Application of rhizoma atractylodis macrocephalae extract in preparation of product with skin barrier repairing function

Abstract

The invention relates to application of a bighead atractylodes rhizome extract in preparation of a skin barrier repairing product, and belongs to the technical field of cosmetics. The bighead atractylodes rhizome extract remarkably repairs the skin barrier by up-regulating the content of ceramide and tight junction protein, inhibiting the abnormal expression of keratin and the activity of inflammatory factors and inhibiting the activity of hyaluronidase, elastase and collagenase, and has the effects of moisturizing, resisting allergy, resisting inflammation and resisting aging. The method not only improves the utilization function and additional value of the bighead atractylodes rhizome plant resource, widens the source of natural skin care product plant raw materials, but also brings more reliable skin care choices for people with sensitive skin, establishes a new benchmark for the skin care product industry, promotes the whole industry to develop towards a safer and more effective direction, and has wide application prospects. The blank of the market in the aspect of natural and efficient sensitive skin repairing products is filled, and great potential is shown in research and market application.

Description

Technical Field

[0001] The invention belongs to the technical field of cosmetic development, and particularly relates to application of atractylodes macrocephala extract in preparing a product with the function of repairing skin barrier. Background Art

[0002] The skin is the largest organ in the human body. It can not only effectively prevent excessive loss of water in the body and maintain the skin's moisture, but also resist various harmful factors from the outside world, such as ultraviolet rays, pollutants, microorganisms, and irritants. It is the first line of defense for the human body against external invasions and is vital to human health. Once the skin barrier is damaged, its function will be greatly reduced. A large amount of transepidermal water will be lost, making the skin dry and dehydrated. At this time, the skin's resistance to external stimuli drops sharply, becoming extremely fragile and sensitive, further increasing the risk of damage to sensitive skin, causing a vicious cycle, and then symptoms such as dryness, desquamation, roughness, wrinkles, erythema, burning, and swelling appear. What's more serious is that these damages may cause epidermal thickening and itching. Over time, they may also lead to other more serious skin diseases, greatly affecting people's quality of life.

[0003] At present, there are a wide variety of products on the market that repair the skin barrier. Among them, although chemically synthesized skin care ingredients can improve skin symptoms to a certain extent, they are often irritating. Some people may experience skin allergic reactions after use. Long-term use may also interfere with the normal metabolism of the skin itself, posing a potential threat to skin health. Ingredients extracted from natural plants have greater advantages in skin care safety, which meets the current consumer demand for "natural and safe" skin care. However, due to its complex ingredients, unstable content of active ingredients, and unclear mechanism of action, the skin care effects of some natural plant extracts are uneven, making it difficult to quickly and significantly relieve the symptoms of damaged skin barriers, and unable to meet consumers' demand for efficient skin care. Therefore, it is urgent to develop a new skin care ingredient that is both safe and can efficiently repair the skin barrier. Summary of the invention

[0004] The purpose of the present invention is to provide an application of an Atractylodes macrocephala extract in preparing a product having the function of repairing the skin barrier. The Atractylodes macrocephala extract of the present invention can repair the skin barrier safely and efficiently.

[0005] The invention provides application of atractylodes macrocephala extract in preparing a product with skin barrier repairing function.

[0006] The invention also provides application of the Atractylodes macrocephala extract in preparing a product with the function of restoring skin elasticity.

[0007] The invention also provides application of the Atractylodes macrocephala extract in preparing products with anti-aging function.

[0008] The present invention also provides the use of Atractylodes macrocephala Koidz. extract in the preparation of a product with the function of inhibiting skin inflammation.

[0009] The present invention also provides the use of Atractylodes macrocephala Koidz. extract in the preparation of a product with the function of anti-sensitivity.

[0010] The present invention also provides the use of Atractylodes macrocephala Koidz. extract in the preparation of a product with the function of moisturizing.

[0011] The present invention also provides the use of Atractylodes macrocephala Koidz. extract in the preparation of a product with any one or more of the functions shown in ① - ⑦:

[0012] ① Up-regulating the content of ceramide in cells;

[0013] ② Up-regulating the content of tight junction proteins in cells;

[0014] ③ Inhibiting the abnormal expression of keratin;

[0015] ④ Inhibiting the activity of inflammatory factors;

[0016] ⑤ Inhibiting the activity of hyaluronidase;

[0017] ⑥ Inhibiting the activity of elastase;

[0018] ⑦ Inhibiting the activity of collagenase.

[0019] Preferably, the product includes cosmetics.

[0020] The present invention also provides the Atractylodes macrocephala Koidz. extract in the above-mentioned use, and the extraction solvent of the Atractylodes macrocephala Koidz. extract is ethyl acetate.

