CN116898765A

CN116898765A - Compound cosmetic raw material composition with synergistic anti-inflammatory effect and application thereof

Info

Publication number: CN116898765A
Application number: CN202310609011.9A
Authority: CN
Inventors: 陈真; 夏金凤; 马任钟; 赵波; 赵琼; 李璠; 韩毅
Original assignee: Yunnan Qumei Biotechnology Co ltd; Kunming Medical University
Current assignee: Yunnan Qumei Biotechnology Co ltd; Kunming Medical University
Priority date: 2023-05-26
Filing date: 2023-05-26
Publication date: 2023-10-20

Abstract

The invention provides a composition prepared by compounding total saponins of pseudo-ginseng and purslane polysaccharide according to a specific proportion, which has anti-inflammatory synergistic effect, has better anti-inflammatory activity than the independent use of two components, and can obviously inhibit macrophage from releasing inflammatory factors in inflammatory reaction. The compound composition can be used as a raw material of cosmetics for preparing skin care products with relieving, anti-inflammatory or repairing effects.

Description

Compound cosmetic raw material composition with synergistic anti-inflammatory effect and application thereof

Technical Field

The invention relates to the field of cosmetic raw materials, and relates to preparation of a composition with synergistic anti-inflammatory effect after being compounded by pseudo-ginseng total saponins and purslane polysaccharide, and a skin care product with functions of relieving anti-inflammatory and repairing skin barrier.

Background

The active ingredients of natural plant sources are used as raw materials of cosmetics in the hot spot field of current cosmetic research and development, about 82.64% of cosmetics in China are added with plant extracts, and the ratio of the cosmetics compounded by two or more than two plant extracts is 80.17%. Compounding of several raw materials of similar efficacy may produce synergistic effects with enhanced activity towards each other and may also produce antagonistic effects with reduced activity towards each other. However, many formulators simply empirically formulate each plant extract in a ratio that ignores the possible synergistic or antagonistic interactions between ingredients with similar activities in designing cosmetics.

The purslane polysaccharide and the total arasaponin are two common plant-source cosmetic raw materials with the same effect of relaxing and anti-inflammatory. The purslane polysaccharide can increase the expression level of anti-inflammatory factors IL-10 by reducing the expression level of serum pro-inflammatory factors IL-6, IL-17 and TNF-alpha, thereby playing an anti-inflammatory role; the Notoginseng radix total saponin can inhibit NF- κBp65 phosphorylation, prevent p65 translocation into nucleus, and reduce expression of iNOS-NO and NF- κB signal paths, thereby achieving antiinflammatory effect. The purslane polysaccharide and the total arasaponin can reduce the expression of inflammatory factors to exert anti-inflammatory effect, and the purslane polysaccharide and the total arasaponin have synergistic effect, but no research report exists on the synergistic effect of the combination of the purslane polysaccharide and the total arasaponin at present.

The invention provides a composition prepared by compounding total saponins of pseudo-ginseng and purslane polysaccharide according to a specific proportion, which has anti-inflammatory synergistic effect, has better anti-inflammatory activity than the independent use of two components, and can obviously inhibit macrophage from releasing inflammatory factors in inflammatory reaction. The compound composition can be used as a cosmetic raw material for preparing skin care products with relieving, anti-inflammatory or repairing effects, and provides a new thought for designing a compound scheme of various cosmetic components with similar activities in the future.

Disclosure of Invention

The invention provides a preparation method of a composition prepared by compounding total saponins of pseudo-ginseng and purslane polysaccharide according to a certain proportion, wherein the compound composition has better anti-inflammatory activity than the independent use of two components, and can obviously inhibit macrophage from releasing inflammatory factors in inflammatory reaction. The compound composition can be used as a raw material of cosmetics and is used for preparing cosmetics with relieving, anti-inflammatory or repairing effects.

In order to achieve the above purpose, the invention is realized by the following technical scheme:

the composition for relieving inflammation is characterized by comprising total arasaponin and purslane polysaccharide, wherein the weight ratio of the total arasaponin to the purslane polysaccharide is 20% -40%:60% -80%.

The total saponins of Notoginseng radix contain ginsenoside Rg1, ginsenoside Rb1, and Notoginseng radix saponin R1.

The purslane polysaccharide contains neutral polysaccharide, acidic polysaccharide and pectic polysaccharide.

The composition for relieving inflammation is characterized by comprising a total arasaponin pre-prepared solution and a purslane polysaccharide pre-prepared solution, wherein the total arasaponin pre-prepared solution and the purslane polysaccharide pre-prepared solution are in a weight ratio of 25% -35%:65% -75%.

The pre-prepared liquid of the total saponins of panax notoginseng contains 70 to 90 percent of propylene glycol, 5 to 20 percent of PEG-40 hydrogenated castor oil and 1 percent of ginsenoside Rg

2% -5%, ginsenoside Rb11% -4% and notoginsenoside R10.1% -1%, wherein the sum of the raw materials is hundred percent.

The total arasaponin pre-prepared solution is a mixed solution containing 78.5% of propylene glycol, 20% of PEG-40 hydrogenated castor oil, 13% -4% of ginsenoside Rg, 12% -3% of ginsenoside Rb and 10.5% of arasaponin R, wherein the sum of the raw materials is hundred percent.

The purslane polysaccharide pre-prepared solution is a mixed solution containing 60% -80% of water, 15% -39% of 1, 3-propanediol, and 0.1% -5% of neutral polysaccharide, acidic polysaccharide and pectic polysaccharide, wherein the sum of the raw materials is hundred percent.

The purslane polysaccharide pre-prepared solution is a mixed solution containing 65-70% of water, 25-35% of 1, 3-propanediol, 0.2% -0.4% of neutral polysaccharide, acidic polysaccharide and pectic polysaccharide, wherein the sum of the raw materials is hundred percent.

Further, the following scheme is provided:

1. anti-inflammatory active raw material compound preparation for relieving anti-inflammatory compound composition

TABLE 1 compounded composition ingredients

Further, the total arasaponin pre-prepared solution is a mixed solution containing propylene glycol (78.5%), PEG-40 hydrogenated castor oil (20%), ginsenoside Rg1 (3% -4%), ginsenoside Rb1 (2% -3%), and arasaponin R1 (0.5%).

Further, the purslane polysaccharide pre-prepared solution is a mixed solution containing water (65-70%), 1, 3-propanediol (25-35%), neutral polysaccharide, acidic polysaccharide and pectic polysaccharide (0.2% -0.4%).

The preparation method of the compound composition comprises the following steps:

(1) The raw materials of the panax notoginseng saponins and the purslane polysaccharide are purchased.

(2) Weighing Notoginseng radix total saponin raw material for use; propylene glycol is heated for standby; PEG-40 hydrogenated castor oil is heated for later use. Adding propylene glycol into hydrogenated castor oil, stirring, adding Notoginseng radix total saponin, and cooling to room temperature. Obtaining the notoginsenoside pre-preparation liquid.

(3) Weighing purslane polysaccharide, mixing with 1, 3-propylene glycol, adding into a stirring pan, adding deionized water, and stirring to dissolve completely. After complete dissolution, the solution is transparent and uniform. Filtering and discharging after complete dissolution to obtain a purslane polysaccharide pre-prepared liquid;

further, the method comprises the following steps:

(1) The raw materials of the panax notoginseng saponins and the purslane polysaccharide are obtained by commercial purchase.

(2) Weighing 5-10g of total saponins of Notoginseng radix for use; adding 65-70mL of propylene glycol into a beaker, heating and stirring to 80 ℃ for standby; adding 18-23mL of hydrogenated castor oil into another beaker, and heating to dissolve for later use. Adding propylene glycol into hydrogenated castor oil, stirring for 5min, adding Notoginseng radix total saponin, and cooling to room temperature. Obtaining the notoginsenoside pre-preparation liquid.

(3) Weighing 0.2-0.4g of purslane polysaccharide, uniformly mixing with 30ml of 1, 3-propanediol, adding into a stirring pot, adding 70ml of deionized water, and fully stirring for complete dissolution (stirring speed 60r/min, stirring time 30 min). After complete dissolution, the solution is transparent and uniform. Filtering and discharging with 200-mesh filter cloth after the dissolution is completed, and obtaining the purslane polysaccharide pre-prepared liquid.

(4) And (3) performing CCK-8 cytotoxicity verification on the pre-prepared liquid (2) and the pre-prepared liquid (3), screening out a safe concentration range, and performing anti-inflammatory efficacy verification on 16 compound groups. The experiment divides the pre-prepared solution (2) into 4 concentrations (1, 5, 25, 125 mug/mL) and the pre-prepared solution (3) into 4 concentrations (2, 10, 50, 250 mug/mL) for two-by-two compounding, and the anti-inflammatory curative effect is evaluated by evaluating the inhibition rate of the 16 compounding combinations on lipopolysaccharide-induced macrophage RAW264.7 to release inflammatory factor TNF-alpha. And quantitatively evaluating interaction of the two components in the compounding with different concentrations by adopting a synergy score value system. When the (2) pre-prepared solution with the concentration of 125 mug/mL is compounded with the (3) pre-prepared solution with the concentration of 50 mug/mL or 250 mug/mL, the inhibition rates are respectively 45.18% and 61.42% and have no obvious difference with dexamethasone with the concentration of 100 mug/mL (the inhibition rate is 53.17%), and P is more than 0.05 through single factor variance analysis); the synergy score values were 15.58 and 26.44, respectively. It was demonstrated that 125. Mu.g/mL of the (2) pre-formulation may exhibit synergistic anti-inflammatory effects in combination with 50. Mu.g/mL or 250. Mu.g/mL of the (3) pre-formulation. The remaining 14 lower dose combination combinations only showed additive anti-inflammatory effects and no synergy.

