CN113274334B

CN113274334B - Pineapple extract composition with anti-aging effect, preparation method and application thereof

Info

Publication number: CN113274334B
Application number: CN202110412291.5A
Authority: CN
Inventors: 刘继涛; 吴迪; 安全; 刘平平; 权强华; 王昌涛
Original assignee: Beiqingjiahua Huangshan Technology Co ltd; Yunnan Baiyao Group Co Ltd
Current assignee: Yunnan Baiyao Group Health Products Co ltd; Yunnan Baiyao Group Shanghai Technology Co ltd
Priority date: 2021-04-16
Filing date: 2021-04-16
Publication date: 2022-07-22
Anticipated expiration: 2041-04-16
Also published as: CN113274334A; WO2022218170A1

Abstract

The invention provides a pineapple extract composition with an anti-aging effect, which is characterized by being prepared by extracting the following raw materials: pineapple, oat, pawpaw, fructus momordicae, kudzu root powder, vegetable oil and water, and also provides a preparation method and application thereof. The inventor proves through experiments that the pineapple extract composition can increase degradation of related peroxides in cells, reduce MDA content in the cells after UVA irradiation, and increase degradation of the related peroxides in the cells, thereby reducing damage of the peroxides to the cells and achieving the anti-aging effect.

Description

Pineapple extract composition with anti-aging effect, preparation method and application thereof

Technical Field

The invention belongs to the field of cosmetics, and particularly relates to a pineapple extract composition with an anti-aging effect, and a preparation method and application thereof.

Background

Natural cosmetics are nowadays more and more favored by people. The natural cosmetics are natural in formula components, and are not added with potential irritant components such as essence, preservative and the like.

Enzymolysis is a technology widely applied to the fields of food, medicine and cosmetics, and can modify macromolecular components into small molecules and generate other active substances such as polysaccharide, polypeptide and the like. The enzymes contained in plants are various and comprise protease, cellulase, amylase, esterase and the like, and macromolecular protein can be enzymolyzed into oligopeptide, starch and cellulase are enzymolyzed into oligosaccharide, and fat is enzymolyzed into glycerol and fatty acid by utilizing the enzymes contained in the plants. The products are applied to cosmetics, so that the functional components can be absorbed by the skin more conveniently, and a certain effect is finally achieved.

Disclosure of Invention

Therefore, the invention aims to overcome the defects in the prior art and provide the pineapple extract composition with the anti-aging effect, the preparation method and the application thereof.

In order to achieve the above object, a first aspect of the present invention provides an anti-aging pineapple extract composition, wherein the pineapple extract composition is prepared by extracting the following raw materials: pineapple, oat, pawpaw, fructus momordicae, radix puerariae powder, vegetable oil and water; and the pineapple extract composition is prepared by mixing oat pretreatment liquid prepared from oat and water with mixed pretreatment liquid containing pineapple, pawpaw and momordica grosvenori, and then adding kudzu root powder and vegetable oil.

The pineapple extract composition of the first aspect of the present invention, wherein the vegetable oil is selected from one or more of: olive oil, prinsepia utilis royle oil, macadamia nut oil, peony seed oil and ailanthus altissima oil; and/or

The pineapple extract composition comprises the following components in parts by weight: 0.01-5 parts of pineapple, 0.1-10 parts of oat, 0.01-5 parts of pawpaw, 0.01-5 parts of momordica grosvenori, 0.5-10 parts of kudzu root powder and 100 parts of water;

preferably, 0.05-2 parts of pineapple, 0.5-5 parts of oat, 0.1-1 part of pawpaw, 0.1-1 part of momordica grosvenori, 1-8 parts of kudzu root powder and 100 parts of water;

more preferably, the pineapple is 0.1-1 part by weight, the oat is 0.5-2 parts by weight, the pawpaw is 0.1-0.5 part by weight, the momordica grosvenori is 0.1-0.5 part by weight, the kudzu root powder is 2-5 parts by weight, and the water is 100 parts by weight.

In a second aspect, the present invention provides a method for preparing the pineapple extract composition of the first aspect, the method comprising the steps of:

(1) mixing and standing oat and water to obtain oat pretreatment solution;

(2) respectively cleaning pineapple, pawpaw and momordica grosvenori, peeling, cutting into blocks, mixing with water, pulping, and standing at room temperature to obtain mixed pretreatment liquid;

(3) mixing the oat pretreatment liquid obtained in the step (1) with the mixed pretreatment liquid obtained in the step (2), heating and stirring, cooling and filtering to obtain a supernatant;

(4) and (4) adding kudzu root powder and vegetable oil into the supernatant obtained in the step (3), and homogenizing to obtain the pineapple extract composition.

