WO2022227286A1 - Preparation method for pyracantha fruit extract, and use - Google Patents

Abstract

A preparation method for a pyracantha fruit extract, which comprises the following steps: (1) enzymolysis: acquire a pyracantha fruit, add a compound enzyme and perform enzymolysis, and obtain an enzymolysis mixture; (2) alcohol extraction: add ethanol to the enzymolysis mixture and perform extraction, filtration, and concentration, and obtain an ethanol extract; (3) macroporous adsorption resin purification: load the ethanol extract on the macroporous adsorption resin and perform purification, and collect a desorption solution; and (4) drying: perform concentration and drying on the desorption solution, thereby obtaining the pyracantha fruit extract. Advantageous characteristics of the present invention are: a flavonoid within the pyracantha fruit can be effectively extracted and industrial production is easy to implement, and a pyracantha fruit extract having antioxidant, brightening, and whitening effects is obtained.

Description

Preparation method and use of Pyracantha fruit extract technical field

The application belongs to the field of daily chemical products, and particularly relates to a preparation method and application of a Pyracantha fruit extract.

Background technique

Human beings' concern for self-image and pursuit of beauty have increased the demand for cosmetics, and with the progress of the times and the improvement of human living standards, people are paying more and more attention to the effectiveness and safety of cosmetics. The focus of current cosmetics. Safe and effective natural plant cosmetic raw materials have a good competitive advantage in the cosmetic market.

Fair skin is a topic of common concern for the majority of oriental women. However, due to environmental pollution, UV damage, work pressure and other factors, skin pigmentation, chloasma, skin aging and other problems appear. Traditional whitening agents (such as hydroquinone, mercury and its compounds, α-arbutin) have good whitening effects, but because of their cytotoxicity, irritation, sensitization and other adverse reactions, they have been banned or limited. Used in cosmetics. In recent years, cosmetics containing naturally derived plant extracts have been favored by consumers, but there are still problems such as low content of active ingredients and insignificant efficacy.

The depth of human skin color mainly depends on the content and distribution of melanin. Melanin is synthesized by melanocytes in the basal layer of the skin, and then transferred to the basal cells. With the migration of epidermal cells, it is brought to the full thickness of the epidermis, and finally shedding with keratinocytes. and lost. Although melanin can reduce the damage of ultraviolet rays to the skin, excessive accumulation can easily lead to the appearance of dark yellow skin and pigmentation, which seriously affects the health and beauty of the face. Current research suggests that skin whitening can be achieved through the following mechanisms: ①inhibiting the activity of melanin synthesis-related mediators such as tyrosinase and dopa; ②inhibiting melanocyte proliferation and melanogenesis; ③inhibiting the transfer of melanocytes to the skin surface 4. Resist the damage to the skin caused by external factors such as ultraviolet rays and oxygen free radicals.

Pyracantha, including Pyracantha fortuneana, Pyracantha atalantioides, Pyracantha koidzumii, Pyracantha crenulata, Pyracantha angustifolia, Pyracantha inermis, Pyracantha densiflora , Pyracantha rogersiana, Pyracantha mekongensis, and Pyracantha heterophylla.

Pyracantha, also known as torch fruit, life-saving food, life-saving food, red seeds, etc., is the fruit of the Rosaceae Pyracantha genus Pyracantha P. fortuneana. The fruit, roots and leaves can be used as medicine. It is a multi-purpose flower and fruit plant with edible and ornamental value. It was first recorded in the herbal monograph "Southern Yunnan Materia Medica" (Ming. Lanmao), sweet, sour, flat in nature, can eliminate accumulation and stop dysentery, promote blood circulation and stop bleeding. For the treatment of dyspepsia, enteritis, dysentery, infantile malnutrition, metrorrhagia, leucorrhea, postpartum abdominal pain and other diseases. Modern research shows that Pyracantha fruit is rich in flavonoids, polyphenols, carbohydrates, proteins and various vitamins, and has multiple functions such as significant antioxidant, anti-tumor, anti-aging, liver protection and stomach protection. It has great application value in other fields, and further research is needed on Pyracantha.

SUMMARY OF THE INVENTION

The present application provides a preparation method of Pyracantha fruit extract, which can effectively extract flavonoids in Pyracantha fruit.

First aspect, the application provides a kind of preparation method of Pyracantha fruit extract, comprising the following steps:

(1) Enzymolysis: take Pyracantha fruit, add compound enzyme for enzymolysis, and obtain an enzymolysis mixture;

(2) alcohol extraction: adding ethanol to the enzymatic hydrolysis mixture for extraction, filtering, and concentrating to obtain an ethanol extract;

(3) macroporous adsorption resin purification: the ethanol extract is loaded on the macroporous adsorption resin for purification, and the analytical solution is collected; and

(4) Drying: Concentrate and dry the analytical solution to obtain the solution.

Preferably, in the step (1), the composite enzyme is composed of polygalacturonase, cellulase and pectinase, the temperature of enzymatic hydrolysis is 37°C to 42°C, and the time of enzymatic hydrolysis is 1 to 3 hours , the pH value of enzymatic hydrolysis is 4.5～6.5.

