WO2010107294A1

WO2010107294A1 - Antifungal compound and its production

Info

Publication number: WO2010107294A1
Application number: PCT/MY2009/000037
Authority: WO
Inventors: Heng Fong Seow; Jap Meng Jee; Coy Choke Ho; Hwen Yee Cheah
Original assignee: Universiti Putra Malaysia
Priority date: 2009-03-16
Filing date: 2009-03-16
Publication date: 2010-09-23

Abstract

The invention relates to a compound for the inhibition of fungal infection, The present invention also relates to the use of one or more compounds for the preparation of a medicament, and methods of obtaining the compound a product of the microorganism. In particular, the present invention relates to the anti-fungal effects pharmaceutical composition, medicament or method of treatment related to said compound of the invention are for the treatment of diseases associated with fungus or yeast.

Description

TITLE

ANTIFUNGAL COMPOUND AND ITS PRODUCTION

FIELD OF INVENTION

The present invention also embraces using a microorganism isolated from soil to produce the antifungal agent. The present invention also relates, to methods of preparing the compound as a product of the microorganism. The present invention includes within its scope a novel strain of ^' Streptomyces sp. Strain H7372

BACKGROUND OF THE INVENTION

Fungal infections are becoming a major health concern for a number of reasons, including the limited number of anti-fungal agents available, the increasing incidence of species resistant to older anti-fungal agents, and the growing population of immunocompromised patients at risk for opportunistic fungal infections. The most common clinical isolate is Candida albicans (comprising about 19% of all isolates). In one study, it was reported that 40% of all deaths from hospital-acquired infections were due to fungi (Sternberg, 1994, Science 266: 1632-1634). Microscopic fungi and fungal spores are ubiquitous in nature, unfortunately also in the materials manufactured by humans as consumer items e. g. clothing, shoes, furniture, carpets, wallpaper, and also more basic materials used for construction purposes. Fungal spores are spread in many ways, airborne through the wind, through droplets or running water, and by touch. A number of different problems are associated with the occurrence of fungal growths (molds) in human habitats. Molds often leave stains which are aesthetically not pleasing, and very often they give off unpleasant earthy mildewy odors. However, more importantly, mold growths may lead to potential health problems for the occupants of a building where they occur, since many molds are allergenic and/or produce toxins. Toxic compounds that have been identified to be produced by various common indoor molds e. g. of the genera Aspergillus, Stachybotrys, Penicillium, Fusarium, or Alternaria, include Aflatoxin, Satratoxin H, Patulin, Vomitoxin, Fumonisin, Zearalone, Patulin, Ochratoxin, and Alternariol. It is thought that these toxins may act synergistically to produce a wide variety of symptoms and disease states. The symptoms of exposure to allergenic or toxic molds may range from allergenic sinusitis, asthma, bronchiopulmonary aspergillosis, and hypersentivity pneumonitis, to lethargy, gastrointestinal disturbances, liver toxicity and varieties of cancers. The infections caused by fungi and yeasts are commonly associated with signs of erythema and scaling and with symptoms of itching or painful burning. Clinical treatment for fungal disease requires at least two to four weeks for complete relief of symptoms. Candida belongs to yeasts. The transmittal of yeasts is possible via contaminated food but also by direct contact with other human beings and animals, particularly pets. Besides the affection of the mucosa in mouth, esophagus or vagina, a Candida infection can also result in- a spread into liver, kidney, spleen and other organs. Candida albicans is the most commonly found species, but lately Candida glabrata and Candida krusei are found more often in isolates, often exhibiting resistance against common antifungal agents. Apart from infections by Candida, Aspergillus spp. (moulds), in particular Aspergillus fumigatus, can cause the life threatening disease invasive aspergillosis. The fungus grows from the lung, the location of the primary infestation following the inhalation of fungal spores, into the tissue and can than affect all other organs of the body. Only modest possibilities are available for therapy of an invasive infection by A. fumigatus. Consequently, the disease leads to the death of the patient in more than 70 percent of the cases. But even if the infection spreads only on the surface of the body, the fungal infections are not only unpleasant but also dangerous in such location. Fungi take in glucose from the blood with their radices and segregate partially toxic metabolites, so called mycotoxins, damaging the liver. Thus, fungal infections have to be treated. Candidiasis and aspergillosis are two increasing invasive opportunistic mycoses among the hospitalised immunocompromised and immunosuppressed patients over the past two decades. Despite many advances in antifungal therapies, the infection is associated with high mortality and morbidity with increasing resistance of pathogens to currently available therapy regiments.

The needs for antifungal compounds have increased considerably in the last decades. The increasing number of high risk patients such as immunocompromised and imrnunosuppreased have led to a strong proliferation of fungal systemic infections within the population. Antifungal drug resistance is not as common as bacterial drug resistance and outbreak has never been reported so far but one long term concern, adding to the scarcity of unique drag targets in fungi, is the limitation of fundamentally different types of antifungal agents that are available for treatment.

