CN115463056A - Swertia mileensis extract and preparation method and application thereof - Google Patents

Abstract

The invention provides a swertia mileensis extract and a preparation method and application thereof. In particular, the invention provides the use of an extract of swertia mileensis for the preparation of a composition against a flora associated with skin problems. The invention also provides a swertia mileensis extract and a preparation method thereof, and the swertia mileensis extract has high safety and good antibacterial effect.

Description

Swertia 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

The herba Swertiae Mileensis EXTRACT (Swertia Mileensis EXTRACT) is dried whole plant of Swertia Mileensis T.N.Hoet W.L.Shi of Gentianaceae. The mile swertia herb is a special plant in Yunnan, is mainly distributed in the counties of Maitreya, kaiyuan and the like in the red river of Yunnan province, has the effects of clearing liver and benefiting gallbladder, clearing heat and promoting diuresis, and is a famous medicinal material for treating acute and chronic hepatitis in the folk of Yunnan. The herba Swertiae Mileensis contains abundant chemical components, and mainly contains iridoid glycosides, flavonoids, oroxanthones and triterpenes.

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

Malassezia serrata is a resident bacterium on the skin surface, is mainly distributed on the parts of the skin of human beings and warm-blooded animals with vigorous oil secretion, and is in a symbiotic state with the body; however, a large number of studies have shown that malassezia bacteria, if it is excessively proliferated, cause various inflammatory skin problems such as dandruff/seborrheic dermatitis, atopic dermatitis, psoriasis, etc., and studies have also shown that it can also act together with propionibacterium acnes to cause the occurrence of acne. Ketoconazole, fluconazole and Zinc Pyrithione (ZPT) are acknowledged effective malassezia inhibitors in the market at present, can effectively inhibit dandruff and relieve skin inflammation, but can ensure that dermatophyte generates obvious drug resistance to azole medicaments after long-term use, greatly reduces the treatment effect of the azole medicaments on fungus-induced seborrheic dermatitis, dandruff and the like, and can not completely eradicate the problem of inflammatory skin; zinc Pyrithione (ZPT) is also subject to ban due to the problems of high skin irritation and potential reproductive toxicity, and therefore, the development of a malassezia inhibitor having both efficacy and safety has made a problem to be solved urgently.

Propionibacterium acnes, a species of Propionibacterium, belongs to anaerobic bacteria, mainly inhabits human and animal skin, sebaceous glands, intestinal tracts and dairy products, and causes no disease under aerobic conditions, but in places where sebum secretion is vigorous, when the pilosebaceous canal is blocked and an anaerobic environment is locally formed, the growth of the bacteria is facilitated, resulting in the occurrence of acne, which further aggravates acne by participating in inflammation, promoting keratinization of the pilosebaceous glands and promoting sebum secretion. At present, acne treatment means mainly comprise drug therapy, including external drugs of tretinoin, benzoyl peroxide, azelaic acid, antibiotics and the like, and skin irritation reactions such as local erythema and desquamation, tightness and burning sensation are easy to occur during the use; secondly, the oral administration of antibiotics, isotretinoin, hormones and other medicines and traditional Chinese medicine compounds is adopted, but the problems of drug resistance, liver and kidney injury and the like are easy to occur after long-term administration, and the auxiliary treatment can be realized by some physical treatment methods, such as red and blue light irradiation, laser treatment and the like, but the treatment cannot be thoroughly cured, and the personal image and the life quality are seriously influenced.

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

Disclosure of Invention

The invention aims to provide application of a swertia mileensis extract in preparing a composition for resisting flora related to 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 of the invention, the invention provides the use of a swertia mileensis extract in the preparation of an antibacterial cosmetic or pharmaceutical composition.

In another preferred embodiment, the swertiamarin content in the swertia mileensis extract is more than or equal to 10wt%, preferably more than or equal to 20wt%, more than or equal to 30wt% or more than or equal to 40wt%, based on the total weight of the swertia mileensis extract.

In another preferred embodiment, the content of the swertia mileensis extract in the cosmetic or pharmaceutical composition is more than or equal to 0.1wt%, preferably more than or equal to 0.2wt% or more than or equal to 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 acnes, or Propionibacterium granulatus), malassezia (Pityrosporum) (e.g., malassezia furfur (m.furfur), malassezia symptomatica (m.sympatholis), malassezia globosa (m.globosa), malassezia restriction (m.restrata), malassezia pachydermata (m.pachydermatis), malassezia schwarfarensis (m.sloofiae), malassezia concha meretrix (m.obtusa)), or combinations 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 bacterial induced skin problems, such as dermatitis, folliculitis, acne, or combinations thereof.

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

In another preferred embodiment, the cosmetic or pharmaceutical composition is for one or more uses 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-inflammation.

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 mileensis extract is from flowers, stems, leaves, or a combination thereof, such as whole herbs, of swertia mileensis herbs.

In another preferred embodiment, the swertia mileensis extract is prepared by the following method, comprising the following steps:

(i) Heating and extracting swertia mileensis medicinal material in 0-85v/v% ethanol water mixed solution, and separating to obtain extract I;

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

(iii) Washing the resin I with water to obtain a 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 from the eluate to obtain the swertia mileensis extract.

In another preferred embodiment, step (i) includes 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 mileensis medicinal material to the ethanol-water mixed solution is 1; preferably, 1:5-25, such as 1;

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

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

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

In another preferred embodiment, step (ii) includes 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) The concentration is to concentrate the filtrate to 0.05-1g medicinal material/mL, preferably 0.1-0.5g medicinal material/mL, calculated by the dosage;

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

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

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

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

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

(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 cosmetic or pharmaceutical composition is in a dosage form selected from the group consisting of: liquid, suspension, semi-solid or solid formulations.

In another preferred embodiment, the cosmetic or pharmaceutical composition is in the form of a skin external preparation.

