CN115006340B - Antioxidant composition, and cosmetics and application thereof - Google Patents

Abstract

The invention relates to the technical field of cosmetics, in particular to an antioxidant composition, and cosmetics and application thereof. The antioxidant composition of the invention comprises the following components: 0.1 to 10 weight parts of lactobacillus fermentation product; 0.01 to 2 parts by weight of nano lipoic acid wrapped by liposome; 0.01 to 0.5 weight portion of VC ethyl ether; 0.001 to 0.1 part by weight of reduced glutathione. Compared with the prior art, the invention organically combines four functional components of lactobacillus fermentation products, liposome-coated nano lipoic acid, VC ethyl ether and reduced glutathione to form a double antioxidation mode mainly with endogenous antioxidation and secondarily with exogenous antioxidation, and has remarkable antioxidation effect.

Description

Antioxidant composition, and cosmetics and application thereof

Technical Field

The invention relates to the technical field of cosmetics, in particular to an antioxidant composition, and cosmetics and application thereof.

Background

Skin aging is an external manifestation of aging in the human body, and one of the factors causing skin aging is: oxidative stress. The sum of the nonspecific responses generated by the very stimulation of the high oxidative activity of the human body towards the outside or inside is called oxidative stress, often accompanied by an increase in free radicals. The nature of this can be thought of as a series of oxidative damage mediated by free radicals, a condition that results in oxidative/antioxidant imbalance in the body. Free radicals are also referred to as "radicals" and refer to atoms or groups that are unpaired electrons. The unpaired electrons make the free radical active and unstable in property, have high oxidation activity, and are very easy to abstract one electron from adjacent molecules to become a stable state, so that new free radicals are mediated. However, both the cells that are deprived of electrons and the cells that acquire electrons, the active ingredients are damaged, and even directly necrotize and die. Free radical mediated oxidative stress is widely involved in numerous pathophysiological changes, ischemia reperfusion injury is an acute manifestation of oxidative stress, and redox state imbalance caused by chronic oxidative stress is an important cause in the occurrence process of tumors, inflammations, aging and the like.

Aging of skin not only affects the appearance and image of human body, but also seriously hampers the physiological function of human skin, and even causes serious pathological changes of skin, so that antioxidant skin care products are attracting attention. At present, the cosmetic at home and abroad has general effect due to unreasonable selection and compatibility of antioxidant components, and has limitation in application of preventing skin from being oxidized.

Disclosure of Invention

In view of the above, the invention aims to provide an antioxidation composition, cosmetics and application thereof, and the antioxidation composition organically combines four functional components of lactobacillus fermentation products, nano lipoic acid, VC ethyl ether and reduced glutathione to form a double antioxidation mode mainly with endogenous antioxidation and secondarily with remarkable antioxidation effect.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

An antioxidant composition comprising the following components:

0.1 to 10 weight parts of lactobacillus fermentation product;

0.01 to 2 parts by weight of nano lipoic acid wrapped by liposome;

0.01 to 0.5 weight portion of VC ethyl ether;

0.001 to 0.1 part by weight of reduced glutathione.

In the present invention, the antioxidant composition comprises the following components:

1-10 parts by weight of lactobacillus fermentation product;

0.1 to 2 parts by weight of nano lipoic acid wrapped by liposome;

0.05 to 0.5 weight portion of VC ethyl ether;

0.02 to 0.1 part by weight of reduced glutathione.

In the present invention, the antioxidant composition comprises the following components:

2.5 to 10 weight portions of lactobacillus fermentation product;

1-2 parts by weight of liposome-coated nano lipoic acid;

0.1 to 0.5 weight portion of VC ethyl ether;

0.05 to 0.1 weight portion of reduced glutathione.

In the present invention, the antioxidant composition comprises the following components:

5-10 parts by weight of lactobacillus fermentation product;

2 parts by weight of nano lipoic acid;

0.3 to 0.5 weight portion of VC ethyl ether;

0.1 part by weight of reduced glutathione.

In one embodiment of the invention, the antioxidant composition comprises the following components:

10 parts by weight of lactobacillus fermentation product;

2 parts by weight of liposome-encapsulated nano lipoic acid;

0.5 parts by weight of VC ethyl ether;

0.1 part by weight of reduced glutathione.

The antioxidant composition comprises four components of lactobacillus fermentation products, liposome-encapsulated nano lipoic acid, VC ethyl ether and reduced glutathione.

1. Lactic acid bacteria fermentation products

Lactic acid bacteria are used as probiotics because they function to break down fibers and complex proteins to produce important nutrients when living in the human digestive system. Lactic acid bacteria have been reported to have effects of maintaining normal intestinal flora, improving intestinal flora, anti-diabetes and anti-hyperlipidemia, inhibiting canceration, inhibiting colitis, inhibiting nonspecific activities of host immune system, and the like. Among these lactic acid bacteria, lactobacillus is a major member of the normal microflora living in the human intestinal tract, and has long been considered to be extremely important for maintaining a healthy digestive tract environment.

The lactobacillus fermentation product of the invention is prepared by the following method: and (3) activating the lactobacillus, culturing in a shaking flask, and culturing in a fermentation tank to obtain a fermentation product of the lactobacillus. The strain activation preferably includes: mixing the culture medium with lactobacillus strain; shake flask culture preferably comprises: mixing the culture medium with the activated strain, wherein strain activation and shaking culture are preferably carried out under shaking conditions, the temperature of shaking culture is preferably 37 ℃, and the time is preferably 8-10h; the fermenter culture preferably comprises: placing the strains and the culture medium after shake flask culture into a fermentation tank for culture; in the process of fermentation tank culture, the pressure is controlled to be in the range of 0.03-0.04MPa, the temperature is preferably controlled to be 37 ℃, the control time is preferably controlled to be 20-24h, the fermentation tank culture is preferably carried out under the condition of stirring, and the stirring rotating speed is preferably 60-100rpm; the culture mediums used in the process of preparing the lactobacillus fermentation product are MRS culture mediums, wherein the MRS culture mediums comprise peptone, raw meat extract powder, yeast extract powder, glucose, dipotassium hydrogen phosphate, diammonium hydrogen citrate, sodium acetate, magnesium sulfate, manganese sulfate and Tween-80, and the pH value of the MRS culture mediums is 5.7; after the fermentation tank culture is completed, the supernatant, namely the fermentation product, is preferably obtained by centrifugal separation.

The lactobacillus fermentation product is rich in various amino acids, water-soluble proteins, polysaccharides, organic acids and other active substances, and has excellent capability of promoting Nrf2 secretion. The technician uses hydrogen peroxide (H ₂O₂) to oxidize damaged cells, and then uses the lactobacillus fermentation product to treat the damaged cells, and the experimental process is as follows:

(1) Cell plating: the cell suspension density was adjusted to 8500 cells/well, inoculated in 96-well plates at 100. Mu.L/well, and incubated in a 5% CO ₂ cell incubator at 37℃for 24 hours with cell negative wells and oxidative damage positive wells.