[0021] Preferably, the mass-volume ratio of the ethyl acetate to Atractylodes macrocephala Koidz. for mixing and soaking is (10 - 20):1;

[0022] The time for mixing and soaking the ethyl acetate and Atractylodes macrocephala Koidz. is 12h - 36h.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] (a) The Atractylodes macrocephala Koidz. extract prepared by the use of the present invention has no obvious toxic effect on HaCaT cells as verified by examples, and has good safety. Moreover, the preparation method is simple, environmentally friendly and low in cost, can meet the requirements of industrial large-scale production, and has broad application prospects, and is an excellent natural raw material for cosmetics.

[0025] (b) Through cosmetic efficacy evaluation experiments, the present invention has clarified that the Atractylodes macrocephala extract has the efficacy of repairing the skin barrier and improving the symptoms of sensitive skin. It synergistically exerts this efficacy mainly through the following aspects: First, the Atractylodes macrocephala extract described in the present invention can significantly up-regulate the contents of ceramide and tight junction proteins; Second, the Atractylodes macrocephala extract described in the present invention can inhibit the abnormal expression of skin keratin; Third, the Atractylodes macrocephala extract described in the present invention can significantly inhibit the expression of inflammatory factors such as IL-6 and IL-8; Finally, the Atractylodes macrocephala extract described in the present invention can inhibit the activities of hyaluronidase, elastase and collagenase, and thus exhibits good cosmetic activities of moisturizing, anti-sensitivity, anti-inflammatory and anti-aging.

[0026] (c) The present invention deeply develops the Yunnan characteristic medicinal plant Atractylodes macrocephala and prepares the Atractylodes macrocephala extract for application in the field of skin care products, which is of great significance. On the one hand, it improves the utilization function and added value of Atractylodes macrocephala plant resources, broadens the source of natural skin care product plant raw materials, and injects new vitality into the development of the natural skin care product industry. On the other hand, for people with sensitive skin, the Atractylodes macrocephala extract brings them a more reliable skin care option. At the same time, its safety and effectiveness set a new benchmark for the skin care product industry, promote the entire industry to develop in a safer and more effective direction, fill the gap in the market for natural and highly effective sensitive skin repair products, and show great potential in both research and market application. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0028] Figure 1 It is a flow chart for preparing the Atractylodes macrocephala extract provided by the present invention;

[0029] Figure 2 It is a schematic diagram of the toxic effect of the Atractylodes macrocephala extract provided by the present invention on HaCaT keratinocytes;

[0030] Figure 3 It is a schematic diagram of the ability of the Atractylodes macrocephala extract provided by the present invention to up-regulate the ceramide level in HaCaT keratinocytes;

[0031] Figure 4 It is a schematic diagram of the ability of the Atractylodes macrocephala extract provided by the present invention to up-regulate the level of tight junction protein (Occludin) in HaCaT keratinocytes;

[0032] Figure 5Schematic diagram of the ability of the Atractylodes macrocephala Koidz. extract provided by the present invention to down-regulate the keratin level in HaCaT keratinocytes;

[0033] Figure 6 Schematic diagram of the anti-inflammatory activity of the Atractylodes macrocephala Koidz. extract provided by the present invention;

[0034] Figure 7 Schematic diagram of the inhibition rate of the Atractylodes macrocephala Koidz. extract provided by the present invention on hyaluronidase;

[0035] Figure 8 Schematic diagram of the inhibition rate of the Atractylodes macrocephala Koidz. extract provided by the present invention on elastase;

[0036] Figure 9 Schematic diagram of the inhibition rate of the Atractylodes macrocephala Koidz. extract provided by the present invention on collagenase;

[0037] Figure 10 Schematic diagram of the repair function of the Atractylodes macrocephala Koidz. extract provided by the present invention on the skin index of barrier-damaged mice;

[0038] Figure 11 Actual shot diagram of the skin recovery of skin barrier-damaged mice by the Atractylodes macrocephala Koidz. extract provided by the present invention;

[0039] Figure 12 Morphological analysis diagram of the skin pathological tissue of mice by the Atractylodes macrocephala Koidz. extract provided by the present invention;

[0040] Figure 13 Determination result diagram of the influence of the Atractylodes macrocephala Koidz. extract provided by the present invention on the ceramide content in mouse skin;

[0041] Figure 14 Determination result diagram of the influence of the Atractylodes macrocephala Koidz. extract provided by the present invention on the content of tight junction proteins in mouse skin;

[0042] Figure 15 Determination result diagram of the influence of the Atractylodes macrocephala Koidz. extract provided by the present invention on the keratin content in mouse skin. Detailed implementation mode

[0043] The present invention provides the application of the Atractylodes macrocephala Koidz. extract in the preparation of a product with a skin barrier repair function. In a specific embodiment, the product includes cosmetics. In a specific embodiment, the cosmetics include cream cosmetics. In a specific embodiment, the addition amount of the Atractylodes macrocephala Koidz. extract in the cream cosmetics can be 5% to 20%, and further can be 10% of the mass of the cream cosmetics.

