TW201440780A - Antrodia cinnamomea fermentation composition and uses thereof - Google Patents

Abstract

The invention discloses an Antrodia camphorate fermentation composition comprising A. cinnamomea fermentation broth and tea leaf extract. It is shown that the composition has the pharmaceutical effects of anti-tumor and immunity enhancement by quantum resonance spectrometry (QRS) measurement.

Description

Antrodia camphorata fermentation broth and its application

The present invention relates to a burdock fermented composition. In particular, the present invention relates to an anti-tumor and immune-enhancing burdock fermented composition.

Antrodia camphorata, also known as Antrodia camphorata, and Astragalus membranaceus, which grows on the inner wall of Taiwan's endemic tree species Cinnamomum kanehirai Hayata . It belongs to Basidiomycetes, the genus Basidiomycetes, Pleurotus ostreatus, Polyporus It has a scent of yellow musk and its scientific name can be Antrodia cinnamomea , Ganoderma comphoratum or Antrodia comphoratum . Antrodia camphora is known to be rich in a variety of active ingredients beneficial to the human body, such as triterpenoids, β-D-glucan, sterols, superoxide dismutase, adenosine, protein, vitamin B group, niacin and Trace elements such as calcium, phosphorus, strontium, zinc, etc., have the effects of enhancing immunity, anti-cancer, anti-oxidation, anti-inflammatory, and protecting the liver.

Although Antrodia camphorata has the above-mentioned multiple functions, it has been grown on Taiwan's high-altitude burdock tree. After artificial indiscriminate mining, the wild Antrodia camphorata resources have been increasingly exhausted. Therefore, in recent years, the traditional cultivation method has been used to obtain the Antrodia camphorata fruit body. Or use the fermentation method to obtain the mycelium or mash of A. glabrata as a health care product.

For example, according to the method of the Chinese Patent No. I226370 " Antrodia camphorata isolate for producing a culture of the isolate and the product obtained by the method", the strain of the Antrodia camphorata disclosed in the patent is specific Under liquid culture conditions, the mash has better anti-cancer activity than other strains of Antrodia camphorata. On the other hand, the application of this mash is also found to effectively reduce the oxidative damage of endothelial cells. Ethnopharmacol., 118: 237-245, 2008), reduces the inflammatory response of macrophages (Int Immunopharmacol., 5: 1914-192, 2005), and has the effect of significantly inhibiting tumor proliferation in heterologous transplanted human breast cancer cells ( Food Chem Toxicol., 46: 2680-2688, 2008) and protection of liver damage caused by carbon tetrachloride (World J Gastroenterol., 12: 2369-2374, 2006). On the other hand, the liquid lysate active ingredient produced by Antrodia camphorata is simpler and more effective, and it has been pointed out that it contains a variety of quinine-based ingredients (J Chilean Chemical Society., 52: 1338-1340, 2007), which is used in animal experiments. The results of the cell test showed anti-inflammatory (Int Immunopharmacol., 5: 1914-1925, 2005), inhibition of tumor cell proliferation (J Ethnopharmacol., 100: 158-167, 2005).

However, the taste of the burdock is not popular with consumers. It has bitterness, sourness or astringency. After drinking, it has an unpleasant aftertaste. Most of the burdock drinks on the market are combined with Chinese herbal medicines. Many of the ingredients with excellent curative taste are bitter. The taste is even less suitable for regular drinking, so that consumers' willingness to drink is reduced, and their efficacy cannot be enjoyed.

Tea is one of the most popular beverages. It refers to the beverages made from the leaves of evergreen shrub tea trees that can be used for making tea. The drinking methods are also very diverse. With the increasing popularity of healthy foods in recent years, the health care function of tea has received much attention. The tea leaves picked from the tea tree can be divided into different types depending on the degree of fermentation. For example, green tea is not a yeast tea; oolong tea is a semi-fermented yeast tea; black tea is a full-fermented yeast tea. About 40% to about 60% of the soluble solids in the tea phthalocyanine are polyphenolic compounds such as flavonoids, which have special physiological functions and are an important source of taste, aroma and color of the tea soup. Flavonoids in tea can be further divided into flavanols, flavonols, phenolic acids, depsides and other polyphenols, among which flavanols The catechins are the main polyphenolic compounds in the tea phthalocyanine, accounting for about 75% to about 80% of the total polyphenols. Epigallocatechin gallate (EGCG) is the most abundant, accounting for about 50% of total catechins. It is confirmed to be the most potent catechin with antioxidant activity, with weight loss and anti-tumor. Reduce blood fat, lower blood pressure, enhance immunity and other effects.

