CN115463056B

CN115463056B - Herba Swertiae Mileensis extract and preparation method and application thereof

Info

Publication number: CN115463056B
Application number: CN202110656080.6A
Authority: CN
Inventors: 郑晓琼; 马占林; 王早; 王媛; 冯秋红; 黄灿; 周戟; 罗慧
Original assignee: Yunnan Yinge Biotechnology Co ltd
Current assignee: Yunnan Yinge Biotechnology Co ltd
Priority date: 2021-06-11
Filing date: 2021-06-11
Publication date: 2023-09-29
Anticipated expiration: 2041-06-11
Also published as: CN115463056A; WO2022257932A1

Abstract

The invention provides a swertia Japonica Makino extract, and a preparation method and application thereof. In particular, the invention provides the use of a swertia Japonica Makino extract in the preparation of a composition against skin problem related flora. The invention also provides the swertia Japonica Makino extract and a preparation method thereof, and the swertia Japonica Makino extract has high safety and good antibacterial effect.

Description

Herba Swertiae Mileensis extract and preparation method and application thereof

Technical Field

The invention belongs to the technical field of daily cosmetics, and particularly relates to a swertia mileensis extract and a preparation method and application thereof.

Background

Herba Swertiae Mileensis (SWERTIA MILEENSIS EXTRACT) is dried whole plant of herba Swertiae Mileensis Swertia mileensis T.N.Hoet W.L.shi of Gentianaceae. The swertia mileensis is a special plant in Yunnan, is mainly distributed in the counties of Maitreya, kaifeng, and the like in red river of Yunnan province, has the effects of clearing liver and promoting bile, and clearing heat and promoting diuresis, and is a famous medicinal material for treating acute and chronic hepatitis in Yunnan folks. The swertia Japonica Makino has rich chemical components, and mainly contains iridoid glycosides, flavonoids, xanthones and triterpenes.

The swertiamarin is a secoiridoid compound, is a component with high content in gentiaceae plants, and has multiple pharmacological effects of clearing liver and promoting bile flow, relieving spasm and pain, resisting inflammation, reducing blood lipid and the like.

The malassezia is resident bacteria on the skin surface, is mainly distributed on the parts of human beings and warm-blooded animals where the skin grease secretion is vigorous, and is in a symbiotic state with the organism; however, a great deal of research has demonstrated that malassezia, if overgrowing, can cause various inflammatory skin problems such as dandruff/seborrheic dermatitis, atopic dermatitis, psoriasis, etc., and that it can also co-act with propionibacterium acnes to cause acne to occur. Ketoconazole, fluconazole and Zinc Pyrithione (ZPT) are currently recognized as effective malassezia inhibitors on the market, can effectively inhibit dandruff and reduce skin inflammation, but can cause obvious drug resistance of dermatophytes to azole drugs after long-term use, so that the treatment effect of the azole drugs on fungal-induced seborrheic dermatitis, dandruff and the like is greatly reduced, and the inflammatory skin problem can not be thoroughly eradicated; zinc Pyrithione (ZPT) is also being banned by the problems of high skin irritation and potential reproductive toxicity, and therefore, development of a malassezia inhibitor having both efficacy and safety makes the current problem to be solved urgently.

Propionibacterium acnes is a species of propionibacterium, belongs to anaerobic bacteria, mainly lodges in skin, sebaceous glands, intestinal tracts and dairy products of humans and animals, does not cause diseases under aerobic conditions, but is beneficial to the breeding of the bacteria when a pilosebaceous canal is blocked and an anaerobic environment is locally formed in places where sebum secretion is vigorous, and causes acne to occur, and the bacteria further aggravate acne by participating in inflammation, promoting the keratinization of the pilosebaceous glands and promoting sebum secretion. At present, the acne treatment means mainly comprises medicine treatment, including external medicines such as tretinoin, benzoyl peroxide, azelaic acid, antibiotics and the like, and skin irritation reactions such as local erythema and desquamation are easy to occur during the use, and tightening and burning feel occur; the traditional Chinese medicine compound is orally taken with antibiotics, isotretinoin, hormones and other medicines, but the problems of drug resistance, liver and kidney injury and the like are easy to occur after long-term administration, and the traditional Chinese medicine compound can be used for auxiliary treatment by a plurality of physical treatment methods, such as red and blue light irradiation, laser treatment and the like, but the traditional Chinese medicine compound cannot be radically cured, and the personal image and the life quality are seriously influenced.

Accordingly, there is a great need in the art to provide a safe, effective antimicrobial agent for the skin problem-related flora.

Disclosure of Invention

The invention aims to provide an application of a swertia mileensis extract in preparing a composition for resisting related flora of skin problems.

The invention also aims to provide the swertia mileensis extract with high safety and good antibacterial effect and the preparation method thereof.

In a first aspect, the invention provides the use of a swertia Japonica Makino extract for the preparation of a cosmetic or pharmaceutical composition for use in antibacterial.

In another preferred embodiment, the swertiamarin content in the swertiamarin extract is not less than 10wt%, preferably not less than 20wt%, not less than 30wt% or not less than 40wt%, based on the total weight of the swertiamarin extract.

In another preferred embodiment, the content of the swertia herb extract in the cosmetic or pharmaceutical composition is not less than 0.1wt%, preferably not less than 0.2wt% or not less than 0.5wt%, such as 0.6wt%, 1wt%, 2wt%, 5wt%, 10wt% or 20wt%, based on the total weight of the cosmetic or pharmaceutical composition.

In another preferred embodiment, the bacteria are selected from the group consisting of: propionibacterium (e.g., propionibacterium freudenreichii (Propionibacterium freudennreichii), propionibacterium acnes (Propionibacterium acnes), or Propionibacterium graininosum (Propionibacterium granulosum)), malassezia (Pityrosporum) (e.g., malassezia furfur (m. Furfurf), malassezia armigera (m. Sympodialis), malassezia globosa (m. Globosa), malassezia restrictum (m. Restrictum), malassezia thickii (m. Pachdermatis), shi Luobang malassezia (m. Sloofibae), malassezia oyster (m. Obtusa)), or a combination thereof.

In another preferred embodiment, the bacteria are selected from the group consisting of: malassezia furfur, propionibacterium acnes, or combinations thereof.

In another preferred embodiment, the cosmetic or pharmaceutical composition is used for skin problems caused by bacteria, such as dermatitis, folliculitis, acne, or combinations thereof.

In another preferred embodiment, the cosmetic or pharmaceutical composition is for use in one or more selected from the group consisting of: dandruff, seborrheic dermatitis, atopic dermatitis, folliculitis, alopecia, acne, psoriasis caused by malassezia, or a combination thereof.

In another preferred embodiment, the cosmetic or pharmaceutical composition is for use in one or more selected from the group consisting of: acne caused by propionibacterium acnes, such as seborrheic acne, closed mouth, comedones, or combinations thereof.

In another preferred embodiment, the cosmetic or pharmaceutical composition is also used for anti-inflammatory.

In another preferred embodiment, the cosmetic or pharmaceutical composition is also used for the secretion of NO and/or COX-2.

In another preferred embodiment, the cosmetic or pharmaceutical composition further comprises a cosmetically or pharmaceutically acceptable carrier.

