KR102007198B1 - Composition for antiinflammation and antioxidation comprising medicinal herb extract - Google Patents

Abstract

The present invention relates to the inhibition of inflammation and oxidation using a medical herb extract which contains red ginseng, Angelica gigas, Astragalus propinquus, Cnidium officinale, and Paeonia lactiflora. The present invention can provide various compositions excellent in anti-inflammatory and antioxidant effects through a special combination of medicinal stuffs by using medical herbs in which red ginseng is mixed with Angelica gigas, Astragalus propinquus, Cnidium officinale, and Paeonia lactiflora.

Description

Composition for Antiinflammation and Anti-Inflammation comprising Medicinal Herb Extracts

The present invention relates to herbal extracts that can effectively inhibit and regulate oxidation and inflammation occurring in vivo.

The occurrence of oxidation and inflammation is related to various diseases and traumas such as general tissue damage, aging, cancer and neurological diseases, so researches to effectively control them are active. Inflammation is an immune action to repair damage to major tissues or cells in vivo, and natural defense substrates or excessive or non-ideal inflammatory reactions that occur to protect the living body can cause unexpected damage to the living body. In vivo oxidation, which is mainly caused by free radicals, is related to the development of an inflammatory response, and promotes aging as well as inflammation, and may also cause various metabolic and neurological diseases including diabetes.

There are a wide variety of known pharmaceutical substances for the control and inhibition of oxidation and inflammation. Among the typical herbal ingredients, red ginseng and ginseng are known to have an inhibitory effect on inflammation and oxidation, and are widely used in health functional foods and beauty products. For example, Korean Unexamined Patent Publication No. 10-2015-0019337 discloses an antioxidant and anti-inflammatory composition utilizing ginseng or red ginseng.

Accordingly, the present inventors have conducted research using red ginseng and various other herbal ingredients, and have developed a composition that shows a remarkably superior effect to the composition having the control and inhibitory effect of oxidation and inflammation using red ginseng.

Korean Patent Publication No. 10-2015-0019337

The present invention provides an herbal extract and a method for producing the same having an excellent effect on the control and inhibition of oxidation and inflammation.

The present invention provides an anti-inflammatory pharmaceutical composition comprising the extract of the herbal medicine including red ginseng, Angelica, Astragalus, Cheongung and Peony as an active ingredient.

The present invention provides an anti-inflammatory health functional food comprising the extract of the herbal medicine including red ginseng, Angelica, Astragalus, Cheongung and Peony as an active ingredient.

The present invention provides an anti-inflammatory cosmetic composition comprising an extract of the herbal medicine including red ginseng, Angelica, Astragalus, Cheongung and Peony as an active ingredient.

The present invention provides an anti-inflammatory quasi-drug composition comprising the extract of the herbal medicine containing red ginseng, Angelica, Astragalus, Cheongung and Peony as an active ingredient.

The present invention provides an antioxidant health functional food comprising the extract of the herbal medicine including red ginseng, Angelica, Astragalus, Cheongung and Peony as an active ingredient.

The present invention provides an antioxidant cosmetic composition comprising the extract of the herbal medicine including red ginseng, Angelica, Astragalus, Cheongung and Peony as an active ingredient.

The present invention provides a quasi-drug composition for antioxidant comprising the extract of the herbal medicine including red ginseng, Angelica, Astragalus, Cheongung and Peony as an active ingredient.

The present invention can provide a variety of compositions remarkably excellent in the control and suppression of antioxidant and anti-inflammatory by using red ginseng herb, Astragalus, Cheongung and Peony.

Figure 1 shows the DPPH radical scavenging activity (DPPH free radical scavenging activity) according to the concentration of the red ginseng extract and the herbal medicine extract of the present invention.
Figure 2 shows the total antioxidant capacity (Total Antioxidant Capacity) of the red ginseng extract and the herbal medicine extract of the present invention.
Figure 3 shows the total flavonoid content (Total flavonoid content) according to the concentration of the red ginseng extract and the herbal medicine extract of the present invention.
Figure 4 shows the hydrogen peroxide scavenging activity (Hydrogen peroxide scavenging activity) according to the concentration of the red ginseng extract and the herbal medicine extract of the present invention.
Figure 5 shows the degree of inhibition of nitric oxide (NO) production by concentration of the red ginseng extract and the herbal medicine extract of the present invention.
Figure 6 shows the degree of inhibition of reactive oxygen species (ROS) production by the concentration of red ginseng extract and the herbal medicine extract of the present invention.
Figure 7 shows the degree of inhibition of TNF-α production by concentration of the red ginseng extract and the herbal medicine extract of the present invention.
Figure 8 shows the degree of inhibition of IL-6 production by the concentration of the red ginseng extract and the herbal medicine extract of the present invention.

