KR101978850B1 - A functional cosmetic composition comprising high purity melittin, Ginsenoside Rb1, Rg3 and extracts from fruits of Rosa davurica pall and preparing method thereof - Google Patents

Abstract

The present invention relates to a functional cosmetic composition containing high-purity melittin, ginsenoside Rb1 and Rg3, and raw nuts extract as an active ingredient, and a process for producing the same. More specifically, fermented red ginseng extract containing melitin, ginsenoside Rb1 and Rg3 components having a purity of 90% or more, which is separated from purified bee venom using a fermented alcohol or edible vegetable ethanol such as prtanol A as a mobile phase, The present invention relates to a cosmetic composition for antioxidant, anti-inflammatory or antimicrobial activity and a process for producing the same, which comprises an extract of A. natto fruit containing an butanol fraction of a natto fruit hot water extract as an active ingredient.
Melitin having a purity of 90% or more, which is one of the active ingredients of the cosmetic composition according to the present invention, is a single substance separated from purified bee venom by using vegetable ethanol such as edible fermented alcohol or food additive, But also the antibacterial and anti-inflammatory effects are remarkable. In addition, the addition of the raw nodule fruit extract as an active ingredient of the cosmetic composition can enhance antioxidative and anti-inflammatory functions. In addition, by adding the fermented red ginseng extract containing the ginsenoside Rb1 and Rg3 components as an active ingredient of the cosmetic composition, various effects such as cell regeneration, wound restoration, waste elimination, fatigue mitigation and anti-aging can be expected. Thus, it can be usefully used for the production of functional cosmetics having antioxidative, anti-inflammatory or antibacterial effects by using high purity melittin, ginsenosides Rb1 and Rg3, and raw nuts extract as active ingredients.

Description

TECHNICAL FIELD The present invention relates to a functional cosmetic composition containing high-purity melittin, ginsenoside Rb1 and Rg3, and raw nuts extract as an active ingredient, and a process for producing the functional cosmetic composition and a process for producing the same. davurica pall and preparing method thereof}

The present invention relates to a functional cosmetic composition containing high-purity melittin, ginsenoside Rb1 and Rg3, and raw nuts extract as an active ingredient, and a process for producing the same. More specifically, fermented red ginseng extract containing melitin, ginsenoside Rb1 and Rg3 components having a purity of 90% or more, which is separated from purified bee venom using a fermented alcohol or edible vegetable ethanol such as prtanol A as a mobile phase, The present invention relates to a cosmetic composition for antioxidant, anti-inflammatory or antimicrobial activity and a process for producing the same, which comprises an extract of A. natto fruit containing an butanol fraction of a natto fruit hot water extract as an active ingredient.

Melittin is the most abundant ingredient in bee venom and is also a major component of the antibacterial and antiinflammatory effects of bee venom and is composed of about 40-50% of bee venom. It is medically reported to have arthritis relief, atherosclerosis relief, relief of back pain, treatment of skin wounds, strengthening of the immune system, antibacterial activity, and anti-inflammatory action. And recently it is known that it is involved in the whitening action and the wrinkle-reducing action of the skin, and thus it is known that it can be used for the purpose of beauty.

Korean Patent No. 10-1608045 discloses a method in which pure melittin is separated using a cation exchange resin. However, in the above conventional techniques, it is difficult to recycle the solution, the reagent and the resin because the buffer solution is prepared and used, and the desalination process is performed later, so that the production cost is high and it is not suitable for mass production.

In addition, Korean Patent No. 10-0744755 discloses a gel filtration method. However, melitin (2.8 kD), acanthin (2.02 kD), mCD (2.0 to 3.0 kD) Secapine) are very difficult to separate depending on the molecular weight, and thus have a great influence on the purity and yield of melittin.

On the other hand, most of the bee venom cosmetics are being studied to solve the safety problem due to the allergic stimulation reaction, but the stability problem of the active ingredient is underestimated. (Jung Keun Park et al. , Analytical Science & Technology. , 29 (4): 194-201, 2016) compared the stability of purified bee venom (PBV) and bee venom melitin (BVM) It is a phenomenon of peptide denaturation which is derived from the conductivity of the aqueous solution and the enzymatic component of the bee venom component. Therefore, there arises a problem that the activity and stability of melitin can not be secured.

Recently, as the function of red ginseng or fermented red ginseng is known, the field of cosmetics has been mentioned, and red ginseng cosmetics are being actively developed. This is suggested as medicinal effect and nutritional effect as application value of red ginseng. However, this does not apply to all skin, for example, if it is applied to bacterial infected skin, sensitive skin, or wounded skin, it affects the propagation of bacteria, allergic irritation, and slow wound healing.

The Rosa davurica pall is distributed in Korea, Japan, China, Manchuria, Siberia and Korea. It grows in 200 ~ 1,200m above sea level in Kangwon Province, Hwacheon, Jeongseon, and mountainous valleys. It has been reported that the raw nuts have an ascorbic acid content of about 10 to 30 times that of lemon and a beta-carotene content of 8 to 10 times that of carrots. Ascorbic acid content of the fruit was reported to be 5.3% at the altitude of 500m and 6% at the altitude of 1,000m above sea level. It is known that the pharmacological action of the fruit is an effect of ascorbic acid, and it has an excellent effect on prevention of cancer and anti-aging, so that it has an effect of prevention and treatment of adult diseases.

Korean Patent No. 10-1043813 discloses a skin damaging protective effect against ozone of a cosmetic composition containing a green ginger extract, and Korean Patent No. 10-0460133 discloses a cosmetic composition containing a catechin Is disclosed. When extracted from alcoholic beverages, the pectin component of the fruit has a large molecular weight of about 4-8% and the nonpolar oil component of 5-10%. Therefore, when the pectin ingredient of the raw nuts extract is used as a raw material for cosmetics due to its large molecular weight, it is not absorbed well on the skin and acts as a cause of thin film formation on the skin surface. Therefore, it is necessary to separate and purify the pectin ingredient to minimize the pectin ingredient .

