KR101625474B1 - Manufacturing method for mass-production of ginsenoside Rh4 - Google Patents
Manufacturing method for mass-production of ginsenoside Rh4 Download PDFInfo
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Abstract
The present invention relates to a mass production method of ginsenoside Re, and more particularly, to a method for producing ginsenoside Rh4 by mixing an organic acid with ginsenoside Re and subjecting it to high temperature and high pressure treatment. Ginsenoside Rh4 does not exist in white ginseng but exists in a small amount in red ginseng or black ginseng. Since ginsenosides having a similar chemical structure and molecular weight are present in various forms through processing of existing ginseng extract or red ginseng extract, It is not only easy to purify the side Rh4, but also contains a trace amount of ginsenoside Rh4 in the extract. However, in the present invention, since ginsenoside Rh4 is produced using only ginsenoside Re which is easily separable from ginseng leaf, purification of ginsenoside Rh4 is easier than in the case of using extract, and mass production This is very easy. In addition, since the price of the standard purified ginsenoside Rh4 is also high, it is possible to produce high added value through mass production of such ginsenoside Rh4 through the mass production method of ginsenoside Re of the present invention.
Description
The present invention relates to a method for mass-producing ginsenoside Rh4, and more particularly, to a method for mass-producing ginsenoside Rh4 by mixing an organic acid with ginsenoside Re and subjecting the mixture to high-temperature high-pressure treatment.
Ginseng (Panax ginseng) has been widely used as a drug or health food in the East, including the country the past few thousand years ago, a herbaceous perennial belonging to the genus Araliaceae senticosus (Aralaiaceae) ginseng. In addition, ginseng can be used in various forms such as ginseng and red ginseng. Especially, it is known that red ginseng processed by steaming steam ginseng contains various ginsenosides useful for human body.
Saponin, which represents the major efficacy of ginseng, is composed of more than 30 kinds of ginsenosides. Ginsenosides are used to enhance immunity, antiinflammatory action, antiallergic action, anticancer effect, effect on erectile dysfunction, Action, anticholesterol action, antithrombotic action, adult disease and aging.
Saponin of ginseng is a neutral glycoside in which glucose, arabinose, xylose, rhamnose and the like are bonded to a triterpenoid-based dammarane skeleton. When ginseng is extracted with water and alcohol, it is confirmed that Rb1, Rb2, Rc, Rg1 and the like exist in a large amount among the ginsenosides of ginseng. However, these ginsenosides are not well absorbed in the body and are not suitable for use as pharmaceutical compositions.
Meanwhile, it has been confirmed that ginsenosides in ginseng are converted into ginsenosides which do not exist in the natural world, through the heating process through the production process of red ginseng and black gins through heating reaction, and the heat extraction with ginseng, It is easily absorbed in the body and is easily used as a pharmaceutical composition for various diseases.
Through the heat treatment process, ginsenosides Rg2, Rg3, Rh2, Rh1 and the like were generated from Rb1, Rb2, Rc and Rg1 among the ginsenosides of the ginseng through the separation of the glycosyl residues of the ginsenosides, Rk5, Rk1, Rg6, Rk3, F4, and Rh4 are produced through the dehydration process of the water.
In addition, the ginsenosides are hydrolyzed by the addition of organic acid, and the sugar is removed and the side chain portion is cyclized to form panaxatriol or panaxadiol, It has also been known that a senoside conversion reaction occurs (Kor. J. Pharm. 3 (4). 211-212, 1972; Chem. Pharm. Bull. 11. 759-761, 1963; Chem. Pharm. Bull. 22 Chem. Pharm Bull, 22: 421-428, 1974).
Such converted specific ginsenosides have been reported to have physiological activities such as antioxidant activity, anti-cancer activity, and platelet aggregation inhibition (American Family Physician, 68 (8), 1539-1542, 2003). In particular, Rh4, a protopanaxatriol system, has been shown to inhibit the proliferation of cancer cells.
