Application of torreya grandis polypeptide in scalp care

Abstract

The invention provides application of torreya grandis polypeptide in scalp care, and belongs to the field of plant extracts. The Chinese torreya polypeptide is prepared by taking Chinese torreya cake meal as a raw material and adopting water extraction and acid precipitation, composite enzymolysis and polypeptide separation. The torreya grandis polypeptide has a good anti-inflammatory effect, meanwhile, the growth of restrictive malassezia is effectively inhibited, the growth of propionibacterium acnes and malassezia globosa is promoted, the scalp micro-ecological balance is adjusted, the generation of scurf is improved, and the scalp health is favorably maintained.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant extracts, in particular to a Torreya grandis polypeptide and its application in scalp care. BACKGROUND

[0002] Dandruff is a common, chronic, mild inflammatory, persistent and recurrent scalp health disease that affects about half of the world's population, and its main manifestations are scalp flaking and itching, and damaged hair. In most cases, dandruff is a mild seborrheic dermatitis. Over time, the dead skin, sebum and other impurities that fall off accumulate on the scalp and deposit inside the hair follicles, clog the hair follicles and promote fungal infections such as dandruff, and also hinder hair follicle growth and destroy the ability to produce strong hair, thereby causing hair loss.

[0003] Skin microecological disorder is one of the main factors inducing dandruff. When the balance of skin microecology is destroyed, it can cause excessive proliferation of microorganisms, lead to pathogenic microbial infection, abnormal immune response of skin and a series of phenomena, thereby causing or aggravating dandruff. It is generally believed that Propionibacterium acnes in the scalp of healthy adults plays a beneficial role to the host by secreting antibacterial peptides and participating in immune regulation. Metabolites of Malassezia such as indole [3, 2-b] carbazole, tryptophanone and 6-formylindole can act as ligands of human cell aromatic receptor and play an important role in scalp abnormalities and cell apoptosis and cell cycle. Recent studies have shown that the occurrence of dandruff is not only related to the overgrowth of Malassezia, but also found that there are differences in species level between Malassezia on healthy scalp and dandruff scalp, and there are more Malassezia globosa on healthy scalp, while more Malassezia restricta is gathered on dandruff scalp. Recent studies have found that not only the fungal abundance but also the bacterial abundance changes in dandruff population. The relative abundance of Propionibacterium acnes and Malassezia globosa decreases in the dandruff part, and the relative abundance of Malassezia restricta increases. Therefore, looking for natural active substances that can effectively regulate the relative abundance of bacteria and fungi on the scalp can purposefully regulate the balance of flora.

[0004] Torreya grandis is a coniferous evergreen tree of Taxaceae Torreya, which is a special product in China and a rare economic tree species in the world. It mainly distributes in Zhejiang, Anhui, Fujian, Jiangsu, Guizhou, Hunan and Jiangxi provinces south of the Yangtze River. Torreya grandis cake meal is a by-product of Torreya grandis processing. About 50% of cake meal by-product is produced after Torreya grandis oil production. The protein content in Torreya grandis cake meal is high, which can be further developed into polypeptide products with biological efficacy to improve the added value of products. At present, the research on Torreya grandis polypeptide focuses on the activities of reducing blood pressure and antioxidant, and there is no report on the development and application of other functions.

[0005] Therefore, the torreya grandis cake is used as a raw material to prepare torreya grandis polypeptides, and the functions and applications of the torreya grandis polypeptides are researched, which has great development prospects. SUMMARY

[0006] The application aims to provide an application of torreya grandis polypeptides in scalp care. The torreya grandis polypeptides are prepared by a specific method, which not only has good anti-inflammatory effect, but also effectively limits the growth of Malassezia restricta, promotes the growth of Propionibacterium acnes and Malassezia globosa, regulates the scalp microflora, and thus improves dandruff, which is beneficial to scalp health.

[0007] The specific technical solutions are as follows:

[0008] The application of torreya grandis polypeptides in preparing the following products:

[0009] 1) a product for improving dandruff;

[0010] 2) a product with anti-inflammatory effect;

[0011] 3) a product for regulating the balance of dandruff scalp flora;

[0012] 4) a product for inhibiting the growth of Malassezia restricta;

[0013] 5) a product for promoting the growth of Propionibacterium acnes and / or Malassezia globosa.

[0014] In some embodiments, the concentration of the torreya grandis polypeptides is 0.1-40 mg / mL, preferably 1.25-20 mg / mL, and further preferably 5-20 mg / mL.

[0015] The torreya grandis polypeptides are prepared from torreya grandis cake, including three steps of water extraction and acid precipitation, complex enzymolysis and polypeptide separation, wherein,

[0016] The water extraction and acid precipitation includes extracting effective components from torreya grandis cake powder under alkaline conditions with water, and then precipitating torreya grandis protein under acidic conditions;

[0017] The complex enzymolysis includes enzymolysis of the torreya grandis protein with a complex protease to obtain an enzymolysis product;

[0018] The polypeptide separation includes separating the enzymolysis product into a part with larger molecular weight and a part with smaller molecular weight, preferably, the part with smaller molecular weight is used as the torreya grandis polypeptide, and more preferably, the part with smaller molecular weight has a molecular weight less than 7000 Da.