In another preferred embodiment, the cosmetic or pharmaceutical composition is in a dosage form selected from the group consisting of: solutions, gels, lotions, ointments, creams, pastes, cakes, powders, patches, etc.

In another preferred embodiment, the cosmetically acceptable carrier or excipient is selected from the group consisting of: a humectant, an antioxidant, an anti-ultraviolet agent, a preservative, a film-forming agent, an oil-soluble gelling agent, an organically modified clay mineral, a resin, an antibacterial agent, a fragrance, a salt, a pH-adjusting agent, a chelating agent, a cooling agent, an anti-inflammatory agent, a skin-beautifying ingredient, a vitamin, an amino acid, a nucleic acid, a hormone, an inclusion compound, or a combination thereof.

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

(i) Heating and extracting swertia mileensis medicinal material in 0-85v/v% ethanol water mixed solution, and separating to obtain extract I;

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

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

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

(v) Optionally removing the solvent from the eluate to obtain the swertia mileensis extract.

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

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

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

(ii) Concentrating the extracting solution I, and adsorbing with LS-305 type resin to obtain adsorbed resin I;

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

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

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

In another preferred embodiment, the swertiamarin content in the swertia mileensis extract is more than or equal to 10wt%, preferably more than or equal to 20wt%, more preferably more than or equal to 30wt% or more than or equal to 40wt%, based on the total weight of the swertia mileensis extract.

In a third aspect of the present invention, there is provided a cosmetic or pharmaceutical composition comprising an extract of swertia mileensis as defined in the second aspect of the present invention; and a cosmetically or pharmaceutically acceptable carrier.

In another preferred embodiment, the content of the swertia mileensis extract in the cosmetic or pharmaceutical composition is more than or equal to 0.1wt%, preferably more than or equal to 0.2wt% or more than or equal to 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 cosmetically acceptable carrier or excipient is selected from the group consisting of: a humectant, an antioxidant, an anti-ultraviolet agent, a preservative, a film forming agent, an oil-soluble gelling agent, an organically modified clay mineral, a resin, an antibacterial agent, an essence, a salt, a pH adjusting agent, a chelating agent, a refreshing agent, an anti-inflammatory agent, a skin beautifying ingredient, a vitamin, an amino acid, a nucleic acid, a hormone, an inclusion compound, or a combination thereof.

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

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

name(s)	wt％
		Guar hydroxypropyl trimethyl ammonium chloride	0.24～0.36
Polyquaternary ammonium salt-10	0.04～0.06
		Citric acid	0.16～0.24
Sodium laureth sulfate	8～12
		Ammonium lauryl sulfate	5.6～8.4
Pentaerythritol distearate	0.4～0.6
		Ethylene glycol stearate	1.2～1.8
Cocoamidopropyl 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
Extract of SWERTIA MILEENSIS (SWERTIA MILEENSIS)	0.48～0.72
		Water (I)	1.6～2.4
Hydantoin compounds	0.16～0.24
		Water (W)	Balance of

In a fourth aspect of the present invention, there is provided a method for preparing the swertia mileensis extract according to the second aspect of the present invention, the method comprising the steps of:

(i) Heating and extracting swertia mileensis medicinal material in 0-85v/v% ethanol water mixed solution, and separating to obtain extract I;

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

(iii) Washing the resin I with water to obtain a 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 from the eluate to obtain the swertia mileensis extract.

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

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

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

(ii) Concentrating the extracting solution I, and adsorbing with LS-305 type resin to obtain adsorbed resin I;

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

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

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

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

In a sixth aspect of the present invention, there is provided a method for skin care, comprising the steps of: administering an effective amount of the swertia mileensis extract of the second aspect of the invention or the composition of the third aspect of the invention to a subject in need thereof to combat skin-associated bacteria and thereby achieve skin care.

In another preferred embodiment, the mode of administration is topical.

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

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 is a chromatogram of swertiamarin reference substance, swertia mileensis water extract, swertia mileensis 60% ethanol extract and swertia mileensis 80% ethanol extract;

FIG. 2 is a liquid chromatogram of a water washing solution and an ethanolysis solution after purification of swertiamarin reference substance, D101, AB-8, HPD-400, HPD-100, LS-305, and LS-300B resins;

FIG. 3 is a liquid chromatogram of swertiamarin reference substance and swertia mileensis extract;

FIG. 4 shows the growth of Malassezia furfur under different concentrations of the swertia mileensis extract, and the inhibition of Malassezia furfur by the swertia mileensis extract is gradually increased with the increase of the concentration. The concentration of 1.5 percent can completely inhibit the growth of malassezia furfur, namely the minimum inhibitory concentration of the mile swertia extract on the malassezia furfur is 1.5 percent.

FIG. 5 shows the growth of Propionibacterium acnes at different concentrations of the swertia mileensis extract, and the inhibition of Propionibacterium acnes by the swertia mileensis extract is gradually increased with the increase of the concentration. The 5% concentration 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 swertia mileensis extract on the viability of Raw264.7 cells at different concentrations, and the cell viability is greater than or equal to 90% within the concentration range of less than or equal to 500 μ g/mL.

Figure 7 shows that swertia mileensis extract 500 μ g/mL and 200 μ g/mL (P < 0.01) significantly inhibited the secretion of inflammatory mediator NO by macrophage raw264.7 model of inflammation.

FIG. 8 shows that 500. Mu.g/mL and 200. Mu.g/mL (. About.P. < 0.05) of the swertia mileensis extract can obviously inhibit secretion of COX-2, an inflammatory mediator in an inflammation model of macrophage Raw264.7.

FIG. 9 shows that the skin tissue activity was > 50% by 1% of swertia mileensis extract, and it was judged to be a non-irritant substance.

Fig. 10 shows that the malassezia bacteria in the petri dishes with the shampoo containing 0.6% of the swertia mileensis extract was significantly less compared to the blank control, demonstrating that it had a significant inhibitory effect on the malassezia bacteria.

