KR20230039856A - Anti-inflammatory composition comprising complex ginsenoside composition - Google Patents

Abstract

The present invention is to provide a natural substance composition having few or no side effects and exhibiting anti-inflammatory effects. The present inventors performed an anti-inflammatory activity test on a material obtained by using a solvent to extract complex ginsenosides from red ginseng, adsorbing same to an adsorption resin, then desorbing same by means of a solvent to so as to partially purify same, and mixing same with red ginseng concentrate, and thus identified remarkable anti-inflammatory activity and used the material to prepare an anti-inflammatory composition. The composition can be used in food, external-use skin preparations, cosmetic compositions and the like.

Description

Anti-inflammatory composition comprising complex ginsenoside composition {Anti-inflammatory composition comprising complex ginsenoside composition}

The present invention relates to an anti-inflammatory composition comprising a complex ginsenoside composition extracted and processed from red ginseng.

Inflammation refers to the body's response to injury, infection, or molecules recognized by the immune system as foreign substances. Inflammation is characterized by pain, swelling, or changes in function of tissues. Inflammatory diseases are characterized by abnormal levels of activation of the immune system in a tissue or organ, which can lead to dysfunction or disease in the tissue or organ.

Inflammatory diseases are one of the leading causes of death worldwide. Inflammatory diseases affect many organs and tissues, such as blood vessels, heart, brain, nerves, joints, skin, lungs, eyes, gastrointestinal tract, kidneys, thyroid, adrenal glands, pancreas, liver and muscle. Treatment of inflammatory diseases is of interest to pharmaceutical companies and researchers. Despite many recent studies in this field, current therapies for inflammatory diseases are alleviating symptoms with non-specific drugs, reducing inflammation, and slowing disease progression, but these therapies have side effects and resistance to drugs. There is a serious problem.

Currently, non-steroidal anti-inflammatory drugs (NSAIDS) are widely used as anti-inflammatory agents. However, nonsteroidal anti-inflammatory drugs are known to have serious side effects such as renal impairment, gastrointestinal disturbance, and liver failure (Rainsford KD., Subcell biochem., 42:3-27, 2007; Guruprasad P. Aithal., Rheumatology., 7:139-150, 2011; Praveen P. N. Rao et al., Pharmaceuticals., 3:1530-1549, 2010).

Therefore, research is being actively conducted to find new drugs that exhibit anti-inflammatory activity from natural substances and have little or no side effects.

The present invention aims to provide a natural substance composition that exhibits anti-inflammatory activity with little or no side effects, and reveals that ginsenosides extracted from ginseng and mixtures containing them exhibit anti-inflammatory effects, and based on this, provide strong anti-inflammatory substances. is to do

The present inventors extracted complex ginsenosides from ginseng using a solvent, adsorbed them to an adsorbent resin, desorbed them with a solvent, partially purified them, and tested anti-inflammatory activity by mixing red ginseng concentrates. As a result, the anti-inflammatory activity was remarkable. It was confirmed that, and an anti-inflammatory composition was prepared using this. This composition can be used for preventing or treating oral inflammation such as stomatitis and gingivitis, toothpaste composition, skin external preparation, cosmetic composition, food, and the like.

the present invention

1) obtaining red ginseng concentrate by extracting and concentrating red ginseng;

2) dissolving some of the red ginseng concentrate in water, adsorbing it through an adsorption resin column, and then desorbing it with ethanol to obtain a complex ginsenoside containing ginsenosides Rb1, Rb2, Rc and Rd;

3) treating complex ginsenosides with alpha-galactosidase to obtain fermented complex ginsenosides; and

4) obtaining a complex ginsenoside composition by adding the fermented complex ginsenoside obtained in step 3) to the red ginseng concentrate obtained in step 1);

Ginsenoside Rg3 (R+S), ginsenoside F2, ginsenoside Rh2, and ginsenoside Rb1 are each 7 parts by weight or more, preferably 30 parts by weight or more, based on 100 parts by weight of ginsenoside compound K. It relates to an anti-inflammatory composition containing, as an active ingredient, a complex ginsenoside composition containing 10 parts by weight or more and 30 parts by weight or less, more preferably 30 parts by weight or less.

