KR20220095075A - Anti-inflammation composition, Health functional food containing the same and Pharmaceutical composition containing the same - Google Patents

Abstract

The present invention relates to an anti-inflammatory composition including a black ginseng extract and/or a black ginseng fraction extract as an active ingredient and an applied product using the same. The present invention provides a health functional food, cosmetics, and a pharmaceutical composition capable of preventing and reducing an inflammatory disease by providing the anti-inflammatory composition that removes inflammatory substances such as intracellular NO and ROS and the like and inhibits generation of inflammatory proteins.

Description

Anti-inflammatory composition, health functional food and pharmaceutical composition containing the same {Anti-inflammation composition, Health functional food containing the same and Pharmaceutical composition containing the same}

The present invention relates to an anti-inflammatory composition comprising a black ginseng extract and/or a black ginseng extract fraction as an active ingredient and an application product using the same.

Ginseng (Panax ginseng C.A. Meyer. Ginseng radix) is a perennial herb belonging to the genus Panax of the Araliaceae family. For this reason, it is rated as the best health food in the world.

Examples of representative species of ginseng include Korean ginseng (Panaxginseng C.A.Meyer), which grows wild in the Far East of Asia (N. 33 to 48: Korea, Northern Manchuria, and parts of Russia) and has excellent medicinal effects; American ginseng (Panax quinquefolium L.) that is native to and cultivated in the United States and Canada; Panax notoginseng F.H.Chen, wild or cultivated from southeastern Yunnan to southwestern Guangxi, China; And, there is a bamboo japonicus (Panaxjaponicus C.A. Meyer) distributed to Japan, southwestern China, and Nepal.

Ginseng saponins are known as active ingredients of ginseng, and ginseng saponins are protopanaxadiol (PPD-based), protopanaxatriol (PPD)-based, and oleanane-based saponins depending on the structure of the non-sugar part. classified as Among them, PPD and PPT are saponins unique to ginseng and are known to have strong effects such as strengthening immunity and anti-cancer effects. Although each of these chemical components of ginseng has efficacy, it has been reported that a combination of several components exhibits remarkably excellent efficacy for specific medicinal uses.

On the other hand, depending on the processing method of ginseng, new ingredients that do not exist in ginseng are created, and new processed ginseng can be developed that shows better effects on various physiological activities, including antioxidant, compared to ginseng.

According to research studies so far, the active ingredient present in ginseng is generally known as ginsenoside. Ginseng saponin is an alcoholic OH of R1, R2, and R3 of a triterpene, non-sugar (protopanaxadiol or protopanaxatriol). It is a compound having a structure in which saccharides such as glucose, rhamnose, xylose, and arabinose are ester-bonded in the group, and more than 30 types have been identified so far, and have anticancer action, antidiabetic action, central nerve inhibitory action, arteriosclerosis and hypertension. It is known to exhibit pharmacological effects such as prevention, liver function promotion and hangover removal effect, anti-fatigue and anti-stress action, antioxidant action, anti-inflammatory action, protein synthesis promoting action, and immune enhancing action. Among them, general saponins (primary products) such as ginsenosides Rb1, Rb2, Rc, Rd, and Re are the most abundant in the living body of ginseng or wild ginseng, and are not absorbed directly into the body, and some of them are However, it is absorbed and its efficacy is expressed only after it is decomposed by internal enzymes and converted into specific saponins (secondary metabolites) such as ginsenosides F1, F2, Rg3, and compound-K. It is reported that the active effect is very good.

There are fermented ginseng, red ginseng, black ginseng, etc. as a product of processed ginseng fortified with active ingredients beneficial to the human body. There are many studies and various products on fermented ginseng or red ginseng, but research using black ginseng is relatively insufficient. Accordingly, there are few products using black ginseng.

Korean Patent Publication No. KR 10-2015-0059345 A (published on: 2015.06.01.) Korean Patent Publication No. KR 10-2016-0121626 A (published date: 2016.10.20.)

The present invention was devised by discovering that black ginseng extract and/or black ginseng extract fraction prepared by a specific method has excellent anti-inflammatory effects through various studies and application development studies on black ginseng, and has no cytotoxicity and inflammation of NO, ROS, etc. An object of the present invention is to provide an anti-inflammatory composition having an excellent anti-inflammatory effect through inhibition of inducing substances and application products using the same.

In order to solve the above problems, the present invention includes black ginseng extract as an active ingredient as an anti-inflammatory composition.

As a preferred embodiment of the present invention, the black ginseng extract contains ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rh4, ginsenoside Rg3(S), ginsenoside Rk1 and ginsenoside Rg5 do.

As a preferred embodiment of the present invention, the black ginseng extract contains ginsenoside Rg1 0.10 to 0.30 mg/g, ginsenoside Rb1 1.50 to 3.20 mg/g, ginsenoside Rh4 4.50 to 6.00 mg/g, ginsenoside Rg3 (S) 4.50 to 6.20 mg/g, ginsenoside Rk1 3.00 to 4.50 mg/g, and ginsenoside Rg5 5.00 to 6.50 mg/g.

As a preferred embodiment of the present invention, the black ginseng extract contains a total of 19.0 mg/ It may be included in an amount of g or more.

As a preferred embodiment of the present invention, the black ginseng extract may include ginsenoside Rh4, ginsenoside Rk1 and ginsenoside Rg5 in a total amount of 12.0 mg/g or more.

As a preferred embodiment of the present invention, the black ginseng extract may contain benzopyrene in an amount of 0.05 μg/kg or more.

As a preferred embodiment of the present invention, the black ginseng extract fraction may include two or more selected from ginsenoside Rk1, ginsenoside Rb1, and ginsenoside Rg3(S).

As a preferred embodiment of the present invention, the black ginseng extract fraction may include ginsenoside Rk1 and ginsenoside Rg3.

As a preferred embodiment of the present invention, the black ginseng extract fraction may contain ginsenoside Rk1 and ginsenoside Rg3 in an amount of 7.5 mg/g or more.

As a preferred embodiment of the present invention, the black ginseng extract fraction may include ginsenoside Rb1 and ginsenoside Rg3.

As a preferred embodiment of the present invention, the black ginseng extract fraction may contain ginsenoside Rb1 and ginsenoside Rg3 in an amount of 7.5 mg/g or more.