Under these circumstances, the inventors of the present invention have made extensive efforts to develop functional, safe and stable functional natural cosmetics with various functionalities, and as a result, they have found that the use of an edible vegetable ethanol such as fermented alcohol or ptanol A as a mobile phase, Which has an antioxidant, anti-inflammatory, or antimicrobial effect, is used as an active ingredient and fermented red ginseng extract containing melittin, ginsenoside Rb1 and Rg3 components having a purity of 90% Functional cosmetics, thereby completing the present invention.

Korean Patent No. 10-1608045 Korean Patent No. 10-0744755 Korean Patent No. 10-1043813 Korean Patent No. 10-0460133

Jung Keun Park et al., Analytical Science & Technology, 29 (4): 194-201, 2016

It is an object of the present invention to provide a method for producing a cosmetic composition for antioxidant, anti-inflammation or antimicrobial containing high-purity melittin, ginsenoside Rb1 and Rg3, and raw nuts extract as an active ingredient.

Another object of the present invention is to provide a cosmetic composition for antioxidant, anti-inflammation or antimicrobial, which comprises high purity melittin, ginsenoside Rb1 and Rg3, will be.

In order to achieve the above object, the present invention provides a cosmetic composition for antioxidant, anti-inflammation or antimicrobial containing high purity melittin, ginsenoside Rb1 and Rg3, and raw nuts extract as an active ingredient and a method for producing the same.

Hereinafter, the present invention will be described in detail.

In one aspect, the present invention provides a method for producing a cosmetic composition for antioxidant, anti-inflammation or antimicrobial containing high purity melittin, ginsenoside Rb1 and Rg3, and raw nuts extract as active ingredients, comprising the following steps do:

A first step of injecting an aqueous solution of purified bee venom (PBV) into a column containing a fixed bed, adsorbing the purified bee venom, and separating melittin having a purity of 90% or more from purified bee venom using vegetable ethanol as a mobile phase;

A second step of obtaining a fermented red ginseng extract containing ginsenoside Rb1 and Rg3 components from fermented red ginseng powder; And

A third step of obtaining an extract of the raw nevus fruit containing the butanol fraction of the hydrothermal extract of the nasal sprout fruit.

In the present invention, the first step may include, but is not limited to, an adsorption step in which an aqueous solution of purified bee venom (PBV) is injected and adsorbed on a column including a stationary phase; And an elution step of eluting melittin by changing the proportion of mobile phase such that the distilled water: vegetable ethanol is 45:55 to 30:70 (v / v).

In the present invention, the vegetable ethanol may be a fermented alcohol or a prethanol A, and is not limited to any edible vegetable ethanol.

Also, the stationary phase may be a C4 resin though not limited thereto.

In the present invention, the step (2) is not limited to this. However, the method may further include the steps of: adding an aqueous solution of fermented red ginseng to the fermented red ginseng powder; Sequentially adding hexane, chloroform, ethyl acetate and butanol to the extract to obtain respective fractions; And isolating and purifying the butanol fraction layer of the obtained fractions using a chromatographic method to obtain a fermented red ginseng extract containing 8 to 15% of ginsenoside Rb1 component and 35 to 45% of Rg3 component; May include.

The ginsenoside Rb1 component may be included in the fermented red ginseng extract in an amount of 8 to 15%, in an amount of 9 to 14%, in an amount of 10 to 13% 11 to 12%.

In addition, the ginsenoside Rg3 component may be contained in 35 to 45%, 37 to 44%, 38 to 42% in the fermented red ginseng extract, though not limited thereto , And 39 to 41%.

Specifically, the ginsenoside Rg3 component may be 20S-Rg3 and 20R-Rg3, though not limited thereto, and the 20S-Rg3 component is contained in the fermented red ginseng extract in an amount of 15 to 20%, or 17 to 18% , And the 20R-Rg3 component may comprise 20 to 25% or 22 to 23%.

In the present invention, the third step may include, but is not limited to, adding purified water to the raw nodulus powder to obtain a hot water extract; Obtaining respective fractions obtained by sequentially adding hexane, chloroform, ethyl acetate and butanol to the hot-water extract; And separating and purifying the butanol fraction layer of the obtained fractions using a chromatographic method to obtain a bioartificial fruit extract.

In the present invention, the method for producing a cosmetic composition for antioxidant, anti-inflammation or antimicrobial containing the high-purity melittin, ginsenoside Rb1 and Rg3, and raw nuts extract as an active ingredient is not limited thereto, And may be sequentially performed in accordance with the first step, the second step, and the third step, and may be performed in a non-sequential manner without being limited to the respective steps.

In another aspect, the present invention provides a cosmetic composition for antioxidant, anti-inflammation or antimicrobial, which comprises high-purity melittin, ginsenoside Rb1 and Rg3, to provide.

The term " antioxidant " of the present invention means an antioxidant action. The human body has a balance between prooxidant and antioxidant, but due to various factors, And leaning towards oxidative stimulation leads to oxidative stress leading to potential cellular damage and pathological disease. Reactive oxygen species (ROS), which is a direct cause of oxidative stress, is unstable and highly reactive. It reacts easily with various bio-materials, attacks irreversible damage to cells and tissues, Cytotoxicity and carcinogenesis. Therefore, when the high purity melittin, ginsenoside Rb1 and Rg3, and the raw nuts extract of the present invention are incorporated into cosmetics, foods or medicines, they can contribute to anti-aging and health promotion by achieving antioxidant effect.

In an embodiment of the present invention, in order to confirm the antioxidative effect of the raw nuts extract obtained by isolating and purifying from the n -butanol fraction of the raw nuts, the DPPH free radical scavenging activity was measured, It was confirmed that the extract showed an eradication effect of DPPH free radicals in a concentration-dependent manner (FIG. 9). Therefore, the antioxidant effect of the present invention may be achieved by free radical scavenging activity.

The term " anti-inflammatory " of the present invention means an action to suppress inflammation. It is known that the control of the inflammatory reaction is extremely complicated, which is known to reduce the enhancement and damage of the in vivo restoration system. During inflammation, large amounts of pro-inflammatory cytokines, nitric oxide (NO), and prostaglandin (prostaglandin E2, PGE2) are induced by inducible nitric oxide synthase (iNOS) It is produced by cyclooxygenase-2 (COX-2). The inflammation is a cause of various inflammatory diseases. The composition comprising the high-purity melittin, ginsenoside Rb1 and Rg3, and the extract of Ganoderma lucidum fruit of the present invention is effective for preventing and improving various inflammatory diseases through anti- Lt; / RTI >

In one embodiment of the invention, the melittin or raw yeolgwi nut n - separated from the butanol fraction, purified in order to confirm the anti-inflammatory effect of the raw yeolgwi nut extract obtained, RAW 264.7 murine macrophages (RAW 264.7 murine macrophage cells, nitric oxide (NO) production inhibitory activity was measured. As a result, it was confirmed that melittin or raw nuts extract exhibited NO production inhibitory activity in a concentration-dependent manner (FIGS. 5 and 8) . Therefore, the anti-inflammatory effect of the present invention can be achieved by the NO production inhibitory activity.

The term " antibacterial " of the present invention means an action of inhibiting or controlling the growth and proliferation of microorganisms such as bacteria or fungi. The microorganism of the present invention is not limited to this, S. aureus or P. acnes . Many kinds of skin diseases are mainly caused by skin fungi, and acne bacteria and dandruff are typical. The onset of acne has been reported to be the main cause of inflammation reaction of acne bacteria, and dandruid is ideally proliferating on skin such as back and neck, causing seborrheic dermatitis. The cause of atopic dermatitis has not been elucidated yet, but it has been reported as staphylococcus as a cause of atopic dermatitis. Preservatives and antimicrobial agents are essential to reduce the incidence of skin diseases caused by such skin diseases and other bacteria, and to protect the skin. However, synthetic substances used in the past may cause allergic reactions to the skin. It is important to do. Therefore, when the high-purity melittin, ginsenoside Rb1 and Rg3, and the raw virgin fruit extract of the present invention are incorporated into cosmetics, foods or medicines, they can contribute to prevention and improvement of skin diseases by achieving microorganism, .

In one embodiment of the present invention, in order to confirm the antibacterial effect of melitin , antimicrobial activity against Propionibacterium acnes or Staphylococcus aureus strain, As a result of the measurement, it was confirmed that melittin had excellent growth inhibitory activity against both strains (FIGS. 6 and 7). Therefore, the antimicrobial effect of the present invention can be achieved by the activity of inhibiting growth of the skin microorganism.

In the present invention, the high-purity melitin having a purity of 90% or more may be contained in an amount of 0.001 to 0.002% by weight based on the total weight of the cosmetic composition, though not limited thereto.

In the present invention, the fermented red ginseng extract may be contained in an amount of 0.002 to 0.005% by weight based on the total weight of the cosmetic composition, though not limited thereto.

Specifically, in the present invention, the fermented red ginseng extract may contain 8-15% of the ginsenoside Rb1 component, 9-14% of the ginsenoside Rb1 component, 10-13% , And may be comprised between 11 and 12%.

In addition, the fermented red ginseng extract may contain 35 to 45% of the ginsenoside Rg3 component, 37 to 44%, and 38 to 42% of the ginsenoside Rg3 , And 39 to 41%.

In the present invention, the raw virgin fruit extract may be contained in an amount of 0.045 to 0.06% by weight based on the total weight of the cosmetic composition, though not limited thereto.

In the present invention, the cosmetic composition may be formulated into various forms such as cream, lotion, skin, essence, emulsion soap, foam cleansing, pack, shampoo, rinse, body wash and the like.

Specifically, the cosmetic composition of the present invention can be produced in the form of a general emulsified formulation and a solubilized formulation. Examples of the emulsified formulations include nutritive lotions, creams, essences, and the like, and the solubilization formulations include softening longevity. Suitable formulations include, but are not limited to, solutions, gels, solid or paste anhydrous products, emulsions obtained by dispersing the oil phase in water, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes) A cream, a skin, a lotion, a powder, an ointment, a spray, or a conical stick. It may also be in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

The cosmetic composition may further contain at least one selected from the group consisting of fatty substances, organic solvents, solubilizers, thickening and gelling agents, softening agents, antioxidants, suspending agents, stabilizers, foaming agents, perfumes, surfactants, water, ionic or nonionic emulsifiers, Auxiliaries such as ionic sequestering agents, chelating agents, preservatives, vitamins, blocking agents, moisturizers, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredients conventionally used in cosmetic compositions ≪ / RTI >

Melitin having a purity of 90% or more, which is one of the active ingredients of the cosmetic composition according to the present invention, is a single substance separated from purified bee venom by using vegetable ethanol such as edible fermented alcohol or food additive, But also the antibacterial and anti-inflammatory effects are remarkable. In addition, the addition of the raw nodule fruit extract as an active ingredient of the cosmetic composition can enhance antioxidative and anti-inflammatory functions. In addition, by adding the fermented red ginseng extract containing the ginsenoside Rb1 and Rg3 components as an active ingredient of the cosmetic composition, various effects such as cell regeneration, wound restoration, waste elimination, fatigue mitigation and anti-aging can be expected. Thus, it can be usefully used for the production of functional cosmetics having antioxidative, anti-inflammatory or antibacterial effects by using high purity melittin, ginsenosides Rb1 and Rg3, and raw nuts extract as active ingredients.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a chart showing the chromatogram peak of high-purity melittin separated from a purified beaked aqueous solution using a fermented alcohol (vegetable ethanol) (A) or prtanol A (B) according to an embodiment of the present invention.
FIG. 2 is a graph showing a chromatogram peak of an active ingredient fraction of ginsenosides Rb1 and Rg3 through fractional extraction from fermented red ginseng powder according to an embodiment of the present invention. FIG.
FIG. 3 is a graph showing the contents of ginsenoside Rb1 and Rg3 components obtained from n -butanol fraction of fermented red ginseng powder according to an embodiment of the present invention.
FIG. 4 is a flow chart schematically illustrating a process for obtaining n -butanol fraction of Biofungi fruit through fractional extraction from the raw hot water extract, and separating and purifying the same, according to an embodiment of the present invention.
FIG. 5 is a graph comparing the anti-inflammatory effect of purified pills (PBV), which is a reference substance of melittin and high purity (over 90%) of melittin, according to an embodiment of the present invention Graph.
Figure 6 and antibacterial effect (A) of high purity (over 90%), melittin (melittin) for the liquid medium sensitivity Experimental Method Staphylococcus Cocos aureus (S. aureus) by using a strain in accordance with an embodiment of the invention This photograph shows antibacterial effect (B) of bee venom as a reference substance against P. acnes strain. Here, NC is a negative control group and melitin non-treated group, PC is a positive control group and treated with an antibiotic gentamicin.
FIG. 7 is a graph showing a high purity (over 90%) melittin concentration of a P. acnes strain using an MTT solution or an Alamar blue solution according to an embodiment of the present invention. Of the present invention. Here, NC is a negative control group and melitin non-treated group, PC is a positive control group and treated with an antibiotic gentamicin.
Figure 8 is a graph showing the inhibition of NO production by the extract of purified naturally-derived Bovine Saccharomyces cerevisiae isolated and isolated from the n -butanol fraction of the present invention against LPS-induced NO production in RAW 264.7 cells according to an embodiment of the present invention Fig.
FIG. 9 is a graph showing concentration-dependent DPPH free radical scavenging effect of the raw nematode fruit extract isolated and purified from the raw nematode n -butanol fraction of the present invention according to an embodiment of the present invention.

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example  1: Use of edible vegetable ethanol Tablet beige  Derived Mellie Melittin separation

(Purity 95.1 ~ 96.9 v / v%, Gyeongin KFDA No 662-9) or Duksan Chemical Industries Co., Ltd. (Korea) for the separation of high purity melittin from purified water and Samseon Pure Chemical Industries, (V / v, containing 0.2% TFA) to 30:70 (v / v, 50% purity, 95% 0.2% TFA) to remove the high-purity melittin from the mobile phase by a gradient method.

Specifically, purified bee venom [melitin content 64%, Chungjin Biotech Co., Ltd.] was dissolved in distilled water to prepare an aqueous solution of tablets beads at a concentration of 10 to 50 mg / ml. A C4 resin [YMC-Pack C4-HG (S-10 탆 / 12 ㎚ / 50 횞 20 mm I.D.)] column was connected to Prep HPLC to prepare a mobile phase. At this time, it is preferable that water [containing 0.2% TFA (Trifluoroacetic acid)]: fermented alcohol (vegetable ethanol) (including purity 95.1 to 96.9%, 0.2% TFA) is 45:55 % TFA (Trifluoroacetic acid)]: The food additive, Pratanol A (vegetable ethanol) (prethanol A, purity 95%), was 45:55 (v / v) as the mobile phase and stabilized. The stabilized column was automatically injected with 1 ml of a purified beaked aqueous solution sample. Thereafter, the mobile phase ratio (water: fermented alcohol (vegetable ethanol) or water: prtanol A (vegetable ethanol)) was maintained at 45:55 (v / v) in the 0 to 11 min interval and in the 12 to 13 min The polarity of the mobile phase was decreased to 30:70 (v / v), and the elution was carried out at a flow rate of 6 ml / min. During the elution process, melittin eluted at 24.4 ~ 26 min. The time required for the elution process is not limited to that depending on various conditions such as the concentration of the sample, the specification of the column, the condition, the flow rate of the solution, and the temperature.

The mobile phase recovery liquid containing melittin, which was separated from the beeswax component adsorbed on the C4 resin column as described above, was separated and concentrated at low temperature as follows. The fermented alcohol (vegetable ethanol) or the phytanol A (vegetable ethanol) was recovered from the melittin recovery solution at 45 ° C using a reduced pressure rotary condenser, and the remaining distilled water layer was freeze-dried to obtain melittin powder. According to the above procedure, melittin isolated from purified bee venom (45-65%) aqueous solution having a concentration of 10-50 mg / ml was confirmed by chromatogram using Prep HPLC (FIG. 1).

Through the confirmation of the above chromatogram, it was confirmed that high-purity melitin having a purity of 90% or more can be obtained by using a fermented alcohol (vegetable ethanol) or a plant ethanol (edible vegetable) I could.

Example  2: Ginsenoside ( Ginsenoside ) Rb1  And Rg3  Of fermented red ginseng extract

10 kg of 6-year-old fresh ginseng which had been washed was steamed at 95 to 98 ° C for 3 hours to obtain a starch component. The starch component was gelled and then dried at 25 ° C for 10 hours. The dried seaweed was immersed in a soil microbial culture solution at 25 캜 for 2 hours to infiltrate soil microorganisms into the soil tissue. The soil impregnated with the soil microorganisms was injected into the fermentation chamber, and primary fermentation was carried out for 2 days while maintaining the temperature condition at 45 ° C and the humidity condition at 85%. After the first fermentation, the temperature was changed from 70 to 85 캜, and the second fermentation was carried out for 7 days while maintaining the humidity condition at 85 to 90%. After the second fermentation, fermented red ginseng was prepared by drying at a room temperature of 25 ° C. for two days to a water content of 10.8 wt%.

The fermented red ginseng powder thus prepared was pulverized and the fermented red ginseng extract containing the high content of ginsenoside Rb1 and Rg3 components was prepared from the fermented red ginseng powder. At this time, a 70% aqueous alcohol solution was used as an extraction solvent.

More specifically, 150 g of fermented red ginseng powder was added with 1 L of 70% aqueous alcohol solution, and the mixture was extracted with ultrasonic waves at 40 to 50 ° C for 40 minutes. The extract was filtered using a vacuum filtration apparatus, and the filtrate was separated from the filtrate by repeating the same procedure two more times. Then, the filtrate obtained by filtration was concentrated with a rotary evaporator under reduced pressure to obtain a 70% fermented red ginseng extract. Were suspended the obtained 70% fermented ginseng ethanol extract in distilled water, and sequentially fractionated by the polarity order by using a separatory funnel n - hexane (n-hexane, n -Hex) , chloroform (chloroform), ethyl acetate (ethyl acetate, EtOAc), and n - butanol (n -butanol, n -BuOH) in order to obtain a total of 4 fractions solvent layer. A n obtained after fractional extraction - to give the butanol fraction-butanol (n -butanol, n -BuOH) layer fraction was concentrated in a vacuum rotary concentrator 65 ℃ with n-butanol to remove the (n -butanol), and fermented ginseng n. The obtained n - butanol fraction of fermented red ginseng was purified by SPE column (solid phase extraction column) and ODS resin.

ODS resin C18 (40 탆, 200 Å) was mixed in a 60% alcohol solution, left at room temperature for 30 minutes, and then the upper layer solution was removed. By repeating this procedure three more times, the lower layer resin was packed in an SPE column (?: 20 mm, L: 80 mm). After packing, it was stabilized as a mobile phase by connecting with a flow system of Fast protein liquid chromatography (FPLC). At this time, a 10% alcohol was stabilized using a mobile phase. When the baseline was stabilized, the obtained fermented red ginseng n - butanol fraction was loaded on the prepared column. After loading, the n - butanol fraction of fermented red ginseng was eluted with 25% alcohol and 70% alcohol, and the final 70% alcohol extract was recovered. Thereafter, the filtrate was concentrated under reduced pressure and freeze-dried to remove the alcohol and water. Finally, fermented red ginseng extract containing ginsenoside Rb1 (including 11%) and Rg3 (including 40%) was obtained.

According to the above procedure, ginsenoside Rb1 (including 11%) and Rg3 (including 40%) components obtained from the fermented red ginseng powder were confirmed by chromatogram (FIG. 2).

That is, it was confirmed through chromatogram confirmation that the fermented red ginseng extract having a high content of ginsenoside Rb1 and Rg3 components can be obtained through fractionation from fermented red ginseng powder according to the above procedure I could.

Example  3: Fermented red ginseng powder n - Butanol From the fraction  The obtained ginsenoside Rb1  And Rg3  Content analysis of ingredients

In order to analyze the content of ginsenosides Rb1 and Rg3 finally obtained from the n -butanol fraction through extraction, separation and purification from the fermented red ginseng powder in Example 2, the procedure was carried out based on the method of KSH2153 (2013) .

Specifically, a sample of n - butanol fraction obtained by fractionation from fermented red ginseng powder was precisely weighed, and was prepared at a concentration of 1 mg / ml and measured by HPLC (high performance liquid chromatography). At this time, the column used was Agilent HPLC Column XDB-C18, 4.6 × 250 mm, and 5 μm for analysis. Acetonitrile was increased from 20% to 32% during the period of 0 to 40 minutes and increased to 50% during the period of 40 to 55 minutes. It was also increased to 65% during the 55-70 min interval and to 90% at 71 min. At 81 minutes, it was reduced to 20% and maintained at 20% for 110 minutes. The flow rate of HLPC was set at 1 ml / min and the detection wavelength was set at 203 nm.

On the other hand, the qualitative analysis part was confirmed by the peak time of the standard water. As a result, as shown in Fig. 3, the indicator component Rb1 appeared at 41.74 minutes, Rg3 (S) at 57.11 minutes, and Rg3 (R) at 57.79 minutes. The quantitative analysis is expressed as the integral area percentage of the surface component at the peak area (peak width 30s, threshold 50 or more) of all the peaks in the analysis time as the relative content. As a result, it was analyzed that Rb1 contained 11.14%, Rg3 (S) contained 17.61% and Rg3 (R) contained 22.44%.

Example  4: A nostalgic tree  Fruit Heat number  Extracts and their solvents Fraction  Produce

The dried and pulverized Rosa davurica Pall fruit was hydrolyzed with purified water at 90 ° C for 30 minutes. The hot-water extract was cooled to room temperature and then concentrated to 1/3 of the total volume of the extract using a vacuum rotary condenser. The fractions in sequence n in accordance with the polarity order of the obtained raw wood yeolgwi hot water extract using a separatory funnel-hexane (n-hexane, n -Hex) , chloroform (chloroform), ethyl acetate (ethyl acetate, EtOAc), and n - butanol (n -butanol, n -BuOH) in order to obtain a total of 4 fractions solvent layer. A n obtained after fractional extraction-concentration-butanol (n -butanol, n -BuOH) fraction layer at a reduced pressure by rotary concentrator n-butanol to remove the (n -butanol), and raw nuts yeolgwi n-butanol fraction was obtained. The obtained n - n - butanol fraction was purified by SPE column (solid phase extraction column) and ODS resin.

An SPE column was prepared in the same manner as the SPE column packing method of Example 2, and the n - n -butanol fraction prepared above was loaded. After loading, the n - butanol fraction of the raw nuts was eluted with a purified water solution containing 5% alcohol as mobile phase. Thereafter, the obtained eluate was dehydrated by using a rotary evaporator under reduced pressure to obtain a water-soluble component of naturally-occurring nuts nuts which was considered to have antioxidative and anti-inflammatory effects.

As shown in Fig. 4, according to the above-mentioned series of steps, the n -butanol fraction obtained from the raw nodules obtained from the raw nodules obtained by considering the antioxidative and antiinflammatory effects was obtained and separated and purified, Nuts extract could be obtained.

Example  5: Melittin  Analysis of anti-inflammatory effect

To analyze the anti-inflammatory effect of melittin having a purity of 93% isolated in Example 1, nitric oxide (NO) was measured using RAW 264.7 murine macrophage cells (RAW 264.7 murine macrophage cell) ) Production inhibitory activity was measured.

Specifically, RAW 264.7 cells were cultured in DMEM medium (Dulbecco's modified Eagle medium, Gibco, USA) containing 10% FBS (fetal bovine serum), 100 μg / ml penicillin, and 100 U / ml streptomycin Co., USA) at 37 ° C and 5% CO 2. The amount of NO produced from RAW 264.7 cells was measured using a Griess reagent reaction method.

(0, 1, 2, and 4 / / ml) of melittin (purified bee venom) and purified purity of bee venom (PBV) And cultured for 24 hours. At this time, 1 μg / ml of lipopolysaccharide (LPS), which is an inflammatory reaction inducer, was pre-treated 2 hours before the tablet beeing and melittin treatment. After 24 hours of culture, the amount of NO produced from RAW 264.7 cells was measured using a Griess reagent kit as the form of NO ^{2 -} present in the cell culture.

Specifically, the cells were cultured on a 96-well plate for 24 hours. The obtained cell culture medium was mixed with a grease reagent and reacted for 10 minutes. Then, the absorbance was measured at a wavelength of 540 nm.

As a result, as shown in FIG. 5, it was confirmed that the high-purity melittin had better NO production inhibitory activity than the reference bee venom.

Therefore, it was found from the above results that the high purity (90% or more) of melitin isolated from tablets bee venom has an excellent anti-inflammatory effect.

Example  6: Melittin  Antibacterial effect analysis

In order to analyze the antibacterial effect of melittin having a purity of 93% in Example 1, broth microdilution susceptibility tests were conducted to determine the microbial minimum inhibitory concentration (MIC) ) Were measured and the degree of antimicrobial effect was analyzed. Meanwhile, Korean J Vet Serv (2006) 29 (1): 19-26), the bee venom as a reference substance has been reported to have an MIC value of ≥64 μg / ml.

Specifically, the strains of a bacterium Propionibacterium sludge skin flora, causing acne Solarium arc Ness (propionibacterim acnes , P. acnes (KCTC 3314)) and staphylococcus aureus ( staphylococcus aureus) aureus , S. aureus (KCTC 1926)) was used. On the other hand, S. aureus strain was preliminarily cultured for 1 to 3 days so that the inoculation strain was 1 x 10 ⁶ to 1 x 10 ⁷ cfu / ml.

As a test sample for analyzing the antibacterial effect, high-purity melittin isolated in Example 1 and gentamicin (20 μg / ml) as an antibiotic as a positive control were dissolved in distilled water and filtered through a 0.45 μm filter. After that, melitin was diluted to concentration (16, 32, 64, 128, and 256 μg / ml) and 3 ml of nutrient broth was taken in a round bottom tube Respectively. After mixing, the tube was inoculated to each of the said pre Cocos Staphylococcus aureus (S. aureus) strain placed by culturing ^{1 × 10 6 ~ 1 × 10} 7 cfu of strain number / ㎖. After inoculation, overnight incubation was carried out in an incubator, and the turbidity of the medium was visually confirmed (MIC ≥ 16 μg / ml, FIG. 6A).

The bee venom (BV), which is a standard substance, was diluted by 4, 8, 16, 32, 64, 128, 256, and 512 μg / ml by the same method as described above, Turbidity was confirmed (MIC ≥ 32 μg / ml, FIG. 6B).

In addition, high-purity melittin prepared by diluting with a concentration of (16, 32, 64, 128, and 256 / / ml) as described above on a 96-well plate was diluted with Propionibacterium acnes P. acnes (KCTC 3314)) and incubated overnight in an incubator. After incubation, 20 μl of 5 mg / ml MTT (tetrazoliumsalt [3- (4,5-dimethylthiazole-2-yl) -2-5-diphenylterazolium bromide]) solution or 10 × Alamar blue solution Lt; / RTI > After staining, the cells were reacted in an incubator for 1 hour, and then the color change was observed (MIC ≥ 16 μg / ml, FIG. 7).

Therefore, it was found from the above results that the antibacterial effect of high purity (90% or more) melittin isolated from tablets bee venom was remarkably superior to bean poisoning as a reference substance.

Example  7: A nostalgic tree  Analysis of anti-inflammatory effect of fruit extract

In order to analyze the anti-inflammatory effect of the crude extract obtained by isolating and purifying n -butanol fraction from the raw novrebo fruit fraction in Example 4, RAW 264.7 murine macrophage (RAW 264.7 murine macrophage cell) was used to measure nitric oxide (NO) production inhibitory activity.

A specific experimental method was performed in the same manner as in Example 5 above. RAW 264.7 cells were isolated from the n -butanol fractions of Example 4, and then treated with the extracts of the raw nuts obtained from the n -butanol fractions (1, 5, 10, and 20 mg / Lt; / RTI > At this time, LPS (lipopolysaccharide) of 1 / / ml as an inflammatory reaction inducer was pre-treated 2 hours before the treatment of the raw nuts extract. After 24 hours of culture, the amount of NO produced from RAW 264.7 cells was measured using a Griess reagent kit as the form of NO ^{2 -} present in the cell culture.

As a result, as shown in Fig. 8, it was confirmed that the NO production inhibitory activity of the raw nuts extract was better.

Thus, the results showed that the extract of the raw nevus extract obtained from the n - butanol fractions was superior in anti - inflammatory effect.

Example  8: A nostalgic tree  Antioxidative Effect Analysis of Fruit Extracts

In order to analyze the antioxidant effect of the naturally derived nuts extract obtained by isolating and purifying n -butanol fractions from the raw nerves of Example 4, DPPH (2,2-Diphenyl-1-picrylhydrazyl ) Free radical scavenging activity was measured. At this time, DPPH was dissolved in ethanol so as to have a concentration of 0.5 mM, and 1 ml of 0.05 M Tris-HCl buffer (pH 7.4) and 1 ml of ethanol were added and used.

Concretely, serial dilution was performed twice in the concentration range of 0.5 / / ml to 200 / / ㎖ to prepare the raw nematode fruit extract finally obtained in Example 4 by concentration. Then, the DPPH solution prepared as described above and a diluted sample solution of the raw virgin fruit extract prepared above were mixed in a 96-well plate and allowed to react for 30 minutes. Then, using an ELISA reader Absorbance was measured at a wavelength of 517 nm.

As a result, as shown in FIG. 9, the DPPH free radical scavenging activity of the raw nuts extract obtained by isolating and purifying from the n -butanol fraction of the raw nuts was confirmed as a strong antioxidant substance Which is similar to that of

Therefore, it was found that the antioxidative effect of the raw nuts extract obtained by isolating and purifying from the n - butanol fractions of the naturally derived ginseng extract was excellent.

Manufacturing example  1: High purity Melitin , Ginsenoside Rb1  And Rg3 , And A nostalgic tree  Antioxidant, anti-inflammatory or antimicrobial containing fruit extract as active ingredient Cosmetic  Produce

Fermented red ginseng extract containing the high purity melittin and the ginsenoside Rb1 and Rg3 components obtained from the n -butanol fraction of the fermented red ginseng powder isolated from tablets bee venom in Examples 1, 2 and 4, respectively, and The crude extract obtained by isolating and purifying from n - butanol fractions was finally obtained. The final obtained melittin, ginsenoside Rb1 and Rg3, and raw nuts extract were used as active ingredients to prepare functional natural cosmetics.

Manufacturing example  1-1: Toner Manufacturing

The final obtained melittin, ginsenoside Rb1, and Rg3, and a raw material containing the raw nuts extract as an active ingredient were prepared as shown in Table 1 below.

Specifically, in order to prepare toner-type natural cosmetics, Aloe vera gel, Hydrolite-5, 1,3-butylene glycol (1 , 3-Butylene Glycol) and GHDO-6 (1,2-Hexanediol) components (No. 2 to 5 components) were weighed and then heated to 70 ° C. After heating, DI water (No. 1 component) was mixed and mixed for 10 minutes at a speed of 5,000 ~ 5,500 rpm with a homo mixer. After 10 minutes, the heating was stopped and the temperature was lowered to 50 ° C. Adenosine, arbutin, ginseng extract, red ginseng extract Rb1 (including 11%), Rg3 (including 40% Tin (purity of 90% or more) and perfume (No. 6 to 11 components) were added. After the addition, the mixing was maintained and cooled to room temperature.

No. Raw material name Content (% by weight) One Purified water (DI-Water) 56.7388 2 Aloe vera gel (Aloe vera gel) 34.0433 3 Hydrolite-5 2.8369 4 1,3-Butylene Glycol (1,3 Butylene Glycol) 2.8369 5 GHDO-6 (1,2-Hexanediol) 3.4043 6 Adenosine 0.0454 7 Arbutin 0.0454 8 Bergwood extract 0.0454 9 Red ginseng extract Rb1 (including 11%), Rg3 (including 40%) 0.0024 10 Melitin (90% purity or more) 0.0011 11 Spices 2 to 3 drops Sum 100

Manufacturing example  1-2: Lotion manufacturing

Lotions containing the final obtained melittin, ginsenoside Rb1 and Rg3, and raw nuts extract as an active ingredient were prepared as shown in Table 2 below.

Specifically, in order to prepare a lotion for improving the trouble, the raw material No. A (water) of the components shown in Table 2 below was used. After the components A2 to A9 are weighed, Was added to the component A1 and heated to 80 deg. On the other hand, the B (oil phase) raw material No. 3 was separately prepared. The ingredients B1 to B7 were weighed and then heated to 80 DEG C while stirring with a homomixer. Thereafter, the temperature of the raw material A (water phase) and the temperature of the raw material B (oil phase) were raised to 80 DEG C, and the B (oil phase) raw material was added to the raw material A (water phase) after the components were completely dissolved. After the addition, the mixture was emulsified by mixing with a homomixer for 10 minutes at a speed of 5,000 to 6,000 rpm. After emulsification, The B8 component was added and the mixture was cooled to 50 to 55 캜 while maintaining the mixing, and the components C, D and E were added. Thereafter, the mixture was stirred for 10 to 20 minutes, cooled to 45 캜, added with the F-component, and mixed by continuous mixing for 10 minutes.

group
No. Raw material name Content (% by weight) A1 Purified water (DI-Water) 76.157 A2 1,3-Butylene Glycol (1,3-Butylene Glycol) 3.949 A3 Nicotinamide 0.049 A4 EDTA-2Na 0.019 A5 Allantion 0.099 A6 DL-Panthenol (DL-Panthenol) 0.100 A7 Carbopol ultrez 21 0.197 A8 MSM (Methyl Sulfonyl Methane) 0.099 A9 Hydrolite-5 0.987 B1 Squalane 0.211 B2 Silicone oil (PMX-200) 0.211 B3 Lanette O 0.215 B4 Arlacel 165 (Rheodol MS-165V) 0.338 B5 LANETTE® 16 0.127 B6 Olive oil wax (Olivem 1000) 0.632 B7 Monta Wax 68 (Montanov 68) 0.423 B8 DC345 2.106 C1 Purified water (DI-Water) 12.163 C2 L-arginine 0.197 D1 Aloe vera gel (Aloe vera gel) 0.098 D2 Natto fruit extract 400 mg / ml 0.041 D3 80 mg / ml of red ginseng extract containing Rb1 (including 11%) and Rg3 (including 40%) 0.003 D4 Melitin 0.002 E1 1,3-Butylene Glycol (1,3-Butylene Glycol) 0.197 F1 Sensiva® SC 50 0.493 F2 Phenoxyethanol 0.493 F3 Spices 0.049 F4 Lavender Oil 0.049 F5 Vitamin E acetate 0.296 TOTAL 100

Manufacturing example  1-3: Manufacture of water cream

The moisture cream containing the final obtained melittin, ginsenoside Rb1 and Rg3, and raw nuts extract as an active ingredient was prepared as shown in Table 3 below.

Specifically, in order to prepare a water cream, the raw material No. A of the constituent components shown in Table 3 below was used. The components A2 to A11 were weighed and counted. Al component and mixed with a homomixer at a rate of 5,000 rpm. On the other hand, The components B1 and B2 were weighed and then heated to 40 to 45 DEG C until the B2 component completely dissolved and slowly added to the raw material A. Mixing was continued and components C were added after metering. After mixing for about 5 minutes, the mixture was replaced with an impeller stirrer and stirred at a speed of 1,500 rpm to add a preliminarily dissolved D raw material. Thereafter, the mixture was cooled by stirring for 5 to 10 minutes to complete the reaction.

group
No. Raw material name Content (% by weight) A1 Purified water (DI-Water) 78.050 A2 Glycerin 5.174 A3 Allantion 0.155 A4 EDTA-2Na 0.021 A5 Carbopol ultrez 21 0.621 A6 DL-Panthenol (DL-Panthenol) 0.103 A7 Lubrajel ^TM CG 0.517 A8 Plucan-S20 3.104 A9  Aloe vera gel (Aloe vera gel) 0.103 B1 Purified water (DI-Water) 3.104 B2 Dow Corning® 2501 1.035 C1 Natto fruit extract 400 mg / ml 0.06 C2 80 mg / ml of red ginseng extract containing Rb1 (including 11%) and Rg3 (including 40%) 0.005 C3 Melitin 0.002 C4  Aloe vera gel (Aloe vera gel) 1.035 C5 1,3-Butylene Glycol (1,3-Butylene Glycol) 2.069 C6 Centella asiatica 0.103 C7 Sensiva® SC 50 0.517 C8 Phenoxyethanol 0.517 C9 Spices 0.026 C10 Lavender Oil 0.026 C11 Nikkol HCO 60 0.828 C12 Vitamin E acetate 0.052 C13 Hydrolite-5 0.500 D1 Purified water (DI-Water) 2.066 D2 L-arginine 0.207 TOTAL 100

Claims (8)

A first step of injecting an aqueous solution of purified bee venom (PBV) into a column containing a fixed bed, adsorbing the purified bee venom, and separating melittin having a purity of 90% or more from purified bee venom using vegetable ethanol as a mobile phase;
A fermented red ginseng extract was obtained by sequentially adding hexane, chloroform, ethyl acetate and butanol to fermented red ginseng extract and obtaining the butanol fraction of fermented red ginseng extract containing senosides Rb1 and Rg3 A second step; And
And a third step of obtaining a butanol fraction obtained by sequentially adding hexane, chloroform, ethyl acetate and butanol to the raw hot water extract of nuts. A method for producing a cosmetic composition for antioxidant, anti-inflammation or antimicrobial, comprising as an active ingredient ginsenoside Rb1 and Rg3 and a butanol fraction of a raw hot-water extract.
2. The method according to claim 1,
An adsorption step in which an aqueous solution of purified bee venom (PBV) is injected and adsorbed on a column containing a fixed bed; And
The elution step of eluting melittin by varying the proportion of the mobile phase such that the distilled water: vegetable ethanol is 45:55 to 30:70 (v / v), and high purity melittin, ginsenosides Rb1 and Rg3, A method for preparing a cosmetic composition for antioxidant, antiinflammation or antimicrobial containing an butanol fraction of hot water extract of Eucalyptus japonica as an active ingredient.
The method according to claim 1, wherein the vegetable ethanol is fermented alcohol or high purity melitin, ginsenoside Rb1 or Rg3, and a butanol fraction of raw hot water extract Gt; antioxidant, anti-inflammatory or antimicrobial < / RTI >
The antioxidant, anti-inflammatory or antibacterial agent according to claim 1, wherein the fixed phase is a C4 resin, which comprises as an active ingredient a high purity melittin, ginsenoside Rb1 and Rg3, and a butanol fraction of a raw hot- Gt;
delete delete An antioxidant, anti-inflammatory, anti-inflammatory, anti-inflammatory, anti-inflammatory, anti-inflammatory, anti-inflammatory, anti-inflammatory, anti-inflammatory, anti-inflammatory, anti- Or an antibacterial cosmetic composition,
Wherein the butanol fraction of fermented red ginseng extract having 0.001 to 0.002% by weight of melitin, 8 to 15% of ginsenoside Rb1, and 35 to 45% of Rg3 component based on the total weight of the cosmetic composition is in the range of 0.002 - 0.005% by weight, and the butanol fraction of the raw nodulus fruit hydrothermal extract is 0.045% to 0.06% by weight. delete

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