WO2022218171A1 - Application of pineapple extract composition in whitening - Google Patents

Abstract

An application of a pineapple extract composition in the preparation of a product having a whitening effect, the pineapple extract composition being prepared and obtained by extracting raw materials of the following components: pineapples, oat, highland barley, coix seeds, tremella polysaccharides and water. It has been proven by means of experimentation that the pineapple extract composition has an inhibiting effect on the activity of tyrosinase after acting on B16-F10 melanoma cells, and has a significant inhibitory effect on melanin synthesis of B16 cells.

Description

A kind of whitening application of pineapple extract composition

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority of Chinese Invention Patent Application No. CN202110413436.3 filed on April 16, 2021, which is incorporated herein by reference in its entirety.

technical field

The invention belongs to the field of cosmetics, and particularly relates to a whitening application of a pineapple extract composition.

Background technique

Natural cosmetics are more and more popular nowadays. Natural cosmetics, in addition to natural ingredients, do not add flavors, preservatives and other potentially irritating ingredients.

As a technology widely used in the fields of food, medicine and cosmetics, enzymolysis can transform macromolecular components into small molecules, which are accompanied by the production of other active substances such as polysaccharides and polypeptides. Plants contain a variety of enzymes, including protease, cellulase, amylase, esterase, etc. The enzymes contained in plants can be used to hydrolyze macromolecular proteins into oligopeptides, and starch and cellulose into oligosaccharides. , lipolysis into glycerol, fatty acid. The application of these products in cosmetics is more conducive to the skin's absorption of functional ingredients, and finally achieves a certain effect.

SUMMARY OF THE INVENTION

Therefore, the object of the present invention is to overcome the defects in the prior art and provide a whitening application of the pineapple extract composition.

Before describing the content of the present invention, the terms used herein are defined as follows:

The term "alpha-MSH" refers to: melanin.

The term "PBS" refers to: Phosphate Buffered Saline.

The term "t-test" refers to: T-test.

The term "B16 cells" refers to: B16 mouse melanoma cells.

The term "ITA" refers to: Skin Color Measurement.

The term "VISIA CR" refers to: Facial Image Analyzer.

In order to achieve the above object, the first aspect of the present invention provides the application of a pineapple extract composition in the preparation of a product with a whitening effect, the pineapple extract composition is prepared by extracting the raw materials of the following components: pineapple, oat , highland barley, barley, tremella polysaccharide and water;

Wherein, the parts by mass of each component in the pineapple extract composition are: 0.1-10 parts of pineapple, 0.1-10 parts of oats, 0.1-5 parts of highland barley, 0.1-5 parts of coix seed, 0.05-2 parts of Tremella polysaccharide, water 100 copies;

Preferably, 0.5-5 parts of pineapple, 0.5-5 parts of oats, 0.2-2 parts of highland barley, 0.2-2 parts of coix seed, 0.1-1.5 parts of Tremella polysaccharide, and 100 parts of water;

More preferably, 0.5-3 parts of pineapple, 0.5-2.5 parts of oats, 0.5-1.5 parts of highland barley, 0.5-1.5 parts of barley, 0.2-1 part of Tremella polysaccharide, and 100 parts of water.

According to the application of the first aspect of the present invention, wherein, the pineapple extract composition is prepared by the following method:

(1) oats, highland barley and barley are crushed and sieved;

(2) pineapple cleaning, peeling, dicing, and beating after mixing with water to obtain pineapple pretreatment liquid;

(3) in step (1), gained oat, highland barley, coix seed and water are mixed and left standstill to obtain mixed pretreatment solution;

(4) the pineapple pretreatment solution obtained in step (2) and the mixed pretreatment solution obtained in step (3) are mixed, heated and stirred, and the supernatant is left by cooling and filtration;

(5) adding Tremella polysaccharide to the supernatant obtained in step (4), and homogenizing to obtain the pineapple extract composition.

According to the application of the first aspect of the present invention, wherein, in step (1), the oats, highland barley, and barley are pulverized and passed through an 80-mesh sieve.

According to the application of the first aspect of the present invention, wherein, in step (2), the ratio of pineapple to water is 1:0.1～20g/mL, preferably 1:0.2～10g/mL, more preferably 1:0.5～5g/mL mL, more preferably 1:1 g/mL.

According to the application of the first aspect of the present invention, in step (3), the standing time is 12-60 hours, preferably 12-48 hours, and most preferably 24 hours.

According to the application of the first aspect of the present invention, wherein, in step (4), the addition ratio of the pineapple treatment solution obtained in step (2) and the mixed pretreatment solution obtained in step (3) is 1:1 to 500 g/mL, preferably 1 : 5 to 200 g/mL, more preferably 1:10 to 100 g/mL, still more preferably 1:20 to 50 g/mL. According to the application of the first aspect of the present invention, wherein, in step (4), the heating temperature is 20-90°C, preferably 25-85°C, more preferably 30-80°C, further preferably 40-75°C;

The heating time is 0.5 to 5 hours, preferably 0.5 to 4 hours, more preferably 1 to 3 hours, further preferably 1 to 2 hours; and/or

The stirring rate is 100-800 r/min, preferably 100-500 r/min, more preferably 100-400 r/min, further preferably 200-300 r/min.

According to the application of the first aspect of the present invention, wherein, in step (5), the ratio of the Tremella polysaccharide to the supernatant obtained in step (4) is 1:1～1000g/mL, preferably 1:100～800g/mL ; more preferably 1:100 to 500 g/mL, most preferably 1:200 to 400 g/mL; and/or

The homogenization time is 1 to 60 minutes, preferably 5 to 50 minutes, more preferably 5 to 30 minutes, and further preferably 10 to 20 minutes.

According to the application of the first aspect of the present invention, the method further comprises the following steps:

(6) sterilizing the pineapple extract composition obtained in step (5);

Preferably, the sterilization treatment method is heat sterilization;

More preferably, the heating sterilization temperature is 60-90°C, preferably 70-80°C, most preferably 75°C; and/or the heating sterilization time is 1-5 hours, preferably 1-3 hours , most preferably 2 hours.

