Application of orientin in preparation of cosmetics with oil control effect

Abstract

The invention discloses application of orientin in preparation of cosmetics with an oil control effect, and researches find that orientin has a remarkable effect in the aspect of oil control and can be used for preparing the cosmetics with the oil control effect. And the effect is better when the compound is used in binary combination with resveratrol and in ternary combination with resveratrol and isoorientin.

Description

Technical Field

[0001] This invention relates to a novel application of purslane, and more particularly to the application of purslane in the preparation of cosmetics with oil-controlling effects. Background Technology

[0002] Sebaceous glands are components of the pilosebaceous unit that secrete sebum. Embryonic development of sebaceous glands occurs from the 13th to 16th week of fetal development, coinciding with the development of hair follicles and epidermal tissue. New sebaceous glands typically do not develop after birth, but their size increases with age. Sebaceous glands are multilobar, holosecretive accessory organs of the epidermis. Sebaceous gland cells are primarily responsible for synthesizing neutral lipids. Terminally differentiated sebaceous gland cells lyse and secrete lipids onto the skin surface through the hair follicle pores. In addition to the production and release of sebum, sebaceous glands also lubricate the skin and hair, provide thermoregulation, and possess antibacterial activity. While the full functions of sebum in human skin remain to be fully elucidated, sebum is an indispensable component of the epidermal barrier and the skin's immune system. Sebaceous gland cells express various hormone receptors, and sebum synthesis is regulated by hormones, particularly androgens. Sebaceous gland dysfunction mainly includes sebaceous hyperplasia, sebaceous adenoma, sebaceous carcinoma, sebaceous nevus, and pilosebaceous cystic hamartoma. Sebaceous glands are also involved in acne vulgaris, seborrheic dermatitis, and androgenetic alopecia. Current clinical treatment for abnormal sebum secretion combines topical medications such as isotretinoin and benzoyl peroxide with phototherapy. While isotretinoin is currently the most effective sebum secretion inhibitor, it also carries teratogenic risks. In recent years, many naturally derived active substances that inhibit lipid secretion have become research hotspots due to their milder nature and lack of side effects. Polyphenols, flavonoids, and many plant extracts have shown certain effects in inhibiting lipid secretion. Poria cocos is also a naturally derived active substance found in various plants. Its known effects include antioxidant, anti-inflammatory, cardiovascular protective, and neuroprotective activities; however, its effectiveness in inhibiting sebum secretion is currently unknown. Summary of the Invention

[0003] The purpose of this invention is to provide an application of arbutin in the preparation of cosmetics with oil-controlling effects. This invention, through research, has discovered that arbutin has significant oil-controlling effects and can be used to prepare cosmetics with oil-controlling effects. Furthermore, its effects are enhanced when used in a binary combination with resveratrol and a ternary combination with resveratrol and isoarbutin.

[0004] The technical solution of this invention: the application of purslane in the preparation of cosmetics with oil-controlling effects. In the aforementioned applications, the concentration of the herbal extract in cosmetics is 1~10 μg / mL.

[0005] In the aforementioned applications, the concentration of the herbal extract in cosmetics is 1 μg / mL, 2 μg / mL, or 10 μg / mL.

[0006] In the aforementioned applications, the cosmetic contains resveratrol.

[0007] In the aforementioned applications, the ratio of erythritol to resveratrol is between 9.5:0.5 and 8.5:1.5.

[0008] In the aforementioned applications, the ratio of erythritol to resveratrol is up to 9:1.

[0009] In the aforementioned applications, the total concentration of the herbal extract and resveratrol in the cosmetics is 1~10 μg / mL.

[0010] In the aforementioned applications, the cosmetic contains resveratrol and isohypericin.

[0011] In the aforementioned applications, the ratio of resveratrol, scutellarin, and isoscutellarin is (0.95~0.75):(0.0165~0.0825):(0.0335~0.1675).

[0012] In the aforementioned applications, the total concentration of resveratrol, strychnine, and isostrychnine in cosmetics is 0.01-10 μg / mL.