Hereinafter, the present invention will be described in detail. Unless otherwise defined, terms used herein should be interpreted as those generally understood by those skilled in the art. The drawings and embodiments of the present specification are intended to easily understand and practice the present invention by those skilled in the art that may obscure the gist of the invention in the drawings and embodiments, the present invention is limited to the drawings and embodiments It doesn't happen.

The present invention relates to various compositions using herbal extracts having excellent effects on anti-inflammation and anti-oxidation, and products using the same.

Herbal medicine in the present invention means a drug (藥劑) is a combination of two or more medicines that can be used in Oriental medicine.

Herbal extract of the present invention is red ginseng ( Panax ginseng, red ginseng ), Angelica gigas , Astragalus membranaceus ), Cnidium officinale MAKINO) and Peony (Paeonia It is an extract of herbal medicine containing lactiflora ).

In the present invention, red ginseng is steamed and dried after steaming using steam, etc., and can be manufactured and used directly according to a general method known in the art, and those sold can be used.

Various medicines used as herbal medicines have different pharmacological effects on different medicines, and most of them produce beneficial effects in vivo. However, when combining two or more medicines may not occur all of the pharmacological effects of each single medicine, rather the effects of each medicine may be inhibited by the combination of medicines. This phenomenon may be due to the interaction between the components exhibiting pharmacological activity contained in the medicinal herbs, or the result of the opposite pharmacological mechanisms in vivo. In other words, the types of medicinal herbs used in the manufacture of herbal medicines and their combination methods are very diverse, and simply combining several medicinal herbs results in all of the pharmacological components of each medicinal herb used in the combination or the effects cannot be enhanced. There are many.

The present invention utilizes the anti-inflammatory and antioxidant effects of a special combination of red ginseng, Angelica, Astragalus, Cheongung and Peony among various medicines that can be used as a herbal medicine, each of which is a single component of Red Ginseng, Angelica, Astragalus, Astragalus or Peony It is remarkably superior to the effect by some combination of the. In particular, according to an embodiment of the present invention, the herbal extract of the present invention may have a remarkably superior effect than the anti-inflammatory and antioxidant effects of the red ginseng single extract. According to an embodiment of the present invention, the herbal extract of the present invention has a flavonoids (Flavonoids) content of more than 2 to 2.5 times higher than the red ginseng extract, and can effectively eliminate free radicals, hydrogen peroxide and free radicals. According to another embodiment of the present invention, the herbal extract of the present invention is nitric oxide (NO) and reactive oxygen species (ROS) and inflammation-induced cytokines TNF-α and IL produced in cells Production of -6 can be effectively suppressed.

Herbal medicine of the present invention may be prepared by mixing 25 to 75 parts by weight, 25 to 75 parts by weight, 25 to 75 parts by weight and 25 to 75 parts by weight of peony and 25 to 75 parts by weight based on 100 parts by weight of red ginseng, but is not limited thereto. Preferably from 100 parts by weight of red ginseng 35 to 65 parts by weight, 35 to 65 parts by weight of Astragalus, 35 to 65 parts by weight and 35 to 65 parts by weight peony. More preferably, it is 45 to 55 parts by weight, 45 to 55 parts by weight of Astragalus, 45 to 55 parts by weight, and 45 to 55 parts by weight of Peony, relative to 100 parts by weight of red ginseng. Most preferably, red ginseng, Angelica, Astragalus, Cheongung and Peony can be used by mixing red ginseng: Angelica: Astragalus: Cheongung: Peony = 2: 1: 1: 1: 1.

Herbal medicine extract of the present invention is a hot water extract extracted with an extraction solvent as water, but is not limited thereto.

The composition containing the herbal medicine extract of the present invention can be utilized in various products in various fields, including various fields of medicine, food and beauty.

