TWI748553B - Skin repairing extract, composition containing the same and use thereof - Google Patents

Abstract

The present invention relates to skin repairing extract and the composition containing the same and the use thereof. The extract is extracted from fermented Lactobacillus plantarum, which is provided with the effects of whitening and anti-aging. The extract can prevent skin dullness caused by melanin production of skin cells, and prevent skin cells from aging due to oxidation. In addition, the extract can be used as a skin repairing composition together with other substances with whitening or anti-oxidant effects. The composition can be made into external use skin products such as cosmetics, skin care products, sunscreens, etc.

Description

Extract used to repair skin, composition containing the extract and its use

The present invention relates to an extract for repairing the skin and a composition containing the extract. The extract is an extract obtained from a fermented lactic acid bacteria. Specifically, it relates to a Lactobacillus plantarum , BCRC Number 10069) The extract obtained from the fermented product, and the skin repairing composition containing the extract.

Human skin can protect the body from various oxidative substances, heavy metals, ultraviolet rays (UV) and air pollution, and it can prevent viruses, fungi and bacteria from invading the body and act as a protective barrier against water loss.

The structure of the skin is roughly divided into three layers, the epidermis, the dermis and the subcutaneous fat. The dermis contains 90% of the skin, which is composed of collagen, elastin, glycoprotein and hyaluronic acid. Collagen is the main component of the skin and is an important fibrin, accounting for about 25% of the total body protein (by weight). Collagen constitutes most of the connective tissue, and plays important roles such as intercellular matrix function, structural support function, inducing cell division and differentiation function, and providing skin tensile strength function. It is known that ultraviolet rays and active oxygen will reduce the production of collagen and damage its structure. Therefore, it is closely related to the formation of skin aging and melanin precipitation.

In recent years, deteriorating environmental factors (such as excessive ultraviolet rays, pollutant gases discharged from factories or cars, etc.) have caused damage to the skin barrier, which has continued to promote skin aging and dullness. Therefore, it is necessary to develop a cost-effective material that can prevent skin aging and melanin precipitation without side effects, and can be made into a composition that is convenient to use in various forms.

Lactic acid bacteria produce lactic acid through the fermentation of carbohydrates or fiber, and they are bacteria with beneficial effects in the human body. Up to now, about 400 species of lactic acid bacteria have been discovered, and about 18 species have been developed into commercial products.

Lactic acid bacteria enter the human intestines and secrete specific materials through their own metabolic activity. Since these materials are beneficial to the human body, many studies have been conducted on lactic acid bacteria. The effects of lactic acid bacteria in antibacterial, antioxidant, and immune activities have been reported. In recent years, research has focused on the whitening and moisturizing effects of lactic acid bacteria, and the possibility of being used as a cosmetic composition, a skin care composition or a sunscreen composition has greatly increased.

Among the lactic acid bacteria, many studies have been conducted on the effects of lactic acid bacteria, and have been reported for immunity, antibacterial effect, enhancement of skin whitening, and improvement of dermatitis. Among them, plant-derived lactic acid bacteria (POLAB) are lactic acid bacteria isolated from fermented foods of plant raw materials (such as pickles, pickles, or kimchi), which are different from dairy-derived lactic acid bacteria.

Compared with animal lactic acid bacteria, plant-derived lactic acid bacteria not only contain at least 10 times more types, but also have excellent adaptability to the external environment. Especially plant-derived lactic acid bacteria isolated from kimchi have high content of salt, low pH and high content of natural antibacterial materials, which can grow in extreme environments, so plant-derived lactic acid bacteria have excellent productivity of a variety of different physiologically active materials .

Representative physiologically active materials include antibacterial peptides, immunostimulants, and γ-aminobutyric acid (GABA), and their performance includes various physiological functions such as anti-inflammatory and immunosuppressive effects. In addition, lactic acid bacteria are hot Stabilize peptides and produce bacteriocins as antibacterial agents. Bacteriocins are natural antimicrobial proteins or protein materials produced by various microorganisms. They usually refer to materials with sterilization mechanisms against bacterial strains, which are similar in morphology and genealogy For microorganisms that produce bacteriocins. Therefore, we are actively developing materials for cosmetic compositions, skin care products, or sunscreen compositions that use lactic acid bacteria culture extracts or lactic acid bacteria fermented plant extracts containing these materials, and can meet recent preferences for environmentally friendly and safe materials. trend.

At present, consumers not only expect general care effects in cosmetic compositions, skin care products or sunscreen compositions, but also various functions, such as anti-aging, improving fine lines, anti-irritation, anti-inflammatory and whitening effects. In recent years, based on the above-mentioned purposes, various plant extracts have been used in cosmetics, and expectations for products based on new concepts have increased. The new concepts show improved performance characteristics by combining various characteristics. Scope.

In order to comply with the era of environmental protection, the inventor of the present invention has worked hard to study the effects of lactic acid bacteria culture extracts in cosmetics, skin care products or sunscreens, which are safe for the skin without any side effects, and are effectively used to prevent aging and Improve dull skin.