Vinegar is a typical seasoning having an acidic taste. It is fermented and brewed, and the sour taste of fruit is used. It stimulates appetite, and it is effective for restoring fatigue and beauty. The English vinegar comes from vinaigre, a combination of French wine (vin) and sour tastes (aigre). It was originally called vinegar because it was made by fermenting acetic acid to wine. Vinegar has a wide variety of varieties, because vinegar is relatively easily produced when acetic acid bacteria are propagated to alcoholic ingredients. Acetic acid bacteria that cause acetic acid fermentation are oxygenated (aerobic) acid bacteria that form a clean membrane on the surface of a fermentation tank. A method of continuously producing acetic acid fermentation while venting has been developed. Vinegar, which is mainly used in each country, is deeply related to alcoholic beverages that are widely produced in the country, and fruits that are cultivated and harvested many times. For example, cider vinegar in the US fermented with apple juice fermented vinegar, wine vinegar in France fermented grape juice, malt vinegar in England, fermented malt juice, , Japanese sake vinegar made from sake (sake), alcohol vinegar (spirit vinegar) fermented with pure alcohol, and distilled vinegar in the United States with distilled fermented vinegar are well known. Synthetic vinegar is obtained by diluting glacial acetic acid or acetic acid with water and adding amino acids or sugars to it. There are lemon vinegar, apricot vinegar, and vinegar processed vinegar which are processed using sour taste of fruit juice. Vinegar is most commonly used in the pH range from 6 to 7 degrees. 3 to 5 degrees of low acidity vinegar, 13 to 14 degrees of double vinegar, 18 to 19 degrees of triple vinegar is also used.
On the other hand, the inventors of the present invention, while studying a mass production method of ginsenoside Rh4 which is highly likely to be used as a useful anticancer agent, have found that by mixing ginsenoside Re with an organic acid and subjecting it to high temperature and high pressure treatment, ginsenoside Rh4 is produced, The inventors of the present invention have completed the present invention by confirming a method capable of easily producing a large amount of ginsenoside Rh4 which was obtained in a trace amount or was not easy to produce as a purified product with high purity.
An object of the present invention is to provide a method for mass-producing ginsenoside Rh4, and more specifically, a method capable of mass-producing ginsenoside Rh4 by mixing an organic acid with ginsenoside Re and subjecting the mixture to high- In order to solve the problem.
The present invention relates to a process for preparing ginsenoside Rh4 of the following formula 2 from a ginsenoside mixture obtained by mixing an organic acid with ginsenoside Re of the following formula 1 and subjecting it to high temperature and high-pressure treatment.
[Chemical Formula 1]
(2)
Preferably, the organic acid is a vinegar having a pH of 2.0 to 3.0 or a diluted solution of an organic acid selected from the group consisting of acetic acid, lactic acid, citric acid and malic acid.
The high-temperature and high-pressure treatment may be carried out at 110 to 140 ° C under 0.11 to 0.16 MPa for 2 to 4 hours.
By using the method of the present invention, the ginsenoside mixture obtained from the ginsenoside Re can be chromatographically purified to purify the ginsenoside Rh4 preferably having a purity of 90% or more.
Hereinafter, the present invention will be described in detail.
The present invention relates to a method for preparing ginsenoside Rh4 by mixing an organic acid with ginsenoside Re and subjecting the ginsenoside Re to high temperature and high pressure treatment as described in the following Reaction Scheme 1, The conversion step is described in detail in the following Reaction Scheme 2, and the step of converting the ginsenoside Re into various ginsenosides through the above Reaction Scheme 2 can be confirmed.
[Reaction Scheme 1]
[Reaction Scheme 2]
The ginsenoside Re used in the present invention can be obtained from ginseng extract. The ginseng extract can be prepared by using all the ginseng-derived roots, fruits, leaves, stems and the like derived from ginseng, and preferably a large amount of ginsenoside Re can be easily obtained from the ginseng fruit.
Ginseng used in the extraction of the ginsenoside Re is a perennial plant belonging to the genus Panax (Panax), ginseng (Panax ginseng), hwagisam (Panax quinquefolia), jeonchilsam (thirty-seven, Panax notoginseng), jukjeol three (Panax japonicus), Panaxa pseudoginseng . And one species selected from Panax vietnamensis , Panax elegatior , Panax wangianus and Panax bipinratifidus , Panax angustifolium , It is most preferable to use Korean ginseng, which is known to have a high saponin content, but the type of ginseng is not particularly limited thereto. These ginsengs may be used alone or in combination of two or more thereof.
The ginseng extract may be extracted from ginseng with water, C1-C4 alcohol or a mixed solution thereof as a solvent. The C1-C4 alcohol may be selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol and isobutanol .
The water, the C1-C4 alcohol, or the mixed solution thereof used in the production of the ginseng extract may be 1 to 40 times as much as the weight of the ginseng used (1 to 40 L based on 1 kg), preferably 5 to 40 times . The ginseng extract may be extracted at 20 to 100 ° C for 1 to 48 hours. The above process can be repeated 1 to 4 times.