Amphotericin B is a broadband antimycotic agent whose effect and function is amply described in patent US 2,908,611. However, its use is limited by its high kidney, liver and myelotoxicity. Because of its poor solubility it can be applied in form of colloids or liposome formulations (US 4,663,167; EP 0 421 733). In a further patent the use of complexing substances is recommended to combat fungi (GB 2033220). Complexing substances deprived fungi of zinc ions, required for their metabolism. New approaches of antimycotic therapy are based on activating the body's own defence. By activation of human dendritic cells by fungal proteins or nucleic acids a cellular immune response is meant to be caused. The method has the disadvantage of being species specific (Bozza et al., 2004) and not having a broad antimycotic reaction. New options for therapy are therefore more than desirable, in particular as amongst pathogenic fungi, antibiotic resistance is increasing. For example, amongst the Candida species an increasing number of C. glabrata and C. krusei strains is resistant primarily to fluconazole (13th European Congress of Clinical Microbiology and Infectious Diseases, Glasgow, June 2003).

The present invention relates to novel compounds having antifungal activity; to a process for preparation of said compounds, comprising isolation of said compounds from the fermentation product of a microorganism producing said compounds; to medicaments containing said compounds as an active ingredient; to pharmaceutical compositions containing said compounds as an active ingredient; to therapeutic or prophylactic agents for fungal infectious diseases containing said compounds as an active ingredient; to a microorganism producing said compounds; to uses of said compounds; and to methods for treating or preventing fungal infectious diseases, comprising administering a pharmaceutically effective amount of said compounds to an animal. The problem to be solved by the present invention is to provide a pharmaceutical composition useful to treat dermal diseases, caused by fungi and yeasts with inflammation and/or associated with a bacterial infection, which provides a fast relief of the symptomatology and reduces side effects The inventors have found novel compound having antifungal activity in products of a microorganism, Streptomyces sp. H7372 strain that was obtain from a mangarove soil sample collected in Malaysia. Moreover, the present invention provides an objective of utilizing the novel compound for being effective against human pathogen yeasts Candida spp, moulds Aspergillus spp and Ctγptococcus spp. More particularly, antifungal compound is effective against Candida spp ATCC and clinical isolates, the fluconazole- resistant Candida clinical isolates, Aspergillus spp ATCC strains and Cryptococcus clinical isolates.

It is another objective of the present invention to provide a process of preparation antifungal compound J5 by fermenting a nutrient medium under aerobic condition by a novel Streptomyces, more specifically Streptomyces sp. H7372, Yet, another objective of the present embodiment was to provide a high performance liquid chromatography (HPLC) purified antifungal compound J5, from the fermentation of Streptomyces sp. H7372. It is another objective of the present invention to identify a novel Streptomyces, Streptomyces sp. H7372 which produced compound J5, by means of cell wall chemistry and molecular phylogenetic analyses. It is another object o the present invention to provide information on antifungal property, by inhibiting the growth of microorganism, particularly fungi, by employing the antifungal compound J5 obtained as a fermentation metabolite of Streptomyces sp. H7372.

SUMMARY OF THE INVENTION

The present invention relates to a biologically pure culture of Streptomyces sp. H7372 or a mutant thereof is obtained from a microorganism isolated from mangrove soil, wherein the microorganism is deposited under the accession number V07/019103 at National Measurement Institute, Australia. Preferably, the biologically pure culture of Streptomyces sp. H7372 or a mutant thereof is capable of producing an antifungal compound J5. It is understood that the culture having the capability to inhibit the growth of fungi including Candida spp, molds Aspergillus spp and Cryptococcus spp. Preferably, the Candida species includes Candida albicans, Candida krusei, Candida glabrala, Candida rugosa, Candida parapsilosis, Candida lusitaniae and Candida tropicalis and the Candida species are of fluconazole-resistant strains.

Preferably, the Aspergillus spp includes Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus lentulus, Aspergillus niger and Aspergillus nidulans and the Cryptococcus includes Cryptococcus neoformans and Cryptococcus humicolus.

Moreover, the present invention also relates to a method of preparing antifungal compound, the compound J5, wherein the method includes; obtaining the microorganism from mangrove soil,characterizing and indentifying the microorganism, obtaining the

Streplomyces sp. H7372, verifying the Streptomyces sp. H7372 by using molecular biology methods, preparing a culture containing the Streptomyces sp. H7372 to obtain a culture for crude cell extract and isolation of bioactive compound, obtaining the antifungal compound from the crude cell extract, verifying and purifying the antifungal compound. It is understood that, the culture having a working temperature between 2O⁰C and 40°C and a working pH between 5 and 8 and the antifungal compound having a purity at least 90%.