As a preferred embodiment of the present invention, the black ginseng extract is obtained by repeating the process of steaming fresh ginseng at 85 ~ 105 ℃ and drying it at 50 ~ 70 ℃ 3 to 7 times to obtain black ginseng; and extracting and concentrating the black ginseng to prepare a black ginseng extract; may be prepared by performing a process comprising a.

As a preferred embodiment of the present invention, in the anti-inflammatory composition of the present invention, when the black ginseng extract concentration is 200 ug/mL, the NO (nitrite) removal rate measured according to Equation 1 below may be 80.0% or more.

[Equation 1]

NO removal rate (%) = 100% - (anti-inflammatory composition treated intrainflammatory intracellular NO concentration/inflammatory induced intracellular NO concentration)×100%

In Equation 1, the intracellular NO concentration induced by inflammation is the RAW264.7 intracellular NO concentration (μM) measured 24 hours after the RAW264.7 cells were treated with 100 ng/ml LPS, and treated with the anti-inflammatory composition The inflamed intracellular NO concentration was measured 24 hours after RAW264.7 cells were treated with an anti-inflammatory composition at a concentration of 200 ug/mL in RAW264.7 cells 24 hours after treatment with LPS. concentration (μM).

As a preferred embodiment of the present invention, the anti-inflammatory composition of the present invention includes ginsenoside Rk1 and ginsenoside Rg3, and when the black ginseng extract fraction concentration is 25 ug/mL, it is measured according to Equation 1 One NO removal rate may be greater than or equal to 80.0%.

As a preferred embodiment of the present invention, in the anti-inflammatory composition of the present invention, when the black ginseng extract concentration is 200 ug/mL, the ROS (Reactive oxygen species) inhibition rate measured based on Equation 2 below may be 60% or more.

[Equation 2]

ROS inhibition rate (%) = 100% - (ROS concentration in inflammatory cells treated with anti-inflammatory composition / ROS concentration in inflammatory cells) × 100%

In Equation 2, the intracellular ROS concentration induced by inflammation is the ROS concentration (%) for RAW264.7 intracellular LPS measured 24 hours after the RAW264.7 cells were treated with 100 ng/ml LPS, The ROS concentration (%) of LPS in the inflammatory composition-treated cells was treated with the LPS and 24 hours after the RAW264.7 cells were treated with the anti-inflammatory composition at a concentration of 200 ug/mL after 24 hours had elapsed, It is the ROS concentration (%) with respect to the measured RAW264.7 intracellular LPS.

Another object of the present invention is to provide a health functional food comprising the anti-inflammatory composition of various aspects described above.

Another object of the present invention is to provide a cosmetic comprising the anti-inflammatory composition of various aspects described above.

In addition, it is another object of the present invention to provide a pharmaceutical composition comprising the anti-inflammatory composition of various aspects described above as an active ingredient, and inhibiting NO (nitrite) and ROS (Reactive oxygen species).

The anti-inflammatory composition of the present invention can inhibit intracellular NO, ROS, and effectively inhibit the expression of iNOS, COX-2, NFkB, IKkB, etc., without cytotoxicity, and the anti-inflammatory composition of the present invention has various forms It can be applied as a functional material for health functional foods and cosmetics, and can be applied to various pharmaceutical compositions.

1a is a result of measuring the viability (toxicity) of RAW264.7 cells for fresh ginseng (Comparative Example 1), white ginseng (Comparative Example 2), red ginseng concentrate powder (Comparative Example 3) and black ginseng extract (Example 1), FIG. 1b is the cytotoxicity measurement result of RAW264.7 cells induced by LPS inflammation.
Figure 2a is the result of NO measurement in cells induced by LPS of fresh ginseng (Comparative Example 1), white ginseng (Comparative Example 2), red ginseng concentrate powder (Comparative Example 3) and black ginseng extract (Example 1) performed in Experimental Example 1 .
Figure 2b is the result of ROS measurement in cells induced by LPS of fresh ginseng (Comparative Example 1), white ginseng (Comparative Example 2), red ginseng concentrate powder (Comparative Example 3) and black ginseng extract (Example 1) performed in Experimental Example 1 .
Figure 3a is fresh ginseng (Comparative Example 1), white ginseng (Comparative Example 2), red ginseng concentrate powder (Comparative Example 3) and black ginseng extract (Example 1) for cell cytotoxicity measurement results and inflammation-induced cells performed in Experimental Example 2; ) is the measurement result of each TNF-α inhibition.
Figure 3b is a measurement result confirming the expression of the inflammatory biomarker carried out in Experimental Example 2.
4A is a measurement result for the inflammatory protective effect performed in Experimental Example 3, A is a cytotoxicity measurement result, B is a NO inhibitory effect measurement result, and C is a TNF-α inhibition measurement result.
Figure 4b is a measurement result confirming the expression of the inflammatory biomarker carried out in Experimental Example 3.
5a is a cytotoxicity measurement result for each of the black ginseng extract fractions carried out in Experimental Example 4.
Figure 5b is the NO inhibition measurement results for each of the black ginseng extract fractions carried out in Experimental Example 4.
5c is a measurement result of ROS inhibition for each of the black ginseng extract fractions carried out in Experimental Example 4.

Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily implement them. The present invention may be embodied in many different forms and is not limited to the embodiments described herein.

The anti-inflammatory composition of the present invention may include a black ginseng extract, a black ginseng extract fraction, and a mixture thereof.

The black ginseng extract is a step 1 of producing black ginseng by repeating the process of steaming fresh ginseng at 85 ~ 105 ℃ and drying it at 50 ~ 70 ℃ 3 to 7 times; and a second step of extracting and concentrating the black ginseng to prepare a black ginseng extract; may be prepared by performing a process comprising a.

First, the first step of manufacturing the black ginseng; will be described.

In step 1, the steaming may be performed by steaming fresh ginseng at 85 to 105° C. for 3 to 7 hours, preferably at 90 to 100° C. for 4 to 6 hours. If the temperature and time conditions of the steaming are not satisfied, the ginsenosides Rg1, Rb1, Rh4, Rk1, Rg3(S) and/or Rg5, preferably Rk1, Rg3(S) and/or Rg5 are increased It may be difficult to obtain the black ginseng extract contained in the content.