(5) According to the experimental conclusion of (4), 4.5-5.5kg of the (2) pre-prepared liquid is taken, 1.5-5.5kg of the (3) pre-prepared liquid is taken, and the mixture is uniformly mixed to obtain the compound composition.

The invention relates to the characteristics of a compound composition:

synergistic anti-inflammatory effect of the compound combination. The natural anti-inflammatory raw materials extracted from the plants can play a better synergistic treatment role on inflammation through the combined action of different targets and multiple ways, and the same even better treatment effect is realized with lower drug dosage. The combination of the pseudo-ginseng and the purslane can regulate and control inflammatory reaction through a plurality of ways of participating in leukocyte migration, oxidative stress reaction, transcription factor activity and the like, and purslane polysaccharide can mainly play a leading role in the release of TNF-alpha inflammatory factors; the Panax notoginseng saponins play a leading role in inhibiting NF- κBp65 target points, and when the Panax notoginseng saponins and the purslane polysaccharide are compounded, the Panax notoginseng saponins and the purslane polysaccharide play a synergistic effect not by interfering with the same target together, but are focused on regulating and controlling different target points to influence different molecular networks, and complementarily regulate inflammatory reaction so as to play a network synergistic effect. According to the invention, the pseudo-ginseng saponin pre-prepared liquid (1, 5, 25, 125 mug/mL) and the purslane polysaccharide pre-prepared liquid (2, 10, 50, 250 mug/mL) are compounded in pairs at different concentrations, and the anti-inflammatory effect is evaluated by evaluating the inhibition rate of 16 compound combinations on lipopolysaccharide-induced macrophage RAW264.7 to release inflammatory factor TNF-alpha. And quantitatively evaluating interaction of the two components in the composition with different concentrations by adopting a Synergy Score value system. When the arasaponin pre-prepared solution with the concentration of 125 mug/mL is compounded with the purslane polysaccharide pre-prepared solution with the concentration of 50 mug/mL or 250 mug/mL, the arasaponin pre-prepared solution has obvious inhibition effects on TNF-alpha, the inhibition rates are 45.18% and 61.42%, respectively, and the inhibition effects have no obvious difference (P > 0.05) with dexamethasone with the concentration of 100 mug/mL (the inhibition rate is 53.17%). The synergy score values were 15.58 and 26.44, respectively. It is proved that 125 mug/mL of the total arasaponin pre-prepared solution can show synergistic anti-inflammatory effect when being used together with 50 mug/mL or 250 mug/mL of the purslane polysaccharide pre-prepared solution. The remaining 14 lower dose combination combinations only showed additive anti-inflammatory effects and no synergy.

2. Preparation of moisturizing water with relieving and repairing effects by using compound composition as active raw material

Further, the moisturizing lotion with the effects of relieving and repairing is characterized by comprising the compound composition.

Adding 1-10% of the compound composition into an aqueous phase solvent to prepare the moisturizing water.

The other ingredient formulation information of the moisturizing lotion according to the present invention is shown in table 2.

TABLE 2 moisture retention water composition information table

The PHL is a mixed solution of 65% of 1, 3-propanediol, 30% of 1, 2-hexanediol and 5% of octanoyl hydroxamic acid.

further, provided is moisturizing water, which comprises 0.5-5kg of a compound composition, 40-50kg of water, 0.01-0.025kg of dipotassium glycyrrhizinate, 1-1.5kg of 4-tertiary butyl cyclohexanol, 0.75-1kg of trehalose, 0.015-0.035kg of tocopherol, 0.01-0.025kg of hyaluronic acid, 0.04-0.06kg of chitosan amine, 0.045-0.1kg of ethylhexyl glycerol, 0.25-0.4kg of octanoyl hydroxamic acid, 0.25-0.5kg of fennel extract, 0.015-0.025kg of extreme feasibility, 90100.03-0.05kg of PE, 0.25-0.5kg of PHL and 0.005-0.015kg of essence.

The preparation method of the moisturizing water comprises the following steps:

(1) Adding water with the formula dosage into an emulsifying pot, adding dipotassium glycyrrhizinate and 4-tertiary butyl cyclohexanol into the emulsifying pot, and stirring until the dipotassium glycyrrhizinate and the 4-tertiary butyl cyclohexanol are completely dissolved. The temperature during this process was room temperature and the stirring speed was 20rpm.

(2) Dispersing trehalose, tocopherol, hyaluronic acid, chitosan amine and sea fennel extract with the formula dosage, adding the dispersed mixture into the emulsifying pot in the step (1), and stirring the mixture until the mixture is uniform. The temperature during this process was room temperature and the stirring speed was 20rpm.

(3) The compound composition prepared in the first example, the ethylhexyl glycerol, the octanoyl hydroxamic acid, the extreme surplus, the PE9010 and the PHL with the dosage of the formula are added into the emulsifying pot in the step (2), and are stirred until the mixture is uniform. The temperature during this process was room temperature and the stirring speed was 20rpm.

(4) Adding the solution prepared by the spice into the emulsifying pot in the step (3), and stirring and mixing for 30min to obtain water. The temperature in the process is room temperature, the stirring speed is 20rpm, and the vacuum degree is-0.06 MPa.

(5) Filtering the water prepared in the step (4) by using 200-mesh filter cloth, and discharging to prepare the moisturizing water.

(6) And (5) detecting indexes of sensory, physicochemical and microorganism of the moisturizing water prepared in the step (5). The product is not greasy, not sticky, comfortable to use and free of foreign matters; meets the specific flavor type; the pH value is 5.5-6.5; the total colony number is less than or equal to 500CFU/; the mould yeast is less than or equal to 50CFU/g.

(7) And (3) filling the moisturizing water which is qualified in the step (6) into a hose by adopting a semi-automatic filling machine, and sealing the hose by using a sealing machine to prepare the repairing moisturizing water.

The invention relates to the characteristics of moisturizing water:

1. synergistic anti-inflammatory effect of moisturizing water pseudo-ginseng total saponins and purslane polysaccharide affect different molecular networks through intervention and regulation of different targets, and complementary regulation of inflammatory reaction is achieved, so that a network synergistic effect is exerted.

2. Repairing barrier tocopherol and chitosan to repair skin barrier, promoting cell regeneration, resisting oxidation, protecting skin blood vessel, stabilizing skin cell membrane, etc., and reducing inflammatory factor expression to relieve skin redness and dryness.

3. The sea fennel extract acts on the cutaneous sensory nerve receptor to relieve pain and itch, and the skin pain and itch are relieved. Can relieve "irritation sensation", promote skin beta endorphin release, inhibit neuropeptide CGRP release, and rapidly relieve pain/itching caused by inflammation.

3. The cream suitable for sensitive skin people is further prepared by adopting the compound composition as an active raw material, and the cream with the soothing and repairing effects is characterized by comprising the compound composition.

Further, 1-10% of the compound composition is added into a milk phase solvent to prepare the cream.

The other ingredient formulation information of the cream according to the present invention is shown in table 3.

TABLE 3 information Table of cream ingredients

Further, the water is deionized water.

Further, the BUTANEDIOL is 1,2-BUTANEDIOL (1, 2-BUTANEDIOL)

Further, the sodium hyaluronate is sodium hyaluronate (sodium hyaluronate)

Further, the BETAINE is BETAINE (BETAINE)

Further, the dipotassium glycyrrhizinate is dipotassium glycyrrhizinate (dipotassium glycyrrhizinate).

Further, the cetostearyl glucoside is cetostearyl glucoside (cetearyl glucoside)

Further, the sorbitan olive oleate is sorbitan olive oleate (sorbitan olive ate)

Further, the CAPRYLIC/CAPRIC triglyceride is CAPRYLIC/CAPRIC succinic triglyceride (capryl ic/CAPRIC/SUCCINIOTRILITILDERIDE)

Further, the polydimethylsiloxane is PEG-17 polydimethylsiloxane (PEG-17 DIMETHICONE).

