HK1056746B - Process for producing a culture of antrodia camphorata and product obtained thereby - Google Patents

Description

Method for producing culture of antrodia camphorata and product obtained by the method

Technical Field

The present invention relates to establishing a culture condition suitable for the large-scale production of pharmacologically active filtrate from a culture of Antrodia camphorata, in particular by optimizing the shaking rate and/or pH during the culture. The invention also relates to a method for obtaining pharmacologically active compositions from a culture of A. camphorata through a series of partial isolation processes. The invention also relates to the use of the aforementioned composition for the preparation of a pharmaceutical composition.

Background

Antrodia camphorata (Antrodia camphorata) [ (Zang & Su) s. — h.wu, Ryvarden & t.t.chang ], also known as "Antrodia camphorata" or "Antrodia camphorata" in taiwan, has recently been reported as a new fungal species characterized by cylindrical basidiospores (basidiospores) appearing on fruit bodies (fruitbodies), micro-amyloid skeletal hyphae, bitter-tasting, light cinnamon-coloured flat to umbelliform basidiocarpores (basidiocarps), and thick sporophytes (chlamydospores) and arthrospores (artrosconidia) in pure media. The growth of this fungus is very slow and limited to the species Cinnamomum kanehirai Hay (Lauraceae), a tree species specific to Taiwan. The detailed characteristics and classification of A.camphorata are described in Chang, T.T.et al, Antrodia cinnamomea sp.nov.onCinnamomum kanehirai in Taiwan, Mycol. Res.99 (6): 756 (1995) and Wu, S. -H., et al, Antrodia camparata ("niu-chang-ch"), newcombination of a medical funcus in Taiwan, bot. Bull. Acad. sin.38: 273-275(1997), the entire disclosures of which are incorporated herein by reference.

In Taiwan folk medicine, the fruiting body of A. camphorata is considered to have some medicinal effects on symptoms caused by poisoning, diarrhea, abdominal pain, hypertension, skin pruritus and liver cancer. However, "A. camphorata" is very expensive in the market due to its severe host specificity and rarity in nature, and failure in artificial cultivation. Undoubtedly, it would be of great industrial value to develop a low cost process for large scale artificial culture of this fungus.

The present inventors have now found that a. camphorata exhibits desirable pharmacological, in particular anti-tumor, activity when cultured in submerged fermentation. As disclosed in U.S. patent application No. 09/566,834, A.camphorata has been successfully cultured on a small scale in synthetic media such as Potato Dextrose Broth (PDB) and fructose as the major carbon source. The resulting cultures exhibited a dark red appearance with a redness index a.gtoreq.3, as measured using the Hunter's coordinate system, and this color correlated with a significant inhibitory effect on growth for certain tumor cell products. More importantly, the active ingredients acting on tumor cells, although not yet identified, have been found to be secreted from fungal mycelia into the liquid phase of the culture, so that pharmacologically active compositions can be easily obtained from the culture for industrial use.

Therefore, it would be highly desirable if such an advantageous method could be optimized for the mass production of fungal cultures. More preferably, the crude filtrate is partially separated for a desired pharmacological activity to obtain a useful composition enriched in the desired activity.

Disclosure of Invention

To meet the above-mentioned industrial needs, the inventors have conducted extensive research and development work. It has now been unexpectedly found that an optimal condition for the cultivation of a. camphorata on an industrial scale can be obtained by judiciously setting certain parameters within specific ranges. The present inventors have found that the pH and the shaking rate are critical factors during the cultivation.

Accordingly, a first embodiment of the present invention provides a method for producing a pharmacologically active culture of A. camphorata, comprising:

(a) inoculating a mycelium inoculum of an isolated strain of a strain of Antrodia camphorata in a medium suitable for growth of the isolated strain to obtain a first culture;

(b) subjecting the first culture resulting from the culturing in step (a) to a first shaking phase set at a first predetermined rate for a period of time allowing further growth of the inoculated isolate to obtain a second culture with proliferated mycelium; and

(c) subjecting the second culture from step (b) to a second shaking phase set at a second predetermined rate different from the first predetermined rate, subjecting the isolate to physiological stress.

According to a second embodiment of the present invention, there is provided a method for producing a pharmacologically active culture of A. camphorata, comprising:

(a) inoculating a mycelium inoculum of an isolated strain of Antrodia camphorata in a medium suitable for growth of the isolated strain; and

(b) culturing the culture obtained from step (a) by adjusting the pH of the culture to a pH in the range of 4.5 to 5.4 throughout step (b).

Preferably, the pH of the antrodia camphorata culture is adjusted to a pH in the range of 4.6 to 5.3, and more preferably in the range of 4.7 to 5.2, throughout step (b).

The invention also provides a method for obtaining a series of liquid fractions isolated from a culture of A. camphorata for a desired pharmacological activity, such as anti-tumor activity. Accordingly, a third embodiment of the present invention provides a method for obtaining a pharmacologically active composition from a culture of A. camphorata, comprising:

(a) inoculating a mycelium inoculum of an isolated strain of Antrodia camphorata in a medium suitable for growth of the isolated strain;

(b) culturing the culture obtained in step (a); and

(c) removing a substantial portion of the insoluble material from the culture, thereby obtaining a pharmacologically active solution; and

(d) treating the solution obtained in step (c) to obtain a pharmacologically active composition comprising fungal molecules having a molecular weight of no more than about 10 kDa.