In order to extract ginsenoside Re from the ginseng extract, water, a C1-C4 alcohol, or a mixed solution thereof is dissolved in water and extracted with n-hexane, methylene chloride, acetone, chloroform, Ethyl acetate, diethyl ether, water saturated butanol, and n-butanol, or sequentially fractionated by combining these solvents, and then purified by column chromatography can do. The production temperature of the extract or its fractions may be 20 to 50 ° C, but is not limited thereto. The extraction time is not particularly limited, but it is preferable to perform extraction within 10 minutes to 1 day. As the extraction apparatus, a conventional extraction apparatus, an ultrasonic pulverization extractor, or a fractionator may be used.
On the other hand, the ginseng extract may be isolated or isolated by known methods used for the separation and extraction of plant components, such as extraction with an organic solvent (alcohol, ether, acetone, etc.), distribution with hexane and water, The ginsenoside Re can be purified by fractionation or purification using a suitable combination method.
The chromatography can be carried out using silica gel column chromatography, LH-20 column chromatography, ion exchange resin chromatography, medium pressure liquid chromatography chromatography, thin layer chromatography (TLC), silica gel vacuum liquid chromatography, and high performance liquid chromatography.
The organic acid is mixed with the purified ginsenoside Re and subjected to high temperature and high pressure treatment to obtain ginsenoside Rh4.
The mixing ratio of the ginsenoside Re and the organic acid is not particularly limited, but it is preferably 0.5 to 100 ml, more preferably 0.5 to 50 ml based on 1 g of ginsenoside Re.
When the organic acid is mixed with the ginsenoside Re, the ginsenoside Re is suspended or precipitated in the organic acid in the solution state, and the high-temperature high-pressure treatment can be performed in this state.
Preferably, the organic acid is a diluted solution of organic acid selected from the group consisting of vinegar, acetic acid (glacial acetic acid), lactic acid, citric acid and malic acid. The diluted solution of vinegar or organic acid may be in a pH of 2.0 to 3.0.
Or a diluted solution of an organic acid selected from the group consisting of vinegar, acetic acid (glacial acetic acid), lactic acid, citric acid and malic acid may be selected and used in combination. As the vinegar, natural fermented vinegar such as rice vinegar, brown rice vinegar, persimmon vinegar, apple vinegar, grape vinegar, and plum vinegar can be used. It is also possible to use commercially available brewed vinegar. Any edible vinegar prepared using all kinds of grains or fruits can be used. Instead of the organic acid, an acidic substance such as dilute hydrochloric acid, diluted sulfuric acid or the like may be used, but it is preferable to use an organic acid used for edible purposes.
The high-temperature and high-pressure treatment after the organic acid mixture is preferably carried out for 120 minutes to 240 minutes. The high-temperature and high-pressure treatment is preferably carried out at 110 to 140 ° C under 0.11 to 0.16 MPa. If the above condition is exceeded, the yield of ginsenoside Rh4 can be remarkably lowered. Or 240 minutes or more, 140 ° C or more, or 0.16 MPa or more, the high-temperature and high-pressure treatment is performed in a state in which the amount of ginsenoside Rh4 is not substantially increased, leading to cost loss in production.
The ginsenoside Re mixed with the organic acid is mixed with the ginsenoside Re, and the ginsenoside mixture subjected to the high-temperature and high-pressure treatment is fractionated or purified by a chromatographic method in a conventional manner to easily purify the ginsenoside Rh4 have. As the chromatography, it is preferable to use reversed phase column chromatography (C18) or HPLC. The purified ginsenoside Rh4 through the chromatography may preferably have a purity of 90% or more, preferably 90 to 99%, and most preferably 95 to 99%.
In the present invention, Rh4 having a purity of 90% or more may be used as an additive for various medicines, cosmetic compositions, and health functional foods. Further, on the other hand, even if the separate purification is not carried out, it is possible to mix the organic acid with ginsenoside Re, which contains a high content of ginsenoside Rh4, . That is, it is possible to provide a ginsenoside mixture containing 10 to 30% by weight or more of ginsenoside Rh4 obtained by mixing an organic acid with ginsenoside Re and subjecting it to high temperature and high pressure treatment, preferably using the method of the present invention , The ginsenoside mixture may be used as an additive for various pharmaceutical compositions, cosmetic compositions and health functional foods.
