WO2022257932A1 - 青叶胆提取物及其制备方法和应用 - Google Patents

青叶胆提取物及其制备方法和应用 Download PDF

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WO2022257932A1
WO2022257932A1 PCT/CN2022/097430 CN2022097430W WO2022257932A1 WO 2022257932 A1 WO2022257932 A1 WO 2022257932A1 CN 2022097430 W CN2022097430 W CN 2022097430W WO 2022257932 A1 WO2022257932 A1 WO 2022257932A1
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extract
resin
gallbladder
water
ethanol
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PCT/CN2022/097430
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English (en)
French (fr)
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郑晓琼
马占林
王早
王媛
冯秋红
黄灿
周戟
罗慧
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云南英格生物技术有限公司
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Publication of WO2022257932A1 publication Critical patent/WO2022257932A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/51—Gentianaceae (Gentian family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/10—Anti-acne agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/005—Antimicrobial preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00—Preparations for care of the hair
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/55—Liquid-liquid separation; Phase separation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95

Definitions

  • the invention belongs to the technical field of daily cosmetics, and in particular relates to an extract of gallbladder leaf and its preparation method and application.
  • Swertia MILEENSIS EXTRACT is the dry whole herb of Swertia mileensis T.N.Hoet W.L.Shi, a plant of Gentianaceae.
  • Qingyedan is a unique plant in Yunnan. It is mainly distributed in Maitreya and Kaiyuan counties in Honghe Prefecture, Yunnan province. It has the effects of clearing liver and gallbladder, clearing heat and dampness.
  • the chemical composition of A. japonicus is rich, mainly containing iridoid glycosides, flavonoids, ketones and triterpenoids.
  • Swertiacroside is a kind of split ring iridoid glycoside compound, which is a kind of component with more content in Gentianaceae plants. pharmacological effects.
  • Malassezia is a resident bacterium on the surface of the skin. It is mainly distributed in the parts of human beings and warm-blooded animals with strong skin oil secretion, and is in a symbiotic state with the body; however, a large number of studies have confirmed that if Malassezia overgrows, it will cause scalp Psoriasis/seborrheic dermatitis, atopic dermatitis, psoriasis and other inflammatory skin problems, and studies have also shown that it can also interact with Propionibacterium acnes to cause acne.
  • Ketoconazole, fluconazole, and zinc pyrithione are currently recognized as effective Malassezia inhibitors in the market, which can effectively inhibit dandruff production and reduce skin inflammation, but long-term use can make skin fungi resistant to azoles Drugs produce obvious drug resistance, which greatly reduces the therapeutic effect of azoles on fungal-induced seborrheic dermatitis, dandruff, etc., and cannot completely eradicate inflammatory skin problems; zinc pyrithione (ZPT) is also due to skin irritation Therefore, the development of a Malassezia inhibitor with both efficacy and safety is an urgent problem to be solved.
  • Propionibacterium acnes is a species of Propionibacterium, which belongs to anaerobic bacteria. It mainly resides in the skin, sebaceous glands, intestinal tract and dairy products of humans and animals. In places where the pilosebaceous duct is blocked and an anaerobic environment is formed locally, it is conducive to the growth of the bacteria, leading to the occurrence of acne. The bacteria further aggravate acne by participating in inflammation, promoting keratinization of the pilosebaceous glands, and promoting sebum secretion.
  • the treatment of acne is mainly drug treatment, including topical retinoic acid, benzoyl peroxide, azelaic acid, antibiotics, etc.
  • the object of the present invention is to provide a kind of application of gallbladder extract in the preparation of the composition of resisting skin problem-related flora.
  • Another object of the present invention is to provide a kind of gallbladder extract with high safety and good antibacterial effect and its preparation method.
  • the first aspect of the present invention provides the use of the gallbladder extract, in the preparation of antibacterial cosmetic or pharmaceutical compositions.
  • the content of swertiamarin is ⁇ 10wt%, preferably ⁇ 20wt%, ⁇ 30wt% or ⁇ 40wt%. of total weight.
  • the content of the extract of the gallbladder leaf is ⁇ 0.1wt%, preferably ⁇ 0.2wt% or ⁇ 0.5wt%, such as 0.6wt%, 1wt% , 2wt%, 5wt%, 10wt% or 20wt%, based on the total weight of the cosmetic composition or pharmaceutical composition.
  • the bacterium is selected from the group consisting of: Propionibacterium (Propionibacterium) (for example, Propionibacterium freudennreichii), Propionibacterium acnes (Propionibacterium acnes) or particle Propionibacterium (Propionibacterium) granulosum)), Malassezia (Pityrosporum) (e.g., M. furfur, M. sympodialis, M. globosa, restricted Malassezia (M. restricta, M. pachydermatis, M. sloofiae, M. obtusa), or combination.
  • Propionibacterium Propionibacterium
  • Propionibacterium acnes Propionibacterium acnes
  • particle Propionibacterium Propionibacterium granulosum
  • Malassezia Pityrosporum
  • Malassezia e.g., M. furfur, M. sympodialis, M. globosa
  • the bacteria are selected from the group consisting of Malassezia furfur, Propionibacterium acnes, or a combination thereof.
  • the cosmetic composition or pharmaceutical composition is used for skin problems caused by bacteria, such as dermatitis, folliculitis, acne, or a combination thereof.
  • the cosmetic composition or pharmaceutical composition is used for one or more purposes selected from the group consisting of dandruff caused by Malassezia, seborrheic dermatitis, atopic dermatitis, folliculitis , hair loss, acne, psoriasis, or a combination thereof.
  • the cosmetic composition or pharmaceutical composition is used for one or more purposes selected from the group consisting of acne caused by Propionibacterium acnes, such as seborrheic acne, closed mouth, acne, or a combination thereof.
  • the cosmetic composition or pharmaceutical composition is also used for anti-inflammation.
  • the cosmetic composition or pharmaceutical composition is also used to inhibit the secretion of NO and/or COX-2.
  • the cosmetic or pharmaceutical composition further includes a cosmetically or pharmaceutically acceptable carrier.
  • the extract of Gallbladder is from the flower, stem, leaf, or a combination thereof, such as the whole plant.
  • the extract of Gallbladder Folium is prepared by the following method, including the following steps:
  • step (i) include one or more technical features selected from the following group:
  • the mixed solution is 20-80v/v% ethanol-water mixed solution, such as 40v/v%, 50v/v%, 60v/v%, 70v/v% or 75v/v%;
  • the dosage ratio (g/mL) of the gallbladder medicinal material to the mixed solution of ethanol and water is 1:2-30; preferably, 1:5-25, such as 1:10, 1:15 or 1:20;
  • the number of extractions is 1-3 times, such as 1, 2 or 3;
  • the extraction temperature is independently 50-100°C, such as 60°C, 70°C or 80°C, preferably, the reflux temperature; and/or
  • the extraction time is independently 0.5-3h, preferably 1-2h;
  • step (ii) include one or more technical features selected from the following group:
  • the resin is selected from the group consisting of AB-8, HPD-400, LS-305, LS-300B, or a combination thereof, preferably, LS-305;
  • the concentration is to concentrate the filtrate to 0.05-1g medicinal material/mL, preferably 0.1-0.5g medicinal material/mL, based on dosage;
  • the dosage of the resin is, based on the dosage of 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.
  • step (iii) include one or more technical features selected from the following group:
  • the amount of water used for cleaning is 2-8BV, preferably, 3-6BV;
  • the flow rate of water used for washing is 6-12BV/h, preferably 8-10BV/h.
  • step (ii) include one or more technical features selected from the following group:
  • the ethanol-water mixed solution is 75-85v/v% ethanol-water mixed solution, such as 80v/v%;
  • the dosage of the ethanol-water mixed solution is 2-10BV, preferably, 3-5BV;
  • the flow rate of the ethanol-water mixed solution is 1-5BV/h, preferably 2-4BV/h.
  • the dosage form of the cosmetic composition or pharmaceutical composition is selected from the group consisting of liquid preparation, suspension preparation, semi-solid preparation or solid preparation.
  • the dosage form of the cosmetic composition or pharmaceutical composition is an external dosage form for skin.
  • the dosage form of the cosmetic composition or pharmaceutical composition is selected from the group consisting of solutions, gels, emulsions, ointments, creams, pastes, cakes, powders, patches and the like.
  • the cosmetically acceptable carrier or excipient is selected from the group consisting of moisturizing agents, antioxidants, anti-ultraviolet agents, preservatives, film-forming agents, oil-soluble gelling agents, organic Modified clay minerals, resins, antibacterial agents, flavors, salts, pH regulators, chelating agents, cooling agents, anti-inflammatory agents, ingredients for skin beautification, vitamins, amino acids, nucleic acids, hormones, inclusion compounds, or combinations thereof.
  • gallbladder extract is provided, and the gallbladder extract is prepared by the following method, and the method comprises the steps of:
  • the resin is LS-305.
  • the method includes the steps of:
  • the content of swertiamarin is ⁇ 10wt%, preferably ⁇ 20wt%, ⁇ 30wt% or ⁇ 40wt%. of total weight.
  • a cosmetic composition or a pharmaceutical composition comprising the extract of Gallbladder of the present invention as described in the second aspect of the present invention; and a cosmetically or pharmaceutically acceptable carrier.
  • the content of the extract of the gallbladder leaf is ⁇ 0.1wt%, preferably ⁇ 0.2wt% or ⁇ 0.5wt%, such as 0.6wt%, 1wt% , 2wt%, 5wt%, 10wt% or 20wt%, based on the total weight of the cosmetic composition or pharmaceutical composition.
  • the cosmetically acceptable carrier or excipient is selected from the group consisting of moisturizing agents, antioxidants, anti-ultraviolet agents, preservatives, film-forming agents, oil-soluble gelling agents, organic Modified clay minerals, resins, antibacterial agents, flavors, salts, pH regulators, chelating agents, cooling agents, anti-inflammatory agents, ingredients for skin beautification, vitamins, amino acids, nucleic acids, hormones, inclusion compounds or combinations thereof.
  • the cosmetic composition is selected from the group consisting of shampoo, hair care spray, scalp care solution, skin care water, skin care lotion, cream, and facial cleanser.
  • the cosmetic composition includes the following components, based on the total weight of the composition:
  • the resin is LS-305.
  • the method includes the steps of:
  • the fifth aspect of the present invention provides an in vitro antibacterial method, comprising the step of: contacting the composition of the gallbladder extract of the second aspect of the present invention and the composition of the third aspect with bacteria, thereby inhibiting the bacteria.
  • a method of skin care comprising the step of: giving an effective amount of the extract of the gallbladder of the second aspect of the present invention or the composition of the third aspect of the present invention to a subject in need, thereby resisting the skin Related bacteria, thus achieving skin care.
  • the administration is external.
  • the subject is a mammal, such as a human, a rat or a mouse.
  • Fig. 1 is the chromatogram of swertiacroside reference substance, Folium Folium L. water extract, C. Folium Fructus 60% ethanol extract and Folium Fructus Folium 80% ethanol extract;
  • Fig. 2 is the liquid chromatogram of water washing liquid and ethanol analysis liquid after purification of swertiacroside reference substance, D101, AB-8, HPD-400, HPD-100, LS-305, LS-300B resin;
  • Fig. 3 is the liquid phase chromatogram of swertiacroside reference substance and the extract of Folium bile;
  • Figure 4 shows the growth of Malassezia furfur under the action of different concentrations of Gallbladder extract. As the concentration increases, the inhibitory effect of Gallbladder extract on Malassezia furfur gradually increases. The concentration of 1.5% can completely inhibit the growth of Malassezia furfur, that is, the minimum inhibitory concentration of the extract of Gallbladder Fructus to Malassezia furfur is 1.5%.
  • Figure 5 shows the growth of Propionibacterium acnes under the action of different concentrations of Gallbladder extract. With the increase of concentration, the inhibitory effect of Gallbladder extract on Propionibacterium acnes is gradually enhanced. 5% concentration can completely inhibit the growth of Propionibacterium acnes, that is, the minimum inhibitory concentration of P. acnes extract to Propionibacterium acnes is 5%.
