TW201634055A - Ophiocordyceps formosana isolate and uses thereof - Google Patents

Abstract

The present invention relates to a novel isolate of Ophiocordyceps formosana, which exhibits excellent anti-oxidation and anti-cancer activities. The invention also relates to use of Ophiocordyceps formosana, in particular the isolate disclosed herein, in manufacturing a medicament for treating diseases associated with reactive oxygen species, such as cancers.

Description

Taiwan Cordyceps sinensis isolate and use thereof

The present invention relates to a novel Taiwan Cordyceps isolate which exhibits superior antioxidant and cancer cell inhibiting properties. The present invention also relates to the use of Taiwan Cordyceps sinensis for the manufacture of a pharmaceutical composition for treating active oxygen-related diseases in one body.

Cordyceps sl ( sensu lato ) in entomopathogenic fungi includes species such as Ophiocordyceps sinensis and Cordyceps militaris , which are highly regarded traditional Chinese medicines. It has been used as a tonic and folk prescription for many years, and it is also called "three treasures of traditional Chinese medicine" with ginseng and velvet. In addition to safety in use, Cordyceps is in the treatment of respiratory and cerebrovascular diseases (Wang, et al. , Metab. Brain Dis. 27, p . 159-165 (2012)), promoting immune regulation (Shi, et al. , Int. Immunopharmacol . 9, p . 582-586 (2009)), regulation of hepatic metabolism (Manabe, et al. , Br. J. Nutr. 83, p . 197-204 (2000)) and treatment of cancer (Jayakumar, The high pharmaceutical and therapeutic value of et al. , J. Cancer Res. Ther. 10, p . 43-49 (2014)) has also received attention. In this regard, in recent years, researchers have also devoted a lot of efforts to discover the bioactive components, pharmacological effectiveness and the second metabolites of various species of Cordyceps. In addition, reactive oxygen species (ROS) promote cell necrosis and contribute to cancer (Faisal, et al. , Urology 84, p. 1434-1441 (2004)), neurodegenerative diseases (Tiurenkov, et al. , Eksp . Klin . Farmakol 77, p . 16-19 (2014)), liver disease (Matsui, et al. , JPEN. J. Parenter. Enteral. Nutr. 31, p . 373-380 (2007)), atherosclerosis (Park, Et al. , Cardiovasc. Toxicol . 14, p . 1-9 (2014)) and other pathological conditions, and even cause aging (Ji, et al. , Phytother . Res. 23, p . 116-122 (2009); Lee , et al. , Arch. Biochem. Biophys. 490, p . 103-109 (2009)). It has been reported that Cordyceps sinensis can scavenge free radicals and can be used as an antioxidant (Yu, et al. , J. Agric. Food Chem. 54, p . 3132-3138 (2006); Li, et al. , Life Sci. 73 , p.2503-2513 (2003)).

In terms of taxonomy, the general Cordyceps is a large paraphyletic group of the generalized Claviciptaceae sl , which contains many species and has a wide range of hosts for insects, spiders and even truffle-like fungi. Recently, molecular markers such as ITS (internal transcribed spacer), RPB (RNA polymerase II), and EF (elongation factor) have been widely used in species identification and phylogenetic studies (Gardes, M. and Bruns, TD, Mol. Ecol. 2, 113-118 (1993); Schoch, et al. , Proc. Natl. Acad. Sci. 109, p . 6241-6246 (2012); Tanabe, et al. , Mol. Phylogenet. Evol. P.438-449 (2004)). Based on the results of multiple genes phylogenetic analysis, the current general Cordyceps has been divided into several genera, including Cordyceps ss , Elaphocordyceps , and green grass . Genus ( Metacordyceps ) and Cordyceps ( Ophiocordyceps ).

Taiwanese Cordyceps sinensis ( Ophiocordyceps formosana ) was first discovered in 1981 by Japanese Kobayashi and Shimizu in the Xitou area of Nantou County, Taiwan (Kobayasi, Y., Bull. Natn. Sci. Mus., Tokyo. Ser. B 7, p. 113-122 (1981)), which was originally identified as a native species of Taiwan. AD 2001, Anhui University professor Li as Huangshan Mountain in Anhui Province to Taiwan also collected Cordyceps sinensis, Cordyceps distribution means is not limited to Taiwan Taiwan (Li, et al., Mycosystema 24, p.349-355 (2005)). However, before the application was filed in this case, the identification of Cordyceps sinensis in Taiwan was limited to the morphological description. The physiology and biochemical analysis of Taiwan Cordyceps sinensis, as well as the application of medicine, are all paid for. The growth habits and morphological characteristics of Cordyceps sinensis in Taiwan are similar to those of other species in the general Cordyceps, making its medicinal potential attractive. However, before the application was filed in this case, there were few reports on the Japanese Cordyceps sinensis, and there was not enough material for further identification and discussion.

Due to the safety and effectiveness of various species of Cordyceps and its related products, it has developed a wide range of complementary and traditional medicines. However, due to its limited supply sources, high prices, excessive collection, and difficulty in artificial cultivation, the species of Cordyceps are on the verge of extinction. Therefore, in the related art, it is urgent to establish a typical strain of Taiwan Cordyceps sinensis based on the phylogenetic relationship among various species of Cordyceps. Further, there is a need in the related art for further research on the cancer cell inhibition potential, antioxidant activity, and safety of the individual to be administered to individuals in Taiwan.

According to the present invention, the inventors of the present invention have identified a novel isolate of Taiwan Cordyceps sinensis which is morphologically and phylogenetically identical to the known characteristics of Taiwan Cordyceps sinensis, but different from other species in the general Cordyceps genus. . In particular, by identifying and analyzing the isolates of Taiwan Cordyceps sinensis disclosed in this case, the inventors of the present invention unexpectedly discovered that Taiwan Cordyceps sinensis is superior to other species in the general Cordyceps genus in terms of antioxidant activity and cancer cell inhibitory activity, and It is less toxic to normal cells. This means that Taiwan Cordyceps can exhibit higher efficacy and greater safety in treating cancer than other species in the general Cordyceps. The disclosure of this case shows that Taiwan Cordyceps sinensis, especially the novel Taiwan Cordyceps sinensis isolates disclosed in this case, has extremely high application value in medical use.

