KR102751859B1 - Anti-inflammatory composition comprising complex ginsenoside composition - Google Patents

Abstract

Task: To provide a composition of natural substances exhibiting anti-inflammatory activity with little or no side effects.
Solution: The inventors of the present invention extracted complex ginsenosides from red ginseng using a solvent, adsorbed them onto an adsorption resin, desorbed them with a solvent, partially purified them, mixed them with a red ginseng concentrate, and tested their anti-inflammatory activity. As a result, they confirmed that the anti-inflammatory activity was remarkable, and prepared an anti-inflammatory composition using this. This composition can be used in foods, external skin preparations, cosmetic compositions, etc.

Description

{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 is the body's response to damage, infection, or molecules recognized as foreign by the immune system. Inflammation is characterized by pain, swelling, or changes in the function of tissues. Inflammatory diseases are characterized by abnormal levels of immune system activation in tissues or organs, 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 muscles. Treatment of inflammatory diseases is a subject of interest for pharmaceutical companies and researchers. Despite much recent research in this area, current treatments for inflammatory diseases are nonspecific drugs that relieve symptoms, reduce inflammation, and slow the progression of the disease, but these treatments have serious problems such as drug side effects and drug resistance.

Currently, non-steroidal anti-inflammatory drugs (NSAIDS) are widely used as anti-inflammatory agents. However, NSAIDs are known to cause serious side effects such as renal dysfunction, gastrointestinal dysfunction, and liver dysfunction (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, active research is being conducted to find new drugs from natural substances that exhibit anti-inflammatory activity and have few or no side effects.

The present invention aims to provide a natural substance composition having little or no side effects and exhibiting an anti-inflammatory activity, and it is intended to provide a powerful anti-inflammatory substance based on the discovery that ginsenosides extracted from ginseng and a mixture containing the same exhibit anti-inflammatory activity.

The present inventors extracted complex ginsenosides from ginseng using a solvent, adsorbed them onto an adsorption resin, desorbed them with a solvent, partially purified them, mixed them with red ginseng concentrate, and tested their anti-inflammatory activity. As a result, they confirmed that the composition had significant anti-inflammatory activity, and prepared an anti-inflammatory composition using this. This composition can be used for preventing or treating oral inflammation such as stomatitis and gingivitis, toothpaste compositions, external skin preparations, cosmetic compositions, foods, etc.

The present invention

1) A step of extracting and concentrating red ginseng to obtain red ginseng concentrate;

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

3) a step of treating the complex ginsenoside with alpha-galactosidase to obtain a fermented complex ginsenoside; and

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

The present invention relates to an anti-inflammatory composition comprising a complex ginsenoside composition as an effective ingredient, wherein ginsenoside Rg3 (R+S), ginsenoside F2, ginsenoside Rh2 and ginsenoside Rb1 are each contained in an amount of 7 parts by weight or more, preferably 7 parts by weight or more and 30 parts by weight or less, more preferably 10 parts by weight or more and 30 parts by weight or less, per 100 parts by weight of ginsenoside compound K.

In addition, the present invention is characterized in that 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 or more, preferably 8 mg/g or more and 30 mg/g or less, and more preferably 10 mg/g or more and 30 mg/g or less.

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

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

In addition, the present invention relates to a composition for external application for skin treatment of 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 in one or more dosage forms selected from the group consisting of oral dosage forms, topical preparations, suppositories, sterile injectable 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 an oral hygiene 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 has gone through a certain degree of processing to become directly edible, and in its conventional sense may include all of foods, food additives, functional foods, health functional foods, health supplements, and beverages. The food composition of the present invention can be manufactured in the form of, but not limited to, various beverages, gum, tea, confectionery, vitamin complexes, health supplements, etc. The preferred intake amount of the food composition of the present invention varies depending on the condition, weight, degree of symptoms, food type, and intake period of the person consuming it, and may be appropriately selected. For optimal effect, it is preferred that the food composition of the present invention be ingested at 0.2 mg/kg to 200 mg/kg of the active ingredient per day.