Another embodiment of the present invention relates to a use of a pharmaceutically effective amount of a compound for the manufacture of pharmaceutical compositions for treating or preventing fungal infections to an animal or human in need of such treatment.

In addition, the present invention also states a pharmaceutical composition containing a compound or a salt thereof as an active ingredient and a pharmaceutically acceptable carrier and the composition is suitable for topical, enteral, or parental administration to a subject. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows colonies of pure culture of Streptomyces sp. H7372 on oatmeal agar.

Figure 2 shows positively gram stained mycelia of Streptomyces sp. H7372.

Figure 3 shows spiral characteristic of aerial mycelia of Streptomyces sp. H7372.

Figure 4 shows spores produced by Streptomyces sp. H7372.

Figure 5 shows phylogenetic standing of Streptomyces sp. H7372 in Actinomycetales.

Figure 6 shows standing of Streptomyces sp. H7372 with its closely related Streptomyces strains obtained from BLAST result.

Figure 7 shows chromatogram for isolation of antifungal containing fraction, J5.

BRIEF DESCRIPTION OF THE INVENTION Additional advantages and novel features of the invention will be set forth in part in the description that follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

The present invention provides novel compounds having antifungal activity; a process for preparation of said compounds; medicaments containing said compounds as an active ingredient; pharmaceutical compositions containing said compounds as an active ingredient; therapeutic or prophylactic agents for fungal infectious diseases containing said compounds as an active ingredient; a microorganism producing said compounds; uses of said compounds; and methods for treating or preventing fungal infectious diseases comprising administering an effective amount of said compounds to an animal. (1) The present invention includes a new antifungal compound or a salt thereof. (2) The present invention includes a compound having the following physicochemical properties, or a salt thereof.

The invention provides compound that is capable of inhibiting the growth of fungal cells and are useful as anti-fungal agents. The invention further provides the methods of inhibiting the growth of fungal cells and methods of treating a fungal infection in an animal or human by administering to the animal or human an effective amount of a compound, either alone or in combination with another anti-fungal agent.

The present invention relates to an antifungal compound designated as J5 as well as an crude acetone extract which effective against human pathogen yeasts Candida spp, molds Aspergillus spp and Cryptococcus spp. The Candida species which found susceptible to antifungal compound J5 included Candida krusei, Candida rugosa, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida lusitaniae. The Aspergillus species which susceptible to active crude extract are Aspergillus niger, Aspergillus terreus, Aspergillus fumigatus, Aspergillus flavus, Aspergillus lentulus and Aspergillus nidulans. The Cryptococcus species susceptible are Cryptococcus neoformans and Cryptococcus humicolus. The MICs of crude acetone extract and antifungal compound J5 were showed in Table 1. Table 1. Minimum Inhibitory Concentrations Determination. Results were obtained from at least three independent experiments performed in duplicate except for * which obtained from one experiment. CI= Clinical isolate, FIc-R= fluconazole resistant, ^ Prominent decrease in turbidity, NT= not tested.

