KR101577111B1 - Pharmaceutical compositions for anti-inflammation or anti-oxidation containing ginsenoside rh4-enriched extraction - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for anti-inflammation containing as an active ingredient a ginsenoside fortification material comprising ginsenoside Rh4. In the production of a ginsenoside fortification material containing ginsenoside Rh4, ginseng or red ginseng extract And extracting the product after adsorbing it on a column. In contrast to the conventional method for producing ginseng extract or the method for producing only a specific ginsenoside contained in ginseng, the production of a fraction enriched with a specific component , The manufacturing method is simple, and it proceeds in a relatively low unit price, thus meeting the commercial aspect. In addition, since the ginsenoside reinforcing material according to the present invention exhibits excellent antioxidative and anti-inflammatory activity, the reinforcing material according to the present invention can be widely used in related industries.

Description

FIELD OF THE INVENTION The present invention relates to a pharmaceutical composition for anti-inflammation or antioxidation, comprising a ginsenoside Rh4-enriched fraction as an active ingredient,

The present invention relates to a pharmaceutical composition for anti-inflammation or antioxidation, and more particularly to a pharmaceutical composition for anti-inflammation or antioxidation containing a ginsenoside Rh4-fortified product as an active ingredient.

Inflammation is a localized immune response that minimizes the damage of a cell or tissue when it is damaged or destroyed by various factors, such as harmful substances or organisms from the outside, and restores the damaged area to its original state. The inflammation is a useful defense mechanism for protecting the living body and removing the products produced by the tissue damage, but it also causes dysfunction due to pain, edema, redness or fever due to the defense mechanism as described above. Therefore, when an inflammatory reaction occurs excessively, a medicament for suppressing it is needed.

Lipoxygenase inhibitors have been attracting attention as one of the drugs that inhibit the inflammatory reaction. Since the lipoxygenase produces various substances acting as various chemical mediators involved in inflammation reaction and allergic reaction in vivo from arachidonic acid, the substances that inhibit the activity of such lipoxygenase are various chemical mediators It is believed that the inflammation reaction can be mitigated as a result.

Apart from such lipoxygenases, active oxygen or free radical scavengers have also received considerable attention as agents that inhibit the inflammatory response. The free radicals and reactive oxygen species are activated by various biological reactions such as normal metabolism by xanthine oxidase or glutathione reductase in the mitochondria, phagocytes or cytoplasm, or inflammation reaction by ultraviolet rays or external stimuli . In addition to this reactive oxygen species, excessive reactive oxygen species and free radicals, especially superoxide anion radicals, which are highly reactive and destructive, accumulate in the body. In addition to inflammatory reactions, they also cause cell death, stroke, myocardial infarction, , Hyperlipemia, diabetes, neurodegenerative diseases, and various other human diseases. Therefore, it has been reported that the above-mentioned active oxygen or free radical scavenging substances have an effect of preventing or treating these diseases, and researches for developing various active oxygen (or free radical) scavenging substances from microorganisms and plants Is continuing.

Meanwhile, ginseng ( Panax Ginseng ) is a medicinal herbal medicine in ginseng and ginseng that has been widely used in Korean medicine as well as folk remedies. It has been used for a long time as a medicinal material and proved its safety and efficacy. It has been reported that the ginseng has a protective effect against radiation, a chromosome abnormality and micro-nuclear defense effect by an alkylating agent, mutation reduction in cultured cells, and reduction of cell deformation. Ginseng contains an acidic polysaccharide including a ginsenoside, a phenol component and the like. Although the ginsenoside contained in the ginseng itself exhibits such excellent effects as described above, the effect can be doubled by strengthening a specific ginsenoside component .

As described above, the derivation and enrichment of the desired ginsenoside utilizes the physicochemical or biological conversion technique of ginsenoside. Most of the biological methods of converting and inducing ginsenosides by hydrolysis of saponins of saponin using physico-chemical methods and glucosidase of saponin are the most common biological methods. These methods are ginseng-free or ginsenoside Induce the side.

In addition to these methods, various techniques for inducing and strengthening specific ginsenoside components have been studied and developed. However, the ginsenosides that exhibit anti-inflammatory and antioxidative activities at a low cost by simplifying and simplifying these induction and enhancement processes Techniques that can be economically strengthened have not been reported.

Korean Patent No. 10-0361187

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of an inflammatory disease and an antioxidative composition containing a ginsenoside fortified material as an active ingredient.

A further object of the present invention is to provide a process for obtaining the ginsenoside fortification product simply and economically.

In order to solve the above problems, the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases and an antioxidative composition containing a ginsenoside fortification material containing ginsenoside Rh4 as an active ingredient.

The present invention also relates to a method for treating ginseng or red ginseng, which comprises treating an extract of ginseng or red ginseng with an acid having a pH of 1 to 4 and heating and hydrolyzing the extract; Adsorbing the hydrolyzed material to a column; Extracting a substance adsorbed on the column with an organic solvent; And a pharmaceutical composition for antioxidation, which comprises a ginseng fortified product prepared by concentrating the extracted substance under reduced pressure and purified, as an active ingredient, for the prophylaxis or treatment of inflammatory diseases.

Although the ginsenoside fortification material containing the ginsenoside Rh4 of the present invention exhibits superior anti-inflammatory or antioxidative effects than the ginsenoside Rh4 single compound, it can be obtained at a much lower unit cost than the ginsenoside Rh4 single compound The ginsenoside fortification according to the present invention can be used not only pharmacologically but also economically as a pharmaceutical for anti-inflammation and antioxidation.

