阅读说明：本技术 一种以滇橄榄鲜果果肉为主原料的美白淡斑复方制剂及其制备方法 (Whitening and spot-fading compound preparation taking fresh fructus phyllanthi pulp as main raw material and preparation method thereof ) 是由 李玉叶 王睿睿 于 2021-08-11 设计创作，主要内容包括：提供了一种以滇橄榄鲜果果肉为主原料的美白淡斑的复方制剂及其制备方法,其主要活性成分由滇橄榄鲜果果肉浆和青刺果油组成。本发明提供的复方制剂以新鲜滇橄榄鲜果果肉浆为主要原料,可以有效地抑制酪氨酸酶的活性,从而抑制黑色素的形成,使其可以改善肌肤暗沉、提亮肤色,能够改善肤质、滋养肌肤。本发明可应用于色素性疾病,如：黄褐斑、黑变病、炎症后色素沉着、脂溢性角化症,黑棘皮病、雀斑、晒斑等。还可应用于日常美白护肤、日晒后皮肤发黑等。本发明提供的复方制剂成分天然健康、质地温和安全,且制备方法简单易操作,适合应用于大规模生产。(A compound preparation for whitening and removing speckle is prepared from pulp of fresh fructus Phyllanthi and Prinsepia utilis Royle oil as main active ingredients. The compound preparation provided by the invention takes the fresh fructus phyllanthi pulp as the main raw material, and can effectively inhibit the activity of tyrosinase, thereby inhibiting the formation of melanin, improving the skin darkness and brightening the skin color, improving the skin quality and nourishing the skin. The invention can be applied to pigmentary diseases, such as: chloasma, melanosis, pigmentation after inflammation, seborrheic keratosis, acanthosis nigricans, freckle, sunburn and the like. It can also be used for daily whitening and skin care, and blackening skin after sun exposure. The compound preparation provided by the invention has natural and healthy components, mild and safe texture, and the preparation method is simple and easy to operate, and is suitable for large-scale production.)

1. A compound preparation taking Yunnan olive fresh fruit pulp as a main raw material comprises the following components in parts by weight: 5-8 parts of Yunnan olive fresh fruit pulp, 0.5-1 part of prinsepia utilis royle oil and auxiliary materials.

2. The compound preparation of claim 1, which comprises the following components in percentage by weight: 50-80% of fructus phyllanthi pulp, 5-10% of prinsepia utilis royle oil, 0.1-0.3% of sodium hyaluronate, 1-4% of glycerol, 1-2% of hexadecanol, 1-2% of octadecanol, 0.5-1% of lanolin, 0.5-2% of stearic acid, 2-4% of butanediol, 3-5% of 1, 2-pentanediol, 1-2% of glyceryl monostearate, 1-2% of poloxamer 1881-2%, 1-2% of polydimethylsiloxane, 0.1-0.5% of xanthan gum, 0.3-1% of phenoxyethanol and 0.05-0.1% of triethanolamine, wherein the total amount of the components is 100%.

3. The compound preparation of claim 1 or 2, wherein the preparation method of the pulp of fresh fructus phyllanthi comprises the following steps: crushing fresh fructus Phyllanthi to obtain pulp, adding water into the pulp, pulping in a wall-breaking pulping machine, and sieving.

4. The compound preparation of claim 3, wherein the preparation method of the pulp of fresh fructus phyllanthi comprises the following steps: cleaning fresh fructus Phyllanthi, removing core, and pulverizing into small pieces; taking 250g of processed Yunnan olive fresh fruit pulp, and mixing the pulp with the pulp according to the proportion of 1: 1-1.5, adding water at 20-30 ℃, and pulping in a wall breaking beater for 3-8 min; filtering with 600 mesh sieve to obtain pulp of fructus Phyllanthi.

5. The preparation method of the compound preparation of claim 1 or 2, which is characterized in that the skin-care emulsion is prepared by the following steps:

(1) adding Prinsepia utilis Royle oil, cetyl alcohol, stearyl alcohol, stearic acid, lanolin, glyceryl monostearate, poloxamer 188 and polydimethylsiloxane into an oil phase pot, heating to 70-75 deg.C, stirring uniformly, and maintaining for 0.5-1 h;

(2) adding glycerol, butanediol, 1, 2-pentanediol, xanthan gum, sodium hyaluronate, and pulp of fructus Phyllanthi into water phase pot, heating to 70-75 deg.C, stirring, and melting and mixing completely;

(3) slowly adding the oil phase into the water phase under stirring at the rotation speed of 400-;

(4) cooling the emulsion to 40 deg.C, adding triethanolamine and phenoxyethanol, and stirring for 3-5 min;

(5) homogenizing with a high-pressure homogenizer at a homogenizing pressure of 200bar-500bar and a homogenizing frequency of 20kHz for 1-2 times;

(6) aging, filling and packaging.

6. The combination preparation of claim 1 or 2, which has tyrosinase inhibitory activity.

7. The compound preparation of claim 1 or 2, which has a long-acting moisturizing effect on skin.

8. The compound preparation according to claim 1 or 2, which has whitening, spot-lightening, antioxidant and antiaging effects, and can be used for treating hyperpigmentation diseases including chloasma, freckle, seborrheic keratosis, post-inflammatory pigmentation, melanosis, acanthosis nigricans, and sunburn.

