Rosa roxburghii tratt extract composition for resisting oxidation and whitening and multiple whitening emulsions prepared from Rosa roxburghii tratt extract composition

Abstract

The invention discloses a rosa roxburghii tratt extract composition for resisting oxidation and whitening and whitening multiple emulsions thereof, and relates to the field of skin care products. The whitening composition comprises the following components: a rosa roxburghii tratt fruit extract rich in Vc; the rosa roxburghii tratt fruit extract is rich in SOD. The whitening multiple emulsion comprises the following components: a whitening matching substance; glycerol; propylene glycol; 26 # white oil; caprylic / capric triglyceride; an EM90; sP-80 is selected from the group consisting of 1, 3 Silicone oil; 6, 16, 18 alkyl glucoside (68); tW-80, TW-80; sorbitol is added; xanthan gum; a poloxamer; water soluble azone; and water. The raw material of the whitening composition is roxburgh rose, the preparation process of the extract is green and safe, and the whitening composition is non-irritant to skin after being used for a long time. Pharmacological test research shows that the composition is beneficial to synergistically scavenging free radicals, inhibiting tyrosinase and scavenging melanin. The multi-emulsion is beneficial to improving the stability of Vc and SOD in the mixture, and when the multi-emulsion is applied to the field of skin whitening, not only can the additional value of roxburgh rose products be improved, but also more choices can be provided for the source of natural whitening active matters.

Description

Technical Field

[0001] The invention belongs to the technical field of skin care products, and particularly relates to a roxburghii roxburghii extract formulation for anti-oxidation and whitening and a whitening multiple emulsion thereof. Background Art

[0002] The public is increasingly concerned about the negative effects of excessive free radical accumulation and abnormal melanin proliferation on the skin. Although the synthetic antioxidants and whitening ingredients that are flooding the market have demonstrated their effectiveness to a certain extent, the hidden cytotoxicity and sensitization risks behind them are like undercurrents, causing deep concerns about the safety of cosmetics in all walks of life. Against this background, the cosmetics industry is actively turning to a new research track: exploring and developing natural, harmless and highly effective active ingredients in order to replace traditional synthetics and bring a green revolution to skin care. Researchers are digging deep into the treasures of nature with unprecedented enthusiasm, improving and innovating traditional natural products, and trying to transform them into high-performance raw materials in the cosmetics field. Natural treasures such as vitamin C, vitamin E, superoxide dismutase (SOD), coenzyme Q10, lycopene and lutein are gradually coming to the forefront of cosmetic formulas, leading the upgrading of the cosmetics industry with their natural safety and excellent skin care effects. However, on the journey of whitening and skin care, a single active ingredient can no longer meet the growing and diversified needs of consumers. Therefore, the concept of multi-ingredient synergy came into being and became a new trend in cosmetics research and development. In addition to the recognized whitening star ingredients such as niacinamide, vitamin C, and arbutin, plant extracts from the depths of nature, such as the gentle nourishment of licorice, the elegant purification of lotus, and the abundant nourishment of longan, are gradually winning the favor and praise of the cosmetics industry with their unparalleled safety, rich active ingredient combinations, and unique synergistic effects. These natural extracts not only inject new vitality into cosmetics, but also meet consumers' ultimate pursuit of health, beauty, and nature with their comprehensive skin care effects. However, there is great unpredictability in the interactions between natural active ingredients from different sources. These interactions may affect the stability and efficacy of the product and even produce adverse reactions. In addition, the synergistic effects of multiple ingredients may increase the complexity of the formula, resulting in a decrease in the stability of the formula, etc. These are all problems that often need to be solved in the research and development of multi-active ingredient skin care products.

[0003] Rosa roxburghii, the fruit of the genus Rosa of the Rosaceae family, is widely planted in southwestern China and is rich in vitamins, polyphenols, flavonoids, terpenes, sugars and other beneficial ingredients. Among them, vitamin C, SOD, polyphenols and flavonoids show strong antioxidant, anti-aging and free radical scavenging abilities. Rosa roxburghii is known as the "king of vitamin C" and the "king of SOD", and has great application potential in the development of whitening cosmetics. Unfortunately, despite the rich active ingredients of Rosa roxburghii, its application in the field of whitening cosmetics has not been widely popularized. This situation is mainly attributed to several key issues: First, the vitamin C and SOD in Rosa roxburghii are unstable and easily inactivated by the external environment, which places extremely high demands on the product formulation design and production process. Second, although studies have shown that vitamin C can enhance the stability of SOD and there is a mutual promotion effect, there is also a complex relationship that high concentrations of vitamin C may inhibit the activity of SOD, which further increases the difficulty of product development. Therefore, how to effectively stabilize these active ingredients, ensure their efficacy in cosmetics, and solve the problem of balancing high-concentration vitamin C and SOD activity are the main challenges currently facing the development of sea buckthorn whitening cosmetics.

[0004] The preparation of natural extracts into emulsions is one of the common forms of their application in cosmetics, but there are many instability problems in traditional emulsion structures, mainly including: due to gravity, solid particles or oil droplets in the emulsion will gradually settle to the bottom of the container or float to the surface of the emulsion, resulting in emulsion stratification; oil droplets or solid particles in the emulsion gather together due to mutual attraction to form floccules; two or more oil droplets merge into a single large oil droplet, resulting in an increase in the particle size of the emulsion and a decrease in stability; small oil droplets will gradually dissolve and diffuse into large oil droplets, resulting in the continuous increase of large oil droplets, and the continuous decrease or even disappearance of small oil droplets; under certain conditions, the water-in-oil structure of the emulsion will be transformed into an oil-in-water structure, or vice versa. This phase transition will cause a serious decrease in the stability of the emulsion, etc. These instability problems will affect the appearance, texture and performance of the emulsion, and even cause the emulsion to deteriorate and fail. In addition, Vc has poor stability in aqueous solution. Therefore, different emulsion preparation methods need to be further studied in the preparation of emulsions to improve their stability. Summary of the invention

[0005] In order to solve the above problems, the present invention uses roxburghii fruit juice as a raw material, and respectively enriches the roxburghii active parts mainly composed of vitamin C and SOD by macroporous resin column chromatography and ammonium sulfate fractionation precipitation, and studies the ratio, antioxidant and whitening activities of the two extracts, in order to obtain the best ratio of roxburghii Vc and SOD, and apply it as the main active raw material to the preparation of multiple emulsions for cosmetics, and optimizes the formula of the multiple emulsions. The specific scheme is as follows:

[0006] Firstly, the present invention provides a roxburghii extract formulation for anti-oxidation and whitening, wherein the formulation comprises one or both of a roxburghii extract rich in Vc and a roxburghii extract rich in SOD.

[0007] Preferably, the formulation consists of a Rosa roxburghii extract rich in Vc and a Rosa roxburghii extract rich in SOD, and the content ratio of the two is 5:32 (μg:U).

[0008] Preferably, auxiliary materials are added to the formulation to prepare a solid preparation, a semisolid preparation or a liquid preparation.

[0009] Preferably, the semisolid preparation is an emulsion.

[0010] Preferably, the emulsion is a multiple emulsion comprising the following excipients: glycerol; propylene glycol; No. 26 white oil; octanoic acid glyceryl ester; EM90; SP-80; silicone oil; 16,18 alkyl glucoside (68); TW-80; sorbitol; xanthan gum; poloxamer; water-soluble azone; and water.

[0011] Preferably, the preparation method of the above-mentioned multiple emulsion (emulsion) comprises the following steps:

[0012] (1) adding the roxburghii extract and polyol to the oil phase and homogenizing to obtain a P / O emulsion;

[0013] (2) adding the obtained P / O emulsion to the aqueous phase, stirring and cooling to room temperature after uniform mixing, thereby obtaining a P / O / W multi-structure emulsion;

[0014] The polyol in step (1) includes glycerol and propylene glycol; the oil phase includes silicone oil, a first emulsifier and lipids; and the water phase in step (2) includes glycerol, sorbitol, xanthan gum, poloxamer, water-soluble azone, deionized water and a second emulsifier.

[0015] Preferably, in step (1), the first emulsifier comprises EM90 and SP-80, wherein the mass ratio of EM90 to SP-80 is 5:5-8:2, and more preferably 8:2; in step (1), the lipid comprises white oil and capric triglyceride, wherein the mass ratio of white oil to capric triglyceride is 5:5-8:2, and more preferably 6:4; in step (1), the amount of the roxburghii extract added to the system is 0.5-2wt%, and more preferably 2wt%; in step (2), the second emulsifier comprises 16,18 alkyl glucoside and TW-80, wherein the mass ratio of 16,18 alkyl glucoside to TW-80 is 5:5-4:6, and more preferably 5:5.

[0016] Preferably, in step (1), the amount of the first emulsifier added to the system is 0.5-2wt%, more preferably 0.5wt%; in step (2), the amount of the second emulsifier added to the system is 1-2wt%, more preferably 1wt%.

[0017] Preferably, the mass ratio of the oil phase to the polyol in step (1) is 6.5:3.5-5:5, more preferably 5.5:4.5; the mass ratio of the water phase to the P / O emulsion in step (2) is 8:2-9.5:0.5, more preferably 8:2.