Detailed Description

The present inventors have made extensive and intensive studies and, as a result of extensive screening and testing, have provided a swertia mileensis extract and a method for producing the same and use thereof. The invention provides a swertia mileensis extract with high content of swertiamarin and a preparation method thereof. The invention also unexpectedly discovers that the swertiamarin has remarkable antibacterial effect under the condition of low concentration, particularly on malassezia and propionibacterium acnes, so that the swertiamarin is very suitable for preparing antibacterial cosmetic compositions or pharmaceutical compositions. The present invention has been completed based on this finding.

Term(s)

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

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of or" consisting of 823030A ".

As used herein, the term "room temperature" or "ambient temperature" means a temperature of 4 to 40 ℃, preferably 25 ± 5 ℃, and the operating temperature may be ambient temperature, if not specifically stated.

Herba Swertiae Mileensis

Herba Swertiae Mileensis EXTRACT (Swertia MILEENSIS EXTRACT) is dried whole plant of Swertia MILEENSIS T.N.Hoet W.L.Shi of Gentianaceae. The swertia mileensis is a special plant in Yunnan, and is mainly distributed in the counties of Maitreya, kaiyuan and the like in the red river of Yunnan province. The invention has no special requirements on the swertia mileensis medicinal material, and can be purchased commercially or obtained by processing the swertia mileensis medicinal material by the conventional method in the field. The medicinal material can be whole herb or chopped or pulverized.

Swertia mileensis extract

In the invention, the active component is swertia mileensis extract, which is an extract extracted from swertia mileensis medicinal materials and taking swertiamarin as a main active component.

In another preferred embodiment, the swertiamarin content in the swertia mileensis extract is more than or equal to 10wt%, preferably more than or equal to 20wt%, more than or equal to 30wt% or more than or equal to 40wt%, such as 20-50wt% or 35-45wt%, based on the total weight of the swertia mileensis extract.

Preparation method

In the present invention, there is no particular requirement on the preparation method of the swertia mileensis extract, and the swertia mileensis extract can be prepared by the extraction method 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 mileensis extract comprises the following steps:

(i) Heating and extracting swertia mileensis medicinal material in 0-85v/v% ethanol water mixed solution, separating to obtain extract I;

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

(iii) Washing the resin I with water to obtain a 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 from the eluate to obtain the swertia mileensis extract.

In another preferred embodiment, step (i) includes 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 mileensis medicinal material to the ethanol-water mixed solution is 1; preferably, 1:5-25, such as 1;

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

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

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

In another preferred embodiment, step (ii) includes 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) The concentration is to concentrate the filtrate to 0.05-1g medicinal material/mL, preferably 0.1-0.5g medicinal material/mL, calculated by dosage;

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

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

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

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

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

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

(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.

Particularly preferably, the method comprises the steps of:

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

(ii) Concentrating the extracting solution I, and adsorbing with LS-305 type resin to obtain adsorbed resin I;

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

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

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

Composition and use

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

The invention surprisingly finds that the swertia mileensis 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 associated bacteria, such as including but not limited to: propionibacterium (Propionibacterium) (e.g., propionibacterium freudenreichii, propionibacterium acnes, or Propionibacterium granulatus), malassezia (Pityrosporum) (e.g., malassezia furfur (m.furfur), malassezia furfur (m.sympodialis), malassezia globosa (m.globosa), malassezia restriction (m.resticta), malassezia pachydermatis (m.papyridematis), malassezia schnikoensis (m.slooeae), malassezia concha (m.isobuta)

In addition, the swertia mileensis extract of the invention is not easy to generate resistance. The bacteria useful in the present invention may be malassezia that are resistant to ketoconazole, fluconazole, and/or Zinc Pyrithione (ZPT); and/or Propionibacterium acnes resistant to erythromycin, cephalosporins and/or clindamycin.

In another preferred embodiment, the compositions of the present invention are suitable for use in bacterial induced skin problems such as dermatitis, folliculitis, acne, or combinations thereof. For example, including, but not limited to, malassezia-induced dandruff, seborrheic dermatitis, atopic dermatitis, folliculitis, alopecia, acne, psoriasis, or combinations thereof; and/or acne caused by 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, the secretion of NO and/or COX-2, which are inflammatory mediators, can be inhibited, and thus, the invention also provides the use of the swertia mileensis extract as an NO and/or COX-2 inhibitor.

It is possible to prepare the swertia mileensis extract of the invention into pharmaceutical compositions such as ointments, creams, gels, pastes, patches, etc. The drug can be prepared by a generally known preparation technique, and a suitable pharmaceutical additive can be added to the drug.

Examples of the pharmaceutical additives include excipients, binders, disintegrating agents, lubricants, flow aids, suspending agents, emulsifiers, stabilizers, warming (wetting) agents, preservatives, solvents, solubilizers, preservatives, flavoring agents, sweeteners, dyes, flavors, propellants and the like, and these pharmaceutical additives may be selected and added in an appropriate amount within a range not affecting the effect of the present invention.

It is possible to prepare the swertia mileensis 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, a gel, a cream, an emulsion, a spray, an ointment, a cream, a paste, a cake, a powder, a patch, etc.

Other ingredients used in general cosmetics, for example, film-forming agents, oil-soluble gelling agents, organically modified clay minerals, resins, moisturizing agents, preservatives, antibacterial agents, fragrances, salts, antioxidants, pH adjusting agents, chelating agents, cooling agents, anti-inflammatory agents, skin beautifying ingredients (whitening agents, cell activating agents, skin roughness improving agents, blood circulation promoting agents, skin astringents, anti-seborrheic agents, etc.), vitamins, amino acids, nucleic acids, hormones, inclusion compounds, and the like may be added to the cosmetic of the present invention within a range that does not impair the effects of the present invention.