Crude Extract J5 Fluconazole

Tester Strains MIC" value μg/ml MIC^a value μg/ml MIC^a value μg/ml

24H 48H 24H 48H 24H 48H

Strain

Candida albicans ATCC 14053 313 313 > 20 > 20 1- 0.5^φ 1*

CI 2696 313 625 >20 >20 j t j Φ

CI 3072 313 625 >20 >20 j Φ j Φ

Candida glabrata ATCC 2001 78 78 2.5 5 8 >128

CI 29907 FIc-R 39 78 0.625 1.25 >128 >128

CI 30547 FIc-R 39 39 0.625 1.25 >128 >128

CI 32338 FIc-R 39 39 0.625 0.625 >128 >128

Candida kriisei ATCC 6258 20 78 0.313 1.25 16 32

CI 2638 39 78 0.313 1.25 32 64

CI 2722 39 78 0.313 1.25 32 64

CI 2748 39 78 0.313 1.25 16 64

CI 3109 FIc-R 39 78 0.625 1.25 64 64

Candida lusitaniae ATCC 66035 39 39-78 1.25 2.5 0.025 0.025

Candida

ATCC 22019 39 78 2.5 10 2 4 parapsilosis

Candida ritgosa ATCC 10571 78 78 0.625 2.5 2 4

CI 2610 78 156 0.625 2.5 4 4

CI 2715 78 78 1.25 2.5 4 4

CI 2745 78 156 0.625 2.5 4 4

5000-

Candida tropicalis ATCC 750 156 5 >20 8 64 2500

Aspergillus flavus ATCC 10124 313 > 20

Aspergillus

ATCC 36607 156 >20 fumigatus

Aspergillus ATCC MYA- V) π lentulus 3566

Aspergillus

ATCC 10074 625 >20 nidulam

Aspergillus niger ATCC 16888 313 >20 NT

Aspergillus

ATCC 1012 1250 terreus >20

Cryptococcus neoformans* CI- Jl 39 78 0.313 1.25

Crptococcus

CI- J2 313 313 humicolus* 10 20 All pathogenic fungi tested and showed generally susceptible to crude acetone extract of aerobic fermentation culture from Streptomyces sp. H7372, antifungal compound J5 and the reference antifungal drug fluconazole. The growth inhibition confirmed the presence of antifungal compound in the crude extract which produced by the strain H7372 in the fermentation medium as a secondary compound. When MICs were read, each well was thoroughly re-suspended to produce homogenous cell suspension and only wells with inhibitor concentration which produced clear vision or light transparent culture broth viewed from the bottom of 96- wells under normal light were read as the MIC. All Candida species tested were susceptible to crude extract (MIC_24H from 20 to 313μg/ml) and antifungal compound J5 (MIC₂₄H range from 0.313 to 5μg/ml) except C. albicans which did not susceptible at 20μg/ml of J5. MIC values of J5 revealed a good susceptibility of several Candida species to this active fraction with readings at 24H of <5μg/ml. The susceptibility patterns of Candida species to J5 were interesting and differed from the susceptibility patterns of other currently available clinically used azoles, echinocandins and polyenes as the Candida albicans, which are normally susceptible at low concentration to those compounds were not susceptible to J5 at the highest concentration tested, which was 20μg/ml. Some non-albicans were highly susceptible to J5. C. krusei, C. rugosa and C. glabrata including the fiuconazole- resistant strains of those species were susceptible at <0.625μg/ml and the most susceptible fungi were C. krusei and Cryptococcus neoformans with MIC as low as 0.313μg/ml.

In one embodiment, the present invention is to provide a purified antifungal compound J5 using high performance liquid chromatography (HPLC) technique. The process comprises of steps of (a) production of crude acetone extract from the aerobic fermentation broth of novel Streptomyces sp. H7372 in which the antifungal compound J5; recovering antifungal compound J5 from the crude extract using HPLC technique. Initially, the fermentation broth is combined with acetone at volume range from 0.5-1.5 times the volume of the total collected fermentation broth, preferably equal volume of acetone. In a preferred embodiment, the resulting extraction mixture is kept in chiller with cooling temperature of 2-8C, preferably 4⁰C, for 3-7days, preferably 3 days, more preferably 5 days, and most preferably 7 days, before proceeded for further purification process. The mixture is then filtered through Whatman filter paper [Whatman 1] followed by removal of acetone using rotary evaporator at ambient temperature. The resulting product is subjected to freeze dry to obtain dry powder material. A defined amount of crude acetone extract of Streptomyces sp. H7372 fermentation broth is dissolved in ultrapure water at ambient temperature. An Alliance HPLC instrument (Waters) with an octadecyl silica reverse phase column (250mm X 4mm; Merck) was used at ambient temperature. The mobile phase consisted of solvent A (100% water) and solvent B (acetonitrile) was used at flow rate of 5ml/minute. The active fraction containing antifungal compound J5 was detected at UV-spectra 200nm and isolated at retention time (RT) of 20-21 minutes with ACN/H2O of 37/63%, revealing the slightly hydrophobic property of this compound. The collected fraction containing J5 is freeze-dried to obtain white cotton like powder. It is this cotton like powder which processes antifungal activity.

In another embodiment, the present invention provides a fermentation process for preparing the antifungal compound J5. In accordance with the present invention, the cultures were performed in a culture medium that comprises a carbon source and a nitrogen source. The carbon source and the nitrogen source and additional nutritional requirements may be conveniently determined by one skilled of the art. The carbon source used in the described experiments for Streptomyces sp. H7372 was mannitol. Illustrative examples of the other suitable supplemental carbon sources include, but are not limited to, other carbohydrates, such as glucose, fructose, starch or starch hydrolysate, cellulose hydrolysate and molasses; organic acids, such as acetic acid, propionic acid, lactic acid, formic acid, malic acid, citric acid; and alcohols, such as glycerol, inositol, mannitol and sorbitol.