[0018] Preferably, the mass ratio of glycerol to propylene glycol in the polyol of step (1) is 4.0:3.5-4.5:3.0; the mass ratio of silicone oil to lipid in step (1) is 0.8:3.5-0.5:4.2; the mass ratio of glycerol to emulsifier in step (2) is (0.44-0.57):1.00, the mass ratio of sorbitol to emulsifier is (0.34-0.38):1.00, the mass ratio of xanthan gum to emulsifier is (0.31-0.38):1.00, the mass ratio of poloxamer to emulsifier is (0.45-0.47):1.00, the mass ratio of water-soluble azone to emulsifier is (0.30-0.38):1.00, and the deionized water accounts for 79.50-80.59wt% of the overall reaction system of step (2).

[0019] Preferably, in step (1), the homogenization is carried out at 15500 r / min for 2 min; in step (2), the homogenization is carried out at 15500 r / min for 3 min; and in step (2), the stirring is carried out at a speed of 500 r / min.

[0020] In some embodiments of the present invention, the amount of each component added in the system in step (1) can be: 4.50wt% glycerol, 3.00wt% propylene glycol, 4.20wt% lipid, 0.50wt% emulsifier, and 0.50wt% silicone oil; the amount of each component added in the system in step (2) is: 1.00wt% emulsifier, 0.57wt% glycerol, 0.38wt% sorbitol, 0.38wt% xanthan gum, 0.47wt% poloxamer, 0.38wt% water-soluble azone, and the balance is 80.59wt% deionized water.

[0021] The present invention also provides application of the roxburghii roxburghii extract formulation in preparing whitening skin care products.

[0022] In some embodiments of the present invention, the method for preparing the above-mentioned roxburghii extract comprises the following steps:

[0023] S1, take the concentrated juice of roxburghii, add ethanol aqueous solution and stir thoroughly, let it stand for treatment, and separate the supernatant and the precipitate after treatment;

[0024] S2, taking the obtained supernatant and concentrating it under reduced pressure, and then applying it to an HPD macroporous resin chromatography column, collecting the filtrate, concentrating the filtrate under reduced pressure to an extract, and freeze-drying it to obtain a Rosa roxburghii dry powder rich in Vc (RRVC);

[0025] S3, take the obtained precipitate, add water to dissolve to obtain an aqueous solution, the obtained aqueous solution is mixed with PBS buffer, and the supernatant is taken as a crude enzyme solution after the first refrigerated centrifugation, and ammonium sulfate is added to the obtained crude enzyme solution for the first time to mix, and the solution is allowed to stand for treatment. After the treatment, a second refrigerated centrifugation is performed, and the supernatant is taken. Ammonium sulfate is added to the supernatant for the second time to mix, and a precipitate is taken after the third refrigerated centrifugation. The obtained precipitate is dissolved in PBS buffer, and a refined enzyme solution of Rosa roxburghii SOD is obtained after dialysis and desalination. The refined enzyme solution is freeze-dried to obtain a Rosa roxburghii dry powder (RRSOD) rich in SOD.

[0026] Preferably, the ethanol concentration in the ethanol aqueous solution added in S1 is 90-95 vol%, and after the ethanol aqueous solution is added to the roxburghii concentrated juice, the final ethanol concentration is 80-85 vol%; the standing treatment in S1 is carried out at 4°C for 12-18 h; the reduced pressure concentration in S2 is at 50-55°C; the supernatant is concentrated under reduced pressure until the obtained concentrate has the same mass as the roxburghii concentrated juice in step S1; the mass of water added in S3 is 2-3 times the mass of the precipitate, the aqueous solution is mixed with PBS buffer, the volume ratio of the two is 1:(3-4), and the pH of the PBS buffer is 7.6; the first refrigerated centrifugation is performed at a speed of 5000-6000r pm, and the time is 10-15min; ammonium sulfate is added to S3 for the first time, and the final concentration is 50-55wt%; the static treatment is performed at a temperature of 4°C and the time is 2-2.5h; the second refrigerated centrifugation is performed at a speed of 5000-6000rpm and the time is 20-25min; ammonium sulfate is added to S3 for the second time, and the final concentration is 75-80wt%; the third refrigerated centrifugation is performed at a speed of 5000-6000rpm and the time is 20-25min; the precipitate is dissolved in PBS buffer, wherein the volume of PBS buffer is 5%-10% of the volume of the crude enzyme solution; the dialysis desalination is performed in deionized water at a temperature of 4°C and the time is 12-48h.

[0027] In some embodiments of the present invention, the preparation method of the above-mentioned roxburghii extract comprises: diluting the roxburghii dry powder rich in Vc with pure water to the IC values ​​of DPPH free radical and tyrosinase respectively. 50 Concentration: 6.67μg / mL, 79.70μg / mL, to obtain Rosa roxburghii Vc solutions a and b; the Rosa roxburghii dry powder rich in SOD was diluted with pure water to the IC values ​​of DPPH free radical and tyrosinase, respectively. 50Concentration: 2.88U / mL, 72.09U / mL concentration, to obtain Rosa roxburghii SOD solutions A and B; respectively mix Rosa roxburghii Vc solution a with Rosa roxburghii SOD solution A to obtain a ratio solution I, and mix Rosa roxburghii Vc solution b with Rosa roxburghii SOD solution B to obtain a ratio solution II; wherein the volume ratio of Rosa roxburghii Vc solution a to Rosa roxburghii SOD solution A is 1:9, and the volume ratio of Rosa roxburghii Vc solution b to Rosa roxburghii SOD solution B is 1:9; the obtained ratio solution I or ratio solution II is concentrated under reduced pressure and then freeze-dried to obtain a ratio dry powder of Rosa roxburghii extract.

[0028] Beneficial effects of the present invention:

[0029] 1. The whitening activity of the extracts and formulations was evaluated by in vitro free radical scavenging test and tyrosinase inhibition test, and the whitening ability of the three was compared by B16 cell model and UVB-induced melanin deposition model. The results showed that the IC values ​​of RRVC on ABTS, DPPH, OH, and tyrosinase were 50 The IC values ​​of RRSOD for the above four whitening evaluation factors were 13.74μg / mL, 6.67μg / mL, 31.42μg / mL, and 79.70μg / mL, respectively. 50 They were 15.79U / mL, 2.88U / mL, 1.33U / mL and 72.09U / mL respectively. Both extracts showed strong whitening activity, and the combination (RRVS) had better activity. The optimal ratio of RRVC to RRSOD was 5:32 (μg:U).

[0030] 2. Within a certain concentration range, the inhibitory effect of this compound on B16 melanin production and tyrosinase activity is stronger than that of the uncompounded extract.

[0031] 3. Both extracts and mixtures can significantly reduce UVB-induced melanin deposition in mouse ears. The melanin clearance rates of RRSOD, RRVC, and RRVS are 55%, 62%, and 74%, respectively. The melanin clearance effect of the mixture is significantly stronger than that of the unmixed extract.

[0032] 4. The multiple emulsions containing the above-mentioned formulations are stable under low temperature, room temperature, high temperature, centrifugation and other conditions; the encapsulation rate reaches 80%; the formulations dispersed in the emulsions have high stability, and the tyrosinase inhibition and free radical scavenging capabilities are relatively stable.

[0033] 5. The roxburghii extracts rich in Vc and SOD have a mutually reinforcing effect when used in combination. The combination of the two in a certain proportion can achieve stronger antioxidant and whitening effects. Applying them to the field of skin whitening can not only increase the added value of roxburghii products, but also provide more choices for the source of natural whitening active ingredients. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Results for the free radical scavenging and tyrosinase inhibitory abilities of the two extracts.

[0035] Figure 2 The results show the effects of different ratios on free radical scavenging and tyrosinase inhibition capabilities.

[0036] Figure 3 The results are the effects of extracts and formulations on the proliferation ability of B16 cells.

[0037] Figure 4 These are the results of the effects of extracts and formulations on the melanin-producing ability of B16 cells.

[0038] Figure 5 The results are the effects of extracts and formulations on tyrosinase activity in B16 cells.

[0039] Figure 6 The results show the changes in pigmentation of mouse ear skin.

[0040] Figure 7 This is the result of melanin staining of the ear skin tissue of C57BL / 6J mice.

[0041] Figure 8 The results of lipid screening. A: Effects of different oils on multi-structured emulsions. Aa: Octinutyl triglyceride; Ab: 26 white mineral oil; Ac: sweet almond oil; Ad: evening primrose oil. Ae-Ag: Stability of emulsions at different temperatures. Ae: room temperature; Af: 4℃; Ag: 50℃, from left to right: Octinutyl triglyceride, 26 white mineral oil, sweet almond oil, evening primrose oil. B: Effects of different oil ratios (26 white oil: GTCC) on multiple emulsions. Ba: 5:5, Bb: 6:4, Bc: 7:3, Bd, 8:2. Be-Bg: Stability of emulsions at different temperatures. Be: room temperature; Bf: 4℃; Bg: 50℃, from left to right: 5:5, 6:4, 7:3, 8:2.