In another preferred embodiment, the swertia herb extract is derived from flowers, stems, leaves of swertia herb, or a combination thereof, such as whole grass.

In another preferred embodiment, the swertia herb extract is prepared by a method comprising the steps of:

(i) Heating herba Swertiae Mileensis in 0-85v/v% ethanol water mixed solution for extraction, and separating to obtain extractive solution I;

(ii) Concentrating the extract I, and then adsorbing with nonpolar or weak polar resin to obtain adsorbed resin I;

(iii) Washing the resin I with water to obtain washed resin II;

(iv) Eluting the resin II by using a 70-85% ethanol water mixed solvent, and collecting an eluent; and

(v) Optionally removing the solvent of the eluent, thereby obtaining the swertia mileensis extract.

In another preferred embodiment, in step (i), one or more technical features selected from the group consisting of:

(a) The mixed solution is 20-80v/v% ethanol water mixed solution, such as 40v/v%, 50v/v%, 60v/v%, 70v/v% or 75v/v%;

(b) The dosage ratio (g/mL) of the swertia herb medicinal material to the ethanol water mixed solution is 1:2-30; preferably, 1:5-25, such as 1:10, 1:15, or 1:20;

(c) The number of times of extraction is 1-3, such as 1, 2 or 3;

(d) The extraction temperature is independently 50-100deg.C, such as 60deg.C, 70deg.C or 80deg.C, preferably reflux temperature; and/or

(e) The extraction time is independently 0.5-3 hours, preferably 1-2 hours; .

In another preferred embodiment, in step (ii), one or more technical features selected from the group consisting of:

(a) The resin is selected from the group consisting of: AB-8, HPD-400, LS-305, LS-300B, or a combination thereof, preferably LS-305;

(b) Concentrating the filtrate to 0.05-1g medicinal material/mL, preferably 0.1-0.5g medicinal material/mL according to the dosage amount;

(c) The resin is used in an amount of 0.2-1g of the medicinal material/g of the wet resin, preferably 0.4-0.8g of the medicinal material/g of the wet resin, such as 0.5g of the medicinal material/g of the wet resin or 0.6g of the medicinal material/g of the wet resin, based on the dosage amount of the medicinal material.

In another preferred embodiment, in step (iii), one or more technical features selected from the group consisting of:

(a) The amount of water used for cleaning is 2-8BV, preferably 3-6BV; and/or

(b) The flow rate of the water used for washing is 6-12BV/h, preferably 8-10BV/h.

(a) The ethanol water mixed solution is 75-85v/v% ethanol water mixed solution, such as 80v/v%; and/or

(b) The dosage of the ethanol water mixed solution is 2-10BV, preferably 3-5BV;

(c) The flow rate of the ethanol-water mixed solution is 1-5BV/h, preferably 2-4BV/h.

In another preferred embodiment, the formulation of the cosmetic or pharmaceutical composition is selected from the group consisting of: liquid, suspension, semi-solid or solid formulations.

In another preferred example, the formulation of the cosmetic composition or the pharmaceutical composition is a skin external formulation.

In another preferred embodiment, the formulation of the cosmetic or pharmaceutical composition is selected from the group consisting of: solutions, gels, emulsions, ointments, creams, pastes, cakes, powders, patches and the like.

In another preferred embodiment, the cosmetically acceptable carrier or excipient is selected from the group consisting of: moisturizers, antioxidants, anti-uv agents, preservatives, film formers, oil-soluble gelling agents, organically modified clay minerals, resins, antimicrobial agents, fragrances, salts, pH modifiers, chelating agents, cooling agents, anti-inflammatory agents, skin-beautifying ingredients, vitamins, amino acids, nucleic acids, hormones, inclusion compounds, or combinations thereof.

In a second aspect of the present invention, there is provided a swertia herb extract prepared by a method comprising the steps of:

(iii) Washing the resin I with water to obtain washed resin II;

In another preferred embodiment, the resin is LS-305.

In another preferred embodiment, the method comprises the steps of:

(i) Reflux-extracting herba Swertiae Mileensis with 60-65v/v% ethanol water mixed solution, and separating to obtain extractive solution I;

(ii) Concentrating the extract I, and then adsorbing with LS-305 resin to obtain adsorbed resin I;

(iii) Washing the resin I with water to obtain washed resin II;

(iv) Eluting the resin II by using an 80-85% ethanol water mixed solvent, and collecting an eluent; and

(v) Removing the solvent of the eluent to obtain the swertia mileensis extract.

In a third aspect, the present invention provides a cosmetic or pharmaceutical composition comprising a swertia herb extract according to the second aspect of the invention; and a cosmetically or pharmaceutically acceptable carrier.

In another preferred embodiment, the cosmetically acceptable carrier or excipient is selected from the group consisting of: moisturizers, antioxidants, anti-uv agents, preservatives, film formers, oil-soluble gelling agents, organically modified clay minerals, resins, antimicrobial agents, fragrances, salts, pH adjusters, chelating agents, cooling agents, anti-inflammatory agents, skin-beautifying ingredients, vitamins, amino acids, nucleic acids, hormones, inclusion compounds, or combinations thereof.

In another preferred embodiment, the cosmetic composition is selected from the group consisting of: shampoo, hair care spray, scalp care solution, skin care lotion, skin care emulsion, cream and facial cleanser.

In another preferred embodiment, the cosmetic composition comprises the following components, based on the total weight of the composition:

name of the name	wt％
		Guar hydroxypropyl trimethyl ammonium chloride	0.24～0.36
Polyquaternium-10	0.04～0.06
		Citric acid	0.16～0.24
Sodium laureth sulfate	8～12
		Ammonium lauryl sulfate	5.6～8.4
Pentaerythritol diStearic acid ester	0.4～0.6
		Glycol stearate	1.2～1.8
Cocamidopropyl betaine	1.6～2.4
		Cocoamide MEA	0.8～1.2
Sodium lauryl glycolate	1.2～1.8
		Pyridone ethanolamine salt	0.32～0.48
Silicone oil	1.6～2.4
		Oleic acid monoglycerides and alkyl glycosides	1.6～2.4
Extractive of swertia Japonica Makino (SWERTIA MILEENSIS)	0.48～0.72
		Water and its preparation method	1.6～2.4
Hydantoin	0.16～0.24
		Water and its preparation method	Allowance of

In a fourth aspect, the present invention provides a method for preparing the swertia herb extract according to the second aspect, the method comprising the steps of:

(iii) Washing the resin I with water to obtain washed resin II;

In another preferred embodiment, the resin is LS-305.

In another preferred embodiment, the method comprises the steps of:

(iii) Washing the resin I with water to obtain washed resin II;

(v) Removing the solvent of the eluent to obtain the swertia mileensis extract.

In a fifth aspect of the present invention, there is provided a method of inhibiting bacteria in vitro comprising the steps of: contacting the composition of the third aspect of the swertia mileensis extract according to the second aspect of the invention with bacteria, thereby inhibiting said bacteria.

In a sixth aspect of the present invention, there is provided a method of skin care comprising the steps of: the swertia herb extract of the second aspect of the present invention or the composition of the third aspect of the present invention is administered to a subject in need thereof in an amount effective to combat skin-related bacteria, thereby effecting skin care.

In another preferred embodiment, the administration is topical.