In addition, it is necessary to study the possibility of its development in domestic industrialization. The inventor has confirmed that the extract of the fermentation product of Lactobacillus plantarum (BCRC Number 10069) has the activity of preventing aging and improving skin dullness, thus completing the present invention Content.

The object of the present invention is to provide an extract for repairing the skin and a composition containing the extract, the extract is extracted from the plant lactic acid bacteria culture, wherein the bacterial body is removed.

Another object of the present invention is to provide a method for preventing skin aging and dull skin, including applying to an individual an extract for repairing the skin or a composition containing the extract, the extract is made from plant lactic acid bacteria fermentation It is obtained by extraction, in which the bacterial body is removed.

The first picture is the analysis diagram of mushroom tyrosinase activity; the second picture is the analysis diagram of intracellular melanin content; the third picture is the analysis diagram of intracellular tyrosinase activity; the fourth picture is Use Western blot method for protein quantitative test analysis diagram; Figure 5 is the cell viability test analysis diagram; Figure 6 is the IBMX (c-AMP phosphodiesterase inhibitor) test analysis diagram; Figure 7 is the analysis diagram SP600125 (JNK inhibitor) test analysis diagram; the 8th picture is the PD98059 (MEK1/2 inhibitor) test analysis diagram; the 9th picture is the DPPH scavenging ability test analysis diagram; the 10th picture is the ABTS ⁺ free radical scavenging ability Test analysis chart; Picture 11 is the analysis chart of the reduction ability test; Picture 12 is the analysis chart of the ferrous ion chelating ability test; Picture 13 is the analysis chart of the total phenol content test; Picture 14 is the fibroblast Fig. 15 is the analysis chart of the survival rate test of fibroblasts after H ₂ O ₂ treatment.

In order to achieve the above-mentioned objective, one aspect of the present invention is to provide an extract for repairing skin and a composition containing the extract. The extract is extracted from the fermented material of plant lactic acid bacteria, wherein Body is removed.

In this article, the term "Lactobacillus plantarum" refers to a microorganism belonging to the genus Lactobacillus, which is a Gram-negative bacterium capable of producing lactic acid. The bacteria can be found in fermented foods such as sauerkraut, pickles and kimchi.

Lactobacillus plantarum (Lactobacillus plantarum) strain, with a BCRC number of 10069, purchased from the Culture Center of the Food Industry Development Institute; and to verify the anti-aging and whitening effects of the extract of Lactobacillus plantarum fermentation, the experimental cells used B16F10 mouse skin melanoma cells (Mouse melanoma cells), with a BCRC Number of 60031, purchased from the National Institutes of Health Cell Bank.

As used herein, the term "fermented product" refers to a medium containing by-products produced when a microbial strain is cultivated in a medium, wherein the strain can ingest and metabolize the nutrients contained in the medium. The term "fermented product" includes all formulations formed by using a culture medium (for example, a diluent or concentrate of a culture medium, a dried product obtained by drying a culture medium, a crude product or a purified product of a culture medium, a mixture thereof, etc.).

Specifically, the fermented product may be obtained by culturing a Lactobacillus plantarum strain, wherein the bacterial body is removed after the cultivation. That is, a cultured medium in which bacterial bodies are removed can be used as a fermented product.

More specifically, the extract used to repair the skin can be obtained by the following methods: (a) culturing seed bacteria; (b) preparing fermented product; (c) sterilizing centrifugal filtration; and (d) removing water.

In steps (a) and (b), the medium can be any medium used in the art, as long as the medium can grow strains, and the fermentation product can be recovered after cultivation, preferably a liquid medium, such as MRS medium, etc. ; And the temperature of the culture seed is 35℃, the speed of the incubator is 50~150rpm, preferably 80~120rpm, and the culture time is 8~16 hours, preferably 10~14 hours; and the temperature for preparing the fermented product, The rotation speed of the incubator is basically the same as that of the cultured bacteria, but the fermentation time is 18 to 36 hours, preferably 20 to 30 hours.

In step (c), a high-speed centrifuge is used for centrifugation, with a rotation rate of 1,300 rpm to 3,600 rpm, preferably 2,600 rpm to 3,100 rpm; centrifugation for 15 to 40 minutes, preferably 20 to 35 minutes; Obtain the supernatant, and use a 0.45 μm filter membrane to remove bacterial cells.

In step (d), a vacuum concentrator is used to remove water, or other equipment for removing water can be used.

The method for preparing the extract for repairing skin of the present invention has specific implementations as follows: (a) Cultivation of inoculum: Lactobacillus plantarum is placed in 50 mL of MRS medium and cultured at 35° C. and 100 rpm for 12 hours; (b) ) Preparation of fermented product: Take 1 mL of the Lactobacillus plantarum cultured in step (a), add it to 100 mL of MRS medium, and cultivate it at 35°C and 100 rpm for 24 hours to obtain the Lactobacillus plantarum fermented product; (c) Sterilizing centrifugation Filtration: Centrifuge the fermentation product of Lactobacillus plantarum under the conditions of 3000rpm, 30min and 25℃, and then take the supernatant liquid, and then remove the bacteria of Lactobacillus plantarum with a 0.45μm filter membrane to obtain Lactobacillus plantarum (D) Dewatering: the supernatant obtained in step (c) is completely removed from the water to obtain the extract.

