Traditional Chinese medicine probiotic fermented product as well as preparation method and application thereof

Abstract

The invention discloses a traditional Chinese medicine probiotic fermented product which is obtained by probiotic fermentation of an extracting solution of a traditional Chinese medicine composition. The traditional Chinese medicine composition comprises the following raw materials in parts by weight: 1-50 parts of red ginseng, 1-50 parts of aloe, 1-50 parts of folium ginkgo, 1-50 parts of rhizoma polygonati, 1-50 parts of Chinese wolfberry, 1-50 parts of salvia miltiorrhiza, 1-50 parts of ampelopsis japonica, 1-50 parts of bighead atractylodes rhizome, 1-50 parts of poria cocos, 1-50 parts of bletilla striata, 1-50 parts of silkworm larva, 1-50 parts of platycodon grandiflorum, 1-50 parts of moutan bark, 1-50 parts of rhizoma nardostachyos and 1-50 parts of liquorice. The invention also discloses a preparation method of the traditional Chinese medicine probiotic fermentation product. The invention also discloses application of the traditional Chinese medicine probiotic fermentation product in preparation of anti-aging, whitening and melanoma growth inhibition preparations and beauty products. The traditional Chinese medicine probiotic fermentation product has good whitening, anti-aging, anti-oxidation and melanoma growth inhibition effects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine probiotic fermentation product and a preparation method and application thereof. BACKGROUND

[0002] For humans or animals, melanin is produced by melanocytes, and the biochemical pathway of melanin synthesis starts from L-tyrosine, and the key enzyme is tyrosinase. Tyrosinase catalyzes two reactions, one is the hydroxylation of L-tyrosine to generate L-dopa, and the other is the oxidation of tyrosine by tyrosinase to generate dopaquinone. Melanin contains phenolic hydroxyl, carboxyl and amino active groups, and can absorb ultraviolet rays, thus having activities such as scavenging free radicals, antibacterial, chelating metal, anti-tumor and anti-radiation. Melanin plays an important role in human skin, but abnormal melanin production can cause excessive melanin formation, which can lead to pigmentation disorders such as freckles, blemishes and age spots. These excessive melanin expressions can be inhibited by effective tyrosinase inhibitors.

[0003] Free radicals are highly reactive substances with strong oxidizing action. Excessive free radicals can damage proteins, nucleic acids and cell membranes, leading to damage to normal cells and tissues of the human body. In addition, excessive free radicals in the body are also closely related to the occurrence, development and aging of many diseases of the human body, and are the main cause of human diseases.

[0004] Traditional Chinese medicine active ingredients have the characteristics of small side effects and green nature. According to the data of 8972 kinds of cosmetic raw materials in use, it can be seen that the proportion of traditional Chinese medicine varieties is about 30%. Therefore, the pursuit of green, natural, safe and stable traditional Chinese medicine active ingredients has become a trend. However, the anti-aging and whitening effects of traditional Chinese medicine or its formula are low at present, and the efficacy needs to be improved. SUMMARY

[0005] Based on the technical problems existing in the background art, the present application provides a traditional Chinese medicine probiotic fermentation product and a preparation method and application thereof. The traditional Chinese medicine probiotic fermentation product has good whitening, anti-aging, antioxidant and melanoma growth inhibition effects.

[0006] The present application provides a traditional Chinese medicine probiotic fermentation product, which is obtained by fermenting an extract of a traditional Chinese medicine composition by probiotics; wherein the raw materials of the traditional Chinese medicine composition comprise, by weight fraction, 1-50 parts of red ginseng, 1-50 parts of aloe, 1-50 parts of ginkgo leaves, 1-50 parts of yellow ginseng, 1-50 parts of wolfberry, 1-50 parts of salvia miltiorrhiza, 1-50 parts of white yam, 1-50 parts of white atractylodes, 1-50 parts of poria cocos, 1-50 parts of bletilla striata, 1-50 parts of white mulberry silk worm, 1-50 parts of platycodon grandiflorum, 1-50 parts of cortex moutan, 1-50 parts of sweetgrass, and 1-50 parts of licorice.

[0007] Preferably, the raw materials of the traditional Chinese medicine composition include, by weight: red ginseng 13-17 parts, aloe 10-14 parts, ginkgo leaves 8-12 parts, polygonatum 18-22 parts, medlar 18-22 parts, salvia miltiorrhiza 10-14 parts, white yam 8-12 parts, atractylodes 10-14 parts, poria 13-17 parts, bletilla 18-22 parts, white muscardine 8-12 parts, platycodon 13-17 parts, cortex moutan 8-12 parts, gancao 13-17 parts, licorice 8-10 parts.

