KR20190114233A - Method of manufacturing natural whitening agent through Citrus Junos Seed embryo fermentation - Google Patents

Abstract

The inventors of the present invention, in order to improve the problems appearing in the conventional whitening agent, extract components of Citrus junos seed embryo by a low temperature reduction method, generate bioconversion of the extracted components using a lactic acid bacterium fermentation method, and improve the penetration into the skin by making into ethosome. The present invention also shows an excellent whitening effect due to the synergistic action of the melanin migration inhibitory effect and melanin production inhibitory effect by the tyrosinase inhibitory effect.

Description

Method for manufacturing natural whitening agent through Citrus Junos Seed embryo fermentation

The present invention relates to a natural whitening agent manufacturing method which is harmless to the human body and has a whitening effect by the method of preparing a citron seed fermentation extract having a whitening effect.

      The extraction method implemented in this invention is a low temperature pressure extraction method. For example, water: glycerin (6: 4 weight ratio) at about 4 to 10 ° C. may be used as a solvent to immerse the raw material for about 48 to 120 hours under reduced pressure. The extract can then be filtered and concentrated.

     Lactic acid bacteria fermentation method is used lactic acid bacteria (lactic acid bacteria) or a specific microorganism used to bioconvert the extract is not limited to the type of bacteria that are harmless to the human body, specifically, Lactobacillus (Lactobacillus), Streptococcus (Streptococcus) , Pediococcus, Leuconostoc and the like.

The method for preparing an etosome is a step of producing an aqueous bioactive substance solution, generating a lipid dissolving solution in which lipid is dissolved in ethanol, stirring the biologically active substance solution and the lipid dissolving solution together to hydrate the liquid crystal in which the lipid dissolving solution is hydrated. Generating a phase and adding purified water to the hydrated liquid crystal and stirring to generate an etosome solution, and encapsulating a bioactive substance.

In general, the skin functions to protect the body from ultraviolet rays. That is, when ultraviolet rays are irradiated, the formation of tyrosinase increases in melanocytes (melanoocytes), and black or red melanin pigments are produced and released and distributed to epidermal cells to block ultraviolet rays. It will be a screening function. At this time, ultraviolet rays and tyrosinase play a key role in the melanin production pathway. In particular, the activity of tyrosinase is promoted in melanocytes, thereby increasing melanin production and eventually blackening the skin and blocking ultraviolet rays. However, excessive production and deposition of melanin pigments causes appearance problems such as blackening of the skin, blemishes, and freckles. Accordingly, a whitening agent and a whitening cosmetic containing the same are required to prevent their skin symptoms.

Conventional whitening ingredients are mainly used for skin whitening, ascorbic acid, kojic acid, kojic acid derivatives, hydroquinone, arbutin, and gourd extracts, cluster extracts, upper lichen extracts, Some of the plant extracts are used, such as the extracts of the mulberry tree, but these conventional whitening cosmetics have poor stability, and thus, the whitening effect of the skin is unclear, and the safety of the skin is also lowered. In particular, the whitening effect rapidly increases over time. There was a problem falling. Ascorbic acid and kojic acid are easily decomposed and very unstable by air, heat, and light. Cosmetics containing small amounts of it can cause browning if stored for a few weeks at room temperature, cause skin cancer, and arbutin is known to be cytotoxic. There are problems such as this poor one. In addition, the whitening agent containing a plant extract component has a limit in its efficacy or in addition, the above-mentioned problems were often present in some cases.

In addition, whitening using physical and chemical methods include chemical peeling, laser treatment, and electro-ionography, but chemical peeling is effective for the epidermal layer but not for the dermal layer. In addition, the laser procedure is expensive, it is difficult to remove the blemishes at one time, there is a concern that the pigmentation is concerned that it can be used only locally.

The mushrooms used as the whitening efficacy raw materials so far known are Snow Cordyceps sinensis (Patent Registration No. 10-0340185-0000), Skirt Mushroom (Patent Application Nos. 10-1999-0004497, No. 10-1999-0076537, Patent Registration No. 10-) 0295623-0000), oyster mushroom (patent application No. 10-2000-0007339), woody mud mushroom (patent registration 10-0320743-0000), white cloud mushroom (patent registration 10-0530593) They were also weak in air, heat, light, pH (hydrogen ion concentration), and it was difficult to expect the desired effect.

