阅读说明：本技术 粉防己提取物的抗痤疮应用 (Anti-acne application of tetrandra root extract ) 是由 张鑫 陆泽安 陈园园 于 2019-09-29 设计创作，主要内容包括：本发明公开了粉防己提取物在非治疗目的的抗痤疮方面的应用。本发明所述粉防己提取物采用乙醇进行提取。本发明还涉及粉防己提取物在制备具有抗痤疮功效的个人护理产品中的应用。所述个人护理产品包括护肤霜,护肤乳,精华液等。(The invention discloses application of a tetrandra root extract in the aspect of non-treatment-purpose anti-acne. The tetrandra root extract is extracted by ethanol. The invention also relates to the use of a tetrandra root extract in the preparation of a personal care product having anti-acne efficacy. The personal care products include skin creams, skin lotions, serums, and the like.)

1. Use of Stephania tetrandra extract for treating acne is provided.

2. Use according to claim 1, wherein said anti-acne is achieved by anti-Propionibacterium acnes.

3. The use as claimed in claim 1, wherein the extract of Stephania tetrandra is prepared by solvent extraction.

4. Use according to claim 3, wherein the solvent is ethanol at a concentration of 60-80% by volume.

5. Use of Stephania tetrandra extract in the preparation of a personal care product having anti-acne effect.

6. Use according to claim 5, the anti-acne efficacy being achieved by anti-Propionibacterium acnes.

7. The use as claimed in claim 5, wherein the Stephania tetrandra extract is prepared by solvent extraction.

8. The use according to claim 7, wherein the solvent is ethanol at a concentration of 60-80% by volume.

9. The use of claim 5, wherein the personal care product comprises Stephania tetrandra extract at a concentration of at least 0.016 g/ml.

10. The use according to claim 5, wherein the personal care product is selected from the group consisting of: skin cream, skin milk and essence.

Technical Field

The invention belongs to the field of phytochemistry, and particularly relates to an antibacterial effect of a tetrandra root extract, in particular to an application of the tetrandra root extract in the field of resisting propionibacterium acnes.

Background

At present, most of the antibacterial additives at home and abroad are mainly synthesized compounds. Although these compounds have the advantages of quick killing of pathogenic microorganisms, low effective concentration and the like, the toxicity and the irritation of the compounds influence the human health to different degrees. The abuse of antibiotics also leads to the emergence of drug-resistant bacteria, which leads to the increasingly difficult cure of diseases such as bacterial infections. Along with the requirements of nature, safety and environmental protection, the plant antibacterial additive is more and more favored by people.

Dried root of Stephania tetrandra (Stephania tetrandra S.Moore) of Stephania of Menispermaceae has effects of dispelling pathogenic wind, relieving pain, inducing diuresis and relieving swelling. Can be used for treating rheumatalgia, edema, loempe, dysuresia, eczema, and skin sore. Modern analysis shows that tetrandrine contains various alkaloids, wherein tetrandrine is effective in treating rheumatoid arthritis and hypertension.

The main effects of tetrandra root include: a calcium channel blocker; hypertension; anti-myocardial ischemia effect; anti-arrhythmic effect; reducing blood pressure; anti-allergic effect; hypertensive crisis; angina pectoris.

Acne is a common condition caused by blackheads, whiteheads, papules, pustules, cysts, and various nodules and scars. Acne usually occurs on the face and upper arms, back. Acne is theoretically caused at least in part by bacteria, such as Propionibacterium acne.