The present invention can provide an anti-inflammatory pharmaceutical composition comprising the herbal extract of the present invention as an active ingredient.

The present invention can provide an anti-inflammatory health functional food comprising the herbal extract of the present invention as an active ingredient.

The present invention can provide an anti-inflammatory quasi-drug composition comprising the herbal extract of the present invention as an active ingredient.

The present invention can provide an anti-inflammatory cosmetic composition comprising the herbal extract of the present invention as an active ingredient.

The present invention can provide an antioxidant health functional food comprising the herbal extract of the present invention as an active ingredient.

The present invention can provide an antioxidant cosmetic composition comprising the herbal extract of the present invention as an active ingredient.

The present invention can provide an antioxidant pharmaceutical composition comprising the herbal extract of the present invention as an active ingredient.

The present invention can provide a quasi-drug composition for antioxidants using the herbal extract of the present invention as an active ingredient.

The present invention can provide a pharmaceutical composition and health functional food composition for the treatment or prevention of inflammatory diseases comprising the herbal extract of the present invention as an active ingredient. Inflammatory diseases may include diseases in which acute, chronic or excessive inflammation develops or is caused by it. The inflammatory disease may be a disease selected from the group consisting of atopic, rhinitis, otitis media, pneumonia, gastritis, conjunctivitis, periodontitis, sore throat, gastric ulcer, hemorrhoids, gout, arthritis, tendinitis, myositis, hepatitis, cystitis, nephritis and multiple sclerosis. It may also be a disease selected from the group consisting of myocardial infarction, hyperlipidemia, dementia, Parkinson's disease, epilepsy, stroke, diabetes and diabetes complications associated with inflammation resulting from increased free radicals in the cell.

In the present invention, the anti-inflammatory pharmaceutical composition may include a pharmaceutically acceptable carrier. The carrier may be used without limitation as long as it is a carrier generally used in the art such as ethanol, saline, buffer and glycerol. And according to the administration method, it can be formulated in a specific form using a diluent or excipient, and the preparation form can be freely selected, such as a solid or liquid, such as powder or a pill, for convenience.

In the present invention, the health functional food is a food which is effective for supplying nutrients or improving biological functions to organisms such as humans, non-human animals, and plants, and may be prepared according to methods commonly used in the art. The dietary supplement may include various food additives such as sweeteners, flavors, emulsifiers, seasonings, vitamins, minerals, amino acids, preservatives, antioxidants, and colorants. The form of the health functional food is not particularly limited, and preferably various forms such as beverages, pills, sweets, gums, breads, animal feeds, plant nutrients and the like can be freely selected.

In the present invention, the cosmetic composition is a composition that can be used as an active ingredient in cosmetics or mask packs as a whole, and includes various additives such as water, alcohols, esters, glycols, humectants, waxes, and pigments commonly used in the art. can do. The cosmetic composition may be used without being limited to the form of the desired product, and various carriers may be used depending on the product form such as skin, cream, gel, pack, spray, powder, and the like.

In the present invention, the quasi-drug is a product that is used to treat or prevent a wound or a disease of a human or a non-human animal, and may include a product that is not a device or a machine. Skin external preparations or hygiene products are representative and specific examples, but are not limited thereto, such as soaps, detergents, detergents, disinfectants, dressing agents, wipes, and the like. The quasi-drug composition of the present invention is a substance that can be added to the quasi-drug, and may be used with various additives or carriers usable in the quasi-drug.

Extract of the herbal medicine of the active ingredient of the present invention can be extracted by the hot water extraction method of heating and putting red ginseng, Angelica, Astragalus, Cheonggung and Peony into the purified water. After hot water extraction, the process of removing impurities through filtration may be further performed, and further concentration may be performed after filtration. The hot water extraction process may use a variety of devices generally used in the field of the present invention, such as a concentrator, a decoction machine, a dropping machine, a filter (filter) is not particularly limited.

EMBODIMENT OF THE INVENTION Hereinafter, the Example for implementing this invention is demonstrated. The examples correspond to one example for carrying out the present invention, and the present invention should not be construed as limited by the examples.

Antioxidant Effect of Herbal Extracts

The manufacturing method of the herbal medicine extract is as follows.

