CN113576970A

CN113576970A - Anti-aging plant extract composition and preparation and application thereof

Info

Publication number: CN113576970A
Application number: CN202110908238.4A
Authority: CN
Inventors: 曹茜; 周秋娜; 金荣熙; 姚清; 申彦晟; 金延埈
Original assignee: Cosmax China Cosmetics Co Ltd
Current assignee: Kesimeshi Shanghai Testing Technology Co ltd; Cosmax China Cosmetics Co Ltd
Priority date: 2021-08-09
Filing date: 2021-08-09
Publication date: 2021-11-02
Anticipated expiration: 2041-08-09
Also published as: CN113576970B

Abstract

The invention discloses an anti-aging plant extract composition, and a preparation method and application thereof. The invention adopts the following steps: flavescent sophora root: the kelp is prepared by crushing, primarily extracting and concentrating the kelp according to the mass ratio of (1-3) to (1-3). The extract is composed of the extracting solutions of perilla, sophora flavescens and kelp, the plant extract composition is prepared by the processes of crushing, extracting, concentrating, formulating, sterilizing and the like, the synergistic effect of the components is good, the autophagy process of skin aging cells can be influenced, the effect is achieved, the property is mild, the absorption is easy, no stimulation is generated, the requirements of people on safety, effectiveness and zero burden of cosmetics are particularly met, and the plant extract composition can be widely applied to skin care cosmetics.

Description

Anti-aging plant extract composition and preparation and application thereof

Technical Field

The invention belongs to the field of cosmetics, and relates to an anti-aging plant extract composition, and preparation and application thereof.

Background

With the continuous change of human living environment and life style and the accelerated aging of the whole human society, the topics of health and longevity are more and more concerned by people. Some studies suggest that aging is closely related to cardiovascular diseases, visceral degenerative diseases, malignant tumors, and the like. Therefore, it is important to adopt appropriate methods to slow down aging.

Autophagy is an evolutionarily conserved important process in eukaryotes for turnover of intracellular material. In the process, some damaged proteins or organelles are wrapped by autophagy vesicles with double-layer membrane structures, and then are delivered into lysosomes (animals) or vacuoles (yeasts and plants) for degradation and recycling. The autophagy is a process of eating oneself, is an important mechanism for maintaining the turnover of materials of cells, degrades and recycles aged proteins, damaged organelles and other wastes, thereby ensuring the metabolic demand of the cells and the renewal of certain organelles and being vital to the survival of the cells.

Perilla frutescens (L.) Britt., a Labiatae, Perilla genus annual herbaceous plant. The Perillae herba can be used for preparing medicine and spice. The medicinal part mainly comprises stem leaves and fruit, and the leaves are diaphoretic, antitussive, aromatic, stomach-invigorating, diuretic, and have analgesic, tranquilizing, and toxic materials clearing away effects, and can be used for treating common cold. Luteolin (luteolin) is a natural flavonoid compound, and is abundantly present in plants such as perilla, honeysuckle, etc. Has various pharmacological activities such as anti-inflammation, anti-allergy, uric acid reduction, anti-tumor, antibacterial and antivirus, and is mainly used for relieving cough, eliminating phlegm, diminishing inflammation, reducing uric acid, treating cardiovascular diseases, treating amyotrophic lateral sclerosis, SARS, hepatitis and the like in clinic. Luteolin induces the increase of ROS in HCC cell strain HepG2 in a concentration-dependent mode and induces autophagy, and Cao 1 and the like find that luteolin resists the multiplication of HCC cell strain SMMC-7721, the number of intracellular autophagosomes is increased, autophagy protein LC 3-I is converted into LC 3-II, the expression of Beclin-1 is increased, apoptosis is reduced after the intervention of an autophagy inhibitor chloroquine, and the fact that the autophagy induced by luteolin promotes apoptosis is shown.

Sophora flavescens ait is a plant of the family Leguminosae, genus Sophora, herb or sub-shrub, which is thin and shrubby, and usually as high as 2 m. Can be used for treating dysentery with heat, hematochezia, jaundice, anuria, leucorrhea with red and white discharge, pudendal swelling, pudendal pruritus, eczema, skin pruritus, scabies, tinea, leprosy, and trichomonas vaginitis. Matrine (murine) is the main active component of radix Sophorae Flavescentis, and has antiviral, antiallergic and antitumor effects. Yang et al [2] found that matrine inhibited MHCC97L graft tumor growth in vivo, promoted up-regulation of LC 3-II, Beclin-1 and PI3KC3 and down-regulation of autophagy substrate p62 in MHCC97L and Huh-7 cells.