9. The compound preparation of claim 1 or 2, which has the whitening and synergistic effects with sunscreen emulsion, and can be used for daily whitening and skin care and skin blackening after sunburn.

10. The use of the fresh fruit pulp of Yunnan olive as claimed in claim 3 or 4 in preparing whitening synergistic compound preparation in combination with Prinsepia utilis oil.

Technical Field

The invention relates to the technical field of whitening, spot-fading and skin care, in particular to a compound preparation taking fresh fructus phyllanthi pulp as a main raw material and a preparation method thereof.

Background

With the rapid development of the cosmetic market and the improvement of the living standard of the substances of people, people have more and more strong pursuit of the beauty, and the cosmetics become daily consumer goods of the national people. In the cosmetic market, the proportion of skin care products is the largest, especially whitening products. However, some whitening cosmetics have high safety hazards such as high contents of hormones, lead and mercury, and even can cause more serious damage to the skin. Skin-care products are forbidden because of the problems of cytotoxicity, carcinogenicity, local irritation reaction, and easy decomposition by heat in the presence of light, such as hydroquinone, kojic acid, etc. The compound preparation protected by the patent has the advantages of nutrition, health, mildness, safety, no stimulation to skin, wide consumer market and necessarily great development potential.

Skin color is mainly determined by the content of melanin in the skin, while tyrosinase is the main enzyme for the synthesis of melanin, playing a key role in the various reaction steps of the synthesis process. Research shows that the inhibition of the activity of tyrosinase can reduce the generation of melanin, thereby achieving the effect of whitening. Therefore, the plant raw pulp is added into the skin moisturizing cream, so that the skin moisturizing cream can inhibit the activity of tyrosinase while keeping the skin moisture for a long time, and has the effects of whitening, fading spots, resisting oxidation, delaying aging and the like.

Fructus phyllanthi, also known as phyllanthus emblica, is a mature fruit of phyllanthus emblica of euphorbiaceae, is a medicinal and edible catalogue variety in China, and is a main production area in Yunnan province. The fresh fruit is rich in functional active ingredients such as phenols, alkaloids, terpenoids, sterols, glycosides, vitamins, amino acids, unsaturated fatty acids and the like, wherein the content and the activity of vitamin C and superoxide dismutase SOD are high. Because the components such as vitamin C, superoxide dismutase SOD, amino acid and the like are easy to decompose and oxidize in the sun-drying and storage process, the effective components of the fresh fructus phyllanthi are better preserved compared with the dry product. The skin care products containing the Yunnan olive in the market all adopt a method of adding the Yunnan olive extract, so that the rich nutrient substances and active ingredients of the Yunnan olive are further destroyed, the ingredients are single, the extraction process is complex, and the internal quality and functions of the Yunnan olive products are reduced to a certain extent. The whitening and spot-fading compound preparation taking the fresh fruit pulp of the phyllanthus emblica as the main raw material not only reflects the nutritive value of a single extract, but also fully reflects the overall value of the extract, and maximally reserves the components of natural vitamin C, superoxide dismutase (SOD), amino acid and the like which have obvious effects on the skin in the phyllanthus emblica.

The Prinsepia utilis Royle is named as the Prinsepia utilis Royle and is the oil-removed fruit. Is fruit of Prinsepia utilis Royle of Rosaceae. Mainly produced in Yunnan, Guizhou, Sichuan and Tibet. Picking mature fruits in summer, removing clean pulp, and air drying. Bitter and sour in taste, warm in nature. Has the functions of invigorating spleen and nourishing stomach, retaining youthful looks and moistening skin, dispelling wind and removing nebula.

Disclosure of Invention

The invention aims to provide a compound preparation taking fresh fructus phyllanthi pulp as a main raw material and a preparation method thereof, so that the compound preparation has multiple effects of whitening, spot-fading, oxidation resistance, aging delay and the like, is natural, safe and nonirritating.

In order to achieve the purpose, the invention adopts the following technical scheme:

a compound preparation taking Yunnan olive fresh fruit pulp as a main raw material comprises the following components in parts by weight: 5-8 parts of Yunnan olive fresh fruit pulp, 0.5-1 part of prinsepia utilis royle oil and auxiliary materials.

A compound preparation taking Yunnan olive fresh fruit pulp as a main raw material comprises the following components in percentage by weight: 50-80% of fructus phyllanthi pulp, 5-10% of prinsepia utilis royle oil, 0.1-0.3% of sodium hyaluronate, 1-4% of glycerol, 1-2% of hexadecanol, 1-2% of octadecanol, 0.5-1% of lanolin, 0.5-2% of stearic acid, 2-4% of butanediol, 3-5% of 1, 2-pentanediol, 1-2% of glyceryl monostearate, 1-2% of poloxamer 1881-2%, 1-2% of polydimethylsiloxane, 0.1-0.5% of xanthan gum, 0.3-1% of phenoxyethanol and 0.05-0.1% of triethanolamine, wherein the total amount of the components is 100%.

