KR100602174B1 - Treatment method of cultivated sansam for heightening absorption rate - Google Patents

Abstract

The present invention relates to a method for treating ginseng cultured roots to increase the body absorption rate of effective ginsenosides present in wild ginseng cultured roots.

The method of treating wild ginseng cultured root of the present invention is characterized by fermenting the wild ginseng cultured root to a desired level using a specific microorganism after preparing the wild ginseng cultured root as a dry powder, wherein the specific microorganism is the genus Bacteroidacea Lactobacillus. ) Saccharomyces strain (Saccharimyces) strains or a mixture of at least one or more of the above strains.

The wild ginseng cultured root according to the treatment method of the present invention has a high pharmacological effect even in a small amount due to high body absorption rate.

Wild ginseng, wild ginseng cultured roots, wild ginseng cultured roots

Description

Treatment method of cultivated sansam for heightening absorption of effective ginsenosides

1 is a flow chart briefly showing the wild ginseng cultured muscle treatment method of the present invention.

Figure 2 is a thin-film chromatography (TLC) results of the analysis of the effective ginsenosides present in wild ginseng cultured roots according to the method for treating wild ginseng cultured according to the present invention.

3 and 4 are HPLC results of analyzing the amount of effective ginsenosides present in wild ginseng cultured roots according to the wild ginseng cultured muscle treatment method of the present invention.

5 is a view showing the absorption of wild ginseng culture roots for each organ after ingesting the wild ginseng cultured muscle labeled with ¹⁴ C isotope treated according to the wild ginseng culture root treatment method of the present invention.

FIG. 6 is a distribution chart of absorbed wild ginseng cultured muscle per unit weight after ingestion of wild ginseng cultured muscle labeled with ¹⁴ C isotope treated according to the wild ginseng cultured muscle treatment method of the present invention.

Wild ginseng is generally classified into three types: cheonjong, jijong, and camphor, and cheonjong and jijong are wild ginseng. Birds and animals eat wild ginseng seeds and excrete them with excrement in the mountains. It means that it is collected and sprayed deep in the mountains and then grown into the wild.

Although wild ginseng has a better pharmacological effect than ginseng, its rareness has limited research and use. Recently, however, as a result of the development of cell culture techniques, wild ginseng cells can be cultured to make callus or, as disclosed in Korean Patent Laid-Open Publication No. 2003-0030348, the single cells derived from callus can be cultured to differentiate the organs of wild ginseng. It is now possible to study or use wild ginseng.

However, the major pharmacological effects of wild ginseng are the central nervous system, protein synthesis, corticosteroid secretion, insulin-like action, detoxification, anti-inflammatory, and platelet aggregation. Phosphorus ginsenoside has a low absorption rate in the body, and according to the known rat experiment, the absorption rate is only 3 to 5% (paper auto radiograph, Seoul National University, 1983 paper).

The reason is that a large amount of ginsenosides present in wild ginseng constitutes a polymer complex, and as a human without the decomposing enzyme of the polymer complex, the effective ginsenoside component having a good effect on the human body can be effectively decomposed and absorbed from the polymer complex. It is because it can not be. In addition, a small amount of effective ginsenosides absorbed by the human body are known to be absorbed in the small intestine, which is known to be caused by the action of bacteria or lactic acid bacteria that are inherent in the small intestine. It can be considered that the kinds or amounts of the native bacteria or lactic acid bacteria are different.

Therefore, the present invention has been made to solve the above problems, there is a pharmacological effect of various ginsenosides present in wild ginseng culture roots by treating the wild ginseng culture roots by a predetermined method using bacteria or lactic acid bacteria that grow in the small intestine of humans. The present invention provides a method for treating ginseng cultured roots to increase the absorption rate of effective ginsenosides in the body and to provide a wild ginseng cultured root composition having high absorption rate of effective ginsenosides.

The method of treating ginseng cultured roots to increase the body absorption rate of the effective ginsenosides according to the present invention for achieving the above object comprises the steps of preparing a wild ginseng cultured root as a dry powder; and a ginseng cultured root prepared by the dry powder at a predetermined mass ratio And fermenting the wild ginseng cultured root by inoculating and culturing the specific microorganism after adding to a medium which is a nutrient source required by the specific microorganism; and separating the wild ginseng cultured root undergoing the fermentation step from the by-product. do. In addition, in order to further increase the absorption rate of the effective ginsenoside in the body further comprising the step of drying the ginseng cultured root isolated from the by-products at 30 ~ 120 ℃ and pulverizing the dried ginseng cultured root.