The oil-soluble gelling agent is selected from metal soaps such as aluminum stearate, magnesium stearate, and zinc myristate; amino acid derivatives such as N-lauroyl-L-glutamic acid, alpha, gamma-di-N-butylamine, and the like; 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; one or two or more gelling agents such as an organically modified clay mineral such as dimethylbenzyldodecylammonium montmorillonite clay or dimethyloctacosylammonium montmorillonite clay may be used as necessary.

The humectant comprises: glycerin, sorbitol, propylene glycol, dipropylene glycol, 1, 3-butylene glycol, 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 parabens, benzoic acid, sodium benzoate, sorbic acid, potassium sorbate, phenoxyethanol, and the like, and antibacterial agents such as: benzoic acid, salicylic acid, carbolic acid, sorbic acid, alkyl parabens, parachloro-metacresol, hexachlorophene, benzalkonium chloride, chlorhexidine chloride, trichloro-carbanilide, triclosan, a photosensitizer, phenoxyethanol, and the like.

The antioxidant comprises the following components: tocopherol, butyl hydroxy anisole, dibutyl hydroxy toluene, phytic acid and the like, and the pH regulator comprises: lactic acid, citric acid, glycolic acid, succinic acid, tartaric acid, dl-malic acid, potassium carbonate, sodium bicarbonate, ammonium bicarbonate, and the like, as chelating agents, alanine, sodium ethylenediaminetetraacetate, sodium polyphosphate, sodium metaphosphate, phosphoric acid, and the like, as cooling agents: l-menthol, camphor, etc., and the anti-inflammatory agents include: allantoin, glycyrrhetinic acid, glycyrrhizic acid, tranexamic acid, and Azulene (Azulene).

The skin beautifying components are as follows: whitening agent such as placenta extract, arbutin, glutathione, and herba Saxifragae extract; cell activator such as Lac Regis Apis, photosensitizer, cholesterol derivative, calf blood extractive solution, etc.; an agent for improving rough skin; blood circulation promoters such as valerian nonanoate, benzyl nicotinate, beta-butoxyethyl nicotinate, capsaicin, zingerone, cantharides tincture, ichthammol, caffeine, tannic acid, alpha-borneol, tocopherol nicotinate, inositol hexanicotinate, cyclamate, cinnarizine, tolazoline, acetylcholine, verapamil, cepharanthin, and gamma-oryzanol; skin astringents such as zinc oxide and tannic acid; sulfur, and antilipidemic agents, and the like, and vitamins include: vitamin A oil, rosin oil, acetic acid rosin oil, palmitic acid rosin oil, and the like; vitamin B2 compounds such as riboflavin, riboflavin butyrate, and flavin adenine nucleotide; vitamin B6 such as pyridoxine hydrochloride, pyridoxine dioctanoate, and pyridoxine tripalmitate, vitamin B12 and its derivatives, and vitamin B15 and its derivatives; vitamin C compounds such as L-ascorbic acid, L-ascorbic acid dipalmitate, L-ascorbic acid-2-sodium sulfate, and L-ascorbic acid phosphoric acid diester dipotassium; vitamin D compounds such as ergocalciferol and cholecalciferol; vitamin E compounds such as alpha-tocopherol, beta-tocopherol, gamma-tocopherol, dl-alpha-tocopherol acetate, dl-alpha-tocopherol nicotinate, and dl-alpha-tocopherol succinate; vitamin H; a vitamin P; nicotinic acids such as nicotinic acid, benzyl nicotinate and nicotinamide; pantothenic acids such as calcium pantothenate, D-panthenol, panthenyl ethyl ether, and acetyl panthenyl ethyl ether; biotin, and the like.

The amino acids are: glycine, valine, leucine, isoleucine, serine, threonine, phenylalanine, arginine, lysine, aspartic acid, glutamic acid, cystine, cysteine, methionine, tryptophan and the like, nucleic acids include deoxyribonucleic acid and the like, and hormones include estradiol, vinylestradiol and the like.

Preferred examples of the cosmetic of the present invention include: skin care cosmetics, hair care cosmetics, color cosmetics, and ultraviolet protection cosmetics. Such as hair care products such as shampoo, hair conditioner, hair essence, hair mask, etc.; basic cosmetics such as lotion, cream, dew, sunscreen cream, facial mask material, facial cleanser, essence, etc.; makeup cosmetics such as foundation, powdery, blush, and the like.

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

The present invention also provides a method of skin (including scalp) care, the method comprising the steps of: administering said swertia mileensis extract of the invention or said composition of the invention to a subject in need thereof.

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

As used herein, the term "effective dose" refers to any amount that, when used alone or in combination with another therapeutic agent or cosmetic, promotes disease regression as a result of reduced severity of disease symptoms, increased frequency and duration of disease symptom-free periods, or prevention of a disorder or disability resulting from the disease. The "therapeutically effective dose" of the drug of the present invention also includes a "prophylactically effective dose", which is any amount of the drug 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 applications of the antibacterial and anti-inflammatory agents include both preventive and post-improvement applications. For example, for antimicrobial purposes, it is contemplated that the inventive swertia mileensis extract or composition can be administered before, during, and/or after infection to prevent and/or repair bacterial infection.

Without wishing to limit the present invention, the antibacterial and anti-inflammatory effects of the swertia mileensis extract may be due to the synergistic effect of a single component (such as swertiamarin) or components of the extract.

The main advantages of the present invention include:

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

2. The inventive mile swertia herb extract has obvious inhibiting effect on malassezia furfur and propionibacterium acnes, and can completely inhibit under the action of low concentration, thus the inventive extract has high antibacterial activity.

4. The swertia mileensis extract has the inhibition effect on NO and COX-2 which are inflammatory mediators;

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

6. The swertia mileensis extract and the composition containing the same 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 mileensis 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 flora associated with skin problems, which provides a novel solution for flora that are resistant to other antimicrobial agents.