[0019] In some embodiments, the water extraction and acid precipitation comprises: crushing and sieving the torreya grandis cake, mixing the obtained torreya grandis powder with water, adjusting the pH value to alkaline, oscillating, centrifuging to obtain the supernatant, adjusting the pH value to acidic, centrifuging to obtain the precipitate, and obtaining the torreya grandis protein.

[0020] In some embodiments, the complex enzymolysis comprises: preparing a protein aqueous solution by adding water to the obtained torreya grandis protein, adjusting the pH value and temperature after ultrasonic treatment, adding complex protease, mixing thoroughly, performing enzymolysis, performing enzyme inactivation treatment on the enzymolysis solution after the enzymolysis is completed, and centrifuging to obtain the supernatant.

[0021] In some embodiments, the method for separating the polypeptide is dialysis extraction, comprising: dialyzing the obtained supernatant, and freeze-drying the dialysate and the retentate to obtain the torreya grandis polypeptide, preferably the torreya grandis polypeptide obtained by freeze-drying the dialysate.

[0022] In some embodiments, the torreya grandis cake is crushed and sieved to 40 mesh in the water extraction and acid precipitation.

[0023] In some embodiments, the water used is distilled water.

[0024] In some embodiments, the weight / volume ratio of the torreya grandis powder to water in the water extraction and acid precipitation is 1:5-15, preferably 1:10.

[0025] In some embodiments, the pH value in the first time in the water extraction and acid precipitation ranges from 8.5 to 10.0, preferably 9.0.

[0026] In some embodiments, the oscillation temperature in the water extraction and acid precipitation is 35-50°C, preferably 40°C, and the time is 1.5-3h, preferably 2h.

[0027] In some embodiments, the pH value in the second time in the water extraction and acid precipitation ranges from 3.5 to 4.5, preferably 4.0.

[0028] In some embodiments, the complex protease in the complex enzymolysis is selected from neutral protease and / or papain, and the ratio of enzyme activity is 2-5:5-8, preferably 4:6.

[0029] In some embodiments, the concentration of the protein aqueous solution in the complex enzymolysis is 15-20mg / mL.

[0030] In some embodiments, the pH value in the complex enzymolysis ranges from 5.0 to 9.0, preferably from 6.0 to 8.0, and more preferably 7.0.

[0031] In some embodiments, the temperature in the complex enzymolysis ranges from 40 to 55°C, preferably 50°C.

[0032] In some embodiments, the conditions of the ultrasonic treatment in the complex enzymatic hydrolysis are 100-200W, 10-20min, preferably, the conditions are 150W ultrasonic treatment for 15min.

[0033] In some embodiments, the method of enzyme inactivation in the complex enzymatic hydrolysis is high-temperature enzyme inactivation, preferably, 80-90℃ enzyme inactivation.

[0034] In some specific embodiments, the high-temperature enzyme inactivation is specifically water bath enzyme inactivation, and the enzyme inactivation conditions are 85℃ water bath enzyme inactivation for 25min.

[0035] In some embodiments, the dialysis temperature in the polypeptide separation is 2-8℃, and the dialysis time is 72h-96h, preferably, the dialysis temperature is 4℃, and the dialysis time is 72h.

[0036] In some embodiments, the dialysis molecular weight in the polypeptide separation is 7000Da.

[0037] In some embodiments, the product is an external product or an internal product, the external product is selected from skin care products, cosmetics, hair care products, and the internal product is selected from solid beverages, liquid beverages, dairy products, seasoning powders, nutritional products, and internal medical health care products.

[0038] In some embodiments, the external product is selected from emulsions, gels, creams, microneedle patches, and masks.

[0039] In some embodiments, the external product further contains an auxiliary material, and the auxiliary material contains a preservative ingredient selected from one or more of nipagin methyl ester, nipagin ethyl ester, nipagin propyl ester, formaldehyde releasers, methyl chloroisothiazolinone, methyl isothiazolinone, phenoxyethanol, imidazolidinyl urea, isothiazolinone, phenol, benzoic acid, benzyl alcohol, potassium sorbate, bronopol, p-hydroxyacetophenone, chlorphenesin, triclosan, quaternary ammonium salt-15, dehydroacetic acid and salts thereof.

[0040] In some embodiments, the external product further contains an auxiliary material, and the auxiliary material contains an emulsifying ingredient selected from one or more of polyethylene glycols, glycerol esters, sugar derivatives, anionic emulsifiers, cationic emulsifiers, and phospholipids.

[0041] In some embodiments, the external product further contains an auxiliary material, and the auxiliary material contains a modifying auxiliary material selected from fragrances and / or pigments.