  • Figure 6 shows the effect of different concentrations of Gallbladder extracts on the viability of Raw264.7 cells. In the concentration range of ⁇ 500 ⁇ g/mL, the cell viability is ⁇ 90%.
  • Fig. 7 shows that 500 ⁇ g/mL and 200 ⁇ g/mL (**P ⁇ 0.01) of the extract of Gallbladder can significantly inhibit the secretion of inflammatory mediator NO in the macrophage Raw264.7 inflammation model.
  • Fig. 8 shows that 500 ⁇ g/mL and 200 ⁇ g/mL (*P ⁇ 0.05) of Gallbladder extracts can significantly inhibit the secretion of inflammatory mediator COX-2 in macrophage Raw264.7 inflammation model.
  • Figure 9 shows that the activity of skin tissue under the action of 1% gallbladder extract is more than 50%, and it is judged as a non-irritating substance.
  • Figure 10 shows that compared with the blank control, in the petri dish of the shampoo added with 0.6% gallbladder extract, Malassezia is significantly less, which proves that it has a significant inhibitory effect on Malassezia.
  • the present inventor provides an extract of Gallbladder and its preparation method and application.
  • the invention provides an extract of bile gall with high content of swertiamarin and a preparation method thereof.
  • the present invention also unexpectedly finds that swertamaroside has significant antibacterial effect on antibacterial, especially Malassezia and Propionibacterium acnes at low concentration, so it is very suitable for the preparation of cosmetic compositions for antibacterial or pharmaceutical compositions.
  • the present invention has been accomplished on this basis.
  • 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.
  • the expression “about 100” includes all values between 99 and 101 and in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the term “comprises” or “includes (comprising)” can be open, semi-closed and closed. In other words, the term also includes “consisting essentially of”, or “consisting of”.
  • room temperature or "normal temperature” refers to a temperature of 4-40°C, preferably 25 ⁇ 5°C. Unless otherwise specified, the operating temperature can be normal temperature.
  • Swertia MILEENSIS EXTRACT is the dry whole herb of Swertia mileensis T.N.Hoet W.L.Shi, a plant of Gentianaceae.
  • Foliage gall is a unique plant in Yunnan, mainly distributed in Mile, Kaiyuan and other counties in Honghe Prefecture, Yunnan province.
  • the present invention has no special requirement on the gallbladder medicinal material, which can be purchased commercially or processed by conventional methods in the field.
  • the medicinal material can be whole herb or chopped or pulverized.
  • the active ingredient is gallbladder extract
  • the gallbladder extract refers to the extract extracted from gallbladder medicinal material, with swerbrotomarin as the main active ingredient.
  • the content of swertiamarin is ⁇ 10wt%, preferably ⁇ 20wt%, ⁇ 30wt% or ⁇ 40wt%, such as 20-50wt% or 35- 45wt%, based on the total weight of the extract of Gallbladder.
  • the preparation method of the extract of Gallbladder Folium there is no special requirement for the preparation method of the extract of Gallbladder Folium. It can be prepared by common extraction methods in this field, including but not limited to: solvent extraction, extraction, supercritical extraction and/or chromatography. .
  • 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.
  • alcohols preferably C1-C4 alcohols, such as methanol, ethanol, propanol
  • a preparation method of Gallbladder extract comprises the following steps:
  • step (i) include one or more technical features selected from the following group:
  • the mixed solution is 20-80v/v% ethanol-water mixed solution, such as 40v/v%, 50v/v%, 60v/v%, 70v/v% or 75v/v%;
  • the dosage ratio (g/mL) of the gallbladder medicinal material to the mixed solution of ethanol and water is 1:2-30; preferably, 1:5-25, such as 1:10, 1:15 or 1:20;
  • the number of extractions is 1-3 times, such as 1, 2 or 3;
  • the extraction temperature is independently 50-100°C, such as 60°C, 70°C or 80°C, preferably, the reflux temperature; and/or
  • the extraction time is independently 0.5-3h, preferably 1-2h;
  • step (ii) include one or more technical features selected from the following group:
  • the resin is selected from the group consisting of AB-8, HPD-400, LS-305, LS-300B, or a combination thereof, preferably, LS-305;
  • the concentration is to concentrate the filtrate to 0.05-1g medicinal material/mL, preferably 0.1-0.5g medicinal material/mL, based on dosage;
  • the dosage of the resin is, based on the dosage of 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.
  • the resin before the adsorption, is treated with 90-100% ethanol water (for example, 24-96h).
  • step (iii) include one or more technical features selected from the following group:
  • the amount of water used for cleaning is 2-8BV, preferably, 3-6BV;
  • the flow rate of water used for washing is 6-12BV/h, preferably 8-10BV/h.
  • step (ii) include one or more technical features selected from the following group:
  • the ethanol-water mixed solution is 75-85v/v% ethanol-water mixed solution, such as 80v/v%;
  • the dosage of the ethanol-water mixed solution is 2-10BV, preferably, 3-5BV;
  • the flow rate of the ethanol-water mixed solution is 1-5BV/h, preferably 2-4BV/h.
  • the method comprises the steps of:
  • composition of the present invention includes the above-mentioned gallbladder extract; and a cosmetically or pharmaceutically acceptable carrier.
  • the present invention unexpectedly finds that the extract of Gallbladder of Foliage has antibacterial activity and can be used to prevent and/or treat diseases and symptoms caused by bacterial infection.
  • the antibacterial refers to inhibiting the growth, reproduction and/or killing of bacteria.
  • Bacteria that can be used in the present invention include Gram-positive bacteria and/or Gram-negative bacteria, especially bacteria related to skin problems, such as including but not limited to: Propionibacterium (Propionibacterium) (for example, P. (Propionibacterium freudennreichii, Propionibacterium acnes or Propionibacterium granulosa), Malassezia (Pityrosporum) (e.g., M. furfur, M.
  • the gallbladder extract of the present invention is not easy to produce resistance.
  • Bacteria that can be used in the present invention can be Malassezia resistant to ketoconazole, fluconazole and/or zinc pyrithione (ZPT); and/or resistant to erythromycin, cephalosporin antibiotics and/or gram Lindamycin-resistant Propionibacterium acnes.
  • the composition of the present invention is suitable for skin problems caused by bacteria, such as dermatitis, folliculitis, acne, or a combination thereof.
  • bacteria such as dermatitis, folliculitis, acne, or a combination thereof.
  • dermatitis such as dermatitis, folliculitis, acne, or a combination thereof.
  • dandruff caused by Malassezia, seborrheic dermatitis, atopic dermatitis, folliculitis, alopecia, acne, psoriasis, or combinations thereof
  • Propionibacterium acnes Acne such as seborrheic acne, acne, comedones, or a combination thereof.
  • the present invention also found that the extract of gallbladder has anti-inflammatory effect. For example, it can inhibit the secretion of inflammatory mediators NO and/or COX-2. Therefore, the present invention also provides the use of the bile gall extract as an inhibitor of NO and/or COX-2.
  • gallbladder extract of the present invention into pharmaceutical compositions such as ointments, creams, gels, pastes, patches and the like.
  • Medicines can be produced by generally known production techniques, and suitable pharmaceutical additives can be added to the medicines.
  • Examples of 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, etc., these pharmaceutical additives can be selected and added in appropriate amounts within the range not affecting the effects of the present invention.
  • gallbladder extract of the present invention into a cosmetic composition, solid dosage form, semi-solid dosage form, or liquid dosage form, such as solution, gel, cream, emulsion, spray, ointment, cream, paste, cake , powder, patch, etc.
  • ingredients commonly used in cosmetics such as film-forming agents, oil-soluble gelling agents, organically modified clay minerals, resins, and moisturizing agents may be added to the extent that the effects of the present invention are not hindered.
  • preservatives antibacterial agents, flavors, salts, antioxidants, pH regulators, chelating agents, cooling agents, anti-inflammatory agents, ingredients for skin beautification (whitening agents, cell activating agents, rough skin improving agents, blood circulation promoters , skin astringent, anti-fat leakage agent, etc.), vitamins, amino acids, nucleic acids, hormones, inclusion compounds, etc.
  • the oil-soluble gelling agent is selected from metal soaps such as aluminum stearate, magnesium stearate, and zinc myristate; amino acids such as N-lauroyl-L-glutamic acid, ⁇ , ⁇ -di-n-butylamine, etc.
  • cyclodextrin fatty acid esters such as cyclodextrin palmitate, cyclodextrin stearate, cyclodextrin 2-ethylhexanoic acid palmitate
  • sucrose such as sucrose palmitate and sucrose stearate Fatty acid esters
  • benzylidene derivatives of sorbitol such as monobenzylidene sorbitol and dibenzylidene sorbitol
  • gelling agents such as ammonium montmorillonite clay and other organically modified clay minerals, one type or two or more types may be used as needed.
  • Moisturizers are: Glycerin, 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, etc.
  • Antibacterial preservatives include: alkyl p-hydroxybenzoate, benzoic acid, sodium benzoate, sorbic acid, potassium sorbate, phenoxyethanol, etc.
  • Antibacterial agents include: benzoic acid, salicylic acid, carbolic acid, sorbic acid, p-hydroxyl Alkyl benzoate, p-chloro-m-cresol, hexachlorophene, benzalkonium chloride, chlorhexidine chloride, trichloro-N-carbanilide, triclosan, photosensitizer, phenoxyethanol, etc.
  • Antioxidants include: tocopherol, butylhydroxyanisole, dibutylhydroxytoluene, phytic acid, etc.
  • pH regulators include: lactic acid, citric acid, glycolic acid, succinic acid, tartaric acid, dl-malic acid, potassium carbonate, carbonic acid Sodium bicarbonate, ammonium bicarbonate, etc.
  • Chelating agents include alanine, sodium ethylenediaminetetraacetic acid, sodium polyphosphate, sodium metaphosphate, phosphoric acid, etc.
  • Cooling agents include: L-menthol, camphor, etc.
  • Anti-inflammatory agents include: : Allantoin, glycyrrhetinic acid, glycyrrhizic acid, tranexamic acid, azulene, etc.
  • Skin beautification ingredients include: placenta extract, arbutin, glutathione, saxifrage extract and other whitening agents; royal jelly, photosensitizer, cholesterol derivatives, calf blood extract and other cell active agents; rough skin Improving agents: valerenamide nonanoate, benzyl nicotinate, ⁇ -butoxyethyl nicotinate, capsaicin, zingerone, cantharidin tincture, ichthyosis, caffeine, tannic acid, ⁇ -borneol, tocopherol nicotinic acid , Inositol hexanicotinate, cyclomandelate, cinnarizine, tolazoline, acetylcholine, verapamil, stephenin, ⁇ -oryzanol and other blood circulation promoters; zinc oxide, tannic acid and other skin Astringents; sulfur, anti-fat leakage agents, etc., vitamins include: vitamin A oil, rosin
  • Type B vitamin C such as L-ascorbic acid, L-ascorbyl dipalmitate, L-ascorbic acid-2-sulfate sodium, dipotassium L-ascorbyl phosphodiester; vitamin D such as ergocalciferol and cholecalciferol; ⁇ - Tocopherol, beta-tocopherol, gamma-tocopherol, dl-alpha-tocopheryl acetate, dl-alpha-tocopherol nicotinic acid, dl-alpha-tocopherol succinate and other vitamin E; vitamin H; vitamin P; Niacin such as niacin, benzyl nicotinate, nicotinamide, etc.; pantothenic acid such as calcium pantothenate, D-panthenol, pantothenyl ethyl ether, acetyl pantothenyl ethyl ether, etc.; biotin,
  • Amino acids are: glycine, valine, leucine, isoleucine, serine, threonine, phenylalanine, arginine, lysine, aspartic acid, glutamic acid, cystine , cysteine, methionine, tryptophan, etc.
  • nucleic acids include deoxyribonucleic acid, etc.
  • hormones include estradiol, vinyl estradiol, etc.
  • the cosmetics of the present invention include skin care cosmetics, hair care cosmetics, color makeup cosmetics, and UV protection cosmetics.