[0042] Fig. 9The results of screening of lipophilic emulsifiers. A: Effects of different lipophilic emulsifiers on the stability of multiple emulsions. Aa: EM90; Ab: SP-80; Ac: EM90+SP-80; Ad: Effects of different lipophilic emulsifiers on the stability of multiple emulsions at 60°C. B: Effects of different proportions of lipophilic emulsifiers on multiple emulsions. Ba: EM90:SP-80=5:5, Bb: EM90:SP-80=6:4, Bc: EM90:SP-80=7:3, Bd: EM90:SP-80=8:2. C: Effects of different proportions of lipophilic emulsifiers on multiple emulsions at different temperatures. Ca: room temperature; Cb: 4°C; Cc: 50-60°C. From left to right: Ba:EM90:SP-80=5:5, Bb:EM90:SP-80=6:4, Bc:EM90:SP-80=7:3, Bd:EM90:SP-80=8:2.

[0043] Fig.10 The results of screening of hydrophilic emulsifiers. A: Effects of different hydrophilic emulsifiers on the stability of multiple emulsions. Aa: 68; Ab: 68 + TW-80; Ac: TW-80; Ad: Effects of different hydrophilic emulsifiers on the stability of multiple emulsions at 60 ° C. B: Effects of different proportions of hydrophilic emulsifiers on multiple emulsions. Ba: 68: TW-80 = 5: 5; Bb: 68: TW-80 = 6: 4; Bc: 68: TW-80 = 7: 3; 68: TW-80 = 8: 2. C: Effects of different proportions of hydrophilic emulsifiers on multiple emulsions at different temperatures. Ca: room temperature; Cb: 4 ° C; Cc: 50-60 ° C. From left to right: 68:TW-80=5:5; Bb:68:TW-80=6:4; Bc:68:TW-80=7:3; 68:TW-80=8:2.

[0044] Fig.11 The results of screening of emulsifier ratios. A: Effects of different lipophilic emulsifier ratios on multiple emulsions. Aa: 0.5%; Ab: 1%; Ac: 1.5%, Ad: 2%, Ae: Effects of different ratios of lipophilic emulsifiers on multiple emulsions at room temperature; Af: Effects of different ratios of lipophilic emulsifiers on multiple emulsions at 4°C; Ag: Effects of different ratios of lipophilic emulsifiers on multiple emulsions at 60°C. B: Effects of different ratios of lipophilic aqueous emulsifiers on multiple emulsions. Ba: 0.5%; Bb: 1%; Bc: 1.5%; Bd: 2%; Be: Effects of different ratios of hydrophilic emulsifiers on multiple emulsions at room temperature; Bf: Effects of different ratios of hydrophilic emulsifiers on multiple emulsions at 4°C; Bg: Effects of different ratios of hydrophilic emulsifiers on multiple emulsions at 60°C. From left to right: 0.5%, 1%, 1.5%, 2%.

[0045] Fig.12Results of screening of oleyl alcohol ratio and water-to-emulsion ratio. A: Screening of oleyl alcohol ratio (lipid: polyol). 6.5:3.5 (Aa), 6:4 (Ab), 5.5:4.5 (Ac), 5:5 (Ad), Ae: Effect of different oleyl alcohol ratios on multiple emulsions at room temperature, Af: Effect of different oleyl alcohol ratios on multiple emulsions at 4°C, Ag: Effect of different oleyl alcohol ratios on multiple emulsions at 60°C. From left to right: 6.5:3.5, 6:4, 5.5:4.5, 5:5. B: Effect of different water-to-emulsion ratios (external aqueous phase: colostrum) on multiple structural emulsions. 8:2 (Ba), 8.5:1.5 (Bb), 9:1 (Bc), 9.5:0.5 (Bd), Be: the effect of different water-emulsion ratios on multiple emulsions at room temperature, Bf: the effect of different water-emulsion ratios on multiple emulsions at 4°C, Bg: the effect of different water-emulsion ratios on multiple emulsions at 60°C. From left to right: 8:2, 8.5:1.5, 9:1, 9.5:0.5.

[0046] Fig.13 The results of screening for the proportion of compound addition. A: The effect of different proportions of compound addition on the multi-structure emulsion. Aa: 0.5%; Ab: 1%; Ac: 1.5%; Ad: 2%. B: The stability of multiple emulsions at different temperatures. Ba: room temperature; Bb: 4℃; Bc: 50-60℃. The proportions of compound addition from left to right are: 0.5%, 1%, 1.5%, 2%.

[0047] Fig.14 Microscopic observation results of multiple emulsions obtained from 9 orthogonal experimental groups.

[0048] Fig.15 The stability test diagram of multiple emulsions obtained from 9 orthogonal experimental groups, where A is at room temperature, B is at 50°C, and C is centrifuged at 4000 r / min (total time 120 min).

[0049] Fig.16 The stability changes of multiple emulsions under different storage conditions.

[0050] Fig.17 The results are the changes in free radical scavenging and tyrosinase inhibitory abilities of the mixture in different media at different time periods.

[0051] Fig.18 Shown are the microscopic changes of the multiple emulsion after one month of storage and the finished emulsion product. DETAILED DESCRIPTION

[0052] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0053] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only and are not intended to limit the content of this application.

[0054] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0055] Experimental materials: Fresh roxburghii fruits (plants from roxburghii were collected from rural areas near Bijie City, Guizhou Province, southwest China in September 2022). Mouse melanoma cell line (B16) (Shanghai Enzyme Research Biotechnology Co., Ltd.); 36 male C57BL / 6J mice, weighing 28-30 g, were provided by the Experimental Animal Center of Guizhou Medical University with license number SCXK(Zhe)2019-0004 and were routinely kept in the animal room. 2,2'-hydrazine-bis(3-ethylbenzothiazoline-6-sulfonamide) diamine salt reagent (ABTS) and 2,2-biphenyl-1-phenylhydrazine reagent (DPPH), salicylic acid were purchased from MacLean Biochemical Technology Co., Ltd. (Shanghai, China). L-DOPA was purchased from G-GLONE Biotechnology Co., Ltd. (Beijing, China). Ascorbic acid standards and arbutin standards were purchased from Solebold Biotechnology Co., Ltd. (Beijing, China). All other reagents were of analytical grade. Glycerol, cosmetic grade, Sinopharm Chemical Reagent Co., Ltd. Propylene glycol, cosmetic grade, Junnuo Chemical Co., Ltd. Dipropylene glycol, cosmetic grade, Junnuo Chemical Co., Ltd. Linear polydimethylsiloxane, cosmetic grade, Jinan Shanhai Chemical Co., Ltd. Cetyl polyoxyethylene / polyoxypropylene-10 / 1 dimethicone, cosmetic grade, Qianhai Jishengya Technology Co., Ltd. Evening primrose oil, food grade, Sinopharm Chemical Reagent Co., Ltd. Sweet almond oil, food grade, Sinopharm Chemical Reagent Co., Ltd. Caprylic / capric triglyceride, cosmetic grade, Jinan Jinbang Environmental Protection Technology Co., Ltd. Sorbitan fatty acid ester, cosmetic grade, Sinopharm Chemical Reagent Co., Ltd. No. 26 white mineral oil, cosmetic grade, Shenzhen Zhongruntong Chemical Co., Ltd. Polyoxyethylene-60 hydrogenated castor oil, cosmetic grade, Shenzhen Zhongruntong Chemical Co., Ltd. 1,2,3,4,5,6-hexanehexaol, cosmetic grade, Sinopharm Chemical Reagent Co., Ltd. Xanthan gum, food grade, Sinopharm Chemical Reagent Co., Ltd. Poloxamer 407, cosmetic grade, Hubei Yamade Biopharmaceutical Co., Ltd. Cetearyl alcohol (and) octadecyl glucoside, cosmetic grade, Hubei Yamade Biopharmaceutical Co., Ltd. Sorbitan monooleate polyoxyethylene ether, cosmetic grade, Xiongda Chemical Co., Ltd.

[0056] Example 1 Preparation of Rosa roxburghii extract:

[0057] S1, take the concentrated juice of roxburghii, add ethanol aqueous solution and stir thoroughly, let it stand for treatment, and separate the supernatant and the precipitate after treatment;

[0058] S2, taking the obtained supernatant and concentrating it under reduced pressure, and then applying it to an HPD macroporous resin chromatography column, collecting the filtrate, concentrating the filtrate under reduced pressure to an extract, and freeze-drying it to obtain Rosa roxburghii Vc dry powder;

[0059] S3. Take the obtained precipitate, add water to dissolve to obtain an aqueous solution, mix the obtained aqueous solution with PBS buffer, take the supernatant after the first refrigerated centrifugation as the crude enzyme solution, add ammonium sulfate to the obtained crude enzyme solution for the first time and mix, let it stand for treatment, perform a second refrigerated centrifugation after the treatment, take the supernatant, add ammonium sulfate to the supernatant for the second time and mix, perform a third refrigerated centrifugation, take the precipitate, dissolve the obtained precipitate in PBS buffer, obtain Rosa roxburghii SOD refined enzyme solution after dialysis and desalination, and freeze-dry the refined enzyme solution to obtain Rosa roxburghii SOD dry powder.

[0060] The ethanol concentration in the ethanol aqueous solution added in S1 is 90-95 vol%. After the ethanol aqueous solution is added to the roxburghii concentrated juice, the final ethanol concentration is 80-85 vol%.

