KR102488533B1 - Preparing method of highly functional anti-inflammatory component drived from Glycine max Merr. fermentation extracts and cosmetic composition using the same - Google Patents

Abstract

Disclosure of a fermentation and separation and purification method that increases the content of highly functional substances in the fermented product through an effective fermentation process and separation and purification process of black soybean extract, and a plan to utilize it as a cosmetic composition with excellent anti-inflammatory and anti-aging effects do. The present invention black bean ( Glycine max Merr. ) Steps of inoculating and fermenting bacteria in the extract; Filtering the fermented product to remove microorganisms; and isolating and purifying the lignan analogues.

Description

Manufacturing method of highly functional anti-inflammatory component derived from fermented black soybean filtrate and cosmetic composition using the same {Preparing method of highly functional anti-inflammatory component driven from Glycine max Merr. fermentation extracts and cosmetic composition using the same}

The present invention relates to a fermentation process and separation and purification by microorganisms, and more particularly, to a fermentation process and separation and purification by microorganisms using black soybeans.

Fermentation is a process in which enzymes possessed by microorganisms change raw materials to create beneficial components. During the fermentation process, active ingredients are extracted and the content of functional substances increases.

Fermentation technology has semantically redundancy with terms such as bioconversion, biosynthesis, and biocatalyst, and refers to a technology for producing a desired product from a precursor by using the enzymatic function of microorganisms. Therefore, fermentation technology that uses biological functions in the cell or enzyme stage can be said to be a major process that constitutes the current bioindustry with the main purpose of producing useful substances. If fermented foods use one-dimensional fermentation technology that utilizes naturally occurring microorganisms, fermented cosmetics require more advanced technology. Bioconversion fermentation technology, which laid the foundation for the cosmetic functional material industry through advanced fermentation production technology that can produce only useful physiologically active substances in large quantities, is establishing itself as a third-generation fermentation technology.

Since Koreans use a lot of fermented foods such as kimchi and soybean paste, the concept of fermentation is very familiar, and cosmetics using fermented ingredients are also used without resistance. In addition, the general public has a high preference for fermented cosmetics because it is known that when a new material is developed using fermentation technology, it shows a completely new effect compared to the existing material or changes many harmful ingredients into safe ingredients. level that is not far behind. However, it is known that many fermented materials currently applied to cosmetics use natural fermentation or a fermentation method close to natural aging.

In other words, it is known that the recent trend in research on fermentation technology in Korea is to use natural fermentation or a fermentation method close to natural aging. Although it takes a long time, various substances can be obtained, so it is possible to obtain effects such as moisturizing, anti-oxidation, wrinkle improvement, cell activation, and skin texture improvement. However, fermentation by natural fermentation or natural aging method has disadvantages in that it is difficult to obtain a fermentation product of constant quality for each production batch, and it is difficult to control the quality.

Korea’s fermented cosmetics market has been growing at a rate of 40% over the past 3 to 4 years, and is expected to reach approximately KRW 450 billion in 2012, KRW 650 billion in 2013, and approximately KRW 1 trillion in 2014 (estimated). are looking forward

Fermentation products can be largely classified into fermentation filtrate and fermentation lysate. Fermentation filtrate utilizes the supernatant from which microorganisms are removed through a filtration device after fermentation is completed. The lysate is a method of using heat, pH, enzyme (Lysozyme), high pressure, etc. to kill microorganisms after fermentation is completed.

On the other hand, black soybean ( Glycine max Merr. ) does not refer to one type of bean, but refers to black beans such as black bean, seoritae, and seomoktae. It is known to contain Isoflavone, a vegetable female hormone contained in black soybeans, acts similarly to estrogen, a female hormone in our body, and is good for the health of postmenopausal women. Although it is not found in the skin of black beans, it is known to be a substance that is detected in large amounts only in the skin of black beans.

In addition, the lecithin component of black soybeans inhibits the decrease of acetylcholine, which is a brain active substance that actively helps cerebral activity, develops memory and learning ability, and improves blood circulation by removing cholesterol in blood vessels.

In particular, black soybean quickly restores destroyed human tissue, and vitamin E and unsaturated fatty acids expand blood vessels to facilitate blood circulation in peripheral blood vessels. This is because nutrients that did not reach the scalp are delivered properly when black soybeans are consumed, preventing hair loss and gray hair, and rich in vitamin E. This protein becomes a material for collagen, a skin elastic fiber. In addition, due to the antioxidant effect of vitamin E contained in black soybeans, it has anti-aging effects, so its use in cosmetics is expanding.

Lignan is a plant hormone present in plants, especially legumes, and is one of the plant female hormones with a chemical structure similar to estrogen, derived from cholesterol. Therefore, it has a structure similar to estrogen, and is known as a component that has preventive effects such as reducing osteoporosis and breast cancer.

In addition, lignans bind to estrogen receptors to suppress estrogen action, thereby balancing hormones with the male hormone progesterone to prevent various diseases caused by excess female hormones such as premenstrual syndrome, uterine fibroids and breast fibroids. In particular, it has been reported that lignans have an effect on prostatic hyperplasia caused by a drop in male hormones that may occur after middle age through inhibition of estrogen activity, and also have a good effect on preventing prostate cancer.

The present invention is a fermentation and separation and purification method for increasing the content of high-functional substances in the fermented product through an effective fermentation process and separation and purification process of black soybean extract, and through this, to be used as a cosmetic composition with excellent anti-inflammatory and anti-aging effects I would like to suggest a plan.