In this article, the term "aging" refers to the oxidation phenomenon of the skin caused by internal genetic inheritance and damage from the external environment; among them, the oxidation phenomenon caused by internal genetic inheritance is mainly due to reactive oxygen species (ROS, reactive oxygen species). The accumulation of by-products of cell metabolism, ROS can cause damage to key cell components such as membranes, enzymes and deoxyribonucleic acid (DNA).

In addition, oxidation caused by damage to the external environment, such as ultraviolet light, will cause cell DNA changes under excessive ultraviolet radiation, causing cumulative damage to the skin; or excessive ultraviolet radiation will cause melanin precipitation, resulting in dull skin, or Further evolved into melanoma (Melanoma).

In the present invention, the composition containing the above-mentioned skin-repairing extract can be cosmetics, skin care products, sunscreens and other skin external products, and these skin external products can be formulated into the following forms: solution, external ointment, cream, Foam, nutritious skin cream, soft skin cream, dressing, soft water, lotion, base cream, essence, soap, liquid detergent, bath lotion, sunscreen, sunscreen oil, suspension, emulsion, paste, gel, lotion , Powders, soaps, detergents containing surfactants, oils, powdered foundations, emulsified foundations, waxy foundations, patches and sprays, but not limited to the above.

In the present invention, the composition containing the above-mentioned skin-repairing extract may further contain at least one acceptable carrier for cosmetics, skin care products or sunscreens, and the carrier may be a general conventional component, such as a surfactant, Humectants, lower alcohols, thickeners, chelating agents, pigments, preservatives, fragrances and combinations thereof, etc., but the components are not limited to the above. In addition, the carrier can be adjusted in an appropriate proportion according to the form of the external skin products.

When the composition of the present invention containing the above-mentioned skin repair extract is an ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, purple cloud can be used as the carrier component English gum, cellulose derivatives, polyethylene glycol, silica resin, bentonite, silica, talc, zinc oxide, etc., but not limited to the above. The carrier component may be used alone or in combination of two or more.

When the composition of the present invention containing the above-mentioned skin repair extract is powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. can be used as carrier components; Especially in the case of spraying, the formulation may further contain propellants, such as chlorofluorocarbons, propane/butane and dimethyl ether, but not limited to the above. The carrier component may be used alone or in a combination of two or more.

When the composition of the present invention containing the above-mentioned skin repair extract is a solution or an emulsion, a solvent, cosolvent or emulsifier (for example, water, ethanol, propylene glycol, 1,3-butane) can be used as a carrier component. Glycol and glycerol, etc.), preferably cottonseed oil, peanut oil, corn seed oil, olive oil, castor oil and sesame oil, glycerol fatty ester, and polyethylene glycol or sorbitan fatty acid ester, but not Limited to the above. The carrier component may be used alone or in a combination of two or more.

When the composition of the present invention containing the above-mentioned skin repair extract is a suspension, a liquid diluent (for example, water, ethanol or propylene glycol), a suspending agent (for example, ethoxylated isostearyl) can be used as a carrier component. Alcohol, polyoxyethylene sorbitol and polyoxyethylene sorbitan), microcrystalline cellulose, aluminum oxyhydroxide, bentonite, agar, and milk vetch gum, etc., but not limited to the above. The carrier component may be used alone or in a combination of two or more.

When the composition of the present invention containing the above-mentioned skin repairing extract is soap, the alkali metal salt of fatty acid, the half ester salt of fatty acid, fatty acid protein hydrolysate, isethion acid, wool can be used as the carrier component. Ester derivatives, aliphatic alcohols, vegetable oils, glycerin and sugars, etc., but not limited to the above. The carrier component may be used alone or in a combination of two or more.

Hereinafter, the content of the present invention will be disclosed in more detail with reference to the following examples. However, for those skilled in the art to which the present invention belongs, these embodiments are only for illustrative purposes, and the present invention is not intended to be limited to these embodiments.

Whitening ability test

Example 1: Analysis of mushroom tyrosinase activity

Mushroom Tyrosinase (Mushroom Tyrosinase) was used for the initial screening. Levodopa (L-dopa) is used as a substrate, and when the product after the reaction of tyrosinase and levodopa substrate is reduced, it can be slightly judged that the extract sample of the fermentation product of Lactobacillus plantarum may be inhibited. The catalytic effect has the possibility of whitening effect.

When keratinocytes are exposed to sunlight, it activates the activity of tyrosinase in melanocytes, which initiates a series of chemical reactions and finally produces melanin. At this time, the melanosomes containing a large amount of melanin are released into the keratinocytes, making the skin dark. Therefore, inhibition of tyrosinase activity is regarded as a key step in reducing melanin production (whitening).