The preparation method of the pulp of the fresh fructus phyllanthi comprises the following steps:

crushing fresh fructus Phyllanthi to obtain pulp, adding water into the pulp, pulping in a wall-breaking pulping machine, and sieving.

Preferably, the preparation method of the pulp of the fresh fructus phyllanthi comprises the following steps:

cleaning fresh fructus Phyllanthi, removing core, and pulverizing into small pieces; taking 250g of processed Yunnan olive fresh fruit pulp, and mixing the pulp with the pulp according to the proportion of 1: 1-1.5, adding water at 20-30 ℃, and pulping in a wall breaking beater for 3-8 min; filtering with 600 mesh sieve to obtain pulp of fructus Phyllanthi.

The invention relates to a compound preparation taking Yunnan olive fresh fruit pulp as a main raw material and a preparation method thereof, which comprises the following steps:

(1) adding Prinsepia utilis Royle oil, cetyl alcohol, stearyl alcohol, stearic acid, lanolin, glyceryl monostearate, poloxamer 188 and polydimethylsiloxane into an oil phase pot, heating to 70-75 deg.C, stirring uniformly, and maintaining for 0.5-1 h;

(2) adding glycerol, butanediol, 1, 2-pentanediol, xanthan gum, sodium hyaluronate, and pulp of fructus Phyllanthi into water phase pot, heating to 70-75 deg.C, stirring, and melting and mixing completely;

(3) slowly adding the oil phase into the water phase under stirring at the rotation speed of 400-;

(4) cooling the emulsion to 40 deg.C, adding triethanolamine and phenoxyethanol, and stirring for 3-5 min;

(5) homogenizing with a high-pressure homogenizer at a homogenizing pressure of 200bar-500bar and a homogenizing frequency of 20kHz for 1-2 times;

(6) aging, filling and packaging.

A compound preparation containing fructus Phyllanthi pulp as main material has tyrosinase inhibiting activity.

A compound preparation containing fructus Phyllanthi pulp as main material has long-acting moisturizing effect on skin.

A compound preparation containing fructus Phyllanthi pulp as main material has effects of whitening skin, removing speckle, resisting oxidation, and delaying aging, and can be used for treating pigment-increasing diseases such as chloasma, melanosis, pigmentation after inflammation, seborrheic keratosis, acanthosis nigricans, freckle, sunburn, etc.

A compound preparation containing fructus Phyllanthi pulp as main material has whitening and synergistic effects with sunscreen emulsion, and can be used for daily whitening and skin care, and blackening skin after sun exposure.

A pulp of fructus Phyllanthi fresh fruit has whitening and synergistic effects with Prinsepia utilis Royle oil.

Fructus phyllanthi, also known as fructus phyllanthi, is the mature fruit of fructus phyllanthi of Euphorbiaceae, and is rich in functional active ingredients such as superoxide dismutase SOD, gallic acid, corilagin, myrobalan tannin, ellagic acid, vitamin C, vitamin B, vitamin E and various vitamins, trace elements, amino acids, unsaturated fatty acid and the like, wherein the content and activity of the vitamin C and the superoxide dismutase SOD are high. The fructus phyllanthi can effectively remove free radicals in vivo, delay skin aging, enhance the moisture retention capacity of the skin, improve skin laxity and wrinkles, and prevent color spots from forming.

The Prinsepia utilis Royle oil is special high-grade pure natural nutritional vegetable oil in Yunnan, contains rich unsaturated fatty acid, multiple vitamins such as vitamin A, vitamin D, vitamin E and vitamin K, and also contains rich major and trace elements such as calcium, sulfur, manganese, magnesium, potassium, phosphorus, zinc and iron. Prinsepia utilis Royle oil has strong skin penetration ability, can regenerate and repair skin, and also has strong moisture keeping effect.

Compared with the prior art, the invention has the following advantages:

the invention directly adds the pulp of the fresh fructus phyllanthi, maintains the nutrient substances and effective components of the fructus phyllanthi to the maximum extent, and has better effect compared with the addition of the extract of the dried fructus phyllanthi.

The main effective components of the skin-care cream are pure natural plants, are mild, safe and nonirritant, and not only can improve the skin and nourish the skin, but also can whiten and lighten the spots, resist oxidation and delay aging.

The invention utilizes the local characteristic traditional Chinese medicine resources in Yunnan province, converts the potential advantages of natural resources into industrial economic advantages, and improves the economic benefits and the comprehensive utilization rate of the Yunnan traditional Chinese medicine resources. The preparation process is simple and is suitable for large-scale and industrial production.