[0042] In some embodiments, the oral product further contains a foodstuff-acceptable auxiliary material containing a flavoring agent: one or more selected from the group consisting of fructooligosaccharide, lactose, stevioside, xylitol, honey, sugar syrup, peppermint oil, instant tea powder, fruit juice powder, edible essence, citric acid.

[0043] In some embodiments, the oral product further contains a foodstuff-acceptable auxiliary material containing a dosage form auxiliary material: one or more selected from the group consisting of maltodextrin, calcium stearate, magnesium stearate, hydroxypropyl methylcellulose, microcrystalline cellulose, silicon dioxide.

[0044] The present application has the beneficial effects of:

[0045] 1) Protease is a general term for a class of enzymes that hydrolyze protein peptide chains. Through action with substrates, different proteases obtain vastly different products. The present application obtains the most effective torreya grandis polypeptide by experimentally combining various proteases and adjusting different parameters.

[0046] 2) The torreya grandis polypeptide of the present application has an effect on the scalp microbial flora. The obtained torreya grandis polypeptide II has an inhibition rate of 60.77% on the restrictive Malassezia CCSM1060, a proliferation rate of 36.33% on Propionibacterium acnes CCSM0331, and a proliferation rate of 50.36% on Malassezia globosa CCSM1050, and has a significant effect of regulating the ratio between bacteria and fungi and restoring the microbial balance of the dandruff population.

[0047] 3) The torreya grandis polypeptide also has an anti-inflammatory effect. The obtained torreya grandis polypeptide II can reduce the level of the inflammatory factor IL-6 produced by LPS (lipopolysaccharide)-induced macrophages RAW264.7, with an inhibition rate of 47.02%.

[0048] 4) The torreya grandis polypeptide of the present application comprehensively treats dandruff from the perspective of maintaining scalp microecological balance and repairing inflammation, and has a wide application prospect in the development of scalp care products for the dandruff population. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 Inhibition rates of torreya grandis polypeptide I, torreya grandis polypeptide II, and torreya grandis polypeptide III on Propionibacterium acnes CCSM0331 at various concentrations;

[0050] Figure 2 Inhibition rates of torreya grandis polypeptide I, torreya grandis polypeptide II, and torreya grandis polypeptide III on Malassezia globosa CCSM1050 at various concentrations;

[0051] Figure 3 Inhibition rates of torreya grandis polypeptide I, torreya grandis polypeptide II, and torreya grandis polypeptide III on restrictive Malassezia CCSM1060 at various concentrations;

[0052] Figure 4Toxicity of Torreya grandis Polypeptide II on macrophage RAW264.7 cells at various concentrations;

[0053] Figure 5 Effect of Torreya grandis Polypeptide II at a concentration of 5 mg / mL on expression of inflammatory factor IL-6 by LPS-induced macrophage RAW264.7. DETAILED DESCRIPTION

[0054] The present application will be described in detail below with reference to the accompanying drawings and specific examples. The present embodiment is implemented on the premise of the technical solution of the present application, and detailed implementation and specific operation processes are given, but the protection scope of the present application is not limited to the following examples.

[0055] The purpose of the present application is to provide an application of Torreya grandis Polypeptide in preparing products as follows:

[0056] 1) products for improving dandruff effect;

[0057] 2) products with anti-inflammatory effect;

[0058] 3) products for regulating dandruff Malassezia flora balance;

[0059] 4) products for inhibiting growth of Malassezia restricta;

[0060] 5) products for promoting growth of Propionibacterium acnes and / or Malassezia globosa.

[0061] In some embodiments, the concentration of the Torreya grandis Polypeptide is 0.1-40 mg / mL, preferably 1.25-20 mg / mL, and further preferably 5-20 mg / mL.

[0062] The Torreya grandis Polypeptide is prepared from Torreya grandis cake meal, including three steps of water extraction and acid precipitation, complex enzymolysis, and polypeptide separation, wherein,

[0063] The water extraction and acid precipitation includes extracting effective components from Torreya grandis cake meal powder under alkaline conditions with water, and then precipitating Torreya grandis protein under acidic conditions;

[0064] The complex enzymolysis includes enzymolysis of the Torreya grandis protein with complex protease to obtain an enzymolysis product;

[0065] The polypeptide separation includes separating the enzymolysis product into a part with larger molecular weight and a part with smaller molecular weight, preferably, the part with smaller molecular weight is used as the Torreya grandis Polypeptide, and more preferably, the part with smaller molecular weight has a molecular weight less than 7000 Da.

[0066] In some embodiments, the water extraction and acid precipitation comprises: crushing the Torreya seed cake and sieving, mixing the obtained Torreya seed powder with water, adjusting the first pH value to alkaline, centrifuging and taking the supernatant after oscillation, adjusting the second pH value to acidic, centrifuging and taking the precipitate, and obtaining the Torreya protein.