In addition, in step 1, the drying may include drying steamed fresh ginseng at 50 to 70° C. for 6 to 10 hours, preferably, steamed fresh ginseng at 55 to 65° C. for 7 to 9 hours. If the drying temperature and time conditions are not satisfied, ginsenoside may not be included in a high content.

And, in step 1, the steaming and drying process is repeated 3 to 7 times, preferably 4 to 6 times in total. If the number of repetitions of the steaming and drying process is small, the ginsenoside content in the black ginseng extract may be small, and even if the repetition number exceeds 7 times, there may be no further increase in the ginsenoside content, so repeat the steaming and drying within the above range It is good to do

Meanwhile, according to an embodiment of the present invention, it may further include a pretreatment step of washing and drying fresh ginseng before the first step. In the pretreatment step, washing and drying may be performed by a method that can be commonly used in the art.

Next, the second step is, step 2-1 to obtain an extract by repeating the extraction process of a mixture containing black ginseng and an organic solvent; and 2-2 steps of preparing a black ginseng extract by concentrating the extract under reduced pressure.

In step 2-1, the extraction process may be performed by repeating the mixed solution containing black ginseng and an organic solvent 2 to 4 times at 60 to 80° C. for 20 to 28 hours, preferably, the mixture is heated at 65 to 75° C. 22 The extraction process can be performed by repeating 3 to 4 times for ~26 hours. If the extraction temperature, time, and/or the number of repetitions are not satisfied, the ginsenoside content in the black ginseng extract may be small, so it is better to repeat the extraction process within the above range.

On the other hand, the organic solvent can be used without limitation as long as it is a material that can be used as an extraction solvent in general, and preferably at least one organic solvent selected from the group consisting of methanol, ethanol, fermented ethanol and ethyl acetate can be used. , More preferably, ethanol may be used. When ethanol is used as the organic solvent, an aqueous ethanol solution having a concentration of 60 to 80% by volume can be used, preferably an aqueous ethanol solution having a concentration of 65 to 75% by volume, more preferably 67 to 73% by volume. An aqueous ethanol solution may be used. If the concentration of the aqueous ethanol solution is out of the above range, the ginsenoside content in the extracted extract may be small.

In addition, the mixed solution may include the black ginseng and the organic solvent in a weight ratio of 1: 7 to 13, preferably 1: 8 to 12 by weight. If the weight ratio of the black ginseng and the organic solvent is out of the above range, it is impossible to obtain an extract containing the extracted ginsenosides Rh4, Rk1 and Rg5 in a high content, and the anti-inflammatory effect may be reduced.

And, the step 2-2 is a step of preparing a black ginseng extract by concentrating the extract under reduced pressure, and as the conditions for the concentration under reduced pressure can be used without limitation as long as the conditions of concentration under reduced pressure that can be used normally, in the present invention, it is specially do not limit

In addition, the step of filtering the extract between steps 2-1 and 2-2 may be further included, but is not limited thereto. In addition, freeze-drying and/or vacuum-drying may be optionally further performed after the concentration under reduced pressure, but the present invention is not limited thereto.

In the present invention, the black ginseng extract prepared by the above-described manufacturing method may contain a specific ginsenoid in a high content.

The black ginseng extract contains ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rh4, ginsenoside Rg3(S), ginsenoside Rk1 and ginsenoside Rg5, preferably ginsenoside Rg1 0.10 ~ 0.30 mg/g, ginsenoside Rb1 1.50 ~ 3.20 mg/g , ginsenoside Rh4 4.50 ~ 6.00 mg/g, ginsenoside Rg3(S) 4.50 ~ 6.20 mg/g, ginsenoside Rk1 3.00 ~ 4.50 mg/g and ginsenoside Rg5 may be included in an amount of 5.00 to 6.50 mg/g, more preferably ginsenoside Rg1 0.15 to 0.30 mg/g, ginsenoside Rb1 1.65 to 3.15 mg/g, ginsenoside Side Rh4 5.00 ~ 6.00 mg/g, ginsenoside Rg3(S) 4.90 ~ 6.10 mg/g, ginsenoside Rk1 3.20 ~ 4.45 mg/g and ginsenoside Rg5 5.40 ~ 6.30 mg/g have.

In addition, the black ginseng extract may have a total amount of ginsenosides (Rg1, Rb1, Rh4, Rg3(S), Rk1, Rg5) of 19.0 mg/g or more, preferably 19.40 mg/g or more, more preferably 21.00 It can contain ~　24.50　mg/g.

In addition, the black ginseng extract may include ginsenoside Rk1, ginsenoside Rg3(S) and ginsenoside Rg5 in a total amount of 12.0 mg/g or more, preferably 12.50 to 16.80 mg/g.

In addition, the black ginseng extract may contain benzopyrene in an amount of 0.05 μg/kg or more, preferably 0.05 to 0.15 μg/kg, more preferably 0.06 to 0.12 μg/kg.

In addition, the black ginseng extract may include a total phenol content of 650 mg/100g or more, preferably 670 mg/100g or more, and more preferably 680 mg/100g or more.

The anti-inflammatory composition of the present invention may include a fraction (a black ginseng extract fraction) of the black ginseng extract prepared by the above-described manufacturing method as an active ingredient.

The black ginseng extract fraction may be obtained by using a known fractionation method of the black ginseng extract prepared by the above-described method to obtain a black ginseng extract fraction containing a large amount of ginsenoside components. In addition, by performing a separation process of specific ginsenoside components from the black ginseng extract fraction, specific ginsenoside components such as Rg1, Rb1, Rh4, Rg3(S), Rk1, Rg5, preferably Rb1, Rg3(S) ), Rk1 and/or Rg5 may be obtained at high concentrations.

The separation may be used without limitation as long as it is a conventional separation method, and preferably may be performed by any one or more methods selected from silica gel column chromatography and reverse phase silica gel column chromatography, but is not limited thereto. In addition, a process of purifying the black ginseng extract fraction or a specific ginsenoside component obtained by separation therefrom may be performed.

For a preferred example, the anti-inflammatory composition of the present invention includes the black ginseng extract fraction obtained by fractionation from the black ginseng extract by the preceding method, and the black ginseng extract fraction is at least two selected from ginsenosides Rk1, Rb1 and Rg3(S). may include, preferably ginsenosides Rk1 and Rg3 in an amount of 7.5 mg/g or more in total, and more preferably, ginsenosides Rk1 and Rg3 in an amount of 8.0 to 11.50 mg/g in total. .