In order to solve the above problem, the present invention, black bean ( Glycine max Merr. ) Steps of inoculating and fermenting bacteria in the extract; Filtering the fermented product to remove microorganisms; and isolating and purifying the lignan analogues.

In addition, the fermentation is performed at a temperature of 30 to 50 ° C. for 2 to 4 days, characterized in that it provides a method for producing lignan analogues.

In addition, it provides a method for preparing a lignan analog, characterized in that the bacteria is a Bacillus sp.

In addition, the separation and purification are performed through solvent fractionation and size exclusion chromatography to provide a method for preparing lignan analogues.

In addition, the lignan analog provides a method for preparing a lignan analog, characterized in that it has an ability to inhibit inducible nitric oxide synthase (iNOS) production and an ability to inhibit nitric oxide (NO) production in macrophages.

In addition, the lignan analogues provide a method for preparing lignan analogues characterized in that they have skin cell proliferation and skin cell regeneration inducing activities.

In addition, the lignan analogue is obtained from the butanol fractionation layer of the solvent fraction, and the molecular weight is 324 to 326.

In order to solve the above another problem, the present invention provides a cosmetic composition containing a lignan analogue prepared by the above method.

According to the present invention, it was confirmed that the concentration of lignans and lignan analogues in black soybeans increased through fermentation of black soybeans, and it was also confirmed that anti-inflammatory activity increased as the content of lignan analogues increased through separation and purification.

In particular, lignan analogs showing excellent anti-inflammatory efficacy were finally isolated to confirm the toxicity, efficacy, and physical properties of a single substance, and from this, fermentation and separation and purification methods that increase the content of highly functional substances in fermented products can be provided, Through this, it can be used as a cosmetic composition with excellent anti-inflammatory and anti-aging effects.

1 is a photograph showing the TLC results of fermented black soybeans purified through solvent fractionation and open column in Examples of the present invention.
Figure 2 is a graph showing the HPLC results of the fermented black soybean fraction after Prep LC purification.
3 is a schematic diagram showing a separation example in the present invention.
Figure 4 is a graph showing the NO production inhibition ability of Preparation Examples 1 to 4 of the present invention.
5 is a graph showing the TLC results of fractions separated in purification examples of the present invention.
6 is a graph showing the results of HPLC analysis of the butanol layer separated in the purification example of the present invention.
7 is a graph showing the content of lignan analogues for each separation in the separation examples according to the present invention.
8 is a graph showing the results of HPLC analysis of the lignan analogues isolated in the present invention.
9 is a graph showing the results of mass analysis of lignan analogues in the experimental example of the present invention.
10 and 11 are graphs showing the results of NMR analysis of lignan analogs in experimental examples of the present invention.
12 is a graph showing the results of iNOS expression level analysis according to the degree of separation in the experimental example of the present invention.
13 and 14 are graphs showing the results of measuring the cell proliferation ability of Experimental Example 8 of the present invention in HDF cells and HACAT cells.
15 is a photograph showing the wound healing ability of skin cells of lignan analogues in the experimental example of the present invention.
16 and 17 are graphs showing light stability and thermal stability measurement results of Experimental Example 10 of the present invention.

Hereinafter, the present invention will be described in detail through preferred embodiments. Prior to this, the terms or words used in this specification and claims should not be construed as being limited to the usual or dictionary meaning, and the inventor appropriately uses the concept of the term in order to explain his/her invention in the best way. Based on the principle that it can be defined, it should be interpreted as meaning and concept consistent with the technical spirit of the present invention. Therefore, since the configurations of the embodiments described in this specification are merely the most preferred embodiments of the present invention and do not represent all of the technical spirit of the present invention, various equivalents and modifications that can replace them at the time of the present application It should be understood that there may be Also, throughout the specification, when a part "includes" a certain component, it means that it may further include other components, not excluding other components, unless otherwise stated.

The present inventors conducted research to develop fermentation and separation and purification methods that increase the content of highly functional substances derived from black soybeans that can be used as cosmetic compositions that effectively prevent or improve skin inflammatory reactions and improve skin barriers. As a result, when fermenting black soybean extract or using lignan analogues derived from fermented black soybean, the activity of inducible nitric oxide synthase (iNOS) is inhibited and nitric oxide (Nitric oxide, NO) production was increased, and it was confirmed that there was a significantly improved anti-inflammatory effect compared to the prior art, leading to the present invention.

Therefore, the present invention black bean ( Glycine max Merr. ) Steps of inoculating and fermenting bacteria in the extract; Filtering the fermented product to remove microorganisms; and isolating and purifying the lignan analogues; a method for producing a lignan analogue having an anti-inflammatory function in a fermented black soybean product is disclosed.

In the present invention, the black soybean is an annual plant belonging to the soybean tree and legumes, and is widely distributed in Asia, Africa, Australia, and the like. The place of origin is estimated to be from the Tumen River basin in the northern part of the Korean Peninsula to China's Huabei, centered in the Dongbei Province (Manchuria) of China. In particular, it is cultivated a lot as an edible crop, and black soybeans have been found to have various pharmacological actions, and are developed as cosmetics and used for skin beauty and anti-aging.

In the present invention, the fermented black soybean product refers to a fermented product obtained by drying and extracting black soybeans and then fermenting them. Black soybean extract has been used as a medicinal material effective for various diseases, and is rich in ingredients with various effects such as lecithin and isoflavones, so anticancer, antioxidant, and anticancer effects have been confirmed.