Drawings

FIG. 1: chromatogram of vitamin C content in reference substance

FIG. 2: chromatogram of vitamin C content in sample

FIG. 3: UPLC spectrum of mixed reference solution (A) fructus Phyllanthi emulsion test solution (B) 1, gallic acid 2, corilagin 3, fructus Chebulae tannin 4, ellagic acid

FIG. 4: clearance rate of 4 phenolic acid components to DPPH free radical

From left to right in sequence: gallic acid 0.54, gallic acid 0.27, gallic acid 0.135, corilagin 0.72, corilagin 0.36, corilagin 0.18, chebulan tannin 0.8, chebulan tannin 0.4, chebulan tannin 0.2, mashup-hua acid 0.72, mashup-hua acid 0.36, mashup-hua acid 0.18

FIG. 5: inhibition of reactive oxygen species ROS by samples

FIG. 6: the emulsion has skin sensory effect on human body

FIG. 7: effects of lotion on skin elasticity of human body

FIG. 8: the effect of lotion on human skin oil

FIG. 9: the effect of the lotion on human skin moisture

FIG. 10: VISIA detection product use effect graph

Detailed Description

The following claims are presented in further detail in connection with the detailed description of the invention, but not to be construed as limiting the invention in any way, and any limited number of modifications that can be made by anyone within the scope of the claims are still within the scope of the invention.

Example 1:

a skin care lotion with Yunnan olive fresh fruit pulp as main raw material and a preparation method thereof are disclosed, which comprises the following steps:

(1) preparing the following raw materials in percentage by mass: 10% of prinsepia utilis royle oil, 70.7% of fresh phyllanthus emblica pulp, 0.2% of sodium hyaluronate, 4% of glycerol, 1% of hexadecanol, 1% of octadecanol, 0.5% of lanolin, 0.5% of stearic acid, 3% of butanediol, 4% of 1, 2-pentanediol, 1.5% of glyceryl monostearate, 1% of polydimethylsiloxane, 1881.5% of poloxamer, 0.3% of xanthan gum, 0.5% of triethanolamine and 0.3% of phenoxyethanol;

(2) adding Prinsepia utilis Royle oil, cetyl alcohol, stearyl alcohol, stearic acid, lanolin, polydimethylsiloxane, glyceryl monostearate, and poloxamer 188 into oil phase pot, heating to 70 deg.C, stirring well, and melting and mixing completely;

(3) adding glycerol, butanediol, xanthan gum, sodium hyaluronate, and pulp of fructus Phyllanthi into water phase pot, heating to 70 deg.C, stirring, and melting and mixing completely;

(4) slowly adding the oil phase into the water phase under the stirring state, wherein the rotating speed is 400r/min, the emulsifying time is 10min, and the emulsifying temperature is 65-70 ℃;

(5) cooling the emulsion to 40 deg.C, adding triethanolamine and phenoxyethanol, and stirring for 5 min;

(6) homogenizing twice by a high-pressure homogenizer, wherein the first homogenizing pressure is 200bar, the homogenizing frequency is 20kHz, the second homogenizing pressure is 400bar, and the frequency is 20 kHz;

(7) aging, filling and packaging.

The preparation method of the pulp of the fresh fructus phyllanthi comprises the following steps:

cleaning fresh fructus Phyllanthi, removing core, and pulverizing into small pieces; taking 200g of processed Yunnan olive fresh fruit pulp, and mixing the pulp according to the weight ratio of 1: 1, adding water of 20 ℃, and pulping for 5min in a wall breaking beater; filtering with 600 mesh sieve to obtain pulp of fructus Phyllanthi.

Example 2:

a skin care lotion with Yunnan olive fresh fruit pulp as main raw material and a preparation method thereof are disclosed, which comprises the following steps:

(1) preparing the following raw materials in percentage by mass: 8% of prinsepia utilis royle oil, 70.7% of fresh phyllanthus emblica pulp, 0.3% of sodium hyaluronate, 4% of glycerol, 2% of hexadecanol, 2% of octadecanol, 0.5% of lanolin, 0.5% of stearic acid, 3% of butanediol, 4% of 1, 2-pentanediol, 1.5% of glyceryl monostearate, 1% of polydimethylsiloxane, 1881.5% of poloxamer, 0.2% of xanthan gum, 0.5% of triethanolamine and 0.3% of phenoxyethanol;

(2) adding Prinsepia utilis Royle oil, cetyl alcohol, stearyl alcohol, stearic acid, lanolin, polydimethylsiloxane, glyceryl monostearate, and poloxamer 188 into oil phase pot, heating to 70 deg.C, stirring well, and melting and mixing completely;

(3) adding glycerol, butanediol, xanthan gum, sodium hyaluronate, and pulp of fructus Phyllanthi into water phase pot, heating to 70 deg.C, stirring, and melting and mixing completely;

(4) slowly adding the oil phase into the water phase under stirring at a rotation speed of 400r/min for 10min at an emulsification temperature of 70 ℃;

(5) cooling the emulsion to 40 deg.C, adding triethanolamine and phenoxyethanol, and stirring for 5 min;

(6) homogenizing twice by a high-pressure homogenizer, wherein the first homogenizing pressure is 200bar, the homogenizing frequency is 20kHz, the second homogenizing pressure is 400bar, and the frequency is 20 kHz;

(7) aging, filling and packaging.

The preparation method of the pulp of the fresh fructus phyllanthi comprises the following steps:

cleaning fresh fructus Phyllanthi, removing core, and pulverizing into small pieces; taking 200g of processed Yunnan olive fresh fruit pulp, and mixing the pulp according to the weight ratio of 1: 1.5, adding 50 deg.C water, and pulping in a wall-breaking pulping machine for 5 min; filtering with No. 9 sieve to obtain fresh fructus Phyllanthi pulp.

