A composition with whitening and anti-aging effects, preparation method and application thereof

Abstract

The present invention provides a composition with whitening and anti-aging effects, a preparation method and application thereof, and belongs to the technical field of medicine or cosmetics. The composition comprises: cucurbitacin F, sutherlandin-5-trans-p-coumarate, quercetin, rutin, wogonin, trifoliol, quercetin-3-O-β-D-xyloside, scutellariae, hyperoside, cinchona sucretin Ib, kaempferol-3-furanarabinoside, arbutin and astragalus glycoside. The composition has significant whitening and anti-freckle effects, anti-aging effects, and is suitable for various skin types such as dry, oily, mixed and sensitive skin, and has high biological application value in the field of cosmetics and medicine.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine or cosmetics, and provides a composition with whitening and anti-aging effects, a preparation method and application thereof. Background Art

[0002] The active ingredients in natural plant extracts can inhibit the production of melanin, reduce pigmentation, and achieve a whitening effect. Active ingredients such as polyphenols and flavonoids have strong antioxidant effects, can neutralize free radicals, and delay the aging process of the skin. At present, the application of natural plant extracts in medicines and cosmetics has been widely studied. Different types of natural plant extracts have different effects and can be used for moisturizing, whitening, anti-aging, sun protection and other aspects.

[0003] Chinese invention patent CN11823385B discloses a composition with anti-aging and whitening effects and its application, including: verbascoside, isovalascoside, forsythiaside, plantagoside, luteolin, baicalin, chlorogenic acid, ursolic acid, palmitic acid, linoleic acid, 2,6-di-tert-butyl-p-cresol and 3-tert-butyl-4-hydroxyanisole. The composition has the effects of whitening, removing spots and resisting radiation, with low dosage and safe and reliable.

[0004] Chinese invention patent publication number CN108653426A discloses the use of an extract of Uncaria rhynchophylla in the preparation of antioxidant drugs and whitening and anti-aging oxidative skin care products, and verifies the anti-inflammatory, whitening and antioxidant activities of alkaloids such as 18,19-dehydrocorynoxinic acid, isorhynchophyllic acid, carbendazim, 18,19-dehydrorhynchophylline, 3β-isodihydrocarbendazim, 3α-isodihydrocarbendazim, rhynchophylline, isorhynchophylline, dehydrorhynchophylline, isodehydrorhynchophylline and hygoside in the extract, and non-alkaloids including chlorogenic acid, hyperoside, rutin and epicatechin.

[0005] Sorbaria sorbifolia belongs to the Rosaceae family. Its main components are polyphenols, triterpenes, polysaccharides, and volatile components, including: pearlwood glycoside, kaempferol-3-furanosyl glycoside, astragaloside, quercetin-3-glucuronide, isorhamnetin-3-glucoside, baicalin, chlorogenic acid, arbutin, baicalin-7-rhamnoside, flavonoid compound IX, as well as trifoliol, quercetin-s-xyloside and kaempferol-3-xyloside, etc. According to the "Handbook of Commonly Used Chinese Herbal Medicines in Northeast China", pearlwood has the effects of promoting blood circulation, removing blood stasis, reducing swelling and relieving pain. It is mainly used to treat fractures, traumatic injuries, joint sprains, redness, swelling and pain, and rheumatoid arthritis.

[0006] Relevant studies in recent years have shown that plants of the genus Pearlwood have pharmacological effects such as inhibiting melanin, anti-aging, scavenging oxygen free radicals, inhibiting lipid peroxidation, anti-inflammatory, bactericidal, protecting the liver, and anti-tumor, and have potential whitening and anti-aging skin care effects. However, the application of its effective active ingredients as a whole in the field of whitening and anti-aging drugs and skin care products has not been reported. The study of the efficacy of plant effective active ingredients is one of the hot topics in the current research of drugs, health products, and skin care products. Therefore, it is necessary to screen the effective active ingredients of Northeast Pearlwood so that they can maximize the efficacy of their corresponding components. Summary of the invention

[0007] In view of the problems existing in the prior art, the inventors found that the Northeast Pearlwood extract has excellent antioxidant activity, melanin and tyrosinase inhibitory activity under the specific active ingredient composition and ratio. Based on this, a composition with whitening and anti-aging effects and its preparation method and application are provided. The composition has significant whitening and anti-freckle effects, delays aging, and is suitable for various skin types such as dry, oily, mixed and sensitive, and has high biological application value in the field of cosmetics and medicine.

[0008] The technical solution of the present invention is as follows:

[0009] The invention provides a composition with whitening and anti-aging effects, characterized in that the composition comprises, by weight, 1-6 parts of cucurbitacin F, 45-83 parts of sutherlandin-5-trans-p-coumarate, 0.1-3.9 parts of quercetin, 0.1-0.9 parts of rutin, 0.01-0.19 parts of wogonin, 1-6 parts of trifolioside, 0.1-3.9 parts of quercetin-3-O-β-D-xyloside, 0.1-3.9 parts of scutellariae, 0.5-2.5 parts of hyperoside, 0.5-2.5 parts of cinchonaside Ib, 1-8 parts of kaempferol-3-arabinofuranoside, 2-12 parts of arbutin and 1-6 parts of astragaloside.