  • hair care products such as shampoo, conditioner, hair care essence, and hair mask
  • basic cosmetics such as lotion, cream, lotion, sunscreen, mask materials, facial cleanser, and essence
  • makeup cosmetics such as foundation, white powder, and blush Wait.
  • the form of the product is not particularly limited, and may be liquid, emulsion, cream, solid, paste, gel, powder, multilayer, mousse, spray, etc.
  • the present invention also provides a skin (including scalp) care method, the method comprising the step of: administering the extract of the gallbladder of the present invention or the composition of the present invention to the individual in need.
  • the effective concentration range of the gallbladder extract is 1 ⁇ g/ml-500 mg/ml, such as 5 ⁇ g/ml, 10 ⁇ g/ml, 20 ⁇ g/ml, 50 ⁇ g/ml, 100 ⁇ g/ml or 200 ⁇ g/ml ml.
  • the term "effective dose” refers to any amount described below that can promote Regression of disease is manifested by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of impairment or disability resulting from the disease.
  • the "therapeutically effective dose” of the medicament of the present invention also includes the “prophylactically effective dose”, and the “prophylactically effective dose” is any amount of the medicament described below, when the composition of this amount is administered alone or in combination with another therapeutic agent or cosmetic When administered to a subject at risk of developing a disease or symptom, or suffering from recurrence of a disease or symptom, the combination inhibits the occurrence or recurrence of the disease or symptom.
  • the application of the antibacterial and anti-inflammatory aspects includes both preventive application and post-improvement application.
  • antibacterial including administering the extract or composition of the present invention before, during, and/or after infection to prevent and/or repair bacterial infection.
  • the antibacterial and anti-inflammatory effects of the extract of Gallbladder may be due to a single component (such as swertiamarin) or the synergistic effect of the components in the extract.
  • the present invention provides a green leaf bile extract, wherein the content of swerbromoside can be as high as more than 40%.
  • the gallbladder extract of the present invention has a significant inhibitory effect on Malassezia furfur and Propionibacterium acnes, and can be completely inhibited under the action of low concentration. It can be seen that the extract of the present invention has high antibacterial activity.
  • the gallbladder extract of the present invention has inhibitory effect on inflammatory mediators NO and COX-2;
  • the gallbladder extract of the present invention is non-irritating to the skin, has high safety, and can be applied to daily cosmetic raw materials.
  • the gallbladder extract of the present invention and the composition containing it can have both antibacterial and anti-inflammatory effects, and are very suitable for preparing antibacterial cosmetic or pharmaceutical compositions.
  • the preparation method of the gallbladder extract of the present invention is simple, the yield is high, the purity of the active ingredient is high, and the obtained extract has high activity.
  • the present invention also provides a new method for killing bacteria related to skin problems, which can provide a new solution for bacteria resistant to other antibacterial agents.
  • the preparation process of the gallbladder extract is determined through steps such as extracting solvent screening, purification resin screening, etc., using gallbladder japonicus as a raw material and swerbrotomarin as a target component, and its efficacy is verified through efficacy testing.
  • the chromatographic column is Hypersil BDS C18 (250mm ⁇ 4.6mm, 5 ⁇ m); the mobile phase is methanol-0.05% phosphoric acid solution (22:78); the detection wavelength is 237nm; the flow rate is 1mL/min; the injection volume is 10 ⁇ L , column temperature 30°C;
  • Swertamarin reference substance Weigh 5mg of swertiamarin reference substance (accurate to 0.01mg), put it in a 100mL volumetric flask, dilute to the mark with 50% methanol, shake well, and prepare a concentration of 50 ⁇ g/mL reference substance solution;
  • test solution dilute the crude extract obtained in 1.2 with 50% methanol to an appropriate multiple, and set aside;
  • the chromatographic column is Hypersil BDS C18 (250mm ⁇ 4.6mm, 5 ⁇ m); the mobile phase is methanol-0.05% phosphoric acid solution (22:78); the detection wavelength is 237nm; the flow rate is 1mL/min; the injection volume is 10 ⁇ l , column temperature 30°C;
  • Swertamarin reference substance Weigh 5mg of swertiamarin reference substance (accurate to 0.01mg), put it in a 100mL volumetric flask, dilute to the mark with 50% methanol, shake well, and prepare a concentration of 50 ⁇ g/mL reference substance solution;
  • Adsorption rate% (C 0 -C 1 )/C 0 *100%
  • Freeze-drying place the extract obtained in 3.7 in a freeze dryer to freeze-dry, and collect the freeze-dried powder; the yield of the freeze-dried powder is 11.56%;
  • the chromatographic column is Hypersil BDS C18 (250mm ⁇ 4.6mm, 5 ⁇ m); the mobile phase is methanol-0.05% phosphoric acid solution (22:78); the detection wavelength is 237nm; the flow rate is 1mL/min; the injection volume is 10 ⁇ l , column temperature 30°C;
  • Swertamarin reference substance Weigh 5mg of swertiamarin reference substance (accurate to 0.01mg), put it in a 100mL volumetric flask, dilute to the mark with 50% methanol, shake well, and prepare a concentration of 50 ⁇ g/mL reference substance solution;
  • Malassezia furfur (Pityrosporum orbitale) was used as the strain, 1% zinc pyrithione (ZPT) was used as the positive control, and the anti-malassezia activity of the extract of the green leaf bile was tested. Inhibition, the specific test method is as follows:
  • Test strain Malassezia furfur (ATCC44344, purchased from Guangdong Microbial Culture Collection Center);
  • Test procedure when preparing the culture medium, add the samples at concentrations of 2%, 1.5%, 1.2%, 1%, 0.8%, and 0.6%, and sterilize at 121°C for 15 minutes.
  • culture Pour the substrate into a sterile petri dish to make a plate for use; scrape the colony of Malassezia furfur grown on the culture medium for 5 days and mix it in sterile saline; take 0.1mL of bacterial suspension (10 5 ⁇ 10 6 CFM/mL) on the prepared agar plate, use the olive oil medium plate that has been inoculated without adding samples as a blank control, and take another olive oil medium plate and apply 1% ZPT100 ⁇ L as a positive control; Place in an incubator at 36°C for 72 hours, and compare and observe the growth of the colonies;
  • Propionibacterium acnes was used as the bacterial species to test the inhibitory effect of the extract of Folium Foliae on Propionibacterium acnes.
  • the specific test method was as follows:
  • Test bacteria Propionibacterium acnes (ATCC6919, purchased from Guangdong Microbial Culture Collection Center);
  • Test procedure when preparing the culture medium, add the samples at concentrations of 5%, 2.5%, and 1.25%, sterilize at 121°C for 15 minutes, add 2mL of sheep blood when the culture medium is cooled to about 45°C, mix well and pack to Make a flat plate for use; scrape the P. acnes colony grown on the medium for 3 days, mix it in sterile saline, take 0.1mL of the bacterial suspension (10 6 ⁇ 10 8 CFM/mL) and spread it on On the prepared blood plate, use the blood medium that has been inoculated without adding samples as a blank control, place it in an incubator at 36°C for 72 hours in an anaerobic environment, and compare and observe the growth of the colony;
  • LPS is the main component of the cell wall of Gram-negative bacteria and the main inflammatory substance, which can induce inflammation in the body, and release inflammatory factors such as TNF- ⁇ and IL-6 and inflammatory mediators such as NO and COX-2.
  • the invention uses LPS to stimulate macrophages to produce an inflammation model, and detects the influence of samples on the inflammatory mediators NO and COX-2 produced by the cells.
  • the specific test method is as follows:
  • mice mouse macrophage Raw264.7 (Cell Bank of Chinese Academy of Sciences), high-glucose DMEM medium (Gibco), PBS (Boster), MTT (Sigma), DMSO, fetal bovine serum (Gibco), lipopolysaccharide LPS (Sigma), Dexamethasone (China Search), NO Detection Kit (Beiyuntian), RNAiso Plus (TaKaRa), Isopropanol (Sinopharm Group), Chloroform (Sinopharm Group), Absolute Ethanol (Sinopharm Group), DEPC Water (Beyotime), reverse transcription kit (TaKaRa), fluorescent dye (TaKaRa).
  • Cell inoculation Inoculate Raw264.7 cells into 96-well plates according to the specified cell inoculation density, and incubate overnight in an incubator (37° C., 5% CO 2 ).
  • Administration is performed when the cell plating rate in the 96-well plate reaches 40%-60%. Add 200 ⁇ L of cell culture solution to each well of the blank control group; add 200 ⁇ L of culture solution containing 10% DMSO to each well of the positive control group; add 200 ⁇ L of culture solution containing the corresponding concentration of the test substance to each well of the sample group; Add 200 ⁇ L of cell culture medium. After administration, the 96-well plate was placed in an incubator (37° C., 5% CO 2 ) for cultivation.
  • Cell inoculation Inoculate the cells into a 6-well plate according to the specified cell inoculation density, and incubate overnight in an incubator (37° C., 5% CO 2 ).
  • LPS stimulation After culturing for 2 hours, add 200 ⁇ L of LPS working solution prepared from the corresponding working solution of the test substance to the well-treated well plate according to the group design, shake the well plate left and right, so that the drug in the well plate is mixed evenly, and the LPS The final concentration was 1 ⁇ g/mL. Continue culturing for 24 hours in an incubator with 5% CO2 at 37°C.
  • Detection of NO content detect according to the operating instructions of the NO content detection kit.
  • the cell supernatant was collected and the NO content was detected.
  • the results are shown in Table 8 and Figure 7.
  • the NO content in the NC group increased significantly, indicating that the LPS stimulation condition was effective in this experiment.
  • the NO content of the positive control group decreased significantly, indicating that the positive control in this experiment was effective.
  • the NO content of the samples of Gallbladder extracts decreased significantly at two concentrations of 500 ⁇ g/mL and 200 ⁇ g/mL.
  • the treated macrophages were treated with RNAiso Plus and the samples were collected. According to the kit instructions, RNA extraction, reverse transcription and fluorescent quantitative PCR operations were carried out. The test results are shown in Table 9 and Figure 8.
  • the expression of COX-2 gene in the NC control group was significantly increased, indicating that the LPS stimulation was effective in this experiment.
  • the expression of COX-2 gene in the positive control group decreased significantly, indicating that the positive control in this experiment was effective.
  • the expression of COX-2 gene was significantly decreased at the two dosage concentrations of 500 ⁇ g/mL and 200 ⁇ g/mL of the samples of Gallbladder extract.
  • the extract of Gallbladder Folium has a significant inhibitory effect on the inflammatory mediator NO induced by LPS; at the same time, it has a significant inhibitory effect on the expression of the inflammation-related gene COX-2.
  • Test method refer to OECD TG439
  • Pre-cultivation transfer the received skin model to a 12-well plate containing 2 mL of test medium, and culture in a CO2 incubator for 24 hours;
  • Post-incubation transfer the processed sample into a new maintenance medium, and put it back into the CO2 incubator for incubation;
  • Tissue activity test After incubation for 42 hours, the skin tissue was transferred to 0.3 mg/mL MTT solution and incubated at 37°C for 3 hours. Observe and record the color of the skin tissue after culture. Cut off the skin tissue and transfer it to a 1.5mL centrifuge tube, add 500 ⁇ l of acidified isopropanol and mix well, place it in the dark at 4°C for 72h, then use acidified isopropanol as a blank, and measure the absorbance value at a wavelength of 560nm;
  • test sample tissue activity > 50% it is a non-irritating substance
  • test sample tissue activity ⁇ 50% is an irritating substance to judge the results.
  • tissue corresponding to 1% Folium Folium extract The activity is 84.5%, which is a non-irritating substance.
