Preparation method of composition for improving activity of bakuchiol and application of composition in products for delaying skin aging

Abstract

The invention provides a preparation method of a composition for improving the activity of bakuchiol and application of the composition in products for delaying skin aging, and belongs to the field of cosmetic additives. According to the preparation method of the saponin composition, the adopted strain is lactobacillus tarda, and the saponin composition is prepared through the steps of inoculation, fermentation culture, induced enzyme production, conversion and post-treatment. In the step of inducing enzyme production, an inducer is added into the fermentation tank, and the adding amount of the inducer is 1-2% of the mass of the basic culture medium. And mixing the prepared saponin composition powder with bakuchiol to prepare the product for delaying skin aging. Capsaicin receptors and metal matrix protease in cells can be effectively inhibited, and the expression level of sirt1 is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cosmetic additives, in particular to a preparation method of a composition for improving the activity of bakuchiol and application of the composition in skin aging delaying products. BACKGROUND

[0002] Bakuchiol component is a compound of terpene phenol. It was first isolated from the seeds of Psoralea corylifolia in 1966, and was named "bakuchiol" according to the Sanskrit name of the plant "bakuchi". Bakuchiol is also called "plant A-alcohol", which is a similar component that can replace real "A-alcohol". The advantage of bakuchiol is that it is mild and non-irritating, and has no light source sensitivity. Bakuchiol component is an effective selective component for improving skin photoaging problems, and is a more tolerant alternative component than vitamin A alcohol. It can be expected that bakuchiol care products have broad development potential in future skin care.

[0003] TRPV1 is a unique ion channel and a molecular sensor that widely accepts various physical and chemical signals in the body. When TRPV1 is activated, the conformation of TRPV1 changes, the channel opens, and the permeability to cations (mainly Ca2+) increases. In turn, it causes COX-2 expression in epithelial cells. Ca+ influx can directly or indirectly activate NF-κB as a messenger.

[0004] Sirt belongs to the class of deacetylases, and the sirt family plays an important role in energy metabolism, material metabolism and maintenance of metabolic homeostasis, and is also called longevity protein. It is closely related to aging-related diseases, and its expression decreases will lead to aging.

[0005] Matrix metalloproteinases (MMPs) are key enzymes for the degradation of type I, II and III collagen, which accelerates skin aging. UVB irradiation stimulates the secretion of MMP-1, MMP-3 and MMP-9. Therefore, if the secretion of MMP-1, MMP-3 and MMP-9 is inhibited, the degradation of collagen can be reduced, and the skin aging can be slowed down.

[0006] Effective and natural source inhibitors of TRPV1, Sirt and MMPs have been expected by the industry. Bakuchiol has a certain inhibitory effect on the production of the above pro-inflammatory factors, reactive oxygen species and MMPs, but the inhibitory effect is not ideal; in recent years, technical personnel in the industry have been committed to seeking more ideal natural source inhibitors to achieve the purpose of delaying skin aging. SUMMARY

[0007] To address the aforementioned problems in the existing technology, this invention provides a method for preparing a composition that enhances the activity of bakuchiol and its application in anti-aging skin products, and achieves the following objectives: to prepare a naturally derived anti-aging skin composition containing ginsenosides, and to use the above composition to prepare anti-aging skin products, effectively inhibiting the production of TRPV1 and matrix metalloproteinases by cells and improving Sirt expression.

[0008] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: A method for preparing a composition that enhances the activity of psoralen includes the following steps: Step 1: Selecting the strain The selected strain was *Lactobacillus fragrans*. The strain was commercially available, and its accession number is CICC 6131.

[0009] Step 2, vaccination Add the liquid basal medium to the fermenter, filling it to one-third of its volume. Inoculate *Lactobacillus fragrans* into the basal medium at room temperature, using an inoculation amount of 1.2-1.8% of the basal medium's mass. The basal medium formula used is as follows: 5.0 g beef peptone, 15.0 g beef extract powder, 8.0 g yeast extract, 10.0 g lactose, 5.0 g malt extract, 5.0 g NaAc, 1.0 g chondroitin sulfate, 2.0 g diammonium citrate, 1.0 g monosodium glutamate, 2.0 g K2HPO4, 0.2 g MgSO4·7H2O, 0.05 g vitamin C, 0.05 g MnSO4·H2O, 0.5 g calcium carbonate, 0.8 g Tween 80, 1000.0 mL distilled water, pH 6.4-6.8.

[0010] Step 3: Fermentation culture After inoculation, start heating and stirring to carry out fermentation culture. The fermentation culture temperature is 34-35℃, the stirring rate is 60-80rpm, and the fermentation culture time is 45-50 hours.