In the present invention, fermented black soybean or purified black soybean can be used as a raw material that acts on the skin to help remove skin inflammatory reactions and improve skin barrier, and by purifying a specific component with efficacy, compared to before purification The effect is further enhanced. At this time, the fermented black soybean product and the fermented black soybean refined product may be used together.

In this fermented black soybean product or fermented black soybean purified product, the highly functional active ingredient that exerts efficacy on the skin is lignan or lignan analogue, and the method for increasing the content of the highly functional substance in the present invention is It is performed through the steps of inoculating and fermenting microorganisms to prepare fermented products and removing the microorganisms from the fermented products.

In the present invention, the step of preparing a fermented product by inoculating the black bean fruit extract with microorganisms and fermenting it is a step for increasing the content of functional substances of the black bean fruit.

The black bean fruit extract may be prepared using a conventional extraction method known in the art, for example, a solvent extraction method. The extraction solvent used in the preparation of the mixed extract using the solvent extraction method is water, a lower alcohol having 1 to 4 carbon atoms (eg, methanol, ethanol, propanol and butanol) or a mixture thereof, such as hydrous lower alcohol, propylene glycol, 1, It may be selected from the group consisting of 3-butylene glycol, glycerin, acetone, diethyl ether, ethyl acetate, butyl acetate, dichloromethane, dichloromethane, hexane, and mixtures thereof, among which is selected from water, alcohol, or mixtures thereof Preferably, when alcohol is used as the extraction solvent, the alcohol is preferably a lower alcohol having 1 to 4 carbon atoms, and the lower alcohol is more preferably selected from methanol or ethanol, and even more preferably ethanol. there is.

Here, when ethanol is used as the extraction solvent of the black bean fruit, for example, 99.5% (v / v) ethanol can be added in a weight of 5 to 20 times that of the black bean, and extracted at room temperature for 18 to 30 hours. In this case, the sugar content of the black bean extract may be 4 to 10 brix, preferably 6 to 8 birx.

In addition, when the extraction of the black bean fruit is performed by hot water extraction, for example, purified water may be added in an amount of 5 to 20 times the weight of the black bean fruit and extracted at 100 to 140 ° C. for 4 to 8 hours. , The sugar content of the black bean fruit extract may be 4 to 10 brix, preferably 6 to 8 birx.

The microorganism used for fermentation in the present invention may be a Bacillus sp. strain, a Lactobacillus sp. strain, or a mixed strain thereof, and the Bacillus sp. strain is Bacillus methyl. It may be Bacillus methylotrophicus , Bacillus subtilis or Bacillus licheniformis , and the Lactobacillus sp. strain may be Lactobacillus paracasei . there is. In the present invention, in terms of maximizing the content of the highly functional active ingredient, preferably, mixed strains of Bacillus subtilis, Bacillus methylotropycus and Bacillus licheniformis may be used in a weight ratio of 1: 0.8 to 1.2: 0.8 to 1.2.

In the present invention, fermentation refers to a process in which enzymes possessed by microorganisms change raw materials to produce beneficial ingredients. During the fermentation process, active ingredients are extracted and the content of functional substances increases. In addition, in the present invention, the fermentation is adjusted to 10 ⁷ to 10 ¹⁰ cfu / ml of the culture medium of the microbial strain, and 0.1 to 10% by weight, preferably 0.5 to 5% by weight of the black bean extract is inoculated. It may be carried out at 30 to 50 °C, preferably 35 to 45 °C for 1 to 5 days, preferably 2 to 4 days.

In addition, the composition of the medium during the microbial culture and fermentation is 0.5 to 2% by weight of beef extract, 1 to 3% by weight of enzyme extract, 3 to 7% by weight of peptone, and 3 to 7% by weight of sodium chloride in terms of maximizing the content of highly functional active ingredients and distilled water (remaining amount).

The step of preparing a fermented black bean product by removing microorganisms from the fermented product is a step of removing microorganisms to prevent further fermentation of the fermented black bean product, and the removal of the microorganisms may be removed through centrifugation and filtration, e.g. For example, after separating the microorganisms from the fermented product through a centrifuge, the microorganisms may be removed by filtering with a 0.1 to 0.3 μm filter.

In the present invention, separation and purification of the fermented black soybean product may be performed through solvent fractionation and chromatography. Solvent fractionation of the fermented black bean product is a step of purifying the fermented black bean product to extract a target component, and the solvent fractionation may be sequentially performed through hexane, dichloromethane, ethyl acetate, butanol and water. In addition, it may be purified through a column after the solvent fractionation.

In addition, in the present invention, the anti-inflammatory component identified through the fermented purification of black soybean may be a lignan analogue derived from black soybean fruit, and the molecular weight of the lignan analogue may be around 325.

In the present invention, the cosmetic composition may include 1 to 90% by weight, preferably 1 to 20% by weight of the fermented black soybean product or the fermented black bean refined product. In addition, the cosmetic composition may include both the fermented black soybean product and the fermented black soybean refined product, respectively, in an amount of 1 to 20% by weight.

In another aspect, the present invention provides a cosmetic composition containing the lignan analog prepared by the above method.

The cosmetic composition according to the present invention contains fermented black soybeans with increased lignan content, thereby inhibiting the activity of inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO). It was confirmed to be very good, and also the effect of promoting the production of lagen through skin cell regeneration and the effect of improving the regeneration ability of skin cells were also confirmed to be very good.