[0044] The present invention also provides the application of the Atractylodes macrocephala Koidz. extract in the preparation of a product having the function of restoring skin elasticity. In a specific embodiment, the product includes cosmetics. In a specific embodiment, the cosmetics include cream cosmetics. In a specific embodiment, the addition amount of the Atractylodes macrocephala Koidz. extract in the cream cosmetics may be 5% to 20%, and further may be 10% of the mass of the cream cosmetics.

[0045] The present invention also provides the application of the Atractylodes macrocephala Koidz. extract in the preparation of a product having the anti-aging function. In a specific embodiment, the product includes cosmetics. In a specific embodiment, the cosmetics include cream cosmetics. In a specific embodiment, the addition amount of the Atractylodes macrocephala Koidz. extract in the cream cosmetics may be 5% to 20%, and further may be 10% of the mass of the cream cosmetics.

[0046] The present invention also provides the application of the Atractylodes macrocephala Koidz. extract in the preparation of a product having the function of inhibiting skin inflammation. In a specific embodiment, the product includes cosmetics. In a specific embodiment, the cosmetics include cream cosmetics. In a specific embodiment, the addition amount of the Atractylodes macrocephala Koidz. extract in the cream cosmetics may be 5% to 20%, and further may be 10% of the mass of the cream cosmetics.

[0047] The present invention also provides the application of the Atractylodes macrocephala Koidz. extract in the preparation of a product having the anti-sensitivity function. In a specific embodiment, the product includes cosmetics. In a specific embodiment, the cosmetics include cream cosmetics. In a specific embodiment, the addition amount of the Atractylodes macrocephala Koidz. extract in the cream cosmetics may be 5% to 20%, and further may be 10% of the mass of the cream cosmetics.

[0048] The present invention also provides the application of the Atractylodes macrocephala Koidz. extract in the preparation of a product having the water replenishing function. In a specific embodiment, the product includes cosmetics. In a specific embodiment, the cosmetics include cream cosmetics. In a specific embodiment, the addition amount of the Atractylodes macrocephala Koidz. extract in the cream cosmetics may be 5% to 20%, and further may be 10% of the mass of the cream cosmetics.

[0049] In a specific embodiment, the Atractylodes macrocephala Koidz. extract in the application has no obvious cytotoxicity to HaCaT cells, meeting the safety conditions for application in cosmetics.

[0050] The present invention also provides the application of the Atractylodes macrocephala Koidz. extract in the preparation of a product having any one or more of the functions shown in ① to ⑦:

[0051] ① Up-regulating the ceramide content in cells;

[0052] ② Up-regulating the content of tight junction proteins in cells;

[0053] ③ Inhibiting the abnormal expression of keratin;

[0054] ④ Inhibit the activity of inflammatory factors;

[0055] ⑤ Inhibit the activity of hyaluronidase;

[0056] ⑥ Inhibit the activity of elastase;

[0057] ⑦ Inhibit the activity of collagenase.

[0058] In specific embodiments, the product includes cosmetics. In specific embodiments, the cosmetics include cream cosmetics. In specific embodiments, the addition amount of the Atractylodes macrocephala extract in the cream cosmetics can be 5% - 20%, and further can be 10% of the mass of the cream cosmetics. In specific embodiments, the inhibition of the activity of inflammatory factors can be the inhibition of the production of inflammatory factors IL-6 and IL-8 in HaCaT cells. In specific embodiments, the Atractylodes macrocephala extract upregulates the content of tight junction proteins in cells, which can restore skin elasticity and prevent the formation of skin wrinkles. In specific embodiments, the Atractylodes macrocephala extract inhibits the abnormal expression of keratin, and the inhibition of the abnormal expression of keratin can inhibit the abnormal hyperplasia of skin keratin. In specific embodiments, the Atractylodes macrocephala extract has the function of inhibiting the activities of hyaluronidase, elastase, and collagenase, and can be added to cosmetics as ingredients for moisturizing, anti-sensitivity, anti-inflammation, and anti-aging.