[0008] Preferably, the raw materials of the traditional Chinese medicine composition include, by weight: red ginseng 15 parts, aloe 12 parts, ginkgo leaves 10 parts, polygonatum 20 parts, medlar 20 parts, salvia miltiorrhiza 12 parts, white yam 10 parts, atractylodes 12 parts, poria 15 parts, bletilla 20 parts, white muscardine 10 parts, platycodon 15 parts, cortex moutan 10 parts, gancao 15 parts, and licorice 9 parts.

[0009] The red ginseng polysaccharide in red ginseng has a moisturizing effect, and the maltol contained therein not only has the effects of scavenging free radicals and resisting oxidation, but also has the effects of inhibiting bacteria, inhibiting lipid peroxidation, and inhibiting cell mutation.

[0010] The amino acids and proteins contained in aloe have the effects of moisturizing and softening the skin, the carotenoids help prevent the formation of free radicals and reduce skin aging caused by free radicals, and the vitamin C and phenolic substances contained therein have the effects of whitening, resisting oxidation, and promoting collagen proliferation.

[0011] The ginkgolides in ginkgo leaves have an anti-inflammatory effect, and the four types of components, ginkgo flavones, ginkgolides, procyanidins, and organic acids, have a synergistic effect and jointly play an antioxidant role.

[0012] The baicalein in polygonatum belongs to flavonoid active ingredients and can effectively scavenge free radicals, playing the role of an antioxidant, and the polygonatum polysaccharide has the pharmacological effect of delaying aging.

[0013] The polysaccharide in medlar, as the main effective component of medlar, has various pharmacological effects such as radiation protection and anti-aging.

[0014] The active ingredients in salvia miltiorrhiza mainly include two categories: one is liposoluble abietane-type diterpene quinone compounds, such as tanshinone ⅡA, cryptotanshinone, and tanshinone I, which can play the roles of antibacterial and anti-inflammatory; and the other is water-soluble polyphenolic acid components, such as danshensu, rosmarinic acid, and salvianolic acid B, which have an antioxidant effect.

[0015] The components such as emodin methyl ether, chrysophanol, emodin, fumaric acid, and gallic acid contained in white yam have the effects of resisting bacteria and fungi.

[0016] Atractylodes has significant anti-inflammatory activity in vivo and in vitro, which may be related to the sesquiterpenes, especially atractylodes lactone I, contained therein.

[0017] Pachyman in Poria cocos has certain antioxidant capacity.

[0018] Bletilla striata has DPPH, ABTS radical scavenging activity and iron ion reduction activity.

[0019] Polysaccharide in white mulberry silkworm has the effects of anti-radiation, antibacterial and antiviral, beauty and anti-aging, etc.

[0020] Platycodon grandiflorum in Platycodon grandiflorum is a fructose-connected platycodon polysaccharide and inulin, which has the effect of anti-aging.

[0021] Paeonol in Paeonia suffruticosa has antibacterial and anti-inflammatory activity, and can produce strong bactericidal and inhibitory effects on more than 20 pathogenic bacteria such as Escherichia coli, hemolytic streptococcus, Staphylococcus aureus and Salmonella typhi.

[0022] Polysaccharide in Nardostachys chinensis has in vitro antioxidant activity, and has the ability to scavenge superoxide anion free radicals and hydroxyl free radicals.

[0023] Glycyrrhizin in Glycyrrhiza has certain inhibitory effect on DPPH free radicals, hydroxyl free radicals, and yolk lipid peroxidation and linoleic acid peroxidation, and has in vitro antioxidant capacity; at the same time, it also has certain inhibitory effect on the oxidative stress damage of SH-EP1 cell strain induced by hydrogen peroxide.

[0024] Preferably, the extract is a water extract.

[0025] Preferably, the density of the extract is 1.00-1.50g / cm 3 .

[0026] Preferably, the density of the extract is 1.00-1.15g / cm 3 .

[0027] Preferably, the probiotic bacteria are Lactobacillus plantarum and Bifidobacterium bifidum.

[0028] The above Lactobacillus plantarum and Bifidobacterium bifidum are purchased from Runying Biological Engineering (Shanghai) Co., Ltd.

[0029] Preferably, after fermentation by probiotic bacteria, solid-liquid separation is performed, the liquid is taken and sterilized to obtain the traditional Chinese medicine probiotic fermentation product.