In order to illustrate the function of the complex enzyme of the present application, the following experiments are specially made:

Weigh two parts of 10 grams of Pyracantha fruit, put them in a round-bottomed flask, add 10 ml of water respectively, and set aside.

One part was added with 0.03 g of compound enzyme and hydrolyzed at 40°C for 2 hours, filtered to obtain a filtrate; the other part was not added with compound enzyme, left standing at 40°C for 2 hours, filtered to obtain a filtrate. The content of flavonoids in the two filtrates was determined respectively. The results are shown in Table 1.

Table 1 Effects of different enzymolysis methods on the extraction of flavonoids from Pyracantha

As can be seen from Table 1, the content of flavonoids was determined by ultraviolet spectrophotometer, and it was found that the flavonoids could be more extracted from Pyracantha fruit after enzymolysis with compound enzymes.

Preferably, the mass ratio of the polygalacturonase, cellulase and pectinase is 1:1:1.

Preferably, the method of ethanol extraction in the step (2) is heating and reflux extraction, the mass percentage concentration of ethanol during ethanol extraction is 50% to 80%, and the number of times of ethanol extraction is 1 to 4 times. The time is 1 to 3 hours.

In order to illustrate the scientific rationality of the technical solution of the present application, the following experiments are specially made:

Weigh two 10g Pyracantha fruit, put them in a round-bottomed flask, add 10mL of water to immerse, the pH is 5.32, and add 0.03g of compound enzyme for enzymolysis. After enzymolysis at 40°C for 2 hours, add 90mL of mass The percentage concentration is 60% ethanol for extraction, wherein, one part is heated to reflux for 1 hour, allowed to cool, weighed again, and the weight is made up with 60% ethanol by mass percentage, shaken, filtered, and the filtrate is obtained. The content of flavonoids in the Pyracantha fruit extract; the other part was extracted with a homogenizer, supplemented with ethanol with a concentration of 60% by mass, shaken, filtered, and the filtrate was obtained. flavonoid content. The test results are shown in Table 2.

Table 2 Effects of different extraction methods on the extraction of flavonoids from Pyracantha

As can be seen from Table 2, the content of flavonoids was determined by ultraviolet spectrophotometer, and it was found that the flavonoids can be extracted from Pyracantha fruit by heating and refluxing extraction.

As preferably, the resin model selected by the macroporous adsorption resin in the step (3) is ADS-7, LS-300B, NKA-9, HPD300, HPD600, HPD100, LS46, LS32, LS308, AB-8, LS305 or D101 one of the.

In order to illustrate the scientific rationality of the technical solution of the present application, the following experiments are specially made:

Weigh 500.00g of Pyracantha fruit, put it in a round-bottomed flask, add 500mL of water for immersion, the pH is 5.72, add 0.15g of compound enzyme for enzymolysis, and after enzymolysis at 40°C for 2 hours, add 3.0L of 60% ethanol Heat under reflux for 2 hours, twice, filter, and concentrate to 500 g. Select macroporous resin ADS-7, LS-300B, NKA-9, HPD300, HPD600, HPD100, LS46, LS32, LS308, AB-8, LS305, D101, and then screen by static adsorption. The test results are shown in Table 3.

Table 3 Effects of different types of resins on the extraction of flavonoids from Pyracantha

Resin model	Adsorption rate	Resolution	extraction rate
ADS-7	86.04%	8.86%	7.63%
LS-300B	72.98%	26.03%	19.00%
NKA-9	89.82%	26.97%	24.23%
HPD300	54.07%	87.88%	47.52%
HPD600	94.92%	95.63%	90.77%
HPD100	65.01%	66.09%	42.96%
LS46	76.58%	25.84%	19.79%
LS32	85.34%	3.72%	3.17%
LS308	83.57%	83.31%	69.62%
LS305	79.62%	28.87%	22.99%
D101	67.66%	67.75%	45.84%
AB-8	89.72%	61.32%	55.02%

As can be seen from Table 3, the content of flavonoids was determined by UV spectrophotometer, and it was found that HPD600 had the highest transfer rate of flavonoids, followed by LS308.

Preferably, when purifying in the step (3), water is first used for elution, and then ethanol with a mass percentage concentration of 60% to 85% is used for analysis.

Preferably, in the step (4), concentration under reduced pressure at 40° C.˜60° C. is used for concentration, and freeze-drying is used for drying.

In a second aspect, the present application also provides a use of the Pyracantha fruit extract in the preparation of antioxidant and/or brightening and/or whitening cosmetics.

In a third aspect, the present application also provides a whitening cream containing the Pyracantha fruit extract. In some specific embodiments, the whitening cream contains (in parts by weight):

Phase A: 2 parts of cetearyl alcohol, 2 parts of isononyl isononanoate, 2 parts of neopentyl glycol diheptanoate, 1 part of dimethicone, 2 parts of squalane, 2.5 parts of A165 , 0.5 part of tocopherol acetate;

Phase B: 68 parts of water, 0.1 part of xanthan gum, 0.1 part of carbomer, 2 parts of trehalose, 4 parts of glycerin;

Phase C: 2 copies of A-EG;

Phase D: 0.3 parts of Pyracantha fruit extract, 4 parts of butanediol, 5 parts of water;

Phase E: 2 parts of Dian Gentian extract;

Phase F: 0.05 part of citric acid; and

Phase G: 0.5 part PE9010.