According to the application of the first aspect of the present invention, wherein, the product with whitening effect comprises:

The pineapple extract composition;

one or more cosmetic ingredients selected from plants;

Preferably, the cosmetic ingredients are selected from one or more of the following: olive oil, matsutake mushrooms, green thorn fruit oil, betaine, Ganoderma lucidum, and Rhodiola rosea.

According to the application of the first aspect of the present invention, wherein,

The whitening effect is inhibition of tyrosinase activity; and/or

The whitening effect is to inhibit the synthesis of melanin.

According to the application of the first aspect of the present invention, wherein, the dosage form of the product with whitening effect is selected from one or more of the following: cream, lotion, water, gel, oil, powder, mud, aerosol , paste, film;

Preferably, the cosmetic product is a cream, mask, lotion or essence.

According to the application of the first aspect of the present invention, wherein, when the product with whitening effect is cream, its preparation method comprises the following steps:

(a) adding an aqueous phase component to the sterilized pineapple extract composition, heating and stirring to obtain an aqueous phase mixture;

(b) mixing oil phase components, heating and dissolving to obtain oil phase mixture;

(c) adding the oil phase mixture prepared in step (b) to the water phase mixture prepared in step (a), and homogenizing to obtain the cosmetic product.

According to the application of the first aspect of the present invention, wherein, in the step (a), the water phase ingredients include vegetable oil and an antibacterial agent; and/or in the step (b), the oil phase ingredients include vegetable oil and emulsification agent;

Preferably, the vegetable oil is selected from one or more of the following: glycerin, green thorn fruit oil, olive oil, peony seed oil, macadamia nut oil, babassu oil, toona japonica oil;

Preferably, the antibacterial agent is selected from one or more of the following: hexanediol, methylparaben, propylparaben, phenoxyethanol; and/or

Preferably, the emulsifier is selected from one or more of the following: wheat emulsifier, polyglycerol-3 diisostearate, dipolyhydroxystearic acid, and yeoleyl glucoside.

According to the application of the first aspect of the present invention, wherein, in the step (a), the ratio of the sterilized pineapple extract composition, vegetable oil and antibacterial agent is 80～95:1～10:0.01～1g/ g/g, preferably 89～93:2～5:0.05～0.2g/g/g;

In the step (b), the ratio of the vegetable oil to the emulsifier is 1～10:2～15g/g, preferably 2～5:5～10g/g; and/or

In the step (c), the ratio of the oil phase mixture to the water phase mixture is 2-25:0.5-5g/g, preferably 5-10:1-2g/g;

Preferably, in the step (a), the step (b) and the step (c), the heating temperature is 70-120°C, more preferably 70-100°C, further preferably 80-90°C; and /or

Preferably, in the step (c), the homogenization time is 15-45 minutes, more preferably 20-40 minutes, and even more preferably 25-35 minutes.

The preparation method of the pineapple extract composition of the present invention comprises the following steps:

(1) The highland barley and the barley seed are physically crushed for later use, and mixed with oat and water to be beaten to prepare a pretreatment solution. Let stand at room temperature for 24 hours.

(2) Mix the pineapple and water, add it to (1), heat and stir. filter.

Tremella polysaccharide is added, and the mask liquid is obtained by homogenization.

(3) Sterilize at 100℃ for 2h and fill under aseptic conditions.

In step (1), the ratio of the oats to water is 1:1～500g/mL, preferably 1:10～300g/mL, more preferably 1:20～150g/mL, most preferably 1:80g/mL mL;

The ratio of highland barley to water is 1:1～400g/mL, preferably 1:10～350g/mL, more preferably 1:30～200g/mL, most preferably 1:100g/mL;

The ratio of coix seed and water is 1:1～400g/mL, preferably 1:20～300g/mL, more preferably 1:50～200g/mL, most preferably 1:100g/mL;

In step (2), the ratio of the pineapple extract to (1) extract is 1:1～200g/mL, preferably 1:1～150g/mL, more preferably 1:1～100g/mL, most preferably 1:1～100g/mL. Preferably 1:50g/mL;

The heating temperature is 1-150°C, preferably 1-120°C, more preferably 20-90°C, most preferably 75°C;

The heating time is 0.1 to 4 hours, preferably 0.5 to 3 hours, more preferably 0.5 to 2 hours, and most preferably 1 hour

The stirring rate is 10-1000 r/min, preferably 10-800 r/min, more preferably 20-500 r/min, most preferably 300 r/min.

In step (3), the ratio of Tremella polysaccharide and extract is 1:1～2000g/mL, preferably 1:1～1000g/mL, more preferably 1:1～800g/mL, most preferably 1:1～1000g/mL 400g/mL

The homogenization speed is 1000～20000r/min, preferably 2000～15000r/min, more preferably 2500～10000r/min; the best is 6000r/min;

The homogenization time is 1 to 60 minutes, preferably 1 to 40 minutes, more preferably 1 to 30 minutes, and most preferably 20 minutes.

The application of the present invention can have but is not limited to the following beneficial effects:

The invention provides an application of a pineapple extract composition in preparing a product with a whitening effect. Tests show that the pineapple extract composition of the invention has an inhibitory effect on B16-F10 tyrosinase activity.

Brief Description of Drawings

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, wherein:

Figure 1 shows the HPLC profile of sample 1 at 220 nm in Example 1.

FIG. 2 shows the HPLC profile of sample 2 at 220 nm in Example 1. FIG.

FIG. 3 shows the HPLC profile of the blank group at 220 nm in Example 1. FIG.

FIG. 4 shows the tyrosinase activity of the pineapple extract composition in Test Example 1 acting on B16-F10.

FIG. 5 is a graph showing an example of the change in pigmentation of the volunteer 1 after using the cream 1 prepared in Example 9 in Test Example 4. FIG.

