KR100813997B1 - Processes for preparing jinseng extracts - Google Patents

Abstract

A method for preparing an extract of ginseng is provided to increase the content of ginsenoside Rg3 and Delta20-ginsenoside Rg3 known to have excellent anti-tumor and anti-cancer effect compared with other ginsenoside derivatives and utilize lactic acid bacteria and/or fermentation enzyme existing in enterobacteria by ripening a culture solution at a specific temperature for a certain time, and obtain the ginseng extract without heat-treatment, neutralization and extraction purification. A method for preparing an extract of ginseng comprises the steps of: (a) fermenting ginseng with lactic acid bacteria or enterobacteria; and (b) ripening a culture solution obtained from the step(a) at a temperature of 50-85 deg.C for 4-12 hours. The method further comprises a step of concentrating or freeze-drying a ripened solution obtained from the step(b).

Description

Process for preparing ginseng extract {Processes for preparing jinseng extracts}

1 is a flow chart showing an example of a process according to the manufacturing method of the present invention.

Figure 2 is a result of measuring the change over time of the saponin and saponin metabolites in the blood appearing when orally administered ginseng extract prepared according to the method of the present invention.

The present invention relates to a method for preparing ginseng extract, and more particularly, to a method for preparing ginseng extract having a high content of ginsenoside Rg3 and Δ20-ginsenoide Rg3, which are known to have excellent antitumor and anticancer effects. .

Ginseng is a perennial root of perennial ginseng belonging to the genus Oga ginseng according to the plant taxonomy.Panax ginseng C. A. Meyer)Panax quinquefolium L.), Whole Chilsam (Panax notoginseng F. H. Chen)Panax japonicus C. A. Meyer). Korean ginsengPanax ginseng C. A. Meyer is native to the Far East of Asia (33-48 North latitudes, Korea, Northern Manchuria, and parts of Russia) and has very good efficacy. American hemp (Panax quinquefolium L.) grows and grows in the United States and Canada.Panax notoginseng F. H. Chen) is grown wild or in southwestern region of Guangxi province from southeastern Yunnan Province of China. Also, bamboo shoots (Panax japonicus C. A. Meyer) extends to Japan, southwest China, and Nepal.

As research on the major ingredients and pharmacological effects of ginseng has been published, it has been spotlighted as a natural health food, its demand is gradually increasing, and it has been accelerated by economic growth and improvement of living standard, and widely used as a popular natural health food. It is becoming. In addition, ginseng has been used for various diseases such as psychiatry, nervous system diseases and diabetes in the form of herbal medicine mainly in Asian countries such as Korea, and saponins, the main components of the ginseng, are tonic, tonic, soothing, hematopoietic and antihypertensive It is reported to have an effect.

In general, ginseng is used in the form of red ginseng, which is produced by washing fresh ginseng, white ginseng or fresh ginseng dried at room temperature with purified water, and then steaming it with steam at about 98 to 100 ° C. Of these, red ginseng is known to be much stronger than white ginseng.

Republic of Korea Patent Application No. 1980-4291 enzymatically decomposes ginseng residues from the intrinsic fragrance of ginseng to produce organic pH when the organic nitrogen concentration is 0.2-0.8% and the glucose content is 3% or more suitable for lactic acid bacteria fermentation. Disclosed is a method for preparing a lactic acid bacteria ginseng beverage, comprising the step of adjusting the concentration of 3.8 to 4.8 to remove insoluble polymer protein and fiber, culturing the lactic acid bacteria in the eluate, and then adding a flavor component unique to ginseng.

Korean Patent Publication No. 1997-061909 discloses a compound as an intestinal bacterial metabolite of ginseng saponin and a metabolite of ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, and ginsenoside Rd, which are protofaanaxadiol-based saponins. [20-0-[[alpha] -L-arabinofuranosyl (1 → 6)-[beta] -D-glucopyranosyl] -20 (S) -protopanaxadai, designated K, Compound Y and Ginsenoside Mc Has also been isolated and identified as 20 (S) -protopanaxatriol as a metabolite of ginsenoside Rg1 and ginsenoside-Re, which are protoparnax triol saponins.