Thallus laminariae (Ecklonia kurome) is also known as "Hecai", "Gymnema japonica", etc. Phaeophyceae, Alariaceae. Kun Bu is cold in nature and salty in taste. Has the functions of softening hardness and dissipating stagnation, reducing swelling and inducing diuresis, moistening lower energizer and eliminating phlegm, is clinically used for treating goiter, cervical lymphadenectasis, bronchitis, tuberculosis, cough, senile cataract and the like, and is also used for treating cancer. Fucoxanthin (fucoxanthin) is one of the active ingredients of laminaria, and experiments of Liao politan et al [3] found that the fucoxanthin induces autophagy and apoptosis of HepG2 cells, and the autophagosome is formed by increasing Beclin-1 and LC3 proteins, and the activation of Akt is inhibited. 3-MA intervention increased apoptosis rates and promoted caspase-3 activation, suggesting that fucoidin might induce protective autophagy by inhibiting Akt pathways.

The peroxide (O2-) molecule is an important component of Reactive Oxygen Species (ROS), and is a byproduct of ATP synthesis by mitochondria, and there is increasing evidence that ROS are a significant cause of cell damage and even death, and are involved in various physiological processes. Autophagy is a physiological process occurring under metabolic conditions such as cell lack of energy and oxygen, and can reduce the level of ROS and avoid further damage to cells.

The existing research almost rarely mentions the application of the compound of the perilla, the radix sophorae flavescentis and the kelp in the field of cosmetics.

Disclosure of Invention

The invention aims to provide an anti-aging plant extract composition, and preparation and application thereof, and solves the problems in the prior art. The invention uses the compounding ratio of the three components to carry out relevant tests on the autophagy process of cells influencing skin aging as an entry point, and finally obtains the plant extract composition with small dosage and strong effect.

The invention provides an anti-aging plant extract composition, which is prepared by the following method: flavescent sophora root: the kelp is prepared by crushing, primarily extracting and concentrating the kelp according to the mass ratio of (1-3) to (1-3).

Preferably, the perilla: flavescent sophora root: the kelp is (2-3) to (1-2) to (2) by mass ratio, preferably 3:1:2 or 2:1:2 or 3:2: 2.

Preferably, the preparation method specifically comprises the following steps:

(1) pulverizing Perillae herba, radix Sophorae Flavescentis and thallus laminariae respectively, and mixing at a certain proportion;

(2) adding deionized water for extraction, wherein the mass ratio of the raw materials to the deionized water is 1:20-1:50, performing water bath at 40-50 ℃ for 3-5 hours, adding cellulase, stirring for 30 minutes, heating to 40-60 ℃, performing microwave treatment for 5-15 minutes, and performing ultrasonic water bath reflux extraction for 1-3 hours to obtain an extracting solution;

(3) cooling the extracting solution obtained in the step (2) to 20-30 ℃, and filtering with 100-200-mesh gauze to obtain a first filtrate;

(4) extracting the first filtrate and n-butanol at a volume ratio of 1:1, and separating an n-butanol layer; extracting for 3-4 times, mixing n-butanol layer solutions, and concentrating under reduced pressure at 85-100 deg.C;

(5) adding a clarifying agent into the n-butanol layer solution obtained in the step (4), standing, centrifuging for 30-60 minutes, and filtering with a microfiltration membrane to obtain a secondary filtrate;

(6) and (5) concentrating the secondary filtrate obtained in the step (5) under reduced pressure to obtain a concentrated solution.

(7) And (4) filtering the solution obtained in the step (6) by using an ultrafiltration membrane, and collecting filtrate to obtain the plant extract composition.

Further preferably, the total mass of the cellulase added accounts for 5-10% of the total mass of the raw materials.

Further preferably, the clarifying agent in the step (5) is one or more of a natural clarifying agent, chitosan, a fruit juice clarifying agent, gelatin and egg white.

Further preferably, the pore diameter of the microfiltration membrane in the step (5) is 0.1-1.0 μm;

the aperture of the ultrafiltration membrane in the step (7) is 0.01-0.05 μm.

The invention also provides a cosmetic, which comprises the plant extract composition and an auxiliary agent which can be used in the cosmetic; such cosmetics include, but are not limited to, lotions, creams, sprays, creams, gels, masks.

Preferably, the composition accounts for 0.5-5% of the cosmetic by mass.

The invention also provides an application of the composition in preparing cosmetics with anti-aging effect, which influence the autophagy process of aging cells.