Further, the shea butter is Butyrospermum parkii (Butyrospermum parkii) fruit butter

Further, the jojoba oil is jojoba (SIMMONDSIACHINENSIS) seed oil

Further, the plant squalane is squalane

Further, the Prinsepia utilis royle oil is Prinsepia utilis (Prinsepia utilis) oil

Further, the Besterol-E is a phytosterol oleate

Further, the INS is isononyl isononanoate

further, the provided cream for sensitive muscles contains 0.5-5kg of a compound composition, 32.5-35kg of deionized water, 1.5-3kg of butanediol, 0.03-0.04kg of sodium hyaluronate, 0.5-1.5kg of betaine, 0.01-0.03kg of dipotassium glycyrrhizinate, 0.25-1kg of cetostearyl glucoside, 0.5-1.5kg of sorbitan olive oleate, 1-2.5kg of caprylic/capric triglyceride, 0.5-1.5kg of cetylstearyl alcohol, 0.5-1.5kg of polydimethylsiloxane, 1-3kg of shea butter, 0.4-0.8kg of jojoba oil, 0.5-1.5kg of plant squalane, 0.2-0.5kg of prinsepia utilis royle oil, 0.25-0.75kg of Besterol-E and 1-2kg of INS. The preparation method of the cream comprises the following steps:

(1) After adding the water with the formula dosage into an emulsifying pot, adding butanediol, sodium hyaluronate, betaine and dipotassium glycyrrhizinate into the emulsifying pot, and stirring until the butanediol, the sodium hyaluronate, the betaine and the dipotassium glycyrrhizinate are completely dissolved. The temperature during this process was room temperature and the stirring speed was 20rpm.

(2) Dispersing the cetostearyl glucoside, the sorbitan olive oleate, the caprylic/capric triglyceride, the cetostearyl alcohol and the polydimethylsiloxane with the formula dosage, adding the mixture into the emulsifying pot in the step (1), and stirring the mixture until the mixture is uniformly mixed. The temperature during this process was room temperature and the stirring speed was 20rpm.

(3) Adding the shea butter, jojoba oil, plant squalane, prinsepia utilis royle oil, besterol-E and INS with the formula dosage into the emulsifying pot in the step (2), and stirring until the materials are uniformly mixed. The temperature during this process was room temperature and the stirring speed was 20rpm.

(4) Adding the solution prepared in the step (3) into the emulsifying pot prepared in the step (2), and stirring and mixing for 30min to obtain the cream. The temperature in the process is room temperature, the stirring speed is 20rpm, and the vacuum degree is-0.06 MPa.

(5) Filtering the cream prepared in the step (4) by using 200-mesh filter cloth, and discharging to prepare the cream.

(6) And (5) detecting indexes of sensory, physicochemical and microorganism of the cream prepared in the step (5). The appearance is white or milky white, has luster, fine texture and no foreign matter; meets the specific flavor type; the pH value is 5.5-6.5; the total colony number is less than or equal to 500CFU/; the mould yeast is less than or equal to 50CFU/g.

(7) And (3) filling the qualified cream in the step (7) into a hose by adopting a semi-automatic filling machine, and sealing the hose by using a sealing machine to obtain the cream.

The invention relates to the characteristics of cream:

(1) The synergistic anti-inflammatory effect of the compound composition in the first embodiment affects different molecular networks by intervening and regulating different targets, and complementarily regulates inflammatory reaction so as to exert network synergistic effect. Dipotassium glycyrrhizinate, auxiliary anti-inflammatory;

(2) The skin cell butanediol and sodium hyaluronate are protected from locking moisture. Betaine enhances the moisture activity of skin, improves the skin elasticity and makes the skin smoother; inhibit cell necrosis caused by high osmotic pressure in external environment, and maintain skin horny layer balance. Plant squalane lubricates skin and alleviates the symptoms of dryness and desquamation. Is fused with the sebaceous membrane of the skin to form a protective layer, and promotes the generation of new cells.

(3) The anti-inflammatory and antibacterial shea butter, jojoba oil and prinsepia utilis royle oil are used for moisturizing, promoting the rehydration of skin horny layer cells, diminishing inflammation and inhibiting bacteria.

Drawings

FIG. 1 influence of a notoginsenoside Pre-formulation solution and a Portulaca oleracea polysaccharide pre-formulation solution on RAW264.7 cell survival

FIG. 2A contour plot of the synergy score values of various concentrations of notoginsenoside pre-formulation (PNS) and purslane polysaccharide pre-formulation (POP) after combination

FIG. 3 contour plot of the synergy score values after various concentrations of notoginsenoside pre-formulation (PNS) and purslane polysaccharide pre-formulation (POP) were combined

FIG. 4RAW264.7 cell groups under microscope (20 Xobjective)

A: blank control group

B: negative control group

C: positive control group

D: test sample group (12.50. Mu.L/mL)

E: test sample group (6.25. Mu.L/mL)

FIG. 5 comparison of TNF- α secretion levels of test samples on LPS-induced RAW264.7 cells

FIG. 6 comparison of IL-1. Beta. Secretion by LPS-induced RAW264.7 cells

FIG. 7 comparison of IL-6 secretion from LPS-induced RAW264.7 cells

FIG. 8RAW264.7 cell groups under microscope (20 Xobjective)

A: blank control group

B: negative control group

C: positive control group

D: test sample group (1.00 mg/mL)

E: test sample group (0.50 mg/mL)

FIG. 9 comparison of TNF- α secretion levels of LPS-induced RAW264.7 cells from test samples

FIG. 10 comparison of IL-1. Beta. Secretion by LPS-induced RAW264.7 cells

FIG. 11 comparison of IL-6 secretion from LPS-induced RAW264.7 cells

Detailed Description

Example 1 preparation of a Complex composition (10 kg)

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

reagent:

the Notoginseng radix total saponin and herba Portulacae polysaccharide raw materials are provided by Yunnan Bai Nilan biological resource limited company;

instrument:

electronic analytical balance Metretolidol instruments (Shanghai Co., ltd.)

Constant temperature magnetic stirrer Hangzhou instruments and motors Co., ltd

Low speed centrifuge Anhui middle science and scientific instruments Co., ltd

This example is illustrative of the method of preparing the compounded composition of the present invention.

(1) The Notoginseng radix total saponin and herba Portulacae polysaccharide are provided by Yunnan Bai Nilan biological resource limited company.

(2) Weighing 0.1-0.15kg of total saponins of Notoginseng radix for use; adding 7.6-7.9L propylene glycol into a beaker, heating and stirring to 80 ℃ for standby; adding hydrogenated castor oil 18-22L into another beaker, and heating for dissolving. Adding propylene glycol into hydrogenated castor oil, stirring for 5min, adding Notoginseng radix total saponin, and cooling to room temperature. Obtaining the pseudo-ginseng total saponin pre-prepared liquid.

(3) Weighing 0.02-0.04kg of purslane polysaccharide, weighing 2.5-3.5L of 1, 3-propanediol, and putting into a stirring pot to mix uniformly; adding 6.5-7L deionized water, and stirring uniformly (stirring speed 60r/min, stirring time 30 min). And (5) completely dissolving to obtain transparent uniform liquid, thus obtaining the purslane polysaccharide pre-prepared liquid.

(4) Taking 4.5-5.5kg of the (2) pre-prepared liquid, and then taking 1.5-5.5kg of the (3) pre-prepared liquid, and uniformly mixing to obtain the compound composition.

EXAMPLE 2 preparation of soothing moisturizing Water Using a Complex combination as active Material (50 kg)

reagent:

example 1 a compounded composition was prepared. Deionized water, dipotassium glycyrrhizinate, 4-tert-butylcyclohexanol, trehalose, tocopherol, hyaluronic acid, chitosan, sea fennel extract, ethylhexyl glycerol, octanoyl hydroxamic acid, super-profitable, PE9010, PHL, and essence were purchased from Yunnan Bai Nilan biological resource development Co.

Instrument:

laboratory vacuum homogenizer available from Shanghai scintillation Intelligent devices Co., ltd., from Mettler Toledo, model PL1002E/02

The sealing machine, guangzhou city common information packaging material limited company, is used for explaining the preparation of the moisturizing water by adopting the compound combination as an active raw material.

Further, there is provided a moisturizing water comprising 3.8kg of the compound composition of example 1, 42.5kg of water, 0.13kg of dipotassium glycyrrhizinate, 1.5kg of 4-t-butylcyclohexanol, 0.88kg of trehalose, 0.03kg of tocopherol, 0.03kg of hyaluronic acid, 0.06kg of chitosan, 0.5kg of sea fennel extract, 0.02kg of super-coix seed, 0.04kg of PE90100.04kg of PHL and 0.15kg of essence.

The preparation method comprises the following steps:

Example 3 preparation of soothing and repairing cream Using a Complex combination as active Material (50 kg)

Reagent:

example 1 a compounded composition was prepared. Deionized water, butylene glycol, sodium hyaluronate, betaine, dipotassium glycyrrhizinate, cetostearyl glucoside, sorbitan olive oleate, caprylic/capric triglyceride, cetostearyl alcohol, polydimethylsiloxane, shea butter, jojoba oil, plant squalane, prinsepia utilis royle oil, besterol-E and INS were purchased from Yunnan Bai Nilan biological resource development Co.

Instrument:

laboratory vacuum homogenizer available from Shanghai scintillation Intelligent devices Co., ltd., model 1

Electronic balance, available from Mettler Toledo, model PL1002E/02

Sealing machine, guangzhou city common information packaging material Co., ltd

This example is used to illustrate the preparation of a soothing and repairing cream using the compounded composition of the present invention as an active ingredient.