Preferably, the resulting composition contains fungal molecules having a molecular weight of no more than about 3kDa, and more preferably no more than about 1 kDa.

In a fourth embodiment of the present invention, there is provided a method for obtaining a pharmacologically active composition from a culture of A. camphorata, comprising:

(a) inoculating a mycelium inoculum of an isolated strain of Antrodia camphorata in a medium suitable for growth of the isolated strain;

(b) culturing the culture obtained in step (a);

(c) removing a substantial portion of the insoluble material from the culture, thereby obtaining a pharmacologically active solution;

(d) treating the solution obtained in step (c) to obtain a separated fraction containing fungal molecules having a molecular weight of no more than about 1 kDa; and

(e) passing the separated portion obtained in step (d) through a water-immiscible phase and obtaining the pharmacologically active composition from the water-immiscible phase.

The water-immiscible phase of step (e) is preferably a stationary phase containing an effective amount of an adsorbent capable of selectively adsorbing hydrophobic fungal molecules. Eluting the stationary phase to obtain a pharmacologically active fraction.

In a preferred embodiment of the invention, the eluate obtained in step (e) is further subjected to a reverse phase partition chromatography, such as in a LichrosorbR RP-18 column (Merck), to obtain several pharmacologically active fractions.

The invention further provides pharmaceutical compositions for treating cancer or neoplastic disease, comprising a product obtained according to any one of the methods of the invention.

The invention further provides a method for treating cancer or neoplastic disease in a patient in need thereof by administering to said patient a composition comprising a product obtained according to any of the methods of the invention.

Drawings

The foregoing and other objects and features of the invention will be apparent from the following description of the preferred embodiments, taken in conjunction with the accompanying drawings, in which:

FIG. 1 shows the antitumor activity of the filtrate derived from the culture of A. camphorata cultured under two different shaking conditions;

FIG. 2 shows the pH shift of three A.camphorata cultures during the cultivation period;

FIG. 3 shows the antitumor activity of the filtrate derived from the culture of Antrodia camphorata of FIG. 2, wherein Antrodia camphorata is cultured at a pH controlled in three different zones;

FIG. 4 shows the antitumor activity of the filtrate from the enlarged scale A. camphorata culture;

FIG. 5 is a flow chart showing a process for purifying the filtrate of Antrodia camphorata with respect to molecular weight;

FIG. 6 is a bar graph showing the anti-tumor activity of the culture filtrate isolated according to FIG. 5, wherein the test cell lines include MRC-5, HeLa, AGS, Hep G2 and MCF-7;

FIG. 7 is a bar graph comparing the antitumor activity of an isolate isolated with Amberlite XAD-4 from a filtrate isolate containing fungal molecules having a molecular weight of no more than about 1kDa, wherein the test cell lines include MRC-5, HeLa, AGS, Hep G2 and MCF-7;

FIG. 8 is a spectral plot of the ethyl acetate eluate of FIG. 7 partially separated in a LichrosorbR RP-18 column; and

FIGS. 9-11 show the anti-tumor activity of several isolated fractions isolated in FIG. 8.

Detailed description of the preferred embodiments

The present invention relates generally to the establishment of culture conditions suitable for the large-scale production of pharmacologically active isolated fractions from a culture of A. camphorata. According to the present invention, the fungus Antrodia camphorata is cultured in a suitable liquid medium to maintain its vegetative reproduction in the mycelial state and to promote its pharmacological activity.

In the present specification, the term "suitable medium" means any medium that can provide an artificial environment suitable for the growth of A. camphorata and maintain its pharmacological activity. Preferably, the culture medium used in the present invention is suitable for promoting the production of pharmacologically active substances in the mycelium and for promoting the secretion of said substance(s) extracellularly.

Suitable media for use in the present invention include natural media known as "potato dextrose broth (potato dextrose broth"), as well as any synthetic media containing fructose as the principal carbon source. A potato dextrose broth can be prepared in the laboratory by, for example, moist heat sterilization of a mixture of 300.0 grams of diced potatoes, 20.0 grams of dextrose, and 1.0 liter of distilled water, or from commercial sources such as DIFCO.

The most preferred medium is any synthetic medium containing fructose as the major carbon source. If desired, other carbon sources such as glucose, sucrose, galactose, fructose, corn starch and malt extract, and combinations thereof, may also be included in the synthetic medium as adjuvants. Preferably, the carbon source is preferably in the range of 1.5 to 2.5 wt%, and more preferably in the content of 1.5 to 2 wt%, based on the total volume of the synthetic medium.

In addition to the carbon source, the synthetic medium may contain a nitrogen source, trace elements (such as inorganic salts), and optionally vitamins or other growth factors. Nitrogen sources include, but are not limited to, ammonium sulfate, ammonium nitrate, sodium nitrate, casamino acids (casamino acids), yeast extract, peptones, and tryptones, and combinations thereof. Preferably, according to the invention, the synthetic medium contains yeast extract as nitrogen source. The nitrogen source is preferably in the range of 0.2 to 2.0% by weight, and more preferably in a content of 0.5% by weight, based on the total volume of the synthetic medium.

