters of variously substituted orthoric acids and are the primary source of what are termed lichen acids. Lichen plants are valued in traditional medicine, but it is difficult to establish conditions for aseptic culture and rapid growth, eliminating their routine use in most conventional screening methods, Its value has been largely ignored by the modern pharmaceutical industry.

  The relationship between fungi and algae is specific and selective. The names of the fungal components are given to all lichens and there are> 13500 listed species, including almost one fifth of all known fungi (Hawcksworth and hill, 1984; The Lichen Forming Fungi, Mecorquodale Ltd). Individual mycobiot and photobiot (fungi, and photosynthetic algae or cyanobacteria, respectively) are small and unnoticeable when cultured in laboratory dishes, but they coexist naturally with a wide range of various beautiful forms Ingredients, such as Ramalina menziessi ("fishnet" lichen) can cover the whole tree and form a profound appearance (Arvis, WO, 2000; Lichens, Smithsonian Situation Press). They play a variety of environmental roles, such as settlement in marginal habitats in Antarctica, establishing soil in the semi-arid desert of Australia, and contributing to nitrogen turnover in North American North Pacific forests.

  They produce characteristic secondary metabolites that are unique with respect to those of higher plants. Lichens produce a wide range of chemical compounds, of which about 350 secondary metabolites have been identified. These mycobiont-derived products usually accumulate as extracellular crystals in the symbiotic cell wall and count up to 10% (up to 40% in exceptional cases) of the dry weight of the fronds (Galun, M. and Shomer-llan , A. (1988) CRC Handbook of Lichenology, Vol. III; 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 are derived from the shikimate and mevalonate pathways, which are key pathways for secondary metabolism in all organisms. Some lichen plant extracts are used as various therapeutic agents in folk remedies, and by screening tests on lichens, antibiotic properties, antimycobacterial properties, antiviral properties, antialgal properties, and antipyretic properties A frequent appearance of metabolites with characteristics has been shown.

  In addition, a separate class of lichen metabolites are depsides. These classes of compounds are formed by the esterification of the carboxyl group of one molecule with the phenolic hydroxyl group of a second molecule by condensation of two or more hydroxybenzoic acids. Because of the phenolic nature of its chemical structure, the main class of inhibitors often contains hydroxylated aromatic rings (Fitzsimmons et al., 1989), making them interesting candidates for assessing their effects on leukotriene biosynthesis. In addition, two small molecule lichen plant-derived metabolites, protrihesteric acid, have been reported to inhibit porcine leukocyte-derived 5-LO (Ogmundsdottir et al., 1998). The latter has also been shown to inhibit peptide leukotriene formation (Gissurarson et al., 1997). Lichen plant depsides have also been described to inhibit prostaglandin biosynthesis (Sankawa et al., 1982).

  Lichen plants and their products have been used in traditional medicine for centuries and are still of considerable interest as alternative treatments throughout the world. In fact, today, a variety of lichen-based tonics, lotions, and lozenges are available for purchase in Iceland and they are also medicinal. However, despite the existence of studies where lichens produce a large number of low molecular weight molecules with diverse structures and provide evidence of biological activity in extracts from whole lichens (Table 1). Lichens are essentially ignored by the modern pharmaceutical industry. Two reasons contributed to this: (1) lichens are naturally slow growing, and (2) lichens are difficult to grow and re-synthesize in culture ( Ahmadjian, 1993; The Lichen Symniosis, Blaisdell Publishing Company). Industrial scale harvesting is economically meaningless, not sustainable, and not easy for many species. Even if lichen plant cultures are established in vitro, they do not produce typical lichen plant material, and the techniques encountered are still unknown.

  It is understood that most secondary metabolites of lichens are formed by mycobiont (Huneck and Yoshimura, (1996) Identification of Lichen Substances, Springer-Verlag). This is not surprising since fungal compounds are well known in medicine (eg penicillin and cyclosporine). However, photobiants can also contribute to the repertoire of lichen metabolites. Cyanobacteria produce many biologically active secondary metabolites (Namikoshi, M. and Rinehart, KL (1996) Bioactive compounds produced by cyanobacteria. J. Ind. Microbiol. 17, 373-143) and are isolated from lichens. There are examples of patented antifungal compounds produced by isolated Nostoc strains (US Pat. No. 4,946,835, Merck & Co).