(2) H ₂O₂ induces oxidative damage: old medium was discarded, H ₂O₂ (50. Mu.M), 100. Mu.L/well, cell negative control wells were added to complete medium, and the cells were incubated in a 5% CO ₂ cell incubator at 37℃for 4 hours and discarded.

(3) Lactobacillus dilution and sample addition: and (3) taking the complete culture medium as a diluent, setting a concentration gradient in a lactobacillus concentration range which enables the cell survival rate to be more than 90% according to a safety experiment concentration test result, adding 100 mu L/hole of the lactobacillus to cells, adding the same amount of complete culture medium to a cell control hole and an oxidative damage positive hole, and placing the cells in a 5% CO ₂ cell incubator at 37 ℃ for culturing for 24 hours.

(4) MTT detection: under the condition of avoiding light, adding 10 mu L of MTT solution (5 mg.mL-1) into each well, placing a 96-well plate into a 37 ℃ and 5% CO ₂ cell incubator for full reaction for 4 hours, discarding supernatant in the well plate, adding DMSO (dimethyl sulfoxide), 150 mu L/well, shaking and dissolving for 10 minutes, detecting absorbance value at 570nm of each well by using an enzyme-labeling instrument, and taking 630nm as a reference wavelength.

(5) And (3) calculating: after the background absorption is subtracted from each group of absorbance values, the cell viability percentage of the cells in each concentration sample addition group is calculated by taking the absorbance value of the cell control group as 100%, and the calculation formula is as follows:

percent cell viability (%) = average OD value of loading wells/average OD value of cell control wells x 100%.

As shown in the experimental results in FIG. 1, it can be seen that the Nrf2 secretion amounts of the 0.5%, 2.5% and 5% lactobacillus fermentation product treated groups are respectively increased by 61.84%, 77.93% and 64.83% compared with the model group, and that the lactobacillus fermentation products exhibit excellent efficacy of promoting the Nrf2 secretion.

In the present invention, the lactobacillus may be selected lactobacillus, which is selected as follows: diluting a lactobacillus strain sample solution, transferring the lactobacillus strain sample solution into a plate, injecting a culture medium into the plate, performing anaerobic culture, picking single bacterial colonies with typical lactobacillus characteristics from the plate for gram staining, and continuing streaking the G+ strain on the culture medium until the strain is determined to be a pure culture; the temperature of the medium is preferably 50 ℃; the culture medium is preferably a calcium carbonate modified MRS culture medium or bromocresol purple modified MRS culture medium.

The screening process specifically comprises the following steps: preparing a probiotic powder solution with proper concentration to form a strain sample solution, selecting 2-3 continuous dilutions with proper concentration, sucking 1mL of sample homogenate from each dilution into a sterilization plate, and making two plates for each dilution. Secondly, after the diluent is transferred into a plate, the calcium carbonate modified MRS culture medium or bromocresol purple modified MRS culture medium cooled to about 50 ℃ is poured into about 15mL of the plate, and the plate is rotated to uniformly mix. Anaerobic culture is carried out at 36+/-1 ℃ for 72 hours+/-2 hours. Dilution from sample to plate pour is required to be completed within 15 min. Finally, single colonies with typical lactic acid bacteria characteristics were picked from the plates for gram staining and the g+ strain continued streaking on MRS solid medium until pure cultures were identified.

2. Liposome-encapsulated nano lipoic acid

Lipoic acid (C ₈H₁₄O₂S₂) has a molecular formula of C ₈H₁₄O₂S₂, can be used as coenzyme to participate in acyl transfer in metabolism of substances in organism, and can eliminate free radicals which cause accelerated aging and pathopoiesia. Lipoic acid is a cofactor for the pyruvate dehydrogenase complex, ketoglutarate, and amino acid hydrogenase complex. Can inhibit lipid oxidation of nerve tissue, inhibit glycosylation of protein, inhibit aldose reductase, and inhibit conversion of glucose or galactose into sorbitol. Animal experiments show that lipoic acid can prevent the development of diabetes, promote the utilization of glucose and prevent neuropathy caused by hyperglycemia. The lipoic acid is easy to reduce into dihydrolipoic acid after entering human body, and both can promote the regeneration of vitamin C and vitamin E to play an antioxidant role. Lipoic acid also increases intracellular reduced glutathione and coenzyme Q10 levels and sequesters certain metal ions.

In order to solve the stability problem of lipoic acid and further expand the application range, the invention adopts a liposome wrapping method to prepare nano lipoic acid; in the liposome-coated nano lipoic acid, the content of nano lipoic acid is 1.0-5.0 wt%, preferably 2.0wt%; the average grain diameter of the nano lipoic acid is 50-100 nm; the liposome-encapsulated nano lipoic acid is prepared from the following raw materials: lipoic acid 1.0-5.0 wt%; 15-35 wt% of liposome; the balance being water, the lipoic acid is preferably alpha-lipoic acid, the content is preferably 2.0wt%, and the content of the liposome is preferably 25wt%; the liposome is preferably one or more of glucose ester, phosphate ester, fatty acid, aliphatic hydrocarbon, ketone compound and alcohol compound, and more preferably glucose ester, phosphate ester, fatty acid, aliphatic hydrocarbon, ketone compound and alcohol compound; the content of glucose ester is preferably 3-6wt%, more preferably 3.6wt%, the content of phosphate ester is preferably 0.5-2wt%, more preferably 1.0wt%, the content of fatty acid is preferably 0.5-2wt%, more preferably 1.3wt%, the content of aliphatic hydrocarbon is preferably 5-10wt%, more preferably 6.5wt%, the content of ketone compound is preferably 0.1-1wt%, more preferably 0.5wt%, the content of alcohol compound is preferably 5-15wt%, more preferably 12wt% of raw material; the glucose ester is preferably PEG-20 methyl glucose sesquistearate and/or methyl glucose sesquistearate, the phosphate ester is preferably hydrogenated lecithin, the fatty acid is preferably stearic acid, the aliphatic hydrocarbon is preferably squalane, the ketone compound is preferably p-hydroxyacetophenone, and the alcohol compound is preferably one or more of butanediol, glycerol, 1, 2-hexanediol and cetostearyl alcohol.