According to the method of the second aspect of the invention, in the step (1), the oat is ground and then sieved by a sieve with 60-100 meshes, preferably 80 meshes;

the mass ratio of the oats to the water is 1: 1-500, preferably 1: 5-200, more preferably 1: 20-100, and most preferably 1: 50; and/or

The standing time is 8-60 hours, preferably 12-48 hours, and most preferably 24 hours.

The method according to the second aspect of the invention, wherein in the step (2), the mass ratio of the pineapple to the water is 1: 1-150, preferably 1: 10-100, more preferably 1: 20-80, and most preferably 1: 20-50;

the mass ratio of the pawpaw to the water is 1: 1-200, preferably 1: 10-150, more preferably 1: 20-100, and most preferably 1: 50-80;

the mass ratio of the momordica grosvenori to the water is 1: 1-200, preferably 1: 10-150, more preferably 1: 20-100, and most preferably 1: 50-80.

The method according to the second aspect of the present invention, wherein, in the step (3), the mass ratio of the mixed pretreatment liquid obtained in the step (2) to the oat pretreatment liquid obtained in the step (1) is 1:1 to 100, preferably 1:1 to 50, more preferably 1:1 to 20, and most preferably 1: 10. the method according to the second aspect of the present invention, wherein, in the step (3), the heating temperature is 20 to 90 ℃, preferably 25 to 85 ℃, more preferably 30 to 80 ℃, and further preferably 40 to 60 ℃;

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

The stirring speed is 50-500 r/min, preferably 50-300 r/min, more preferably 50-200 r/min, and further preferably 50-150 r/min.

The method according to the second aspect of the invention, wherein in the step (4), the mass ratio of the pueraria powder to the supernatant obtained in the step (3) is 1: 10-200, preferably 1: 10-100, more preferably 1: 20-80, and most preferably 1: 25-50; and/or

The homogenization time is 1-60 minutes, preferably 5-50 minutes, more preferably 5-30 minutes, and further preferably 15-20 minutes.

The method according to the second aspect of the invention, wherein the method further comprises the steps of:

(5) sterilizing the pineapple extract composition obtained in the step (4);

preferably, the sterilization treatment mode is heat sterilization;

more preferably, the heating sterilization temperature is 60-100 ℃, preferably 70-90 ℃, and most preferably 80 ℃; and/or the heating sterilization time is 1-5 hours, preferably 1-3 hours, and most preferably 2 hours.

In a third aspect of the invention, the application of a pineapple extract composition in preparing an anti-aging product is provided, wherein the pineapple extract composition comprises pineapple, oat, pawpaw, momordica grosvenori and pueraria powder;

preferably, the pineapple extract composition is the pineapple extract composition of the first aspect or the pineapple extract composition prepared by the method of the second aspect.

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

(1) mixing oat and water to prepare a pretreatment solution. The mixture was allowed to stand at room temperature for 24 hours.

(2) Mixing fructus Ananadis Comosi, fructus Chaenomelis, and fructus Siraitiae Grosvenorii with water, pulping, adding into the mixture obtained in step (1), heating under stirring, and filtering.

(3) Adding the kudzu root powder into the extracting solution in the step (2). Adding vegetable oil, and homogenizing to obtain the pineapple extract composition.

(4) Sterilizing at 100 deg.C for 2 hr, and packaging under aseptic condition.

In the step (1), the ratio of the oat to the water is 1: 1-500 g/mL, preferably 1: 5-300 g/mL, more preferably 1: 20-200 g/mL, and most preferably 1:100 g/mL;

in the step (2), the proportion of the pineapple to the water is 1: 1-150 g/mL, preferably 1: 10-100 g/mL, more preferably 1: 20-80 g/mL, and most preferably 1:50 g/mL;

the ratio of the pawpaw to the water is 1: 1-200 g/mL, preferably 1: 10-150 g/mL, more preferably 1: 20-100 g/mL, and most preferably 1:80 g/mL;

the ratio of the momordica grosvenori to the water is 1: 1-200 g/mL, preferably 1: 10-150 g/mL, more preferably 1: 50-100 g/mL, and most preferably 1:80 g/mL;

the heating temperature is 1-150 ℃, preferably 10-100 ℃, more preferably 20-100 ℃, and most preferably 40 ℃;

the heating time is 1 to 10 hours, preferably 1 to 8 hours, more preferably 1 to 5 hours, and most preferably 4 hours

The stirring speed is 10-600 r/min, preferably 10-500 r/min, more preferably 10-300 r/min, and most preferably 150 r/min.

