KR101086528B1 - New isoflavonoids of Cordyceps militaris grown in the germinated soybean - Google Patents

Abstract

The present invention germinated soybean cordyceps ( Coldyceps militaris grown in the germinated soybean) is a new natural product, and more specifically, Cordyceps militaris inoculated with soybean germination was isolated by gel filtration chromatography by separating the soluble part of ethyl acetate in 80% methanol extract The present invention relates to an isoflavone-based compound and a method for separating the same, and the present invention relates to the germinated soybean Cordyceps sinensis extract and the compound represented by the formulas (1), (2), (3) and (4) according to the present invention. It is useful for the development of a drug using a novel functional bioactive substance as a compound.

Cordyceps militaris, soybean, isoflavone

Description

New isoflavonoids of Cordyceps militaris grown in the germinated soybean}

The present invention germinated soybean cordyceps ( Coldyceps militaris grown in the germinated soybean) and isoflavone-based compounds isolated from the soybean, and more particularly, gel filtration chromatography of the ethyl acetate soluble part of the extract extracted with 80% methanol from the inoculation of Cordyceps militaris in soybean germination The present invention relates to a novel isoflavone-based compound separated by grafting and a method for separating the same.

In general, isoflavone-based compounds in soybeans are compounds that function similar to estrogen, a female hormone in the body, and have the effect of blocking the action of estrogen in the body, and activate it in a situation of lack of estrogen. It is known to be possible. In particular, isoflavone-based compounds present in soybeans, among which typical Dadezein and Genistein, have been reported to significantly reduce the risk of breast cancer, and have been reported to inhibit the incidence of prostate cancer. In addition, the isoflavone-based compound of soybean has an antioxidant effect and lowers bad cholesterol in the blood, thereby preventing atherosclerosis and heart disease.

Cordyceps sinensis is known as a elixir of immortality in China, Japan, etc., and has been recognized for its value as an opioid antidote, post-care and tonic, pulmonary disease treatment, and immune booster. Overseas, as interest in fungi as a food source for the future has been concentrated, research on higher fungi, especially Cordyceps fungus, is being conducted not only as a basic research but also as a food, medicine, and natural pesticide product. In Korea, Cordyceps militaris and Snowflower Cordyceps ( Paecilomyces tenupis ) have been successfully used in pupae or silkworms and have been successfully applied. Recently, prevention and improvement of adult diseases such as cancer, stroke and heart disease have been found. It is spotlighted as a material with infinite potential. It has been reported that various bioactive components such as cordycepin and polysaccharide derived from Cordyceps sinensis have anti-cancer effects. These soybeans and various fungus and based on physiological activity produced by culturing the fungus inoculated seed germination in soybean Soybean Germination of Cordyceps (Cordyceps militaris) (Cordyceps militaris grown in the germinated It is convinced that improved bioactive substances can be developed in soybean), and it is necessary to identify, identify and evaluate various useful substances.

Accordingly, it is an object of to solve the problems as described above, the present invention is Cordyceps (Cordyceps militaris germinated soybeans grown in the germinated Soybean) was used to isolate novel high-functional physiologically active substances, and as a result, new isoflavones isolated from germinated soybean cordyceps were extracted and purified to identify their structure. To provide a compound.

In addition, another object of the present invention is germinated soybean Cordyceps militaris grown in the germinated soybean) and 80% methanol extraction and ethyl acetate soluble part of the extract to provide a separation method to separate the novel isoflavone-based compounds by gel filtration chromatography.

In order to achieve the above object, the novel isoflavone-based compound isolated from germinated soybean cordyceps according to the present invention is an isoflavone-based compound represented by the following formulas (1), (2), (3) and (4).

(Formula 1)

(Formula 2)

(Formula 3)

(Formula 4)

In addition, the method for isolating a novel isoflavone-based compound isolated from germinated soybean Cordyceps sinensis according to the present invention, Germinated soybean Cordyceps sinensis ( Cordyceps militaris grown in the germinated soybean) extracted with 80% lower methanol, the second step of extracting the lower alcohol extract with ethyl acetate to recover the ethyl acetate soluble part, and the ethyl acetate soluble part by gel filtration chromatography with active fractions A third step of obtaining, a fourth step of concentrating the active fraction under reduced pressure, and a fifth step of obtaining the concentrated powder under reduced pressure as a single pure compound using high performance liquid chromatography (High Perfomance Liquid Chromatography) .

Germinated soybean Cordyceps sinensis extract and the compounds represented by the formulas (1), (2), (3) and (4) according to the present invention are novel isoflavone-based compounds that have not been reported to date, and are useful for the development of drugs using novel functional bioactive substances.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

The present invention is a novel isoflavone-based compound represented by the formula (1), (2), (3), (4), and germinated soybean cordyceps ( Cordyceps militaris grown in the germinated) soybean) to extract and isolate such a novel compound.