  • a component is uniformly dispersed at 75-80°C;
  • Test strain Malassezia furfur (ATCC44344, purchased from Guangdong Microbial Culture Collection Center);
  • the culture medium After the culture medium is prepared, it is sterilized at 121°C for 15 minutes, and divided into plates, each plate is about 15-20ml. After cooling and solidifying, it is ready for use;
  • Bacterial suspension preparation Scrape the Malassezia colonies grown on olive oil medium for 72 hours and mix them in sterile normal saline;
  • Sample preparation and inoculation take 1g of sample and add 5ml of normal saline to dilute, after mixing, take 4.5ml of the mixed solution and put it in an empty test tube, add 0.5ml of bacterial suspension, and take 1ml of normal saline for ten-fold dilution after 10 minutes of action. Then take 0.1ml of diluent to coat the plate. Replace the test sample with physiological saline, and follow the above steps at the same time, as a control sample;
  • Cultivation culture in an incubator at 36°C for 72 hours, record the number of colonies, and calculate the antibacterial effect
  • Inhibition rate (%) (average number of colonies in control - average number of colonies in sample)/average number of colonies in control * 100
  • the present invention provides the application of Folium Fructus extract in inhibiting Malassezia, Propionibacterium acnes and anti-inflammatory effects. While propionibacterium grows, it can also effectively reduce skin itching, redness and other inflammatory symptoms caused by bacterial reproduction, and can effectively solve dandruff/seborrheic dermatitis, atopic dermatitis, folliculitis/acne, psoriasis, etc.
  • a variety of skin problems, and the extract of Gallbladder Folium has low irritation and high safety, which is very suitable for application in the field of daily cosmetics.
  • the present invention also provides a specific extract of Gallbladder and its preparation method.
  • the extract of Gallbladder prepared by the method of the present invention can obtain up to 45wt% swertiamarin.

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Abstract

本发明提供了一种青叶胆提取物及其制备方法和应用。具体地，本发明提供了青叶胆提取物在制备抵抗皮肤问题相关菌群的组合物中的应用。本发明还提供了一种青叶胆提取物及其制备方法，所述青叶胆提取物安全性高、抗菌效果好。

Description

青叶胆提取物及其制备方法和应用 技术领域
本发明属于日用化妆品技术领域，具体涉及一种青叶胆提取物及其制备方法和应用。
背景技术
青叶胆(SWERTIA MILEENSIS EXTRACT)是龙胆科植物青叶胆Swertia mileensis T.N.Hoet W.L.Shi的干燥全草。青叶胆为云南特有植物，主要分布于云南省红河州境内的弥勒、开远等县，具有清肝利胆、清热利湿的功效，是云南民间用于治疗急、慢性肝炎的著名药材。青叶胆化学成分丰富，主要含有环烯醚萜苷类、黄酮类、口山酮类和三萜类化合物。
獐芽菜苦苷是一类裂环环烯醚萜苷类化合物，是龙胆科植物中含量较多的一类成分，具有清肝利胆、解痉镇痛、抗炎、降血脂等多重药理作用。
马拉色菌是皮肤表面的常驻菌，主要分布于人类及温血动物皮肤油脂分泌旺盛的部位，与机体处于共生状态；但是大量研究证实马拉色菌如果发生过度繁殖，则会引起头皮屑/脂溢性皮炎、特应性皮炎、银屑病等多种炎症性皮肤问题，同时也有研究表明其还可与痤疮丙酸杆菌共同作用引起痤疮的发生。酮康唑、氟康唑、吡硫鎓锌(ZPT)是目前市场上公认有效的马拉色菌抑制剂，可有效抑制头屑产生、减轻皮肤炎症，但是长期使用可使皮肤真菌对唑类药物产生明显的耐药性，使得唑类药物对真菌诱发的脂溢性皮炎、头皮屑等的治疗效果大大降低，无法彻底根除炎症性皮肤问题；吡硫鎓锌(ZPT)也因为皮肤刺激性大和潜在生殖毒性的问题而面临禁用，因此，开发一种兼具功效性和安全性的马拉色菌抑制剂使目前亟需解决的问题。
痤疮丙酸杆菌是丙酸杆菌属的一个种，属于厌氧菌，主要寄居于人和动物的皮肤、皮脂腺、肠道以及乳制品中，在有氧条件下不会引起疾病，但是在皮脂分泌旺盛的地方，当毛囊皮脂腺导管被阻塞，局部形成厌氧环境时则有利于该菌的滋生，导致痤疮的发生，该菌通过参与炎症、促进毛囊皮脂腺角化以及促进皮脂分泌来进一步加重痤疮。目前痤疮治疗手段主要是药物治疗，包括外用药物维A酸类、过氧苯甲酰、壬二酸、抗生素等，使用期间易出现皮肤刺激反应，如局部红斑、脱屑，出现紧绷和烧灼感；其次是口服抗生素、异维A酸、激素类等药物及中药复方，但长期服用易出现耐药和肝肾损伤等问题，还可通过一些物理治疗的方法来辅助治疗，如照射红蓝光、激光治疗等，但均无法彻底根治，严重影响个人形象及生活质量。
因此，本领域急需提供一种安全、有效的皮肤问题相关菌群的抗菌剂。
发明内容
本发明的目的是提供一种青叶胆提取物在制备抵抗皮肤问题相关菌群的组合物中的 应用。
本发明的另一目的在于提供一种安全性高、抗菌效果好的青叶胆提取物及其制备方法。
本发明第一方面，提供了一种青叶胆提取物的用途，在制备用于抗菌的化妆品组合物或药物组合物中的用途。
在另一优选例中，所述青叶胆提取物中，獐芽菜苦苷的含量≥10wt％，较佳地≥20wt％、≥30wt％或≥40wt％，以所述青叶胆提取物的总重量计。
在另一优选例中，化妆品组合物或药物组合物中，所述青叶胆提取物的含量≥0.1wt％，较佳地≥0.2wt％或≥0.5wt％，如0.6wt％、1wt％、2wt％、5wt％、10wt％或20wt％，以所述化妆品组合物或药物组合物总重量计。
在另一优选例中，所述菌选自下组：丙酸杆菌属(Propionibacterium)(例如，费氏丙酸杆菌(Propionibacterium freudennreichii)、痤疮丙酸杆菌(Propionibacterium acnes)或颗粒丙酸杆菌(Propionibacterium granulosum))、马拉色菌属(Pityrosporum)(例如，糠秕马拉色菌(M.furfur)、合轴马拉色菌(M.sympodialis)、球形马拉色菌(M.globosa)、限制马拉色菌(M.restricta)、厚皮马拉色菌(M.pachydermatis)、史洛邦马拉色菌(M.sloofiae)、蛎壳马拉色菌(M.obtusa))，或其组合。
在另一优选例中，所述菌选自下组：糠秕马拉色菌、痤疮丙酸杆菌，或其组合。
在另一优选例中，所述化妆品组合物或药物组合物用于细菌引起的皮肤问题，如皮炎、毛囊炎、痤疮，或其组合。
在另一优选例中，所述化妆品组合物或药物组合物用于一个或多个选自下组的用途：马拉色菌引起的头皮屑、脂溢性皮炎、特应性皮炎、毛囊炎、脱发、痤疮、银屑病，或其组合。
在另一优选例中，所述化妆品组合物或药物组合物用于一个或多个选自下组的用途：痤疮丙酸杆菌引起的痤疮，如溢脂性痤疮、闭口、粉刺，或其组合。
在另一优选例中，所述化妆品组合物或药物组合物还用于消炎。
在另一优选例中，所述化妆品组合物或药物组合物还用于抑制NO和/或COX-2的分泌。
在另一优选例中，所述化妆品组合物或药物组合物还包括化妆品或药学上可接受的载体。
在另一优选例中，所述青叶胆提取物来自青叶胆药材的花、茎、叶，或其组合，如全草。
在另一优选例中，所述青叶胆提取物通过如下方法制备，包括如下步骤：
(i)将青叶胆药材在0-85v/v％的乙醇水混合溶液中加热提取，分离得提取液I；
(ii)将提取液I浓缩后用非极性或弱极性树脂吸附，得吸附后的树脂I；
(iii)用水清洗所述树脂I，得清洗后的树脂II；
(iv)用70-85％的乙醇水混合溶剂洗脱所述树脂II，收集洗脱液；和
(v)任选地去除所述洗脱液的溶剂，从而得所述青叶胆提取物。
在另一优选例中，步骤(i)中，包括一个或多个选自下组的技术特征：
(a)所述混合溶液为20-80v/v％的乙醇水混合溶液，如40v/v％、50v/v％、60v/v％、70v/v％或75v/v％；
(b)所述青叶胆药材与所述乙醇水混合溶液的用量比(g/mL)为1:2-30；较佳地，1：5-25，如1:10、1:15或1:20；
(c)所述提取的次数为1-3次，如1、2或3；
(d)所述提取的温度独立地为50-100℃，如60℃、70℃或80℃，较佳地，回流温度；和/或
(e)所述提取的时间独立地为0.5-3h，较佳地，1-2h；。