Tyrosinase inhibition rate formula (%)=(B-A)/B×100%

Tyrosinase relative activity formula (%)=A/B×100%

A: The absorbance of the sample group at OD490 B: The absorbance of the control group at OD490

Among them, OD490 refers to the absorbance value at a wavelength of 490nm, which can be measured by a spectrophotometer. Qualitative and quantitative analysis.

Refer to Figure 1, the control group is the inhibition rate calculated by the formula without adding any tyrosinase inhibitor, and then adding the plant lactic acid rods with the concentration of 1.32mg/mL, 6.6mg/mL and 13.2mg/mL The inhibition rate of the extract of the fermentation product after 24 hours of reaction, and the inhibition rate after adding kojic acid and arbutin.

It can be seen from Figure 1 that when the extract of the fermentation product of Lactobacillus plantarum is added, there is indeed an inhibitory effect. When the concentration is increased to 13.2mg/mL, the inhibition rate can reach 40%; although the extraction of the fermentation product of Lactobacillus plantarum Compared with kojic acid and arbutin, a higher concentration is required to achieve a higher inhibition rate. However, the use of high-concentration extracts of Lactobacillus plantarum fermented products has much milder side effects on human skin than kojic acid and arbutin.

Example 2: Analysis of intracellular melanin content

Utilizing the strong alkali properties of sodium hydroxide and high temperature to dissolve the melanin in the cells, and measuring the absorbance value of its OD405, in order to analyze the effect of various treatments on the melanin content in B16F10 mouse skin melanoma cells.

Formula of relative melanin content (%)=A/B×100%

A: The absorbance of the sample group at OD405 B: The absorbance of the control group at OD405

Referring to Figure 2, the control group is the melanin content calculated by the formula without adding any tyrosinase inhibitor, and then adding the fermented product of Lactobacillus plantarum at the concentration of 1.32mg/mL, 6.6mg/mL and 13.2mg/mL The melanin content of the extract after 24 hours of reaction, and the melanin content after adding kojic acid and arbutin.

It can be seen from Figure 2 that when the extract of Lactobacillus plantarum fermented product is added, the melanin content does decrease. When the concentration is increased to 13.2mg/mL, the melanin content decreases up to 40%; although the content of Lactobacillus plantarum fermented Compared with kojic acid and arbutin, the extract requires a higher concentration to achieve a higher melanin reduction. However, as mentioned above, the use of high-concentration Lactobacillus plant fermented extract has side effects on human skin compared with kojic acid, Arbutin is much milder.

Example 3: Analysis of intracellular tyrosinase activity

Using the Freeze-thaw cycle method, the B16F10 mouse skin melanoma cells after the reaction with the extract sample of the fermentation product of Lactobacillus plantarum were frozen at a low temperature of -80°C, and the lysis solution was added to make the cells The moisture forms frost, and then the cells are taken out and thaw at room temperature to restore the frost to a liquid state. This process ruptures the cell and releases intracellular material, where the tyrosinase to be measured is in it.

By adding tyrosinase to catalyze the reaction of levodopa receptor (L-dopa), and analyzing the production of dopachrome, the relative activity of its tyrosinase can be inferred.

The relative activity formula of tyrosinase (%)=A/B×100%

A: The absorbance of the sample group at OD490 B: The absorbance of the control group at OD490

Refer to Figure 3, the control group is the tyrosinase activity calculated by the formula without adding any tyrosinase inhibitor, and then adding the concentration of 1.32mg/mL, 6.6mg/mL and 13.2mg/mL of Lactobacillus plantarum The tyrosinase activity of the extract of the fermented product after 24 hours of reaction, and the tyrosinase activity after adding kojic acid and arbutin.

It can be seen from Figure 3 that when the extract of the fermentation product of Lactobacillus plantarum is added, the tyrosinase activity does decrease. When the concentration is increased to 13.2 mg/mL, the tyrosinase activity decreases by up to 80%; The extract of the fermentation product of Lactobacillus plantarum requires a higher concentration than kojic acid and arbutin to achieve a higher decrease in tyrosinase activity. The side effects of human skin are much milder than those of kojic acid and arbutin.

Example 4: Protein quantitative test analysis

Proteins related to melanin synthesis, such as the first type melanocortin receptor (MC1R), microphthalmia transcription factor (microphthalmia transcription factor) factor, MITF), tyrosinase-related protein 1 (tyrosinase-related protein 1, TRP-1), tyrosinase-related protein 2 (tyrosinase-related protein 1, TRP-2) and tyrosinase (tyrosinase) On the other hand, the phosphorylation of p38 protein and cAMP-response element binding protein (CREB) will promote the expression of ocular-related transcription factor (MITF) and promote melanin production; c- Jun amino terminal kinase (c-Jun N-terminal kinase, JNK) and extracellular signal-regulated kinase (ERK) are phosphorylated, which will promote the degradation of MITF and inhibit melanin production.