[0059] The present invention also provides the Atractylodes macrocephala extract in the application described in any one of the above technical solutions. The extraction solvent of the Atractylodes macrocephala extract is ethyl acetate. In specific embodiments, the Atractylodes macrocephala in the Atractylodes macrocephala extract can be the rhizome of Atractylodes macrocephala, and further can be the dried rhizome of Atractylodes macrocephala. In specific embodiments, the Atractylodes macrocephala extract can be obtained by soaking and extracting Atractylodes macrocephala raw materials with ethyl acetate. In specific embodiments, the Atractylodes macrocephala raw materials can be Atractylodes macrocephala powder after powder processing. In specific embodiments, the mass-volume ratio of the ethyl acetate to the Atractylodes macrocephala for mixing and soaking can be (10 - 20):1, and further can be 10:1; the time for mixing and soaking the ethyl acetate and the Atractylodes macrocephala can be 12h - 36h, and further can be 24h. In specific embodiments, the Atractylodes macrocephala can be extracted repeatedly, and further can be 3 times. In specific embodiments, the extraction liquid obtained from the repeated extraction operation is collected and dried by distillation to obtain the Atractylodes macrocephala extract. In specific embodiments, the distillation drying can be vacuum distillation drying.

[0060] In order to further illustrate the present invention, the application of the Atractylodes macrocephala extract provided by the present invention in the preparation of a product with a skin barrier repair function will be described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0061] Example 1

[0062] 1. Preparation of Atractylodes macrocephala extract.

[0063] The dried rhizomes of Atractylodes macrocephala were purchased from the Xinluoshuiwan Medicinal Materials Market in Kunming in April 2023, and the voucher specimen (YNU20230405) was stored in the School of Chemical Science and Engineering of Yunnan University. The dried rhizomes of Atractylodes macrocephala (300 g) were crushed and then cold-extracted with ethyl acetate at room temperature according to the ratio of ethyl acetate to Atractylodes macrocephala liquid to material of 10:1 for 3 times (3 L each time, 24 h), the extracts were combined, and the ethyl acetate was removed by vacuum concentration to obtain the Atractylodes macrocephala extract (60.1 g).

[0064] 2. Preparation of skin cream matrix.

[0065] The preparation formula of the skin cream base is as follows: 80.3% purified water, 15% capric acid / capric triglyceride, 2% stearyl alcohol, 1% glyceryl monostearate, 1% PEG-100 stearate, 0.25% phenoxyethanol, 0.25% ethylhexylglycerin, and 0.2% ammonium acryloyldimethyltaurate / VP copolymer.

[0066] 3. Preparation of skin care cream containing Atractylodes macrocephala extract.

[0067] Preparation of 10% Atractylodes macrocephala extract skin care cream: Weigh an appropriate amount of Atractylodes macrocephala extract, add it to the skin care cream base and stir it thoroughly to obtain a 10% (w / w) Atractylodes macrocephala skin care cream for standby use.

[0068] Example 2

[0069] Toxic effect of Atractylodes macrocephala extract on HaCaT cells.

[0070] Experimental method: HaCaT cells (1×10 5 10 μL of Atractylodes macrocephala extracts were inoculated into 96-well plates and incubated for 24 hours. The Atractylodes macrocephala extracts prepared in Example 1 were dissolved in dimethyl sulfoxide (DMSO) and the final concentration of DMSO was adjusted to 2%. Different concentrations of Atractylodes macrocephala extracts were added to the cells and incubated for 24 hours. Each concentration was used in a group of 5 parallel wells. 10 μL of CCK-8 reagent was added to each well and incubated at 37°C for 2 hours. The absorbance was measured at 450 nm using an enzyme reader. The control group was untreated cells.

[0071] The cell proliferation rates after 24 h, 48 h and 72 h of incubation were measured using the same method. The results are shown in Table 1 and Figure 1 shown.

[0072] Table 1 Effect of Atractylodes macrocephala extract on cell proliferation rate of HaCaT cells

[0073]

[0074] Experimental results: The results showed that the Atractylodes macrocephala extract at a concentration of 0.5 - 62.5 μg / mL had no toxic effect on HaCaT cells, meeting the prerequisite for the safety and non-toxicity of cosmetics.

[0075] Example 3

[0076] Regulatory effects of Atractylodes macrocephala extract on ceramide, tight junction proteins, and keratin in keratinocytes.

[0077] Experimental method: HaCaT cells (1×10 5 cells / well) were seeded in 96-well plates. When the cell growth density reached about 80%, HaCaT cells were treated with Atractylodes macrocephala extract at different concentrations (6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL). After 24 h of incubation, the cells were collected, and proteins were extracted using RIPA lysis buffer. The protein concentration was measured by the BCA method, and then the contents of ceramide, tight junction proteins, and keratin were quantified according to the instructions provided by the ELISA kit. The same method was used to evaluate the changes in the levels of ceramide, tight junction proteins, and keratin after treatment with Atractylodes macrocephala extract for different times (24 h, 48 h, and 72 h). The results of the effect of Atractylodes macrocephala extract on ceramide in HaCaT keratinocytes are shown in Table 2 and Figure 3 as shown, the results of the effect of Atractylodes macrocephala extract on tight junction protein (Occludin) in HaCaT keratinocytes are shown in Table 3 and Figure 4 as shown, and the results of the effect of Atractylodes macrocephala extract on keratin (Keratin) in HaCaT keratinocytes are shown in Table 4 and Figure 5 as shown.