在另一优选例中，步骤(ii)中，包括一个或多个选自下组的技术特征：
(a)所述树脂选自下组：AB-8、HPD-400、LS-305、LS-300B，或其组合，较佳地，LS-305；
(b)所述浓缩为将滤液浓缩至0.05-1g药材/mL，较佳地0.1-0.5g药材/mL，以投药量计；
(c)所述树脂的用量为，以药材投药量计，0.2-1g药材/g湿树脂，较佳地0.4-0.8g药材/g湿树脂，如0.5g药材/g湿树脂或0.6g药材/g湿树脂。
在另一优选例中，步骤(iii)中，包括一个或多个选自下组的技术特征：
(a)用于清洗的水的用量为2-8BV，较佳地，3-6BV；和/或
(b)用于清洗的水的流速为6-12BV/h，较佳地，8-10BV/h。
在另一优选例中，步骤(ii)中，包括一个或多个选自下组的技术特征：
(a)所述乙醇水混合溶液为75-85v/v％的乙醇水混合溶液，如80v/v％；和/或
(b)所述乙醇水混合溶液的用量为2-10BV，较佳地，3-5BV；
(c)所述乙醇水混合溶液的流速为1-5BV/h，较佳地，2-4BV/h。
在另一优选例中，所述化妆品组合物或药物组合物的剂型选自下组：液体制剂、混悬制剂、半固体制剂或固体制剂。
在另一优选例中，所述化妆品组合物或药物组合物的剂型为皮肤外用剂型。
在另一优选例中，所述化妆品组合物或药物组合物的剂型选自下组：溶液、凝胶、乳液、膏剂、霜剂、糊剂、饼、粉剂、贴剂等。
在另一优选例中，所述化妆品学上可接受的载体或赋形剂选自下组：保湿剂、抗氧化剂、抗紫外线剂、防腐剂、成膜剂、油溶性凝胶化剂、有机改性粘土矿物、树脂、抗菌剂、香精、盐类、pH调节剂、螯合剂、清凉剂、抗炎剂、皮肤美化用成分、维生素、氨基酸、核酸、激素、包合化合物，或其组合。
本发明第二方面，提供了一种青叶胆提取物，所述青叶胆提取物通过如下方法 制备，所述方法包括步骤：
(i)将青叶胆药材在0-85v/v％的乙醇水混合溶液中加热提取，分离得提取液I；
(ii)将提取液I浓缩后用非极性或弱极性树脂吸附，得吸附后的树脂I；
(iii)用水清洗所述树脂I，得清洗后的树脂II；
(iv)用70-85％的乙醇水混合溶剂洗脱所述树脂II，收集洗脱液；和
(v)任选地去除所述洗脱液的溶剂，从而得所述青叶胆提取物。
在另一优选例中，所述树脂为LS-305。
在另一优选例中，所述方法包括步骤：
(i)将青叶胆药材在60-65v/v％的乙醇水混合溶液回流提取，分离得提取液I；
(ii)将提取液I浓缩后用LS-305型树脂吸附，得吸附后的树脂I；
(iii)用水清洗所述树脂I，得清洗后的树脂II；
(iv)用80-85％的乙醇水混合溶剂洗脱所述树脂II，收集洗脱液；和
(v)去除所述洗脱液的溶剂，从而得所述青叶胆提取物。
在另一优选例中，所述青叶胆提取物中，獐芽菜苦苷的含量≥10wt％，较佳地≥20wt％、≥30wt％或≥40wt％，以所述青叶胆提取物的总重量计。
本发明第三方面，提供了一种化妆品组合物或药物组合物，所述组合物包括如本发明第二方面所述的青叶胆提取物；和化妆品或药学上可接受的载体。
在另一优选例中，化妆品组合物或药物组合物中，所述青叶胆提取物的含量≥0.1wt％，较佳地≥0.2wt％或≥0.5wt％，如0.6wt％、1wt％、2wt％、5wt％、10wt％或20wt％，以所述化妆品组合物或药物组合物总重量计。
在另一优选例中，所述化妆品学上可接受的载体或赋形剂选自下组：保湿剂、抗氧化剂、抗紫外线剂、防腐剂、成膜剂、油溶性凝胶化剂、有机改性粘土矿物、树脂、抗菌剂、香精、盐类、pH调节剂、螯合剂、清凉剂、抗炎剂、皮肤美化用成分、维生素、氨基酸、核酸、激素、包合化合物或其组合。
在另一优选例中，所述化妆品组合物选自下组：洗发水、护发喷雾、头皮护理液、护肤水、护肤乳液、膏霜、洗面奶。
在另一优选例中，所述化妆品组合物包括如下组分，以组合物总重量计：
名称 wt％
瓜尔胶羟丙基三甲基氯化铵 0.24～0.36
聚季铵盐-10 0.04～0.06
柠檬酸 0.16～0.24
月桂醇聚醚硫酸酯钠 8～12
月桂醇硫酸酯铵 5.6～8.4
季戊四醇二硬脂酸酯 0.4～0.6
乙二醇硬脂酸酯 1.2～1.8
椰油酰胺丙基甜菜碱 1.6～2.4
椰油酰胺MEA 0.8～1.2
月桂基甘醇羧酸钠 1.2～1.8
吡啶酮乙醇胺盐 0.32～0.48
硅油 1.6～2.4
油酸单甘油酯和烷基糖苷 1.6～2.4
青叶胆(SWERTIA MILEENSIS)提取物 0.48～0.72
水 1.6～2.4
乙内酰脲 0.16～0.24
水 余量
本发明第四方面，提供了一种制备如本发明第二方面所述的青叶胆提取物的制备方法，所述方法包括如下步骤：
(i)将青叶胆药材在0-85v/v％的乙醇水混合溶液中加热提取，分离得提取液I；
(ii)将提取液I浓缩后用非极性或弱极性树脂吸附，得吸附后的树脂I；
(iii)用水清洗所述树脂I，得清洗后的树脂II；
(iv)用70-85％的乙醇水混合溶剂洗脱所述树脂II，收集洗脱液；和
(v)任选地去除所述洗脱液的溶剂，从而得所述青叶胆提取物。
在另一优选例中，所述树脂为LS-305。
在另一优选例中，所述方法包括步骤：
(i)将青叶胆药材在60-65v/v％的乙醇水混合溶液回流提取，分离得提取液I；
(ii)将提取液I浓缩后用LS-305型树脂吸附，得吸附后的树脂I；
(iii)用水清洗所述树脂I，得清洗后的树脂II；
(iv)用80-85％的乙醇水混合溶剂洗脱所述树脂II，收集洗脱液；和
(v)去除所述洗脱液的溶剂，从而得所述青叶胆提取物。
本发明第五方面，提供了一种体外抑菌的方法，包括步骤：将本发明第二方面的青叶胆提取物第三方面的组合物与细菌接触，从而抑制所述细菌。
本发明第六方面，提供了一种护肤的方法，包括步骤：将有效量的本发明第二方面的青叶胆提取物或本发明第三方面的组合物给予有需要的对象，从而抵抗皮肤相关细菌，从而实现护肤。
在另一优选例中，所述给予的方式为外用。
在另一优选例中，所述对象为哺乳动物，如人、大鼠或小鼠。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具 体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1为獐芽菜苦苷对照品、青叶胆水提液、青叶胆60％乙醇提取液和青叶胆80％乙醇提取液的色谱图；
图2为獐芽菜苦苷对照品、D101，AB-8，HPD-400，HPD-100，LS-305，LS-300B树脂纯化后水洗液和乙醇解析液液相色谱图；
图3为獐芽菜苦苷对照品和青叶胆提取物液相色谱图；
图4为青叶胆提取物不同浓度作用下糠秕马拉色菌的生长情况，随浓度增加，青叶胆提取物对糠秕马拉色菌的抑制作用逐渐增强。1.5％浓度能完全抑制糠秕马拉色菌的生长，即青叶胆提取物对糠秕马拉色菌的最小抑菌浓度为1.5％。
图5为青叶胆提取物不同浓度作用下痤疮丙酸杆菌的生长情况，随浓度增加，青叶胆提取物对痤疮丙酸杆菌的抑制作用逐渐增强。5％浓度能完全抑制痤疮丙酸杆菌的生长，即青叶胆提取物对痤疮丙酸杆菌的最小抑菌浓度为5％。
图6为不同浓度青叶胆提取物对Raw264.7细胞的活力影响，在浓度≤500μg/mL范围内，细胞活力≥90％。
图7示出了青叶胆提取物500μg/mL和200μg/mL(**P＜0.01)可显著抑制巨噬细胞Raw264.7炎症模型分泌炎症介质NO。
图8示出了青叶胆提取物500μg/mL和200μg/mL(*P＜0.05)可明显抑制巨噬细胞Raw264.7炎症模型分泌炎症介质COX-2。
图9示出了1％青叶胆提取物作用下皮肤组织活性＞50％，判定其为非刺激性物质。
图10示出了相较于空白对照，添加了0.6％青叶胆提取物的洗发水的培养皿中，马拉色菌明显较少，证明其对马拉色菌具有明显抑制作用。
具体实施方式
本发明人经过广泛而深入的研究，通过大量筛选和测试，提供了一种青叶胆提取物及其制备方法和应用。本发明提供了一种具有高含量的獐芽菜苦苷的青叶胆提取物，及其制备方法。本发明还意外地发现獐芽菜苦苷在抗菌，尤其是对马拉色菌、痤疮丙酸杆菌在低浓度下即具有显著的抗菌效果，从而非常适合用于制备用于抗菌的化妆品组合物或药物组合物。在此基础上完成了本发明。
术语
除非另有定义，否则本文中所用的全部技术术语和科学术语均具有如本发明所属领域普通技术人员通常理解的相同含义。
如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列 举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
如本文所用，术语“室温”或“常温”是指温度为4-40℃，较佳地，25±5℃，如未特别说明时，操作温度可为常温。
青叶胆
青叶胆(SWERTIA MILEENSIS EXTRACT)是龙胆科植物青叶胆Swertia mileensis T.N.Hoet W.L.Shi的干燥全草。青叶胆为云南特有植物，主要分布于云南省红河州境内的弥勒、开远等县。本发明对青叶胆药材没有特别要求，可商业购买或通过本领域常规方法处理得到。药材可以为全草或经切碎或粉碎。
青叶胆提取物
本发明中，活性成分为青叶胆提取物，所述青叶胆提取物指提取自青叶胆药材，且以獐芽菜苦苷为主要活性成分的提取物。
在另一优选例中，所述青叶胆提取物中，獐芽菜苦苷的含量≥10wt％，较佳地≥20wt％、≥30wt％或≥40wt％，如20-50wt％或35-45wt％，以所述青叶胆提取物的总重量计。
制备方法
本发明中，对所述青叶胆提取物的制备方法没有特别要求，可采用本领域常用提取方法制备，包括但不限于：溶剂提取法、萃取法、超临界萃取法和/或色谱法进行。
优选地，所述提取物的提取剂选自下组：水、醇类(优选C1-C4醇，如甲醇、乙醇、丙醇)、或其混合物。
优选地，一种青叶胆提取物的制备方法包括如下步骤：
(i)将青叶胆药材在0-85v/v％的乙醇水混合溶液中加热提取，分离得提取液I；
(ii)将提取液I浓缩后用非极性或弱极性树脂吸附，得吸附后的树脂I；
(iii)用水清洗所述树脂I，得清洗后的树脂II；
(iv)用70-85％的乙醇水混合溶剂洗脱所述树脂II，收集洗脱液；和
(v)任选地去除所述洗脱液的溶剂，从而得所述青叶胆提取物。
在另一优选例中，步骤(i)中，包括一个或多个选自下组的技术特征：
(a)所述混合溶液为20-80v/v％的乙醇水混合溶液，如40v/v％、50v/v％、60v/v％、70v/v％或75v/v％；
(b)所述青叶胆药材与所述乙醇水混合溶液的用量比(g/mL)为1:2-30；较佳地，1：5-25，如1:10、1:15或1:20；
(c)所述提取的次数为1-3次，如1、2或3；
(d)所述提取的温度独立地为50-100℃，如60℃、70℃或80℃，较佳地，回流温度；和/或
(e)所述提取的时间独立地为0.5-3h，较佳地，1-2h；。
在另一优选例中，步骤(ii)中，包括一个或多个选自下组的技术特征：
(a)所述树脂选自下组：AB-8、HPD-400、LS-305、LS-300B，或其组合，较佳地，LS-305；