In addition, in the present invention, the composite ginsenoside composition contains ginsenoside compound K of 100 mg/g or more, ginsenoside Rg3 (R+S), ginsenoside F2, ginsenoside Rh2, and ginsenoside Rb1 of 8 mg/g, respectively. g or more, preferably 8 mg/g or more and 30 mg/g or less, more preferably 10 mg/g or more and 30 mg/g or less.

The adsorption resin column is not particularly limited, but is preferably a porous adsorption resin column, more preferably at least one of HP2MG and HP-20.

In addition, the present invention relates to an anti-inflammatory composition characterized in that the red ginseng concentrate in steps 1), 2) and 4) is in powder form or liquid form.

In addition, the present invention relates to a composition for external application for skin for treating skin inflammation comprising the anti-inflammatory composition.

In addition, the present invention relates to a cosmetic composition comprising the anti-inflammatory composition.

In addition, the present invention relates to a pharmaceutical composition for preventing or treating inflammation comprising the anti-inflammatory composition. In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group consisting of oral formulations, external preparations, suppositories, sterile injection solutions, and sprays.

In addition, the present invention relates to a toothpaste composition for preventing or improving oral inflammation comprising the anti-inflammatory composition.

In addition, the present invention relates to a mouthwash composition for preventing or improving oral inflammation comprising the anti-inflammatory composition.

In addition, the present invention relates to a food composition for preventing or improving inflammation comprising the anti-inflammatory composition.

The term "food" used in the present invention means a natural product or processed product containing one or more nutrients, and preferably means a product that can be eaten directly through a certain degree of processing process. As a meaning, it may include all foods, food additives, functional foods, health functional foods, health supplement foods, and beverages. The food composition of the present invention may be prepared in the form of various beverages, chewing gum, tea, sweets, vitamin complexes, health supplements, and the like, without limitation. The preferred intake amount of the food composition of the present invention varies depending on the condition, body weight, severity of symptoms, food type, and intake period of the person who consumes it, and may be appropriately selected. In the food composition of the present invention, it is preferable for optimal effect that the active ingredient be ingested at 0.2 mg/kg to 200 mg/kg per day.

The anti-inflammatory composition containing the complex ginsenoside composition as an active ingredient can be formulated with a carrier commonly accepted in the pharmaceutical field and formulated into an injection form, oral administration, or coating form by conventional methods. For example, it can be formulated into a liquid, syrup, capsule, granule, powder, ointment, emulsion, gel, cream, etc., and can be administered through various routes. Among the various dosage forms, for example, injectable compositions, preferably isotonic aqueous solutions or suspensions, the mentioned compositions are sterile and/or contain additives such as preservatives, stabilizers, wetting agents or emulsifiers, solution accelerators, salts and/or buffers for regulating osmotic pressure. ) contains In addition, they may contain other therapeutically useful substances. Pharmaceutically acceptable carriers include lactose, glucose, sucrose, sorbitol, mannitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, These include methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The composition may further include lubricants, wetting agents, flavoring agents, emulsifying agents and preservatives. The composition of the present invention may be formulated into oral formulations such as granules, powders, solutions, tablets, capsules, or dry syrups, or parenteral formulations such as injections, but is not limited to these formulations.

The pharmaceutical preparation prepared as described above may be administered by subcutaneous administration, intramuscular administration or oral administration, intravenous administration, skin application, oral application, gum application, etc., depending on the purpose, and the daily dosage is 0.001 μg / kg ~ 100 The amount of mg/kg may be administered in one to several divided doses. The dosage level for a specific patient may vary depending on the patient's weight, age, sex, health condition, administration time, administration method, severity of disease, and the like.

In addition, the anti-inflammatory composition containing the complex ginsenoside composition as an active ingredient is characterized in that it is an external preparation for skin inflammation treatment. A coating agent containing the anti-inflammatory composition of the present invention as an active ingredient can be easily prepared in any form according to a conventional manufacturing method. As an example, in preparing a cream-type coating agent, the anti-inflammatory composition of the present invention is contained in a general oil-in-water (O/W) or water-in-oil (W/O) cream base, and there are fragrances, chelating agents, pigments, antioxidants, While preservatives are used as needed, synthetic or natural materials such as proteins, minerals, and vitamins may be used in combination for the purpose of improving physical properties.

In addition, the present invention relates to a cosmetic composition containing an anti-inflammatory composition containing the composite ginsenoside composition as an active ingredient as a main component. It can be formulated into lotion, gel, water-soluble powder, fat-soluble powder, water-soluble liquid, cream, essence, etc. by adding a pH adjusting agent, fragrance, emulsifier, preservative, etc. as needed.

In addition, the present invention relates to a toothpaste composition for preventing or improving oral inflammation containing the complex ginsenoside composition as an active ingredient. Mastic oil, propolis extract, sodium pyrophosphate, sodium fluoride, tocopherol acetate, hydroxyapatite, sweetener, thickener, abrasive, humectant, or surfactant may be added as necessary to formulate a toothpaste composition in a conventional method. can

In addition, the present invention relates to an oral composition for preventing or improving oral inflammation containing the complex ginsenoside composition as an active ingredient. A solubilizer, a wetting agent, a surfactant, a lubricant, a flavoring agent, a sweetener, a preservative or a medicinal agent may be added as necessary and formulated according to a conventional oral composition manufacturing method, and gum, mouthwash, mouthwash, It may be prepared as a formulation selected from the group consisting of an oral spray, an oral ointment, an oral patch, or a combination thereof.

The composite ginsenoside composition of the present invention exhibited inhibition of NO production, inhibition of pro-inflammatory cytokine expression, and excellent anti-inflammatory activity.

Therefore, the complex ginsenoside composition of the present invention can be applied to an anti-inflammatory pharmaceutical composition, a toothpaste composition for preventing or treating oral inflammation, an oral composition including a mouthwash, an external skin composition for treating skin inflammation, a cosmetic composition, a food composition, and the like. .

Figure 1 shows the results of NO production after 48 hours by inducing inflammation with LPS after treating BTEX-K samples by concentration (**p<0.01, ***p<0.001 compared to the control group).
Figure 2 shows the results of pro-inflammatory cytokine production after 48 hours by inducing inflammation with LPS after treating BTEX-K samples by concentration (*p<0.05, **p<0.01 compared to the control group).

Hereinafter, a specific method of the present invention will be described in more detail with reference to examples. However, it is apparent to those skilled in the art that the scope of the present invention is not limited only to the description of these embodiments.

Example 1: Preparation of complex ginsenosides

Domestic dried red ginseng (4-year-old) was purchased and used as an experimental raw material (Geumsan, Chungcheongnam-do, Deokwon ginseng). Complex ginsenosides were extracted from dried red ginseng. 10 kg of dried red ginseng was repeatedly extracted and concentrated with 50% ethanol at 60 ° C for 12 hours 3 times to obtain red ginseng concentrate.

A total of 5.6kg (63brix) of red ginseng concentrate was dissolved, of which 5.5kg was dissolved in water and passed through a column filled with synthetic adsorption resin (HP2MG, Misvisi Chemical or HP-20, Diaion) to be adsorbed and then desorbed with 95% ethanol. Thus, 460 g of complex ginsenosides including ginsenosides Rb1, Rb2, Rc and Rd were obtained.

Example 2: Preparation of red ginseng concentrate powder

100 g of 5.6 kg of red ginseng concentrate prepared in Example 1 was separately taken and concentrated under reduced pressure to obtain 71 g of red ginseng concentrate powder.

Example 3: Preparation of fermentation complex ginsenoside

Using 100 g of the complex ginsenoside obtained in Example 1 as a substrate, alpha-galactosidase (0.75 g) derived from the genus Aspergillus in 50 mM sodium acetate buffer (pH 4.5) and 84 hours in a constant temperature water bath at 60 ° C. Through the above reaction, a fermentation complex ginsenoside (41 g) was prepared.

The fermented complex ginsenoside prepared in this way includes ginsenoside compound K, ginsenoside F2, ginsenoside Rh2, and the like.

Example 4: Preparation of a composite ginsenoside composition (hereinafter mixed with "BTEX-K")

A complex ginsenoside composition BTEX-K was prepared by mixing 40 g of the red ginseng concentrate powder prepared in Example 2 with 100 g of the fermented complex ginsenoside prepared in Example 3.

The composite ginsenoside composition BTEX-K contains various ginsenosides such as ginsenoside F2 and ginsenoside Rh2 in addition to ginsenoside compound K = 100 mg/g.

Content of major ginsenosides in BTEX-K (more than 10mg/g) ginsenoside Rb1 F2 Rg3 (R+S) Rh2 Compound K Content (mg/g) 11.5 11.5 25.5 15.4 100

Experimental Example 1: Cell viability analysis (MTT assay)

1) Cells (Raw264.7) were seeded in 8×10 ³ per well in a 96-well plate, and cultured in a CO ₂ incubator for 24 hours.

2) The medium was removed, replaced with 100 ul of a new medium in which sample extracts using ethanol were added according to concentration, and cultured for 24 hours.

number sample density One Ginsenoside Compound K 1, 5, 10, 20 μg/ml 2 BTEX-K 5, 10, 20, 30, 40, 50, 100 μg/ml

3) MTT solution was added and reacted for 4 hours.

4) Absorbance was measured at a wavelength of 450 nm (420 ~ 480 nm) using a spectrophotometer (reference is 600 nm).

Experimental Example 2: Measurement of NO production

1) Cells (Raw 264.7) were suspended in DMEM medium containing 10% fetal calf serum.

2) After seeding the suspended cells in a well plate at 8×10 ³ per well, they were cultured in a CO ₂ incubator for 24 hours.

3) After 24 hours of culture, the attached cells were washed twice with PBS, then a new medium was added, and samples diluted to the concentrations shown in the table below were pretreated for 24 hours, and then 100 ng/ml LPS ( Escherichia coli 0111; B4 ) was processed. After that, it was cultured for 48 hours.

number sample density One Ginsenoside Compound K 1, 5, 10 μg/mL 2 BTEX-K 5, 10, 20 μg/mL

4) After 48 hours, the supernatant was centrifuged and the absorbance at 540 nm was measured using a Griess reagent (0.2% N-(1-naphthyl group) ethylenediamine solution: 0.2% sulfanilamide solution = 1:1). was measured.

5) A calibration curve was prepared using sodium nitrite solution, and the concentration of nitrite was calculated through absorbance.

6) Activity was evaluated by comparing the group treated with LPS only.

Experimental Example 3: Cytokine measurement (ELISA)

1) Cells (Raw 264.7) were suspended in DMEM medium containing 10% fetal calf serum.

2) The suspended cells were seeded in 8×10 ³ per well in a 96-well plate and then cultured in a CO ₂ incubator for 24 hours.

3) After 24 hours, the attached cells were washed twice with PBS, and the search sample diluted to the concentration shown in Table 3 in a new medium was pretreated for 24 hours, and then treated with 100 ng/ml LPS ( Escherichia coli 0111; B4 ) and incubated for 48 hours thereafter.

number sample density One Ginsenoside Compound K 1, 5, 10 μg/mL 2 BTEX-K 5, 10, 20 μg/mL

4) After 48 hours, the supernatant was centrifuged and IL-1β and IL-6 were quantified using a mouse ELISA kit (R&D Systemes, Inc., USA).

Result 1: cell viability

Since the solvent in which the sample is dissolved is ethanol, the cell viability of Raw 264.7 cells against ethanol was investigated. Ethanol was added to the Raw 264.7 cell culture solution by concentration, followed by culturing for 24 hours, and as a result of MTT analysis, it was confirmed that at an ethanol concentration of 2% or more, more than 40% of the Raw264.7 cells were killed. Therefore, the ethanol concentration of the sample solvent was fixed to 1% or less.

In addition, the cell viability of Raw 264.7 for each concentration of ginsenoside compound K, which is a positive control group, was confirmed. It was confirmed that the viability of Raw 264.7 cells decreased from the concentration of ginsenoside compound K of 20 ug/ml. Therefore, the following experiments were performed by fixing the sample concentrations of ginsenoside compound K to 1, 5, and 10 ug/ml.

In addition, sample BTEX-K was administered according to concentration to confirm the cell viability of Raw 264. It was confirmed that the viability of Raw 264.7 cells decreased from the BTEX-K concentration of 30 ug/ml. Therefore, the following experiments were performed by fixing the BTEX-K sample concentration to 5, 10 and 20 ug/ml.

Raw 264.7 cells were treated with LPS, an inflammation-inducing reagent, at each concentration, and cell viability and NO production were tested. Cell viability increased at LPS concentrations of 100 ng/ml and 200 ng/ml, and NO production increased only at 100 ng/ml LPS concentration. Therefore, the experiment was conducted by confirming the concentration of LPS as 100 ng/ml.