Propionibacterium acnes (c. acnes), also known as acnes and propionibacterium acnes, are the main bacteria responsible for acne. Acne is a common chronic inflammatory skin disease, and the morbidity of the acne reaches 70 to 87 percent (commonly called whelk) in adolescents. Studies have shown that the occurrence of acne is closely related to propionibacterium acnes. Therefore, acne removing effects are often achieved by using certain drugs that inhibit or kill propionibacterium acnes. Benzoyl peroxide and salicylic acid are well known for use as anti-acne actives. However, formulations of these chemical components (e.g., benzoyl peroxide or salicylic acid) are often physically unstable and tend to precipitate out of solution. Alternatively still, formulations of these chemical ingredients (e.g., benzoyl peroxide or salicylic acid) often result in severe skin irritation. Therefore, there is still a need in the art to find products from the Chinese herbal medicine field that can prevent and treat acne. For example, the literature "experimental research on in vitro antibacterial activity of traditional Chinese medicine on acne pathogenic bacteria" (Jiangli, Lemna minor, Shen national Qing, etc.) and the literature "determination of activity of traditional Chinese medicine in vitro inhibition of Propionibacterium acnes" (Zhu Yao Fang, Zhao Hao, etc.) disclose that the traditional Chinese medicine extract has complex components, various action mechanisms and small side effects, and the treatment of acne becomes a new direction.

The inventor unexpectedly finds that the tetrandra root extract has an antibacterial effect, particularly has an anti-propionibacterium acnes effect, and can be used as a functional additive to be applied to personal care products such as skin cream, skin milk, essence and the like.

Disclosure of Invention

The invention discovers that the tetrandra root extract has the effect of resisting propionibacterium acnes for the first time. Therefore, the tetrandra root extract can be used as a functional additive to be applied to personal care products such as skin cream, skin milk, essence and the like.

In one aspect, the present invention provides the use of an extract of Stephania tetrandra for the treatment of acne in non-therapeutic applications.

In a preferred embodiment, the anti-acne described herein is achieved by anti-propionibacterium acnes.

In a preferred embodiment, the tetrandra root extract of the present invention is prepared by a solvent extraction method. In a more preferred embodiment, the extraction solvent is ethanol at a concentration of 60-80% by volume.

In another aspect, the present invention is also directed to the use of tetrandra root extract in the preparation of a personal care product having anti-acne efficacy.

In a preferred embodiment, the anti-acne efficacy of the present invention is achieved by anti-propionibacterium acnes.

In a preferred embodiment, the tetrandra root extract of the present invention is prepared by a solvent extraction method. In a more preferred embodiment, the extraction solvent is ethanol at a concentration of 60-80% by volume.

In a preferred embodiment, the personal care product comprises tetrandra root extract at a concentration of at least 0.016 g/ml.

In a preferred embodiment, the personal care product is selected from the group consisting of: skin cream, skin milk and essence.

Detailed Description

Acne is a chronic inflammatory skin disease of pilosebaceous unit, mainly occurs to teenagers, and has great influence on the psychology and the social interaction of the teenagers. The clinical manifestations are marked by the polymorphic skin lesions of face, such as acne, papule, pustule, and nodule. The occurrence of acne is often closely associated with bacterial (e.g., propionibacterium acnes) infection. A number of microorganisms in the hair follicle, particularly propionibacterium acnes, multiply in number, and lipases produced by propionibacterium acnes break down sebum to produce free fatty acids, while chemotactic inflammatory cells and mediators, ultimately inducing and exacerbating the inflammatory response.

Stephania tetrandra is a name of a traditional Chinese medicine, and is a dried root of Stephania tetrandra (Stephania tetrandra S.Moore) belonging to Stephania of Menispermaceae. Most regions in south China are distributed, and the regions are mainly produced in Zhejiang, Anhui, Hubei, Jiangxi and other provinces. Has the effects of dispelling wind, relieving pain, inducing diuresis and relieving swelling. Can be used for treating rheumatalgia, edema, loempe, dysuresia, eczema, and skin sore. To date, no literature has reported that tetrandra root has bacteriostatic efficacy, especially against propionibacterium acnes.

Any suitable means of preparing the tetrandra root extract for use according to the present invention may be used. Suitable extracts may be obtained using conventional methods including, but not limited to, direct extraction from the starting material by: grinding, macerating, squeezing, mashing, centrifuging, and/or methods such as cold percolation, stirring/distillation, microwave assisted extraction, sonication, supercritical/subcritical CO with or without polar modifier₂Compressed gas extraction, pressurized solvent extraction, accelerated solvent extraction, surfactant-assisted pressurized hot water extraction, oil extraction, membrane extraction, soxhlet extraction, or by other methods such as solvent extraction, and the like. In particular, the extract according to the present invention is preferably a solvent-based extract prepared by grinding or impregnating a plant material in a solvent, typically an organic solvent such as alcohol, acetone, liquid carbon dioxide with or without a polar modifier, hexane or chloroform.