(b)所述浓缩为将滤液浓缩至0.05-1g药材/mL，较佳地0.1-0.5g药材/mL，以投药量计；
(c)所述树脂的用量为，以药材投药量计，0.2-1g药材/g湿树脂，较佳地0.4-0.8g药材/g湿树脂，如0.5g药材/g湿树脂或0.6g药材/g湿树脂。
在另一优选例中，在吸附之前，所述树脂用90-100％的乙醇水处理(如24-96h)。
在另一优选例中，步骤(iii)中，包括一个或多个选自下组的技术特征：
(a)用于清洗的水的用量为2-8BV，较佳地，3-6BV；和/或
(b)用于清洗的水的流速为6-12BV/h，较佳地，8-10BV/h。
在另一优选例中，步骤(ii)中，包括一个或多个选自下组的技术特征：
(a)所述乙醇水混合溶液为75-85v/v％的乙醇水混合溶液，如80v/v％；和/或
(b)所述乙醇水混合溶液的用量为2-10BV，较佳地，3-5BV；
(c)所述乙醇水混合溶液的流速为1-5BV/h，较佳地，2-4BV/h。
特别优选地，所述方法包括步骤：
(i)将青叶胆药材在60-65v/v％的乙醇水混合溶液回流提取，分离得提取液I；
(ii)将提取液I浓缩后用LS-305型树脂吸附，得吸附后的树脂I；
(iii)用水清洗所述树脂I，得清洗后的树脂II；
(iv)用80-85％的乙醇水混合溶剂洗脱所述树脂II，收集洗脱液；和
(v)去除所述洗脱液的溶剂，从而得所述青叶胆提取物。
组合物及用途
本发明的组合物包括如上所述的青叶胆提取物；和化妆品或药学上可接受的载体。
本发明意外地发现，青叶胆提取物具有抗菌活性，可用于预防和/或治疗细菌感染引发的疾病和症状。本发明中，所述抗菌指抑制细菌生长、繁殖和/或杀灭细菌。可用于本发明的细菌包括革兰氏阳性菌和/或革兰氏阴性菌，尤其是皮肤问题相关细菌，如包括但并不限于：丙酸杆菌属(Propionibacterium)(例如，费氏丙酸杆菌(Propionibacterium freudennreichii)、痤疮丙酸杆菌或颗粒丙酸杆菌)、马拉色菌属(Pityrosporum)(例如，糠秕马拉色菌(M.furfur)、合轴马拉色菌(M.sympodialis)、球形马拉色菌(M.globosa)、限制马拉色菌(M.restricta)、厚皮马拉色菌(M.pachydermatis)、 史洛邦马拉色菌(M.sloofiae)、蛎壳马拉色菌(M.obtusa))
此外，本发明的青叶胆提取物不易产生抗性。可用于本发明的细菌可以为对酮康唑、氟康唑和/或吡硫鎓锌(ZPT)产生抗性的马拉色菌；和/或对红霉素、头孢类抗生素和/或克林霉素产生抗药性的痤疮丙酸杆菌。
在另一优选例中，本发明的组合物适合用于细菌引起的皮肤问题，如皮炎、毛囊炎、痤疮，或其组合。例如，包括但并不限于马拉色菌引起的头皮屑、脂溢性皮炎、特应性皮炎、毛囊炎、脱发、痤疮、银屑病，或其组合；和/或痤疮丙酸杆菌引起的痤疮，如溢脂性痤疮、闭口、粉刺，或其组合。
此外，本发明还发现，青叶胆提取物具有抗炎效果。例如，可以抑制炎症介质NO和/或COX-2的分泌，因此，本发明还提供了青叶胆提取物作为NO和/或COX-2抑制剂的用途。
有可能将本发明的青叶胆提取物制备成药物组合物，如膏剂、霜剂、凝胶剂、糊剂、贴剂等。药物能够由通常已知的制备技术来制备，并且合适的药物添加剂能够被添加到该药物中。
药物添加剂的例子包括赋形剂、粘结剂、分解剂、润滑剂、流动助剂、悬浮剂、乳化剂、稳定剂、保温(润湿)剂、防腐剂、溶剂、增溶剂、防腐剂、调味剂、增甜剂、染料、香料、推进剂等，这些药物添加剂可以进行选择并以在不影响本发明的效果的范围内的合适量添加。
有可能将本发明的青叶胆提取物制备成化妆品组合物，固体剂型、半固体剂型、或液体剂型，如溶液、凝胶、膏霜、乳液、喷雾、膏剂、霜剂、糊剂、饼、粉剂、贴剂等。
在不防碍本发明的效果的范围内，本发明的化妆品中可以添加通常的化妆品中使用的其它成分，例如成膜剂、油溶性凝胶化剂、有机改性粘土矿物、树脂、保湿剂、防腐剂、抗菌剂、香精、盐类、抗氧化剂、pH调节剂、螯合剂、清凉剂、抗炎剂、皮肤美化用成分(美白剂、细胞活性剂、皮肤粗糙改善剂、血液循环促进剂、皮肤收敛剂、抗脂漏剂等)、维生素类、氨基酸类、核酸、激素、包合化合物等。
油溶性凝胶化剂是选自硬脂酸铝、硬脂酸镁、肉豆蔻酸锌等金属皂；N-月桂酰基-L-谷氨酸、α，γ-二-正丁基胺等氨基酸衍生物；环糊精棕榈酸酯、环糊精硬脂酸酯、环糊精2-乙基己酸棕榈酸酯等环糊精脂肪酸酯；蔗糖棕榈酸酯、蔗糖硬脂酸酯等蔗糖脂肪酸酯；一亚苄基山梨醇、二亚苄基山梨醇等山梨醇的亚苄基衍生物；二甲基苄基十二烷基铵蒙脱石粘土、二甲基二十八烷基铵蒙脱石粘土等有机改性粘土矿物等的凝胶化剂，可以根据需要使用一种，或者使用二种或以上。
保湿剂有：甘油、山梨醇、丙二醇、双丙甘醇、1，3-丁二醇、葡萄糖、木糖醇、麦芽糖醇、聚乙二醇、透明质酸、硫酸软骨素、吡咯烷酮羧酸盐、聚氧乙烯甲基葡糖苷、聚氧丙烯甲基葡糖苷等。
抗菌防腐剂有：对羟基苯甲酸烷基酯、苯甲酸、苯甲酸钠、山梨酸、山梨酸钾、苯氧基乙醇等，抗菌剂有：苯甲酸、水杨酸、石炭酸、山梨酸、对羟基苯甲酸烷基酯、对氯间 甲酚、六氯酚、苯扎氯铵、氯化洗必泰、三氯-N-碳酰苯胺、三氯生、感光素、苯氧基乙醇等。
抗氧化剂有：生育酚、丁基羟基茴香醚、二丁基羟基甲苯、植酸等，pH调节剂有：乳酸、柠檬酸、乙醇酸、琥珀酸、酒石酸、dl-苹果酸、碳酸钾、碳酸氢钠、碳酸氢铵等，螯合剂有丙氨酸、乙二胺四乙酸钠盐、多磷酸钠、偏磷酸钠、磷酸等，清凉剂有：L-薄荷醇、樟脑等，抗炎剂有：尿囊素、甘草亭酸、甘草酸、凝血酸、甘葡环烃(Azulene)等。
皮肤美化用成分有：胎盘提取液、熊果苷、谷胱甘肽、虎耳草提取物等美白剂；蜂王浆、感光素、胆甾醇衍生物、小牛血液提取液等细胞活性剂；皮肤粗糙改善剂；壬酸缬草酰胺、烟酸苄酯、烟酸β-丁氧基乙酯、辣椒素、姜油酮、斑蝥酊、鱼石脂、咖啡因、鞣酸、α-冰片、烟酸生育酚、六烟酸肌醇脂、环扁桃酯、桂利嗪、妥拉唑啉、乙酰胆碱、维拉帕米、千金藤素、γ-谷维醇等血液循环促进剂；氧化锌、鞣酸等皮肤收敛剂；硫、等抗脂漏剂等，维生素类有：维生素A油、松香油、乙酸松香油、棕榈酸松香油等维生素A类；核黄素、丁酸核黄素、黄素腺嘌呤核苷酸等维生素B2类；吡多辛盐酸盐、吡多辛二辛酸酯、吡多辛三棕榈酸酯等维生素B6类，维生素B12及其衍生物，维生素B15及其衍生物等维生素B类；L-抗坏血酸、L-抗坏血酸二棕榈酸酯、L-抗坏血酸-2-硫酸钠、L-抗坏血酸磷酸二酯二钾等维生素C类；麦角钙化醇、胆钙化醇等维生素D类；α-生育酚、β-生育酚、γ-生育酚、乙酸dl-α-生育酚、烟酸dl-α-生育酚、琥珀酸dl-α-生育酚等维生素E类；维生素H；维生素P；烟酸、烟酸苄酯、烟酰胺等烟酸类；泛酸钙、D-泛醇、泛酰基乙基醚、乙酰基泛酰基乙基醚等泛酸类；生物素等。
氨基酸类有：甘氨酸、缬氨酸、亮氨酸、异亮氨酸、丝氨酸、苏氨酸、苯丙氨酸、精氨酸、赖氨酸、天冬氨酸、谷氨酸、胱氨酸、半胱氨酸、甲硫氨酸、色氨酸等，核酸有脱氧核糖核酸等，激素有雌二醇、乙烯基雌二醇等。
本发明的化妆品的优选例子包括：皮肤护理化妆品、毛发护理化妆品、彩妆化妆品、防紫外线化妆品。例如有洗发水、护发素、护发精华、发膜等护发产品；乳液、霜、露、防晒霜、面膜材料、洗面奶、精华液等基础化妆品；粉底、白粉、腮红等彩妆化妆品等。
对于产品的形态没有特别限定，可以是液状、乳液状、霜状、固体状、糊状、凝胶状、粉末状、多层状、摩丝状(mousse)、喷雾状等。
本发明还提供了一种皮肤(包括头皮)护理方法，所述方法包括步骤：对需要的个体施用本发明的所述的青叶胆提取物或本发明所述的组合物。
在另一优选例中，所述青叶胆提取物的有效浓度范围为1μg/ml–500mg/ml，如5μg/ml、10μg/ml、20μg/ml、50μg/ml、100μg/ml或200μg/ml。
如本文所用，术语“有效剂量”是指任何如下所述的量，当单独使用或与另一种治疗剂或化妆品组合使用该量的本发明的青叶胆提取物或组合物时，可促进疾病消退，疾病消退表现为疾病症状的严重度降低、无疾病症状期的频率和持续时间增加、或者防止由患病导致的障碍或失能。本发明药物的“治疗有效剂量”也包括“预防有效剂量”，“预防有效剂量”是药物的任何如下所述的量，当将该量的组合物单独施 用或者与另一种治疗剂或化妆品组合施用于具有发生疾病或症状的风险或者遭受疾病或症状复发的受试者时，可抑制疾病或症状的发生或复发。
在本发明中，对所述抗菌、消炎等方面的应用，既包括预防性的应用，也包括事后改善性的应用。例如，对于抗菌，包括在感染之前、之中、和/或之后施用本发明的青叶胆提取物或组合物，以预防和/或修复细菌感染。
不希望限制本发明，青叶胆提取物的抗菌、消炎等功效可能是由于其中的某一单一组分(如獐牙菜苦苷)或提取物中的各组分协同作用的结果。
本发明的主要优点包括：
1.本发明提供了一种青叶胆提取物，其中獐芽菜苦苷含量可高达40％以上。
2.本发明的青叶胆提取物对糠秕马拉色菌、痤疮丙酸杆菌具有显著抑制作用，低浓度作用下可实现完全抑制，可见本发明的提取物具有高抑菌活性。
4.本发明的青叶胆提取物对炎症介质NO和COX-2具有抑制作用；
5.本发明的青叶胆提取物对皮肤无刺激性，安全性高，可应用于日用化妆品原料中。
6.本发明的青叶胆提取物及包含其的组合物可同时具有抗菌和消炎效果，非常适合用于制备用于抗菌的化妆品组合物或药物组合物。
7.本发明的青叶胆提取物制备方法简单，收率高，活性成分纯度高，且所得提取物具有高活性。
8.本发明还提供了一种杀灭皮肤问题相关菌群的新方法，可为对其他的抗菌剂产生抗性的菌群提供新的解决方案。
下面结合具体实施，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
本发明以青叶胆为原料，以獐芽菜苦苷为目标成分，通过提取溶剂筛选、纯化树脂筛选等步骤确定青叶胆提取物的制备工艺，并通过功效测试验证其功效。
实施例1
1.提取溶剂筛选
1.1提取：称取三份干燥的青叶胆药材全草(拉丁名Swertia mileensis T.N.Ho et W.L.Shi，产地云南)，每份50g，分别加入1000mL的纯化水、60％乙醇和80％乙醇，加热回流提取2h，300目筛网过滤分出滤液，再次往滤渣中分别加入1000mL纯化水、60％乙醇和80％乙醇，加热回流提取1.5h，300目筛网过滤分出滤液，合并两次所得 滤液；
1.2浓缩：滤液分别浓缩至100g，得纯化水、60％乙醇和80％乙醇提取粗提浸膏；
1.3高效液相色谱法测定3种溶剂对应浸膏中獐芽菜苦苷的含量。
1)色谱条件：色谱柱为Hypersil BDS C18(250mm×4.6mm，5μm)；流动相为甲醇－0.05％磷酸溶液(22：78)；检测波长为237nm；流速为1mL/min；进样量10μL，柱温30℃；
2)獐芽菜苦苷对照品：称取獐牙菜苦苷对照品5mg(精确至0.01mg)，置于100mL容量瓶中，用50％甲醇定容至刻度，摇匀，配制成浓度为50μg/mL的对照品溶液；
3)供试品溶液制备：用50％甲醇将1.2所得粗浸膏分别稀释适当倍数，待用；
4)测定：分别取适量对照品和供试样品溶液用0.45μm滤膜过滤后置于液相色谱仪样品瓶中，测定，结果如图1所示。