Result 2: Production of Nitric Oxide

As shown in Figure 1, Raw 264.7 cells were treated with BTEX-K at different concentrations, cultured for 24 hours, and then 100 ng/ml LPS was added to confirm the NO concentration after 48 hours. Significance at the concentration of 20 ug/ml BTEX-K A decrease was confirmed (**p<0.01).

Result 3: Measurement of pro-inflammatory cytokines

Raw 264.7 cells were treated with BTEX-K at different concentrations, cultured for 24 hours, and 100 ng/ml of LPS was added to confirm cytokine production after 48 hours. A significant decrease in -1β was confirmed respectively (*p<0.05, **p<0.01).

Before the anti-inflammatory experiment, since both samples of ginsenoside compound K and BTEX-K were dissolved in ethanol, the cell viability of Raw 264.7 cells used in the anti-inflammatory experiment against ethanol was first confirmed. Up to about 1% ethanol had little effect on cell viability, but at higher concentrations it was confirmed that the viability of Raw 264.7 cells was affected. Therefore, it was found that it was not appropriate for the concentration of ethanol to exceed 1%.

In addition, the viability of Raw 264.7 cells was confirmed according to the concentrations of ginsenoside compound K and BTEX-K, and appropriate concentrations to be used in the experiment were determined. As a result, it was confirmed that ginsenoside compound K decreased the viability of Raw 264.7 cells at a concentration of 20 ug/ml, and BTEX-K decreased viability of Raw 264.7 cells at a concentration of 30 ug/ml. Therefore, the appropriate concentrations of the anti-inflammatory test to be performed thereafter were fixed at 1, 5, and 10 ug/ml for ginsenoside compound K and 5, 10, and 20 ug/ml for BTEX-K.

In addition, as a result of confirming the optimal concentration of LPS for inflammation by NO production, NO production was optimal at a concentration of 100 ng/ml of LPS. Therefore, as a result of treating Raw 264.7 cells with ginsenoside compound K and BTEX-K at the concentrations determined above and then treating them with 100 ng/ml of LPS to confirm NO production, significance at the concentration of BTEX-K 20 ug/ml A decrease in NO production was confirmed.

In addition, in the cytokine production experiment, it was confirmed that the cytokines of IL-6 and IL-1β were significantly reduced when BTEX-K was treated with 20 ug/ml. However, in ginsenoside compound K, NO and cytokine production tended to decrease and increase, respectively, and there was no significance.

Claims (9)

1) obtaining red ginseng concentrate by extracting and concentrating red ginseng;
2) dissolving red ginseng concentrate with water, adsorbing it through a synthetic adsorption resin column, and then desorbing it with ethanol to obtain complex ginsenosides including ginsenosides Rb1, Rb2, Rc and Rd;
3) treating complex ginsenosides with alpha-galactosidase to obtain fermented complex ginsenosides; and
4) obtaining a complex ginsenoside composition by adding the fermented complex ginsenoside obtained in step 3) to the red ginseng concentrate obtained in step 1);
A composite ginsenoside composition containing 7 parts by weight or more of ginsenoside Rg3 (R+S), ginsenoside F2, ginsenoside Rh2, and ginsenoside Rb1, respectively, based on 100 parts by weight of ginsenoside compound K, as an active ingredient Anti-inflammatory composition containing as.
The method of claim 1,
The composite ginsenoside composition contains 100 mg/g or more of ginsenoside compound K, ginsenoside Rg3 (R+S), ginsenoside F2, ginsenoside Rh2, and ginsenoside Rb1 in an amount of 8 mg/g or more, respectively. Characterized by an anti-inflammatory composition.
The method of claim 1,
The anti-inflammatory composition, characterized in that the red ginseng concentrate in steps 1), 2) and 4) is in powder form or liquid form.
A composition for external application for skin for treating skin inflammation comprising the anti-inflammatory composition of claim 1 or 2.
A cosmetic composition comprising the anti-inflammatory composition of claim 1 or claim 2.
A pharmaceutical composition comprising the anti-inflammatory composition of claim 1 or claim 2.
Toothpaste composition for preventing or improving oral inflammation comprising the anti-inflammatory composition of claim 1 or claim 2.
A mouthwash composition for preventing or improving oral inflammation comprising the anti-inflammatory composition of claim 1 or 2.
A food composition comprising the anti-inflammatory composition of claim 1 or claim 2.

Images