Fig. 1 is a graph showing the relationship between the content of ginsenoside enhancer (hereinafter referred to as "enhancer of Example 1") prepared in Example 1 and the content of paraxaxyl trihydrate ginsenoside of Rh4 ginsenoside 98% And Rh4 ginsenoside content: PTC; The tricolored ginsenoside.
FIG. 2 is a chart comparing the amount of nitric oxide produced from the red ginseng crude saponin, the reinforcing material of Example 1, and the Rhg_4cinoside 98% standard product.
3 is a graph showing the efficacy of inhibiting the expression of TNF-a in the red ginseng crude saponin, the fortified material of Example 1, and the Rhginsinoside 98% standard product.
Figure 4 shows the anti-inflammatory, anti-allergic potency of the panaxadiol type ginsenosides and other ginsenosides and the fortifier of Example 1 as determined by the degree of inhibition of 5-lipoxygenase activity: PDC; Pyrazine diol type ginsenoside mixture; And Example 1; The reinforcing material of Example 1.
Fig. 5 shows the results of confirming the p-38 phosphorylation inhibiting effect of the reinforcing material of Example 1. Fig.
Figure 6 shows the results of confirming the free radical scavenging activity of DPPH (2, 2-diphenyl-1-picrylhydrazyl radical) scavengers of other ginsenosides, including panaxydiol type ginsenosides, and the reinforcement of Example 1: PDC; Pyrazine diol type ginsenoside mixture; And Example 1; The reinforcing material of Example 1.
FIG. 7 shows the results of confirming peroxide anion radical scavenging ability of other ginsenosides including paraxaxidol ginsenoside and the reinforcing material of Example 1 by NBT (Nitro Blue Tetrazolium) scavenging degree.

First, terms used in the specification of the present invention will be described.

The term " ginsenoside " Enhancer "means an extract of ginseng or red ginseng extract which is specifically induced and fortified and contained in a high content compared to other ginsenoside components.

The term " extract " used herein refers to a substance extracted from raw materials by all known methods, and includes the extracted extract, the concentrate obtained therefrom, the dried product and the powder of the concentrate.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases, comprising as an active ingredient a ginsenoside fortification comprising ginsenoside Rh4.

In addition, the present invention provides a pharmaceutical composition for antioxidant containing, as an active ingredient, a ginsenoside fortification including ginsenoside Rh4.

The pharmaceutical composition of the present invention comprises a ginsenoside fortification comprising ginsenoside Rh4.

The ginsenoside fortification comprises ginsenoside Rh4, and in particular contains a higher content of ginsenoside Rh4 than other ginsenoside components.

The ginsenoside Rh4 may be contained in an amount of 10 to 90% by weight based on the total weight of the pyrazine trihydrate ginsenosides contained in the ginsenoside fortification, and the ginsenoside Rh4 may be included in the ginsenoside fortification, Based on the total weight of the triacylglycinosides, and is preferably contained in an amount of 20 to 70 wt% based on the total weight of the triol-based ginsenosides, and is preferably 30 to 50 wt% , But the present invention is not limited thereto. When the content of ginsenoside Rh4 is less than 10% by weight based on the total weight of the pyrazine trihydrate ginsenosides contained in the ginsenoside fortified water, the anti-inflammatory and antioxidative activity of the ginsenoside fortifier is lowered to the ginsenoside Rh4 The content of ginsenoside Rh4 is less than 90 weight% based on the total weight of the pyrenthroid triplet contained in the ginsenoside fortification, %, The cost of obtaining the ginsenoside-reinforced material increases sharply, which is economically inefficient. The ginsenoside fortification of the present invention is free from cytotoxicity, inhibits the production of nitrogen monoxide and 5-lipoxygenase activity which are involved in inflammation induction, inhibits the expression of inflammatory cytokines such as TNF-a, It has an effect of inhibiting phosphorylation. Thus, the ginsenoside fortifier can be used as a medicament for the treatment and prevention of edema, dermatitis, allergy, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, Sjogrens syndrome, multiple sclerosis, acute inflammatory disease, or chronic inflammatory disease, including, but not limited to, inflammatory bowel disease, diabetes mellitus, fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, Inflammatory diseases and diseases such as inflammatory diseases such as aging of cells, stroke, myocardial infarction, diabetic vascular disorders, hyperlipemia, acute inflammation, rheumatic diseases, alcoholic hepatitis, diabetes, epilepsy, Parkinson's disease and dementia And can be usefully used for prevention or treatment of various human diseases that can be caused.

The ginsenoside fortification comprises: 1) treating the extract of ginseng or ginseng product with an acid and heating to hydrolyze; 2) adsorbing the hydrolyzed material to a column; And 3) extracting the substance adsorbed on the column with an organic solvent. However, the present invention is not limited thereto.

The step 1) above is a step of obtaining crude saponin from the processed ginseng or ginseng product and strengthening the ginsenoside Rh4 component in the obtained crude saponin by treating the extract of the ginseng or ginseng product with an acid and heating and hydrolyzing do.

Ginseng of step 1) is goryeosam (Panax ginseng , P. quiquefolius , P. notoginseng , P. japonicus , P. trifolium , P. pseudoginseng , P. vietnamensis , But are not limited to, P. quinquefolium . Preferably, the ginseng used in the present invention is Korean ginseng.

In addition, the ginseng processed product of step 1) includes various kinds of processed ginseng, such as ginseng, misham, white ginseng, taegeuk ginseng, black ginseng, ginseng ginseng, enzyme treated ginseng, fermented ginseng, red ginseng, fermented red ginseng, But is not limited thereto. Preferably, the ginseng work product used in the present invention is red ginseng or fermented red ginseng. The red ginseng is manufactured by heating ginseng through steam or sun drying, preferably steam.

The extract of step 1) may be extracted from the processed ginseng or ginseng product or the dried product thereof, or the commercially available extract itself may be used directly, and the raw material of the extract may be cultivated or commercially available.

When the extract of step 1) is obtained by extracting from the raw material, the extract may be any of conventional extraction methods known in the art such as solvent extraction, ultrasonic extraction, filtration and reflux extraction, and preferably solvent extraction or reflux extraction . ≪ / RTI > The extraction process may be repeated several times, followed by further concentrating, lyophilizing, and the like. Specifically, the obtained extract is concentrated under reduced pressure to obtain a concentrated liquid, the concentrated liquid is lyophilized, and a concentrated powder can be produced using a pulverizer.