[0013] Compared with existing technologies, the beneficial effects of this invention are: providing the application of purslane in the preparation of cosmetics with oil-controlling effects. Model experiments show that purslane has significant oil-controlling effects. Purslane is a mild, naturally derived active substance with minimal toxic side effects on the human body. Furthermore, further experiments have revealed that the binary combination of purslane and resveratrol within a specific ratio range, and the ternary combination of purslane, resveratrol, and isopurslane within a specific ratio range, exhibit significantly better effects than the single addition of purslane. The compositions demonstrate a synergistic effect and should possess broad market application prospects. Attached Figure Description

[0014] Figure 1 This is a bar chart showing the effect of different concentrations of LA on the viability of SZ95 cells.

[0015] Figure 2 This is a graph showing the results of detecting the viability of SZ95 cells at different concentrations of a single herbal extract.

[0016] Figure 3 This is a graph showing the results of SZ95 cell viability assays at different concentrations of isochorin alone.

[0017] Figure 4 These are comparison images of the staining effects obtained from a single herbal extract.

[0018] Figure 5 This is a comparison chart of fluorescence intensities of different groups of single purslane.

[0019] Figure 6 This is a comparison chart of the staining effects obtained from different groups of single isochorine.

[0020] Figure 7 This is a comparison chart of fluorescence intensities of different groups of single isochorine.

[0021] Figure 8 This is a comparison chart of the staining effects obtained by different proportions of resveratrol, erythritol, and isohythritol.

[0022] Figure 9 This is a comparison chart of fluorescence intensity for different proportions of resveratrol, erythritol, and isohyritol.

[0023] Figure 10 This is a comparison chart of the staining effects obtained by different concentrations of resveratrol, erythritol, and isohythritol.

[0024] Figure 11 This is a comparison chart of fluorescence intensity at different concentrations of resveratrol, erythritol, and isohythritol.

[0025] Figure 12 Comparison of staining effects of resveratrol and erythritol in different proportions.

[0026] Figure 13 Comparison of fluorescence intensity of resveratrol and erythritol in different proportions.

[0027] Figure 14 Comparison of staining effects of different concentrations of resveratrol and erythritol.

[0028] Figure 15 Comparison of fluorescence intensity at different concentrations of resveratrol and erythritol.

[0029] Figure 16 It is an AR electrophoresis image.

[0030] Figure 17 This is a comparison chart of the relative expression levels of AR proteins.

[0031] Figure 18 This is a 24-hour electrophoresis image of HSD17B1.

[0032] Figure 19 This is a comparison of the relative protein expression levels of HSD17B1 over 24 hours.

[0033] Figure 20 This is an electrophoresis image of HSD17B1 after 48 hours.

[0034] Figure 21This is a comparison of the relative expression levels of HSD17B1 protein over 48 hours.

[0035] Figure 22 This is a 24-hour electrophoresis image of p-AKT.

[0036] Figure 23 This is a comparison of the relative expression levels of p-AKT protein over 24 hours.

[0037] Figure 24 This is a comparison chart of the relative protein expression levels of AKT over 24 hours.

[0038] Figure 25 This is a 48-hour electrophoresis image of p-AKT.

[0039] Figure 26 This is a comparison of the relative expression levels of p-AKT protein at 48 hours.

[0040] Figure 27 This is a comparison chart of the relative expression levels of AKT protein at 48h.

[0041] Figure 28 This is an electrophoresis image of ACSL1.

[0042] Figure 29 This is a comparison chart of the relative protein expression levels of ACSL1. Detailed Implementation

[0043] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0044] Example 1: I. Oil-controlling active ingredient - erythritol (used alone).

[0045] Step 1.1): After reviving sebaceous gland cells (SZ95), observe cell growth. Count the cells when the cell coverage reaches 80% or more, and seed them into multiple 96-well and 24-well plates. The 96-well plates are used to detect cell viability, and the 24-well plates are used to detect Nile Red staining of sebaceous gland cells. Incubate the plates overnight in a CO2 incubator (37°C, 5% CO2).