The cosmetic composition according to the present invention may be applied in a gel type, skin type, cream type, ointment type, etc., but is not limited thereto. The above composition can be appropriately prepared by a known method by adding suitable conventional softeners, emulsifiers, thickeners or other materials known in the art according to its type.

The gel-type composition may be prepared by adding a softening agent such as trimethylolpropane, polyethylene glycol or glycerin, a solvent such as propylene glycol, ethanol, or isocetic alcohol, purified water, or the like.

The skin type composition contains fatty alcohols such as stearyl alcohol, myristyl alcohol, behenyl alcohol, arachidyl alcohol, isostearyl alcohol and isocetyl alcohol, butylene glycol, glycerin, allantoin, methyl paraben, EDTA- It can be prepared by adding 2-sodium, xanthan gum, dimethicone, polyethylene glycol-60 hydrogenate, castol oil, polysorbate 60, and purified water.

The cream-type composition includes fatty alcohols such as stearyl alcohol, myristyl alcohol, behenyl alcohol, arachidyl alcohol, isostearyl alcohol, and isocetyl alcohol, lecithin, phosphatidylcholine, phosphatidylethanolamine, sofphatidylserine, sofphatidyl Lipids such as inositol, derivatives thereof, emulsifiers such as glyceryl stearate, sorbitan palmitate, and soribitan stearate, natural fats or oils such as avocado oil, apricot oil, babassu oil, vitreous borage oil, camellia oil, It can be prepared by adding a solvent such as propylene glycol and purified water.

The ointment-type composition may be prepared by adding a softener, an emulsifier, and a wax such as microcrystalline wax, paraffin, ceresin, beeswax, spermaceti, and vaseline.

The cosmetic composition of the present invention may further contain at least one anti-inflammatory component exhibiting the same or similar function in addition to the active ingredient. The anti-inflammatory component may be at least one selected from Portulaca oleracea extract, folding screen extract, dipotassium glycyrrhizate, and ammonium glycyrrhizate, anti-inflammatory functional peptides, but is not limited thereto. In addition, the cosmetic composition of the present invention may further contain excipients including fluorescent substances, fungicides, hydrotropes inducing substances, moisturizers, fragrances, fragrance carriers, proteins, solubilizers, sugar derivatives, sunscreens, vitamins, plant extracts, and the like. can

In the present invention, the cosmetic composition is softening lotion, astringent lotion, nutrient lotion, eye cream, serum, nutrient cream, massage cream, cleansing cream, cleansing lotion, cleansing foam, cleansing water, powder, essence, pack, hair tonic, hair treatment It can be formulated as a ment, shampoo or rinse.

The cosmetic composition may be formulated by conventional methods. In the formulation of external skin preparations, reference may be made to the contents disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA, and in the formulation of cosmetic compositions, International Cosmetic Ingredient Dictionary, 6th ed (The cosmetic, Toiletry and Fragrance Association , Inc, Washington, 1995) may be referred to.

Specifically, the cosmetic composition may be prepared in a general emulsified formulation and solubilized formulation. For example, cosmetic lotion, such as softening lotion or nourishing lotion; emulsions such as facial lotion and body lotion; creams such as nourishing creams, moisture creams, and eye creams; essence; cosmetic ointment; spray; gel; pack; sunscreen; makeup base; foundations such as liquid type, solid type or spray type; powder; makeup removers such as cleansing creams, cleansing lotions, and cleansing oils; Alternatively, it may be formulated as a cleanser such as a cleansing foam, soap, body wash, etc., but is not limited thereto. In addition, the skin external preparation may be formulated as an ointment, patch, gel, cream or spray, but is not limited thereto.

In addition to the essential components in each formulation, the cosmetic composition may be appropriately mixed with other components within a range that does not impair the purpose of the present invention depending on the type of formulation or purpose of use.

The cosmetic composition may include a conventionally acceptable carrier, and for example, oil, water, a surfactant, a moisturizer, a lower alcohol, a thickener, a chelating agent, a colorant, a preservative, a flavoring agent, and the like may be appropriately blended, but are not limited thereto. not.

The acceptable carrier may vary depending on the formulation. For example, when formulated into an ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanthate, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide or any of these as a carrier component Mixtures may be used.

When the cosmetic composition is formulated as a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, or a mixture thereof may be used as a carrier component, and in the case of a spray, chlorofluoro It may further contain a propellant such as hydrocarbon, propane, butane or dimethyl ether.

When the cosmetic composition is formulated as a solution or emulsion, a solvent, solubilizing agent, or emulsifying agent may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil may be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, fatty acid esters of glycerol, polyethylene glycol or sorbitan may be used.

When the cosmetic composition is formulated as a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth and the like can be used.

The cosmetic composition includes fatty substances, organic solvents, solubilizers, thickeners, gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, and fragrances commonly used in the industry depending on the quality or function of the final product. , surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives commonly used in cosmetics It may additionally contain adjuvants commonly used in cosmetology or dermatology, such as any other ingredient. However, the adjuvant and its mixing ratio may be appropriately selected so as not to affect desirable properties of the cosmetic composition according to the present invention.

The cosmetic composition containing the fermented black soybean product or fermented black soybean product prepared according to the present invention as an active ingredient inhibits intracellular NO production and induces nitrogen monoxide It has the effect of inhibiting the activity of inducible nitric oxide synthase (iNOS) and regenerating the skin barrier.

Hereinafter, specific embodiments according to the present invention will be described.