FIG. 6 is a graph showing an example of the change in pigmentation of Volunteer 2 after using Cream 1 prepared in Example 9 in Test Example 4. FIG.

FIG. 7 is a graph showing an example of the change in pigmentation of Volunteer 3 after using Cream 1 prepared in Example 9 in Test Example 4. FIG.

FIG. 8 is a graph showing an example of the change in pigmentation of volunteer 1 after using the cream 2 prepared in Example 10 in Test Example 4. FIG.

FIG. 9 is a graph showing an example of the change in pigmentation of Volunteer 2 after using the cream 2 prepared in Example 10 in Test Example 4. FIG.

FIG. 10 is a graph showing an example of the change in pigmentation of Volunteer 3 after using Cream 2 prepared in Example 10 in Test Example 4. FIG.

Figure 11 shows the effect of the sample (facial cream 1) prepared in Example 9 in Test Example 4 on the skin melanin value (time point comparison), wherein, **, p<0.01, indicating that compared with the background, it has extremely high Significant difference; n.s., p>0.05, indicating no significant difference compared with the background.

Figure 12 shows the effect of the sample (cream 2) prepared in Example 10 in Test Example 4 on the skin ITA value (time point comparison), wherein, **, p<0.01, indicating that compared with the background, it has a very high Significant difference; n.s., p>0.05, indicating no significant difference compared with the background.

Figure 13 shows the proportion of consumers who are satisfied with the efficacy evaluation of consumers in Test Example 4.

Figure 14 shows the proportion of the overall number of satisfied consumers in Test Example 4.

Best way to implement your invention

The present invention is further illustrated by specific examples below, but it should be understood that these examples are only used for more detailed description, and should not be construed as being used to limit the present invention in any form. If no specific technique or condition is indicated in the examples, the technique or condition described in the literature in the field or the product specification is used. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased through regular channels.

This section provides a general description of the materials and test methods used in the tests of the present invention. While many of the materials and methods of operation used for the purposes of the present invention are known in the art, the present invention is described in as much detail as possible. It will be clear to those skilled in the art that, in the context, if not specifically stated, the materials and methods of operation used in the present invention are well known in the art.

The reagents and instruments used in the following examples are as follows:

Material:

Pineapple, purchased from Hongxing Farm, Zhanjiang City, Guangdong Province;

Oatmeal (fried and cooked), purchased from Zhangjiakou Jianjun Oat Food Co., Ltd.;

Highland barley and barley, purchased from Beijing Tongrentang (Group) Co., Ltd., China;

Olive oil, purchased from Yunnan Yuchenglong Economic and Trade Co., Ltd.

Reagents:

B16 cells, Subacom (Shanghai) Biotechnology Co., Ltd.;

T25, 96-well plate, 6-well plate, sterile PBS, purchased from Corning Life Sciences Co., Ltd.;

Tyrosinase, purchased from Sigma Ltd.

instrument:

ELISA instrument, purchased from Thermo Fisher Scientific OY Co., Ltd., model 1510-00662C;

Homogenizer, purchased from IKA Co., Ltd., model T25;

Medical centrifuge, purchased from Wuxi Ruijiang Analytical Instrument Co., Ltd., model RJ-TGL-10B;

Skin elasticity test instrument, purchased from Beijing Jinhongfan Trading Co., Ltd., model MPA580 multi-probe;

Facial image analyzer, purchased from Beijing Jinhongfan Trading Co., Ltd., model VISIA CR.

Example 1

This example is used to illustrate the selection of the pineapple enzyme solution of the present invention.

In this experiment, when pineapple was used as the enzyme solution, and oat, highland barley and barley were used as the substrate, the effect of the enzyme solution obtained from different varieties of pineapple on the molecular weight of the product polypeptide was investigated. The experimental method is as follows:

(1) After cleaning, peeling and dicing pineapples of different varieties, they are mixed and beaten with water according to a mass ratio of 1:1 to obtain pineapple enzyme solution 1 as sample 1, and pineapple enzyme solution 2 as sample 2. Among them, sample 1 is Barry pineapple, and sample 2 is golden diamond pineapple.

(2) The oat is physically pulverized and mixed with water in a mass ratio of 1:25.

(3) Add (1) to the pretreatment solution (2) according to the mass ratio of enzyme solution:substrate=1:50. The numbers of the obtained mixed solutions are sample 1 and sample 2 respectively, and the blank group is the pretreatment solution (2) without adding the pineapple enzyme solution.

(4) The mixed solution in (3) was incubated at 35°C for 1 hour, and then placed in a 95°C water bath for 0.5 hour to inactivate the enzyme.

(5) Centrifuge the mixed solution in (4) at 5000 r/min for 20 minutes to obtain a supernatant.

(6) Determination of the polypeptide molecular weight of the supernatant by high performance liquid chromatography.

(7) The specific measurement method is as follows:

(a) Chromatographic conditions

Chromatographic column: TsK gel 2000 SWXL 300mm×7.8mm

Mobile phase: 0.05mol/L phosphate buffer (pH 7)+0.3mol/LNaCl

Detection wavelength: UV 220nm

Flow rate: 1ml/min

Column temperature: 25℃

Sample preparation: use the mobile phase as the solvent to prepare a sample with a concentration of 5 mg/ml, and then filter it with a microporous membrane (0.45 μm) for injection.

Standard sample: bovine serum albumin (Mr 67000), B12 (Mr 1335), and oxidized glutathione (Mr 614) were prepared into mixed standards, and the content of each substance was 5mg/ml.

(b) Determination of standard curve

The three standard samples were prepared at 5 mg/ml, and the standard curve was prepared according to the above chromatographic conditions. The least square method was used to obtain the regression equation of the straight line: y=-0.3913x+7.4563, and its regression coefficient R ² =0.9982.

It can be seen from the regression coefficient R ² =0.9982 of the equation that the standard curve of the mixed standard sample has a better linear relationship, which can improve the accuracy of the calculation. In the relationship, X represents the elution time, and Y represents the logarithm of the molecular weight. In this way, when the mask sample is analyzed by HPLC, its corresponding molecular weight can be calculated by the appearance time of the elution peak of each substance in the elution pattern.