The invention is further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally according to conventional conditions, or according to conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight.

The preparation process of the swertia mileensis extract is determined by taking swertiamarin as a target component and swertiamarin as a raw material through steps of extraction solvent screening, purified resin screening and the like, and the efficacy of the swertia mileensis extract is verified through efficacy test.

Example 1

1. Screening of extraction solvent

1.1, extraction: weighing three parts of dry Swertia mileensis medicinal material whole grass (Latin name Swertia mileensis T.N.Ho et W.L.Shi, yunnan of China), adding 1000mL of purified water, 60% ethanol and 80% ethanol into each part of 50g of the medicinal material, heating and refluxing for 2h, filtering by using a 300-mesh screen to separate filtrate, adding 1000mL of purified water, 60% ethanol and 80% ethanol into filter residue, heating and refluxing for 1.5h, filtering by using a 300-mesh screen to separate filtrate, and combining the filtrates obtained by two times;

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

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

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

2) Swertiamarin reference substance: weighing swertiamarin 5mg (accurate to 0.01 mg) in a 100mL volumetric flask, adding 50% methanol to constant volume to scale, shaking up, and making into reference solution with concentration of 50 μ g/mL;

3) Preparing a test solution: diluting the crude extract obtained by 1.2 by proper times respectively by 50% methanol for later use;

4) And (3) determination: an appropriate amount of each of the control and test sample solutions was filtered through a 0.45 μm filter and then placed in a sample bottle of a liquid chromatograph, and the results were measured as shown in fig. 1.

5) And (4) result processing and analysis: calculating the content of swertiamarin in crude extract obtained by extracting with different solvents according to A1/A2= C1/C2, as shown in the following table 1.

Table 1: sorting of screening results of extraction solvent

The results show that the content of the swertiamarin in the extractum extracted by 3 different solvents is 60% ethanol more than 80% ethanol more than purified water from high to low.

Example 2

2. Purified resin screening

2.1 taking 100g of each of D101, AB-8, HPD-400, HPD-100, LS-305 and LS-300B resin, soaking the resin in 95% ethanol for 48h, weighing 15g (based on wet resin) of each resin type, washing the resin with purified water until no ethanol smell exists, and placing the resin in a 100mL beaker for later use

2.2 weighing 100g of dried swertia mileensis medicinal material, and carrying out heating reflux extraction by using 60% ethanol as a solvent for 2 times, wherein the dosage of the solvent is 2000mL each time, and the extraction time is 2h and 1h respectively. Collecting and combining the liquid medicines obtained by 2 times of extraction, and concentrating to 200g for later use;

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

2.4 measuring 60mL of purified water, washing the resin for 4 times with 15mL of purified water each time, and collecting the water washing liquid after washing the resin; then measuring 6 parts of 80% ethanol with the volume of 80mL, respectively pouring the 80% ethanol into a beaker filled with the resin, uniformly stirring the mixture, and then putting the mixture into a shaking table to shake the mixture for 15min so as to ensure that the resin is fully contacted with the extract; filtering the resin with a 300-mesh screen, and collecting 80% ethanol solution;

2.5 respectively supplementing the filtrate obtained in step 2.3, the water washing solution obtained in step 2.4 and the 80% ethanol solution to 100g by using purified water;

2.6 high performance liquid chromatography is used for measuring the contents of swertiamarin in filtrate, water washing liquid and 80% ethanol analytic liquid corresponding to 6 kinds of resin;

1) Chromatographic conditions are as follows: the chromatographic column is Hypersil BDS C18 (250 mm multiplied by 4.6mm,5 μm); the mobile phase was methanol-0.05% phosphoric acid solution (22; the detection wavelength is 237nm; the flow rate is 1mL/min; the sample amount is 10 mu l, and the column temperature is 30 ℃;

2) Swertiamarin reference substance: weighing 5mg (accurate to 0.01 mg) of swertiamarin reference substance, placing in a 100mL volumetric flask, fixing the volume to scale with 50% methanol, shaking up, and preparing into reference substance solution with concentration of 50 μ g/mL;

3) Preparing a test solution: diluting the crude extract obtained in 2.5 with 50% methanol by proper times respectively for later use;

4) And (3) determination: the appropriate amount of the control and test sample solutions were filtered through 0.45 μm filter membranes and placed in a sample bottle of a liquid chromatograph, and the results of the measurements are shown in FIG. 2.

5) And (4) result processing and analysis: calculating the content of swertiamarin in crude extract obtained by extracting with different solvents according to A1/A2= C1/C2, as shown in the following table 2:

TABLE 2 resin screening assay results collated

The adsorption rate, the resolution ratio and the yield of each resin to swertiamarin were calculated according to the following formulas, 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 Content of swertiamarin (%) before adsorption

C ₁ -swertiamarin content (% in water washing solution)

C ₂ -swertiamarin content (% in ethanol solution)

Table 3: influence of different types of resins on separation and purification of swertiamarin

Resin type	Adsorption rate%	The resolution ratio%	The yield is percent
				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 the LS-305 are all higher than those of other resin models, and the yield of the swertiamarin is 74.24%.