Preparation Example 1

After rinsing the dried black beans with purified water and soaking for a day, 1 kg of purified water was added to 1 kg of black beans, hot water extraction was performed at 121 ° C. for 30 minutes, and after extraction, a black bean extract having a sugar content of 6 to 8 birx was prepared. After preparing the extract, Bacillus licheniformis (purchased from the Korean Cell Line Bank) strain, Bacillus methylotrophicus ( Bacillus methylotrophicus , Apep microrganism S001, purchased from the Korean Cell Line Bank) and Bacillus subtilis ( Bacillus subtilis , Apep microrganism) K007, purchased from the Korea Cell Line Bank) at a weight ratio of 1:1:1 by culturing a mixed strain, 1% by weight of a culture medium having a concentration of 1.0 × 10 ⁹ cfu / ml measured by a spectrophotometer in the black bean extract was inoculated and fermented at 40 ° C for 3 days, and the medium used for culture was a medium composed of 1% by weight of beef extract, 2% by weight of enzyme extract, 5% by weight of peptone, 5% by weight of sodium chloride and distilled water (remaining amount). did After fermentation, the fermentation broth was centrifuged at 4,000 rpm for 10 minutes to separate the bacteria and the culture medium, and then the culture medium was filtered through a 0.2 μm filter to prepare a fermented black soybean product.

Preparation Example 2

2 kg of 80% (v / v) ethanol was added to 200 g of dried black beans, extracted at 25 ° C. for 24 hours, and after extraction, a black bean extract having a sugar content of 6 to 8 birx was prepared. After preparing the extract, Bacillus licheniformis (purchased from Korea Cell Line Bank) strains, Bacillus methylotrophicus ( Bacillus methylotrophicus , Apep microrganism S001, purchased from Korea Cell Line Bank) and Bacillus subtilis ( Bacillus subtilis , Apep microrganism K007, purchased from the Korea Cell Line Bank) at a weight ratio of 1:1:1 by culturing a mixed strain, 1% by weight of a culture medium having a concentration of 1.0 × 10 ⁹ cfu / ml measured by a spectrophotometer in the black bean extract was inoculated and fermented at 37 ° C for 3 days, and the medium used for culture was a medium composed of 1% by weight of beef extract, 2% by weight of enzyme extract, 5% by weight of peptone, 5% by weight of sodium chloride and distilled water (remaining amount). did After fermentation, the fermentation broth was centrifuged at 4,000 rpm for 10 minutes to separate the bacteria and the culture medium, and then the culture medium was filtered through a 0.2 μm filter to prepare a fermented black soybean product.

Preparation Example 3

2 kg of purified water was added to 200 g of dried black beans, and hot water extraction was performed at 120 ° C. for 6 hours to prepare a black bean extract having a sugar content of 6 to 8 birx.

Production Example 4

2 kg of 80% (v / v) ethanol was added to 200 g of dried black beans and extracted at 25 ° C. for 24 hours to prepare a black bean extract having a sugar content of 6 to 8 birx.

purification example

(1) n-hexane, dichloromethane, ethyl acetate and butanol were sequentially added to the fermented black bean prepared in Preparation Example 2 to separate n-hexane fraction, dichloromethane fraction, ethyl acetate fraction, butanol fraction and water fraction to prepare fractions.

(2) After concentrating the separated butanol fraction under reduced pressure, silica column chromatography was performed. At this time, a silica column was used for the column, and the mobile phase solvent was changed by 1 L in the order of distilled water CH ₂ Cl ₂ : MeOH = 4:1, 3:1, 2:1, and proceeded 1) No.3 2 ) No.4~5 3) No.6~7 4) No.8~11 5) No.12~15 6) No.16~19 7) No.20~22 8) No.23~30 9) No.31~35 10) A total of 10 fr.s were separated into No.36~41, and the results are shown in FIG.

(3) Prep the separated fr.1. It was purified by HPLC (C18 ODS column, 3 ml/min). Solvent A: Water in 0.1% TFA, Solvent B: Acetonitrile (ACN) in 0.1% TFA, 85:15 conditions were carried out to purify Compound 1 from fermented black soybean products. The results of HPLC analysis after purification are shown in FIG. 2 . In addition, the overall separation process through the above-described fractionation and purification is shown in a schematic diagram of FIG.

Experimental Example 1

In order to confirm the anti-inflammatory effect of the fermented black soybean product or black soybean extract prepared in Preparation Examples 1 to 4, NO inhibition experiments were performed according to the following method, and the results are shown in Table 1 and FIG. 4 below.

[Experiment method]

LPS (lipopolysaccharide), a substance that induces inflammation, is treated with Raw264.7 cells, which are immune cells, to increase the concentration of NO within the cells. measured. The detailed experimental method is as follows.

(1) After thawing raw cells, prepare cells by culturing HDFn in DMEM-FBS10%. (1.5×10 ⁶ cells/100mm dish)

(2) Seeding the cultured Raw264.7. (1.5×10 ⁵ cells/48well)

(3) Using DMEM-FBS10% medium, after 0.5 ml/48well, culture in DMEM-FBS10% medium for 24h. Prepare DMEM to be used for drug treatment and washing by CO ₂ -37℃-incubation. (O/N)

(4) Washing: Completely remove DMEM-FBS10%, replace with DMEM, and put in incubator.

(5) Drug treatment (DMSO 0.2% in DMEM): 0.3 ml/48well

1) 1 μg/ml LPS treatment: 30 μl/48 well (0.3 ml) of 10 μg/ml LPS in DMEM-FBS10% is added.