[0030] The application further provides a preparation method of the Chinese medicine probiotic fermentation product, which comprises the following steps: soaking the Chinese medicine composition in water, then performing reflux extraction, performing solid-liquid separation, taking the liquid, and concentrating to obtain an extraction liquid; inoculating the probiotic into the extraction liquid to perform fermentation, then performing solid-liquid separation, taking the liquid, and sterilizing to obtain the Chinese medicine probiotic fermentation product.

[0031] The water is used in an amount of 1-20 times the weight of the Chinese medicine composition.

[0032] Preferably, the Chinese medicine composition is soaked in water for 30-240 min.

[0033] Preferably, the reflux extraction is performed at 100-250 ℃ for 30-120 min.

[0034] Preferably, the anaerobic fermentation is performed at 20-37 ℃ for 24-48 h.

[0035] Preferably, at the end of the fermentation, the concentration of Lactobacillus plantarum is 1×10 8 -10×10 8 CFU / mL, and the concentration of Bifidobacterium bifidum is 1×10 8 -10×10 8 CFU / mL.

[0036] Preferably, the probiotic is activated and seed-cultured before use.

[0037] Preferably, the volume ratio of the probiotic to the extraction liquid is 0.8-1.2:10.

[0038] The activation and seed-culturing of the probiotic comprises the following steps: streak-inoculating Lactobacillus plantarum into MRS solid culture medium, streak-inoculating Bifidobacterium bifidum into TPY solid culture medium, and culturing at 35-37 ℃ in an anaerobic box for 24-48 h; then picking single colonies and inoculating into MRS and TPY liquid culture medium respectively, and culturing anaerobically at 35-37 ℃ for 24-48 h, then centrifuging at 4000-10000 rpm for 10-20 min, and discarding the supernatant to obtain bacterial cells.

[0039] The bacterial cells are inoculated into the extraction liquid to perform fermentation.

[0040] The MRS culture medium and the TPY culture medium are conventional microbial culture media.

[0041] The application further provides application of the Chinese medicine probiotic fermentation product in preparation of anti-aging, whitening and melanoma growth inhibiting preparations and cosmetic products.

[0042] The melanoma cell is human melanoma A375 cell.

[0043] Preferably, the preparation is an external preparation.

[0044] Preferably, the cosmetic product is an external cosmetic product.

[0045] The dosage form of the preparation or the cosmetic product can be a smearing preparation or a patching preparation.

[0046] The smearing preparation can be a film powder, a paste, a cream, a gel, a jelly, etc.

[0047] The raw materials of the preparation or the cosmetic product include adjuvants and the traditional Chinese medicine probiotic fermentation product, wherein the adjuvants can be a base, a metal chelating agent, an antibacterial agent, a pH regulator, etc.

[0048] Beneficial effects:

[0049] The present application screens each traditional Chinese medicine raw material and its ratio, and selects a suitable probiotic for fermentation, which can greatly improve the whitening, anti-aging, antioxidant, and melanoma growth inhibition effects; and can be used for preparing a drug preparation or a cosmetic product for whitening, anti-aging, and inhibiting melanoma growth. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 DPPH scavenging rate results of the traditional Chinese medicine probiotic fermentation product prepared in Example 1.

[0051] Figure 2 DPPH scavenging rate results of tocopherol.

[0052] Figure 3 DPPH half-scavenging concentration (IC50 value) results of the traditional Chinese medicine probiotic fermentation product prepared in Example 1.

[0053] Figure 4 DPPH half-scavenging concentration (IC50 value) results of tocopherol.

[0054] Figure 5 Inhibition rate results of the traditional Chinese medicine probiotic fermentation product prepared in Example 1 on tyrosinase activity.

[0055] Figure 6 Inhibition rate results of arbutin on tyrosinase activity.

[0056] Figure 7 Half-inhibition concentration (IC50 value) results of the traditional Chinese medicine probiotic fermentation product prepared in Example 1 on tyrosinase.

[0057] Figure 8The results of the half-inhibitory concentration (IC50 value) of arbutin on tyrosinase.

[0058] Figure 9 The results of the relative inhibition rate of the Chinese medicine probiotic fermentate prepared in Example 1 on A375 cells.

[0059] Figure 10 The effect of the Chinese medicine probiotic fermentate prepared in Example 1 on the migration ability of A375 cells, with a scale of 100 μm. Figure 11 The results of the Chinese medicine probiotic fermentate prepared in Example 1 on the migration rate of A375 cells, wherein treat is Example 1 and control is the control group. DETAILED DESCRIPTION

[0060] Hereinafter, the technical solutions of the present application will be described in detail through specific examples, but it should be made clear that these examples are used for illustration, but not to be construed as limiting the scope of the present application.