[0010] Preferably, the composition comprises, by weight: 1.5-5 parts of cucurbitacin F, 56-72 parts of sutherlandin-5-trans-p-coumarate, 1-3 parts of quercetin, 0.2-0.8 parts of rutin, 0.05-0.15 parts of wogonin, 1.5-5.5 parts of trifolioside, 1-3 parts of quercetin-3-O-β-D-xyloside, 1-3 parts of scutellariae, 0.7-2.3 parts of hyperoside, 0.7-2.3 parts of cinchonaside Ib, 2-7 parts of kaempferol-3-furanosyl glycoside, 4-10 parts of arbutin and 1.5-5.5 parts of astragaloside.

[0011] More preferably, the composition comprises, by weight: 2-4 parts of cucurbitacin F, 60-68 parts of sutherlandin-5-trans-p-coumarate, 1.5-2.5 parts of quercetin, 0.3-0.7 parts of rutin, 0.08-0.12 parts of wogonin, 3-4 parts of trifolioside, 1.5-2.5 parts of quercetin-3-O-β-D-xyloside, 1.5-2.5 parts of baicalin, 1-2 parts of hyperoside, 1-2 parts of cinchonaside Ib, 4-5 parts of kaempferol-3-furanosyl glycoside, 5-9 parts of arbutin and 3-4 parts of astragaloside.

[0012] Further preferably, the composition comprises, by weight: 3 parts of cucurbitacin F, 64 parts of sutherlandin-5-trans-p-coumarate, 2 parts of quercetin, 0.5 parts of rutin, 0.1 parts of wogonin, 3.5 parts of trifolioside, 2 parts of quercetin-3-O-β-D-xyloside, 2 parts of baicalin, 1.5 parts of hyperoside, 1.5 parts of cinchonaside Ib, 4.5 parts of kaempferol-3-furanosyl glycoside, 7 parts of arbutin and 3.5 parts of astragaloside.

[0013] The present invention also provides a method for preparing any of the above-mentioned compositions, which specifically comprises: uniformly mixing cucurbitacin F, sutherlandin-5-trans-p-coumarate, quercetin, rutin, wogonin, trifolioside, quercetin-3-O-β-D-xyloside, scutellaria baicalensis, hyperoside, cinchonadin Ib, kaempferol-3-furanarabinoside, arbutin and astragaloside, and sieving to obtain the composition.

[0014] The present invention also provides the use of any of the above-mentioned compositions or the compositions prepared by the above-mentioned preparation methods in medicines having whitening and anti-aging effects.

[0015] Furthermore, the drug also includes pharmaceutically acceptable excipients; the excipients include: one or more of fillers, binders, wetting agents, disintegrants, flavoring agents, preservatives, pH adjusters, and antioxidants.

[0016] Furthermore, the pharmaceutical dosage form includes any one of tablets, granules, capsules, ointments, suspensions and injections.

[0017] The present invention also provides the use of any of the above-mentioned compositions or the compositions prepared by the above-mentioned preparation methods in skin care products with whitening and anti-aging effects.

[0018] Furthermore, the skin care product also includes the drug as an auxiliary material acceptable to skin care products; the auxiliary material includes: one or more of a surfactant, a moisturizer, a binder, a preservative, an antioxidant and an ultraviolet absorber.

[0019] Furthermore, the skin care product dosage form includes any one of toner, lotion, essence water, cream, eye mask and facial mask.

[0020] Further, the composition removes DPPH and ABTS + Free radicals, inhibit tyrosinase activity, improve the ability of HaCaT cells to resist oxidative damage and / or reduce the content of melanin and tyrosinase in B16-F10 cells, which has the effect of whitening and anti-aging.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] (1) The present invention has carried out in-depth screening and analysis of the effective active ingredients in the Northeast Pearlwood extract, and studied the ratio of each active ingredient. The obtained composition has good effects on removing DPPH and ABTS. + It has significant effects on reducing free radicals, inhibiting tyrosinase activity, improving the ability of HaCaT cells to resist oxidative damage and / or reducing the content of melanin and tyrosinase in B16-F10 cells. It has potential application in whitening and anti-aging drugs or cosmetics, filling the gap in the overall application of active ingredients of Northeast Pearl Plum extract in the field of whitening and anti-aging, and further provides a theoretical basis for the research of whitening and anti-aging drugs and cosmetics.

[0023] (2) The composition of the present invention is obtained by directly mixing the active ingredients according to the formula amount, and the preparation method is simple;

[0024] (3) The active ingredients of the composition of the present invention are all derived from the extract of Northeast Pearlwood, which is green, safe and has no toxic side effects. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. It is worth noting that the raw materials used in the present invention are all common commercial products, and their sources are not specifically limited; the specific raw material information is shown in Table 1.

[0026] Table 1 Raw material information

[0027]

[0028] Example 1

[0029] A composition with whitening and anti-aging effects, comprising: 1 part of cucurbitacin F, 60 parts of sutherlandin-5-trans-p-coumarate, 0.1 part of quercetin, 0.1 part of rutin, 0.01 part of wogonin, 1 part of trifolioside, 0.1 part of quercetin-3-O-β-D-xyloside, 0.1 part of scutellaria baicalensis, 0.5 part of hyperoside, 0.5 part of cinchona ib, 1 part of kaempferol-3-furanarabinoside, 2 parts of arbutin and 1 part of astragaloside.