The nitrogen source used in the described experiments for Streptomyces sp. H7372 was soybean flour Type 1. Illustrative examples of suitable nitrogen sources include, but are not limited to, ammonia, including ammonia gas and aqueous ammonia; ammonium salts of inorganic or organic acids, such as ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium sulfate and ammonium acetate; urea; nitrate or nitrite salts, and other nitrogen- containing materials, including amino acids as either pure or crude preparations, meat extract, peptone, fish meal, fish hydrolysate, corn steep liquor, casein hydrolysate, soybean cake hydrolysate, yeast extract, dried yeast, ethanol-yeast distillate, soybean flour, cottonseed meal, and the like. The amount of each of these ingredients to be employed is preferably selected to maximize the production of antimicrobial agent. Such amounts may be determined empirically by one skilled in the art according to the various methods and techniques known in the art. The fermentation culture conditions employed, including temperature, pH, aeration rate, agitation rate, culture duration, and the like, may be determined empirically by one of skill in the art to maximize the production. The selection of specific culture conditions depends upon factors such as the medium composition and type, culture technique, and similar considerations. In a preferred embodiment of the present invention, cultivation takes place at a temperature in the range of about 20°C to 4O⁰C, preferably at 28⁰C, and at a pH in the range of 5 to 9, preferably in the range of 6.5 to 7.5. Preferably, the time of fermentation is about 3 days to about 12 days, preferably about 5 days. Preferably, the volume of fermentation broth is about 10ml to 25ml in a 125ml conical flask, most preferably, 10ml of fermentation in 125ml conical flask. The culture conditions employed can, of course, be varied by known methods at different time-points during cultivation, as appropriate, to maximize production of the antimicrobial compound. Fermentation culture of the microorganism strain may be accomplished using any of the submerged fermentation techniques known to those skilled in the art, such as airlift, traditional sparged-agitated designs, or in shaking culture.

In another embodiment, the objective of the present is to identify the novel species level of Streptomyces sp. H7372. Streptomyces sp. H7372 was isolated using HV agar without artificial sea water. The strain forms milky-white chalk-like appearance colonies within the first week on oatmeal agar. The aerial mycelia turned brownish-grey on the third week. Dark brownish spore were observed which cover all colonies on the agar on the forth week. Streptomyces sp. H7372 tolerated 6% of saline as compared to terrestrial actinomycete which normally tolerate up to 4% saline, a unique physiological characteristic of actinomycete isolated from mangrove swamp near to sea shore. As shown in Table 2, Streptomyces sp. H7372 produced well-developed (colours of colonies) sporophores on most media listed and generally showed good growth on most media.

Table 2. Cultural characteristics of Strain H7372 on different media. Well sporulation was observed in all test media. Diffusible pigments and melanin were not produced on any of the media listed.

Medium .. ψ Aerial mycelia* Sporulation

NaCl (6%) agar medium White Beige ++ Yeast extract-malt extract agar (ISP medium 2) White Brownish beige -H- Oatmeal Agar (OA) (ISP medium 3) Whitish Gray Beige ++ Inorganic salts-starch agar (ISP medium 4) Grayish white Beige + Glycerol-asparagine agar (ISP medium 5) White Yellowish white ++ Melanin production test medium : Tyrosine agar White Beige ++ (ISP medium 7)

- No sporulation; + Moderate sporulation ; ++ Good sporulation φ Colours taken from Nippon Paint Catalogue 9000

No extracellular pigments and diffusible pigments were produced. Aerial hyphae were developed on most media tested and well sporulated on most media except on inorganic salts- starch agar (ISP medium 4). Strain H7372 utilized all simple carbon source tested such as (Table T). The cell wall of Streptomyces sp. H7372 was detected to contain only LL-DAP. A nearly full sequence of amplified 16S rDNA sequence of Strain H7372 (1439 nucleotides) was subjected to NCBI BLAST. The BLAST result showed Streptomyces sp. H7372 shared high level of sequence homology (97 to 98.45%) to members of Streptomyces shown in Figure 6. Streptomyces sp. H7372 was clustered in the true Streptomyces clade (Figure 5 and 6) forming a distinct clade from its related members. Strain H7372 has been deposited at the National Measurement Institute in Australia (Accession number: V07/019103). The almost complete sequence of 16S RNA gene from Strain H7372 was deposited in GenBank with nucleotide accession number FJ014942. Streptomyces sp. H7372 formed a distinct monophletic clade with members of the genus true Streptomyces from the members of Kitasatospora and Streptacidiphilus (Figure 5). The association in the true Streptomyces clade was further revealed in the maximum-likelihood tree shown in Figure 6 where Strain H7372 was placed in a distinct subclade from the related & kasugaensis clade which supported with high bootstrap value (80%). The ability to tolerate 6% saline and the distinct clade formed in phylogenetic trees (Figure 5 and 6) by Strain H7372T has provided strong evidences on the new species status of this Streptomyces. DETAILED DESCRIPTION OF THE INVENTION

BEST MODE TO CARRYO OUT THE INVENTION

Examples

The following examples are provided for illustrative purposes only, it is to be understood that both the foregoing description and the detailed description are not intended to limit the scope of the present invention. The following experimental scheme is provided in detailed hereinafter for illustrating the: a) isolation of pure culture of Streptomyces sp. H7372; b) preparation of active crude acetone extract; and c) isolation and purification of antifungal compound J5. All procedures were done in ambient temperature unless stated otherwise.