(c) Molecular weight measurement results

Figures 1 to 3 show the HPLC chromatograms of sample 1, sample 2 and blank group in Example 1, respectively. The peak time, area and molecular weight of sample 1, sample 2 and blank group are shown in Table 1 below.

Table 1 Peak time, area and molecular weight of sample 1, sample 2 and blank group

Experimental results: In the blank sample, the content of the polypeptide with a molecular weight of 262946.30Da was 6.14%, the content of the polypeptide with a molecular weight of 69342.58Da was 71.11%, the content of the polypeptide with a molecular weight of 2266.84Da was 6.95%, and the content of the polypeptide with a molecular weight of 1661.12Da was 81.29% , the content of the polypeptide with a molecular weight of 142.01Da is 11.76%; in sample 1, the content of the polypeptide with a molecular weight of 1357.09Da is 40.55%, the content of the polypeptide with a molecular weight of 962.50Da is 43.80%, and the content of the polypeptide with a molecular weight of 145.33Da is 15.65%; 2. The content of the polypeptide with a molecular weight of 1495.12 Da is 88.49%, and the content of the polypeptide with a molecular weight of 148.08 Da is 11.51%. The results showed that compared with the blank group, the proportion of peptides with molecular weight below 1600 Da in the supernatant obtained by adding pineapple enzyme solution increased, especially in sample 1, under the action of protease in pineapple, the peptides with molecular weight below 1000 Da The content is 59.45%. This shows that bromelain can degrade high-molecular-weight polypeptides in oats into lower-molecular-weight polypeptides, and by comparing the molecular weight distribution results of sample 1 and sample 2, the pineapple enzyme solution used to prepare sample 1 was finally selected in the subsequent experiments. .

Example 2

This example is used to illustrate the preparation method of the pineapple extract composition of the present invention.

1. Raw material processing (food grade)

Oats, highland barley, and barley: crush the oats, barley, and barley to 80 mesh for use.

Pineapple: Wash, peel, and dice. 1:1 (g/ml) was mixed with water and beaten to obtain a pineapple pretreatment solution for later use.

2. Preparation of mask liquid

(1) The addition ratio of oat, highland barley, barley and water is shown in Table 2. After mixing, let stand for 24 hours at room temperature.

Table 2 Addition ratio of mixed pretreatment solution

raw material	parts by mass
water	100
oat	2
highland barley	1.25
barley	1.25

(2) The pineapple pretreatment solution and the mixed pretreatment solution obtained in step (1) are mixed according to the mass ratio of 1:20 (g/ml), heated at 40° C. for 2 hours, and stirred at 300 rad/min; cooled to room temperature, filtered to remove impurities, Leave the supernatant (opaque).

(3) adding Tremella polysaccharide in a mass ratio of 1:200 (g/mL), and homogenizing with a homogenizer for 20 minutes to obtain a pineapple extract composition.

3. Sterilization

Fill the pineapple extract composition into a mask bag (containing mask cloth).

Sterilize at 75°C for 2 hours.

Example 3

This example is used to illustrate the preparation method of the pineapple extract composition of the present invention.

1. Raw material processing (food grade)

Oats, highland barley, and barley: crush the oats, barley, and barley to 80 mesh for use.

Pineapple: Wash, peel, and dice. 1:1 (g/ml) was mixed with water and beaten to obtain a pineapple pretreatment solution for later use.

2. Preparation of mask liquid

(1) The addition ratio of oat, highland barley, barley and water is shown in Table 3. After mixing, let stand for 24 hours at room temperature.

Table 3 Addition ratio of mixed pretreatment solution

raw material	parts by mass
water	100
oat	1
highland barley	0.5
barley	0.5

(2) The pineapple pretreatment solution and the mixed pretreatment solution obtained in step (1) are mixed according to the mass ratio of 1:50 (g/ml), heated at 75°C for 1 hour, and stirred at 200rad/min; cooled to room temperature, filtered to remove impurities, Leave the supernatant (opaque).

(3) adding the Tremella polysaccharide in a mass ratio of 1:400 (g/mL), and homogenizing with a homogenizer for 10 minutes to obtain a pineapple extract composition.

3. Sterilization

Fill the pineapple extract composition into a mask bag (containing mask cloth).

Sterilize at 75°C for 2 hours.

Example 4

This example is used to illustrate the preparation method of the pineapple extract composition of the present invention.

1. Raw material processing (food grade)

Oats, highland barley, and barley: crush the oats, barley, and barley to 80 mesh for use.

Pineapple: Wash, peel, and dice. 1:1 (g/ml) was mixed with water and beaten to obtain a pineapple pretreatment solution for later use.

2. Preparation of mask liquid

(1) The addition ratio of oats, highland barley, barley and water is shown in Table 4. After mixing, let stand for 24 hours at room temperature.

The addition ratio of table 4 mixed pretreatment liquid

raw material	parts by mass
water	100
oat	2.5
highland barley	1.5
barley	1.5

(2) the pineapple pretreatment solution and the mixed pretreatment solution obtained in step (1) are mixed according to the mass ratio of 1:30 (g/ml), heated at 50 ° C for 2 hours, and stirred at 250 rad/min; cooled to room temperature, filtered to remove impurities, Leave the supernatant (opaque).

(3) adding Tremella polysaccharide in a mass ratio of 1:300 (g/mL), and homogenizing with a homogenizer for 10 minutes to obtain a pineapple extract composition.

3. Sterilization

Fill the pineapple extract composition into a mask bag (containing mask cloth).

Sterilize at 75°C for 2 hours.

Example 5

This example is used to illustrate the preparation method of the pineapple extract composition of the present invention.

1. Raw material processing (food grade)

Oats, highland barley, and barley: crush the oats, barley, and barley to 80 mesh for use.