Thus, the pharmaceutical composition may contain purely isolated ginsenoside Rh4 and a pharmaceutical excipient. Or the pharmaceutical composition may contain a ginsenoside mixture and pharmaceutical excipient containing 10 to 30% by weight or more of the ginsenoside Rh4 prepared by the method of the present invention.
The pharmaceutical compositions may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method. Examples of pharmaceutical excipients which can be included in the pharmaceutical composition include carriers, other excipients and diluents, and preferably lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, , Gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient, such as starch, calcium carbonate, sucrose, Lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.
The dosage of the pharmaceutical composition will vary depending on the age, sex, body weight, the specific disease or condition to be treated, the severity of the disease or condition, the route of administration, and the judgment of the prescriber. Dosage determinations based on these factors are within the level of ordinary skill in the art and generally the dosage ranges from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferable dosage is 1 mg / kg / day to 500 mg / kg / day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.
The pharmaceutical composition may be administered to mammals such as rats, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection. Since the compound of the present invention has little toxicity and side effects, it can be safely used even for long-term administration for preventive purposes.
The health functional food may contain purely isolated ginsenoside Rh4 and a pharmaceutically acceptable food-aid additive. Alternatively, the health functional food may contain a ginsenoside mixture and a pharmaceutically acceptable food-aid additive containing 10 to 30% by weight or more of the ginsenoside Rh4 prepared by the method of the present invention.
The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids. Examples of the foods to which the extract of the present invention can be added include various foods, beverages, gums, tea, vitamins , And health functional foods.
The cosmetic composition may contain, in addition to purely separated ginsenoside Rh4, all components commonly used in cosmetics. Alternatively, the cosmetic composition may contain all of the components commonly used in cosmetics in addition to the ginsenoside mixture containing 10 to 30% by weight or more of the ginsenoside Rh4 prepared by the method of the present invention.
For example, emulsifying agents, thickeners, emulsions, surfactants, lubricants, alcohols, water soluble polymers, gelling agents, stabilizers, vitamins, inorganic salts, emulsifiers, perfumes. The above-mentioned components can be selected within a range that does not impair the effect inherent in the cosmetic, depending on the purpose of formulation or use. The addition amount of the above components may be, for example, 0.1 to 10% by weight, preferably 0.1 to 6% by weight based on the total weight of the composition, but is not limited thereto. The kind of the cosmetic material is not particularly limited, and examples thereof include skin care cosmetic materials such as lotion, milky lotion, gel, cream, essence, pack, ampoule, lotion, cleanser, soap, body products, soap, oil, Makeup cosmetics such as foundation, and cosmetics for hair, and the formulations thereof are not particularly limited.
The present invention relates to a mass production method of ginsenoside Re, and more particularly, to a method for producing ginsenoside Rh4 by mixing an organic acid with ginsenoside Re and subjecting it to high temperature and high pressure treatment.
Ginsenoside Rh4 does not exist in white ginseng but exists in a small amount in red ginseng or black ginseng. It can be processed by conventional ginseng extract or red ginseng extract using organic acid treatment method such as heat extraction, Korean Patent No. 1441002, Korean Laid-Open Patent No. 2013-0074118, etc.), and it has been known that these extracts appear in trace amounts in red ginseng and black ginseng. However, in the extracts of ginseng, red ginseng or black ginseng, there are various types of ginsenosides having similar chemical structure and molecular weight, so that it is not easy to purify ginsenoside Rh4 purely, and a small amount of ginsenoside Rh4 It was not easy to use it because the amount of purified was very small. However, in the present invention, since ginsenoside Rh4 is produced using only ginsenoside Re, which is easily separable from ginseng leaves, ginsenoside Rh4 is relatively purified compared to using ginseng, red ginseng, And the mass production is very easy. In addition, since the price of standard purified ginsenoside Rh4 is also high at 450,000 won per 10 mg as of October 2014, mass production of such ginsenoside Rh4 through the mass production method of ginsenoside Re of the present invention It is possible to create added value.
Fig. 1 shows spectral results obtained by HPLC analysis of the results obtained by adding vinegar to ginsenoside Re and boiling at 121 캜 for 4 hours.
Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, the intention is to provide an exhaustive, complete, and complete disclosure of the principles of the invention to those skilled in the art.