5)结果处理分析：根据A1/A2＝C1/C2，计算不同溶剂提取所得粗浸膏中獐牙菜苦苷含量，如下表1所示。
表1：提取溶剂筛选结果整理
Figure PCTCN2022097430-appb-000001
结果显示，3种不同溶剂提取所得浸膏中獐芽菜苦苷含量由高到低的顺序为60％乙醇＞80％乙醇＞纯化水。
实施例2
2.纯化树脂筛选
2.1 取D101，AB-8，HPD-400，HPD-100，LS-305，LS-300B树脂各100g，用95％乙醇浸泡48h，每种树脂型号各称取15g(以湿树脂计)，纯化水洗至无乙醇味，置于100mL烧杯中待用
2.2 称取100g干燥的青叶胆药材，以60％乙醇为溶剂进行加热回流提取，共提取2次，每次溶剂用量为2000mL，提取时间分别为2h和1h。收集并合并2次提取所得药液，浓缩至200g，待用；
2.3 分别称取6份质量为15g的2.2所得浸膏，加入装有树脂的烧杯中，搅拌均匀后置于摇床振摇15min，使树脂与浸膏充分接触；300目筛网过滤树脂，收集滤液；
2.4 量取60mL纯化水，每次15mL，洗树脂4次，收集洗过树脂的水洗液；然后量取6份体积为80mL的80％乙醇分别倒入装有树脂的烧杯中，搅拌均匀后置于摇床振摇15min，使树脂与浸膏充分接触；300目筛网过滤树脂，收集80％乙醇解析液；
2.5 用纯化水将2.3所得滤液和2.4所得水洗液及80％乙醇解析液分别补足至100g；
2.6 高效液相色谱法测定6种树脂对应的滤液、水洗液及80％乙醇解析液中獐芽菜苦苷的含量；
1)色谱条件：色谱柱为Hypersil BDS C18(250mm×4.6mm，5μm)；流动相为甲醇－0.05％磷酸溶液(22：78)；检测波长为237nm；流速为1mL/min；进样量10μl，柱温30℃；
2)獐芽菜苦苷对照品：称取獐牙菜苦苷对照品5mg(精确至0.01mg)，置于100mL容量瓶中，用50％甲醇定容至刻度，摇匀，配制成浓度为50μg/mL的对照品溶液；
3)供试品溶液制备：用50％甲醇将2.5所得粗浸膏分别稀释适当倍数后，待用；
4)测定：分别取适量对照品和供试样品溶液用0.45μm滤膜过滤后置于液相色谱仪样品瓶中，测定，结果如图2所示。
5)结果处理分析：根据A1/A2＝C1/C2，计算不同溶剂提取所得粗浸膏中獐牙菜苦苷含量，如下表2所示：
表2树脂筛选含量测定结果整理
Figure PCTCN2022097430-appb-000002
根据下述公式计算每种树脂对獐芽菜苦苷的吸附率、解析率及得率，如下表3所示，。
吸附率％＝(C 0-C 1)/C 0*100％；
解析率％＝C 2/(C 0-C 1)*100％
得率％＝吸附率％*解析率％
C 0——吸附前獐芽菜苦苷含量(％)
C 1——水洗液中獐芽菜苦苷含量(％)
C 2——乙醇解析液中獐芽菜苦苷含量(％)
表3：不同型号树脂对分离纯化獐芽菜苦苷的影响
树脂型号 吸附率％ 解析率％ 得率％
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
结果显示，LS-305吸附率、解析率及得率均高于其他树脂型号，其獐芽菜苦苷得率为74.24％。
实施例3
青叶胆提取物制备
3.1提取：称取50g干燥的青叶胆药材，以60％乙醇为溶剂进行加热回流提取，共提取2次，每次溶剂用量为1000mL，提取时间分别为2h和1h；
3.2浓缩：收集并合并2次提取所得药液，浓缩至500g，待用；
3.3装柱：将LS-305树脂用95％乙醇浸泡48h，然后称取100g湿树脂装入树脂柱中，用纯化水洗至无乙醇味；
3.4上样：将6.3.2所得浸膏倒入树脂柱中并将流速控制在4BV/h，收集上样流出液；
3.5水洗：待上样结束后，往树脂柱中加入4BV纯化水，并将流速控制在8BV/h，收集水洗液；
3.6乙醇解析：待水洗结束后，往树脂柱中加入3BV浓度为80％的乙醇，将流速控制在2.5BV/h，收集乙醇解析液；
3.7浓缩：将3.6所得乙醇解析液除去酒精并浓缩至100g；
3.8冷冻干燥：将3.7所得浸膏置于冷冻干燥机中冻干，收集冻干粉；冻干粉收率为11.56％；
3.9高效液相色谱法测定青叶胆提取物中獐芽菜苦苷的含量；
1)色谱条件：色谱柱为Hypersil BDS C18(250mm×4.6mm，5μm)；流动相为甲醇－0.05％磷酸溶液(22：78)；检测波长为237nm；流速为1mL/min；进样量10μl，柱温30℃；
2)獐芽菜苦苷对照品：称取獐牙菜苦苷对照品5mg(精确至0.01mg)，置于100mL 容量瓶中，用50％甲醇定容至刻度，摇匀，配制成浓度为50μg/mL的对照品溶液；
3)供试品溶液制备：称取适量3.8所得青叶胆提取物，用50％甲醇稀释适当倍数后，待用；
4)测定：分别取适量对照品和供试样品溶液用0.45μm滤膜过滤后置于液相色谱仪样品瓶中，测定，结果如图3所示。
5)结果处理分析：根据A1/A2＝C1/C2，计算青叶胆提取物中獐牙菜苦苷含量，结果为45.60％，如表4所示。
表4：青叶但提取物含量测定结果
样品名称 出峰时间(min) 峰面积 浓度(ug/ml) 含量％
獐牙菜苦苷对照品 10.466 787.186 49.49 /
冻干粉-LS-305 10.465 702.111 44.14 45.60
综上，最终以60％乙醇为提取溶剂、LS-305为纯化树脂为工艺条件制备青叶胆提取物，所得成品收率为11.56％，獐芽菜苦苷含量为45.60％。
实施例4
青叶胆提取物抑菌功效测试
4.1马拉色菌抑制实验：以糠秕马拉色菌(Malassezia furfur(Pityrosporum orbiculare)为菌种，1％吡啶硫酮锌(ZPT)为阳性对照，测试青叶胆提取物对马拉色菌的抑制作用，具体测试方法如下：
1)实验试剂：橄榄油培养基
2)测试菌种：糠秕马拉色菌(ATCC44344，购自广东微生物菌种保藏中心)；
3)测试步骤：培养基配置时将样品按2％、1.5％、1.2％、1％、0.8％、0.6％浓度加入，121℃灭菌15min，待培养基冷却至45℃左右时，将培养基浇注入无菌培养皿中，制成平板备用；刮取在培养基上生长5天的糠秕马拉色菌菌落于无菌生理盐水中混匀；取0.1mL菌悬液(10 5～10 6CFΜ/mL)涂布于制备好的琼脂平板上，用已接菌未加样品的橄榄油培养基平板做空白对照，另取1块橄榄油培养基平板涂布1％ZPT100μL作阳性对照；置于培养箱中36℃培养72h，对比观察菌落生长情况；
4)实验结果：结果如图4所示，青叶胆提提取物对马拉色菌具有明显抑制作用，随浓度增加，抑菌作用逐渐增强，含1.5％样品的培养基能够完全抑制住糠秕马拉色菌的生长，证明该样品对糠秕马拉色菌的最小抑菌浓度为1.5％。
实施例5
痤疮丙酸杆菌抑制实验：
以痤疮丙酸杆菌(Propionibacterium acnes)为菌种，测试青叶胆提取物对痤疮丙酸杆菌的抑制作用，具体测试方法如下：
1)实验试剂：胰酪大豆胨琼脂培养基(TSA培养基)，绵羊血，无菌生理盐水；
2)测试菌种：痤疮丙酸杆菌(ATCC6919，购自广东微生物菌种保藏中心)；
3)测试步骤：培养基配置时将样品按5％、2.5％、1.25％的浓度加入，121℃灭菌15min，待培养基冷却至45℃左右时加2mL羊血，混匀后分装至平板，制成平板备用；刮取在培养基上生长3天的痤疮丙酸杆菌菌落于无菌生理盐水中混匀，取0.1mL菌悬液(10 6～10 8CFΜ/mL)涂布于制备好的血平板上，用已接菌未加样品的血培养基做空白对照，置于培养箱中36℃厌氧环境中培养72h，对比观察菌落生长情况；
4)实验结果：结果如图5所示，青叶胆提取物对痤疮丙酸杆菌具有一定抑制作用，随着浓度增加抑制作用逐渐增强。含5％样品的培养基能够完全抑制住痤疮丙酸杆菌的生长，证明该样品对痤疮丙酸杆菌的最小抑菌浓度为5％。
实施例6
青叶胆提取物对NO和COX-2的影响实验
LPS是革兰氏阴性菌细胞壁的主要组成成分，是主要的致炎物质，可诱导机体产生炎症，出现TNF-α、IL-6等炎症因子和NO、COX-2等炎症介质的释放。本发明以LPS刺激巨噬细胞产生炎症模型，检测样品对细胞产生的炎症介质NO和COX-2的影响。具体测试方法如下：
实验材料：小鼠巨噬细胞Raw264.7(中国科学院细胞库)，高糖DMEM培养基(Gibco)、PBS(博士德)、MTT(Sigma)、DMSO、胎牛血清(Gibco)、脂多糖LPS(Sigma)、地塞米松(中检索)、NO检测试剂盒(碧云天)、RNAiso Plus(TaKaRa)、异丙醇(国药集团)、氯仿(国药集团)、无水乙醇(国药集团)、DEPC水(Beyotime)、反转录试剂盒(TaKaRa)、荧光染料(TaKaRa)。
仪器：CO2培养箱(Thermo)、酶标仪(BioTek)、超净工作台(苏州安泰)、倒置显微镜(Olympus)、PCR仪(BIO-RAD)、荧光定量PCR仪(BIO-RAD)。
测试方法
6.1青叶胆提取物最大安全浓度测定
1)细胞接种：按规定的细胞接种密度接种Raw264.7细胞至96孔板中，培养箱(37℃、5％CO2)中孵育过夜。
2)实验分组：实验设置空白对照组、阳性对照组与样品组。样品组中，每个样品设置8个浓度梯度，每个浓度梯度下设置3个重复孔。
3)配液：按实验设计(表5)配制不同浓度的受试物工作液。
表5测试浓度设定表
Figure PCTCN2022097430-appb-000003
4)给药：待96孔板中细胞铺板率达到40％～60％时进行给药。空白对照组每孔加入200μL细胞培养液；阳性对照组每孔加入200μL含10％DMSO的培养液；样品组每孔加入200μL含有相应浓度受试物的培养液；调零孔无细胞接种，仅加入 200μL细胞培养液。给药完成后将96孔板放置在培养箱(37℃、5％CO2)中培养。
5)检测：细胞孵育培养24h后，弃掉上清，加入MTT工作液(0.5mg/mL，现配现用)，37℃避光孵育4h，孵育结束后，弃掉上清，每孔加150μL DMSO，在490nm处读取OD值。
6)细胞相对活力计算：根据公式计算，细胞相对活力(％)＝(样品-调零孔)/(空白对照孔-调零孔)*100％
6.2青叶胆提取物对NO和COX-2分泌的影响实验
1)细胞接种：按规定的细胞接种密度接种细胞至6孔板，培养箱(37℃、5％CO2)中孵育过夜。
2)配液：按照实验设计(表6)配置受试物工作液。
表6实验设计
Figure PCTCN2022097430-appb-000004
3)给药：根据表6实验具体设计，待6孔板中细胞铺板率达到40％～50％时，进行分组给药，每孔给药量为1.8mL，每组设3个复孔。37℃，5％CO2培养箱继续培养2h。
4)LPS刺激：培养2h后，根据组别设计向已给药的孔板中加入200μL由相应受试物工作液配制的LPS工作液，左右摇晃孔板，使得孔板内药物混匀，LPS终浓度为1μg/mL。在37℃，5％CO2的培养箱继续培养24h。
5)收集细胞上清液：培养24h后，收集细胞培养上清液于EP管中。
6)NO含量的检测：根据NO含量检测试剂盒的操作说明书进行检测.
