KR100902669B1 - A method for isolation and purification of calycosin from Astragali Radix - Google Patents

Abstract

The present invention relates to a method for isolating and / or purifying an isoflavone compound, calicosin, from sulfuric acid, comprising: (S1) extracting sulfuric acid (Astragali Radix) using an alcohol or an aqueous alcohol solution having 1 to 4 carbon atoms; (S2) treating the extract of step (S1) with carbohydrate and pectin hydrolase; And (S3) adding an organic solvent for fractionation to the resultant of step (S2) and fractionating the resultant. The process of the present invention is very useful for the separation and / or purification of the isoflavone compound, Calicosin, from Astragalus.

Astragali Radix, Pectin Hydrolase, Carbohydrate Hydrolase, Calicosin

Description

A method for isolation and purification of calycosin from Astragali Radix}

The present invention relates to a method for separating and / or purifying calicosin from astragalus.

Astragali Radix is a perennial herb belonging to Leguminosae , which is widely distributed in Asia such as Korea, Japan, and Europe such as Russia, Bulgaria, and Europe, and has been widely used as an herbal medicine since ancient times. Anorexia, antiperspirant, tonic, etc. (Sun T. et al ., Chin. Med. J. 61, 97, 1981; Tu GS, Pharmacopoeia of the People's Republic of China, p. 109, 1988). In addition, Hwanggi is listed as a medicinal herb in the new agricultural plant, so in the Korean medicine, hundreds of prescriptions such as Gukgiikgitang, Hwanggi Kunjung, Hwanggi Geobangbang, Jeopjeondaebotang, Gamidaebotang, Hwanggiyukiltang, and Palbohoechuntang, It is a commonly used example (Song et al ., Planta Med 70 (12): 1222-1227, 2004; Chiang Su, Dictionary of Chinese Crude drugs, Shanghai Scientific Technologic Publisher, Shanghai, p. 2036, 1977).

The known pharmacological actions of Astragalus have been reported to lower blood pressure, cardiac activity, hepatoprotective action, hypoglycemic action, and immunopotentiation (Zhang et al ., Acta Pharm Sin 19, 333, 1984; Zhang et al. ., J Ethnopharmacol 30, 145, 1990; Kim et al , Kor J. Pharmacogn. 26, 51, 1995; Park et al ., Yakhak Hoeji 40, 52, 1996; Kim et al ., Yakhak Hoeji 46, 52, 2002 ). In addition, triterpenoid glycosides and flavonoids are mainly physiologically active components of Astragalus, and other saponin and polysaccharide components are included (Fang et al ., Org. Chem 26, 1982; Kitagawa). et al ., Chem Pharm Bull 31, 709, 1983; Chen et al ., Acta Pharm Sin 23, 218, 1988; Subarnas et al ., Planta Med. 57, 590, 1991; Cui et al ., Chem Pharm Bull, 40, 3330, 1992).

Flavonoids are plant pigments widely distributed in nature and consist of two benzene rings connected through heterocyclic pyron rings. In the pyron rings, the presence or absence of double bonds and ketone groups, the hydroxyl group and the benzoid B ring Therefore, it is divided into flavone, flavonol, flavanol, flavanonone and isoflavone (Han Myung Kyu, Food Chemistry, Hyungseol Publisher, p. 236-240, 2002; Ahn Myung-soo, Food Chemistry, Shin Gwang Publisher, p. 266-269, 2006 Fennema OR, Food Chemistry, Marcel Dekker, Inc., New York, USA, 681-696, 1996). Of these, isoflavones have a very limited distribution in nature, unlike other flavonoids that are widely distributed throughout plants, and there is a significant nutritional dose only in soybeans (Cornwell et al., Phytochemistry 65: 995-1016, 2004 Havsteen B. Biochem Pharmacol 32: 1141-1148, 1983).

Isoflavones are primarily members of the legumes family (Leguminosae family), and more than 1,000 isoflavones have been identified. 65: 995-1016, 2004; Alekel et al ., Am. J. Clin. Nutr. 72: 844-852, 2000). In nature, isoflavones mostly exist as glycosides, but slightly as aglycones. In plants, isoflavones combine with glucose to form physiologically inactivated glycosides (Setchell KD, Soy Connection Newslatter 6, Spring, 1998).

