Use of long-chain acylalanine-containing composition for inhibiting malassezia and / or propionibacterium acnes

Abstract

Provided is use of a long-chain acylalanine-containing composition for inhibiting malassezia and / or propionibacterium acnes. The present invention also relates to related anti-dandruff shampoo and anti-acne composition, and a non-therapeutic cosmetic method.

Description

Use of compositions containing long-chain acyl alanine to inhibit Malassezia and / or Propionibacterium acnes. Technical Field

[0001] This invention relates to the field of skin and hair care, and particularly to compositions containing long-chain acylalanine for inhibiting Malassezia and / or Propionibacterium acnes. Background Technology

[0002] Malassezia is a yeast-like fungus, an opportunistic pathogen, and a normal parasite on the skin of warm-blooded animals and humans. It primarily grows in the stratum corneum of human skin and is lipophilic. Clinically common conditions such as tinea versicolor, Malassezia folliculitis, and seborrheic dermatitis are all related to it. Recent studies suggest that excessive proliferation of Malassezia on the scalp is a significant cause of seborrheic dermatitis of the scalp.

[0003] The reduction and disappearance of Malassezia species on the scalp directly translates to a reduction in dandruff. Therefore, the inhibitory effect on Malassezia is used as an indicator in the efficacy evaluation of many anti-dandruff agent ingredients. Malassezia furfur is considered one of the main Malassezia species causing dandruff.

[0004] With the increasing incidence of seborrheic dermatitis (dandruff), antifungal shampoos are often used to treat it. For example, shampoos containing 1% zinc thiopyridine can be used to treat seborrheic dermatitis of the scalp. However, the active ingredient of zinc thiopyridine can deposit and remain deep in the hair follicle for a relatively long time.

[0005] Most chemical dandruff-reducing agents commonly used in shampoos are broad-spectrum antibacterial agents, such as piroctone olamine, zinc pyrithione, and ketoconazole. Intermittent use can effectively inhibit the growth of bacteria and fungi, but long-term use can easily lead to a decrease in transepidermal moisture and lipids in the scalp, causing skin irritation and sensitization. Furthermore, the toxicity and safety of zinc pyrithione itself cannot be ignored; the European Union banned its use as an antidandruff agent in shampoos and conditioners in May 2022 (CN117338639A).

[0006] In addition, some teams have studied the inhibitory effect of plant essential oils on Malassezia, but plant essential oils have disadvantages such as skin irritation, volatility which affects efficacy, and unpleasant odors, all of which affect the practical application of plant essential oils in dandruff removal products.

[0007] In summary, most dandruff treatments on the market rely on potent antifungal chemicals or irritating ingredients, which can cause significant damage to the scalp with long-term use. There is an urgent need to find safe dandruff-reducing ingredients to replace or partially replace antifungal chemical medications.

[0008] Acne is a chronic inflammatory skin condition that commonly affects the cheeks and forehead, followed by the chest, back, and shoulders. It often manifests as comedones (whiteheads), and in severe cases, can present as inflammatory papules, pustules, nodules, cysts, and even scarring. Acne is a very common skin condition among adolescents, typically starting before puberty and continuing into adulthood. It not only affects appearance but also has a profound negative impact on mental health and social interactions.

[0009] Clinical studies have shown that the total bacterial detection rate in acne-affected facial lesions is higher than that in normal facial skin. The main bacteria isolated from acne-affected facial lesions is *Propionibacterium acnes*, followed by *Staphylococcus epidermidis*. This indicates a certain relationship between the pathogenesis of acne and *Propionibacterium acnes*. Currently, clinical treatment mainly involves isotretinoin and antibiotics, but these have adverse reactions.

[0010] Acne treatment is one of the most popular cosmetic efficacy claims in the current skincare market. Acne-reducing cosmetics can be used as daily care products for consumers with acne problems, providing some auxiliary improvement. Therefore, exploring safe and effective methods for preventing / delaying / treating / and daily care of acne is of great significance. Summary of the Invention

[0011] The present invention aims to provide a composition that can inhibit Malassezia and / or Propionibacterium acnes, which is effective and very safe with no side effects.

[0012] Specifically, the present invention provides the following solution.

[0013] [1]. The non-therapeutic use of compositions containing N-long-chain acylalanine and / or its salts for inhibiting Malassezia and / or Propionibacterium acnes when applied to a surface, especially for cosmetic purposes.

[0014] [2]. According to the use described in [1], the surface is the surface of human skin and / or scalp.

[0015] [3]. The composition is a composition for topical application according to any one of [1]-[2].

[0016] [4]. The composition is a cosmetic composition, preferably a skin or hair care composition, according to any one of [1]-[3].

[0017] [5]. The use according to any one of [1]-[4] is for preventing the occurrence of dandruff and / or reducing dandruff.

[0018] [6]. The use according to any one of [1]-[4] is for inhibiting acne; further, for treating acne.

[0019] [7]. The use according to any one of [1]-[4] is for achieving microbiome balance, which means selectively reducing the number of Malassezia and / or Propionibacterium acnes.

[0020] [8]. A non-therapeutic cosmetic method comprising topically applying a cosmetic composition to the skin or hair for the prevention / reduction of skin defects caused by acne or the reduction of the number of pimples / erythema or the prevention / reduction of dandruff, said cosmetic composition comprising N-long-chain acylalanine and / or its salts.

[0021] [9]. Use of compositions comprising N-long-chain acylalanine and / or its salts in the preparation of medicaments for treating acne.

[0022]

[0010] . Use of compositions comprising N-long-chain acylalanine and / or its salts in the preparation of anti-dandruff medicaments / creams.