The extract can be extracted with water, an organic solvent or a mixture thereof as an extraction solvent. The organic solvent may be any of alcohols, hexane (n-hexane), ether, glycerol, propylene glycol, butylene glycol, ethyl acetate, methyl acetate, dichloromethane, chloroform, ethyl acetate, benzene, The solvent is preferably C1 to C4 alcohol, and the organic solvent is more preferably ethanol, but is not limited thereto. The acid of step 1) may have a pH of 1 to 4, and the pH of the acid is preferably 2 to 3.8, more preferably 2.5 to 3.5. If the pH of the acid is lower than 1, there is a problem that the ginsenoside is excessively hydrolyzed in the extract of the ginseng or ginseng work product to lose the active ingredient or produce other byproducts other than ginsenoside Rh4, , There is a problem that the ginsenoside in the extract is not hydrolyzed properly or takes a long time to hydrolyze. In particular, malic acid, tartaric acid, lactic acid, citric acid, succinic acid or acetic acid can be used as the acid having a pH of 1 to 4 as described above, and preferably lactic acid, citric acid or acetic acid can be used, Acetic acid can be used.

The concentration of the acid in step 1) may be 1 to 15% (v / v) and the concentration of the acid is preferably 3 to 13% (v / v) v), but is not limited thereto. If the concentration of the acid is lower than 1% (v / v), the ginsenoside is not hydrolyzed properly or the hydrolysis takes a long time in the extract of the processed ginseng or ginseng. (v / v), the ginsenoside is excessively hydrolyzed in the extract to lose the active ingredient or produce a by-product other than ginsenoside Rh4.

The heating in step 1) is carried out at a temperature of 80 to 120 캜, preferably 90 to 110 캜, more preferably 95 to 105 캜 for 4 to 12 hours, preferably 6 to 10 hours, But is not limited thereto. If the heating temperature is higher than 120 ° C, it does not help increase the content of ginsenoside Rh4 and unnecessary cost is caused to maintain a high temperature. When the heating temperature is lower than 80 ° C, And the anti-inflammatory and antioxidant effects of the ginsenoside fortified product are lowered. Further, if the heating time is longer than 12 hours, it does not help to increase the content of ginsenoside Rh4 and unnecessary cost is caused by continuing the heating reaction. If the heating time is shorter than 4 hours, ginsenoside Rh4 Is not sufficiently generated.

Wherein said step 2) and step 3) comprise separating the ginsenoside fortification comprising the ginsenoside Rh4 component enriched in said step 1), wherein the hydrolyzate is adsorbed to the column in step 1) , And extracting the substance adsorbed on the column with an organic solvent.

The column of step 2) may be any kind of column capable of separating ginsenoside fortification. Therefore, in the column of step 2), a filler such as silica gel, activated alumina, synthetic polymer, magnesium silicate, activated carbon, cellulose, ion exchange resin and the like may be used and aromatic synthetic resin is preferably used as a filler. It is more preferable that the -20 synthetic adsorbent is used as the filler, but it is not limited thereto. The separation using the column can be carried out once to several times until the fraction of the desired purity is purified, and concentration and recrystallization can be carried out if necessary.

The organic solvent in step 3) may be any one of alcohols, hexane (n-hexane), ether, glycerol, propylene glycol, butylene glycol, ethyl acetate, methyl acetate, dichloromethane, chloroform, ethyl acetate, benzene, Preferably, the organic solvent is a C1 to C4 alcohol, and the organic solvent is more preferably methanol, but is not limited thereto.

The ginsenoside Rh4 enrichment method may further include 4) a step of concentrating the extracted material of the step 3) at a reduced pressure, and the step 4) may be carried out by any of the known purification methods . The purification methods known in the art include fractional concentration using an organic solvent, concentration under reduced pressure, freeze-drying and centrifugation, but it is more preferable to use reduced-pressure concentration in order to obtain the ginsenoside fortification of the present invention .

In a specific embodiment of the present invention, dried and cut 6-year old red ginseng is firstly extracted with 30 L of 70% ethanol at room temperature, and the extracted liquid is secondly extracted with 10 L of water saturated butanol to obtain a red ginseng extract. The red ginseng extract was treated with 8% (v / v) of acetic acid having a pH of 3.0 or less and heated at 100 ° C for 8 hours for hydrolysis. The hydrolyzed substance was adsorbed on a Diaion HP-20 column, The resulting material was extracted with methanol and then concentrated under reduced pressure to give a purified ginenoside-fortified material. The obtained ginsenoside fortified material contained about 21% of pyrazine triazinecinoside, and Rh4 was found to account for more than half of Rh4 (see FIG. 1).

In a specific experimental example of the present invention, the activity of the ginsenoside fortified product thus obtained was confirmed. As a result, the ginsenoside fortification was confirmed to be a safe substance free of cytotoxicity to human fibroblasts regardless of the concentration (see Table 3), and the ginsenoside fortification showed higher levels of monoclonal (See FIGS. 3 and 5), as well as inhibiting the expression of inflammatory cytokines such as TNF-a (tumor necrosis factor-a). In addition, it was confirmed that the activity of lipoxygenase, which is a factor involved in inflammation, was inhibited (see FIGS. 4 and 6), and the effect of inhibiting phosphorylation of p-38 involved in the regulation of the synthesis of inflammatory mediators 5). (Fig. 6, Fig. 7, Table 7 and Table 8) as compared with other ginsenosides such as Rg3, Rh1, Rh2 and the like.

The single compound of ginsenoside Rh4 has a problem that its purification method is very strict and its production cost is high and it is not suitable for use as an active ingredient of a pharmaceutical composition or health functional food. On the other hand, the ginsenoside fortification of the present invention can be obtained by a simple method and a low cost that is only 1/3000 to 1/5000 as compared with the method of purifying the ginsenoside Rh4 single compound , And the enhanced ginsenoside Rh4 exhibits synergistic action with other pyruvate triazine ginsenosides and thus exhibits superior anti-inflammatory or antioxidative effects compared to the conventional ginsenoside Rh4 single compound. As a result, Which can be advantageously utilized.

The pharmaceutical composition of the present invention may contain, in addition to the above-mentioned ginsenoside-fortified material, other ingredients that can give a synergistic effect to the effect of the ginsenoside-fortified material, May be further contained. For example, conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavoring agents, or carriers.

The administration route of the pharmaceutical composition includes oral, intravenous, intramuscular, intraarterial, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal administration, preferably parenteral administration, more preferably Is applied in a topical application manner by application. The parenteral route includes subcutaneous, intradermal, intravenous, intramuscular, intralesional injection or infusion techniques.

The pharmaceutical composition may contain at least one active ingredient which exhibits the same or similar function in addition to the ginsenoside fortification. The compositions may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, oral preparations, external preparations, suppositories and sterilized injection solutions according to a conventional method.