In a fourth aspect, the application also provides a method for preparing the whitening cream, comprising the following steps:

(1) Weigh phase B according to the amount, heat and stir to dissolve completely, then keep stirring at 75℃～80℃ for 30 minutes;

(2) Weigh phase A by quantity, heat and dissolve at 75℃～80℃, add it to phase B, stir while adding, and homogenize for 10 minutes after mixing;

(3) when the above-mentioned A-phase and B-phase mixed components are cooled to 60° C., add C-phase and stir evenly;

(4) Pre-preparing phase D, adding the Pyracantha fruit extract to butanediol, dispersing and dissolving at 40°C to 60°C, adding water and stirring evenly, until the above-mentioned phases A, B and C are mixed. When the components are cooled to 50°C, add the Phase D and mix; and

(5) Add the E phase, F phase and G phase in turn, and stir evenly.

This application has the following beneficial effects:

(1) In this application, the Pyracantha fruit material is treated with a compound enzyme to destroy the Pyracantha fruit cell wall, and then combined with heating and reflux extraction, the cell membrane permeability of the Pyracantha fruit material is increased, and finally the effective substances in the cell membrane flow out. It can maximize the dissolution rate of the effective substances of the Pyracantha fruit, and effectively improve the extraction rate of the flavonoids from the Pyracantha fruit, which is 43.13% higher.

(2) The method of the present application uses ethanol as the extraction solvent, and the solvent is safe and non-toxic.

(3) The extraction method of the present application is a heating and refluxing extraction method, which is simple, low in equipment requirements, and low in cost, and is suitable for industrial production

(4) Macroporous resin purification method, the yield of flavonoids is high after purification by HPD600 or LS308 resin.

(5) Pyracantha fruit extract has the application of anti-oxidation, brightening and whitening in daily chemicals.

Description of drawings

Figure 1 is the skin irritation test;

Fig. 2 is the inhibitory effect of Pyracantha fruit extract on tyrosinase;

Fig. 3 is the scavenging rate % of DPPH free radical by Pyracantha fruit extract;

Fig. 4 is a graph of reducing power (%) of Pyracantha fruit extract;

Figure 5 is a 3D melanin skin model - apparent chromaticity;

Figure 6 is a 3D melanin skin model - apparent brightness map;

Fig. 7 is D melanin skin model - melanin content map;

Figure 8 is a schematic diagram of the phenotype of Pyracantha fruit extract concentration exploration;

Figure 9 is a typical diagram of melanin on the head of zebrafish after treatment with Pyracantha fruit extract;

Fig. 10 is the sum (pixel) diagram of zebrafish melanin optical density after treatment with Pyracantha fruit extract;

Figure 11 is a comparison diagram of the whitening effect of Pyracantha fruit extract on zebrafish in the normal control group.

Detailed ways

The specific embodiments of the present application will be further described below. It should be noted here that the description of these embodiments is used to help the understanding of the present application, but does not constitute a limitation to the present application. In addition, the technical features involved in the various embodiments of the present application described below can be combined with each other as long as they do not conflict with each other.

Example 1

Preparation of Pyracantha Fruit Extract:

(1) Enzymatic hydrolysis: take Pyracantha fruit, add compound enzyme for enzymatic hydrolysis, and obtain an enzymatic hydrolysis mixture; wherein, the compound enzyme is composed of polygalacturonase, cellulase and pectinase, and the temperature of enzymatic hydrolysis is At 40°C, the enzymatic hydrolysis time was 2 hours, and the pH value of the enzymatic hydrolysis was 5. The mass ratio of polygalacturonase, cellulase and pectinase was 1:1:1.

(2) Alcohol extraction: adding ethanol to the enzymatic hydrolysis mixture, heating and refluxing extraction, filtering, and concentrating to obtain an ethanol extract; wherein, the mass percentage concentration of ethanol during ethanol extraction is 60%, and the number of ethanol extractions is 2 times. The time for ethanol extraction was 2 hours.

(3) Purification of macroporous adsorption resin: The ethanol extract was loaded on a macroporous adsorption resin with resin type HPD600 for purification, and the analytical solution was collected; wherein, during purification, firstly use water to elute, and then use a mass percentage concentration of 70 % ethanol resolved.

(4) Drying: Concentrate the analytical solution under reduced pressure at 50° C. and freeze-dry to obtain it.

Example 2

Preparation of Pyracantha Fruit Extract:

(1) Enzymatic hydrolysis: take Pyracantha fruit, add compound enzyme for enzymatic hydrolysis, and obtain an enzymatic hydrolysis mixture; wherein, the compound enzyme is composed of polygalacturonase, cellulase and pectinase, and the temperature of enzymatic hydrolysis is At 42°C, the enzymatic hydrolysis time was 3 hours, and the pH value of the enzymatic hydrolysis was 6.5. The mass ratio of polygalacturonase, cellulase and pectinase was 2:1:1.

(2) Alcohol extraction: add ethanol to the enzymatic hydrolysis mixture and heat under reflux for extraction, filter, and concentrate to obtain an ethanol extract; wherein, the mass percentage concentration of ethanol during ethanol extraction is 80%, and the number of times of ethanol extraction is 4 times. The time for ethanol extraction was 1 hour.