[0023]

[0011] . According to any one of the uses or methods described in [1]-

[0010] , the weight percentage of N-long-chain acylalanine and / or its salt in the composition is 0.3% or more, preferably 1% or more.

[0024]

[0012] . According to any one of the uses or methods described in [1]-

[0011] , the weight percentage of N-long-chain acylalanine and / or its salt in the composition is 2%-30%, preferably 3%-20%, more preferably 4%-15%.

[0025]

[0013] . According to any one of the uses or methods described in [1]-

[0012] , the composition comprises N-long-chain acylalanine and a basic amino acid, or comprises a salt formed by N-long-chain acylalanine and a basic amino acid.

[0026]

[0014] . According to any one of the uses or methods described in [1]-

[0012] , the composition comprises a sodium, potassium or ammonium salt of N-long-chain acylalanine.

[0027]

[0015] . According to any one of the uses or methods described in [1]-

[0014] , the N-long-chain acyl group in the N-long-chain acyl alanine is derived from a saturated or unsaturated straight-chain or branched fatty acid with 8 to 22 carbon atoms; preferably, the N-long-chain acyl group is selected from one or more of octanoyl, decanoyl, undecanoyl, lauroyl, myristoyl, pentadecanoyl, palmitoyl, stearyl, oleyl, linoleyl, isostearyl, coconut oil fatty acyl, and palm oil fatty acyl.

[0028]

[0016] . According to any one of the uses or methods described in [1]-

[0015] , the N-long-chain acylalanine is selected from one or more of N-octanoyl-L-alanine, N-decanoyl-L-alanine, N-undecanoyl-L-alanine, N-lauroyl-L-alanine, N-cocoyl-L-alanine, N-myristoyl-L-alanine, N-pentadecanoyl-L-alanine, N-palmitoyl-L-alanine, N-stearoyl-L-alanine, N-isostearoyl-L-alanine, N-oleoyl-L-alanine, and N-linoleoyl-L-alanine, preferably N-lauroyl-L-alanine.

[0029]

[0017] . According to any one of the uses or methods described in [1]-

[0016] , the basic amino acid is selected from one or more of arginine, lysine, citrulline, ornithine, creatine, histidine, diaminobutyric acid, and diaminopropionic acid, preferably arginine and / or lysine, and most preferably L-arginine.

[0030]

[0018] . An anti-dandruff shampoo, wherein the shampoo contains N-long-chain acylalanine and basic amino acids, and the shampoo is free or substantially free of clomiphene, pyrrolidone ethanolamine salt, zinc pyrithione, ketoconazole, sulfur, selenium disulfide and salicylic acid; and the shampoo is free or substantially free of sulfate surfactants, ethoxylate surfactants and fatty acid salt surfactants.

[0031]

[0019] . An acne-removing composition, the composition comprising N-long-chain acylalanine and basic amino acids, the composition being free of or substantially free of retinoic acid, benzoyl peroxide, antibiotics, vitamins, salicylic acid, fruit acids, prednisone, dexamethasone; and the composition being free of sulfate surfactants, ethoxylate surfactants and fatty acid salt surfactants.

[0032]

[0020] . According to

[0018] or

[0019] , the N-long-chain acyl group in the N-long-chain acyl alanine is derived from a saturated or unsaturated straight-chain or branched fatty acid with 8 to 22 carbon atoms; preferably, the N-long-chain acyl group is selected from one or more of octanoyl, decanoyl, undecanoyl, lauroyl, myristoyl, pentadecanoyl, palmitoyl, stearoyl, oleoyl, linoleoyl, isostearoyl, coconut oil fatty acyl, and palm oil fatty acyl.

[0033]

[0021] . According to

[0018] or

[0019] , the N-long-chain acylalanine is selected from one or more of N-octanoyl-L-alanine, N-decanoyl-L-alanine, N-undecanoyl-L-alanine, N-lauroyl-L-alanine, N-cocoyl-L-alanine, N-myristoyl-L-alanine, N-pentadecanoyl-L-alanine, N-palmitoyl-L-alanine, N-stearoyl-L-alanine, N-isostearoyl-L-alanine, N-oleoyl-L-alanine, and N-linoleoyl-L-alanine, preferably N-lauroyl-L-alanine.

[0034]

[0022] . According to

[0018] or

[0019] , the basic amino acid is selected from one or more of arginine, lysine, citrulline, ornithine, creatine, histidine, diaminobutyric acid, and diaminopropionic acid, preferably arginine and / or lysine, and most preferably L-arginine.

[0035]

[0023] . According to any one of

[0018] -

[0022] , the N-long-chain acylalanine in the composition has a weight percentage of 2% or more, preferably 4% or more; the basic amino acid in the composition has a weight percentage of 1% or more, preferably 2% or more.

[0036] Compared with the prior art, the present invention has the following beneficial technical effects:

[0037] 1. The present invention unexpectedly discovers that compositions containing N-long-chain acylalanine and / or its salts can effectively inhibit Malassezia and / or Propionibacterium acnes.

[0038] 2. The present invention has unexpectedly discovered that compositions containing N-long-chain acylalanine and / or its salts can prevent the occurrence of dandruff and / or reduce dandruff.

[0039] 3. The present invention unexpectedly discovered that compositions containing N-long-chain acylalanine and / or its salts can be used to treat acne. Detailed Implementation

[0040] In one embodiment, there is a non-therapeutic use of compositions comprising N-long-chain acylalanine and / or its salts for inhibiting Malassezia and / or Propionibacterium acnes when applied to a surface. Preferably, the surface is a human skin and / or scalp surface.