In addition, the black ginseng extract fraction may include Rb1 and Rg3(S) in an amount of 7.5 mg/g or more, more preferably ginsenoside Rk1 and Rg3 in an amount of 8.0 to 11.50 mg/g.

The anti-inflammatory composition of the present invention comprising the black ginseng extract (and / or black ginseng extract fraction) described above inhibits intracellular NO and ROS without cytotoxicity, and inhibits the expression of iNOS, COX-2, NFkB, IKkB, etc. can

When the anti-inflammatory composition of the present invention contains black ginseng extract at a concentration of 200 ug/mL, the NO (nitrite) removal rate measured based on Equation 1 below is 80.0% or more, preferably 84.5 to 95.0%, more Preferably, it may be 87.0 to 92.5%.

[Equation 1]

NO clearance (%) = 100% - {(concentration of inflammatory intracellular NO (μM) treated with anti-inflammatory composition)/(concentration of NO in inflammatory cells)×100%}

In Equation 1, the intracellular NO concentration induced by inflammation is the RAW264.7 intracellular NO concentration (μM) measured 24 hours after the RAW264.7 cells were treated with 100 ng/ml LPS, and treated with the anti-inflammatory composition The inflamed intracellular NO concentration was measured 24 hours after RAW264.7 cells were treated with an anti-inflammatory composition at a concentration of 200 ug/mL in RAW264.7 cells 24 hours after treatment with LPS. concentration (μM).

In addition, when the anti-inflammatory composition of the present invention contains black ginseng extract at a concentration of 200 ug/mL, the ROS (Reactive oxygen species) inhibition rate measured based on Equation 2 below is 60% or more, preferably 62.0 to 80.0% , more preferably 64.0 to 75.0%.

[Equation 2]

ROS inhibition rate (%) = 100% - (ROS concentration in inflammation-induced cells / ROS concentration in inflammation-induced cells) × 100%}

In Equation 2, the intracellular ROS concentration induced by inflammation is the ROS concentration (%) with respect to the RAW264.7 intracellular LPS measured 24 hours after the RAW264.7 cells were treated with 100 ng/ml LPS, The ROS concentration (%) in the anti-inflammatory composition-treated cells was measured after 24 hours had elapsed after 24 hours had elapsed since RAW264.7 cells were treated with an anti-inflammatory composition at a concentration of 200 ug/mL after treatment with LPS. ROS concentration (%) for LPS in one RAW264.7 cell.

In addition, when the anti-inflammatory composition of the present invention contains black ginseng extract at a concentration of 200 ug/mL, the TNF-α inhibition rate measured based on Equation 3 below is 35% or more, preferably 36.0 to 50.0%, more preferably For example, it may be 38.0 to 46.0%.

[Equation 3]

TNF-α inhibition rate (%) = 100% - (TNF-α concentration in inflammation-induced cells / TNF-α concentration in inflammation-induced cells after treatment with anti-inflammatory composition)×100%}

In Equation 3, the TNF-α concentration in the cells induced by inflammation is the TNF-α concentration in RAW264.7 cells measured 24 hours after the induction treatment of the RAW264.7 cells with 100 ng/ml LPS ), and the TNF-α concentration (%) in the cells induced by the anti-inflammatory composition was treated with 100 ng/ml LPS and 24 hours after the RAW264.7 cells were treated with an anti-inflammatory concentration of 200 ug/mL. It is the TNF-α concentration (%) in RAW264.7 cells measured 24 hours after treatment with the composition.

In addition, the anti-inflammatory composition of the present invention includes black ginseng extract fractions Rk1 and Rg3, and when the black ginseng extract fraction concentration is 25 ug/mL, the NO removal rate measured according to Equation 1 may be 80.0% or more, , preferably 85.0 to 95.0%, more preferably 86.5 to 93.0%.

The anti-inflammatory composition of the present invention may be provided as a functional health functional food, cosmetic, or pharmaceutical composition capable of preventing and improving inflammation.

The present invention will be described in more detail through the following examples, but the following examples are not intended to limit the scope of the present invention, which should be construed to aid understanding of the present invention.

[Example]

Preparation Example 1: Preparation of black ginseng extract

(1) Production of black ginseng

After steaming 873 kg of 5-year-old fresh ginseng (produced in March, 2020) at 95° C. for 5 hours under no pressure conditions, and then drying it with hot air at 60° C. for 8 hours, the steaming and hot air drying were repeated 5 times. to prepare 203 kg of black ginseng (yield 23.3%).

(2) Preparation of black ginseng extract

The mixture obtained by mixing 90 kg of black ginseng with 70% ethanol in a weight ratio of 1:10 was extracted three times at 70° C. for 24 hours, filtered, and then concentrated under reduced pressure to obtain 36.92 kg of black ginseng concentrate powder (yield 41.02%). ), and then freeze-drying it to prepare a black ginseng extract.

Comparative Preparation Example 1: Preparation of fresh ginseng extract

A mixture of 90 kg of 5-year-old fresh ginseng (produced in March, 2020) used in the preparation of the black ginseng extract of Preparation Example 1 with 70% ethanol and 1:10 weight ratio was repeated 3 times at 70° C. for 24 hours. After extraction, filtration, and concentration under reduced pressure, a fresh ginseng concentrate powder was obtained, which was then freeze-dried to prepare a fresh ginseng extract.

Comparative Preparation Example 2: Fresh Ginseng Extract

A fresh ginseng extract prepared at the Domestic International Phosphoric Herb Research Institute was prepared as Comparative Preparation Example 2.

Comparative Preparation Example 3: Red Ginseng Concentrate Powder

Red ginseng concentrate powder of Geumsan Deokwon was prepared in Comparative Preparation Example 3.