  There is a compelling reason to expand research on natural product drugs as previously trusted standard antibiotics are gradually becoming less effective against strains of new multidrug-resistant pathogens . It is suggested that the end of the antibiotic era is approaching quickly. In part, the search for pharmaceutically active molecules has focused on microbial products that can be cultured in the laboratory. Much attention has been focused on more recent synthetic chemistry methodologies, and combinatorial chemistry has advanced as a molecular source for automated high-throughput screening methods. While these approaches have provided some lead molecules, there is still a great demand for discovering new chemical molecules for therapeutic applications.

Systemic and superficial fungal infections affect millions of people worldwide. Most of these diseases are caused by Candida albicans, Cryptococcus neoformans, Aspergillus species, Trichophyton species, Microsporum gypseum, Epidermophyton floccossum, which are naturally infectious. In India, a large number of people are involved in agriculture, and most of them live in villages where fungal infections are common due to generally unsanitary conditions. Fungal infections are also presumed to be of increasing importance, especially due to organ transplantation, cancer chemotherapy, and a decline in the immune system due to acquired immune deficiency syndrome (AIDS). In addition, skin infections are spreading rapidly across the entire population due to lack of hygienic conditions and increased levels of environmental pollution. Although the available antibacterial agents are abundant, only a few antifungal agents such as griseofulvin, amphotericin, and nystatin are available on the market to combat these infections. Most of these antifungal agents are synthetic derivatives with known side effects in humans and animals. Around this problem, resistance to commonly used drugs is being formed, and chemotherapy has become less useful. Therefore, new antifungal substances of natural origin need to be produced to eliminate the resistance phenomenon. Between 1990-96, the global market for antifungals reached US $ 1,500,000,000, accounting for 1.5% of the global antiinfective market. Current antifungal agents (both local and systemic) account for more than 6% of all antiinfectives. The global market for antifungal agents is expanding at a rate of 20% per year and is estimated to reach US $ 600,000,000. However, many synthetic drugs cause side effects in patients who are stressed by immunity. On the other hand, formulations made from natural products and herbal sources will be more receptive than anti-fungal agents.
Hawcksworth and hill, 1984; The Lichen Forming Fungi, Mecorquodale Ltd Arvis, WO, 2000; Lichens, Smithsonian Situation Press Galun, M. and Shomer-llan, A. (1988) CRC Handbook of Lichenology, Vol. III; Galun, M., ed., Pp. 3-8. CRC Press Ahmadjian, 1993; The Lichen Symniosis, Blaisdell Publishing Company Huneck and Yoshimura, (1996) Identification of Lichen Substances, Springer-Verlag Namikoshi, M. and Rinehart, KL (1996) Bioactive compounds produced by cyanobacteria. J. Ind. Microbiol. 17, 373-143 U.S. Pat.No. 4,946,835, Merck & Co

  The main objective of the present invention is to identify lichen plant extracts that can specifically kill human polyene drug resistant fungal infections.

  The object of the present invention is also to isolate, characterize and establish the properties of bioactive molecules from active lichen plant extracts by bioactive guided fractionation.

  Yet another object of the invention is to test the ergosterol binding activity of bioactive molecules using in vitro assays.

Accordingly, the present invention is to provide an antifungal / anticancer composition comprising a pharmaceutically effective amount of methyl-β-orcinol carboxylate of formula I and a pharmaceutically acceptable carrier.

  In one embodiment of the invention, the composition is antifungal and the methyl-β-orcinol carboxylate of formula I is present at a concentration in the range of 10-400 μg / ml.

  In another embodiment of the invention, the composition is anticancer and the methyl-β-orcinol carboxylate of formula I is present in a concentration in the range of 1-10 μg / ml.

  In another embodiment of the invention, the fungus is derived from a group of yeast consisting of the Candida species exemplified by Candida albicans.

  In another embodiment of the invention, the cancer is human liver, colon, ovary, or mouth (oral) cancer.

The present invention relates to fungal infections in a patient comprising administering to the patient an antifungal composition comprising a pharmaceutically effective amount of methyl-β-orcinol carboxylate of formula I and a pharmaceutically acceptable carrier. Relates to a method of treatment.

  In one embodiment of the invention, the methyl-β-orcinol carboxylate of formula I is isolated from the lichen Everniastrum cirrhatum.

  In another embodiment of the invention, the fungus comprises multiple or single drug resistant strains.

  In another embodiment of the invention, the methyl-β-orcinol carboxylate of formula I is present at a concentration in the range of 10-400 μg / ml.

  In a further embodiment of the invention, the fungus is derived from the group of yeasts consisting of the Candida species exemplified by Candida albicans.