(3) Purification of macroporous adsorption resin: The ethanol extract was loaded on a macroporous adsorption resin with resin model HPD600 for purification, and the analytical solution was collected; wherein, during purification, firstly use water to elute, and then use a mass percentage concentration of 85 % ethanol resolved.

(4) Drying: Concentrate the analytical solution under reduced pressure at 60° C. and freeze-dry it.

Example 3

Preparation of Pyracantha Fruit Extract:

(1) Enzymatic hydrolysis: take Pyracantha fruit, add compound enzyme for enzymatic hydrolysis, and obtain an enzymatic hydrolysis mixture; wherein, the compound enzyme is composed of polygalacturonase, cellulase and pectinase, and the temperature of enzymatic hydrolysis is At 37°C, the enzymatic hydrolysis time was 1 hour, and the pH value of the enzymatic hydrolysis was 4.5. The mass ratio of polygalacturonase, cellulase and pectinase was 1:2:1.

(2) Alcohol extraction: adding ethanol to the enzymatic hydrolysis mixture, heating and refluxing extraction, filtering, and concentrating to obtain an ethanol extract; wherein, the mass percentage concentration of ethanol during ethanol extraction is 50%, the number of ethanol extraction is 1 time, and the ethanol extraction The time is 3 hours.

(3) Purification of macroporous adsorption resin: The ethanol extract was loaded on a macroporous adsorption resin with resin type HPD600 for purification, and the analytical solution was collected; wherein, during purification, firstly use water to elute, and then use a mass percentage concentration of 60 % ethanol resolved.

(4) Drying: Concentrate the analytical solution under reduced pressure at 40° C., freeze-dry it, and obtain it.

Examples 4-14

Replace the macroporous adsorption resin with resin type HPD600 with one of ADS-7, LS-300B, NKA-9, HPD300, HPD100, LS46, LS32, LS308, AB-8, LS305 or D101, and the rest are completely Example 1 is the same to obtain Examples 4-14.

Example 15

For the Pyracantha fruit extract prepared by the above-mentioned preparation method, carry out anti-oxidation, brightening and whitening efficacy tests, and the specific embodiments are as follows:

1. Security testing

Skin irritation test: Episkin 3D skin model test (OECD TG439, criterion: cell viability greater than 50% is a non-irritating substance)

According to OECD TG439 test results: Pyracantha fruit extract is a non-irritating substance

2. Phototoxicity test

2.1 Experimental materials

2.1.1 Test System

The immortalized mouse fibroblast cell line BALB/c 3T3 used in this test was purchased from the American Type Collection (ATCC).

2.1.2 Main reagents

DMEM medium (1897086), newborn bovine serum (SLBJ5032V), 0.25% trypsin (1859509), chlorpromazine hydrochloride (E7X5M-LH), DPBS buffer (1902146), neutral red (20190613), absolute ethanol ( 20191002), acetic acid (SZBE0640V).

2.1.3 Main Equipment

CO ₂ incubator (2015SW0055), microplate reader (2015SW0006), incubator (Tester), biological safety cabinet (2015SW0004), fluorescence inverted microscope (2015SW0056), sunlight simulator (2015SW0035), UV illuminator (2015SW0069) )

2.2 Experimental method

2.2.1 Solution: The sample Pyracantha fruit extract is an alcohol-soluble sample, which is dissolved in absolute ethanol and diluted 100 times with DPBS, and then diluted with DPBS containing 1% ethanol to the concentration shown in Table 4 and then filtered and sterilized for use.

Table 4 Sample concentration design table

2.2.2 For BALB/c 3T3 cells, supplement 10% newborn calf serum with DMEM medium, and incubate in an incubator (37° C., 5% CO ₂ , 95% RH). The cells in logarithmic growth phase were collected and seeded into a 96-well plate at a cell density of 1×10 ⁴ cells/well, and each well contained 100 μL of culture medium.

2.2.3 After culturing in the incubator for 24 hours, randomly select a 96-well plate for the detection of cytotoxicity (-Irr), the control plate; the other for the detection of photocytotoxicity (+lrr), the treatment plate. Samples were prepared and added according to Table 4, and untreated cells were used as negative controls, and chlorpromazine hydrochloride (CPZ) with different concentrations was used as positive controls, and three replicate wells were set in each group.

2.2.4 Continue to incubate for 60min in the incubator after adding the sample. The treated plate is exposed to +lrr and irradiated with an irradiance of 1.7mW/ ^cm2 for 50min. The control plate is placed in a dark box for 50min at the same temperature. After treatment, the medium was replaced and cultured in an incubator for 18-22 h.

2.2.5 Neutral red uptake: use serum-free medium containing 50 μg/mL neutral red, and incubate for 3 h. After washing, 150 μL of neutral red eluent (water: ethanol: acetic acid = 49:50:1) was added to each well, and after shaking for 10 min, the absorbance OD value was read at 540 nm. Calculate cell viability.