Experimental Example 1

By performing HPLC chromatograms for each of Preparation Examples 1 and 3, the ginsenoside (Rg1, Rb1, Rh4, Rg3(S), Rk1 and Rg5) content and benzopyrene content were measured, and as a result, is shown in Table 1 below.

division Ginsenosides (mg/g) Total (mg/g) Benzopyrene (ug/kg) Rg1 Rb1 Rh4 Rg3(S) Rk1 Rg5 Comparative Preparation Example 1 (FG) 6.09 11.04 non-detection 0.07 non-detection non-detection 17.2 - Comparative Preparation Example 2 (WG) 8.91 16.92 non-detection 0.11 non-detection non-detection 25.94 - Comparative Preparation Example 3 (RG) 2.81 13.98 1.21 1.64 0.93 1.3 21.87 - Preparation Example 1 (BG) 0.14 1.84 5.4 4.97 3.89 5.79 22.03 0.07

As can be seen in Table 1, the contents of ginsenosides Rg1, Rb1, Rh4, Rg3(S), Rk1 and Rg5 in the black ginseng extract prepared in Preparation Example 1 are very high, and the content of benzopyrene is also very high at 0.05 ug/kg or more. could check In contrast, Comparative Preparation Example 2 had the highest total ginsenosides, but as in Comparative Preparation Example 1, Rh4, Rk1 and Rg5 were not detected. In particular, in the case of Preparation Example 1, as compared to Comparative Preparation Example 3, the content of Rh4, Rk1, Rg3 and Rg5 was significantly higher.

Example 1: Anti-inflammatory composition

The black ginseng extract of Preparation Example 1 was prepared as an anti-inflammatory composition.

Comparative Examples 1 to 3

Comparative Preparation Example 1, Comparative Preparation Example 2 and Comparative Preparation Example 3 were each prepared as an anti-inflammatory composition, respectively, and Comparative Examples 1 to 3 were performed (see Table 2).

division extract Example 1 (BG) Preparation Example 1 Comparative Example 1 (FG) Comparative Preparation Example 1 Comparative Example 2 (WG) Comparative Preparation Example 2 Comparative Example 3 (RG) Comparative Preparation Example 3

Experimental Example 1: Cell viability (toxicity) test

After treating the inflammation-induced cells with each of Example 1 (BG), Comparative Example 1 (FG), Comparative Example 2 (WG) and Comparative Example 3 (RG), a cytotoxicity experiment was performed.

(1) Cell distribution and culture

The mouse macrophage line RAW264.7 cells were distributed from ATTC and an experiment was performed using them.

(2) Evaluation of cell viability (toxicity)

(2-1) Inflammation induction untreated group

RAW264.7 cells were inoculated into a 96-well cell culture plate at 5×10 ³ cells/well, and cultured at 37° C., 5% CO ₂ in an incubator for 24 hours.

The cultured cells were treated with 50 μg/ml, 100 μg/ml, and 200 μg/ml in Example 1 and Comparative Examples 1 to 3, respectively, and then after 6 hours, 12 hours and 24 hours, EZ-cytox was added to the media ( media) treated at 10 μL per 200 μL, and reacted for 1 hour in a CO ₂ incubator. Then, 100 μL of each was transferred, and then the absorbance was measured at 450 nm using an ELISA reader (BIO-RAD 450, USA), and the results are shown in FIG. 1a.

(2-2) Inflammation induction treatment group

In addition, separately, 5×10 ³ cells/well of RAW264.7 cells were inoculated in a 96-well cell culture machine, treated with 100 ng/ml LPS to induce inflammation, and 24 hours later, Example 1 , Comparative Examples 1 to 3 were treated at 50 μg/ml, 100 μg/ml, and 200 μg/ml, respectively. Next, after the lapse of 6 hours, 12 hours and 24 hours, 10 μL of EZ-cytox per 200 μL of media was treated, and the reaction was performed in a CO ₂ incubator for 1 hour. Then, 100 μL of each was transferred, and then the absorbance was measured at 450 nm using an ELISA reader (BIO-RAD 450, USA), and the results are shown in FIG. 1B.

The cont of FIG. 1a is a control, which is not treated with both LPS and the extract, and the LPS of FIG. 1b is that the cultured cells are treated with 100 ng/ml of LPS to induce inflammation.

Looking at the cell viability measurement results of Figures 1a and 1b, Preparation Example 1 and Comparative Preparation Examples 1 to 3 showed no toxicity or very low results.

Experimental Example 2: Inflammation improvement effect analysis experiment 1

(1) Measurement of intracellular NO removal effect

After measuring the intracellular NO content of cells subjected to inflammation-induced cell viability (toxicity) evaluation with the LPS of Experimental Example 1, the NO removal rate (%) was calculated by Equation 1 below, and the result is shown in FIG. 2a and Table 3 below. Cont in FIG. 2a is a control that was not treated with both LPS and the extract, and LPS was induced by treating the cultured cells with 100 ng/ml of LPS to induce inflammation.

[Equation 1]

NO removal rate (%) = 100% - (concentration of inflammatory intracellular NO treated with anti-inflammatory composition/concentration of inflammatory intracellular NO)×100%

In Equation 1, the intracellular NO concentration induced by inflammation is the RAW264.7 intracellular NO concentration (μM) measured 24 hours after the RAW264.7 cells were treated with 100 ng/ml of LPS, and treated with the anti-inflammatory composition The inflamed intracellular NO concentration was measured after a specific time elapsed after the RAW264.7 cells were treated with an anti-inflammatory composition at a concentration of 200 ug/mL for 24 hours after treatment with LPS. concentration (μM).

division
(NO concentration, μM) Comparative Example 1
(FG) Comparative Example 2
(WG) Comparative Example 3
(RG) Example 1
(BG) 6 hours lapse control (cont) 79.22 79.22 79.22 79.22 LPS 100 100 100 100 50 μg/ml 91.56 93.83 93.18 86.69 100 μg/ml 89.61 101.62 89.61 99.13 200 μg/ml 87.34 99.68 84.42 107.14 12 hours lapse control (cont) 17.83 17.83 17.83 17.83 LPS 100 100 100 100 50 μg/ml 82.97 84.54 82.75 68.36 100 μg/ml 69.80 87.19 67.58 52.05 200 μg/ml 48.97 71.59 44.39 35.08 24 hours lapse control (cont) 3.43 LPS 100 50 μg/ml 76.51 85.39 71.06 48.43 100 μg/ml 56.82 80.20 48.83 27.35 200 μg/ml 31.13 62.45 25.25 12.40 At 200 μg/ml,
NO Removal Rate (%) 68.9% 37.6% 63.6% 87.6%

Referring to the results of the NO removal effect in Table 3 and FIG. 2A, it can be confirmed that the NO removal effect increases in a concentration-dependent manner in both Examples 1 and Comparative Examples 1 to 3 from the time point after 12 hours.