In another preferred embodiment, the subject is a mammal, such as a human, rat or mouse.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 is a chromatogram of a control swertiamarin, a green She Danshui extract, a green leaf liner 60% ethanol extract, and a green leaf liner 80% ethanol extract;

FIG. 2 shows liquid-liquid chromatography of water-soluble liquid and ethanol-soluble liquid after purification of swertiamarin control, D101, AB-8, HPD-400, HPD-100, LS-305, LS-300B resin;

FIG. 3 is a liquid chromatogram of swertiamarin control and swertia Japonica Makino extract;

FIG. 4 shows the growth of malassezia furfur under the action of different concentrations of the swertia extract, wherein the inhibition effect of the swertia extract on malassezia furfur gradually increases with the increase of the concentration. The concentration of 1.5% can completely inhibit the growth of malassezia furfur, namely the minimum inhibitory concentration of the swertia furfur extract on malassezia furfur is 1.5%.

FIG. 5 shows the growth of Propionibacterium acnes with different concentrations of the swertia Japonica Makino extract, with increasing concentration, the inhibition of the swertia Japonica Makino extract on Propionibacterium acnes gradually increasing. The concentration of 5% can completely inhibit the growth of Propionibacterium acnes, i.e. the minimum inhibitory concentration of the swertia mileensis extract on Propionibacterium acnes is 5%.

FIG. 6 shows the effect of different concentrations of swertia Japonica Makino extract on the viability of Raw264.7 cells, wherein the cell viability is greater than or equal to 90% in the concentration range of less than or equal to 500 μg/mL.

Fig. 7 shows that swertia Japonica Makino extract 500 μg/mL and 200 μg/mL (< 0.01) can significantly inhibit macrophage raw264.7 inflammation model from secreting inflammatory mediator NO.

FIG. 8 shows that the swertia Japonica Makino extract 500 μg/mL and 200 μg/mL (< 0.05P) significantly inhibited secretion of inflammatory mediator COX-2 by macrophage Raw264.7 inflammation model.

FIG. 9 shows that 1% of the swertia Japonica Makino extract has skin tissue activity > 50% and is judged to be non-irritating.

Fig. 10 shows that malassezia is significantly less in the dishes with shampoo added with 0.6% swertia herb extract than the blank, demonstrating a significant inhibition of malassezia.

Detailed Description

Through extensive and intensive studies, the present inventors have provided a swertia mileensis extract, a preparation method and applications thereof, through a large number of screening and testing. The invention provides a swertiamarin extractive with high content and a preparation method thereof. The invention also surprisingly finds that swertiamarin has remarkable antibacterial effect at low concentration, especially on malassezia and propionibacterium acnes, so that the swertiamarin is very suitable for preparing a cosmetic composition or a pharmaceutical composition for antibacterial. The present invention has been completed on the basis of this finding.

Terminology

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 invention belongs.

As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

As used herein, the term "room temperature" or "normal temperature" refers to a temperature of 4 to 40 ℃, preferably 25±5 ℃, and the operating temperature may be normal temperature, as not specifically described.

Herba Swertiae Mileensis

Herba Swertiae Mileensis (SWERTIA MILEENSIS EXTRACT) is dried whole plant of herba Swertiae Mileensis Swertia mileensis T.N.Hoet W.L.shi of Gentianaceae. The swertia mileensis is a special plant in Yunnan, and is mainly distributed in Miler, kaifeng and other counties in red river of Yunnan province. The invention has no special requirement on the swertia herb medicinal material, and can be purchased commercially or obtained by processing by a conventional method in the field. The medicinal materials can be whole herb or chopped or crushed.

Herba Swertiae Mileensis extract

In the invention, the active ingredient is a swertia herb extract, wherein the swertia herb extract refers to an extract extracted from swertia herb medicinal materials and takes swertiamarin as a main active ingredient.

In another preferred embodiment, the swertiamarin content in the swertiamarin extract is not less than 10wt%, preferably not less than 20wt%, not less than 30wt% or not less than 40wt%, such as 20-50wt% or 35-45wt%, based on the total weight of the swertiamarin extract.

Preparation method

In the present invention, the preparation method of the swertia Japonica Makino extract is not particularly limited, and can be prepared by extraction methods commonly used in the art, including but not limited to: solvent extraction, supercritical extraction and/or chromatography.

Preferably, the extractant of the extract is selected from the group consisting of: water, alcohols (preferably C1-C4 alcohols such as methanol, ethanol, propanol), or mixtures thereof.

Preferably, the preparation method of the swertia Japonica Makino extract comprises the following steps:

(iii) Washing the resin I with water to obtain washed resin II;

(c) The number of times of extraction is 1-3, such as 1, 2 or 3;

(e) The extraction time is independently 0.5-3 hours, preferably 1-2 hours; .

In another preferred embodiment, the resin is treated with 90-100% ethanol water (e.g., 24-96 hours) prior to adsorption.

(a) The amount of water used for cleaning is 2-8BV, preferably 3-6BV; and/or

(b) The dosage of the ethanol water mixed solution is 2-10BV, preferably 3-5BV;

Particularly preferably, the method comprises the steps of:

(iii) Washing the resin I with water to obtain washed resin II;

(v) Removing the solvent of the eluent to obtain the swertia mileensis extract.

Composition and use

The composition of the invention comprises the swertia Japonica Makino extract as described above; and a cosmetically or pharmaceutically acceptable carrier.

The present invention surprisingly found that the swertia Japonica Makino extract has antibacterial activity and can be used for preventing and/or treating diseases and symptoms caused by bacterial infection. In the present invention, the antibacterial means inhibiting the growth, reproduction and/or killing of bacteria. Bacteria useful in the present invention include gram positive and/or gram negative bacteria, particularly skin problem-related bacteria, such as including but not limited to: propionibacterium (e.g., propionibacterium freudenreichii (Propionibacterium freudennreichii), propionibacterium acnes or Propionibacterium granatum), malaromyces (Pityrosporum) (e.g., mallotus furfur (M. Fursurface), mallotus axostoma (M. Sympodialis), mallotus globosum (M. Globosa), mallotus restrictus (M. Restrictum), mallotus thickii (M. Pachydermatides), shi Luobang Mallotus (M. Sloofibae), mallotus oyster (M. Obtusa))

In addition, the swertia Japonica Makino extract of the invention is not easy to generate resistance. Bacteria useful in the present invention may be malassezia which are resistant to ketoconazole, fluconazole and/or Zinc Pyrithione (ZPT); and/or propionibacterium acnes that develop resistance to erythromycin, cephalosporins, and/or clindamycin.

In another preferred embodiment, the composition of the present invention is suitable for use in skin problems caused by bacteria, such as dermatitis, folliculitis, acne, or combinations thereof. For example, including but not limited to, malassezia-induced dandruff, seborrheic dermatitis, atopic dermatitis, folliculitis, hair loss, acne, psoriasis, or combinations thereof; and/or propionibacterium acnes, such as seborrheic acne, closed mouth, comedones, or combinations thereof.

In addition, the invention also discovers that the swertia mileensis extract has anti-inflammatory effect. For example, secretion of NO and/or COX-2, which are inflammatory mediators, can be inhibited, and thus the present invention also provides the use of swertia Japonica Makino extract as NO and/or COX-2 inhibitor.