In one embodiment of the present invention, the method for preparing liposome-encapsulated nano lipoic acid comprises the following steps: mixing lipoic acid, liposome and water, homogenizing; the mass ratio of lipoic acid to liposome is preferably 1 (3-35), more preferably 1 (10-15), and even more preferably 1:12.5; the liposome is preferably glucose ester, phosphate ester, fatty acid, aliphatic hydrocarbon, ketone compound and alcohol compound, and the alcohol compound is preferably butanediol, glycerol, 1, 2-hexanediol and cetostearyl alcohol; the preparation method further preferably comprises mixing lipoic acid with glucose ester, phosphate ester, ketone compound, butanediol, glycerol and 1, 2-hexanediol, mixing with fatty acid, aliphatic hydrocarbon, cetostearyl alcohol and water, and homogenizing; the pressure for homogenization is preferably 600Bar.

3. VC ethyl ether

Vitamin C ethyl ether is a very useful vitamin C derivative, known as 3-O-ethyl ascorbate ether, the alias VC ethyl ether, and has a molecular weight of 204.18. The vitamin C derivative is very stable in chemical substances, is a vitamin C derivative without color change, is a lipophilic and hydrophilic amphiprotic substance, and greatly expands the application range of the vitamin C derivative, and is especially applied to daily chemicals. The 3-O-ethyl ascorbate can easily enter the dermis layer through the stratum corneum, and can be easily decomposed by biological enzymes in the body after entering the body, so that the biological effect of the vitamin C can be exerted.

The VC ethyl ether directly participates in the synthesis of collagen after entering the dermis layer, and recovers the activity of skin cells, so that the collagen is increased, and the skin becomes full and elastic, and is fine and smooth. Inhibiting tyrosinase activity, inhibiting melanin formation, reducing melanin to colorless, and whitening skin with high efficiency. The VC ethyl ether has excellent antioxidant effect in cosmetics, is very stable and does not change color compared with VC, and is light-resistant, heat-resistant, acid-resistant, alkali-resistant, salt-resistant and air-oxidizing.

Researchers select healthy experimental mice, perform different interferences according to experimental groups, determine the expression influence of external interferences on mouse lung cells Nrf2 (a one-step polymer detection system) by using an immunohistochemical method, and perform statistical analysis by adopting SPSS software, and the result is shown in figure 2. From FIG. 2, it is clear that the PM 2.5-interfered group (PM group) Nrf2 was statistically different from the control group, and that VC ethyl ether promoted the expression of each protein, and that PM group Nrf2 nuclear protein was statistically different from LVC group (low dose group, experimental dose 10 mL/kg) and HVC group (high dose group, experimental dose 30 mL/kg). The vitamin C can effectively prevent external pollution and avoid the influence and inhibition of external stimulus on Nrf2 channels. The VC ethyl ether is easily decomposed by biological enzymes in the body after entering the body, so that the biological effect of the vitamin C is exerted, and therefore, the vitamin C has similar effect.

4. Glutathione

Glutathione (GSH), a peptide compound containing glutamic acid, cysteine and glycine, is a natural active peptide with rich body content, widely exists in tissues and organs of mammals, and is higher in liver content than other organs. GSH has two forms in the body, oxidized glutathione (oxidized glutathione, GSSG) and reduced glutathione (reduced glutathione, r GSH). The reduced glutathione has the characteristics of scavenging free radicals and lipid peroxides in the body and maintaining stable oxidation-reduction balance in the body exogenously, and the active group sulfhydryl (-SH) of the reduced glutathione has the functions of detoxification and cell protection. 2 molecules of GSH can be oxidized to GSSG, which under the action of NADPH and GR, can be reduced to GSH, wherein rGSH can bind with oxygen radicals and hydrogen radicals, thereby reducing oxidative damage within the cell. The mechanism of the antioxidant action of glutathione is shown in figure 8.

As can be seen from fig. 8, GSH synthesis has two main pathways: ① Catalyzing synthesis of glutamic acid, cysteine and glycine under the action of gamma-GCL and GS. gamma-GCL is composed of GCLC and GCLM, wherein GCLC has the effect of catalyzing gamma-GCL, while GCLM has the effect of modulating gamma-GCL, GCLC is encoded by mRNA of GCLC, and GCLM is encoded by mRNA of GCLM. ② Under the action of glutathione reductase and reduced coenzyme II, GSSG is catalyzed to be reduced to rGSH. Glutathione mostly appears in rGSH forms in cells, the content of GSSG is very low, the ratio of rGSH/GSSG is about 100/1, the maintenance of the steady state of rGSH/GSSG in cells is the key for maintaining the normal physiological state of cells, the maintenance of the stability of an intracellular oxidation-reduction resisting system is one of the key indexes of oxidative damage in cells, and GSH/GSSG is an indicator of the oxidative damage degree and the regulation change degree of oxidation-reduction reaction genes. Studies have shown that intracellular GSH levels are associated with the development of diseases such as cancer, cystic fibrosis, senile dementia, etc., GSH is commonly found in mammalian cells, especially in large amounts in organs such as liver, kidney, etc.

The invention also provides an antioxidant cosmetic, which comprises the antioxidant composition and cosmetically acceptable auxiliary materials.

The antioxidant cosmetic of the present invention is preferably a astringent, an emulsion, a foundation, an essence, a mask, a sun cream, a face cream, an eye cream, a hand cream or a body cream; the mask is preferably a patch type mask or a smearing type mask; the essence is preferably essence water, essence oil or essence gel.

The auxiliary materials are acceptable auxiliary materials in cosmetics, and are preferably one or more of solvents, thickeners, moisturizers, film forming agents, liposomes, preservatives, colorants, pH regulators and essence; the solvent is preferably water or an alcohol solvent. The auxiliary material is preferably water.

In a specific embodiment of the invention, the antioxidant cosmetic consists of the following components:

0.1 to 10 percent of lactobacillus fermentation product;

0.01 to 2 percent of nano lipoic acid;

0.01 to 0.5 percent of VC ethyl ether;

reduced glutathione 0.001-0.1%;

The balance being water.

In a specific embodiment of the invention, the antioxidant cosmetic consists of the following components:

1-10% of lactobacillus fermentation product;

0.1 to 2 percent of nano lipoic acid;

0.05 to 0.5 percent of VC ethyl ether;

Reduced glutathione 0.02-0.1%;

The balance being water.

In a specific embodiment of the invention, the antioxidant cosmetic consists of the following components:

2.5 to 10 percent of lactobacillus fermentation product;

1-2% of nano lipoic acid;

0.1 to 0.5 percent of VC ethyl ether;

Reduced glutathione 0.05-0.1%;

The balance being water.

In a specific embodiment of the invention, the antioxidant cosmetic consists of the following components:

lactobacillus fermentation product 5-10%;

Nano lipoic acid 2%;

0.3 to 0.5 percent of VC ethyl ether;

reduced glutathione 0.1%;

The balance being water.

In a specific embodiment of the invention, the antioxidant cosmetic consists of the following components:

10% of lactobacillus fermentation product;

Nano lipoic acid 2%;

0.5% of VC ethyl ether;

reduced glutathione 0.1%;

The balance being water.