Example 3

Preparation method of swertia mileensis extract

3.1, extraction: weighing 50g of dried swertia mileensis medicinal material, and carrying out heating reflux extraction by using 60% ethanol as a solvent for 2 times, wherein the dosage of the solvent is 1000mL each time, and the extraction time is 2h and 1h respectively;

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

3.3, column filling: 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 loading: pouring the extract obtained in 6.3.2 into a resin column, controlling the flow rate at 4BV/h, and collecting the effluent liquid of sample loading;

3.5, washing with water: after the sample loading is finished, adding 4BV of 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 ethanol with the concentration of 80 percent into the resin column, controlling the flow rate at 2.5BV/h, and collecting an ethanol analysis solution;

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

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

3.9 high performance liquid chromatography is used for measuring the content of swertiamarin in swertia mileensis extract;

1) Chromatographic conditions are as follows: the chromatographic column is Hypersil BDS C18 (250 mm multiplied by 4.6mm,5 μm); the mobile phase was methanol-0.05% phosphoric acid solution (22; 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) Swertiamarin reference substance: weighing swertiamarin 5mg (accurate to 0.01 mg) in a 100mL volumetric flask, adding 50% methanol to constant volume to scale, shaking up, and making into reference solution with concentration of 50 μ g/mL;

3) Preparing a test solution: weighing a proper amount of the swertia mileensis extract obtained by 3.8, diluting the swertia mileensis extract by a proper multiple with 50% methanol for later use;

4) And (3) determination: the appropriate amount of the control and test sample solutions were filtered through 0.45 μm filter membranes and placed in a sample bottle of a liquid chromatograph, and the results of the measurements are shown in FIG. 3.

5) And (4) result processing and analysis: the swertiamarin content in swertia mileensis extract was calculated according to A1/A2= C1/C2, and the result was 45.60%, as shown in table 4.

Table 4: determination of the content of green leaves but extract

Sample name	Time to peak (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, the yield of the obtained finished product is 11.56%, and the content of swertiamarin is 45.60%.

Example 4

Swertia mileensis extract bacteriostatic efficacy test

4.1 Malassezia inhibition assay: the inhibition effect of the swertia mileensis extract on the Malassezia is tested by taking Malassezia furfur (Pityrosporum orbiculare) as a strain and 1 percent Zinc Pyrithione (ZPT) as a positive control, and the specific test method is as follows:

1) Experimental reagent: olive oil culture medium

2) And (3) testing strains: malassezia furfur (ATCC 44344, available from guangdong collection of microorganisms);

3) The testing steps are as follows: adding the samples according to the concentration of 2%, 1.5%, 1.2%, 1%, 0.8% and 0.6% when preparing the culture medium, sterilizing at 121 ℃ for 15min, 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 a culture medium for 5 days, and uniformly mixing in sterile normal saline; 0.1mL of bacterial suspension (10) was taken ⁵ ～10 ⁶ CF μm/mL) on prepared agar plates, using inoculated olive oil medium plates without samples as blank control, taking 1 additional olive oil medium plate coated with 1% zptl 100 μ L as positive control; culturing at 36 deg.C for 72 hr in incubator, and observing colony growthA long condition;

4) The experimental results are as follows: the result is shown in figure 4, the swertia mileensis extract has obvious inhibiting effect on malassezia furfur, the inhibiting effect is gradually enhanced along with the increase of the concentration, the culture medium containing 1.5 percent of the sample can completely inhibit the growth of malassezia furfur, and the minimum inhibiting concentration of the sample on malassezia furfur is proved to be 1.5 percent.

Example 5

Propionibacterium acnes inhibition experiments:

the method for testing the inhibition effect of the swertia mileensis extract on the Propionibacterium acnes by taking the Propionibacterium acnes as a strain comprises the following specific test methods:

1) The experimental reagent: trypticase soy peptone agar medium (TSA medium), sheep blood, sterile saline;

2) And (3) testing strains: propionibacterium acnes (ATCC 6919, available from guangdong collection of microorganisms);

3) And (3) testing: adding the samples according to the concentration of 5%, 2.5% and 1.25% when preparing the culture medium, sterilizing at 121 deg.C for 15min, adding 2mL of sheep blood when the culture medium is cooled to about 45 deg.C, mixing, and packaging to plate; scraping Propionibacterium acnes colony growing on the culture medium for 3 days, mixing in sterile physiological saline, and collecting 0.1mL of bacterial suspension (10) ⁶ ～10 ⁸ CF mu/mL) is coated on a prepared blood plate, a blood culture medium without a sample of inoculated bacteria is used as a blank control, the blood culture medium is placed in an incubator at 36 ℃ in an anaerobic environment for culturing for 72 hours, and the growth condition of bacterial colonies is observed in a contrast manner;

4) The experimental results are as follows: the results are shown in fig. 5, where the swertia mileensis extract has a certain inhibitory effect on propionibacterium acnes, and the inhibitory effect is gradually increased with the increase of the concentration. The culture medium containing 5% of the sample can completely inhibit the growth of the propionibacterium acnes, and the minimal inhibitory concentration of the sample on the propionibacterium acnes is proved to be 5%.

Example 6

Effect of swertia mileensis extract on NO and COX-2

LPS is the main component of gram-negative bacteria cell wall, is the main inflammatory substance, can induce organism to produce inflammation, and release of inflammatory factors such as TNF-alpha and IL-6 and inflammatory mediators such as NO and COX-2 appears. The invention uses LPS to stimulate macrophage to generate an inflammation model, and detects the influence of a sample on inflammation 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 (Boshide), MTT (Sigma), DMSO, fetal bovine serum (Gibco), lipopolysaccharide LPS (Sigma), dexamethasone (middle search), NO detection kit (Biyuntian), RNAioso Plus (TaKaRa), isopropanol (national group), chloroform (national group), absolute ethanol (national group), DEPC water (Beyotime), reverse transcription kit (TaKaRa), fluorescent dye (TaKaRa).

The instrument comprises the following steps: CO2 incubator (Thermo), enzyme labeling instrument (BioTek), clean bench (Suzhou Antai), inverted microscope (Olympus), PCR instrument (BIO-RAD), and fluorescence quantitative PCR instrument (BIO-RAD).

Test method

6.1 determination of maximum safe concentration of Chondrus crispus extract

1) Cell inoculation: raw264.7 cells were seeded at a predetermined cell seeding density into 96-well plates and incubated overnight in an incubator (37 ℃ C., 5% CO2).