2) Prepare a test sample by diluting compound 1 in an appropriate concentration range (at least 3 concentration ranges).

3) Take out the 48 well seeded with Raw264.7, remove the DMEM, and exchange it with the DMEM to which the test sample prepared in 3) is added. (0.5 ml/48 well)

4) Incubate for 24 h.

(6) NO measurement

1) After mixing 50 μl of Griess reagent R1 with 100 μl of the recovered medium supernatant, react at room temperature for 5 minutes.

2) After 5 minutes, 50 μl of R2 solution was mixed and reacted at room temperature for 10 minutes.

3) Measure absorbance at a wavelength of 540 nm.

division NO inhibition ability (%) Control group (untreated group) 0 Preparation Example 1 41 Preparation Example 2 38 Preparation Example 3 11 Production Example 4 8 LPS treatment group 100

Referring to Table 1 and FIG. 4, the NO inhibitory ability can be confirmed in all fermented products or extracts (Preparation Examples 1 to 4), and when fermented (Preparation Examples 1 and 4) than when not fermented (Preparation Examples 3 and 4). It can be confirmed that 2) has excellent NO suppression ability.

In addition, it can be seen that the NO inhibition ability is more excellent in the case of ethanol extraction (Production Example 2) than in the case of hot water extraction (Production Example 1).

Experimental Example 2

The total lignan content of the fractions isolated from the black bean extract of Preparation Example 4, the black bean fermented product of Preparation Example 2 and the purification example was analyzed, and a calibration curve was prepared using Sesamol, Sesaminol, Sesamin and Sesamolin as standards. In order to measure the total lignan content of the sample, the experiment was conducted using a C18 column as basic equipment using HPLC. The mobile phase was measured at MeOH:Water = 8:2, Flow 0.7 ml/min, confirmed at 285 nm, and the measurement results are shown in Table 2 below.

division Total lignan quantitative value (mg/ml) Total amount of lignans in the fraction (mg) total (%) Production Example 4
(black bean extract) 0.195 47.775 - Preparation Example 2
(fermented black soybean) 0.215 52.675 - purification example
(Hexane, n-Hex) 0.002 0.49 0.207 purification example
(Dichloromethane, CH2Cl2) 0.0915 22.418 9.469 Purification example (ethyl acetate) 0.105 25.725 10.866 purification example
(Butanol, n-BuOH) 0.512 135.68 57.312 purification example
(water) 0.214 52.43 22.147 total - 236.74 100

Referring to Table 2, it can be confirmed that the lignan content of the fermented black soybean product (Preparation Example 2) is increased by about 10% in quantitative value compared to the black bean extract (Preparation Example 4), and the fermented black bean fraction (purification example ) can confirm that the lignan content of the butanol fraction is the highest.

In addition, it can be confirmed that the lignan content (mg / ml) of the butanol fraction fermented with black soybean was increased by 2.38 times compared to the fermented black soybean product (Preparation Example 2).

Experimental Example 3

Silica gel thin layer chromatography (TLC) was performed on the fr.1 to 10 isolated in the above purification example in a dichloromethane: 99.5% (v / v) methanol (2: 1) mixed solvent, and the resulting TLC pattern is shown in Figure 5. 5, 3 to 12 denote fr.1 to 10, respectively.

Referring to FIG. 5, as a result of confirming the separated material, it can be confirmed at 254 UV, and as a result of confirming through HPLC, it can be confirmed that it is lignan, and accordingly, the lignan purification conditions can be confirmed.

Experimental Example 4

The content of lignan analogues contained in butanol (butanol layer) fr.3 prep2 separated in the purification example was measured using HPLC, and the measurement results are shown in Table 3 and FIG. 6 below.

division lignan quantitative value
(Lignan content, mg/ml) Total lignans (mg) Purification example (butanol layer) 0.512 135.68 Purification example (Slica column -fr.3) 0.615 215.12 Purification example (HPLC Prep2) 0.695 241.12

Referring to Table 3 and FIG. 6, it was confirmed that the lignan content increased during separation and purification through HPLC (High Pressure Liquid Chromatography), but it could be confirmed that the lignan content did not increase beyond a certain level.

Experimental Example 5

The black soybean fermented product according to Preparation Example 2, the butanol layer of the purification example, and the content of lignan analogues contained in fr.3 prep.2 to 3 were measured using the same HPLC as in the purification example, and the results are shown in Table 4 and FIG. 7.

HPLC analysis result Lignan analogue content (%) black bean extract - fermented black bean Unknown BuOH layer 1.543 Silica gel column 3.15 HPLC prep.1 16.12 prep.2 95.162 prep.3 0.152 prep.4 -

Referring to Table 4 and FIG. 7, in the case of black bean extract (Preparation Example 4) and prep.4 (Purification Example), no lignan analogues (Compound 1) were found, and the fermented black soybean product (Preparation Example 2) case could not be confirmed. In the case of prep 2 (purification example), it was confirmed that the content of lignan analogues was improved to 95% through HPLC content confirmation, and this result is shown in FIG. 7. In addition, when the black soybean extract (Preparation Example 4) was separated and purified in the same step, the lignan analog (compound 1) could not be confirmed. Therefore, it can be confirmed that the lignan analog was produced through fermentation.

Experimental Example 6

In order to confirm the structure of the lignan analogue (compound1) obtained after HPLC purification, experiments were conducted according to the following method, and the results are shown in FIGS. 8 and 9.