  In a further embodiment of the invention, the fungus is a polyene drug resistant strain and the polyene drug is exemplified by nystatin and amphotericin.

  In yet another embodiment of the invention, the fungus comprises an azole resistant strain, and the azole drug is exemplified by clotrimazole, flucananoazole, itracanazole, and micanazole.

  In yet another embodiment of the invention, the fungus is simultaneously resistant to both polyene and azole classes of antibiotics.

  The patient is preferably a human.

The present invention also includes administering to a patient a pharmaceutically effective amount of methyl-β-orcinol carboxylate of formula I and a pharmaceutically acceptable carrier, in a patient, such as a human, Provided are methods for treating any of the cancers of the large intestine, ovary, and mouth (oral).

  In one embodiment of the invention, the concentration of methyl-β orcinol carboxylate of formula I is present in the range of 1-10 μg / ml.

The invention also relates to the use of methyl-β-orcinol carboxylate of formula I for the treatment of fungal infection or cancer in a patient.

The present invention relates to a biomolecule of formula I methyl-β isolated from lichens (Everniastrum cirrhatum) for treating human pathogenic fungal infections resistant to polyene antibiotics such as amphotericin B, nystatin etc. -Regarding the use of orcinol carboxylate. However, the biomolecule does not have ergosterol binding properties.

  Infection with Candida species accounts for approximately 80% of all major systemic fungal infections. Candida is now the fourth most common organism found in bloodstream infections and is the most common cause of fungal infection in immunocompromised people. Intravaginal Candida commonly affects women, including humans with normal immunity, especially after the use of antibiotics.

Lichen plants were collected from Narayan Ashram, Pithoragarh; Uttaranchal, India during January 2002. Later, the lichen was taxonomically identified as Everniastrum cirrhatum. The collected lichens were air-dried in the shade and crushed into fine powder. Powdered lichen plant material was further used for chemical analysis. Ethanol extracts were prepared and tested against Candida albicans MTCC 1637 (equivalent to ATCC 18804). The fungus produces various forms of Candida species in humans, resulting in fungal drug-resistant mutants. Amphotericin and nystatin are standard polyene antifungal agents used in chemotherapy. Candida albicans isolates that are resistant to these polyene antibiotics have already been reported. The high level of resistance to amphotericin B observed in all major Candida species is most common in neutropenic patients undergoing the long-term course of amphotericin B. Such drug-resistant infections are clinically difficult to treat and cause doctors' nightmares. We therefore developed such polyene resistant strains of Candida albicans in vitro and evaluated the anti-Candida effect of lichen extracts / compounds on them. The extract and subsequent solvent (hexane and ethyl acetate) fractions were found to be active against amphotericin and nystatin resistant Candida. Isolation of the active compound was obtained by column chromatography by bioactivity-guided fractionation of the active fraction. The active compound could be crystallized from a 96% hexane: 4% ethyl acetate fraction. The resulting compound was analyzed by spectrophotometry using ¹ H and ¹³ C NMR, LC-MS, etc. to reveal the chemical structure. The compound was identified as methyl-β-orcinol carboxylate of formula I. The compound is a colorless crystal having a melting temperature of 137 ° C. Caccamese et al. (1985) have already found that methyl-β-orcinol carboxylate inhibits the growth of yeast strains such as Saccharomyces cerevisiae. In this study, however, we have shown the unique property that methyl-β-orcinol carboxylate specifically inhibits the growth of Candida albicans and Saccharomyces cerevisiae polyenes and azole-resistant strains. The main sterol in the fungal cell membrane is the target site of action of amphotericin B and azole. Amphotericin B, a polyene, binds irreversibly to ergosterol, destroying membrane integrity and ultimately leading to cell death. Therefore, the ability of lichen compounds to bind ergosterol was also investigated using an in vitro ergosterol binding assay (Antonio & Molinski 1993; J. Natl. Prod. 56: 54-61). As a result, it was shown that the compound does not have any properties of ergosterol in wild type and drug resistant strains of Candida species.

  The present invention therefore provides an antifungal / anticancer composition comprising a pharmaceutically effective amount of methyl-β-orcinol carboxylate of formula I and a pharmaceutically acceptable carrier. Concentrations of methyl-β-orcinol carboxylate of formula I in the range of 10-400 μg / ml provide antifungal activity against a group of yeasts including Candida species exemplified by Candida albicans. A concentration of methyl-β-orcinol carboxylate of formula I in the range of 1-10 μg / ml provides anticancer activity against human liver, colon, ovary, or mouth (oral) cancer.

  Methyl-β-orcinol carboxylate of formula I is isolated from the lichen Everniastrum cirrhatum.

  The fungus may be either a multidrug resistant strain or a single drug resistant strain. For example, the fungus may be derived from a group of yeasts including Candida species exemplified by Candida albicans. The drug in question may be a polyene drug exemplified by nystatin and amphotericin, or an azole drug exemplified by clotrimazole, flucanazole, itracanazole, and micanazole.

  The following examples are illustrative and should not be considered as limiting the scope of the invention in any way.

1. Isolation of Candida albicans MTCC 1637 (equivalent to ATCC 18804) polyene drug resistant mutant strain
C. albicans were grown in logarithmic growth phase in Sabouraud dextrose broth (5 ml) for 48 hours at 37 ° C. in a shaker at 250 rpm. The cells were pelleted by centrifugation at 5000 rpm at 4 ° C. and the pellet was lysed in 5 ml phosphate buffered saline PBS (6.8 pH). The cultures were divided into groups of 5 with 1 ml each in Eppendorf tubes.

  Ethyl methanesulfonate (EMS) was added to each tube at 0.1% (v / v) and grown at 40 ° C. The mutagen was then washed thoroughly twice by repeatedly pelleting the cells and resuspended in PBS. Mutagenized stocks were diluted 2-fold with Sabouraud dextrose broth and grown for 6 hours at 37 ° C. in a shaker at 250 rpm. The titer of cells before and immediately after EMS treatment was calculated to obtain the kill percentage in each of the 5 tubes. Mutagenized and immobilized cultures were placed in Sabouraud dextrose agar containing various concentrations of amphotericin, nystatin, and clotrimazole.

  Colonies that were found to grow after day 5 from each of the 5 mutagenized stocks were purified separately three times by streaking in the same medium containing antibiotics.

2. Drug resistance of mutant strains to polyenes and azoles The drug resistance properties of mutants were studied by a standardized disc diffusion assay (Bauer et al., 1966, American Journal of Clinical Pathology 45: 493-496) with minor modifications. Disks were prepared (5 mm diameter formed with Whatman # 3 filter paper) by immersing 8 μl of test compound and placing them on the pre-inoculated agar surface.

  A disc containing only solvent was used as a control. The growth inhibition area surrounding the disk is an indicator of the strain's resistance to antibiotics. As is apparent from the results of this example, all mutant strains were very resistant to amphotericin and nystatin, and the growth inhibition region was considerably smaller in the mutant than in the wild type parent strain. However, only Amph C7R, Amph C6R, Clo 31R, and Clo 28R were only resistant to clotrimazole, indicating a small area of growth inhibition.

3. Collection and extraction of lichen plant materials
2 kg lichen (Everniastrum cirrhatum) material was collected from Narayan Ashram, Pithoragargh, Uttaranchal during the month of April 2002. They were separated and air-dried in the shade at room temperature (35 ° C-40 ° C). After air drying, they were pulverized and sieved to a fine powder with a mixer pulverizer. 1.5 kg of powdered material was immersed in absolute ethanol with a percolator at room temperature (35 ° C.-40 ° C.) for 72 hours.

  The ethanol extract was filtered using Whatman filter paper No. 1 and concentrated under reduced pressure at 60 ° C. The ethanol extract was then lyophilized to give 15.5 g of crude extract. A 100 mg / ml stock was made in DMSO and tested for bioactivity.

4). Bioactive guided fractionation of lichen plant material Solvent fractionation of the active crude extract was performed to isolate the active ingredients. The ethanol extract was dissolved in 500 ml hexane. It was then filtered using Whatman No. 1 filter paper. The insoluble part was dissolved in 500 ml of ethyl acetate. All solvent fractions were concentrated at 40 ° C. under reduced pressure to obtain an extract of 3 g hexane and 1.5 g ethyl acetate and tested. As a result, it was shown that the fractions of both ethyl acetate and hexane obtained from the crude extract have bioactivity against drug-resistant strains of C. albicans. The hexane fraction was much more active than the ethyl acetate extract.