[0078] Table 2 Effects of Atractylodes macrocephala extract (12.5 μg / mL) on ceramide secretion

[0079]

[0080] Table 3 Effects of Atractylodes macrocephala extract (50 μg / mL) on tight junction protein (Occludin) secretion

[0081]

[0082] Table 4 Effects of Atractylodes macrocephala extract (50 μg / mL) on keratin (Keratin) secretion

[0083]

[0084] Experimental results: The results showed that in the concentration range of 6.25 μg / mL to 50 μg / mL, the ability of Atractylodes macrocephala extract at 12.5 μg / mL to up-regulate ceramide was 1.61 times that of the control group; the Atractylodes macrocephala extract at 12.5 μg / mL could up-regulate ceramide at 24 h, 48 h, and 72 h, and there were significant differences (P < 0.05). In the concentration range of 6.25 μg / mL to 50 μg / mL, the ability of Atractylodes macrocephala extract at 50 μg / mL to up-regulate tight junction proteins was 2.16 times that of the control group; the Atractylodes macrocephala extract at 50 μg / mL could up-regulate tight junction proteins at 24 h, 48 h, and 72 h, and there were significant differences (P < 0.05). In the concentration range of 6.25 μg / mL to 50 μg / mL, the ability of Atractylodes macrocephala extract at 50 μg / mL to down-regulate keratin was 0.62 times that of the control group; the Atractylodes macrocephala extract at 50 μg / mL could down-regulate keratin at 24 h, 48 h, and 72 h, and there were significant differences (P < 0.01).

[0085] Example 4

[0086] Anti-inflammatory activity of Atractylodes macrocephala extract.

[0087] Experimental method: HaCaT cells (1×10 5 cells / well) were seeded in 96-well plates. When the cell growth density reached about 80%, the cells were treated with Atractylodes macrocephala extract at different concentrations (6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL), and HaCaT cells treated with an equal volume of cell culture medium were used as the negative control. After 24 h of incubation, the six-well plates were removed, and the supernatants were collected and centrifuged. Subsequently, according to the manufacturer's instructions, an ELISA kit was used to quantitatively detect the levels of IL-6 and IL-8 in the supernatants, and the results are shown in Table 5 and Figure 6 as follows.

[0088] Table 5 Effects of Atractylodes macrocephala extract on the secretion of inflammatory factors IL-8 and IL-6

[0089]

[0090] Experimental results: The results showed that in the concentration range of 6.25 μg / mL to 50 μg / mL, for the Atractylodes macrocephala extract, the extract could significantly inhibit the production of inflammatory factors IL-6 and IL-8 in keratinocytes (P < 0.001); it can be seen from this that the Atractylodes macrocephala extract has significant anti-inflammatory activity and can inhibit the occurrence of skin inflammation.

[0091] Example 5

[0092] Inhibitory activity of Atractylodes macrocephala extract on hyaluronidase.

[0093] Experimental method: The inhibitory effect on hyaluronidase was determined by measuring the level of N-acetylglucosamine produced by the interaction between sodium hyaluronate and hyaluronidase. Briefly, a detection solvent system composed of 30 μL of samples with different concentrations (0.125 mg / mL, 0.250 mg / mL, 0.500 mg / mL, 1.00 mg / mL, 1.50 mg / mL, 2.00 mg / mL, 2.50 mg / mL, 5.00 mg / mL) and 30 μL of hyaluronidase (10 U / mL) was incubated at 37 °C for 10 min, and then 10 μL of phosphate buffer (300 mM, pH 5.35) was added and incubated at 37 °C for 10 min. Subsequently, 30 μL of hyaluronic acid substrate (300 mM sodium phosphate at 0.03%, pH 5.35) was added to the incubated mixture and further incubated at 37 °C for 45 min to start the detection. The decomposition reaction of hyaluronic acid was stopped by adding 100 μL of acidic albumin solution composed of 0.1% BSA in 24 mM sodium acetate and 79 mM acetic acid (pH 3.75). After incubation at 37 °C for 10 min, the absorbance of the reaction mixture was measured at 600 nm. Using commercially available ursolic acid (purchased from Shanghai Titan Technology Co., Ltd., ≥98%, CAS: 77-52-1) as a control and 5% DMSO as a control, the inhibitory activity of hyaluronidase was calculated, and the calculation formula is shown in Equation I:

[0094] Enzyme inhibitory activity (%) = [(OD 空白组 - OD 实验组 ) / OD 空白组 × 100%, Equation I.