[0061] The standing treatment in S1 is carried out at 4° C. for 12-18 hours.

[0062] The S2 is concentrated under reduced pressure at a temperature of 50-55° C.; the supernatant is concentrated under reduced pressure, and the concentrated product obtained is of the same quality as the concentrated roxburghii juice produced in step S1.

[0063] The mass of water added to the S3 is 2-3 times the mass of the precipitate, the aqueous solution is mixed with the PBS buffer solution, the volume ratio of the two is 1:(3-4), and the pH of the PBS buffer solution is 7.6; the first refrigerated centrifugation has a rotation speed of 5000-6000rpm and a time of 10-15min.

[0064] In the S3, ammonium sulfate is added for the first time, and the final concentration is 50-55wt%. The temperature of the static treatment is 4°C, and the time is 2-2.5h. The speed of the second refrigerated centrifugation is 5000-6000rpm, and the time is 20-25min.

[0065] The second addition of ammonium sulfate to S3 has a final concentration of 75-80wt%. The third refrigerated centrifugation is performed at a speed of 5000-6000rpm for 20-25min. The precipitate is dissolved in PBS buffer, wherein the volume of PBS buffer is 5-10% of the volume of the crude enzyme solution. The dialysis desalination is performed in deionized water at a temperature of 4°C for 12-48h.

[0066] Example 2 Preparation of Rosa roxburghii extract formulation (using two Rosa roxburghii extract formulations prepared in Example 1):

[0067] The roxburghii vitamin C was diluted with pure water to the IC values ​​of DPPH free radical and tyrosinase. 50Concentration: 6.67μg / mL, 79.70μg / mL, to obtain Rosa roxburghii Vc solutions a and b; Rosa roxburghii SOD was diluted with pure water to the IC values ​​of DPPH free radical and tyrosinase, respectively. 50 Concentration: 2.88U / mL, 72.09U / mL concentration, to obtain Rosa roxburghii SOD solutions A and B; respectively mix Rosa roxburghii Vc solution a with Rosa roxburghii SOD solution A to obtain a ratio solution I, and mix Rosa roxburghii Vc solution b with Rosa roxburghii SOD solution B to obtain a ratio solution II; wherein the volume ratio of Rosa roxburghii Vc solution a to Rosa roxburghii SOD solution A is 1:9, and the volume ratio of Rosa roxburghii Vc solution b to Rosa roxburghii SOD solution B is 1:9; the obtained ratio solution I or ratio solution II is concentrated under reduced pressure and then freeze-dried to obtain a ratio dry powder of Rosa roxburghii extract.

[0068] The obtained mixture includes roxburghii vitamin C and roxburghii superoxide dismutase (SOD), and the content ratio of the two is 5:32 (μg:U).

[0069] Example 3 Preparation of multiple emulsions including the above-mentioned roxburghii extract:

[0070] (1) adding the roxburghii extract and polyol to the oil phase and homogenizing to obtain a P / O emulsion;

[0071] (2) Add the obtained P / O emulsion to the water phase, homogenize, and then stir and cool to a dark temperature at room temperature to obtain a P / O / W multi-structure emulsion.

[0072] In step (1), the polyol comprises glycerol and propylene glycol; the oil phase comprises silicone oil, an emulsifier and lipids; and the homogenization is carried out at 15500 r / min for 2 minutes.

[0073] The emulsifier in step (1) includes EM90 and SP-80, wherein the mass ratio of EM90 to SP-80 is 5:5-8:2, and more preferably 8:2; the lipid includes white oil and capric triglyceride, wherein the mass ratio of white oil to capric triglyceride is 5:5-8:2, and more preferably 6:4.

[0074] The aqueous phase in step (2) includes glycerol, sorbitol, xanthan gum, poloxamer, water-soluble azone, deionized water, and an emulsifier; the homogenization in step (2) is performed at 15500 r / min for 3 minutes; the stirring is performed at a stirring speed of 500 r / min.

[0075] The emulsifier in step (2) comprises 16,18 alkyl glucoside and TW-80, wherein the mass ratio of 16,18 alkyl glucoside to TW-80 is 5:5-4:6, more preferably 5:5.

[0076] The amount of the emulsifier added to the system in step (1) is 0.5-2wt%, more preferably 0.5wt%; the amount of the emulsifier added to the system in step (2) is 1-2wt%, more preferably 1wt%.

[0077] The mass ratio of the oil phase to the polyol in step (1) is 6.5:3.5-5:5, and more preferably 5.5:4.5; the mass ratio of the water phase to the P / O emulsion in step (2) is 8:2-9.5:0.5, and more preferably 8:2.

[0078] In step (1), the amount of the roxburghii extract added to the system is 0.5-2wt%, more preferably 2wt%.

[0079] The amounts of the components added in the system in step (1) are: 4.50 wt% glycerol, 3.00 wt% propylene glycol, 4.20 wt% lipid, 0.50 wt% emulsifier, and 0.50 wt% silicone oil; the amounts of the components added in the system in step (2) are: 1.00 wt% emulsifier, 0.57 wt% glycerol, 0.38 wt% sorbitol, 0.38 wt% xanthan gum, 0.47 wt% poloxamer, 0.38 wt% water-soluble azone, and the balance is 80.59 wt% deionized water.

[0080] Using roxburgh roxburgh juice as raw material, active parts of roxburgh roxburgh, mainly VC and roxburgh roxburgh SOD, were respectively enriched, and the ratio, antioxidant and whitening activity were studied, so as to obtain the best ratio of roxburgh roxburgh VC extract and roxburgh roxburgh SOD extract, and optimize the formula and preparation method of the whitening multiple emulsion, as follows:

[0081] 1. Preparation of extracts

[0082] Weigh 200g of roxburghii concentrated juice, add 95% ethanol to make the ethanol concentration reach 85%, stir well, place in a refrigerator at 4℃ overnight, separate the supernatant and the precipitate. Take the supernatant and concentrate it to 200g under reduced pressure at 50℃, apply it to HPD macroporous resin chromatography column, collect the filtrate and concentrate it to extract, freeze-dry to obtain roxburghii extract powder rich in Vc, and set aside;

[0083] Collect the precipitate, add an appropriate amount of pure water to dissolve it, add 3 times the amount of PBS buffer with a pH of 7.6, stir and mix, and centrifuge at 6000r / min for 10 minutes to obtain the supernatant as the crude enzyme solution; add ammonium sulfate powder to make its saturation reach 50%, stir well, let it stand in a refrigerator at 4°C for 2 hours, centrifuge at a speed of 6000r / min for 20 minutes, add ammonium sulfate to the supernatant to make it reach 80%, centrifuge at a speed of 6000r / min for 20 minutes, redissolve the precipitate in a buffer solution with a volume of 1 / 20 of the original volume of the crude enzyme solution, dialyze and desalt in a refrigerator at 4°C to obtain a Rosa roxburghii SOD refined enzyme solution, and freeze-dry to obtain a Rosa roxburghii extract powder rich in SOD for standby use.

[0084] 2. ABTS free radical

[0085] Use pH 7.4 phosphate buffer to prepare 7.4 mM ABTS stock solution and 2.6 mM potassium persulfate stock solution. Mix the two stock solutions in equal volumes and react in the dark for 12 h to obtain ABTS free radical working solution. Take an appropriate amount of methanol to dilute the ABTS working solution to an absorption of 0.700±0.02 at 734 nm.

[0086] Take 4.0mL ABTS free radical working solution and 100μL sample solution in a brown sample bottle, mix vigorously, place in the dark for 20min, and measure the absorbance at 734nm. Each sample was measured in parallel 3 times, and ascorbic acid standard was used as a positive control. Calculate the ABTS free radical scavenging rate according to the formula, draw the correlation curve between the extract concentration gradient and the DPPH free radical scavenging rate, and calculate the IC value of the sample. 50 Concentration (the sample concentration required to scaveng 50% of DPPH free radicals).

[0087] ABTS free radical scavenging rate = [1-(A1-A2) / A0] × 100% (1)

[0088] Where A1 is the absorbance of the sample plus ABTS working solution; A2 is the absorbance of the sample plus methanol; A0 is the absorbance of ABTS working solution plus methanol.

[0089] 3. DPPH free radical

[0090] Take 0.4mL of sample solution in a stoppered test tube, add 1.2mL of 0.32mmol / L DPPH solution to the test tube, and adjust the total volume to 4mL with anhydrous ethanol. After thorough shaking and mixing, react in a dark place away from light for half an hour, and measure the absorbance of DPPH at 517nm for each group of solutions by ultraviolet spectrophotometry. Each sample was measured in parallel 3 times, with ascorbic acid as a positive control. Calculate the DPPH free radical scavenging rate according to the formula, draw the correlation curve between the extract concentration gradient and the DPPH free radical scavenging rate, and calculate the IC of the sample 50 Concentration (the sample concentration required to scaveng 50% of DPPH free radicals).

[0091] DPPH free radical scavenging rate (%) = [1-(B1-B2) / B0] × 100% (2)

[0092] Wherein B1 is the absorbance of the sample in the reaction system, B2 is the absorbance of the sample solution plus anhydrous ethanol, and B0 is the absorbance of the DPPH solution plus anhydrous ethanol.