 Chinese ginseng 100g, Angelica 50g, Astragalus 50g, Cheongung 50g, Peony 50g was mixed into 1500ml of purified water and heated by using a decoction machine for 150-200 minutes at a temperature of 100-120 ° C. to prepare a hydrothermal extract. Then, the impurities were filtered, concentrated and freeze-dried to prepare an extract in powder form, and then stored and used.

The manufacturing method of the red ginseng extract is as follows.

100 g of red ginseng was added to 500 ml of purified water, and heated water was then heated at a temperature of 100 to 120 ° C. for 150 to 200 minutes to prepare a hot water extract. Then, the impurities were filtered, concentrated and freeze-dried to prepare an extract in powder form, and then stored and used.

Example  1-1. Of extract DPPH  Free radical scavenging measurement

Free radical scavenging activity of the medicinal herb extract was measured by modifying the DPPH (1,1-diphenyl-2-picrylhydrazyl) method of Blois (Blois, M. S., Nature, 181: 1190-1201, 1958). In the experimental method, 100 μl of 0.3 mM DPPH ethanol solution was added to 100 μl of the sample prepared by concentration in ethanol, and the absorbance was measured at 517 nm using a spectrophotometer after 15 minutes of treatment at room temperature. The magnitude of the activity was the control (control) when no sample was added and the experimental group (experimental) with the sample represented the inhibition rate of DPPH activity by the following equation.

Inhibition% = [(A _control -A _sample ) / A _control ]

Experimental results, the herbal extract extract group was a single red ginseng extract at all concentrations (20 ㎍ / ㎖, 50 ㎍ / ㎖, 100 ㎍ / ㎖, 200 ㎍ / ㎖, 500 ㎍ / ㎖, 1000 ㎍ / ㎖, 2000 ㎍ / ㎖) Compared to the treatment group, DPPH free radical scavenging ability was remarkably superior. In the 200 ㎍ / ㎖ red ginseng extract and medicinal herb extract treatment group, the red ginseng extract treatment group showed 9% and the medicinal herb extract treatment group showed 9%, and the high concentration 500㎍ / ㎖ treatment group of the red ginseng extract 23 %, Herbal medicine extract treatment group showed more than 75% free radical scavenging activity, herbal medicine extract has a superior free radical scavenging ability compared to the control group in the concentration range of 150 ~ 750㎍ / ㎖ including 200 ~ 500㎍ / ㎖ compared to the control group red ginseng alone 3 times or more free radical scavenging ability was confirmed (Fig. 1).

Example  1-2. Total of extract Antioxidant activity  Measure

Total antioxidant capacity was measured by the total antioxidant capacity (TAC) by modifying the Troloc equivalent antioxidant capacity (TEAC) method. At acidic pH, the colorless, reduced ABTS (2,2'-azinobis 3-ethylbenzothiazoline-6-sulfonate) is oxidized to blue-green ABTS. + By H ₂ O ₂ and, if there is an antioxidant in the extract, ABTS. + Decolorizes in proportion to the concentration, and the result of this color change reaction can be measured by absorbance at 600 nm. For the TAC measurement of sample extracts, standard curves were prepared using 20, 50, 100, 200, 500, 1000, 2000 μg / ml and 100 μM gallic acid (Gallic acid) as standard reagents.

As a result, the herbal extract treatment group was treated with single extract of red ginseng at all concentrations (20µg / ml, 50µg / ml, 100µg / ml, 200µg / ml, 500µg / ml, 1000µg / ml and 2000µg / ml). Compared to the group, total antioxidant capacity was remarkably superior. In particular, 4, 7, 17.5, 18, 21, 23.5, 24μM in the red ginseng extract treatment group, 10, 12.5, 19.8, 20, 24, 25, 25.2μM gallic acid equivalent in the herbal extract treatment group. . At high concentrations, there was no significant difference, but the herbal extracts were slightly superior, especially at low concentrations, the herbal extracts were remarkably excellent (FIG. 2).

Example  1-3. Flavonoids flavonoid Gun Antioxidant activity  Measure

The total flavonoid content of red ginseng and mixed extracts was tested by modifying Zhuang et al. (Zhuang XP, LuYY, Yang GS (1992) Extraction and determination of flavonoid in ginkgo. Chinese Herb Med, 23, 122-124). In 0.1 ml of the sample extract, 0.3 ml of distilled water, 0.03 ml of 5% NaNO ₂ and 10% AlCl ₃ were mixed, and then reacted for 5 minutes. Then, 0.2 ml of 1 mM NaOH was added and reacted for 15 minutes and measured at 510 nm. Total flavonoid content was calculated according to the calibration curve prepared by Quercetin standard.