However, until now, no whitening agent has been reported using citron seed germ components using the lactic acid bacteria fermentation method.

The cosmetics industry is continuously researching to absorb the ingredients of cosmetics as much as possible to make the ingredients penetrate the cell membrane and affect the cell mechanism by utilizing the yuja seed embryo fermentation extract with whitening effect. To increase transdermal absorption, drug delivery formulations are most commonly used, the most representative of which is liposomes. Liposomes are drug carriers that are composed of double lipid membranes similar to cell membranes and can contain water-soluble bioactive substances. Liposomes have been widely used in medicine and cosmetics because they can deliver various active substances in vivo and have high biocompatibility.

However, in recent years, various modified forms of liposomes have been actively studied due to problems such as physical instability, low emulsification stability, low collection efficiency of active ingredients, and the like. Representative examples are etosomes and elastic liposomes. Etosome is a method to enhance skin absorption effect than liposomes. It contains ethanol, which reduces interfacial tension of phospholipids and softens the membrane of vesicle itself.

The problem of etosome, which is used directly on the skin as a cosmetic raw material, is the low encapsulation rate and ethanol content. The amount of ethanol contained in the developed etosome so far is 20 to 40%, which is very high in ethanol.

For example, Korean Patent Laid-Open Publication No. 10-2014-0101570 relates to an etosome containing evergreen extract, which has an ethanol content of 20% or more. In addition, US Patent No. 5,540,934 was also a composition containing 20 to 46% of the content of ethanol. However, the high ethanol content of etosome is not suitable as a cosmetic raw material, it is difficult to apply a high content of the raw material and has high skin irritation, and ethanol promotes moisture evaporation of the skin to dry the skin and is not suitable as a cosmetic raw material. In addition, the etosome has a disadvantage that the sealing rate is low because of the flexibility of the membrane of the vesicle.

For example, the above-described Korean Patent Publication No. 10-2014-0101570 shows that the encapsulation rate is about 54% as a result of capturing 0.1% of isoquercitrin with etosome. This means that about half of the substance encapsulated in the etosome is dissolved in the solution without being trapped in the carrier. That is, even if the transdermal absorption rate of the etosome is very high, it means that the drug delivery efficiency is not high.

Therefore, it is necessary to develop the etosome having the highest transdermal absorption rate as a carrier having a high encapsulation rate and controlling the content of ethanol to be suitable for cosmetic raw materials.

The present inventors have made diligent efforts to improve the problems shown in the conventional whitening agents, and extracts the citron seed embryos by low temperature reduction method, and the extracted components are subjected to etosome by generating bioconversion (BIO CONVERTION) using the lactic acid bacteria fermentation method. The present invention was completed by discovering that the skin whitening effect is improved due to the synergistic action of the melanin production inhibitory effect by the tyrosinase activity inhibitory effect, and the melanin migration inhibitory effect.

Therefore, it is an object of the present invention to provide a natural whitening agent with enhanced whitening effect. Other objects and advantages of the invention will be apparent from the following examples and claims.

The present invention not only contains and selects a substance that inhibits the production of melanin pigments while blocking ultraviolet rays for skin whitening, and also whitens the skin through decomposition of melanin pigments already produced and distributed in the epidermal and dermal layers and suppression of pigment dispersion. In order to improve the stability while exerting the multi-functional function for the development, it extracts the components of the citron seed embryos and ferments them by applying the bioconversion technology through the lactic acid bacterium fermentation method to develop natural whitening agent and penetrate into the skin as this whitening agent. In order to increase the effect of the etosome to solve the problem (problem) for conventional skin whitening.

1: DPPH radical scavenging activity (% of control)
[FIG. 2] ABTS radical cation decolorization
[Figure 3] Tyrosinase inhibition activity (%)

The liquid fermentation process is for example a bacterium such as Lactobacillus fermentum, Lactobacillus brevis, Lactobacillus plantarum or Saccharomyces cerevisiae, Leucono Yeasts such as leuconostoc mesenteroites can be used.

This liquid fermentation process, for example, the liquid extract may be run with the bacteria or yeast at a temperature of 20 to 35 ℃ for about 1 to 10 days, or 2 to 6 days, the fermentation broth is again in the centrifuge 10,000 to 20,000 Centrifugation can be performed for 10 to 30 minutes at rpm. After the centrifuged supernatant can be separated and concentrated.