After 24 hours, looking at the measurement results, there was no change in NO removal efficiency in Comparative Example 2 (WG) compared to the measurement results when 12 hours had elapsed. And, Example 1 (BG), Comparative Example 1 (FG), and Comparative Example 3 (RG) showed an increase in the NO removal effect after 24 hours rather than 12 hours, in particular, Example 1 compared to Comparative Example 1 and Comparative Example 1 Compared to Example 3, the NO removal effect was relatively excellent, and when 200 μg/ml of Example 1 was treated, it showed an excellent NO removal efficiency of 80% or more of 87.6%.

(2) Measurement of intracellular ROS inhibition effect

After measuring the intracellular ROS content of the cells subjected to the cell viability (toxicity) evaluation, the ROS inhibition rate (%) was calculated by Equation 2 below, and the results are shown in FIG. 2B and Table 4 below. The cont of FIG. 1c is a control, which is not treated with both LPS and the extract, and LPS is a cultured cell treated with 100 ng/ml of LPS to induce inflammation.

[Equation 2]

ROS inhibition rate (%) = 100% - (ROS concentration in inflammation-induced cells treated with anti-inflammatory composition / ROS concentration in inflammation-induced cells)×100%

In Equation 2, the intracellular ROS concentration induced by inflammation is the ROS concentration (%) for RAW264.7 intracellular LPS measured 24 hours after the RAW264.7 cells were treated with 100 ng/ml LPS, The ROS concentration (%) of LPS in the inflammatory composition-treated cells was treated with the LPS and 24 hours after the RAW264.7 cells were treated with the anti-inflammatory composition at a concentration of 200 ug/mL after 24 hours had elapsed, ROS concentration (%) for LPS in RAW264.7 cells as measured.

division
(ROS production, % of LPS) Comparative Example 1
(FG) comparative ratio 2
(WG) Comparative Example 3
(RG) Example 1
(BG) control (cont) 23.67 23.67 23.67 23.67 LPS 100% 100% 100% 100% 50 μg/ml 93.22% 82.60% 77.50% 63.55% 100 μg/ml 72.72% 83.52% 65.80% 53.33% 200 μg/ml 53.73% 86.67% 45.54% 34.80% At 200 μg/ml,
ROS inhibition rate (%) 46.37% 13.33% 54.46% 66.2%

Looking at the measurement results in Table 4 and FIG. 2B, Comparative Example 2 showed not only no increase in the ROS removal effect according to the increase in concentration, but also insignificant ROS removal effect. Comparative Example 1, Comparative Example 3 and Example 1 all increased the ROS removal effect in a concentration-dependent manner, Example 1 showed a relatively significant ROS removal effect than Comparative Examples 1 and 3, in particular, Example 1 200㎍ / In the case of ㎖ treatment, it showed excellent ROS removal efficiency of 66.2%, which is more than 60%.

Experimental Example 3: Inflammation improvement effect experiment 2

(1) Measurement of TNF-α level in inflammation-inducing cells

In a 96-well cell culture plate, 5 × 10 ³ cells/well were inoculated with RAW264.7 cells, and the RAW264.7 cells were treated with 100 ng/ml LPS to induce inflammation, and then at 37°C, Incubated in a 5% CO ₂ incubator for 24 hours.

Next, the cultured cells were treated with 50 μg/ml, 100 μg/ml, and 200 μg/ml in Examples 1 and Comparative Examples 1 to 3, respectively. Next, after 24 hours, EZ-cytox was treated at 10 μL per 200 μL of media, and reacted in a CO ₂ incubator for 1 hour. Then, 100 μL of each was transferred and then the absorbance was measured at 450 nm using an ELISA reader (BIO-RAD 450, USA), and the results are shown in FIG. 3a and Table 5 below.

division
(TNF-α level, % of LPS) Comparative Example 1
(FG) Comparative Example 2
(WG) Comparative Example 3
(RG) Example 1
(BG) Control (Cont) 0% 0% 0% 0% LPS 100% 100% 100% 100% 50 μg/ml 84.77% 85.19% 89.34% 72.79% 100 μg/ml 78.73% 97.26% 83.62% 65.99% 200 μg/ml 74.26% 85.84% 74.64% 61.47% At 200 μg/ml,
TNF-α inhibition rate (%) 25.74% 14.16% 25.36% 38.53%%

Looking at the experimental results of Figure 3a and Table 5, Example 1 can be confirmed that a relatively small decrease in TNF-α level, it was possible to confirm the excellent anti-inflammatory effect through this.

(2) Evaluation of inflammation expression through biomarkers

Measuring the expression of biomarkers for the non-inflammatory induction group sample (control group, Cont), the inflammation induction treatment group sample (LPS), and the group treated with the anti-inflammatory composition of 200 μg/ml after induction of inflammation as for TNF-α level measurement In order to perform a western blot evaluation experiment, the results are shown in Figure 3b.

Referring to FIG. 3b , it can be confirmed that there is an expression inhibitory effect on iNOS and COX-2 in Comparative Example 1 (FG), Comparative Example 3 (RG) and Example 1 (BG). It was confirmed that there is an excellent expression inhibitory effect.

Experimental Example 4: Inflammation protective effect test

After the cells were treated with each of Example 1 (BG), Comparative Example 1 (FG), Comparative Example 2 (WG) and Comparative Example 3 (RG), inflammation was induced, and then cytotoxicity was measured by the following method, intracellular NO (nitrie) concentration and TNF-α level by performing an experiment, the inflammatory protective effect of the anti-inflammatory composition was measured.

(1) Cell distribution and culture

The mouse macrophage line RAW264.7 cells were distributed from ATTC and an experiment was performed using them.

(2) Evaluation of cell viability (toxicity)

RAW264.7 cells were inoculated at 5×10 ³ cells/well each in a 96-well cell incubator, and each of Examples 1 and Comparative Examples 1 to 3 were treated with 50 μg/ml, 100 μg/ml, and 200 μg/ml. Then, it was incubated for 24 hours at 37° C., 5% CO ₂ incubator.