[0093] 4. Hydroxyl free radicals

[0094] The Fenton reaction was used to determine the scavenging rate of samples at different concentrations for hydroxyl radicals (·OH). In a 10mL stoppered test tube, 0.5mL of 9mmol·L-1 salicylic acid, 0.5mL of 9mmol·L-1 ferrous sulfate solution, and 3mL of 1mmol·L-1 hydrogen peroxide were added, and 1mL of the test solution was added. After mixing, the mixture was placed in a water bath at 37°C for 1h, and the absorbance was measured at a wavelength of 510nm. Formaldehyde was used as the blank control, and the absorbance was measured as A0; sample solutions of different concentrations and ascorbic acid standard solutions were used as test solutions, and the absorbance was measured as Ai. Each sample was measured three times in parallel, and the hydroxyl radical scavenging rate was calculated according to the formula. The correlation curve between the extract concentration gradient and the hydroxyl radical scavenging rate was drawn, and the IC of the sample was calculated. 50 Concentration (the sample concentration required to scaveng 50% of hydroxyl radicals).

[0095] OH removal rate (%) = (As-An) / As×100% (3)

[0096] Where: As is the absorbance of added formaldehyde, An is the absorbance of added test solution.

[0097] 5. Tyrosinase inhibition test

[0098] Take 200 μL of each sample with different concentration gradients, add 1 mL of 0.03% levodopa solution, add phosphate buffer to 5 mL, add 0.4 mL of tyrosinase extract, react at 37°C for 10 minutes, and measure the absorbance at 475 nm. Each sample was measured in parallel 3 times, with ascorbic acid as a positive control. Calculate the tyrosinase inhibition rate according to the formula, draw a correlation curve between the extract concentration gradient and the tyrosinase inhibition rate, and calculate the IC 50 Concentration (the sample concentration required to inhibit 50% of tyrosinase).

[0099] Tyrosinase inhibition rate % = [(AB) - (CD)] / (AB) × 100 (4)

[0100] Wherein A is the absorbance value of the reaction system containing tyrosinase but not containing sample; B is the absorbance value of the reaction system containing neither tyrosinase nor sample; C is the absorbance value of the reaction system containing tyrosinase and sample; D is the absorbance value of the reaction system containing neither tyrosinase nor sample.

[0101] 6. Screening of the best ratio

[0102] Take the obtained Rosa roxburghii extract rich in Vc and SOD, dilute it with pure water to a concentration where the scavenging rate or inhibition rate reaches about 50%, calculate it by volume of the extract, and make the proportions according to Table 1. According to the above method, the proportioned liquid is tested for ABTS, DPPH and hydroxyl free radical scavenging and tyrosinase inhibition ability, and the whitening effect score of the proportion is calculated according to the formula.

[0103]

[0104] Where: A 测 Indicates the ABTS free radical scavenging ability measurement value, A min Indicates the minimum ABTS free radical scavenging capacity in the system; D 测 Indicates the DPPH free radical scavenging ability measurement value, D min Indicates the minimum DPPH free radical scavenging capacity in the system; 测 represents the hydroxyl free radical scavenging ability measurement value, O min Indicates the minimum hydroxyl radical scavenging capacity in the system; L 测 Indicates the tyrosinase inhibition ability test value, L min It indicates the minimum tyrosinase inhibition ability in this system.

[0105] Table 1 Proportion of roxburghii extracts rich in Vc and SOD

[0106]

[0107] 7. Cell culture and grouping

[0108] Culture conditions: temperature: 37°C, humidity: >85%, CO2 concentration: 5%, DMEM culture medium containing 10% FBS and 1% penicillin-streptomycin mixture, cells in the logarithmic phase were used for subsequent experiments.

[0109] Different RRVC concentration groups of 25-400 μg / mL were set up, and ascorbic acid at a concentration of 50 μg / mL was used as a positive control.

[0110] 8. Determination of B16 cell proliferation rate

[0111] When the cells grow to 80% to 90% confluence, digest them with 0.25% trypsin, add fresh culture medium and mix thoroughly, and adjust the cell concentration to 5×10 4 / mL, the cells were inoculated in a 96-well culture plate, 100μL per well, placed in a CO2 incubator for 24h, the supernatant was discarded, rinsed once with PBS, 100μL of fresh culture medium containing different concentrations of the test substance was added, placed in a CO2 incubator for 48h, the supernatant was discarded, and 10μL of 5mg / mL MTT solution and 90μL of fresh serum-free medium were added to each well under light-proof conditions. The cells were placed in an incubator for 4h, the culture medium and MTT were discarded, 100μL of dimethyl sulfoxide (DMSO) was added to each well, oscillated for 15min, and the OD value of each well at 570nm was detected with an enzyme marker to calculate the cell proliferation rate.

[0112] 9. Determination of melanin content in B16 cells

[0113] The NaOH lysis method was used to determine the intracellular melanin content of B16 cells. 5 B16 cells of different concentrations were inoculated in a 12-well cell plate, 1 mL per well, and cultured normally for 24 h. The old culture medium was discarded, and 1 mL of culture medium containing different mass concentrations of the test substance was added. Three replicate wells were set for each group and cultured for another 48 h. 200 μL of trypsin was added to each well for digestion, and then 600 μL of PBS buffer was added to collect the cell fluid, centrifuged at 5000 r / min for 10 min, the supernatant was discarded, 200 μL of 1 mol / L NaOH solution (containing 10% dimethyl sulfoxide) was added to each well, and the plates were heated in a water bath at 80°C until the cell clusters were completely lysed and the melanin particles were completely dissolved, and the absorbance was measured at a wavelength of 405 nm.

[0114] 10. Determination of tyrosinase activity in B16 cells

[0115] Collect cell precipitates as in "9. Determination of melanin content in B16 cells", add 200 μL 1% TritonX-100 solution to each well. Freeze at 80℃ for 1 hour, thaw at room temperature to lyse the cells, repeat the freezing and thawing steps several times, centrifuge the cell lysate, take 100 μL of the supernatant in a 96-well plate, add 60 μL of 2 mmol / L L-dopa solution, incubate at 37℃ in the dark for 30 minutes, and measure the absorbance at a wavelength of 475 nm.

[0116] 11. Animal Testing

[0117] Thirty-six C57BL / 6J mice weighing 20-25g were randomly divided into a blank control group, a model group, an arbutin positive control group, a RRVC group, a RRSOD group, and a RRVS group after one week of adaptive feeding. Except for the blank control group, the ears of each group of mice were exposed to ultraviolet light for 10 minutes every day for 3 weeks using an ultraviolet light therapy device. After the formation of stable melanin deposition, the model group, except the blank control group, was smeared with propylene glycol, and the other groups were smeared with the corresponding extracts or ratios once a day for 3 weeks. Finally, after the application, the ears of the mice in each group were photographed to compare the amount of melanin deposition. After recording, the mice were anesthetized to death, and the ears on both sides of the mice were cut off for sectioning and staining observation.

[0118] 12. Establishment of comprehensive evaluation model for multiple emulsions

[0119] Based on the experts’ scores, the relative importance of the five first-level indicators was finally determined, and a judgment matrix was constructed (see Table 2).

[0120] Table 2 Multiple emulsion formula importance rating table

[0121]

[0122] Substitute the constructed judgment matrix into the calculation model of SPSSAU online data analysis software to calculate the weight values ​​of the five basic indicators. The AHP analysis results are shown in Table 3. The consistency test result is CR<0.1, which passes the one-time test. The same method is used to perform AHP analysis on the basic indicators under the first-level indicators.

[0123] Table 3 Results of importance analysis of multiple emulsion formulas

[0124]

[0125] (1) AHP analysis of emulsion appearance

[0126] A judgment matrix was constructed for the appearance of the lotion, as shown in Table 4. The constructed judgment matrix was substituted into the calculation model of the SPSSAU online data analysis software to calculate the weights of the three basic indicators. The AHP analysis results are shown in Table 5. The calculation results show that CR < 0.1, passing the one-time test.

[0127] Table 4 Multiple emulsion appearance importance rating table

[0128]

[0129] Table 5 Analysis results of the importance of multiple emulsion appearance

[0130]

[0131] (2) AHP analysis of lotion odor

[0132] A judgment matrix was constructed for the lotion odor, as shown in Table 6. The constructed judgment matrix was substituted into the calculation model of SPSSAU online data analysis software to calculate the weights of the three basic indicators. The AHP analysis results are shown in Table 7. The calculation results show that CR < 0.1, passing the one-time test.

[0133] Table 6 Multiple emulsion odor importance rating table

[0134]

[0135] Table 7 Analysis results of odor importance of multiple emulsions

[0136]

[0137] (3) AHP analysis of the experience of using lotion

[0138] A judgment matrix was constructed for the experience of using lotion, as shown in Table 8. The constructed judgment matrix was substituted into the calculation model of SPSSAU online data analysis software to calculate the weights of the three basic indicators. The AHP analysis results are shown in Table 9. The calculation results show that CR < 0.1, passing the one-time test.