[0011] Step 4: Induction of enzyme production Add an inducer to the fermenter. The amount of inducer added should be 1-2% of the mass of the basal culture medium.

[0012] The inducing agent is a mixture of ginseng root total saponins and anemarrhena asphodeloides extract in a mass ratio of 7:3. After adding the inducing agent, fermentation continues for 48-60 hours at a temperature of 34-35℃ and a stirring speed of 60-80 rpm. The ginseng root total saponins have a mass percentage of 80%; the anemarrhena asphodeloides extract has a mass percentage of 95%, and 98% of the particles pass through an 80-mesh sieve.

[0013] Step 5, Conversion Add substrate and conversion-promoting matrix to the fermenter; continue fermentation for 60-72 hours at a temperature of 35-37℃ and a stirring rate of 80-120 rpm.

[0014] The amount of substrate added is 2-3% of the mass of the basal culture medium; the amount of the transformation-promoting matrix added is 5-8% of the mass of the basal culture medium. The substrate is 90% total saponins from ginseng root. The formulation of the transformation-promoting matrix is: 0.2g vitamin B5, 0.07g inositol, 0.1g glycine, and 1000ml water.

[0015] Step 6, Post-processing After the transformation culture is completed, the material is discharged. At the same time, the liquid phase is passed through a 300-mesh sieve and retained. The liquid phase is then concentrated to 10% of its original volume to obtain a concentrated solution.

[0016] The concentrate was added to ethanol and stirred for 4-5 minutes. Insoluble matter was removed by centrifugation, and the mixture was concentrated until the water content reached 24-26%, yielding the crude extract. The volume ratio of the concentrate to ethanol was 1:4; the stirring speed was 200-280 rpm; and the ethanol concentration was 95%.

[0017] The crude extract was purified by macroporous adsorption resin; after the saponins were adsorbed by the resin, the residue was washed with water for 5 column volumes to remove impurities.

[0018] The saponins were eluted with 90% alcohol, and the eluent was evaporated to dryness to obtain a saponin composition powder.

[0019] Application of a saponin composition in a product for delaying skin aging: The prepared saponin composition powder is mixed with psoralen at a mass ratio of 1-2:0.5-2 to obtain a product for delaying skin aging. Beneficial effects

[0020] (1) The present invention provides a saponin composition that can effectively inhibit the production of TRPV1 and matrix metalloproteinases by cells.

[0021] (2) The saponin composition of the present invention can produce a synergistic effect with bakuchiol, significantly enhance the activity of bakuchiol, and enhance the effect of delaying skin aging.

[0022] (3) The prepared anti-aging skin products can significantly improve the expression level of sirt1. Attached Figure Description

[0023] Appendix Figure 1 The images show the electrophoresis results of TRPV1 expression levels in the blank group, model group, and experimental groups 1-3 in Example 3. AppendixFigure 2 This is a graph showing the content analysis of TRPV1 expression in the blank group, model group, and experimental groups 1-3 in Example 3; Appendix Figure 3 The image shows the electrophoresis results of detecting sirt1 expression levels in the blank group, model group, and experimental groups 1-3 in Example 4. Appendix Figure 4 This is a graph showing the content analysis of sirt1 expression in the blank group, model group, and experimental groups 1-3 in Example 4; Appendix Figure 5 This is a graph showing the content analysis of MMP-1 in the cell supernatant of the blank group, model group, and experimental groups 1-3 in Example 5; Appendix Figure 6 This is a graph showing the content analysis of MMP-3 in the cell supernatant of the blank group, model group, and experimental groups 1-3 in Example 5; Appendix Figure 7 This is a graph showing the content analysis of MMP-9 in the cell supernatant of the blank group, model group, and experimental groups 1-3 in Example 5. Detailed Implementation

[0024] Example 1: A method for preparing a composition that enhances the activity of psoralen. Step 1: Selecting the strain The selected strain was *Lactobacillus fragrans*. The strain was commercially available, and its accession number is CICC 6131.

[0025] Step 2, vaccination Add liquid basal culture medium to the fermenter, filling it to one-third of its volume. Inoculate *Lactobacillus branii* into the basal culture medium at room temperature, using an inoculation amount of 1.5% of the basal culture medium's mass. The basal culture medium formula used is as follows: 5.0 g beef peptone, 15.0 g beef extract powder, 8.0 g yeast extract, 10.0 g lactose, 5.0 g malt extract, 5.0 g NaAc, 1.0 g chondroitin sulfate, 2.0 g diammonium citrate, 1.0 g monosodium glutamate, 2.0 g K2HPO4, 0.2 g MgSO4·7H2O, 0.05 g vitamin C, 0.05 g MnSO4·H2O, 0.5 g calcium carbonate, 0.8 g Tween 80, 1000.0 mL distilled water, pH 6.5.