[0041] Preferably, the non-therapeutic use is a cosmetic use. The term "cosmetic use" is intended to mean a use that is not therapeutic, pharmaceutical, or dermatological, i.e., that the use does not require therapeutic treatment and is intended for use on healthy skin. The term "healthy skin" is intended to mean skin that, as described by experts in the art (such as dermatologists), is non-pathological, i.e., skin that does not show any infection, scarring, skin disease or condition (such as candidiasis, impetigo, psoriasis), wounds or injuries, and / or other skin conditions. In this cosmetic use, the composition is a cosmetic composition, particularly a skin or hair care composition.

[0042] The composition can be a composition for topical application. The composition can be formulated into dosage forms such as aqueous solutions, ointments, liniments, gels, creams, powders, emulsions, or sprays. Specific product forms may include, for example, shampoos, conditioners, serums, hair creams, and facial cleansers. Application methods may include, for example, applying the composition to hair, optionally massaging it in for a certain period, and then rinsing it off with water; or applying the composition to the skin, optionally massaging it in for a certain period, and then rinsing it off with water.

[0043] On the one hand, the reduction and elimination of Malassezia on the scalp directly reflects the reduction of dandruff. Therefore, the present invention relates to compositions containing N-long-chain acylalanine and / or its salts for the prevention of dandruff and / or reduction of dandruff.

[0044] On the other hand, the bacteria isolated from facial lesions in acne are mainly Propionibacterium acnes, and there is a certain relationship between the pathogenesis of acne and Propionibacterium acnes. Therefore, this invention relates to compositions containing N-long-chain acylalanine and / or its salts for inhibiting acne; further, for treating acne.

[0045] On the other hand, by effectively controlling the large-scale proliferation of Malassezia and / or Propionibacterium acnes, the skin / scalp microbiome balance can be well maintained. Therefore, the present invention relates to compositions comprising N-long-chain acylalanine and / or its salts for achieving microbiome balance, wherein the balance refers to the selective reduction of the number of Malassezia and / or Propionibacterium acnes.

[0046] In each embodiment, the weight percentage of N-long-chain acylalanine and / or its salt in the composition is 0.3% or more. If the content is too low, the effect of inhibiting Malassezia and / or Propionibacterium acnes is insufficient. Further, the weight percentage of N-long-chain acylalanine and / or its salt in the composition is 1%, 2%, 3%, 4%, 5%, 6% or more, and 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15% or less.

[0047] For N-long-chain acyl alanine, the N-long-chain acyl group in N-long-chain acyl alanine is derived from a saturated or unsaturated straight-chain or branched fatty acid with 8 to 22 carbon atoms. Further, the N-long-chain acyl group in the N-long-chain acyl alanine is selected from one or more of octanoyl, decanoyl, undecanoyl, lauroyl, myristoyl, pentadecanoyl, palmitoyl, stearoyl, oleoyl, linoleoyl, isostearoyl, coconut oil fatty acyl, and palm oil fatty acyl, preferably coconut oil fatty acyl or lauroyl, and most preferably lauroyl.

[0048] Furthermore, the N-long-chain acylalanine is selected from one or more of N-octanoyl-L-alanine, N-decanoyl-L-alanine, N-undecanoyl-L-alanine, N-lauroyl-L-alanine, N-cocoyl-L-alanine, N-myristoyl-L-alanine, N-pentadecanoyl-L-alanine, N-palmitoyl-L-alanine, N-stearoyl-L-alanine, N-isostearoyl-L-alanine, N-oleoyl-L-alanine, and N-linoleoyl-L-alanine, preferably N-lauroyl-L-alanine.

[0049] The synthesis process of N-long-chain acylalanine can refer to the general synthesis methods of N-acyl amino acid type surfactants, and is divided into direct and indirect methods. Direct synthesis of fatty acid raw materials includes enzymatic synthesis and dehydration condensation. Indirect synthesis methods include acylation of fatty acid acyl chlorides, hydrolytic acylation of fatty nitriles, acylation of fatty acid anhydrides, and amide carbonylation reactions. A preferred method is preparation via the amino reaction of fatty acyl chlorides with amino acids (Shotten-Baumann condensation reaction or Shotten-Baumann reaction).

[0050] A typical preparation process suitable for this invention is as follows: amino acids and sodium hydroxide are dissolved in water or a mixture of water and acetone to obtain an amino acid salt solution; then lauroyl chloride and sodium hydroxide solution are slowly added dropwise to the amino acid salt solution, controlling the pH of the reaction system; after the addition is complete, post-processing of the product is performed. Representative methods are disclosed in CN1798821A, US6703517B2, CN102875409B, JPH0570418A, etc.

[0051] For the post-processing of N-long-chain acylalanine products, conventional methods such as recrystallization, water washing, and drying can be used. A preferred post-processing step includes the following steps: mixing the crude N-long-chain acylalanine with a solvent, optionally stirring, and controlling the temperature T of the mixed system to be above the melting point of the long-chain fatty acids and below the melting point of N-long-chain acylalanine. The solvent is water, an organic solvent, or a mixture of water and an organic solvent. After temperature control, solid-liquid separation is performed. Related methods are disclosed by the inventors in CN202210867760.7 and PCT / CN2022 / 107270, the contents of which are incorporated herein by reference.

[0052] For N-long-chain acylalanine and / or its salts, it refers to one or more combinations of N-long-chain acylalanine and N-long-chain acylalanine salts. The N-long-chain acylalanine salt is formed by N-long-chain acylalanine and a base. The base is selected from one or more of inorganic bases, organic amines, and basic amino acids; the inorganic base is selected from one or more of sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate, preferably sodium hydroxide or potassium hydroxide; the organic amine is selected from amines, alkanolamines, etc.