[0030] The preparation method is as follows:

[0031] The above-mentioned weight portions of cucurbitacin F, sutherlandin-5-trans-p-coumarate, quercetin, rutin, wogonin, trifolioside, quercetin-3-O-β-D-xyloside, scutellaria baicalensis, hyperoside, cinchonadin Ib, kaempferol-3-furanarabinoside, arbutin and astragaloside are mixed and passed through an 80-mesh sieve to obtain the product.

[0032] Example 2

[0033] A composition with whitening and anti-aging effects, comprising: 6 parts of cucurbitacin F, 68 parts of sutherlandin-5-trans-p-coumarate, 3.9 parts of quercetin, 0.9 parts of rutin, 0.19 parts of wogonin, 6 parts of trifolioside, 3.9 parts of quercetin-3-O-β-D-xyloside, 3.9 parts of scutellaria baicalensis, 2.5 parts of hyperoside, 2.5 parts of cinchona ib, 8 parts of kaempferol-3-furanarabinoside, 12 parts of arbutin and 6 parts of astragaloside.

[0034] The preparation method is the same as Example 1.

[0035] Example 3

[0036] A composition with whitening and anti-aging effects, comprising: 2 parts of cucurbitacin F, 56 parts of sutherlandin-5-trans-p-coumarate, 1 part of quercetin, 0.2 parts of rutin, 0.05 parts of wogonin, 1.5 parts of trifolioside, 1 part of quercetin-3-O-β-D-xyloside, 1 part of scutellaria baicalensis, 0.7 parts of hyperoside, 0.7 parts of cinchona ib, 2 parts of kaempferol-3-furanarabinoside, 4 parts of arbutin, and 1.5 parts of astragaloside.

[0037] The preparation method is the same as Example 1.

[0038] Example 4

[0039] A composition with whitening and anti-aging effects, comprising: 4 parts of cucurbitacin F, 72 parts of sutherlandin-5-trans-p-coumarate, 3 parts of quercetin, 0.8 parts of rutin, 0.15 parts of wogonin, 5.5 parts of trifolioside, 3 parts of quercetin-3-O-β-D-xyloside, 3 parts of scutellaria baicalensis, 2.3 parts of hyperoside, 2.3 parts of cinchona ib, 7 parts of kaempferol-3-furanarabinoside, 10 parts of arbutin and 5.5 parts of astragaloside.

[0040] The preparation method is the same as that of Example 1.

[0041] Example 5

[0042] A composition with whitening and anti-aging effects, comprising: 3 parts of cucurbitacin F, 64 parts of sutherlandin-5-trans-p-coumarate, 2 parts of quercetin, 0.5 parts of rutin, 0.1 parts of wogonin, 3.5 parts of trifolioside, 2 parts of quercetin-3-O-β-D-xyloside, 2 parts of scutellaria baicalensis, 1.5 parts of hyperoside, 1.5 parts of cinchona ib, 4.5 parts of kaempferol-3-furanarabinoside, 7 parts of arbutin and 3.5 parts of astragaloside.

[0043] The preparation method is the same as that of Example 1.

[0044] Comparative Example 1

[0045] The difference between this comparative example and Example 5 is that the dosage of sutherlandin-5-trans-p-coumarate, trifoliate glycoside, baicalin, hyperoside and astragalin is different; specifically: 3 parts of cucurbitacin F, 16 parts of sutherlandin-5-trans-p-coumarate, 2 parts of quercetin, 0.5 parts of rutin, 0.1 parts of baicalin, 14 parts of trifoliate glycoside, 2 parts of quercetin-3-O-β-D-xyloside, 8 parts of baicalin, 0.1 parts of hyperoside, 1.5 parts of cinchona ib, 4.5 parts of kaempferol-3-furanosyl glycoside, 7 parts of arbutin and 0.8 parts of astragalin.

[0046] The preparation method is the same as that of Example 1.

[0047] Comparative Example 2

[0048] The difference between this comparative example and Example 5 is that the dosage of trifolioside, baicalin, hyperoside, arbutin and astragaloside is different; specifically: 3 parts of cucurbitacin F, 64 parts of sutherlandin-5-trans-p-coumarate, 2 parts of quercetin, 0.5 parts of rutin, 0.1 parts of baicalin, 0.8 parts of trifolioside, 2 parts of quercetin-3-O-β-D-xyloside, 0.5 parts of baicalin, 6 parts of hyperoside, 1.5 parts of cinchonaside Ib, 4.5 parts of kaempferol-3-furanarabinoside, 0.1 parts of arbutin and 14 parts of astragaloside.

[0049] The preparation method is the same as that of Example 1.