The anti-inflammatory composition containing the above complex ginsenoside composition as an active ingredient can be formulated into an injection form, oral administration form, topical application form, etc. by a conventional method by mixing it with a carrier conventionally accepted in the pharmaceutical field. For example, it can be formulated into a liquid, syrup, capsule, granule, powder, ointment, emulsion, gel, cream, etc., and can be administered via various routes. Among various formulations, for example, an injectable composition is preferably an isotonic aqueous solution or suspension, and the aforementioned composition is sterilized and/or contains auxiliary agents (e.g., preservatives, stabilizers, wetting agents or emulsifiers, solution accelerators, salts for osmotic pressure control, and/or buffers). In addition, they can contain other therapeutically useful substances. Pharmaceutically acceptable carriers include lactose, glucose, sucrose, sorbitol, mannitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. The composition may further contain a lubricant, a wetting agent, a flavoring agent, an emulsifier, and a preservative. The composition of the present invention may be formulated into oral formulations such as granules, powders, liquids, tablets, capsules, or dry syrups, or parenteral formulations such as injections, but is not limited to such formulations.

The pharmaceutical preparation manufactured in this way can be administered subcutaneously, intramuscularly, orally, intravenously, on the skin, orally, or on the gums, depending on the intended purpose, and the dosage can be administered in one to several divided doses of 0.001 μg/kg to 100 mg/kg per day. The dosage level for a specific patient can vary depending on the patient's weight, age, sex, health condition, administration time, administration method, and severity of the disease.

In addition, the anti-inflammatory composition containing the above-mentioned complex ginsenoside composition as an effective ingredient is characterized as an external skin preparation for treating skin inflammation. An application agent containing the anti-inflammatory composition of the present invention as an effective ingredient can be easily manufactured in any form according to a conventional manufacturing method. For example, in manufacturing a cream-type application 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 a fragrance, a chelating agent, a pigment, an antioxidant, a preservative, etc. are used as needed, while synthetic or natural materials such as proteins, minerals, and vitamins can be used together for the purpose of improving physical properties.

In addition, the present invention relates to a cosmetic composition having as its main ingredient an anti-inflammatory composition containing the above complex ginsenoside composition as an effective ingredient. A pH regulator, a fragrance, an emulsifier, a preservative, etc. may be added as needed, and the composition may be formulated into a toner, gel, water-soluble powder, fat-soluble powder, water-soluble liquid, cream, or essence, etc., using a conventional cosmetic manufacturing method.

In addition, the present invention relates to a toothpaste composition for preventing or improving oral inflammation, which contains the above-mentioned complex ginsenoside composition as an effective ingredient. Mastic oil, propolis extract, sodium pyrophosphate, sodium fluoride, tocopherol acetate, hydroxyapatite, a sweetener, a thickener, an abrasive, a humectant, or a surfactant may be added as needed, and the toothpaste composition may be formulated using a conventional toothpaste composition manufacturing method.

In addition, the present invention relates to an oral composition for preventing or improving oral inflammation, containing the above complex ginsenoside composition as an effective ingredient. The composition can be formulated according to a conventional oral composition manufacturing method by adding a solubilizer, a wetting agent, a surfactant, a lubricant, a flavoring agent, a sweetener, a preservative, or a medicinal agent, as needed, and can be manufactured in a dosage form selected from the group consisting of a gum, a mouthwash, a mouthwash, an oral spray, an oral ointment, an oral patch, or a combination thereof.

The complex 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, etc.

Figure 1 shows the results of NO production 48 hours after BTEX-K samples were treated at various concentrations and inflammation was induced with LPS (**p<0.01, ***p<0.001 compared to the control group).
Figure 2 shows the results of pro-inflammatory cytokine production 48 hours after BTEX-K samples were treated at different concentrations and then induced with LPS to induce inflammation (*p<0.05, **p<0.01 compared to the control group).