Pineapple: Wash, peel, and dice. 1:1 (g/ml) was mixed with water and beaten to obtain a pineapple pretreatment solution for later use.

2. Preparation of mask liquid

(1) The addition ratio of oat, highland barley, barley and water is shown in Table 5. After mixing, let stand for 24 hours at room temperature.

The addition ratio of table 5 mixed pretreatment liquid

raw material	parts by mass
water	100
oat	2
highland barley	1
barley	1

(2) the pineapple pretreatment solution and the mixed pretreatment solution obtained in step (1) are mixed according to the mass ratio of 1:40 (g/ml), heated at 60 ° C for 1 hour, and stirred at 300 rad/min; cooled to room temperature, filtered to remove impurities, Leave the supernatant (opaque).

(3) adding Tremella polysaccharide in a mass ratio of 1:300 (g/mL), and homogenizing with a homogenizer for 15 minutes to obtain a pineapple extract composition.

3. Sterilization

Fill the pineapple extract composition into a mask bag (containing mask cloth).

Sterilize at 75°C for 2 hours.

Example 6

This example is used to illustrate the preparation method of the pineapple extract composition of the present invention.

1. Raw material processing (food grade)

Oats, highland barley, and barley: crush the oats, barley, and barley to 80 mesh for use.

Pineapple: Wash, peel, and dice. 1:5 (g/ml) was mixed with water and beaten to obtain a pineapple pretreatment solution for later use.

2. Preparation of mask liquid

(1) The addition ratios of oats, highland barley, barley and water are shown in Table 6. After mixing, let stand for 36 hours at room temperature.

Table 6 Addition ratio of mixed pretreatment solution

raw material	parts by mass
water	100

oat	5
highland barley	2
barley	2

(2) the pineapple pretreatment solution and the mixed pretreatment solution obtained in step (1) are mixed according to the mass ratio of 1:100 (g/ml), heated at 80° C. for 3 hours, and stirred at 400 rad/min; cooled to room temperature, filtered to remove impurities, Leave the supernatant (opaque).

(3) adding Tremella polysaccharide in a mass ratio of 1:500 (g/mL), and homogenizing with a homogenizer for 30 minutes to obtain a pineapple extract composition.

3. Sterilization

Fill the pineapple extract composition into a mask bag (containing mask cloth).

Sterilize at 80°C for 3 hours.

Example 7

This example is used to illustrate the preparation method of the pineapple extract composition of the present invention.

1. Raw material processing (food grade)

Oats, highland barley, and barley: crush the oats, barley, and barley to 80 mesh for use.

Pineapple: Wash, peel, and dice. 1:10 (g/ml) was mixed with water and beaten to obtain a pineapple pretreatment solution for later use.

2. Preparation of mask liquid

(1) The addition ratios of oats, highland barley, barley and water are shown in Table 7. After mixing, let stand for 48 hours at room temperature.

The addition ratio of table 7 mixed pretreatment liquid

raw material	parts by mass
water	100
oat	3
highland barley	1
barley	1

(2) the pineapple pretreatment solution and the mixed pretreatment solution obtained in step (1) are mixed according to the mass ratio of 1:200 (g/ml), heated at 85 ° C for 4 hours, and stirred at 500 rad/min; cooled to room temperature, filtered to remove impurities, Leave the supernatant (opaque).

(3) adding Tremella polysaccharide in a mass ratio of 1:800 (g/mL), homogenizing with a homogenizer for 50 minutes, to obtain a pineapple extract composition.

3. Sterilization

Fill the pineapple extract composition into a mask bag (containing mask cloth).

Sterilize at 80°C for 2 hours.

Example 8

This example is used to illustrate the preparation method of the pineapple extract composition of the present invention.

1. Raw material processing (food grade)

Oats, highland barley, and barley: crush the oats, barley, and barley to 80 mesh for use.

Pineapple: Wash, peel, and dice. 1:20 (g/ml) was mixed with water and beaten to obtain a pineapple pretreatment solution for later use.

2. Preparation of mask liquid

(1) The addition ratio of oats, highland barley, barley and water is shown in Table 8. After mixing, let stand for 60 hours at room temperature.

Table 8 Addition ratio of mixed pretreatment solution

raw material	parts by mass
water	100
oat	10
highland barley	5
barley	5

(2) the pineapple pretreatment solution and the mixed pretreatment solution obtained in step (1) are mixed according to the mass ratio of 1:500 (g/ml), heated at 90 ° C for 5 hours, and stirred at 800 rad/min; cooled to room temperature, filtered to remove impurities, Leave the supernatant (opaque).

(3) adding Tremella polysaccharide in a mass ratio of 1:1000 (g/mL), and homogenizing with a homogenizer for 60 minutes to obtain a pineapple extract composition.

3. Sterilization

Fill the pineapple extract composition into a mask bag (containing mask cloth).

Sterilize at 90°C for 1 hour.

Example 9

This example is used to illustrate the method for preparing a whitening cream using the pineapple extract composition of Example 3 of the present invention.

1. Prepare a whitening cream, wherein the ingredients and proportions of the cream are shown in Table 9.

Table 9 Cream ingredients and addition ratio

2. Mix the water phase components in Table 9, heat at 85° C. for 30 min, and stir at 250 rad/min to obtain mixture 1.

3. Mix the oil phase components in Table 9 and heat at 85° C. until dissolved to obtain mixture 2.

4. Add mixture 2 to mixture 1 at 85° C., and homogenize for 30 minutes to obtain whitening cream 1.

Example 10

This example is used to illustrate the method for preparing a whitening cream using the pineapple extract composition of Example 8 of the present invention.

1. Prepare an anti-whitening cream, wherein the ingredients and proportions of the cream are shown in Table 10.

Table 10 Cream ingredients and addition ratio

2. Mix the water phase components in Table 10, heat at 85° C. for 30 min, and stir at 250 rad/min to obtain mixture 1.

3. Mix the oil phase components in Table 10, and heat at 85° C. until dissolved to obtain mixture 2.

4. Add mixture 2 to mixture 1 at 85° C., and homogenize for 30 minutes to obtain whitening cream 2.

Example 11

This example is used to prepare the whitening cream 3 which is compared with Example 9 and Example 10.

The difference is that the composition of the pineapple extract is replaced with water, and the other ingredients, proportions and operation steps remain unchanged, and the face cream 3 is prepared.

Test Example 1

This test example is used to illustrate the tyrosinase activity of the pineapple extract composition (pineapple 1) in Example 2 on B16-F10.

B16 melanoma cells were plated in 6-well plates, and the number of cells was 5× ^{10 4} , and cultured overnight. Discard the old medium, replace with fresh serum-free medium, add 1 μM α-MSH for stimulation, establish a cell model, and add pineapple extract composition (pineapple 1) at 1.25%, and test after 48 hours. As a positive control, kojic acid was selected at a concentration of 100 μg/ml. The results are shown in Figure 4.

Figure 4 shows the tyrosinase activity of the pineapple extract composition acting on B16 melanoma cells, α-MSH is the model group, kojic acid is the positive control, and pineapple 1 is the pineapple extract composition, after statistical difference analysis Pineapple 1 sample can be obtained, P<0.05, which is significant. Table 11 shows the significant difference analysis of tyrosinase activity after the pineapple extract composition acts on B16 melanoma cells in Test Example 1. There is a significant statistical difference between the model group and the blank group. The model is successfully established. The extract composition had a reduced effect on tyrosinase in the model group.

Table 11 Significant difference analysis of tyrosinase activity of pineapple extract composition acting on B16-F10

	blank group	α-MSH	Kojic acid		Pineapple 1
average value	63.87281751	100	57.83918195	88.45995329
error	3.563226519	3.386418359	2.327785138	6.308142069

Test Example 2

This test example is used to illustrate the tyrosinase inhibition rate of the pineapple extract composition (pineapple 1) in Examples 3 to 5 on B16.

The specific method and steps are the same as those in Test Example 1, except that the pineapple extract composition prepared in Examples 3 to 5 is used.

Results: Table 12 shows the analysis of significant differences in tyrosinase activity after the pineapple extract compositions prepared in Examples 3 to 5 acted on B16. There was a significant statistical difference between the model group and the blank group, and the model was successfully established. The pineapple extract composition prepared by the invention has a significant inhibitory effect on tyrosinase activity.

Table 12 Significant difference analysis of tyrosinase activity after pineapple extract composition acts on B16-F10

sample name	Tyrosinase activity	SD	P-value
blank group	63.87	3.56	/
α-MSH	100	3.39	0.007##
Alpha Arbutin	58.20	2.97	0.000**
Example 3	69.76	4.31	0.016*
Example 4	60.23	3.11	0.001**
Example 5	79.05	4.07	0.022*

Remarks: When using t-test method for statistical analysis, the significance of NC group compared with BC group is indicated by #, P-value<0.05 is indicated as #, P-value＜0.01 is indicated as ##; PC group, sample group Compared with the NC group, significance is indicated by *, P-value<0.05 is indicated by *, and P-value<0.01 is indicated by **.

Test Example 3

This test example is used to illustrate the inhibitory effect of the pineapple extract composition in Examples 2 to 8 on melanin synthesis on B16 cells.

B16 cells with good logarithmic growth phase were inoculated into 6-well plates at a density of 1×10 ⁵ cells/mL, and placed in an incubator at 37°C and cultured overnight in a 5% CO ₂ environment; the medium was replaced with 1.5% Concentrations of the compositions prepared in Examples 2 to 8, the negative control group was untreated, and 1 mg/mL arbutin was used as a positive control. After culturing for 48 hours, the culture medium was aspirated, washed with PBS, and then digested with 250 μL trypsin. For 4 min, add 1 mL of culture solution to stop digestion, centrifuge at 1500 rmp for 3 min, aspirate the supernatant, add 1 mL of 1 M sodium hydroxide solution containing 10% DMSO, ultrasonically disperse for 3 min, take 20 μL of the above solution for BCA assay, and the remaining solution in a water bath at 80°C 30min; shake and mix, centrifuge at 10000rmp for 2min, pipette 200 μL of the supernatant to a 96-well plate and read the absorbance value at 405nm.

Changes in melanin content=(OD value of assay well-OD value of blank control)/(OD value of cell control group-OD value of blank control). Each group of components was replicated with 3 samples, and the rate of change in melanin content needed to be corrected for BCA content.

Results: Table 13 shows the results of the inhibitory effect of the pineapple extract composition prepared by the present invention on melanin synthesis acting on B16. It can be seen from the table that the pineapple extract composition has a significant inhibitory effect on melanin synthesis.

Table 13 Significant difference analysis of melanin inhibition rate after pineapple extract composition acts on B16-F10

sample name	Tyrosinase inhibition rate (%)	SD	P-value
control group	0	0	/
Alpha Arbutin	41.88	1.56	0.000**
Example 2	25.76	1.44	0.002**
Example 3	24.51	2.21	0.011*
Example 4	24.19	1.43	0.011**
Example 5	25.64	1.52	0.01**
Example 6	17.65	1.3	0.032*
Example 7	15.38	2.63	0.021*
Example 8	13.74	1.71	0.165*

Remarks: When the t-test method is used for statistical analysis, the NC group is compared with the BC group, the significance is represented by #, P-value<0.05 is represented as #, P-value<0.01 is represented as ##; PC group, sample group Compared with the NC group, significance is indicated by *, P-value<0.05 is indicated by *, and P-value<0.01 is indicated by **.

Test Example 4 (Comparative Example)

This test example is used to compare the whitening efficacy of the cream prepared in Example 9, Example 10 and the cream prepared in Example 11 on human body.

1. Basic principles

According to the Declaration of Helsinki, the selection of volunteers follows the medical and ethical standards for human testing, and all volunteer testing must be done voluntarily by the volunteers and signed informed consent before testing. Before the volunteers sign the informed consent form, the testers need to inform the volunteers of the purpose of the test, possible benefits, potential risks and problems, and related rights and obligations.