[0053] For basic amino acids, they are selected from one or more of arginine, lysine, citrulline, ornithine, creatine, histidine, diaminobutyric acid, and diaminopropionic acid, with arginine and / or lysine being preferred, and L-arginine being the most preferred.

[0054] Preferably, the base is present in an amount that completely or partially neutralizes N-long-chain acylalanine, preferably with a degree of neutralization of 85% or more and less than or equal to 100%, more preferably with a degree of neutralization of 88% or more and less than 96%.

[0055] In one example, the composition comprises N-long-chain acylalanine and a basic amino acid, or comprises a salt formed by N-long-chain acylalanine and a basic amino acid. In another example, the composition comprises a sodium, potassium, or ammonium salt of N-long-chain acylalanine.

[0056] In one embodiment, an anti-dandruff shampoo is disclosed, comprising N-long-chain acylalanine and basic amino acids. The shampoo is free of or substantially free of clomiphene citrate, piroctone olamine, zinc pyrithione, ketoconazole, sulfur, selenium disulfide, and salicylic acid; and is free of or substantially free of sulfate surfactants, ethoxylate surfactants, and fatty acid salt surfactants. The solution provided by this invention achieves excellent dandruff removal without the use of conventional anti-dandruff agents.

[0057] In another embodiment, an acne-removing composition is disclosed, comprising N-long-chain acylalanine and a basic amino acid, and containing little or no retinoic acid, benzoyl peroxide, antibiotics, vitamins, salicylic acid, fruit acids, prednisone, or dexamethasone; and containing little or no sulfate surfactants, ethoxylated surfactants, or fatty acid salt surfactants. The solution provided by this invention achieves very good acne-removing effects without using conventional acne-removing ingredients.

[0058] The term "substantially free" in this invention means that the content of the substance in the composition will not significantly or substantially affect the performance of the product. Further, "substantially free" means that the weight percentage of the substance in the composition is less than 0.5 wt%, preferably less than 0.1 wt% or less than 0.01 wt%, and more preferably less than the detectable limit.

[0059] Sulfate surfactants: Examples include sodium lauryl sulfate (SLS), ammonium lauryl sulfate (ALS), sodium laureth sulfate (SLES) or ammonium laureth sulfate (ALES). Considering that they may cause irritation to the skin or scalp and / or damage to hair fibers, the compositions of the present invention are preferably substantially free of sulfate surfactants, and more preferably completely free of sulfate surfactants.

[0060] Ethoxylated surfactants: typically sodium lauryl ether sulfate (SLES or AES), but considering their moderate irritant properties, the large amount of non-renewable carbon from ethylene oxide, and safety concerns (the dioxane impurities they contain are carcinogenic), the compositions of the present invention are preferably substantially free of ethoxylates, and more preferably completely free of ethoxylates.

[0061] Fatty acid salts: Examples include laurate, myristic acid salt, palmitate, and stearate. Considering the tension, dryness, and tightness that these raw materials cause after washing, the composition of the present invention is preferably substantially free of fatty acid salts, and more preferably completely free of fatty acid salts.

[0062] The N-long-chain acylalanine and basic amino acids are defined as described in the foregoing embodiments and will not be repeated here. Regarding the component content, the weight percentage of N-long-chain acylalanine in the composition is preferably 2%, 3%, 4%, 5%, or 6% or more, and less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, or 15%. The weight percentage of basic amino acids in the composition is preferably 1%, 1.5%, 2%, 2.5%, or 3% or more, and less than 25%, 22.5%, 20%, 17.5%, 15%, 12.5%, 10%, or 7.5%.

[0063] In one embodiment, a non-therapeutic cosmetic method is involved, comprising topically applying a cosmetic composition to the skin or hair for the prevention / reduction of skin defects caused by acne, or for reducing the number of pimples / redness, or for the prevention / reduction of dandruff, said cosmetic composition comprising N-long-chain acylalanine and / or its salts.

[0064] Furthermore, the composition comprises N-long-chain acylalanine and a basic amino acid, or comprises a salt formed from N-long-chain acylalanine and a basic amino acid, or comprises a sodium, potassium, or ammonium salt of N-long-chain acylalanine. The definitions of the N-long-chain acylalanine and / or its salts and basic amino acids in the foregoing embodiments are provided and will not be repeated here.

[0065] In one embodiment, the use of a composition comprising N-long-chain acylalanine and / or its salt in the preparation of a medicament for treating acne is discussed.

[0066] In another embodiment, the use of a composition comprising N-long-chain acylalanine and / or its salts in the preparation of an anti-dandruff medicated lotion / cream is discussed.

[0067] Furthermore, the composition comprises N-long-chain acylalanine and a basic amino acid, or comprises a salt formed from N-long-chain acylalanine and a basic amino acid, or comprises a sodium, potassium, or ammonium salt of N-long-chain acylalanine. The definitions of the N-long-chain acylalanine and / or its salts and basic amino acids in the foregoing embodiments are provided and will not be repeated here.

[0068] In the above embodiments, the compositions of the present invention may optionally further comprise other ingredients, such as fragrances, dyes, thickeners, humectants, chelating agents, extracts, amino acids, nucleic acids, vitamins, enzymes, anti-inflammatory agents, antioxidants, ultraviolet absorbers, antiperspirants, pH adjusters, pearlescent agents, etc.

[0069] In each implementation, details such as the selection of types and dosages not discussed can be referred to the relevant guidance of other implementations if there is no conflict in the content. For the sake of brevity, each implementation has not been described in detail.