2.2.6 Take the sample concentration as the x-axis and the cell viability as the y-axis, fit the concentration-response curve and refer to the EU Directive EU 67/548/EEC Annex VB.41 "In vitro 3T3 neutral red uptake phototoxicity test method" light Stimulating Factor (PIF) Value:

1) Calculate the PIF value if a complete concentration response curve is obtained in both cases with light (+lrr) and without light (-Irr).

2) If a compound is only cytotoxic in the presence of light (+lrr) but not in the absence of light (-Irr), even if the test results indicate that it may have potential phototoxicity, PIF cannot be calculated. In this case, if the highest concentration of the test substance (Cmax) is used for cytotoxicity experiments under no light (-Irr), Cmax can be used to calculate the ">PIF" value by the following formula:

3) If the test substance does not show cytotoxicity at the highest allowable concentration, so that the IC ₅₀ (+lrr) and IC ₅₀ (-lrr) values cannot be calculated, it indicates that the test substance has no potential phototoxicity. At this time, "PIF=*1" describes the result. which is:

4) The numerical value obtained from the prediction model, if: the PIF of the test substance is less than 2, it is predicted that "no phototoxicity"; Predict "phototoxicity". But in the case of using the predictive model PIF: if only one ">PIF" is obtained, then any value greater than 1 indicates that the test substance has potential phototoxicity; if only one "PIF=*1" is obtained, then the test substance is No potential phototoxicity.

2.2.7 Test acceptance criteria: According to GB/T 21769-2008, test acceptance criteria are shown in Table 5.

Table 5 Test acceptance criteria

2.3 Experimental results

The analysis of cell viability results is shown in Table 6, and the evaluation of phototoxicity results is shown in Table 7.

Table 6 Analysis of cell viability results

Table 7 Phototoxicity result evaluation

The results showed that the Pyracantha fruit extract had no phototoxicity.

3. Skin sensitization test

3.1 Experimental materials

3.1.1 Cell line: THP-1 (source Guangzhou | Customs Technology Center)

3.1.2 Complete medium: RPMI-1640 medium containing 10% FBS, containing 0.05 mM β-mercaptoethanol

(FBS was purchased from Gibco, Lot: 2045512CP).

3.1.3 Culture conditions: 37°C, 5% CO ₂ , 95% relative humidity.

3.1.4 Blank control (Control): complete medium.

3.1.5 Negative control (NC): lactate LA (1000 μg/mL).

3.1.6 Positive control (PC): NiS04 (100 μg/mL).

3.1.7 Test sample (TA): Weigh 0.5013g of sample, add 2.0mL of 50% DMSO aqueous solution, dissolve by oscillating ultrasonic, and obtain 250mg/mL stock solution after 0.22μM filtration, and use the complete culture solution to dilute to the required level during the test. Test concentration.

3.1.8 Antibody and blocking agent: CD86-FITC, CD54..PE, 7AAD and Fc segment receptor blocker were purchased from BD Company.

3.2 Experimental method

3.2.1 Cytotoxicity detection

Cell plating: Centrifuge the pre-cultured THP-1 cells and remove the supernatant, resuspend the cells in fresh complete medium at a concentration of 2×10 ⁶ cells/mL, and inoculate 500 μL into a 24-well cell culture plate.

Test substance exposure: Dilute the test substance to a specific concentration, add 500 μL of the test substance to each well, test 8 concentrations of each test substance, and incubate in an incubator for 24 hours.

Activity analysis: After the cells were collected, the cells were pelleted by centrifugation, the supernatant was discarded, the cells were washed once with FACS buffer, and the cells were collected by centrifugation again. 7AAD cell dye was added, and the cells were used for 10 min at room temperature in a dark room. The cells were cleared twice with FACS buffer, and the cells were resuspended and measured by flow cytometry.

3.2.2 Cell activation test

Cell plating: Centrifuge the pre-cultured THP-1 cells and remove the supernatant, resuspend the cells in fresh complete medium at a concentration of 2×10 ⁶ cells/mL, and inoculate 500 μL into a 24-well cell culture plate.

Test substance exposure: the highest concentration is the concentration that causes CV75, and 8 test concentrations are set. Pipette 500 μL of the prepared test substance liquid into the above-mentioned lower 24-well cell culture plate to be exposed. Incubate in an incubator for 24h.

Collection of cells: The cells under the action of each test substance were transferred from the correct-well cell culture plate to the corresponding 1mL EP tubes, and the cells were collected by centrifugation. After washing with FACS buffer once, the cells were collected by centrifugation again under the same conditions.

Fluorescent labeling: The cells obtained by centrifugation were added to FcR blocker for blocking. Divide the above cells into 4 1mL EP tubes at a rate of 50μL per tube, about 2.5x 10 ⁵ cells in each tube, add 50μL of the corresponding antibody, and stain the column for 10min under 2-8℃ environment and dark room conditions. After washing twice with FACS buffer and resuspending the cells, the cells were measured by flow cytometry.

3.3. Data Analysis and Prediction

Data is saved in electronic format for data analysis.

3.3.1 Cell viability analysis: 10,000 cells need to be accumulated in one flow injection experiment, and 30,000 cells can be accumulated when the cell viability is too small, and the time of each injection is controlled within 1 min. Cell viability can be obtained using software analysis.