[0139] Table 8 Multiple emulsion usage experience importance rating table

[0140]

[0141] Table 9 Analysis results of importance of multiple emulsion usage experience

[0142]

[0143] (4) AHP analysis of emulsion stability

[0144] A judgment matrix was constructed for emulsion stability, as shown in Table 10. The constructed judgment matrix was substituted into the calculation model of SPSSAU online data analysis software to calculate the weights of the three basic indicators. The AHP analysis results are shown in Table 11. The calculation results show that CR < 0.1, passing the one-time test.

[0145] Table 10 Multiple emulsion stability importance score table

[0146]

[0147] Table 11 Results of importance analysis of multiple emulsion stability

[0148]

[0149] (5) AHP analysis of multiple structures of emulsions

[0150] A judgment matrix was constructed for the multiple structures of the emulsion, as shown in Table 12. The constructed judgment matrix was substituted into the calculation model of the SPSSAU online data analysis software to calculate the weights of the three basic indicators. The AHP analysis results are shown in Table 13. The calculation results show that CR < 0.1, passing the one-time test.

[0151] Table 12 Multiple emulsion multiple structure importance score table

[0152]

[0153] Table 13 Results of importance analysis of multiple structures of multiple emulsions

[0154]

[0155] The weight values ​​of all first-level indicators that passed the consistency test were multiplied by the weight values ​​of the corresponding indicators to calculate the comprehensive weight values ​​of each indicator, and the importance of each basic indicator was ranked, as shown in Table 14. The results showed that the most important basic indicators among the five categories were texture, light matrix odor, weak stickiness, high temperature resistance and clear phase interface.

[0156] Table 14 Comprehensive weights of various evaluation indicators

[0157]

[0158]

[0159] 13. Preparation of multiple emulsions

[0160] Table 15 Matrix formulation

[0161]

[0162] In order to effectively encapsulate the polyol in the inner phase, a two-step method was used to prepare the P / O / W multi-structure emulsion. In the first step, the extract and polyol phase were added to the oil phase, homogenized at 15500r / min for 2min to prepare the P / O emulsion; in the second step, the P / O emulsion (colostrum) was added to the outer water phase, homogenized at 15500r / min for 3min, and then stirred at 500r / min using a stirrer to cool to room temperature to obtain the P / O / W multi-structure emulsion. Microscope observation and stability test were used to evaluate the excellence of the multiple emulsion during the single factor experiment.

[0163] 13.1 Formulation Optimization of Multiple Emulsions

[0164] The single-factor experimental method was used to screen the factors such as the type of oil and the proportion of composite oil, the type of lipophilic emulsifier and the proportion of composite lipophilic emulsifier, the type of hydrophilic emulsifier and the proportion of composite hydrophilic emulsifier, the amount of emulsifier added, the oil-alcohol ratio, the water-emulsion ratio, and the amount of compounding material added in the multiple emulsion according to the formula in the table.

[0165] 13.2 Orthogonal Experiment

[0166] Based on the results of single factor experiments, three factors with significant influence were selected to ensure the formation of emulsions, and a three-factor three-level orthogonal experiment was designed to obtain the best experimental scheme. The optimal formula of multiple emulsions was determined through verification experiments.

[0167] Table 16 Orthogonal test factor level table

[0168]

[0169] Table 17 Orthogonal test method table

[0170]

[0171] 13.3 Stability Investigation

[0172] (1) Alternating cold and heat resistance test

[0173] Take 20g of sample and place it in a -5℃ refrigerator for 24h, then take it out and store it at room temperature for 24h and observe it. Then place it in a 40℃ constant temperature box for 24h, then take it out and store it at room temperature for 24h. Observe for no less than 7 days to see if there is any water-oil stratification, and observe whether there are obvious changes in properties and microscopic changes compared with before the test.

[0174] (2) pH test

[0175] During the cold and heat resistance alternating test, the pH value of the sample is tested and recorded every 24 hours. Take 1g of the sample and place it in a beaker, add 10g of deionized water, stir it in a 40℃ water bath until it is uniform, and then let it stand and cool at room temperature to obtain the solution to be tested. According to the instrument manual, use three standard solutions to calibrate the instrument at the specified temperature. After calibration, wash the electrode with water, and wait for the filter paper to absorb it dry before measuring the sample.

[0176] 13.4 Encapsulation efficiency determination

[0177] The encapsulation efficiency of the multiple emulsion was determined by ultrafiltration centrifugation. Specifically, 1 mL of the multiple emulsion loaded with Vc was accurately drawn into an ultrafiltration centrifuge tube, and its weight was determined as W1. The free Vc in the emulsion was separated from the multiple emulsion into the lower aqueous phase of the ultrafiltration centrifuge tube by centrifugation (5000 r / min centrifugation for 25 min), and the lower aqueous phase of the ultrafiltration centrifuge tube was diluted and analyzed by HPLC injection to obtain W2. Another 1 mL of the emulsion was accurately drawn into a volumetric flask, an equal amount of dimethyl sulfoxide was added as a demulsifier, and ultrasonicated for 10 minutes until it was transparent, and then diluted and analyzed by HPLC to obtain W3. The encapsulation efficiency and drug loading were calculated according to the following equation:

[0178] Encapsulation efficiency (%) = (W2-W1) / W2×100

[0179] Wherein, W2 is the total mass of the Vc-loaded multiple emulsion, and W1 is the mass of free Vc in the multiple emulsion.

[0180] 13.5 Free Radical Scavenging Activity Test

[0181] The free radical scavenging activity test of the extracts and formulations is the same as that described above.

[0182] 13.6 Tyrosinase inhibition test

[0183] The free radical scavenging activity test of the extracts and formulations is the same as that described above.

[0184] 13.7 Security Testing

[0185] Twenty volunteers were selected and the prepared multiple emulsion was applied on the back of their hands evenly, once in the morning and once in the evening every day for one month. The skin conditions of the volunteers were recorded at four nodes after 1d, 7d, 14d and 30d to see if there was erythema or swelling.

[0186] 14. Data processing and analysis

[0187] The experimental data were expressed as mean ± standard deviation. SPSS software was used to perform one-way analysis of variance on the data. Multiple comparisons were performed and p < 0.05 was considered statistically significant. GraphPad Prism was used to plot the data.

[0188] 14.1 Free Radical Scavenging and Tyrosinase Inhibition Ability

[0189] Both RRVC and RRSOD showed strong free radical scavenging and tyrosinase inhibitory abilities ( Figure 1 The scavenging rates of the two extracts on the three free radicals and tyrosinase were concentration-dependent and increased with increasing concentration. GraphPadPrism was used to simulate the IC under each reaction system 50 IC values ​​of RRVC and ascorbic acid against ABTS free radical, DPPH free radical, hydroxyl free radical and tyrosinase 50 The order is: 13.74μg / mL, 27.86μg / mL (Aa); 6.67μg / mL, 6.98μg / mL (Ab); 31.42μg / mL, 37.63μg / mL (Ac); 79.70μg / mL, 103.90μg / mL (Ad). IC 50 The smaller the value, the stronger the scavenging ability. It can be seen that compared with the ascorbic acid standard, RRVC has stronger scavenging ability and tyrosinase inhibition ability for the above three free radicals than ascorbic acid, especially the scavenging / inhibiting ability for ABTS free radicals and tyrosinase. In addition, RRSOD also showed good free radical scavenging activity and tyrosinase inhibition ability, with IC values ​​of ABTS free radical, DPPH free radical, hydroxyl free radical and tyrosinase being 0. 50 They are: 15.79U / mL(Ba), 2.88U / mL(Bb), 1.33U / mL(Bc), and 72.09U / mL(Bd).

[0190] 14.2 Screening of the Optimal Ratio

[0191] Dilute the extract to IC 50Then, 9 proportions were obtained, and the results showed that when the volume ratio between RRVC and RRSOD was 7:3, the scavenging rate of ABTS free radicals was the highest, which was 25.71% higher than before the proportion; the scavenging effect on DPPH free radicals showed an overall increasing trend, and the scavenging ability was the strongest when the volume ratio was 1:9, which was 36.02% higher than the two extracts; the scavenging effect on hydroxyl free radicals was regular as a whole. As the proportion of RRVC increased, its scavenging rate decreased, and the scavenging ability was the strongest when the volume ratio was 1:9, which was 21.30% higher than the two extracts; the inhibitory effect on tyrosinase showed an overall increasing trend, except for the volume ratios of 3:7, 7:3, and 9:1, which were similar to the inhibitory abilities of the two extracts, the other ratios were stronger than when acting alone, among which the inhibitory ability was the strongest when the volume ratio was 1:9, which was 35.24% higher than the two extracts. According to the comprehensive scores, the whitening efficacy of each ratio is ranked as 1:9>2:8>4:6>5:5>8:2>3:7>6:4>9:1>7:3. The optimal volume ratio was finally determined to be 1:9, which is equivalent to 5:32 (μg:U).

