JP2006288405A - Process for producing a culture of antrodia camphorata and product obtained thereby - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop a cost-effective process on an artificial culturing of Antrodiac camphorata. <P>SOLUTION: This process for producing the Antrodiac camphorata culture having pharmacologically activity comprises (a) inoculating a mycelium inoculum of an Antrodiac camphorata isolate into a liquid culture medium suitable for the proliferation of the isolate to obtain the first culture product, (b) supplying the first culture from the process (a) to the first step stirring treatment set to the first period and the first prescribed rate enabling the furthermore culturing of the inoculated isolate to obtain the second culture having the proliferated mycelia, and (c) supplying the second culture obtained from the process (b) to the second step stirring treatment set to the larger second prescribed rate than that of the first prescribed rate to subject the isolate to a treatment under a physiological stress. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

This application claims priority from US patent application serial number 10 / 113,903, filed March 29, 2002, the disclosure of which is incorporated herein by reference. The

FIELD OF THE INVENTION The present invention provides a method for A.D., particularly by optimizing agitation speed and / or pH value during culture. It relates to the establishment of culture conditions that are suitable for large-scale production of pharmacologically active filtrates from camphorata cultures. The present invention also provides A. It relates to a method for obtaining a pharmacologically active composition from a culture of camphorata. The present invention is further directed to the use of the above composition in the manufacture of a pharmaceutical composition.

Description of Related Art A. also known as “niu-chang-chih” or “niu-chang-ku” in Taiwan, camphorata [(Zang & Su) S. -H. Wu, Ryvarden & T. T.A. Chang] recently described its basilar spores found in fruiting bodies, weak amyloid skeletal mycelium, bitter taste and light cinnamon reversed to pyrite basidiomycetes, and thick spore and segmental conidia in pure culture. (Arthroconidia) has been reported as a novel fungus. A. The detailed characterization and taxonomic location of Camphorata is described in Chang, T .; T.A. Et al. cinnamomea sp. nov. on Cinnamum Kanehirai in Taiwan, Mycol. Res. 99 (6): 756-758 (1995) and Wu, S .; -H. Et al. cinnamomea ("niu-chang-chih"), New combination of amedical fungus in Taiwan, Bot. Bull. Acad. Sin. 38: 273-275 (1997), the entire disclosure of which is incorporated herein by reference.

  In Taiwanese folk medicine, Camphorata is believed to have certain medical effects on symptoms caused by addiction, diarrhea, abdominal pain, hypertension, skin itches and liver cancer. However, “niu-chang-chin” is very expensive in the local market due to severe host specificity and unusualness in nature, as well as failure of artificial cultures. Clearly there is great industrial value in developing a cost effective process for the artificial culture of this fungus on a large scale.

  Recently, the inventors have described A.I. Camphorata has been found to exhibit the desired pharmacological activity, particularly antitumor activity, when subjected to submerged fermentation. As disclosed in USSN 09 / 566,834, Camphorata was successfully cultured on a small scale in natural media (ie, potato dextrose broth (PDB)) and synthetic media containing fructose as the main carbon source. The resulting culture exhibits a deep dark red color appearance with a red index a ≧ 3 as measured using the Hunter's coordinate system, which is significant for the growth of certain tumor cell lines. It has been shown to occur simultaneously with the inhibitory effect. More importantly, active substances that act on tumor cells have not been identified but have been found to be secreted from the fungal mycelium into the liquid phase of the culture, resulting in pharmacologically active The composition can be easily harvested from the culture for industrial use.

  Therefore, it would be desirable if such advantageous methods were optimized for large scale production of the fungal culture. More preferably, the crude filtrate is further fractionated for the desired pharmacological activity in order to obtain a useful composition enriched in the desired activity.

SUMMARY OF THE INVENTION Extensive research has been conducted to meet the industrial requirements described above. Here, surprisingly, A.I. It has been found that optimal conditions for industrial scale cultivation of Camphorata can be achieved by carefully setting specific parameters within specific ranges. In this invention, the pH value and stirring speed have been found to be very important during the culture.

Therefore, the first aspect of the present invention is a method for producing an Anthrocamera culture having pharmacological activity,
(A) inoculating a mycelial inoculum of an isolate of Antrodia camphorata into a medium suitable for growth of the isolate to produce a first culture;
(B) a first stage agitation of the first culture cultured from step (a) set for a first period and at a first predetermined speed allowing further growth of the inoculated isolate. And (c) setting the second culture obtained from step (b) at a second predetermined speed different from the first predetermined speed. Subjecting it to a second stage of agitation and subjecting the isolate under physiological stress,
Providing a method comprising:

According to a second aspect of the present invention, there is provided a method for producing an Anthrodia camphorata culture having pharmacological activity comprising:
(A) inoculating the mycelium inoculum of an isolate of Anthrodia camphorata into a medium suitable for growth of the isolate; and (b) the culture obtained from step (a) Culturing by adjusting the pH value of the culture to a range between 4.5 and 5.4,
Providing a method comprising:

  Preferably, A.I. The pH value of the camphorata culture is adjusted during the step (b) within the range of 4.6 to 5.3, and more preferably 4.7 to 5.2.