2. Volunteer screening

Volunteers who meet all of the following criteria will be selected.

(1) Volunteers aged 18 to 45 years old, both male and female, with an ITA between 20 and 41°, except for women who are pregnant or breastfeeding, or women who are planning to become pregnant within six months

(2) The test site has not undergone skin treatment, cosmetic or other tests that may affect the results;

(3) Those who have not used hormone drugs and immunosuppressants in the past month;

(4) Those who have not participated in other clinical trials at the test site at present or in the last three months.

(5) Voluntarily participate and sign the informed consent.

(6) The specified content can be completed according to the requirements of the test plan.

3. Experimental design

(1) Test environment: temperature: 20±2°C; humidity: (50±10)%, and real-time dynamic monitoring is performed.

(2) Valid number of people: 60 people aged 18 to 45, 30 people use Cream 1 and Cream 3, and 30 people use Cream 2 and Cream 3.

(3) Experimental equipment: MPA580 (Germany CK company) multi-probe, VISIA CR.

(4) Test area: face

(5) Test time points and indicators:

Use for 4 consecutive weeks, once in the morning and once in the evening. The left face uses the example sample, and the right face uses the comparative sample. The skin melanin index, ITA, and VISIA CR of both cheeks were collected at 0d, 14d, and 28d, respectively. And fill in the questionnaire in Annex 1 at 28d. Monitoring is required to ensure that subjects correctly distinguish between the test product and the control product on both sides and the continuous use sample.

(6) Data analysis:

Statistical methods: Normal data were described by mean and standard deviation, and t-test was used for comparison; non-normal data were described by median and interquartile range, and non-parametric test was used for comparison. The difference was statistically significant at p<0.05.

result:

Figure 11 shows the effect of the sample (cream 1) prepared in Example 9 in Test Example 4 on the skin melanin index (time point comparison), wherein, **, p<0.01, indicating that compared with the background, it has a very high Significant difference; n.s., p>0.05, indicating no significant difference compared with the background. It can be seen from Figure 11 that after the 28-day sample use cycle, the skin melanin value of the sample group was extremely significantly lower than the background. There was no significant difference between the skin melanin value of the control group and the background.

Figure 12 shows the effect of the sample (cream 2) prepared in Example 10 in Test Example 4 on the skin ITA value (time point comparison), wherein, **, p<0.01, indicating that compared with the background, it has a very high Significant difference; n.s., p>0.05, indicating no significant difference compared with the background. It can be seen from Figure 12 that after 14 days of sample use cycle, the skin ITA value of the sample (cream 2) group is extremely significantly higher than the background. There was no significant difference between the skin ITA value of the control group and the background.

Figure 13 shows the proportion of consumers who are satisfied with the efficacy evaluation of consumers in Test Example 4. As can be seen from Figure 13: after the 28-day sample use period, 30 subjects compared the satisfaction of the sample and the control in four aspects of moisturizing, whitening effect, brightening skin tone, and lightening spots, and the results showed that the sample group (cream 1 ) was more satisfied than the control group in three aspects: whitening effect, brightening skin tone and lightening spots.

Figure 14 shows the proportion of the overall number of satisfied consumers in Test Example 4. It can be seen from Figure 14 that after the 28-day sample use period, 30 subjects were satisfied with the overall use effect, and the proportion of satisfied people in the sample group (cream 2) reached 80%, which was higher than that in the control group.

FIG. 5 is a graph showing an example of the change in pigmentation of the volunteer 1 after using the cream 1 prepared in Example 9 in Test Example 4. FIG. FIG. 6 is a graph showing an example of the change in pigmentation of Volunteer 2 after using Cream 1 prepared in Example 9 in Test Example 4. FIG. FIG. 7 is a graph showing an example of the change in pigmentation of Volunteer 3 after using Cream 1 prepared in Example 9 in Test Example 4. FIG. It can be seen from Figures 5-7 that the pigmentation was improved 14d and 28d after using Cream 1.

FIG. 8 is a graph showing an example of the change in pigmentation of volunteer 1 after using the cream 2 prepared in Example 10 in Test Example 4. FIG. FIG. 9 is a graph showing an example of the change in pigmentation of Volunteer 2 after using the cream 2 prepared in Example 10 in Test Example 4. FIG. FIG. 10 is a graph showing an example of the change in pigmentation of Volunteer 3 after using Cream 2 prepared in Example 10 in Test Example 4. FIG. It can be seen from Figures 8-10 that the pigmentation was improved 14d and 28d after using Cream 1.

It is shown that the facial cream prepared by using the pineapple extract composition of the present invention has a significant whitening effect compared to the base cream prepared by adding only water in Example 11 (comparative example). It showed a good whitening effect in terms of ITA value and stain area.

Although this invention has been described to a certain extent, it will be apparent that suitable changes in various conditions may be made without departing from the spirit and scope of the invention. It is to be understood that the invention is not limited to the embodiments described, but is to be included within the scope of the claims, which include equivalents for each of the elements described.

Claims (15)

1. An application of a pineapple extract composition in preparing a product with a whitening effect, characterized in that the pineapple extract composition is prepared by extracting the following raw materials: pineapple, oats, highland barley, barley, tremella polysaccharide and water ;

   Wherein, the parts by mass of each component in the pineapple extract composition are: 0.1-10 parts of pineapple, 0.1-10 parts of oats, 0.1-5 parts of highland barley, 0.1-5 parts of coix seed, 0.05-2 parts of Tremella polysaccharide, water 100 copies;

   Preferably, 0.5-5 parts of pineapple, 0.5-5 parts of oats, 0.2-2 parts of highland barley, 0.2-2 parts of coix seed, 0.1-1.5 parts of Tremella polysaccharide, and 100 parts of water;