In the present invention, the tetrandra root extract is prepared by a solvent extraction method. The extraction solvent employed in the extraction process may be any solvent known in the art, such as polar and non-polar solvents. The polar or non-polar solvent may comprise a solvent selected from the group consisting of: liquid carbon dioxide, hydrous ethanol, C with or without polar modifier₁-C₈Alcohols (such as methanol, ethanol, propanol and butanol), C₁-C₈Alkanes (such as pentane, hexane and heptane), C₂-C₈Diols/polyols (such as glycerol, butylene glycol and propylene glycol), C₅-C₈Cycloalkanes (such as cyclopentane, cyclohexane and cycloheptane), C₁-C₈Alkyl ethers, C₁-C₈Aliphatic compounds, ketones, methylene chloride, ethyl acetate, xylene, toluene, vegetable oils, mineral oils, and combinations thereof.

In certain preferred embodiments, the extract of the present invention is an extract prepared by grinding the stephania tetrandra raw material into powder and extracting with a solvent having a dielectric constant value of between about 1 and about 80 at 20 ℃, preferably between about 2 and about 60 at 20 ℃, more preferably between about 2 and about 40 at 20 ℃, and even more preferably between about 2 and 35 at 20 ℃.

In a preferred embodiment, the extraction solvent may be ethanol. In a preferred embodiment, the extraction solvent is ethanol with a volume concentration of 60-80%. In a more preferred embodiment, the extraction solvent is ethanol at a concentration of 70% by volume.

The bacteriostatic action of the extract of Stephania tetrandra has many advantages over chemically synthesized compounds. Firstly, the tetrandra root extract obtained by extracting natural Chinese herbal medicines as raw materials better meets the requirements of nature, safety and environmental protection. In consumer groups, the Chinese herbal medicine extract as the bacteriostatic agent has wide application prospect and huge market value. Secondly, the tetrandra root extract obtained by extracting the Chinese herbal medicines as the raw materials is obviously superior to the chemical bacteriostatic agent used conventionally in the prior art in the aspects of toxicity and irritation. Thirdly, the abuse of antibiotics causes the emergence of bacterial drug resistance, so the tetrandra root extract taking Chinese herbal medicines as raw materials is more and more accepted by consumers.

The research of the prior art on the tetrandra root extract is mainly focused on various alkaloids in the tetrandra root extract. The medicinal value of tetrandrine is mainly reflected in the curative effect of tetrandrine (an alkaloid) on rheumatoid arthritis and hypertension at present. However, the present inventors have unexpectedly found that the extract of tetrandra root has bacteriostatic effects, and is particularly effective against propionibacterium acnes.

In another aspect, the present invention is also directed to the use of tetrandra root extract in the preparation of a personal care product having anti-acne efficacy. In some embodiments, such personal care products comprise the tetrandra root extract of the present invention and a suitable carrier. In some embodiments, the personal care product comprises tetrandra root extract at a concentration of at least 0.016 g/ml. In a preferred embodiment, the personal care product comprises tetrandra root extract at a concentration of at least 0.02 g/ml. In a preferred embodiment, the personal care product comprises tetrandra root extract at a concentration of at least 0.05 g/ml. In a preferred embodiment, the personal care product comprises tetrandra root extract at a concentration of at least 0.1 g/ml. In a preferred embodiment, the personal care product comprises tetrandra root extract at a concentration of 0.016 to 0.5 g/ml.

Any suitable carrier may be used in the personal care product. Preferably, the carrier is a cosmetically acceptable carrier. As will be appreciated by those skilled in the art, cosmetically acceptable carriers include those suitable for use in contact with the body, particularly the skin, without undue toxicity, incompatibility, instability, irritation, allergic response, and the like. A safe and effective amount of carrier comprises from about 50% to about 99.999%, preferably from about 80% to about 99.9%, more preferably from about 99.9% to about 95%, and most preferably from about 98% to about 99.8% of the personal care product.