The protein quantification test analysis system quantifies the protein concentration of the protein extract by the BCA method (bicinchoninic acid assay). Prepare a standard curve with Bovine serum albumin (BSA), and pipette 25μL of BSA and the protein extract of different concentrations of samples containing dimethylsulfoxide (DMSO) solvent into a microcentrifuge tube, and add 200μL Pierce ^® BCA Protein Assay Kit (A:B=50:1), mix evenly, and place it at 37°C for 30 minutes. After 30 minutes, 70 μL was pipetted to a 96-well plate, and then western blotting assay was performed on each mixed sample.

In the protein quantitative test analysis, by adding α-melanocyte-stimulating hormone (α-MSH), the concentration is 0.264mg/mL, 1.32mg/mL, 6.6mg/mL, 13.2mg/mL And 19.8mg/mL of the extract of the fermentation product of Lactobacillus plantarum, and adding arbutin to the proteins related to melanin synthesis, and observe the protein expression related to melanin synthesis. In addition, because glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is affected by melanin and its performance is imbalanced, its signal strength is used as a reference standard, and the proteins related to the synthesis of melanin are The expression level after adding the extract of the fermentation product of Lactobacillus plantarum was used as a comparison.

Refer to Figure 4, which shows the results of the Western blot analysis method and the comparison chart of the proteins related to melanin synthesis using GAPDH as the reference standard. In order to keep the diagram concise, the diagrams are all represented by English abbreviations; by As can be seen in Figure 4, as the concentration of the extract of the fermentation product of Lactobacillus plantarum increases, the expression level of these proteins related to melanin synthesis decreases significantly, and targets such as tyrosinase, TRP-1, and TRP-2. The effect is particularly significant. The specific data is shown in Table 1. In order to keep the table concise, the English abbreviations are used in the table.

Example 5: Analysis of cell viability

(formazan)結晶而分辨細胞是否存活，由於死亡細胞之粒線體中不具有活性之脫氫酶，所以MTT的顏色仍然為黃色不會產生藍紫色之甲

結晶。而甲

結晶於波長570nm時具有最大吸光值，因此可經由吸光值的測定來計算細胞的存活率。 Cell viability analysis is based on the ability of succinate dehydrogenase in the mitochondria of B16F10 mouse skin melanoma cells to MTT([3-(4,5)-dimethylthiazol-2-yl)-2, The tetrazolium ring of 5-diphenyltetrazolium bromide) in the pale yellow aqueous solution is cut to produce a blue-violet nail

(formazan) crystallizes to distinguish whether the cells are alive or not. Since there is no active dehydrogenase in the mitochondria of dead cells, the color of MTT is still yellow and does not produce blue-violet nails.

crystallization. And A

The crystal has the maximum absorbance at a wavelength of 570nm, so the survival rate of the cells can be calculated by measuring the absorbance.

Cell survival rate formula (%)=A/B×100%

A: The absorbance value of the sample group at OD570 B: The absorbance value of the control group at OD570

Refer to Figure 5, the control group is the cell survival rate calculated by the formula without adding any Lactobacillus plantarum fermentation extract, and then adding the concentration of 1.32mg/mL, 6.6mg/mL, 13.2mg/mL, 19.8mg/ The cell survival rate after 24 hours of reaction with the extract of the fermentation product of Lactobacillus plantarum at mL and 26.4 mg/mL.

It can be seen from Figure 5 that the survival rate of the cells still does not change significantly after adding the extract of the fermentation product of Lactobacillus plantarum, but when the concentration of the extract of the fermentation product of Lactobacillus plantarum is increased to 19.8 mg/mL, the cell survival rate The survival rate began to decrease; when the concentration reached 26.4mg/mL, the survival rate of the cells was only 40%. It can be seen that the highest concentration range of the extract using Lactobacillus plantarum fermentation is about 13.2mg/mL, and the lowest The effective concentration range is about 1.32mg/mL.

Example 6: Analysis of whitening mechanism

By mixing the extracts of IBMX (c-AMP phosphodiesterase inhibitor), SP600125 (JNK inhibitor), PD98059 (MEK1/2 inhibitor) and the fermentation product of Lactobacillus plantarum with cells separately, these inhibitors It can promote the production of melanin; in addition, the above-mentioned inhibitors were mixed with the extract of the fermentation product of Lactobacillus plantarum and then mixed with the cells as a control to observe the mechanism of the extract of the fermentation product of Lactobacillus plantarum on inhibiting melanin production.

First, B16F10 mouse skin melanoma cells with a cell density of 5×10 ⁴ cells/well were mixed with a medium containing 100nM α-MSH into a 24-well culture dish, and placed in a 37°C, 5% CO ₂ incubator for 24 hours ; Then replaced with a medium containing different types of 10μM inhibitors (the control group did not contain inhibitors), placed in an incubator for 1 hour; then replaced with different types of 10μM inhibitors and different concentrations of Lactobacillus plantarum The extract of the fermented product was placed in an incubator for 23 hours; then the medium was removed, washed twice with PBS, 100μL of 1N NaOH solution (containing 10% DMSO) was added, and reacted at 60°C for 1 hour; finally 70μL to 96well, The OD405 absorbance value was measured using an immunoenzyme analyzer (ELISA).