Accordingly, in the first aspect disclosed in the present case, a Taiwan Cordyceps sinensis isolate is provided, which was deposited at the Republic of China Food Industry Development Research Institute (FIRDI) on January 16, 2015 under the registration number of BCRC 930172. The Center for the Conservation and Research of Biological Resources (BCRC), and based on the international recognition of the microbial deposits for patent procedures based on the Budapest Treaty, was deposited with the German National Collection of Microorganisms on January 28, 2015 under the registration number of DSM 32000. Center (Deutsche Sammlung von Mikroorganismen und Zellkulturen; DSMZ).

In a second aspect disclosed in the present invention, there is provided a cordyceps sinensis composition comprising the dried pulverized granule of the aforementioned Taiwan Cordyceps sinensis isolate or the aqueous extract of the aforementioned Taiwan Cordyceps sinensis isolate, and optionally comprising an acceptable one. excipient.

In the third aspect disclosed in the present invention, there is provided a health food comprising the aforementioned Taiwan Cordyceps composition.

In a fourth aspect disclosed in the present invention, there is provided a use of Taiwan Cordyceps sinensis for the manufacture of a medicament for treating a reactive oxygen-related disease in a body, preferably for use in the manufacture of a medicament for treating cancer. Pharmaceuticals, for example, for the treatment of lung cancer, breast cancer, liver cancer and leukemia.

Figure 1A-1G shows the morphological characteristics of Taiwan Cordyceps collected in Example 1, wherein Figure 1A shows that the sub-seat of the Taiwan Cordyceps sinensis grows from the host and has a long columnar handle, and Figure 1B shows the sub-seat head. It has a long elliptical shape, and the 1C figure shows that the ovate-shaped ascospore shell is buried in the sub-seat. The first DD shows that the capsular shell has a constricted neck surrounded by the thick wall of the ascospore shell, and the first E-picture shows that the ascospore spores The rod-shaped secondary spores formed by the break, the 1F figure shows that the colonies of the isolates disclosed in the present case are orange to white on the potato dextrose agar medium, and the outer surface is fluffy with small water drops, and the 1G image shows The asexual generation of Taiwan Cordyceps sinensis ( Hirsutella ); Figure 2 shows the phylogenetic tree of the general Cordyceps genus, which is linked by three sets of genes ( RPB1 , RPB2 , EF1 - α ) and pushed by the maximum approximation rule It is derived from three newly created subjects: Ophiocordycipitaceae, Cordycipitaceae, and Clavicipitaceae ss. The scale in the figure represents 50 mutations; 3A- 3F picture for various insects HPLC chromatogram of the aqueous extract of grass, where Figure 3A shows the residence time of D-mannitol (labeled D), adenosine (labeled A), and cordycepin (labeled C) in the HPLC column, Figures 3B to 3F are Taiwan Cordyceps sinensis mycelium (OF _MY ), Tibetan Cordyceps sinensis mycelium (OS _MY ), Tibetan Cordyceps sinensis (OS _FB ), Cordyceps sinensis mycelium (CM _MY ) and Cordyceps sinensis fruit bodies. HPLC elution profile of aqueous extracts of (CM _FB ); Figure 4A shows exposure of various cancer cell lines to Taiwan Cordyceps mycelium (OF _MY ), Tibetan Cordyceps sinensis mycelium (OS _MY ), Tibetan Cordyceps sinensis ( Survival rate of hot water extracts of OS _FB ), Cordyceps sinensis mycelium (CM _MY ) and Cordyceps sinensis fruit body (CM _FB ); Figure 4B shows survival of normal cell strains exposed to various Cordyceps extracts Rate; 5A-5D shows the antioxidant activity of each Cordyceps extract in vitro and in vivo, wherein Figure 5A shows Taiwan Cordyceps mycelium (OF _MY ), Tibetan Cordyceps mycelium (OS _MY ) and hot pupa Cordycepts (CM _MY) of an extracted object DPPH antioxidant activity in vitro of , FIG. 5B show Taiwan Cordycepts (OF _MY), fruiting body of Cordyceps sinensis (OS _FB) and pupae Cordyceps Grain (CM _FB) hot water the extract of DPPH in vitro antioxidant activity of 5C- Panel D shows the ROS scavenging efficacy and quantitative results of various Cordyceps extracts in vivo by flow cytometry ( p <0.05); and Figures 6A and 6B show Taiwan Cordyceps isolate O.formosana MUCHO 815 The anti-tumor activity exhibited by xenograft nude mice model inoculated with MDA-MB-231 human breast cancer cells, wherein Figure 6A is a histogram showing 1 time and 5 times the amount of Taiwan Cordyceps isolate O. The hot water extract of formosana MUCHO 815 (labeled with 1×OF _MY and 5×OF _{MY, respectively} ) inhibited tumor development in a nude mouse model in a dose-dependent manner, data expressed as mean ± standard deviation, and 6B The graph shows tumor size photographs of each group of nude mice at 20 days of treatment.

Unless otherwise stated, the following terms used in the patent specification and claims of this case have the definitions given below. Please note that the singular expression "a" used in the description and the claims is intended to cover one or more items, such as at least one, at least two, or at least three, A matter of inclusion. In addition, the open-ended conjunctions such as "including" and "including" used in the claims of the present invention are combinations of elements or components described in the claims, and do not exclude other elements or components not specified in the claims.

The term " Tianjin Cordyceps sinensis" as used in this specification is intended to cover all isolates classified as Cordyceps formonsana and classified in Ophiocordyceps formosana , in particular based on the nucleotide sequences of the RPB1 , RPB2 and EF1 -α genes. Identity , and is classified as all isolates of this species in Ophiocordyceps formosana . These strains of Cordyceps sinensis in Taiwan include strains of Cordyceps sinensis that are readily available to those of ordinary skill in the art, such as isolates that can be purchased from domestic or foreign depository institutions or obtained through application for distribution, or use in the art. A strain of Taiwan Cordyceps sinensis isolated from a natural source by a conventional microbial separation method. In a preferred embodiment, the Taiwan Cordyceps sinensis is the Taiwan Cordyceps sinensis isolate disclosed in the present case, which is deposited under the registration number of BCRC 930172 in the Food Industry Research and Development Institute (FIRDI). The Biosource Collection and Research Center (BCRC), and the DSM 32000 registration number are deposited with the German National Collection of Microorganisms (DSMZ).