[0061] Example 1

[0062] A preparation method of a Chinese medicine probiotic fermentate, comprising the following steps:

[0063] The raw materials of the Chinese medicine composition include: 15 g of red ginseng, 12 g of aloe, 10 g of ginkgo leaves, 20 g of polygonatum, 20 g of medlar, 12 g of salvia miltiorrhiza, 10 g of white yam, 12 g of atractylodes, 15 g of poria cocos, 20 g of bletilla striata, 10 g of white mulberry silk worm, 15 g of platycodon, 10 g of peony, 15 g of sweetgrass, and 9 g of licorice root, which are mixed to obtain the Chinese medicine composition;

[0064] 2 L of water is added to the Chinese medicine composition, soaked for 180 min, and then heated to liquid boiling by using an electric heating jacket, and the reflux extraction is started to be timed for 60 min. The filter is filtered by using a 100-mesh filter screen, and the filtrate is concentrated by high-heat cooking to obtain an extract with a density of 1.15 g / cm 3 ;

[0065] Lactobacillus plantarum is streaked on MRS solid culture medium, and Bifidobacterium bifidum is streaked on TPY solid culture medium, and cultured at 37℃ for 48 h in an anaerobic box; then single colonies are picked and inoculated in MRS liquid culture medium and TPY liquid culture medium, respectively, and cultured at 37℃ for 24 h in an anaerobic state, and then centrifuged at 20℃ and 4000 rpm for 10 min, and the supernatant is discarded, and Lactobacillus plantarum and Bifidobacterium bifidum are obtained, respectively.

[0066] Lactobacillus plantarum and Bifidobacterium bifidum were mixed at a weight ratio of 1:1 and inoculated at a total cell to extract volume ratio of 1:10. The mixture was then anaerobic fermented at 37°C for 48 hours (at the end of fermentation, the concentration of Lactobacillus plantarum was 5.6 × 10⁻⁶). 8 The concentration of Bifidobacterium bifidum (CFU / mL) was 5.2 × 10⁻⁶. 8 The mixture was centrifuged at 4000 rpm for 10 min, and the supernatant was sterilized to obtain the fermented product of traditional Chinese medicine probiotics.

[0067] Example 2

[0068] A method for preparing a fermented product of traditional Chinese medicine probiotics includes the following steps:

[0069] Take 13g of red ginseng, 14g of aloe vera, 8g of ginkgo leaves, 22g of polygonatum, 18g of wolfberry, 14g of salvia miltiorrhiza, 8g of white peony root, 14g of atractylodes macrocephala, 13g of poria cocos, 22g of bletilla striata, 8g of silkworm pupae, 17g of platycodon grandiflorus, 8g of peony bark, 17g of nardostachys chinensis, and 8g of licorice root, mix them evenly to obtain a traditional Chinese medicine composition;

[0070] Add 2L of water to the herbal composition and soak for 240 minutes. Then, heat the liquid with an electric heating mantle until it boils, and start timing. Reflux and extract for 120 minutes. Filter through a 100-mesh filter, and concentrate the filtrate by boiling over high heat to obtain a density of 1.30 g / cm³. 3 The extract was obtained; other steps were the same as in Example 1, to obtain the fermented product of Chinese herbal probiotics.

[0071] Example 3

[0072] A method for preparing a fermented product of traditional Chinese medicine probiotics includes the following steps:

[0073] Take 17g of red ginseng, 10g of aloe vera, 12g of ginkgo leaves, 18g of polygonatum, 22g of wolfberry, 10g of salvia miltiorrhiza, 12g of white peony root, 10g of atractylodes macrocephala, 17g of poria cocos, 18g of bletilla striata, 12g of silkworm pupae, 13g of platycodon grandiflorus, 12g of peony bark, 13g of nardostachys chinensis, and 10g of licorice root, mix them evenly to obtain a traditional Chinese medicine composition;

[0074] Add 2L of water to the herbal composition and soak for 120 minutes. Then, heat the liquid with an electric heating mantle until it boils, and start timing. Reflux and extract for 90 minutes. Filter through a 100-mesh filter, and concentrate the filtrate by boiling over high heat to obtain a density of 1.20 g / cm³. 3 The extract was obtained; other steps were the same as in Example 1, to obtain the fermented product of Chinese herbal probiotics.