Example 3:

determining the content of the vitamin C in the emulsion by an HPLC method:

preparation of control solutions:

0.0041g of a vitamin C reference substance (Beijing Zhongke quality testing biology, Ltd.) (50-81-7) (the purity is more than or equal to 98%) is precisely weighed, and a mixed solution of acetonitrile-water (2: 1) is added to a volumetric flask with the capacity of 100 mL.

The control solution concentrations were:preparation of sample solution:

precisely weighing 2g of emulsion, placing the emulsion in a 250mL conical flask, precisely adding 50mL of acetonitrile-water (2: 1) mixed solution, weighing, carrying out ultrasonic treatment for 30min, weighing again, complementing weight loss with acetonitrile-water (2: 1) mixed solution, shaking up, filtering, and taking a subsequent filtrate.

The determination method comprises the following steps:

chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile-0.01 mol/L potassium dihydrogen phosphate water solution (80: 20) is used as a mobile phase; the detection wavelength is 246 nm; the flow rate was 1.0 mL/min. The theoretical plate number is not less than 3000 calculated according to vitamin C peak.

And (3) measuring results:

TABLE 1 Retention time, Peak area, sample weight, concentration of control and sample

	Retention time	Peak area	Sample weighing	Concentration of
					Reference substance	2.118	862340	0.0041g	40.18(ug/mL)
20200326	2.116	3024470	2.0758g	132.55(ug/mL)
					20200406	2.117	2027540	2.0008g	88.85(ug/mL)
20200512	2.116	2523480	2.0326g	110.59(ug/mL)

Vitamin C content of the emulsion sample:

20200326 batch number content 3.32mg/g20200406 batch number content 2.22mg/g202004512 batch number content 2.77 mg/g.

Example 4:

determining the content of amino acid in the emulsion by using an amino acid analyzer:

preparation of control solutions:

a40 uL solution of the amino acid control (Wako pure chemical industries, Ltd.) (013-.

Preparation of sample solution:

precisely weighing 2.0g of emulsion sample, placing the emulsion sample in a 250mL conical flask with a plug, precisely adding 50mL of 0.02mol/L hydrochloric acid solution, weighing, carrying out ultrasonic treatment for 30min, cooling, weighing again, complementing weight loss with 0.02mol/L hydrochloric acid solution, shaking up, filtering, and taking the subsequent filtrate.

The determination method comprises the following steps:

the species and content of amino acid in the sample are determined by adopting an amino acid automatic analyzer LA 8080 and an ion exchange chromatography-ninhydrin column derivation method.

The results for the three samples (20200326, 2020406, 20200512) averaged as follows:

TABLE 2 amino acid component content of Phyllanthus emblica emulsion

Example 5:

1. extraction and activity determination of Yunnan olive fresh fruit emulsion SOD

1.1 extraction of SOD from Yunnan olive fresh fruit emulsion

Weighing 1g of fresh phyllanthus emblica fruit milk, placing the fresh phyllanthus emblica fruit milk into a test tube, adding 7mL of distilled water, shaking up, fully dissolving, transferring into a 10mL centrifuge tube, washing a mortar by using a small amount of water, adding the mortar into the centrifuge tube, adding water to scale, centrifuging for 15min at 4000r/min, and taking 1mL of supernatant to fix the volume to 100 mL.

1.2 determination of autoxidation rate of Dian olive emulsion SOD activity is carried out by adding the reagents in the following table into 10ml colorimetric tube, keeping temperature at 25 deg.C for 20min, adding preheated pyrogallol (prepared by 3mmol/l hydrochloric acid solution, replacing control tube with HCL), shaking up quickly and pouring into 1cm colorimetric tube, determining initial value at 325nm and absorbance value after 1min, respectively, the difference between the two values is pyrogallol autoxidation rate ODA, controlling autoxidation rate at about 0.07OD/min, controlling enzyme liquid amount to make the inhibiting pyrogallol autoxidation rate 1/2ODA, ODB.

SOD activity unit is defined by the unit enzyme activity calculation of phyllanthus emblica emulsion, wherein in 1mL of reaction solution, the enzyme amount for inhibiting the pyrogallol autoxidation rate to 50% per minute is defined as an activity unit.

TABLE 3 dosage of reagents for determining autoxidation rates

SOD activity calculation formula

In the formula: OD_AThe rate of auto-oxidation of the pyrogallol; OD_BA rate of change of optical density value of the sample; d, sample dilution times; v-determine sample volume, mL.

TABLE 4 SOD Activity in three batches of Dian Olive emulsions (u/g)

Example 6:

content determination of 4 phenolic acid components in phyllanthus emblica emulsion

1. Chromatographic conditions

An Agilent ZORBAX SB-Aq (C18100 mm X2.1 mm, 1.8 μm) chromatographic column was used; mobile phase: 0.3% formic acid solution (a) -acetonitrile: methanol (1: 5) (B), gradient elution (0-4 min, 5-10% A, 4-8 min, 10-20% A, 8-9 min, 20-25% A, 9-21 min, 25-50% A, 21-24 min, 50-50% A, 24-25 min, 50-5% A, 25-26 min, 5% A.), flow rate: 0.2 mL/min; detection wavelength: 254 nm; column temperature: 30 ℃; sample introduction volume: 1 μ L. Under the chromatographic conditions, the components to be detected, namely gallic acid, corilagin, myrobalan tannin and ellagic acid, are well separated without interference.