According to one embodiment, the solid phase fermentation may be performed by fermenting the extract in powder form with Lactobacillus bacteria, preferably Lactobacillus plantarium, in MRS medium for 2 to 6 weeks. Such solid fermentation may be, for example, at 20 to 35 ° C. The fermented material can be extracted by a method suitable for food standards (purified water, alcohol, etc. solvent extraction) and then applied to a whitening agent having a whitening effect.

As described above, the yuza seed embryo fermentation extract obtained by various extraction methods is non-toxic to human body since it is derived from natural ingredients. Therefore, it has the advantage that it is possible to eat.

According to one embodiment, the extract may be etosome (liposomal) to improve the skin absorption of the citron seed embryo fermentation extract. Citron seed germ extract, lecithin and ethanol are mixed to 0.1 to 10.0% by weight, respectively, and then heated to 60 ° C. to dissolve completely.

The mixture is slowly added to 60 ° C. purified water and stirred at 2,000 rpm for 5 minutes using an azimixer and then quenched to 30 ° C. The microfluidizer is passed three times at 1,000 bar to obtain a uniform etosome.

According to one embodiment, such citron seed embryo extract may be used as a whitening agent in an amount of about 0.1 to 10% by weight, respectively, based on the total weight of the composition. The whitening effect less than the content is not sufficient, if there is more than the content there is a fear that the economic efficiency is lowered.

According to one embodiment, the whitening agent according to the present invention may or may not include, in addition to the extract, a binder excipient of dextrin, glycine, and starch in an amount of 0.1 to 10% by weight based on the total weight of the composition.

According to one embodiment, the sunscreen cosmetic whitening agent of the present invention may have a formulation according to a conventional cosmetic, it is not particularly limited. For example, the cosmetic composition of the present invention can be prepared in a liquid, solid, powder, or ointment formulation. More specifically, the whitening cosmetic composition of the present invention is any one selected from suspensions, solutions, emulsions, pastes, gels, creams, lotions, powders, oils, powder foundations, emulsion foundations, wax foundations, packs, massage creams and sprays It can be one.

These various formulations can be prepared by conventional methods used in the art. That is, while containing the citron seed embryo fermentation extract of the present invention in a predetermined amount may include a variety of components.

Preparation Example 1 Preparation of Citron Seed Embryo Extract

After washing and drying 1,000 g of citron seed purchased commercially, the shell was separated, 600 g of embryos were separated and used for extraction. The citron seed embryos were added to water: glycerine at 6: 4 (weight ratio) by a low temperature extraction method at 4 ° C., and then the active ingredient was extracted under reduced pressure immersion conditions for 72 hours. Subsequently, the resultant was filtered using a filter paper having a pore size of 0.1 micron to remove residual solids to obtain an active ingredient, which was then concentrated under reduced pressure, frozen, and dried to obtain 10 g of a powdery solid.

Preparation Example 2 Preparation of Fermented Extract of Citron Seed Embryonic Lactobacillus

1L of LB variable medium (Luria-Bertani media) and 94L of extract were added to the fermenter, and the fermenter was sterilized by steam. The temperature of the sterilized fermenter was maintained at 40 ° C, and 5 L of lactic acid bacteria culture solution was added to the fermenter to inoculate. At this time, the inoculated lactic acid bacteria medium was precultured with Lactobacillus bacteria in LB variable medium (Luria-Bertani media), and the cell count concentration of lactic acid bacteria was 1 × 10 ⁷ cfu (ColonyFormingUnit) / l. As the LB medium, 10 g / L of tryptone, 10 g / L of sodium chloride (NaCl), and 5 g / L of yeast extract were used on a distilled water base.

The temperature of the fermenter inoculated with the lactic acid bacteria culture medium was adjusted to about 35 ° C. and fermented for about 12 hours to bioconvert the extract. Thereafter, the fermentation broth of the fermenter was filtered sequentially using a filter cloth and a membrane filter to obtain 100 kg of the fermentation conversion extract in liquid form. In addition, it was concentrated and dried to obtain a fermentation extract in the form of a powder.

DPPH assay

In order to examine the antioxidant effect of each extract prepared in Preparation Example 2, free radical scavenging of each extract of different concentration using DPPH (1,1-diphenyl-2-picryl-hydrazyl) assay Activity was measured, ascorbic acid (L-ascobic acid) was used as a positive control, the results are shown in FIG.