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

Example 1: Preparation of complex ginsenosides

Domestically produced dried red ginseng (4 years old) was purchased and used as experimental raw material (Geumsan, Chungnam, Deokwon ginseng). Complex ginsenosides were extracted from dried red ginseng. 10 kg of dried red ginseng was extracted and concentrated three times for 12 hours at 60℃ with 50% ethanol to obtain red ginseng concentrate.

The red ginseng concentrate was 5.6 kg in total (63 brix), of which 5.5 kg was dissolved in water and passed through a column filled with synthetic adsorption resin (HP2MG, Mitsubishi Chemical or HP-20, Diaion) for adsorption, followed by desorption with 95% ethanol to obtain 460 g of complex ginsenosides including ginsenosides Rb1, Rb2, Rc, and Rd.

Example 2: Preparation of red ginseng concentrate powder

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

Example 3: Production of fermented complex ginsenosides

Using 100 g of the complex ginsenoside obtained in Example 1 as a substrate, a fermented complex ginsenoside (41 g) was produced by reacting with Aspergillus spp.-derived alpha-galactosidase (0.75 g) in a 50 mM sodium acetate buffer solution (pH 4.5) in a constant temperature water bath at 60°C for more than 84 hours.

The fermented complex ginsenoside manufactured in this way contains ginsenoside compound K, ginsenoside F2, and ginsenoside Rh2.

Example 4: Preparation of complex ginsenoside composition (hereinafter mixed with “BTEX-K”)

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

The complex ginsenoside composition BTEX-K contains various ginsenosides, including ginsenoside compound K = 100 mg/g, ginsenoside F2, and ginsenoside Rh2.

Major ginsenoside content in BTEX-K (10mg/g or more) 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 at 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 μl of new medium containing the sample extract using ethanol at various concentrations, and cultured for 24 hours.

number Sample density 1 Ginsenoside compound K 1, 5, 10, 20 ㎍/㎖ 2 BTEX-K 5, 10, 20, 30, 40, 50, 100 ㎍/㎖

3) After adding MTT solution, reaction was performed for 4 hours.

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

Experimental Example 2: Measurement of NO production

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

2) The suspended cells were seeded at 8× ¹⁰³ per well in a well plate and cultured in a _CO2 incubator for 24 hours.

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

number Sample density 1 Ginsenoside compound K 1, 5, 10 ㎍/㎖ 2 BTEX-K 5, 10, 20 ㎍/㎖

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

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

6) Activity was evaluated by comparing it with 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 bovine serum.

2) The suspended cells were seeded at 8× ¹⁰³ per well in a 96-well plate and cultured in a _CO2 incubator for 24 hours.

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

number Sample density 1 Ginsenoside compound K 1, 5, 10 ㎍/㎖ 2 BTEX-K 5, 10, 20 ㎍/㎖

4) After 48 hours, centrifugation was performed, and the supernatant was collected 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 was dissolved was ethanol, the cell viability of Raw 264.7 cells in response to ethanol was investigated. After adding ethanol at various concentrations to the Raw 264.7 cell culture and culturing for 24 hours, the results of the MTT assay confirmed that more than 40% of Raw 264.7 cells died at ethanol concentrations higher than 2%. Therefore, the ethanol concentration of the sample solvent was fixed at 1% or less.

In addition, the cell viability of Raw 264.7 was confirmed for various concentrations of ginsenoside compound K as a positive control. It was confirmed that the viability of Raw 264.7 cells decreased starting from a ginsenoside compound K concentration of 20 ug/ml. Therefore, the following experiments were conducted by fixing the concentration of ginsenoside compound K samples to 1, 5, and 10 ug/ml.

In addition, the cell viability of Raw 264.7 was confirmed by administering sample BTEX-K at various concentrations. It was confirmed that the viability of Raw 264.7 cells decreased from a BTEX-K concentration of 30 ug/ml. Therefore, the following experiments were conducted by fixing the BTEX-K sample concentrations at 5, 10, and 20 ug/ml.