Although tea beverages are widely used today, consumer demand for functional tea beverages continues to increase, and in response to the trend of diversified tastes, the development of new tea beverages remains an important issue in the food and beverage sector.

In summary, there is an urgent need in the art to develop beverage materials that take into consideration the taste and efficacy of the beverage, and to make the application of Antrodia camphorata and tea more extensive.

The invention provides a burdock and yoghurt fermentation composition, which can eliminate the unpleasant aftertaste remaining after the bitterness, sourness or astringency of the fermentation product of the burdock, and obviously improve the taste of the burdock medicinal glutinous protein, is suitable for daily drinking, and has anti-tumor and immune lifting functions. The increase in adultness can also be expanded into functional tea drinks and pharmaceuticals.

The present invention provides a composition comprising: Antrodia camphorata; and a tea extract.

The invention further provides the use of a composition as described above for the preparation of a medicament for the treatment of a tumor.

The invention also provides the use of a composition as described above for the preparation of a medicament for boosting immunity.

Figure 1 shows a high pressure liquid chromatogram of the extract of Antrodia camphorata.

The present invention provides a composition comprising: Antrodia camphorata; and a tea extract.

Unless otherwise defined herein, the scientific terms used in connection with the present invention shall have the meaning commonly understood by one of ordinary skill. The meaning and scope of the terms should be clear; however, if there is any potential ambiguity, the definitions provided herein take precedence over any dictionary or foreign definition.

Unless the context requires otherwise, singular terms shall include the plural and plural terms shall also include the singular.

Unless otherwise indicated, it should be understood that the following terms as used in this case have the following meanings:

The scope of the invention is generally indicated by the <RTI ID=0.0>" </ RTI> </ RTI> <RTIgt; Ranges may be expressed in the above-described manner, and if there is a range from a particular value and/or to another particular value, it is another embodiment. Similarly, a numerical value may be referred to by the term "about" to mean an approximation, and when the value is a particular value, it will be understood to be another embodiment. It can also be understood from this that when referring to other endpoints and other endpoints themselves, the endpoints of each range are meaningful.

"As appropriate" or "as appropriate" means that the subsequently described event or condition may or may not occur, and that the described event or condition contains or does not include an example. For example, the phrase "optionally contains a medicament" means that the medicament may or may not be present.

The term "treating" as used herein means reducing and/or improving the symptoms of a rickety individual.

The term "individual" as used herein refers to an animal, particularly a mammal. According to a preferred aspect of the invention, "individual" refers to a human.

The term "therapeutically effective amount" as used herein means an amount of an active ingredient, either alone or in combination with other treatments/drugs, which is sufficient to demonstrate therapeutic efficacy.

The term "carrier" or "pharmaceutically acceptable carrier" as used herein refers to diluents, excipients, acceptors or the like which are well known to those of ordinary skill in the art of making pharmaceutical compositions. .

According to the present invention, "Bagba lucidum" is also known as Antrodia camphorata, and Astragalus membranaceus, belonging to the genus Basil, the genus Basidiomycetes, the genus Pleurotus, the Polyporaceae, and the genus Phaeocystis, and its scientific name may be Antrodia cinnamomea , Ganoderma. Comphoratum or Antrodia comphoratum ; preferably Antrodia cinnamomea TTChang & WNChou. The Antrodia camphorata according to the present invention may be a natural source, such as a strain of the inner wall of the heart of the Taiwanese burdock, or a strain that has been isolated; on the other hand, the Antrodia camphorata according to the present invention may be a wild strain or a modified mutant, Commercially available strains can be used. In a preferred embodiment of the present invention, the strain of Antrodia camphorata is the BCC 930032, 35396, 35398, 35716, 36401, 36611, 36795, 37848, 37849 or 37850 of the Hsinchu Food Industry Development Institute of the Republic of China.