The invention also provides a preparation method of the anti-aging plant extract composition, which comprises the following steps:

(6) concentrating the secondary filtrate obtained in the step (5) under reduced pressure to obtain a concentrated solution;

(7) filtering the solution obtained in the step (6) by using an ultrafiltration membrane, and collecting filtrate to obtain the plant extract composition;

the purple perilla: flavescent sophora root: the kelp is (2-3) to (1-2) to (2) by mass ratio, preferably 3:1:2 or 2:1:2 or 3:2: 2.

The invention provides an anti-aging plant extract composition influencing autophagy of aging cells, a preparation method of the composition and application of the composition. The extract is composed of perilla, radix sophorae flavescentis and kelp, the plant extract composition is prepared by processes of crushing, extracting, concentrating, formulating, sterilizing and the like, the synergistic effect of the components is good, the autophagy process of skin aging cells can be influenced, the composition plays a role, the composition is mild in property, easy to absorb and free of stimulation, the requirements of people on safety, effectiveness and zero burden of cosmetics are particularly met, and the composition can be widely applied to skin care cosmetics.

Drawings

FIG. 1 is a graph comparing the effect of different compositions on HHDPC cells in Experimental example 2.

Detailed Description

Hereinafter, the present invention is described in more detail and specifically with reference to examples, but the following examples are not intended to limit the present invention.

In the present invention, all the equipment and materials are commercially available or commonly used in the art, and the methods in the following examples are conventional in the art unless otherwise specified.

Example 1: preparation of plant extract composition capable of affecting autophagy effect of skin aging cells

In the examples and comparative examples of the present invention described below, plant extract compositions capable of affecting autophagy effects of skin aging cells were prepared by the following processes.

(1) Respectively crushing the raw materials according to the mass ratio;

(2) adding deionized water for extraction, wherein the mass ratio of the raw material to the deionized water is 1:30, performing water bath at 48 ℃ for 4 hours, adding cellulase, the total mass of the added enzyme accounts for 8 percent of the total mass of the raw material, stirring for 30 minutes, heating to 54 ℃, performing microwave treatment for 15 minutes, and performing ultrasonic water bath reflux extraction for 3 hours;

(3) cooling the extracting solution obtained in the step (2) to room temperature, and filtering with 200-mesh gauze to obtain a filtrate 1;

(4) extracting the filtrate 1 and n-butanol at a volume ratio of 1:1, shaking with strong force, standing for 3 hr, separating n-butanol layer, extracting for 3 times, mixing n-butanol layer solutions, and recovering n-butanol at 85 deg.C under reduced pressure in a rotary evaporator;

(5) adding a clarifying agent into the n-butanol layer solution obtained in the step (4), standing, centrifuging for 30 minutes, and filtering with a microfiltration membrane to obtain a secondary filtrate;

(6) characterized in that the rotating speed in the step (4) is 8000 rpm;

(7) and (5) carrying out reduced pressure concentration on the secondary filtrate obtained in the step (5), and recycling n-butyl alcohol through reduced pressure concentration to obtain a concentrated solution.

(8) And (4) filtering the solution obtained in the step (7) by using an ultrafiltration membrane, and collecting filtrate to obtain the anti-aging plant extract composition influencing the autophagy of the aged cells.

Example 2: different proportions of the composition are compounded with peroxide (O)₂ ^-) Cleaning test of

The three plant extracts were compounded according to the proportions in table 2 below, following the procedure in example 1, to give compositions 1-15, comparative examples 1-3.

TABLE 1

In the above-mentioned composition of the present invention and the comparative example, the plant extract composition was treated with peroxide (O)₂ ^-) The clearance test method and results are as follows:

O₂ ^-free radical scavenging test

4.0mL of Tris-HCl buffer (pH8.2, 50.0mmol/L) was added to 1.0mL of the test solution, and the mixture was incubated in a 25 ℃ water bath for 10 min. Adding 0.1mL pyrogallol solution (25.0mmol/L), mixing, shaking thoroughly, keeping the temperature for 5min, adding several drops of hydrochloric acid solution (10.0mmol/L) to terminate the reaction, measuring absorbance A at 325nm, and measuring in parallel for 3 times. Vitamin C was used as a positive control.

The following formula is used to calculate the O of each test solution pair₂ ^-Radical clearance rate:

O₂ ^-clearance (%) ([ A0- (A1-A2)]/A0*100％

In the formula: a0 is the absorbance of the blank control solution;

a1 is the absorbance of the reaction after the addition of the test solution;

a2 is the absorbance of the test solution without pyrogallol.