Raw 264.7 cells were treated with LPS, an inflammatory agent, at various concentrations, 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 LPS concentration of 100 ng/ml. Therefore, the experiment was conducted by setting the concentration of LPS to 100 ng/ml.

Result 2: Nitric oxide production

As shown in Fig. 1, Raw 264.7 cells were treated with BTEX-K at various concentrations, cultured for 24 hours, and then 100 ng/ml of LPS was added. The NO concentration was checked 48 hours later, and a significant decrease was confirmed at a concentration of 20 ug/ml of BTEX-K (**p<0.01).

Result 3: Measurement of pro-inflammatory cytokines

Raw 264.7 cells were treated with various concentrations of BTEX-K, cultured for 24 hours, and then 100 ng/ml of LPS was added. Cytokine production was checked 48 hours later. A significant decrease in IL-6 and IL-1β was confirmed at a concentration of 20 ug/ml of BTEX-K (*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 was first confirmed in response to ethanol. It was confirmed that up to about 1% ethanol had little effect on cell viability, but at higher concentrations, it affected the viability of Raw 264.7 cells. Therefore, it was found that the concentration of ethanol exceeding 1% was not appropriate.

In addition, the viability of Raw 264.7 cells was confirmed according to the concentration of ginsenoside compound K and BTEX-K, and the appropriate concentration to be used in the experiment was 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 the viability of Raw 264.7 cells starting from a concentration of 30 ug/ml. Therefore, the appropriate concentrations for the subsequent anti-inflammatory experiments 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, when the optimal concentration of LPS to induce inflammation was confirmed by NO production, NO production was optimal at a concentration of LPS 100 ng/ml. Therefore, when Raw 264.7 cells were treated with two samples, ginsenoside compound K and BTEX-K, at the concentrations determined above, and then treated with LPS 100 ng/ml to confirm NO production, a significant decrease in NO production was confirmed at a concentration of BTEX-K 20 ug/ml.

In addition, in the cytokine production experiment, a significant decrease in the cytokines IL-6 and IL-1β was confirmed when BTEX-K 20 ug/ml was treated. However, in the case of ginsenoside compound K, the production of NO and cytokines showed a tendency to decrease and increase, respectively, but there was no significance.

Claims (10)

1) A step of extracting and concentrating red ginseng to obtain red ginseng concentrate;
2) A step of dissolving a portion of the red ginseng concentrate in water, passing it through a synthetic adsorption resin column for adsorption, and then desorbing it with ethanol to obtain a complex ginsenoside including ginsenosides Rb1, Rb2, Rc, and Rd;
3) a step of treating the complex ginsenoside with alpha-galactosidase to obtain a fermented complex ginsenoside; and
4) A step of obtaining a complex ginsenoside composition by adding the fermented complex ginsenoside obtained in step 3) to a portion of the red ginseng concentrate obtained in step 1) above;
An anti-inflammatory composition comprising a complex ginsenoside composition as an effective ingredient, wherein the complex ginsenoside composition comprises 7 to 30 parts by weight of ginsenoside Rg3 (R+S), 7 to 30 parts by weight of ginsenoside F2, 7 to 30 parts by weight of ginsenoside Rh2, and 7 to 30 parts by weight of ginsenoside Rb1 per 100 parts by weight of ginsenoside compound K.
delete delete In claim 1,
An anti-inflammatory composition characterized in that the red ginseng concentrate of steps 1), 2) and 4) is in a powder or liquid state.
A cosmetic composition comprising the anti-inflammatory composition of claim 1.
A pharmaceutical composition for preventing or treating an inflammatory disease, comprising the anti-inflammatory composition of claim 1.
A toothpaste composition for preventing or improving oral inflammation, comprising the anti-inflammatory composition of claim 1.
An oral hygiene composition for preventing or improving oral inflammation, comprising the anti-inflammatory composition of claim 1.
A food composition comprising the anti-inflammatory composition of claim 1.
A composition for external application to the skin for treating skin inflammation, comprising the anti-inflammatory composition of claim 1.