In the step (3), the ratio of the radix puerariae powder to the extracting solution is 1: 1-100 g/mL, preferably 1: 5-80 g/mL, more preferably 1: 10-70 g/mL, and most preferably 1:50 g/mL;

the addition amount of the vegetable oil is 1-30%, and the preferable amount is 1-20%; more preferably 1 to 10%, most preferably 3%;

the homogenizing speed is 1000-8000 r/min, preferably 1000-7000 r/min, and more preferably 2000-6000 r/min; optimally 5000 r/min;

the homogenization time is 1 to 60 minutes, preferably 1 to 50 minutes, more preferably 10 to 40 minutes, and most preferably 15 minutes.

The pineapple extract composition of the present invention can have the following beneficial effects, but not limited to:

the invention provides application of a pineapple extract composition in preparation of a product with an anti-aging effect.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 shows the effect on glutathione peroxidase (GSH-Px) in fibroblasts in test example 1.

FIG. 2 shows the effect of the content of lipid peroxide in fibroblasts in Experimental example 2.

Fig. 3 shows a standard curve of hydrogen peroxide in experimental example 3.

FIG. 4 shows the effect of catalase enzyme activity in test example 3.

Detailed Description

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

This section generally describes the materials used in the testing of the present invention, as well as the testing methods. Although many materials and methods of operation are known in the art for the purposes of this invention, the invention is nevertheless described herein in as detail as possible. It will be apparent to those skilled in the art that the materials and methods of operation used in the present invention are well within the skill of the art in this context, if not specifically mentioned.

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

reagent:

pineapple, papaya and momordica grosvenori purchased from Hongxing farms in Zhanjiang city, Guangdong province;

oat (fried) was purchased from Zhangjiakou Jianjun oat food Co., Ltd;

radix Puerariae (dried powder) is purchased from Tongrentang big pharmacy;

the instrument comprises:

homogenizer available from IKA.

Fibroblasts, melanocytes, purchased from the institute of mesocarp stem cell bank;

red blood cells purchased from Beijing Xinglong animal farming;

SDS, purchased from alatin;

sodium chloride, purchased from national medicine;

the instrument comprises the following steps:

enzyme-linked immunoassay instrument, purchased from Thermo Fisher Scientific OY, model 1510-00662C.

Homogenizer available from IKA.

Example 1

This example illustrates a method for preparing a pineapple extract composition of the present invention.

1. Raw material treatment

Pineapple: cleaning, peeling and cutting into blocks.

Pawpaw: cleaning, peeling and cutting into blocks.

Momordica grosvenori: cleaning, peeling and cutting into blocks.

Oat: pulverizing, and sieving with 80 mesh sieve.

2. Preparation of pineapple extract composition

(1) The oat powder and water are mixed according to the mass ratio of 1:50, and standing at room temperature for 24 hours to obtain the oat pretreatment solution.

(2) Preparing a mixed pretreatment solution, wherein the adding ratio of the pineapple, the pawpaw and the momordica grosvenori to water is shown in table 1, mixing the pineapple, the pawpaw and the momordica grosvenori with water, pulping, and standing for 2 hours to obtain the mixed pretreatment solution.

TABLE 1 addition ratio of the mixed pretreatment solution

Raw materials	Parts by mass
		Water (I)	100
Pineapple	5
		Pawpaw	2
Momordica grosvenori	2

(3) And (2) mixing the pretreatment solution in the step (2) with the oat pretreatment solution in the step (1) according to the mass ratio of 1:10, mixing, heating at 40 ℃ for 2 hours, and stirring at 50 rad/min; cool to room temperature and filter to remove impurities, leaving a supernatant (opaque).

(4) Adding the mixture, wherein the mass ratio of the mixture to the supernatant is 1:25 of kudzuvine root powder with the mass ratio of 2 percent of olive oil, and homogenizing for 15 minutes by a homogenizer to obtain the pineapple extract composition.