The present invention also provides A.A. for desired pharmacological activity (eg, antitumor activity). A method for obtaining a series of liquid fractions separated from Camphorata is provided. Therefore, the third aspect of the present invention is the following. A method for obtaining a pharmacologically active composition from a culture of camphorata comprising:
(A) A. inoculating the mycelium inoculum of a camphorata isolate into a medium suitable for growth of the isolate;
(B) culturing the culture obtained from step (a);
(C) removing most of the insoluble material from the culture, thereby harvesting a pharmacologically active solution; and (d) treating the solution from step (c) to obtain about Obtaining a pharmacologically active composition containing a fungal molecule having a molecular weight of 10 kDa or less,
Providing a method comprising:

    Preferably, the resulting composition contains fungal molecules having a molecular weight of about 3 kDa or less, more preferably about 1 kDa or less.

A fourth aspect of the present invention relates to A. A method for obtaining a pharmacologically active composition from a culture of camphorata comprising:
(A) A. inoculating the mycelium inoculum of a camphorata isolate into a medium suitable for growth of the isolate;
(B) culturing the culture obtained from step (a);
(C) removing most of the insoluble material from the culture, thereby harvesting the aqueous solution;
(D) treating the aqueous solution from step (c) to obtain a fraction containing fungal molecules having a molecular weight of about 1 kDa or less; and (e) converting the fraction from step (d) into a water-immiscible phase. Passing through and obtaining a pharmacologically active composition therefrom,
Providing a method comprising:

  The water immiscible phase from step (e) above is preferably a stationary phase containing an effective amount of adsorbent capable of selectively adsorbing hydrophobic fungal molecules. The stationary phase is eluted so that a pharmacologically active fraction is obtained.

  In a preferred embodiment of the present invention, the eluate from step (e) is further subjected to reverse phase partition chromatography (eg, Lithosorb® RP-18 column to obtain multiple fractions with pharmacological activity. (Merck)).

  The present invention further provides a pharmaceutical composition for the treatment of cancer or tumor diseases comprising a product obtained from any one of the methods of the present invention.

  The invention further relates to a method of treating cancer or tumor disease in a patient in need of treatment of cancer or tumor disease, comprising a composition comprising a product obtained from any one of the methods according to the invention. To provide a method by prescribing.

DETAILED DESCRIPTION OF THE INVENTION The present invention generally includes A. It relates to the establishment of culture conditions suitable for the large-scale production of pharmacologically active fractions from Camphorata cultures. According to the present invention, fungi A. Camphorata is cultured in a liquid medium suitable to maintain its vegetative growth in the mycelium state and promote its pharmacological activity.

  The term “suitable medium” as used herein refers to A.I. Any medium that provides an artificial environment suitable for the growth of Camphorata. Preferably, the medium used in the present invention is suitable for promoting the production of pharmacologically active substance (s) in the mycelium and promoting its secretion into the environment.

  Suitable culture media for use in the present invention include natural media called “potato dextrose broth” and any synthetic media containing fructose as the main carbon source. Potato dextrose broth can be prepared in the laboratory, for example by autoclaving a mixture of 300.0 g chopped potato, 20.0 g dextrose and 1.0 L distilled water, or from a commercial source (eg DIFCO). Can be purchased. Most preferred is a synthetic medium containing fructose as the main carbon source. If necessary, other carbon sources such as glucose, sucrose, galactose, fructose, corn starch, malt extract and combinations thereof can be included in the synthetic medium as supplements. Preferably, the carbon source is present in the range of 1.5 to 2.5 wt%, and more preferably 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 vitamins or other growth factors as required. Nitrogen sources include, but are not limited to, ammonium sulfate, ammonium nitrate, sodium nitrate, casamino acid, yeast extract, peptone, tryptone, and combinations thereof. Preferably, according to the invention, the synthetic medium contains yeast extract as a nitrogen source. The nitrogen source is preferably present in an amount ranging from 0.2 to 2.0% by weight, and more preferably 0.5% by weight, based on the total volume of the synthetic medium.

  A. Any available isolate of Camphorata can be used in the culture method according to the present invention, so long as the microorganism used retains the ability to produce a detectable amount of a pharmacologically active metabolite. A. Useful isolates of Camphorata include CCRC 35396 (the Culture Collection and Research Center of the Culture Collection and Research. Of the Food Industry Research and Development Institute (FIRD), December 1, 1994). 35398 (December 1, 1994), 35716 (May 3, 2000), 36401 (January 27, 2000), 36695 (January 27, 2000) and 930032 (2000) January 27)), but is not limited thereto. According to a preferred embodiment of the present invention, A. Camphorata CCRC 930032 (also deposited with the American Type Culture Center (ATCC) under the accession number PTA-1233 for patent procedures) was used for the preparation of the culture filtrate.

  In order to evaluate the ability in inhibiting the growth of tumor cells, The camphorata crude filtrate and its fractions were subjected to MTT colorimetric assay.

The term “MTT colorimetric assay”, also known as “MTT-tetrazolium assay,” as used herein, is described in U.S. Pat. S. Refers to anticancer drug screening scheme established in 1980s by National Cancer Institute's Developmental Therapeutics Program, Division of Cancer Treatment (for example, Alley, MC et al., Feasibility of drug screening with a panel of human tumor cell lines using Assay Res.48: 589-601,1988; Scudiero, DA et al., Evaluation of a soluble tetrazolium / formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines.Cancer Res. 48: 4827-4833 , 1988; Vistica, DT et al., Tetrazolium-based assays for cellular viability: a critical examination of selected parameters affecting formazan. Cancer Res. 51: 251-2520, 1991; and Monks, A. et al., Feasibility of a high-flux anticancer. drug
screen using a diverse panel of cultured human tumor cell lines. See J. Nat. Cancer Inst. 83: 757-766, 1991).