[0026] Step 3: Fermentation culture After inoculation, start heating and stirring to carry out fermentation culture. The fermentation culture temperature is 34.5℃, the stirring rate is 70rpm, and the fermentation culture time is 48 hours.

[0027] Step 4: Induction of enzyme production Add an inducer to the fermenter at a rate of 1.5% of the mass of the basal culture medium.

[0028] The inducing agent is a mixture of ginseng root total saponins and anemarrhena asphodeloides extract in a mass ratio of 7:3. After adding the inducing agent, fermentation continues for 48 hours at a temperature of 34.5℃ and a stirring speed of 70 rpm. The ginseng root total saponins have a mass percentage of 80%; the anemarrhena asphodeloides extract has a mass percentage of 95%, and 98% of the particles pass through an 80-mesh sieve.

[0029] Step 5, Conversion Substrate and conversion-promoting matrix were added to the fermenter; fermentation was continued for 72 hours at a temperature of 36°C and a stirring rate of 120 rpm.

[0030] The substrate was added at 2.5% of the mass of the basal culture medium; the conversion-promoting matrix was added at 6% of the mass of the basal culture medium. The substrate was ginseng root total saponins with a mass percentage of 90%. The conversion-promoting matrix formula was: vitamin B5 0.2g, inositol 0.07g, glycine 0.1g, and water 1000ml.

[0031] Step 6, Post-processing After the transformation culture is completed, the material is discharged. At the same time, the liquid phase is passed through a 300-mesh sieve and retained. The liquid phase is then concentrated to 10% of its original volume to obtain a concentrated solution.

[0032] The concentrate was added to ethanol and stirred for 4 minutes. Insoluble matter was removed by centrifugation, and the mixture was concentrated until the water content reached 25%, yielding the crude extract. The volume ratio of the concentrate to ethanol was 1:4; the stirring speed was 260 rpm; and the ethanol concentration was 95%.

[0033] The crude extract was purified by macroporous adsorption resin; after the saponins were adsorbed by the resin, the residue was washed with water for 5 column volumes to remove impurities.

[0034] The saponins were eluted with 90% alcohol, and the eluent was evaporated to dryness to obtain a saponin composition powder, which is a composition that enhances the activity of psoralen.

[0035] Example 2: Application of a saponin composition in an anti-aging skin product The prepared saponin composition powder was mixed with psoralen at a mass ratio of 1:1 to obtain a product that delays skin aging.

[0036] The content of psoralen is 98%.

[0037] Example 3: Experiment on the effect of TRPV1 expression level Animal feeding, grouping, and modeling: After being fed at room temperature and relative humidity of 40% to 50% for 4 days, rats were randomly divided into 5 groups: blank group, model group, experimental group 1, experimental group 2, and experimental group 3.

[0038] Rats in experimental groups 1-3 were administered the drugs by gavage for 10 consecutive days, once daily. Specifically, experimental group 1 was given Bakuchiol 100 mg / kg; experimental group 2 was given the saponin composition (Ginsenosides) prepared in Example 1 100 mg / kg; and experimental group 3 was given the product prepared in Example 2 (Bakuchiol + Ginsenosides) 100 mg / kg.

[0039] Two hours after administration on day 10, rats in the model group and experimental groups 1-3 were injected intravenously with LPS (bacterial lipopolysaccharide) at a dose of 6 mg / kg to establish an acute pneumonia model.

[0040] Rats in the blank group, model group, and experimental groups 1-3 were fed for another 3 days, and lung tissue was harvested from each group. The expression of TRPV1 protein in the lung tissue of the five groups of rats was detected by Western blot.

[0041] The Western blot method for detecting the expression of the target protein is as follows: The tissue to be tested is added to RIPA tissue lysis buffer and thoroughly homogenized. After homogenization, it is centrifuged at 4°C for 5 min at a speed of 12,000 r / min. -1 Collect the supernatant and determine the protein concentration using the BCA protein quantification method. Then perform SDS-PAGE with polyacrylamide gel electrophoresis. Prepare a 10% separating gel and a 5% stacking gel. After loading the sample, initially maintain a constant voltage of 70 V. Once the indicator enters the separating gel, adjust the voltage to 90 V and continue electrophoresis. After electrophoresis, transfer the membrane to a PVDF membrane and place it in a blocking chamber. Place the blocking chamber on a decolorizing shaker and shake at low speed for 3 hours at room temperature. Wash the PVDF membrane 2-3 times to remove residual blocking solution. Dilute the primary antibody for the target protein according to the dilution ratio in the antibody instructions. Place the cut bands into the corresponding antibody tubes and incubate overnight on a shaker at 4°C. Wash the membrane, add the secondary antibody, and incubate for another 2 hours at room temperature. Wash the membrane again, and then use the enhanced ECL method to develop the protein color. Calculate the relative expression level of TRPV1 protein based on the results.