2) Grouping experiments: the experiment was set up with a blank control group, a positive control group and a sample group. In the sample set, 8 concentration gradients were set for each sample, and 3 replicate wells were set under each concentration gradient.

3) Preparing a liquid: the test substance working solutions with different concentrations were prepared according to the experimental design (table 5).

TABLE 5 test concentration setting table

4) Administration: and (3) administration is carried out when the cell plating rate in the 96-well plate reaches 40-60%. Adding 200 mu L of cell culture solution into each hole of the blank control group; adding 200 μ L of culture medium containing 10% DMSO per well of the positive control group; adding 200 mu L of culture solution containing the test object with corresponding concentration into each hole of the sample group; wells were zeroed for cell-free seeding and only 200 μ L of cell culture medium was added. After completion of the administration, the 96-well plates were placed in an incubator (37 ℃ C., 5% CO2) and cultured.

5) And (3) detection: after 24h of cell incubation culture, the supernatant was discarded, MTT working solution (0.5 mg/mL, ready for use) was added, incubation was carried out at 37 ℃ in the dark for 4h, after incubation was completed, the supernatant was discarded, 150. Mu.L of DMSO was added to each well, and the OD was read at 490 nm.

6) Calculating the relative activity of the cells: calculated according to the formula, relative cell viability (%) = (sample-wells to zero)/(blank control wells to zero) × 100%

6.2 Effect of swertia mileensis extract on NO and COX-2 secretion

1) Cell inoculation: cells were inoculated into 6-well plates at a predetermined cell inoculation density and incubated overnight in an incubator (37 ℃ C., 5% CO2).

2) Preparing a liquid: the test article working solution was prepared according to the experimental design (table 6).

TABLE 6 Experimental design

3) Administration: according to the experimental concrete design of table 6, when the cell plating rate in the 6-well plate reaches 40% -50%, the administration amount of each well is 1.8mL, and each group is provided with 3 multiple wells. 37 ℃ C., 5% CO2 incubator for 2h.

4) Stimulation by LPS: after 2h of culture, 200. Mu.L of LPS working solution prepared from the corresponding test substance working solution was added to the administered well plate according to the group design, and the well plate was shaken from side to mix the drugs in the well plate uniformly, with a final concentration of LPS of 1. Mu.g/mL. The cultivation was continued at 37 ℃ for 24h in an incubator with 5% CO2.

5) Collecting cell supernatant: after 24h of culture, cell culture supernatants were collected in EP tubes.

6) Detection of NO content: and detecting according to the operation instruction of the NO content detection kit.

7) Collecting cells: washing twice with 2 mL/well PBS, adding 1mL RNAioso Plus per well, blowing to crack cell, and collecting sample.

8) Gene detection: RNA extraction, reverse transcription and fluorescent quantitative PCR detection are carried out according to the kit specification, and a 2-delta CT method is adopted for calculating results.

Statistical analysis of test results

The graphs were generated using GraphPad Prism Program software, and statistical analysis of t-test between groups, with p < 0.05 indicating significant differences and p < 0.01 indicating significant differences.

6.3 determination of maximum safe concentration of swertia mileensis extract

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

TABLE 7 determination of the maximum safe concentration of the swertia mileensis extract

6.4 measurement of NO content

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

TABLE 6-8 measurement results of NO content

Sample name	Mean concentration (μ M)	SD	p-value
				BC	0.99	0.10	/
NC	47.45	1.72	0.000##
				PC	22.30	0.72	0.000**
Swertia mileensis extract-500. Mu.g/mL	5.92	0.17	0.000**
				Herba Swertiae Mileensis extract-200 μ g/mL	25.39	1.72	0.000**

Remarking: when the statistical analysis is carried out by using a t-test method, compared with a BC group, the significance of an NC group is represented by # and p-value < 0.05 is represented by #, and p-value < 0.01 is represented by # #; the significance of the PC group and the sample group was expressed as p-value < 0.05 and p-value < 0.01, respectively, compared to the BC group.

6.5COX-2 Gene expression assay results

Based on the experimental method 6.5.3.2, the affected macrophages are processed by RNAioso Plus and then collected, and RNA extraction, reverse transcription and fluorescent quantitative PCR operation are carried out according to the instruction of the kit. The test results are shown in table 9 and fig. 8, and the expression level of COX-2 gene in the NC control group was significantly increased compared to that in the BC group, indicating that LPS stimulation was effective in this experiment. Compared with the NC group, the COX-2 gene expression level of the positive control group is obviously reduced, which indicates that the positive control of the experiment is effective. Compared with the NC group, the COX-2 gene expression level of the swertia mileensis extract is remarkably reduced under two dosing concentrations of 500 mu g/mL and 200 mu g/mL.

TABLE 9 result of COX-2 gene expression assay

Remarking: when the statistical analysis is carried out by using a t-test method, compared with a BC group, the significance of an NC group is represented by # and p-value < 0.05 is represented by #, and p-value < 0.01 is represented by # #; the significance of the PC group and the sample group was expressed as p-value < 0.05 and p-value < 0.01, respectively, compared to the BC group.

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

Example 7

Swertia mileensis extract skin irritation test

7.1 Experimental materials: 3D skin model kit (Shanghai Si Anfuno Biotechnology Co., ltd.), MTT (Shanghai Yuan Ye), DPBS (BI), isopropyl alcohol (Tianjin Feng)

7.2 Experimental facilities: enzyme-linked immunosorbent assay (Thermo), clean bench (Suzhou Antai), CO2 incubator (Memmert)

7.3 test methods: reference OECD TG439

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

2) Sample adding treatment: after the skin model is cultured for 24, the skin model is taken out, the skin model is divided into groups according to the experimental design of the table 10, each group is provided with 3 compound holes, and 10 mu L of corresponding samples are uniformly coated on the surface of skin tissues;

table 10 skin irritation test experimental design

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

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

5) Tissue viability assay: 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 into 1.5mL centrifuge tube, adding 500 μ l acidified isopropanol, mixing, standing at 4 deg.C in dark for 72 hr, taking acidified isopropanol as blank, and measuring absorbance at 560nm wavelength;

6) And (4) result processing and analysis: viability of the corresponding tissue of each sample was calculated according to = (experimental OD value-blank OD value)/(negative control OD value-blank OD value) × 100% skin tissue viability.