  In the assay, potential anti-cancer drugs or natural products derived from plants or microorganisms (in this case from A.Camphorata) to a group of cell line panels, each representing a major clinical category of human malignancies Were tested. The number of viable cells per well is directly proportional to formazan production, which can be measured spectrophotometrically by solubilization. In principle, a biologically active substance or a natural product containing the substance can inhibit or even stop cell growth and as a result hardly forms formazan.

  Using the MTT colorimetric assay, important parameters in fungal cultures, i.e., agitation speed and pH value, are measured over time for the culture. Their effects on the pharmacological activity of Camphorata were evaluated.

  In the first set of experiments, A. Camphorata cultures are duplicated, each experiencing two different processes of agitation. Here, one process is carried out to give the fungus constant but vigorous stirring, and the other relates to gentle to vigorous two-stage stirring. In comparison, the two-stage stirring according to the present invention results in the early generation of pharmacological activity. Gentle agitation at an earlier stage is provided to allow nutritious growth of the inoculated fungus, so that a culture in which the mycelium is grown can be obtained. Increased size mycelium pellets harvested at the end of the culture indicate success with a gentle agitation step. The appropriate timing for the transition from gentle to vigorous agitation speed can vary widely depending on the fungal isolate selected. Typically, if the fungal isolate is inoculated at 10% v / v based on the final volume of the culture, gentle agitation lasts for about 3 days (ie, about 72 hours) and then the agitation rate is increased. Vigorous agitation at a later stage is provided to place the fungus under physiological stress. Under such stress, A. Camphorata undergoes multiple physiological changes in response, including promoting the production of secondary metabolites that are not expressed in a constitutive manner. It is believed that some fungal molecules of pharmacological activity can be “stressed out” by this method. A. Other culture parameters applicable to impart physiological stress to Camphorata (eg, aeration rate, nutritional modification and thermal stress) can also be used alone or in combination with agitation rate parameters according to the present invention. Suitable agitation rates can be determined empirically as described above or, for example, in Bioprocess Engineering Principles, (1995), p. 150-151 edited by Pauline M. Doran and published by Academic Press Ltd. According to the formula described, it can be estimated based on the experiments provided below. In some cases, when two-stage agitation is suitably applied, the fungal culture appears red on day 6.

  In a preferred embodiment of the invention, the two-stage agitation is performed in a 5 L fermentor (B. Braun) pre-loaded with 3 L of medium, where the agitation is initially about 300 rpm or less, preferably about 200 rpm or less. The speed was set and then increased to a speed of about 400 rpm or more, preferably about 500 rpm or more. In another preferred embodiment of the present invention, the two-stage agitation is performed in a 250 L fermentor (Bio-Top) preloaded with 160 L of medium, where the agitation speed is initially set at about 40 rpm, and Thereafter, the pressure was increased to about 150 rpm.

  In the second set of experiments, A. Camphorata cultures are triplicated and each is cultured at adjusted pH values at three different intervals over the entire duration of the culture. In comparison, it can be seen that culturing performed at pH 4.5-5.4 results in early production of pharmacological activity. Preferably, A. The pH value of the camphorata culture is adjusted within the range of 4.6 to 5.3, and more preferably 4.7 to 5.2 over the entire period of culture.

  Utilizing useful parameters for agitation and pH values as described above, A. Camphorata culture methods are successfully scaled up to 160 liter volumes while maintaining the desired pharmacological activity derived from fungi.

  According to the method of the present invention, A.A. A pharmacologically active filtrate from Camphorata can be obtained in an economical, efficient and time-saving manner.

  The present invention also relates to the establishment of an effective purification method, whereby novel compositions rich in pharmacologically active substances are available for various medical applications. According to the present invention, the purification method comprises: This is done by selectively separating the crude Camphorata filtrate to obtain a fraction containing fungal molecules having a molecular weight of about 10 kDa or less, preferably about 3 kDa or less, and more preferably about 1 kDa or less. The separation method can be performed by any conventional method that can separate molecules based on molecular weight (ie, acting as a molecular sieve), examples of which include gel filtration, density gradient purification, ultrafiltration, ultracentrifugation Examples include separation and methods known in the art.

  According to the present invention, a fraction containing molecules having a molecular weight of about 1 kDa or less is further fractionated based on polarity to obtain a water immiscible phase, from which a pharmacologically active fraction is obtained. . The water immiscible phase can be an organic phase that is immiscible with water or an insoluble solid phase. In a preferred embodiment of the invention, the ≦ 1 kDa fraction is passed through a stationary phase containing an effective amount of adsorbent capable of selectively adsorbing hydrophobic fungal molecules. Subsequently, the stationary phase is eluted to obtain a fraction having the desired pharmacological activity. Briefly, the stationary phase selectively adsorbs and concentrates a hydrophobic solute, possibly containing the desired pharmacologically active substance, from the ≦ 1 kDa fraction, resulting in a flow through. The inert material present can be easily removed. Suitable adsorbents included in the stationary phase can be any adsorbent with functional groups adapted to capture hydrophobic material from the mobile phase. An example of an adsorbent is Amberlite® XAD-4 (Sigma) and its equivalents. Fractions can be incubated in any conventional manner, for example by incubating a ≦ 1 kDa fraction with a batch of adsorbent, as long as the amount of pharmacologically active substance retained on the surface of the adsorbent is sufficient, or This can be done by passing a ≦ 1 kDa fraction through a chromatography column packed with an adsorbent. Suitable eluents for eluting bound material from the stationary phase are well known to those skilled in the art and can therefore be readily selected by those skilled in the art. Preferably, the eluent is an organic solvent having a lower polarity than water, and more preferably a lower polarity than methanol. Most preferred eluents include ethyl acetate and ethanol.