According to the present invention, the method of cultivating or fermenting the aforementioned Antrodia camphorata may comprise a strain of Antrodia camphorata or a strain of Antrodia camphorata, and the method for removing the strain of Antrodia camphorata may be a conventional sterilization method; preferably, The Antrodia camphorata strain is used to remove the strain of Antrodia camphorata.

The term "sterilization" as used herein refers to a measure of killing or removing microorganisms and bacterial spores on an object. Sterilization methods commonly used in the art include a pressure sterilization method, an ultraviolet irradiation sterilization method, a filtration sterilization method, and a chemical sterilization method; preferably, a pressure sterilization method. The pressure sterilization method is generally carried out in a sealed high-pressure steam pot. When the vapor pressure in the pot is raised to 1.05 kg/cm 2 , the steam temperature can reach 121 ° C. At this high temperature, it is preferably about 15 minutes or more. Sterilization can be accomplished for about 15 to 120 minutes, more preferably about 30 to 60 minutes.

According to the present invention, the culture or culture medium for the fermentation of Antrodia camphorata can provide the energy required for the cultivation or fermentation of Antrodia camphorata, and it can be a natural source or a synthetic culture or a culture medium, and its natural source such as Taiwan beef calf decays. The inner wall of the heartwood is preferably a synthetic culture or culture medium.

The synthetic medium according to the present invention comprises a carbon source, a nitrogen source, and a salt; wherein the carbon The source is monosaccharide and disaccharide or a mixture thereof, and the monosaccharide may use glucose, fructose, galactose, mannose or a mixture thereof; the disaccharide may use sucrose, maltose and lactose or a mixture thereof; wherein the nitrogen source is mainly composite As the nitrogen source, for example, yeast powder, yeast extract, soy protein, beef mash or a mixture thereof can be used. The salts include magnesium salts and potassium salts or mixtures thereof. In a preferred embodiment of the invention, the ratio of carbon source, nitrogen source, magnesium salt and potassium salt is (about 10 to about 30): (about 1 to about 10): (about 0.1 to about 2): (about 0.1 to about 2).

Those of ordinary skill in the art to which the present invention pertains can select the temperature conditions for fermentation between about 20 ° C and about 35 ° C according to their knowledge. In a preferred embodiment of the invention, the fermentation is incubated at about 30 °C. On the other hand, those having ordinary knowledge in the technical field to which the present invention pertains may select other conditions or fermentation forms of fermentation, such as liquid fermentation or solid fermentation, according to their own knowledge; preferably, the fermentation is liquid fermentation. .

The liquid fermentation process according to the present invention preferably comprises a multi-stage culture. In a preferred embodiment of the present invention, the culture is carried out by cultivating the flask at a rotation speed of about 50 rpm to about 300 rpm for about 3 days to about 7 days. And culturing in a 50 liter liquid fermentation tank at a speed of about 50 rpm to about 500 rpm for about 3 days to about 7 days, followed by culturing at a flow rate of about 20 rpm to about 150 rpm for about 3 days to about 7 days in a 500 liter liquid fermentation tank. Finally, the culture is carried out at a temperature of about 10 rpm to about 100 rpm in about 5,000 liters of liquid fermentation tank for about 3 days to about 7 days, and then the rotation speed is from about 20 rpm to about 100 rpm for about 9 days to about 14 days.

The method for obtaining the extract of Antrodia camphorata according to the present invention may be determined by those having ordinary knowledge in the technical field of the present invention, such as liquid fermentation, which may be directly used after the medium or the medium is treated; For fermenting, the fermented medium may be extracted with hot water or a solution containing methanol, ethanol, propylene glycol, isopropanol or ethyl acetate; preferably hot water or ethanol, and the water-soluble portion thereof is taken. The step of obtaining the mash from the solid medium can be carried out by any conventional method such as boiling, soaking, autoclaving, ultrasonic extraction, stirring, shaking, or a combination thereof.

The Astragalus membranaceus mash according to the present invention can be used to remove the solid part from the mash. In a sub-step, one of ordinary skill in the art can select suitable removal conditions that can be removed by any conventional method, such as by gauze filtration, centrifugation, or a combination thereof; Precipitation filtration removed.