The carrier can exist in a wide variety of forms. For example, carriers in the form of emulsions, including but not limited to oil-in-water, water-in-oil-in-water, and oil-in-water-in-silicone emulsions, can be used herein. These emulsions can cover a wide range of viscosities, for example from about 100 centipoise to about 200,000 centipoise.

Examples of suitable cosmetically acceptable carriers include cosmetically acceptable solvents and materials for cosmetic solutions, suspensions, lotions, creams, essences, gels, toners, sticks, sprays, ointments, lotions and soap bars, shampoos, hair conditioners, pastes, foams, mousses, powders, shaving creams, wipes, patches, tapes, power patches, microneedle patches, bandages, hydrogels, film-forming products, masks and skin patches, foundations, droplets, and the like. These product types may contain several types of cosmetically acceptable carriers including, but not limited to, solutions, suspensions, emulsions such as microemulsions and nanoemulsions, gels, solids, liposomes, other encapsulation techniques, and the like.

The following are non-limiting examples of vectors. Other carriers can be formulated by one of ordinary skill in the art. In one embodiment, the carrier comprises water. In another embodiment, the carrier may further comprise one or more aqueous or organic solvents. Examples of organic solvents include, but are not limited to: dimethyl isosorbide; isopropyl myristate; cationic, anionic and nonionic surfactants; a vegetable oil; mineral oil; a wax; a gum; synthetic and natural gelling agents; an alkanol; a diol; and a polyol. Examples of diols include, but are not limited to, glycerol, propylene glycol, butylene glycol, pentylene glycol, hexylene glycol, polyethylene glycol, polypropylene glycol, diethylene glycol, triethylene glycol, octanediol, glycerol, butylene glycol, and hexanetriol, and copolymers or mixtures thereof. Examples of alkanols include, but are not limited to, those having from about 2 carbon atoms to about 12 carbon atoms (e.g., from about 2 carbon atoms to about 4 carbon atoms), such as isopropanol and ethanol. Examples of polyols include, but are not limited to, those having from about 2 carbon atoms to about 15 carbon atoms (e.g., from about 2 carbon atoms to about 10 carbon atoms), such as propylene glycol. The organic solvent can be present in the carrier in an amount of from about 1% to about 99.99% (e.g., from about 20% to about 50%), based on the total weight of the carrier. Water may be present in the carrier (prior to use) in an amount of from about 5% to about 95% (e.g., from about 50% to about 90%), based on the total weight of the carrier. The solution can comprise any suitable amount of solvent, including from about 40% to about 99.99%. Certain preferred solutions comprise from about 50% to about 99.9%, from about 60% to about 99%, from about 70% to about 99%, from about 80% to about 99%, or from about 90% to 99% solvent.

Lotions may be made from such solutions. In addition to the solvent, the lotion typically contains at least one emollient. The lotion formulation can comprise from about 1% to about 20% (e.g., from about 5% to about 10%) of an emollient and from about 50% to about 90% (e.g., from about 60% to about 80%) of water.

Another type of product that can be formulated from a solution is a cream. Creams typically comprise from about 5% to about 50% (e.g., from about 10% to about 20%) of an emollient and from about 45% to about 85% (e.g., from about 50% to about 75%) of water.

Another type of product that can also be formulated from a solution is a paste. The ointment may comprise a simple base of animal, vegetable or synthetic oil or semi-solid 10 hydrocarbons. The cream may comprise from about 2% to about 10% of an emollient and from about 0.1% to about 2% of a thickener.

The products useful in the present invention may also be formulated as emulsions. If the carrier is an emulsion, about 1% to about 10% (e.g., about 2% to about 5%) of the carrier comprises an emulsifier. The emulsifier may be nonionic, anionic or cationic.

Lotions and creams may be formulated as emulsions. Typically such lotions will contain from 0.5% to about 5% of an emulsifier, while such creams will typically contain from about 1% to about 20% (e.g., from about 5% to about 10%) of an emollient; from about 20% to about 80% (e.g., from 30% to about 70%) water; and from about 1% to about 10% (e.g., from about 2% to about 5%) emulsifier.