Next, the cultured cells were treated with 100 ng/ml LPS and then cultured for 24 hours.

Next, EZ-cytox was treated at 10 μL per 200 μL of media, and reacted for 1 hour in a CO ₂ incubator. Then, 100 μL of each was transferred and then the absorbance was measured at 450 nm using an ELISA reader (BIO-RAD 450, USA), and the results are shown in A of FIG. 4A. Cont in FIG. 4a is a control that is not treated with LPS and an anti-inflammatory composition, and LPS is an inflammation-induced cell without treatment with an anti-inflammatory composition.

Referring to FIG. 4a, Comparative Examples 1 to 3 and Example 1 showed no toxicity.

(3) NO (nitrite) concentration measurement

Cells were treated with an anti-inflammatory composition in the same manner as the cell viability and cultured for 24 hours, then treated with LPS to induce inflammation and after 24 hours, the intracellular NO concentration was measured, and the results are shown in FIG. 4B and Table 6 below. shown in

division
(NO concentration, μM) Comparative Example 1
(FG) comparative ratio 2
(WG) Comparative Example 3
(RG) Example 1
(BG) control (cont) 2.26 2.26 2.26 2.26 LPS 100 100 100 100 50 μg/ml 64.54 85.11 60.83 41.50 100 μg/ml 45.91 78.07 36.41 14.29 200 μg/ml 29.93 64.87 12.30 3.67

Referring to Figure 4b and Table 6, Example 1 and Comparative Example 3 showed a significantly lower NO concentration compared to Comparative Examples 1 and 2 in the overall concentration, and also, Example 1 was relatively lower than Comparative Example 3 NO concentration was shown. In particular, when the anti-inflammatory composition of Example 1 was 200㎍ / ㎖, NO generation inhibition rate (%) was 95% or more 96.4%, it was confirmed that there is a very good anti-inflammatory effect.

(4) TNF-α level measurement

TNF-α level was measured in the same manner as in Experimental Example 3 for the anti-inflammatory composition treatment and inflammation-induced cells in the same manner as the NO concentration measurement, and the results are shown in C of FIG. 4A and Table 7 below.

division
(TNF-α level, % of LPS) Comparative Example 1
(FG) Comparative Example 2
(WG) Comparative Example 3
(RG) Example 1
(BG) Control (Cont) 0% 0% 0% 0% LPS 100% 100% 100% 100% 50 μg/ml 82.62 73.01 68.24 58.67 100 μg/ml 57.43 71.84 50.99 43.50 200 μg/ml 30.93 67.04 27.86 25.38 At 200 μg/ml,
TNF-α inhibition rate (%) 39.07% 32.96 72.14 74.62%

Looking at the experimental results shown in C of FIG. 4A and Table 7, it can be seen that Example 1 has a relatively low TNF-α level, and through this, an excellent anti-inflammatory effect can be confirmed.

(5) Evaluation of inflammation expression through biomarkers

Biomarker expression for the inflammation-inducing group sample after treatment with the same inflammation-inducing untreated group sample (control group, Cont), inflammation-inducing treatment group sample (LPS), and an anti-inflammatory composition of 200 μg/ml as measured for TNF-α level To measure, a western blot evaluation experiment was performed, and the results are shown in FIG. 4B .

Referring to Figure 4b, it can be confirmed that there is an expression inhibitory effect on iNOS and COX-2 in Comparative Example 1 (FG), Comparative Example 3 (RG) and Example 1 (BG), in particular, in Example 1 very It was confirmed that there is an excellent expression inhibitory effect.

Preparation Example 2-1 ~ Preparation Example 2-6: Preparation of black ginseng extract fraction

From the black ginseng extract of Preparation Example 1, each of the ginsenosides was fractionated as shown in Table 8 to obtain each of the black ginseng extract fractions.

division Black ginseng extract fraction Preparation Example 2-1 Ginsenoside Rb1 Preparation Example 2-2 Ginsenoside Rg1 Preparation Example 2-3 Ginsenoside Rg3 Preparation Example 2-4 Ginsenoside Rg5 Preparation Example 2-5 Ginsenoside Rk1 Preparation Example 2-6 Ginsenoside Rh4

Examples 2 to 5

Some of the black ginseng extract fraction ginsenosides obtained in Preparation Examples 2-1 to 2-6 were mixed as shown in Table 9 below to prepare an anti-inflammatory composition in which two types of black ginseng extract fractions were mixed, respectively, in Examples 2 to 5 were performed respectively.

division Black ginseng extract fraction mix Example 2 (M1) Rb1 (50uM) + Rg1 (50uM) Example 3 (M2) Rb1 (50uM) + Rg3 (50uM) Example 4 (M3) Rk1 (50uM) + Rg3 (50uM) Example 5 (M4) Rk1 (50uM) + Rg5 (50uM)

Experimental Example 4: Cell viability (toxicity) test of black ginseng extract fraction

In the same manner as in Experimental Example 1, cells induced by LPS were treated with the black ginseng extract fractions of Preparation Examples 2-1 to 2-6 and Examples 2 to 5, respectively, and then cytotoxicity measurement, intracellular NO removal rate and An experiment was performed on the intracellular ROS removal rate as follows.

At this time, the treatment concentration of the black ginseng extract fraction was 5 μg/ml, 10 μg/ml, 25 μg/ml and 50 μg/ml in Preparation Examples 2-1 to 2-6, and the measurement results are shown in A of FIG. 5a it was

In addition, Examples 2 to 5 were treated with 12.5 μg/ml and 25 μg/ml, and the measurement results are shown in FIG. 5A B .

Through the measurement result of FIG. 5a, it was confirmed that all of the black ginseng extract fractions had no cytotoxicity.