  An eluate exhibiting the desired pharmacological activity can be subjected to further purification procedures based on several other physical, chemical or biological characteristics. In a preferred embodiment of the present invention, the eluate is further separated based on the degree of hydrophobicity, and more preferably the separation is performed, for example, in a Lichlorosorb® RP-18 column (Merck). The numerous fractions obtained in this way prove to be pharmacologically active.

  The above findings are as follows. It strongly suggests that the pharmacological activity in Camphorata is mainly derived from hydrophobic compounds having a molecular weight of 1 kDa or less. This is because polysaccharides with an average molecular weight of 500-2,000 kDa are regarded as a major source of antitumor activity possessed by mushrooms (Mizuno et al., Antitumor-active substances from food rev. Int. 11 (1). : 23-61), contrary to previous hypothesis. A. Camphorata is reported to be rich in low molecular weight substances such as triterpenoids, flavinoids, flavonoids, steroids, sesquiterpene lactones, and phenyl and biphenyl compounds (Chang, supra; Cherng et al., Triterpenoids from Antrodia cinnamomea. Pytochem. 41 (1): 263-267 (1996); Chiang et al., A sesquiterpene lactone, phenyl and biphenyl compounds from Antrodia cinnamomea. Pytochem. 39 (1): 613-616 (1995); and Yang et al., Steroids and Triterpenoids of Antrodia cinnamomea-a fungus parasitic on Cinnamomum Micranthum. Pytochem. 41 (5): 1389-1392 (1996)), but no teaching has been reported linking these substances to the pharmacological activity of the fungal species.

  The purification method according to the invention provides a number of compositions in which the active substance is concentrated and the inert substance is extensively removed. The composition clearly has an enhanced pharmacological effect on human or animal subjects and is therefore suitable for various industrial applications (eg production of pharmaceutical compositions or nutritional supplements) . Accordingly, the present invention is also formulated as a medicament for treating a disease (especially cancer or tumor disease) in a human or animal patient in need of such treatment or in the form of, for example, food, beverage and / or feed The use of the new composition as a nutritional supplement.

In accordance with a preferred embodiment of the present invention, the following aspects of the invention are provided.
Item 1. A method for producing an Anthrodia camphorata culture having pharmacological activity comprising:
(A) inoculating a mycelium inoculum of an isolate of Anthrodia camphorata into a medium suitable for growth of the isolate to produce a first culture;
(B) a first stage cultivated from step (a) for a first period allowing a further growth of the inoculated isolate and at a first predetermined rate; Subjecting to agitation to obtain a second culture growth having a mycelium; and (c) the second culture obtained from step (b) is set at a second predetermined speed different from the first predetermined speed. Subjecting the isolate to a second stage of agitation under physiological stress,
Including a method.
Item 2. Item 2. The method according to Item 1, wherein the second predetermined speed is faster than the first predetermined speed.
Item 3. In step (b) and step (c), the first culture from step (a) and the second culture from step (b) are adjusted to a pH value in the range of 4.5 to 5.4. Item 2. The method according to Item 1, wherein the method is cultured.
Item 4. In step (b) and step (c), the first culture from step (a) and the second culture from step (b) are adjusted to a pH value in the range of 4.6 to 5.3. Item 4. The method according to Item 3, wherein the method is cultured.
Item 5. In step (b) and step (c), the first culture from step (a) and the second culture from step (b) are adjusted to a pH value in the range of 4.7 to 5.2. Item 5. The method according to Item 4, which is cultured.
Item 6. Item 2. The method according to Item 1, wherein the medium is selected from the group consisting of potato dextrose broth and a synthetic medium containing fructose as the main carbon source.
Item 7. Item 7. The method according to Item 6, wherein the medium is a synthetic medium containing fructose as a main carbon source.
Item 8. The isolates are CCRC 930032 (ATCC PTA-1233), CCRC
The method of paragraph 1 selected from the group consisting of 35396, 35398, 35716, 36401 and 36795.
Item 9. A method for producing an Anthrodia camphorata culture having pharmacological activity comprising:
(A) inoculating the mycelium inoculum of an isolate of Anthrodia camphorata into a medium suitable for growth of the isolate; and (b) the culture obtained from step (a) Culturing by adjusting the pH value of the culture to a range between 4.5 and 5.4,
Including a method.
Item 10. Item 10. The method according to Item 9, wherein the culture from step (a) is cultured by adjusting the pH value of the culture to a range of 4.6 to 5.3 throughout step (b).
Item 11. Item 11. The method according to Item 10, wherein the culture from step (a) is cultured by adjusting the pH value of the culture to a range of 4.7 to 5.2 throughout step (b).
Item 12. Item 10. The method according to Item 9, wherein the medium is selected from the group consisting of potato dextrose broth and a synthetic medium containing fructose as a main carbon source.
Item 13. Item 13. The method according to Item 12, wherein the medium is a synthetic medium containing fructose as a main carbon source.
Item 14. Item 10. The method according to Item 9, wherein the step (b) is performed by stirring at a predetermined speed.
Item 15. The isolates are CCRC 930032 (ATCC PTA-1233), CCRC
Item 10. The method according to Item 9, wherein the method is selected from the group consisting of 35396, 35398, 35716, 36401, and 36795.
Item 16. A. A method for obtaining a pharmacologically active composition from a culture of camphorata comprising:
(A) A. inoculating the mycelium inoculum of a camphorata isolate into a medium suitable for growth of the isolate;
(B) culturing the culture obtained from step (a);
(C) removing most of the insoluble material from the culture, thereby harvesting a pharmacologically active solution; and (d) treating the solution from step (c) to obtain about 10 kDa or less. Obtaining a pharmacologically active composition containing a fungal molecule having a molecular weight,
Including a method.
Item 17. Item 17. The method according to Item 16, wherein the composition obtained in step (d) contains a fungal molecule having a molecular weight of about 3 kDa or less.
Item 18. Item 18. The method according to Item 17, wherein the composition obtained in the fraction of step (d) contains a fungal molecule having a molecular weight of about 1 kDa or less.
Item 19. A. A method for obtaining a pharmacologically active composition from a culture of camphorata comprising:
(A) A. inoculating the mycelium inoculum of a camphorata isolate into a medium suitable for growth of the isolate;
(B) culturing the culture obtained from step (a);
(C) removing most of the insoluble material from the culture, thereby harvesting the aqueous solution;
(D) treating the aqueous solution from step (c) to obtain a fraction containing fungal molecules having a molecular weight of about 1 kDa or less; and (e) converting the fraction from step (d) into a water-immiscible phase. Passing through and obtaining a pharmacologically active composition therefrom,
Including a method.
Item 20. The water immiscible phase in step (e) is a stationary phase containing an effective amount of adsorbent capable of selectively adsorbing hydrophobic fungal molecules, and eluting the stationary phase to pharmacologically active Item 20. The method according to Item 19, wherein a composition is obtained.
Item 21. Item 21. The method according to Item 20, wherein the stationary phase includes Amberlite (registered trademark) XAD-4 resin as an adsorbent.
Item 22. Item 21. The method according to Item 20, wherein the stationary phase is eluted by an eluent that is an organic solvent having a lower polarity than water.
Item 23. Item 23. The method according to Item 22, wherein the eluate is an organic solvent having a lower polarity than methanol.
Item 24. Item 24. The method according to Item 23, wherein the eluate is selected from the group consisting of ethyl acetate and ethanol.
Item 25. Furthermore, performing reverse phase partition chromatography on the composition from step (e) to obtain a pharmacologically active fraction,
Item 20. The method according to Item 19, comprising:
Item 26. Item 2. The method according to Item 1, wherein the pharmacological activity is an inhibitory activity against tumor or cancer cells.
Item 27. Item 10. The method according to Item 9, wherein the pharmacological activity is an inhibitory activity against tumor or cancer cells.
Item 28. Item 17. The method according to Item 16, wherein the pharmacological activity is inhibitory activity against tumor or cancer cells.
Item 29. Item 20. The method according to Item 19, wherein the pharmacological activity is an inhibitory activity against tumor or cancer cells.
Item 30. Item 26. The method according to Item 25, wherein the pharmacological activity is inhibitory activity against tumor or cancer cells.
Item 31. Item 26. A product obtained from the method according to any one of Items 1, 9, 16, 19, and 25.
Item 32. Item 26. A pharmaceutical composition comprising a product obtained from the method according to any one of Items 1, 9, 16, 19, and 25.
Item 33. Item 33. A pharmaceutical composition according to Item 32 for use in the treatment of cancer or tumor diseases.
Item 34. Item 26. A method for treating cancer or tumor disease in a patient in need of treatment for cancer or tumor disease, comprising a product obtained from the method according to any one of items 1, 9, 16, 19 and 25. Administering an article to the patient.

Preferred embodiments for practicing the invention The following examples are given by way of illustration only and are not intended to limit the scope of the invention.

Example 1: A.1. Preparation of a camphorata liquid culture A stock culture of Camphorata CCRC 930032 was maintained at −80 ° C., from which a small aliquot of fungus was picked and placed on a plate of potato dextrose agar (PDA, purchased from DIFCO). After harvesting, the culture was then transferred to a potato dextrose agar slant tube. Slant cultures were incubated at 25 ° C. and subcultured every 2 months. Slant cultures were used as working cultures. For the preparation of mycelium inoculum, PDA plates were inoculated with a culture from PDA slant and incubated at 28 ° C. for 15-20 days.

Preparation of mycelium inoculum Incubation was carried out until a mycelial colony with a diameter of 15-30 mm was observed. A. The mycelial characteristics of Camphorata were studied under a light microscope to ensure that no contamination occurred. The whole mycelium was cut into fine pieces and then aseptically homogenized with 50 ml of sterile water in a homogenizer (Osterizer) for 30 seconds. An aliquot of mycelium suspension was used as the inoculum for submerged shake cultures. The inoculum was synthesized in pre-loaded Erlenmeyer flasks (2% fructose, 0.5% (w / v) yeast extract (DIFCO), 0.1% (w / v) KH2PO4 (Merck), And 0.05% (w / v) MgSO 4 .7H 2 O (Merck)) at a volume ratio of 1: 9. The submerged culture is mixed with constant agitation (orbital rotary shaker (orbital purchased from Hotech)
in a rotary shaker) at 50 rpm) for 5 days at 30 ° C. The resulting culture was used as an inoculum for subsequent large scale culture.

Example 2: Effect of stirring speed on the pharmacological activity of fungal filtrate A. camphorata CCRC 930032 inoculum was loaded with 2.7 liters of synthetic medium (2% fructose, 0.5% (w / v) yeast extract (DIFCO BRL)) preloaded into a 5 L fermentor (B. Braun), 0.1% (w / v) KH 2 PO 4 (Merck) and 0.05% (w / v) MgSO 4 .7H 2 O (Merck)) were added at a volume ratio of 1: 9. The culture was incubated at 30 ° C. with an aeration rate of 0.6 L / min. The agitation speed for the culture was initially set at about 200 rpm and increased to about 500 rpm after 74 hours incubation. Samples are sampled from the culture at 48, 113, 170, 217 and 259 hours after mycelial inoculation, and then a simple filtration assembly of a suction filter funnel, flask and vacuum to remove most of the insoluble material Was passed. The filtrate thus harvested was adjusted to pH 7 with NH4OH and sterilized by autoclaving. The resulting sample was stored at 4 ° C. for subsequent MTT colorimetric assay.

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

  The HepG2 tumor cell line was selected for the MTT colorimetric assay. In preparation for the assay, cells were maintained as stock in α-MEM medium (GIBCO BRL) supplemented with 10% fetal calf serum (Hyclone) just like ATCC medium. Tumor cell lines were passaged once or twice a week using trypsin-EDTA (GIBCO BRL) to separate cells from cell culture flasks. Tumor cells were harvested, counted and seeded into 96-well microtiter plates at a concentration of 3,000 cells per well. The total volume of cell culture medium in each well was 180 μl and incubation was performed overnight at 37 ° C. in an incubator aerated with 5% CO 2.

  A 20 μl aliquot of sample was applied in triplicate to the culture wells and the resulting culture was incubated for 72 hours at the incubation conditions described above. Subsequently, 20 μl of MTT stock solution (Merck), previously prepared at 5 mg / ml in PBS solution (GIBCO BRL), was added to each well.

  After an additional 4 hours incubation at 37 ° C. in a CO 2 incubator, the supernatant was removed from each well and 100 μl of 100% DMSO (dimethyl sulfoxide, available from Sigma) was added to dissolve the MTT-formazan product. . After thorough mixing with a mechanical plate mixer, the absorbance at 540 nm was measured with an ELISA reader (MRX, Dynex). The relative viability of tumor cells in the test filtrate was calculated by dividing the absorbance of each experimental sample by that of the corresponding non-inoculated control.

Comparative Example 1:
Example 2 was repeated except that the fungal culture was incubated at a constant rate of about 500 rpm for the entire duration of the culture.

  The relative viability of tumor cells after treatment of the filtrates from Example 2 and Comparative Example 1 is compared in Table 1 and FIG.

In Table 1, the filtrate sampled at the indicated time points
The inhibitory effect on G2 tumor cells is compared. Regarding the filtrate picked up at 170 hours after the mycelial inoculation, A. Camphorata CCRC 930032 can be seen to provide 16% relative survival in the MTT assay when experiencing two-step agitation from 200 rpm to 500 rpm, which is observed to be 35% higher relative survival. This is far more effective than a culture cultured at a constant speed of 500 rpm. The anti-tumor activity of Example 2 and Comparative Example 1 is closer at the later time points (at 217 and 259 hours) and initial stirring at a slower rate in combination with a faster stirring rate at the slower stage is Suggests achieving fast production of the pharmacologically active substance (s) in the culture medium.

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

Example 3: Effect of pH value on pharmacological activity of fungal filtrate Three batches of synthetic medium (1.5% fructose, 0.5% (w / v) yeast extract (DIFCO BRL), 0. 1% (w / v) KH2PO4 (Merck) and 0.05% (w / v) MgSO4 · 7H2O (Merck)) were added at about 4.5 (test A) and 5.0 (test B), respectively. ) And 5.5 (test C) were prepared to have an initial pH, to which the inoculum prepared in Example 1 was added in a ratio of 1: 9. The resulting cultures were cultured as described in Example 2, except that the pH value of each culture was monitored at predetermined time points and carefully adjusted to near the initial pH value by addition of NaOH solution. (Table 2). Incubation continued for 336 hours. The timing for the addition of the NaOH solution varied depending on the selected pH value. For example, Test B and Test C have aliquots of NaOH solution from 192 and 168 hours to maintain a pH value between about 4.9-5.1 and about 5.4-5.6, respectively. While NaOH solution was not added to Test A until 240 hours.

  Samples are sampled from cultures at 96, 144, 192, 240, 288 and 336 hours after mycelial inoculation, and then a simple vacuum filter funnel, flask and vacuum to remove most of the insoluble material. The filter assembly was passed through. The filtrate thus harvested was adjusted to pH 7 with NH4OH and sterilized by autoclaving. The resulting sample was subjected to an MTT colorimetric assay as shown in Example 2. Non-inoculated medium was used as a negative control for the MTT assay.

As shown in Table 3, A. The Camphorata filtrate was added to Hep
The inhibitory effect on G2 tumor cells is compared. The filtrate picked up at 192 hours after inoculation of mycelium was cultured in Test A and Test B. It can be seen that Camphorata CCRC 930032 produces a relative survival rate lower than that observed in test C. Such differences in anti-tumor effects are maximal at 240 hours and gradually decrease at later time points, pH 4.5-5.4, preferably 4.6-5.3, and more preferably 4 Cultures performed at .7 to 5.2 suggest that rapid production of the pharmacologically active substance (s) in the culture medium is achieved.