[0095] The final results of the inhibitory rate of the Atractylodes macrocephala extract on hyaluronidase are shown in Table 6 and Figure 7 as follows.

[0096] Table 6 Inhibitory rate of the Atractylodes macrocephala extract on hyaluronidase (%)

[0097]

[0098]

[0099] Experimental results: The results showed that the Atractylodes macrocephala extract had an inhibitory effect on hyaluronidase and showed a dose-dependent behavior. The inhibitory activity on the enzyme exceeded 50% at the highest concentration of 5 mg / mL. Compared with the positive control group of ursolic acid (IC 50 = 0.6975 mg / mL), the Atractylodes macrocephala extract (IC 50 = 0.5544 mg / mL) had a stronger inhibitory effect on hyaluronidase and a stronger inhibitory degree than ursolic acid. Therefore, the Atractylodes macrocephala extract has cosmetic activities of moisturizing, anti-sensitivity and anti-inflammation.

[0100] Example 6

[0101] Inhibitory activity of Atractylodes macrocephala Koidz. extract against elastase.

[0102] Experimental method: The elastase inhibition experiment was carried out according to the method reported in relevant literature. For details, see: Zixuan Han, Weiwei Ren, Xiaojuan Liu, Nan Lin, Jialin Qu, Xuchang Duan, Bin Liu, Hypoglycemic activity of immature persimmon (Diospyros kaki Thunb.) extracts and its inhibition mechanism for α - amylase and α - glucosidase, International Journal of Biological Macromolecules, Volume 257, Part 2, 2024, 128616, ISSN 0141 - 8130, https: / / doi.org / 10.1016 / j.ijbiomac.2023.128616. Briefly, this experiment was carried out in a mixed solution containing 50 μL of samples with different concentrations (0.125 mg / mL, 0.250 mg / mL, 0.500 mg / mL, 1.00 mg / mL, 1.50 mg / mL, 2.00 mg / mL, 2.50 mg / mL, 5.00 mg / mL), 50 μL of porcine pancreatic elastase (0.5 U / mL), and 50 μL of Tris - HCl buffer (0.1 mM, pH 8.0), and then incubated at 37 °C for 15 min. Finally, 50 μL of 1 mM N - Sucanyl - Ala - Ala - Ala - p - Nitroanilide substrate (purchased from Shanghai Titan Scientific Co., Ltd., Aladdin #S357870 - 5mg) was added to the reaction mixture and incubated at 37 °C for 20 min. The absorbance at a wavelength of 410 nm was measured on a microplate reader. The negative control was 5% DMSO. Under exactly the same experimental conditions, ursolic acid was used as a reference to prove the performance of this assay method, and the inhibition rate results of Atractylodes macrocephala Koidz. extract against elastase are shown in Table 7 and Figure 8 as follows. Among them, the calculation formula for the inhibitory activity of elastase is the same as Formula I in Example 5.

[0103] Table 7 Inhibition rate of Atractylodes macrocephala Koidz. extract against elastase (%)

[0104]

[0105]

[0106] Experimental results: The results showed that the inhibition of the atractylodes macrocephala extract on elastase activity was concentration-dependent. The atractylodes macrocephala extract had the highest inhibitory activity on elastase, and its IC 50 value was 0.4304 mg / mL, while the IC 50 value of ursolic acid was 1.434 mg / mL. The results indicated that, compared with ursolic acid, the atractylodes macrocephala extract had a significant inhibitory effect on elastase, which was helpful for restoring skin elasticity and preventing skin wrinkles.

[0107] Example 7

[0108] Inhibitory activity of atractylodes macrocephala extract on collagenase.

[0109] Experimental method: The measurement method used was based on the spectrophotometry reported in the literature with some modifications. The mixed solution included 50 μL of Clostridium histolyticum collagenase (1 U / mL), 50 μL of Tris-HCl buffer (0.05 mM, pH 7.5), and 50 μL of samples at different concentrations (0.125 mg / mL, 0.250 mg / mL, 0.500 mg / mL, 1.00 mg / mL, 1.50 mg / mL, 2.00 mg / mL, 2.50 mg / mL, 5.00 mg / mL). After incubation at 37 °C for 15 min, 50 μL of N-[3-(2-furyl)acryloyl]-leu-gly-pro-ala substrate at 0.48 mg / mL (purchased from Shanghai Titan Technology Co., Ltd., S28032-5 mg) was added and incubated at 37 °C for 20 min. The absorbance was measured at a wavelength of 335 nm. For the control group, 50 μL of 5% DMSO was added instead of the sample. Using ursolic acid as the reference substance, the inhibition rate results of the atractylodes macrocephala extract on collagenase are shown in Table 8 and Figure 9 as follows. Among them, the calculation formula for the inhibitory activity of collagenase is the same as Formula I in Example 5.