Contains 3.8kg of compound composition, 33.75kg of deionized water, 2kg of butanediol, 0.035kg of sodium hyaluronate, 1kg of betaine, 0.025kg of dipotassium glycyrrhizinate, 0.5kg of cetostearyl glucoside, 0.75kg of sorbitan olive oleate, 1.25kg of caprylic/capric triglyceride, 0.75kg of cetostearyl alcohol, 0.75kg of polydimethylsiloxane, 1.75kg of shea butter, 0.6kg of jojoba oil, 0.5kg of plant squalane, 0.4kg of prinsepia utilis oil, 0.5kg of Besterol-E0.5kg and 1.5kg of INS.

The preparation method comprises the following steps:

(4) Adding the solution prepared in the step (3) into the emulsifying pot prepared in the step (2), stirring and mixing for 30min to obtain the cream. The temperature in the process is room temperature, the stirring speed is 20rpm, and the vacuum degree is-0.06 MPa.

(7) And (3) filling the qualified frost in the step (7) into a hose by adopting a semi-automatic filling machine, and sealing the hose by using a sealing machine to obtain the cream.

Test example one skin cytotoxicity test

The skin is divided into epidermis, dermis and subcutaneous tissue from outside to inside. Cytotoxicity test was performed on human immortalized epidermal cells HaCaT of the epidermal layer of skin and human dermal fibroblast HSF of the dermal layer with reference to the guidelines for implementation of cosmetic evaluation alternative method standard, compiled by Cheng Shujun, to evaluate the cytotoxicity effect of the anti-inflammatory component on human skin cells. When the relative cell activity is not less than 80%, the test substance is considered to be non-cytotoxic at this concentration. The pre-prepared solutions of the extracts of the two plants (2) and (3) prepared in example 1 are diluted to a specified concentration by a culture medium, co-cultured with the two cells for 24 hours, 100 mu L of the culture medium and 10 mu LCCK-8 solution mixture are added to each well, incubated for 2 hours, and the absorbance OD value at 450nm is measured by an enzyme-labeled instrument. Cell viability was calculated for each test group using the following formula, and each group of experiments was independently repeated 3 times, data to give And (3) representing. As can be seen from tables 4 and 5, the concentrations of Notoginseng radix extract and herba Portulacae extract were less than 250ug/mL, and the HSF cell survival rate was low>80%; at concentrations below 500ug/mL, hacat cell viability was demonstrated>80% of the total amount of the sample, it is considered that the test sample has no effect on cell proliferation at this concentration.

TABLE 4 influence of notoginsenoside Pre-formulation on HSF and Hacat cell viability

TABLE 5 influence of Portulaca oleracea polysaccharide pre-formulation on HSF and Hacat cell viability

Test example two evaluation of synergistic anti-inflammatory efficacy of the Compound composition

Purpose of testing

The anti-inflammatory soothing efficacy of the composition (prepared from example 1) after the compounding of the two soothing RAW materials was evaluated using an anti-inflammatory efficacy model in which Lipopolysaccharide (LPS) induced macrophage inflammatory cells (RAW 264.7) to secrete the inflammatory factor TNF- α content.

Principle of

LPS-induced RAW264.7 is a classical cell model for the study of inflammatory factors. LPS and macrophage surface antigen recognition receptor are combined, so that the macrophage can be induced to secrete various inflammatory factors such as TNF-alpha and the like. TNF-alpha can activate three signal paths of Caspase protease, JNK and transcription factor NF- κB, and can implement biological functions of cytotoxicity, antivirus, immunoregulation and cell apoptosis.

The method evaluates the effect of the compound composition on inhibiting TNF-alpha secretion by comparing the difference of the content of the secreted TNF-alpha of RAW264.7 cells after the administration of the negative control and the compound composition. The content measurement adopts an enzyme-linked immunosorbent assay (ELISA) method, and the specific principle is as follows: after the inflammatory factor TNF-alpha is specifically combined with the TNF-alpha antibody coated on the ELISA plate, the anti-TNF-alpha antibody with a substrate label is combined, a colored product is generated after the substrate is catalyzed, and the TNF-alpha content and the colored product are measured in optical density (OD value) at the wavelength of 450nm to calculate the TNF-alpha content.

Experimental part

Main materials, reagents and instruments

The 16-group compound combination of the total saponins of panax notoginseng and the purslane polysaccharide is prepared by mixing the components (2) and (3) of the embodiment 1 according to the concentration proportion; TNF-alpha ELISA detection kit purchased from Xinbo biotechnology Co., ltd; CCK-8 detection kit, dexamethasone, available from Soy technologies Co., ltd; dimethyl sulfoxide (DMSO), LPS (e.coli source), purchased from merck life science, german; the mouse mononuclear macrophage leukemia cell line RAW264.7 is derived from a Kunming cell bank of the China academy of sciences typical culture Collection, and is selected from a model KCB200603YJ; DMEM medium, available from white shark biotechnology limited; fetal bovine serum, purchased from sammer feishier technologies limited; phosphate Buffered Saline (PBS), available from Jiangsu Kaiki Biotechnology Co.

96-well plate, 24-well plate, T25 flask, T75 flask, purchased from Corning Life sciences Co., ltd; the enzyme-labeled instrument is purchased from America millet company; CO ₂ Incubator, available from the biological company of America Mo Erte, germany; an incubator at 37 ℃ purchased from Shanghai forest communications instruments; centrifuges, purchased from Shanghai scientific instrumentation plants; automatic autoclave, purchased from middle inspection southwest metering limited; pipetting guns and tips, purchased from white shark biotechnology limited; vortex mixer, available from its ringer instruments, inc.

Experimental method

1. Preparation of test article

Stimulus: 10mgLPS was precisely weighed, dissolved in 5mLPBS to prepare a LPS mother solution having a mass concentration of 2mg/mL, and 100. Mu.L of the LPS mother solution was dispensed into 0.5mL EP tubes and stored in a refrigerator at-20 ℃. The culture medium was diluted to a mass concentration of 5. Mu.g/mL as a stimulator before use.

Experimental group: respectively accurately weighing 10mg of notoginsenoside pre-prepared liquid and purslane polysaccharide pre-prepared liquid, respectively dissolving with 0.5mL and 1mL of LDMSO, and preparing into a mother solution of the notoginsenoside pre-prepared liquid and a mother solution of the purslane polysaccharide pre-prepared liquid with the mass concentration of 20 mg/mL. 100. Mu.L and 200. Mu.L of each were dispensed into 0.5mLEP tubes and stored in a refrigerator at-20 ℃. The test solution is diluted into a test solution with target concentration by using a culture medium gradient before use, and is used for detecting the cell viability and evaluating the anti-inflammatory activity of different compound combinations.

Positive control group: accurately weighing 5mg dexamethasone, dissolving with 1mL of LDMSO, subpackaging 100 μl into 0.5mL EP tube, storing at-20deg.C, diluting with culture medium to 100 μg/mL, and adding stimulator as positive control.

2. Cell culture

RAW264.7 cells were added to DMEM high-sugar culture medium containing 10% fetal calf serum by volume fraction, and placed in an incubator (37 ℃ C., volume fraction 5% CO) ₂ ) Conventional culture in the medium. When the cell fusion degree reaches 80-90%, scraping off the adherent cells at the bottom of the bottle by using the cell to digest and passaging, and passaging or changing the liquid for 2-3 times per week.

RAW264.7 cell viability assay

When the cell fusion degree reaches 80-90%, obtaining cell suspension by using the method of cell passage, and adjusting the cell density to 6.5X10 ⁴ mu.L of cell suspension per well was added to a 96-well plate at a rate of 200 mL/mL, and the mixture was further placed in an incubator (37 ℃ C., 5% CO) ₂ ) Is cultured for 24 hours. Old culture solution in 96-well plates was aspirated first, and then 200. Mu.L of complete culture solution containing different mass concentrations of the test substance (experimental group) and 200. Mu.L of complete culture medium containing no test substance (negative control group) were added to each well, and 3 duplicate wells were set as parallel groups. Culturing in incubator at constant temperature for 24 hr. The supernatant was aspirated and discarded, 100. Mu.L of the medium and 10. Mu.LCCK-8 solution mixture were added to each well, incubated for 2h, and the absorbance OD at 450nm was measured with a microplate reader. Cell viability was calculated for each test group using the following formula, and each group of experiments was independently repeated 3 times, data to giveAnd (3) representing. When cell viability>At 80%, the test sample was considered to have no effect on RAW264.7 cell proliferation at this concentration.

4. In vitro cell test to verify anti-inflammatory efficacy of test substance

Cell culture as described aboveThe method comprises taking cell suspension when cell fusion degree reaches 80-90%, and adjusting cell density to 9×10 ⁴ mu.L of the cell suspension per well, added to a 24-well plate, and placed in an incubator (37 ℃,5% CO) ₂ ) Culturing at medium constant temperature for 24h. When the cell plating rate in the pore plate meets the test requirement, the model group and the blank group are replaced by new complete culture media, a positive control group is added with the complete culture media containing 100 mug/mL dexamethasone, an experimental group is added with culture media containing arasaponin pre-prepared liquid (1, 5, 25, 125 mug/mL) and purslane polysaccharide pre-prepared liquid (2, 10, 50, 250 mug/mL) and culture media containing 16 compound combinations, and cells are pre-treated for 2 hours. Except for the blank, both the model and experimental groups were incubated with complete medium supplemented with LPS (5. Mu.g/mL) for an additional 16h. Collecting supernatant, centrifuging at 3000r/min for 10min, and collecting supernatant for storage. TNF- α content was measured according to ELISA kit instructions. Each set of experiments was independently repeated 3 times, data to And (3) representing.

5. Synergistic analysis

And analyzing the synergistic effect of the combination of the notoginsenoside pre-formulation and the purslane polysaccharide pre-formulation by using a synergy Finder analysis tool (https:// synergyfnder. Fimm. Fi). The tool is a publicly accessible drug combination analysis network platform for interactive analysis and visualization of multi-drug combination response data. The drug combination (combination) is quantified to quantify the interaction of the two components used in combination. Synergy score >10, demonstrating that the combination has a synergistic effect; -10. Ltoreq.synergy score. Ltoreq.10, indicating that the combination is additive; synergyScare < -10, indicates that the combination is antagonistic.

6. Data processing

Experimental data were statistically analyzed using SPSS17.0 software and metrology data were collectedBy addition or subtraction of standard deviation by mean The data comparison among multiple groups adopts single factor analysis of variance to obtain P<0.05 indicates that the difference is statistically significant.

Test results

1. Influence of pre-prepared liquid with different proportioning concentrations on RAW264.7 cell viability

The cell viability of RAW264.7 cells after 24h intervention with different concentrations of notoginsenoside pre-formulation or purslane polysaccharide pre-formulation is shown in FIG. 1. When the dosage concentration of the arasaponin pre-preparation is less than 125 mug/mL and the dosage concentration of the purslane polysaccharide pre-preparation is less than 250 mug/mL, the cell activity is more than 80%, which indicates that the concentrations are safe dosage ranges without influencing the cell survival and proliferation. When the concentration of the purslane polysaccharide pre-prepared solution is 62.5-250 mug/mL, the activity of RAW264.7 cells is more than 100%, which shows that the proliferation of cells is promoted. In the subsequent test, the concentration is selected to be the maximum concentration of the notoginsenoside pre-preparation liquid and the purslane polysaccharide pre-preparation liquid in the compound combination design, and a series of anti-inflammatory activity tests are carried out after the equal ratio dilution and the pairwise compounding are carried out on the basis.

2. Anti-inflammatory efficacy results of pre-formulated liquids of different ratios

(1) Inflammation model

In the test example, after the RAW264.7 cells are stimulated by LPS for 16 hours, the TNF-alpha content of a model group (stimulated by LPS) is increased by 133.61 times compared with that of a blank group (without LPS), and the test example meets the requirement of validity verification of the group standard of the cosmetic soothing efficacy test-in vitro TNF-alpha inflammatory factor content determination lipopolysaccharide-induced macrophage RAW264.7 test method, and proves that under the test condition, a research model of producing inflammatory factor TNF-alpha by the RAW264.7 cells stimulated by 5 mug/mL LPS is established.

(2) Analysis of anti-inflammatory efficacy results and synergy

Both the notoginsenoside pre-formulation and the purslane polysaccharide pre-formulation with different concentrations can inhibit the production of TNF-alpha by RAW264.7 induced by LPS (see Table 6). According to the T/SHRH034-2021 standard, when the TNF-alpha inhibition rate is more than or equal to 25%, the test component is judged to have the effect of relieving. TNF-alpha inhibition greater than 25% at concentrations of 25 μg/mL and 125 μg/mL with notoginsenoside pre-formulation alone; the inhibition rate of TNF-alpha is also more than 25% when the purslane polysaccharide pre-prepared solution is singly used at the concentration of 250 mug/mL. When the notoginsenoside pre-formulation (125 mug/mL) and the purslane polysaccharide pre-formulation (250 mug/mL) are compounded, the inhibition rate of TNF-alpha is 61.42 percent, which is larger than the sum of the inhibition rates of the notoginsenoside pre-formulation and the purslane polysaccharide pre-formulation which are independently used at the dosage, and the synergy score value is 26.44 (see figure 2); when the notoginsenoside pre-preparation solution (125 mug/mL) and the purslane polysaccharide pre-preparation solution (50 mug/mL) are compounded, the synergy score value is 15.58, which indicates that the compounded combination has a synergistic effect. The other 14 compound combinations have calculation results within the range of-10 less than or equal to the synergy score value less than or equal to 10, which indicates that the compound combinations only have additive effect and have no synergistic effect.

TABLE 6 comparison of inhibition (%) of LPS-induced RAW264.7 cells to release TNF-alpha after pairwise compounding of pre-prepared solutions with different proportioning concentrations

Synergy score >10, demonstrating that the combination has a synergistic effect; -10. Ltoreq.synergy score. Ltoreq.10, indicating that the combination is additive; synergyScare < -10, indicates that the combination is antagonistic.

Dexamethasone is a clinically common anti-inflammatory drug, and in the experiment, dexamethasone (100 mug/mL) is used as a positive control group, and the inhibition rate of TNF-alpha is 53.17%. As can be seen from fig. 3, both 125 μg/mL of the arasaponin pre-formulation and 50 μg/mL of the purslane polysaccharide pre-formulation (synergistic group 1) and 250 μg/mL of the arasaponin pre-formulation (synergistic group 2) which produce synergistic effects can significantly reduce (P < 0.01) the TNF- α content of LPS-induced RAW264.7 cells, and the difference has no statistical significance (P > 0.05) compared to the positive control group.

The natural anti-inflammatory raw materials extracted from the plants can play a better synergistic treatment role on inflammation through the combined action of different targets and multiple ways, and the same even better treatment effect is realized with lower drug dosage. Network pharmacological studies show that the combination of pseudo-ginseng and purslane can regulate and control inflammatory reaction through various ways such as participation in leukocyte migration, oxidative stress reaction, transcription factor activity and the like, and purslane polysaccharide can mainly play a leading role in the release of TNF-alpha inflammatory factors; the Panax notoginseng saponins play a leading role in inhibiting NF- κBp65 target points, which suggests that the Panax notoginseng saponins and the purslane polysaccharide do not play a synergistic effect by interfering with the same target together when the Panax notoginseng saponins and the purslane polysaccharide are compounded, but focus on regulating different targets to influence different molecular networks, and complementarily regulate inflammatory reaction to play a network synergistic effect, which is probably the essence of compounding the raw material of the cosmetic plant.

Conclusion 3

When the notoginsenoside pre-prepared solution with the concentration of 125 mug/mL and the purslane polysaccharide pre-prepared solution with the concentration of 50 mug/mL or 250 mug/mL are compounded, the synergistic anti-inflammatory effect can be shown, macrophage RAW264.7 induced by LPS stimulation can be effectively inhibited from releasing inflammatory factor TNF-alpha, and the inhibition effect is equivalent to that of dexamethasone with the concentration of 100 mug/mL. However, when the notoginsenoside pre-formulation and the purslane polysaccharide pre-formulation are compounded at a lower concentration, the synergistic anti-inflammatory effect is not achieved, and only the additive effect is exhibited.

Test example three evaluation of anti-inflammatory efficacy of moisturizing Water

Purpose of testing

The anti-inflammatory efficacy of moisturizing water (prepared from example 2) was evaluated according to the standard method of "T/SHRH 034-2021 cosmetic soothing efficacy test-in vitro TNF- α inflammatory factor content assay lipopolysaccharide-induced macrophage RAW264.7 test method" (2021 version).

Principle of:

LPS-induced RAW264.7 is a classical cell model for the study of inflammatory factors. LPS and macrophage surface antigen recognition receptor are combined, so that the macrophage can be induced to secrete various inflammatory factors such as TNF-alpha, IL-1 beta, IL-6 and the like.

TNF-alpha can activate three signal paths of Caspase protease, JNK and transcription factor NF- κB, and can implement biological functions of cytotoxicity, antivirus, immunoregulation and cell apoptosis.

IL-1β is an important mediator of inflammatory responses and can be involved in a variety of cellular activities including cell proliferation, differentiation, apoptosis, etc. when the body is in an inflammatory state or an immune response occurs. IL-1β can also cause increases in acute phase reactive proteins such as C-reactive proteins, etc. by activating endothelial cells, causing bone marrow neutrophil mobilization (leukocytosis), etc., thereby causing systemic inflammation.

IL-6 produces different patterns in different inflammations. In the early stages of infectious inflammation, different pathogens stimulate the production of IL-6 by monocytes and macrophages through Toll-like receptors (TLRs) of the relevant molecular pattern (PAMP). In non-infectious inflammatory conditions (e.g., burns or traumatic injury), IL-6 production by injured cells is stimulated by the TLR of the injury-associated molecular pattern (DAMP). This acute IL-6 expression plays a central role in host defense by stimulating various cell populations.

The method evaluates the effect of the test sample on inhibiting TNF-alpha, IL-1 beta and IL-6 secretion by comparing the difference of the contents of the secreted TNF-alpha, IL-1 beta and IL-6 of RAW264.7 cells after the administration of the negative control and the test sample. The content measurement adopts an enzyme-linked immunosorbent assay (ELISA) method, and the specific principle is (taking TNF-alpha as an example): after the inflammatory factor TNF-alpha is specifically combined with the TNF-alpha antibody coated on the ELISA plate, the anti-TNF-alpha antibody with a substrate label is combined, a colored product is generated after the substrate is catalyzed, and the TNF-alpha content and the colored product are measured in optical density (OD value) at the wavelength of 450nm to calculate the TNF-alpha content.

Study test method:

1. and (3) cells: the mouse mononuclear macrophage leukemia cell line RAW264.7 is derived from the Kunming cell bank of the typical culture preservation committee of China academy of sciences, and the model is KCB200603YJ.

2. Culture conditions: DMEM medium, fetal bovine serum, trypsin/EDTA solution (0.25% pancreatin solution mixed with 0.02mol/LEDTA solution 1:1), phosphate Buffer (PBS), cell culture flasks.

3. Stimulus: lipopolysaccharide (from LPS E.coli) U.S. Sigma Co., batch number: 0000126449.

4. positive control: dexamethasone (soribao D8040).

5. Materials, instruments: a 96-well plate; TNF-alpha ELISA detection kit, IL-1 beta ELISA detection kit and IL-6ELISA detection kitELISA kit); CCK-8 cytotoxicity detection kit (MF 128-01, beijing Polymer Biotechnology Co., ltd.); an enzyme-labeled instrument; an ultra-clean workbench; dimethyl sulfoxide (DMSO).

6. Test packets

TABLE 7 test packet design

7. Test procedure

7.1 cell and test sample preparation

(1) Cell preparation:

a. cell resuscitation:

1) Preheating a water bath kettle to 37 ℃, preparing clean disposable PE gloves, and adding 9mL of sterile DMEM culture medium into a sterile centrifuge tube;

2) Taking out the cells from the liquid nitrogen tank, putting the cells into PE gloves, quickly immersing the cells into a water bath, and shaking the freezing tube to accelerate dissolution, wherein the cells are dissolved completely within 1 minute;

3) Adding the resuscitated cell sap into a centrifuge tube filled with a DMEM culture medium in an ultra-clean bench, centrifuging at 1000rpm/min for 4 minutes, and removing the supernatant after centrifugation;

4) Cells were resuspended in 6mL of DMEM medium containing 10% serum, inoculated into T25 flasks, supplemented with 5mL of medium, and placed in an incubator for culture.

b. Cell digestion and passaging:

cells were digested with trypsin/EDTA (preferably 0.25%) in 1mL of T25 flask and 3mL of T75 flask. When most cells are rounded and in a suspension state, adding DMEM medium containing 10% serum, which is about 2-3 times of the volume of pancreatin, stopping digestion, collecting the cells into a centrifuge tube, centrifuging for 4min at 1000r/min, removing supernatant after centrifugation, adding the DMEM medium containing 10% serum into the centrifuge tube, blowing the cells uniformly by a suction tube, and transferring the cells into two T25 culture flasks.

(2) Test sample solution preparation

Prior to testing, the test samples were diluted with medium to the test dose concentration.

7.2CCK-8 cell viability assay

The in vitro toxicity of the test samples against RAW264.7 cells was examined with CCK-8 (CellCounting kit 8). At 6.5X10 ⁴ Cell density of cells per mL cells were seeded into 96-well plates at 200 μl of cell suspension per well. Culturing in an incubator for 24 hours, and discarding old culture medium; adding substances to be tested with different concentrations diluted by a culture medium into a 96-well plate, incubating for 24 hours, sucking the supernatant and discarding; 100. Mu.L of the medium and 10. Mu.LCCK-8 solution mixture were added to each well, incubated for 2 hours, and absorbance at 450nm was measured using an ELISA reader. Cell viability was calculated based on the negative control. When the cell viability was ∈ 80%, it was considered that the test sample at this concentration had no effect on cell proliferation, i.e., a safe cell concentration.

7.3ELISA kit for detecting inflammatory factor content

(1) Stimulus solution preparation

Preparation of LPS stock solution (12.5. Mu.L/mL): 10mgLPS was dissolved in 5000. Mu.LPBS, vortexed and mixed for 5min, and after 30 min of standing, 100. Mu.L was dispensed into 0.5ml centrifuge tubes and stored frozen at-20 ℃.

Preparation of LPS working solution (5. Mu.g/mL): 100. Mu.L of LPS at a concentration of 12.5. Mu.L/mL was added to 1900. Mu.L of the medium to obtain 100. Mu.g/mL of a working solution. After 2h of pretreatment, 106uL of LPS working solution was added to the 6-well plate.

(2) Positive control solution preparation:

preparation of dexamethasone stock solution (100 mg/mL): dissolving 100mg dexamethasone with 1000 mu LDMSO, shaking for 2h, mixing, subpackaging according to 20 mu L/branch volume, storing to-80deg.C, and filtering with 0.22 mu m filter membrane for sterilization.

Preparation of dexamethasone working solution (100. Mu.g/mL): 20. Mu.L of dexamethasone stock solution with the concentration of 100mg/mL is diluted to 2mL by using a culture medium, and 1000. Mu.g/mL of dexamethasone working solution 1 is obtained. 250 mu L of dexamethasone working solution 1 with the concentration of 1000 mu g/mL is diluted to 2.5mL by a culture medium, 100 mu g/mL of dexamethasone working solution 2 is obtained for standby, and 2000 mu L of dexamethasone working solution 2 is taken to a 6-hole plate for administration.

(3) Cell seeding

Cells were diluted with cell culture medium to seeding density (24 h confluence reached 45% -60%, 10 ten thousand/mL after seeding) and seeded into 6-well plates with a liquid volume per well of 2000 μl. After inoculation is finished, the mixture is placed in CO ₂ Culturing in an incubator for 24 hours. The fusion degree of the cells after 24 hours of inoculation is ensured to be in the range of 45-60%.

(4) Induction and administration

After the cell fusion degree reaches 45-60%, the old culture medium is sucked and removed, and the culture medium is replaced by a serum-free culture medium (high-sugar DMEM) for continuous culture for 12 hours; old culture solution in a 6-well plate is sucked and removed, a culture medium containing a certain concentration of a test substance is added into a test substance hole, a cell culture medium is added into a negative control hole, a culture medium containing dexamethasone is required to be added into a positive control hole, a cell culture medium is added into a blank/solvent control hole, 2000 mu L of the cell culture medium is added into each hole, and after administration, the mixture is placed into a 37 ℃ and 5% incubator for 2 hours. After 2h, except for the blank/solvent control wells, 106 μl of LPS working solution at a concentration of 100ug/ml was added to the remaining wells, the 6-well plate was gently shaken and mixed well. Put back to 37 ℃,5% CO ₂ Culturing in an incubator for 24 hours.

(5) Cell supernatant collection

After the incubation, 2000. Mu.L of the cell culture supernatant was collected in a 2.0mL sterile centrifuge tube and centrifuged at 3000r/min for 10min before the supernatant was stored. Placing in a low temperature refrigerator at-20deg.C, and freeze preserving.

(6) ELISA detection

a. Preparation before detection:

taking TNF-alpha kit as an example

1) Please remove the kit from the refrigerator 20 minutes in advance to equilibrate to room temperature.

2) The 20 Xconcentrated wash was diluted with double distilled water to a 1 Xworking solution. The unused was returned to 4 ℃.

3) Standard substance: centrifuge at 1000rpm for 1min before uncapping. Adding 0.5mL of standard substance and specimen universal diluent into the freeze-dried standard substance, standing for 15 minutes, and after the standard substance and specimen universal diluent are fully dissolved, gently mixing uniformly (the concentration is 2000 pg/mL). Dilutions were then performed as needed (e.g., using the following concentrations: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81pg/mL for the assay standard curve).

4) Biotinylated antibody working solution: the 30 Xconcentrated biotinylated antibody was diluted with biotinylated antibody diluent to a 1 Xworking solution 20 minutes prior to use in the amounts required for the current assay. Is used on the same day.

5) Enzyme conjugate working solution: the 30 Xconcentrated enzyme conjugate was diluted with enzyme conjugate diluent to a 1 Xworking solution 20 minutes prior to use in the amounts required for the current assay. Is used on the same day.

b. The operation steps are as follows:

1) The strips required for the test were removed from the sealed bag equilibrated to room temperature, unused strips and desiccant were returned to the aluminum foil bag to compress the self-seal, seal the bag, and return to 4 ℃.

2) Blank Kong Jiabiao standard substance and sample general diluent, sample or standard substance with different concentration (100 mu L/hole) is added into the other corresponding holes, the reaction holes are sealed by sealing plate gummed paper, and the incubator is incubated for 90 minutes at 37 ℃ in the dark.

3) Biotinylated antibody working solution was prepared 20 minutes in advance.

4) The plate was washed 5 times.

5) Blank wells were filled with biotinylated antibody dilution and the remaining wells were filled with biotinylated antibody working solution (100 μl/well). The reaction holes are sealed by new sealing plate gummed paper, and incubated for 60 minutes at 37 ℃ in a dark place.

6) The enzyme conjugate working solution was prepared 20 minutes in advance. Placed in the dark at room temperature (22-25 ℃).

7) The plate was washed 5 times.

8) Blank wells were added with enzyme conjugate dilutions and the remaining wells were added with enzyme conjugate working solution (100 μl/well). The reaction holes are sealed by new sealing plate gummed paper, and incubated for 30 minutes at 37 ℃ in a dark place.

9) And (3) turning on a power supply of the enzyme label instrument, preheating the instrument, and setting a detection program.

10 Plate washing 5 times.

11 100. Mu.L/well of chromogenic substrate (TMB) was added and incubated at 37℃for 15 minutes in the absence of light.

12 Adding reaction stop solution (100. Mu.L/well), mixing, and measuring OD immediately _450nm Value (within 3 minutes).

c. And (3) judging results:

OD values for each standard and specimen should be subtracted from OD values for blank wells. And (3) drawing and selecting a best fit curve by using the standard substance concentration as an abscissa and the OD value as an ordinate, and recommending to use a quadratic polynomial equation for fitting. The concentration can be found on a standard curve from the OD value of the specimen.

Calculation of TNF-alpha, IL-1 beta and IL-6 content and inhibition Rate

(1) Content of

Preparing standard substances of TNF-alpha, IL-1 beta and IL-6 into standard solutions, respectively, proportionally and stepwise diluting into a series of solutions with known concentrations, and measuring OD (OD) at 450nm by ELISA method ₄₅₀ ) Fitting regression equation for standard curve to obtain OD of each test sample ₄₅₀ The value is given by equation, and the results of the TNF-alpha, IL-1 beta and IL-6 content in the test sample are calculated. 3 duplicate wells were tested in parallel for each group and mean and Standard Deviation (SD) were calculated. The T-test analysis is carried out on the TNF-alpha, IL-1 beta and IL-6 contents of each sample group measured by the test and the negative control group by using SPSS statistical software, and the difference of P < 0.05 is statistically significant.

(2) TNF-alpha, IL-1 beta and IL-6 inhibition rates

Inhibition (%) = (1-T/C) ×100%, where T is the average of TNF- α, IL-1β or IL-6 content for each sample group and C is the average of TNF- α, IL-1β or IL-6 content for the negative control group.

Study test results:

CCK-8 cell viability assay

The effect of different doses of test samples (moisturizing water) on RAW264.7 cell viability using CCK-8 kit is shown in table 8:

TABLE 8 comparison of the Effect of test samples at different concentrations on RAW264.7 cell viability

The dosage concentration of the test sample is selected to ensure that the cell viability is not less than 80%, so 12.50 mu L/mL and 6.25 mu L/mL are selected as the dosage concentrations for the subsequent inflammatory factor content test.

RAW264.7 cell morphology characterization

The morphology of each group of RAW264.7 cells is shown in FIG. 4, the normal RAW264.7 cells are round or oval (FIG. 4A), LPS stimulation is induced for 24 hours, the morphology of the cells in the negative group is changed, the volume is increased, and the cells are stretched to be fusiform (FIG. 4B). Compared to the negative group, the test samples at different concentrations were co-stimulated with LPS, and it was found that most of the cells remained round and the morphology tended to normal cells (FIGS. 4D, 4E).

3. Detection of content and inhibition rate of inflammatory factor TNF-alpha

TABLE 9 inhibition of LPS-induced RAW264.7 cells to secrete inflammatory factor TNF-alpha by test samples

Remarks: inhibition (%) = (1-T/C) ×100%

T-average value of TNF-alpha content in sample group

Average value of TNF-alpha content in C-negative control group

FIG. 5 and Table 9 compare the secretion of TNF- α by LPS-induced RAW264.7 cells and the inhibition of the same in each test group. LPS-induced RAW264.7 cells secreted TNF- α at 1325.06pg/mL and 1599.81pg/mL, respectively, using 12.5. Mu.L/mL and 6.25. Mu.L/mL of test sample (moisturizing water), significantly lower than negative control (2060.50 pg/mL, p < 0.05), at 35.69% and 22.36% inhibition, respectively; the positive control group using 100. Mu.g/mL dexamethasone showed a content of 1278.31pg/mL for TNF- α secretion by LPS-induced RAW264.7 cells, which was also significantly lower than that of the negative control group (p < 0.05), with a TNF- α inhibition of 37.96%.

4. Detection of content and inhibition rate of inflammatory factor IL-1 beta

TABLE 10 inhibition of LPS-induced RAW264.7 cells to secrete inflammatory factor IL-1. Beta. By test samples

Remarks: inhibition (%) = (1-T/C) ×100%

Average value of IL-1 beta content in T-sample group

Average value of IL-1 beta content in C-negative control group

FIG. 6 and Table 10 compare the IL-1β content and inhibition of LPS-induced RAW264.7 cells by each test group. LPS-induced RAW264.7 cells secreted IL-1β at 17.84pg/mL and 21.41pg/mL, respectively, using 12.5. Mu.L/mL and 6.25. Mu.L/mL of test sample (moisturizing water), significantly lower than the negative control group (95.01 pg/mL, p < 0.05), with IL-1β inhibition rates of 81.22% and 77.46%, respectively; the positive control group using 100 mug/mL dexamethasone has 18.36pg/mL IL-1β content of LPS-induced RAW264.7 cells, and the IL-1β inhibition rate is 80.68% that of the negative control group (p < 0.05).

5. Detection of content and inhibition rate of inflammatory factor IL-6

Table 11 test samples for inhibition of secretion of inflammatory factor IL-6 by LPS-induced RAW264.7 cells

Remarks: inhibition (%) = (1-T/C) ×100%

Average value of IL-6 content in T-sample group

Average value of IL-6 content in C-negative control group

FIG. 7 and Table 11 compare the IL-6 secretion and inhibition of LPS-induced RAW264.7 cells by each test group. LPS-induced RAW264.7 cells secreted IL-6 at 147.04pg/mL and 354.81pg/mL, respectively, using 12.5. Mu.L/mL and 6.25. Mu.L/mL of test sample (moisturizing water), with IL-6 inhibition rates of 70.59% and 38.18% lower than negative control (544.04 pg/mL, p < 0.05), respectively; the positive control group using 100 mug/mL dexamethasone had 49.78pg/mL IL-6 content of LPS-induced RAW264.7 cells, which was also significantly lower than that of the negative control group (p < 0.05), and the IL-1 beta inhibition rate was 91.12%.

The small knot:

under the conditions of the test, 12.5 mu L/mL of the test product (moisturizing water) has inhibition effects on secretion of TNF-alpha, IL-1 beta and IL-6 by RAW264.7 cells induced by LPS, and the inhibition rates are 35.69%, 81.22% and 70.59%, respectively; 6.25. Mu.L/mL of the test product (moisturizing water) had an inhibitory effect on secretion of TNF- α, IL-1β and IL-6 by LPS-induced RAW264.7 cells, with inhibition rates of 22.36%, 77.46% and 38.18%, respectively. In conclusion, the test product has the efficacy of relieving inflammation.

Test example four efficacy evaluation of preparing cream with Compound composition

Purpose of testing

The anti-inflammatory efficacy of moisturizing water (prepared from example 3) was evaluated according to the standard method of "T/SHRH 034-2021 cosmetic soothing efficacy test-in vitro TNF- α inflammatory factor content assay lipopolysaccharide-induced macrophage RAW264.7 test method" (2021 version).

Principle research and test method is the same as test example III

Study test results:

CCK-8 cell viability assay

The effect of different doses of test samples (creams) on RAW264.7 cell viability using CCK-8 kit is shown in table 2:

TABLE 12 comparison of the Effect of test samples at different concentrations on RAW264.7 cell viability

The dosage concentration of the selected test sample should be capable of ensuring that the cell viability is not less than 90%, so that 1.00mg/mL and 0.50mg/mL are selected as the dosage concentrations for the subsequent inflammatory factor content test.

RAW264.7 cell morphology characterization

The morphology of each group of RAW264.7 cells is shown in FIG. 8, the normal RAW264.7 cells are round or oval (FIG. 8A), LPS stimulation is induced for 24 hours, the morphology of the cells in the negative group is changed, the volume is increased, and the cells are stretched to be fusiform (FIG. 8B). Compared to the negative group, the test samples at different concentrations were co-stimulated with LPS, and it was found that most of the cells remained round and the morphology tended to normal cells (FIGS. 8D, 8E).

3. Detection of content and inhibition rate of inflammatory factor TNF-alpha

TABLE 13 inhibition of LPS-induced RAW264.7 cells to secrete inflammatory factor TNF-alpha by test samples

Remarks: inhibition (%) = (1-T/C) ×100%

T-average value of TNF-alpha content in sample group

Average value of TNF-alpha content in C-negative control group

FIG. 9 and Table 13 compare the TNF- α levels and inhibition of LPS-induced RAW264.7 cells in each test group. The LPS-induced RAW264.7 cells secreted TNF- α content was 852.67pg/mL and 1042.33pg/mL, respectively, significantly lower than that of the negative control group (1725.77 pg/mL, p < 0.05), with 50.39% and 39.60% inhibition of TNF- α, respectively, using 1mg/mL and 0.5mg/mL of the test sample (cream); the positive control group using 100. Mu.g/mL dexamethasone showed a content of 957.75pg/mL for TNF- α secretion by LPS-induced RAW264.7 cells, which was also significantly lower than that of the negative control group (p < 0.05), with a TNF- α inhibition of 44.50%.

4. Detection of content and inhibition rate of inflammatory factor IL-1 beta

TABLE 14 test samples for inhibition of secretion of inflammatory factor IL-1. Beta. By LPS-induced RAW264.7 cells

Remarks: inhibition (%) = (1-T/C) ×100%

Average value of IL-1 beta content in T-sample group

Average value of IL-1 beta content in C-negative control group

FIG. 10 and Table 14 compare the IL-1β content and inhibition of LPS-induced RAW264.7 cells by each test group. The content of IL-1 beta secreted by LPS-induced RAW264.7 cells by using 1mg/mL and 0.5mg/mL of test sample (cream) is 9.16pg/mL and 10.97pg/mL respectively, which is obviously lower than that of a negative control group (27.99 pg/mL, p < 0.05), and the IL-1 beta inhibition rate is 67.26% and 61.88% respectively; the positive control group using 100 mug/mL dexamethasone had IL-1 beta content of 16.39pg/mL for LPS-induced RAW264.7 cells, which was also significantly lower than that of the negative control group (p < 0.05), and the IL-1 beta inhibition rate was 40.24%.

5. Detection of content and inhibition rate of inflammatory factor IL-6

TABLE 15 inhibition of LPS-induced RAW264.7 cells to secrete inflammatory factor IL-6 by test samples

Remarks: inhibition (%) = (1-T/C) ×100%

Average value of IL-6 content in T-sample group

Average value of IL-6 content in C-negative control group

FIG. 11 and Table 15 compare the IL-6 secretion and inhibition of LPS-induced RAW264.7 cells by each test group. IL-6 secretion levels for LPS-induced RAW264.7 cells were 729.15pg/mL and 652.22pg/mL, respectively, lower than that of the negative control group (777.00 pg/mL, p > 0.05), with IL-6 inhibition rates of 16.06% and 6.16%, respectively, using 1mg/mL and 0.5mg/mL of the test sample (cream); the positive control group using 100. Mu.g/mL dexamethasone had 275.81pg/mL IL-6 secretion by LPS-induced RAW264.7 cells, which was also significantly lower than the negative control group (p < 0.05), and the IL-1. Beta. Inhibition was 64.50%. The small knot:

Under the conditions of the test, 1mg/mL of the test product (cream) has inhibition effects on secretion of TNF-alpha, IL-1β and IL-6 by LPS-induced RAW264.7 cells, and inhibition rates are 50.59%, 61.88% and 16.06%, respectively; the test product (cream) at 0.5mg/mL had inhibitory effects on secretion of TNF- α, IL-1β and IL-6 by LPS-induced RAW264.7 cells at 39.60%, 67.26% and 6.16%, respectively. In conclusion, the test product has the efficacy of relieving inflammation. Test example five moisturizing water sensitive skin safety test and subject self-assessment

Purpose of testing

Asian adult subjects of sensitive skin normally use a test sample (moisturizing water) for 28 consecutive days, and evaluate the efficacy and safety of the test sample in soothing and moisturizing, and whether it meets the claim of being applicable to sensitive skin, by subject self-evaluation and dermatologist evaluation.

Test results

1. Subject self-assessment

After 28 days of use of the test samples, the overall satisfaction of 30 subjects with the product (moisturizing water) was 100.0%.

After 14 days of application of the sample

After 28 days of use of the sample

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2. Dermatological doctor safety assessment

Summarizing adverse reaction:

project	Adverse event proportion
		Adverse events observed by dermatologists	0％(0/30)
Adverse events rated by dermatologists as being associated with trial samples	0％(0/30)
		Serious adverse events rated by dermatologists as being associated with trial samples	0％(0/30)

Subject self-assessment

Throughout the test, no subject felt discomfort among 30 subjects.

Conclusion of the test

30 adult subjects of sensitive skin aged 21 to 36 years use the test sample (moisturizing water) for 28 consecutive days under normal conditions, and the test sample (moisturizing water) is shown to have a soothing and moisturizing effect after 28 days of use by the subject self-evaluation.

Under the control of the dermatologist, the potential for causing adverse reactions to human skin was evaluated by observing 30 adult subjects with sensitive skin aged 21 to 36, under normal conditions, using test samples continuously for 28 days. The dermatologist evaluated that 30 subjects did not develop adverse reactions after use, and the subject self-evaluation results were that 30 subjects did not feel discomfort during and after use, and the test sample (moisturizing water) was satisfied with the statement "applicable sensitive skin".

Evaluation of human body efficacy of test example Liu cream

Purpose of testing

Asian adult female subjects of sensitive skin normally use test samples (creams) for 28 consecutive days, the skin stratum corneum moisture content and the skin transepidermal moisture loss TEWL value were measured by an instrumental probe, and the subjects self-assessed, evaluating the efficacy of the test samples in moisturizing and repairing, and whether "applicable sensitive skin claims are met.

Test results

1. Instrument measurement

2. Subject self-assessment

After 28 days of use of the test samples, 31 subjects had an overall satisfaction of 100.0% with the test samples (cream), and 100.0% of the subjects considered the product to be milder and less irritating, and did not cause allergic phenomena.

Conclusion of the test

31 Asian adult female subjects with sensitive skin aged 41 to 60 years old use test samples under normal conditions for 28 days continuously, and the moisture content of the skin cuticle and the TEWL value of the skin trans-epidermal moisture loss are measured by an instrument probe, so that the test samples have the effects of moisturizing and repairing after 28 days of use; the results, by subject self-assessment, showed that the test sample had soothing efficacy after 28 days of use and was consistent with the claim of "applicable sensitive skin".

Claims

1. The composition for relieving inflammation is characterized by comprising total arasaponin and purslane polysaccharide, wherein the weight ratio of the total arasaponin to the purslane polysaccharide is 20% -40%:60% -80%.

2. The composition of claim 1, wherein the total saponins of panax notoginseng contains ginsenoside Rg1, ginsenoside Rb1 and ginsenoside R1.

3. The composition of claim 1, wherein the purslane polysaccharide comprises neutral polysaccharide and acidic polysaccharide and pectic polysaccharide.

4. The composition for relieving inflammation is characterized by comprising a total arasaponin pre-prepared solution and a purslane polysaccharide pre-prepared solution, wherein the total arasaponin pre-prepared solution and the purslane polysaccharide pre-prepared solution are in a weight ratio of 25% -35%:65% -75%.

5. The composition of claim 4, wherein the total arasaponin pre-formulation is a mixed solution containing 70% -90% of propylene glycol, 5% -20% of PEG-40 hydrogenated castor oil, 12% -5% of ginsenoside Rg, 11% -4% of ginsenoside Rb and 10.1% -1% of arasaponin R, wherein the sum of the raw materials is hundred percent.

6. The composition of claim 4, wherein the purslane polysaccharide pre-formulation is a mixed solution containing 60% -80% of water, 15% -39% of 1, 3-propanediol, 0.1% -5% of neutral polysaccharide and acidic polysaccharide and pectin polysaccharide, wherein the sum of the raw materials is hundred percent.

7. Composition according to any one of claims 1 to 6, characterized in that it is formulated as a cosmetic product.

8. The composition of claim 7, wherein the cosmetic is moisturizing water or cream.

9. A method of preparing the composition of claim 4, comprising the steps of:

(1) Purchasing raw materials of panax notoginseng saponins and purslane polysaccharide;

(2) Weighing Notoginseng radix total saponins for use; propylene glycol is heated for standby; heating PEG-40 hydrogenated castor oil for later use; adding propylene glycol into hydrogenated castor oil, stirring, adding Notoginseng radix total saponin raw material, and cooling to room temperature to obtain Notoginseng radix saponin pre-preparation solution;

(3) Weighing purslane polysaccharide for later use, uniformly mixing with 1, 3-propanediol, adding into a stirring pot, adding deionized water, and fully stirring and dissolving; transparent uniform liquid is obtained after complete dissolution; filtering and discharging after complete dissolution to obtain a purslane polysaccharide pre-prepared liquid;

(4) Uniformly mixing the notoginsenoside pre-prepared solution and the purslane polysaccharide pre-prepared solution according to the weight ratio.

10. Use of a composition according to any one of claims 1 to 6 for the preparation of a synergistic anti-inflammatory product.