[Experiment method]

After preparing a sample at 100 ppm, Mass was measured using Mass (ESI-Mass). Thereafter, using 600 MHz NMR (Agilent Technologies (DD2 600 MHz FT NMR)), the sample was dissolved in MeOD in an NMR-only solvent, and 1H, 13C, HMBC, HMQC, and DEPT were measured to confirm the structure.

8 and 9, in the case of the lignan analogue (compound 1), the exact structure was identified through 1H NMR, 13C NMR, and 2D NMR, and the peak of CH2 was confirmed at δ 1.173 in 1H NMR, and CH2 was found at δ 1.934. The peak of was confirmed. In addition, several peaks of CH and C were confirmed at δ3.575. In addition, C * 6, CH * 5, CH2 * 1, and CH3 * 0 were checked through DEPT to determine the total number of CH, CH2, and CH3, and the combination of each C, CH, CH2, and CH3 was determined through 2D-NMR. confirmed. Through this, when compared with the molecular weight of 325.2274 confirmed through ESI-Mass, it was found that the molecular weight was higher than the number of carbon atoms, and it was confirmed that the C peak overlapped in C NMR. In addition, using HMBC and HMQC of FIG. 8, it was confirmed that the structure had a symmetrical structure around carbon 3. In view of this information, the characteristics of overlapping peaks in the δ7 band and specific peaks appearing across δ3 to 4, which are NMR characteristics of lignans, were confirmed through the NMR of FIG. 10. However, it is understood that it is not a perfectly symmetrical structure like lignans. Therefore, compound 1 was confirmed to be an analogue having a lignan structure.

Experimental Example 7

Raw264.7 macrophages were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 50 U/ml penicillin, and 50 μg/ml streptomycin at 37°C and 5% CO ₂ conditions.

After RNA was isolated from macrophages using Trizol reagent (Invitrogen), cDNA was synthesized by adding AMV reverse transcriptase (Promega) and an oligo dT16 primer. To determine the expression level of iNOS (inducible nitric oxide synthase) mRNA, two primers of 5'CCTCCTCCACCCTACCAAGT3' and 5'CACCCAAAGTGCTTCAGTCA3' were used. In the case of TNF-α, two primers of 5'GGTGAGGAGCACGTAGTCGG3' and 5'TCCCAGGTTCTCTTCAAGGGA3' were used. The amount of each mRNA was quantified by performing real-time PCR.

12 is a graph showing the results of iNOS protein concentration reduction by black bean extract and black bean fermented product according to an embodiment of the present invention.

Referring to FIG. 12, when macrophage Raw264.7 is treated with LPS (Lipopolysaccharide), the macrophage is activated and the production of iNOS protein, NO, and iNOS mRNA expression, which are inflammatory indicators, increase, whereas the fermented black bean according to the present invention When treated with LPS-treated macrophages, it can be confirmed that the protein expression of iNOS decreases according to the degree of isolation of the fermented black bean.

Experimental Example 8

In order to confirm the skin cell proliferative ability of the purified lignan analog (compound 1), the experiment was conducted in the following manner, and the results are shown in FIGS. 13 and 14. Cell proliferation ability test was measured using MTT assay.

[Experiment method]

Human Dermal Fibroblast (HDF) and HACAT cells were seeded in a 24-well plate at a cell number of 4×10 ⁴ cells/24 well per well, and then incubated at 37° C. and 5% CO ₂ conditions for 24 hours. . After washing twice with PBS (phosphate buffer saline), each sample was treated with concentration (1 mg to 1 ug/mL) and incubated for 24 hours. After cultivation, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) 0.5% in DPBS (Dulbecco's Phosphate-Buffered Saline) was mixed with the culture medium at a ratio of 1:9 (v/v). After addition, incubate in a CO ₂ incubator for 2 hours. Then, the produced formazan was dissolved in dimethyl sulfoxide (DMSO), and absorbance was measured at 570 nm using ELISA.

13 and 14, for HACAT cells and HDF cells, it was confirmed that the lignan analogue (compound 1) treated from low to high concentrations had cell proliferation ability in a concentration-dependent manner, and no significant changes were observed even in microscopic observation of the cells. not observed

Through this, it can be seen that the cosmetic composition of the present invention has no toxicity to skin-related cells and appears to have a skin cell proliferation effect. Therefore, it can be predicted that even if the cosmetic composition of the present invention is treated on the skin, it will not have a significant effect.

Experimental Example 9

The effect of inducing skin cell regeneration of a lignan analogue (compound 1) was verified through wound healing assay, which is an experiment that can promote the growth of damaged skin cells and improve cell recovery ability.

Human keratinocytes (HACAT, Human Keratinocyte) to be used in the test were cultured at 37° C. and 5% CO ₂ conditions in a medium containing 10% FBS in DMEM medium. After uniformly scratching the middle of the human skin keratinocyte cell line grown to 100% in a 6 well plate with a tip, washing twice with PBS to remove floating cells, and then adding Purification Example 1 to the medium, respectively 100, 50 and 50 It was treated to be 25 μg/ml (w/v).

The experimental time was based on a total of 24 and 48 hours, and the degree of recovery of the wound was observed through image equipment for each time to confirm the degree of skin cell regeneration, and the results are shown in FIG. 15.