[0070] The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalents also fall within the scope defined by the appended claims.

[0071] Example 1

[0072] 1. Experimental Materials

[0073] MIC & Long-lasting Antibacterial Test: According to standard requirements, accurately weigh 20.0g of lauroyl alanine, 12.20g of arginine, and 167.80g of sterile water into a sterile glass bottle, stir to dissolve and mix well to prepare a sample solution. Dilute sequentially as needed for the test, adding the test concentration group and culture medium at a 1:1 ratio to form the final sample solution. Short-lasting Antibacterial & Antibacterial Rate Test: Accurately weigh 10.0g of lauroyl alanine, 6.10g of arginine, and 83.9g of sterile water into a sterile glass bottle, stir to dissolve and mix well to prepare a 10% sample solution, and dilute as needed for the test.

[0074] Control: Sterile ultrapure water.

[0075] Control: A mixture of MIT (2-methyl-5-chloro-4-isothiamazole-3-one) and CMIT (2-methyl-4-isothiamazole-3-one). Accurately weigh 250 mg of the MIT and CMIT mixture, add 750 mg of sterile water to prepare a 25% (w / w) solution of the MIT and CMIT mixture, and mix well for later use.

[0076] Strain: BNCC 337308 Malassezia furfur

[0077] Culture conditions: 30±1℃

[0078] Reagents:

[0079] Culture medium: Leeming & Notman agar Modified (MLNA) medium, Lot: 20231018

[0080] Leeming and Notman culture medium basic, Lot: 20220915;

[0081] Glycerin, Guangzhou Chemical Reagent Factory, Lot: C14784798;

[0082] Glyceryl monostearate, Yuanye Biotechnology, Lot: L28N10S104297;

[0083] Tween-60, Yuanye Biotechnology, Lot: J17GS155145;

[0084] Olive oil, Yuanye Bio, Lot: F27HS176630;

[0085] Actinomycete ketone solution, Qingdao Rishui Biotechnology Co., Ltd., Lot: 20231103;

[0086] Chloramphenicol solution, Qingdao Rishui Biotechnology Co., Ltd., Lot: 20231103.

[0087] Equipment: Biochemical incubator (CALT / 2020E002)

[0088] 2. Experimental Principle

[0089] MIC test: Different concentrations of antibacterial agents are mixed and dissolved in agar medium using the agar dilution method. Bacteria are then inoculated, and the minimum concentration at which the antibacterial (antimicrobial) substance inhibits the growth of the test bacteria is determined by observing whether the bacteria grow or not.

[0090] Long-lasting antibacterial test: Different concentrations of antibacterial agents were mixed and dissolved in agar medium using the agar dilution method. Bacteria were then inoculated and their growth was observed at multiple time points to determine whether the antibacterial (antimicrobial) substances inhibited the growth of the test bacteria.

[0091] Short-acting antibacterial test: This test utilizes the continuous dissolution of the antibacterial agent, diffused through agar to create different concentration gradients, to demonstrate its antibacterial effect. The size of the inhibition zone is used to determine whether it possesses antibacterial ability. This test is applicable to the identification of antibacterial agents and leaching antibacterial (antimicrobial) products.

[0092] Quantitative inhibition rate test of suspension: A certain amount of bacterial suspension is added to different concentrations of antibacterial agents, and after a certain period of time, it is inoculated onto plates. The bactericidal rate is calculated by comparing the residual bacterial count with the initial bacterial count.

[0093] 3. Experimental Procedure

[0094] Strain revival and subculturing: Under aseptic conditions, the strain was inoculated and subcultured using an inoculation loop, and the third generation strain was used as the test strain.

[0095] Bacterial suspension preparation: The original bacterial suspension was appropriately diluted with sterile physiological saline. The working bacterial suspension concentration for MIC testing and long-term antibacterial testing was 5.69 × 10⁻⁶. 5 CFU / mL. The concentration of the short-acting antibacterial working solution was 7.61 × 10⁻⁶ CFU / mL. 7 CFU / mL. The working solution concentration for quantitative inhibition rate of the suspension was 1.0 × 10⁻⁶. 4 ~9.0×10 4 Between CFU / mL.

[0096] Preparation of culture medium plates:

[0097] Preparation of sample culture medium plates: Measure 10 mL of diluted sample solution and 10 mL of double-concentration MLNA agar medium and add them to the petri dish. Mix the diluted sample solution and the medium thoroughly and let it solidify before use.

[0098] Preparation of positive control culture medium plates: Measure 10 mL of sterile distilled water and 10 mL of double-concentration MLNA agar medium and add them to the petri dish. Mix the sterile distilled water and the culture medium thoroughly and let it solidify before use.

[0099] Preparation of negative control culture medium plates: Measure 10 mL of diluted sample solution and 10 mL of double-concentration MLNA agar medium into a petri dish, mix the diluted sample solution and the medium thoroughly, and let it solidify before use.

[0100] Inoculate the working bacterial solution: Use a pipette to inoculate 10 μL of the working bacterial solution into the center of the sample culture medium plate and the positive control culture medium plate. The diameter of the bacterial suspension formed after inoculation is about 5 mm to 8 mm.

[0101] MIC test: Place the inoculated plates in a 30°C incubator and incubate for 96 hours. Observe and record whether there is colony growth in each plate.

[0102] Long-lasting antibacterial test: After inoculation, the plates were placed upside down in a 30℃ incubator and cultured. The presence or absence of colony growth was observed and recorded at 18h, 24h, 48h, 72h, 96h, and 120h.