3. Sterilizing treatment

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

Sterilizing at 80 deg.C for 2 hr.

Example 2

1. Raw material processing (food grade)

Pineapple: cleaning, peeling and cutting into blocks.

Pawpaw: cleaning, peeling and cutting into blocks.

Momordica grosvenori: cleaning, peeling and cutting into blocks.

Oat: pulverizing, and sieving with 80 mesh sieve.

2. Preparation of pineapple extract composition

(1) Mixing the oat flour and water according to the mass ratio of 1:100, and standing for 24 hours at room temperature to obtain the oat pretreatment liquid.

(2) Preparing a mixed pretreatment solution, wherein the adding ratio of the pineapple, the pawpaw and the momordica grosvenori to water is shown in table 2, mixing the pineapple, the pawpaw and the momordica grosvenori with water, pulping, and standing for 2 hours to obtain the mixed pretreatment solution.

TABLE 2 addition ratio of pretreatment solution

Starting materials	Parts by mass
		Water (I)	100
Pineapple	2
		Pawpaw	1.25
Momordica grosvenori	1.25

(3) Mixing the pretreatment solution in the step (2) with the oat pretreatment solution in the step (1) according to the mass ratio of 1:10, heating at 60 ℃ for 1 hour, and stirring at 150 rad/min; cool to room temperature and filter to remove impurities, leaving a supernatant (opaque).

(4) Adding the mixture into the mixed solution, wherein the mass ratio of the mixture to the supernatant is 1:50 portions of kudzuvine root powder and 3 portions of olive oil are homogenized for 20 minutes by a homogenizer.

3. Sterilization treatment

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

Sterilizing at 80 deg.C for 2 hr.

Test example 1

This test example is intended to illustrate the effect of the preparation of an extract according to example 1 on the activity of glutathione peroxidase (GSH-Px) in fibroblasts.

1) Preparation of supernatant of cell lysate

Counting fibroblasts in good logarithmic growth phase, inoculating the cells in 6-well culture plate, and controlling the number of cells per well to be 5x10⁵And (4) cells. At 37 ℃ 5% CO₂The cells were incubated overnight under ambient conditions, the medium was discarded, a small amount of PBS (pH 7.4) was added to cover the cells just enough, followed by stimulation of the cells with UVA, and UV irradiation at 7J/cm²Blank groups were not irradiated. PBS was aspirated off and samples at different concentrations (sample concentrations to achieve fibroblast viability of 50%, 80%, 100%) were added to the cells for 24 hours (serum-free DMEM medium was added to the model and blank controls). Taking out, placing on ice, and washing with PBS for 2 times; scraping cells with a cell scraper, collecting into a centrifuge tube, centrifuging at 5000r/min for 5min, discarding the supernatant to obtain cell precipitate, adding 200 μ L lysate to lyse cells, centrifuging at 12000g at 4 deg.C for 5min, and collecting the supernatant to obtain cell lysis supernatant.

2) Preparation of the kit:

a. 30mM NADPH was prepared. 600 microliters of ultrapure water is added into 15mg of NADPH provided by the kit, dissolved and mixed evenly.

b.preparation of a solution of 84mM GSH. To 14mg of GSH, 550. mu.l of ultrapure water was added, dissolved and mixed well. And (3) solution.

And c, preparing GPx detection working solution.

Table 3 formulation of GPx assay working solution

The number of samples (including control) can be determined	1 sample
		30mM NADPH	5μL
84mM GSH	5μL
		Glutathione reductase	1.6μL
GPx detection of working fluid volume	11.6μL

d.15mM peroxide reagent solution preparation. 21.5. mu.l of peroxide reagent (t-Bu-OOH) was added to 10 ml of ultrapure water and mixed to prepare a 15mM peroxide reagent solution.

3) Sample assay

Referring to table 4 below, the detection buffer, the sample to be detected, and the GPx detection working solution were sequentially added using a 96-well plate, and mixed well. The reaction started after the addition of 4. mu.l of 15mM peroxide reagent solution. It should be mixed well properly.

TABLE 4 addition of each solution

	Blank control (blank)	Sample background control	Sample (I)
				Glutathione reductase	185μL	179-187μL	175-183μL
Sample to be tested	—	2-10μL	2-10μL
				GPx detection working solution	11μL	11μL	11μL
15mM peroxide reagent solution	4μL	—	4μL
				Total volume	200μL	200μL	200μL

The A340 values were recorded every 2 minutes using a fluorescent microplate reader for 10 minutes continuously to obtain data at 6 points.