Referring to FIG. 15, it was confirmed that cell growth was active in a concentration-dependent manner in the lignan analogue (compound 1) isolated according to the present invention compared to the untreated control group (Control), and the wound healing effect of skin cells was excellent. was observed to be Through this, it was found that the cosmetic composition according to the present invention has a cell regeneration inducing effect.

Experimental Example 10

In order to confirm the thermal and photostability of the purified lignan analog (compound 1), experiments were conducted in the following manner, and the results are shown in FIGS. 16 and 17.

[Experiment method]

Each sample was dissolved in 50 mM Tris-HCl (pH 8.0) buffer solution so that each sample was 10 mg/mL, dispensed into glass vials, and stored at 50°C for 4 weeks to prevent loss of target substances in the composition due to heat. and the stability was measured under daylight conditions in the same manner.

16 and 17, as a result of measuring the loss of material due to heat and measuring the stability in daylight conditions, all showed a loss of less than 10% of the content up to 4 weeks, indicating high stability in the storage period of heat and sunlight. confirmed that it has.

Manufacturing example of cosmetic composition

A cream-type composition was prepared in the following manner. The cream-type composition was formulated as shown in Table 5 below, including the above-mentioned fermented product in a weight ratio of 1%.

(1) [Phase B] Manufacturing

While heating water to 60 to 70 ° C. while stirring, powdery raw materials are gradually added in sequence, stirred with a disper mixer until thickening, and then evenly dissolved, and then cooled. Prepare in advance for ease of processing.

(2) [Water phase A] manufacturing

After sufficiently impregnating the powdery raw material in glycerin, water is added and heated to 50 to 60° C. to dissolve evenly with a disper mixer.

(3) [Phase A] + [Phase B] Mixed Stirring Preparation

Water phase B prepared in advance is added to water phase A and warmed while mixing evenly. Prepare so that 65 to 75 ° C can be maintained.

(4) [Oil Phase] Mixing and Stirring Manufacturing

Each raw material is weighed in an individual beaker and melted by heating and stirring. (Above 80℃) The melted phase is prepared so that the temperature can be maintained above 75℃.

(5) whole cream preparation

The oil phase is gradually added to the prepared water phase mixture, and the emulsification process is performed by vigorously stirring (6,000 rpm) for about 10 minutes. At this time, the temperature is maintained at 65 ~ 75 ℃.

After completion, additive A is prepared to proceed with pH adjustment and neutralization process. At this time, since the viscosity of the contents rises, stir vigorously (6,000 rpm) evenly for 5 minutes or more.

When the contents cool down below 50℃, add additive B and mix slowly for 2 minutes (3,000~4,000 rpm). After stirring, the contents are collected through a degassing/filtration process.

After completion, the pH adjustment and neutralization process proceeds. At this time, since the viscosity of the contents rises, stir vigorously (6,000 rpm) evenly for 5 minutes or more.

When the contents cool down below 50℃, add a preservative and mix slowly for 2 minutes (3,000~4,000 rpm). ) After stirring, the contents are collected through a degassing/filtration process.

The prepared cream was stored at low temperature (4℃), high temperature (50℃), room temperature (25℃), and sunlight conditions to check the stability of the cream. Discoloration, odor change, phase change, and pH change of the cream for 4 weeks under all conditions. The same phenomenon was not found, and it was confirmed that the stability of the cream was maintained through this. In particular, the stability of the black soybean-derived lignan analogue (compound 1) was maintained, and as a result of HPLC analysis, it was confirmed that the content was maintained at 90% or more for 4 weeks under all conditions. Table 6 below shows the change in content (%) of the black soybean lignan analogue (compound 1) for each formulation storage condition for 4 weeks.

division low temperature (4℃) room temperature (25℃) high temperature (50℃) daylight 1 week later 99 98 97 97 2 weeks later 98 95 95 96 3 weeks later 97 95 92 93 4 weeks later 97 93 90 91

Above, preferred embodiments of the present invention have been described in detail. The description of the present invention is for illustrative purposes, and those skilled in the art will understand that other specific forms can be easily modified without changing the technical spirit or essential features of the present invention.

Therefore, the scope of the present invention is indicated by the following claims rather than the above detailed description, and all changes or modifications derived from the meaning, scope and equivalent concept of the claims are included in the scope of the present invention. should be interpreted

Claims (8)

Black bean ( Glycine max Merr. ) Step of inoculating and fermenting bacteria in the extract;
Filtering the fermented product to remove microorganisms; and
A method for producing a lignan analog having an anti-inflammatory function in fermented black soybeans, comprising the steps of isolating and purifying the lignan analog,
The separation and purification are performed through solvent fractionation and size exclusion chromatography,
The method for producing a lignan analogue, characterized in that the lignan analogue is obtained from the butanol fractionation layer of the solvent fraction, and has a molecular weight of 324 to 326. According to claim 1,
The method for producing a lignan analog, characterized in that the fermentation is carried out at a temperature of 30 to 50 ℃ for 2 to 4 days. According to claim 1,
The method for producing a lignan analogue, characterized in that the bacteria is a Bacillus sp. delete According to claim 1,
The method for producing a lignan analog, characterized in that the lignan analog has an ability to inhibit inducible nitric oxide synthase (iNOS) production and an ability to inhibit nitric oxide (NO) production in macrophages. According to claim 1,
The method for producing a lignan analogue, characterized in that the lignan analogue has the ability to induce skin cell proliferation and skin cell regeneration. delete A cosmetic composition containing a lignan analogue prepared by the method of any one of claims 1 to 3, 5 and 6.

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