[0103] Short-acting antibacterial test: Under aseptic conditions, place Oxford cups directly on a petri dish. Heat sterilized single-dose MLNA medium until completely melted. Cool 200 mL of melted MLNA medium to approximately 50°C and mix in 4 mL of the test bacterial suspension. Slowly pour the mixture along the side of the petri dish to ensure even distribution of the medium. Place the dish on a horizontal plate to allow it to solidify. Once solidified, gently apply pressure with sterile forceps to remove the Oxford cups.

[0104] Add 0.2 mL of the sample solution to each well in sequence. Incubate the plate in a 30°C incubator for 96 h, observe whether an inhibition zone forms, and measure and record the size of the inhibition zone using calipers.

[0105] Quantitative suspension inhibition rate test: Take 5.0 mL of the original sample solution or its dilution and place it in a sterile test tube. Incubate at 20℃ for 5 min. Add 0.1 mL of the test bacterial solution to the test tube containing 5.0 mL of the sample, mix quickly, and start timing immediately. After 6 h of incubation, add 0.5 mL of the mixed solution to a test tube containing 4.5 mL of sterilized PBS, mix thoroughly, and place 1 mL of the solution in a Petri dish. Add culture medium cooled to 40℃-45℃ to the Petri dish, ensuring thorough mixing. Place the solidified plates in an incubator at 37℃ and invert them for 96 h. Observe and record whether colonies grow on each plate, and count the viable colonies.

[0106] 4. Test Results

[0107] Table 1 MIC Experiment Results Note: "+" indicates the presence of bacterial colonies, and "-" indicates the absence of bacterial colonies.

[0108] As can be seen from the data in Table 1, the minimum inhibitory concentration of "lauroyl alanine" against Malassezia furfur is <0.3wt%.

[0109] Table 2. Results of the Long-Acting Antibacterial Experiment Note: "+" indicates the presence of bacterial colonies, and "-" indicates the absence of bacterial colonies.

[0110] Under the experimental conditions, no colonies grew on plates of lauroyl alanine at any concentration after 120 hours, while colonies grew on the positive control plates after 24 hours.

[0111] Table 3 Comparison of inhibition zone diameters (mm) among different groups Note: "-" indicates that no inhibition zone was formed.

[0112] In the short-acting antibacterial test, lauroyl alanine exhibited inhibition zone formation against Malassezia furfur. A mixture of MIT and CMIT also showed inhibition zone formation against Malassezia furfur. This indicates that lauroyl alanine has an inhibitory effect on Malassezia furfur.

[0113] Table 4. Results of the quantitative antibacterial rate experiment of suspension.

[0114] In the quantitative antibacterial rate test of suspension, according to the judgment criteria of QB / T 2738-2023 7.3 Test Method for Antibacterial Effect of Antibacterial Daily Chemical Products, under the experimental conditions, the test sample "lauroyl alanine" showed an antibacterial rate of >99.97% against Malassezia furfur at concentrations of 10%, 5%, and 2.5% for 6 hours, indicating a strong antibacterial effect.

[0115] Example 2

[0116] 1. Experimental Materials

[0117] MIC & Long-lasting Antibacterial Test: According to standard requirements, accurately weigh 20.0g of lauroyl alanine, 12.20g of arginine, and 167.80g of sterile water into a sterile glass bottle, stir to dissolve and mix well to prepare a sample solution. Dilute sequentially as needed for the test, adding the test concentration group and culture medium at a 1:1 ratio to form the final sample solution. Short-lasting Antibacterial & Antibacterial Rate Test: Accurately weigh 10.0g of lauroyl alanine, 6.10g of arginine, and 83.9g of sterile water into a sterile glass bottle, stir to dissolve and mix well to prepare a 10% sample solution, and dilute as needed for the test.

[0118] Control: Sterile ultrapure water.

[0119] Control: A mixture of MIT (2-methyl-5-chloro-4-isothiamazole-3-one) and CMIT (2-methyl-4-isothiamazole-3-one). Accurately weigh 250 mg of the MIT and CMIT mixture, add 750 mg of sterile water to prepare a 25% (w / w) solution of the MIT and CMIT mixture, and mix well for later use.

[0120] Bacterial strain: BNCC 336443 Propionibacterium acnes

[0121] Culture conditions: 37±1℃

[0122] reagents

[0123] Culture medium: GAM basal medium.

[0124] Equipment: Biochemical incubator (CALT / 2020E002)

[0125] 2. Experimental Procedure

[0126] Strain revival and subculturing: Under aseptic conditions, the strain was inoculated and subcultured using an inoculation loop, and the third generation strain was used as the test strain.

[0127] Bacterial suspension preparation: The original bacterial suspension was appropriately diluted with sterile physiological saline. The working bacterial suspension concentration for MIC testing and long-term antibacterial testing was 6.59 × 10⁻⁶. 5 CFU / mL. The concentration of the short-acting antibacterial working solution was 7.61 × 10⁻⁶ CFU / mL. 7 CFU / mL.

[0128] Preparation of culture medium plates:

[0129] Preparation of sample culture medium plates: Measure 10 mL of diluted sample solution and 10 mL of double-concentration GAM agar medium and add them to the petri dish. Mix the diluted sample solution and the medium thoroughly and let it solidify before use.

[0130] Preparation of positive control culture medium plates: Measure 10 mL of sterile distilled water and 10 mL of double concentration GAM agar medium and add them to the petri dish. Mix the sterile distilled water and the medium thoroughly and let it solidify before use.

[0131] Preparation of negative control culture medium plates: Measure 10 mL of diluted sample solution and 10 mL of double-concentration GAM agar medium and add them to the petri dish. Mix the diluted sample solution and the medium thoroughly and let it solidify before use.

