CN111961702A

CN111961702A - Cytological evaluation method for anti-skin infectious inflammatory reaction of plant extract and application thereof

Info

Publication number: CN111961702A
Application number: CN202010708424.9A
Authority: CN
Inventors: 卢伊娜; 周利丹; 田军
Original assignee: Shanghai Jiabei Biotechnology Co ltd
Current assignee: Shanghai Jiakai Biological Co ltd
Priority date: 2020-07-22
Filing date: 2020-07-22
Publication date: 2020-11-20

Abstract

The invention discloses a cytological evaluation method for plant extract resisting skin infectious inflammatory reaction, which comprises the following steps: treating cells with the stimulator and the plant extract, collecting supernatant, detecting cell membrane integrity and/or inflammatory factor expression level, and evaluating the effect of the plant extract on resisting skin infectious inflammatory response. The method establishes an inflammation model including specific stimulators and specific detection indexes, is a high-throughput, low-cost and easily-repeated cytological detection method, is used for evaluating the efficacy of the plant extract on resisting skin infectious inflammatory reactions, and greatly shortens the research period of products of the plant extract on resisting skin infection.

Description

Cytological evaluation method for anti-skin infectious inflammatory reaction of plant extract and application thereof

Technical Field

The invention relates to the technical field of cytological detection, in particular to a cytological evaluation method for anti-skin infectious inflammatory response of a plant extract and application thereof.

Background

Skin infection refers to skin diseases caused by microorganisms such as bacteria, fungi, viruses and the like, and after an infection source invades the skin, skin cells are damaged, the cells release inflammatory mediators, dendritic cells, lymphocytes and mast cells are further promoted to be activated, inflammatory reaction is caused, symptoms such as skin redness, itching, swelling and pain and even ulceration are shown, the appearance is not only influenced, but also various inconveniences are brought to patients.

At present, many plant extracts on the market are declared to have strong anti-skin infection activity and can resist skin inflammation, but the efficacy is not evaluated by a quantifiable laboratory method, and tests on animals or human bodies generally have long test period, low efficiency and objective results, so that the development speed of the plant extracts applied to skin care products and cosmetics is limited.

Disclosure of Invention

In order to solve the above problems, a first aspect of the present invention provides a cytological evaluation method of an anti-infectious inflammatory reaction of skin of a plant extract, comprising the steps of: treating cells with the stimulator and the plant extract, collecting supernatant, detecting cell membrane integrity and/or inflammatory factor expression level, and evaluating the effect of the plant extract on resisting skin infectious inflammatory response.

As a preferred technical scheme, the stimulant is selected from one or more of fungi, bacteria and virus RNA analogues.

As a preferred technical scheme, the fungus is malassezia furfur.

As a preferred technical scheme, the bacteria are selected from one or more of Propionibacterium acnes, staphylococcus, streptococcus and bacterial mural lipopolysaccharide.

In a preferred embodiment, the viral RNA analog is polyinosinic acid cytidylic acid.

As a preferred embodiment, the fungi or bacteria are inactivated prior to treating the cells.

As a preferred technical solution, the inactivation treatment comprises the following steps: culturing fungi or bacteria for not less than 48h, determining the final concentration of the bacteria, inactivating at 80-90 ℃ for 0.5-1.5 h, cooling, subpackaging, and freezing.

As a preferred technical scheme, the detection method of the integrity of the cell membrane is selected from one or more of MTT staining method, neutral red staining method, LDH staining method and microscopic photography method.

In a preferred embodiment, the inflammatory factor is selected from one or more of nitric oxide, prostaglandin, and interleukin.

The second aspect of the invention provides the application of the cytological evaluation method, which is applied to the fields of cosmetics, dermatology and biomedicine.

Has the advantages that: the invention provides a cytological evaluation method for plant extract anti-skin infection inflammatory reaction, establishes an inflammation model including specific stimulators and specific detection indexes, is a high-throughput, low-cost and easily-repeated cytological detection method, is used for evaluating the efficacy of plant extract on resisting skin infection inflammatory reaction, and greatly shortens the research period of plant extract anti-skin infection products.

Drawings

To further illustrate the advantages of the cytological evaluation of the anti-inflammatory response of skin infection of the plant extract provided in the present invention and the use thereof, the accompanying drawings are provided, it should be noted that the drawings provided in the present invention are only selected as individual examples in all drawings and are not intended to limit the claims, and all other corresponding maps obtained by the drawings provided in the present application should be considered within the scope of protection of the present application.