Solid form preparations for oral administration include powders, granules, tablets, capsules, soft capsules, and the like. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. As the agent for parenteral administration, the external preparation such as powders, granules, tablets, capsules, sterilized aqueous solutions, liquid preparations, non-aqueous solutions, suspensions, emulsions, syrups, suppositories and aerosols, The composition may be formulated in the form of a cream, a gel, a patch, a spray, an ointment, a warning agent, a lotion, a liniment, a pasta or a cataplasma, , But is not limited thereto. 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.

Such compositions may additionally contain preservatives, stabilizers, wettable or emulsifying accelerators, adjuvants such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances and may be prepared by conventional methods of mixing, It can be formulated according to the coating method.

The dosage of the pharmaceutical composition may vary depending on various factors including the age, body weight, general health, sex, administration time, route of administration, rate of excretion, drug combination and severity of the specific disease. In addition, the pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers. The pharmaceutical composition may be administered at a dose within the range of 0.001 to 100 mg / kg on an adult basis. When the pharmaceutical composition is an external preparation, it may be administered once to 5 It is preferable to apply it for one month or longer, but the dose does not limit the scope of the present invention.

When the pharmaceutical composition is formulated in unit dosage form, it is preferable that the ginsenoside fortification of the present invention as an active ingredient is contained in a unit dose of about 0.01 to 1,500 mg, The range of about (1 to 500) mg per day is usually, but not limited to, depending on the frequency and intensity of administration, and in some patients a higher daily dose may be desirable.

The pharmaceutical composition may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

In addition, the present invention provides a health functional food for antioxidant and anti-inflammation containing, as an active ingredient, a ginsenoside fortification including ginsenoside Rh4.

The ginsenoside fortification may be utilized as an active ingredient of a health functional food. The ginsenoside fortification is preferably contained in an amount of 0.005 to 20.0% by weight, more preferably 0.01 to 1.0% by weight, based on the total weight of the health functional food, but is not limited thereto. If the effective content of the ginsenoside reinforced material is less than 0.005% by weight, the required anti-inflammatory effect can not be obtained. If the content is more than 20% by weight, There is a problem that can not be changed.

In addition to the ginsenoside fortification of the present invention, the above-mentioned health functional food may contain other ingredients which can give a synergistic effect to the effect of the ginsenoside fortification within the range not impairing the intended effect of the present invention May be further contained. For example, conventional adjuvants such as stabilizers, solubilizers, vitamins, pigments and flavoring agents, or carriers.

It may also include components that are typically added in the manufacture of foods, including, for example, proteins, carbohydrates, fats, nutrients, flavoring agents, and flavoring agents. Examples of such carbohydrates are monosaccharides, such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. As the flavor, natural flavoring agents such as tautatin, stevia extract and synthetic flavor can be used. For example, when the health functional food of the present invention is prepared as a drink, it may further contain citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, natural extract, etc., in addition to the ginsenoside fortification of the present invention.

There is no particular limitation on the kind of the health functional food. Examples of foods to which the ginsenoside fortification of the present invention can be added include meat products, sausages, breads, chocolates, candies, snacks, confectionery, pizza, ramen noodles, dairy products including gums, ice cream, Drinks, tea, drinks, alcoholic beverages, and vitamin complexes, all of which include health functional foods in a conventional sense.

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

However, the following examples and experimental examples are provided only for illustrating the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

< Example  1> Gin Senocide Reinforcement  Manufacturing and refining

After 6 kg of red ginseng was dried, 1 ㎏ of red ginseng was dried, and 30 ℓ of 70% ethanol was used at room temperature for 5 days. The residue was removed by filtration and extracted with 10 L of water saturated butanol and concentrated to obtain crude saponin .

100 g of crude saponin was dissolved in 8% acetic acid, and the mixture was placed in a reflux condenser. The mixture was heated at 100 ° C. for 8 hours to effect acid hydrolysis and adsorbed onto a Diaion HP-20 column (2.8 × 22 cm, wet volume 80 ml) (Mitsubishi Chemical Industry, Yokohama, Japan). The adsorbed column was sufficiently washed with distilled water and then extracted with 99.8% methanol (Sigma, USA) and concentrated under reduced pressure to obtain a fraction. The obtained fraction was purified to obtain 23.35 g of ginsenoside fortification.

< Example  2> Crude saponin  And process conditions Gin Senocide Fortified Gin Senocide  Content analysis

< Example  2-1> red ginseng Crude saponin  And Example  1 &lt; / RTI &gt; Gin Senocide Reinforcement  undergarment Gin Senocide  Content analysis

(MoaCam, Seoul, Korea) and ginsenoside prepared by the method of Example 1 were used to confirm that the ginsenoside content was changed when the ginsenoside fortification product was prepared by the method of Example 1, The content of ginsenosides in water (hereinafter referred to as "reinforcing material of Example 1") was confirmed by HPLC, respectively. A Rh4 standard (98% purity) (Natural Products Chemistry, Daejeon, Korea) was used as a control.

As a result, in the case of commercially available crude saponin, Rh4 was not detected, and when crude PTC was strengthened through acid treatment and column purification as in Example 1, crude P And Rh4 was found to account for more than half of the total. In addition, the purity of the control Rh4 standard product was measured to be 98%, indicating that the above Rh4 content measurement result is correct (FIG. 1).

< Example  2-2> Depending on the hydrolysis time Gin Senocide Fortified  Content analysis

The same procedure as in Example 1 was carried out except that only the hydrolysis time was changed to prepare a gynecoid strengthened product and the content of PTC and ginsenoside Rh4 in these reinforcements was measured in the same manner as in Example < Respectively.

Hydrolysis time 1 hours 2 hours 3 hours 4 hours 5 hours 6 hours 7 hours 8 hours Rh4 (mg / g) 12.25 15.06 17.14 21.59 25.86 28.64 34.09 36.23 PTC (mg / g) 50.93 53.27 54.21 60.68 64.91 67.27 74.87 76.37 Content of Rh4 (%) 24.05 28.27 31.62 35.58 39.84 42.57 45.53 47.44

As a result, as shown in Table 2, the content of PTC increased as the hydrolysis time became longer, and the content of ginsenoside Rh4 in the various ginsenosides constituting PTC also increased (Table 1). It was also confirmed that when the hydrolysis was proceeded for 4 hours, the content of ginsenoside Rh4 was at least 35% by weight based on the total weight of PTC in the reinforcing material of Example 1 (Table 1).