[0132] Inoculate the working bacterial solution: Use a pipette to inoculate 10 μL of the working bacterial solution into the center of the sample culture medium plate and the positive control culture medium plate. The diameter of the bacterial suspension formed after inoculation is about 5 mm to 8 mm.

[0133] MIC test: After inoculation, the plates were placed in an incubator at 37°C for anaerobic culture. After 48 hours, the presence or absence of colony growth was observed and recorded on each plate.

[0134] Long-lasting antibacterial test: After inoculation, the plates were placed in an anaerobic incubator at 37℃ and inverted. The presence or absence of bacterial growth on each plate was observed and recorded at 18h, 24h, 48h, 72h, 96h, and 120h.

[0135] Short-acting antibacterial test: Under aseptic conditions, place Oxford cups directly on a petri dish. Heat sterilized single-dose GAM medium until completely melted. Cool 200 mL of melted GAM medium to approximately 50°C and mix in 4 mL of the test bacterial solution. Slowly pour the mixture along the side of the petri dish to ensure even distribution of the medium. Place the dish on a horizontal platform to allow it to solidify. After solidification, gently press with sterile forceps to remove the Oxford cups. Add 0.2 mL of the test sample solution to each well in sequence. Incubate the plates anaerobically at 37°C for 48 hours. Observe whether an inhibition zone forms on the plate. Measure and record the size of the inhibition zone using calipers.

[0136] 3. Test Results

[0137] Table 5 MIC Experiment Results Note: "+" indicates the presence of bacterial colonies, and "-" indicates the absence of bacterial colonies.

[0138] As can be seen from the data in Table 5, the minimum inhibitory concentration of "lauroyl alanine" against Propionibacterium acnes is <2.5 wt%.

[0139] Table 6. Results of the Long-Lasting Antibacterial Experiment Note: "+" indicates the presence of bacterial colonies, and "-" indicates the absence of bacterial colonies.

[0140] Under the experimental conditions, no colonies grew on plates of lauroyl alanine at any concentration after 120 hours, while colonies grew on the positive control plates after 24 hours.

[0141] Table 7 Comparison of inhibition zone diameters (mm) among different groups Note: "-" indicates that no inhibition zone was formed.

[0142] In the short-acting antibacterial test, lauroyl alanine formed an inhibition zone against Propionibacterium acnes. A mixture of MIT and CMIT also formed an inhibition zone against Propionibacterium acnes. This indicates that lauroyl alanine has an antibacterial effect against Propionibacterium acnes.

[0143] Furthermore, those skilled in the art will understand that although some embodiments described herein include certain features but not others included in other embodiments, combinations of features from different embodiments are intended to be within the scope of the invention and form different embodiments. For example, in the claims, any of the claimed embodiments can be used in any combination.