   More preferably, 0.5-3 parts of pineapple, 0.5-2.5 parts of oats, 0.5-1.5 parts of highland barley, 0.5-1.5 parts of barley, 0.2-1 part of Tremella polysaccharide, and 100 parts of water.
2. The application according to claim 1, wherein the pineapple extract composition is prepared by the following method:

   (1) oats, highland barley and barley are crushed and sieved;

   (2) pineapple cleaning, peeling, dicing, and beating after mixing with water to obtain pineapple pretreatment liquid;

   (3) in step (1), gained oat, highland barley, coix seed and water are mixed and left standstill to obtain mixed pretreatment solution;

   (4) the pineapple pretreatment solution obtained in step (2) and the mixed pretreatment solution obtained in step (3) are mixed, heated and stirred, and the supernatant is left by cooling and filtration;

   (5) adding Tremella polysaccharide to the supernatant obtained in step (4), and homogenizing to obtain the pineapple extract composition.
3. The application according to claim 2, characterized in that, in step (1), the oats, highland barley, and barley are pulverized and passed through an 80-mesh sieve.
4. The application according to claim 2 or 3, wherein in step (2), the ratio of pineapple to water is 1:0.1～20g/mL, preferably 1:0.2～10g/mL, more preferably 1:0.2～10g/mL 0.5 to 5 g/mL, more preferably 1:1 g/mL.
5. The application according to any one of claims 2 to 4, wherein in step (3), the standing time is 12-60 hours, preferably 12-48 hours, and most preferably 24 hours.
6. The application according to any one of claims 2 to 5, wherein in step (4), the addition ratio of the pineapple treatment solution obtained in step (2) and the mixed pretreatment solution obtained in step (3) is 1:1 ~500 g/mL, preferably 1:5 to 200 g/mL, more preferably 1:10 to 100 g/mL, still more preferably 1:20 to 50 g/mL.
7. The application according to any one of claims 2 to 6, wherein in step (4), the heating temperature is 20-90°C, preferably 25-85°C, more preferably 30-80°C, More preferably, it is 40～75℃;

   The heating time is 0.5 to 5 hours, preferably 0.5 to 4 hours, more preferably 1 to 3 hours, further preferably 1 to 2 hours; and/or

   The stirring rate is 100-800 r/min, preferably 100-500 r/min, more preferably 100-400 r/min, further preferably 200-300 r/min.
8. The application according to any one of claims 2 to 7, wherein in step (5), the ratio of the Tremella polysaccharide to the supernatant obtained in step (4) is 1:1 to 1000 g/mL, preferably 1:100-800 g/mL; more preferably 1:100-500 g/mL, most preferably 1:200-400 g/mL; and/or

   The homogenization time is 1 to 60 minutes, preferably 5 to 50 minutes, more preferably 5 to 30 minutes, and further preferably 10 to 20 minutes.
9. The application according to any one of claims 2 to 8, wherein the method further comprises the following steps:

   (6) sterilizing the pineapple extract composition obtained in step (5);

   Preferably, the sterilization treatment method is heat sterilization;

   More preferably, the heating sterilization temperature is 60-90°C, preferably 70-80°C, most preferably 75°C; and/or the heating sterilization time is 1-5 hours, preferably 1-3 hours , most preferably 2 hours.
10. The application according to any one of claims 1 to 9, wherein the product with whitening effect comprises:

    the pineapple extract composition; and/or

    one or more cosmetic ingredients selected from plants;

    Preferably, the cosmetic ingredients are selected from one or more of the following: olive oil, matsutake mushrooms, green thorn fruit oil, betaine, Ganoderma lucidum, and Rhodiola rosea.
11. The application according to any one of claims 1 to 10, characterized in that:

    The whitening effect is inhibition of tyrosinase activity; and/or

    The whitening effect is to inhibit the synthesis of melanin.
12. The application according to any one of claims 1 to 10, characterized in that: the dosage form of the product with whitening effect is selected from one or more of the following: cream, lotion, water, gel, oil , powder, mud, aerosol, paste, film;

    Preferably, the cosmetic product is a cream, mask, lotion or essence.
13. Application according to claim 12, is characterized in that: when described product with whitening effect is cream, its preparation method comprises the following steps:

    (a) adding an aqueous phase component to the sterilized pineapple extract composition, heating and stirring to obtain an aqueous phase mixture;

    (b) mixing oil phase components, heating and dissolving to obtain oil phase mixture;

    (c) adding the oil phase mixture prepared in step (b) to the water phase mixture prepared in step (a), and homogenizing to obtain the cosmetic product.
14. The application according to claim 13, wherein:

    In the step (a), the water phase component includes vegetable oil and an antibacterial agent; and/or in the step (b), the oil phase component includes vegetable oil and an emulsifier;

    Preferably, the vegetable oil is selected from one or more of the following: glycerin, green thorn fruit oil, olive oil, peony seed oil, macadamia nut oil, babassu oil, toona japonica oil;

    Preferably, the antibacterial agent is selected from one or more of the following: hexanediol, methylparaben, propylparaben, phenoxyethanol; and/or

    Preferably, the emulsifier is selected from one or more of the following: wheat emulsifier, polyglycerol-3 diisostearate, dipolyhydroxystearic acid, and yeoleyl glucoside.
15. The application according to claim 13 or 14, characterized in that: in the step (a), the ratio of the sterilized pineapple extract composition, vegetable oil and antibacterial agent is 80～95:1～10: 0.01～1g/g/g, preferably 89～93:2～5:0.05～0.2g/g/g;

    In the step (b), the ratio of the vegetable oil to the emulsifier is 1～10:2～15g/g, preferably 2～5:5～10g/g; and/or

    In the step (c), the ratio of the oil phase mixture to the water phase mixture is 2-25:0.5-5g/g, preferably 5-10:1-2g/g;

    Preferably, in the step (a), the step (b) and the step (c), the heating temperature is 70-120°C, more preferably 70-100°C, further preferably 80-90°C; and /or

    Preferably, in the step (c), the homogenization time is 15-45 minutes, more preferably 20-40 minutes, and even more preferably 25-35 minutes.

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