2. Preparation of test solution

Precisely weighing 1g of fructus Phyllanthi emulsion, placing in a 100mL triangular flask, adding 50mL of 50% methanol, weighing, performing ultrasonic treatment for 60min, cooling, supplementing the original weight, filtering, collecting the filtrate, and filtering with 0.22 μm microporous membrane to obtain test solution.

3. Preparation of control solutions

Precisely weighing gallic acid, corilagin and myrobalan tannin reference substances 6.40mg, 6.41mg and 8.81mg, respectively placing in 10mL volumetric flasks, respectively adding 50% methanol for dissolving and diluting to scale, filtering with 0.22 μm microporous membrane to obtain gallic acid, corilagin and myrobalan tannin reference substance solution; precisely weighing 11.81mg of ellagic acid reference substance, placing in a 5mL volumetric flask, adding dimethyl sulfoxide to dissolve and dilute to scale, and filtering with 0.22 μm microporous membrane to obtain ellagic acid reference substance solution. Precisely sucking 1mL of each control solution, and mixing to obtain mixed control solution of gallic acid, corilagin, chebular tannin and ellagic acid.

4. Measurement results

As shown in figure 3, under the chromatographic conditions, the components to be detected, namely gallic acid, corilagin, myrobalan tannin and ellagic acid, are well separated without interference.

TABLE 5 content (mg/g) of 4 phenolic acid components in different batches of Dian olive emulsions

(II) evaluation of antioxidant ability of 4 phenolic acid components in Phyllanthus emblica emulsion

Determination of DPPH free radical scavenging activity of 4 phenolic acid components

Adding 2mL of 0.0399mg/mL of DPPH solution and 2mL of methanol into a test tube, uniformly mixing, reacting for 30min, measuring the absorbance at 517nm, and calculating as D0, taking 2mL of control solution with series concentrations and 2mL of DPPH solution, uniformly mixing, reacting for 30min, measuring the absorbance at 517nm, and calculating as Di; taking 2mL of control solution with series concentration and 2mL of methanol, mixing uniformly, reacting for 30min, measuring absorbance at 517nm, calculating the clearance according to a formula as Dj: the radical scavenging ability was evaluated by the scavenging rate (%) -1- (Di-Dj)/D0.

Drawing of DPPH Standard Curve

And (3) drawing a standard curve according to the absorbance and the mass concentration to obtain a linear regression equation, wherein Y is 28.28X +0.033(r is 0.9995), and the DPPH solution has a good linear relation in the mass concentration range of 0.0066-0.0528 mg/mL.

1.4 measurement of DPPH radical scavenging Activity by phenolic acid Components

As shown in the figure, 4 phenolic acid components in the phyllanthus emblica have stronger antioxidant capacity, and the DPPH free radical scavenging capacity is enhanced along with the increase of the concentration. At high concentrations, the DPPH radical scavenging capacity of all 4 phenolic compounds was greater than 70%. In contrast, myrobalan tannin is relatively weak at low concentrations to scavenge DPPH radicals.

(III) inhibition experiment on tyrosinase

The experimental method comprises the following steps:

mixing the drug to be detected with levodopa (L-Dopa), adding tyrosinase (final concentration 25U/mL) to start reaction, setting 3 repeated holes, setting blank control without drug and Kojic Acid (Kojic Acid) positive control, measuring OD value with enzyme labeling instrument at room temperature for 5min, and detecting wavelength at 490 nm. And calculating to obtain the tyrosinase activity inhibition rate.

Tyrosinase activity inhibition (%) [ 1-sample OD_{490 nm}Experimental control well OD_{490 nm}Results of experiment # 100: as shown in Table 6, the fresh fructus phyllanthi fruit juice has significant tyrosinase inhibiting activity, the effect is better than that of positive control kojic acid, and the activity of the fruit juice prepared from the dried fructus phyllanthi fruit is obviously reduced.

TABLE 6 inhibition of tyrosinase by the samples

(IV) inhibition of Reactive Oxygen Species (ROS)

The experimental method comprises the following steps:

reference T/SHRH 032-2020 cosmetic tightening and anti-wrinkle

Efficacy test-in vitro keratinocyte Reactive Oxygen Species (ROS) inhibition test method, a fluorescent probe DCFH-DA with a concentration of 2mg/mL is added to a cell suspension to a final concentration of 40ug/mL, and vortex oscillation is uniform. Incubate in a 30 ℃ incubator for 30min in the dark. The mixture was inverted every 3-5min to bring the probe into full contact with the cells. Cells were harvested by centrifugation and washed three times with PBS and resuspended in PBS buffer. Each well was 100. mu.L, and three duplicate wells were set for each sample. Placing in a multifunctional microplate reader, exciting at 488nm, emitting at 525nm, scanning for 0.5s, and measuring fluorescence intensity of each well.