[0192] 14.3 Effects of extracts and formulations on the proliferation of B16 cells

[0193] The proliferation toxicity of each concentration gradient extract and the ratio of the extract on B16 cells is shown in Figure 3 . In the concentration range of 25-200 μg / mL of RRVC, the relative cell viability reached more than 90%, indicating that RRVC had no toxicity to B16 cells within this range. When the concentration increased to 400 μg / mL, the cell viability decreased to 70%-80% (3A). RRSOD and its matching substances had no toxicity to B16 cells in the activity range of 25-200 U / mL, and the relative cell viability reached more than 90%. When the concentration increased to 400 U / mL, RRSOD and its matching substances showed certain toxicity to B16 cells, and the relative cell viability decreased to less than 50% (3B-3C).

[0194] 14.4 Effects of extracts and formulations on the ability of B16 cells to produce melanin

[0195] After the B16 cell melanin high expression model was given a certain concentration of extracts and mixtures, its melanin expression was significantly reduced (4A). Figure 4As shown in Figure B, compared with the model group, RRVC and positive drug vitamin C can significantly reduce the content of melanin in B16 cells (P < 0.01). Among them, the ability of RRVC to inhibit the production of melanin in B16 cells is concentration-dependent. The higher the concentration, the stronger the inhibition ability. When the concentration reaches 200 μg / mL, the inhibition rate of melanin is about 53.66%. Compared with the positive drug vitamin C, the inhibition of melanin by RRVC at the same concentration is stronger than ascorbic acid (P < 0.01). Compared with the model group, RRSOD and the matching substance can significantly reduce the content of melanin in B16 cells. The ability of both to inhibit the production of melanin in B16 cells is concentration-dependent. The higher the concentration, the stronger the inhibition ability. When the SOD enzyme activity reaches 200U / mL, the inhibition rate of melanin reaches more than 70%. Compared with RRSOD, in the range of 50-200U / mL, the ability of the matching substance at the same concentration to inhibit the production of melanin in B16 cells is stronger than the former. Compared with ascorbic acid, when the activity of RRSOD was greater than 100 U / mL and the activity of RRRVS was greater than 50 U / mL, the inhibitory ability on melanin was significantly stronger than that of ascorbic acid (4Bb-4Bc).

[0196] Effects of 14.5 extracts and formulations on tyrosinase activity in B16 cells

[0197] like Figure 5 As shown in A, compared with the model group, RRVC and the positive drug vitamin C can significantly reduce the tyrosinase activity in B16 cells (P < 0.01). The ability of RRVC to inhibit the tyrosinase activity in B16 cells is concentration-dependent. The higher the concentration, the stronger the inhibition ability. When the concentration reaches 200 μg / mL, the inhibition rate is about 42.66%. Compared with the positive drug vitamin C, the same concentration of RRVC has a similar inhibitory ability on tyrosinase, and the inhibition rates of both on tyrosinase activity are about 20%. Figure 5 As shown in B-5C, compared with the model group, RRSOD and the matching substance can significantly inhibit the tyrosinase activity in B16 cells, and the ability of both to inhibit tyrosinase activity is concentration-dependent. The higher the concentration, the stronger the inhibition ability. When the SOD enzyme activity reaches 200U / mL, the inhibition rate of RRSOD on tyrosinase activity reaches 45.78%, and the matching substance reaches 63.66%. Compared with RRSOD, in the range of 25 to 100U / mL, the inhibitory ability of the matching substance with the same concentration on the tyrosinase activity in B16 cells is significantly stronger than that of the former (P<0.01).

[0198] 14.6 Effects of Melanin Deposition on Mouse Ear Skin

[0199] like Figure 6As shown, the Model group mice had obvious melanin deposition in the ear skin after 21 days of UVB radiation, and the use of extracts and formulas could significantly inhibit UVB-induced pigment deposition in the ear skin of C57BL / 6J mice. In order to visually observe the changes in melanin in the ear skin tissue of C57BL / 6J mice, we used a melanin staining kit to stain the tissue sections. It is worth noting that this staining method (ferrous sulfate staining) will not stain lipofuscin with trivalent iron, and the identification of melanin is more accurate. Figure 7 As shown in the figure, the epidermis of the model group was significantly thickened after UVB exposure, and a large amount of melanin deposition appeared in the epidermis and dermis, and this phenomenon was significantly improved after the positive drug arbutin and sample treatment. Compared with arbutin, RRSOD has the same ability to reduce melanin deposition, while melanin deposition is further reduced after RRVC and RRVS treatment, among which RRVS shows the best ability to reduce melanin deposition.

[0200] In summary, the above experimental results show that RRVC and RRSOD obtained by macroporous resin column chromatography and ammonium sulfate fractionation precipitation respectively have strong in vitro free radical scavenging and tyrosinase inhibitory activities, among which RRVC has stronger ability than ascorbic acid standard. The optimal ratio of RRVC and RRSOD was screened out as 5:32 by the comprehensive ability of free radical scavenging and tyrosinase inhibition. Within a certain concentration range, the inhibitory effect of this ratio on B16 melanin production and tyrosinase activity is stronger than that of the unproportioned extract. Both extracts and ratios can significantly reduce UVB-induced melanin deposition in mouse ears, among which the ratio has a stronger scavenging effect on melanin than the extract. Therefore, exploring the use effect of the ratio of the two sea buckthorn extracts is conducive to improving the application value of sea buckthorn in the fields of medicine, health care and cosmetics, and can also provide more choices for people's demand for natural whitening.

[0201] 14.7 Screening of Oils and Fats

[0202] Depend on Figure 8 A shows that no multiple structures were observed in the emulsions prepared by sweet almond oil, evening primrose oil, and GTCC. Only No. 26 white oil can form multiple structures in the emulsion. From the perspective of stability, each emulsion is stable at room temperature and 4°C, but stratification occurs at 50°C. The stability of the emulsion formed by GTCC is better than that of the other three oils. No. 26 white oil and GTCC were selected as lipids for preparing multiple emulsions, and the ratio between the two was screened. Figure 8 B shows that when No. 26 white oil:GTCC>5:5, more multiple structures can be observed in the formed emulsion, but as the proportion of No. 26 white oil increases, the stability of the emulsion decreases, so the oil ratio of 6:4 was selected for subsequent experiments.

[0203] 14.8 Screening of Water-in-Oil Emulsifiers

[0204] Depend on Fig. 9 As shown in A, a relatively regular multi-structured emulsion was prepared using emulsifier EM90, while the emulsion system using SP-80 was more stable than that using EM90, and the emulsion stability was better when the two were mixed. Therefore, EM90 and SP-80 were mixed and used as oil-in-water emulsifiers, and the optimal ratio of the two was investigated. Fig. 9 As shown in Figures B and 9C, as the proportion of EM90 increases, the stability of the emulsion improves. When the ratio of EM90:SP-80 is 8:2, the multi-structured emulsion phase interface is clear and the stability is optimal. Therefore, the oil-in-water emulsifier EM90:SP-80 is selected as 8:2 for subsequent experiments.

[0205] 14.9 Screening of Oil-in-Water Emulsifiers

[0206] like Fig.10 As shown in A, the effects of TW-80, 68 and their combination on the formation of multiple emulsions were compared. The use of emulsifiers TW-80 or 68 in the external water phase can form a water-in-oil-in-alcohol structure, and the combination of the two has more and more uniform droplets in the emulsion multiple structure, and the emulsion formed by emulsifier 68 has better stability. Therefore, the two are used in combination as water-in-oil emulsifiers, and the optimal ratio of the two is investigated. Fig.10 As shown in Figures B and 10C, when the ratio of 68:TW-80 is 3:7 and 2:8, a clear phase interface can be formed, but the droplets are large, the roundness is low, and the stability is poor. When the ratio is 5:5 and 4:6, the multi-structured emulsion phase interface is clear, the droplets are uniform, and the roundness is high. The emulsion stability is best when the ratio is 5:5, so the water-in-oil emulsifier 68:TW-80 is 5:5 for subsequent experiments.

[0207] 14.10 Screening of emulsifier addition ratio

[0208] Depend on Fig.11 As shown in Figure A, the effect of different addition ratios of water-in-oil emulsifier on multiple emulsions was investigated. When the addition ratio reached 2%, the emulsion stability was good, but as the addition ratio increased, the observed multiple structures decreased. When the addition ratio was 0.5%, a better multiple structure could be formed, so the addition amount of water-in-oil emulsifier was selected to be 0.5%. Fig.11 As shown in Figure B, the effects of different addition ratios of water-in-oil emulsifier on multiple emulsions were investigated. When the addition ratio was 0.5%, the oil-water phase interface of the multiple emulsion was blurred. At the other addition ratios, multiple emulsions with clear phase interfaces, uniform droplets, and high roundness could be formed. When the addition ratio was 1%, the emulsion stability was optimal. Therefore, the addition ratio of water-in-oil emulsifier was selected as 1% for subsequent experiments.

[0209] 14.11 Screening of Oil-to-Alcohol Ratio and Water-to-Emulsion Ratio

[0210] Depend on Fig.12 A shows that a high or low oleyl alcohol ratio cannot form a good multiple emulsion structure. When the oleyl alcohol ratio is 5.5:4.5, the number of multiple structure particles and the number of internal phase particles in the system increase significantly. The emulsion stability under each oleyl alcohol ratio is not much different, so the oleyl alcohol ratio is selected to be 5.5:4.5. Fig.12 B shows that the emulsions at each water-to-emulsion ratio can form good multi-structured particles. As the water-to-emulsion ratio increases, the emulsion stability decreases. This is because there is too much external aqueous phase and the emulsifying capacity of the emulsifier is limited, which leads to a decrease in the viscosity of the multiple emulsions and a decrease in the emulsion stability. However, when the water-to-emulsion ratio is 8:2, the emulsion has a large viscosity and is not easy to spread. Therefore, a water-to-emulsion ratio of 8:8.5 was selected for subsequent tests.