According to the present invention, any available isolate of A. camphorata may be used in the cultivation process, as long as the microorganism used has the ability to produce a detectable amount of a pharmacologically active metabolite. Useful isolates of A.camphorata include, but are not limited to, CCRC35396 (deposited at the food industry research and development institute, New bamboo, Taiwan, 12.1.1994), CCRC 35398 (deposited at the food industry research and development institute, 5.3.2000), 35716 (deposited at the food industry research and development institute, Taiwan), and 930032 (deposited at the 1.27.2000). according to a preferred embodiment of the present invention, A.camphorata CCRC930032, also deposited at the American Type Culture Collection (ATCC), as deposited at PTA-1233, is used as a patent program.

To assess the ability to inhibit tumor cell growth, the crude filtrate and isolated fractions of A. camphorata were subjected to MTT colorimetric assay.

The term "MTT colorimetric assay" or "MTT-tetrazolium assay" as used herein refers to an anticancer drug screening procedure constructed by the developmental treatment program of the cancer treatment department of the national cancer institute in the 1980's (see, for example, Alley, M.C., et al, Feasibility of drug discovery with wires of human tumor use a microscopic diagnosis assay. cancer Res.48: 589-601, 1988; Scudience, D.A., cell, Evaluation of drug discovery/drug discovery for use in a clinical diagnosis system, cancer diagnosis of cancer research, C.10. filtration, C.32. and C. filtration, C.31. cell, et al, Evaluation of cell discovery, cancer diagnosis of cell discovery, C.31. filtration, C.31. filtration, filtration : 757-766, 1991).

In this assay, potential anticancer agents or natural products derived from plants or microorganisms (in this case, from the five A. camphorata isolates) were tested for their ability to fight against several groups of cell lines, each of which represents a major clinical classification of human malignancies. The number of viable cells per well was proportional to the amount of formazan * (formazan) produced, and formazan * was measured by the spectrometer via lysis. In principle, biologically active substances or natural products containing these substances can inhibit or even stop cell growth, so that only small amounts of formazan * are formed, which can be examined by means of MTT colorimetric analysis for important parameters in fungal cultures, such as the rate of oscillation and the pH value, in order to evaluate the effect of these parameters on the pharmacological activity of A. camphorata during the culture.

In the first set of experiments, an A.camphorata culture was divided into two portions and subjected to two different shaking procedures, one of which was performed to apply a constant and vigorous shaking to the fungus and the other to shake from a mild to vigorous two stages. By comparison, the two-stage oscillation according to the present invention can lead to early generation of pharmacological activity. The gentle shaking provided for the first stage is to obtain vegetative reproduction of the inoculated fungus to obtain mycelium of the cultured reproduction. The increased size of the mycelial pellets (mycelial pellets) obtained at the end of the culture can be used to indicate the success or failure of the temperature and shaking phase. The appropriate timing for switching the oscillation rate from mild to vigorous will vary widely depending on the fungal isolate selected. In general, when a fungal isolate is inoculated at 10% v/v based on the final volume of the culture, gentle shaking can continue for about 3 days (or about 72 hours) followed by an increase in the shaking rate. The vigorous shaking provided for the subsequent stage is to place the fungus under physiological stress. Under such pressure, a number of physiological changes are forced on a response by a. camphorata, including the promotion of the production of secondary metabolites that do not behave in a constant manner. It is believed that certain physiologically active fungal molecules can be "" stressed "" by this method to yield. Physiological pressure may be applied to other culture parameters of A. camphorata, such as aeration rate, nutrient adjustment, and heat pressure, either alone or in combination with the oscillation rate of the present invention. Suitable oscillation rates may be measured experimentally as described above or estimated based on data described below, such as in bioprocess engineering Principles, pages 150 to 151 of this book, compiled by Pauline M.Doran and published by Academic Press Ltd. In some cases, the fungal culture turned red on day 6 when the two-stage shaking was properly applied.

In a preferred embodiment of the invention, the two-stage shaking is carried out in a 5 liter fermenter (b.braun) with 3 liters of medium prepared, wherein the shaking is initially set at a rate of not more than about 300rpm, preferably about 200rpm, and subsequently raised to a rate of not more than about 400rpm, preferably about 500 rpm. In another preferred embodiment of the invention, the two-stage shaking is carried out in a 250 liter fermentation tank (Bio-Top) with a pre-set of 160 liters of medium, wherein the shaking rate is initially set at about 40rpm and subsequently raised to about 150 rpm.

In a second set of experiments, an A.camphorata culture was divided into three portions and cultured at pH controlled in three different zones throughout the culture period. By comparison, it was found that incubation at pH4.5 to 5.4 resulted in early generation of pharmacological activity. Preferably, the pH of the A.camphorata culture is adjusted to a pH in the range of 4.6 to 5.3 and preferably 4.7 to 5.2 throughout the culture period.

By means of the aforementioned useful parameters regarding shaking and pH, the method for culturing Antrodia camphorata according to the present invention can be successfully scaled up to a volume of 160 liters while maintaining the desired pharmacological activity derived from the fungus.

According to the process of the present invention, a pharmacologically active filtrate for various industrial uses can be obtained from Antrodia camphorata in an economical, efficient and time-saving manner.