The experimental results are as follows: the Yunnan olive fresh fruit solution has obvious active oxygen inhibition effect, which shows that the fresh fruit solution has the active oxygen elimination effect, and the main components of the Yunnan olive fresh fruit, namely the corilagin and the gallic acid, also have certain inhibition effect.

(V) inhibitory Effect on melanogenesis in melanoma cells (B16)

The experimental method comprises the following steps:

referring to a test method for whitening and freckle removing effects of T/ZHCA 001-2018 cosmetics, a sample to be tested is used for treating a-melanocyte stimulating hormone (a-MSH) -induced B16 melanoma cells for 72h, the MTT method is used for detecting cell proliferation activity, and the enzyme-linked immunosorbent assay is used for determining melanin content.

The inhibition of melanoma cell proliferation by the samples is shown in table 7: the fresh fructus phyllanthi fruit solution, the dried fruit solution and the like have no obvious inhibition effect on B16 melanoma cells, which indicates that the fresh fructus phyllanthi fruit, the dried fruit and the main components have no influence on the proliferation of the melanoma cells.

TABLE 7 inhibition of melanoma cell proliferation by samples

Sample (concentration)	Melanoma cell survival (%)
		Kojic acid (10 mug/mL)	90.54±3.54
Yunnan olive fresh (10%)	98.17±6.21
		Dried Yunnan olive (10%)	92.37±8.39
Corilagin (0.72 mu g/mL)	95.78±1.28
		Gallic acid (0.54. mu.g/mL)	93.68±5.77
Myrobalan tannin (0.8 μ g/mL)	89.86±9.87

(VI) inhibition of melanin content

As shown in Table 8, the fresh fructus Phyllanthi solution also has obvious melanin generation effect, and the inhibition rate of the fresh fructus Phyllanthi solution on melanin can reach 55.60%, which is superior to the inhibition rate of the positive control tranexamic acid on melanin, which is 49.39%; the inhibition rate of the dry fruit solution with the same concentration is only 11.92 percent; the main components of the fresh fruit, corilagin and gallic acid, also have weak inhibitory effects (24.52% and 12.65%, respectively). In conclusion, the fresh fructus phyllanthi has better activity of inhibiting melanin generation.

TABLE 8 inhibition of melanoma cells by samples

(VII) human skin detection experiment

And (3) inclusion standard:

12 healthy women aged 20-40 years with symmetrical skin and little hair on the inner side of the forearm. Excluding standard forearm with any skin disease or receiving any skin cosmetic treatment for nearly 3 months; engaging outdoor workers for a long time; active allergic diseases or other systemic diseases; those who use glucocorticoids or immunosuppressive agents within approximately 1 month; participate in other clinical trials in the last 3 months; pregnant or lactating.

The experimental conditions are as follows: room temperature is 20-24 ℃, humidity is 40-60%, natural light is provided, and sunlight direct irradiation is avoided. The subject left their forearm in the temperature controlled room for 30min to stabilize the skin blood flow. The skin surface to be tested of the test subject is not contacted with water within 12 hours before the test is started, and any cosmetics, medicines and other excipients cannot be applied. The experimental method comprises the following steps: the inner side of the forearm selects 8 test areas, the left and right are respectively 4, the test areas are numbered in sequence, the area of each area is 3cm multiplied by 3cm, and the interval between the areas is 1 cm. The 8 regions were randomly subjected to 4 different treatments (two regions for each treatment). The 4 treatments were respectively, and emulsion 1 (fresh fruit formula), emulsion 2 (fresh fruit formula), and emulsion 3 (dried fruit formula). The smearing amount of the test sample is (2.0 +/-0.1) mg/cm². The cosmetics are applied by using rubber finger sleeves, and are required to be uniformly applied to the greatest extent until the cosmetics are absorbed. The moisture, oil and elasticity values of each area and the moisture, oil and elasticity values of 0.5h, 1h, 2h, 6h and 24h before and after the cosmetic is applied are sequentially measured by using a skin moisture tester (Real Bubee). Each area was measured 3 times in duplicate and averaged.

Sensory evaluation: the evaluation index includes the fragrance, appearance, spreadability, skin absorption speed, skin refreshing feeling, smoothness, moisturizing feeling, irritation, and the like of the product. Each index was evaluated in terms of its degree from 1 point (dislike) to 5 points (very like), and the moisture retention degree of the product was compared by the degree of feeling (difference in score) after using different products, and the evaluation criteria were: the user likes and dislikes 1,2, 3, 4, and 5.

As a result: as shown in figure 6, the emulsion 1 and the emulsion 2 of the fresh fructus phyllanthi formula are superior to the emulsion 3 of the dried fructus phyllanthi formula in terms of moisture retention, smoothness and refreshing feeling, and as shown in figure 7, the emulsion 1 and the emulsion 2 of the fresh fructus phyllanthi formula are similar to the positive control Arselia emulsion. As shown in fig. 8, the effect on skin oil was similar for fresh fruit formula emulsion 1 and emulsion 2 to the positive control ashiramate emulsion, with lower oil, while dry fruit formula emulsion 3 increased skin oil secretion. As shown in fig. 9, the effect on skin moisture was similar for fresh fruit formula emulsion 1 and emulsion 2 to the positive control ashiramate emulsion, with a higher moisture content, while for dry fruit formula emulsion 3, the skin moisture content was the lowest, consistent with the moisturizing effect of fig. 8. Therefore, the moisturizing effect of the fresh fruit formula is obviously better than that of the dry fruit formula.