As a result: the effect of pineapple extract composition on glutathione peroxidase enzymatic activity in fibroblasts is shown in fig. 1. The model group is a cell group which is irradiated only by UVA and is not added with a sample, the blank group is a cell group which is not irradiated and is not added with the sample, tests show that the activity of glutathione peroxidase of cells is reduced by UVA irradiation, and statistical differences are made between the sample group and the model group, so that the pineapple extract composition can remarkably improve the activity of glutathione peroxidase of fibroblasts, even higher than that of a positive control VC, and the extract composition can increase the degradation of related peroxides in cells, thereby reducing the damage of peroxidation on the cells and achieving the anti-aging effect.

Test example 2

This test example is intended to illustrate the effect of the preparation of an extract according to example 1 on the content of lipid peroxides in fibroblasts.

1) Preparation of samples:

a. the cell lysate is lysed. 0.1ml of lysate or homogenate per 100 million cells was used. After lysis, the supernatant was centrifuged at 10,000g-12,000g for 10 minutes for subsequent assays. The sample preparation steps such as lysis are preferably performed in an ice bath or at 4 ℃.

2) Preparation of the kit:

preparing TBA stock solution: weighing a proper amount of TBA, and preparing TBA stock solution with the concentration of 0.37% by using TBA preparation solution. For example, 18.5mg of TBA was prepared in 5ml of TBA preparation to a final concentration of 0.37%. The TBA preparation is used after it is completely dissolved, and may be heated to 70 ℃ to facilitate dissolution. TBA stock solutions are more difficult to dissolve, and are heated to 70 ℃ and passed through a vigorous Vortex to facilitate dissolution. The prepared TBA storage liquid is stored at room temperature in a dark place and is effective within at least 3 months.

And b, preparing an appropriate amount of MDA detection working solution according to the number of samples to be detected (including a control) and by referring to the following table 5 before detection.

TABLE 5 preparation of MDA assay working solutions

Number of detections	1 time of	10 times of	20 times (twice)	50 times
					TBA diluent	150μl	1500μl	3000μl	7500μl
TBA stock solution	50μl	500μl	1000μl	2500μl
					Antioxidant agent	3μl	30μl	60μl	150μl

c. Dilution of the standard: and taking a proper amount of standard substance, diluting the standard substance to 1, 2, 5, 10, 20 and 50 mu M with distilled water, and using the diluted standard substance for subsequent preparation of a standard curve.

3) And (3) sample determination:

a. adding 0.1ml of appropriate solution such as homogenate, lysate or PBS as blank control into a centrifuge tube or other appropriate container, adding 0.1ml of the standard substance with different concentrations for preparing standard curve, and adding 0.1ml of sample for determination; subsequently, 0.2ml of MDA assay working solution was added. The assay reaction system was set up with reference to the following table:

TABLE 6 setup of the reaction System

b. After mixing, the mixture was heated in a boiling water bath at 100 ℃ for 15 minutes. Care must be taken to avoid bumping and splashing liquid during heating. When a boiling water bath is used, a centrifuge tube with a cover locked or a screw cap centrifuge tube is used, or a Parafilm is used to seal the opening of the centrifuge tube, and a small hole is punctured by a needle.

c. The water bath was cooled to room temperature and 1000g was centrifuged at room temperature for 10 min. 200. mu.l of the supernatant was added to a 96-well plate, followed by measurement of absorbance at 532nm with a microplate reader. If it is not convenient to measure the absorbance at 532nm, the absorbance between 530 nm and 540nm may also be measured.

d, calculating the MDA content: the molar concentration of MDA can be directly calculated from the standard curve, and after the MDA content in the sample solution is calculated, the MDA content in the original sample can be expressed by the protein content per unit weight, such as mu mol/mg protein.

As a result: the effect of pineapple extract composition on the content of lipid peroxides in fibroblasts is shown in fig. 2. The model group is a cell group which is irradiated by only UVA and is not added with a sample, the blank group is a cell group which is not irradiated and is not added with a sample, tests show that the content of MDA in cells can be increased by UVA irradiation, and statistical difference analysis between the sample group and the model group shows that the pineapple extract composition can remarkably reduce the content of MDA in cells after UVA irradiation, reduce the damage of excessive lipid peroxide to the cells and achieve the anti-aging effect.

Test example 3

This test example is intended to illustrate the effect of the extract preparation mask of example 2 on the activity of catalase in fibroblasts.

1) Preparation work:

a. a250 mM hydrogen peroxide solution was prepared. The kit provides a hydrogen peroxide concentration of about 1M. Since hydrogen peroxide is not very stable, the actual concentration of hydrogen peroxide needs to be self-determined before use. Hydrogen peroxide at a concentration of about 1M was diluted 100-fold with catalase detection buffer provided in the kit to a hydrogen peroxide concentration of about 10 mM. Measurement A240. The hydrogen peroxide concentration (mM) was 22.94X A240. Thereby calculating the actual concentration of hydrogen peroxide provided by the kit. A250 mM hydrogen peroxide solution was then prepared based on the actual hydrogen peroxide concentration.

b. A5 mM hydrogen peroxide solution was prepared. A5 mM hydrogen peroxide solution was prepared based on the actual hydrogen peroxide concentration determined.

c. And preparing a color developing working solution. Dissolving the chromogenic substrate in ice bath, packaging properly and then using, and avoiding repeated freeze thawing as much as possible. The other reagents were placed on an ice bath until use. An appropriate amount of peroxidase was diluted with a chromogenic substrate at a ratio of 1:1000 to prepare a chromogenic working solution. For example, 5. mu.l of peroxidase is added to 5ml of a chromogenic substrate, and the mixture is mixed to obtain 5ml of a chromogenic working solution.

2) Sample preparation: the cells or tissue are lysed with an appropriate lysis solution to obtain a sample. 3) And (3) standard curve determination:

a. 0, 12.5, 25, 50 and 75 microliters of prepared 5mM hydrogen peroxide solution is put into a 1.5ml plastic centrifuge tube, catalase detection buffer solution is respectively added until the final volume is 100 microliters, and the mixture is uniformly mixed.

b. Add 4 μ l each to one well of a 96-well plate. Add 200. mu.l of color developing working solution. A520 is measured after at least 15 minutes incubation at 25 ℃ but preferably the incubation time is not longer than 45 minutes.

4) Sample assay

TABLE 7 addition amount of each solution

	Blank control (blank)	Sample (sample)
			Sample volume	0μl	4μl
Catalase detection buffer	40μl	36μl
			250mM hydrogen peroxide solution	10μl	10μl

a. Referring to Table 7, 4. mu.l of sample was placed in a 1.5ml plastic centrifuge tube, and catalase detection buffer was added to a volume of 40. mu.l and mixed well. An additional 10. mu.l of 250mM hydrogen peroxide solution was added and quickly mixed using a pipette. Referring to Table 1, the reaction was carried out at 25 ℃ for 5 minutes.

b. 450. mu.l of catalase reaction terminator was added, and the reaction was terminated by mixing by inversion or by mixing by Vortex. The following steps c and d were completed within 15 minutes after the termination of the reaction.

c. Add 40. mu.l of catalase assay buffer to a clean plastic centrifuge tube, add 10. mu.l of the above reaction system which had been quenched and mixed well, and mix well.

d. From the 50. mu.l system of the previous step, 10. mu.l was added to one well of a 96-well plate. Add 200 μ l of color developing working solution.

A520 is measured after incubation at 25 ℃ for at least 15 minutes, but the incubation time is preferably not longer than 45 minutes.

5) Calculation of catalase enzyme activity in the sample:

a. a standard curve is calculated. A520 ═ k [ micromoles of hydrogen peroxide ] + b, the values of k and b were calculated from a standard curve. Fig. 3 shows a standard curve of hydrogen peroxide in experimental example 3.

b. The micromolar residual hydrogen peroxide in the sample was calculated.

Micromole number of residual hydrogen peroxide (A520-b)/k

c. Definition of catalase enzyme activity unit: 1unit of enzyme activity (1unit) can catalyze and decompose 1 micromole of hydrogen peroxide in 1 minute at the temperature of 25 ℃ and the pH value of 7.0.

d. Catalase activity for cell or tissue samples was calculated:

[ sample catalase activity ] - (micromole hydrogen peroxide consumed ] X [ dilution ]/([ reaction minutes ] X [ sample volume ] X [ protein concentration ])

[ Catalase Activity of sample ] is in units of units/mg protein

[ micromoles of hydrogen peroxide consumed ] - [ micromoles of residual hydrogen peroxide for blank ] - [ micromoles of residual hydrogen peroxide for sample ]

[ dilution factor ] ═ 250

[ reaction minutes ] means the actual reaction minutes

[ sample volume ] is X. mu.l in Table 2, expressed in ml as X/1000 ml.

[ protein concentration ] the protein concentration in a sample is in mg/ml when X. mu.l of the sample is taken.

As a result: the effect of pineapple extract composition on catalase enzyme activity in fibroblasts is shown in fig. 4. The model group is a cell group which is irradiated only by UVA and is not added with a sample, the blank group is a cell group which is not irradiated and is not added with the sample, tests show that after UVA is irradiated, the enzymatic activity of catalase in cells can be reduced, and after statistical difference analysis is carried out on the sample group and the model group, the pineapple extract composition can remarkably increase the enzymatic activity of catalase in cells, and the effect is equivalent to that of positive control VC. The pineapple extract composition can increase the degradation of related peroxides in cells, thereby reducing the damage of the peroxides to the cells and achieving the anti-aging effect.

Although the present invention has been described to a certain extent, it is apparent that appropriate changes in the respective conditions may be made without departing from the spirit and scope of the present invention. It is to be understood that the invention is not limited to the described embodiments, but is to be accorded the scope consistent with the claims, including equivalents of each element described.

Claims

1. The pineapple extract composition with the anti-aging effect is characterized by being prepared by extracting the following raw materials: pineapple, oat, pawpaw, fructus momordicae, radix puerariae powder, vegetable oil and water; the pineapple extract composition is prepared by mixing oat pretreatment liquid prepared from oat and water with mixed pretreatment liquid containing pineapple, pawpaw and momordica grosvenori, and then adding kudzu root powder and vegetable oil; wherein the content of the first and second substances,

the pineapple extract composition comprises the following components in parts by weight: 0.01-5 parts of pineapple, 0.1-10 parts of oat, 0.01-5 parts of pawpaw, 0.01-5 parts of momordica grosvenori, 0.5-10 parts of kudzu root powder and 100 parts of water; and also,

the mass ratio of the mixed pretreatment liquid to the oat pretreatment liquid is 1: 10.

2. The pineapple extract composition of claim 1, wherein the vegetable oil is selected from one or more of the following: olive oil, prinsepia utilis royle oil, macadamia nut oil, peony seed oil and ailanthus altissima oil.

3. The pineapple extract composition according to claim 2, wherein the pineapple extract composition comprises 0.05 to 2 parts of pineapple, 0.5 to 5 parts of oat, 0.1 to 1 part of papaya, 0.1 to 1 part of momordica grosvenori, 1 to 8 parts of kudzu root powder and 100 parts of water.

4. The pineapple extract composition according to claim 3, wherein the pineapple extract composition comprises 0.1 to 1 part of pineapple, 0.5 to 2 parts of oat, 0.1 to 0.5 part of papaya, 0.1 to 0.5 part of momordica grosvenori, 2 to 5 parts of pueraria powder and 100 parts of water.

5. The method for preparing the pineapple extract liquid composition of any one of claims 1 to 4, wherein the pineapple extract liquid composition is prepared by:

(1) mixing and standing oat and water to obtain oat pretreatment liquid;

(2) respectively cleaning, peeling and cutting pineapple, pawpaw and momordica grosvenori, mixing with water in proportion, pulping and standing to obtain mixed pretreatment liquid;

6. The method for preparing the pineapple extract composition of claim 5, wherein in the step (2): the standing time is 1-5 hours.

7. The method for preparing a pineapple extract composition according to claim 6, wherein the standing time is 1 to 3 hours.

8. The method for preparing the pineapple extract liquid composition of claim 7, wherein the standing time is 2 hours.

9. The method according to claim 5, wherein in the step (1), the oat is ground and sieved by a sieve of 60-100 meshes;

the mass ratio of the oats to the water is 1: 1-500; and/or

The standing time is 8-60 hours.

10. The method according to claim 9, wherein in step (1), the oat is ground and sieved through a 80-mesh sieve;

the mass ratio of the oats to the water is 1: 5-200; and/or

The standing time is 12-48 hours.

11. The method according to claim 10, wherein, in step (1),

the mass ratio of the oats to the water is 1: 20-100; and/or

The standing time was 24 hours.

12. The method according to claim 11, wherein the oat and water are present in a mass ratio of 1: 50.

13. The method according to claim 5, wherein in the step (2), the mass ratio of the pineapple to the water is 1: 1-150;

the mass ratio of the pawpaw to the water is 1: 1-200; and/or

The mass ratio of the momordica grosvenori to the water is 1: 1-200.

14. The method of claim 13, wherein, in step (2),

the mass ratio of the pineapple to the water is 1: 10-100;

the mass ratio of the pawpaw to the water is 1: 10-150; and/or

The mass ratio of the momordica grosvenori to the water is 1: 10-150.

15. The method of claim 14, wherein, in step (2),

the mass ratio of the pineapples to the water is 1: 20-80;

the mass ratio of the pawpaw to the water is 1: 20-100;

the mass ratio of the momordica grosvenori to the water is 1: 20-100.

16. The method of claim 15, wherein, in step (2),

the mass ratio of the pineapple to the water is 1: 20-50;

the mass ratio of the pawpaw to the water is 1: 50-80;

the mass ratio of the momordica grosvenori to the water is 1: 50-80.

17. The method according to claim 5, wherein in the step (3), the mass ratio of the mixed pretreatment solution obtained in the step (2) to the oat pretreatment solution obtained in the step (1) is 1: 1-100.

18. The method according to claim 17, wherein in the step (3), the mass ratio of the mixed pretreatment solution obtained in the step (2) to the oat pretreatment solution obtained in the step (1) is 1: 1-50.

19. The method according to claim 18, wherein in the step (3), the mass ratio of the mixed pretreatment solution obtained in the step (2) to the oat pretreatment solution obtained in the step (1) is 1: 1-20.

20. The method according to claim 5, wherein in the step (3), the heating temperature is 20-90 ℃;

the heating time is 0.5-5 hours; and/or

The stirring speed is 50-500 r/min.

21. The method of claim 20, wherein, in step (3),

the heating temperature is 25-85 ℃;

the heating time is 0.5-4 hours; and/or

The stirring speed is 50-300 r/min.

22. The method of claim 21, wherein, in step (3),

the heating temperature is 30-80 ℃;

the heating time is 1-3 hours; and/or

The stirring speed is 50-200 r/min.

23. The method of claim 22, wherein, in step (3),

the heating temperature is 40-60 ℃;

the heating time is 1-2 hours; and/or

The stirring speed is 50-150 r/min.

24. The method according to claim 5, wherein in the step (4), the mass ratio of the kudzu root powder to the supernatant obtained in the step (3) is 1: 10-200; and/or

The homogenizing time is 1-60 minutes.

25. The method as claimed in claim 24, wherein in the step (4), the mass ratio of the kudzu root powder to the supernatant obtained in the step (3) is 1: 10-100; and/or

The homogenization time is 5-50 minutes.

26. The method as claimed in claim 25, wherein in the step (4), the mass ratio of the kudzu root powder to the supernatant obtained in the step (3) is 1: 20-80; and/or

The homogenizing time is 5-30 minutes.

27. The method as claimed in claim 26, wherein in the step (4), the mass ratio of the kudzu root powder to the supernatant obtained in the step (3) is 1: 25-50; and/or

The homogenizing time is 15-20 minutes.

28. The method of claim 5, further comprising the steps of:

(5) and (4) sterilizing the pineapple extract composition obtained in the step (4).

29. The method of claim 28, wherein the sterilization treatment is heat sterilization.

30. The method of claim 29,

the heating and sterilizing temperature is 60-100 ℃; and/or

The heating sterilization time is 1-5 hours.

31. The method of claim 30,

the heating and sterilizing temperature is 70-90 ℃; and/or

The heating sterilization time is 1-3 hours.

32. The method of claim 31,

the heating sterilization temperature is 80 ℃; and/or

The heat sterilization time is 2 hours.

33. The application of a pineapple extract composition in preparing an anti-aging product is characterized in that the pineapple extract composition comprises pineapple, oat, pawpaw, fructus momordicae and kudzu root powder; wherein the content of the first and second substances,

the pineapple extract composition is the pineapple extract composition of any one of claims 1 to 4 or the pineapple extract composition prepared by the method of any one of claims 5 to 32.