Isolation of pure culture of Strain H7372'

A) Soil sample collection and preparation

Actinomycete Strain H7372 is a novel strain isolated from dark brown dry mud (mud pH 7.31) under the root of mangrove tree (Bruguiera sp), located at sea shore near mouth of river into mangrove swamp, Kampung Termunong, Tuaran, Sabah, Borneo, Malaysia. Mud samples were air dried for 48 hours at 30⁰C before isolation of soil microoganisms. The soil or mud sample (0.05g) was suspended in 9.95ml of sterile Artificial Sea Water (ASW) and mixed using a vortex mixer for 30s. The suspension was diluted to 10-3. A 0.1ml aliquot was spreaded onto isolation media using a L-shape spreader. Four types of isolation media were used. There were (1) Humic acid -vitamin agar without ASW, (2) Humic acid -vitamin agar with ASW, (3) Starch Casein agar without ASW, and (4) Starch Casein agar with ASW. Each of these media was incubated at 28°C for 7 -14days. Compact, dry and chalk like colonies of different colours were picked out and transferred onto Oatmeal agar (ISP medium 3) for purification and growth.

B) Characterization of Actinomycete Strain H7372

Cultural characteristic: There were 4 types of cultural media used for this test. There were yeast extract -malt extract agar (ISP medium 2), Inorganic salts-starch agar (ISP medium 4), Glycerol-asparagine agar (ISP medium 5) and Nutrient Agar (Waksman medium 14). The plates were inoculated by streaking with 7 days old oatmeal agar (ISP medium 3) culture of actinomycetes strains. The colour of aerial mycelium, substrate mycelium and diffusible soluble pigments were observed under normal daylight by using Nippon Paint Catalogue 9000 as a standard colour guide. The ability of sporulation was also recorded. The physiological characteristics investigated comprise of a) Carbon utilization; b) melanin/pigment production; and c) salt tolerance.

Carbon utilization: Spore suspension of actinomycetes was used as inoculums for this test. 0.05ml of spore suspension pipetted onto one edge of the basal agar medium surface. The drop of spore suspension was streaked across the petri dish. A second drop was repeated. Two plates were prepared for each strain. The plates were observed at 10-16 days. The growth was compared on a given carbon source with the two controls: growth on basal medium alone and growth on basal medium plus glucose. The results were recorded according as followings: Strong positive utilization (++): when growth on tested carbon in basal medium is equal to or greater than growth on basal medium plus glucose. Positive utilization (+): when growth on tested carbon is significantly better than on basal medium without carbon, but somewhat less than on glucose. Utilization doubtful (±): when growth on tested carbon is only slightly better than on the basal medium without carbon and significantly less than with glucose. Utilization negative (-): when growth is similar to or less than growth on basal medium without carbon. Table 3 shows carbon utilization profile of Streptomyces sp. H7372.

Table 3. Carbon utilization of Strain H7372

Carbon source Utilization

D- Fructose ^bPositive utilization.

D- Glucose ^aStrong positive utilization

I- Inositol ^bPositive utilization

D- Mannitol ^aStrong positive utilization

D- Sucrose ^bPositive utilization.

'When growth on tested carbon in basal medium is equal to or greater than growth on basal medium p plluuss gglluuccoossee

' WWhheenn g grroowwtthh oonn tteesstteedd ccaarrbon is significantly better than on basal medium without carbon, but somewhat less than on glucos Melanin Production: Production of melanoid pigments was determined on Tyrosine agar (ISP medium 7). Inoculums source was 10-14 days old of actinomycetes culture on oatmeal agar. Heavy inoculum of spores and aerial mycelium were picked up with an inoculating wire loop. The inoculum was streaked on the surface of Tyrosine agar plate. Each of experimental culture was inoculated onto two agar plates of Tyrosine agar. Melanoid pigments on Tyrosine agar was observed after 4 days. The inoculated plates were compared with uninoculated controls. Cultures forming a greenish brown to brown to black diffusible pigment or a distinct brown pigment modified by other colour would be recorded as positive (+). Absence of brown to black colours, or total absence of diffusible pigment would be recorded as negative (-) for melanoid pigment production. If diffusible soluble pigment other than melanins was produced on culture medium, response of colour to pH change was determined by addition of a drop of 0.05N NaOH and 0.05N HCl to the coloured agar. Observation on whether or not soluble colour affected by pH change was recorded after 10 to 15 minutes.

Salt tolerance test: A range of 0 to 15% (at 1% increments) NaCl-incorporated Nutrient Agar (NA) medium were prepared, autoclaved and dispensed into petri dishes. The plates were inoculated by streaking with 7 days old oatmeal agar (ISP medium 3) culture of Strain H7372. The 0% NaCl plate was used as control in this invention. The plates were incubated at 28°C for 5-7 days. The growth was observed visually and recorded. The growth was compared to the growth in control plate. The highest NaCl percentage which Strain H7372 could tolerate was recorded. The ability of sporulation at different salinities was also recorded.