[0067] In some embodiments, the complex enzymolysis comprises: preparing a protein aqueous solution by adding water to the obtained Torreya protein, adjusting the pH value and temperature after ultrasonic treatment, adding complex protease, mixing thoroughly, performing enzymolysis, performing enzyme inactivation treatment on the enzymolysis solution after the enzymolysis is completed, and obtaining the supernatant by centrifugation.

[0068] In some embodiments, the method for separating the polypeptide is dialysis extraction, which comprises: dialyzing the obtained supernatant, and freeze-drying the dialysate and the retentate after dialysis to obtain the Torreya polypeptide, preferably the Torreya polypeptide obtained by freeze-drying the dialysate.

[0069] The term "Torreya" refers to a perennial evergreen tree of the Taxaceae family. Common species include the wild Torreya (Torreya grandis Fort. et Lindl.) produced in China, also known as Xiangjiao, Yangjiaojiao, Xiaojiao, Yujiao, Yeshu, and so on; Florida Torreya (Torreya taxifolia Am.) and California Torreya (Torreya californica Torrey) produced in North America; and Japanese Torreya (Torreya nucifera (L.) Sieb. et Zucc.) produced in Japan.

[0070] In some specific embodiments, the Torreya is Torreya grandis Fort. et Lindl.

[0071] The term "Torreya seed cake" refers to a processing byproduct of Torreya. After Torreya oil is extracted, a byproduct of about 50% cake is produced, and the Torreya seed cake has a high protein content.

[0072] In some embodiments, the Torreya seed cake is crushed to pass through a 40-mesh sieve to obtain Torreya seed powder in the water extraction and acid precipitation.

[0073] In some embodiments, the water used is distilled water.

[0074] In some embodiments, the weight-to-volume ratio of the Torreya seed powder to water in the water extraction and acid precipitation is 1:5-15, preferably 1:10.

[0075] In some embodiments, the first pH value in the water extraction and acid precipitation ranges from 8.5 to 10.0, preferably 9.0.

[0076] In some embodiments, the temperature of the water extraction and acid precipitation is 35-50°C, preferably 40°C, and the time is 1.5-3h, preferably 2h.

[0077] In some embodiments, the second pH value in the water extraction and acid precipitation is 3.5-4.5, preferably 4.0.

[0078] In some embodiments, the complex protease in the complex enzymolysis is selected from neutral protease and / or papain, and the ratio of enzyme activity is 2-5:5-8, preferably 4:6.

[0079] The term "enzyme activity" also referred to as enzymatic activity, refers to the ability of an enzyme to catalyze a chemical reaction, and under optimal conditions, the amount of enzyme required to convert 1 micromole of substrate per minute is one unit of enzyme activity (U).

[0080] In some embodiments, the concentration of the protein aqueous solution in the complex enzymolysis is 15-20mg / mL.

[0081] In some embodiments, the pH value in the complex enzymolysis is 5.0-9.0, preferably 6.0-8.0, and further preferably 7.0.

[0082] In some embodiments, the temperature in the complex enzymolysis is 40-55°C, preferably 50°C.

[0083] In some embodiments, the ultrasonic treatment in the complex enzymolysis is 100-200W for 10-20min, preferably 150W for 15min.

[0084] In some embodiments, the enzyme inactivation method in the complex enzymolysis is high-temperature enzyme inactivation, preferably 80-90°C enzyme inactivation.

[0085] The currently commonly used enzyme inactivation methods mainly include two categories of physical methods and chemical methods. The physical method refers to inactivating the enzyme by physical treatment, which usually includes high-temperature treatment, low-temperature treatment, radiation treatment and ultrasonic treatment. The chemical method refers to inactivating the enzyme by adding chemical reagents or changing the chemical environment, which usually includes acid-base treatment, metal ion treatment, reducing agent treatment and enzyme inhibitor treatment, etc. In order to prevent the introduction of impurities, the physical enzyme inactivation method is preferred in the present application.

[0086] In some specific embodiments, the high-temperature enzyme inactivation is specifically water bath enzyme inactivation, and the enzyme inactivation condition is 85°C water bath enzyme inactivation for 25min.

[0087] In some embodiments, the dialysis temperature in the polypeptide separation is 2-8°C, and the dialysis time is 72h-96h, preferably the dialysis temperature is 4°C, and the dialysis time is 72h.

[0088] In some embodiments, the polypeptide is isolated with a dialysis molecular weight of 7000 Da.

[0089] In some specific embodiments, the product with the above-mentioned efficacy is a product for external use or a product for internal use.

[0090] The product for external use is selected from the group consisting of a skin care product, a cosmetic product, a hair care product, preferably a skin care product and / or a cosmetic product;

[0091] The product for internal use is selected from the group consisting of a solid beverage, a liquid beverage, a dairy product, a seasoning powder, a nutritional product, a medical health care product for internal use.

[0092] In some embodiments, the product for external use is selected from the group consisting of an emulsion, a gel, a cream, a microneedle patch, a mask.