[0075] The formulations of the traditional Chinese medicine compositions in Comparative Examples 1-5 are shown in Table 1, and their preparation methods are the same as those in Example 1.

[0076] Table 1. Formulas of Traditional Chinese Medicine Compositions in Comparative Examples 1-5

[0077] Comparative Example 1 Without mulberry silk worm, white yam, white atractylodes, poria, white yam, the others are the same as Example 1 Comparative Example 2 Without red ginseng, salvia miltiorrhiza, ginkgo leaf, the others are the same as Example 1 Comparative Example 3 Without liquorice, jujube, aloe, medlar, the others are the same as Example 1 Comparative Example 4 Without polygonatum, cortex moutan, gancao, the others are the same as Example 1 Comparative Example 5 Traditional Chinese medicine composition extract of Example 1

[0078] Example 4

[0079] The products obtained in Example 1 and Comparative Examples 1-5 were tested for their DPPH free radical scavenging ability; the specific steps are as follows:

[0080] 1. Testing the free radical scavenging performance of DPPH

[0081] Preparation of DPPH test solution: Dissolve 1 mg of DPPH solid in 24 mL of 95% ethanol, sonicate for 5 min, and shake thoroughly to obtain DPPH test solution;

[0082] Preparation of sample solutions: Take an appropriate amount of each group of products, evaporate to dryness in a water bath, and dissolve them in 95% ethanol to prepare six sample solutions with concentrations of 0.0504 g / mL, 0.1520 g / mL, 0.2520 g / mL, 0.5040 g / mL, 0.7540 g / mL, and 1.0100 g / mL, respectively.

[0083] Preparation of tocopherol positive control solutions: Weigh 1 mg of tocopherol reference standard, dissolve it in 95% ethanol, and dilute it to prepare six positive control solutions with concentrations of 0.02 mg / mL, 0.04 mg / mL, 0.06 mg / mL, 0.10 mg / mL, 0.30 mg / mL, and 0.50 mg / mL;

[0084] Take 500 μL of sample solutions of different concentrations (0.0504, 0.1520, 0.2520, 0.5040, 0.7540, 1.0100 g / mL), add 1 mL of DPPH test solution, mix well, and let stand at room temperature for 30 min. Measure the absorbance at 517 nm. Use tocopherol as a positive control. Calculate the DPPH free radical scavenging rate using the formula: Scavenging rate % = [A0 - (A1 - A2)] / A0 × 100%.

[0085] Among them, the absorbance of A0—500μL 95% ethanol + 1mL DPPH solution;

[0086] A1—Absorbance of 500μL sample solution / positive control solution + 1mL DPPH solution;

[0087] A2—Absorbance of 500 μL sample solution / positive control solution + 1 mL 95% ethanol.

[0088] 2. Detect the half-maximal clearance concentration (IC50 value).

[0089] Plot a curve with the concentration of the sample solution / tocopherol positive control solution on the x-axis and the corresponding DPPH clearance rate on the y-axis, and fit the curve to obtain the regression equation (R²). 2 (≥0.9). Based on the regression equation, calculate the sample concentration corresponding to a DPPH clearance rate of 50%, which is the IC50 value of the test sample.

[0090] The results are shown in Table 2, and typical images are shown below. Figures 1-4 As shown.

[0091] Table 2 IC50 Value Results

[0092] Grouping IC50 Tocopherol 0.03047mg / ml Traditional Chinese medicine probiotic ferment of Example 1 0.3077g / ml Traditional Chinese medicine probiotic ferment of Comparative Example 1 0.6135g / ml Traditional Chinese medicine probiotic ferment of Comparative Example 2 0.6348g / ml Traditional Chinese medicine probiotic ferment of Comparative Example 3 0.4666g / ml Traditional Chinese medicine probiotic ferment of Comparative Example 4 0.4013g / ml Comparative Example 5 (traditional Chinese medicine composition extract of Example 1) 0.6054g / ml

[0093] As can be seen from Table 2, the fermented Chinese herbal probiotics of the present invention have a strong ability to eliminate free radicals and resist oxidation, which is much higher than that of comparative examples 1-4 and the extracts of unfermented Chinese herbal compositions.

[0094] Figure 1 The results show the DPPH scavenging rate of the probiotic fermentation product prepared in Example 1.

[0095] Figure 2 The results show the DPPH clearance rate of tocopherol.

[0096] Figure 3 The results are the DPPH half-maximal scavenging concentration (IC50 value) of the fermented Chinese herbal probiotics prepared in Example 1.