Taxonomy Analyses of Strain H7372

One Dimentional Thin Layer Chromatography (TLC) for DAP determination

Single colony of each strain inoculated in 10 ml fermentation medium (Soybean- mannitol: for 100ml, soybean flour, 2g; D-mannitol, 2g; pH7.2). Aerobic growth was done at 28 ⁰C at 220 rpm for 5 days. The cell culture was centrifuged for 10 minutes at 1 l,000rpm. The supernatant was discarded and cell pellet was weighed (the wet weight of cell pellet must be more than 300mg where 10% of the wet weight is considered dry weight). The cell pellet was hydrolysed with 1 ml of 6N HCl in a screw capped tube at 100°C for 18hrs. After cooling, the hydrolysate was filtered by a wet filter paper (Whatman No. 1 Filter paper). The filtrate was evaporated to dryness in boiling water bath, redissolved with 1 ml distilled water, and taken to dryness again. The residue was redissolved in distilled water (0.3ml) and transferred to an Eppendorf tube. Sample of residue was applied as aliquots (2 μl) onto the baseline of a cellulose TLC plate (20 x 20 cm, Merck 5716). As a standard, 2 μl of 2, 6- diaminopimelic acid (Riken) also applied. TLC was developed with the solvent system methanol-water-6N HCl-pyridine (80:26:4:10, v/v). Ascending chromatography was performed for approximately 4 hrs with the solvent system that was until the solvent front had nearly reached the top of the plate. The plates was dried in a fume cupboard and spots visualized by spraying with ninhydrin in acetone (0.2%, w/v) followed by heating at 100°C for 5 minutes.

Ribosomal (16S) DNA Sequence

To determine if actinomycete Strain H7372 was a novel species of Streptomyces, 5 days old actinomycete Strain H7372 on oatmeal agar were grown in 25ml fermentation broth (Soybean-mannitol) shake at 200rpm, 280C for three days. Cells (2ml) were harvested by centrifuging (10000 rpm for 2 minutes), washed twice with ImI of sterile distilled water. The actinomycete pellet was resuspended in 500μl lysis buffer (5OmM Tris HCl pH 7.5, 1OmM EDTA and 0.5 % β-mercaptoethanol). The cell suspension was incubated in 50°C dry incubator for 60 minutes with constant inversion of the tubes. SDS and Proteinase K were added into the suspension to final concentration of 1% v/v and 0.4mg/ml, mixed gently and the mixture was incubated at 56° C for 60 minutes. Then, the tubes were boiled at 100° C for 5 minutes to inactivate the Proteinase K. For removal of residual proteins, RNAs and cell debris, the mixture was extracted twice with 250μl of phenol/chloroform/isoamylalcohol (25:24:1). Then, ethanol precipitation was carried out and the DNA pellet was air dried. PCR primers and condition were adapted from work by Cook and Meyers [29]. Briefly, the primer sequences were Fl (5'- AGAGTTTGATCITGGCTCAG-S'; I= inosine) and R5 (5'- ACGGITACCTTGTTACGACTT-3'). The primers were used to amplify nearly full sequence of the 16S rRNA gene of Streptomyces ambofaciens ATCC 23877^T (GenBank accession number: M27245). Sequence alignment and phylogenetic studies

Reference strains used in this present embodiment of the invention were chosen from

BLAST search result in NCBI database. Multiple alignment of 16S rDNA sequence of actinomycete Strain H7372 and other reference strains and manual calculation of levels of sequences similarity were carried out using ClustalW 1.81. Phylogenetic trees were constructed using Neighbour- Joining method by using Kimura 2 parameter and

Maximum Composite- Likelihood algorithm contained in the treeing software MEGA version 4. The topology of trees was evaluated by using bootstrap resampling method of Felsenstein with 1000 replicates.

Preparation of crude cell free extract and isolation of bioactive compound

A) Fermentation culture of Strain H7372

Strain H7372 was maintained on oatmeal agar. 10ml of Medium C (2% D-mannitol [Fluka, Biochemika 63566], 2% Soybean flour Type 1 [Sigma S9633] at ρH7) in 25ml conical flask was inoculated with a loopful of fresh five days old culture and shaked at 200rpm for 5 days in 28⁰C incubator [ DAIKI, DKS 1020].