5). Purification and characterization of the active molecule The hexane and ethyl acetate fractions thus obtained were mixed together and further fractionated on a glass column having a 3.0 cm inner diameter and a 72.0 cm length. Hexane was used as the initial mobile phase and silica gel (particle size 60-120 mesh) was used as the stationary phase. Approximately 100 ml of various fractions were collected and dried under vacuum. The concentrated fractions were then run on the TLC phase and fractions with the same TLC pattern were pooled together. After collecting about 3 liters of hexane fraction, the mobile phase polarity was increased from fraction No. 36 by adding ethyl acetate to hexane (final volume of 4% ethyl acetate). Similarly, fractions Nos. 64 to 78 were combined together based on the same 7-spot band as it appeared in TLC. The above fraction was dissolved in 50 ml of acetone and kept at room temperature (25-30 ° C.) for crystallization of the compound. The crystals thus obtained were washed again with acetone and a TLC of the crystals was performed using a mobile phase of 98% benzene + 2% acetone. The TLC plate showed a single spot when exposed to iodine gas. When immersed in bacopa reagent (3.5 g vanillin, 17.8 g H ₂ SO ₄ , 332.5 ml absolute alcohol) and heated at 120 ° C. for 5 minutes, these TLC spots showed a single dark red spot. Approximately 40 mg of crystals could be recovered from the above operation. The melting temperature of the crystals thus obtained was found to be 137 ° C.

The active spot obtained by TLC was further purified by iterative column chromatography, which could be performed by ^one skilled in the art and analyzed by ¹ H & ¹³ C NMR, LC-MS to determine the structure of the active pure compound. Based on the spectroscopic data, the isolated compound was identified as methyl-β-orcinol carboxylate.

6). Specific anti-candida activity of methyl-β-orcinol carboxylate against polyene and azole resistant strains Isolated pure compounds were tested against polyene and azole resistant strains of Candida albicans. The data described below show that the compound methyl-β-orselic acid can inhibit the growth of drug-resistant strains in a dose-dependent manner while being inactive in the wild-type strain. In another experiment, a well-defined amphotericin and nystatin resistant strain of Saccharomyces cerevisiae was used in the assay. These strains named erg2 and erg6 have mutations in the ergosterol biosynthetic pathway and therefore cannot synthesize ergosterol, the binding site for polyene drugs. Therefore, the absence of ergosterol leads to polyene resistance. This result suggests that methyl-β-orcinol carboxylase could specifically inhibit the growth of polyene drug resistant Saccharomyces cerevisiae.

7). Anti-cancer properties of methyl β-orselic acid against human cancer cell lines
In vitro cytotoxicity tests were performed by the method of Woerdenbag et al., 1993; J. Nat. Prod. 56 (6): 849-856. 2 × 10 ³ cells / well were incubated for 24 hours in a CO ₂ incubator and allowed to adhere exactly to 96-well polystyrene microplates (Grenier, Germany). Test compounds dissolved in 100% DMSO (Merck, Germany) at a dose of at least 5 are added and left for 6 hours, after which the compound + medium is replaced with fresh medium and the cells are placed in a CO ₂ incubator at 37 ° C. Incubated for a further 48 hours. The concentration of DMSO used in this experiment did not exceed 1.25% and was found to be non-toxic to the cells. Then 10 μl of MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide; Sigma M 2128] was added and the plate was incubated at 37 ° C. for 4 hours. 100 μl of dimethyl sulfoxide (DMSO, Merck, Germany) was added to all wells and mixed thoroughly to dissolve the dark blue crystals. After several minutes at room temperature, the plate was read at 570 nm with a SpectraMax 190 Microplate Elisa reader (Molecular Devices Inc., USA) to confirm that all crystals had dissolved. Plates were read usually within 1 hour after addition of DMSO. Experiments were performed in triplicate and inhibitory concentration (IC) values were calculated as follows:% inhibition = [1-OD of sample well (570 nm) / OD of control well (570 nm)] × 100. IC90 is the concentration μg / ml required for 90% inhibition of cell growth compared to that of the untreated control. The described results show that the crude raw ethanol extract of lichens and the isolated pure compound methyl-β-orcinol carboxylate are liver (WRL-68); large intestine (Caco-2); ovary (MCF- 7 and PA-1) and mouth (KB403) were active against human cancer cell lines.

[References]

Claims (3)

An anti-cancer composition comprising a pharmaceutically effective amount of methyl-β-orcinol carboxylate of formula I and a pharmaceutically acceptable carrier.

The composition according to claim 1 , wherein the methyl-β-orcinol carboxylate of formula I is present in a concentration in the range of 1-10 µg / ml. The composition according to claim 1 or 2 , wherein the cancer is human liver, large intestine, ovary, or mouth (oral) cancer.