[0050] Comparative Example 3

[0051] The difference between this comparative example and Example 5 is that there is no quercetin and kaempferol-3-furanosyl glycoside; specifically, it is composed of 3 parts of cucurbitacin F, 64 parts of sutherlandin-5-trans-p-coumarate, 0.5 parts of rutin, 0.1 parts of wogonin, 3.5 parts of trifolioside, 2 parts of quercetin-3-O-β-D-xyloside, 2 parts of baicalin, 1.5 parts of hyperoside, 1.5 parts of cinchonaside Ib, 7 parts of arbutin, and 3.5 parts of astragaloside.

[0052] The preparation method is as follows:

[0053] The above-mentioned weight portions of cucurbitacin F, sutherlandin-5-trans-p-coumarate, rutin, wogonin, trifolioside, quercetin-3-O-β-D-xyloside, scutellaria baicalensis, hyperoside, cinchonadin Ib, arbutin and astragalus glycoside are mixed and passed through an 80-mesh sieve to obtain the whitening and anti-aging composition.

[0054] Test example: Experimental study on the efficacy of whitening and anti-aging composition

[0055] 1. Experimental Materials

[0056] (1) Experimental drugs:

[0057] Positive drugs: Vitamin A (VA), kojic acid;

[0058] Samples to be tested: compositions prepared in Examples 1-5 and Comparative Examples 1-3 of the present invention.

[0059] (2) Experimental materials:

[0060] CCK8 reagent was purchased from Beijing Bioson Biotechnology Co., Ltd.; SOD kit and MDA kit were purchased from Nanjing Jiancheng Reagent Co., Ltd.; DMEM medium, RPMI 1640 medium, and PBS buffer were all purchased from Gibco; K2S2O8 and ABTS were purchased from Shanghai Aladdin Technology Co., Ltd.; DPPH was purchased from Shanghai McLean Biochemical Technology Co., Ltd., and DMSO and tyrosinase were purchased from Sigma.

[0061] (3) Experimental cells and experimental animals:

[0062] Human immortalized keratinocytes (HaCaT cells) and mouse melanoma cells (B16-F10 cells) were obtained from the Central Laboratory of Xiangya School of Medicine, Central South University. Wild-type (WT) zebrafish of AB strain used in zebrafish experiments were purchased from the Zebrafish Platform of the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences.

[0063] (4) Experimental equipment:

[0064] Infinite M200 PRO microplate reader, centrifuge, balance, cell disruptor, constant temperature water bath.

[0065] 2. Data Processing

[0066] SPSS 23.0 software was used for statistical analysis and the data were expressed as mean ± standard deviation ( ) indicates that the data analysis conformed to the normal distribution, and the comparison of means among multiple groups was performed using the One-Way ANOVA method; if P <0.05, the difference was considered statistically significant.

[0067] 3. In vitro antioxidant and whitening ability determination

[0068] (1) DPPH, ABTS + Antioxidant capacity and tyrosinase inhibition ability assay

[0069] The antioxidant capacity was determined using DPPH and ABTS working solutions. The positive drug (VA), the compositions prepared in Examples 1-5 and Comparative Examples 1-3, and the DPPH and ABTS + The free radical scavenging rate was tested in the concentration range of 0.025-1 mg / mL.

[0070] 1.1 Determination of DPPH free radical scavenging ability:

[0071] The compositions prepared by different concentrations of positive drugs (VA), samples of Examples 1-5 and Comparative Examples 1-3 (prepared with anhydrous ethanol to concentrations of 0.025, 0.05, 0.1, 0.2, 0.5 and 1 mg / mL, respectively) were reacted with 0.2 mM DPPH ethanol solution, and the reaction mixture contained 50 μL of the sample to be tested and 100 μL of the reaction solution. The blank group was added with 50 μL of sample solvent and then 100 μL of anhydrous ethanol. After sufficient mixing, the reaction was allowed to react at room temperature for 30 min, and the change in absorbance at 517 nm was measured, and the determination was repeated three times. The DPPH free radical scavenging rate was calculated according to the following formula:

[0072] DPPH clearance rate (%) = [(A1-A2)-(A3-A4)] / (A1-A2)×100%

[0073] Wherein: A1: blank group, 50 μL of sample solvent was added to the reaction system instead of the sample solution; A2: blank control group, 100 μL of anhydrous ethanol and 50 μL of sample solvent were added to the reaction system instead of the DPPH ethanol solution and the sample solution respectively; A3: sample group; A4: blank absorbance of the sample group, 100 μL of anhydrous ethanol was added to the reaction system instead of the DPPH ethanol solution.

[0074] 1.2 ABTS + Free radical scavenging ability determination:

[0075] Preparation of ABTS working solution: 7.04 mM ABTS + solution and 2.6 mM K2S2O8 solution; ABTS + The solution was mixed with K2S2O8 solution in equal volumes and placed in the dark at room temperature for 12-16 h to obtain ABTS working solution. Before use, it was diluted 39 times with anhydrous ethanol to an absorbance of 0.70±0.02 at 734 nm and stored in the dark until tested.