B) Preparation of cell free extracts (Acetone crude extract) At the end of fermentation, fermentation broth was prepared as follow. The resulting 300ml of fermented culture media containing the actinomycete Strain H7372 and the associated bioactive antifungal compound were transferred to a IL Erlenmeyer flask. The said fermented culture was mixed with an equal volume of acetone [Merck]. The resulting mixture was kept in 4OC chiller for 5 days before proceeded for further extraction process. The mixture (~600ml) was then filtered through Whatman filter paper [Whatman 1] followed by removal of acetone at temperature 42 C using rotary evaporator set to rotate at 50 rpm for 40-60 minutes. The resulting product (-280- 300ml) was subjected to freeze dryer [TELSTAR Cryodos 50], set at -50⁰C, to obtain dry powder material which contains antifungal compound J5. C) HPLC isolation of Bioactive Fraction

The freeze-dried crude cell free extract which containing antifungal compound J5 was dissolved in water and filtered through 0.22um filter unit [Millex GS, Millipore]. An Alliance HPLC instrument (Waters) with an octadecyl silica reverse phase column

(250mm X 4mm; Merck) was used at ambient temperature. The mobile phase consisted of solvent A (100% water) and solvent B (acetonitrile) was used at flow rate of 5ml/min. The column was pre-condition with 95% water and 5% of acetonitrile.

After injection of the sample, the solvents gradient was programmed from 5% to 50% solvent B over 30 minutes. The eluates were detected at 200nm by a dual absorbance detector (Waters) and collected with fraction collector.

Antifungal Susceptibility Testing

Tester isolates were subcultured onto Sabouraud dextrose agar plates for 24 hours at 37⁰C. The Minimum Inhibitory Concentrations, MICs of fluconazole and crude cell free extract and active fraction were determined by broth microdilution method from National Committee for Clinical Laboratory Standards (NCCLS) Document M27-A2 (NCCLS 1997) and M38-A (NCCLS 2002) but with higher concentration for yeast cell (105cell/ml). Fluconazole was purchased from Sigma Inc (USA) and dissolved in DMSO at 100 times of the final concentrations used in the assay while crude extract and HPLC fractions were dissolved solely in ultrapure water at 2 times of final assay concentration, prefiltered through 0.22μm filter (Millex-GS, Millipore) prior to use in assay. The final concentration of fluconazole ranged from 1 to 128 μg/ml and HPLC fractions were ranged from 0.5 to 20μg/ml while crude extract ranged from 10- 5000μg/ml. The MIC endpoints were read visually and defined as lowest concentration which produced clear well or no visible turbidity compared with that of drug-free growth control following 24 and 48 hours incubation. Prominent growth reduction is defined as >90% turbidity reduction in treatment wells compare to growth control wells measured by optical density at 530nm. The quality control strains, Candida krusei ATCC 6258 and Candida parapsilosis ATCC 22091 were included in each test runs. MICs reading were determined from duplicate readings of three occasions.

Claims

1. A biologically pure culture of Streptomyces sp. H7372 or a mutant thereof is obtained from a microorganism isolated from mangrove soil, wherein the microorganism is deposited under the accession number V07/019103 at National Measurement Institute, Australia.

2. The biologically pure culture of Streptomyces sp. H7372 or a mutant thereof is capable of producing an antifungal compound J5.

3. The biologically pure culture as claimed in claim 1, wherein the culture having the capability to inhibit the growth of fungi including Candida spp, molds Aspergillus spp and Cryptococcus spp.

4. The biologically pure culture as claimed in claim 3, wherein Candida species includes Candida albicans, Candida b'usei, Candida glabrata, Candida rugosa, Candida par apsilosis, Candida lusitaniae and Candida tropicalis.

5. The biologically pure culture as claimed in claim 4, wherein the Candida species are of fluconazole-resistant strains.

6. The biologically pure culture as claimed in claim 3, wherein Aspergillus spp includes Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus lentulus, Aspergillus niger and Aspergillus nidulans.

7. The biologically pure culture as claimed in claim 3, wherein the Cryptococcus includes Cryptococcus neoformans and Cryptococcus humicolus.

8. A method of preparing antifungal compound, the compound J5, wherein the method comprising the steps of:

a) obtaining the microorganism from mangrove soil, b) characterizing and indentifying the microorganism, c) obtaining the Streptomyces sp. H7372 from step (b), d) verifying the Streptomyces sp. H7372 from step (c) by using molecular biology methods, e) preparing a culture containing the Streptomyces sp. H7372 to obtain a culture for crude cell extract and isolation of bioactive compound, f) obtaining the antifungal compound from the crude cell extract from step (e), g) verifying and purifying the antifungal compound from step (f).

9. The method as claimed in claim 8(e), wherein the culture having a working temperature between 2O⁰C and 40°C and a working pH between 5 and 8.

10. The method as claimed in claim 8(g), wherein the antifungal compound having a purity at least 90%.

11. The use of a pharmaceutically effective amount of a compound according to any of claims 1 to 10 for the manufacture of pharmaceutical compositions for treating or preventing fungal infections to an animal or human in need of such treatment.

12. A pharmaceutical composition containing a compound according to any of claims 1 to 11 or a salt thereof as an active ingredient and a pharmaceutically acceptable carrier.

13. The pharmaceutical composition comprising as claimed in claim 12, wherein the composition is suitable for topical, enteral, or parental administration to a subject.