[0042] Results: The protein expression results of TRPV1 in the lung tissues of the five groups of rats are shown in the attached figure. Figure 1 and attached Figure 2As shown in the figure, compared with the control group, the expression level of TRPV1 protein in the lung tissue of rats in the model group was significantly increased (P < 0.01), indicating that the modeling was successful.

[0043] The expression level of TRPV1 protein in the lung tissue of rats in Experiment 1-3 (P < 0.01) was significantly lower than that in the model group. Among them, the expression level of TRPV1 protein in the lung tissue of rats in Experiment 3 was the lowest, which was significantly better than that in Experiment 1 and Experiment 2. This indicates that the saponin composition prepared in Example 1 can produce a significant synergistic effect with psoralen.

[0044] The saponin composition prepared in Example 1 can effectively inhibit the expression of TRPV1 protein.

[0045] Example 4: Experiment on the effect on sirt1 protein expression Detection method: After 7 days of adaptive feeding, Wistar rats were randomly divided into 5 groups, one of which was a blank control group and the other 4 groups were used for modeling.

[0046] The blank group was fed with ordinary feed for 6 weeks; after 6 weeks, the blank group was injected intraperitoneally with 35 mg / kg solvent as a control.

[0047] Four groups of rats were fed a high-fat diet for 6 weeks. After 6 weeks, these four groups of rats were intraperitoneally injected with STZ in sodium citrate buffer (pH 4.5) solution (35 mg / kg) under fasting conditions to establish the T2DM model. Three days after injection, a blood glucose level ≥16.7 mol / L in the model group was considered a successful model. The successfully modeled rats were randomly divided into the model group, experimental group 1, experimental group 2, and experimental group 3.

[0048] Experimental groups 1-3 were administered oral medication as follows: Experimental group 1: Bakuchiol was administered orally at a dose of 100 mg / kg; Experimental group 2: The saponin composition (Ginsenosides) prepared in Example 1 was administered orally at a dose of 100 mg / kg; Experimental group 3: The product prepared in Example 2 (Bakuchiol + Ginsenosides) was administered orally at a dose of 100 mg / kg.

[0049] The oral administration frequency is once a day for 6 consecutive weeks.

[0050] Then, livers were collected from rats in the blank group, model group, and experimental groups 1-3, and sirt1 protein expression was detected by Western blot (the specific method is the same as the detection method in Example 3). The detection results are attached.Figure 3 and attached Figure 4 As shown.

[0051] Analysis shows that the Sirt1 protein content in the model group was significantly lower than that in the blank group, indicating that the modeling was successful.

[0052] The Sirt1 content in the livers of rats in experimental groups 1-3 was significantly higher than that in the model group (p < 0.01). In particular, the Sirt1 expression level was highest in experimental group 3 (the group that received the anti-aging product from Example 2), even exceeding that of the blank group. This indicates that the combined use of psoralen and saponin significantly enhanced Sirt1 expression compared to using either alone. Therefore, the saponin composition prepared in Example 1 significantly promoted Sirt1 protein expression. Regarding the inhibition of Sirt1 protein expression, the saponin composition prepared in Example 1 exhibited a significant synergistic effect with psoralen.

[0053] Example 5: Experiment on the effect on matrix metalloproteinases Detection method: HaCaT cells (human immortalized keratinocytes) were cultured in DMEM medium containing 10% FBS and 1% penicillin and streptomycin at 37°C in an incubator containing 5% carbon dioxide (CO2). When the cells reached 80% confluence, HaCaT cells were washed three times with phosphate-buffered saline (PBS).

[0054] Cells were divided into four groups: blank control group, control group, experimental group 1, experimental group 2, and experimental group 3. The following treatments were then applied: The saponin composition (Ginsenosides) prepared in Example 1 was added to the cell culture medium of Experiment 1. Bakuchiol was added to the cell culture medium of Experiment 2. The anti-aging product (Bakuchiol + Ginsenosides) prepared in Example 2 was added to the cell culture medium of Experiment 3.

[0055] The control group received no drug treatment.

