CN111358802A

CN111358802A - Composition with anti-inflammatory, anti-allergy and soothing effects

Info

Publication number: CN111358802A
Application number: CN202010280866.8A
Authority: CN
Inventors: 贾霆涵; 朱丽萍; 庄琳懿; 贺来健
Original assignee: Pigeon Mother And Baby Products Shanghai Co ltd
Current assignee: Pigeon Mother And Baby Products Shanghai Co ltd
Priority date: 2020-04-10
Filing date: 2020-04-10
Publication date: 2020-07-03

Abstract

The invention provides a composition with anti-inflammatory, anti-allergy and soothing effects, which comprises hot spring water and a licorice extract. The invention also provides the application of the composition in preparing cosmetics or medicines with anti-inflammatory, anti-allergy and relieving effects.

Description

Composition with anti-inflammatory, anti-allergy and soothing effects

Technical Field

The present invention relates to a composition having anti-inflammatory, anti-allergic and soothing effects comprising spa water and licorice extract.

Background

Skin inflammation diseases caused by various factors such as environmental pollution, life pressure, use of cosmetics and the like are continuously generated, the incidence rate of skin irritation and allergic phenomena is gradually increased, people who feel sensitive skin are more and more, frequent skin inflammation reactions and related diseases thereof cause a plurality of hazards to the health of people, and meanwhile, great troubles are brought to the life of people. Therefore, the research and development of cosmetics with anti-inflammatory, anti-allergy and soothing effects for people with inflammatory skin are receiving more and more attention and favor of industries and consumers.

The following are commonly used in the present anti-inflammatory compositions:

the first is zinc oxide + licorice extract. Zinc oxide is a common drug in dermatology, and can be used as astringent, skin protectant, and sunscreen agent, and zinc ion can promote wound healing and has antiinflammatory activity. The Glycyrrhrizae radix extract such as dipotassium glycyrrhizinate has multiple effects of inhibiting bacteria, diminishing inflammation, removing toxic substance, resisting allergy, deodorizing, etc., and can be widely used in pharmaceutical, cosmetic daily chemical, food, etc. However, zinc oxide is insoluble in water and has low activity, and the powder needs to be ground and dispersed in advance, so that the zinc oxide is not stable easily in the formula. According to the legislation, when zinc oxide is used as a sunscreen, it must be added in the formulation in an amount not exceeding 25%.

The second is allantoin + licorice extract. Allantoin is an active agent claimed as a facial emollient for many people with sensitive skin, has physiological functions of promoting cell growth, accelerating wound healing, softening keratin and the like, and is a good healing agent and an anti-ulcer agent for skin wounds. The Glycyrrhrizae radix extract such as dipotassium glycyrrhizinate has multiple effects of inhibiting bacteria, diminishing inflammation, removing toxic substance, resisting allergy, deodorizing, etc., and can be widely used in pharmaceutical, cosmetic daily chemical, food, etc. However, because the content of natural allantoin extracted from natural animals and plants by the traditional process is very low, the process is complex, the cost is too high, and the traditional process cannot be applied in a large scale, the conventional chemical synthesis method is mainly adopted to extract and synthesize the allantoin, and the requirements of the market on natural source components cannot be met.

The third is bisabolol and ginger root extract, chamomile (with the chemical name: Anthemis tinctoria L.) is perennial herb of Compositae and Chamomilla, the anti-inflammatory effect is mainly from bisabolol, the ginger root extract contains gingerdiol, zingiberedione, dehydrozingiberedione and other active substances, and in vitro tests prove that the composition can effectively inhibit IL-1 α and PGE2, and has anti-inflammatory and anti-irritation effects.

Therefore, the above three compositions are not used too much in the actual product for various reasons.

The current anti-inflammatory mechanism mainly comprises the following aspects:

1. targeting pattern recognition receptor proteins, such as TLRs, located on the cell surface;

2. inhibiting the production of inflammatory-related cytokines, mainly selected from IL 1 α, IL 1 β, TNF α, IL 6, IL 8;

3. the expression of inflammation-related pathway proteins is mainly inhibited, and the inflammation-related pathway proteins mainly comprise NF kappa B and MAPKs, wherein the MAPKs comprise three pathway proteins including ERK1/2, p38 and c Jun, and the mainly detected proteins comprise NF kappa B, ERK1/2, p38 and c Jun.

4. Enzymes involved in the synthesis of inflammatory mediators: COX 2 and 5LOX, where COX 2 is an inducible enzyme, COX 2 is generally rarely expressed in normal tissues because endogenous corticosteroids exert an inhibitory effect on it. When inflammation occurs, COX 2 is highly expressed in inflamed tissues, and the expression level is closely related to the degree of inflammation. Most anti-inflammatory drugs mainly take enzymes and end products related to the metabolism process of arachidonic acid as targets.

5. Inhibit the secretion of inflammatory mediators, mainly selected from metabolites of arachidonic acid such as PGE2 and LTB4, amines such as histamine, 5HT and peptide inflammatory mediators such as bradykinin.

Aiming at the anti-allergy and relieving effects, the prior industry mainly adopts the following modes:

1. improving skin barrier, such as promoting synthesis of three major lipids and regulating balance of three major lipids;

2. modulating the reactivity of dendritic cells, thereby enhancing tolerance of the skin;

3. nerve calming effects, namely nerve calming effects are achieved by inhibiting release of inflammatory mediators such as PGE2 and histamine, and nerve calming effects are achieved by using antagonists of a thermal induction receptor TRPV 1;

4. anti-inflammatory action, since inflammation is the main symptom of skin irritation, allergy and sensitization, the main means of allergy relief at present is anti-inflammation, and the allergy relief effect is achieved by inhibiting the production of inflammatory factors such as IL 1 α, TNF α, IL8, IL 6, inflammatory mediators such as PGE2 and histamine.

Thus, there remains a need for compositions that have anti-inflammatory, anti-allergic, and soothing effects simultaneously.

Disclosure of Invention

The applicant unexpectedly found that hot spring water can synergistically act with licorice extract to elevate skin barrier, inhibit inflammatory pathway and inhibit expression level of TRPV1 gene. Accordingly, the applicant developed a composition comprising spa water and licorice extract and demonstrated that the composition of the present invention has better anti-inflammatory, anti-allergy and soothing effects in vitro 3D skin models than compositions using prior art licorice extract and zinc oxide or allantoin.

The hot spring water contains rich mineral substances, trace elements, mineral salts and proteins, particularly mineral hot springs, combines the moisturizing factors and the mineral trace elements, forms an elastic water activating network on the surface of skin, has the beautifying effects of relieving the skin, resisting sensitivity and strengthening the natural protective barrier of the skin, and enables the skin to be tender and white. The hot spring water can be directly used, has a long history in the treatment of various skin diseases, and shows higher safety.

Accordingly, in one aspect, the present invention provides a composition having anti-inflammatory, anti-allergy and soothing effects comprising spa water and licorice extract.

According to a preferred embodiment, the hot spring water is contained in an amount of 0.001 to 100% by weight, and wherein the licorice extract is contained in an amount of 0.001 to 1% by weight.

According to a preferred embodiment, the hot spring water is contained in an amount of 0.01 to 50 wt%, and wherein the licorice extract is contained in an amount of 0.01 to 0.8 wt%.

According to a preferred embodiment, the hot spring water is contained in an amount of 0.1 to 20 wt%, and wherein the licorice extract is contained in an amount of 0.03 to 0.5 wt%.

According to a preferred embodiment, the hot spring water is contained in an amount of 0.5 to 5 wt%, and wherein the licorice extract is contained in an amount of 0.05 to 0.2 wt%.

According to a preferred embodiment, said licorice extract is selected from dipotassium glycyrrhizinate.

According to a preferred embodiment, the composition with anti-inflammatory, anti-allergy and soothing effects further comprises at least one of a humectant, a fat, a thickener, an emulsifier, a neutralizing agent and a preservative.

According to a particularly preferred embodiment, the composition with anti-inflammatory, anti-allergic and soothing action according to the invention comprises as active substance only hot spring water and licorice extract.

In the invention, the hot spring water and the licorice extract are used as active ingredients for improving skin barriers, inhibiting inflammatory pathways and inhibiting the expression level of TRPV1 gene, thereby having better anti-inflammatory, anti-allergy and relieving effects. The composition can be prepared into different dosage forms according to different requirements, and preferably skin external preparations, including liquid, gel, emulsion, cream, foam, patch and the like. However, it is understood by those skilled in the art that the composition of the present invention may be added with various desired additives, including but not limited to humectants, fats and oils, thickeners, emulsifiers, neutralizing agents, preservatives, and the like, depending on the various dosage forms of the composition. In the present invention, the humectant is selected from at least one of glycerin, butylene glycol, 1, 3-propanediol, 1, 2-pentanediol, caprylyl glycol, and sodium hyaluronate. The oil is at least one selected from squalane, caprylic/capric triglyceride and shea butter. The thickening agent is at least one selected from carbomer, acrylic acid/C10-30 alkanol acrylate cross-linked polymer and xanthan gum. The emulsifier is at least one selected from polysorbate-60, sorbitan stearate, glyceryl stearate SE, cetearyl olive oleate and sorbitan olive oleate. The neutralizing agent is at least one selected from sodium hydroxide and potassium hydroxide. The preservative is selected from phenoxyethanol.

The composition of the present invention may further comprise various required cosmetically or pharmaceutically acceptable excipients depending on various formulations of the composition. For example, when the composition of the present invention is prepared as a liquid, the excipient may be selected from water, physiological saline, etc.; when the composition of the present invention is prepared into a gel, the excipient may be selected from cellulose derivatives, starch, gelatin, agar, polysaccharides, and the like; when the composition of the present invention is prepared into a cream, the excipient may be selected from glycerin, vaseline, paraffin, etc.; when the composition of the present invention is prepared into a foaming agent, the excipient may be selected from hydroxypropyl methyl cellulose, sodium lauryl sulfate polyethylene glycol, sodium fatty alcohol-polyoxyethylene ether sulfonate, etc.; when the composition of the present invention is prepared as a patch, the excipient may be selected from cellulose derivatives, starch, gelatin, agar, polysaccharides, and the like.

According to a most preferred embodiment of the present invention, the anti-inflammatory, anti-allergic and soothing composition of the present invention comprises only spa water and licorice extract as active substances and consists of the following components in weight percent:

the compositions and various dosage forms of the present invention having anti-inflammatory, anti-allergic and soothing effects can be prepared according to conventional methods known to those skilled in the art.

In yet another aspect, the present invention provides a cosmetic or pharmaceutical composition comprising a composition according to the present invention and a cosmetically or pharmaceutically acceptable excipient.

In another aspect, the present invention provides the use of the composition comprising spa water and licorice extract for the preparation of a medicament having anti-inflammatory, anti-allergy and soothing effects.

Unexpectedly, the inventors unexpectedly found that hot spring water can synergistically cooperate with licorice extract, particularly dipotassium glycyrrhizinate, to thereby enhance skin barrier, inhibit inflammatory pathway and inhibit expression level of TRPV1 gene, thereby having anti-inflammatory, anti-allergy and soothing effects.

The composition with the functions of resisting inflammation, resisting allergy and relieving has the advantages that:

the composition of the invention has better anti-inflammatory, anti-allergy and soothing effects in an in vitro 3D skin model than compositions using prior art licorice extracts and zinc oxide or allantoin.

Drawings

Figure 1 shows the effect of hot spring water, dipotassium glycyrrhizinate + zinc oxide, dipotassium glycyrrhizinate + allantoin, dipotassium glycyrrhizinate + hot spring water, and placebo on the relative expression level of TRPV1 gene in skin models.

Figure 2 shows the effect of hot spring water, dipotassium glycyrrhizinate + zinc oxide, dipotassium glycyrrhizinate + allantoin, dipotassium glycyrrhizinate + hot spring water, and placebo on the relative expression levels of COX 2 in skin models.

FIG. 3 shows the effect of hot spring water, dipotassium glycyrrhizinate + zinc oxide, dipotassium glycyrrhizinate + allantoin, dipotassium glycyrrhizinate + hot spring water, and placebo on the expression level of VEGF in a skin model.

FIG. 4 shows the effect of hot spring water, dipotassium glycyrrhizinate + zinc oxide, dipotassium glycyrrhizinate + allantoin, dipotassium glycyrrhizinate + hot spring water, and placebo on the relative expression levels of VEGF in skin models.

FIG. 5 shows the effect of hot spring water, dipotassium glycyrrhizinate + zinc oxide, dipotassium glycyrrhizinate + allantoin, dipotassium glycyrrhizinate + hot spring water, and placebo on the expression level of IL-1 α in a skin model.

FIG. 6 shows the effect of hot spring water, dipotassium glycyrrhizinate + zinc oxide, dipotassium glycyrrhizinate + allantoin, dipotassium glycyrrhizinate + hot spring water, and placebo on the relative expression level of IL-1 α in a skin model.

FIG. 7 shows a flow chart of the luminex operation described in example 5.

Detailed Description

Example 1:

creams A1, A2 and A3 comprising the hot spring water of the invention and dipotassium glycyrrhizinate, and creams A4, A5 and A6 comprising prior art compositions were prepared according to the ingredients listed in the following table, where the percentages are by weight relative to the weight of the composition.

The preparation method comprises the following steps:

1) mixing water, glycerol, acrylic acid (ester)/C10-30 alkanol acrylate crosslinked polymer, and EDTA disodium uniformly with a stirrer, and heating to 80 deg.C to obtain water phase.

2) Mixing cetearyl olive oleate, sorbitan olive oleate, shea butter, and caprylic/capric triglyceride, heating to 80 deg.C to obtain oil phase.

3) Adding the water phase into a blending kettle, adding the oil phase, mixing, and vacuum emulsifying at 80 deg.C for 30 min. Cooling to 60 deg.C, adding potassium hydroxide, and cooling to below 40 deg.C. Adding phenoxyethanol, dipotassium glycyrrhizinate, hot spring water, zinc oxide, allantoin, bisabolol, and rhizoma Zingiberis recens extract, stirring for 10min, and discharging to obtain skin cream A1-A6.

Example 2:

creams B1, B2 and B3 comprising spa water according to the invention and dipotassium glycyrrhizinate, and creams B4, B5 and B6 comprising prior art compositions were prepared according to the ingredients listed in the table below, wherein the percentages are by weight relative to the weight of the composition.

The preparation method comprises the following steps:

1) mixing water, glycerol, butanediol, acrylic acid (ester)/C10-30 alkanol acrylate crosslinked polymer with a stirrer, and heating to 80 deg.C to obtain water phase.

2) Mixing glyceryl stearate SE, polysorbate-60, and caprylic/capric triglyceride, heating to 80 deg.C to obtain oil phase.

3) Adding the water phase into a blending kettle, adding the oil phase, mixing, and vacuum emulsifying at 80 deg.C for 30 min. Cooling to 60 deg.C, adding potassium hydroxide, and cooling to below 40 deg.C. Adding phenoxyethanol, caprylyl glycol, dipotassium glycyrrhizinate, hot spring water, zinc oxide, allantoin, bisabolol, and rhizoma Zingiberis recens extract, stirring for 10min, and discharging to obtain cream B1-B6.

Example 3:

lotions C1, C2 and C3 comprising the hot spring water of the invention and dipotassium glycyrrhizinate, and lotions C4, C5 and C6 comprising prior art compositions were prepared according to the ingredients listed in the table below, wherein the percentages are percentages by weight relative to the weight of the composition.

The preparation method comprises the following steps:

1) mixing water, glycerol, 1, 2-pentanediol and carbomer with a stirrer, and heating to 80 deg.C to obtain water phase.

2) Mixing sorbitan stearate, glyceryl stearate SE, caprylic/capric triglyceride, and squalane uniformly, and heating to 80 deg.C to obtain oil phase.

3) Adding the water phase into a blending kettle, adding the oil phase, mixing, and vacuum emulsifying at 80 deg.C for 30 min. Cooling to 60 deg.C, adding sodium hydroxide, and continuously cooling to below 40 deg.C. Adding phenoxyethanol, sodium hyaluronate, disodium EDTA, dipotassium glycyrrhizinate, hot spring water, zinc oxide, allantoin, bisabolol, and rhizoma Zingiberis recens extract, stirring for 10min, and discharging to obtain skin caring cream C1-C6.

Example 4:

creams D1, D2 and D3 comprising the hot spring water of the invention and dipotassium glycyrrhizinate, and creams D4, D5 and D6 comprising prior art compositions were prepared according to the ingredients listed in the following table, where the percentages are by weight relative to the weight of the composition.

The preparation method comprises the following steps:

1) mixing water, 1, 3-propylene glycol, glycerol, carbomer and xanthan gum with a stirrer, and heating to 80 deg.C to obtain water phase.

2) Mixing sorbitan stearate, polysorbate-60, shea butter and squalane uniformly, and heating to 80 deg.C to obtain oil phase.

3) Adding the water phase into a blending kettle, adding the oil phase, mixing, and vacuum emulsifying at 80 deg.C for 30 min. Cooling to 60 deg.C, adding sodium hydroxide, and continuously cooling to below 40 deg.C. Adding phenoxyethanol, sodium hyaluronate, dipotassium glycyrrhizinate, hot spring water, zinc oxide, allantoin, bisabolol, and rhizoma Zingiberis recens extract, stirring for 10min, and discharging to obtain cream D1-D6.

Example 5: VEGF angiogenesis assay

Inflammatory factors and the like are detected on the culture supernatant by adopting a luminex method. After extraction of mRNA from the skin model using Trizol, genes of the relevant proteins were semi-quantitatively determined using RT-qPCR.

Graph Pad Prism was used for mapping and results are expressed as mean + -SD. Multiple comparisons between groups were performed using one-way ANOVA.

And (5) carrying out one-way variance statistical analysis. All statistical analyses were two-tailed. p <0.05 was considered to be of differential significance.

The specific luminex operation is as follows:

cell supernatant samples: 10,000rpm, centrifugation for 10min, supernatant was taken, 2-fold dilution was performed with RD6-52, and 50. mu.L of diluted sample was finally applied for testing, and all samples were set for duplicate testing. To the standard bottle, the corresponding amount of RD6-52 was added as specified, turned upside down several times, and placed on a low speed horizontal shaker for 15 min. The standard was then diluted as shown in FIG. 7.

Taking the microbeads, shaking for 30s at 1,400rpm on a shaker, and diluting the microbeads by using RD 2-1; shaking the diluted microbeads for 30s again by using an oscillator at 1,400rpm, and adding 50 mu L of the microbeads into a 96-well plate per well; 50 μ L of the prepared standard, sample and Blank were added to the corresponding wells, a sealing film was applied, and the mixture was placed on a plate shaker at 850rpm and shaken under dark conditions at room temperature for 2 h. Discarding the sample, and washing 3 times by using a plate washing machine; biotin Antibody Cocktail was diluted as required by the instructions using RD 2-1; mu.L of diluted Biotin Antibody Cocktail was added to each well, and a sealing membrane was attached, and the mixture was shaken at 850rpm on a plate shaker, protected from light, and incubated at room temperature for 1 hour. The Biotin Antibody Cocktail was discarded and washed 3 times using a plate washer; diluting Streptavidin-PE by using Wash Buffer according to the instruction; adding 50 μ L of diluted Streptavidin-PE into each well, attaching a sealing membrane, placing on a flat shaking table, oscillating at 850rpm, keeping out of the sun, incubating at room temperature for 30 min; washing for 3 times by using a plate washing machine; adding 100 μ L Wash Buffer into each well, resuspending, attaching sealing membrane, placing on a flat plate shaker at 850rpm, room temperature, keeping out of the sun, and shaking for 2 min; the corrected readings are sent to a Luminex 200 machine. After the sample and the standard substance detected by the experiment are detected by a Luminex 200 detector, the obtained fluorescence is automatically calculated and optimized by software.

1mL of cell culture supernatant obtained by co-culturing the emollient cream A2 of the present invention prepared in example 1 and the prior art emollient creams A4, A5 and A6, and water as a blank control with a recombinant human epidermal model were added to the luminex liquid phase chip, respectively. The culture method comprises the following steps: experimental grouping and processing conditions were grouped and labeled. The samples were treated by surface administration in a volume of 25. mu.L/model. The 6-well plate of the treated skin model sample was incubated at 37 ℃ in an incubator with 5% CO2 and 95% relative humidity for 24 h. After incubation for 24h, 6-well plates were taken out from the incubator, the media in the 6-well plates were taken out and stored at-80 ℃ for downstream luminex liquid chip quantitative experiments, while different experimental groups of skin models were washed with sterile PBS. The washing time of each model is controlled to be about 1min, so that the time for contacting each model with PBS is the same. After 15 times of cleaning, gently absorbing water inside and outside the model by using a sterile cotton swab, then placing the model in a 6-hole plate, and adding cell lysate into the skin model to perform nucleic acid extraction.

In the identification of the proteins secreted after the action of the respective cream products, we found that cream a2 of the present invention resulted in a significant reduction in the inflammatory factors of VEGF by erythropoiesis, relative to the prior art creams a4, a5 and a6 and the blank control (see fig. 3 and fig. 4).

Example 6 IL-1 α inflammatory stimulus assay

The IL-1 α inflammation stimulation test was performed as described in example 5, 1mL of cell culture supernatant obtained by grouping and labeling the experimental grouping and treatment conditions with the skin cream A2 of the present invention prepared in example 1, the prior art skin creams A4, A5 and A6, and water as a blank, and incubating with a reconstituted human epidermal model, by grouping and labeling the experimental grouping and treatment conditions, the samples were treated by surface administration at a volume of 25. mu.L/model, the 6-well plate of the treated skin model samples was placed in an incubator at 37 ℃, 5% CO2, and a relative humidity of 95%, incubated for 24h and incubated for 24h, the 6-well plate was removed from the incubator, the 6-well medium was removed from the 6-well plate and stored at-80 ℃ for the downstream luminex liquid phase chip quantitative test, while the skin models of different experimental groups were washed with sterile PBS, the washing time for each model was controlled at about 1min to ensure that the time for each model was in contact was the same PBS 15 times, the skin models were washed with sterile PBS, and the cell samples were gently lysed by adding sterile PBS, and the cotton swab was added to the cell samples.

In the identification of the proteins secreted after the action of the respective cream products, we found that the cream a2 of the present invention resulted in a significant reduction in the inflammatory factors secreted by the cells of inflammation-related IL-1a, relative to the prior art creams a4, a5 and a6 and the blank control (see fig. 5 and 6).

Example 7: TRPV1 Gene expression level assay

The quantitative and qualitative analysis of the initial template is realized by real-time detection of fluorescent signals corresponding to PCR products amplified in each cycle of real-time quantitative PCR, the experiment adopts an SYBR fluorescent dye method, SYBR fluorescent dye is added into a PCR reaction system, and after being specifically doped into a DNA double strand, the SYBR fluorescent dye emits fluorescent signals, but SYBR dye molecules not doped into the strand do not emit any fluorescent signals, so that the increase of the fluorescent signals and the increase of PCR products are completely synchronous. The specific operation steps of the fluorescent quantitative PCR detection gene semiquantitative analysis are as follows:

the RNA extraction procedure was as follows:

1. after adding 1ml of Trizol, the sample is placed for 5min at room temperature, fully cracked, and can be stored for a long time at-80 ℃ (if the sample is tissue, liquid nitrogen grinding is needed, and then Trizol is added for cracking).

2.12000 g were centrifuged at 4 ℃ for 5min and the supernatant was transferred to a new 1.5ml centrifuge tube.

3. Chloroform was added at 1/5 chloroform/ml Trizol, inverted for 30s, left at room temperature for 5min, and the shaker was disabled to avoid fragmentation of genomic DNA.

Centrifuge at 12000g for 15min at 4.4 ℃.

5. The upper aqueous phase was pipetted as much as possible into another centrifuge tube (here a 1.5ml EP tube). Note: the intermediate interface is aspirated, and if simultaneous extraction of DNA and protein is desired, the lower liquid is kept at 4 ℃.

6. 0.5-1 times of isopropanol/ml Trizol, adding isopropanol, inverting, and standing at room temperature for 10 min.

Centrifuging at 7.4 deg.C and 12000g for 10min, discarding supernatant, and precipitating RNA at the bottom of the tube. (in the centrifugal process, the large head end of the ep tube cover faces outwards, the RNA is pasted on the bottom of the surface after centrifugation, and the other surface can be sucked when the supernatant is sucked, so that the RNA is prevented from being sucked away)

8. The pellet was washed with 75% ethanol in equal amounts to Trizol. 7500g, 4 deg.C, centrifugating for 5min, discarding supernatant and retaining precipitate.

9. Repeating the step 8;

10. air drying at room temperature or vacuum drying for 5-10 min.

11. The RNA sample can be solubilized by treatment with an appropriate amount of DEPC.

The procedure for reverse transcription of Trizol-extracted RNA into cDNA using reverse transcription reagent was as follows:

first Strand cDNA Synthesis

1. RT reaction solution was prepared according to the following composition (the reaction solution was prepared on ice).

Reagent	Amount of the composition used	Final concentration
			5×PrimeScript RT Master Mix(Perfect Real Time)	2μl	1X
Total RNA	-	500ng
			RNase Free dH2O	up to 10μl

2. After gentle and uniform mixing, carrying out reverse transcription reaction under the following conditions:

15min at 37 ℃ (reverse transcription)

5sec at 85 ℃ (inactivation reaction of reverse transcriptase)

And (4) preserving at the temperature of 4 ℃.

The cDNA was semi-quantitatively detected using fluorescent quantitative PCR, the procedure was as follows:

quantitative fluorescence QPCR assay

The template used on the machine is the dilution multiple: 5 times of

qPCR reaction system:

reagent	Dosage per ul
		TB Green Premix Ex Taq II(Tli RNaseH Plus)(2×)	5
Primer F	0.4
		Primer R	0.4
ROX Reference Dye(50×)	0.2
		DNA Sample	1
ddH2O	3
		Total Volume	10

qPCR reaction conditions:

95℃ 30s 1cycle

95℃ 5s 40cycles

60℃ 30s 40cycles

CT detection limit: 40 cycles;

1mL of cell tissue lysate was used, which was obtained by co-culturing the emollient cream A2 of the present invention prepared in example 1 and the prior art emollient creams A4, A5 and A6, and water as a blank control, with a human recombinant epidermal model. The culture method comprises the following steps: experimental grouping and processing conditions were grouped and labeled. The samples were treated by surface administration in a volume of 25. mu.L/model. The 6-well plate of the treated skin model sample was incubated at 37 ℃ in an incubator with 5% CO2 and 95% relative humidity for 24 h. After incubation for 24h, 6-well plates were taken out from the incubator, the media in the 6-well plates were taken out and stored at-80 ℃ for downstream luminex liquid chip quantitative experiments, while different experimental groups of skin models were washed with sterile PBS. The washing time of each model is controlled to be about 1min, so that the time for contacting each model with PBS is the same. After 15 times of cleaning, gently absorbing water inside and outside the model by using a sterile cotton swab, then placing the model in a 6-hole plate, and adding cell lysate into the skin model to extract nucleic acid for a downstream fluorescent quantitative PCR experiment.

In the above fluorescent quantitative PCR results we found that emollient a2 of the present invention resulted in a significant reduction of TRPV1, a thermosensitive receptor that modulates neurosedation, relative to prior art emollients a4, a5 and a6 and the blank control (see fig. 1).

Example 8: COX 2 inflammatory protein assay

The COX 2 inflammatory protein assay was performed as described in example 7 for the fluorescent quantitative PCR procedure.

1mL of a tissue lysate obtained by co-culturing the emollient cream A2 of the present invention prepared in example 1 and the prior art emollient creams A4, A5 and A6 and water as a blank control with a human reevaluated epidermal model was used. The culture method comprises the following steps: experimental grouping and processing conditions were grouped and labeled. The samples were treated by surface administration in a volume of 25. mu.L/model. The 6-well plate of the treated skin model sample was incubated at 37 ℃ in an incubator with 5% CO2 and 95% relative humidity for 24 h. After incubation for 24h, 6-well plates were taken out from the incubator, the media in the 6-well plates were taken out and stored at-80 ℃ for downstream luminex liquid chip quantitative experiments, while different experimental groups of skin models were washed with sterile PBS. The washing time of each model is controlled to be about 1min, so that the time for contacting each model with PBS is the same. After 15 times of cleaning, gently absorbing water inside and outside the model by using a sterile cotton swab, then placing the model in a 6-hole plate, and adding cell lysate into the skin model to extract nucleic acid for a downstream fluorescent quantitative PCR experiment.

In the fluorescent quantitative PCR results we found that emollient A2 of the present invention resulted in a significant reduction of COX-2, represented by the inflammatory pain, relative to prior art emollients A4, A5 and A6 and the blank control (see FIG. 2).

And (4) conclusion: the above examples 5-8 show that the compositions of the present invention have better anti-inflammatory, anti-allergic and soothing effects in vitro 3D skin models than compositions using prior art licorice extracts and zinc oxide or allantoin.

Claims

1. A composition with antiinflammatory, antiallergic and soothing effects comprises hot spring water and Glycyrrhrizae radix extract.

2. The composition of claim 1, wherein said thermal spring water is present in an amount of 0.001-100% by weight, and wherein said licorice extract is present in an amount of 0.001-1% by weight.

3. The composition of claim 2 wherein said thermal spring water is present in an amount of 0.01 to 50% by weight, and wherein said licorice extract is present in an amount of 0.01 to 0.8% by weight.

4. The composition according to claim 3, wherein the hot spring water is contained in an amount of 0.1-20 wt%, and wherein the licorice extract is contained in an amount of 0.03-0.5 wt%.

5. The composition according to claim 4, wherein the hot spring water is contained in an amount of 0.5-5 wt%, and wherein the licorice extract is contained in an amount of 0.05-0.2 wt%.

6. The composition of claim 1, wherein the licorice extract is selected from dipotassium glycyrrhizinate.

7. The composition of any one of claims 1-6, further comprising at least one of a humectant, a grease, a thickener, an emulsifier, a neutralizer, and a preservative.

8. The composition of claim 7, wherein the humectant is selected from at least one of glycerin, butylene glycol, 1, 3-propanediol, 1, 2-pentanediol, caprylyl glycol, and sodium hyaluronate.

9. The composition of claim 7, wherein the oil is selected from at least one of squalane, caprylic/capric triglyceride, and shea butter.

10. The composition of claim 7, wherein the thickener is selected from at least one of carbomer, acrylic/C10-30 alkanol acrylate crosspolymer, and xanthan gum.

11. The composition according to claim 7, wherein the emulsifier is selected from at least one of polysorbate-60, sorbitan stearate, glyceryl stearate SE, cetearyl olive oleate and sorbitan olive oleate.

12. The composition of claim 7, wherein the neutralizing agent is selected from at least one of sodium hydroxide and potassium hydroxide.

13. The composition of claim 7, wherein the preservative is selected from phenoxyethanol.

14. The composition according to claim 1, comprising only spa water and licorice extract as active substances.

15. A cosmetic or pharmaceutical composition comprising a composition according to any one of claims 1 to 13 and a cosmetically or pharmaceutically acceptable excipient.

16. Use of a composition according to any one of claims 1 to 14 for the preparation of a cosmetic or pharmaceutical product with anti-inflammatory, anti-allergic and soothing effects.