In the invention, the preparation method of the antioxidant cosmetic comprises the following steps: mixing lactobacillus fermentation product, nanometer lipoic acid, VC ethyl ether, reduced glutathione and water to obtain antioxidant cosmetic; the preparation method specifically comprises the following steps: mixing VC ethyl ether, reduced glutathione and water to obtain a mixed solution, and mixing the mixed solution, lactobacillus fermentation products, nano lipoic acid and water; mixing VC ethyl ether and reduced glutathione with water, preferably mixing VC ethyl ether and reduced glutathione with water respectively to obtain VC ethyl ether aqueous solution and reduced glutathione aqueous solution; after mixing the four components, the system is preferably filter sterilized, preferably with a 0.22 μm membrane; the mode of mixing is not particularly limited in the present invention, and stirring mixing is preferable.

The invention also provides application of the antioxidant composition and/or the antioxidant cosmetic in improving the antioxidant capacity of cells.

In particular, the invention provides the application of the antioxidant composition and/or the antioxidant cosmetic in promoting the expression of cells Nrf 2.

In particular, the invention provides the use of the above antioxidant composition and/or antioxidant cosmetic in promoting cell T-AOC.

In particular, the invention provides the application of the antioxidant composition and/or the antioxidant cosmetic in reducing the ROS content of cells.

The antioxidant composition and the antioxidant cosmetic comprise four antioxidant components of lactobacillus fermentation products, liposome-coated nano lipoic acid, VC ethyl ether and reduced glutathione, free radicals are eliminated based on different mechanisms, the components are synergistically enhanced, and the antioxidant effect is better exerted.

Antioxidation is a long-lasting research subject, and raw materials and products with antioxidation effect are also endless. However, most raw material oxidation resistance studies are mainly conducted by results, and the studies on specific action mechanisms are not sufficient. In recent years, the mechanism of organism antioxidation is gradually uncovered with the development and the deep research of medicine, but the application of the mechanism in skin care products is still rare. Compared with the prior art, the invention organically combines four functional components of lactobacillus fermentation products, nano lipoic acid, VC ethyl ether and reduced glutathione to form a double antioxidation mode which takes endogenous antioxidation as a main component and exogenous antioxidation as an auxiliary component, realizes the improvement of antioxidation capacity in multiple dimensions, has the excellent characteristics of raw materials, and gives higher safety performance to the antioxidation composition. The antioxidant composition and the cosmetic thereof have the characteristics of clear mechanism, combined treatment and prevention, high safety performance, high temperature and no irritation, and have wide application prospect, and the beneficial effects of the invention are further described below.

1. The mechanism is clear:

In the normal state Nrf2 is present in the cytoplasm in an inactive state, whereas Keap1 binds to dimers formed by Nrf2 and actin in the cytosol and is anchored in the cytosol. It can be rapidly decomposed by ubiquitin proteasome pathway to maintain Nrf2 with low transcriptional activity. Decoupling of the Nrf2-Keap1 dimer is a key step of the pathway for resisting oxidative stress, and the mechanism for regulating the decoupling mainly comprises Keap1 conformational change and Nrf2 phosphorylation.

(1) Activation of the Nrf2-Keap1/ARE Signal pathway

① Keap1 conformational change mechanism. When cells are stimulated to generate oxidative stress, ROS and RNS modify the cysteine residue of Keap1 to change the conformation of Keap1, further inhibit ubiquitination of Nrf2, promote phosphorylation of Nrf2, reduce the recognition capacity of 26S proteasome to Nrf2, decouple Nrf2-Keap1 dimer and promote Nrf2 to enter the nucleus. Nrf2 steadily increases and binds to ARE, which is the classical way of uncoupling Nrf2-Keap1 dimer. The Nrf2 entering the cell nucleus is combined with the Maf protein to form a heterodimer, and then is identified and combined with the ARE gene, so that the transcription of downstream antioxidant stress kinase and phase II detoxification enzyme genes is started, and the antioxidation protein is encoded and generated, thereby achieving the action of antioxidation stress.

② Nrf2 phosphorylation mechanism. Many protein kinases mediate transcriptional regulatory activity of Nrf2 by inducing phosphorylation of Nrf2, such as Protein Kinase C (PKC), protein kinase R-like endoplasmic reticulum kinase (PERK), extracellular signal-regulated kinase (ERK), phosphatidylinositol-3 kinase (PI 3K), mitogen-activated protein kinase (MAPKs), and the like, all of which are involved in uncoupling of Nrf2-Keap1 dimers, induction of Nrf2 phosphorylation, and regulation of transcriptional activity of Nrf 2.

(2) Closure of Nrf2-Keap1/ARE Signal pathway

When the body returns from oxidative stress to normal physiological conditions, nrf2 dissociates from the ARE and returns to the cytosol, degrading through ubiquitination, thereby actively closing the Nrf2 pathway, with Nrf2 again remaining at a relatively low level. The dissociated Nrf2 is combined with other proteins to complete the final regulation and control of the target gene transcription, and the cell homeostasis is restored.

(3) Target protein for regulating Nrf2-Keap1/ARE signal path

Under normal conditions, the binding of Keap1 to Nrf2 places Nrf2 in an inhibited state, whereas under external oxidative stress stimuli (e.g. ROS or RNS), uncoupled Nrf2 enters the nucleus and binds to ARE, initiating downstream antioxidant enzymes including superoxide dismutase (superoxide dismutase, SOD), quinone oxidoreductase, reduced glutathione peroxidase (glutathione peroxidase, GSHPx), catalase (catalase, CAT) and the like.

The invention mainly takes the Keap1-Nrf2-ARE pathway antioxidation mechanism as a main realization path, and innovatively subdivides antioxidation into endogenous and exogenous two dimensions. Endogenous antioxidation is mainly improved by activating an Nrf2 channel, and exogenous antioxidation is mainly further ensured by adding reduced glutathione. On one hand, starting from an antioxidant stress critical path Kelch-like epichlorohydrin related protein 1 (Keap 1) -nuclear factor E2 related factor 2 (Nrf 2) -Antioxidant Reaction Element (ARE), the autonomous antioxidant capacity of the organism is improved by activating and promoting the expression of the Nrf2 factor; on the other hand, the self oxidation resistance is further improved through the protection of the Nrf2 pathway and the introduction of reduced glutathione. Specifically, lactobacillus fermentation products ARE selected to promote the increase of the secretion amount of Nrf2, and activate an Nrf2-ARE antioxidant pathway; nano lipoic acid is selected to activate Nrf2 phosphorylation, so that the nano lipoic acid is promoted to transfer from cytoplasm to nucleus, the regeneration of reduced glutathione is realized, and the endogenous antioxidant capacity is activated; the VC ethyl ether is used for protecting the Nrf2 passage, so that the damage and inhibition of external stimulus to the Nrf2 passage are avoided; in addition, reduced glutathione has the characteristics of scavenging free radicals and lipid peroxides in the body, maintaining stable oxidation-reduction balance in the body exogenously, and has the functions of detoxification and protecting cells by active group sulfhydryl (-SH), and the activity substances such as amino acid, water-soluble protein, polysaccharide, organic acid and the like in lactobacillus fermentation products also have the promotion effect on the secretion of Nrf2 by cells, and the tree-like thinking diagram is shown in figure 9.