In addition, Korean Patent Registration Nos. 479,803, 497,895, 517,899, 618,171, etc., ferment the ginseng or ginseng extract with lactic acid bacteria or enteric bacteria, 20-0-β-D-glucopyranosyl-20 (S Fermented ginseng (i.e. ginseng extract) with high content of) -protopanaxadiol (Compound K), ginsenoside F, 20 (S) -protopanaxadiol, 20 (S) -protopanaxatriol, and the like, and The preparation method thereof has been disclosed.

Meanwhile, Korean Patent Registration No. 228,510 heats ginseng at a temperature of 110-180 ° C. for 0.5-20 hours, and processes the obtained ginseng with alcohol, hexane, ether, dichloromethane, chloroform, ethyl acetate, and mixed solvents thereof. After the extraction with an organic solvent selected from the group consisting of chromatographed the extract and ginsenosides Rg3 and / or Rg5 comprising a step of separating and obtaining a method for preparing ginsenosides Rg3 and / or Rg5. According to Korean Patent Registration No. 228,510, the ginsenoside Rg3 and / or Rg5 is a metabolite of saponin having an excellent vasorelaxant effect.

The ginsenosides Rg3 and Δ20-ginsenoide Rg3 are known to have excellent anti-tumor and anticancer effects as well as excellent vasodilating effects (Kim et al., Eur. J. Pharmacol. 1999, 367 (1): 51 -57; Kim et al., Arch. Pharm. Res. 2004, 27 (4): 429-435; Li X. et al., Sichuan Da Xue Xue Bao Yi Xue Ban. 2005, 36 (2): 217- 220).

However, as disclosed in Korean Patent Registration No. 228,510, when preparing ginseng extracts having a high content of ginsenoside Rg3 and Δ20-ginsenoide Rg3, carcinogens such as HMF (Hydroxylmethylfurfual) produced during the heat treatment process, etc. Production is inevitable, and it is difficult to use directly as a food because it must inevitably undergo a neutralization process according to heat and / or acid treatment process and a separate extraction and purification process.

In addition, when ginseng extract is prepared by fermenting ginseng with lactic acid bacteria or enteric bacteria as disclosed in Korean Patent Registration Nos. 479,803, 497,895, 517,899, 618,171, Ginsenoside Rg3 and △ Very low content of 20-ginsenoide Rg3 makes it difficult to expect vasorelaxant, antitumor, and anticancer effects.

The present inventors have sought to develop a method for producing a ginseng extract having a high content of ginsenoside Rg3 and Δ20-ginsenoide Rg3 by a simple process, excluding a heat treatment and / or an acid treatment. As a result, when ginseng was fermented with lactic acid bacteria or enteric bacteria and aged under specific conditions, it was found that ginseng extracts having a high content of ginsenoside Rg3 and Δ20-ginsenoide Rg3 can be obtained.

Accordingly, an object of the present invention is to provide a method for preparing ginseng extract having a high content of ginsenoside Rg3 and Δ20-ginsenoide Rg3 including a aging process.

According to one aspect of the invention, (a) fermenting ginseng with lactic acid bacteria or enterobacteriaceae having saponin degrading capacity; And (b) aging the culture solution obtained in step (a) at 50 to 85 ° C. for 4 to 12 hours.

Hereinafter, the present invention will be described in detail.

In the present invention, in the preparation of ginseng extract by fermenting ginseng with lactic acid bacteria and / or enteric bacteria having saponin-degrading ability, by culturing the culture at a specific temperature for a certain period, the fermentation enzymes present inside the lactic acid bacteria and / or enteric bacteria are utilized. At the same time, it is possible to increase the content of ginsenoside Rg3 and Δ20-ginsenoide Rg3 which are known to have superior antitumor and anticancer effects as compared to other ginsenoside derivatives in the extract obtained. In addition, the ginseng extract obtained according to the production method of the present invention may exclude the generation of carcinogens such as HMF (Hydroxylmethylfurfual) generated in the conventional heat treatment process, and must be subjected to neutralization process inevitably according to the heat and / or acid treatment process It can also be excluded and can be used as a food directly without extracting tablets.

The production method of the present invention includes the step of fermenting ginseng with lactic acid bacteria or enteric bacteria having saponin resolution.

The lactic acid bacteria or enteric bacteria having the saponin degrading ability may use all microorganisms commonly used in the field of ginseng fermentation.

As the lactic acid bacteria having saponin resolution, lactic acid bacteria such as Lactobacillus genus, Streptococcus genus, and Lactococcus genus can be used. For example, Lactobacillus rhamnose ATCC 7469, Lactobacillus gasser DSM 20243 strain ( Lactobacillus gasseri DSM 20243), Lactobacillus mali (Lactobacillus gmali ATCC 27304), Lactobacillus Planta Room ATCC 14947 (Lactobacillus plantarum ATCC 14947), Lactobacillus plantarum ATCC 10241 ( Lactobacillus plantatum ATCC 10241), or Lactobacillus Lactobacillus genus lactic acid bacteria such as lactis ) can be used. Further, Streptomyces Cocos write a brush loose (Streptococcus Lactobacillus of Streptococcus genus such as thermophilus ) can be used, Lactococcus lactis ), Lactococcus lactis ATCC 15577 ( Lactococcus) Lactococcus lactic acid bacteria such as lactis ATCC 15577) can be used.

Examples of intestinal bacteria having saponin degrading ability include Bifidobacterium infantis infantis ), Bifidobacterium B. bifidum , Bifidobacterium K-103 (Kim, Kyung Hee University College of Medicine, Dong-Hyun Kim, Arch. Pharm. Res., 21, p54-61, 1988), Bifidobacterium K -506 (Kim, Hee-Dong, Kyung Hee University, Arch. Pharm. Res., 21, p 54-61, 1988) Bifidobacterium K-513 (Kim, Hee-Dong, University of Kyung Hee University, Arch. Pharm. Res., 21, p 54-61) , 1988), Bifidobacterium K-525 (Professor Dong-Hyun Kim, Kyung Hee University, Arch. Pharm. Res., 21, p54-61, 1988), Bifidobacterium KK-1 (Accession No .: KCCM 10364), Bifidogam Bifidobacterium such as Terium KK-2 (Accession Number: KCCM 10365), Bifidobacterium adolescents ATCC 15703 (Bifidobacterium adolescentis ATCC 15703), Bifidobacterium bifidium JCM 7002 (Bifidobacterium bifidum JCM 7002) Lactic acid bacteria of the genus Bifidobacterium . In addition, the intestinal bacteria having saponin-degrading ability include IFO 0309 strain ( Saccharomyces ) in Saccharomyces cerevisiae. microorganism of the genus Saccharomyses , such as cerevisiae IFO 0309, Clostridium butyricum ) Clostridium butyricum ) Microorganism, Bacterioides genus Microorganisms of Bacterioides , such as Bacterioides JY-6 (Professor Dong-Hyun Kim, Kyung Hee University, Biol. Pharm. Bull., 23, pp1481-1485, 2000) Dong-Hyun Kim, Ph.D., Biol. Pharm. Bull., 23, pp1481-1485, 2000), etc. Microorganisms in Fusobacterium , Eubacterium L-8 (Kim, Dong-Hyun Kim, Ph.D., Biol. Pharm. Bull., 23, pp1481-1485, 2000) microorganisms of the genus Eubacterium can also be used.

Lactic acid bacteria or intestinal bacteria having the resolution of saponin is used by itself or may be used as a mixed strain, preferably Lactobacillus Planta column (Lactobacillus plantarum), Lactobacillus casei (Lactobacillus casei ), L actobacillus acidophillus , Bifidobacterium long gum ( Bifidobacterium) longum ) and Bifidobacterium infantis can be used.

The ginseng used as a substrate of lactic acid bacteria and / or enteric bacteria in the preparation method of the present invention is not particularly limited, and both ginseng itself and ginseng processed materials may be used, for example, ginseng, red ginseng, red ginseng, white ginseng, rice ginseng, Ginseng fruit, ginseng leaves, etc. may be used, or ginseng extract and ginseng powder. The origin of the ginseng is not limited, and for example, Korean ginseng, samchil ginseng, American ginseng, bamboo shoot ginseng, Himalayan ginseng, Vietnamese ginseng and the like can be used without limitation. In addition, the ginseng may be used by sterilization in a conventional manner as needed.

In addition, ginseng in the form of a dry powder, acid treated ginseng, high temperature treated ginseng, and pressurized ginseng disclosed in Korean Patent Registration No. 497,895 may also be used. However, when the acid treatment is lowered the pH of the culture medium may lower the activity of lactic acid bacteria and / or intestinal bacteria, and when treated at high temperature may cause carcinogens such as HMF during heat treatment, ginseng or ginseng powder as it is It is preferable to use. The ginseng powder may have a particle size of at least 100 mesh or more to increase the surface area of ginseng to increase the speed of fermentation of microorganisms.

The method of fermenting ginseng with the lactic acid bacteria or enteric bacteria may be performed according to conventional methods (Korean Patent Registration Nos. 479,803, 497,895, 517,899, 618,171, etc.). For example, ginseng powder or extract of ginseng (for example, alcohol or alcohol aqueous solution extract, hot water extract, etc.) is suspended in water to add lactic acid bacteria and / or enteric bacteria, 20 to 50 ° C., preferably 25 to 40 It can carry out by incubating at 30 degreeC, More preferably, 30 to 37 degreeC for 12 hours-15 days, Preferably it is 24 hours-5 days.

The process of fermenting ginseng with the lactic acid bacteria or enteric bacteria is a pretreatment process, before inoculating the lactic acid bacteria or enteric bacteria, the incubator (or fermenter) is removed by depressurizing air and then supplying a mixed gas for microbial culture, ginseng (for example, ginseng) By removing the dissolved oxygen contained in the extract of powder or ginseng), it may include a step of facilitating the permeation of the seed fermentation broth. The mixed gas for culturing the microorganism may be a mixed gas of 75 to 90% nitrogen, 3 to 10% carbon dioxide, and 5 to 15% hydrogen.

By going through the above fermentation process, ginsenosides Rb1, Rb2, Rc, Rd, etc. contained in the ginseng raw materials are ginsenosides Rg3, Δ20-ginsenosides Rg3, panaxytriol, panaxidol (panaxydol), panaxynol, ginsenoside Rc, ginsenoside Rb2, ginsenoside Re, compound K (Compound K), 20 (R) -ginsenoside Rh2, 20 (R) -protopa Naxadiol (20 (R) -protopanaxadiol), Δ20-ginsenoside Rh2, 20 (S) -protopanaxadiol), 20 (S) -ginsenoside Rh1, ginsenoside Bioconverted to Rh2, 20 (S) -protopanaxatriol and the like.

The preparation method of the present invention comprises the step of aging the culture solution obtained at 50 to 85 ℃ for 4 to 12 hours, ginsenoside Rg3 and Δ20-jin which is known to have excellent antitumor and anticancer effects in the extract obtained The content of cenide Rg3 can be increased.

The aging may be performed at 50 to 85 ℃, more preferably may be carried out at about 55 ℃. In addition, the aging may be performed for 4 to 12 hours, more preferably 4 to 6 hours. If necessary, the step of removing the carbon dioxide gas in the incubator may be performed before the aging process, that is, after performing the process of step (a) and before performing the process of step (b). The carbon dioxide gas removing process in the incubator may be performed, for example, by removing the air by depressurizing the incubator and replacing the air in the incubator with a mixed gas for microbial culture.

The ginseng extract obtained according to the manufacturing method of the present invention may exclude the generation of carcinogens such as HMF produced in the conventional heat treatment process, and may also exclude the neutralization process that inevitably undergoes according to the heat and / or acid treatment process. In addition, it can be used as a food directly without extracting tablets.

Therefore, the ginseng extract obtained according to the production method of the present invention may be concentrated or lyophilized and powdered according to a conventional method and applied to food as it is. That is, the concentrated or lyophilized aging liquid obtained through the aging process may be applied to the food as it is, or the supernatant taken by centrifugation of the aging liquid may be applied to the food as it is. .

1 shows an example of a process according to the manufacturing method of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example  One.

250 g of water was added to 50 g of Korean ginseng powder, and sterilized at 85 ° C., and then Lactobacillus plantarum KCTC 3108 ( Lactobacillus plantarum KCTC 3108) was inoculated at a concentration of about 2%, and the oxygen of the fermentor was removed by air using a reduced pressure pump, and then dissolved in a mixed gas for culture (85% nitrogen, 5% carbon dioxide, and 10% hydrogen). After removing oxygen, the mixture was incubated with stirring at about 37 ° C. for 2 days. After depressurizing the incubator, the internal air was replaced with the mixed gas for microbial culture, and then aged at about 55 ° C. for 5 hours. The obtained aging solution was vacuum dried at 60 ° C. to prepare ginseng extract (ie, fermented ginseng).

Example  2.

American ginseng is used instead of Korean ginseng, and Bifidobacterium long gum KCCM 11953 ( Bifidobacterium ) instead of Lactobacillus plantarum fermented ginseng was prepared in the same manner as in Example 1 using longum KCCM 11953).

Example  3.

Bamboo ginseng is used instead of Korean ginseng and Saccharomyces cerevisiae KCCM 50549 ( Sacharomyses ) instead of Lactobacillus plantarum cerevisiae KCCM 50549), fermented ginseng was prepared in the same manner as in Example 1.

Example  4.

Instead of Korean ginseng, use Himalayan ginseng, and Lactobacillus plantarum Fermented ginseng was prepared in the same manner as Example 1 using Streptococcus thermophillus KCTC 2185 instead of plantarum .

Example  5.

Using ginseng instead of Korean ginseng, fermented ginseng was prepared in the same manner as in Example 1.

Test Example  One.

As a comparative example, as disclosed in Korean Patent Registration No. 228,510 (Example 1) and Korean Patent Registration No. 479,803 (Example 4), ginseng extract was prepared (Comparative Examples 1 and 2, respectively). The column chromatography process for obtaining the Rg3 fraction in Comparative Example 1 was omitted for direct comparison with the extract.

The change in the components of the ginseng extract obtained in Examples and Comparative Examples was measured. That is, 5 g of the ginseng extract obtained is precisely weighed, taken in a 100 ml concentrated flask, and then concentrated under reduced pressure, 50 ml of water-saturated butanol is added, a reflux condenser is added and heated and extracted at 70 to 80 ° C. in a water bath for about 1 hour. The same operation was repeated twice for water. The filtrate was washed with 10 ml of saturated butanol, and the filtrate and the tax solution were combined and placed in a 250 ml separatory funnel and washed well by shaking with 20 ml of water. Transfer the entire saturated saturated butanol extract to a concentrated flask with a constant amount, remove it under reduced pressure in a water bath, remove 50 mL of butanol, add 50 mL of ether to the residue, attach it to a reflux condenser, heat it to 36 ° C for 30 minutes, and degrease the ether. Removed. 10 times methanol was added to the obtained residue to completely dissolve it, and then filtered using a filter (0.45 μm), followed by quantitative analysis of ginsenoside components by high performance liquid chromatography (HPLC). same. The HPLC conditions are as follows.

Organization: Shimadzu Prominence

Column: Gromsil ODS 4.6 (i, d) × 250 mm

Mobile phase A: acetonitrile: distilled water = 15: 85

Mobile phase B: acetonitrile: distilled water = 80: 20

Flow rate: 1.0ml / min

Detector: ELSD (Alltech)

division Ginsenoside (%) Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Ginsenoside Rb1 0.3 0.4 0.3 2.7 2.8 Ginsenoside Rb2 0.3 0.2 0.3 1.5 2.1 Ginsenoside Rc 0.2 0.2 0.3 1.6 2.0 Ginsenoside Rd 0.4 0.5 0.6 0.8 1.1 Ginsenoside Rg3 26.4 26.1 25.9 15.2 23.4 Δ20-ginsenoside Rg3 10.2 10.8 11.2 5.8 0.8 Ginsenoside Rh2 0.2 0.2 0.2 <1 0.2 Compound K 0.1 0.1 0.1 0 0