Oil-in-water and water-in-oil single emulsion skin care formulations, such as lotions and creams, are well known in the art and can be used in the present invention. Multiple emulsion products, such as water-in-oil-in-water or oil-in-water-in-oil, can also be used in the present invention. Typically, such single or multiple emulsions comprise water, emollient, and emulsifier as major ingredients.

The products of the invention may also be formulated as gels (e.g., aqueous, alcoholic/aqueous or oleogels using suitable gelling agents). Suitable gelling agents for hydrogel-containing and/or alcogel include, but are not limited to, natural gums, acrylic acid and acrylate polymers and copolymers, and cellulose derivatives (e.g., hydroxymethyl cellulose and hydroxypropyl cellulose). Suitable gelling agents for oils (such as mineral oil) include, but are not limited to, hydrogenated butylene/ethylene/styrene copolymers and hydrogenated ethylene/propylene/styrene copolymers. Such gels typically comprise between about 0.1 and 5 wt% of such gelling agents.

The products of the invention may also be formulated as solid formulations (e.g., wax-based sticks, soap bar products, powders or wipes). The products of the present invention may also be combined with a solid, semi-solid, or dissolvable substrate (e.g., a wipe, mask, pad, glove, or tape).

Products used in accordance with the principles of the present invention may also contain any of a variety of additional cosmetically active agents. Examples of suitable additional active agents include: skin lightening agents, tanning agents, additional anti-aging agents, tropoelastin promoters, collagen promoters, anti-acne agents, oil control agents, antimicrobial agents such as anti-yeast agents, antifungal and antibacterial agents, anti-inflammatory agents, antiparasitic agents, external analgesics, sunscreens, photoprotective agents, antioxidants, keratolytic agents, detergents/surfactants, moisturizers, nutrients, vitamins, energy enhancers, antiperspirants, astringents, deodorants, depilatories, hair growth enhancers, hair growth retardants, firming agents, moisturizers, synergists, anti-sclerosants, skin conditioning agents, anti-cellulite agents, odor control agents such as odor masking agents or pH modifiers, and the like.

Detailed Description

The inventors of the present invention have unexpectedly found that: the tetrandra root extract has the function of resisting propionibacterium acnes, and can be used as a functional additive to be applied to personal care products such as skin cream, skin milk, essence and the like. There is no mention in the literature of the bacteriostatic efficacy of the tetrandra root extract, nor is it a mention of its incorporation as a functional bacteriostatic additive into personal care products for the treatment and management of acne.

Therefore, the invention firstly provides that the tetrandra root extract has the function of resisting the propionibacterium acnes. The tetrandra root extract is added into a personal care product as a functional additive to effectively prevent and treat acne.

The invention is further illustrated below with reference to specific examples. It is to be understood, however, that these examples are illustrative only and are not to be construed as limiting the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the manufacturer. All percentages and parts are by weight unless otherwise indicated.

Example 1: preparation of Stephania tetrandra extract

Taking radix Stephaniae Japonicae, pulverizing, weighing 100g, pouring into a glass beaker, adding 800mL of 70% ethanol, stirring thoroughly, extracting at room temperature for 3h, stirring intermittently during the period, filtering the extractive solution with filter paper (qualitative filter paper of Xinxing company, diameter of 9cm, medium speed, pore diameter of 15-20 μm) after 3h, adding 800mL of 70% ethanol into the residue, extracting at room temperature, stirring intermittently, and filtering the extractive solution with filter paper after 3 h; adding 800mL of 70% ethanol into the residue, stirring, standing at room temperature overnight, filtering the extractive solution with filter paper, mixing the extractive solutions for 3 times, and recovering ethanol under reduced pressure to volume of about 100 mL.

Example 2: stephania tetrandra extract with bacteriostatic effect

1. Culture medium and reagent

1.1 Standard strains: propionibacterium acnes (ATCC 11827).

1.2 culture Medium:

brain Heart Infusion Agar Medium (Brian Heart Infusion Agar; trade name: brain Heart Infusion Agar Medium, manufacturer: BD) (bacterial culture) [ prepared according to the instructions, 121 deg.C (151b), 15min after autoclaving ] used

2. Procedure for the preparation of the

2.1 preparing the bacteriostatic tablets: the bacteriostatic agent carrier is 7mm diameter circular Xinhua I qualitative filter paper sheet, and is dried at 120 deg.C for 2h after pressure steam sterilization, and stored for use.