It is possible to prepare the swertia herb extract of the present invention into a pharmaceutical composition such as a paste, cream, gel, paste, patch, etc. The medicament can be prepared by generally known preparation techniques, and suitable pharmaceutical additives can be added to the medicament.

Examples of the pharmaceutical additives include excipients, binders, disintegrants, lubricants, flow aids, suspending agents, emulsifiers, stabilizers, heat-retaining (wetting) agents, preservatives, solvents, solubilizers, preservatives, flavoring agents, sweeteners, dyes, fragrances, propellants and the like, which may be selected and added in suitable amounts within a range not affecting the effect of the present invention.

It is possible to prepare the swertia herb extract of the present invention into a cosmetic composition, a solid dosage form, a semisolid dosage form, or a liquid dosage form, such as a solution, gel, cream, emulsion, spray, paste, cream, paste, cake, powder, patch, etc.

Other ingredients used in usual cosmetics, such as film forming agents, oil-soluble gelling agents, organically modified clay minerals, resins, moisturizers, preservatives, antibacterial agents, fragrances, salts, antioxidants, pH adjusting agents, chelating agents, cooling agents, anti-inflammatory agents, ingredients for beautifying the skin (whitening agents, cell active agents, skin roughness improving agents, blood circulation promoters, skin astringents, antilipemic agents, etc.), vitamins, amino acids, nucleic acids, hormones, inclusion compounds, etc., may be added to the cosmetics of the present invention within the range that does not impair the effects of the present invention.

The oil-soluble gelling agent is selected from metallic soap such as aluminum stearate, magnesium stearate, zinc myristate, etc.; amino acid derivatives such as N-lauroyl-L-glutamic acid, α, γ -di-N-butylamine; cyclodextrin fatty acid esters such as cyclodextrin palmitate, cyclodextrin stearate, and cyclodextrin 2-ethylhexanoate palmitate; sucrose fatty acid esters such as sucrose palmitate and sucrose stearate; benzylidene derivatives of sorbitol such as monobenzylidene sorbitol and dibenzylidene sorbitol; the gelling agent for organomodified clay minerals such as dimethylbenzyl dodecylammonium montmorillonite clay and dimethyl dioctadecyl ammonium montmorillonite clay may be used singly or in combination of two or more kinds as required.

The humectant comprises: glycerol, sorbitol, propylene glycol, dipropylene glycol, 1, 3-butanediol, glucose, xylitol, maltitol, polyethylene glycol, hyaluronic acid, chondroitin sulfate, pyrrolidone carboxylate, polyoxyethylene methyl glucoside, polyoxypropylene methyl glucoside, and the like.

The antibacterial preservative comprises: alkyl p-hydroxybenzoates, benzoic acid, sodium benzoate, sorbic acid, potassium sorbate, phenoxyethanol and the like, and the antibacterial agents are: benzoic acid, salicylic acid, carbolic acid, sorbic acid, alkyl p-hydroxybenzoates, p-chlorometacresol, hexachlorophene, benzalkonium chloride, chlorhexidine chloride, trichloro-carbanilide, triclosan, photoreceptors, phenoxyethanol, and the like.

The antioxidants include: tocopherol, butylated hydroxyanisole, dibutylhydroxytoluene, phytic acid, etc., and the pH adjusting agents are: lactic acid, citric acid, glycolic acid, succinic acid, tartaric acid, dl-malic acid, potassium carbonate, sodium bicarbonate, ammonium bicarbonate and the like, and the chelating agent is alanine, ethylenediamine tetraacetic acid sodium salt, sodium polyphosphate, sodium metaphosphate, phosphoric acid and the like, and the cooling agent is: l-menthol, camphor, etc., and anti-inflammatory agents are: allantoin, glycyrrhetinic acid, glycyrrhizic acid, tranexamic acid, azulene (Azulene), and the like.

The skin beautifying components are: whitening agents such as placenta extract, arbutin, glutathione, herba Saxifragae extract, etc.; cell activator such as Lac Regis Apis, photoreceptor, cholesterol derivative, calf blood extractive solution, etc.; a skin roughness improver; blood circulation accelerators such as valeramide, benzyl nicotinate, beta-butoxyethyl nicotinate, capsaicin, zingiberone, cantharidin, ichthyol, caffeine, tannic acid, alpha-borneol, tocopheryl nicotinate, inositol hexanicotinate, cyclic mandelate, cinnarizine, tolazoline, acetylcholine, verapamil, cepharanthine, gamma-oryzanol and the like; skin astringents such as zinc oxide and tannic acid; sulfur, and other antilipemic agents, and vitamins include: vitamin a oil, rosin acetate oil, rosin palmitate oil and the like; vitamin B2 such as riboflavin, riboflavin butyrate, flavin adenine nucleotide, etc.; vitamin B6 such as pyridoxine hydrochloride, pyridoxine dioctate, pyridoxine tripalmitate, vitamin B12 and its derivatives, vitamin B15 and its derivatives, etc.; vitamin C such as L-ascorbic acid, L-ascorbyl dipalmitate, L-ascorbic acid-2-sodium sulfate, and L-ascorbyl phosphodiester dipotassium; vitamin D such as ergocalciferol and cholecalciferol; vitamin E such as alpha-tocopherol, beta-tocopherol, gamma-tocopherol, dl-alpha-tocopherol acetate, dl-alpha-tocopherol nicotinate, dl-alpha-tocopherol succinate, etc.; vitamin H; vitamin P; nicotinic acid, nicotinic acid benzyl ester, nicotinamide and other nicotinic acids; pantothenic acid compounds such as calcium pantothenate, D-panthenol, pantetheine, and acetyl pantetheine; biotin, and the like.

Amino acids are: glycine, valine, leucine, isoleucine, serine, threonine, phenylalanine, arginine, lysine, aspartic acid, glutamic acid, cystine, cysteine, methionine, tryptophan, etc., nucleic acids such as deoxyribonucleic acid, etc., and hormones such as estradiol, vinyl estradiol, etc.

Preferred examples of the cosmetic of the present invention include: skin care cosmetics, hair care cosmetics, make-up cosmetics, and ultraviolet-proof cosmetics. Hair care products such as shampoos, conditioners, hair care essences, hair films, etc.; basic cosmetics such as emulsion, cream, dew, sun cream, facial mask material, facial cleanser, essence, etc.; make-up cosmetics such as foundation, white powder, blush, etc.

The form of the product is not particularly limited, and may be liquid, emulsion, cream, solid, paste, gel, powder, multilayer, mousse, spray, or the like.

The present invention also provides a method of skin (including scalp) care, the method comprising the steps of: the swertia herb extract of the present invention or the composition of the present invention is administered to an individual in need thereof.

In another preferred embodiment, the effective concentration of the swertia Japonica extract is in the range of 1 μg/ml to 500mg/ml, such as 5 μg/ml, 10 μg/ml, 20 μg/ml, 50 μg/ml, 100 μg/ml or 200 μg/ml.

As used herein, the term "effective dose" refers to any amount that, when used alone or in combination with another therapeutic or cosmetic agent, promotes disease regression as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free symptoms, or prevention of disorders or disability caused by the illness. "therapeutically effective dose" of a medicament of the present invention also includes a "prophylactically effective dose," which is any amount of a medicament that, when administered alone or in combination with another therapeutic agent or cosmetic, inhibits the occurrence or recurrence of a disease or symptom in a subject at risk of developing the disease or symptom or suffering from the recurrence of the disease or symptom.