As a result of the experiment, the herbal extract extract group was a single extract of red ginseng at all concentrations (20 µg / ml, 50 µg / ml, 100 µg / ml, 200 µg / ml, 500 µg / ml, 1000 µg / ml and 2000 µg / ml). The total flavonoid content was significantly higher than the treatment group. In particular, the total flavonoid content at concentrations of 200, 500, 1000, 2000 ㎍ / mL was 19, 54, 137, 266 ㎍ / mL in the red ginseng extract alone treatment group, and 44, 131, 295, 624 in the herbal extract treatment group. It was confirmed that the total flavonoid content in the medicinal herb extract was marked as ㎍ / ㎖ quercetin equivalent (Quercetin equivalent) (Fig. 3).

Example  1-4. Hydrogen peroxide Scatters  Measure

The hydrogen peroxide scavenging ability was performed according to a known method. Hydrogen peroxide scavenging ability was measured by adding 100 μl of PBS (phosphate buffered saline) and 20 μl of extract to a 96-well microplate and preliminarily reacting with 1 mM H ₂ O ₂ for 5 minutes, followed by 30 μl of 1.25 mM ABTS. 30 μl of peroxidase (1 unit / ml) dissolved in PBS was added thereto, followed by reaction at 37 ° C. for 10 minutes, and the absorbance was measured at 405 nm. When the sample was not added as a control and the sample was added as an experimental group, the hydrogen peroxide scavenging ability was expressed by the following equation.

Inhibition% = [(A _control -A _sample ) / A _control ]

As a result of the experiment, the herbal extract extract group was a single extract of red ginseng at all concentrations (20 µg / ml, 50 µg / ml, 100 µg / ml, 200 µg / ml, 500 µg / ml, 1000 µg / ml and 2000 µg / ml). The hydrogen peroxide scavenging ability was remarkably superior to the treatment group. In particular, the results of treatment of the red ginseng alone group and the red ginseng-Herbal medicinal mixed group of 200㎍ / ㎖ concentration were 38.5% in the red ginseng extract treatment group and 82.7% in the medicinal herb extract treatment group compared to the control group. It could be confirmed (Fig. 4).

Example  2-1. Confirmation of inhibition of nitric oxide (NO) production

The amount of NO produced was quantified by measuring NO ₂ ⁻ , a reaction product of NO, using a cell culture filtrate. 50 μl of the culture filtrate was added to the same volume of Greases reagent (0.5% sulfanilyamide, 0.05% N- (1-naphthyl) ethylene diamine dihydrochloride / 2.5% H ₃ PO ₄ ) in a 96-well tissue culture plate (96-well). The mixture was mixed in a tissue culture plate and reacted for 10 minutes in a dark place at room temperature, and the absorbance of the sample was measured with a plate reader at 540 nm. The concentration of NO ₂ ⁻ was made using NaNO ₃ to create a standard curve, and the amount of NO produced was measured. All experiments were repeated three more times and each was quantified using Dunnet'st-test.

As a result, when the LPS 1㎍ / ㎖ treated control was increased by more than 5 times the NO, the two samples were able to confirm the NO production inhibitory effect. Through cell experiments, the red ginseng extract prevented the activation of macrophages, and the herbal medicine extract was superior to the red ginseng extract alone treatment group. In particular, the red ginseng extract alone treatment group showed a 40% NO production inhibitory effect, while the herbal medicine extract treatment group showed a 40% NO production inhibitory effect was found to be markedly excellent NO production inhibitory effect (Fig. 5) .

Example  2-2. Reactive oxygen species (Reactive oxygen species, ROS ) Confirm generation suppression

To view the generation of reactive oxygen species induced by lipopolysaccharides in RAW264.7 cells, see Hayakawa et al. (Hayakawa M, Miyashita H, Sakamoto I, Kitagawa M, Tanaka H, Yasuda H, Karin M, Kikugawa K. Hydroxyl radical using 2 ′, 7′-dichlorofluorescein diacetate (DCFH2-DA, Molecular Probes) according to the method of Evidence that reactive oxygen species do not mediate NF-kappaB activation.EMBO J. 2003; 22: 3356-66). (hydroxyl radical) was measured. That is, the amount of hydroxy peroxidase generated by external stimulation and accumulated in the cells was compared. When grown in 96-well plate (96-well plate) 1x10 ⁴ cells to be filled more than 80% was processed red ginseng extract and herbal extracts in the order of increasing concentration. Thereafter, the cells were stimulated with 1 μg / ml LPS 16 hours before and after, and then reacted with DCFH2-DA at 10 μM for 1 hour. After the reaction, washing with PBS five times and using a fluorescence microplate reader (1420 VICTOR, Perkin-Elmer, Brussels, Belgium) at 485-nm excitation and 530-nm emission wavelengths Measured.

As a result, in the control group treated with LPS only, it was confirmed that the fluorescence value of DCF, which is an indicator of ROS, increased by 4.5 times with time. Red ginseng extract alone treatment was inhibited with increasing concentration, showing a suppression effect of about 10% at the maximum concentration. Compared to the red ginseng extract alone treatment group, the herbal medicine extract treatment group showed an inhibitory effect of 37.5% at the maximum concentration, thereby effectively inhibiting the oxidative stress of macrophages, which can be used to treat inflammatory diseases more effectively than the red ginseng extract (Fig. 6).

Example  2-3. TNF -Inhibition of α and IL-6 Production

To measure the amount of representative TNF-α in the increased intracellular cytokine during LPS treatment was measured using a commonly used mouse TNF-α or IL-6 Immunoassay kit (R & D systems, Minneapolis, MN) . After raising appropriate amounts of RAW264.7 cells in 6 wells or 12 wells, the desired compound was treated at a cluster degree of about 80%, and then activated by LPS for about 16 hours. Thereafter, all of the medium was collected and stored, and the cells were allowed to react by adding a lysis solution, followed by centrifugation, and only the supernatant was collected and stored for use in the experiment. After diluting about 1/5 ~ 1/10 when using the experiment was carried out, absorbance was measured using a 450nm wavelength Microplate reader (Bio-Rad, Co., Hercules, CA, USA).

As a result, the production of IL-1β in the cells was 180.4 ± 76.3pg / ml in the normal group and 2,065.5 ± 127.9 pg / ml in the control group. Red ginseng extract alone showed 1,802 ± 74.9 pg / mL and 160.5 ± 40.3 pg / mL at 100 and 200µg / mL treatment groups, respectively, and 1,625.5 ± 38.9 pg / mL and 1,404 ± 73.5 pg / mL in Chinese herbal medicine extract treatment groups. Compared with the red ginseng extract alone treatment group, it was confirmed that the herbal extract showed a better TNF-α production inhibitory effect (**: p <0.01) (Fig. 7).

For IL-6 cytokines, the normal group was 65.7 ± 15.1 pg / ml and the control group was 563.5 ± 44.9 pg / ml. Both red ginseng extract treatment group and Chinese herbal medicine extract treatment group showed significant inhibitory effect. In particular, the herbal extract extract showed the inhibitory effect of 60% IL-6 production at the highest concentration of 200㎍ / ㎖, whereas the red ginseng extract alone showed a 30% inhibitory effect, inhibiting IL-6 production of the herbal extracts It was confirmed that the effect is significantly superior to the red ginseng extract (Fig. 8).

Claims (10)

Anti-inflammatory pharmaceutical composition comprising the extract of the herbal medicine containing red ginseng, Angelica, Astragalus, Cheongung and Peony as an active ingredient.
The method of claim 1,
The herbal medicine is anti-inflammatory pharmaceutical composition containing 25 to 75 parts by weight, 25 to 75 parts by weight, 25 to 75 parts by weight, 25 to 75 parts by weight and peony 25 to 75 parts by weight relative to 100 parts by weight of red ginseng.
The method of claim 1,
The extract is an anti-inflammatory pharmaceutical composition of hot water extract.
Anti-inflammatory health functional food comprising the extract of Chinese herbal medicine including red ginseng, Angelica, Hwanggi, Cheongung and Peony as an active ingredient.
Anti-inflammatory cosmetic composition comprising the extract of the herbal medicine containing red ginseng, Angelica, Astragalus, Cheongung and Peony as an active ingredient.
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