7)细胞收样：2mL/孔PBS清洗两次，每孔加入1mL RNAiso Plus，吹打裂解细胞后，收样。
8)基因检测：依据试剂盒说明书，开展RNA提取、反转录及荧光定量PCR检测，采用2-△△CT方法进行结果计算。
测试结果统计分析
应用GraphPad Prism Program软件作图，各组间采用t-test统计分析，*p＜0.05表示明显显著，**p＜0.01表示差异显著。
6.3青叶胆提取物最大安全浓度测定
样品由高到低设定8个给药浓度，在巨噬细胞上开展细胞毒性测试，测试结果见表7，细胞活力变化趋势如图6所示。根据MTT结果，认为样品青叶胆提取物基于巨噬细胞，在500μg/mL的浓度范围内无明显细胞毒性，可作为最大安全浓度
表7青叶胆提取物最大安全浓度测定结果
Figure PCTCN2022097430-appb-000005
6.4 NO含量测定结果
基于实验方法6.2，收集细胞上清，进行NO含量检测，结果如表8和图7所示。与BC组相比，NC组NO含量显著上升，说明本次实验LPS刺激条件有效。与NC组相比，阳性对照组NO含量显著下降，说明本次实验阳性对照有效。与NC组相比，样品青叶胆提取物在500μg/mL和200μg/mL两个给药浓度下，NO含量均显著下降。
表6-8 NO含量测定结果
样品名称 平均浓度(μM) SD p-value
BC 0.99 0.10 /
NC 47.45 1.72 0.000##
PC 22.30 0.72 0.000**
青叶胆提取物-500μg/mL 5.92 0.17 0.000**
青叶胆提取物-200μg/mL 25.39 1.72 0.000**
备注：用t-test方法进行统计分析时，NC组与BC组相比，显著性以#表示，p-value＜0.05表示为#，p-value＜0.01表示为##；PC组、样品组与BC组相比，显著性以*表示，p-value＜0.05表示为*，p-value＜0.01表示为**。
6.5 COX-2基因表达测定结果
基于实验方法6.5.3.2，对作用后的巨噬细胞进行RNAiso Plus处理后收样，依据试剂盒说明书，开展RNA提取、反转录及荧光定量PCR操作。测试结果见表9和图8所示，与BC组相比，NC对照组COX-2基因表达量显著上升，说明本次实验LPS刺激有效。与NC组相比，阳性对照组COX-2基因表达量显著下降，说明本次实验阳性对照有效。与NC组相比，样品青叶胆提取物在500μg/mL和200μg/mL两个给药浓度下，COX-2基因表达量均显著下降。
表9 COX-2基因表达测定结果
Figure PCTCN2022097430-appb-000006
备注：用t-test方法进行统计分析时，NC组与BC组相比，显著性以#表示，p-value＜0.05表示为#，p-value＜0.01表示为##；PC组、样品组与BC组相比，显著性以*表示，p-value＜0.05表示为*，p-value＜0.01表示为**。
由上可知，青叶胆提取物对LPS诱导产生的炎症介质NO具有显著抑制作用；同时对炎症相关基因COX-2表达具有显著抑制作用。
实施例7
青叶胆提取物皮肤刺激性测试
7.1实验材料：3D皮肤模型试剂盒(上海斯安肤诺生物科技有限公司)，MTT(上海源叶)，DPBS(BI),异丙醇(天津风)
7.2实验设备：酶标仪(Thermo)，超净工作台(苏州安泰)，CO2培养箱(Memmert)
7.3测试方法：参照OECD TG439
1)预培养：将收到的皮肤模型转移至装有2mL测试用培养基的12孔板中，CO2培养箱培养24h；
2)加样处理：皮肤模型培养24后取出，按照表10实验设计进行分组，每组3个复孔，取对应样品10μL均匀涂布于皮肤组织表面；
表10皮肤刺激性测试实验设计
Figure PCTCN2022097430-appb-000007
3)样品处理与冲洗：15min后用DPBS彻底冲洗以去除产品并擦干皮肤组织表面水分；
4)后孵育：将处理好的样品转入新的维持培养基中，放回CO2培养箱中孵育；
5)组织活性测试：孵育42h后，将皮肤组织转移至0.3mg/mL的MTT溶液中，37℃培养3h。观察培养后的皮肤组织颜色并记录。切下皮肤组织，并转移至1.5mL离心管中，加入500μl酸化异丙醇混匀，4℃避光放置72h后以酸化异丙醇为空白，于560nm波长处测定吸光度值；
6)结果处理分析：根据皮肤组织活性＝(实验组OD值-空白OD值)/(阴性对照组OD值-空白OD值)*100％计算各样品对应组织的活力。
测试结果
根据测试样品组织活性＞50％为非刺激性物质，测试样品组织活性≤50％则为刺激性物质对结果进行判定，如表11和图9所示，1％青叶胆提取物对应的组织活性为84.5％，为非刺激性物质。
表11组织活性结果
分组 产品名称 组织活性 结果判定
阴性对照 DPBS(NgC) 100％ 非刺激性物质
阳性对照 5％SDS(PC) 8.4％ 刺激性物质
实验组 青叶胆提取物 84.5％ 非刺激性物质
实施例8
青叶胆洗发水组合物
8.1按照下表12配方制备青叶胆洗发水，并进行马拉色菌抑制实验。
表12青叶胆洗发水配方表
Figure PCTCN2022097430-appb-000008
制备方法：
1、A组分75-80℃分散均匀；
2、A组分搅拌均匀后，依次加入B组分，保温搅拌30-45min；
3、加入C组分；
4、缓慢降温至60℃，加入C组分；
5、常温加入D、E、F、G组分。
8.2青叶胆洗发水马拉色菌抑制实验：以糠秕马拉色菌为菌种，参照《QBT 2738-2012日化产品抗菌抑菌效果的评价方法》，测试青叶胆洗发水对马拉色菌的抑制作用，具体测试方法如下：
1)实验试剂：橄榄油培养基
2)测试菌种：糠秕马拉色菌(ATCC44344，购自广东微生物菌种保藏中心)；
3)测试步骤：
培养基的准备：将培养基配制好后高温121℃灭菌15分钟，分装至平板，每个平板大约15～20ml，待冷却凝固后，备用；
菌悬液制备：刮取在橄榄油培养基上生长72h的马拉色菌菌落于无菌生理盐水中混匀；
样品配制及接种：取1g样品加5ml生理盐水进行稀释，混匀后取4.5ml混匀液置空试管中，加入0.5ml菌悬液，作用10min后取1ml用生理盐水进行十倍稀释后，再取0.1ml稀释液涂布平板。以生理盐水代替试验样品，同时按照以上步骤操作，作为对照样品；
培养：置于培养箱中36℃下培养72h，记录菌落数，计算抑菌效果；
4)测试结果：根据下述公式进行计算，结果下表13所示
抑菌率(％)＝(对照平均菌落数-样品平均菌落数)/对照平均菌落数*100
表13青叶胆洗发水抑菌率
Figure PCTCN2022097430-appb-000009
结果表明相较于空白对照组，青叶胆洗发水能显著抑制马拉色菌的繁殖(图10)。
综上所述，本发明提供了青叶胆提取物抑制马拉色菌、痤疮丙酸杆菌和抗炎功效方面的应用，实验证明，青叶胆提取物在显著抑制糠秕马拉色菌和痤疮丙酸杆菌生长的同时，还可有效减轻细菌繁殖引起的皮肤发痒、发红等炎症症状，能够有效解决头屑/脂溢性皮炎、特应性皮炎、毛囊炎/痤疮、银屑病等多种皮肤问题，而且青叶胆提取物刺激性低，安全性高，非常适合在日用化妆品领域应用。
此外，本发明还提供了一种特定的青叶胆提取物及其制备方法，本发明的方法制备的青叶胆提取物能够获得高达45wt％的獐牙菜苦苷。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 青叶胆提取物在制备用于抗菌的化妆品组合物或药物组合物中的用途。
  2. 如权利要求1所述的用途，其特征在于，所述菌选自下组：丙酸杆菌属(Propionibacterium)(例如，费氏丙酸杆菌(Propionibacterium freudennreichii)、痤疮丙酸杆菌(Propionibacterium acnes)或颗粒丙酸杆菌(Propionibacterium granulosum))、马拉色菌属(Pityrosporum)(例如，糠秕马拉色菌(Malassezia.furfur)、合轴马拉色菌(M.sympodialis)、球形马拉色菌(M.globosa)、限制马拉色菌(M.restricta)、厚皮马拉色菌(M.pachydermatis)、史洛邦马拉色菌(M.sloofiae)、蛎壳马拉色菌(M.obtusa))，或其组合。
  3. 如权利要求1所述的用途，其特征在于，所述化妆品组合物或药物组合物用于一个或多个选自下组的用途：马拉色菌引起的头皮屑、脂溢性皮炎、特应性皮炎、毛囊炎、脱发、痤疮、银屑病，或其组合。
  4. 如权利要求1所述的用途，其特征在于，所述化妆品组合物或药物组合物还用于抑制NO和/或COX-2的分泌。
  5. 如权利要求1所述的用途，其特征在于，所述青叶胆提取物来自青叶胆药材的花、茎、叶，或其组合，如全草。
  6. 如权利要求1所述的用途，其特征在于，所述青叶胆提取物通过如下方法制备，包括如下步骤：
    (i)将青叶胆药材在0-85v/v％的乙醇水混合溶液中加热提取，分离得提取液I；
    (ii)将提取液I浓缩后用非极性或弱极性树脂吸附，得吸附后的树脂I；
    (iii)用水清洗所述树脂I，得清洗后的树脂II；
    (iv)用70-85％的乙醇水混合溶剂洗脱所述树脂II，收集洗脱液；和
    (v)任选地去除所述洗脱液的溶剂，从而得所述青叶胆提取物。
  7. 如权利要求1所述的用途，其特征在于，所述化妆品组合物或药物组合物还包括化妆品或药学上可接受的载体。
  8. 一种青叶胆提取物，其特征在于，所述青叶胆提取物通过如下方法制备，所述方法包括步骤：
    (i)将青叶胆药材在0-85v/v％的乙醇水混合溶液中加热提取，分离得提取液I；
    (ii)将提取液I浓缩后用非极性或弱极性树脂吸附，得吸附后的树脂I；
    (iii)用水清洗所述树脂I，得清洗后的树脂II；
    (iv)用70-85％的乙醇水混合溶剂洗脱所述树脂II，收集洗脱液；和
    (v)任选地去除所述洗脱液的溶剂，从而得所述青叶胆提取物。
  9. 如权利要求8所述的提取物，其特征在于，所述树脂为LS-305。
  10. 一种化妆品组合物或药物组合物，其特征在于，所述组合物包括如权利要求8所述的青叶胆提取物；和化妆品或药学上可接受的载体。
  11. 如权利要求10所述的组合物，其特征在于，化妆品组合物或药物组合物中，所述青叶胆提取物的含量≥0.1wt％，较佳地≥0.2wt％或≥0.5wt％，如0.6wt％、1wt％、2wt％、5wt％、10wt％或20wt％，以所述化妆品组合物或药物组合物总重量计。
  12. 如权利要求10所述的组合物，其特征在于，所述化妆品组合物或药物组合物的剂型选自下组：液体制剂、混悬制剂、半固体制剂或固体制剂。
  13. 如权利要求10所述的组合物，其特征在于，所述化妆品组合物包括如下组分，以组合物总重量计：
    名称 wt％ 瓜尔胶羟丙基三甲基氯化铵 0.24～0.36 聚季铵盐-10 0.04～0.06 柠檬酸 0.16～0.24 月桂醇聚醚硫酸酯钠 8～12 月桂醇硫酸酯铵 5.6～8.4 季戊四醇二硬脂酸酯 0.4～0.6 乙二醇硬脂酸酯 1.2～1.8 椰油酰胺丙基甜菜碱 1.6～2.4 椰油酰胺MEA 0.8～1.2 月桂基甘醇羧酸钠 1.2～1.8 吡啶酮乙醇胺盐 0.32～0.48 硅油 1.6～2.4 油酸单甘油酯和烷基糖苷 1.6～2.4 青叶胆(SWERTIA MILEENSIS)提取物 0.48～0.72 水 1.6～2.4 乙内酰脲 0.16～0.24 水 余量
    。
  14. 一种制备如权利要求5所述的青叶胆提取物的制备方法，其特征在于，所述方法包括如下步骤：
    (i)将青叶胆药材在0-85v/v％的乙醇水混合溶液中加热提取，分离得提取液I；
    (ii)将提取液I浓缩后用非极性或弱极性树脂吸附，得吸附后的树脂I；
    (iii)用水清洗所述树脂I，得清洗后的树脂II；
    (iv)用70-85％的乙醇水混合溶剂洗脱所述树脂II，收集洗脱液；和
    (v)任选地去除所述洗脱液的溶剂，从而得所述青叶胆提取物。
  15. 如权利要求14所述的方法，其特征在于，所述树脂为LS-305。
PCT/CN2022/097430 2021-06-11 2022-06-07 青叶胆提取物及其制备方法和应用 WO2022257932A1 (zh)

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