2. Dual antioxidant: in general, antioxidant compositions are composed of several materials having antioxidant properties to achieve the antioxidant properties of the antioxidant composition. However, most of the antioxidant properties of the antioxidant compositions are exogenous, and few antioxidant compositions are realized by promoting the endogenous antioxidant properties of the organism, and the normal functions of the organism are affected when the exogenous antioxidant substances excessively scavenge free radicals. The invention goes deep into the study of oxidation resistance, realizes endogenous oxidation resistance by activating and stimulating an oxidation resistance channel of Nrf2, and ensures the realization of oxidation resistance by assisting with exogenous oxidation resistance substances, thereby forming double oxidation resistance mainly of endogenous oxidation resistance and secondarily of exogenous oxidation resistance.

3. Prevention and treatment combination: the traditional antioxidant composition realizes the antioxidant function mainly by exogenously adding components with antioxidant effect. However, most of these antioxidant compositions are aimed at solving the existing skin oxidation problem, and cannot improve the oxidation resistance of the organism. The invention combines exogenous antioxidation and endogenous antioxidation, provides antioxidation components and promotes the improvement of the self antioxidation performance of the organism, thereby realizing the realization of double antioxidation. The improvement of endogenous oxidation resistance is also beneficial to maintaining the health state of the skin, preventing the oxidation of the skin in the past, and realizing the effect of combination of prevention and treatment.

4. The safety performance is high: with the rise of the efficacy type antioxidant cosmetics, the safety performance of the products is also receiving more and more attention. This is mainly because the skin care products of the efficacy class do not avoid irritation problems, and the skin tolerance level is related to the specific situation of the individual, and cannot be summarized. Security is thus a focus of increasing consumer and brand concern. The invention focuses on the research of an Nrf2-ARE pathway, realizes the improvement of endogenous oxidation resistance by activating the pathway, can not disturb the function of an organism while realizing oxidation resistance, and has no stimulation and extremely high biological safety because four functional components in the oxidation resistance composition belong to skin conditioning agents.

Drawings

FIG. 1 is a diagram showing experiments of stimulating Nrf2 secretion by lactobacillus fermentation products;

FIG. 2 is a graph showing the effect of VC ethyl ether on cell secretion of Nrf2 under PM2.5 conditions;

FIG. 3 is a graph of the Nrf2 standard curve in Experimental example 3.1;

FIG. 4 is a graph showing the relative growth rate of Nrf2 in Experimental example 3.1;

FIG. 5 is a graph of the relative growth rate of T-AOC in Experimental example 3.2;

FIG. 6 is a plot of the relative improvement rate trend of ROS in Experimental example 3.3;

FIG. 7 is a photograph showing the comparison of the positive control group in experimental example 3.3 with that of example 3;

FIG. 8 is a diagram showing the mechanism of antioxidation action of reduced glutathione;

Fig. 9 is a tree-like mind map of the beneficial effects of the present invention.

Detailed Description

The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

In order to further illustrate the present invention, the following examples are provided. The raw materials used in the following examples of the present invention are all commercially available.

The liposome-encapsulated nano lipoic acid in the embodiment of the invention is prepared by the following method:

(1) PEG-20 methyl glucose sesquistearate, hydrogenated lecithin, butanediol, glycerol, p-hydroxyacetophenone and 1, 2-hexanediol are added into a water phase pot according to the proportion and part of water in the table 1, and the mixture is heated and stirred until the solid is completely dissolved, so as to obtain a water phase solution for standby.

(2) Adding stearic acid, cetostearyl alcohol and squalane into an oil pan, heating and stirring until the mixture is completely and uniformly dissolved, adding alpha-lipoic acid, and continuously stirring until the mixture is uniformly dispersed. And (3) after starting a homogenizer, pumping the oil phase into the aqueous phase solution obtained in the step (1), homogenizing, and stirring uniformly to obtain the emulsion containing the alpha-lipoic acid.

(3) Homogenizing the emulsion obtained in the step (2) under the pressure of 600Bar for 3 times, and cooling to obtain the nano liposome emulsion coated with alpha-lipoic acid.

TABLE 1 Liposome-coated nano lipoic acid raw material composition table

The lactobacillus fermentation product in the embodiment of the invention is prepared by the following method:

lactobacillus rhamnosus (Lactobacillus rhamnosus), strain number: ACCC 61771, deposit institution: china center for culture Collection of microorganisms. After strain activation, shake flask culture and fermenter culture, a fermentation product is obtained, and the specific experimental process is as follows:

1. Strain activation

1.1 Preparation of Medium

The formula of the MRS broth culture medium is as follows: 10g/L of peptone, 8g/L of raw meat extract powder, 4g/L of yeast extract powder, 20g/L of glucose, 2g/L of dipotassium hydrogen phosphate, 2g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, 0.2g/L of magnesium sulfate, 0.04g/L of manganese sulfate and 1g/L of tween-80.

The preparation flow of the culture medium is as follows: 180ml of MRS broth culture medium was prepared according to the formulation, the pH was adjusted to 5.7.+ -. 0.2, and sterilized at 118℃for 15min.

1.2 Strain activation

Ultraviolet sterilizing for 15min on an ultra-clean workbench, taking out 30ml of sterilized MRS culture medium, and loading into a sterile 50ml centrifuge tube. Placing a strain into an ultra-clean bench, naturally thawing, inoculating into a centrifuge tube according to a proportion of 3%, screwing a cover, sealing with a sealing film, and standing and culturing for 1 day at 37 ℃ in a shaking table.

2. Shake flask culture

Opening an ultra-clean bench, sterilizing for 15min by ultraviolet, loading 150ml of sterilized MRS culture medium into a sterile 250ml shake flask, inoculating 7.5ml of activated strain, sealing with a sealing film on a breathable bottle opening film, and standing and culturing at 37 ℃ for 8-10 hours by a shaking table.

3. Fermentation tank culture

3.1 Preparation of fermentation Medium and lye

1) Description of Medium preparation

The 5L liquid amount was 3L, the culture medium was MRS broth, and the pH was adjusted to about 5.7.

2) Preparing sodium hydroxide solution

500Ml of 10% sodium hydroxide solution was prepared and sterilized at 121℃for 20 minutes.