2.2 taking a sterile and dried filter paper sheet, dripping 10ul of the radix stephaniae tetrandrae extracting solution prepared in the embodiment 1 into each filter paper sheet, then placing the filter paper sheet in a clean sterile plate, and naturally drying for 5min at room temperature for later use.

2.3 negative control samples for liquid bacteriostatic agents: sterile dry filter paper sheets can be taken, 10ul of sterile deionized water is dripped into each sheet, and the sheets are dried for later use.

2.4 Positive control samples: the stock solution of the drip-dew (EXP 2019-01-04, product of Jieji Jia Hua (China) Co., Ltd., manufacturer's Li) was used, 10ul of each piece of the aseptic dried filter paper was dropped, and the aseptic dried filter paper was dried for use.

2.5 solvent control sample: taking a given solvent sample, taking a sterile dry filter paper sheet, dropwise adding 10ul of the sterile dry filter paper sheet into each sheet, and drying for later use; if the solvent is deionized water, the control sample can be omitted.

2.6 inoculation of test bacteria:

2.6.1 sterile medium plates (sterile plate manufacturer Qingdao Jindian Biochemical instruments Co., Ltd. specification 90X 15mm culture dish (ethylene oxide sterilization)) were prepared, about 15ml of medium was injected into each medium plate, and the medium plate was left at room temperature and naturally dried and solidified.

2.6.2 test bacterial suspension preparation:

the test bacteria liquid requires fourth or fifth generation fresh bacteria liquid, i.e. bacteria are beveled 24h in advance, yeast are beveled 48h in advance, and during the test, a picking rod is used to pick 2-3 rings of fresh bevel bacteria in 9ml sterilized physiological saline (compared with 0.5 wheat turbidimetric tube, the actual concentration should be slightly more concentrated than the turbidimetric tube, and the actual concentration should be 10)⁸cfu/ml), then carrying out 1: 100 are diluted to the bacterial suspension required by the test.

2.6.3 the concentration is 5 × 10⁵cfu/ml～5×10⁶cfu/ml test bacterial suspension, sucking 50 μ L of test bacterial suspension with a micropipette to the surface of a plate prepared in advance, smearing the bacterial solution uniformly with a sterile L-rod, and covering the plate. Drying at room temperature for 5 min.

2.6.4 test bacteria liquid count: the test bacterial suspensions were counted by plate counting.

2.7 sticking bacteriostatic agent sample pieces:

each test is stuck with 1 contamination flat plate, each flat plate is stuck with 2 parallel test sample plates, 1 negative control sample plate, 1 positive control sample plate and 1 solvent control sample plate (if any), and 4-5 test sample plates are all stuck. And (3) using sterile forceps to take samples and paste the samples on the surface of the flat plate, wherein the distance between the centers of the samples is more than 25mm, and the distance between the centers of the samples and the periphery of the flat plate is more than 15 mm. After the sample is placed, the sample is lightly pressed by using sterile forceps to be tightly attached to the surface of the flat plate, the flat plate is covered, the culture box is placed at 37 ℃ under an anaerobic condition for culturing for 48 hours, and the result is observed (after the first test, the sample placing rule is properly adjusted according to the size of the bacteriostatic zone).

3. Measurement of the diameter of the antibacterial ring:

3.1 the counting result of the test bacterial suspension is within the required concentration range of the required bacterial liquid (5X 10)⁵cfu/ml～5×10⁶cfu/ml)。

3.2 when measuring the bacteriostatic ring, should choose the bacteriostatic ring of even and complete aseptic growth to carry on, measure its diameter should regard bacteriostatic ring outer edge as the boundary.

4. Determination of bacteriostatic action:

4.1 the diameter of the bacteriostatic ring is larger than 7.00mm, and the product is judged to have bacteriostatic action.

4.2 the diameter of the bacteriostatic ring is less than or equal to 7.00mm, and the product is judged to have no bacteriostatic action.