[0097] Figure 4 The results are the DPPH half-maximal scavenging concentration (IC50 value) of tocopherol.

[0098] Depend on Figures 1-2 It can be seen that the DPPH clearance rate of both the fermented Chinese herbal probiotics and tocopherol increased in a concentration-dependent manner. There was a significant difference between the high concentration group (1.01 g / mL) and the low concentration group (0.152 g / mL, 0.0504 g / mL) of the fermented Chinese herbal probiotics (p < 0.05); there were also significant differences among the different concentrations of tocopherol (p < 0.05).

[0099] Example 5

[0100] The products obtained in Example 1 and Comparative Examples 1-5 were used to test their ability to inhibit tyrosinase activity; the specific steps are as follows:

[0101] 1. Assay for inhibition of tyrosinase activity (in vitro method)

[0102] The testing steps are as follows:

[0103] Preparation of L-tyrosine solution: Weigh 0.025 g L-tyrosine, dissolve it in PBS buffer (pH = 6.8, 0.1 mol / L), and bring the volume to 50 mL;

[0104] Preparation of tyrosinase solution: Prepare mushroom tyrosinase to 500 U / mL using PBS buffer;

[0105] Preparation of sample solutions: Take an appropriate amount of product from each group, evaporate to dryness in a water bath, dissolve in PBS buffer, and prepare six sample solutions with concentrations of 0.0252 g / mL, 0.0420 g / mL, 0.0630 g / mL, 0.1575 g / mL, 0.3150 g / mL, and 0.6300 g / mL, respectively.

[0106] Preparation of arbutin positive control solutions: Weigh 1 mg of arbutin reference standard, dissolve it in PBS buffer, and dilute it to prepare six positive control solutions with concentrations of 0.050 mg / mL, 0.125 mg / mL, 0.200 mg / mL, 0.250 mg / mL, 0.500 mg / mL, and 1.000 mg / mL.

[0107] According to Table 3, add L-tyrosine solution, sample solution / positive control solution, and PBS buffer to each well of the ELISA plate in sequence, mix thoroughly, incubate at 37℃ for 10 min, then add 20 μL of tyrosinase solution to each well, mix well, react at 37℃ for 5 min, and measure the absorbance at 475 nm. Calculate the inhibition rate of the sample against tyrosinase using the formula: Inhibition rate % = [1 - (Ad - Ac) / (Ab - Aa)] × 100%.

[0108] Where Aa is the absorbance of the solvent background well; Ab is the absorbance of the solvent reaction well; Ac is the absorbance of the sample background well; and Ad is the absorbance of the sample reaction well.

[0109] Table 3. Amounts of each solution added to the ELISA plate

[0110]

[0111] 2. Detect the half-maximal inhibitory concentration (IC50 value).

[0112] Plot a curve with the concentration of the sample solution / arbutin positive control solution on the x-axis and the corresponding tyrosinase inhibition rate on the y-axis, and fit the curve to obtain the regression equation (R²). 2 (≥0.9). Based on the regression equation, calculate the sample concentration corresponding to a tyrosinase inhibition rate of 50%, which is the IC50 value of the test sample.

[0113] The results are shown in Table 4, and typical images are shown below. Figures 5-8 As shown.

[0114] Table 4 IC50 Value Results

[0115]

[0116]

[0117] As can be seen from Table 4, the fermented Chinese herbal probiotics of the present invention have a strong skin whitening effect and a strong binding affinity for tyrosinase, which is much higher than that of comparative examples 1-4 and the extracts of unfermented Chinese herbal compositions.

[0118] Figure 5 The results show the inhibition rate of tyrosinase activity by the fermented herbal probiotics prepared in Example 1.

[0119] Figure 6 The results show the inhibition rate of arbutin on tyrosinase activity.

[0120] Figure 7 The results show the half-maximal inhibitory concentration (IC50) of the fermented Chinese herbal probiotics prepared in Example 1 against tyrosinase.

[0121] Figure 8 The results show the half-maximal inhibitory concentration (IC50) of arbutin against tyrosinase.

[0122] Depend on Figures 5-6 It can be seen that the inhibition rate of tyrosinase by fermented Chinese herbal probiotics and arbutin increases with concentration, and there are significant differences among the concentrations (p<0.05).

[0123] Example 6

[0124] The products obtained in Example 1 and Comparative Examples 1-5 were used to test their effects on cell viability. The specific steps are as follows:

[0125] Preparation of sample solutions: Take an appropriate amount of each group of products, evaporate to dryness in a water bath, dissolve in PBS buffer, and prepare sample solutions of different concentrations.