The isolates of Taiwan Cordyceps sinensis disclosed in this case can be cultured and propagated according to the conventional Cordyceps mycelium culture method. In general, the culture conditions suitable for Cordyceps mycelium are glucose, sucrose, galactose, fructose, corn starch, malt extract or a combination thereof as a carbon source, and ammonium sulfate and ammonium nitrate are used. , sodium nitrate, casein amino acid, casamino acid, yeast extract, peptone and tryptone or a combination of them as a nitrogen source, and adjust the pH of the medium To neutral to slightly acidic, it is preferably pH 5.0-7.0, such as pH 5.5-6.0. In a preferred embodiment, the medium is a liquid medium suitable for shake flask fermentation to obtain Cordyceps mycelium, such as potato dextrose broth. In another preferred embodiment, the medium is a solid medium such as potato dextrose agar (PDA) or Sagittide dextrose agar extract (S-DAY). The culture temperature is preferably controlled at room temperature or slightly below room temperature, for example, 15 ° C to 25 ° C.

According to the MTT analysis described later, the species of Cordyceps sinensis in Taiwan, especially the isolate of Taiwan Cordyceps sinensis disclosed in this case, can exhibit significant cancer cell inhibitory activity. Moreover, compared with the activity of the mycelium of Cordyceps sinensis and Cordyceps sinensis, Taiwan Cordyceps can display stronger cancer cells. It inhibits the effect, and its ability to inhibit cancer cells is even comparable to that of Tibetan Cordyceps sinensis (see Example 7).

In general, Cordyceps sinensis is not easy to produce fruiting bodies by artificial culture, and it is usually only possible to mass produce mycelium by fermentation technology. Although the fruiting body of Cordyceps sinensis has been successfully cultured by artificial culture, the fruiting body takes a long time to grow, and the fruiting rate of the fruiting body is low and the mutation rate is high, so the production of mycelium is still relatively high. Economically beneficial. As is known to those of ordinary skill in the art, the content of active ingredients such as adenosine and cordycepin in Cordyceps sinensis is much higher than that of mycelium, so it is generally recognized that Cordyceps sinensis is compared to mycelium. Has a higher medicinal value. However, according to the disclosure of the present specification, Taiwan Cordyceps sinensis, especially the Taiwan Cordyceps sinensis isolate disclosed in this case, has a cancer cell inhibitory activity exhibited by the mycelium extract, which is equivalent to the fruiting body extract of the Tibetan Cordyceps sinensis. It is much higher than the mycelial excipient exhibited by the mycelium extract of Cordyceps sinensis and Cordyceps sinensis.

On the other hand, the adverse effects of oxidative stress on human health are also widely noted. In fact, exposure of organisms or cells to excess reactive oxygen species (ROS) can significantly lead to homeostasis. Finally, large amounts of ROS can cause damage to cells and DNA, cause cell death, and contribute to the development of harmful complications such as aging, high blood pressure, inflammatory reactions, and even cancer. It is generally believed that the antioxidant activity of Cordyceps is closely related to its pharmaceutical efficacy. In the present specification, the results of DPPH antioxidant analysis (see Example 8) and reactive oxygen species (ROS) clearance analysis (see Example 9) show that the mycelium of Taiwan Cordyceps sinensis is comparable to the mycelium of other Cordyceps species. have It has superior antioxidant capacity, its antioxidant capacity is even comparable to that of Cordyceps sinensis, and it has the lowest toxicity to cells or individuals, so it is suitable for treating, preventing or improving reactive oxygen-related diseases.

In summary of the results obtained by the above analysis, one aspect of the present invention is the use of Taiwan Cordyceps sinensis in the manufacture of a medicament for treating diseases associated with ROS in a body. The term "active oxygen" as used in this specification is as defined in WO 94/13300 A1 and is intended to encompass oxygen-centered free radicals such as hydroxyl radicals (‧OH), superoxide anions (O2‧-), etc. And non-radical oxygenated oxidants such as hydrogen peroxide. As used herein, "reactive oxygen-related disease" means an individual's condition caused, at least in part, by the production of reactive oxygen species or at least in part by exposure to an active oxygen environment, including but not limited to cataracts, diabetes, Aziz. Hyperthermia, heart disease, inflammation, cancer, male infertility, amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, and aging.

In a preferred embodiment, the aforementioned reactive oxygen species-related disease is cancer, including but not limited to solid tumors such as lung cancer (eg, non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC)), lung, head. And squamous cell carcinoma of the neck, breast cancer, ovarian cancer, prostate cancer, stomach cancer, cervical cancer, esophageal cancer, bladder cancer, pancreatic cancer, brain cancer, liver cancer, colon cancer, rectal cancer, skin cancer, hereditary and Sporadic mastoid renal cell carcinoma, melanoma, glioblastoma, neurofibromatosis, mesothelioma, and osteosarcoma; and liquid tumors such as lymphoma, white blood Diseases, such as acute lymphoblastic leukemia, acute myeloid leukemia, B-cell chronic lymphocytic leukemia, and chronic granulocytic leukemia, And multiple myeloma. In a more preferred embodiment, the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, and leukemia.

The terms "treatment" and the like as used herein mean reversing, alleviating, delaying, or inhibiting the progression of the disease described in the present specification or one or more of its symptoms. In some embodiments, the treatment can be performed after developing one or more symptoms. In other embodiments, the treatment can be performed asymptomatically. For example, treatment can be performed to an susceptible individual prior to the onset of symptoms (eg, based on the history of symptoms and/or based on genetic or other susceptibility factors). Treatment can also be continued after the symptoms have been resolved to prevent or delay the recurrence.

According to the disclosure of the present invention, any amount of Taiwan Cordyceps sinensis which can effectively treat active oxygen-related diseases or can effectively alleviate the severity of these diseases can be administered to a subject in need through any administration route. The exact amount required will vary from individual to individual, depending on the species, age and health of the individual, gender and diet, severity of illness, specific dosage form, mode of administration, duration of treatment, and agents used in combination or concurrently. Depending on factors. Those with ordinary knowledge in the related art should be able to understand that the daily total dose of Cordyceps sinensis in Taiwan will be determined by the physician within the scope of sound medical judgment. When given to a human individual, the extract of Cordyceps sinensis in Taiwan is usually one to three times a day, once or three times a week, or every double Monday. The frequency of the second administration is administered in an amount of about 0.01 to 1000 mg/kg of body weight per time, preferably about 0.1 to 100 mg/kg of body weight per time, more preferably about 1 to 50 mg per time. The amount of kilograms of body weight is given.

The term "individual" as used in this description is intended to encompass human or non-human vertebrates, such as non-human mammals. Non-human mammals include livestock animals, companion animals, laboratory animals, and non-human primates. Non-human mammals also include, without limitation, horses, cows, pigs, goats, dogs, cats, mice, rats, guinea pigs, hamsters, rabbits, baboons, and the like. It is preferably human, and more preferably an individual suffering from a reactive oxygen-related disease, such as an individual suffering from cancer.