[0056] HaCaT cells from the control group, experimental group 1, experimental group 2, and experimental group 3 were exposed to UVB (144 mJ / cm²) for 24 hours. After UVB irradiation, the cells were washed with PBS immediately.

[0057] The control group received no UVB irradiation or drug treatment.

[0058] Cells from each group were collected, and RNA was extracted using TRNzol lysis buffer. Following the instructions of the reverse transcription kit, the RNA was reverse transcribed into cDNA. qRT-PCR was then performed using a real-time quantitative PCR kit to detect the levels of MMP-1, MMP-3, and MMP-9 in HaCaT cells from each group. The results are shown in the attached figure. Figures 5-7 As shown.

[0059] From the appendix Figures 5-7 It is evident that the levels of MMP-1, MMP-3, and MMP-9 in the control group were significantly higher than those in the blank group, indicating that UVB irradiation significantly increases the levels of these three enzymes in cells.

[0060] In the cells of experimental groups 1-3, the levels of the three enzymes MMP-1, MMP-3, and MMP-9 were significantly reduced, especially in experimental group 3, where the levels of these three enzymes were significantly lower than in experimental groups 1 and 2. This indicates that the saponin composition prepared in Example 1 has a significant inhibitory effect on the expression of the three enzymes MMP-1, MMP-3, and MMP-9.

[0061] In inhibiting the expression of three enzymes, MMP-1, MMP-3, and MMP-9, the saponin composition prepared in Example 1 exhibits a significant synergistic effect with psoralen.

[0062] Unless otherwise specified, all ratios mentioned in this invention are mass ratios, and all percentages are mass percentages.

[0063] Obviously, there are many other possible implementation methods under the concept of this invention. It should be stated here that any changes made under the inventive concept of this invention will fall within the protection scope of this invention.

Claims

1. A method for preparing a composition that enhances the activity of psoralen, characterized in that: The strain used was Lactobacillus fragrans, and the saponin composition was prepared through inoculation, fermentation culture, induction of enzyme production, transformation, and post-treatment.

2. The method for preparing a composition for enhancing the activity of psoralen according to claim 1, characterized in that: The inoculation process used a basic culture medium comprising the following components: beef peptone, beef extract, yeast extract, lactose, malt extract, NaAc, chondroitin sulfate, diammonium citrate, sodium glutamate, K2HPO4, MgSO4·7H2O, vitamin C, MnSO4·H2O, and calcium carbonate.

3. The method for preparing a composition for enhancing the activity of psoralen according to claim 1, characterized in that: The inoculation amount is 1.2-1.8% of the mass of the basal culture medium.

4. The method for preparing a composition for enhancing the activity of psoralen according to claim 1, characterized in that: The induction of enzyme production: an inducer is added to the fermenter, the amount of which is 1-2% of the mass of the basal culture medium.

5. A method for preparing a composition for enhancing the activity of psoralen according to claim 4, characterized in that: The inducer is a mixture of ginseng root total saponins and anemarrhena extract in a mass ratio of 7:

3.

6. A method for preparing a composition for enhancing the activity of psoralen according to claim 1, characterized in that: The conversion process involves adding substrate and conversion-promoting matrix to the fermenter and continuing fermentation for 60-72 hours.

7. A method for preparing a composition for enhancing the activity of psoralen according to claim 6, characterized in that: The substrate is total ginseng root saponins with a mass percentage of 90%.

8. A method for preparing a composition for enhancing the activity of psoralen according to claim 6, characterized in that: The formula for the conversion-promoting matrix is ​​as follows: Vitamin B5 0.15-0.3g, Inositol 0.05-0.09g, Glycine 0.08-0.15g, and Water 1000ml.

9. A method for preparing a composition for enhancing the activity of psoralen according to claim 2, characterized in that: The basic culture medium formula is as follows: 5.0 g beef peptone, 15.0 g beef extract powder, 8.0 g yeast extract, 10.0 g lactose, 5.0 g malt extract, 5.0 g NaAc, 1.0 g chondroitin sulfate, 2.0 g diammonium citrate, 1.0 g monosodium glutamate, 2.0 g K2HPO4, 0.2 g MgSO4·7H2O, 0.05 g vitamin C, 0.05 g MnSO4·H2O, 0.5 g calcium carbonate, 0.8 g Tween 80, 1000.0 mL distilled water, pH 6.4-6.

8.

10. The application of a saponin composition in a product for delaying skin aging, characterized in that: The prepared saponin composition powder was mixed with psoralen at a mass ratio of 1-2:0.5-2 to obtain a product that delays skin aging.

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