[0046] Step 1.2): Solution preparation: Prepare various sample working solutions, including linoleic acid (LA) working solution, scutellarin working solution, isoscutellarin working solution, binary combination working solution of scutellarin + resveratrol, and ternary combination working solution of scutellarin + resveratrol + isoscutellarin.

[0047] Step 1.3): When the cell deposition rate reaches approximately 60%, administer the drug to the affected groups. Each group has 3 replicates.

[0048] Step 1.3.1): Take linoleic acid (LA) working solution, group it according to Table 1 below, explore the experimental concentration of linoleic acid (LA), and select LA concentration in the range of 5~1000μM for cell viability experiments.

[0049] Table 1 Step 1.3.2): Take the working solution of purslane and group it according to Table 2 below. Explore the experimental concentration of purslane in SZ95 cells and select a concentration of 0.5~100μg / mL for cell viability experiment.

[0050] Table 2 Take the isopropionate working solution and group it according to Table 3 below. Explore the concentration of isopropionate in SZ95 cells and select a concentration of 0.5~100μg / mL for cell viability experiments.

[0051] Table 3 Step 1.3.3): Following step 1.3.1), the optimal LA concentration for modeling was determined to be 100 μM. Following the grouping in Table 4, samples containing erythritol within the safe concentration range of 0.5–100 μg / mL were selected for Nile Red staining and photography to detect lipid droplet secretion in SZ95 cells. Following the grouping in Table 5, samples containing isoerythritol within the safe concentration range of 0.5–100 μg / mL were selected for Nile Red staining and photography to detect lipid droplet secretion in SZ95 cells.

[0052] Table 4 Table 5 Step 1.3.4): The specific steps for Nile Red staining are as follows: Rinse: Discard the culture medium and wash 3 times with PBS; Fixation: Add 4% paraformaldehyde (300 µL) for 30 min; Staining: Prepare Nile Red staining solution (working concentration is 10 μg / mL), discard the fixative, rinse the cells 3 times with PBS, add Nile Red staining solution (300 µL / well) and stain for 15 min; Photography: Observe the staining of cells in each group under an inverted fluorescence microscope and take photos.

[0053] Results analysis: The fluorescence intensity was quantitatively analyzed using Image Pro Plus software.

[0054] II. Data Processing Step 2.1): Cell viability test. For each group in Tables 1, 2, and 3, the specific method is as follows: Discard the supernatant from the 96-well plate, add 100 µL of CCK-8 working solution (prepared as CCK8 stock solution: medium = 1:9) to each well, and incubate in an incubator (37°C, 5% CO2) for 2 hours. Then, measure the absorbance at 450 nm using a microplate reader.

[0055] Step 2.2): Cell viability. For each group in Tables 1, 2, and 3, the inhibition rate is calculated as follows: Cell viability (%) = [(As-Ab) / (Ac-Ab)] x 100% As: Absorbance of experimental wells (including cells, culture medium, CCK-8 solution and drug solution); Ac: Absorbance of control wells (containing cells, culture medium, and CCK-8 solution, but excluding drugs); Ab: Absorbance of blank wells (containing culture medium and CCK-8 solution, but excluding cells and drugs).

[0056] Step 2.3): Nile Red staining: Rinse, fix cells, stain with Nile Red, and then take pictures using a fluorescence microscope.

[0057] Step 2.4): Statistical analysis of results: Quantitative analysis of fluorescence intensity was performed using Image Pro Plus software, and graphs were plotted using GraphPad Prism. The results are presented as Mean+SEM. t-tests were used for comparisons between groups. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0058] III. Experimental Results Step 3.1): Regulation of lipid droplet secretion by erythritol and isohythritol (single): For each group in Table 1, such as Figure 1 As shown in Table 6, SZ95 cells exhibited a viability of over 70% and low toxicity when LA concentrations ranged from 5 to 100 μM, indicating that further experiments could be conducted.