As can be seen in Table 1, the ginseng extract of Comparative Example 1 prepared through the heat treatment and organic solvent extraction process without undergoing fermentation by lactic acid bacteria or enteric bacteria, the content of ginsenoside Rg3 is relatively high but satisfactory. The ginseng extract of Comparative Example 2 prepared through acid treatment and lactic acid bacteria fermentation was not increased, but the content of ginsenoside Rg3 was increased than that of Comparative Example 1, but the Δ20-ginsenoside Rg3 content was still low. On the contrary, ginseng extract prepared by lactic acid bacteria or enteric bacterial fermentation and aging process has very high ginsenoside Rg3 and Δ20-ginsenoside Rg3 content.

Test Example  2. Valid Ginsenoside  Bioavailability Measurement

40 g of the ginseng extract prepared in Example 1 was orally administered to 60 to 70 kg of healthy adults (males, n = 3) once, and then blood samples at 1 hour, 2 hours, 6 hours, 12 hours, and 24 hours. (5 mL) was taken to analyze the saponin and saponin metabolites present in the blood. 5 ml of blood was collected by time, and the analysis conditions were extracted and purified by ginsenosides using solid phase extraction. Then, methanol was added to elute ginsenosides adsorbed on the solid phase extractor adsorbent, followed by a filter (0.45 μm). The ginsenoside component was quantitatively analyzed by filtration using high-performance liquid chromatography (HPLC). As a result of measuring the change over time of blood saponin derivatives are shown in FIG.

Saponin, known as a pharmacological component of ginseng, has a glycoside structure, which means that saponin is not absorbed by itself and is enhanced by the intestinal microorganisms after bioconversion into saponin metabolites (compound K, ginsenoside F1, etc.). (Hasegawa H., J. Pharmacol. Sci., 2004, 95 (2): 153-157; Tawab MA et al., Drug Metab. Dispos., 2003, 31 (8): 1065-1071.). As can be seen in FIG. 2, it can be seen that ginsenoside Rg3 and Δ20-ginsenoside Rg3 are detected at 3-12 hours and 12-24 hours, respectively.

The production method according to the present invention undergoes a step of aging a culture at a specific temperature for a certain period of time, thereby making use of fermenting enzymes present in lactic acid bacteria and / or intestinal bacteria, as well as other ginsenoside derivatives in the resulting extract. Compared with ginsenoside Rg3 and Δ20-ginsenoide Rg3, which are known to have excellent antitumor and anticancer effects, the content of ginsenoside Rg3 and Δ20-ginsenoide Rg3 can be increased. In addition, the ginseng extract obtained according to the manufacturing method of the present invention may exclude the generation of carcinogens such as HMF produced in the conventional heat treatment process, and also to eliminate the necessity of neutralization process that must be inevitably followed by heat and / or acid treatment process. It can be used as a food directly without going through extraction tablets.

Claims (7)

(a) fermenting ginseng with lactic acid bacteria or enterobacteriaceae having saponin degrading ability; And (b) aging the culture solution obtained in step (a) at 50 to 85 ° C. for 4 to 12 hours. The method according to claim 1, wherein the aging is carried out at 55 ° C. The method of claim 1, wherein the aging is performed for 4 to 6 hours. The method according to claim 1, further comprising the step of removing carbon dioxide in the incubator after performing the process of step (a) and before performing the process of step (b). The method of claim 1, wherein the fermentation of step (a) further includes a step of replacing air in the incubator with a mixed gas for microbial culture after removing the air by depressurizing the incubator before inoculating the lactic acid bacteria or enteric bacteria. Manufacturing method characterized in that. The method according to any one of claims 1 to 5, further comprising the step of concentrating or lyophilizing the matured liquid obtained by performing step (b). The method according to claim 6, wherein the supernatant obtained by centrifuging the aging liquid obtained by performing step (b) is concentrated or lyophilized.

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