For the purposes of research, the term "individual" as used in this specification is also intended to encompass cells, tissues and organs. The invention disclosed in the present invention is thus applicable not only to in vivo but also to in vitro.

Taiwan Cordyceps sinensis can be administered to individuals in the form of fruit bodies and mycelia. Since the fruit body is not easily cultured, it is preferably administered to the individual in the form of a mycelium, more preferably in the form of a dried pulverized particle of a mycelium, an aqueous extract or a preparation of an aqueous extract.

The dried Cordyceps sinensis fruit body or mycelium can be directly ground using a grinder to prepare the aforementioned dry pulverized particles. The particle size of the dried pulverized particles is not particularly limited as long as it is suitable for direct administration to an individual and is advantageous for the intake and absorption of the individual, or is advantageous for further processing into other forms suitable for administration to an individual.

Any conventional process can be used to prepare the aqueous extraction of Cordyceps As long as the extraction process can effectively transfer the water-soluble and water-miscible components of Cordyceps from the solid portion of Cordyceps to an aqueous solvent. Generally, the extraction process comprises pulverizing the fruiting body or mycelium of Cordyceps sinensis, and then using water, a lower alcohol having 1 to 4 carbon atoms (for example, methanol, ethanol) or a combination thereof. The solvent is subjected to an extraction operation, and preferably, the extraction operation is performed by applying thermal energy, ultrasonic vibration or mechanical agitation to promote the extraction efficiency, and finally, the solid portion is removed by filtration, centrifugation, etc., thereby obtaining the aqueous extract of Cordyceps. Produce. Preferably, the extraction process is selected from the group consisting of a leaching method, a hot water reflux method, a hot alcohol reflux method, an ultrasonic water extraction method, and an ultrasonic alcohol extraction method. In a preferred embodiment, the aqueous extract of Taiwan Cordyceps sinensis is prepared by hot water reflux method, that is, the quantitative hot water is continuously flowed through the cordyceps material to extract the aqueous extract, preferably the hot water temperature is 30-100 ° C, more preferably 40-60 ° C, most preferably 45-55 ° C, such as 50 ° C, and the extraction operation lasts from 30 minutes to 12 hours, preferably from 1 to 6 hours, for example 2 hours. In a preferred embodiment, the efficiency of the extraction operation can be assessed by the amount of extract of Cordyceps active ingredients such as D-mannitol, adenosine and/or cordycepin.

The aqueous extract of the aforementioned Taiwan Cordyceps sinensis can be formulated into an appropriate concentration and then formulated into an oral form or a parenteral form for administration to an individual. When these administration forms are prepared, a diluent such as a filler, a bulking agent, a binder, a wetting agent, a disintegrating agent, a surfactant, or the like may be added, or other types of excipients may be added.

The form of the liquid preparation for oral administration includes a suspension, a solution (syrup, a drink, etc.), an emulsion, and the like. In addition to the most commonly used diluents such as water or liquid paraffin, wetting agents, sweeteners, flavoring agents and preservatives may be added. oral The form of the solid preparation includes a tablet, a pill, a powder, a granule, a capsule, a lozenge or the like.

Forms for parenteral administration include sterile solutions, non-aqueous solutions, suspensions, emulsions, lyophilizates, and the like. Solvents for use in solvents, suspensions or emulsions for non-aqueous solutions include, but are not limited to, vegetable oils, propylene glycol, polyethylene glycol, olive oil, and ethyl oleate.

In general, these preparations are prepared by adding at least one excipient to the pulverized granules or aqueous extract, if necessary, and then granulating, tableting or encapsulating. The excipients include, but are not limited to, water, lower alcohols having from 1 to 4 carbon atoms, natural or synthetic oils, gelling agents, suspending agents, emulsifiers, thickeners, inert powders, natural and synthetic Polymers, humectants, sweeteners, flavoring agents, fragrances, perfumes, colorants, preservatives, lubricants, surfactants. Preferred excipients include, but are not limited to, water, ethanol, starch, calcium carbonate, sucrose, lactose, gelatin, gums, dextrin, cyclodextrin, microcrystalline cellulose, carboxymethyl cellulose, cross-linked carboxy Methyl cellulose sodium and the like. Further, a lubricant such as magnesium stearate or talc may be added in addition to the excipient alone.

Accordingly, one aspect of the present disclosure is a Cordyceps composition comprising an aqueous extract of a Taiwan Cordyceps isolate isolated as described above or a dry comminuted granule, and optionally comprising an acceptable excipient. The term "acceptable excipient" as used in this context means an inert substance which is used as a carrier of the composition of Cordyceps sinensis in this case, which is not toxic, irritating, pyrogenic, antigenic and hemolytic to the individual to be administered, and No substantial pharmacological activity will not hinder the beneficial effects of the composition of Cordyceps sinensis in this case. Another aspect disclosed in this case is a health food. The product comprises the aforementioned composition of Cordyceps sinensis for direct consumption, incorporation, or blending into various edible materials such as beverages, snacks, animal feed, dietary supplements, other Chinese herbal medicines, or packaging into other suitable materials. In the form of administration, for example, the aforementioned dry pulverized granules are packaged in a tea bag in the form of a tea bag for brewing. In a preferred embodiment, the composition of Cordyceps sinensis disclosed in the present invention may be combined with a composition derived from other Cordyceps species, for example, with a fruiting body or mycelium composition from Cordyceps sinensis and/or Cordyceps sinensis, thereby being formulated into A health food and pharmaceutical that is suitable for individual intake and that combines safety and efficacy.

In another preferred embodiment, the Cordyceps sinensis composition of the present invention consists essentially of an aqueous extract of the Taiwan Cordyceps sinensis isolate as described above or a dry comminuted granule and the aforementioned acceptable excipient. The term "consisting essentially of" as used herein means that the other combinations of the components described above do not exclude other undescribed components which do not substantially affect the properties of the aqueous extract or the dried pulverized particles. In another preferred embodiment, the Cordyceps sinensis composition of the present invention consists solely of the aqueous extract of the aforementioned Cordyceps sinensis isolate or the dried comminuted granules and the aforementioned acceptable excipients.

The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. All data shown in the examples was done via at least three independent experiments and expressed as mean ± standard deviation. Statistical analysis was performed using single factor analysis of variance (ANOVA) followed by Tukey's test and its significance was determined by the manner described in the examples.