[0059] Table 6 For each group in Table 2, such as Figure 2 As shown in Table 7, when erythritol was used alone at concentrations ranging from 0.5 to 10 μg / mL, the viability of SZ95 cells was higher than 90%, and there was no cytotoxicity.

[0060] Table 7 For each group in Table 3, such as Figure 3As shown in Table 8, when isopropionate was used alone at concentrations ranging from 0.5 to 10 μg / mL, the viability of SZ95 cells was higher than 90%, and there was no cytotoxicity.

[0061] Table 8 Step 3.2.1): Based on the staining results of each group in Table 4, the data is processed to obtain... Figure 4 and Figure 5 ,from Figure 4 and Figure 5 It was observed that the LA group significantly increased lipid droplet secretion compared to the blank control group, while finasteride and isosorbide Vate significantly decreased lipid droplet secretion compared to the blank control group. At concentrations of 1 μg / mL, 2 μg / mL, and 10 μg / mL, phorboloidin significantly reduced lipid droplet secretion compared to the blank control group, demonstrating an oil-controlling effect.

[0062] Step 3.2.2): Based on the staining results of each group in Table 5, the data is processed to obtain... Figure 6 and Figure 7 As a result, from Figure 6 and Figure 7 It is evident that the LA group significantly increased lipid droplet secretion compared to the blank control group; finasteride and isosorbide Vate significantly reduced lipid droplet secretion compared to the blank control group; and isosorbide Vate at concentrations of 2 μg / mL, 5 μg / mL, and 10 μg / mL all significantly reduced lipid droplet secretion compared to the blank control group, demonstrating oil-controlling effects.

[0063] As can be seen from Example 1, purslane alone has an oil-controlling effect and can be used alone in the preparation of cosmetics with oil-controlling effects. The concentration range is 1~10μg / mL, preferably 1μg / mL, 2μg / mL or 10μg / mL.

[0064] Example 2: Oil-controlling active ingredients—resveratrol + erythritol + isohythritol (a ternary combination), and the ratio range was determined.

[0065] Following step 1.1) of Example 1, seed plates were prepared, and drugs were administered in groups according to Table 9 below. After drug administration, the 24-well plates were placed in an incubator (37°C, 5% CO2) and incubated for 24 hours. Nile red staining was then performed according to step 1.3.4). Figure 8 and Figure 9 .

[0066] Table 9 from Figure 8 and Figure 9The results showed that the LA group significantly increased lipid droplet secretion compared to the blank control group; finasteride and isosorbide A acid significantly reduced lipid droplet secretion compared to the blank control group; resveratrol + erythritol + isoerythritol in the range of (0.95~0.75):(0.0165~0.0825):(0.0335~0.1675) significantly reduced lipid droplet secretion compared to the blank control group, and had an oil-controlling effect, which was better than that of a single active ingredient.

[0067] As shown in Example 2, the oil-controlling effect of the combination of resveratrol, scutellarin, and isoscutellarin is better than that of scutellarin alone. It can be used in combination in the preparation of cosmetics with oil-controlling effects, with an addition ratio of (0.95~0.75):(0.0165~0.0825):(0.0335~0.1675) and a total addition concentration of 1 μg / mL.

[0068] Example 3: Oil-controlling active ingredients—resveratrol + erythritol + isohythritol (a ternary combination), and the concentration range was determined.

[0069] Following step 1.1) of Example 1, seed plates were prepared, and drugs were administered in groups according to Table 10 below. After drug administration, the 24-well plates were placed in an incubator (37°C, 5% CO2) and incubated for 24 hours. Nile red staining was then performed according to step 1.3.4). Figure 10 and Figure 11 .

[0070] Table 10 like Figure 10 and Figure 11 As shown, the LA group significantly increased lipid droplet secretion compared to the blank control group; finasteride and isosorbide A acid significantly decreased lipid droplet secretion compared to the blank control group; the total concentration of resveratrol, strychnine and isosorbide A (9:0.33:0.67) in the range of 0.01-10 μg / mL significantly reduced lipid droplet secretion and had an oil-controlling effect.