[0093] In some embodiments, the product for external use further contains an auxiliary material containing a preservative ingredient selected from one or more of the group consisting of methylparaben, ethylparaben, propylparaben, formaldehyde releasers, methylchloroisothiazolinone, methylisothiazolinone, phenoxyethanol, imidazolidinyl urea, isothiazolinone, phenol, benzoic acid, benzyl alcohol, potassium sorbate, bronopol, p-hydroxyacetophenone, chlorphenesin, triclosan, quaternary ammonium salt-15, dehydroacetic acid and salts thereof.

[0094] In some embodiments, the product for external use further contains an auxiliary material containing an emulsifying ingredient selected from one or more of the group consisting of polyethylene glycols, glycerol esters, sugar derivatives, anionic emulsifiers, cationic emulsifiers, phospholipids.

[0095] In some embodiments, the product for external use further contains an auxiliary material containing a modifying auxiliary material selected from the group consisting of fragrances and / or pigments.

[0096] The product for internal use according to the present application can include all foods in the conventional sense, and can be used interchangeably with the terms known in the art, such as functional foods and health functional foods.

[0097] When the Torreya grandis polypeptide according to the present application is used as a food additive, it can be directly added or used with other foods or food ingredients, and can be appropriately used according to conventional methods.

[0098] In some embodiments, the product for internal use further contains a food-acceptable auxiliary material containing a flavoring agent selected from one or more of the group consisting of fructooligosaccharides, lactose, steviol glycosides, xylitol, honey, sugar syrup, peppermint oil, instant tea powder, fruit juice powder, food flavor, citric acid.

[0099] In some embodiments, the oral product further contains a food-acceptable adjuvant, which contains a dosage form adjuvant: one or more selected from the group consisting of malt dextrin, calcium stearate, magnesium stearate, hydroxypropyl methyl cellulose, microcrystalline cellulose, and silicon dioxide.

[0100] The present application is further illustrated by the following examples without thereby limiting the present application to the examples described. The raw materials or processing techniques in the following examples, if not specifically stated, are all conventional commercially available raw material products or conventional processing techniques in the art.

[0101] Chinese torreya cake: The source is the cake after the oil extraction of torreya grandis.

[0102] Experimental strains: Propionibacterium acnes CCSM0331: isolated from human healthy facial skin; Malassezia globosa CCSM1050, Malassezia restricta CCSM1060: isolated from scalp skin;

[0103] Culture medium: Tryptic soy agar medium (TSA): tryptone 15 g / L, soybean papain hydrolysate 5 g / L, sodium chloride 5 g / L, agar 15 g / L, 121 ℃ high pressure sterilization for 15 min, pour flat plate when cooled to about 55 ℃;

[0104] Tryptic soy broth (TSB): tryptone 17 g / L, soybean papain hydrolysate 3 g / L, sodium chloride 5 g / L, potassium phosphate dibasic 2.5 g / L, glucose 2.5 g / L, 121 ℃ high pressure sterilization for 15 min, ready for use;

[0105] ATCC medium: 2693 modified Dixon (mDixon): malt extract powder 36 g / L, oxgall 20 g / L, proteose peptone 6 g / L, glycerol 2 g / L, oleic acid 2 g / L, 121 ℃ high pressure sterilization for 15 min, ready for use.

[0106] Protease: alkaline protease, neutral protease and papain were purchased from solarbio company.

[0107] Example 1 Preparation of torreya grandis polypeptide I and torreya grandis polypeptide II

[0108] The torreya grandis polypeptide of the present application is prepared from torreya grandis cake, including three steps of water extraction and acid precipitation, complex enzymolysis and polypeptide separation:

[0109] (1) Water extraction and acid precipitation:

[0110] ①Torreya grandis cake was crushed through a 40-mesh sieve to obtain torreya grandis seed powder;

[0111] ②Mix the Chinese torreya seed powder with distilled water at a ratio of 1:10 (w / v), adjust the pH to 9.0, and then extract at 40°C for 2 hours. Centrifuge at high speed, filter the supernatant, and obtain the filtrate.

[0112] ③Adjust the pH of the supernatant obtained in step ② to 4.0 to precipitate the protein. Collect the protein precipitate by high-speed centrifugation, and obtain Chinese torreya protein.

[0113] (2) Complex enzymolysis:

[0114] Using the Chinese torreya protein obtained in step (1) as the raw material, prepare a 20 mg / mL protein aqueous solution. Ultrasonically treat at 150 W for 15 min, then adjust the pH to 7.0 and the temperature to 50°C. Add neutral protease and papain at a ratio of 4:6 in terms of enzyme activity, and a total of 7000 U / g, to perform enzymolysis. After the enzymolysis is completed, place the container containing the enzymolysis solution in a 85°C water bath to inactivate the enzyme for 25 min. After the enzyme inactivation is completed, centrifuge at high speed, and obtain the supernatant.

[0115] (3) Polypeptide separation:

[0116] Dialyze the obtained supernatant at 4°C, with a molecular weight cut-off of 7000 Da. Freeze-dry the retained liquid and the dialysate after dialysis, respectively. Freeze-dry the retained liquid to obtain Chinese torreya polypeptide I (a part with a molecular weight greater than 7000 Da), and freeze-dry the dialysate to obtain Chinese torreya polypeptide II (a part with a molecular weight less than 7000 Da).

[0117] Example 2 Preparation of Chinese torreya polypeptide III

[0118] Compared with Example 1, the difference is that, in the complex enzymolysis of step (2), the pH is adjusted to 8.0, and alkaline protease and papain are added at a ratio of 3:7 in terms of enzyme activity, and a total of 7000 U / g, to perform enzymolysis. Freeze-dry the dialysate after dialysis to obtain Chinese torreya polypeptide III (a part with a molecular weight less than 7000 Da).

[0119] Example 3 Effect of Chinese torreya polypeptide on the proliferation of Propionibacterium acnes

[0120] (1) Prepare aqueous solutions of Chinese torreya polypeptide I, Chinese torreya polypeptide II, and Chinese torreya polypeptide III at different concentrations, respectively, with the concentrations being 40, 20, 10, 5, and 2.5 mg / mL. After sterilization by passing through a 0.22 μm filter, mix with sterile double-concentration TSB medium at a ratio of 1:1 to obtain Chinese torreya polypeptide solutions with concentrations of 20, 10, 5, 2.5, and 1.25 mg / mL.

[0121] (2) Preparation of P. acnes CCSM0331 bacterial suspension, the preserved P. acnes CCSM0331 strain milk powder freeze-dried tube was taken out from the -80 °C refrigerator, the milk powder was picked up with a disposable inoculation ring on a TSA agar plate, and the three-zone streaking method was used to streak and place in a 37 °C incubator for anaerobic culture for 72 h as a first generation activated plate. Single colonies were picked from the first generation plate and streaked on a second generation plate and placed in a 37 °C incubator for anaerobic culture for 72 h. P. acnes CCSM0331 activated for two generations was picked for single colony inoculation in a conical flask containing 100 mL of TSB liquid medium, and placed in a 37 °C anaerobic incubator for 60 h. The OD 600 value was adjusted to 0.3000-0.4000, and the dilution was continued by 1000 times for standby;

[0122] (3) 96-well plate single hole added the following solution, each group made 4 duplicate wells:

[0123]

[0124] (4) The 96-well plate was incubated at 37 °C for 72 h, and the OD 600 value was measured using an enzyme marker, and the inhibition rate was calculated, and the results are shown in Figure 1 .

[0125] Inhibition rate = 1- (experimental group OD value-experimental blank group OD value) / (growth control hole OD value-sterile control hole OD value) x 100%

[0126] (5) Experimental results

[0127] As can be seen from Figure 1 , the Carya cathayensis polypeptide II inhibits the growth of P. acnes CCSM0331 at low concentrations, and significantly promotes the growth of P. acnes CCSM0331 at a concentration of ≥10 mg / mL (P<0.001), and reaches the maximum at 20 mg / mL, with a proliferation rate of 36.33%. The Carya cathayensis polypeptide I promotes the growth of P. acnes CCSM0331 at low concentrations, and inhibits the growth of P. acnes CCSM0331 at a concentration of ≥5 mg / mL. Although Carya cathayensis polypeptide III promotes the growth of P. acnes CCSM0331 at a concentration of ≥10 mg / mL, the promotion effect is poor.

[0128] Example 4 Effect of Carya cathayensis polypeptide on Malassezia proliferation

[0129] (1) Aqueous solutions of Torreya grandis polypeptide I, Torreya grandis polypeptide II, and Torreya grandis polypeptide III with different concentrations were prepared, with concentrations of 40, 20, 10, 5, and 2.5 mg / mL, respectively. After sterilization through a 0.22 μm filter membrane, they were mixed with sterile double-concentration mDixon medium at a ratio of 1:1 to obtain Torreya grandis polypeptide solutions with concentrations of 20, 10, 5, 2.5, and 1.25 mg / mL;

[0130] (2) Prepare a suspension of Malassezia. Take out the freeze-dried milk powder tubes of the preserved Malassezia globosa CCSM1050 and Malassezia restricta CCSM1060 strains from a -80°C refrigerator, pick the milk powder with a disposable inoculation loop and place it on an mDixon agar plate. Use the three-zone streak method to streak the plate and place it in a 32°C constant temperature incubator for 72 hours to form a first-generation activation plate. Pick a single colony from the colonies grown on the first-generation plate and streak it on a second-generation plate and place it in a 32°C constant temperature incubator for 72 hours. Pick a single colony from the two-generation activated Malassezia and inoculate it into a conical flask containing 100 mL of mDixon liquid culture medium, place it in a 32°C shaker, and shake and culture it at a speed of 150 r / min for 72 hours to adjust the bacterial OD. 600 In the range of 0.7-0.8, continue to dilute 100 times for later use;

[0131] (3) Add the following solution to each well of a 96-well plate, with 4 replicates per group:

[0132]

[0133] (4) The 96-well plate was incubated at 32°C for 72 h and the OD was measured using a microplate reader. 600 The inhibition rate was calculated and the results were as follows: Figure 2 and Figure 3 shown.

[0134] Inhibition rate = 1-(OD value of experimental group-OD value of experimental blank group) / (OD value of growth control well-OD value of sterile control well) × 100%

[0135] (5) Experimental results

[0136] Depend on Figure 2 It can be seen that Torreya grandis polypeptide II has a promoting effect on Malassezia globosa CCSM1050, and it also promotes the growth of Malassezia globosa CCSM1050 at a lower concentration (1.25 mg / mL). It significantly promotes the growth of Malassezia globosa CCSM1050 at a concentration of 20 mg / mL (P<0.001), with an increase rate of 50.36%. Torreya grandis polypeptide I only promotes the growth of Malassezia globosa CCSM1050 at a concentration of ≥5 mg / mL. Although Torreya grandis polypeptide III promotes the growth of Malassezia globosa CCSM1050 at a concentration of ≥2.5 mg / mL, the promoting effect is poor.Figure 3 It can be seen that Torreya grandis polypeptides I, II and III all inhibited the growth of Malassezia restricta CCSM1060, and Torreya grandis polypeptide II had the highest inhibition rate of 70.25% at a concentration of 10 mg / mL.

[0137] Example 5: Test of Torreya grandis polypeptide in reducing LPS-induced IL-6 production in macrophage RAW264.7 cells

[0138] (1) RAW264.7 cell viability assay

[0139] RAW264.7 cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum under the conditions of 5% CO2 and 37°C. When the cells grew to 80-90% confluence, they were digested with trypsin and plated at 8×10 5 Cells were seeded in 96-well plates at a density of 10 cells / mL. After 24 hours of cell attachment, the cells were treated with different concentrations of the test sample for 24 hours. A solvent control group was also established using high-glucose DMEM medium supplemented with 10% fetal bovine serum. Three parallel wells were set up for each experimental group. After washing the cells once with DPBS, cell viability was measured at 450 nm using a CCK-8 kit (Dojindo Laboratories, CK04).

[0140] Relative cell activity % = (OD value of sample group / OD value of solvent control group) × 100%

[0141] (2) LPS-induced IL-6 content in RAW264.7 cells

[0142] The cell lines were cultured in a high-glucose DMEM medium containing 10% fetal bovine serum at 37°C with 5% CO2. When the cells grew to 80-90% confluence, they were digested with trypsin and plated at 8×10 5 Cells were seeded in 96-well plates at a density of 10 cells / ml. After 24 hours of cell attachment, the cells were treated with the sample, with three parallel wells set up for each group. Supernatants were collected 24 hours after treatment and assayed for IL-6 inflammatory factors using an IL-6 ELISA kit.

[0143] IL-6 relative production % = (IL-6 content in sample group / IL-6 content in solvent control group) × 100%.

[0144] (3) Experimental results

[0145] from Figure 4 It can be seen that compared with the solvent control group, the addition of Torreya grandis polypeptide II at a concentration of 0.1-10 mg / mL did not show cytotoxicity, and could significantly promote the growth of macrophage RAW264.7 cells (P<0.01).

[0146] According to the experimental results of cell viability, the highest dose possible was selected to achieve the optimal effect, but the highest concentration of protein can have a pro-inflammatory effect, so 5 mg / mL of Torreya grandis polypeptide II was selected for the LPS (1 μg / mL) induced cell inflammation experiment. The experimental results show that Figure 5 ), normal cells produce a small amount of IL-6 cytokines, and after LPS treatment, the secretion of inflammatory cytokine IL-6 is significantly increased (P<0.001), and after the addition of Torreya grandis polypeptide II, the amount of inflammatory cytokine IL-6 can be significantly reduced (P<0.001), and the inhibition rate of Torreya grandis polypeptide II on inflammatory cytokine IL-6 is 47.02%. It can be seen that Torreya grandis polypeptide II can significantly reduce the expression of inflammatory cytokine IL-6 caused by LPS, and shows good anti-inflammatory effect.

[0147] The above examples are only preferred embodiments of the present application, and are not any form and substantial limitation of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the present application, a number of improvements and supplements can also be made, which should be considered as the protection scope of the present application.

Claims

1. Use of Torreya grandis polypeptide in the preparation of products: 1) products for improving dandruff effect; 2) products with anti-inflammatory effect; 3) products for regulating dandruff scalp flora balance; 4) products for inhibiting growth of limited Malassezia restricta; 5) products for promoting growth of Propionibacterium acnes and / or Malassezia globosa.

2. Use according to claim 1, wherein The concentration of the Torreya grandis polypeptide is 0.1-40 mg / mL, preferably 1.25-20 mg / mL, and further preferably 5-20 mg / mL.