≪ Example 1: Preparation of ginsenoside Re >
Example 1-1. Production of butanol fraction of ginseng leaves
One kilogram of dried ginseng leaves is accurately weighed and leached in 10 liters of 70% (v / v) aqueous methanol solution for one day, shaken ultrasonically for 3 hours and then filtered. 8 liters of 70% (v / v) methanol was added to the leaf of ginseng, and the process of leaching and ultrasonic shaking was repeated 2 times. The filtrate was combined with the filtrate for the first filtration and concentrated under reduced pressure to obtain 416 g of completely dried methanol extract. The methanol extract was completely dissolved in 3 L of water by shaking ultrasonically, and then 2 L of diethyl ether was added thereto, followed by stirring at room temperature for 2 days. After stirring, the solution was transferred to a separatory funnel, and the layer was separated. The diethyl ether layer was removed, and 5 liters of water-saturated butanol was added to the remaining water layer, followed by stirring in a stirrer for 2 days. After stirring, the solution was transferred to a separatory funnel and separated for more than one day. Only the butanol layer was collected and concentrated under reduced pressure to obtain 157 g of a completely dried butanol extract.
Examples 1-2. Isolation and Purification of Ginsenoside Re from Ginseng Leaf Extract
The crude saponin extract of 157 g of the butanol fraction of ginseng leaf obtained in Example 1-1 was dissolved in methanol and mixed with 500 g of silica gel. The mixture was concentrated under reduced pressure to obtain a fine powder. 250-400 mesh silica gel was kneaded in chloroform and poured into a 12 × 50 cm open column to a height of 25 cm. Then, a sea sand (silica sand) And the powder was put in a state of being filled with water. The solution was mixed in a ratio of chloroform: methanol: water = 10: 2: 0.2 (1500 ml: 300 ml: 30 ml), and the solution was mixed in a separatory funnel and used as a development solvent for silica gel column chromatography. The first fractionation was performed to produce 10 fractions, and the fraction 7 containing ginsenoside Re was obtained. C18 reverse phase column chromatography was performed with fraction 7 containing ginsenoside Re. The C18 reversed phase filler kneaded with a 40% (v / v) aqueous methanol solution was filled up to a height of 30 cm in a 3.5 × 60 cm flash column, and then the fraction containing ginsenoside Re was dissolved in a 25% (v / v) aqueous methanol solution The eluted solvents were separated and purified by changing methanol: water at a ratio of 1: 1 (v: v) to 2: 1 (v: v) and 3: 1 (v: v). As a result, 26.4 g of pure ginsenoside Re was finally obtained. The physical and chemical properties of this ginsenoside Re are as follows.
Ginsenoside Re;
White powder;
mp: 201-203 ℃, [α] D 25: 0 (c 0.90, MeOH), C 48 H 82 O 18, MW = 947.165;
1 H-NMR (500 MHz, C 5 D 5 N, ppm)? 0.94 (s, 3H), 0.95 (s, 1H), 1.54 (s, 3H), 1.48 (s, 3H), 1.48 (s, 1H), 1.74 (s, 1H), 1.74 (s, 1H), 1.74 (s, (s, 1H), 2.92 (s, 1H), 2.92 (s, 1H), 1.95 1H), 3.94 (s, 1H), 3.94 (s, 1H), 3.44 (dd, J = 5.3, 11.7 Hz, 1H) 1H), 4.12 (s, IH), 4.16 (s, IH), 4.17 (s, IH), 4.27 1H), 4.75 (s, 1H), 4.45 (s, 1H), 4.45 (s, 1H), 5.14 (d, J = 7.5 Hz, 1H), 5.22 (d, J = 6.9 Hz, 1H), 5.24 (s, 1H), 6.44 (brs, 1H);
13 C-NMR (125㎒, C 5 D 5 N, ppm) δ 17.3, 17.6, 17.7, 17.8 (C-19, C-27), 18.8, 22.4, 23.3, 25.4, 26.7, 27.8, 30.8, 31.0, 32.3, 36.1, 39.5, 39.7, 40.0, 41.2, 46.0, 49.1, 49.7, 51.5, 51.7, 60.9, 63.0, 63.2, 69.5, 70.3, 71.7, 72.3, 72.5, 72.7, 74.2, 75.2, C-5 '), 78.3, 78.5, 78.7, 79.3, 79.4, 83.4, 98.3, 101.9 (C-1', C-1 "), 126.0, 131.0.