Among the various isoflavones contained in Astragalus, calycosin and formononetin and its glycosides (calycosin-7- O -β-D-glycoside and formononetin-7- O -β-D- glycoside) (Wu et al ., Fitoterapia 76 (2): 157-165, 2005; Luo et al ., Zhongguo Zhong Yao Za Zhi 28 (7): 603-606, 2003). Contains calicosin that is lower than the genistein and daidzein content of soybeans, well known as flavone reports (Ma et al ., J Chromatogr A 992, 193, 2003; Wu et. al ., Fitoterapia 76, 157, 2005; Kuwahara et al ., Yakugaku Zasshi 120 (9): 779-785, 2000). These isoflavone glycosides have various physiological activities such as antibacterial, antioxidant, and anticancer (EI-Sebakhy et al ., Phytochemistry 36, 1387, 1994; Toda et al ., Phytother Res 12: 59-63, 1998). However, their content varies considerably between varieties and regions (Ma et al ., Nat Med 54: 213-217, 2000).

Astragalus formmononetin induces estradiol-like reactions in several tissues and cell lines, and in some tissues it can act as an estrogen antagonist (Ji et al ., Gynecol Endocrinol 22 (10): 578-584, 2006). Formononetin inhibits the incidence and growth of breast tumors caused by chemicals in rat-based studies. And formononetin has effects on cell cycle progression in tumorous (MCF-7) and non-tumoral (MCF-10F) human breast cells (Wang et al ., Nutr Cancer 23 (2): 131-140, 1995; Dickinson et al ., J Anim Sci 66 (8): 1969-1973, 1988). In addition, such formmononetin lowers blood pressure, prevents heart disease, inhibits cancer cell differentiation, and exhibits antioxidant activity (Nestel et al ., Atherosclerosis 25: 126-132, 2006; Jung et al ., Arch Pharm Res 28 (5): 534-540, 2005; Yu et al ., J Ethnopharmacol 98 (1-2): 89-94, 2005), and other potent neuronal protective and hydrogen peroxide scavengers (Yu et. al ., Biomed Environ Sci 18 (5): 297-301, 2005; Yu et al ., J Ethnopharmacol 98 (1-2): 89-94, 2005).

Carlycosin, on the other hand, has not been studied much compared to formonenetine, but it has been shown to act not only as an vasodilator but also as an antioxidant (Wuer et al ., Acta Pharmacol Sin 27 (8): 1007-). 1012, 2006; Fan et al., Eur J Pharmacol 481 (1): 33-40, 2003) and also promote allergic reactions (Park et al ., Immunology 116 (1): 71-81, 2005;), Weak estrogen activity.

Thus, phytoestrogens not only inhibit hormone-dependent prostate cancer, breast cancer and colon cancer, but also prevent heart disease and osteoporosis, and have antimicrobial and antiviral properties (Adlercreutz H, Setchell KDR, J. Steroid Biochem., 13) . : 11-15, 1979; Adlercreutz H, Gastroenterology 86: 761-767, 1984; Watanabe S, Koessel S, J. Epidemiol., 3: 47-53, 1993; Yuan JM, Wang QS, Ross RK, Henderson BE, Yu MC, Br. J. Cancer 71: 1353-1358, 1995). In addition, legumes containing soy isoflavones in large amounts (soybean, brown root, Astragalus, licorice, etc.) are now widely used as dietary supplements for cancer and heart disease prevention in Japan, China and Korea.

To date, isolation, identification and quantitation of calicosin and formonenetine glycosides from Astragalus (Ma et al ., Se Pu 23 (3): 299-301, 2005; Wuer et al ., Fitoterapia 76 (2): 157- 165, 2005; Hu et al ., Zhong Yao Cai 26 (9): 634-636, 2003; Ma et al ., J Chromatogr A 992 (1-2): 193-197, 2003; Ma et al ., J In addition to Chromatogr A 962 (1-2): 243-247, 2002, numerous studies on their physiology have been reported (Nestel et al ., Atherosclerosis 25: 126-132, 2006; Jung et al ., Arch Pharm Res 28 (5): 534-540, 2005; Yu et al ., J Ethnopharmacol 98 (1-2): 89-94, 2005; Yu et al ., Biomed Environ Sci 18 (5): 297-301, 2005; Yu et al ., J Ethnopharmacol 98 (1-2): 89-94, 2005; Wuer et al ., Acta Pharmacol Sin 27 (8): 1007-1012, 2006; Fan et al., Eur J Pharmacol 481 (1 ): 33-40, 2003), but the development of technology for converting isoflavone glycosides that are still present in Astragalus into aglycone, Phrase is the situation with little.