[0076] Different concentrations of positive drug (VA), samples of Examples 1-5 and compositions prepared in Comparative Examples 1-3 (prepared with anhydrous ethanol to concentrations of 0.025, 0.05, 0.1, 0.2, 0.5 and 1 mg / mL, respectively) were added to ABTS working solution, and the reaction mixture contained 50 μL of the sample to be tested and 100 μL of the reaction solution; 50 μL of sample solvent was added to the blank group, and then 100 μL of anhydrous ethanol was added. After sufficient mixing, the mixture was allowed to stand at room temperature in the dark for 6 min, and the absorbance at 734 nm was immediately measured, and the measurement was repeated three times. ABTS was calculated according to the following formula + Free radical scavenging rate:

[0077] ABTS +Free radical scavenging rate (%) = [(A1-A2)-(A3-A4)] / (A1-A2)×100%

[0078] In the formula: A1: blank group, 50 μL of sample solvent was added to the reaction system instead of the sample solution; A2: blank control group, 100 μL of anhydrous ethanol and 50 μL of sample solvent were added to the reaction system instead of the ABTS working solution and sample solution respectively; A3: sample group; A4: blank absorbance of the sample group, 100 μL of anhydrous ethanol was added to the reaction system instead of the ABTS working solution.

[0079] 1.3 Tyrosinase inhibition ability determination:

[0080] Preparation of PBS buffer: weigh 71.6 g Na2HPO4·12H2O and dissolve it in 1000 mL ultrapure water; weigh 31.2 g NaH2PO4·2H2O and dissolve it in 1000 mL ultrapure water; take 51 mL Na2HPO4·12H2O solution, 49 mL Na2HPO4·12H2O solution and 100 mL ultrapure water to prepare 0.1 M PBS buffer (pH = 6.8).

[0081] Preparation of 2.5 mM L-DOPA solution: Accurately weigh 49.3 mg of L-DOPA, first dissolve it with the PBS buffer prepared above by ultrasound, and make up to volume in a 100 mL brown volumetric flask.

[0082] Add 50 μL of different concentrations of kojic acid (prepared with fresh serum-free medium to concentrations of 0.3125, 0.625, 1.25, 2.5, 5, and 10 mg / mL), 50 μL of different concentrations of sample solution (prepared with fresh serum-free medium to concentrations of 2.5, 5, 10, 20, 30, 40, and 50 mg / mL) to the ELISA plate, and add 100 μL of 125 U / mL tyrosinase solution and 100 μL of 2.5 mM L-DOPA solution, respectively. Use an oscillator to micro-oscillate, incubate at 37°C for 30 min, measure the absorbance at 475 nm, and measure three times in parallel. Tyrosinase activity inhibition rate is calculated according to the following formula:

[0083] Tyrosinase activity inhibition rate (%) = [(A1-A2)-(A3-A4)] / (A1-A2)×100%

[0084] Wherein: A1: blank group, 10 μL of sample solvent was added to the reaction system instead of the sample solution; A2: blank control group, 10 μL of sample solvent and 50 μL of PBS solution were added to the reaction system instead of the sample solution and L-DOPA substrate solution; A3: sample group; A4: blank absorbance of the sample group, 50 μL of sample solvent was added to the reaction system instead of the L-DOPA substrate solution.

[0085] Calculate the corresponding EC 50 The results are shown in Table 2.

[0086] Table 2 DPPH, ABTS + Antioxidant capacity and tyrosinase inhibitory capacity EC 50 Value (mg / mL)

[0087]

[0088] As can be seen from the above table, compared with the compositions prepared in Comparative Examples 1-3, the EC 50 The lower the concentration of the value, the better the antioxidant activity. It can be seen that the composition of the present invention has good DPPH and ABTS under the specific active ingredient composition and ratio. + Free radical scavenging ability and tyrosinase inhibition ability.

[0089] (2) Evaluation of the anti-aging and whitening activity of the combination based on oxidative stress and α-MSH cell model

[0090] Oxidative stress of skin keratinocytes and melanocytes is an important factor causing skin aging and depigmentation diseases. Next, human keratinocytes (HaCaT) were used for H2O2 modeling, and mouse melanoma cells (B16-F10) were used for α-MSH modeling to verify the whitening and anti-aging effects of the composition of the present invention.

[0091] 2.1 Cell viability assay

[0092] HaCaT cells were cultured in a sterile humidified incubator at 37°C and 5% CO2 in modified RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. When the cells grew to 80%-90%, they could be used for experiments.

[0093] B16-F10 cells were cultured in a sterile humidified incubator at 37°C and 5% CO2 in modified DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. When the cells grew to 80%-90%, they could be used for experiments.

[0094] Discard the old culture medium of HaCaT cells and B16-F10 cells, wash each with PBS twice, add 2 mL of 0.25% trypsin to digest for 5 min, add 2 mL of complete culture medium to dilute, collect the cells into a centrifuge tube, centrifuge to remove the supernatant, dilute the cells with an appropriate amount of complete culture medium, gently pipette until the cells are suspended, take part of the cell suspension to continue diluting, and add 1×10 5 The cells were inoculated into 96-well plates, and 100 μL of cell fluid was added. The plates were cultured in a 37 ℃, 5% CO2 incubator for 24 h to allow the cells to adhere to the wall before administration. Before administration, fresh serum-free culture medium was used to prepare the test sample solutions, positive drug VA solutions (for HaCat cell viability assay) and positive drug kojic acid solutions (for B16-F10 cell viability assay) with concentrations of 0.00625, 0.0125, 0.025, 0.50, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, respectively, and the solutions were filtered with sterile filter membranes for sterilization. After 24 hours of drug administration and culture, 10 μL of CCK8 reagent was added to each well of each group and cultured for 1 hour. After the culture, the absorbance (OD value) of each group of solutions at 450 nm was tested using an ELISA instrument, and the absorbance of the blank group was used as a control; the concentrations with cell survival rates above 90% (0.00625, 0.0125, 0.025, 0.05 mg / mL) were selected for subsequent experiments. The cell survival rate was calculated as follows:

[0095] Cell viability (%) = OD value of drug-treated group / OD value of blank group × 100%.