[0071] Example 4: Oil-controlling active ingredients—resveratrol + erythritol (binary combination), and the ratio range was determined.

[0072] Following step 1.1) of Example 1, seed plates were prepared, and drugs were administered in groups according to Table 11 below. After drug administration, the 24-well plates were placed in an incubator (37°C, 5% CO2) and incubated for 24 hours. Nile red staining was then performed according to step 1.3.4). Figure 12 and Figure 13 .

[0073] Table 11 from Figure 12 and Figure 13 The results showed that the LA group significantly increased lipid droplet secretion compared to the blank control group; finasteride and isosorbide A (VA) significantly decreased lipid droplet secretion compared to the blank control group; resveratrol + erythritol (total concentration 1 μg / mL) significantly reduced lipid droplet secretion compared to the blank control group within the range of 9.5:0.5 to 8:2, exhibiting an oil-controlling effect, and within the range of 9.5:0.5 to 8.5:1.5, the oil-controlling effect was superior to that of a single active ingredient. The effect was best at a ratio of 9:1.

[0074] Example 5: Oil-controlling active ingredients—resveratrol + erythritol (binary combination), concentration range was determined.

[0075] Following step 1.1) of Example 1, seed plates were prepared, and drugs were administered in groups according to Table 12 below. After drug administration, the 24-well plates were placed in an incubator (37°C, 5% CO2) and incubated for 24 hours. Nile red staining was then performed according to step 1.3.4). Figure 14 and Figure 15 .

[0076] Table 12 from Figure 14 and Figure 15 The results showed that the LA group significantly increased lipid droplet secretion compared to the blank control group; finasteride and isosorbide Vate significantly reduced lipid droplet secretion compared to the blank control group; resveratrol: erythritol (9:1) in the range of 1~10μg / mL significantly reduced lipid droplet secretion compared to the blank control group, and had an oil-controlling effect.

[0077] Oil-control target testing was conducted using resveratrol + erythritol (binary combination): Following step 1.1) of Example 1, the cells were seeded and administered drugs according to the groups in Table 13 below. After administration, the 24-well plates were placed in an incubator (37°C, 5% CO2) for incubation, and the cells were collected separately.

[0078] Table 13 Step 6.1.1): Take cells from the corresponding group, add RIPA lysis buffer, lyse on ice, centrifuge at freezing, and collect the supernatant.

[0079] Step 6.1.2): Determine the protein concentration in the supernatant using the BCA method to ensure that the concentrations of samples in each group are consistent.

[0080] Step 6.1.3): Add loading buffer (5×SDS Loading Buffer) and boil at 100°C for 5 minutes to denature the protein.

[0081] Step 6.2): ​​SDS-PAGE electrophoresis: Step 6.2.1): Assemble the electrophoresis gel plate, prepare the separating gel, add a layer of deionized water to seal, and let it solidify.

[0082] Step 6.2.2): Pour off the top layer of water, prepare the concentrated glue, insert the comb, and let it solidify.

[0083] Step 6.2.3): Remove the comb, place the gel plate into the electrophoresis tank, add electrophoresis buffer, and load the samples in sequence.

[0084] Step 6.2.4): Turn on the power: First, maintain a constant voltage. After the sample enters the separating gel, adjust it to 120V until the bromophenol blue indicator reaches the bottom of the gel, then turn off the power.

[0085] Step 6.3): Transfer: Step 6.3.1): Remove the gel, cut off the stacking gel and excess portion, and equilibrate with transfer buffer for 5 minutes.

[0086] Assemble the transfer sandwich in the order of "negative electrode → sponge → filter paper → gel → membrane → filter paper → sponge → positive electrode"; place the sandwich in the transfer tank and add pre-cooled transfer buffer.

[0087] Step 6.3.2): After the transfer is complete, remove the membrane, rinse it with deionized water, and wash away the staining agent with TBST.