Example 1: Collection of isolates and maintenance of micromorphological microscopy

The Taiwan Cordyceps sinensis isolates disclosed in this case were collected in the Lola Mountainous Area of Taoyuan County, Taiwan in August 2013. They were collected from the Tenebrionoidea in the fallen rot trunk, hence the name O.formosana. MUCHO 815. Immediately take an image of the collected sample as shown in Figures 1A and 1B. The samples were taken back to the Applied Fungal Laboratory of the University of Taiwan in Taipei City, where the spores and mycelium were separated under standard decontamination procedures.

Microscopic examination was performed using cryosection and compound microscopy. The sample was fixed using a 4% formaldehyde solution in phosphate buffered saline (PBS) for one day, during which time the solution was changed. After placing the sample in the optimal cutting temperature compound (OCT), it was cryosectioned in a Leica CM3050-S cryostat slicer to obtain 5 micrometers (μm) to 10 micrometers. Thick slices. Each section was examined and imaged under an Olympus BH2 microscope equipped with a Canon-ds126 camera, as shown in Figure 1C-1E.

Figure 1A-1E shows the Taiwan Cordyceps isolate isolated from Example 1, whose sub-seat is grown by the head or abdomen of the host. The handle has a long columnar shape and is orange in color and has a size of 10-30 mm × 0.5-2 mm. It has short hair and no sheath. The head of the sub-seat is oblong and has a size of 4-6 mm x 1-4 mm. The mycelium tissue is a thin-walled tissue, and the ascospore shell is buried, oval, brownish orange, and has a size of 360-480 micrometers x 240-320 micrometers. The capsule shell has a pore width of 60 μm and the ascospore shell wall is 20 μm thick and is buried under 40 μm thick hyphae. The ascus has a long cylindrical shape with a base contracted, 6.5-7.9 microns wide and 160-240 microns long. The ascus has a top width of 3.9-5.3 microns and a length of 3.2-4.6 microns. It has an iron bell-shaped shape and a narrow groove. Ascomycetes There are 8 sub-parts, transparent and colorless. When mature, they are broken into secondary spores. The secondary spores are rod-shaped, and the ends are frustoconical, and the size is 2.6-3.0 micron × 6.5-7.3 micron.

Example 2: Maintenance of isolates

Next, the isolated spores and mycelia were transferred to extracts containing potato dextrose agar (PDA; purchased from Difco, Detroit, Michigan, USA) or S. cerevisiae agar extract ( S-DAY; purchased from a Petri dish at Difco, Detroit, Michigan, USA, and cultured at 25 °C. Colonies were harvested once every 3-4 weeks. The colonies received were first dried at 55 ° C until the next day, and then stored in a desiccator for subsequent analysis or production of extracts. The small agar containing mycelia was frequently transferred to a new PDA or S-DAY medium to maintain the culture of the Taiwan Cordyceps isolate.

As shown in Figures 1F to 1G, the colonies of Taiwan Cordyceps sinensis isolates on the potato dextrose agar medium are orange to white, which are in the form of a mat-like to white button, with white hyphae and small appearance. Water drops. Its asexual type resembles Hirsutella , and the cells that produce conidia are solitary, transparent, and have an ampoule (size 1.5-2.3×8.6-17.0 μm). The conidia are transparent and cylindrical (dimensions of 1.6-2.3 x 3.6-6.9 microns).

Based on the observations shown in Examples 1 and 2, the Cordyceps isolates disclosed in this case have the same macroscopic and microscopic morphology as other previously discovered strains of Taiwan Cordyceps sinensis, indicating that they are indeed isolates of Taiwan Cordyceps sinensis.

Example 3: Extraction of Cordyceps genomic DNA

The extraction method of Cordyceps genus DNA is based on the literature published by Dürr et al. Make further changes ((Doyle and Doyle 1990)). Briefly, the fungal samples are derived from direct cutting of the stroma and stalks of Cordyceps, or from the mycelium in the culture dish. The collected samples were lyophilized, and 500 μl of hot Cetyltrimethyl ammonium bromide (CTAB) buffer (2% CTAB, 1.4 M NaCl, 20 mM EDTA, pH 8.0) was added. 100 mM Tris, 2% PVP-40) and fresh 3 microliters of 2-mercaptoethanol were ground with a mash. After incubation at 60 ° C for 20 minutes, 500 μl of CI (chloroform: isoamyl alcohol = 24:1) was added, and gently mixed for 10 minutes, and then centrifuged at 13,200 × g for 2 minutes. The supernatant was carefully transferred to a new eppendorf tube and 0.6 volumes of isopropanol were added. Next, the mixture was incubated at -20 ° C for 30 minutes to precipitate a DNA. DNA was collected by centrifugation at 13,200 g for 30 minutes and washed once with 75% ice-cold ethanol, vacuum dried, and resuspended in 50 microliters of secondary deionized water.

Example 4: Amplification and sequencing of DNA

Polymerase chain reaction (PCR) is used to perform amplification of specific fragments, as described below. Roughly, the PCR reaction mixture contained 2.5 microliters of 10X reaction buffer, 1 microliter of 10 μM each specific primer, 0.5 microliter of 10 mM dNTPs, and 2 microliters of template DNA in a total volume of 25 microliters. And 0.3 microliter of 1 U Taq polymerase (purchased from Boehringer-Mannheim, Inc., Indiana, USA). Use positive primer cRPB1 (sequence identification number: No. 1) and reverse primer RPB1c-r (sequence identification number: No. 2) (Castlebury, et al. , Mycol. Res. 108, p.864-872 (2004) )), and follow the steps below to amplify the rpb1 sequence: denaturation at 95 ° C for 5 minutes, followed by 40 minutes consisting of 95 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 1 minute. The cycle was followed by an additional extension step at 72 ° C for 10 minutes. The forward primer fRPB2-5f (sequence identification number: No. 3) and the reverse primer fRPB2-7cr (sequence identification number: No. 4) were used (Liu, et al. , Mol. Biol. Evol . 16, p . 1799). -1808 (1999)), and following the steps below to amplify the rpb2 sequence: denaturation at 95 ° C for 5 minutes, followed by a 50 ° consisting of 95 ° C for 50 seconds, 55 ° C for 1 minute and 72 ° C for 90 seconds One cycle followed by an additional extension step of 10 minutes at 72 °C. Use forward primer EF1-2218R (sequence identification number: No. 5) and reverse primer EF1-983F (sequence identification number: No. 6) (Rehner, SA, Buckley, E., Mycologia 97, 84-98 (2005) )), and follow the steps below to amplify the EF1-α sequence: denaturation at 94 ° C for 5 minutes, followed by 40 cycles consisting of 94 ° C for 1 minute, 46 ° C for 30 seconds and 72 ° C for 2 minutes And an additional extension step of 7 minutes at 72 °C. The PCR product was subjected to electrophoretic analysis and eluted for sequencing using the ABI 3730XL system (Applied Biosystems, CA). Next, the resulting sequence was spliced and trimmed using ContigExpress of Vector NTI® software (Invitrogen, Calif.).