The invention also relates to the establishment of an operable purification process whereby several new compositions enriched with pharmacologically active substances can be obtained for various medical uses. According to the invention, the purification process is carried out by selectively separating a crude filtrate of Antrodia camphorata to obtain a separated fraction containing fungal molecules having a molecular weight of no more than about 10kDa, preferably no more than about 3kDa, and more preferably no more than about 1 kDa. The separation method may be carried out by any conventional method for separating molecules on the basis of molecular weight (i.e., molecular sieve), and examples of such methods include gel filtration, density gradient purification, ultrafiltration, ultracentrifugation, and other similar conventional methods.

According to the invention, the fraction containing molecules having a molecular weight of not more than about 1kDa is further partially separated on the basis of polarity to obtain a water-immiscible phase and a pharmacologically active fraction is obtained therefrom. The water immiscible phase may be an insoluble solid phase or a water immiscible organic phase. In a preferred embodiment of the invention, the ≦ 1kDa separating portion is passed through a stationary phase comprising an effective amount of an adsorbent capable of selectively adsorbing the hydrophobic fungal molecule. Subsequently, the stationary phase is eluted to obtain a separated fraction having the desired pharmacological activity. In short, the stationary phase selectively captures and concentrates hydrophobic solutes believed to contain the desired pharmacologically active substance from the ≦ 1kDa separation fraction, so that inactive substances located within the flow through solution (flow through) can be removed. The adsorbent suitable for inclusion in the stationary phase may be any adsorbent bearing functional groups suitable for trapping hydrophobic materials from a mobile phase. An example of such an adsorbent is Amberlite XAD-4(Sigma) and its equivalent. The partial separation can be carried out by any conventional means, such as co-incubating the < 1kDa separation fraction with a batch of adsorbent or passing the < 1kDa separation fraction through a chromatography column packed with adsorbent, provided that the amount of pharmacologically active substance retained on the adsorbent surface is satisfactory. Suitable eluents (eluents) for eluting the bound material from the stationary phase are well known in the art and can be readily selected by one skilled in the art. Preferably, the eluent is an organic solvent having a polarity lower than water, and more preferably an organic solvent having a polarity lower than methanol. Preferred eluents include ethyl acetate and ethanol.

The eluate (eluate) exhibiting the desired pharmacological activity may be subjected to additional purification procedures based on other physical, chemical or biological properties. In a preferred embodiment of the invention, the eluate is further separated on the basis of the degree of hydrophobicity, more preferably the separation is performed in a column such as a LichrosorbR RP-18 column (Merck) and equivalents thereof. The resulting fractions were found to be pharmacologically active.

The foregoing findings strongly suggest that the pharmacological activity of A. camphorata is mainly derived from hydrophobic compounds having a molecular weight of not more than 1 kDa. This finding is in contrast to a previous hypothesis in which polysaccharides having an average molecular weight of 500 to 2,000kDa were identified as the main source of anti-tumor activity possessed by mushrooms (Mizuno, et al., anti-active bases from mushrooms Rev. Intl.11 (1): 23-61). Antrodia camphorata is reported to be rich in low molecular weight substances such as triterpenes, flavonoids (flavanoids), Steroids, sesquiterpene lactones (sesquiterpene lactones) and phenyl and diphenyl compounds (Chang, supra; Cherng, et al; Triterpinenes from Antrodia cindamomea Pyrochem.41 (1): 263 267 (1996); Chiang, et al, A sesquiterpene lactone, phenyl and biphenol compounds from Antrodia cindamomea Pyroluomea.Pythochem.39 (1): 613:. 1995); and Yang, et al, Steroids and Triterpinenes Antrodia cirrha a a a fungap of fungal Minnum.41 (1395)), but no pharmacological activity has been reported in connection with the fungus.

The purification process according to the invention provides compositions in which the active substance is concentrated and the inactive substance is further removed. The compositions are apparently of enhanced pharmacological effectiveness for human or animal subjects and are therefore suitable for use in various industrial applications such as the manufacture of pharmaceutical compositions or nutritional supplements. Accordingly, the invention also relates to the use of the novel composition as a medicament for the treatment of diseases, in particular cancer or tumour diseases, in a human or animal subject in need of such treatment, or as a nutritional supplement formulated in a form such as a food, beverage and/or animal feed.

The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention.

Example 1: preparation of Antrodia camphorata liquid culture

The original culture of A.camphorata isolate CCRC930032 was maintained at-80 deg.C, a small amount of the fungus was removed from the original culture and plated onto potato dextrose agar plate medium (PDA, from Difco). After the fungi had recovered viability, the cultures were transferred to potato dextrose agar slant medium. The slant medium was incubated at 25 ℃ and subcultured every two months. Such slant culture media are used as culture for operation (working cultures). To prepare the mycelium inoculum, cultures derived from PDA slant medium were inoculated onto PDA plates and incubated at 28 ℃ for 15 to 20 days.

Preparation of mycelium inoculum

The fungus is cultured until a colony of hyphae is observed having a diameter of 15 to 30 mm. The mycelia of A. camphorata were inspected under an optical microscope to ensure that it was not contaminated. The whole mycelium was cut into small pieces and then homogenized aseptically in a homogenizer (Osterizer) using 50ml of sterile water for 30 seconds. The amount of mycelium suspension is available as an inoculum for submerged shake flask culture. A500 ml Erlenmeyer flasks (Erlenmeyer flasks) were pre-filled with a synthetic medium (2% fructose, 0.5% (w/v) yeast extract (DIFCO), 0.1% (w/v) KH₂PO₄(Merck) and 0.05% (w/v) MgSO₄·7H₂O (Merck)), and the inoculum is added to the synthetic medium in a volume ratio of 1: 9. The submerged cultures were incubated for 5 days at 30 ℃ and subjected to constant shaking (shaking at 50rpm on a rotary shaker from Hotech). The resulting culture is used as an inoculum for subsequent large-scale cultivation.

Example 2: effect of the Rate of oscillation on the pharmacological Activity of fungal filtrates

A5-liter fermentor (B.Braun) was charged with 2.7 liters of a synthetic medium (2% fructose, 0.5% (w/v) yeast extract (DIFCO), 0.1% (w/v) KH₂PO₄(Merck) and 0.05% (w/v) MgSO₄·7H₂O (Merck)), and the strain of Antrodia camphorata CCRC930032 prepared in example 1 was added to the synthetic medium at a volume ratio of 1: 9. The culture was incubated at 30 ℃ and an aeration rate of 0.6 liter/min. The culture shaking rate was initially set at about 200rpm and after 74 hours of incubation, was again increased to about 500 rpm. At 48, 113, 170, 217 and 259 hours after inoculation of the mycelium, samples were obtained from the culture taken and then passed through a simple filter device consisting of a filter funnel, a conical flask and an air suction device to remove the majority of itAn insoluble substance. With ammonia (NH)₄OH) the pH of the filtrate obtained is adjusted to 7 and moist heat sterilization is carried out. The resulting samples were stored at 4 ℃ for subsequent MTT colorimetric analysis.

The non-inoculated medium was used as a negative control for the MTT assay.

Hep G2 tumor cells were selected for MTT colorimetric analysis. Before performing the analysis, cells were maintained in α -MEM medium (GIBCO BRL) supplemented with 10% calf serum (Hyclone) as the original culture. The tumor cell line is subcultured once or twice a week by detaching cells from a cell culture conical flask by using trypsin-EDTA (GIBCO BRL). Tumor cells were harvested, counted and plated at a concentration of 3,000 cells/well in a 96-well microtiter plate. The total volume of cell culture medium in each well was made up to 180. mu.l and incubated at 37 ℃ in a 5% CO medium bath₂The cultivation is performed in the cultivation box until every other day.

Three sets of samples, each in 20. mu.l portions, were added to the culture wells and the cultures were incubated for 72 hours under the incubation conditions described above. Subsequently, 20. mu.l of stock MTT (Merck) solution prepared beforehand in PBS solution (GIBCO BRL) at a concentration of 5mg/ml were added to each well.

At 37 ℃ in CO₂After an additional 4 hours incubation in the incubator, the supernatant from each well was removed and 100 μ l of 100% DMSO (dimethyl sulfoxide, available from Sigma) was added to dissolve the MTT-formazan * product. After thorough mixing with a mechanical plate mixer, absorbance at 540nm was measured using an ELISA reader (MRX, Dynex). Thus, the relative survival rate of tumor cells in the tested filtrate can be determined by dividing the absorbance of each experimental sample by the absorbance of the corresponding uninoculated control.

Comparative example 1:

example 2 was repeated except that the fungal culture was incubated at a constant rate of about 500rpm throughout the culture.

The relative survival rates of tumor cells treated with the filtrates obtained in example 2 and comparative example 1 are compared in Table 1 and FIG. 1.

TABLE 1

Sampling time (hours)	Relative survival Rate (%)
			Comparative example 1	Example 2
	48	63			79
113			62	48
	170	35			16
217			24	18
	259	19			19

In table 1, the inhibitory effect on Hep G2 tumor cells was compared for filtrates sampled at the indicated time points. For the filtrate obtained at 170 hours after inoculation of the mycelium, it was found that the Antrodia camphorata CCRC930032, which was shaken in two stages at 200 to 500rpm, could obtain a relative survival rate of 16% in the MTT assay, which is much more efficient than the culture cultured at a constant rate of 500rpm, since the latter only observed a higher relative survival rate of 35%. At subsequent time points (i.e., 217 and 259 hours), the antitumor activities of example 2 and comparative example 1 gradually approached, suggesting that the combination of initial low-speed shaking and subsequent high-speed shaking may lead to early production of the pharmacologically active substance in the culture.

Consistent results were observed in MTT assays using tumor cell lines such as AGS, HeLa and MCF-7 (results not shown).

Example 3: effect of pH on the pharmacological Activity of fungal filtrates

Three batches of synthetic medium (1.5% fructose, 0.5% (w/v) yeast extract (DIFCO), 0.1% (w/v) KH) with initial pH values of about 4.5 (test A), 5.0 (test B) and 5.5 (test C), respectively, were prepared₂PO₄(Merck) and 0.05% (w/v) MgSO₄·7H₂O (Merck)), and the inoculum prepared in example 1 was added to the synthetic media in a volume ratio of 1: 9. The resulting cultures were incubated as described in example 2, but the pH of each culture was monitored at predetermined time points and carefully adjusted to around the initial pH by addition of NaOH solution (Table 2). The incubation procedure lasted 336 hours. The timing of the addition of the NaOH solution depends on the selected pH. For example, run B and run C added NaOH solution from 192 and 168 hours, respectively, to maintain the pH at about 4.9 to 5.1 and about 5.4 to 5.6, while run a did not add NaOH solution until 240 hours later.

TABLE 2

Sampling time (hours)	pH
				Test A	Test B	Test C
	0	4.67	5.02				5.58
24				4.71	5.05	5.65
	40	4.74	5.09				5.7
72				4.81	5.15	5.71
	96	4.84	5.16				5.68
120				4.84	5.09	5.61
	144	4.84	5.01				5.52
168				4.84	4.9	5.37
	192	4.85	4.81				5.4
209				4.86	4.9	5.4
	240	4.86	4.9				5.4
264				4.8	4.9	5.4
	288	4.66	4.9				5.4
312				4.52	4.9	5.4
	336	4.37	4.9				5.39

At 96, 144, 192, 240, 288 and 336 hours after inoculation of the mycelium, samples were obtained from the culture samples and then passed through a simple filter device consisting of a filter funnel, a conical flask and a suction device to remove most of the insoluble material. With ammonia (NH)₄OH) the pH of the filtrate obtained is adjusted to 7 and moist heat sterilization is carried out. The resulting samples were stored at 4 ℃ for subsequent MTT colorimetric analysis. The resulting samples were subjected to MTT colorimetric analysis as described in example 2. The non-inoculated medium was used as a negative control for the MTT assay.

TABLE 3

Sampling time (hours)	Phase of Hep G2 cellsFor survival rate (%)
				Test A	Test B	Test C
	96	96	112				123
144				103	102	107
	192	67	85				112
240				8	19	91
	288	9	10				18
336				11	12	8

As shown in Table 3, the Antrodia camphorata filtrate sampled at the indicated time points was compared for its inhibitory effect on HepG2 tumor cells. For the filtrate obtained at 192 hours after mycelium inoculation, it can be seen that the cultured A. camphorata CCRC930032 in test A and test B gave lower relative survival rates than that observed in test C. This difference in antitumor effect is at its highest at 240 hours and decreases gradually at subsequent time points, which suggests that culturing at pH4.5 to 5.4, preferably pH4.6 to 5.3 and more preferably pH4.7 to 5.2, will lead to early production of pharmacologically active substances in the culture.

Consistent results were observed in the MTT assay using corresponding experiments with tumor cell lines such as AGS, HeLa and MCF-7 (results not shown).

Example 4: scale-up culture of Antrodia camphorata in 250L fermenter

160 liters of synthetic medium (1.5% fructose, 0.5% (w/v) yeast extract (DIFCO), 0.1% (w/v) KH) was previously placed in a 250 liter fermentation tank (Bio-Top)₂PO₄(Merck) and 0.05% (w/v) MgSO₄·7H₂O (Merck)), and the strain of Antrodia camphorata CCRC930032 prepared in example 1 was added to the synthetic medium at a volume ratio of 1: 9. The culture was incubated at 30 ℃ and an aeration rate of 0.6 l/min. The shaking rate of the culture was initially set at about 40rpm and after 70 hours of incubation, was again raised to about 150 rpm. The pH of the culture was adjusted to a pH in the range of 4.9 to 5.1 throughout the culture.

At 96, 144, 168, 186.5, 244 and 284 hours after inoculation with mycelium, several samples were obtained from the culture samples, followed by filtration by a conventional method to remove most of the insoluble material. The pH of the filtrate was adjusted to 7 with ammonia water, and wet heat sterilization was performed. The resulting samples were subjected to MTT colorimetric analysis in which HeLa, AGS, Hep G2 and MCF-7 cells were tested at initial concentrations of 1,000, 3,000 and 3,000 cells/well. The non-inoculated medium was used as a negative control for the MTT assay. The results are shown in table 4 and fig. 4.

TABLE 4

Sampling time (hours)	Relative survival Rate (%)
					HeLa	AGS	Hep G2	MCF-7
	96	89	83	91					61
144					70	67	49	38
	168	65	32	36					31
186.5					30	26	33	22
	244	23	34	42					26
284					25	37	43	26

Table 4 and FIG. 4 show that the Antrodia camphorata cultivation method according to the present invention can be successfully scaled up to a volume of 160 liters by setting the shaking rate and pH value within the preferred ranges described in examples 2 and 3.

Example 5: preparation of filtrate of Antrodia camphorata culture in synthetic medium

The whole mycelium of Antrodia camphorata CCRC930032 cultured on PDA plate medium was cut into small pieces, and then the mycelium was homogenized under aseptic conditions in a homogenizer (Osterizer) using 50ml of sterile water for 30 seconds to obtain a mycelium suspension. 200ml of synthetic medium (2% fructose, 0.5% (w/v) yeast) was placed in a 1 liter Erlenmeyer flaskPaste (DIFCO), 0.1% (w/v) KH₂PO₄(Merck) and 0.05% (w/v) MgSO₄·7H₂O (Merck)), and 20ml of the hypha suspension was added thereto. The submerged cultures were incubated for 14 days at 30 ℃ and subjected to constant shaking (shaking at 75rpm on a rotary shaker available from Hotech). After the cultivation is completed, the fungal culture is passed through a simple filter device consisting of a filter funnel, a conical flask and an air-extracting device. The crude filtrate obtained was used for subsequent analysis.

Example 6: preparation and analysis of active fractions derived from Antrodia camphorata filtrate

The crude filtrate (F0) obtained in example 5 was passed through a CerriprepR Concentrator 10 (a commercial mini-column from Amicon with a molecular weight cut-off of 10 kDa) by low speed centrifugation according to the manufacturer's guidelines. The primary stream is referred to as the separated fraction F1. The column was then refilled with deionization and centrifuged again to collect the second pass stream F2. The fungal molecule remaining in the column was harvested and designated F3.

F1 was partially separated by a CerriprepR Concentrator 3 mini-column (Amicon) with a molecular weight cut-off of 3kDa as described above, so as to obtain a first pass-through (F4), a second pass-through (F5) and a separated fraction (F6) still remaining in the column. This purification scheme is shown in figure 5.

The resulting fractions were subjected to MTT colorimetric analysis to assess the ability to inhibit tumor cell growth. In this example, HeLa cells were tested at an initial concentration of 1500 cells/well, while MRC-5, AGS, Hep G2 and MCF-7 cells had an initial loading of 3,000 cells/well. As clearly shown in fig. 6, both F1 and F4 exhibited anti-tumor activity comparable to that exhibited by the crude filtrate (F0). This result suggests that most, if not all, of the pharmacologically active substance present in the filtrate has a low molecular weight, particularly a molecular weight of no more than about 3 kDa.

In addition, the crude filtrate (F0) obtained in example 5 was partially separated through a series of membrane groups to obtain a separated fraction (F7) containing fungal molecules having a molecular weight of not more than 1 kDa. The antitumor activity coexisted with F7 in the same isolated fraction (left-most panel in fig. 7) -the fact suggests that the apparent molecular weight of the pharmacologically active agent may be reduced to no more than about 1 kDa.

In the aforementioned MTT assay, reduced survival was observed in filtrate-treated MRC-5 cells (normal lung fibroblasts), indicating that fungal filtrate and active fractions thereof may exhibit inhibitory effects on normal cells. However, this inhibitory effect is greatly reduced when seeding with MRC-5 cells, such as up to 10,000 cells/well. By comparison, the effectiveness of the fungal filtrate and its active isolated fraction was very unlikely to be affected by the increased tumor cells (data not shown). Such in vitro observations suggest that the compositions according to the invention are less harmful to normal cells when administered in vivo, but give a severe impact to the tumor.

Example 7: isolation of Antrodia camphorata filtrate on Amberlite XAD-4 resin

The filtrate F7 obtained in example 6 was co-incubated with a batch of Amberlite XAD-4(Sigma) and subjected to gentle shaking. After completion of the incubation, the resin particles were precipitated by centrifugation. Supernatant containing unbound substances and resin beads were obtained separately. Subsequently, the resin beads were eluted sequentially with the same volume of deionized water, methanol and ethyl acetate, and the eluates from the three elution procedures were collected separately. The methanol and ethyl acetate eluates were evaporated to dryness at low pressure and then dissolved in a small amount of ethanol. An appropriate amount of sterile water was added to the resulting ethanol solution so that the final concentration of ethanol in the subsequent MTT colorimetric assay was adjusted to not more than 0.5%. The MTT colorimetric assay was performed as described in example 6, using either non-inoculated medium or 0.5% aqueous ethanol as a negative control. The results are shown in FIG. 7.

FIG. 7 shows that the ethyl acetate eluate has superior antitumor activity against all five cells, whereas the water and methanol eluates do not exhibit significant effects on the cells. Residual antitumor activity was observed in the supernatant, presumably due to incomplete adsorption of the hydrophobic substance by the resin. These results suggest that the pharmacological activity present in the filtrate is primarily derived from hydrophobic substances having a molecular weight of no more than about 1 kDa.

Example 8: separation of Antrodia camphorata filtrate in Lichrosorb RP 18 column

The ethyl acetate eluate from example 7 was further separated on a LichrosorbR RP-18 column (Hibar prepacked RT 250-25; 7 μm; from Merck). Gradient elution was performed with acetonitrile/water eluent at a percentage of acetonitrile from 40% to 100% over a period of 200 minutes and a flow rate of 5.7 ml/min. The absorbance at 254nm was measured and is plotted in FIG. 8. Fractions of 12-ml each were collected and combined into several fractions as shown in Table 5.

TABLE 5

Separation section	Number of collecting tube	Separation section	Number of collecting tube	Separation section	Number of collecting tube
						A	1、2	G	23、24、25、26	N	57、58、59、60
B	3、4、5	H	28、29、30、31	P	69、70、71、72、73
						C	6	I	33、34、35	Q	74、75、76、77
C′	7、8、9、10	J	36、37、38	R	80、81
						D	11、12、13、14	K	39、40、41、42	S	82、83、84
E	15、16、17	L	43、44、45、46	T	85、86、87、88
						F	18、19、20、21	M	50、51、52、53

The fractions were evaporated to dryness and prepared as working solution at low pressure as described in example 7. The MTT colorimetric assay was performed as described in example 6. The results are shown in FIGS. 9 to 11.

As shown in FIGS. 9 to 11, isolated fractions G, K and L exhibited significant inhibitory effects on AGS cells, while adjacent isolated fractions G and H suppressed the survival of Hep G2 cells to a level of less than 50%. MCF-7 cells are extensively inhibited by fraction B, E, F, G, H, K, L and R.

However, the inhibitory activity on HeLa cells in the ethyl acetate eluate almost disappeared during purification by reverse phase partition chromatography, indicating that some of the pharmacological activity in the antrodia camphorata filtrate may result from a synergistic effect of many molecules, which is quite susceptible to vigorous purification procedures.

While the invention has been described in conjunction with the specific embodiments set forth herein, it is to be understood that many modifications and variations will be apparent to those skilled in the art without departing from the spirit of the invention and the scope of the appended claims.

Claims (20)

1. A method for obtaining a pharmacologically active composition from a culture of Antrodia camphorata, the method comprising:

(a) inoculating a mycelium inoculum of an isolate of a strain of Antrodia camphorata in a medium suitable for growth of the isolate to form a first culture;

(b) culturing the first culture obtained in step (a) for a period of time;

(c) removing a substantial portion of the insoluble material from the cultured culture obtained in step (b) to obtain a pharmacologically active solution; and

(d) treating the solution from step (c) by a selective separation based on molecular weight to obtain a pharmacologically active composition comprising a fungally produced compound having a molecular weight of no more than about 10 kDa.

2. The method of claim 1, wherein:

the composition obtained in step (d) contains a fungally produced compound having a molecular weight of no more than about 3 kDa.

3. The method of claim 1, wherein:

the composition obtained in step (d) contains a compound produced by the fungus having a molecular weight of no more than about 1 kDa.

4. The method of claim 3, wherein the method further comprises:

(e) subjecting the fraction obtained in step (d) to a polarity-based chromatographic separation, thereby obtaining a pharmacologically active composition comprising a hydrophobic fungus-producing compound.

5. The method of claim 4, wherein:

the chromatographic separation step (e) is carried out by means of a fixed water-immiscible phase containing an effective amount of an adsorbent capable of selectively adsorbing hydrophobic fungal-derived compounds, and the pharmacologically active composition is obtained by eluting the fixed water-immiscible phase with an organic solvent.

6. The method of claim 5, wherein:

the stationary water-immiscible phase used in the chromatographic separation step (e) comprises Amberlite * XAD-4 resin as adsorbent.

7. The method of claim 5, wherein:

in the chromatographic separation step (e), an organic solvent having a polarity lower than water is used to elute the stationary water-immiscible phase.

8. The method of claim 7, wherein:

the organic agent used in the chromatographic separation step (e) has a polarity lower than methanol.

9. The method of claim 8, wherein:

the organic agent used in the chromatographic separation step (e) is selected from the group consisting of ethyl acetate and ethanol.

10. The method of claim 4, wherein the method further comprises:

(f) subjecting the composition obtained in step (e) to reverse phase partition chromatography, and obtaining a pharmacologically active fraction.

11. The method of claim 1, wherein the culturing step (b) comprises the following substeps:

(i) subjecting the first culture obtained in step (a) to a first shaking phase set at a first predetermined rate for a period of time to allow the inoculated isolate to grow, whereby a second culture with proliferated mycelium is obtained; and

(ii) (ii) subjecting the second culture obtained in substep (i) to a second shaking phase set at a second predetermined rate higher than the first predetermined rate, whereby the isolate grown in the second culture is incubated under physiological pressure.

12. The method of claim 11, wherein:

in the substeps (i) and (ii), the first culture obtained in step (a) and the second culture obtained in substep (i) are respectively cultured at a pH value adjusted to a range of 4.5 to 5.4.

13. The method of claim 12, wherein:

in the substeps (i) and (ii), the first culture obtained in step (a) and the second culture obtained in substep (i) are respectively cultured at a pH value adjusted to a range of 4.6 to 5.3.

14. The method of claim 12, wherein:

in the substeps (i) and (ii), the first culture obtained in step (a) and the second culture obtained in substep (i) are respectively cultured at a pH value adjusted to a range of 4.7 to 5.2.

15. The method of claim 1, wherein:

the isolate of Antrodia camphorata used in step (a) is selected from the group consisting of ATCC PTA-1233, CCRC35396, 35398 and 35716.

16. The method of claim 1, wherein:

the medium used in step (a) is potato dextrose broth or a synthetic medium containing fructose as the main carbon source.

17. The method of claim 16, wherein:

the medium used in step (a) is a synthetic medium containing fructose as the main carbon source.

18. Use of a composition obtained by the method of any one of claims 1-17 in the manufacture of a medicament for inhibiting the growth of a tumor or cancer cell.

19. A product obtained by the process of claim 1.

20. A pharmaceutical composition for treating cancer or neoplastic disease, comprising: the composition comprises the product obtained by the process of claim 1.