(VIII) Effect of Using the product

The spot-lightening and whitening effects of the cosmetics can be evaluated by applying VISIA skin detector to perform image analysis. The VISIA skin detector takes a picture through a camera with 1200 ten thousand pixels, takes polarized light and ultraviolet light as light sources respectively, takes an all-round picture of the human face, and uses optical imaging and software to analyze the image, and the obtained image comprises special pictures such as spots, ultraviolet color spots, brown color spots, red areas and the like. The whitening efficacy of emulsion 1 of example 1 was characterized mainly by visual comparison of facial images before and after use of the cosmetic, and statistical analysis of the score changes resulting from image software processing (table 9). A comparative photograph of the back of the treatment is shown in fig. 10.

And (3) inclusion standard: 30 women of 25-50 years old suffering from freckle, chloasma, seborrheic keratosis and post-inflammatory pigmentation. The treatment period is 3 months, and the lotion is used in the morning and evening every day.

As a result: emulsion 1 is effective in treating freckle, chloasma, seborrheic keratosis and pigmentation, and has statistical significance (P is less than 0.05) for the change of speckle, ultraviolet spot, brown spot and red region before and after treatment. Has similar effect on pigmented dermatosis such as melanosis and sunburn. And (4) adverse reaction observation: all patients had no adverse reactions.

TABLE 9 therapeutic effect of emulsion 1 on freckles, chloasma, seborrheic keratosis and post-inflammatory hyperpigmentation

Example 7:

a detection method for the synergistic effect of the pulp of the fresh fruit of fructus phyllanthi and the prinsepia utilis royle oil is disclosed, the experimental methods are shown in the experiment for inhibiting the proliferation of the melanoma B16 cells and inhibiting the generation of melanin, as can be seen from the table 10, the pulp of the fresh fruit of fructus phyllanthi and the prinsepia utilis royle oil have very good synergistic effect, the pulp of the fresh fruit of fructus phyllanthi and the prinsepia utilis royle oil have obvious synergistic effect of inhibiting the secretion of melanin under the content of 2% and 1% and the content of 4% of the prinsepia utilis royle oil, the joint index CI value reaches 0.04, and the strong synergistic effect is achieved. While the dry phyllanthus emblica fruit solution does not show good synergistic effect. The main component of the fresh fruit, corilagin, also has weak synergistic effect, and the combination index CI can reach 0.3. The positive control tranexamic acid showed no synergistic effect with prinsepia utilis royle oil. The fructus phyllanthi fresh fruit pulp and prinsepia utilis royle oil have the potential of being jointly applied to treatment of pigment diseases, such as: chloasma, melanosis, pigmentation after inflammation, seborrheic keratosis, acanthosis nigricans, freckle, etc.

TABLE 10 synergistic effect with Prinsepia utilis oil

Note: when CI is less than or equal to 0.5, the two medicines act in a synergistic manner; when CI is more than 0.5 and less than or equal to 4, the action modes of the two medicines are irrelevant; when CI is greater than 4, the two drugs act in an antagonistic manner.

Example 8:

a detection method for synergism of pulp of fresh fructus Phyllanthi and sunscreen emulsion comprises the following steps: 60 chloasma female patients are recruited, the age is 20-40 years old, the patients are randomly divided into a control group and an experimental group, and the patients are 30 patients; the difference between the two groups of patients is not obvious (P is more than 0.05) through statistical analysis on the general conditions of age, disease course, skin damage and the like. Exclusion criteria: before treatment, all patients are excluded from the discomfort of pregnancy, sensitive skin and the like which are suitable for treatment.

The method comprises the following steps: the patients in the experimental group use the lotion 1 and the Atlantic sunscreen cream to wipe the facial skin every day for 2 times, and the lotion 1 is firstly applied and then the Atlantic sunscreen cream is immediately applied after the lotion is dried. In the control group, the skin of face is applied with the sun cream of Asteris blue for 2 times a day, and the sun cream is used for 3 months. Before treatment and at the end of 3 months treatment VISIA face analysis (canfield, usa) was used for face detection analysis.

As a result: the combination of emulsion 1 and the sun-screening milk group has better curative effect on chloasma than the single sun-screening milk group, P is less than 0.05, and the difference has statistical significance (Table 11). And (4) adverse reaction observation: all patients had no adverse reactions. The composition has similar curative effect for healthy people, and can be used as whitening product in combination with sunscreen product.

Different from hydroquinone cream which can not be used in the daytime by light decomposition, the emulsion 1 has good stability, can be used in the daytime, can be used with sunscreen emulsion simultaneously, and has the unique effects of combined whitening and synergism.

TABLE 11 therapeutic effect of emulsion 1 in combination with sunscreen lotion on chloasma patients

The above embodiments are further detailed for the claims of the present invention, but do not constitute any limitation to the present invention, and any limited number of modifications made by anyone within the scope of the claims of the present invention are still within the scope of the claims of the present invention.