According to the above method, the nucleotide sequences of the RPB1 , RPB2 and EF1 -α genes of the Japanese Cordyceps sinensis isolates disclosed in the present invention were obtained, which have 790, 1062 and 1068 nucleotides, respectively. The blast search was performed in the NCBI database using the obtained sequences, and the three sequences were comprehensively considered. The top three most similar to the Taiwan Cordyceps sinensis isolates disclosed in this case are Taiwan Cordyceps sinensis isolate TNM F13893, and the large group of Cordyceps sinensis isolate CBS. 100239 ( Elaphocordyceps ophioglossoides CBS 100239) and Taiwan Cordyceps isolate TNM F13893 enjoyed 99% (676/679), 89% (939/1061) and 99% (847/849) sequence identity, respectively. As far as RPB2 is concerned, although the closest is not Taiwan Cordyceps sinensis, the RPB2 sequence of the Taiwan Cordyceps sinensis isolate disclosed in this case is 100% identical to the Taiwan Cordyceps isolate TNM F13893 and has a coverage of 47%. These results show that at the molecular level, the Taiwan Cordyceps sinensis isolates disclosed in this case belong to the same species as the traditional Taiwan Cordyceps isolate TNM F13893, as shown in Table 1 below.

Example 5: Analysis of phylogenetics

The nucleotide sequences of the three largest and second largest units of RNA polymerase II ( RPB1 , RPB2 ) and the translation elongation factor 1-α ( EF1 - α ) of each Cordyceps strain are followed by Song et al. The requirements described in the literature (Sung, et al. , Mol. Phylogenet. Evol. 44, p . 1204-1223 (2007)), or the whole-geneoorthologous genes (orthologs) by the Cordyceps sinensis isolate CM01 BlastN (Basic Local Alignment Search Tool Nucleotide) sequencing tool, obtained from the PCR sequencing product of the Taiwan Cordyceps isolate isolated from the case, or from the National Center for Biotechnology Information (NCBI) database. A total of 51 species/isolated strains were included in the study contained in this example. The molecular evolution of the three groups of genes was linked and aligned using Molecular Evolution Genetic Analysis Software Version 6.0 (MEGA 6.0; http://www.megasoftware.net). Use the RAxML blackbox page (RAxML 7.2.7; http://embnet.vital-it.ch/raxml-bb) set up on the CIPRES portal to analyze the completed sequence to find the closest system occurrence. tree. Save the phylogenetic tree and plot it using Adobe® Illustrator® CS3 graphical design software.

The most likely phylogenetic tree is constructed in Figure 2, and its tree shape is very consistent with the literature published by Song et al. (Sung, et al. , Mol. Phylogenet. Evol. 44, p . 1204-1223 (2007)). Moreover, the species of Cordyceps sinensis in Taiwan is located in the branch of Ophiocordycipitaceae, not in the two branches of Cordycipitaceae or Clavicipitaceae ss. In the same way, the phylogenetic tree constructed in this case shows that the relationship between Taiwan Cordyceps sinensis and Tibetan Cordyceps sinensis is closely related, while Cordyceps sinensis is located in Cordyceps. Accordingly, the isolates of Taiwan Cordyceps sinensis disclosed in this case belong to the same branch of the Japanese isolates of Cordyceps sinensis in the phylogeny and belong to the same species. Moreover, according to the phylogenetic tree constructed in this case, the scientific name of Taiwan Cordyceps sinensis should be modified from the original Cordyceps formonsana to Ophiocordyceps formosana .

Example 6: Preparation and HPLC analysis of Cordyceps extracts

The mycelium and fruiting bodies of Cordyceps sinensis used in the following examples were obtained from Taiwan Muqi Biotech Co., Ltd. Mycelium and fruiting bodies of Cordyceps sinensis It was purchased from Beijing Tongrentang Sinopharm Co., Ltd. The mycelium of the Taiwan Cordyceps isolate was obtained from Example 2.

The dried Cordyceps sample was pulverized at a running speed of 1300 rpm using a homogenizer and stored in a moisture-controlled dryer at room temperature for subsequent high performance liquid chromatography (Hitachi D-2000 type) HPLC management system), analysis of de novo secondary metabolites in these Cordyceps samples. Cordyceps extracts were prepared according to standard operating procedures as described below. Briefly, in a 50 ml Falcon® centrifuge tube (purchased from Becton Dickinson and Company, USA), 1 gram of Cordyceps powder was dissolved in deionized water at a ratio of solid to solvent: 1:40. The oscillation was applied for 30 seconds. Next, the hot water was refluxed at 50 ° C for 2 hours to carry out an extraction operation (an ultrasonic wave was applied for the first 30 minutes). After centrifugation at 4000 rpm (about 3200 x g) for 20 minutes, the supernatant was collected and filtered through a 0.22 micron pore size filter. The resulting extract can be directly subjected to HPLC analysis or stored at -20 ° C for subsequent experiments of Examples 7 to 10.

High performance liquid chromatography was performed on an RP-18 column (150252 Purospher® STAR RP-18 endcapped (5 μm) LiChroCART® 250-4; Merck) at a flow rate of 1 ml per minute. The composition of the mobile phase was 20% methanol/H ₂ O. Three components, D-mannitol, adenosine and cordycepin, were detected at 260 nm using a diode array sensor (DAD detector). The results are shown in Figures 3A-3F.

As shown in Figure 3A-3F, the chemical composition of the Japanese Cordyceps sinensis isolate O.formosana MUCHO 815 is very similar to that of Tibetan Cordyceps sinensis and Cordyceps sinensis, which means that the biochemical characteristics of the isolates of Cordyceps sinensis in Taiwan are reasonable and well-known. The general Cordyceps species/isolated strain is similar. In addition, the peak area corresponding to D-mannitol, adenosine, and cordycepin standards in the HPLC profile of each Cordyceps extract sample was measured to uniquely metabolize three species of Cordyceps, D-mannitol, adenosine, and cordycepin. The contents of the materials were compared (r ² >0.999), and the results are shown in Table 2 below. The data in Table 2 indicates that the isolates of Cordyceps sinensis in this case also contain a metabolite content comparable to or even slightly higher than the conventional broad Cordyceps species/isolated strain.