The experimental results are as follows: results table 2 below, comparing compositions 1-15 with comparative examples 1-3, it was found that:

1) peroxide (O) prepared by compounding and combining 3 raw materials₂ ^-) The cleaning effect of the plant is better than that of a single plant;

2) the peroxide clearance rates corresponding to different proportions of the 3 plant compound combinations are different, preferably perilla: flavescent sophora root: kelp (1-3): (1-3): 1-3), more preferably perilla: flavescent sophora root: laminaria ═ 3:1:2 or 2:1:2 or 3:2: 2.

TABLE 2

The results of the above tests show that the compositions of test examples 1-15 all have peroxide (O) scavenging properties relative to the blank₂ ^-) The effect, but the composition proportion is different under the same condition, the removing effect of the peroxide can be influenced, wherein the bacteriostatic effect of the

compositions

7, 12 and 14 is relatively better.

Example 3: expression detection of autophagy gene LC3A

(1) MTT cytotoxicity assay

In 96well Multi plate (corning) as per 1X10⁴cells/well were seeded at a density of 100. mu.L each in DMEM medium containing 10% bovine serum and keratinocytes (HaCaT), and 24 hours later in culture were replaced with serum-free medium. The

compositions

7, 12 and 14 of the above examples were added to serum-free medium, respectively, and the mixture was cultured for 24 hours after treatment. Thereafter, the medium was removed, treated with 20. mu.L of MTT solution, and allowed to react at 37 ℃ for 2 hours. In removing MTTThe solution of formazan was added to cells at 200 μ L isopropanol, gently shaken for 30min to dissolve the crystalline formazan completely, absorbance was measured at 570nm, and cell viability was calculated according to the following formula.

The control group was tested without the addition of sample. The cytotoxicity-related results are shown in table 3 below.

TABLE 3

(2) Autophagy gene expression detection

TABLE 4

Epidermal keratinocytes (HaCaT) were inoculated into a culture flask containing DK-SFM medium and cultured in a CO2(CO2 volume fraction: 5%) incubator at 37 ℃ for 6 d. Taking out the cell culture bottle, adding trypsin for digestion for 4min, inoculating the cells into a 96-well plate, incubating for 24h, adding 200 μ L of cell culture solution into each well of a control group, adding 200 μ L of plant extract composition extract containing 1% of composition 7/12/14 into each well of a drug group, culturing for 24h in an incubator, detecting the expression of LC3A gene by QPCR, and repeating the result for three times to obtain an average value. The results of the detection are shown in FIG. 1.

The experimental results show that the expression rate of the autophagy-related gene LC3A is significantly improved when the composition 7/12/14 is used for culture, so that the combination of perilla, sophora flavescens and kelp can be judged to effectively influence the autophagy process of cells.

Example 4: intracellular ROS level detection

Rat skin fibroblasts were cultured in DMEM medium containing 10% newborn calf serum by volume fraction and placed at 37 ℃ in CO₂(CO₂5% volume fraction) at constant temperatureIn the box, the human skin fibroblasts after subculture are spherical, the cells start adherent growth after 4h of culture and gradually become fusiform, the cells are basically adherent, the 7 th generation cells are adopted for experiment, and the cells are cultured for 48-72 h and used for expression detection of intracellular active oxygen.

Inoculating fibroblast cells into six-well plate at a ratio of 1 × 100/ml, each well having a volume of 2ml, pre-preparing fibroblast cells with 20, 50, 100mg/L plant extract composition for 24 hr, and adding H to final concentration of 200 μmol/L₂O₂Incubate for 4 hours, and add a blank control (without H)₂O₂And drugs) and model sets (plus H only)₂O₂No medicine is added), cells are collected by trypsinization, the operation is carried out according to an active oxygen detection kit, and the fluorescence intensity is detected by an up-flow cytometer.

The results are given in table 5 below:

TABLE 5

The results showed that the compositions of test examples 1 to 15 all had the effect of reducing the intracellular reactive oxygen species level. Of these,

compositions

7, 12 and 14 showed the best results, and composition 12 showed the best results.

Example 5

In a specific embodiment, the invention provides a formula process of a mask muscle base solution capable of effectively resisting, and the specific formula is as follows:

TABLE 6

Water (W)	86.95
		Glycerol	4.55
Butanediol	2.6
		Dipropylene glycol	2
Erythritol and its preparation method	1.5
		Bis-diethoxydiethylene glycol cyclohexane 1, 4-dicarboxylic acid ester	0.5
P-hydroxyacetophenone	0.4
		Arginine	0.11
Polymethylsilsesquioxane	0.1
		Acrylic acid (ester)/C10-30 alkanol acrylate crosspolymer	0.08
Panthenol	0.1
		Ethyl hexyl glycerol	0.04
Carbomer	0.03
		Glycyrrhizic acid dipotassium salt	0.03
EDTA disodium salt	0.01
		Plant extract composition	1

The extract of claim was added to the

compositions

7, 12, 14, below, in the mask muscle base formulation of table 7, at an amount of 1%, to make mask muscle base examples 1-3.