3.3.2 Calculation of 75% cell viability (CV75)

The concentration of the test substance obtained by the above experiments and the cell viability at each dose can be calculated by curve fitting to obtain the CV75 of the corresponding test substance.

3.3.3 CD86/CD54 analysis

Mean fluorescence intensity (MFI) = geometric mean of fluorescence intensity

3.3.4 Prediction model

According to OECD TG442E-ANNEX1 (2018), the test substance is judged to be positive for skin sensitization if at least one of the following conditions is met: at least one test concentration when the average CD86RFI ≥ 150% and the cell viability ≥ 50%, and/or condition 2 : When the average CD54RFI is ≥200% and the cell viability is ≥50% at at least one test concentration.

3.4 Experimental results

3.4.1 Cytotoxicity results

Calculated according to the results of cell viability at different concentrations, no cytotoxicity was caused at the maximum concentration, CV75=532.35μg/mL

Table 8 Cell viability under test substance exposure at different concentrations

Concentration (μg/mL)	active(%)
1000	56.9
500	76.8
250	82.0
125	86.8
62.5	91.0

31.25	91.0
15.625	94.3
7.8125	95.2

3.4.2 CD54/CD86 results

Calculate the mean relative fluorescence intensity (mean RFI) of CD86 and CD54 according to the results of flow cytometry analysis

Table 9 Relative fluorescence intensities of CD86 and CD54 exposed to different concentrations of test substances

3.5 Results

The Pyracantha fruit extract was tested with OECD TG442E-ANNEX1 (2018), and the results showed that the mean RFICD54 at all tested concentrations was less than 200, the mean RFICD86 was less than 150, and the cell activity was greater than 50%. Pyracantha fruit extract was based on H-CLAT skin Prediction of sensitization was negative.

4 In vitro tyrosinase activity inhibition efficacy test

4.1 Main experimental reagents and instruments

Phosphate buffer (PH value 6.8), L-tyrosine, tyrosinase, α-arbutin (positive control), microplate reader, water bath

4.2 Experimental method

Using L-tyrosine as the substrate and α-arbutin as the positive control substance, add the sample solution/α-arbutin and L-tyrosine solution with different concentration gradients into the deep-well plate in turn, and mix well. After 30min in a 37°C water bath, the tyrosinase solution was added, and after mixing, it was placed in a 37°C water bath for 30min, and the absorbance was measured at 470 nm.

4.3 Experimental results

The maximum inhibition rate of Pyracantha fruit extract reached nearly 60%, and at the same concentration, the tyrosinase inhibition rate of Pyracantha fruit extract was significantly higher than that of α-arbutin. See Figure 2.

5 DPPH scavenging efficacy test

5.1 Main experimental reagents and instruments

DPPH, methanol, ascorbic acid (positive control), microplate reader, water bath

5.2 Experimental method

With ascorbic acid as the positive control substance, the sample solution/ascorbic acid solution and DPPH methanol solution with different concentration gradients were added to the deep-well plate in turn.

5.3 Experimental results

The DPPH clearance rate of Pyracantha fruit extract is higher than 95%, which is higher than that of ascorbic acid solution of the same concentration; overall, the DPPH clearance rate of Pyracantha fruit extract is slightly lower than that of ascorbic acid of the same concentration, but its concentration is similar to that of DPPH clearance. There is a good linear relationship between the ratios, and it has a good antioxidant effect. See Figure 3.

6Reducing force test

6.1 Main experimental reagents and instruments

Phosphate buffer (0.2M, pH 6.6), potassium ferricyanide, trichloroacetic acid, ferric chloride, ascorbic acid (positive control), microplate reader, water bath

6.2 Experimental method

Using ascorbic acid as the positive control substance, add sample solutions/ascorbic acid solutions with different concentration gradients into the deep-well plate in turn, add potassium ferricyanide, mix well, place in a 50°C water bath for 20 minutes, take out, add trichloroacetic acid solution, trichloroacetic acid After the ferric chloride solution was mixed, the absorbance was measured at 700 nm.

6.3 Experimental results

Pyracantha fruit extract has obvious reducing power, and the reducing power % increases linearly with the increase of concentration, indicating that it is an antioxidant substance with great potential. See Figure 4.

7 3D skin model melanin suppression efficacy test

7.1 Main experimental reagents and instruments

3D melanin skin model (EpiKutis), KA (positive control), bovine serum albumin, phosphate buffer, trypsin, digital camera, colorimeter, centrifuge, microplate reader, water bath, etc.

7.2 Experimental method

The 3D skin model was stimulated by UVB irradiation for 6 consecutive days, and on the 3rd and 5th days of stimulation, positive control substance and Pyracantha fruit extract were given to the skin model for intervention. The skin model's apparent chroma, brightness and melanin content were measured.

7.3 Experimental results

According to the results of apparent chromaticity photos, apparent brightness (L* value,) and melanin content, after UVB stimulation of the melanin model, the model became significantly darker, and the apparent brightness (L* value) was significantly reduced (p<0.01). ), the melanin content was significantly increased (p<0.01), indicating that the modeling was successful;

Compared with the UVB stimulation group, the Pyracantha fruit extract was obviously whitened, the apparent brightness (L* value) was significantly increased by 42.23%, and the melanin content was significantly decreased by 17.44% at the administration concentration of 0.1%. Pyracantha fruit extract can better inhibit the melanin deposition caused by UVB pathway at the administration concentration of 0.1%. See Figure 5, Figure 6, Figure 7, Figure 8.

8 Zebrafish efficacy test

Zebrafish are developed by in vitro fertilization, and the embryo body is transparent. The embryo develops rapidly, the membrane hatches about 3 days after fertilization, and it eats in about 5 days. It reaches sexual maturity in about 3 months, and its life span can reach more than 2 years. Zebrafish within 5 days is not subject to the EU ban and is a non-animal experiment.

8.1 Main experimental reagents and instruments

Dissecting microscope (SZX7, OLYMPUS, Japan); camera connected to microscope (VertA1); precision electronic balance (CP214, OHAUS, America); 6-well plate (Nest Biotech, China); methylcellulose (Sigma, USA); Dimethyl sulfoxide (DMSO, Sigma, France).

8.2 Experimental methods

8.2.1 Maximum Test Concentration (MTC):

180 wild-type AB strain zebrafish were randomly selected 6 hours after fertilization (6hpf) in a six-well plate, 30 zebrafish were treated in each well (experimental group), and water-soluble 62.5, 125, 250, 500 of Pyracantha fruit extract was given. and 1000 μg/mL concentration, and set up the normal control group (zebrafish treated with water for fish farming) at the same time, and the volume of each well is 3 mL. During the treatment of Pyracantha fruit extract, the number of dead zebrafish in each experimental group was counted every day and removed in time. MTC.

8.2.2 Zebrafish whitening test method:

150 zebrafish of 6hpf wild-type AB strain were randomly selected in six-well plates, and 30 zebrafish were treated in each well (experimental group), and water-soluble were given Pyracantha fruit extract at concentrations of 6.9, 20.8 and 62.5 μg/mL, positive control group The concentration of arbutin was 3000 μg/mL, and a normal control group (zebrafish was treated with water for fish farming) was set at the same time, and the volume of each well was 3 mL. After treating zebrafish with Pyracantha fruit extract and arbutin for 48 hours, 10 zebrafish were randomly selected from each experimental group to take pictures under a dissecting microscope and collect data. The results of statistical analysis of the sum of densities evaluated the whitening effect of Pyracantha fruit extract on zebrafish.

8.3 Experimental results

At the concentration of 62.5μg/mL, the Pyracantha fruit extract did not produce any obvious toxic side effects, nor did it cause death. At the concentration of 125-500μg/mL, it induced zebrafish developmental delay and caudal fin damage, and at 250μg/mL 3.3% (1/30) zebrafish died at the concentration of mL, 6.7% (2/30 fish) at the concentration of 500 μg/mL, and 100% (30/30 fish) at the concentration of 1000 μg/mL Zebrafish die. Therefore, the MTC of Pyracantha fruit extract to normal zebrafish was determined to be 62.5μg/mL.

The sum of the optical density of zebrafish melanin in the positive control group with 3000μg/mL arbutin concentration (2133 pixels) compared with the normal control group (54592 pixels), p<0.001, and its whitening effect on zebrafish was 96%, indicating that arbutin has an effect on zebrafish. Fish has obvious whitening effect. The sum of the optical density of zebrafish melanin in the 6.9, 20.8 and 62.5 μg/mL concentration groups of Pyracantha fruit extract was 38902, 25188 and 2393 pixels, respectively. were 29%, 54% and 96%. It is suggested that the extract of Pyracantha fruit has obvious whitening effect on zebrafish at the concentration of this experiment. Under the same whitening efficiency (96%), the dosage of Pyracantha extract is only 1/48 of that of arbutin. See Figures 9, 10 and 11.

Example 16

A method for preparing a whitening cream, comprising the following steps:

(1) Weigh phase B by weight, heat and stir until completely dissolved, and heat and stir at 78°C for 30 minutes;

(2) Weigh phase A by quantity, heat and dissolve at 78°C, add it to phase B, stir while adding, and homogenize for 10 minutes after mixing;

(3) when the above-mentioned A-phase and B-phase mixed components are cooled to 60° C., add C-phase and stir evenly;

(4) Pre-preparing phase D, adding the Pyracantha fruit extract to butanediol, dispersing and dissolving at 50°C, adding water and stirring evenly, and cooling the components after the above-mentioned A phase, B phase and C phase are mixed At 50°C, add the D phase and mix;

(5) Add the E phase, F phase and G phase in turn, and stir evenly.

Example 17

A method for preparing a whitening cream, comprising the following steps:

(1) Weigh phase B by weight, heat and stir until completely dissolved, and heat and stir at 80°C for 30 minutes;

(2) Weigh phase A by quantity, heat and dissolve at 75℃～80℃, add it to phase B, stir while adding, and homogenize for 10 minutes after mixing;

(3) when the above-mentioned A-phase and B-phase mixed components are cooled to 60° C., add C-phase and stir evenly;

(4) Pre-preparing Phase D, adding Pyracantha fruit extract to butanediol, dispersing and dissolving at 60°C, adding water and stirring evenly, and cooling the components after mixing the above-mentioned Phase A, Phase B and Phase C At 50°C, add the D phase and mix;

(5) Add the E phase, F phase and G phase in turn, and stir evenly.

Example 18

A method for preparing a whitening cream, comprising the following steps:

(1) Weigh phase B by weight, heat and stir until completely dissolved, and heat and stir at 75°C for 30 minutes;

(2) Weigh phase A by quantity, heat and dissolve at 75°C, add it to phase B, stir while adding, and homogenize for 10 minutes after mixing;

(3) when the above-mentioned A-phase and B-phase mixed components are cooled to 60° C., add C-phase and stir evenly;

(4) Pre-preparing phase D, adding the Pyracantha fruit extract to butanediol, dispersing and dissolving at 40°C, adding water and stirring evenly, and cooling the components after the above-mentioned A phase, B phase and C phase are mixed At 50°C, add the D phase and mix;

(5) Add the E phase, F phase and G phase in turn, and stir evenly.

The formulations of the whitening creams in the above Examples 15-18 are shown in Table 10.

Table 10 Recipe of whitening cream

The embodiments of the present application are described above in detail, but the present application is not limited to the described embodiments. For those skilled in the art, without departing from the principle and spirit of the present application, various changes, modifications, substitutions and alterations to these embodiments still fall within the protection scope of the present application.

Claims (10)

1. A preparation method of a Pyracantha fruit extract, comprising the following steps:

   (1) Enzymolysis: take Pyracantha fruit, add compound enzyme for enzymolysis, and obtain an enzymolysis mixture;

   (2) alcohol extraction: adding ethanol to the enzymatic hydrolysis mixture for extraction, filtering, and concentrating to obtain an ethanol extract;

   (3) macroporous adsorption resin purification: the ethanol extract is loaded on the macroporous adsorption resin for purification, and the analytical solution is collected; and

   (4) Drying: Concentrate and dry the analytical solution to obtain the solution.
2. The preparation method of Pyracantha fruit extract according to claim 1, wherein, in the step (1), the composite enzyme is composed of polygalacturonase, cellulase and pectinase, and the temperature of enzymatic hydrolysis is 37℃～42℃, the time of enzymolysis is 1～3 hours, and the pH value of enzymolysis is 4.5～6.5.
3. The preparation method of Pyracantha fruit extract according to claim 2, wherein the mass ratio of the polygalacturonase, cellulase and pectinase is 1:1:1.
4. The preparation method of Pyracantha fruit extract according to claim 1, wherein, in the step (2), the method of ethanol extraction is heating and reflux extraction, and the mass percentage concentration of ethanol during ethanol extraction is 50% to 80%, The number of times of ethanol extraction is 1 to 4 times, and the time of each ethanol extraction is 1 to 3 hours.
5. The preparation method of Pyracantha fruit extract according to claim 1, wherein, the resin model selected by the macroporous adsorption resin in the step (3) is ADS-7, LS-300B, NKA-9, HPD300, HPD600, One of HPD100, LS46, LS32, LS308, AB-8, LS305 or D101.
6. The preparation method of the Pyracantha fruit extract according to claim 5, wherein, when purifying in the step (3), water is first used for elution, and then ethanol with a mass percentage concentration of 60% to 85% is used for analysis.
7. The preparation method of the Pyracantha fruit extract according to claim 5, wherein, in the step (4), concentration under reduced pressure is performed at 40°C to 60°C, and drying is performed by freeze-drying.
8. Use of the Pyracantha fruit extract prepared by the preparation method according to any one of claims 1-7 in the preparation of antioxidant and/or brightening and/or whitening cosmetics.
9. A whitening face cream comprising, calculated in parts by weight:

   Phase A: 2 parts of cetearyl alcohol, 2 parts of isononyl isononanoate, 2 parts of neopentyl glycol diheptanoate, 1 part of dimethicone, 2 parts of squalane, 2.5 parts of A165 , 0.5 part of tocopherol acetate;

   Phase B: 68 parts of water, 0.1 part of xanthan gum, 0.1 part of carbomer, 2 parts of trehalose, 4 parts of glycerin;

   Phase C: 2 copies of A-EG;

   Phase D: 0.3 parts of Pyracantha fruit extract, 4 parts of butanediol, and 5 parts of water prepared according to the preparation method of any one of claims 1-7;

   Phase E: 2 parts of Dian Gentian extract;

   Phase F: 0.05 part of citric acid; and

   Phase G: 0.5 part PE9010.
10. A method of preparing the described whitening cream of claim 9, which comprises the following steps:

    (1) Weigh phase B according to the amount, heat and stir to dissolve completely, then keep stirring at 75℃～80℃ for 30 minutes;

    (2) Weigh phase A by quantity, heat and dissolve at 75℃～80℃, add it to phase B, stir while adding, and homogenize for 10 minutes after mixing;

    (3) when the above-mentioned A-phase and B-phase mixed components are cooled to 60° C., add C-phase and stir evenly;

    (4) Pre-preparing phase D, adding the Pyracantha fruit extract to butanediol, dispersing and dissolving at 40°C to 60°C, adding water and stirring evenly, until the above-mentioned phases A, B and C are mixed. When the components are cooled to 50°C, add the Phase D and mix; and

    (5) Add the E phase, F phase and G phase in turn, and stir evenly.

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