The Antrodia camphorata mash according to the present invention may be concentrated as needed, and the ferment may be concentrated by any conventional method such as freeze drying or evaporation (J. Pharmacol. Sci., 99: 294-300, 2005 and J. Chromatography, 932: 91-95, 2001) to obtain a concentrated leavek extract, and optionally freeze-dried by adding an excipient such as methyl cellulose.

In a preferred embodiment of the invention, the Antrodia camphorata lysate is analyzed by High Pressure Liquid Chromatography (HPLC) using a chromatography column of Inersil ODS-2, 5 μm, (GL Sciences, ToKyo, Japan), the length of the column is 250 mm, the inner diameter is 4.6 mm, the chromatograph used is Hitachi® PDA L2455, the extract (A) is 0.1% phosphoric acid solution, and the extract (B) is 100%. Acetonitrile, the flushing formula is from 0 minutes to 60 minutes, the extract (A) is reduced from about 90% to about 70% and the extract (B) is raised from about 10% to about 30%; 60 minutes to 62 minutes: maintenance The extract (A) is about 70% and the extract (B) is about 30%; from 62 minutes to 65 minutes, the extract (A) is raised from about 70% to about 90% and the extract (B) is About 30% is reduced to about 10%; 65 minutes to 80 minutes: the extract (A) is about 90% and the extract (B) is about 10%, wherein the residence time is about 9.6, about 11.0, about 12.2. About 15.5, about 22.6, about 28.8, about 35.1, about 49.6 minutes have an absorption peak at a wavelength of 220 nm. The active composition of the extract of Antrodia camphorata (J Chilean Chemical Society., 52: 1338-1340, 2007) has been preliminarily identified in the literature, containing the compound 1:2,3-Dimethoxy-5-methyl[1,4]benzoquinone, Compound 2: 2-Methoxy-6-methyl-1,4-benzoquinone, Compound 3: 5-Methyl-benzo[1,3]dioxole-4,7-dione, Compound 4:5-Methyl-benzo[1,3 There are four components such as dioxole-4 and 7-diol. The absorption peak of this analytical method includes one or all of the above four components.

The term "tea" as used in the present invention refers to the leaves of the evergreen shrub tea tree or the leaves containing the stem which can be used for making tea; preferably leaves. The tea of the present invention contains no fermentation depending on the degree of fermentation. Tea, semi-fermented yeast tea and whole yeast tea; preferably whole yeast tea. Depending on the species, the tea leaves may comprise green tea, yellow tea, white tea, green tea, black tea or black tea; preferably black tea.

In a preferred embodiment of the invention, the tea leaves are dried. The specific embodiment of the drying may be a drying method of general tea leaves, such as natural air drying, drying in an oven or freeze drying.

In a preferred embodiment of the invention, the tea leaves are separated into pieces to facilitate extraction. The specific embodiment of the separation into fragments may be a method of separating the common tea leaves into pieces, such as chopping, shredding, chopping, grinding, and the like.

The tea extract according to the present invention is obtained by extracting tea leaves, and the steps may be determined by those having ordinary knowledge in the art to which the present invention pertains, for example, by freeze extraction, water and/or aqueous extraction; For water extraction.

In a specific embodiment of the present invention, the method of freeze extraction is a general food freezing extraction method, for example, the composition according to the present invention is rapidly frozen into a solid state, and the ice is sublimated into water vapor under a suitable vacuum condition. An extract of the composition. In another embodiment of the present invention, the composition is extracted with hot water or an aqueous solution containing methanol, ethanol, propylene glycol, isopropanol or ethyl acetate; preferably hot water or ethanol, and water soluble Part. In a preferred embodiment of the invention, the methanol, ethanol, propylene glycol, isopropanol or ethyl acetate liquid is an aqueous solution having a concentration of from about 10 to about 100 v/v%; preferably about 50 to About 90 v/v%; more preferably from about 50 to about 80 v/v%; more preferably from about 50 to about 70 v/v% solution.

The extraction step such as boiling, soaking, autoclaving, ultrasonic extraction, stirring, shaking or a combination thereof is preferably decoction or soaking. The extraction weight ratio of tea leaves to water is from about 1 time to about 100 times; the extraction time is from about 30 seconds to about 24 hours; preferably from about 1 minute to about 50 minutes; more preferably from about 1 minute to about 30 minutes. . The extraction temperature is from about 0 ° C to about 121 ° C, preferably from about 95 ° C to about 121 ° C.