< Example  2-3> column  Depending on the number of refinements Gin Senocide Fortified  Content analysis

 In the same manner as in Example 1, ginsenoside fortifications were prepared by changing the number of column refinements only, and the contents of PTC and Rh4 in these fortifications were analyzed.

1 column purification Column purification 2 times Column purification 3 times Rh4 (mg / g) 85.3 120.36 130.27 PTC (mg / g) 163.2 214.76 227.62

As shown in Table 3, when the column was refined twice, the reinforcing material of Example 1 had enhanced PTCs such as Rg6, Rk3, F4 and Rh4 in comparison with the case of the one-time purification, In particular, the content of Rh4 was 120.36 mg / g, accounting for 56% of the total amount of PTC (214.76 mg / g) (Table 2).

< Experimental Example  1> Example  1 of Gin Senocide Fortified  Cytotoxicity measurement

To examine the cytotoxicity of the reinforcing material of Example 1, MTT reduction method (3, 4,5-dimethylthiazol-2-yl) -2,5-diphenyltetra zolium bromide reduction method was performed. Human fibroblasts (ATCC 2076) were dispensed into 24-well plates at a density of 1 x 10 ⁵ cells using Iscove's modified Dulbeco's medium (Gibco, USA) containing 10% bovine serum. After 1 day, the fortified material of Example 1 was treated at 0, 0.5, 1.0, 2.5, and 5.0 μg / ml with fresh medium (IMDM, bovine serum 0%) and cultured at 37 ° C for 24 hours. At this time, the sample not treated with the fortified material was used as a control group. The cells were then washed with PBS, and MTT (2.5 mg / ml) was diluted 10-fold in IMDM medium, 1 ml of the diluted solution was added, and cultured at 37 ° C for 4 hours. After that, the medium was discarded, 0.5 ml of DMSO was added, and the resultant was dissolved at room temperature for 10 minutes, completely dissolved, and then absorbance was measured at 570 nm. These experiments were repeated three times to determine cytotoxicity.

As a result, it was confirmed that the reinforcing material of Example 1 was not cytotoxic at all treatment concentrations, and thus it was confirmed that the reinforcing material of Example 1 was a safe material without cytotoxicity (Table 3).

sample Concentration (mg / ml) Cell viability (%) Control group 0 100.0 ± 0.00

The reinforcing material of Example 1
0.5 102.1 ± 0.36 1.0 101.7 ± 0.72 2.5 100.1 ± 0.43 5.0 101.2 ± 0.51

< Experimental Example  2> Example  1 of Fortified  Confirm anti-inflammatory effect

< Experimental Example  2-1> Example  1 of Fortified  Nitrogen monoxide ( nitric oxide ) Production inhibitory effect

In order to evaluate the anti-inflammatory effect of the reinforcing material of Example 1, the inhibitory effect on the production of nitrogen monoxide used as a main index of inflammatory reaction was compared with commercially available red ginseng crude saponin (Moa-Cam). A Rh4 standard (natural product chemistry) having a purity of 98% was used as a control.

RAW 264.7 cell lines derived from macrophages were divided into 48-well plates at a density of 1 x 10 ⁶ cells using DMEM containing 10% bovine serum. After 1 day, 1 占 퐂 / ml of lipopolysaccharide (Sigmaaldrich, USA) derived from Escherichia coli and red ginseng crude saponin, Rh4 standard or the fortification of Example 1 were treated at a concentration of 1 ppm or 10 ppm, respectively. After incubation at 37 ° C for 1 day, 100 μl of the cultured medium was transferred to a 96-well plate, treated with 50 μl each of Griess reagents Ⅰ and Ⅱ (Intron biotech, Korea), reacted at room temperature for 10 minutes , The absorbance was measured at 540 nm to confirm the amount of nitrogen monoxide produced.

Untreated group LPS treatment Crude saponin Rh4 standard (98%) The reinforcing material of Example 1 Treatment concentration - 1 mu g / ml 10ppm 1 ppm 10ppm 1 ppm 10ppm 1 ppm NO production (μM) 8.3 1371.7 781.7 1198.3 951.7 978.3 288.3 881.7 NO production inhibition rate (%) - - 43.0 12.6 30.6 28.7 79.0 35.7

As a result, it was confirmed that, in the case of the Rh4 standard product, the amount of nitrogen monoxide was suppressed by 28.7 to 30.6% depending on the concentration, while the reinforcing material of Example 1 suppressed the amount of 35.7% to 79.0% 4 and FIG. 2).

From the above results, it can be seen that the ginsenoside Rh4 is strengthened through the method of Example 1, and the reinforced ginsenoside Rh4 and the other pyrazine triazine ginsenosides cause a synergistic action, It can be seen that it exhibits an effect of suppressing the formation of nitrogen monoxide, which is superior to that of the single component.

< Experimental Example  2-2> Example  1 of Fortified TNF -a Confirmation of production inhibition

In order to confirm the anti-inflammatory activity of the reinforcing material of Example 1, the expression amount of TNF-a was analyzed and confirmed. RAW 264.7 cells were treated with the enhancer of Example 1 and the amount of TNF-a expressed in the cell culture of the treated cells was quantitated using an ELISA kit. RAW 264.7 cells were seeded at a density of 5 × 10 ⁵ cells / ml in a 96-well plate, and the cells were stabilized for 16 hours. The crude saponin, the Rh4 standard and the enhancer of Example 1 were treated at 1 ppm and 10 ppm, respectively, Lt; / RTI &gt; After cultivation, LPS was treated at a concentration of 0.5 μg / ml and cultured in an incubator at 37 ° C. and 5% CO ₂ for 20 hours. Cytokines were measured in cell cultures of the cultured cells using an ELISA kit (Pierce Endogen, USA).