Accordingly, the present invention is to provide an improved method for effectively separating and / or purifying the calicosin component, which is a phytoestrogens isoflavone component having anticancer, antihypertensive, anti-osteoporosis and anti-aging activity from Astragalus. .

In order to achieve the above technical problem, the present invention (S1) step of extracting Astragali Radix or crushed sulfur group using an alcohol or alcohol solution having 1 to 4 carbon atoms; (S2) treating the extract of step (S1) with carbohydrate and pectin hydrolase; And (S3) it provides a method for separating or purifying calicosin from the sulfur phase comprising the step of fractionating by adding an organic solvent for fractionation to the resultant of the step (S2).

More preferably, the present invention, in the method for separating or purifying calicosin from the Astragalus, degreasing the extract of step (S1) by using an organic solvent for degreasing between steps (S1) and (S2) and the ( It provides a method characterized in that it further comprises the step of separating or purifying the fractions of step S3) using the adsorption ion resin column chromatography.

Hereinafter, a method for separating or purifying calicosin from the sulfur group of the present invention will be described in more detail.

The method for separating or purifying calicosin from the sulfur groups of the present invention includes (S1) extracting Astragali Radix or pulverized sulfur groups using an alcohol or alcohol solution having 1 to 4 carbon atoms; (S2) treating the extract of step (S1) with carbohydrate and pectin hydrolase; And (S3) adding and fractionating an organic solvent for fractionation to the resultant of step (S2).

The method of the present invention includes the step of extracting (S1) sulfuric or dried and pulverized sulfur using an alcohol or aqueous alcohol solution having 1 to 4 carbon atoms, such as an ethanol aqueous solution, an aqueous methanol solution, an isopropanol aqueous solution, butanol aqueous solution, and the like. Considering the enzymatic treatment or toxicity of the phosphorus (S2) step, an ethanol aqueous solution is more preferable.

In addition, the alcohol content of the alcohol aqueous solution is preferably about 75% to about 95% by weight, more preferably about 80% to about 90% by weight.

The extraction of the step (S1) is preferably carried out at 60-90, more preferably 70-80, if the temperature is too low extraction yield is low, the extraction time is long, when extracted at too high temperature There may be denaturation of the extract components.

The method also includes the step (S2) of treating the extract of step (S1) with carbohydrate and pectin hydrolase. This treatment is preferably done at 40-70, more preferably at 40-50.

The method also includes the step of concentrating the extract of step (S1) between step (S1) and (S2) to increase the efficiency of step (S2) using the enzyme, and dissolving the concentrate again with an ethanol aqueous solution. Preferably, the ethanol aqueous solution is preferably 5 to 30% by weight, and more preferably about 10% by weight ethanol aqueous solution, the content of ethanol is lower than the extraction step of (S1) step.

The method of the present invention also comprises the step of (S3) by adding an organic solvent for fractionation to the culture of the step (S2), and the organic solvent for fractionation such as ethyl acetate, diethyl ether, trichloromethane, butanol Etc. may be used alone or in combination, and it is most preferable that the fractional organic solvent is ethyl acetate.

The method of the present invention preferably further comprises the step of degreasing the extract of step (S1) using the organic solvent for degreasing between the steps (S1) and (S2), such a degreasing organic solvent is normal -Hexane, petroleum ether, ether and the like can be used, of which normal-hexane is most preferred.

The method of the present invention preferably further comprises the step of (S4) separating or purifying the fractions of the step (S3) by using an adsorption ion resin column chromatography, and this step is an adsorption ion resin to which calicosin is adsorbed. Preferably, the column is eluted with ethanol or an ethanol aqueous solution, most preferably about 80% ethanol, with an ethanol content of 60 to 100% by weight.

The method of the present invention also comprises the steps of (A) (S2) between the step (S2) and (S3) step (S2) fraction or fractional concentration using an organic solvent for fractionation; And (b) further treating the carbohydrate hydrolase and the pectin hydrolase in the fraction or fraction concentrate of step (a) to further increase the content of useful components in the Astragalus extract. When the steps (a) and (b) are added in the method of the present invention, the content of calicosin, a useful component, is dramatically increased.