As described above, in the present invention, the compounds represented by the following Chemical Formulas 1, 2, 3, and 4 may be synthesized by an organic synthesis method, or may be extracted and separated from germinated soybean cordyceps according to the method of the present invention.

(Formula 1)

(Formula 2)

(Formula 3)

(Formula 4)

The method for extracting and separating novel isoflavone family compounds isolated from germinated soybean cordyceps of the present invention includes the following steps.

Cordyceps militaris grown in the germinated soybean) extracting with 80% lower methanol,

A second step of recovering the ethyl acetate soluble portion by shaking the lower alcohol extract with ethyl acetate;

A third step of obtaining an active fraction by gel filtration chromatography of the ethyl acetate soluble part;

A fourth step of concentrating the active fraction under reduced pressure, and

The fifth step is to obtain the powder concentrated under reduced pressure in the fourth step as a single pure compound by using high performance liquid chromatography (High Perfomance Liquid Chromatography).

Next, germinated soybean cordyceps ( Coldyceps militaris grown in the germinated soybean) from the germinated soybean Cordyceps Sinensis extract or the separation process of the active substance in detail step by step, first, seeded soybean Cordyceps inoculated and cultured germinated soybean Cordyceps soybean germinated soybeans. Then, the mixture was refluxed for 48 to 72 hours with 80% lower methanol and filtered under reduced pressure, and the filtrate was concentrated under reduced pressure at a temperature of 20 to 50 ° C. Then, the concentrated 80% methanol extract was suspended in excess water, put into an aliquot, and the excess ethyl acetate was added to shake extraction. The supernatant is recovered and concentrated under reduced pressure at a temperature of 40-60 ° C. to obtain an ethyl acetate soluble part.

The concentrated ethyl acetate soluble part is coated on a small amount of silica (silica), followed by gel filtration chromatography to collect the active aliquots. At this time, the active liquid is collected by increasing the content of methanol using chloroform and methanol in silica gel column chromatography. The active fractions thus obtained are subjected to liquid chromatography mass spectrometry (LC / MS) to obtain active fractions containing new compounds.

Next, the obtained active fractions are subjected to Sephadex LH-20 gel column chromatography to collect the granular active fractions.

The collected active fractions are concentrated under reduced pressure at a temperature of 40-60 ° C. to obtain a powder, dissolved in a small amount of higher methanol, and then obtained as a single pure compound using high performance liquid chromatography (High Perfomance Liquid Chromatography).

The active compound finally separated by the above extraction separation process is a novel isoflavone-based compound which has not been reported to date. Thus, the present invention provides an extract of germinated soybean Cordyceps sinensis or a novel isoflavone represented by Chemical Formulas 1, 2, 3, and 4. Family compounds and their pharmaceutically acceptable salts.

The compounds represented by the formulas (1), (2), (3) and (4) may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful.

Compounds represented by the formula (1), (2), (3), (4) may form an acid addition salt which is pharmaceutically adhering according to a conventional method in the art.

Free acid and inorganic acid may be used as the free acid, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, etc. may be used as the inorganic acid, and citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, and fumaric acid may be used as the organic acid. (fumaric acid), formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galluxuronic acid, embonic acid, glutamic acid or aspartic acid Can be used.

In the case of using the novel compound of the present invention as a medicine, the extract of germinated soybean Cordyceps sinensis or the compound represented by the above formulas (1), (2), (3) and (4), or a pharmaceutically acceptable salt thereof is conventionally used in the pharmaceutical field. Formulated with a carrier to prepare conventional oral preparations in the pharmaceutical field, such as tablets, capsules, troches, solutions, suspensions, and the like, and solutions or suspensions for injection, or distilled water for injection at the time of injection. It may be formulated into a variety of preparations, such as injectable preparations in the form of dry powders for injection, ready-to-use injections, and ointments.

Pharmaceutical formulations prepared using conventional carriers can be administered orally or parenterally, for example, intravenously, subcutaneously, intraperitoneally or topically.

The dosage of the germinated soybean Cordyceps Sinensis extract of the present invention or the compound represented by Formulas 1, 2, 3, and 4 may vary depending on the age, condition, etc. of the patient, and generally 10 to 500 mg per day, preferably to adults. The amount of 50 to 300mg is to be administered, and may be divided into several times a day, preferably once to six times at regular intervals according to the judgment of the doctor or pharmacist.

When the present invention will be described in detail with reference to Examples, the present invention is not limited to the following Examples.