[0110] Table 8 Inhibition rate of atractylodes macrocephala extract on collagenase (%)

[0111]

[0112] Experimental results: The experimental results showed that the collagenase activity of the atractylodes macrocephala extract was concentration-dependent. In the high-concentration range (2.00 - 5.00 mg / mL), the collagenase inhibitory activity of ursolic acid was lower than that of the atractylodes macrocephala extract. The IC 50 value of ursolic acid was 1.228 mg / mL, which was higher than that of the atractylodes macrocephala extract (1.220 mg / mL). These results indicated that the atractylodes macrocephala extract had an inhibitory effect on collagenase and had good anti-aging activity.

[0113] Example 8

[0114] Repair function of Atractylodes macrocephala Koidz. extract on damaged skin barrier of mice.

[0115] Experimental method: Healthy male SPF CD-1 / ICR mice (6 weeks old, body weight 18 g - 22 g) were selected. Breeding conditions: room temperature 24 ± 2 °C, relative humidity 55 ± 10%, light-dark cycle 12 h, and food and water were available ad libitum. After 1 week of adaptive breeding, they were randomly divided into 5 groups with 3 mice in each group. Before the experiment, the hair on the back of the mice was shaved off with a razor to an area of 2×2 cm. 2 Except for the normal group, the skin epidermal barrier damage model was established in the remaining experimental groups by repeatedly stripping the back skin with adhesive tape for 5 days until the trans-epidermal water loss (TEWL) measured by VapoMeter exceeded 35 mg / cm 2 / h, indicating successful modeling. The normal group was not given any treatment, and the experimental groups were treated with water (200 μL), the skin cream containing Atractylodes macrocephala Koidz. extract prepared in Example 1 (200 μL, Atractylodes macrocephala Koidz. extract content 10%), and the skin cream matrix prepared in Example 1 (200 μL) twice a day for 8 consecutive days. At 0 d, 1 d, 4 d, and 8 d after using the Atractylodes macrocephala Koidz. extract, the recovery of the epidermal barrier was measured, represented by TEWL, pH value, skin elasticity, and water content. All animal experiments were conducted in accordance with international ethical guidelines and the principles of experimental animal care. The calculation formula for the skin recovery rate is shown in Equation II.

[0116] Epidermal barrier recovery rate (100%) = (TEWL after barrier damage - TEWL at the specified time point) / (TEWL after barrier damage - reference TEWL) × 100%, Equation II.

[0117] The results of the effects of Atractylodes macrocephala Koidz. extract on mouse skin indicators are shown in Table 9 and Figure 10 as follows.

[0118] Table 9 Effects of Atractylodes macrocephala Koidz. extract on mouse skin indicators

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[0121] Experimental results: The experimental results showed that using the skin cream added with Atractylodes macrocephala Koidz. extract could significantly reduce skin TEWL, indicating rapid recovery of the epidermal barrier. After 8 days of use, the skin recovery rates of the skin cream added with Atractylodes macrocephala Koidz. extract were 79.7% respectively, while the skin recovery rate of the skin cream matrix group was only 51.8%. The other three skin parameters, pH value, skin elasticity, and hydration, were significantly improved compared with the model group and all recovered close to the normal group. The above results indicated that Atractylodes macrocephala Koidz. extract could significantly accelerate the epidermal barrier repair process.

[0122] Example 9

[0123] Determination of the effects of Atractylodes macrocephala Koidz. extract on the histomorphology analysis of mouse skin, and the contents of skin ceramide, tight junction protein and keratin.

[0124] Experimental method: The back skin of mice was subjected to histological examination according to the method reported in the literature. For details, see: ShuangGao, Yu Chen, Jungang Zhao, Rongrong Jing, KeKe Guo, Lusheng Wang, Xuenan Li, Chengliang Li, Zhenlin Hu, Nuo Xu, Oatβ-glucan ameliorates epidermal barrier disruption by upregulating the expression of CaSR through dectin-1-mediated ERK and p38 signaling pathways, International Journal of Biological Macromolecules, Volume 185, 2021, Pages 876 - 889, ISSN 0141 - 8130, https: / / doi.org / 10.1016 / j.ijbiomac.2021.07.002., and the actual photos of the recovery of the skin of mice with damaged skin barrier by Atractylodes macrocephala Koidz. extract were obtained as Figure 11 shown. After the mouse modeling and drug administration experiment was completed, the animals were sacrificed. The back skin of each mouse was immediately excised and cut into two segments. One was fixed in 10% neutral buffered formalin solution for histological analysis, and the other was used to detect the ceramide content. Histological analysis: The skin samples were embedded in paraffin and cut into 6 - μm thin sections, and stained with hematoxylin - eosin (H&E). The epidermal thickness was measured using a Biorevo integrated microscope, and the histomorphology analysis map of the skin of mice by Atractylodes macrocephala Koidz. extract was obtained as Figure 12 shown. The average value of 5 random measurements was considered as the representative value of the individual animal. Determination of skin ceramide content: The damaged skin tissue was taken and homogenized. Proteins were extracted with RIPA lysis buffer, and the protein concentration was determined by the BCA method. Referring to the operation steps in the kit instruction manual, the ceramide contents in the damaged skin tissues of mice in each group were detected respectively (the kit was purchased from Shanghai Keqiao Biotechnology Co., Ltd., product number: KQ112032, and the detection results are shown in Table 10 and Figure 13 shown), and the contents of tight junction proteins (the kit was purchased from Shanghai Keqiao Biotechnology Co., Ltd., product number: KQ111910, and the detection results are shown in Table 11 and Figure 14as shown) and the content of keratin (the kit was purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd., product number: JK99852, and the test results are shown in Table 12 and Figure 15 as shown).