Referring to Figure 6, Figure 7, and Figure 8, the absorbance value when IBMX, SP600125 or PD98059 was added alone did not show any significant changes compared to the control group, and the extract of the fermentation product of Lactobacillus plantarum was added separately. The light absorption value drops significantly, indicating that it has an inhibitory effect on melanin production. In addition, the light absorption value of these inhibitors separately mixed with the extract of the fermentation product of Lactobacillus plantarum rises again, which represents the whitening mechanism of Lactobacillus plantarum and IBMX, Inhibitors such as SP600125 or PD98059 are related to the mechanism of melanin production.

Determination of antioxidant capacity

Example 7: DPPH free radical scavenging ability test analysis

In the process of self-oxidation, lipids will generate free radicals and cause lipid rancidity. Common antioxidants provide hydrogen (Hydrogen doner) to scavenge lipid peroxide free radicals (Peroxyl free radicals), thereby inhibiting the oxidation chain reaction. It goes on.

DPPH (2,2-Diphenyl-1-picrylhydrazyl) is a stable free radical. It has a strong absorbance value at 517nm with ethanol (Methanol) solution, so if the absorbance value is lower, it indicates the antioxidant capacity of hydrogen supply The stronger, the better the antioxidant capacity.

DPPH free radical scavenging rate (%)=(1-A/B)×100%

A: The absorbance value of the sample group at OD517 B: The absorbance value of the control group at OD517

Refer to Figure 9, the blank test is without any antioxidants, followed by the addition of extracts of the fermentation product of Lactobacillus plantarum at concentrations of 0.176mg/mL, 0.88mg/mL, 1.76mg/mL, 4.4mg/mL and 8.8mg/mL The DPPH scavenging ability after 24 hours of reaction, and the addition of vitamin C (Vit C) and butyl hydroxy The DPPH scavenging ability after BHA is shown in English abbreviations for vitamins and butylated hydroxyanisole in order to make the diagram concise.

It can be seen from Figure 9 that when the extract of Lactobacillus plantarum fermented product is added, the DPPH scavenging ability does increase. When the concentration is increased to 8.8mg/mL, the DPPH scavenging ability can reach about 60%; although Lactobacillus plantarum Compared with vitamin C and butylated hydroxyanisole, the fermented extract needs a higher concentration to achieve higher DPPH scavenging ability. However, the use of high-concentration Lactobacillus plantarum fermented extract has side effects on human skin compared with vitamins C. Butylated hydroxyanisole is much lighter.

Example 8: ABTS ⁺ free radical scavenging ability test analysis

ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) is a chemical free radical initiator and also a color developing agent in the reaction. After ABTS is oxidized by active oxygen, it can generate stable blue-green cation ABTS ⁺ . When it is added with a substance with antioxidant activity, it can react with ABTS ⁺ to make the color lighter. Therefore, the lower the absorbance value after the reaction, the stronger the ability of the ^{sample to scavenge ABTS+ free radicals.}

ABTS ⁺ free radical scavenging rate (%)=(1-AB)×100%

A: The absorbance of the sample group at OD410 B: The absorbance of the control group at OD410

Refer to Figure 10, the blank test is that no antioxidant is added, and then the extract of the fermentation product of Lactobacillus plantarum at the concentration of 0.211mg/mL, 1.056mg/mL, 5.28mg/mL, 10.56mg/mL and 15.84mg/mL is added The DPPH scavenging ability after 24 hours of reaction, and the ABTS ⁺ scavenging ability after adding vitamin C (VitC) and butyl hydroxyanisole (BHA). To keep the diagram simple, the vitamins and butyl hydroxyanisole in the figure are all in English Abbreviation.

It can be seen from Figure 10 that when the extract of Lactobacillus plantarum fermentation is added, the ABTS ⁺ scavenging ability does increase. When the concentration is increased to 5.28mg/mL, the DPPH scavenging ability can reach about 100%; although plant lactic acid Compared with vitamin C and butylated hydroxyanisole, the extract of Bacillus fermented product needs a higher concentration to achieve higher ABTS ⁺ removal ability. However, the use of high-concentration extract of Lactobacillus plantarum fermented product has side effects on human skin. It is much milder than vitamin C and butylated hydroxyanisole.

Example 9: Analysis of reduction ability test

According to the production of Prussian blue Fe ₄ [Fe(CN) ₆ ] ₃ as an indicator, the principle is to reduce red blood salt K ₃ Fe(CN) ₆ to yellow blood salt K ₄ Fe(CN) ₆ , and then yellow blood salt again Using Fe ^{3+ to} form Prussian blue, the reducing power is detected by the change in absorbance at a wavelength of 700 nm. The higher the absorbance, the stronger the reducing power.

Reducing power (%)=(A/B)×100%

A: The absorbance of the sample group at OD700 B: The absorbance of the control group at OD700

Refer to Figure 11, the blank test is that no antioxidant is added, and then the extract of the fermentation product of Lactobacillus plantarum at the concentration of 0.042mg/mL, 0.211mg/mL, 1.056mg/mL, 2.112mg/mL and 3.168mg/mL is added The reducing power after 24 hours of reaction, as well as the reducing power after adding vitamin C (Vit C) and butyl hydroxyanisole (BHA), in order to make the diagram concise, the vitamins and butyl hydroxyanisole in the figure are all expressed in English abbreviations .