Example 1 Preparation of Germinated Soybean Cordyceps Sinensis Extract

Inoculate germinated soybean seedlings with germinated soybean seed inoculated and cultured, germinated soybeans, and reflux-extracted with 80% methanol for 48 ~ 72 hours, filter under reduced pressure, and concentrate the filtrate under reduced pressure in a 40 ℃ water bath. It was suspended in 1 L of distilled water. The methanol extract suspended in distilled water was extracted with 2 L of ethyl acetate in a separatory filter, extracted with an ethyl acetate soluble part, and the extract was concentrated under reduced pressure in a 50 ° C. water bath. The extract obtained after concentration was azeotropically concentrated three times with distilled water of 10 times the total amount of the extract, and the same amount of distilled water was added to suspend homogeneously.

Example 2 Isolation of Active Compound from Germinated Soybean Cordyceps Sinensis Extract

After coating 20g of the Champsacaceae extract obtained in Example 1 on an appropriate amount of silica, silica gel column chromatography was performed by changing the content of methanol using chloroform and methanol to collect the active fraction. Separation of the active fraction by Sepadex LH-20 gel chromatography using 85% methanol as the elution solvent, and removal of the solvent using a reduced pressure dryer, the residue obtained by dissolving in high-performance methanol (High Perfomance Liquid Chromatography) was performed to obtain the following active substances (Formula 1, 2, 3, 4).

For each active material, physicochemical properties were analyzed using LC-ESI-MS (Liquid Chromatography Electron Spray Ionization Mass Spectrometer) and nuclear magnetic resonance spectra. .

(Formula 1)

① Material Property: Powder

② Molecular Weight: 430.3

③ Molecular Formula: C ₂₂ H ₂₂ O ₉

④ Mass Spec. [MH] ^- : 429 ( m / z )

In addition, hydrogen nuclear magnetic resonance spectra and carbon nuclear magnetic resonance spectra measured using methanol as a solvent were found to be compounds represented by Chemical Formula 1 (see FIGS. 1A and 1B).

(Formula 2)

① Material Property: Powder

② Molecular Weight: 460.14

③ Molecular Formula: C ₂₃ H ₂₄ O ₁₀

④ Mass Spec. [MH] ^- : 459 ( m / z )

In addition, hydrogen nuclear magnetic resonance spectra and carbon nuclear magnetic resonance spectra measured using methanol as a solvent, were found to be compounds represented by Chemical Formula 2 (see FIGS. 2A and 2B).

(Formula 3)

① Material Property: Powder

② Molecular Weight: 446.12

③ Molecular Formula: C ₂₂ H ₂₂ O ₁₀

④ Mass Spec. [MH] ^- : 445 ( m / z )

In addition, hydrogen nuclear magnetic resonance spectra and carbon nuclear magnetic resonance spectra were measured using methanol as a solvent. As a result, it was found to be a compound represented by Chemical Formula 3 (see FIGS. 3A and 3B).

(Formula 4)

① Material Property: Powder

② Molecular Weight: 446.12

③ Molecular Formula: C ₂₂ H ₂₂ O ₁₀

④ Mass Spec. [MH] ^- : 445 ( m / z )

In addition, hydrogen nuclear magnetic resonance spectra and carbon nuclear magnetic resonance spectra measured using methanol as a solvent, were found to be compounds represented by Chemical Formula 4 (see FIGS. 4A and 4B).

Figure 1a shows a hydrogen nuclear magnetic resonance ( ¹ H-NMR) spectrum of the compound represented by Formula 1,

Figure 1b shows the carbon nuclear magnetic resonance ( ¹³ C-NMR) spectrum of the compound represented by Formula 1,

Figure 2a shows a hydrogen nuclear magnetic resonance ( ¹ H-NMR) spectrum of the compound represented by Formula 2,

Figure 2b shows the carbon nuclear magnetic resonance ( ¹³ C-NMR) spectrum of the compound represented by Formula 2,

Figure 3a shows a hydrogen nuclear magnetic resonance ( ¹ H-NMR) spectrum of the compound represented by Formula 3,

Figure 3b shows the carbon nuclear magnetic resonance ( ¹³ C-NMR) spectrum of the compound represented by Formula 3,

Figure 4a shows a hydrogen nuclear magnetic resonance ( ¹ H-NMR) spectrum of the compound represented by Formula 4,

Figure 4b shows the carbon nuclear magnetic resonance ( ¹³ C-NMR) spectrum of the compound represented by the formula (4).

Claims (3)

A novel isoflavone-based compound isolated from germinated soybean cordyceps, characterized in that the isoflavone-based compound represented by the formula (1), (2), (3), (4). (Formula 1)

(Formula 2)

(Formula 3)

(Formula 4)

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