Therefore, the preferred method according to an embodiment of the present invention is as follows. After adding about 80% ethanol aqueous solution to 1 ton / 100kg in dry sulfuric acid and extracting by heating at about 70-80 for about 12 hours, filtration and concentration under reduced pressure to obtain the sulfuric ethanol extract, and dissolved by adding about 80% ethanol aqueous solution to the extract , Add about twice the amount of 100% normal-hexane and fractionate to remove the oil in the upper layer, and partially depressurize the lower organic skim ethanol extract, and then add 10 times the 10% ethanol aqueous solution to the extract. Suspension was added to the degreasing ethanol extract, and about 10% pectinase and about 10% biscozyme mixed enzyme were added to hydrolyze at about 40-50 for about 12 hours, and the obtained hydrolyzate was left at room temperature and then filtered. After concentrating the upper layer obtained by adding ethyl acetate and fractionating, the ethyl acetate fraction was dissolved in about 60% ethanol aqueous solution. After adsorbing to ion resins such as Diaion HP-20, the column was first passed through a column with about 60% ethanol solution, and then eluted and concentrated with about 80% ethanol solution, two isoflavone compounds, calicosin and formono. Netine can be isolated and purified.

As such, the present invention provides a method for effectively separating and / or purifying calicosin, which is a phytoestrogen isoflavone compound from Astragalus. Therefore, the separation and purification method of the present invention can efficiently produce a large amount of active isoflavone aglycone components from sulfur.

Hereinafter, specific examples, such as examples and experimental examples, will be described to help understanding of the present invention. However, the following examples may be modified in many different forms and should not be construed as limited to the examples set forth herein.

Example 1 Isolation and Purification of Calicosin and Formonenetine from Astragalus

In order to separate and purify the calicosin and formmononetin, which are vegetable estrogen isoflavone compounds, from sulfuric acid, sulfuric acid degreasing ethanol extract was subjected to enzyme treatment, ethyl acetate fraction, and Diaion HP-20 adsorption ion resin column chromatography.

First, 1 ton of 80% ethanol solution was added to 100 kg of dried Astragali Radix, and the extract was heated and extracted in a extractor attached with a low temperature condenser for 70 to 12 hours. The mixed ethanol extract obtained by repeating the extraction process twice was partially concentrated under reduced pressure to obtain astragalus ethanol extract (200 kg). Next, 100% ethanol (200 kg) was added to the ethanol extract, solubilized with stirring, and allowed to stand at room temperature overnight, and then the obtained supernatant was separated, and then normal-hexane (400 kg) was added thereto and the mixture was stirred and fractionated. The pigment was removed and the lower layer was concentrated to obtain anhydrous skim ethanol extract (13 kg). This was again suspended in 10% aqueous ethanol solution (100 kg), and then 10 L each of pectin hydrolase (Pectinex, Novozymes, Denmark) and carbohydrate hydrolase (Viscozyme L, Novozymes, Denmark) were added to 40 to 50 to 12. After hydrolysis with stirring for a while, the mixture was allowed to stand for cooling, and then, ethyl acetate (200 kg) was added to the supernatant obtained by filtration. The upper layer was dehydrated twice with anhydrous manganese (10 kg), filtered and concentrated under reduced pressure to obtain ethyl acetate extract ( 1.2 kg). It was suspended in a 60% ethanol aqueous solution (10 kg) and filled into a Diaion HP-20 Adsorption Ion Resin (Mistubishi Chem. Co., Japan) column (25 cm x 200 cm), which was previously equilibrated with 60% ethanol aqueous solution. Elution with 60% ethanol aqueous solution (100 L), 80% ethanol aqueous solution (200 L), and 100% ethanol (100 L), respectively. At this time, the fractions eluted with 80% ethanol solution were collected and concentrated under reduced pressure using a rotary evaporator at 50 or less, and then calicosin (11.8 g, yield: 11.8 mg%) and formmononetin (10.5 g, yield: 10.5 mg). A crude residue powder (150 g, yield: 0.15%) containing the%) component was obtained.

Example 2 Quantitative Analysis of Calicosin and Formonenetine in Astragalus Purified Powder

Quantitative analysis of calicosin, an isoflavone compound, in the sulfur-based purified powder prepared according to the method described in Example 1 was carried out using high performance liquid chromatography (HPLC) as follows.