5. Results of the experiment

TABLE 1

As shown in Table 1, the tetrandra root extract has an inhibition zone of 48.01mm on Propionibacterium acnes (ATCC11827), and has a potent inhibitory effect.

Example 3: minimum Inhibitory Concentration (MIC) determination test

1. The application range is as follows: this example uses broth dilution method, and the method is suitable for determination of the minimum inhibitory concentration of soluble anti (bacteriostatic) bacteria product.

2. Culture medium and reagent

2.1 Standard strains: propionibacterium acnes (ATCC11827) bacterial suspension and other bacterial species used depending on the particular use of the bacteriostatic agent.

2.2 Brain Heart Infusion Broth Medium (Brain Heart Infusion Broth; trade name: Brain Heart Infusion Broth, manufacturer: BD) [ prepared according to product instructions, 121 deg.C (151b), used after 15min autoclaving ]

2.3PBS buffer

Adjusting the pH value to 7.2-7.4 by using HCl, and adding distilled water to a constant volume of 1000m 1.

2.4 physiological saline

The preparation method comprises the following steps: NaCl 8.5g

Distilled water is added to 1000ml

After dissolving, adding into a liquid dispenser with an adjustable bottle top. Adjusting the scale of the liquid preparation device on the top of the adjustable bottle to 9ml of sample adding amount each time, adding physiological saline into a 20ml sampling bottle with 9ml of sample, and sterilizing at high temperature and high pressure. Storing at room temperature for later use.

2.5 brain Heart Infusion Agar Medium (Brian Heart Infusion Agar; trade name: brain Heart Infusion Agar Medium, manufacturer: BD) (bacterial culture) [ prepared as described in the Specification, 121 deg.C (151b), autoclaved for 15min and used.

3. Procedure for the preparation of the

3.1 preparation of a bacterial suspension of the test strains, adjustment of the concentration of the bacterial suspension to 10 with sterile physiological saline⁸cfu/ml。

3.2 preparation of the culture medium containing the antibacterial agent: the antibacterial solution is prepared into test solutions with different concentrations by using distilled water, and 2.5ml of each diluted test solution is added into a test tube containing 2.5ml of brain heart leachate broth culture medium with double concentration.

3.3 taking 0.1ml of the mixture with the bacterial content of 10⁸cfu/ml of the bacterial suspension was inoculated into a test tube containing an anti-bacterial agent (bacteriostatic) brain heart extract broth as a test group sample.

3.4 the bacterial suspension was inoculated into single-ploid brain heart infusion broth tube without anti (bacteriostatic) agent in the same way as positive control group sample.

3.5 2 tubes containing only single-time brain heart extract broth were taken as negative control samples.

3.6 placing the test group sample, the positive control group sample and the negative control group sample in an incubator at 37 ℃ for anaerobic culture until the broth of the positive control group becomes turbid, namely taking 1ml of broth culture solution in each test tube to a plate, carrying out anaerobic culture in the incubator at 37 ℃ based on pouring culture, and observing the result after 24-48 h.

3.7 in the test, the viable bacteria culture count of the test bacterial suspension should be carried out at the same time, and the concentration should be 5 multiplied by 10⁵cfu/ml～5×10⁶cfu/ml。

4. Determination of results

When the positive control tube has bacteria growth (turbidity) and the negative control tube has no bacteria growth (transparency), the active concentration of the test bacterial suspension is 5X 10⁵cfu/ml～5×10⁶The concentration of the anti-bacterial liquid corresponding to the highest dilution for aseptic growth of the test group at cfu/ml is the MIC of the sample to the test bacteria.

5. Results of the experiment

TABLE 2

Note: the above results are obtained by culturing the test group sample, the positive control group sample and the negative control group sample in an incubator at 37 ℃ until the broth of the positive control group becomes turbid, adding 1ml of broth culture solution in each test tube into a plate, performing casting culture in the incubator at 37 ℃, and observing the results after 24-48 h. The Minimum Inhibitory Concentration (MIC) of the tetrandra root extract on Propionibacterium acnes is 0.016 g/ml.