It can be seen from Figure 11 that when the extract of the fermentation product of Lactobacillus plantarum is added, the reducing power does increase. When the concentration is increased to 3.168mg/mL, the reducing power can reach about 70%; although the fermentation product of Lactobacillus plantarum Compared with vitamin C and butylated hydroxyanisole, the extract of butyl hydroxyanisole requires a higher concentration to achieve higher reducing power. However, the use of a high concentration of Lactobacillus plantarum fermented extract has side effects on human skin compared to vitamin C and butyl Glyoxyanisole is slightly more.

Example 10: Test analysis of ferrous ion chelating ability

Among the metal ions, Fe ²⁺ is the most influential pro-oxidant. ^{The bright red complex of Fe 2+} and ferrozine has a strong absorbance at a wavelength of 562 nm, which can be used to determine the sample’s effect on Fe ²⁺ . Chelating ability. When the sample chelates Fe ²⁺ , it will cause a decrease in the OD562 absorbance value. The lower the absorbance value, the stronger its ability to chelate metal ions. Ethylenediaminetetraacetic acid (EDTA) was used as a positive control group.

Ferrous ion chelating capacity (%)=(1-A/B)×100%

A: The absorbance of the sample group at OD562 B: The absorbance of the control group at OD562

Refer to Figure 12, the blank test is that no chelating agent is added, and then the extract of the fermentation product of Lactobacillus plantarum at the concentration of 0.053mg/mL, 0.106mg/mL, 0.264mg/mL and 0.528mg/mL is added after 24 hours of reaction. The chelating ability and the chelating ability of ethylenediaminetetraacetic acid (EDTA) added at concentrations of 0.02mg/mL, 0.04mg/mL, and 0.08mg/mL. In order to simplify the diagram, ethylenediaminetetraacetic acid is in English Abbreviation.

It can be seen from Figure 12 that when the extract of the fermentation product of Lactobacillus plantarum is added, the chelating ability does increase. When the concentration is increased to 0.528mg/mL, the chelating ability can reach about 90%; although Lactobacillus plantarum Compared with ethylenediaminetetraacetic acid, the fermented extract needs a higher concentration to achieve higher chelating ability. However, the use of high-concentration Lactobacillus plantarum fermented extract has side effects on human skin compared with ethylenediaminetetraacetic acid. Acetic acid is much lighter.

Example 11: Test analysis of total phenol content

Folin-Ciocalteu's phenol reagent was used to detect the content of phenolic compounds in the samples. If there are phenolic compounds in the sample, they will react with the phenolic indicator at a wavelength of 750nm to form a color. The higher the absorbance value, the more polyphenols in the sample and the stronger the antioxidant capacity. Configure the reaction of different known concentrations of gallic acid and phenolic indicator as standard products and make a calibration curve to detect the absorption value of the unknown substance after the reaction with the indicator, and the corresponding calibration curve can measure the phenolic compound content.

Refer to Figure 13, the blank test is the extract without any fermentation of Lactobacillus plantarum, and then add the concentration of 0.66mg/mL, 3.3mg/mL, 16.5mg/mL, 33mg/mL and 49.5mg/mL The total phenol content of the extract of the fermentation product of Lactobacillus plantarum after 24 hours of reaction (using gallic acid equivalent as a reference value).

It can be seen from Figure 13 that when the extract of the fermentation product of Lactobacillus plantarum is added, the total phenol content does increase. When the concentration is increased to 49.5 mg/mL, the total phenol content relative to the gallic acid equivalent can reach approximately One equivalent means that the total phenol content at this concentration is similar to gallic acid.

Example 12: Fibroblast survival rate test analysis

In order to confirm the toxicity of the extract of the fermentation product of Lactobacillus plantarum to the cells at different concentrations, a test was carried out using fibroblasts (WS1) to determine the appropriate concentration of the extract of the fermentation product of Lactobacillus plantarum.

(formazan)結晶物，最後使用免疫酵素分析儀(ELISA)測定產物之OD570吸光值。 Firstly, fibroblasts with a cell density of 5×10 ⁴ cells/well were implanted in a 24-well culture dish, and placed in a 37°C, 5% CO ₂ incubator for 24 hours; after that, the medium was removed, and the culture medium was removed according to different concentrations of plants. Lactobacillus fermented extract, hydrogen peroxide (H ₂ O ₂ ) and vitamin E (Trolox) of specific concentration, mixed with DMEM medium, replaced the medium in 24well, placed in an incubator for 24 hours; then removed the medium, Wash twice with PBS, add 500μL of diluted MTT solution (1mg/mL) to each well, cover the well plate with aluminum foil to protect from light, put it in the incubator for 30 minutes; finally remove the medium, wash twice with PBS, each Add 500μL DMSO to the well to dissolve the blue-violet nail

(formazan) crystals, and finally use an immunoenzyme analyzer (ELISA) to determine the OD570 absorbance of the product.