Test results

The results were judged for non-irritating substances when the tissue viability of the test samples was > 50% and irritating substances when the tissue viability of the test samples was < 50%, as shown in table 11 and fig. 9, where the tissue viability of 1% of the swertia mileensis extract was 84.5% and was non-irritating substances.

TABLE 11 tissue Activity results

Grouping	Name of product	Tissue viability	Determination of results
				Negative control	DPBS(NgC)	100％	Non-irritating substance
Positive control	5％SDS(PC)	8.4％	Irritant substance
				Experimental group	Swertia mileensis extract	84.5％	Non-irritating substances

Example 8

Swertia mileensis shampoo composition

8.1 the swertia mileensis shampoo is prepared according to the formula of the following table 12 and the malassezia inhibition experiment is carried out.

TABLE 12 Green leaf and gall shampoo formula table

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, sequentially adding the component B, and keeping the temperature and stirring for 30-45min;

3. adding the component C;

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

5. adding the components D, E, F and G at normal temperature.

8.2 malassezia inhibition experiment of mile swertia: the malassezia furfur is taken as a strain, the inhibition effect of the mile swertia shampoo on the malassezia is tested according to the QBT2738-2012 evaluation method for the antibacterial and bacteriostatic effect of daily chemical products, and the specific test method is as follows:

1) Experimental reagent: olive oil culture medium

2) And (3) testing strains: malassezia furfur (ATCC 44344, available from guangdong collection of microorganisms);

3) The testing steps are as follows:

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

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

sample preparation and inoculation: diluting 1g sample with 5ml normal saline, mixing well, placing 4.5ml mixed solution in empty test tube, adding 0.5ml bacterial suspension, acting for 10min, diluting 1ml with normal saline ten times, and coating 0.1ml diluent on plate. Replacing a test sample with normal saline, and operating according to the steps to serve as a control sample;

culturing: culturing for 72h at 36 ℃ in an incubator, recording the number of colonies, and calculating the bacteriostatic effect;

4) And (3) testing results: the results of the calculation according to the following formula are shown in Table 13 below

Bacteriostatic ratio (%) = (control average number of colonies-sample average number of colonies)/control average number of colonies x 100

TABLE 13 bacteriostasis rate of swertia mileensis shampoo

The results show that the swertia mileensis shampoo can obviously inhibit the reproduction of malassezia compared with the blank control group (figure 10).

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

In addition, the invention also provides a specific swertia mileensis extract and a preparation method thereof, and the swertiamarin with the weight percent as high as 45 percent can be obtained from the swertia mileensis extract prepared by the method.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes or modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.

Claims (10)

1. The application of the swertia mileensis extract is characterized in that the swertia mileensis extract is used for preparing an antibacterial cosmetic composition or a antibacterial pharmaceutical composition.

2. The use of claim 1, wherein the bacteria are selected from the group consisting of: propionibacterium (e.g., propionibacterium freudenreichii, propionibacterium acnes, or Propionibacterium granulatus), malassezia (Pityrosporum) (e.g., malassezia furfur (malassezia. Furfur), malassezia symptomata (m.sympatholis), malassezia globosa (m.globosa), malassezia limited (m.resticta), malassezia pachydermatis (m.papyhermatis), malassezia schnivea (m.smoleae), malassezia concha ostreatus (m.suloderma), or combinations thereof.

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

4. The use according to claim 1, wherein the swertia mileensis extract is prepared by a process comprising the steps of:

(i) Heating and extracting swertia mileensis medicinal material in 0-85v/v% ethanol water mixed solution, separating to obtain extract I;

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

(iii) Washing the resin I with water to obtain a 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 from the eluate to obtain the swertia mileensis extract.

5. A swertia mileensis extract, characterized in that it is prepared by a process comprising the steps of:

(i) Heating and extracting swertia mileensis medicinal material in 0-85v/v% ethanol water mixed solution, and separating to obtain extract I;

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

(iii) Washing the resin I with water to obtain a 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 to obtain the swertia mileensis extract.

6. The extract of claim 5, wherein the resin is LS-305.

7. A cosmetic or pharmaceutical composition comprising the swertia mileensis extract of claim 5; and a cosmetically or pharmaceutically acceptable carrier.

8. The composition according to claim 7, wherein the swertia mileensis extract is present in the cosmetic or pharmaceutical composition in an amount of at least 0.1 wt.%, preferably at least 0.2 wt.% or at least 0.5 wt.%, such as 0.6 wt.%, 1 wt.%, 2 wt.%, 5 wt.%, 10 wt.% or 20 wt.%, based on the total weight of the cosmetic or pharmaceutical composition.

9. The composition of claim 7, wherein the cosmetic composition comprises, based on total composition weight:

name(s) 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 distearate 0.4～0.6 Ethylene glycol stearate 1.2～1.8 Cocoamidopropyl 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 Herba Swertiae Mileensis (SWERTIA MILEENSIS) extract 0.48～0.72 Water (W) 1.6～2.4 Hydantoin compounds 0.16～0.24 Water (W) Balance of

。

10. A method for preparing the swertia mileensis extract as claimed in claim 5, wherein the method comprises the steps of:

(i) Heating and extracting swertia mileensis medicinal material in 0-85v/v% ethanol water mixed solution, separating to obtain extract I;

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

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

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

(v) Optionally removing the solvent from the eluate to obtain the swertia mileensis extract.

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