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

Example 4: A. in a 250 L fermenter. camphorata scaled-up culture A. prepared as in Example 1. camphorata CCRC 930032 was preloaded into a 250 L fermentor (Bio-Top) with 160 liters of synthetic medium (1.5% fructose, 0.5% (w / v) yeast extract (DIFCO
BRL), 0.1% (w / v) KH 2 PO 4 (Merck), and 0.05% (w / v) MgSO 4 .7H 2 O (Merck)) at a volume ratio of 1: 9. The culture was incubated at 30 ° C. with an aeration rate of 0.6 L / min. The agitation speed for the culture was initially set at about 40 rpm and increased to about 150 rpm after 70 hours incubation. The pH value of the culture was adjusted within the range of 4.9 to 5.1 over the whole period of culture.

  Samples were sampled from the cultures at 96, 144, 168, 186.5, 244, and 284 hours after mycelial inoculation and then filtered in a conventional manner to remove most of the insoluble material. The filtrate thus harvested was adjusted to pH 7 with NH4OH and sterilized by autoclaving. The resulting samples were subjected to an MTT colorimetric assay where HeLa, AGS, Hep G2 and MCF-7 cells were initiated at 1,000, 3,000, 3,000 and 3,000 cells per well. Tested at concentration. Non-inoculated medium was used as a negative control for the MTT assay. The results are shown in Table 4 and FIG.

  Table 4 and FIG. FIG. 5 shows that the camphorata culture process is successfully scaled up to a 160 liter volume by setting the agitation speed and pH value within the desired ranges as described in Examples 2 and 3.

Example 5: A. in synthetic medium Preparation of the culture filtrate of camphorata A. cultured on PDA plates. The entire mycelium of Camphorata CCRC 930032 was cut into fine fragments and then aseptically homogenized with 50 ml of sterile water for 30 seconds in an Osterizer, resulting in a mycelium suspension. 1L
200 ml of synthetic medium (2% fructose, 0.5% (w / v) yeast extract (DIFCO), 0.1% (w / v) KH2PO4 (Merck)) preloaded in an Erlenmeyer flask, and To 0.05% (w / v) MgSO4 · 7H2O (Merck)) was added 20 ml of mycelium suspension. The submerged cultures were incubated for 14 days at 30 ° C. with constant agitation (75 rpm in an orbital rotary shaker purchased from Hotech). After incubation, the fungal culture was passed through a simple filtration assembly of a suction filter funnel, flask and vacuum. The crude filtrate was used for subsequent analysis.

Example 6: A.1. Preparation and analysis of active fractions from camphorata filtrate The crude filtrate from Example 5 (F0) was centrifuged at low speed according to the protocol provided by the manufacturer, with the Cripriprep® Concentrator 10 (molecular weight of 10 kDa) A commercial mini column purchased from Amicon) with a cut-off value. The primary passage solution is referred to as fraction F1. The column was then refilled with deionized water and the centrifugation was repeated to recover secondary pass solution F2. Harvest the fungal molecules retained in the column and call it F3.

  F1 is further fractionated by a Certriprep® Concentrator 3 minicolumn (Amicon) with a molecular weight cutoff of 3 kDa, so that it is retained on the first pass (F4), second pass (F5), and minicolumn. Fraction (F6) was obtained as described above. The purification scheme is shown in FIG.

  The resulting fractions were subjected to an MTT colorimetric assay to evaluate their ability to inhibit tumor cell growth. In this case, HeLa cells were tested at a starting concentration of 1500 cells per well, while MRC-5, AGS, Hep G2 and MCF-7 cells were initially loaded to have 3,000 cells per well. . As clearly demonstrated in FIG. 6, the anti-tumor activity exhibited by both F1 and F4 is comparable to that of the crude filtrate (F0). Most, if not all, of the pharmacologically active substances present in the filtrate are suggested to have a low molecular weight, especially a molecular weight of about 3 kDa or less.

  The crude filtrate (F0) from Example 5 was also fractionated with a series of membrane filters to obtain a fraction (F7) containing fungal molecules having a molecular weight of 1 kDa or less. The fact that anti-tumor activity is co-fractionated with F7 (FIG. 7, leftmost panel) indicates that the apparent molecular weight of pharmacologically active substances can drop below about 1 kDa.

  In the MTT assay described above, reduced viability is observed in filtrate-treated MRC-5 cells (normal lung fibroblasts), and the fungal filtrate and its active fraction can exhibit a specific inhibitory effect on normal cells. It shows that. However, when MRC-5 cells are seeded, for example, up to 10,000 cells per well, the suppressive effect is dramatically reduced. In comparison, the efficacy of the fungal filtrate and its active fraction was much less affected by increasing amounts of tumor cells (data not shown). This in vitro observation shows that the composition according to the present invention is pharmacologically favorable to normal tissues when administered to a living subject, but at the same time has severe effects on tumors. This leads to an estimation.

Example 7: A. in Amberlite® XAD-4 resin Separation of camphorata filtrate Fraction F7 from Example 6 was incubated with 1 batch of Amberlite® XAD-4 (Sigma) with gentle agitation. After completion of the incubation, the resin beads were precipitated by centrifugation. Supernatant and resin beads containing non-adsorbed material were harvested separately. The beads were then eluted sequentially with an equal volume of deionized water, methanol and ethyl acetate, and the eluates from the three elution steps were collected separately. The methanol and ethyl acetate eluates were evaporated to dryness under reduced pressure and redissolved in a small amount of ethanol. The ethanol solution thus obtained was added with an appropriate amount of sterile water to adjust the final concentration of ethanol in the subsequent MTT colorimetric assay to 0.5% or less. The MTT colorimetric assay was performed as described in Example 6 using non-inoculated medium or 0.5% ethanol in water as a negative control. The results are shown in FIG.

  In FIG. 7, the ethyl acetate eluate is shown to have excellent antitumor activity against all five types of cells, while the water and methanol eluates show significant effects on the cells. Absent. Residual anti-tumor activity is observed in the supernatant portion, which is thought to result from incomplete adsorption of hydrophobic substances by the resin. The results suggest that the pharmacological activity present in the filtrate is mainly derived from hydrophobic substances with a molecular weight of about 1 kDa or less.

Example 8: A. on a Lithosorb RP 18 column. Separation of camphorata filtrate The ethyl acetate eluate from Example 7 was further separated on a Lichlorosorb® RP-18 column (Hibar, pre-packed column RT 250-25; 7 μm; purchased from Merck). Gradient elution was performed using an acetonitrile / water eluent with an acetonitrile ratio of 40% to 100% over 200 minutes at a flow rate of 5.7 ml / min. Absorbance at 254 nm is measured and plotted in FIG. 12 ml fractions were collected and combined into several fractions as shown in Table 5.

  Fractions were evaporated to dryness under reduced pressure and prepared into working solution as described in Example 7. The MTT colorimetric assay was performed as described in Example 6. The results are shown in FIGS.

  As demonstrated in FIGS. 9-11, fractions G, K and L show a significant suppressive effect on AGS cells, while adjacent fractions G and H reduce the survival rate of Hep G2 cells to less than 50%. To the level of. MCF-7 cells have a broad spectrum that is suppressed by fractions B, E, F, G, H, K, L and R. However, the inhibitory activity against HeLa cells seen in the ethyl acetate eluate was almost eliminated during purification in reverse phase partition chromatography. Some pharmacological activity in the Camphorata filtrate can result from the cooperative action of various molecules, and this cooperation is quite susceptible to aggressive purification.

  Although the present invention has been described with reference to the above specific embodiments, various modifications and variations that will be apparent to those skilled in the art can be made without departing from the spirit and scope of the invention. Should be understood.

The above and other subjects and features of the invention will become apparent with reference to the above description of preferred embodiments made in connection with the accompanying drawings.
FIG. FIG. 2 is a diagram demonstrating the antitumor activity of filtrate derived from a camphorata culture, wherein A. camphorata is cultured under two different stirring conditions. FIG. 2 shows three A.D. Figure 2 is a diagram demonstrating pH fluctuations of a camphorata culture. FIG. 3 shows an A.D. FIG. 3 is a diagram demonstrating the anti-tumor activity of filtrate derived from a camphorata culture, wherein A. camphorata is cultured at controlled pH values at three different intervals. FIG. Figure 2 is a diagram demonstrating the antitumor activity of filtrate derived from a scaled-up culture of camphorata. Based on molecular weight 2 is a flow chart illustrating a purification scheme for camphorata filtrate. FIG. 6 is a bar graph demonstrating the anti-tumor activity of the culture filtrate isolated according to FIG. 5, where the cell lines tested include MRC-5, HeLa, AGS, Hep G2, and MCF-7. FIG. 7 is a bar graph comparing the antitumor activity of fractions separated in Amberlite® XAD-4 from filtrate fractions containing fungal molecules having a molecular weight of 1 kDa or less, where the cell lines tested were , MRC-5, HeLa, AGS, Hep G2, and MCF-7. FIG. 8 is a spectral profile of the ethyl acetate eluate of FIG. 7 fractionated on a Lichlorosorb® RP-18 column. FIGS. 9-11 demonstrate the anti-tumor activity of the fractions separated in FIG. FIGS. 9-11 demonstrate the anti-tumor activity of the fractions separated in FIG. FIGS. 9-11 demonstrate the anti-tumor activity of the fractions separated in FIG.

Claims (10)

A method for producing an Anthrodia camphorata culture having pharmacological activity comprising:
(A) inoculating a mycelium inoculum of an isolate of Anthrodia camphorata into a liquid medium suitable for growth of the isolate to produce a first culture;
(B) The first culture cultured in step (a) is subjected to a first stage of agitation set at a first period and a first predetermined speed allowing further growth of the inoculated isolate. And providing a second culture having the grown mycelium; and (c) setting the second culture obtained from step (b) to a second predetermined speed that is faster than the first predetermined speed. Subjecting the isolate to physiological stress under a second stage of agitation;
Including methods. Step (b) and Step (c) by adjusting the first culture from step (a) and the second culture from step (b) to pH values in the range of 4.5 to 5.4, respectively. The method according to claim 1, wherein the method is cultured in the medium. Step (b) and Step (c) by adjusting the first culture from step (a) and the second culture from step (b) to pH values in the range of 4.6 to 5.3, respectively. The method according to claim 2, wherein the method is cultured. Step (b) and Step (c) by adjusting the first culture from step (a) and the second culture from step (b) respectively to a pH value in the range of 4.7 to 5.2. 4. The method of claim 3, wherein the method is cultured in The method of claim 1, wherein the medium used in step (a) is selected from the group consisting of potato dextrose broth and a synthetic medium containing fructose as the main carbon source. The method according to claim 5, wherein the medium used in step (a) is a synthetic medium containing fructose as a main carbon source. 2. The method of claim 1, wherein the Antrodia camphorata isolate used in step (a) is selected from the group consisting of CCRC 930032 (ATCC PTA-1233), CCRC 35396, CCRC 35398 and CCRC35716. The method according to claim 1, wherein the culture obtained from the step (c) has a tumor or cancer cell growth inhibitory activity. A product obtained from the method according to any one of claims 1-8. A pharmaceutical composition for the treatment of tumors or cancer diseases, comprising a product obtained from the method according to any one of claims 1-8.

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