≪ Example 2: Treatment of ginsenoside Re with organic acid and high temperature and high pressure >
An organic acid was mixed with the ginsenoside Re obtained in Example 1 and subjected to high-temperature high-pressure treatment to prepare ginsenoside Rh4. Each sample contained 1 g of ginsenoside Re in a 125 ml vial bottle, commercial vinegar of pH 2.0 ~ 3.0 (of which pH 2.6 vinegar had an acidity of 6 ~ 7), brown rice vinegar, and diluted acetic acid Ml. No other water or solvent was used at all. After adding apple vinegar to ginsenoside Re, the mixture of ginsenoside Re and apple vinegar was boiled under high temperature and high pressure conditions using a SANYO sterilizer (SANYO autoclave, MLS-3780 model). Steaming conditions are shown in Table 1 below.
time
< Comparative Example 1. Comparison of Gin Senocide Re Of organic acids and high temperature and high pressure treatment>
An organic acid was mixed with ginsenoside Re in the same manner as in Example 2, and subjected to high-temperature high-pressure treatment. Experiments were conducted under the conditions shown in Table 2 below. In the untreated group, the distilled water was treated with the same volume. In the group without the high temperature and high pressure, the reaction was carried out at room temperature (25 ° C).
time
< Test Example 1. Each Ginenoside HPLC Analysis>
The ginsenoside samples obtained in the above Example 2 and Comparative Example 1 were concentrated under reduced pressure and a small amount of water was removed by adding the organic acid. Each of these samples was dissolved in 2 ml of methanol for HPLC, which was then filtered through a 0.50 탆 filter, and used for HPLC analysis.
Ginsenosides standard products were purchased from Ambo Lab (www.ambo.co.kr) (each product number - Ginsenoside F4 + Rg6: G1022; Ginsenoside Rh1- (20R): G1016; Ginsenoside Rh4 + Rk3: G1023).
The HPLC apparatus was Agilent Technologies 1260 infinity and an ACE 5-C 18 column (250 x 4.6 mm) was used. Water (H 2 O) was used as the solvent A and acetonitrile (CH 3 CN) was used as the solvent B as the mobile phase. The solvent was eluted with a gradient elution system for 0-35 minutes (20% B), 35-85 minutes (40% B), 85-105 minutes (50% B), 105-135 minutes (65% 145 to 155 minutes (100% B), 155 to 160 minutes (100% B), 160 to 163 minutes (20% B), 163 to 165 minutes (20% Lt; / RTI > The development temperature was 40 ° C and the flow rate was 1.0 ml / min. The detector was analyzed for the components of
Solvent
(35% B), 85- 105 minutes (50% B), 105-135 minutes (65% B), 135-145 minutes (85% B) 145-155 minutes (100% B), 155-160 minutes (100% B), 160-163 minutes (20% B), 163-165 minutes (20
The types of ginsenosides and the content of each of the thus-analyzed ginsenosides and comparative examples are shown in FIG. 1 and Table 4 below.
Fig. 1 shows spectral results of a ginsenoside mixture of Example 2-1 (obtained by adding apple vinegar to ginsenoside Re and boiled for 120 minutes at 121 캜) by HPLC. Referring to FIG. 1, it can be seen that no ginsenoside Re is present, indicating that the ginsenoside Re is hydrolyzed and converted to another kind of ginsenoside. Each ginsenoside position was confirmed in comparison with the standard ginsenoside, and it was confirmed that Rh1-R / S, Rg6, F4, Rk3 and Rh4 were representative among the resulting ginsenosides.
On the other hand, referring to Table 4, since the samples of Examples 2-1 to 2-3 and Comparative Examples 1-1 to 1-3 differed only in reaction time under conditions of apple vinegar, 121 ° C and 0.13 MPa , Which makes it easy to confirm that the ginsenoside Re changes to another ginsenoside.
As a result, it can be confirmed that (R) -Rh1, (S) -Rh1, Rg6, F4, Rk3 and Rh4 were generated in the sample of Comparative Example 1-1 by disappearance of ginsenoside Re. The results are shown in Table 2. In Comparative Example 1-2, the amount of ginsenoside Rh1 was further reduced and the amount of ginsenoside was further increased. In the case of ginsenoside Rh4, 1-1, compared with the previous study. The samples of Comparative Examples 1-3 have the longest reaction time, but the content of ginsenoside Rh4 is not significantly different from that of Example 2-3, and thus it can be understood that the method of the present invention is a method of increasing the manufacturing cost over time.