In the present invention, the application to the aspects of antibacterial, anti-inflammatory, etc. includes both prophylactic application and post-improvement application. For example, for antibacterial purposes, including administration of the swertia herb extract or composition of the invention before, during, and/or after infection to prevent and/or repair bacterial infection.

Without wishing to limit the invention, the antibacterial, anti-inflammatory, etc. effects of the swertia herb extract may be due to the synergistic effect of a single component thereof (e.g., swertiamarin) or components of the extract.

The main advantages of the invention include:

1. the invention provides a swertiamarin extract, wherein the content of swertiamarin can be up to more than 40%.

2. The swertia mileensis extract has remarkable inhibition effect on malassezia furfur and propionibacterium acnes, can realize complete inhibition under the action of low concentration, and has high antibacterial activity.

4. The swertia Japonica Makino extract has an inhibitory effect on inflammatory mediators NO and COX-2;

5. the swertia Japonica Makino extract of the invention has no irritation to skin, has high safety, and can be applied to daily cosmetic raw materials.

6. The swertia Japonica Makino extract and the composition containing the swertia Japonica Makino extract have antibacterial and anti-inflammatory effects, and are very suitable for preparing antibacterial cosmetic compositions or pharmaceutical compositions.

7. The preparation method of the swertia Japonica Makino extract is simple, the yield is high, the purity of active ingredients is high, and the obtained extract has high activity.

8. The present invention also provides a novel method of killing skin problem-related flora, which provides a novel solution to the flora that is resistant to other antimicrobial agents.

The invention is further described below in conjunction with the specific embodiments. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

The invention uses swertia herb as raw material, swertiamarin as target component, and determines the preparation process of swertia herb extract through the steps of extraction solvent screening, purification resin screening and the like, and verifies the efficacy through efficacy test.

Example 1

1. Extraction solvent screening

1.1 extraction: weighing three parts of dried swertia herb whole herb (Latin name Swertia mileensis T.N.Hoet W.L.shi, yunnan of origin), adding 1000mL of purified water, 60% ethanol and 80% ethanol into each 50g, heating and refluxing to extract for 2h, filtering with 300-mesh screen to obtain filtrate, adding 1000mL of purified water, 60% ethanol and 80% ethanol into filter residues, heating and refluxing to extract for 1.5h, filtering with 300-mesh screen to obtain filtrate, and combining the two filtrates;

1.2 concentrating: concentrating the filtrate to 100g to obtain purified water, 60% ethanol and 80% ethanol crude extract;

1.3 high performance liquid chromatography is used for measuring the content of swertiamarin in the extracts corresponding to the 3 solvents.

1) Chromatographic conditions: the column was a Hypersil BDS C18 (250 mm. Times.4.6 mm,5 μm); the mobile phase is methanol-0.05% phosphoric acid solution (22:78); the detection wavelength is 237nm; the flow rate is 1mL/min; the sample injection amount is 10 mu L, and the column temperature is 30 ℃;

2) A swertiamarin control: weighing 5mg (0.01 mg accurate) of swertiamarin reference substance, placing into a 100mL volumetric flask, fixing volume to scale with 50% methanol, shaking uniformly, and preparing into reference substance solution with concentration of 50 μg/mL;

3) Sample solution preparation: diluting the crude extract obtained in step 1.2 with 50% methanol respectively to proper times for use;

4) And (3) measuring: the appropriate amount of control and sample solutions were filtered with 0.45 μm filter membrane, and then placed in a sample bottle of a liquid chromatograph, and the measurement results were shown in FIG. 1.

5) Result processing analysis: according to A1/a2=c1/C2, the swertiamarin content in the crude extracts obtained by different solvent extractions was calculated as shown in table 1 below.

Table 1: finishing the screening result of the extraction solvent

The results show that the content of swertiamarin in the extracts obtained by extracting with 3 different solvents is 60% ethanol > 80% ethanol > purified water from high to low.

Example 2

2. Purification resin screening

2.1 taking 100g of D101, AB-8, HPD-400, HPD-100, LS-305 and LS-300B resin respectively, soaking in 95% ethanol for 48h, weighing 15g (calculated as wet resin) of each resin model respectively, purifying and washing until no ethanol smell exists, and placing in a 100mL beaker for standby

2.2 weighing 100g of dried swertia Japonica Makino, and extracting with 60% ethanol under reflux for 2 times, wherein the solvent dosage is 2000mL each time, and the extraction time is 2h and 1h respectively. Collecting and combining the liquid medicine obtained by 2 times of extraction, and concentrating to 200g for later use;

2.3 respectively weighing 6 parts of the extractum obtained by 2.2 with the mass of 15g, adding into a beaker filled with resin, uniformly stirring, and then placing into a shaking table to shake for 15min to enable the resin to fully contact with the extractum; filtering the resin with a 300-mesh screen, and collecting filtrate;

2.4 measuring 60mL of purified water, washing resin for 4 times each time by 15mL, and collecting the water washing liquid after washing the resin; then 6 parts of 80% ethanol with the volume of 80mL is measured and respectively poured into beakers filled with resin, and the beakers are placed into a shaking table to shake for 15min after being uniformly stirred, so that the resin is fully contacted with the extractum; filtering the resin with a 300-mesh screen, and collecting 80% ethanol analysis solution;

2.5 adding purified water to 100g of the filtrate obtained in 2.3 and the water washing liquid obtained in 2.4 and 80% ethanol analysis liquid respectively;

2.6 high performance liquid chromatography is used for measuring the content of swertiamarin in the filtrate, the water washing liquid and the 80% ethanol analysis liquid corresponding to 6 resins;

1) Chromatographic conditions: the column was a Hypersil BDS C18 (250 mm. Times.4.6 mm,5 μm); the mobile phase is methanol-0.05% phosphoric acid solution (22:78); the detection wavelength is 237nm; the flow rate is 1mL/min; the sample injection amount is 10 μl, and the column temperature is 30 ℃;

3) Sample solution preparation: diluting the crude extract obtained in step 2.5 with 50% methanol respectively to proper times for later use;

4) And (3) measuring: the control and sample solutions were filtered through 0.45 μm filters, and then placed in a sample bottle of a liquid chromatograph, and the results were shown in FIG. 2.

5) Result processing analysis: according to A1/a2=c1/C2, the swertiamarin content in the crude extracts obtained by different solvent extractions was calculated as shown in the following table 2:

TABLE 2 resin Screen content determination result finishing

The adsorption rate, resolution and yield of swertiamarin by each resin were calculated according to the following formula, as shown in Table 3 below.

Adsorption rate = (C) ₀ -C ₁ )/C ₀ *100％；

Resolution% = C ₂ /(C ₀ -C ₁ )*100％

Yield% = adsorption% = resolution%

C ₀ Swertiamarin content (%)

C ₁ Swertiamarin content (%)

C ₂ Ethanol analysis solution Swertiamarin content (%)

Table 3: effect of different types of resins on isolation and purification of swertiamarin

Resin model	Adsorption rate%	Resolution%	Yield%
				D101	9.25	65.31	6.04
AB-8	77.83	87.03	67.74
				HPD-400	69.34	71.56	49.62
HPD-100	16.04	83.53	13.40
				LS-305	77.92	95.28	74.24
LS-300B	67.26	57.92	38.96

The result shows that the adsorption rate, the resolution rate and the yield of LS-305 are all higher than those of other resin types, and the yield of swertiamarin is 74.24%.

Example 3

Preparation of swertia Japonica Makino extract

3.1 extraction: weighing 50g of dried swertia Japonica Makino medicinal material, performing heating reflux extraction with 60% ethanol as solvent for 2 times, wherein the solvent dosage is 1000mL each time, and the extraction time is 2h and 1h respectively;

3.2 concentrating: collecting and combining the liquid medicine obtained by 2 times of extraction, and concentrating to 500g for later use;

3.3 column packing: soaking LS-305 resin in 95% ethanol for 48h, weighing 100g of wet resin, loading into a resin column, and washing with purified water until no ethanol smell exists;

3.4 sample loading: pouring the extractum obtained in the step 6.3.2 into a resin column, controlling the flow rate at 4BV/h, and collecting a loading effluent;

3.5 washing: after the sample loading is finished, adding 4BV purified water into the resin column, controlling the flow rate at 8BV/h, and collecting water washing liquid;

3.6 ethanol resolution: after the water washing is finished, adding 3BV of 80% ethanol into the resin column, controlling the flow rate at 2.5BV/h, and collecting ethanol analysis liquid;

3.7 concentrating: removing alcohol from the ethanol solution obtained in step 3.6 and concentrating the ethanol solution to 100g;

3.8 freeze drying: freeze-drying the extract obtained in 3.7 in a freeze dryer, and collecting freeze-dried powder; the yield of the freeze-dried powder is 11.56%;

3.9 high performance liquid chromatography for determining the content of swertiamarin in swertia Japonica Makino extract;

3) Sample solution preparation: weighing a proper amount of the swertia Japonica Makino extract obtained by 3.8, diluting with 50% methanol for a proper multiple for use;

4) And (3) measuring: the appropriate amount of control and sample solutions were filtered with 0.45 μm filter membrane, and then placed in a sample bottle of a liquid chromatograph, and the measurement results were shown in FIG. 3.

5) Result processing analysis: according to A1/a2=c1/C2, the swertiamarin content in the swertia herb extract was calculated to be 45.60%, as shown in table 4.

Table 4: results of measurement of content of green leaves but extracts

Sample name	Peak time (min)	Peak area	Concentration (ug/ml)	Content%
					Swertiamarin reference substance	10.466	787.186	49.49	/
Freeze-dried powder-LS-305	10.465	702.111	44.14	45.60

In conclusion, the swertia mileensis extract is finally prepared by taking 60% ethanol as an extraction solvent and LS-305 as a purification resin as process conditions, the yield of the obtained finished product is 11.56%, and the content of swertiamarin is 45.60%.

Example 4

Antibacterial efficacy test of swertia Japonica Makino extract

4.1 malassezia inhibition experiments: the inhibition of the malassezia is tested by using malassezia furfur (Malassezia furfur (Pityrosporum orbiculare) as a strain and 1% Zinc Pyrithione (ZPT) as a positive control, and the specific test method is as follows:

1) Experimental reagent: olive oil culture medium

2) Testing strains: malassezia furfur (ATCC 44344, available from the cantonese collection of microbial species);

3) The testing steps are as follows: adding samples according to concentration of 2%, 1.5%, 1.2%, 1%, 0.8% and 0.6% when preparing the culture medium, sterilizing for 15min at 121 ℃, pouring the culture medium into a sterile culture dish when the culture medium is cooled to about 45 ℃, and preparing a flat plate for later use; scraping malassezia furfur colonies growing on the culture medium for 5 days, and uniformly mixing the malassezia furfur colonies in sterile normal saline; 0.1mL of the bacterial suspension (10) ⁵ ～10 ⁶ CF μm/mL) was coated on the prepared agar plates, a blank control was made from the inoculated olive oil medium plates without the sample, and 1 olive oil medium plate was coated with 1% ZPT100. Mu.L as a positive control; culturing in an incubator at 36 ℃ for 72 hours, and comparing and observing the growth condition of the bacterial colony;

4) Experimental results: as shown in FIG. 4, the extract of herba Swertiae Mileensis has remarkable inhibiting effect on malassezia, and the inhibiting effect is gradually enhanced with increasing concentration, and the culture medium containing 1.5% of the sample can completely inhibit the growth of malassezia furfur, so that the minimum inhibiting concentration of the sample on malassezia furfur is 1.5%.

Example 5

Propionibacterium acnes inhibition experiment:

the inhibition of the swertia pavonica (Propionibacterium acnes) on the propionibacterium acnes is tested by the swertia pavonica extract, and the specific test method is as follows:

1) Experimental reagent: trypticase soy agar medium (TSA medium), sheep blood, sterile normal saline;

2) Testing strains: propionibacterium acnes (ATCC 6919, available from the collection of microorganism strains in the guangdong);

3) The testing steps are as follows: adding samples according to the concentration of 5%, 2.5% and 1.25% when preparing the culture medium, sterilizing for 15min at 121 ℃, adding 2mL sheep blood when the culture medium is cooled to about 45 ℃, mixing uniformly, and then sub-packaging to flat plates to prepare the flat plates for later use; scraping propionibacterium acnes colony growing on the culture medium for 3 days, mixing in sterile physiological saline, and collecting 0.1mL bacterial suspension (10 ⁶ ～10 ⁸ CF μm/mL) is coated on the prepared blood plate, the blood culture medium with no sample added by the inoculated bacteria is used as a blank control, and the blank control is placed in an incubator for culturing for 72 hours in an anaerobic environment at 36 ℃ to compare and observe the growth condition of bacterial colonies;

4) Experimental results: as shown in FIG. 5, the swertia Japonica Makino extract has a certain inhibiting effect on Propionibacterium acnes, and the inhibiting effect is gradually enhanced with increasing concentration. The culture medium containing 5% of the sample can completely inhibit the growth of propionibacterium acnes, and the minimum inhibitory concentration of the sample on propionibacterium acnes is proved to be 5%.

Example 6

Experiment of influence of swertia Japonica Makino extract on NO and COX-2

LPS is the main component of the cell wall of gram-negative bacteria, is the main inflammatory substance, can induce the organism to produce inflammation, and can release inflammatory factors such as TNF-alpha, IL-6 and the like and inflammatory mediators such as NO, COX-2 and the like. The invention uses LPS to stimulate macrophages to generate an inflammation model, and detects the influence of a sample on inflammatory mediators NO and COX-2 generated by cells. The specific test method is as follows:

experimental materials: mouse macrophage raw264.7 (cell bank of chinese academy of sciences), high sugar DMEM medium (Gibco), PBS (bosd), MTT (Sigma), DMSO, fetal bovine serum (Gibco), lipopolysaccharide LPS (Sigma), dexamethasone (medium search), NO detection kit (bi cloud days), RNAiso Plus (TaKaRa), isopropanol (national drug group), chloroform (national drug group), absolute ethanol (national drug group), DEPC water (Beyotime), reverse transcription kit (TaKaRa), fluorescent dye (TaKaRa).

Instrument: CO2 incubator (Thermo), enzyme-labeled instrument (BioTek), ultra clean bench (Antai, suzhou), inverted microscope (Olympus), PCR instrument (BIO-RAD), fluorescent quantitative PCR instrument (BIO-RAD).

Test method

6.1 maximum safe concentration determination of swertia Japonica Makino extract

1) Cell inoculation: raw264.7 cells were seeded at the prescribed cell seeding density into 96-well plates and incubated overnight in an incubator (37 ℃, 5% CO 2).

2) Experimental grouping: the experiment sets a blank control group, a positive control group and a sample group. In the sample group, 8 concentration gradients were set for each sample, and 3 duplicate wells were set for each concentration gradient.

3) Preparing liquid: test object working solutions with different concentrations are prepared according to experimental design (table 5).

TABLE 5 test concentration setting table

4) Administration: and when the cell plating rate in the 96-well plate reaches 40% -60%, the administration is carried out. 200. Mu.L of cell culture medium was added to each well of the blank control group; 200. Mu.L of culture medium containing 10% DMSO was added to each well of the positive control group; 200 mu L of culture solution containing the test substance with corresponding concentration is added into each hole of the sample group; zero-well cell-free inoculation was performed, and only 200. Mu.L of cell culture medium was added. After completion of the administration, the 96-well plate was placed in an incubator (37 ℃, 5% co 2) for culturing.

5) And (3) detection: after incubation of the cells for 24h, the supernatant was discarded, MTT working solution (0.5 mg/mL, as prepared) was added, incubated at 37℃for 4h in the absence of light, after incubation, the supernatant was discarded, 150. Mu.L of DMSO was added to each well, and the OD was read at 490 nm.

6) Cell relative viability calculation: cell relative viability (%) = (sample-zeroed well)/(blank well-zeroed well) ×100% calculated according to the formula

6.2 experiments on the Effect of swertia Japonica Makino extract on NO and COX-2 secretion

1) Cell inoculation: cells were seeded at the prescribed cell seeding density in 6-well plates and incubated overnight in an incubator (37 ℃,5% co 2).

2) Preparing liquid: test working fluids were prepared according to the experimental design (table 6).

TABLE 6 experimental design

3) Administration: according to the specific design of the experiment in Table 6, when the cell plating rate in the 6-hole plate reaches 40% -50%, group administration is carried out, the administration amount of each hole is 1.8mL, and 3 compound holes are arranged in each group. The culture was continued at 37℃in a 5% CO2 incubator for 2 hours.

4) LPS stimulation: after 2h of culture, 200. Mu.L of LPS working solution prepared from the corresponding test object working solution is added into the dosed pore plate according to the group design, and the pore plate is shaken left and right to uniformly mix the medicines in the pore plate, so that the final concentration of LPS is 1. Mu.g/mL. The incubation was continued for 24h at 37℃in an incubator with 5% CO 2.

5) Cell supernatant was collected: after 24h of incubation, the cell culture supernatants were collected in EP tubes.

6) And (3) detecting the NO content: detection is performed according to the operating instructions of the NO content detection kit.

7) Cell collection: 2 mL/well PBS was washed twice, 1mL RNAiso Plus was added to each well, and after lysis of the cells by blowing, the samples were collected.

8) Gene detection: according to the instruction of the kit, carrying out RNA extraction, reverse transcription and fluorescence quantitative PCR detection, and calculating the result by adopting a 2-delta CT method.

Statistical analysis of test results

Plotting using GraphPad Prism Program software, t-test statistical analysis was used between groups, p < 0.05 indicated significant differences, p < 0.01 indicated significant differences.

6.3 maximum safe concentration determination of swertia Japonica Makino extract

The samples were set to 8 dosing concentrations from high to low, cytotoxicity tests were performed on macrophages, the test results are shown in table 7, and the cell viability trend is shown in fig. 6. According to MTT results, the sample swertia Japonica Makino extract is considered to be based on macrophage, has no obvious cytotoxicity in the concentration range of 500 mug/mL, and can be used as the maximum safe concentration

TABLE 7 maximum safe concentration determination results of swertia Japonica Makino extract

6.4 measurement of NO content

Based on experimental method 6.2, cell supernatants were collected for NO content detection, and the results are shown in table 8 and fig. 7. Compared with the BC group, the NO content of the NC group is obviously increased, which indicates that the LPS stimulation condition of the experiment is effective. Compared with NC group, the NO content of positive control group is obviously reduced, which indicates that the positive control of this experiment is effective. Compared with NC group, the NO content of the sample swertia Japonica Makino extract is obviously reduced at two administration concentrations of 500 mug/mL and 200 mug/mL.

Tables 6 to 8NO content measurement results

Sample name	Average concentration (mu M)	SD	p-value
				BC	0.99	0.10	/
NC	47.45	1.72	0.000##
				PC	22.30	0.72	0.000**
Herba Swertiae Mileensis extract-500 μg/mL	5.92	0.17	0.000**
				Herba Swertiae Mileensis extract-200 μg/mL	25.39	1.72	0.000**

Remarks: when the statistical analysis is carried out by a t-test method, compared with a BC group, the significance of the NC group is expressed as # and the p-value < 0.05 is expressed as # and the p-value < 0.01 is expressed as #; compared with the BC group, the significance of the PC group and the sample group is expressed as the x, the p-value < 0.05 is expressed as the x, and the p-value < 0.01 is expressed as the x.

6.5COX-2 Gene expression assay

And (3) carrying out RNAiso Plus treatment on the macrophage after the action based on an experimental method 6.5.3.2, and carrying out RNA extraction, reverse transcription and fluorescent quantitative PCR operation according to a kit instruction. The test results are shown in Table 9 and FIG. 8, and the NC control group showed a significant increase in COX-2 gene expression compared with the BC group, indicating that LPS stimulation was effective in this experiment. Compared with NC group, the positive control group COX-2 gene expression quantity is obviously reduced, which proves that the positive control is effective in the experiment. Compared with NC group, the expression level of COX-2 gene of the sample swertia Japonica Makino extract is obviously reduced at two administration concentrations of 500 mug/mL and 200 mug/mL.

TABLE 9COX-2 Gene expression assay results

From the above, the swertia Japonica Makino extract has remarkable inhibition effect on inflammatory mediator NO induced by LPS; meanwhile, the compound has obvious inhibition effect on the expression of inflammation related gene COX-2.

Example 7

Skin irritation test of swertia Japonica Makino extract

7.1 experimental materials: 3D skin model kit (Shanghai An Funuo Biotechnology Co., ltd.), MTT (Shanghai Yuan Ye), DPBS (BI), isopropanol (Tianjin Feng)

7.2 experimental facilities: enzyme-labeled instrument (Thermo), ultra-clean bench (Suzhou Antai), CO2 incubator (Memmert)

7.3 test method: reference OECD TG439

1) Pre-culturing: transferring the received skin model to a 12-well plate filled with 2mL of test culture medium, and culturing for 24 hours in a CO2 incubator;

2) Sample adding treatment: taking out the skin model after 24 culture, grouping according to the experimental design of table 10, taking 10 mu L of corresponding samples from each group of 3 compound holes, and uniformly coating the corresponding samples on the surface of skin tissue;

TABLE 10 skin irritation test Experimental design

3) Sample treatment and washing: after 15min, thoroughly rinsing with DPBS to remove the product and dry the skin tissue surface moisture;

4) Post-incubation: transferring the treated sample into a new maintenance culture medium, and placing the sample back into a CO2 incubator for incubation;

5) Tissue activity test: after 42h incubation, the skin tissue was transferred to 0.3mg/mL MTT solution and incubated at 37℃for 3h. The color of the cultured skin tissue was observed and recorded. Cutting skin tissue, transferring to a 1.5mL centrifuge tube, adding 500 μl of acidified isopropanol, mixing, standing at 4deg.C in dark for 72 hr, taking acidified isopropanol as blank, and measuring absorbance at 560nm wavelength;

6) Result processing analysis: the viability of the corresponding tissue of each sample was calculated from skin tissue activity = (experimental group OD value-blank OD value)/(negative control group OD value-blank OD value) ×100%.

Test results

According to the test sample, the tissue activity is more than 50% and is not more than 50%, and the test sample is judged to be the stimulating substance, as shown in Table 11 and FIG. 9, the tissue activity corresponding to 1% of the swertia mileensis extract is 84.5%, and the test sample is not more than 50% and is not more than the stimulating substance.

TABLE 11 tissue Activity results

Grouping	Product name	Tissue activity	Result determination
				Negative control	DPBS(NgC)	100％	Non-irritating substances
Positive control	5％SDS(PC)	8.4％	Stimulating substance
				Experimental group	Herba Swertiae Mileensis extract	84.5％	Non-irritating substances

Example 8

Swallow-grass-leaved gentian shampoo composition

8.1A swertia herb shampoo was prepared according to the following Table 12 formulation and a malassezia inhibition experiment was performed.

Table 12 formula table of swertia herb shampoo

The preparation method comprises the following steps:

1. the component A is uniformly dispersed at 75-80 ℃;

2. after the component A is uniformly stirred, the component B is sequentially added, and the temperature is kept for 30-45min;

3. adding the component C;

4. slowly cooling to 60 ℃, and adding the component C;

5. d, E, F, G components are added at normal temperature.

8.2 experiments on inhibition of malassezia by swertia mileensis shampoo: the inhibition effect of the green leaf gall shampoo on malassezia is tested by taking malassezia furfur as a strain and referring to QBT2738-2012 evaluation method of the antibacterial and bacteriostatic effects of daily chemical products, the specific test method is as follows:

1) Experimental reagent: olive oil culture medium

3) The testing steps are as follows:

preparation of the culture medium: sterilizing the culture medium at high temperature 121 ℃ for 15 minutes after preparing the culture medium, subpackaging the culture medium into flat plates, wherein each flat plate is about 15-20 ml, and cooling and solidifying the culture medium for later use;

preparing a bacterial suspension: scraping malassezia colonies growing on the olive oil culture medium for 72 hours, and uniformly mixing the malassezia colonies in sterile normal saline;

sample preparation and inoculation: diluting 1g sample with 5ml physiological saline, mixing, taking 4.5ml mixed solution, placing into a test tube, adding 0.5ml bacterial suspension, taking 1ml physiological saline for ten times dilution after 10min action, and then taking 0.1ml diluted solution for coating on a flat plate. Using normal saline to replace a test sample, and simultaneously operating according to the steps to serve as a control sample;

culturing: culturing in incubator at 36 deg.c for 72 hr, recording colony number and calculating bacteriostasis effect;

4) Test results: the calculation was performed according to the following formula, and the results are shown in Table 13 below

Antibacterial ratio (%) = (average colony count of control-average colony count of sample)/average colony count of control 100

Table 13 antibacterial rate of swertia Japonica Makino shampoo

The results showed that the swertia pavioris shampoo significantly inhibited the proliferation of malassezia compared to the blank control (fig. 10).

In summary, the application provides the application of the swertia mileensis extract in inhibiting malassezia, propionibacterium acnes and anti-inflammatory efficacy, and experiments prove that the swertia mileensis extract can obviously inhibit the growth of malassezia furfur and propionibacterium acnes, simultaneously can effectively relieve the skin itching, redness and other inflammatory symptoms caused by bacterial reproduction, can effectively solve various skin problems such as dandruff/seborrheic dermatitis, atopic dermatitis, folliculitis/acne, psoriasis and the like, has low irritation and high safety, and is very suitable for being applied to the field of daily cosmetics.

In addition, the application also provides a specific swertia Japonica Makino extract and a preparation method thereof, and the swertia Japonica Makino extract prepared by the method can obtain swertiamarin with the weight percent of up to 45%.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims

1. The use of a swertia Japonica Makino extract for the preparation of a cosmetic or pharmaceutical composition for antibacterial purposes,

wherein the bacteria are selected from the group consisting of: propionibacterium, malassezia, or a combination thereof;

the swertia Japonica Makino extract is prepared by a method comprising the following steps:

(i) Heating herba Swertiae Mileensis in 0-85 v/v% ethanol water mixed solution, extracting, and separating to obtain extractive solution I;

(iii) Washing the resin I with water to obtain washed resin II;

(v) Optionally removing the solvent of the eluent, thereby obtaining the swertia Japonica Makino extract; and is also provided with

Wherein the resin is LS-305.

2. The use according to claim 1, wherein the bacteria are selected from the group consisting of: propionibacterium freudenreichii, propionibacterium acnes, propionibacterium granatum, malassezia furfur, malassezia axillaris, malassezia globosa, malassezia restrictum, malassezia thickii, malassezia Shi Luobang, malassezia oyster shell, or combinations thereof.

3. Use according to claim 1, wherein the cosmetic or pharmaceutical composition is for one or more uses selected from the group consisting of: dandruff, seborrheic dermatitis, atopic dermatitis, folliculitis, alopecia, acne caused by malassezia, or a combination thereof.

4. The use according to claim 1, wherein the cosmetic or pharmaceutical composition is for acne caused by propionibacterium acnes.

5. A swertia herb extract, characterized in that the swertia herb extract is prepared by a method comprising the steps of:

(i) Heating herba Swertiae Mileensis in 0-85 v/v% ethanol water mixed solution for extraction, and separating to obtain extractive solution I;

(iii) Washing the resin I with water to obtain washed resin II;

Wherein the resin is LS-305.

6. A cosmetic or pharmaceutical composition for antibacterial use, characterized in that it comprises the swertia herb extract according to claim 5; and a cosmetically or pharmaceutically acceptable carrier; wherein the bacteria are selected from the group consisting of: propionibacterium, malassezia, or combinations thereof.

7. The composition of claim 6, wherein the amount of the swertia herb extract in the cosmetic or pharmaceutical composition is greater than or equal to 0.1wt% based on the total weight of the cosmetic or pharmaceutical composition.

8. The composition of claim 7, wherein the amount of the swertia herb extract in the cosmetic or pharmaceutical composition is greater than or equal to 0.5wt% based on the total weight of the cosmetic or pharmaceutical composition.

9. The composition of claim 6, wherein the cosmetic composition comprises the following components, based on the total weight of the composition:

。

10. a method of preparing the swertia herb extract of claim 5, comprising the steps of:

(iii) Washing the resin I with water to obtain washed resin II;

Wherein the resin is LS-305.