1 is a graph showing the free radical scavenging activity according to the concentration of the extract of Preparation Example 2 (25ppm, 50ppm, 100ppm, 500ppm), L-A means ascorbic acid, respectively.

As shown in Figure 1, the yuzu seed embryo fermentation extract increased free radical scavenging activity in a concentration-dependent manner.

1] DPPH radical scavenging activity (% of control)

ABTS assay

In order to determine the antioxidant effect of each extract prepared in Preparation Example 2, extracts of # 1 to # 5, and # 11 of Example 1 at different concentrations using free radicals using ABTS radical cation decolorization Scavenging activity was measured, and ascorbic acid (L-ascobic acid) was used as a positive control, and the results are shown in FIG. 2.

Figure 2 is a graph showing the free radical scavenging activity according to the concentration of the extract of Example 1 (25ppm, 50ppm, 100ppm, 500ppm), L-A means ascorbic acid, respectively.

As shown in Figure 2, the yuzu seed embryo fermentation extract increased free radical scavenging activity in a concentration-dependent manner.

Figure 2 ABTS radical cation decolorization

Tyrosinase Activity Inhibition Assessment

In order to produce melanin, tyrosinase acts as a substrate of tyrosine in the cell to generate dopaquinone, and spontaneous and enzymatic reactions from dopaquinone produce a copolymer and a black pigment melanin.

Specifically, 760ul of 0.1M Tris-HCl buffer (pH 7.0) was added to the test tube to maintain 37%, 10mM L-DOPA 190ul, enzyme solution (intyrosinase) 50ul, and the whitening activity measurement sample (# 1 to Example 1). Extracts of # 5, and # 11) were added to 40ul and mixed well to measure the absorbance change at 475nm. Ascorbic acid, which is widely used in whitening cosmetics, was used as a control, and the untreated group was used as a control. 3 is shown.

Figure 3 shows the tyrosinase activity inhibitory activity according to the extract of Example 1, tyrosinase inhibition activity (Tyrosinase inhibition activity (%)) is calculated by the following equation (1).

[Figure 3] Tyrosinase inhibition activity (%)

Skin Patch Test

Citron seed embryo extract (SEEDWHITE) was evaluated for safety.

Stimulation was evaluated by applying citron embryo extract (in 20% DW) to 10 subjects at 24 hour intervals. As a control, purified water (DW) used as a vehicle was used.

In each test, cumulative human skin reactions were tested and classified into 0 to 4 grades (No visible reaction, Mild erythema, Intense erythema, Intense erythema with edema, Intense erythema with edema & vesicle). The trends in skin reactions were recorded according to water content, water content and laser blood flow. No test subjects showed a grade 1 or higher response.

Table 1 below shows the measurement results of the human patch experiment.

After removing the patch of the test subjects, skin reaction was observed 30 minutes and 24 hours later.

The evaluation criteria were evaluated according to the following criteria reflecting Frosch & Kligman and International Contact Dermatitis Research Group (ICDRG).

Evaluation standard Display Explanation Remarks - Negative response ± Suspicious reaction + Weak reaction (erythema, papules) ++ Strong reaction (erythema, papule, blister)

The average skin reactivity was calculated after 3 minutes and 24 hours after patch removal, and the degree of skin irritation of the test product was determined according to the criteria of each reactivity.

Skin irritation criteria Average skin reactivity Criteria 0.0-0.9 Non-irritating 1.0-2.9 Light stimulation 3.0-4.9 Heavy stimulation 5.0 or more Strong stimulation

result sample 30 minutes after patch removal 24 hours after patch removal Judgment - ± + ++ +++ - ± + ++ +++ D.W (purified water) 10 - - - - 10 - - - - Non-irritating seedwhite
(Yuzu seed embryo extract)
(in DW 20%) 8 - 2 - - 8 2 - - - Light stimulation

As can be seen from the above results, it can be seen that the composition according to the present invention does not cause irritation to the skin.

Claims (3)

Method for producing natural whitening agent containing 0.1-10 wt% extract of whitening active ingredient obtained from low temperature vacuum extraction method from citron seed embryos The method of claim 1,
Natural whitening agents comprising lactic acid bacteria fermentation products, each of which is in an amount of 0.1 to 30% by weight based on the total weight of the composition The method of claim 1,
Natural whitening agent showing a whitening effect that the composition is a liquid, solid, powder, or ointment formulation

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