[0096] Then the cell viability IC of each group was calculated 50 The results are shown in Table 3.

[0097] Table 3 HaCaT cell viability and B16-F10 cell viability IC 50 Value (mg / mL)

[0098]

[0099] As can be seen from the above table, compared with the compositions prepared in Comparative Examples 1-3, the compositions IC prepared in Examples 1-5 of the present invention 50 The concentration was higher and had no toxicity to HaCaT cells and B16-F10 cells.

[0100] 2.2 Antioxidant damage ability of HaCaT cells

[0101] HaCaT cells were taken at 5×10 5 / mL was inoculated in a 96-well plate, 2 mL per well, and cultured for 24 h. 100 μL of complete medium was added to the blank group, 50 μL of 4 mM H2O2 oxidative damage high expression model was added to the other groups, and then the positive drug VA (prepared with fresh serum-free medium to a concentration of 0.2 mg / mL) was added to the positive group, and 50 μL of the corresponding composition (prepared with fresh serum-free medium to a concentration of 0.0125, 0.025, 0.05, and 0.1 mg / mL) was added to the Example 1-5 group and the Comparative Example 1-3 group, and cultured for 24 h, and the cells of each group were collected and washed twice with PBS buffer, and the MDA and SOD contents were detected using MDA and SOD detection kits; the results are shown in Table 4.

[0102] Table 4 Antioxidant damage ability of HaCaT cells ( )

[0103]

[0104] Note: △ Compared with the blank group P <0.05; △△ Compared with the blank group P <0.01; # Compared with the model group P <0.05; ## Compared with the blank group P <0.01; * Compared with the positive group P <0.05; ** Compared with the positive group P <0.01; & Compared with Example 5 P <0.05; && Compared with Example 5 P <0.01.

[0105] As can be seen from the table above, compared with the blank group, the SOD content of HaCaT cells in the model group was significantly reduced ( P <0.01) MDA content was significantly increased ( P <0.01), indicating that the model was successfully established.

[0106] Compared with the model group, the SOD content of Example 1-5 group and Comparative Example 1-2 group had a very significant difference ( P <0.01), indicating that the compositions provided in the examples and comparative examples of the present invention can significantly increase the SOD content; there is a very significant difference in the MDA content between the examples 1-5 group and the comparative examples 1-3 group ( P<0.01), indicating that both the composition provided in the embodiment of the present invention and the composition provided in the comparative example can reduce the MDA content; and there is no significant difference between the composition provided in the embodiment of the present invention and the positive drug group ( P >0.05).

[0107] Compared with Example 5, the SOD content of HaCaT cells in Comparative Examples 1-3 was lower and the MDA content was higher, and both were significantly different ( P <0.01), indicating that the composition provided in the embodiment of the present invention can repair the oxidative damage of H2O2 to HaCaT cells to a certain extent, reduce the intracellular oxidation level, and improve the antioxidant capacity, thereby better delaying aging.

[0108] 2.3 Effect of melanin content and tyrosinase activity on B16-F10 cells

[0109] Melanin content determination:

[0110] B16-F10 cells were collected at 5×10 5 / mL was inoculated in a 6-well plate, and the liquid volume of each well was 2 mL, and cultured for 24 hours. Incomplete culture medium was added to the blank group, 1.5 μM α-MSH was added to the model group, kojic acid (concentration 0.1 mg / mL) + 1.5 μM α-MSH was added to the positive group, and the corresponding composition 0.050 mg / mL + 1.5 μM α-MSH was added to the Example 1-5 group and the Comparative Example 1-3 group, and cultured for 48 hours, and rinsed twice with pre-cooled PBS buffer, 1 mL of trypsin was added to each well for 2 minutes, and the cells of each group were collected, centrifuged and the supernatant was discarded, and 150 μL 1N NaOH / 10% DMSO was immediately added to the cell pellet of each group, lysed at 80 ° C for 1 hour, and the total content of melanin was determined by measuring the absorbance (OD) at 450 nm with an enzyme reader. The absorbance of the blank group was used as a control to calculate the relative content of melanin in each group of cells. The protein concentration of all samples was determined according to the BCA method, and the melanin content was normalized according to the protein concentration. The formula is as follows:

[0111] Melanin content (%) = OD of drug-treated group / OD of blank group × 100%.