More specifically, the specific microorganism is any one of the strains Bacteroidacea Lactobacillus or Saccharimyces, or a mixture of at least one of the strains. More preferably, the Bacteroidacea Lactobacillus strain is a johnsoni strain (KCTC3141), and the Saccharomyces strain Saccaromyces strain is cerevisiae. It is characterized in that the strain (KCTC7248).

And the addition rate of the wild ginseng cultured root prepared by the dry powder is 1 to 30% by mass with respect to the medium, the fermentation temperature of the specific microorganism is 25 to 40 ℃, the fermentation time of the specific microorganism is 6 to 48 hours It is characterized by.

In order to achieve the above object, the ginseng cultured root composition having high absorption rate of the effective ginsenoside of the present invention is added to the culture medium of wild ginseng cultured as a nutrient source required by a specific microorganism, and then the specific microorganism is added. The fermented wild ginseng culture root was fermented by inoculation and incubation, and the fermented wild ginseng culture root was separated from by-products, dried at 30 to 120 ° C., and then ground.

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

1 is a flow chart showing a method for treating wild ginseng cultured muscle to increase the body absorption rate of the effective ginsenoside of the present invention. Referring to Figure 1, the method of treating ginseng culture root to increase the body uptake rate of the effective ginsenoside of the present invention, first to maximize the surface area of the ginseng culture root to increase the fermentation rate by microorganisms ginseng cultured in the ginseng cultured in the wild ginseng culture period It is dried and prepared by powder (101). And prepare a medium 102 required by the specific microorganism to ferment the wild ginseng cultured root prepared by the dry powder.

The medium refers to a culture medium rich in sugar and the like that can be used to feed a specific microorganism. The composition is different depending on the type of microorganism, the medium conditions used in the present invention will be described in detail in Example 1 to be described below.

Then, the wild ginseng cultured muscle 101 prepared from the dry powder is uniformly mixed to be uniformly distributed in the medium 102 at a predetermined mass ratio, and then inoculated and cultured. Preferred examples of the specific microorganism used in the present invention are any of the strains belonging to the genus Lactobacillus of Bacteroidacea or the strains belonging to the genus Saccharimyces or among the strains. At least one strain is mixed. And more preferred examples are the johnsoni strain belonging to the genus Lactobacillus (Accession No .: KCTC3141) {L. johnsoni} or the cerevisiae strain belonging to the genus Saccharomyces (Accession No .: KCTC7248) {S. cerevisiae}.

In addition, the ratio of wild ginseng cultured roots prepared by the dry powder to the medium is preferably in a mass ratio of 1 to 30%. And the fermentation temperature using the strains is preferably 25 to 40 ℃, fermentation time is preferably 6 to 48 hours. The criterion for determining the fermentation temperature is the optimum growth temperature of the microorganisms, and the criterion for determining the fermentation time is a polymer complex containing effective ginsenosides present in the wild ginseng cultured root ginseng produced by the dry powder by metabolizing specific microorganisms. By comparing the rate at which the effective ginsenosides are separated from the rate at which the specific effective microorganisms are consumed through metabolic action, an appropriate time period for obtaining the effective ginsenosides is obtained.

At the end of the fermentation step, the fermented wild ginseng culture root is separated from the by-products (104, 105).

That is, the fermented wild ginseng culture root is separated from water and other by-products generated during the fermentation of the specific microorganism to prevent the fermented wild ginseng culture root from being fermented more than necessary.

The fermented wild ginseng culture root isolated from the by-product is subjected to a drying step at 30 ~ 120 ℃ (106). The drying step completely blocks the activity of the specific microorganisms that may be present in the fermented wild ginseng culture root separated from the by-product due to evaporation of water, thereby preventing further fermentation. And by evaporating the moisture it is easy to store the fermented wild ginseng culture root.

Fermented wild ginseng cultured root through the drying step is crushed to increase the surface area and to facilitate the absorption in the body (107). Therefore, the wild ginseng cultured muscle 108 with high absorption rate of effective ginsenosides is prepared. The wild ginseng cultured roots, which have undergone each step of the method of treating ginseng cultured roots to increase the absorption rate of the effective ginsenoside in the body, are in a powdered state. It can be used as a raw material.

In addition, the specific microorganism used in the present invention is edible as intestinal bacteria or lactic acid bacteria.

Types and characteristics of the microorganisms used in the embodiment of the present invention are shown in Table 1.

Table 1: Types and Characteristics of Microorganisms

number Type of microorganism Characteristic    One L. casei YIT9018  Human intestinal microflora, commercial growth rate, 2 L. casei KCTC2180 Human intestinal microflora 3 L. johnsoni KCTC3141 Human intestinal microflora 4  L. acidophilus ATCC4356 Probiotics are fast because they are commercial 5 L.acidophilus CU681 Human intestinal microflora 6 L. acidophilus KCTC2182  Probiotics 7 L. brevis ATCC8287 Release Fermentation to Produce Lactic Acid and Acetic Acid 8 L. plantarum KCTC1384 As a kimchi microorganism, it is activated during the initial fermentation, it is strong in flame resistance and grows quickly. 9  L. paracasei ssp paracasei CU580 Isolation and Identification on European Fermented Dairy Products  10 L. rhamnosus GG ATCC53103 Probiotics 11 L. gaserri KCTC3183 Beta-galactose breakdown 12 L. oris KCTC3670 Human saliva microorganism  13 Saccharomyces cerevisiae KCTC7248 Vegetable yeast 14 Microorganisms except 7, 8, 12 ^* of the specific microorganisms 15 14 + 7 ^* 16 15 + 8 ^* 17 15 + 12 ^*

^* : The number indicates the number listed in the first column of <Table 1>.

Hereinafter, the present invention will be described in detail with reference to the following Examples.

Example 1 Inoculation and Fermentation of Specific Microorganisms in Wild Ginseng Cultured Roots Treated with Powder

The ginseng culture root used in this example was first sterilized and sterilized with all materials to induce organ differentiation of wild ginseng, and then modified wood plant medium (hereinafter referred to as 'WPM') (Lloyd and McCown, 1980; Owen and Miller, 1992). 30 g / l of sugar, 7 g / l of agar, and 1-5 ppm of plant growth regulator IAA (3-indole acetic acid) to induce organ differentiation were added to Petri dish. (Petri dish) was incubated for 40 to 60 days (pH 5.8, 25 ℃ dark room). In the next step, the cultured wild ginseng root cultured in a sterile state using WPM as a medium and incubated in a small organism incubator and a large organism incubator, and then passaged and transferred wild ginseng root in large quantities for about 6 weeks.

In addition, the strains listed in <Table 1> used in <Example 1> were cultured using MRS broth (Difco. USA) medium under aerobic conditions at 37 ℃, and enough vitality by two or more passages before the experiment It was used after raising.

First, the cultured wild ginseng cultured root is dried at 30 ° C. for at least 48 hours to increase the surface area, and then ground to prepare a powder. The ginseng cultured root made of the powder was added to the medium No. 178 (medium name; MRS Medium, oxioid CM 359; Difco 0881, pH 6.2-6.6) prepared in a sterile Petri dish, respectively, and then passaged. Inoculate the cultured microorganisms. Thereafter, the wild ginseng cultured root prepared from the powder inoculated with the microorganisms is fermented at 36 ° C. for 24 hours.

The pH of each fermentation broth in which the microorganisms were fermented was measured to determine whether the microorganisms were activated after 24 hours of fermentation.

TABLE 2 pH of each culture medium fermented for 24 hours

number Type of microorganism pH  One L. casei YIT9018 4.25 2 L. casei KCTC2180 3.83 3 L. johnsoni KCTC3141 3.82 4  L. acidophilus ATCC4356 4.33 5 L.acidophilus CU681 4.40 6 L. acidophilus KCTC2182 4.41 7 L. brevis ATCC8287 4.54 8 L. plantarum KCTC1384 4.51 9  L. paracasei ssp paracasei CU580 4.34  10 L. rhamnosus GG ATCC53103 3.94 11 L. gaserri KCTC3183 3.81 12 L. oris KCTC3670 4.22 13 Saccharomyces cerevisiae KCTC7248 5.83 14 Microorganisms except 7, 8, 12 ^* of the specific microorganisms 3.93 15 14 + 7 ^* 3.88 16 15 + 8 ^* 3.91 17 15 + 12 ^* 3.84

^* : The numbers represent the numbers listed in the first column of Table 1.

As shown in Table 2, it can be seen that the microorganisms used in Example 1 of the present invention grow smoothly in each medium to which wild ginseng cultured root is added to perform a fermentation process.

Example 2 Thin layer chromatography (hereinafter referred to as 'TLC') analysis to confirm the presence of effective ginsenosides in microorganism fermented wild ginseng cultured roots

TLC analysis was performed to confirm the presence of effective ginsenosides in wild ginseng cultured roots fermented by microorganisms in <Example 1>. The wild ginseng cultured roots fermented by the microorganisms are separated from the byproduct, dried at 36 ° C. for 24 hours, and then ground to prepare a powder. After mixing the wild ginseng cultured root made of the powder with 70% ethanol and sufficiently shaken, the contents are extracted by keeping at least 1 hour in a 4 ℃ refrigerator. The extracted contents were centrifuged at 3,000 rpm for 10 minutes, and only 3 ml of the separated supernatant was sufficiently mixed with the same amount of 3 ml hexane (Hexane H ₂ O), followed by centrifugation at 3,000 rpm for 10 minutes to remove the supernatant. Conduct the process three times in a row. The lower solids remaining after the above process is added again 3 ml of butanol and then sufficiently mixed, and the protein is extracted in the same manner as the extraction method to take the supernatant of the protein extract. 9 ml of the supernatant of the protein extract and 9 ml of distilled water were added to completely evaporate the water using a concentrator, and 1 g of methanol (methanol) was used to completely dissolve the remaining solids. Prepare a sample solution.

For analysis, cut the TLC coated glass plate into 5 × 6 cm size, draw a line with a pencil under one side, place the sample sample on the bottom, dry it drop by drop with a fine glass tube, and soak the sample at the same spot. Concentrate. Chloroform: Methanol: Distilled water is separated into a band of the concentrated sample with a solvent of 65:25:10, and then dried and irradiated with a UV lamp to separate the non-saponin-based band having a double bond to eliminate the activity. Thereafter, the TLC coated glass plate is dried in 5% sulfuric acid dissolved in ethanol, and heated to a hot plate to develop a saponin series. A control sample containing standard saponin was also prepared and analyzed for comparison.

As a result of the analysis, although there was a slight difference depending on the type of each cultured microorganism, all of them contained effective ginsenosides. Figure 2 shows the results of TLC analysis of John S. strains (L. johnsoni) and S. cerevisiae representatively. In Figure 2, 2-24 columns and 2-48 columns show TLC results after fermenting wild ginseng cultured roots with L. johnsoni for 24 hours and 48 hours, respectively. TLC results after fermentation of wild ginseng cultured roots with S. cerevisiae for 24 and 48 hours. Column 16 is the effective ginsenoside TLC result of the standard.

Example 3 HPLC Analysis for Confirmation of Effective Ginsenoside Content in Microorganism Fermented Wild Ginseng Cultured Roots

Sample preparation is the same as the sample preparation process of <Example 2>.

HPLC analysis was performed by using a UV detector (203 nm) and an ELSD detector (uBondapak C18, 3.9 × 150mm Waters) at 1 ml / min flow rate for 90 minutes using water and acetite solution (ACN). Table 3 is as follows.

Table 3 HPLC Standard Retention Time

Time (min) ACN (%) 0 17 33 33 58 80 70 80 73 17 90 17

 Intrinsic retention time by separate and mixed analysis of standard saponins (Rg1, Re, Rf, s-Rh1, r-Rh3, Rb1, Rc, Rd, s-Rg3, r-Rg3, etc.) for HPLC analysis After the retention time is determined, qualitative analysis is performed by comparing the retention time of the prepared sample. In addition, the quantitative analysis was performed by dividing the concentrations of standard saponins such as Re and Rg3 into 0.025, 0.05, and 0.1 mg / ml, respectively, to quantify the peak amount and compare it with the peak amount of the prepared sample.

<Table 4> and <Table 5> show the results of HPLC analysis after fermenting L. johnsoni and S. cerevisiae strains in wild ginseng culture roots for 24 hours.

3 and 4 are graphs showing the HPLC results according to the <Table 4> and <Table 5>.

Table 4 Contents of ginsenosides in wild ginseng cultured roots fermented with L. johnsoni for 24 hours

; Ginsenoside type and content in total saponin content (approximately 109 ug / g dry matter, 0.109% w / w)

Retension time (min) Kind of ginsenoside Ginsenoside content (ug / ml) 5.887 Rb 8.3 (16.4%) 7.720 Rc 6.8 (13.5%) 11.100 Rd 4.1 (8.1%) 14.060 Re 3.3 (6.5%) 37.613 Rf 2.1 (4.2%) 39.620 Rg3 9.3 (18.4%) 42.120 Rh1 13.9 (27.5%) 44.080 Undefined 1.8 (3.6%) 44.867 Rg5 1.3 (2.6%) 45.420 Undefined 1.8 (3.6%) 46.467 Undefined 0.9 (1.8%)

TABLE 5 Ginsenosides content in wild ginseng cultured roots fermented with S. cerevisiae for 24 hours; Ginsenoside type and content in total saponin content (approximately 162 ug / g dry matter, 0.162% w / w)

Retension time (min) Kind of ginsenoside Ginsenoside content (ug / ml) 38.540 Rf 4.7 (6.8%) 42.113 Rh1 84.6 (87.8%) 45.433 Rg5 7.1 (7.4%)

Example 4 Radioisotope ( ¹⁴ Measurement of Absorption Rate of Wild Ginseng Cultured Roots Cultured by C)

In order to examine the absorption rate of ginsenoside present in the wild ginseng cultured muscle cultured by using radioisotopes, the cultured wild ginseng roots labeled with radioisotopes were first cultured, and some of them were cultured in the same manner as in <Example 1>. Culture treatment and the rest was used as a control without microbial culture treatment.

1. Culture of wild ginseng cultured root labeled with radioisotope ¹⁴ C

First, all materials are sterilized and sterilized to induce organ differentiation of wild ginseng, and 30 g / of sugar is prepared based on a modified wood plant medium (hereinafter referred to as 'WPM') (Lloyd and McCown, 1980; Owen and Miller, 1992). 1, 5 ppm of plant growth regulator IAA (3-indole acetic acid) to induce organ differentiation after the addition of 7 g / l of agar, was used for 40 to 60 days in a petri dish. Incubated (pH 5.8, 25 ℃ dark room).

The cultured wild ginseng was precultured in a 5 liter incubator for 2 weeks to secure populations, followed by the addition of 250 uCi of deoxyglucose ( ¹⁴ C-deoxyglucose, Amersharm, UK) substituted with isotopic carbon under the same aseptic conditions. Incubate more weekly. After culturing until the initially added liquid medium is completely consumed, the cultured wild ginseng is harvested and washed three times with distilled water to remove the isotopes on the epidermis and dried completely at 30 ° C.

The sample, ie, the dried ginseng cultured root and the isotope content of each culture medium, was measured with a beta counter (Beckmann, UK) to confirm the transfer rate (about 82%) of the isotope transferred to the wild ginseng cultured root. The isotope content (CPM) per gram of the dried wild ginseng cultured root is determined based on the transfer rate (13883.5 cpm / g).

The total mass of the dried ginseng cultured root labeled with the isotope is measured and then stored frozen. Some of the frozen ginseng cultured roots undergo microbial culture treatment as in <Example 1>.

2. Measurement of Body Absorption Rate and Distribution of Ginsenosides Using ¹⁴ C-labeled Wild Ginseng Cultured Roots

A. Rat Metabolism Experiment

The animals (ICR rats, 7 weeks old, weighing about 25-30 grams) were acclimated to metabolism cages for 2 weeks, and then repeated 3 times.

After fasting for 24 hours of wild ginseng, molasses was added to wild ginseng powder, agglomerated into lumps, and freely fed for 36 hours, and the actual intake, the amount left behind, and the amount of bowel movements were measured. Urine was collected by drying three layers of filter paper on the bottom of the cage and used for isotope determination. After anesthesia, blood was drawn from the heart (up to 1 ml), and the abdominal cavity was opened to remove each organ, weighed, and used for analysis.

B. Isotope Measurement

After weighing the blood and the total weight of each organ, resample a portion and add 5 ml of each scintillation cocktail solution for measurement on an isotope meter (Beta counter, Beckmann, UK) and shake well after 5 minutes. The content of ¹⁴ C per unit weight of each organ was measured.

<Table 6> Absorption rate of wild ginseng cultured muscle

Microbial fermentation treatment (experimental group) When microorganism fermentation is not done (control) Total salary (total isotope content in the feed, cpm) 111068.9 118023.3 INPUT amount {total salary-(sum, stomach, small intestine, large intestine residual ginseng), cpm} 18880.6 18392.6 OUTPUT amount (dose volume + urination volume, cpm) 5006.4 15006.3 Residual amount in the body (INPUT amount-OUTPUT amount, cpm) 13874.1 3386.3 Net Absorption Rate (Body Residue / INPUT,%) 73.5 28.7 Total Absorption Rate (Internal Residue / Gross Salary,%) 12.5 2.5

As shown in Table 6, the ginseng cultured roots fermented by microorganisms showed a net absorption rate of 73.5%, which is superior to the net absorption rate of 28.7% when the microorganisms were not fermented.

5 and 6 are diagrams showing the distribution in each organ and the amount absorbed in the body per unit weight for each organ when ¹⁴ C-labeled wild ginseng cultured muscle of the present invention was absorbed into the body of the test animal. 5 and 6, it can be seen that wild ginseng components are evenly absorbed and distributed in each organ of the body such as bone, liver, blood, kidneys, and muscle testis.

As described above, the detailed description of the present invention has been made by the embodiments with reference to the accompanying drawings, but the above-described embodiments are only described with reference to the preferred examples of the present invention, and thus the present invention is limited to the above embodiments. It should not be understood as. Therefore, in addition to the above description, a person having ordinary knowledge in the art to which the present invention pertains may easily implement the present invention in another form within the same scope as the above-described embodiments only by describing the embodiment of the present invention, or the present invention. It will be possible to practice the invention in an equivalent area to the

As described above, according to the wild ginseng cultured muscle treatment method of the present invention, first, the high ginsenoside absorption rate in the body of the ginseng according to the treatment method of the present invention can be seen a high pharmacological effect, even if a small amount is ingested,

Second, relatively similar pharmacological effects can be seen regardless of human constitution.

Third, it is possible to produce a high effect even in a small amount is possible in a variety of commercial applications and there is an advantage that can create high added value.

Claims (9)

Preparing a wild ginseng cultured root as a dry powder; Fermenting the wild ginseng culture root by inoculating and culturing the specific microorganism after adding the wild ginseng culture root prepared by the dry powder to a medium which is a nutrient source required by a specific microorganism; And separating the wild ginseng cultured roots from the fermentation step from the by-product. The specific microorganism is any one of the bacteroidacea Lactobacillus strain or Saccharimyces strain or a mixture of at least one of the strains, The Bacteroidacea Lactobacillus strain is a johnsoni strain (KCTC3141) and the Saccharomyces strain is a cerevisiae strain (KCTC7248). A method of treating ginseng cultured roots to increase the body absorption rate of effective ginsenosides. The method of claim 1, The method of treating ginseng culture root to increase the absorption rate of the effective ginsenosides in the body, characterized in that it further comprises the step of drying at 30 ~ 120 ℃ the ginseng cultured root isolated from the by-product. The method of claim 2, The method of treating ginseng culture root to increase the body absorption rate of the effective ginsenoside, characterized in that it further comprises the step of grinding the dried ginseng cultured root. delete delete The method of claim 1, The ginseng culture root treatment method for increasing the body uptake rate of effective ginsenosides, characterized in that the addition ratio of the ginseng cultured root prepared from the dry powder to 1 to 30% by mass with respect to the medium. The method of claim 1, Fermentation temperature of the specific microorganism is a ginseng cultured root treatment method to increase the body absorption rate of the effective ginsenoside, characterized in that 25 ~ 40 ℃. The method of claim 1, Fermentation time of the specific microorganism is wild ginseng cultured root treatment method to increase the body absorption rate of the effective ginsenoside, characterized in that 6 to 48 hours. delete

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