Untreated group LPS
process Crude saponin Rh4 standard
(98%) The reinforcing material of Example 1 Treatment concentration - 0.5 mu g / ml 10ppm 1 ppm 10ppm 1 ppm 10ppm 1 ppm TNF-a production (pg / ml) 0.00 86.60 89.43 86.75 39.58 84.66 26.75 48.24 TNF-a production inhibition rate (%) - - - 0 54.3 2.3 69.12 44.3

As a result, crude saponin had almost no inhibitory effect on TNF-a production, and it was confirmed that the enhancer of Example 1 effectively inhibited the expression level of TNF-a at a concentration of 1 ppm and 10 ppm than that of the Rh4 98% standard product 5 and Fig. 3).

From the above results, it can be seen that the reinforcing material of Example 1 is reinforced by reinforcing ginsenoside Rh4 through the method of Example 1 and causing the reinforced ginsenoside Rh4 to act synergistically with the other pyrazine triazine ginsenosides It is found that TNF-a production inhibitory effect is more excellent than Rh4 alone component.

< Experimental Example  2-3> Example  1 of Fortified  5- Lipoxygenase  Identifying the activity inhibition effect

Lipoxygenase produces chemical mediators involved in inflammatory reactions, so by measuring the activity of lipoxygenase, inflammation inhibition can be confirmed. Specifically, the following method was used to prepare a mixture of the pyrazole diol-based ginsenosides, individual ginsenosides Rg3 (S), Rh2 (R), Rh2 (20s), Rh1 The degree of lipoxygenase activity inhibition was compared and confirmed.

To measure the inhibition of 5-lipoxygenase activity of other ginsenosides and the fortification of Example 1, the pH was adjusted to 8.6 with 200 mM Tris (hydroxymethyl) aminomethane (Sigma, USA) HCl A buffer solution was prepared by dissolving buffer solution and linoleic acid (Sigma, USA) serving as a substrate in ethanol at 300 μM. (Ambo Laboratories, Daejeon, Korea), individual ginsenoside samples Rg3 (S) (Ambo Laboratories), Rh2 (Ambo Laboratories), and the like were mixed with 3 ml of the buffer solution and 100 μl of the substrate solution. (Ambo), Rh2 (20s) (Ambo Lab) and Rh1 (S) (Ambo Lab), the enhancer of Example 1 and nordihydro-guaiaretic acid (NDGA) Respectively. Then, 60 대 of 100,000 units / ml of soybean lipoxygenase (Sigma, USA) was added, followed by enzyme reaction at room temperature for 20 minutes. After the enzyme reaction was completed, the absorbance was measured at 234 nm using a UV / Vis spectrophotometer. The inhibitory effect of 5-lipoxygenase on the activity of each experimental group is shown in Table 6 as a ratio in which the activity of 5-lipoxygenase is suppressed.

(Mg / ml) Rg3 (S) Dioscorea dianosine mixture Rh2 (R) Rh2 (20s) Rh1 (S) The reinforcing material of Example 1 NDGA 0.25 14.8 25.0 21.9 6.0 9.2 55.2
50.2
0.5 19.2 38.2 32.0 9.8 15.0 75.3 One 24.3 39.3 39.3 18.4 24.8 78.0

As a result, it was found that the reinforcing material of Example 1 contained the individual paraxanized diol ginsenosides Rg3 (S), Rh2 (R), Rh2 (20s), the panaxadiol type ginsenoside mixture and ginsenoside Rh1 ) Higher than that of the 5-lipoxygenase activity inhibitor. Specifically, the activity of 5-lipoxygenase was inhibited by 55.2%, 75.3% and 78.0% at the treated concentrations of 1, 0.5 and 0.25 mg / ml, respectively. In the case of the fortification of Example 1 in particular, Which is higher than that of NDGA (Table 6 and Fig. 4).

From the above results, it can be seen that the strengthening of ginsenoside Rh4 through the method of Example 1 resulted in the enhancement of Example 1 being the individual or mixture thereof of the panaxadiol-based ginsenoside, And exhibits a superior anti-inflammatory effect than Rh1 (S) which is a triol-based ginsenoside.

< Experimental Example  2-4> Example  1 of Fortified  Identify the principle of anti-inflammatory effect

Because p-38 is involved in the regulation of the synthesis of inflammatory mediators, the fortification of Example 1 is treated By confirming the phosphorylation activity of p-38, anti-inflammatory activity was confirmed.

Specifically, phosphorylation activity of p-38 was confirmed by Western blotting after treating the reinforcing materials of Example 1 at concentrations of 50 ppm and 100 ppm, respectively. The cultured cells were washed with PBS, dissolved in 5 × SDS reagent buffer, boiled for 10 minutes, and subjected to 10% SDS-polyacrylamide gel electrophoresis. A sample containing the same amount of protein was dissolved in the gel through protein quantification and then transferred to a nitrocellulose reaction membrane (0.45 μm, BioRad lab., Hercules, Calif, USA). The cells were incubated for 1 hour at room temperature in PBST (PBS containing 0.05% Tween 20) at 5%, and washed with PBST for 4 times for 10 min. The primary antibody (1: 1,000- p-38, Cell-signaling Tech., Beverly, Mass., USA) was added to the reaction mixture and incubated overnight at 4 ° C. After washing, the reaction membrane was incubated with a peroxidase- For 1 hour, washed 8 times with PBST, visualized with enhanced chemiluminescence (ECL, Amersham, Arlington Heights, USA) and developed with Kodak XAR5 film.

As a result, it was confirmed that the phosphorylation of p38 was suppressed at the concentrations of 50 ppm and 100 ppm in the fortification treatment of Example 1 (Fig. 5). From the results, it was confirmed that the reinforcing material of Example 1 had the enhanced ginsenoside Rh4 inhibits phosphorylation of p38 more effectively, thereby showing a more improved anti-inflammatory effect (Fig. 5).

< Experimental Example  3> Example  1 of Fortified  Identify antioxidant effect

Free radicals are known to be closely related to inflammatory reactions. Therefore, it was confirmed that other ginsenosides, including the reinforcing material of Example 1, were capable of eliminating free radicals and superoxide anion radicals, such as DPPH (2,2-Diphenyl-1-picrylhydrazyl radical) and NBT (Nitro Blue Tetrazolium) The comparison was confirmed by erasing.