Likewise, in Example 2-1, ginsenoside Re was not confirmed. As a result, ginsenoside Rh1 almost disappeared, and the amount of ginsenosides Rg6 and F4 decreased, and ginsenoside Rh4 was markedly increased. It was confirmed that ginsenoside Re was not confirmed in Examples 2-2 and 2-3, and that ginsenosides Rh1, Rg6, and F4 almost disappeared and ginsenoside Rh4 was remarkably produced.
On the other hand, as can be seen from the sample of Table 4, it can be seen that ginsenoside Rh4 is increased to the greatest amount in 2 to 4 hours (120 minutes to 240 minutes) It is preferable to carry out the reaction at a pressure of 0.16 MPa.
Compound information of ginsenoside Rh4 isolated in the ginsenoside mixtures of Example 2 is as follows.
Ginsenoside Rh4;
White powder;
mp: 160-161 C, [?] D 25 : +28.2 (c 1.0, MeOH), C 38 H 60 O 8 , MW = 621;
1 H-NMR (500 MHz, DMSO, ppm)? 0.81 (s, 4H), 0.85 (s, 6H), 0.94 (s, 1H), 1.21 (s, 4H), 1.29 (s, 1H), 1.31 1H), 1.92 (s, 1H), 1.94 (s, 3H), 1.62 (s, 2H), 3.13 (s, 1 H), 2.91 (s, 2H), 2.91 (dd, J = 4.5, 13.9 Hz, (S, 1H), 3.40 (s, 1H), 3.59 (dd, J = 5.1, 12.0 Hz, 1H), 3.88 , J = 7.5 Hz, IH), 5.02 (t, J = 7.5 Hz, IH), 5.09 (t, J = 7.2 Hz, IH);
13 C-NMR (125㎒, DMSO , ppm) δ 12.7, 15.4 (C-29, C-30), 16.7, 17.1, 17.6, 25.5, 26.6, 26.8, 28.4, 30.6, 31.8 (C-11, C- C-17), 50.2, 61.2, 61.3, 71.0 (C-12, C-4), 74.0, 76.5, 77.1, 77.7, 78.6, 104.3, 121.9, 123.4, 130.4, 138.9.
Claims (4)
[Chemical Formula 1]
(2)
Wherein the organic acid is a vinegar having a pH of 2.0 to 3.0 or a diluted solution of an organic acid selected from the group consisting of acetic acid, lactic acid, citric acid and malic acid.
And chromatographing said ginsenoside mixture to obtain ginsenoside Rh4 having a purity of at least 90%.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101783295B1 (en) | 2016-07-27 | 2017-09-29 | 충남대학교산학협력단 | Manufacturing method for mass-production of ginsenoside Rg4 and Rg6 |
| KR101897811B1 (en) * | 2016-07-22 | 2018-09-12 | 충남대학교산학협력단 | Manufacturing method for mass-production of ginsenoside Rh1-S, Rh1-R, Rg4 and Rg6 |
| KR20200119079A (en) | 2019-04-09 | 2020-10-19 | 아레즈 주식회사 | Manufacturing method of ginsenoside Rg2, Rg4, Rg6 and Rh1 mixture |
| WO2024085659A1 (en) * | 2022-10-20 | 2024-04-25 | 아레즈 주식회사 | Composition for preventing or treating oral disease, containing steamed ginseng berry extract as active ingredient |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101897811B1 (en) * | 2016-07-22 | 2018-09-12 | 충남대학교산학협력단 | Manufacturing method for mass-production of ginsenoside Rh1-S, Rh1-R, Rg4 and Rg6 |
| KR101783295B1 (en) | 2016-07-27 | 2017-09-29 | 충남대학교산학협력단 | Manufacturing method for mass-production of ginsenoside Rg4 and Rg6 |
| KR20200119079A (en) | 2019-04-09 | 2020-10-19 | 아레즈 주식회사 | Manufacturing method of ginsenoside Rg2, Rg4, Rg6 and Rh1 mixture |
| KR20220041812A (en) | 2019-04-09 | 2022-04-01 | 아레즈 주식회사 | Manufacturing method of ginsenoside Rg2, Rg4, Rg6 and Rh1 mixture |
| WO2024085659A1 (en) * | 2022-10-20 | 2024-04-25 | 아레즈 주식회사 | Composition for preventing or treating oral disease, containing steamed ginseng berry extract as active ingredient |
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