FIG. 1 shows the results of MTT staining method of example 1 of the present invention.

FIG. 2 shows the results of experiments on the expression levels of IL-6 and PGE-2 in example 1 of the present invention.

FIG. 3 shows the results of MTT staining method of example 2 of the present invention.

FIG. 4 shows the results of experiments on the expression levels of IL-6 and PGE-2 in example 2 of the present invention.

FIG. 5 shows the results of experiments on the expression levels of IL-6 and PGE-2 in example 3 of the present invention.

Detailed Description

The invention will be further understood by reference to the following detailed description of preferred embodiments of the invention and the examples included therein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. To the extent that a definition of a particular term disclosed in the prior art is inconsistent with any definitions provided herein, the definition of the term provided herein controls.

As used herein, a feature that does not define a singular or plural form is also intended to include a plural form of the feature unless the context clearly indicates otherwise. It will be further understood that the term "prepared from …," as used herein, is synonymous with "comprising," including, "comprising," "having," "including," and/or "containing," when used in this specification means that the recited composition, step, method, article, or device is present, but does not preclude the presence or addition of one or more other compositions, steps, methods, articles, or devices. Furthermore, the use of "preferred," "preferably," "more preferred," etc., when describing embodiments of the present application, is meant to refer to embodiments of the invention that may provide certain benefits, under certain circumstances. However, other embodiments may be preferred, under the same or other circumstances. In addition, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.

The term "plant extract" as used herein refers to a substance extracted or processed from all or a part of a plant using a suitable solvent or method. The detection method in the present application is used for evaluating the efficacy of different plant extracts against infectious inflammatory reactions of the skin, and the type of plant extract is not particularly limited.

In some preferred embodiments, the cell is a keratinocyte.

The term "keratinocytes" as used herein is the main constituent cells of the epidermis, accounting for more than 80% of the epidermal cells, which produce keratin during differentiation and form a protective barrier for the human body, and is divided into five layers according to the differentiation stages and characteristics, the stratum basale, the stratum spinosum, the stratum granulosum, the stratum lucidum and the stratum corneum from the inside to the outside.

In some preferred embodiments, the keratinocytes are human immortalized keratinocytes (HaCaT cells) or normal human epidermal keratinocytes (NHEK cells).

The term "stimulant" as used herein refers to a substance capable of causing an infectious inflammatory response in a cell.

In some preferred embodiments, the stimulus is selected from the group consisting of a fungus, a bacterium, a mixture of one or more of viral RNA analogs.

The term "fungi" as used herein refers to eukaryotic, sporulating, chloroplast-free eukaryotes, some of which cause infectious diseases of human skin, mucous membranes, hair, and the like.

In some preferred embodiments, the fungus is malassezia furfur.

The term "malassezia furfur" (scientific name m.furfur) as used herein is a lipotropic yeast that exists on the skin surface and causes pityriasis versicolor, pityrosporum folliculitis, seborrheic dermatitis, atopic dermatitis, etc.

The term "bacterium" as used herein refers to a prokaryotic organism with a simple cellular structure lacking nuclei, according to the bacterial morphology, there are cocci, bacilli, spirochetes, etc.

In some preferred embodiments, the bacteria are selected from one or more of propionibacterium acnes, staphylococci, streptococci, bacterial mural lipopolysaccharides; further preferably, the bacteria are propionibacterium acnes and/or bacterial mural lipopolysaccharides.

The term "Propionibacterium acnes" as used herein is a species of Propionibacterium, a causative bacterium of acne, whelk, mainly colonizing the skin, sebaceous glands, intestinal tract and dairy products of humans and animals, and genomic studies have shown that several genes of this bacterium are capable of producing several enzymes and immunogenic proteins that break down skin tissue.

The term "Lipopolysaccharide" (abbreviated as LPS) as used herein is a component of the outer wall of the cell wall of gram-negative bacteria, and is composed of lipids and polysaccharides, which exhibit various biological activities by binding to Toll-like receptors on the cell membrane surface of host cells when they act on biological cells such as human or animals, to cause inflammatory reactions.

In some preferred embodiments, the viral RNA analog is polyinosinic acid cytidylic acid.

The term "polyinosinic acid cytidylic acid" as used herein refers to a polynucleotide and polyamine compound synthesized by mimicking viral RNA, which is a viral RNA analog that induces interferon production in various cells in vivo. Meanwhile, the polypeptide can be combined with Toll receptor 3 to activate a TLR3 pathway and induce immune response.

From the viewpoint of improving the detection accuracy, in some preferred embodiments, the fungus or bacterium is subjected to inactivation treatment before treating the cells.

In some preferred embodiments, the inactivation treatment comprises the steps of: culturing fungi or bacteria for not less than 48h, determining the final concentration of the bacteria, inactivating at 80-90 ℃ for 0.5-1.5 h, cooling, subpackaging, and freezing.

In some preferred embodiments, the method for determining the final concentration of bacteria is absorbance, and the determination wavelength is 620 nm.

In some preferred embodiments, the method for detecting the integrity of the cell membrane is selected from a mixture of one or more of MTT staining, neutral red staining, LDH staining, and microscopic photography.

The term "MTT staining method" herein is a method for detecting the survival and growth of cells by detecting the fact that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT (chemical name: 3- (4, 5-dimethylthiazol-2) -2, 5-diphenyltetrazolium bromide, trade name: thiazole blue, a yellow dye) into water-insoluble blue-violet crystalline Formazan (Formazan) and deposit in cells, while dead cells do not have this function, dimethyl sulfoxide (DMSO) can dissolve Formazan in cells, and the light absorption value is measured at 492nm wavelength by an enzyme linked immunosorbent assay, which can indirectly reflect the number of living cells.

The concentration of the MTT solution in the application is 5mg/mL, and the preparation method comprises the following steps: weighing 0.5g of MTT, dissolving in 100mL of Phosphate Buffer Solution (PBS), filtering, and refrigerating for storage; wherein the formulation of Phosphate Buffer Solution (PBS) is 8g of sodium chloride, 0.2g of potassium chloride, 1.44g of disodium hydrogen phosphate and 0.24g of potassium dihydrogen phosphate, the pH is adjusted to 7.4, and the volume is fixed to 1L.

The term "neutral Red" (chemical name: 3-amino-7-methylamino-2-methylphenazine hydrochloride, CAS number: 553-24-2) is used herein as a basic phenazine dye for vital cell staining and for pH indicators, where living cells absorb a large amount of the neutral Red stain without dead cells being stained in a neutral or slightly alkaline environment, and a neutral Red aqueous solution having a concentration of 3.3mg/mL is used for the experiment. The preparation method comprises the following steps: 0.33g of neutral red was weighed, dissolved in 100mL of Phosphate Buffered Saline (PBS), filtered, and stored at room temperature in the dark.

The term "LDH" as used herein refers to lactate Dehydrogenase (England name: Lactic Acid Dehydrogenase), which is a stable cytosolic enzyme, present in all cells and rapidly released into the cell culture solution when the cell membrane is damaged, and the integrity of the cell membrane can be indirectly obtained by detecting the activity of LDH.

In some preferred embodiments, the inflammatory factor is selected from the group consisting of nitric oxide, prostaglandins, interleukins, and mixtures of one or more thereof.

In some preferred embodiments, the prostaglandin is prostaglandin E2 (PGE-2).

In some preferred embodiments, the interleukin is selected from a mixture of one or more of interleukin 6(IL-6), interleukin 1 alpha (IL-1 alpha), interleukin 8 (IL-8).

The term "inflammatory factor" herein refers to various cytokines involved in inflammatory reactions, in which nitric oxide and prostaglandin cause vasodilation, pain, tissue damage, etc., and interleukin (collectively, interleukin) causes fever.

In some preferred embodiments, the method for detecting inflammatory factors is an ELISA method.

The term "ELISA method" as used herein refers to an Enzyme-Linked ImmunoSorbent Assay (Enzyme-Linked ImmunoSorbent Assay) which is based on the principle that an antibody is bound to an Enzyme complex and an analyte is detected by color development.

Examples

The present invention will be specifically described below by way of examples. It should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and that the insubstantial modifications and adaptations of the present invention by those skilled in the art based on the above disclosure are still within the scope of the present invention.

Example 1

Example 1 provides a cytological evaluation method of plant extracts against infectious inflammatory reactions of the skin, wherein the reagent consumables and instruments and equipment mainly used in the method are shown in table 1, and all the plant extracts are commercially available products.

TABLE 1 Primary reagent consumables and instrumentation

The method comprises the following steps:

a. preparation of malassezia furfur m.furfur inactivated culture solution: furfur is cultured in Malassezia LAN medium under aerobic conditions for 48 hours or more, and the final concentration of the bacteria (generally 8X 10) is determined based on the absorbance at 620nm⁸CFU/mL), thermally inactivating at 85 deg.C for 30min, cooling, subpackaging 1.6mL each tube, and freezing at-20 deg.C;

b. evaluation of efficacy of the extracts against infectious inflammatory reactions of the skin: keratinocyte NHEK was cultured to logarithmic growth phase with DMEM medium containing 10 wt% FBS (fetal bovine serum), 1 wt% PS (streptomycin qinghaosu). The first day of the experiment, after 5min of trypsinization, the cell suspension was collected at 1 x 10⁴Spreading the culture medium to a 96-well plate per well, and continuously culturing overnight; the following day of the experiment, after the cell change, 200. mu.l of the diluted medium was added to 0.75X 10⁸CFU/ml Malassezia furfur (M.furfur) culture solution, adding different plant extracts diluted to 0.25 wt% with culture medium, wherein the plant extracts comprise Glycyrrhrizae radix extract, fructus Vitics Simplicifoliae extract, radix Gentianae extract, Polygoni Multiflori radix extract, plant sensitizer (containing radix Sophorae Flavescentis, Glycyrrhrizae radix, Scutellariae radix) solution, and nutrient extract (containing fructus Vitics Simplicifoliae, radix Gentianae, Polygoni Multiflori radix) solution, and continuously treating cells for 20 hr; on the third day of the experiment, the supernatant was collected into a new 96-well plate, the expression levels of PGE-2 and IL-8 were measured by ELISA, 200. mu.l of MTT (medium prepared at a concentration of 500. mu.g/ml) was added to the cell plate and stained for 3 hours, the supernatant was discarded, 200. mu.l of DMSO solution was added, absorbance was measured at 492nm using a microplate reader, and the integrity of the cell membrane was evaluated by microscopic photography.

The results of the m.furfur-treated MTT staining method are shown in fig. 1, the left panel is an untreated control group, the middle panel is an m.furfur-treated group, and a significant reduction in the number of cells in the treated sample was observed. The right panel is the combined treatment group of m.furfur with 0.25 wt% of nutrient concentrate, and it can be observed that the cell number of the treated sample is significantly increased compared to the medium panel m.furfur group. 0.25 wt% of vitex rotundifolia extract also increased cell number to some extent, but was less effective than the nutrient source extract. The licorice extract, gentian extract, fleece-flower root extract and plant sensitizer (containing flavescent sophora root, licorice and scutellaria root) solution have no obvious influence on cell number. The results of the microscopic photography were consistent with the MTT staining method.

The results of expression levels of IL-6 and PGE-2, which are mediators of inflammation, are shown in FIG. 2. Compared with a control group, the M.furfur can promote the increase of the expression levels of IL-6 and PGE-2, different plant extracts have different efficacies, and the expression levels of the plant extracts to the inflammation mediators are ranked according to the results of combining the two inflammation mediators: the control group is a nourishing source extract, a Chinese goldthread extract, a liquorice extract, a polygonum multiflorum extract and a stimulating group.

And then, the efficacy of the plant extract is continuously verified on a human clinical anti-inflammatory model, and through earlier groping, the M.furfur can not induce the generation of inflammation no matter the M.furfur is smeared on a 3D skin model or human skin. However, the inventors found that malassezia is associated with dandruff, scalp itch, and scalp inflammation, so 17 subjects with symptoms of scalp redness, scalp itch, dandruff, scalp oil, etc. were recruited by the inventors to evaluate the association of cytological procedures with clinical evaluation of humans as follows:

17 subjects with seborrheic scalp dermatitis were recruited, of which 5 subjects with severe dandruff symptoms and 8 subjects with severe red and swollen scalp symptoms all had symptoms of itching head. The conditioning shampoo is used in 2 weeks of the pre-conditioning period, the testing shampoo containing 1 wt% of the nutrient source essence extract and 1 wt% of the licorice extract is used in 4 weeks of the testing period, the shampoo is washed 3 times per week, and the shampoo is washed clean after massaging the scalp for at least 1 minute each time. The subjects were followed regularly during the pre-conditioning period T0w and the testing period T4w, respectively, and the degree of dandruff, the degree of red swelling of the scalp, and the degree of itching of the scalp were recorded. The scoring criteria were: score 0-none, score 1-insignificant, score 2-mild, score 3-moderate, score 4-severe, score 5-very severe.

As shown in table 2, the conditioning period was substantially the same as before the use, and after 4 weeks using the shampoo containing 0.5 wt% of the extract of the nutrient source, the redness score, dandruff score, and itching score were all significantly decreased (P <0.01) compared to the conditioning period, and the improvement effect was not strong in the shampoo containing 0.5 wt% of the licorice extract.

Table 2 shampoo efficacy test results with plant extracts

	Before use	Conditioning period	1 wt% of licorice extract	1 wt% of nutrient source essence
					Red and swollen score	2.21±1.00	2.31±0.90	2.06±0.90	0.75±0.40^**
Dandruff scoring	1.85±1.20	1.98±1.40	1.91±1.30	0.47±0.30^**
					Pruritus scoring	3.14±0.90	3.20±1.00	3.03±1.10	1.75±1.20^**

The results are combined to show that the anti-inflammatory efficacy of different plant extracts can be evaluated by using the M.furfur-induced cell damage detection and inflammatory factor expression detection methods, and the cytological evaluation result is basically consistent with the clinical result.

Example 2

Example 2 provides a cytological evaluation method of plant extracts against infectious inflammatory reaction of skin, in which the main reagent consumables and instruments and equipment are shown in table 3, and each plant extract is a commercially available product.

TABLE 3 Primary reagent consumables and instrumentation

The method comprises the following steps:

a. preparation of propionibacterium acnes inactivation culture solution: culturing Propionibacterium acnes under anaerobic condition for more than 48h, and measuring the final concentration of bacteria (generally 5 × 10) according to the absorbance at 620nm⁸CFU/mL), heat-inactivated at 85 ℃ for 1h, cooled, and then portioned 1.0mL per tube, frozen at-20 ℃ for use.

b. Evaluation of efficacy of the extracts against infectious inflammatory reactions of the skin: HaCaT cells were cultured to logarithmic growth phase in DMEM medium containing 10 wt% FBS (fetal bovine serum), 1 wt% PS (streptomycin qinghao). The first day of the experiment, after 5min of trypsinization, the cell suspension was collected at 1 x 10⁴Spreading the culture medium to a 96-well plate per well, and continuously culturing overnight; the following day of the experiment, after the cell change, 200. mu.l of the diluted medium was added to 0.25X 10⁸Adding CFU/ml P.acnes culture solution, adding different plant extracts diluted with culture medium to 0.25 wt%, wherein the plant extracts include Glycyrrhrizae radix extract, radix Sophorae Flavescentis extract, fructus Vitics Simplicifoliae extract, nutrient source essence (containing fructus Vitics Simplicifoliae, radix Gentianae, Polygoni Multiflori radix) solution, fructus Punicae Granati extract, cortex Magnolia officinalis extract, herba Andrographitis extract, and triplicin (containing fructus Punicae Granati and cortex Magnolia officinalis) solution, and further processing cells for 20 hr; on the third day of the experiment, the supernatant was collected in a new 96-well plate, and prostaglandin E2(PGE2) and oxygen were tested by ELISAThe expression levels of Nitric Oxide (NO) and interleukin 8(IL-8) were measured, and 200. mu.l of MTT (medium prepared in a concentration of 500. mu.g/ml) was added to the cell plate and stained for 3 hours, and then the supernatant was discarded, and 200. mu.l of DMSO solution was added thereto, and the cell membrane integrity was evaluated by measuring the absorbance at 492nm using a microplate reader, and also by the LDH staining method.

The results of the p.acnes-treated MTT staining are shown in fig. 3, with the left panel showing the untreated control group and the middle panel showing the p.acnes-treated group, a significant reduction in the number of cells in the treated sample was observed. The right panel is the combined treatment group of p.acnes with 0.25 wt% tripicolin, and a significant increase in cell number of the treated samples was observed compared to the middle panel p.acnes group. 0.25 wt% of pomegranate extract and magnolia extract also increased cell number to some extent, but the effect was weaker than triplicin. The licorice extract, the sophora flavescens extract, the vitex rotundifolia extract, the nutrient source essence extract and the andrographis paniculata extract have no obvious influence on the cell number. The results of the microscopic photography method and the LDH staining method are consistent with the MTT staining method.

The results of expression levels of IL-6 and PGE-2, which are mediators of inflammation, are shown in FIG. 4. Compared with a control group, the P.acnes can promote the increase of the expression levels of IL-6 and PGE-2, different plant extracts have different effects, and the expression levels of the plant extracts to the inflammation mediators are ranked according to the results of combining the two inflammation mediators: the control group (sancortin) is a nutrient extract (Magnolia officinalis extract is less than or equal to pomegranate extract), the andrographis paniculata extract (licorice extract is less than or equal to chastetree extract) is a radix sophorae flavescentis extract is less than or equal to stimulation group.

For the expression quantity of NO, compared with a control group, the P.acnes can promote the expression quantity, but the promotion amplitude is not strong, and the effects of the plant extract except the tripicol and the magnolia bark extract are not strong.

And continuously verifying the efficacy of the plant extract on a human clinical anti-inflammatory model. Through earlier exploration, no matter the P.acnes is smeared on a 3D skin model or human skin, the generation of inflammation cannot be induced. However, the inventors found that propionibacterium acnes was associated with the development of inflammation in acne patients, and therefore the inventors recruited acne patients to evaluate the relevance of cytological procedures to clinical evaluation in humans.

The gel containing 5 wt% of triplicin and 5 wt% of licorice extract is prepared into an anti-acne gel together with a proper amount of water and an auxiliary agent, the anti-acne gel is respectively delivered to 20 class I-II acne patients for use, the gel is used once after face cleaning in the morning and evening, the efficacy of the gel is tracked and tested within 1 month, and the comprehensive curative effect evaluation is carried out by calculating the treatment efficiency according to the total integral of skin lesion degree, skin lesion distribution, symptoms and physical signs by referring to the clinical research guiding principles of new traditional Chinese medicines. The results are shown in Table 4.

Efficacy ═ 100% pre-treatment skin loss count-post-treatment skin loss count/pre-treatment skin loss count.

And (3) curing: the skin lesion is removed by more than 90 percent, and only a little pigmentation exists;

the effect is shown: the skin lesion is reduced by 60 to 90 percent, and the symptom is relieved;

the method has the following advantages: the skin lesion is reduced by 20 to 60 percent, and the symptoms are improved;

and (4) invalidation: the skin lesions resolved by < 20%, the clinical symptoms were not improved;

the total effective rate is the cure rate + significant efficiency + effective rate.

Table 4 acne treatment efficacy test results

The results are combined to show that the anti-inflammatory efficacy of different plant extracts can be evaluated by using a method for detecting P.acnes-induced cell damage and detecting the expression level of inflammatory factors, and the cytological evaluation result is basically consistent with the clinical result.

Example 3

Example 3 provides a cytological evaluation method of plant extracts against infectious inflammatory reaction of skin, in which the main reagent consumables and instruments and equipment are shown in table 5, and each plant extract is a commercially available product.

TABLE 5 Primary reagent consumables and instrumentation

The cytological evaluation method for the anti-skin infectious inflammatory response of the plant extract comprises the following steps: keratinocyte NHEK was cultured to logarithmic growth phase with DMEM medium containing 10 wt% FBS (fetal bovine serum), 1 wt% PS (streptomycin qinghaosu). The first day of the experiment, after 5min of trypsinization, the cell suspension was collected at 1 x 10⁴Spreading the culture medium to a 96-well plate per well, and continuously culturing overnight; on the next day of the experiment, after the cells are changed, 200 μ l of mixed solution (culture medium dilution) of 100 μ g/ml Poly (I: C) +20 μ g/ml LPS is added, and simultaneously different plant extracts which are diluted to 0.5 wt% concentration by using the culture medium are added, wherein the plant extracts comprise licorice extract, sophora flavescens extract, magnolia bark extract, nutrient source essence extract (containing fructus viticis, gentian and polygonum multiflorum) solution, fructus aurantii extract, radix puerariae extract, Jiabeishu (containing fructus aurantii and radix puerariae) solution and plant soothing agent (containing licorice, sophora flavescens and scutellaria baicalensis) solution, and the cells are continuously treated for 20 hours; on the third day of the experiment, the supernatant was collected into a new 96-well plate, the expression levels of prostaglandin E2(PGE2), Nitric Oxide (NO), interleukin 6(IL-6) and interleukin 8(IL-8) were measured by ELISA, 200. mu.l of MTT (medium prepared at a concentration of 500. mu.g/ml) was added to the plate and stained for 3 hours, and then the supernatant was discarded, 200. mu.l of DMSO solution was added, and absorbance was measured at 492nm using a microplate reader to evaluate the integrity of the cell membrane, and the LDH staining method was also used.

After the cells are treated by the Poly (I: C) and LPS, the cell viability is not obviously changed and reaches 97.6 percent compared with the untreated group. The detection results of the LDH method and the microscopic photography method are similar to those of the MTT method.

As shown in FIG. 5, the results of measurement of inflammatory factors were that the levels of IL-6 and PGE-2 were significantly increased, while the expression levels of NO and IL-8 were not significantly increased, when cells were treated with Poly (I: C) in combination with LPS, as compared with the untreated group. Different plant extracts also have different effects, and the expression quantity of the plant extracts to the inflammation mediators is ranked according to the results of combining two inflammation mediators: the contrast group is Gajiabeishu, the kudzu root extract, the bitter orange extract, the plant soothing agent, the nourishing source essence extract, the liquorice extract, the magnolia bark extract, the sophora flavescens extract and the stimulation group.

And continuously verifying the efficacy of the plant extract on a human clinical anti-inflammatory model. Poly (I: C) in combination with LPS treatment of the 3D skin model induced the production of infectious inflammation, but there was no significant effect of smearing on human skin. The inventors found that Poly (I: C) is involved in the mechanism of allergic reaction in eczema and atopic dermatitis, and therefore, the inventors recruited 15 subjects and used histamine to induce allergic reaction to evaluate the correlation of cytological procedures with clinical evaluation in humans.

15 subjects were recruited and selected for testing in the forearm, and the reagents required are detailed in Table 6. The inside of the front arm is cleaned by clear water, and then the smearing area of 3cm multiplied by 3cm is marked. Histamine dihydrochloride of 5 wt%, xanthan gum of 0.5 wt%, hydrogenated castor oil of 0.2 wt% and water were mixed uniformly to prepare a stimulating solution. 5 wt% of Jiabeishu and 5 wt% of plant soothing agent are also mixed with xanthan gum, hydrogenated castor oil and water with the same concentration to prepare sample solution. First 100 mul of stimulating solution is evenly smeared on a marked area, after drying for 8min, 100 mul of sample solution and matrix control solution are respectively dripped into different areas, the fading condition of the sample to the wheal and the red halo is observed and recorded, and VISIA is used for photographing and analyzing the change of the red area.

TABLE 6 list of instrumental reagents used in the experiment

Instrument reagent name	Brand	Model number
			Histamine dihydrochloride	TCI	TCI#H0146-5
Xanthan gum	CP KELCO	-
			PEG-40 hydrogenated Castor oil	CRODA	-
Liquid transfer device	Eppendorf	10-100μl；
			VISIA	CK	The seventh generation

The results are shown in Table 7, with "+" indicating severity and more indicating more severe symptoms. At 60min after stimulation, the feelings of wheal, erythema and itching in the stimulation group all reached above "+ + + + + +", while the feelings of wheal, erythema and itching in the 5 wt% kabeishu sample group all decreased to "+", and the feelings of plant soothing agent in the 5 wt% plant soothing agent group were relieved, and the effect was equivalent to that of kabeishu. Analysis of red zone changes after imaging using VISIA can indicate inflammation deeper in the skin. The stimulation group scored "++++", whereas the 5 wt% Gabeshu or plant soothing agent group was used to reduce the score to "++++". The contrast effect is more significant with time.

TABLE 7 results of the measurement of antiallergic efficacy

Performance index	5 wt% Jiabeishu	5 wt% of plant soothing and sensitizing agent	Stimulating group
				Evaluation of wheal	++	++	++++
Evaluation of erythema	++	++	+++++
				Evaluation of itching feeling	++	++	++++
VISIA Red zone assessment	+++	+++	+++++

The results are combined to show that the anti-inflammatory efficacy of different plant extracts can be evaluated by using a method for detecting the expression level of the cell inflammatory factors induced by the combination of Poly (I: C) and LPS, and the cytological evaluation result is basically consistent with the clinical result.

Comparative example 1

Referring to the method of example 3, different concentrations of LPS, Poly (I: C), TNF- α and Poly (I: C) in combination with TNF- α were used to stimulate keratinocytes NHEK, and cell culture fluid was collected to measure the expression of IL-6 inflammatory factor by ELISA method, while cell viability was measured by MTT method.

The stimulation of the substances alone or in combination does not obviously affect the cell viability, but has a certain effect on the expression quantity of the inflammatory factors. As shown in Table 8, the expression level of IL-6 was also increased in the cells by the action of LPS, Poly (I: C) and TNF-. alpha.at high concentrations, as compared with the control group, and the effect of LPS at 100. mu.g/ml in promoting the expression of IL-6 was most significant. But in comparison with the combined treatment of Poly (I: C) and LPS, the concentration required was higher and the induction effect was worse. Poly (I: C) in combination with TNF-alpha treatment of cells also had a strong effect on IL-6 expression at high concentrations, but 100ng/ml TNF-alpha was more costly and stimulated at a lower amplitude than 20. mu.g/ml LPS.

TABLE 8 Effect of different stimulators on cytokines

Comparative example 2

Referring to the methods of examples 1 and 2, keratinocyte NHEK was stimulated by live P.acnes and M.furfur at different concentrations, and IL-6 inflammatory factors were detected by ELISA method after collecting cell culture solution, and cell viability was detected by MTT method.

As shown in Table 9, although viable bacteria were induced to some extent in the expression level of IL-6, the induction was significantly lower than that of killed bacteria, and the effect on cell viability was also stronger than that of killed bacteria. Because the difference between the culture medium and the culture condition of bacteria and fungi is large, the co-culture of the viable bacteria and the cells is more difficult than the co-culture of the killed bacteria and the cells, and the incubator is easily polluted.

TABLE 9 Effect of different stimuli on cells

The foregoing examples are merely illustrative and serve to explain some of the features of the method of the present invention. The appended claims are intended to claim as broad a scope as is contemplated, and the examples presented herein are merely illustrative of selected implementations in accordance with all possible combinations of examples. Accordingly, it is applicants' intention that the appended claims are not to be limited by the choice of examples illustrating features of the invention. Also, where numerical ranges are used in the claims, subranges therein are included, and variations in these ranges are also to be construed as possible being covered by the appended claims.

Claims

1. A cytological evaluation method for the anti-skin infection inflammatory response of plant extracts is characterized by comprising the following steps: treating cells with the stimulator and the plant extract, collecting supernatant, detecting cell membrane integrity and/or inflammatory factor expression level, and evaluating the effect of the plant extract on resisting skin infectious inflammatory response.

2. The method of claim 1, wherein the stimulus is selected from the group consisting of one or more of fungi, bacteria, viral RNA analogs, and combinations thereof.

3. The cytological evaluation method of the anti-infective inflammatory reaction of the skin by a plant extract according to claim 2, characterized in that the fungus is malassezia furfur.

4. A method according to claim 2, wherein the bacteria are selected from the group consisting of propionibacterium acnes, staphylococci, streptococci, and mixtures of one or more of bacterial parietal lipopolysaccharides.

5. The method of claim 2, wherein the viral RNA analog is polyinosinic-cytidylic acid.

6. The method of claim 2, wherein the fungi or bacteria are inactivated prior to treatment of the cells.

7. The method for cytological evaluation of the anti-infective inflammatory response of the skin by a plant extract of claim 6, wherein said inactivation treatment comprises the steps of: culturing fungi or bacteria for not less than 48h, determining the final concentration of the bacteria, inactivating at 80-90 ℃ for 0.5-1.5 h, cooling, subpackaging, and freezing.

8. A cytological assessment method of anti-inflammatory skin infection by a plant extract according to any of claims 1 to 7, wherein said detection of cell membrane integrity is selected from the group consisting of MTT staining, neutral Red staining, LDH staining, and microscopy.

9. A cytological assessment method according to any one of claims 1 to 7, wherein said inflammatory agent is selected from the group consisting of nitric oxide, prostaglandins, interleukins, and mixtures thereof.

10. Use of the cytological evaluation method of any of claims 1 to 9 in the fields of cosmetics, dermatology and biomedicine.