TABLE 7

Composition 7	Composition 12	Composition 14
			Sample 1	Sample 2	Sample 3

Example 6

1. Safety test (human skin patch test)

Selecting 15 healthy subjects with no allergic history of skin diseases between the ages of 20 and 50, and performing a spot pasting method: selecting a qualified spot tester, dropping about 15 mu L of samples 1-3 into the spot tester in a closed spot test mode, externally sticking a special adhesive tape on the back of a test subject, sticking 20 spot testers on each test subject, respectively sticking muscle base fluid samples of the samples 1-3, removing the test substances after 24 hours, observing skin reactions after 0.5, 6, 12, 24 and 48 hours after removal, and recording the results according to the skin reaction grade standard in skin care product sanitation standard.

And (3) test results: the results of the human skin patch test show that all the subjects pass the patch test, and the skin reaction is observed in 0.5, 6, 12, 24 and 48 hours, wherein 0 case has adverse reactions such as skin erythema, pimple and blister, which indicates that the product of the invention is safe and non-irritant.

2. Crowd test

And (3) testing a sample: the effective mask muscle base solutions of examples 1-3 were used as control groups without plant extracts.

The test population: 60 persons, without a history of skin allergy, were eligible for voluntary enrollment in subjects between 18 and 35 years of age, divided into 4 groups of 15 persons each. The face has obvious dark yellow, rough, wrinkle and other bad conditions.

The test method comprises the following steps: the appropriate amount of test sample is applied to the affected area, 2 times a day, 1 time each in the morning and evening, for 4 weeks. The subjects kept good work and rest time and diet habits during the use period, and the feedback of the subjects was counted after 4 weeks. Wherein the facial texture is improved, and the skin becomes fine and glossy; effective in slightly improving facial texture and slightly smoothing skin; others are not. Specific results are shown in Table 8 below.

TABLE 8

There are many other possible embodiments of the present invention, which are not listed here, and the embodiments claimed in the claims of the present invention can be implemented.

The details not described in the specification of the present application belong to the common general knowledge of those skilled in the art.

In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. "substantially" means within an acceptable error range, and a person skilled in the art can solve the technical problem within a certain error range to substantially achieve the technical effect.

It is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a good or system that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such good or system. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other like elements in a commodity or system that includes the element.

The foregoing description shows and describes several preferred embodiments of the present application, but as aforementioned, it is to be understood that the application is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the application, which is to be protected by the claims appended hereto.

Claims

1. An anti-aging plant extract composition is characterized by being prepared by the following method: flavescent sophora root: the kelp is prepared by crushing, primarily extracting and concentrating the kelp according to the mass ratio of (1-3) to (1-3).

2. The plant extract composition of claim 1, wherein the perilla: flavescent sophora root: the kelp is (2-3) to (1-2) to (2) by mass ratio, preferably 3:1:2 or 2:1:2 or 3:2: 2.

3. The plant extract composition according to claim 1 or 2, wherein the preparation method is specifically as follows:

4. The plant extract composition of claim 3, wherein the total mass of cellulase added is 5-10% of the total mass of the feedstock.

5. The plant extract composition as claimed in claim 3, wherein the clarifying agent in step (5) is one or more of natural clarifying agent, chitosan, fruit juice clarifying agent, gelatin and egg white.

6. The plant extract composition as claimed in claim 3, wherein the pore size of the microfiltration membrane in the step (5) is 0.1-1.0 μm;

the aperture of the ultrafiltration membrane in the step (7) is 0.01-0.05 μm.

7. A cosmetic comprising the plant extract composition of any one of claims 1 to 6 and a cosmetically acceptable adjuvant; such cosmetics include, but are not limited to, lotions, creams, sprays, creams, gels, masks.

8. The cosmetic according to claim 7, wherein the composition is present in an amount of 0.5 to 5% by mass based on the total weight of the cosmetic.

9. Use of a composition according to any one of claims 1 to 6 for the preparation of a cosmetic product with anti-aging effect which affects the autophagy process of senescent cells.

10. The preparation method of the anti-aging plant extract composition is characterized by comprising the following steps: