Sijunzi decoction self-fermentation product and application thereof in preparing cosmetics

Abstract

The invention discloses a Sijunzi decoction self-fermentation product and application of the Sijunzi decoction self-fermentation product to preparation of cosmetics, and a preparation method of the self-fermentation product comprises the following steps: weighing ginseng decoction piece powder, bighead atractylodes rhizome decoction piece powder and liquorice decoction piece powder according to the proportion of decoction pieces in a Sijunzi decoction prescription, and sieving all the powder; uniformly mixing and stirring the powder with a proper amount of water, sterilizing at high temperature to obtain a self-solid fermentation substrate, and cooling for later use; uniformly inoculating fresh poria cocos sclerotia into the self-solid fermentation substrate, and fermenting at 25-35 DEG C for 3-20 days; and after the fermentation is finished, carrying out ultrasonic extraction by using 70-95% ethanol, centrifuging, collecting supernate, and freeze-drying to obtain a self-fermentation product of the sijunzi decoction. Activity researches show that the self-fermentation product provided by the invention has the activities of moisturizing, maintaining skin elasticity, promoting skin injury repair, resisting skin photoaging, delaying skin aging, protecting skin barrier and resisting skin inflammation, and has the prospect of being developed into cosmetics with corresponding activities.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine fermentation, relates to a traditional Chinese medicine compound self-solid fermentation product and an application thereof, and specifically relates to a Sijunzi decoction self-fermentation product and an application thereof in preparing cosmetics. Background Art

[0002] As one of the traditional Chinese medicine prescriptions, Sijunzi Decoction contains ginseng, Atractylodes macrocephala, Poria cocos, and licorice, which are rich in multiple active ingredients and active functions. The Poria cocos in the prescription is the dried sclerotium of the Polyporaceae fungus Poria cocos (Schw.) Wolf, which has the effects of dispelling dampness and promoting diuresis, strengthening the spleen and stomach, and calming the mind. Modern studies have shown that Poria cocos has the effects of regulating immunity, anti-inflammatory, anti-tumor, and lowering blood sugar, but it is generally used after being dried and processed. There is a lack of investigation and exploration of the use of fresh Poria cocos sclerotium as a fungus for two-way solid fermentation with other medicinal materials in Sijunzi Decoction and other applications.

[0003] When studying the bidirectional solid self-fermentation of fresh Poria cocos sclerotia and other three medicinal materials in Sijunzi Decoction, our research group found that the fermentation product had significant improvement in certain biological activities. Based on this discovery, the present invention is proposed. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a self-fermented product of Sijunzi decoction and its use in preparing cosmetics.

[0005] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0006] A self-fermented product of Sijunzi decoction, the preparation method comprising the following steps:

[0007] Step S1: according to the proportion of the decoction pieces in the Sijunzi Decoction, weigh the ginseng decoction piece powder, the white atractylodes decoction piece powder, and the licorice decoction piece powder, and sieve all the powders; use fresh Poria cocos sclerotia, cut into small pieces after ultraviolet sterilization;

[0008] Step S2: Mix the above powder with an appropriate amount of water, sterilize at high temperature to obtain a self-solid fermentation substrate, and cool it for later use;

[0009] Step S3: uniformly inoculating fresh Poria cocos sclerotia into the above self-solid fermentation substrate, and fermenting at 25-35° C. for 3-20 days;

[0010] Step S4: After the fermentation is completed, ultrasonic extraction is performed using 70-95% ethanol, centrifugation is performed, the supernatant is collected, and freeze-dried to obtain the self-fermentation product of Sijunzi Decoction.

[0011] Preferably, the inoculation mass of fresh Poria cocos sclerotia is 10-40% of the sum of the mass of all powders and fresh Poria cocos sclerotia.

[0012] Preferably, the mass ratio of the ginseng slice powder, the atractylodes macrocephala slice powder, and the licorice slice powder is 1.5:1.5:1.

[0013] Preferably, in step S2, the mass ratio of powder to water is 1:1-4.

[0014] Preferably, in step S4, the centrifugal speed is 4000-8000 r / min, and the centrifugal time is 20-40 min.

[0015] Preferably, in step S4, the ultrasonic temperature is 30-60° C., and the ultrasonic time is 100-200 min.

[0016] The self-fermented product of Sijunzi Decoction is used for preparing cosmetics with improved moisturizing effect.

[0017] The self-fermented product of Sijunzi Decoction is used for preparing cosmetics for maintaining skin elasticity.

[0018] The self-fermented product of Sijunzi Decoction is used for preparing cosmetics for promoting skin damage repair.

[0019] The self-fermented product of Sijunzi Decoction is used for preparing cosmetics for resisting skin photoaging.

[0020] The self-fermented product of Sijunzi Decoction is used for preparing cosmetics for delaying skin aging.

[0021] The self-fermented product of Sijunzi Decoction is used for preparing cosmetics for protecting the skin barrier.

[0022] The self-fermented product of Sijunzi Decoction is used for preparing cosmetics for resisting skin inflammation.

[0023] Beneficial effects:

[0024] 1. The present invention provides a self-fermented product of Sijunzi Decoction, which is obtained by solid fermentation of fresh Poria cocos sclerotia and other medicinal materials in the Sijunzi Decoction, and the preparation method is simple and feasible;

[0025] 2. Activity studies have shown that the self-fermented product provided by the present invention has the activities of moisturizing, maintaining skin elasticity, promoting skin damage repair, resisting skin photoaging, delaying skin aging, protecting the skin barrier and resisting skin inflammation, and has the prospect of being developed into cosmetics with corresponding activities. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the expression level of AQP3 and HAS2 in HaCaT cells with different treatments;

[0027] Figure 2is the expression level of Claudin-1 in HaCaT cells with different treatments;

[0028] Figure 3 The expression levels of inflammatory factors IL-6 and TNF-α in HaCaT cells with different treatments. DETAILED DESCRIPTION

[0029] The essential contents of the present invention are described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0030] Example 1: Fermentation product preparation example

[0031] 1. Preparation of fresh Poria cocos sclerotia

[0032] Wash the fresh Poria sclerotium, wipe it dry, and place it under the ultraviolet light of the clean bench for sterilization for 1 hour. Turn the sclerotium every 20 minutes to ensure that it is fully sterilized. After sterilization, cut the Poria sclerotium into small square pieces (about 1cm×1cm×1cm) for later use.

[0033] 2. Preparation of the Self-fermentation Products of Sijunzi Decoction

[0034] Step S1: according to the proportion of the decoction pieces in the Sijunzi Decoction prescription, weigh 18g of ginseng decoction piece powder, 18g of Atractylodes macrocephala decoction piece powder, and 12g of licorice decoction piece powder, all of which can pass through an 8-mesh sieve but cannot pass through a 30-mesh sieve;

[0035] Step S2: Mix the above powder with 120 g of water, sterilize at 121° C. for 30 min to obtain a self-solid fermentation substrate, and cool for later use;

[0036] Step S3: 18 g of fresh Poria cocos sclerotia (the inoculation mass is about 27% of the sum of the mass of all powders and fresh Poria cocos sclerotia in step S1) are uniformly inoculated into the above-mentioned self-solid fermentation substrate, and fermented at 28° C. for 7 days;

[0037] Step S4: After the fermentation is completed, ultrasonic extraction is performed with 95% ethanol (95% volume fraction ethanol aqueous solution, solid-liquid ratio is 1g:10mL) at 40°C for 1h, and extraction is performed 3 times in total. The mixture is centrifuged at 6000r / min for 30min, the supernatant is collected, and freeze-dried to obtain the self-fermentation product of Sijunzi Decoction.

[0038] In the above steps, fresh Poria cocos sclerotia can be fermented using a solid fermentation substrate.

[0039] 3. Preparation of unfermented products of Sijunzi Decoction (as a comparison with the self-fermented products of Sijunzi Decoction)

[0040] Step S1: according to the proportion of the decoction pieces in the Sijunzi Decoction prescription, weigh 18g of ginseng decoction piece powder, 18g of Atractylodes macrocephala decoction piece powder, and 12g of licorice decoction piece powder, all of which can pass through an 8-mesh sieve but cannot pass through a 30-mesh sieve;

[0041] Step S2: Mix the above powder with 120g of water, sterilize at 121°C for 30min to obtain sterilized powder, and cool for later use;

[0042] Step S3: evenly adding 18 g of Poria cocos decoction powder to the sterilized powder, and placing at 28° C. for 7 days;

[0043] Step S4: After 7 days, ultrasonic extraction was performed with 95% ethanol (95% volume fraction ethanol aqueous solution, solid-liquid ratio of 1g:10mL) at 40°C for 1h, for a total of 3 extractions, and centrifuged at 6000r / min for 30min, the supernatant was collected, and freeze-dried to obtain the unfermented product of Sijunzi Decoction.

[0044] In the above steps, the Poria cocos decoction pieces are different from fresh Poria cocos sclerotia and do not have fermentation activity.

[0045] Example 2: Fermentation product activity test example

[0046] In a clean bench, HaCaT cells before F13 were recovered and made into cell suspension using DMEM culture medium containing 10% FBS. 5 Each well was inoculated in a 6-well plate and placed in a 37°C, 5% CO2 incubator until the cells were completely attached (about 70%). The previously plated HaCat cells were removed from the incubator, the old culture medium was discarded, and after washing once with PBS buffer, it was replaced with a complete culture medium containing 12.5, 25, 50 μg / mL of Sijunzi Decoction self-fermentation products or Sijunzi Decoction unfermentation products. At the same time, a blank group without Sijunzi Decoction self-fermentation products and Sijunzi Decoction unfermentation products was set up and placed in a 37°C, 5% CO2 incubator for culture. After 24 hours of culture, the cells were collected, washed, and lysed, and the expression levels of aquaporin 3 (AQP3), hyaluronan synthase 2 (HAS2), and tight junction protein 1 (Claudin-1) genes were determined by RT-QPCR according to the instructions of the kit.

[0047] The RT-QPCR steps are as follows:

[0048] ① After RNA extraction, purity and concentration were tested, genomic DNA was removed, and then gently pipetted to mix, and placed in a gene amplification instrument at 42°C for 2 minutes.

[0049] ②Reverse transcription

[0050] a. Preparation of reverse transcription reaction system:

[0051]

[0052] Mix by gently pipetting.

[0053] b. Perform reverse transcription reaction:

[0054]

[0055]

[0056] ③Real-Time qPCR

[0057] a. Use the obtained cDNA as a template for quantitative PCR detection. The reaction system is as follows:

[0058]

[0059] b. Use Real-Time qPCR instrument for qPCR detection.

[0060] The CT value of each sample was corrected with the internal reference to obtain ΔCt, and then 2 -ΔΔCt Compare the differences between groups.

[0061] Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad Software, Inc, USA). One-way analysis of variance (ANOVA) was used for comparisons between different groups. All data were n ≥ 3, with Compared with the blank group, * P<0.05, ** P<0.01, *** P < 0.001, **** P<0.0001, indicating a statistically significant difference.

[0062] The expression levels of AQP3, HAS2, and Claudin-1 in HaCaT cells treated with different methods are shown in Tables 1 and Figures 1-2 shown.

[0063] Table 1 Expression levels of AQP3, HAS2 and Claudin-1 in HaCaT cells treated with different methods

[0064]

[0065] Table 1 and Figures 1-2The results showed that: (1) Compared with the blank group, the expression levels of AQP3, HAS2, and Claudun-1 in the HaCaT cells of the non-fermented group were not significantly increased; (2) Compared with the blank group, the expression levels of AQP3, HAS2, and Claudun-1 in the HaCaT cells of the fermented group were significantly increased, and the difference was statistically significant.

[0066] Those skilled in the art know that ultraviolet irradiation causes skin moisture loss, causing dry skin and aggravating the progression of photoaging, while improving skin moisture content and promoting moisturizing function help to resist photoaging. AQP-3 is an important transporter for maintaining skin hydration, participating in the absorption and secretion of cellular substances, helping to maintain elasticity and repair damaged stratum corneum (reference: Evaluation of in vitro anti-photoaging activity of ultrafiltration components of Dendrobium candidum and preliminary exploration of mechanisms, master's thesis of Jiangnan University, 2022). Therefore, the above experimental results show that the self-fermented product of Sijunzi Decoction provided by the present invention can improve the moisture of the skin, has moisturizing activity, moisturizing helps maintain skin elasticity and promotes the repair of skin damage, helps resist photoaging, and can be used to prepare cosmetics that moisturize, maintain skin elasticity, promote skin damage repair and / or resist skin photoaging.

[0067] Those skilled in the art know that hyaluronic acid (HA) is the main component of the intercellular matrix and extracellular matrix. HA can interact with other matrix molecules in the human body to maintain the integrity of cell and tissue structure, provide an internal environment for cells, maintain the stability and elasticity of the extracellular matrix, and have an important impact on the physiological function of cells. The carboxyl group and other polar groups in the HA molecule can form hydrogen bonds with water to bind a large amount of water. Its theoretical water retention value is as high as 500mL / g or more, so it has a good moisturizing effect and is widely added to various cosmetics and pharmaceutical products as a natural moisturizing factor. At the same time, increasing the content of HA in the skin can delay cell or tissue aging, which is very popular in skin beauty (reference: Effect of N-acetylglucosamine on hyaluronic acid synthesis of human skin fibroblasts, Biotechnology, 2012). HA can also promote the proliferation and migration of epidermal keratinocytes and promote skin wound healing (Efficacy study of moisturizing and barrier function of rose / iris / lily active composition based on infant 3D skin model, Daily Chemical Science, 2024). Therefore, the above experimental results show that the self-fermentation product of Sijunzi Decoction provided by the present invention can promote the synthesis of HA in the skin, improve the moisturizing ability of the skin, delay the aging of skin cells or tissues, and promote the repair of skin damage, and can be used to prepare cosmetics that moisturize, delay skin aging and / or promote skin damage repair.

[0068] Those skilled in the art know that the skin barrier function is mainly composed of five components: microbiome, stratum corneum, chemical barrier, immune barrier and TJ. TJ includes a variety of transmembrane proteins and intracellular proteins, mainly including claudin family, occludin family, ZO family, JAM family, symplekin protein, etc. These proteins cross-link with each other during cell polarization, eventually forming a dense mesh structure and filling and blocking the intercellular gaps. Claudin protein is the main component of the TJ structure involved in maintaining the TJ barrier function, responsible for regulating the paracellular dispersion of hydrophilic small molecules, and its reduced expression increases transepidermal water loss, leading to damage to the skin barrier function. Claudin-1 and Claudin-4 play a key role in maintaining the epidermal barrier of mammals (references: Post-translational modifications of tight junction transmembrane proteins and their direct effect on barrier function [J]. BiochimBiophys Acta Biomembr, 2020; Dose-dependent role of claudin-1 in vivo inorchestrating features of atopic dermatitis [J]. Proc Natl Acad Sci USA, 2016). Therefore, the above experimental results show that the self-fermented product of Sijunzi Decoction provided by the present invention has a skin barrier protective effect and can be used to prepare cosmetics that protect the skin barrier.

[0069] Example 3: Fermentation product activity test example

[0070] In a clean bench, HaCaT cells before F13 were recovered and made into cell suspension using DMEM culture medium containing 10% FBS. 5 Cells were inoculated into 6-well plates at 37°C and 5% CO2. When the cell density reached 60%-70%, the cells were treated with basal medium for 12 hours. The old medium was discarded and washed with PBS buffer. The corresponding medium was replaced according to the following groups and continued to be cultured:

[0071] Blank group (Control): complete medium;

[0072] Model group: complete medium containing 20 μg / mL LPS;

[0073] Unfermented group (low concentration, 15 μg / mL): 15 μg / mL unfermented product of Sijunzi Decoction was added to the culture medium of the model group;

[0074] Fermentation group (low concentration, 15 μg / mL): 15 μg / mL of Sijunzi decoction fermentation product was added to the culture medium of the model group;

[0075] Unfermented group (high concentration, 30 μg / mL): 30 μg / mL unfermented product of Sijunzi Decoction was added to the culture medium of the model group;

[0076] Fermentation group (high concentration, 30 μg / mL): 30 μg / mL of Sijunzi Decoction fermentation product was added to the culture medium of the model group.

[0077] After continuing to culture for 18 h, the cells were collected, washed, and lysed, and the expression levels of the inflammatory factors IL-6 and TNF-α genes were determined by RT-QPCR according to the instructions of the kit.

[0078] The RT-QPCR steps are as follows:

[0079] ① After RNA extraction, purity and concentration were tested, genomic DNA was removed, and then gently pipetted to mix, and placed in a gene amplification instrument at 42°C for 2 minutes.

[0080] ②Reverse transcription

[0081] a. Preparation of reverse transcription reaction system:

[0082]

[0083] Mix by gently pipetting.

[0084] b. Perform reverse transcription reaction:

[0085]

[0086] ③Real-Time qPCR

[0087] a. Use the obtained cDNA as a template for quantitative PCR detection. The reaction system is as follows:

[0088]

[0089] b. Use Real-Time qPCR instrument for qPCR detection.

[0090] The CT value of each sample was corrected with the internal reference to obtain ΔCt, and then 2 -ΔΔCt Compare the differences between groups.

[0091] Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad Software, Inc, USA). One-way analysis of variance (ANOVA) was used for comparisons between different groups. All data were n ≥ 3, with Compared with the blank group, *** P<0.001; compared with the model group, ## P<0.01, ### P<0.001, indicating a statistically significant difference.

[0092] The expression levels of inflammatory factors IL-6 and TNF-α in HaCaT cells treated with different methods are shown in Tables 2 and Figure 3 shown.

[0093] Table 2 Expression levels of inflammatory factors IL-6 and TNF-α in HaCaT cells treated with different

[0094]

[0095] Table 2 and Figure 3 Results show that compared with the blank group, the expression levels of inflammatory factors IL-6 and TNF-α in HaCaT cells in the model group were significantly increased, indicating that the skin inflammation model was successfully established; compared with the model group, the expression levels of inflammatory factors IL-6 and TNF-α in HaCaT cells in the low-concentration and high-concentration unfermented groups were not significantly downregulated, indicating that no obvious anti-skin inflammation effect was observed in the low-concentration and high-concentration unfermented products of Sijunzi Decoction; compared with the model group, the expression levels of inflammatory factors IL-6 and TNF-α in HaCaT cells in the low-concentration and high-concentration fermented groups were significantly downregulated, and the difference was statistically significant.

[0096] The above experimental results show that the self-fermented product of Sijunzi Decoction provided by the present invention has the effect of inhibiting skin inflammation and can be used to prepare cosmetics against skin inflammation.

[0097] The purpose of the above-mentioned embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.

Claims

1. A self-fermented product of Sijunzi Decoction, characterized in that: The preparation method comprises the following steps: Step S1: according to the proportion of the decoction pieces in the Sijunzi Decoction, weigh the ginseng decoction piece powder, the white atractylodes decoction piece powder, and the licorice decoction piece powder, and sieve all the powders; use fresh Poria cocos sclerotia, cut into small pieces after ultraviolet sterilization; Step S2: Mix the above powder with an appropriate amount of water, sterilize at high temperature to obtain a self-solid fermentation substrate, and cool it for later use; Step S3: uniformly inoculating fresh Poria cocos sclerotia into the above self-solid fermentation substrate, and fermenting at 25-35° C. for 3-20 days; Step S4: After the fermentation is completed, ultrasonic extraction is performed using 70-95% ethanol, centrifugation is performed, the supernatant is collected, and freeze-dried to obtain the self-fermentation product of Sijunzi Decoction.

2. The Sijunzi Decoction self-fermentation product according to claim 1, characterized in that: The inoculation mass of fresh Poria cocos sclerotia is 10-40% of the sum of the mass of all powders and fresh Poria cocos sclerotia.

3. The Sijunzi Decoction self-fermentation product according to claim 1, characterized in that: The mass ratio of the ginseng decoction slice powder, the atractylodes decoction slice powder and the licorice decoction slice powder is 1.5:1.5:

1.

4. Use of the self-fermented product of Sijunzi Decoction according to any one of claims 1 to 3 for preparing cosmetics with improved moisturizing properties.

5. Use of the self-fermented product of Sijunzi Decoction according to any one of claims 1 to 3 for preparing cosmetics for maintaining skin elasticity.

6. Use of the self-fermented product of Sijunzi Decoction according to any one of claims 1 to 3 for preparing cosmetics for promoting skin damage repair.

7. Use of the self-fermented product of Sijunzi Decoction according to any one of claims 1 to 3 for preparing cosmetics for resisting skin photoaging.

8. Use of the self-fermented product of Sijunzi Decoction according to any one of claims 1 to 3 for preparing cosmetics for delaying skin aging.

9. Use of the self-fermented product of Sijunzi Decoction according to any one of claims 1 to 3 for preparing cosmetics for protecting the skin barrier.

10. Use of the self-fermented product of Sijunzi Decoction according to any one of claims 1 to 3 for preparing cosmetics for resisting skin inflammation.

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