3.2 Preparation of Medium and Sterilization

1) Washing the weighing equipment with deionized water for use, wherein the weighing equipment mainly comprises 1 of 2L measuring cups and 1L feeding bottle

2) Preparing 3L MRS culture medium, dissolving, mixing, and transferring into fermentation tank.

3) And calibrating a pH electrode. Before sterilization, the pH electrode is calibrated, and the calibration points are pH=4.01 and pH=7.00, and the two-point calibration is achieved. Note the slope after calibration, the slope value is over the range of 0.8-1.2, and the electrode error is larger.

4) After the calibration of the pH electrode is completed, the electrode is inserted into a fermentation tank to be screwed, and the prepared fermentation medium is poured into the fermentation tank to perform in-situ sterilization.

5) Sterilizing the fermentation tank culture medium according to the in-situ sterilization procedure of the fermentation tank, wherein the sterilization temperature is 118 ℃, the sterilization time is 15min, and cooling for standby. After sterilization, the compressed air is not blown into the tank, and if the air filter element is dried, the compressed air can be blown to the lower row of the filter element. Closing exhaust in the cooling process, manually intermittently feeding air, and ensuring positive pressure in the tank. After the temperature is reduced to 37 ℃ and stabilized, the air inlet valve and the air outlet valve are closed. And (5) inoculating after the seed growth in the shake flask reaches the standard.

3.3 Fermenter inoculation

1) Seed preparation work

After culturing the strain in shake flask for 8-10 hours, sampling and detecting that the OD value is 2.5-5, and preparing for inoculating the fermentation tank.

2) Confirmation of fermenter preparation

The temperature was confirmed to be stabilized at 37℃and the stirring was carried out at 60rpm, the pot pressure was 0.04MPa, and dissolved oxygen was used as a reference only without control.

3) Inoculating in fermentation tank

A) Opening exhaust to reduce the tank pressure, closing the exhaust after the tank pressure drop is 0, igniting an alcohol inoculation loop, placing the alcohol inoculation loop at an inoculation port, adding 150ml of seed liquid into a fermentation tank through the inoculation port under the condition of open flame inoculation, screwing up the inoculation port, and avoiding bacteria infection.

B) After inoculation, the inlet air is opened, the pressure of the tank is increased to 0.04MPa, the inlet air is closed, and the culture is started.

4) Fermenter culture and process control

A) The pH value in the fermentation process is not controlled and is only monitored.

B) The fermentation process ensures positive pressure in the tank and the pressure is 0.03-0.04MPa.

C) Stirring and fixing at 60rpm.

5) Sampling

Sampling twice a day in the morning and evening, and detecting residual sugar and bacterial concentration.

6) Ending the culture

Fermenting for about 20-24 hours, and ending the fermentation after the pH is not reduced any more and the residual sugar is less than 1 g/L.

3.4 Off-tank treatment of fermenter

1) The fermentation broth was centrifuged at 8500rpm at 4℃for 10 min.

2) Separating the supernatant and the thalli by centrifugation, taking the supernatant as a fermentation product, and preserving the supernatant at 4 ℃ for standby, and freezing the thalli at-20 ℃.

1. Examples

Example 1

The antioxidant cosmetic of the embodiment consists of the following components:

0.1% of lactobacillus fermentation product;

liposome-encapsulated nano lipoic acid 0.01%;

0.01% of VC ethyl ether;

Reduced glutathione 0.001%;

The balance being water;

the preparation method comprises the following steps:

(1) The VC ethyl ether and the reduced glutathione which are measured are respectively and accurately weighed, and are respectively dissolved by using a proper amount of water to obtain a mixed solution.

(2) And precisely weighing the lactobacillus fermentation product and the nano lipoic acid wrapped by the liposome, adding the lactobacillus fermentation product, the VC ethyl ether aqueous solution and the reduced glutathione aqueous solution into a stirring kettle, adding the rest water, fully stirring, and then sterilizing (0.22 mu m membrane filtration) under the condition of a biosafety cabinet to obtain the antioxidant cosmetic.

Example 2

The antioxidant cosmetic of the embodiment consists of the following components:

lactobacillus fermentation product 1%;

Liposome-encapsulated nano lipoic acid 0.1%;

0.05% of VC ethyl ether;

Reduced glutathione 0.02%;

The balance being water;

The preparation thereof was carried out with reference to the procedure in example 1.

Example 3

The antioxidant cosmetic of the embodiment consists of the following components:

2.5% of lactobacillus fermentation product;

Liposome-encapsulated nano lipoic acid 1%;

0.1% of VC ethyl ether;

reduced glutathione 0.05%;

The balance being water;

The preparation thereof was carried out with reference to the procedure in example 1.

Example 4

The antioxidant cosmetic of the embodiment consists of the following components:

lactobacillus fermentation product 5%;

Liposome-encapsulated nano lipoic acid 2%;

0.3% of VC ethyl ether;

reduced glutathione 0.1%;

The balance being water;

The preparation thereof was carried out with reference to the procedure in example 1.

Example 5

The antioxidant cosmetic of the embodiment consists of the following components: 10% of lactobacillus fermentation product;

Liposome-encapsulated nano lipoic acid 2%;

0.5% of VC ethyl ether;

reduced glutathione 0.1%;

The balance being water;

The preparation thereof was carried out with reference to the procedure in example 1.

2. Comparative example

Comparative example 1

The antioxidant cosmetic of the comparative example consists of the following components:

Lactobacillus fermentation product 0;

Liposome-encapsulated nano lipoic acid 0;

0.1% of VC ethyl ether;

reduced glutathione 0.05%;

The balance being water;

The preparation thereof was carried out with reference to the procedure in example 1.

Comparative example 2

The antioxidant cosmetic of the comparative example consists of the following components:

2.5% of lactobacillus fermentation product;

Liposome-encapsulated nano lipoic acid 0;

0.1% of VC ethyl ether;

reduced glutathione 0.05%;

The balance being water;

The preparation thereof was carried out with reference to the procedure in example 1.

Comparative example 3

The antioxidant cosmetic of the comparative example consists of the following components:

Lactobacillus fermentation product 0;

Liposome-encapsulated nano lipoic acid 1%;

0.1% of VC ethyl ether;

reduced glutathione 0.05%;

The balance being water;

The preparation thereof was carried out with reference to the procedure in example 1.

Comparative example 4

The antioxidant cosmetic of the comparative example consists of the following components:

2.5% of lactobacillus fermentation product;

Liposome-encapsulated nano lipoic acid 1%;

VC ethyl ether 0;

Reduced glutathione 0;

The balance being water;

The preparation thereof was carried out with reference to the procedure in example 1.

Comparative example 5

The antioxidant cosmetic of the comparative example consists of the following components:

0.001% of lactobacillus fermentation product;

liposome-encapsulated nano lipoic acid 0.001%;

VC ethyl ether 3%;

reduced glutathione 0.01%;

The balance being water;

The preparation thereof was carried out with reference to the procedure in example 1.

3. Experimental example

3.1 Nrf2 cell assay

1) Principle of testing

The experiment uses a double antibody sandwich method to measure the level of a human nuclear factor E2 related factor (Nrf 2) in a specimen, uses a purified human nuclear factor E2 related factor (Nrf 2) antibody to coat a microplate, prepares a solid-phase antibody, sequentially adds the nuclear factor E2 related factor (Nrf 2) into a single antibody-coated microwell, combines the single antibody-coated microwell with an HRP-labeled nuclear factor E2 related factor (Nrf 2) antibody to form an antibody-antigen-enzyme-labeled antibody complex, and then thoroughly washes and develops color by adding a substrate TMB. TMB is converted to blue under the catalysis of HRP enzyme and to final yellow under the action of acid. The shade of the color and the nuclear factor E2-related factor (Nrf 2) in the sample are positively correlated. The absorbance (OD value) was measured at a wavelength of 450nm with a microplate reader, and the concentration of human nuclear factor E2-related factor (Nrf 2) in the sample was calculated from a standard curve.

2) Test method

A. Cell culture supernatant: the secreted components were collected by sterile tube. Centrifuging for 20min (2000-3000 rpm). The supernatant was carefully collected. When detecting the intracellular components, the cell suspension was diluted with PBS (ph=7.2 to 7.4) to a cell concentration of about 100 ten thousand/mL. The freezing and thawing are repeated to destroy cells and release intracellular components. Centrifuging for 20min (2000-3000 rpm). The supernatant was carefully collected. If precipitate is formed during preservation, the solution should be centrifuged again.

B. Dilution of standard: the kit provides one standard of original multiple, and can be diluted in a small test tube according to the following table.

160pg/mL	Standard No. 5	150. Mu.L of original multiple standard substance is added with 150. Mu.L of dilution of the standard substance
			80pg/mL	No. 4 standard substance	150. Mu.L of No. 5 standard is added with 150. Mu.L of standard diluent
40pg/mL	Standard No. 3	150. Mu.L of No. 4 standard is added with 150. Mu.L of standard diluent
			20pg/mL	Standard No. 2	150. Mu.L of standard No. 3 was added to 150. Mu.L of standard diluent
10pg/mL	Standard No. 1	150. Mu.L of No. 2 standard is added with 150. Mu.L of standard diluent

C. Sample adding: blank holes (blank control holes are not added with samples and enzyme-labeled reagents, and the rest steps are the same), standard holes and holes of samples to be tested are respectively arranged, 50 mu L of standard samples are accurately added on an enzyme-labeled coating plate, 40 mu L of sample diluent is firstly added in the holes of the samples to be tested, and then 10 mu L of samples to be tested are added (the final dilution of the samples is 5 times). Sample adding: and (3) adding the sample at the bottom of the ELISA plate hole, and slightly and uniformly shaking without touching the hole wall as much as possible.

D. incubation: sealing the plates by using sealing plates, and then placing the plates at 37 ℃ for incubation for 30min.

E. Preparing liquid: the concentrated 30-fold (20-fold of 48T) washing solution was diluted with distilled water 30-fold (20-fold of 48T) and used.

F. washing: carefully removing the sealing plate film, discarding the liquid, spin-drying, filling each hole with the washing liquid, standing for 30 seconds, discarding, repeating the process for 5 times, and beating.

G. Adding enzyme: 50. Mu.L of enzyme-labeled reagent was added to each well, except for blank wells.

H. incubation: operating as D

I. washing: the operation is the same as F.

J. color development: 550 mu L of color developing agent and 50 mu L of color developing agent B are added into each hole, and the mixture is gently vibrated and mixed uniformly, and developed for 10min at 37 ℃ in a dark place.

K. And (3) terminating: the reaction was stopped by adding 50. Mu.L of stop solution to each well (blue turned to yellow at this time) for 10min.

L, determination: the absorbance (OD value) of each well was measured sequentially at the wavelength of blank Kong Diaoling, 450 nm. The measurement should be performed within 15min after the addition of the stop solution.

3) Test results

Firstly, the Nrf2 content of each group is detected by ELISA, a standard curve is obtained according to the experimental result, as shown in figure 3,

y＝0.0016x+0.0815，R²＝0.9972

(The abscissa x is the Nrf2 content, and the ordinate y is the absorbance).

Secondly, reversely deducing the Nrf2 content according to the absorbance of the standard curve,

Nrf2 relative growth rate calculation formula: n% = (N _x-N₀)/N₀ x 100%

Where N represents the relative growth rate of Nrf2,

N _x represents the number of samples of the set Nrf2 after the cell experiment,

N ₀ represents the value of Nrf2 in the positive control group after the cell experiment.

The data were collated to give table 2,

TABLE 2 relative growth rate of Nrf2

As shown in FIG. 4, it can be seen from FIG. 4 and Table 2 that the antioxidant cosmetic provided by the present invention promotes the increase of the expression level of Nrf2 to a certain extent, indicating that the use of the antioxidant cosmetic provided by the present invention promotes the secretion of cellular Nrf 2. By comparing the relative increase rates of Nrf2 between comparative examples 1,4 and example 3, it is known that the components of the antioxidant cosmetic of the present invention have a synergistic effect, and when some of the components are missing, the effect of the antioxidant cosmetic is significantly reduced.

3.2 T-AOC cell assay

1) Principle of testing

The test uses the kit to detect the total antioxidant capacity (T-AOC) level, and measures the total antioxidant level formed by various antioxidant substances, antioxidant enzymes and the like in the object. In biological and medical pharmaceutical research, the total antioxidant capacity of various body fluids such as blood plasma, serum, saliva, urine, cell or tissue lysate, plant or Chinese herbal medicine extract and various antioxidant (antioxidant) solutions is often detected.

The ability to reduce Fe ³⁺ -tripyridine triazine (Fe ³⁺ -TPTZ) to produce blue colored Fe ²⁺ -TPTZ in an acidic environment reflects the total antioxidant capacity.

2) Test method

A. Sample treatment: cells were collected in centrifuge tubes according to cell number (10 ⁴): adding 1.0mL of precooled extracting solution with the volume (mL) of 500-1000:1, ultrasonically crushing cells, centrifuging at 10000rpm at 4 ℃ for 10min, and taking the supernatant to be tested on ice.

B. The enzyme label instrument is preheated for more than 30min, and the wavelength is regulated to 593nm.

C. Operation table

3) Test results

T-AOC relative growth rate calculation formula: t% = (T _x-T₀)/T₀ x 100%

Wherein T represents the relative growth rate of T-AOC,

T _x represents the T-AOC value of the sample group after the cell experiment,

T ₀ represents the positive control T-AOC value after the cell experiment.

The data were collated to give table 3,

TABLE 3T relative increase rate of AOC

As shown in FIG. 5, it can be seen from FIG. 5 and Table 3 that the antioxidant cosmetic provided by the present invention increases the T-AOC growth rate to some extent, indicating that the use of the antioxidant cosmetic of the present invention promotes the enhancement of T-AOC ability of cells. By comparing the relative increase rates of T-AOC between comparative examples 1,4 and example 3, it is known that the antioxidant cosmetic of the present invention has a synergistic effect between the components, and the effect of the antioxidant cosmetic is significantly reduced when a part of the components are missing.

3.3 ROS cell assay

1) Principle of testing

The Reactive Oxygen species detection (Reactive Oxygen SPECIES ASSAY KIT) is a kit for detecting Reactive Oxygen species by using a fluorescent probe DCFH-DA. DCFH-DA itself has no fluorescence, can pass through cell membrane, and can be hydrolyzed by esterase in the cell after entering the cell to generate DCFH. Whereas DCFH cannot penetrate the cell membrane, thus making the probe easily loaded into the cell. Intracellular reactive oxygen species can oxidize non-fluorescent DCFH to produce fluorescent DCF. The level of intracellular active oxygen can be known by detecting the fluorescence of DCF.

2) Test method

A. Loading a probe: for cells with shorter stimulation times (typically less than 2 hours), the probe is loaded first and then the cells are stimulated with active oxygen positive control or drug of own interest. For cells with longer cell stimulation times (typically more than 6 hours), the cells are stimulated with active oxygen positive control or drug of interest to themselves, and then the probes are loaded.

In situ loading of probes: the method is only applicable to adherent culture of cells. DCFH-DA was pipetted in a serum-free medium at a final concentration of 10. Mu. Mol/L at 1:1000. The cell culture broth was removed and a suitable volume of diluted DCFH-DA was added. The volume of addition is preferably sufficient to cover the cells, and usually no less than 1mL of diluted DCFH-DA is added to one well of a six-well plate. Incubating in a cell culture incubator at 37 ℃ for 20min. The cells were washed three times with serum-free cell culture medium to sufficiently remove DCFH-DA that did not enter the cells. Typically, an active oxygen positive control can significantly increase active oxygen levels 20-30 minutes after stimulation of cells.

After collection of cells, probes were loaded: DCFH-DA was diluted 1:1000 in serum-free medium to a final concentration of 10. Mu. Mol/L. After cell collection, the cells were suspended in diluted DCFH-DA at a cell concentration of one to two million/mL and incubated in a 37℃cell incubator for 20min. The mixture is inverted and evenly mixed every 3-5min to ensure that the probe and the cells are fully contacted. The cells were washed three times with serum-free cell culture medium to sufficiently remove DCF-DA that did not enter the cells. Cells were stimulated directly with active oxygen positive controls or drugs of interest to themselves, or after dividing the cells into several aliquots. Typically, an active oxygen positive control significantly increases active oxygen levels 20-30 minutes after stimulation of cells.

Description: rosup was added as positive control only to the positive control wells, and Rosup was not necessary to be added to the remaining wells.

B. And (3) detection: the sample with the probe loaded in situ can be directly observed by a laser confocal microscope or can be detected by an enzyme-labeled instrument after collecting cells. The sample loaded with the probe after collecting the cells can be detected by a fluorescence enzyme-labeled instrument, and can also be directly observed by a laser confocal microscope.

C. parameter setting: the intensity of fluorescence before and after stimulation was detected in real time or time-point by time using an excitation wavelength of 488nm and an emission wavelength of 525 nm. The fluorescence spectrum of DCF is very similar to FITC, and the DCF can be detected using the parameter settings of FITC.

3) Test results

ROS relative improvement rate calculation formula: r% = (R ₀-R_x)/R₀ x 100%

Wherein R represents the relative improvement rate of ROS,

R _x represents the ROS value of the sample set after the cell experiment,

R ₀ represents ROS values of positive control group after cell experiments.

The data were collated to give table 4,

TABLE 4 ROS relative improvement rate

As shown in fig. 6, it can be seen from fig. 6 and table 4 that the antioxidant cosmetics provided by the present invention improve intracellular ROS levels to some extent, indicating that intracellular ROS content was reduced using the antioxidant cosmetics of the present invention. By comparing the relative improvement rates of ROS between comparative examples 1, 4 and example 3, it is known that the antioxidant cosmetic of the present invention has a synergistic effect between the components, and when a part of the components are missing, the effect of the antioxidant cosmetic is significantly reduced.

In addition, the present invention photographs the positive control group and the fluorescent contrast photograph of example 3, and as shown in fig. 7, it can be seen that the relative improvement rate of ROS of the cells after the antioxidant cosmetic treatment of the present invention is more remarkable.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (10)

1. An antioxidant composition, consisting of the following components:

0.1-10 parts by weight of lactobacillus fermentation product;

0.01-2 parts by weight of nano lipoic acid wrapped by liposome;

0.01-0.5 parts by weight of VC ethyl ether;

0.001-0.1 part by weight of reduced glutathione.

2. The antioxidant composition of claim 1, consisting of:

1-10 parts by weight of lactobacillus fermentation product;

0.1-2 parts by weight of nano lipoic acid wrapped by liposome;

0.05-0.5 parts by weight of VC ethyl ether;

0.02-0.1 parts by weight of reduced glutathione.

3. The antioxidant composition of claim 1, consisting of:

10 parts by weight of lactobacillus fermentation product;

2 parts by weight of liposome-encapsulated nano lipoic acid;

0.5 parts by weight of VC ethyl ether;

0.1 part by weight of reduced glutathione.

4. An antioxidant composition according to any one of claims 1 to 3, wherein the content of nano-lipoic acid in the liposome-encapsulated nano-lipoic acid is 1.0 to 5.0wt%.

5. An antioxidant composition according to any one of claims 1-3, wherein the liposome-encapsulated nano lipoic acid is prepared from the following raw materials:

Lipoic acid 1.0-5.0wt%;

15-35wt% of liposome;

The balance being water.

6. The antioxidant composition of claim 5, wherein the liposome is one or more of glucose esters, phosphate esters, fatty acids, aliphatic hydrocarbons, ketones, and alcohols.

7. The antioxidant composition of claim 5, wherein the liposome-encapsulated nano lipoic acid is prepared by the steps of: mixing lipoic acid, liposome and water, and homogenizing.

8. An antioxidant cosmetic comprising the antioxidant composition of any one of claims 1-7, and cosmetically acceptable adjuvants.

9. The antioxidant cosmetic of claim 8, wherein the adjuvant is water.

10. The use of the antioxidant composition according to any one of claims 1 to 7 for preparing a cosmetic for improving the antioxidant capacity of cells.