Example 7: Cell Culture and MTT Analysis

MTT assay is used to measure the ability of Cordyceps extract to inhibit proliferation of human cancer cells. In a 96-well culture dish containing 200 microliters of medium per well, about 5 x 10 ³ cells were inoculated into each well and treated after 24 hours. After exposure to various concentrations of the designated Cordyceps extract for 96 hours, 20 μl of thiazole blue tetrazolium bromide (MTT; purchased from Sigma-Aldrich Corp., Missouri, USA, catalog number: M2128) was added. The cells were incubated for 4 hours. Cell proliferation was measured using a spectrometer (SpectraMax® 190 UV-Vis Microplate, Molecular Devices, USA) to measure the absorbance at 570 nm. This measurement is repeated three times. The percentage of cell survival was calculated by the following formula: (sample OD _{570 nm} / blank sample OD _{570 nm} ) × 100 (%).

In this example, the hot water extract of the mycelium of Taiwan Cordyceps sinensis isolate, the Cordyceps sinensis and the Cordyceps sinensis prepared by the procedure described in Reference Example 6 was used to treat various human cancer cells at different concentrations. For example, A549 lung cancer cell line, MDA-MB-231 breast cancer cell line, Huh7 liver cancer cell line, and HL-60 leukemia cell line. As shown in Figure 5A, the extract of Taiwan Cordyceps isolate O.formosana MUCHO 815 (labeled as OF _MY ) successfully demonstrated significant cancer cell inhibition and had a half-inhibitory concentration of 1.0 mg/ml for A549 cells. (IC50), having an IC50 of 0.53 mg/ml for MDA-MB-231 cells, an IC50 of 0.44 mg/ml for Huh7 cells, and an IC50 of 0.19 mg/ml for HL-60 cells. Compared with the mycelial extracts (OS _MY and CM _MY ) of Cordyceps sinensis and Cordyceps sinensis, the extract of O. formosana MUCHO 815 from Taiwan Cordyceps sinensis (OF _MY ) showed stronger The cancer cell suppresses the effect. Moreover, its ability to inhibit all tested cancer cell lines is even comparable to the fruiting body extract (OS _MY ) of Tibetan Cordyceps sinensis. The IC ₅₀ values of the respective Cordyceps extracts on the anticancer activity are shown in Table 3 below.

The inventors of the present invention have unexpectedly discovered that the extract of Taiwan Cordyceps isolate O.formosana MUCHO 815 is comparable to other Cordyceps extracts for normal cells, including HCEC normal human corneal epithelial cells and MCF10A normal human mammary epithelial cells (purchased). Since the American Type Culture Collection (ATCC), it exhibits the lowest toxicity (cell survival rate over 90%), as shown in Figure 5B. It is particularly noteworthy that human corneal epithelial cells are extremely sensitive to toxic and irritating substances and are often used to test the toxicity of the substance. This example shows that the extract of Taiwan Cordyceps isolate O.formosana MUCHO 815 is for human cornea. Epithelial cells are completely non-toxic. In addition, mice were fed with Japanese mycelium of Cordyceps sinensis at a dose of 5 grams per kilogram of body weight for 14 days, sacrificed and observed for tissue/organ sections, and no significant damage or biochemical changes were found (data not shown). The results of this example indicate that the isolates of Taiwan Cordyceps sinensis in this case can selectively inhibit the proliferation of cancer cells without causing damage to normal cells, and thus have higher medicinal potential than the conventional Cordyceps.

Example 8: DPPH antioxidant analysis

By mixing an equal amount of Cordyceps extract and 2,2-diphenyl-picrylhydrazyl hydrate (DPPH) solution (0.1 mM in methanol) by vigorous means, DPPH free radicals are removed. This was followed by incubation at room temperature for 50 minutes and then at 517 nm using a spectrometer (SpectraMax® 190 UV-Vis Microplate, Molecular Devices, USA) for detection. The scavenging activity was determined by comparing blank samples containing only DPPH and solvent (set to 100%). The percentage of scavenging activity was calculated by the following formula: (sample OD ₅₁₇ nm / blank sample OD ₅₁₇ nm) × 100 (%).

As shown in Figure 5A, compared to the mycelial extracts of Cordyceps sinensis and Cordyceps sinensis (labeled OS _MY and CM _MY , respectively), the hot water extract of the mycelium of the Cordyceps sinensis isolate from Taiwan was revealed in this case. (OF _MY ) clearly shows superior antioxidant capacity. Moreover, as shown in Fig. 5B, its antioxidant capacity is even comparable to the fruiting body extract (OS _FB ) of Cordyceps sinensis.

Example 9: Analysis of Reactive Oxygen (ROS) Removal

In a 12-well culture dish, each well was inoculated with approximately 5×10 ⁴ CHO-K1 cells (purchased from the American Type Culture Collection (ATCC)) and after 16 hours of serum starvation treatment, Exposure to the designated Cordyceps extract for 1 hour. In this example, treatment with 10 mM N-acetylcysteine was used as a positive control. After treatment with H ₂ O ₂ (1800 M) for 5 minutes, the cells were incubated with a fluorescent probe CM-H2DCFDA (Invitrogen, California, USA) in a 37 ° C incubator under dark room conditions. Finally, the cells were trypsinized cells from the culture dish, and then use a BD FACSCanto ^TM flow cytometer (purchased from U.S. BDBiosciences) 525nm detected dichloro-luciferin (DCF) in the level, and then calculate the The content of active oxygen.

As shown in Figure 5C, compared to the mycelial extracts of Cordyceps sinensis and Cordyceps sinensis (labeled OS _MY and CM _{MY respectively} ), the hot water extract of mycelium of Taiwan Cordyceps sinensis isolates was revealed in this case. (OF _MY ) clearly shows superior antioxidant capacity. It is worth noting that its antioxidant capacity is also comparable to the fruiting body extracts (OS _FB and CM _FB ) of Cordyceps sinensis and Cordyceps sinensis, as shown in Figure 5D.

Example 10: Nude mouse tumor animal model

In order to further investigate the anti-tumorigenic activity of Cordyceps sinensis in Taiwan, this example established a xenograft mouse model. 5×10 ⁶ MDA-MB-231 human breast cancer cells were inoculated into the left thigh of 9 10-week-old nude mice (BALB/cAnN.Cg- Foxn1nu /CrlNarl) via subcutaneous injection. After 10 days, the tumor has grown to the point where it can be detected by hand touch, and then the feeding process begins. The nude mice were divided into three groups, and the hot water extracts of the Japanese Cordyceps sinensis isolate O.formosana MUCHO 815 were separately fed (concentration of 25 mg/ml, 100 μl per day; 1×OF) _MY label group), 5 times the amount of hot water extract of O.formosana MUCHO 815 from Taiwan Cordyceps sinensis isolate (concentration: 125 mg/ml, 100 microliters per day; group marked with 5×OF _MY ), As well as the control group (only 100 μl of PBS was fed). Tumor size was measured and recorded every 4 days, i.e., tumor size = 0.4 x tumor length x (tumor width) ² , and the results are shown in Fig. 6A. After 20 days of treatment, the images of the tumor were imaged as shown in Fig. 6B.

It can be observed from Figures 6A and 6B that the 1×OF _MY group and the 5×OF _MY group reduced the tumor size in a dose-dependent manner compared with the control group, which confirmed that the Taiwan Cordyceps isolate O.formosana MUCHO 815 was able to It exhibits superior anti-tumor activity in vivo.

While the invention has been described in detail hereinabove, it will be apparent to those of ordinary skill in the art. The disclosure of the related art and the disclosure of the references discussed in the Detailed Description of the Invention and the Detailed Description of the Invention are hereby incorporated by reference.

【Biomaterial Storage】

Domestic registration information [please note according to the registration authority, date, number order]

Republic of China Food Industry Development Institute, January 16, 104, BCRC 930172

Foreign deposit information [please note according to the country, organization, date, number order]

German National Collection of Microorganisms (DSMZ), January 28, 104, DSM 32000

<110> Mu Qisheng Technology Co., Ltd.

<120> Taiwan Cordyceps sinensis isolate and its use

<160> Number of sequence identification numbers: 6

<210> Sequence ID: No. 1

<211> Sequence length: 20

<212> Sequence Type: DNA

<213> Organism: Artificial sequence

<220> Features:

<223> RPB1 gene for PCR amplification of Taiwan Cordyceps sinensis

<400> Sequence: 1

<210> Sequence ID: No. 2

<211> Sequence length: 23

<212> Sequence Type: DNA

<213> Organism: Artificial sequence

<220> Features:

<223> RPB1 gene for PCR amplification of Taiwan Cordyceps sinensis

<400> Sequence: 2

<210> Sequence ID: No. 3

<211> Sequence length: 20

<212> Sequence Type: DNA

<213> Organism: Artificial sequence

<220> Features:

<223> RPB2 gene for PCR amplification of Taiwan Cordyceps sinensis

<400> Sequence: 3

<210> Sequence ID: No. 4

<211> Sequence length: 20

<212> Sequence Type: DNA

<213> Organism: Artificial sequence

<220> Features:

<223> RPB2 gene for PCR amplification of Taiwan Cordyceps sinensis

<400> Sequence: 4

<210> Sequence ID: No. 5

<211> Sequence length: 23

<212> Sequence Type: DNA

<213> Organism: Artificial sequence

<220> Features:

<223> EF1-α gene for PCR amplification of Taiwan Cordyceps sinensis

<400> Sequence: 5

<210> Sequence ID: No. 6

<211> Sequence length: 23

<212> Sequence Type: DNA

<213> Organism: Artificial sequence

<220> Features:

<223> EF1-α gene for PCR amplification of Taiwan Cordyceps sinensis

<400> Sequence: 6

Claims (14)

A Taiwanese Cordyceps sinensis ( Ophiocordyceps formosana ) isolate, which is deposited with the Bioresource Conservation and Research Center (BCRC) of the Republic of China Food Industry Development Research Institute (FIRDI) under the registration number of BCRC 930172, and is registered with the registration number of DSM 32000. It is deposited at the German National Collection of Microorganisms (Deutsche Sammlung von Mikroorganismen und Zellkulturen; DSMZ). A Cordyceps sinensis composition comprising an aqueous extract of a Cordyceps sinensis isolate according to claim 1 of the patent application, optionally comprising an acceptable excipient. The composition of the Cordyceps sinensis according to claim 2, wherein the aqueous extract of the Taiwan Cordyceps sinensis isolate is prepared by pulverizing the mycelium of Taiwan Cordyceps sinensis, extracting with an aqueous solvent, and removing the solid portion. . A Cordyceps sinensis composition according to claim 3, wherein the aqueous solvent is selected from the group consisting of water, a lower alcohol having 1 to 4 carbon atoms, and a combination thereof. For example, the Cordyceps sinensis composition of claim 4, wherein the extraction with an aqueous solvent is carried out by refluxing the mycelium of Taiwan Cordyceps sinensis with hot water at a temperature of 40-60 ° C for 30 minutes to 12 hours. A Cordyceps sinensis composition comprising dried pulverized granules of a Taiwan Cordyceps sinensis isolate according to claim 1 of the patent application, optionally comprising an acceptable excipient. A use of Taiwan Cordyceps sinensis for the manufacture of a medicament for treating active oxygen-related diseases in a body. For example, the use of the patent scope of the seventh item, wherein the Taiwan Cordyceps sinensis is the Taiwan Cordyceps isolate isolated in the first application scope of the patent application. For example, the use of the Taiwan Cordyceps isolate is a composition of the Taiwan Cordyceps sinensis contained in any one of claims 2-6. The use of the seventh aspect of the patent application, wherein the reactive oxygen-related disease is selected from the group consisting of cataract, diabetes, Alzheimer's disease, heart disease, inflammation, cancer, male infertility, and muscular atrophic spinal cord A group consisting of sclerosing, multiple sclerosis, Parkinson's disease, and aging. The use of claim 7, wherein the reactive oxygen species-related disease is cancer. The use according to claim 11, wherein the cancer is selected from the group consisting of squamous cell carcinoma, breast cancer, ovarian cancer, prostate cancer, gastric cancer, cervical cancer, and esophagus from lung cancer, lung, head and neck. Cancer, bladder cancer, pancreatic cancer, brain cancer, liver cancer, colorectal cancer, rectal cancer, skin cancer, hereditary and sporadic mastoid renal cell carcinoma, melanoma, glioblastoma, neurofibroma, mesothelial cells A group of tumors and osteosarcoma, lymphoma, leukemia, and multiple myeloma. The use of claim 12, wherein the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, and leukemia. A health food comprising the provisions of any of claims 2-6 Taiwan Cordyceps composition.