[0125] Table 10 Effects of the Atractylodes macrocephala group on the secretion of ceramide in mouse skin

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[0127] Table 11 Effects of the Atractylodes macrocephala extract group on the secretion of tight junction proteins in mouse skin

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[0129] Table 12 Effects of the Atractylodes macrocephala extract group on the secretion of keratin in mouse skin

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[0131] Experimental results: Histological examination of the back skin of mice was performed by H&E staining of skin samples. Severe epidermal hyperplasia-like symptoms and an increased epidermal thickness (70.89 μm) were observed in the skin sections of the model group. Under the intervention of the Atractylodes macrocephala extract, the epidermal thickness was significantly reduced (27.06 μm respectively), and the repair effect on the skin epidermis was better than that of the matrix group (54.48 μm). These experimental results were consistent with the morphological changes of skin lesions observed by photographing the skin at the specified time: The back skin of the model group mice was significantly thickened and reddened. The epidermal barrier repair rate of the Atractylodes macrocephala extract group mice was significantly faster than that of the model group, and the effect was stronger than that of the matrix group because there were still certain erythema symptoms on the back skin of the matrix group mice on the 8th day. By measuring the levels of ceramide, tight junction proteins, and keratin in the dorsal skin of mice, the results showed that the Atractylodes macrocephala extract up-regulated the levels of ceramide and tight junction proteins, and their values were close to those of the normal group, showing significant differences compared with the model group (p < 0.001). At the same time, the abnormal expression of keratin was reduced (p < 0.001), and the hyperplastic keratosis symptoms of mouse skin were alleviated. All these experimental results clearly indicate that the Atractylodes macrocephala extract can repair the damaged skin barrier, relieve skin discomfort symptoms, and has great potential for cosmetic applications.

[0132] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can obtain other embodiments without creative labor based on this embodiment, and these embodiments all belong to the protection scope of the present invention.

Claims

1. Use of Atractylodes macrocephala Koidz. extract in the preparation of a product with skin barrier repair function.

2. Use of Atractylodes macrocephala Koidz. extract in the preparation of a product with skin elasticity restoration function.

3. Use of Atractylodes macrocephala Koidz. extract in the preparation of a product with anti-aging function.

4. Use of Atractylodes macrocephala Koidz. extract in the preparation of a product with skin inflammation inhibition function.

5. Use of Atractylodes macrocephala Koidz. extract in the preparation of a product with anti-sensitivity function.

6. Use of Atractylodes macrocephala Koidz. extract in the preparation of a product with moisturizing function.

7. Use of Atractylodes macrocephala Koidz. extract in the preparation of a product having any one or more of the functions shown in ① to ⑦: ① Up-regulating the content of ceramide in cells; ② Up-regulating the content of tight junction proteins in cells; ③ Inhibiting the abnormal expression of keratin; ④ Inhibiting the activity of inflammatory factors; ⑤ Inhibiting the activity of hyaluronidase; ⑥ Inhibiting the activity of elastase; ⑦ Inhibiting the activity of collagenase.

8. The application according to any one of claims 1 to 6, characterized in that, The product includes cosmetics.

9. The Atractylodes macrocephala Koidz. extract in the application according to any one of claims 1 to 6, characterized in that, The extraction solvent of the Atractylodes macrocephala Koidz. extract is ethyl acetate.

10. The Atractylodes macrocephala Koidz extract according to claim 9, wherein, The mass-volume ratio of the ethyl acetate to the Atractylodes macrocephala Koidz. for mixing and soaking is (10 - 20):1; The time for mixing and soaking the ethyl acetate and the Atractylodes macrocephala Koidz. is 12h - 36h.

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