Experimental Example 5: Inflammation improvement effect analysis experiment of black ginseng extract fraction

In the same manner as in Experimental Example 2, the NO removal and ROS inhibitory effects on the cells treated with the inflammation-induced cells and the black ginseng extract fraction were measured, and the results are shown in FIGS. 5B and 5C. The NO inhibition rates in Tables 10 and 11 below were measured based on Equation 1 above.

division
(NO concentration, μM) Rb1 Rg1 Rg3 Rg5 Rk1 Rh4 control (cont) 5.31 5.31 5.31 5.31 5.31 5.31 LPS 100 100 100 100 100 100 5 μg/ml 93.07 90.32 90.54 91.60 88.95 88.72 10 μg/ml 90.19 90.04 87.46 90.34 86.09 84.68 25 μg/ml 70.79 72.23 63.89 40.44 44.24 69.57 50 μg/ml 35.96 44.97 22.76 8.07 5.03 40.16 At 50 μg/ml,
NO Removal Rate (%) 64.04 55.03 77.24 91.93 94.9% 59.84

division
(NO concentration, μM) Example 2 Example 3 Example 4 Example 5 control (cont) 5.54 5.54 5.54 5.54 LPS 100 100 100 100 12.5 μg/ml 77.56 73.10 71.34 84.37 25 μg/ml 44.86 30.02 13.28 44.16 At 25 μg/ml,
ROS inhibition rate (%) 55.14 69.98 86.72% 55.84

Referring to A of FIG. 5B , it can be confirmed that Rg3, Rg5, and Rk1 have a relatively superior NO inhibitory effect than other ginsenosides having an NO inhibitory effect.

And, looking at B of FIG. 5B , it was confirmed that Example 4 (M3) composed of a combination of Rk1 and Rg3 had the most excellent effect of inhibiting NO of 75% or more.

In addition, looking at A of FIG. 5C , Rk1 and Rg3 have an excellent ROS inhibitory effect, and looking at FIG. 5C , Example 4 (M3) consisting of a combination of Rk1 and Rg3 and Example 3 (M3) consisting of a combination of Rb1 and Rg3 ( It was confirmed that M2) has an excellent ROS inhibitory effect, and in particular, Example 3 (M2) has the most excellent ROS inhibitory effect.

Through this, it can be confirmed that the ginsenosides Rk1, Rb1 and Rg3 among the black acid extracts have excellent anti-inflammatory effects, in particular, the NO inhibitory effect of Rk1 is the best, and Rg3 has the excellent ROS inhibitory effect, and the black ginseng extract It was confirmed that it was advantageous in terms of anti-inflammatory to mix Rk1 and Rg3 or use a mixture of Rb1 and Rg3 when using the two types of mixture.

Through the above Examples and Experimental Examples, it was confirmed that there is an excellent anti-inflammatory effect of the anti-inflammatory composition of the present invention comprising black ginseng extract and/or black ginseng extract fraction, and the anti-inflammatory composition of the present invention prevents and improves inflammation It can be applied to various functional products such as health functional foods, cosmetics, and pharmaceutical compositions.

Although one embodiment of the present invention has been described above, the spirit of the present invention is not limited to the embodiments presented herein, and those skilled in the art who understand the spirit of the present invention can add components within the scope of the same spirit. , changes, deletions, additions, etc. may easily suggest other embodiments, but this will also fall within the scope of the present invention.

Claims (10)

An anti-inflammatory composition comprising at least one selected from black ginseng extract and black ginseng extract fraction as an active ingredient.
The method of claim 1, wherein the black ginseng extract
Ginsenoside Rg1 0.10 ~ 0.30 mg/g, Ginsenoside Rb1 1.50 ~ 3.20 mg/g , Ginsenoside Rh4 4.50 ~ 6.00 mg/g, Ginsenoside Rg3(S) 4.50 ~ 6.20 mg/g, Ginsenoside Rk1 3.00 to 4.50 mg/g and ginsenoside Rg5 5.00 to 6.50 mg/g Anti-inflammatory composition, characterized in that it comprises an amount.
The method of claim 1, wherein the black ginseng extract
Anti-inflammatory composition comprising ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rh4, ginsenoside Rg3(S), ginsenoside Rk1 and ginsenoside Rg5 in a total amount of 19.0 mg/g or more .
According to claim 1, wherein the black ginseng extract
An anti-inflammatory composition comprising ginsenoside Rk1, ginsenoside Rg3(S) and ginsenoside Rg5 in an amount of 12.0 mg/g or more in total.
The anti-inflammatory composition according to claim 1, wherein when the black ginseng extract concentration is 200 ug/mL, the NO (nitrite) removal rate measured according to Equation 1 is 80.0% or more;
[Equation 1]
NO removal rate (%) = 100% - (concentration of inflammatory intracellular NO treated with anti-inflammatory composition/concentration of inflammatory intracellular NO)×100%
In Equation 1, the intracellular NO concentration induced by inflammation is the RAW264.7 intracellular NO concentration (μM) measured 24 hours after the RAW264.7 cells were treated with 100 ng/ml LPS, and treated with the anti-inflammatory composition The inflamed intracellular NO concentration was measured 24 hours after RAW264.7 cells were treated with an anti-inflammatory composition at a concentration of 200 ug/mL after 24 hours of treatment with LPS. (μM).
According to claim 1, wherein the black ginseng extract fraction is
An anti-inflammatory composition comprising two or more selected from ginsenoside Rk1, ginsenoside Rb1, and ginsenoside Rg3(S).
According to claim 1, wherein the black ginseng extract fraction comprises ginsenoside Rk1 and ginsenoside Rg3,
When the black ginseng extract fraction concentration is 25 ug/mL, an anti-inflammatory composition, characterized in that the NO removal rate measured according to the following Equation 1 is 80.0% or more;
[Equation 1]
NO removal rate (%) = 100% - (anti-inflammatory composition treated intrainflammatory intracellular NO concentration/inflammatory induced intracellular NO concentration)×100%
In Equation 1, the intracellular NO concentration induced by inflammation is the RAW264.7 intracellular NO concentration (μM) measured 24 hours after the RAW264.7 cells were treated with 100 ng/ml LPS, and treated with the anti-inflammatory composition The inflamed intracellular NO concentration was measured 24 hours after RAW264.7 cells were treated with an anti-inflammatory composition at a concentration of 200 ug/mL in RAW264.7 cells 24 hours after treatment with LPS. concentration (μM).
A health functional food comprising the anti-inflammatory composition of any one of claims 1 to 7.
A cosmetic comprising the anti-inflammatory composition of any one of claims 1 to 7.
A pharmaceutical composition comprising the anti-inflammatory composition of any one of claims 1 to 7 as an active ingredient, and inhibiting NO (nitrite) and ROS (Reactive oxygen species).

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