[0126] A375 cell (human melanoma cell) suspension (100 μL / well) was seeded into 96-well plates. The plates were pre-cultured in an incubator (37℃, 5% CO2) for 4-6 h to allow cell adhesion. Then, drug solutions were added at concentrations of 200, 100, 50, 25, 12.5, 6.25, and 3.175 mg / mL. Cell viability was then measured at 6 h, 12 h, 24 h, and 48 h. 10 μL of CCK solution was added to each well, and the plates were incubated in an incubator for 1-4 h. The absorbance at 450 nm was measured using a microplate reader. The drug concentration (IC50) at which the inhibitory effect of each sample on A375 cell proliferation reached 50% of the normal proliferation level of A375 cells was calculated.

[0127] The results are shown in Table 5, and typical images are shown below. Figure 9 As shown.

[0128] Figure 9 The results show the relative inhibition rate of the fermented Chinese herbal probiotics prepared in Example 1 on A375 cells.

[0129] Table 5. Drug concentrations (IC50) at which the inhibitory effect on A375 cell proliferation reaches 50% of the normal A375 cell proliferation level.

[0130] Grouping IC50 (mg / mL) Traditional Chinese medicine probiotic ferment of Example 1 13.23 Traditional Chinese medicine probiotic ferment of Comparative Example 1 28.39 Traditional Chinese medicine probiotic ferment of Comparative Example 2 22.41 Traditional Chinese medicine probiotic ferment of Comparative Example 3 17.92 Traditional Chinese medicine probiotic ferment of Comparative Example 4 15.88 Comparative Example 5 (traditional Chinese medicine composition extract of Example 1) 26.45

[0131] As can be seen from Table 5, the fermented Chinese herbal probiotics of the present invention have a strong ability to inhibit the growth of A375 cells, which is much higher than that of comparative examples 1-4 and the extracts of unfermented Chinese herbal compositions.

[0132] Example 7

[0133] The specific steps for the cell scratch assay are as follows:

[0134] Preparation of sample solutions: Take an appropriate amount of the fermented Chinese herbal probiotics obtained in Example 1, evaporate to dryness in a water bath, dissolve in PBS buffer, and prepare sample solutions of different concentrations.

[0135] Marking: First, use a marker to draw at least 5 parallel horizontal lines on the back of the 6-hole board, with each line spaced 0.5-1cm apart;

[0136] Plating: A375 cells (human melanoma cells) in logarithmic growth phase were trypsin-digested into a single-cell suspension and seeded into 6-well culture plates; cell plating was performed at a concentration of 6 × 10⁻⁶ cells / well. 4 Cells per well, seeded according to the principle of achieving 100% confluence after overnight incubation, with a final total culture medium volume of 2 mL per well; cultured at 37°C in a 5% CO2 incubator for 24 h;

[0137] Scratching: On the second day, use a ruler to scratch a line on each well on the front side of the 6-well plate, so that the scratch on the front side is perpendicular to the horizontal line on the back side; then wash the cells 3 times with PBS to remove the scratched cells, and add serum-free culture medium to each well.

[0138] Photography: Erase the horizontal lines on the back of the 6-well plate and take photos under a 4x microscope, ensuring the scratch is centered and vertical, and that the background is consistent. Add sample solution with a concentration of 5 mg / ml (add an equal volume of PBS buffer as a control group), set a concentration gradient, and take samples at 0, 24, 48h, and 72h time points, and take photos. Use ImageJ software to calculate the mean intercellular distance or the mean scratch area. Calculate the cell migration rate using the formula: Cell migration rate (wound healing rate) = (initial scratch area - scratch area at time t) / initial scratch area * 100%.

[0139] Statistical analysis was performed using R language to analyze the test results. One-way ANOVA was used, and p < 0.05 was considered statistically significant.

[0140] The concentration of the above sample solution was set at 5 mg / mL based on the A375 cell toxicity results; at this concentration, no toxicity was observed in A375 cells.

[0141] The results are as follows Figures 10-11 As shown.

[0142] Figure 10 The effect of the fermented Chinese herbal probiotics prepared in Example 1 on the migration ability of A375 cells is shown in the figure. The scale bar is 100 μm.

[0143] Figure 11 The results show the migration rate of A375 cells by the fermented Chinese herbal probiotics prepared in Example 1, where treat represents Example 1 and control represents the control group.