[0211] 14.12 Screening of the proportion of added materials

[0212] like Fig.13 It can be seen that good multiple emulsion structures can be formed at each addition ratio. In the range of 0.5% to 2%, as the amount of the compounding material increases, the stability of the emulsion increases. This may be because the extract is dispersed in the external phase, so that the interfaces of the emulsion droplets do not directly contact each other, reducing the aggregation of the droplets, thereby improving the stability of the emulsion.

[0213] 14.13 Orthogonal Test Results

[0214] (1) Microscopic observation

[0215] Microscopic observation of multiple emulsions obtained from 9 orthogonal experimental groups Fig.14 As shown in the figure, the emulsion droplets increase with the increase of the amount of oil-in-water emulsifier added. The emulsion droplets of experimental groups No. 1, No. 2 and No. 3 are small, No. 4, No. 5 and No. 6 are in the middle, and No. 7, No. 8 and No. 9 are the largest. When the amount of lipophilic emulsifier added is 0.5g (experimental groups No. 1-3), the multiple emulsion interface is not clear. When the amount added is 1g (experimental groups No. 4-6) and 1.5g (experimental groups No. 7-9), multiple emulsions with clear interfaces can be formed. This shows that the lipophilic emulsifier has a great influence on the formation of multiple emulsions.

[0216] (2) Stability test

[0217] The stability of the multiple emulsions obtained from the 9 orthogonal experimental groups was investigated as follows Fig.15 As shown in the following table: At room temperature, the stability of each group is ranked as No. 7 > No. 5 > No. 1 > No. 9 > No. 4 > No. 8 > No. 3 > No. 2 > No. 6. At a high temperature of 50°C, the stability of each group is ranked as No. 7 > No. 4 > No. 5 > No. 6 > No. 8 > No. 9 > No. 2 > No. 1 > No. 3. Under centrifugal conditions, the stability of each group is ranked as No. 7 > No. 5 > No. 9 > No. 1 > No. 8 > No. 6 > No. 4 > No. 2 > No. 3.

[0218] (3) Sensory evaluation such as appearance, smell, and usage experience

[0219] Invite 10 volunteers to score, and use the average value as the score of each item; stability is based on Fig.15 The results are scored, and multiple structures are based on Fig.14 To score, multiply each score by the corresponding weight value and sum them up.

[0220] Table 18 The scores and comprehensive scores of 9 multiple emulsions in orthogonal test

[0221]

[0222]

[0223] From the results of the multiple emulsion orthogonal design experiment in Table 19, it can be seen that the comprehensive score of the 7th group of experiments is the highest, indicating that the comprehensive performance of this group, such as sensory evaluation, usage experience and stability, is better.

[0224] Table 19 Orthogonal test results

[0225]

[0226]

[0227] From the variance analysis results in Table 20, it can be seen that the amount of lipophilic emulsifier added has the greatest impact on the experimental results, followed by the amount of hydrophilic emulsifier added and the amount of extract added. The effects of the two on the comprehensive performance of the multiple emulsions are significantly different (P<0.01, P<0.05).

[0228] Table 20 Orthogonal test results analysis of variance

[0229]

[0230] According to the formula optimization, the final multiple emulsion formula is shown in Table 21.

[0231] Table 21 Final preferred formula of multiple emulsion

[0232]

[0233] 14.14 Encapsulation efficiency

[0234] Encapsulation rate (%) = (encapsulated drug content / total drug content) × 100% = 5.2 / (1.3+5.2) × 100% = 80%

[0235] 14.15 Stability Changes

[0236] (1) Stability changes of multiple emulsions under different storage conditions

[0237] Depend on Fig.16 It can be seen that the optimized multiple emulsion did not show stratification at room temperature, repeated freeze-thaw, and centrifugation conditions, and stratification only began to occur after being placed at a high temperature of 50°C for two weeks, indicating that the emulsion has excellent stability under various storage conditions.

[0238] (2) Results of the latex cold and heat alternating test and pH value test

[0239] Depend on Fig.16 It can be seen that the optimized multiple emulsion did not show stratification after three cycles of alternating cold and heat resistance. During the observation period, the pH value of the emulsion was stable between 6 and 7, indicating that the emulsion has good stability under alternating cold and heat resistance conditions.

[0240] Table 22 Cold and heat resistance alternating test and pH value test results

[0241]

[0242]

[0243] 14.16 Antioxidant and tyrosinase scavenging

[0244] Depend on Fig.17 The results showed that the tyrosinase inhibition rate of the emulsion was relatively stable and remained high during the test period, while the tyrosinase inhibition rate of the sea buckthorn extract aqueous solution decreased rapidly, indicating that the sea buckthorn extract was extremely unstable in water. The ABTS clearance rates of the sea buckthorn extract glycerol solution and the emulsion were not much different and were relatively stable.

[0245] 14.17 Microscopic Changes of Emulsions

[0246] like Fig.18 After one month, the emulsion had no obvious microscopic changes. The obtained emulsion had uniform texture, uniform color and good fluidity.

[0247] 14.18 Security Testing

[0248] The results showed that after using the multiple lotions for one month, the skin condition of the volunteers was observed as shown in Table 23. The skin irritation intensity evaluation standard was 0, the skin remained moisturized, non-irritating, no erythema, edema, itching, pain, allergy and other adverse reactions occurred.

[0249] Table 23 Multiple emulsion safety test table

[0250]

[0251] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

[0252] The present invention and its implementation methods are described above, which is not restrictive. The drawings are only one of the implementation methods of the present invention, and the actual application is not limited thereto. In short, if ordinary technicians in the field are inspired by it and do not deviate from the purpose of the invention, they can creatively design methods and embodiments similar to the technical solution, which should fall within the protection scope of the present invention.

Claims

1. A roxburghii extract for anti-oxidation and whitening, characterized in that: The invention comprises one or both of a roxburghii roxburghii extract rich in Vc and a roxburghii roxburghii extract rich in SOD.

2. The roxburghii extract formulation for anti-oxidation and whitening according to claim 1, characterized in that: The mixture consists of a roxburghii roxburghii extract rich in Vc and a roxburghii roxburghii extract rich in SOD, and the content ratio of the two is 5:32 (μg:U).

3. A roxburghii extract formulation for anti-oxidation and whitening according to claim 1 or 2, characterized in that: Add auxiliary materials to the formulation to prepare solid preparations, semisolid preparations or liquid preparations.

4. The roxburghii extract formulation for anti-oxidation and whitening according to claim 3, characterized in that: The semisolid preparation is an emulsion.

5. The roxburghii extract formulation for anti-oxidation and whitening according to claim 4, characterized in that: The emulsion is a multiple emulsion containing the following auxiliary materials: glycerol; propylene glycol; No. 26 white oil; octanoic acid glyceryl ester; EM90; SP-80; silicone oil; 16,18 alkyl glucoside (68); TW-80; sorbitol; xanthan gum; poloxamer; water-soluble azone; and water.

6. A method for preparing a multiple emulsion as claimed in claim 5, characterized in that: The following steps are involved: (1) adding the roxburghii extract and polyol to the oil phase and homogenizing to obtain a P / O emulsion; (2) adding the obtained P / O emulsion to the aqueous phase, stirring and cooling to room temperature after uniform mixing, thereby obtaining a P / O / W multi-structure emulsion; The polyol in step (1) includes glycerol and propylene glycol; the oil phase includes silicone oil, a first emulsifier and lipids; and the water phase in step (2) includes glycerol, sorbitol, xanthan gum, poloxamer, water-soluble azone, deionized water and a second emulsifier.

7. The method for preparing a multiple emulsion according to claim 6, characterized in that: The first emulsifier in step (1) comprises EM90 and SP-80, wherein the mass ratio of EM90 to SP-80 is 5:5-8:2; the lipid in step (1) comprises white oil and capric triglyceride, wherein the mass ratio of white oil to capric triglyceride is 5:5-8:2; the amount of the roxburghii extract added to the system in step (1) is 0.5-2wt%; the second emulsifier in step (2) comprises 16,18 alkyl glucoside and TW-80, wherein the mass ratio of 16,18 alkyl glucoside to TW-80 is 5:5-4:

6.

8. The method for preparing a multiple emulsion according to claim 7, characterized in that: In step (1), the amount of the first emulsifier added to the system is 0.5-2 wt %; in step (2), the amount of the second emulsifier added to the system is 1-2 wt %.

9. The method for preparing a multiple emulsion according to claim 8, characterized in that: The mass ratio of the oil phase to the polyol in step (1) is 6.5:3.5-5:5; the mass ratio of the water phase to the P / O emulsion in step (2) is 8:2-9.5:0.

5.

10. Use of the roxburghii extract formulation as claimed in claim 1 in the preparation of whitening skin care products.

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