[0088] Step 6.4): Blocking: Place the membrane in the blocking solution and block on a shaker at room temperature for 1 hour. After blocking, wash three times with TBST for 10 minutes each time (shaking).

[0089] Step 6.5): Antibody incubation, performed according to the standard experimental procedure for Western blotting (WB), mainly as follows: Step 6.5.1): Primary antibody incubation: Dilute the primary antibody with blocking buffer, place the membrane in the diluted primary antibody, incubate overnight on a shaker at 4°C, and wash the membrane 3 times with TBST for 10 minutes each time.

[0090] Step 6.5.2) Secondary antibody incubation: Dilute the secondary antibody with blocking buffer, incubate on a shaker at room temperature for 1 hour, wash the membrane 3 times with TBST for 10 minutes each time, and finally wash once with TBS.

[0091] Four types of antibodies were incubated, which were used for the detection of AR, HSD17B1, AKT and ACSL1, respectively.

[0092] Step 6.6) Colorimetric detection: Mix ECL substrate solutions A and B in a 1:1 ratio, and evenly drop the mixture onto the membrane. Incubate for 1-2 minutes. Expose and develop the membrane using an imager (such as ChemiDoc), adjusting the exposure time according to the signal intensity (avoiding overexposure or underexposure), and save the image.

[0093] Step 6.7): Result analysis: Use software such as ImageJ to analyze the gray values ​​of the bands. The relative expression level of the protein is expressed as the ratio of the gray value of the target protein band to the gray value of the internal reference (such as β-actin, GAPDH) band. Statistical analysis is then performed.

[0094] Step 7.1): AR detection index: Resveratrol:Hydroxyalin in a ratio of 9:1, such as Figure 16 and Figure 17 As shown, treatment of sebaceous gland cells for 24 hours can inhibit AR expression and has an oil-controlling effect.

[0095] Step 7.2): HSD17B1 detection index: resveratrol: erythritol = 9:1 ratio, such as Figures 18 to 21 Treatment of sebaceous gland cells for 24 hours and 48 hours can inhibit HSD17B1 expression, thus exhibiting an oil-controlling effect.

[0096] Step 7.3): AKT detection index: Resveratrol:Hydroxylin = 9:1 ratio, such as Figures 22 to 27 As shown, treatment of sebaceous gland cells for 24 h and 48 h both inhibited p-AKT expression, demonstrating an oil-controlling effect. p-AKT is phosphorylated AKT.

[0097] Step 7.4): ACSL1 detection index: resveratrol: erythritol = 9:1 ratio, such as... Figure 28 and Figure 29 As shown, treatment of sebaceous gland cells for 24 hours can inhibit ACSL1 expression and has an oil-controlling effect.

Claims

1. Application of purslane in the preparation of cosmetics with oil-controlling effects.

2. The application according to claim 1, characterized in that: The concentration of the herbal extract in cosmetics is 1~10 μg / mL.

3. The application according to claim 2, characterized in that: The concentration of the herbal extract in the cosmetic is 1 μg / mL, 2 μg / mL or 10 μg / mL.

4. The application according to claim 1, characterized in that: The cosmetic contains resveratrol.

5. The application according to claim 4, characterized in that: The ratio of the herbal extract to resveratrol is between 9.5:0.5 and 8.5:1.

5.

6. The application according to claim 5, characterized in that: The ratio of erythritol to resveratrol is up to 9:

1.

7. The application according to claim 5 or 6, characterized in that: The total concentration of the herbal extract and resveratrol in the cosmetic is 1~10 μg / mL.

8. The application according to claim 1, characterized in that: The cosmetic contains resveratrol and isohypericin.

9. The application according to claim 8, characterized in that: The ratio of resveratrol, scutellarin, and isoscutellarin is (0.95~0.75):(0.0165~0.0825):(0.0335~0.1675).

10. The application according to claim 8 or 9, characterized in that: The total concentration of resveratrol, strychnine, and isostrychnine in the cosmetic is 0.01~10 μg / mL.

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