In a specific embodiment of the invention, the tea extract is removed from the solid portion Steps, one of ordinary skill in the art may select suitable removal conditions, which may be removed by any conventional method, such as by gauze filtration, centrifugation, or a combination thereof; preferably by filtration or precipitation filtration. Remove.

In a preferred embodiment of the invention, the composition according to the invention further comprises a plant extract, wherein the plant extract is a root or stem extract of the family Zingiberaceae; preferably a root extract of ginger.

The components according to the present invention may be adjusted in proportion according to requirements. Preferably, the weight ratio of the Antrodia camphorata: tea extract is from about 1:1 to about 1:20. If the plant extract is included, the weight ratio of the Antrodia camphorata: plant extract is from about 10:1 to about 1:1. According to the composition of the present invention, the burdock mash and the tea extract can be prepared separately and mixed, or can be prepared together, preferably separately prepared and mixed.

In a preferred embodiment of the present invention, the composition is a food composition or a pharmaceutical composition; in a further preferred embodiment of the present invention, the composition is a food composition; In a particularly preferred embodiment, the composition is used as a beverage raw material.

The compositions of the invention may be administered to an individual by any suitable route, such as oral administration. Suitable formulations of the compositions of the present invention include, but are not limited to, lozenges, blister packs, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs. If desired, the composition can be sterilized or mixed with any pharmaceutically acceptable carrier, such as a stabilizer, a wetting agent, and the like.

The compositions of the present invention may be admixed by any conventional method with conventional pharmaceutically acceptable excipients. Thus, the compositions of the invention to be used orally may comprise, for example, one or more color formers, sweeteners, flavor enhancers, and/or preservatives.

The invention further provides the use of a composition as described above for the preparation of a medicament for the treatment of a tumor.

The invention also provides the use of a composition as described above for the preparation of a medicament for boosting immunity.

Those of ordinary skill in the art to which the present invention pertains can select the appropriate route of administration and dosage to treat the individual in need of treatment without difficulty. According to the invention, the preferred route is oral administration. The dose is determined according to the nature of the disease and its condition, the age and health of the individual, the route of administration and pretreatment. Those of ordinary skill in the art to which the invention pertains will appreciate that the dosage will vary with the age, weight, health, and other relevant factors of the individual.

The above-described embodiments are merely illustrative of the principles and effects of the invention, and are not intended to limit the invention. Therefore, modifications and variations of the embodiments described above will be made without departing from the spirit of the invention. The scope of the invention should be as set forth in the appended claims.

Instance Preparation of burdock

The A. sinensis strain BCRC 930032 was inoculated on a PDA-containing plate medium, cultured in a 25 ° C incubator for 7 days, and the well-growth colonies were homogenized in sterile water, and then inoculated to a medium: bacterial solution = 9:1 ratio. The flask containing PDB was shaken for 7 days in a 25 ° C incubator at 150 rpm. Then, the fermented mycelium was inoculated into the 50-liter liquid fermentation tank in the same ratio at a rotation speed of 50 rpm and aeration of 0.2 to 0.3 vvm. After 7 days of culture, inoculate into a 500-liter liquid fermentation tank at a rotation speed of 20 Rpm, aeration of 0.2 to 0.3 vvm, cultured for 7 days, inoculated into a 5000 liter liquid fermentation tank at a speed of 20 rpm, aeration of 0.1 vvm, and after 4 days of culture, the rotational speed was changed to 50 rpm, and the bacteria were sterilized at a high rotational speed. The body produces pressure, induces metabolite production, and is cultured for another 10 to 12 days. The temperature was 30 ° C in each stage of liquid fermentation, and the medium was as described above. After the cultivation is completed, the sorghum sorghum mash is obtained by filtration.

Tea extract preparation

The black tea leaves are mixed with water according to the weight ratio of 1:10, and then heated to boiling for extraction for half an hour. After the extraction, the tea leaves are filtered out, and the obtained filtrate is a tea extract.

Ginger extract preparation

After being planted for 10 months, the ginger is ground into a mud and mixed with water at a weight ratio of 1:10. The mixture is heated to boiling for extraction for half an hour. After the extraction, the slag is filtered off, and the obtained filtrate is extracted by ginger. Things.