[0112] Tyrosinase content determination:

[0113] According to the above melanin content determination method, the plates were plated, the cell pellets were collected, the cells were dissolved in PBS solution containing 1% TritonX-100, and the cells were repeatedly frozen and thawed three times at -80 °C. After centrifugation at 12000 rpm for 10 min, the cell supernatant protein was obtained. After quantification and normalization, 90 uL of cell supernatant and L-DOPA (10 mM 10 μL) were added to the 96-well plate and incubated at 37 °C for 1 h. The absorbance at 405 nm was measured with an enzyme marker. The blank group was used as a control to calculate the relative content of tyrosinase in the cells. Tyrosinase activity was normalized according to protein concentration. The formula is as follows:

[0114] Tyrosinase content (%) = OD treatment group / OD blank group × 100%.

[0115] The results are shown in Table 5.

[0116] Table 5 Relative content of melanin and tyrosinase in B16-F10 cells ( )

[0117]

[0118] Note: △ Compared with the blank group P <0.05; △△ Compared with the blank group P <0.01; # Compared with the model group P <0.05; ## Compared with the blank group P <0.01; * Compared with the positive group P <0.05; ** Compared with the positive group P <0.01; & Compared with Example 5 P <0.05; && Compared with Example 5 P <0.01.

[0119] As can be seen from the table above, compared with the blank group, the B16-F10 cells in the model group secreted a large amount of melanin after being stimulated by α-MSH, and the relative content of intracellular melanin and tyrosinase content increased significantly ( P <0.01), indicating that the model was successfully established.

[0120] Compared with the model group, the relative content of melanin and tyrosinase in Example 1-5 group and Comparative Example 1-3 group were significantly different ( P<0.01), indicating that the compositions provided in the examples of the present invention and the comparative examples can significantly reduce the relative content of melanin and the relative content of tyrosinase; and there is no significant difference between the compositions provided in the examples of the present invention and the positive drug group ( P >0.05).

[0121] Compared with Example 5, the relative content of melanin and tyrosinase in B16-F10 cells in Comparative Examples 1-3 groups were significantly different ( P <0.01), indicating that the composition of the present invention reduces the generation of melanin and tyrosinase to a certain extent compared with the compositions provided in Comparative Examples 1-3, and has a more significant whitening and freckle-removing effect.

[0122] 4. Animal Experiments

[0123] 7 hpf embryos were placed in a 12-well plate, with 25 normally developed embryos in each well, and randomly divided into a blank group (adding an equal volume of culture medium), a positive group (100 μg / mL kojic acid), Example 1-5 group and Comparative Example 1-3 group (each Example and Comparative Example group was added with a concentration of 25, 50, 100, 250, 500, 750, 1000 μg / mL of the test solution, respectively) (the positive group drug, the composition of Example 1-5 and Comparative Example 1-3 were diluted with zebrafish culture medium to prepare the test solution), each group was set up in three parallel groups, the volume of solution added to each well was 3 mL, and the culture was kept at a constant temperature of 28±0.5°C. The test solution was replaced every 24 h. The zebrafish survival rate was observed after 72 h, and the relevant zebrafish survival rate IC was calculated. 50 The results are shown in Table 6.

[0124] Table 6 Zebrafish survival rate IC 50 Value (μg / mL)

[0125]

[0126] It can be seen from the above table that compared with comparative examples 1-3, the zebrafish survival rate IC 50 The values ​​correspond to higher concentrations and stronger cell viability.

[0127] 7 hpf embryos were placed in a 12-well plate, with 25 normally developed embryos in each well, and randomly divided into a blank group (adding equal volume of culture medium), a model group (1.5 μM α-MSH), a positive group (100 μg / mL kojic acid) + 1.5 μM α-MSH, 50 μg / mL corresponding composition solution of Example 1-5 groups + 1.5 μM α-MSH and 50 μg / mL corresponding composition solution of Comparative Example 1-3 groups + 1.5 μM α-MSH, and three groups were set up in parallel for each group. The volume of solution added to each well was 3 mL, and the cells were cultured at a constant temperature of 28±0.5°C. The test solution was replaced every 24 h. After 72 h, the zebrafish in each group were collected in a 1.5 mL EP tube, 150 μL RIPA lysis solution was added, and the zebrafish were homogenized using a handheld small homogenizer. After sufficient homogenization, the cells were centrifuged at 4°C and 12000 rpm for 10 min, the supernatant was discarded, and the precipitate was collected. The melanin content in zebrafish was then measured using the B16-F10 cell melanin content assay method. The melanin content in zebrafish was also normalized after protein quantification using the BCA method.

[0128] The model was then established and administered according to the above method, and the zebrafish precipitate was collected after 72 hours. The collected precipitate was dissolved in a PBS solution containing 1% Triton X-100, and the relative content of tyrosinase in zebrafish was measured according to the B16-F10 cell tyrosinase content determination method. Tyrosinase activity also needs to be normalized after protein quantification according to the BCA method, and the results are shown in Table 7.

[0129] Table 7 Relative content of melanin and tyrosinase in zebrafish ( )

[0130]

[0131] Note: △ Compared with the blank group P <0.05; △△ Compared with the blank group P <0.01; # Compared with the model group P <0.05; ## Compared with the blank group P <0.01; * Compared with the positive group P <0.05; ** Compared with the positive group P <0.01; & Compared with Example 5 P <0.05; && Compared with Example 5 P <0.01.

[0132] As can be seen from the table above, compared with the blank group, the zebrafish in the model group secreted a large amount of melanin after being stimulated by α-MSH, and the relative content of melanin and tyrosinase in the zebrafish increased significantly ( P <0.01), indicating that the model was successfully established.

[0133] Compared with the model group, the relative content of melanin and tyrosinase in zebrafish in Example 1-5 and Comparative Example 1-3 groups were significantly different ( P <0.01), indicating that the compositions provided in the examples of the present invention and the comparative examples can significantly reduce the relative content of melanin; and there is no significant difference between the examples 1-5 groups and the positive drug group ( P >0.05).

[0134] Compared with Example 5, the relative content of melanin and tyrosinase in zebrafish in Comparative Examples 1-3 groups had extremely significant differences ( P <0.01), indicating that the composition of the present invention can reduce the generation of melanin to a certain extent, and has a more significant whitening and freckle-removing effect.

[0135] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A composition with whitening and anti-aging effects, characterized in that: The composition comprises, by weight, 1-6 parts of cucurbitacin F, 45-83 parts of sutherlandin-5-trans-p-coumarate, 0.1-3.9 parts of quercetin, 0.1-0.9 parts of rutin, 0.01-0.19 parts of wogonin, 1-6 parts of trifolioside, 0.1-3.9 parts of quercetin-3-O-β-D-xyloside, 0.1-3.9 parts of scutellariae, 0.5-2.5 parts of hyperoside, 0.5-2.5 parts of cinchonaside Ib, 1-8 parts of kaempferol-3-furanarabinoside, 2-12 parts of arbutin and 1-6 parts of astragaloside.

2. The composition according to claim 1, characterized in that The composition comprises, by weight, 1.5-5 parts of cucurbitacin F, 56-72 parts of sutherlandin-5-trans-p-coumarate, 1-3 parts of quercetin, 0.2-0.8 parts of rutin, 0.05-0.15 parts of wogonin, 1.5-5.5 parts of trifolioside, 1-3 parts of quercetin-3-O-β-D-xyloside, 1-3 parts of scutellariae, 0.7-2.3 parts of hyperoside, 0.7-2.3 parts of cinchonaside Ib, 2-7 parts of kaempferol-3-furanarabinoside, 4-10 parts of arbutin and 1.5-5.5 parts of astragaloside.

3. The composition according to claim 2, characterized in that The composition comprises, by weight, 2-4 parts of cucurbitacin F, 60-68 parts of sutherlandin-5-trans-p-coumarate, 1.5-2.5 parts of quercetin, 0.3-0.7 parts of rutin, 0.08-0.12 parts of wogonin, 3-4 parts of trifolioside, 1.5-2.5 parts of quercetin-3-O-β-D-xyloside, 1.5-2.5 parts of scutellariae scutellariae, 1-2 parts of hyperoside, 1-2 parts of cinchona ib, 4-5 parts of kaempferol-3-furanarabinoside, 5-9 parts of arbutin and 3-4 parts of astragaloside.

4. The composition according to claim 3, characterized in that The composition comprises, by weight, 3 parts of cucurbitacin F, 64 parts of sutherlandin-5-trans-p-coumarate, 2 parts of quercetin, 0.5 parts of rutin, 0.1 parts of wogonin, 3.5 parts of trifolioside, 2 parts of quercetin-3-O-β-D-xyloside, 2 parts of baicalin, 1.5 parts of hyperoside, 1.5 parts of cinchonaside Ib, 4.5 parts of kaempferol-3-furanarabinoside, 7 parts of arbutin and 3.5 parts of astragaloside.

5. The method for preparing the composition according to any one of claims 1 to 4, characterized in that: The method comprises mixing cucurbitacin F, sutherlandin-5-trans-p-coumarate, quercetin, rutin, wogonin, trifolioside, quercetin-3-O-β-D-xyloside, baicalin, hyperoside, cinchonadin Ib, kaempferol-3-furanarabinoside, arbutin and astragaloside uniformly and sieving the mixture to obtain the product.

6. Use of the composition according to any one of claims 1 to 4 or the composition prepared by the preparation method according to claim 5 in the preparation of medicines or skin care products with whitening and anti-aging effects.

7. The use according to claim 6, characterized in that: The drug further includes pharmaceutically acceptable excipients; the excipients include: one or more of fillers, binders, wetting agents, disintegrants, flavoring agents, preservatives, pH regulators, and antioxidants; The pharmaceutical dosage form includes any one of tablets, granules, capsules, ointments, suspensions and injections.

8. The use according to claim 6, characterized in that: The skin care product further comprises an auxiliary material acceptable to skin care products; the auxiliary material comprises: one or more of a surfactant, a moisturizer, a binder, a preservative, an antioxidant and an ultraviolet absorber; The dosage form of the skin care product includes any one of toner, lotion, essence water, cream, eye mask and facial mask.

9. The use according to claim 6, characterized in that: The composition removes DPPH and ABTS + Free radicals, inhibit tyrosinase activity, improve the ability of HaCaT cells to resist oxidative damage and / or reduce the content of melanin and tyrosinase in B16-F10 cells, which has the effect of whitening and anti-aging.

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