First, 0.1 g of Astragalus refined powder was dissolved in 10 mL of 100% methanol, and then, the same solution was used as 100 mL. The solution was diluted 10-fold with the same solvent, 1 mL of the solution was passed through a 0.45 um membrane filter (gelman Sci, USA), and 20 ul was injected into HPLC for quantitative analysis. Gilson 506B high performance liquid chromatography; Column YMC-Pack C ₁₈ (AS-303, 5 um, 250 × 4.6 mm ID, YMC Inc., USA); Mobile phase (solvent gradient eluting with solvent B for 60 min), solvent A (0.05% phosphoric acid), solvent B (100% methanol); Ultraviolet detector UV _{248 nm} ; And flow rate 0.8 mL / min.

In this case, a high-performance liquid chromatogram of calicosin and formmononetin isolated from Astragalus according to Example 1 is shown in FIG. 1, and the chemical structure is represented by the following Chemical Formula 1.

In addition, UV, IR, NMR and MS spectral data of calicosin and formonenetine isolated from Astragalus are shown in Table 1 below.

Instrument analysis result Calicosin Formononetine UV ( _max ) nm in MeOH (loge) 207 (2.9), 231 (2.83), 259 (2.96), 305 (2.40) 205 (3.0), 230 (2.84), 260 (2.98), 306 (2.42) IR (v _max ) cm ^-1 3371 (-OH), 1623 (C = O), 1516, 1447 (aromatic C = C), 1266, 1196, 1173 (C-O) 3373 (-OH), 1623 (C = O), 1515, 1445 (aromatic C = C), 1263, 1197, 1174 (C-O) ¹ H-NMR H-2 8.39 (1 H, s ) 8.39 (1 H, s ) H-5 8.03 (1H, doublet , J = 8.8 Hz) 8.03 (1H, doublet , J = 8.8 Hz) H-6 7.13 (1H, doublet of doublets , J = 8.8, 2.0 Hz) 7.13 (1H, doublet of doublets , J = 8.8, 2.0 Hz) H-8 7.20 (1H, doublet , J = 2.0 Hz) 7.20 (1H, doublet , J = 2.0 Hz) H-2 ' 6.86 (1H, doublet , J = 2.0 Hz) 7.39 (2H, doublet , J = 8.5 Hz) H-3 ' 6.80 (2H, doublet , J = 8.5 Hz) H-5 ' 7.18 (1H, doublet , J = 8.5 Hz) 6.80 (2H, doublet , J = 8.5 Hz) H-6 ' 6.89 (1H, doublet , J = 8.8, 2.0 Hz) 7.39 (2H, doublet , J = 8.5 Hz) OCH ₃ 3.85 3.86 ¹³ C-NMR C-2 153.22 153.11 C-3 124.53 124.45 C-4 175.04 174.86 C-5 127.42 127.46 C-6 115.39 115.34 C-7 162.74 162.75 C-8 102.15 102.23 C-9 157.66 157.65 C-10 116.74 116.84 C-1 ' 131.47 123.37 C-2 ' 114.34 130.26 C-3 ' 146.77 113.75 C-4 ' 148.15 159.18 C-5 ' 112.12 113.76 C-6 ' 130.18 130.23 OCH ₃  55.92  55.25 FABMS [M + H ⁺ ] 165 149

In Table 1, NMR spectra were obtained from DMSO using an AMX-500 spectrometer (Varian Unity ^plus , USA), chemical shifts were expressed as d values, and coupling constants ( J in parentheses). Is given in Hz.

According to the method described in Example 1, the results of measuring the content of two isoflavone compounds, calicosin and formonenetine, among the purified powders prepared from Astragalus, are shown in Table 2 below.

Refined powder Isoflavones Content (%, fine powder 100 g) Primary analysis Secondary analysis 3rd analysis Average I Calicosin 11.74 ± 1.02 12.53 ± 1.20 11.89 ± 0.32 12.05 ± 0.24 Formononetine 10.23 ± 0.72 10.02 ± 0.77 9.83 ± 0.26 10.02 ± 0.46 II Calicosin 11.35 ± 0.42 12.04 ± 0.74 11.90 ± 0.52 11.49 ± 0.56 Formononetine 10.42 ± 0.49 10.13 ± 0.52 9.92 ± 0.32 10.16 ± 0.44 III Calicosin 10.94 ± 1.18 12.82 ± 0.62 10.93 ± 0.51 11.56 ± 0.77 Formononetine 10.19 ± 0.61 10.17 ± 0.62 9.88 ± 0.32 10.08 ± 0.52

As shown in Table 2, the crude degreasing ethanol extract of calicosin and formonenetine in purified powders I, II and III prepared by separating enzyme treatment, ethyl acetate fraction, and diion HP-20 column chromatography The contents were 11.70 ± 0.52% and 10.09 ± 0.47%, respectively, and both components were obtained in about 10% yield.