Antrodia camphora fermentation broth

The boletus edulis fermented composition obtained by centrifugation in Example 1 was thoroughly mixed with a ratio of 3:3:1 by weight in a ratio of 3:3:1 by weight.

Functional analysis of the fermentation composition of Antrodia camphorata

The quantum resonance pattern is used to detect the function of the Antrodia camphorata fermentation composition according to the present invention. The quantum resonance spectrum is an emerging rapid, accurate and non-invasive spectrum detection method, and is particularly suitable for the examination of the contrast of health foods, mainly collected by hand sensors. The frequency and energy of the weak magnetic field of the human body are compared with the standard quantum resonance spectrum of the nutrient index designed by the instrument after being amplified by the instrument and processed by the computer. The waveform of the sample is analyzed by the Fourier analysis method, and the effect of the health food is made according to the waveform analysis result. Out of the analysis, it has been widely accepted, such as Conf Proc IEEE Eng Med Biol Soc., a report on the dynamic monitoring of mouse sacroma-180 cells (Conf Proc IEEE Eng Med Biol Soc., 4:4119- 22, 2005), showing that QE values are directly related to immunity and anti-tumor capabilities. There are also related invention patents, such as the Republic of China Patent Bulletin No. 538351.

The beverages of the combination of Astragalus membranaceus, tea extract and ginger extract in a ratio of 3:3:1 were measured by quantum resonance spectroscopy. The results of quantum resonance experiments are shown in Table 1:

(1) Tumor message part

If the test results are increased by 32% compared with the test sample in which only the burdock sorghum broth is mixed with the black tea extract in a ratio of 1:1, if the ginger extract is added, the value is increased. 64%.

(2) Immune function part

If the test results are increased by 42%, the test value is increased by 42% if the test mixture is mixed with the tea extract in a ratio of 1:1. If the tea extract is mixed, add ginger extract. The value of the object is increased by 83%.

Claims (10)

A composition comprising: Antrodia cinnamomea mash; and a tea extract. The composition according to claim 1, wherein the Astragalus membranaceus lysate is obtained by fermenting a strain of Antrodia camphorata, selected from the Hsinchu Food Industry Development Institute of the Republic of China, BCRC 930032, 35396, 35398, 35716, 36401 a group consisting of 36711, 36795, 37848, 37849 and 37850. According to the composition of claim 1, wherein the Antrodia camphorata lysate is analyzed by High Pressure Liquid Chromatography (HPLC), and the chromatography column used is Inersil ODS-2, 5 μm, (GL Sciences, ToKyo , Japan), the length of the column is 250 mm, the inner diameter is 4.6 mm, the chromatograph used is Hitachi® PDA L2455, the extract (A) is 0.1% phosphoric acid solution, and the extract (B) is 100. % acetonitrile, the flushing formula is from 0 minutes to 60 minutes, the extract (A) is reduced from about 90% to about 70% and the extract (B) is raised from about 10% to about 30%; 60 minutes to 62 minutes: Maintaining the extract (A) is about 70% and the extract (B) is about 30%; from 62 minutes to 65 minutes, the extract (A) is raised from about 70% to about 90% and the extract (B) From about 30% to about 10%; 65 minutes to 80 minutes: maintain the extract (A) is about 90% and the extract (B) is about 10%; 65 minutes to 80 minutes: maintain the extract ( A) 90% with an absorption peak at a wavelength of 220 nm at a residence time of about 9.6, about 11.0, about 12.2, about 15.5, about 22.6, about 28.8, about 35.1, about 49.6 minutes. The composition of claim 1, wherein the tea extract is an extract of whole yeast tea. The composition of claim 1, wherein the tea extract is a black tea extract. The composition of claim 1, further comprising a plant extract, wherein the plant extract is a ginger extract. The composition of claim 1, wherein the weight ratio of the Antrodia camphorata: tea extract is from about 1:1 to about 1:20. The composition of claim 7, wherein the weight ratio of the Antrodia camphorata: plant extract is from about 10:1 to about 1:1. A use of a composition according to any one of claims 1 to 8 for the preparation of a medicament for treating a tumor. A use of a composition according to any one of claims 1 to 8 for the preparation of a medicament for boosting immunity.