< Experimental Example  3-1> DPPH  Confirmation of antioxidative effect by elimination

("Stopped-flow and spectrophotometric study on radical scavenging by tea catechins and model compound", Chem Pharm Bull 47: 1369-1374 (1999)) was conducted in order to confirm DPPH radical scavenging ability. 1.5 ml of DPPH dissolved in ethanol, 0.15 ml of the sample and 1.35 ml of distilled water were added thereto so as to have a concentration of 2.0 × 10 ^-4 M and reacted at 25 ° C. for 30 minutes. Then, the absorbance at 520 nm was measured, , 0.25, 0.125, 0.05 mg / ml. The DPPH radical scavenging capacity (%) was determined from the equation 100 - [(absorbance of the reaction group to which the sample was added / absorbance of the control group to which no sample was added) × 100]. Vitamin C, an antioxidant, was used as a positive control.

(Mg / ml) Rg3 (S) Dioscorea dianosine mixture Rh2 (R) Rh2 (20s) Rh1 (S) The reinforcing material of Example 1 Vitamin C 0.05 5.1 5.0 3.9 1.8 2.0 2.5

50

0.125 4.4 5.7 4.4 2.9 0.0 4.2 0.25 2.7 8.9 4.3 3.3 0.0 8.4 0.5 4.3 17.4 4.2 3.4 0.0 17.2 One 4.0 21.1 5.1 2.2 0.0 37.9

As a result, the reinforcing material of Example 1 exhibited a higher DPPH radical removal effect than the other ginsenosides including paraxaxidol ginsenoside, and the reinforcing material of Example 1 was treated at a concentration of 1 mg / , It was confirmed that DPPH radical scavenging effect closest to the DPPH radical scavenging effect of the positive control vitamin C was exhibited (Table 7 and FIG. 6).

< Experimental Example  3-2> NBT  Confirmation of antioxidative effect by elimination

Peroxide anion radical scavenging activity was confirmed by using SOD activity detection kit. 0.05 ml of each sample (concentration of each sample: 0.5, 0.25, 0.125, 0.05 mg / ml), 0.5 ml of coloring solution (0.4 mM xanthine, 0.24 mM NBT in pH buffer, 0.1 M sodium phosphate buffer, 0.5 ml of 0.048 unit / ml xanthine oxidase, pH 8.0) was added to the buffer, and the mixture was reacted at 37 ° C for 20 minutes. 1 ml of 70 mM sodium dodecyl sulfate was added to the reaction mixture, The reaction was stopped and absorbance was measured at 560 nm. The peroxide anion radical scavenging ability (%) was obtained from the formula of 100 - [(absorbance of the reaction group to which the sample was added / absorbance of the control group to which no sample was added) × 100]. As a positive control, BHA (Butyl hydroxyanisole), which is widely used in cosmetics, was used.

(Mg / ml) Rg3 (S) Dioscorea dianosine mixture Rh2 (R) Rh2 (20s) Rh1 (S) The reinforcing material of Example 1 BHA 0.05 7.0 20.7 9.5 5.0 0.0 24.6

50

0.125 7.1 21.8 11.4 5.0 2.9 30.4 0.25 7.9 29.9 17.6 5.1 5.4 48.1 0.5 7.9 34.6 25.2 6.8 8.7 55.0 One 11.8 40.0 30.3 10.4 14.8 69.7

As a result, the reinforcing material of Example 1 exhibited a higher peroxide removal effect than the other ginsenosides including paraxaxidol ginsenosides, and the reinforcing material of Example 1 was treated at a concentration of 0.25 mg / ml , It was confirmed that DPPH radical scavenging effect most closely resembles the DPPH radical scavenging effect of vitamin C, which is a positive control group. When treated with 0.5 mg / ml, 1 mg / ml concentration, And high peroxide removal effect (Table 8 and Fig. 7).

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

However, the following Production Examples are only for illustrating the present invention, and the content of the present invention is not limited by the following Production Examples.

< Manufacturing example  1> Preparation of pharmaceutical composition

<1-1> Preparation of external ointment for skin

Skin ointments containing the reinforcing material of Example 1 of the present invention as an active ingredient were prepared according to the composition shown in Table 9 below.

Raw material Content (parts by weight) Purified water 100 glycerin 8.0 Butylene glycol 4.0 Liquid paraffin 15.0 Beta Glucan 7.0 Carbomer 0.1 The reinforcing material of Example 1 20 Caprylic capric triglyceride 3.0 Squalane 1.0 Cetearyl glucoside 1.5 Sorbitan stearate 0.4 Cetearyl alcohol 1.0 antiseptic Suitable amount incense Suitable amount Pigment Suitable amount Wax 4.0

<1-2> External application Patchy  Produce

The external skin patch containing the reinforcing material of Example 1 of the present invention as an active ingredient was prepared in accordance with the composition shown in Table 10 below.

Raw material Content (parts by weight) The reinforcing material of Example 1 10.0 Hexylene glycol 20.0 Diethylamine 0.7 Polyacrylic acid 1.0 Sodium sulphate 0.1 Polyoxyethylene lauryl ether (E.0 = 9) 1.0 Polyhydroxyethylene cetyl stearyl ether 1.0 Viscous paraffin oil 2.5 Caprylic acid ester / capric acid ester (cetiol LC) 2.5 Polyethylene glycol 400 3.0 Deionized water 70

<1-3> Sanje  Produce

2 g of the reinforcing material of Example 1

Lactose 1 g

The above components were mixed and packed in airtight bags to prepare powders.

<1-4> Preparation of tablets

100 mg of the reinforcing material of Example 1

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

&Lt; 1-5 > Preparation of capsules

100 mg of the reinforcing material of Example 1

Corn starch 100 mg

Lactose 100 mg

2 mg of magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

&Lt; 1-6 >

1 g of the reinforcing material of Example 1

Lactose 1.5 g

Glycerin 1 g

0.5 g of xylitol

After mixing the above components, they were prepared so as to be 4 g per one ring according to a conventional method.

<1-7> Preparation of granules

150 mg of the fortification of Example 1

Soybean extract 50 mg

200 mg of glucose

600 mg of starch

After mixing the above components, 100 mg of 30% ethanol was added and the mixture was dried at 60 캜 to form granules, which were then filled in a capsule.