Claims

Non-therapeutic use of a composition comprising N-long chain acyl alanine and / or a salt thereof for inhibiting Malassezia and / or Propionibacterium acnes when applied to a surface. Use according to claim 1, characterized in that The surface is a human skin and / or scalp surface. Use according to claim 1, characterized in that The composition is a composition for topical application. Use according to claim 1, characterized in that The composition is a cosmetic composition, preferably a skin or hair care composition. Use according to claim 1, characterized in that The use is for preventing the appearance of dandruff and / or reducing dandruff. Use according to claim 1, characterized in that The use is for inhibiting acne; further, for combating acne. Use according to claim 1, characterized in that The use is for achieving a microbiome balance, which balance is a selective reduction in the number of Malassezia and / or Propionibacterium acnes. Use according to any one of claims 1 to 7, characterized in that The N-long chain acyl alanine and / or a salt thereof is present in the composition in a weight percentage of 0.3% or more, preferably 1% or more. Use according to claim 8, characterized in that, The N-long chain acyl alanine and / or a salt thereof is present in the composition in a weight percentage of 2% to 30%, preferably 3% to 20%, more preferably 4% to 15%. Use according to any one of claims 1 to 9, characterized in that The composition comprises N-long chain acyl alanine and a basic amino acid, or a salt of N-long chain acyl alanine and a basic amino acid. Use according to any one of claims 1 to 9, characterized in that The composition comprises a sodium, potassium or ammonium salt of N-long chain acyl alanine. Use according to any one of claims 1 to 11, characterized in that The N-long chain acyl group of the N-long chain acyl alanine is derived from a saturated or unsaturated, straight-chain or branched-chain fatty acid having 8 to 22 carbon atoms; preferably, the N-long chain acyl group is selected from one or more of octanoyl, decanoyl, undecanoyl, lauroyl, myristoyl, pentadecanoyl, palmitoyl, stearoyl, oleoyl, linoleoyl, isostearoyl, coconut oil fatty acyl, palm oil fatty acyl. Use according to any of claims 1 to 12, characterized in that The N-long chain acyl alanine is selected from one or more of N-octanoyl-L-alanine, N-decanoyl-L-alanine, N-undecanoyl-L-alanine, N-lauroyl-L-alanine, N-cocoyl-L-alanine, N-myristoyl-L-alanine, N-pentadecanoyl-L-alanine, N-palmitoyl-L-alanine, N-stearoyl-L-alanine, N-isostearoyl-L-alanine, N-oleoyl-L-alanine, N-linoleoyl-L-alanine, preferably N-lauroyl-L-alanine. Use according to claim 10, characterized in that, The basic amino acid is selected from one or more of arginine, lysine, citrulline, ornithine, creatine, histidine, diaminobutyric acid, diaminopropionic acid, preferably arginine and / or lysine, most preferably L-arginine. A dandruff shampoo characterized by comprising The shampoo comprises N-long chain acyl alanine and a basic amino acid, the shampoo being free or substantially free of climbazole, piroctone olamine, zinc pyrithione, ketoconazole, sulphur, selenium disulphide and salicylic acid; and the shampoo being free or substantially free of sulphate surfactants, ethoxylate surfactants and fatty acid salt surfactants. The antidandruff shampoo according to claim 15, wherein The N-long chain acyl alanine is selected from one or more of N-octanoyl-L-alanine, N-decanoyl-L-alanine, N-undecanoyl-L-alanine, N-lauroyl-L-alanine, N-palmitoyl-L-alanine, N-myristoyl-L-alanine, N-pentadecanoyl-L-alanine, N-palmitoyl-L-alanine, N-stearoyl-L-alanine, N-isostearoyl-L-alanine, N-oleoyl-L-alanine, N-linoleoyl-L-alanine, preferably N-lauroyl-L-alanine; and the basic amino acid is selected from one or more of arginine, lysine, citrulline, ornithine, creatine, histidine, diaminobutyric acid, diaminopropionic acid, preferably L-arginine. The antidandruff shampoo according to claim 15 or 16, characterized by The N-long chain acyl alanine is present in the composition in a weight percentage of 2% or more, preferably 4% or more; and the basic amino acid is present in the composition in a weight percentage of 1% or more, preferably 2% or more. An acne composition characterized in that, The composition comprises the N-long chain acyl alanine and the basic amino acid, and the composition is free or substantially free of retinoids, benzoyl peroxide, antibiotics, vitamins, salicylic acid, fruit acids, prednisone, dexamethasone; and the composition is free or substantially free of sulfate surfactants, ethoxylate surfactants, and fatty acid salt surfactants. The anti-acne composition according to claim 18, characterized in that, The N-long chain acyl alanine is selected from one or more of N-octanoyl-L-alanine, N-decanoyl-L-alanine, N-undecanoyl-L-alanine, N-lauroyl-L-alanine, N-palmitoyl-L-alanine, N-myristoyl-L-alanine, N-pentadecanoyl-L-alanine, N-palmitoyl-L-alanine, N-stearoyl-L-alanine, N-isostearoyl-L-alanine, N-oleoyl-L-alanine, N-linoleoyl-L-alanine, preferably N-lauroyl-L-alanine; and the basic amino acid is selected from one or more of arginine, lysine, citrulline, ornithine, creatine, histidine, diaminobutyric acid, diaminopropionic acid, preferably L-arginine. The anti-acne composition according to claim 18 or 19, characterized in that, The N-long chain acyl alanine is present in the composition in a weight percentage of 2% or more, preferably 4% or more; and the basic amino acid is present in the composition in a weight percentage of 1% or more, preferably 2% or more. A non-therapeutic cosmetic method comprising topically applying to the skin or hair a cosmetic composition for preventing / reducing skin defects caused by acne or reducing the number of comedones / erythema or preventing / reducing dandruff, the cosmetic composition comprising an N-long chain acyl alanine and / or a salt thereof. The cosmetic method according to claim 21, characterized in that The composition comprises the N-long chain acyl alanine and the basic amino acid, or a salt formed between the N-long chain acyl alanine and the basic amino acid. The cosmetic method according to claim 21, characterized in that, The composition comprises a sodium salt, a potassium salt, or an ammonium salt of the N-long chain acyl alanine. The cosmetic method according to any one of claims 21 to 23, characterized in that The N-long chain acyl alanine is selected from one or more of N-octanoyl-L-alanine, N-decanoyl-L-alanine, N-undecanoyl-L-alanine, N-lauroyl-L-alanine, N- myristoyl-L-alanine, N-pentadecanoyl-L-alanine, N-palmitoyl-L-alanine, N-stearoyl-L- alanine, N-isostearoyl-L-alanine, N-oleoyl-L-alanine, N-linoleoyl-L-alanine, preferably N- lauroyl-L-alanine; the basic amino acid is selected from one or more of arginine, lysine, citrulline, ornithine, creatine, histidine, diaminobutyric acid, diaminopropionic acid, preferably L-arginine. Use of a composition comprising N-long chain acyl alanine and / or a salt thereof for the manufacture of a medicament for the treatment of acne. Use of a composition comprising N-long chain acyl alanine and / or a salt thereof for the manufacture of a medicament for the treatment of acne. Use according to claim 25 or 26, characterized in that, The composition comprises N-long chain acyl alanine and a basic amino acid, or a salt of N-long chain acyl alanine and a basic amino acid. Use according to claim 25 or 26, characterized in that, The composition comprises a sodium salt, a potassium salt or an ammonium salt of N-long chain acyl alanine. Use according to any one of claims 25-28, characterized in that The N-long chain acyl alanine is selected from one or more of N-octanoyl-L-alanine, N-decanoyl-L-alanine, N-undecanoyl-L-alanine, N-lauroyl-L-alanine, N- myristoyl-L-alanine, N-pentadecanoyl-L-alanine, N-palmitoyl-L-alanine, N-stearoyl-L- alanine, N-isostearoyl-L-alanine, N-oleoyl-L-alanine, N-linoleoyl-L-alanine, preferably N- lauroyl-L-alanine; the basic amino acid is selected from one or more of arginine, lysine, citrulline, ornithine, creatine, histidine, diaminobutyric acid, diaminopropionic acid, preferably L-arginine. Use of a composition comprising N-long chain acyl alanine and / or a salt thereof for the manufacture of a medicament for the treatment of acne. Use of a composition comprising N-long chain acyl alanine and / or a salt thereof for the manufacture of a medicament for the treatment of acne.

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