Example 3 Quantitative Analysis of Isoflavone Compounds Contained in Astragalus Extract with and without Enzyme Treatment

Calicosin 7- O -beta-di-glycoside and formononetin-7- O -beta-di-glycoside and their aglycone components, calicosin and isoflavone glycosides contained in the Astragalus extract with or without enzyme treatment As a result of HPLC analysis of the degreasing ethanol extract of the non-enzyme treatment group according to the HPLC conditions in order to quantify formmononetine, four isoflavone compounds were identified as shown in FIG. standard calibration curve was created using a flavone compound, of Columbia -7- potassium O-beta-D-glycoside: y = 10.12x + 0.19 (r 2 = 0.997), formononetin -7- O-beta-D-glycoside Side: y = 10.17x + 0.12 (r ² = 0.998), calicosin: y = 6.78x + 0.08 (r ² = 0.997), and formonenetine: y = 6.89x + 0.10 (r ² = 0.998)] Based on this, the content of four isoflavone compounds in ethanol extracts with and without enzyme treatment was measured. To the results shown in Table 3 below.

Treatment Content (%, Astragalus Ethanol Extract) Calicosin-7- O -beta-di-glycoside Formonenetine-7- O -beta-di-glycoside Calicosin Formononetine Untreated (control) 6.54 ± 0.43 5.25 ± 0.24 6.71 ± 0.72 5.82 ± 0.31 Enzyme treatment Tr ^* Tr ^* 12.13 ± 1.02 10.18 ± 0.79

The values in Table 3 are expressed as the average ± standard deviation after repeated measurements three times, ^* Trace means less than 1 mg%.

As shown in Table 3, in the non-treated group, the contents of the four isoflavone compounds of the sulfuric ethanol extract were 6.54% of calicosin-7- O -beta-di-glycoside and formonenetine-7- O -beta-di. Glycosides 5.25%, Calicosin 6.71%, and Formonenetine 5.82%, which had almost the same level of isoflavone glycosides and aglycone, while in the enzyme treatment, the two isoflavone glycosides were almost absent, while their aglycone components Only two isoflavone aglycones were present as phosphoricincin 12.13% and formmononetin 10.18%. In this way, it was found that the isoflavone glycoside component present in Astragalus was almost converted to aglycone component by enzyme treatment.

Figure 1 shows the high-performance liquid chromatogram results of the two isoflavone compounds separated from the sulfuric acid degreasing ethanol extract prepared by the enzyme treatment.

Figure 2 shows the high-performance liquid chromatogram results of the four isoflavone compounds separated from the sulfur-free skim ethanol extract.

Claims (5)

(S1) extracting Astragali Radix or crushed Astragalus using an alcohol having 1 to 4 carbon atoms or an aqueous alcohol solution, and fractionating the Astragalus extract with an organic solvent for degreasing to remove oil from the upper layer; (S2) treating the degreased extract of step (S1) with carbohydrate and pectin hydrolase; (S3) adding ethyl acetate as an organic solvent for fractionation to the resultant of step (S2) to obtain an ethyl acetate fraction; And (S4) separating or purifying the fractions of the step (S3) by using an adsorption ion resin column chromatography Is integrated and proceeds sequentially, The method for separating or purifying calicosin from the sulfur group comprising the step of eluting the calicosin adsorbed ion resin column with an ethanol aqueous solution or ethanol having an ethanol content of 60 to 100% by weight. The method of claim 1, wherein the organic solvent for degreasing is normal-hexane. According to claim 1, wherein the alcohol solution of step (S1) is characterized in that the ethanol aqueous solution of 75 to 95% by weight of ethanol. The method of claim 1, wherein the method comprises the steps of (A) (S2) between step (S2) and step (S2) fractions or fractional concentration using an organic solvent for fractionation; And (b) treating carbohydrate hydrolase and pectin hydrolase to the fraction or fraction concentrate of step (a). The method of claim 1, wherein the method comprises the steps of concentrating the extract of step (S1) between steps (S1) and (S2), and dissolving the concentrate again with an ethanol solution having an ethanol content of 5 to 30% by weight. Method comprising a.

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