< Manufacturing example  2> Example  1 of Fortified water  Manufacture of Health Functional Foods Containing

The present inventors confirmed that the reinforcing material of Example 1 has anti-inflammatory activity through the above Examples. Thus, a health food containing the reinforcing material of Example 1 as an active ingredient was prepared as follows.

<2-1> Production of beverage

Honey 522 mg

5 mg &lt; RTI ID = 0.0 &gt;

Nicotinic acid amide 10 mg

3 mg of sodium riboflavin hydrochloride

Pyridoxine hydrochloride 2 mg

Inositol 30 mg

Orthoic acid 50 mg

0.48 to 1.28 mg of the reinforcing material of Example 1

Water 200 ml

A beverage was prepared using the above-mentioned composition and content by a conventional method.

<1-2> Of chewing gum  Produce

Gum base 20%

Sugar 76.36 ~ 76.76%

0.24 to 0.64% of the reinforcing material of Example 1,

Fruit flavor 1%

Water 2%

Chewing gum was prepared using the above-mentioned composition and content by a conventional method.

<1-3> Manufacture of candy

Sugar 50 to 60%

Syrup 39.26 ~ 49.66%

0.24 to 0.64% of the reinforcing material of Example 1,

Orange fragrance 0.1%

The composition and the content of the candy were prepared using a conventional method.

<1-4> Biscuit  Produce

First class 88 ㎏ power

Gravity Class 1 76.4 kg

16.5 kg

Salt 2.5 ㎏

Glucose 2.7 kg

Palm shortening 40.5 kg

0.6 kg

Sodium bisulfite 0.55 kg

Rice powder 5.0 kg

Vitamin B1 0.003 kg

Vitamin B2 0.003 kg

Milk flavor 0.16 kg

Water 71.1 kg

Whole milk powder 4 ㎏

1 kg of substitute milk powder

Calcium phosphate 0.1 kg

Spray salt 1 kg

Spray oil 25 kg

0.2 to 0.5 kg of the reinforcing material of Example 1

The biscuits were prepared using the above-mentioned composition and content by a conventional method.

<1-5> Production of ice cream

Fat milk 10.0%

Solid oil content 10.8%

Sugar 12.0%

Starch syrup 3.0%

Emulsion stabilizer (span) 0.5%

Perfume (Strawberry) 0.15%

Water 63.31 ~ 62.91%

0.24 to 0.64% of the reinforcing material of Example 1,

Ice cream was prepared using the above-mentioned composition and content by a conventional method.

<1-6> Of chocolate  Produce

Sugar 34.36 ~ 34.76%

Cocoa Butter 34%

Cocoa mass 15%

Cocoa powder 15%

Lecithin 0.5%

Vanilla flavor 0.5%

0.24 to 0.64% of the reinforcing material of Example 1,

The composition and the content thereof were used to prepare chocolate by a conventional method.

Claims (11)

(1) extracting red ginseng alcohol extract at room temperature again with water saturated butanol to obtain crude saponin;
(2) treating the crude saponin with an acid having a pH of 1 to 4 and heating to hydrolyze the crude saponin;
(3) adsorbing the hydrolyzed substance to a column;
(4) extracting the substance adsorbed on the column with alcohol; And
(5) A pharmaceutical composition for preventing or treating an inflammatory disease, which comprises, as an active ingredient, a panesynthriol ginsenoside fortification product comprising ginsenoside Rh4 produced by concentrating the extracted substance under reduced pressure and purifying. The method according to claim 1,
The ginsenoside Rh4 is added to the ginsenoside fortification in an amount of 10 &lt; RTI ID = 0.0 &gt; To 90% by weight, based on the total weight of the composition. The method according to claim 1,
The pharmaceutical composition for the prevention or treatment of inflammatory diseases, wherein the alcohol in step (1) is ethanol. The method according to claim 1,
A pharmaceutical composition for the prevention or treatment of inflammatory diseases, wherein the acid is acetic acid. The method of claim 4,
Wherein the acetic acid is 6% (v / v) to 10% (v / v) acetic acid. The method according to claim 1,
Wherein the heating is carried out at a temperature of 80 to 120 DEG C for 4 to 12 hours. The method according to claim 1,
The inflammatory disease is selected from the group consisting of edema, dermatitis, allergies, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, ankylosing spondylitis, rheumatic fever, (s) selected from the group consisting of fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, shoulder inflammation, neuritis, periuritis, myositis, hepatitis, cystitis, nephritis, sjogrens syndrome, multiple sclerosis and acute or chronic inflammatory diseases Or a pharmaceutically acceptable salt thereof. (1) extracting red ginseng alcohol extract at room temperature again with water saturated butanol to obtain crude saponin;
(2) treating the crude saponin with an acid having a pH of 1 to 4 and heating to hydrolyze the crude saponin;
(3) adsorbing the hydrolyzed substance to a column;
(4) extracting the substance adsorbed on the column with alcohol; And
(5) A method for treating a subject suffering from stroke, myocardial infarction, diabetic vascular disorder, diabetic neuropathy, diabetic neuropathy, diabetic neuropathy, diabetic neuropathy, Hyperlipidemia, diabetes, epilepsy, Parkinson's disease and dementia. delete (1) extracting red ginseng alcohol extract at room temperature again with water saturated butanol to obtain crude saponin;
(2) treating the crude saponin with an acid having a pH of 1 to 4 and heating to hydrolyze the crude saponin;
(3) adsorbing the hydrolyzed substance to a column;
(4) extracting the substance adsorbed on the column with alcohol; And
(5) A health functional food for preventing or ameliorating an inflammatory disease containing, as an active ingredient, a panesatrazyl-ginsenoside fortified product comprising ginsenoside Rh4 produced by concentrating the extracted substance under reduced pressure and purifying. (1) extracting red ginseng alcohol extract at room temperature again with water saturated butanol to obtain crude saponin;
(2) treating the crude saponin with an acid having a pH of 1 to 4 and heating to hydrolyze the crude saponin;
(3) adsorbing the hydrolyzed substance to a column;
(4) extracting the substance adsorbed on the column with alcohol; And
(5) A method for treating a subject suffering from stroke, myocardial infarction, diabetic vascular disorder, diabetic neuropathy, diabetic neuropathy, diabetic neuropathy, diabetic neuropathy, Hyperlipidemia, diabetes, epilepsy, Parkinson's disease and dementia.

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