Cell survival rate (%)=(A/B)×100%

A: The absorbance of the sample group at OD570 B: The absorbance of the control group at OD570

Referring to Figure 14, the control group is the extract of the fermentation product of Lactobacillus plantarum without addition, followed by the addition of the extract of the fermentation product of Lactobacillus plantarum at a concentration of 1.32mg/mL, 6.6mg/mL, and 13.2mg/mL. The reaction is 24 hours later. Fibroblast survival rate; it can be seen from Figure 14 that when adding plant lactic acid rods The survival rate of fibroblasts did not change significantly after the extract of the bacterial fermentation product. When the concentration was increased to 13.2mg/mL, the survival rate of fibroblasts decreased slightly. Therefore, it can be seen that the cell has not been toxic at these concentrations.

Refer to Figure 15, the control group is not added with hydrogen peroxide (H ₂ O ₂ ), the extract of the fermentation product of Lactobacillus plantarum, and vitamin E (Trolox), followed by the addition of hydrogen peroxide, followed by the addition of 0.264 mg. /mL, 1.32mg/mL, 6.6mg/mL, 13.2mg/mL, 19.8mg/mL of Lactobacillus plantarum fermentation extract for 24 hours and vitamin E (Trolox) cell survival rate, in order to make the diagram concise In the figure, hydrogen peroxide, Lactobacillus plantarum and Vitamin E are all expressed in English abbreviations.

It can be seen from Figure 15 that the cell survival rate after hydrogen oxide treatment decreased slightly, but when the extract of the fermentation product of Lactobacillus plantarum was added, the cell survival rate increased significantly, when the concentration was increased to 6.6 mg/mL , The cell survival rate can be increased up to 150%, and both are higher than the cell survival rate of the control group. It can be seen that at these concentrations, the cell has not yet been toxic.

Example 13: Use of extracts from fermentation products of Lactobacillus plantarum

With reference to the experimental data of the extract of the fermentation product of Lactobacillus plantarum described in Examples 1 to 12, it does have whitening and anti-oxidant effects, so it can be used as an active ingredient in a skin repair composition. The skin-repairing composition can specifically be cosmetics, skin care products, sunscreens and other external skin products. These external skin products can be formulated into the following forms: solution, external ointment, cream, foam, nutritional skin cream, soft skin cream, dressing , Soft water, lotion, base cream, essence, soap, liquid detergent, body wash, sunscreen, sunscreen oil, suspension, emulsion, paste, gel, lotion, powder, soap, cleaning with surfactant Agents, oils, powdered foundations, emulsified foundations, waxy foundations, patches and sprays, but not limited to the above.

From the above results, it can be confirmed that the extract obtained from the fermentation of Lactobacillus plantarum (BCRC Number 10069) of the present invention can effectively prevent the formation of melanin, and has good antioxidant capacity, thereby effectively whitening the skin and preventing Dull skin and prevent skin aging.

Those skilled in the art to which the present invention pertains can understand from the foregoing that the present invention can be implemented in other specific forms without changing the technical concepts or essential features of the disclosure. In this regard, the illustrative aspects disclosed in this article are for illustrative purposes only, and should not be construed as limiting the scope of the disclosure. On the contrary, the content of the present disclosure tends to cover not only these exemplary aspects, but also various changes, modifications, equivalents, and various changes, modifications, equivalents, and the spirit and scope of the content of the present invention that can be included in the definition of the scope of the patent application attached hereafter. Other aspects.

Claims (3)

A composition for repairing the skin, the composition comprising an extract obtained from the fermentation of Lactobacillus plantarum registered by the BCRC number 10069, wherein the extract can inhibit the activity of tyrosine amide and inhibit the skin Medium melanin production or inhibition of skin cell oxidation, and the minimum effective concentration of the extract is 1.32mg/mL, the maximum concentration is 13.2mg/mL; and the extract is obtained by the following preparation method: (a) Cultivation of seed bacteria : Put Lactobacillus plantarum in 50mL of MRS medium and culture it at 35°C and 100rpm for 12 hours; (b) Preparation of fermented product: Take 1mL of Lactobacillus plantarum grown in step (a) and add 100mL of MRS medium Cultivation at 35°C and 100rpm for 24 hours to obtain the fermentation product of Lactobacillus plantarum; (c) Sterilization and centrifugal filtration: Centrifuge the fermentation product of Lactobacillus plantarum at 3000rpm, 30min, and 25°C, and then take the supernatant And then remove the supernatant of the Lactobacillus plantarum with a filter membrane with a pore size of 0.45μm to obtain a supernatant with the removed Lactobacillus plantarum; and (d) water removal: the step (c) The water in the supernatant is completely removed to obtain the extract. The composition according to claim 1, which is used as a raw material for a skin external use product. The composition according to claim 2, wherein the external skin product is selected from the group consisting of cosmetics, skin care products and sunscreen products.

Images