[0144] Depend on Figures 10-11 It can be seen that, compared with the control group, after intervention with the herbal probiotic fermentation product of Example 1, the scratch healing of A375 cells was relatively slower, and the scratches were wider. The scratch width increased significantly after 24 hours, with statistical significance (P < 0.05), and the difference was even more significant after 48 hours and 72 hours (P < 0.01). The results indicate that the herbal probiotic fermentation product of Example 1 can inhibit the migration ability of A375 cells.

[0145] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A fermented product of traditional Chinese medicine probiotics, characterized in that, It is obtained by fermentation of the extract of a traditional Chinese medicine composition with probiotics; wherein, the raw materials of the traditional Chinese medicine composition include, by weight, 1-50 parts of red ginseng, 1-50 parts of aloe vera, 1-50 parts of ginkgo leaves, 1-50 parts of polygonatum, 1-50 parts of wolfberry, 1-50 parts of salvia miltiorrhiza, 1-50 parts of white peony root, 1-50 parts of atractylodes macrocephala, 1-50 parts of poria cocos, 1-50 parts of bletilla striata, 1-50 parts of silkworm pupa, 1-50 parts of platycodon grandiflorus, 1-50 parts of peony bark, 1-50 parts of nardostachys jatamansi, and 1-50 parts of licorice.

2. The herbal probiotic fermentation product according to claim 1, characterized in that, The raw materials of the traditional Chinese medicine composition include, by weight: 13-17 parts red ginseng, 10-14 parts aloe vera, 8-12 parts ginkgo leaves, 18-22 parts polygonatum, 18-22 parts wolfberry, 10-14 parts salvia miltiorrhiza, 8-12 parts white peony root, 10-14 parts atractylodes macrocephala, 13-17 parts poria cocos, 18-22 parts bletilla striata, 8-12 parts silkworm pupa, 13-17 parts platycodon grandiflorus, 8-12 parts peony bark, 13-17 parts nardostachys jatamansi, and 8-10 parts licorice.

3. The fermented herbal probiotic product according to claim 1 or 2, characterized in that, The raw materials of the traditional Chinese medicine composition include, by weight: 15 parts red ginseng, 12 parts aloe vera, 10 parts ginkgo leaves, 20 parts polygonatum, 20 parts wolfberry, 12 parts salvia miltiorrhiza, 10 parts white peony root, 12 parts atractylodes macrocephala, 15 parts poria cocos, 20 parts bletilla striata, 10 parts silkworm pupa, 15 parts platycodon grandiflorus, 10 parts peony bark, 15 parts nardostachys jatamansi, and 9 parts licorice.

4. The fermented herbal probiotic product according to any one of claims 1-3, characterized in that, The extract is an aqueous extract; preferably, the density of the extract is 1.00-1.50 g / cm³. 3 Preferably, the density of the extract is 1.00-1.15 g / cm³. 3 .

5. The fermented herbal probiotic product according to any one of claims 1-4, characterized in that, The probiotics are Lactobacillus plantarum and Bifidobacterium bifidum.

6. The fermented herbal probiotic product according to any one of claims 1-5, characterized in that, After fermentation with probiotics, the solid and liquid components are separated, and the liquid is sterilized to obtain the fermented product of Chinese herbal probiotics.

7. A method for preparing a traditional Chinese medicine probiotic ferment as described in any one of claims 1-6, characterized in that, The process includes the following steps: soaking the traditional Chinese medicine composition in water, then refluxing to extract, separating the solid and liquid, concentrating the liquid to obtain an extract; inoculating probiotics into the extract for fermentation, then separating the solid and liquid, sterilizing the liquid to obtain the fermented product of traditional Chinese medicine probiotics.

8. The method for preparing the probiotic fermentation product of traditional Chinese medicine according to claim 7, characterized in that, The herbal composition is soaked in water for 30-240 min; preferably, it is refluxed for 30-120 min; preferably, it is anaerobic fermented at 20-37℃ for 24-48 h; preferably, at the end of fermentation, the concentration of Lactobacillus plantarum is 1×10⁻⁶. 8 -10×10 8 The concentration of Bifidobacterium bifidum (CFU / mL) was 1×10⁻⁶. 8 -10×10 8 CFU / mL; preferably, the probiotics are activated and seed cultured before use; preferably, the volume ratio of probiotics to extract is 0.8-1.2:

10.

9. The use of a traditional Chinese medicine probiotic ferment as described in any one of claims 1-6 in the preparation of anti-aging, whitening, and melanoma-inhibiting formulations and beauty products.

10. The application according to claim 9, characterized in that, The preparation is a topical preparation; preferably, the beauty product is a topical beauty product.

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