3. Use according to claim 1 or 2, characterized in that, The Torreya grandis polypeptide is prepared from Torreya grandis cake meal, including three steps of water extraction and acid precipitation, complex enzymolysis and polypeptide separation, wherein, The water extraction and acid precipitation comprises extracting effective components from Torreya grandis cake meal powder under alkaline conditions with water, and then precipitating Torreya grandis protein under acidic conditions; The complex enzymolysis comprises enzymolysis of Torreya grandis protein with complex protease to obtain enzymolysis products; The polypeptide separation comprises separating the enzymolysis products into a part with larger molecular weight and a part with smaller molecular weight, and preferably, the part with smaller molecular weight is used as the Torreya grandis polypeptide, and more preferably, the part with smaller molecular weight has a molecular weight less than 7000 Da.

4. The use according to claim 3, wherein 1) the water extraction and acid precipitation comprises: sieving Torreya grandis cake meal after crushing, mixing Torreya grandis seed powder obtained with water, adjusting the first pH value to alkaline, oscillating, centrifuging to obtain supernatant and filtering, adjusting the second pH value to acidic, centrifuging to obtain precipitate, and obtaining Torreya grandis protein; 2) the complex enzymolysis comprises: adding water to Torreya grandis protein prepared in step 1) to prepare a protein aqueous solution, treating with ultrasonic, adjusting the pH value and temperature, adding complex protease, mixing thoroughly, performing enzymolysis, performing enzyme inactivation treatment on the enzymolysis solution after the enzymolysis is completed, and centrifuging to obtain supernatant; 3) the polypeptide separation method is dialysis extraction, comprising: dialyzing the supernatant obtained in step 2), and freeze-drying the dialysate and the retentate obtained after dialysis to obtain the Torreya grandis polypeptide, and preferably, freeze-drying the dialysate to obtain the Torreya grandis polypeptide.

5. Use according to claim 3 or 4, wherein the compound is ###0002### Step 1) The water extraction and acid precipitation has one or more of the following characteristics, 1) the weight / volume ratio of the Torreya grandis seed powder to water is 1:5-15, and preferably 1:10; 2) the first pH value ranges from 8.5 to 10.0, and preferably 9.0; 3) the oscillation temperature is 35-50°C, and preferably 40°C, and the time is 1.5-3 h, and preferably 2 h; 4) the second pH value ranges from 3.5 to 4.5, and preferably 4.

0.

6. Use according to any one of claims 3 to 5, wherein the compound is of formula (I) ###0001### (I) or a pharmaceutically acceptable salt thereof. Step 2) The complex enzymolysis has one or more of the following characteristics, 1) the complex protease is selected from neutral protease and / or papain, and the ratio of enzyme activity is 2-5:5-8, and preferably 4:6; 2) the concentration of the protein aqueous solution is 15-20 mg / mL; 3) the pH value ranges from 5.0 to 9.0, and preferably from 6.0 to 8.0, and further preferably 7.0; 4) the temperature ranges from 40 to 55°C, and preferably 50°C; 5) the ultrasonic treatment is at 100-200 W for 10-20 min, preferably at 150 W for 15 min. 6) the enzyme inactivation treatment is by high temperature, preferably at 80-90 °C, more preferably at 85 °C.

7. Use according to any one of claims 3 to 6, wherein the compound is of formula (I) ###00002### (I) or a pharmaceutically acceptable salt thereof. Step 3) the polypeptide is isolated by one or more of the following characteristics, 1) the dialysis is at 2-8 °C for 72-96 h, preferably at 4 °C for 72 h; 2) the dialysis is at a molecular weight of 7000 Da.

8. Use according to any one of claims 1 to 7, wherein The product is an external product or an internal product, the external product is selected from skin care products, cosmetics, hair care products; the internal product is selected from solid beverages, liquid beverages, dairy products, seasoning powders, nutritional products, internal medical health products.

9. Use according to claim 8, wherein the compound is ###0002### The external product is selected from emulsions, gels, creams, microneedle patches, masks; Preferably, the external product further contains an auxiliary material, which has any one or more of the following characteristics: 1) contains a preservative ingredient: the preservative ingredient is selected from one or more of the following: methylparaben, ethylparaben, propylparaben, formaldehyde releasers, methylchloroisothiazolinone, methylisothiazolinone, phenoxyethanol, imidazolidinyl urea, isothiazolinone, phenol, benzoic acid, benzyl alcohol, potassium sorbate, bronopol, p-hydroxyacetophenone, chlorphenesin, triclosan, quaternary ammonium salt-15, dehydroacetic acid and its salts; 2) contains an emulsifying ingredient: the emulsifying ingredient is selected from one or more of the following: polyethylene glycol, glycerol ester, sugar derivative, anionic emulsifier, cationic emulsifier, phospholipid; 3) contains a modified auxiliary material: the modified ingredient is selected from fragrance and / or pigment.

10. The use according to claim 7, wherein the compound is ###00007### The internal product further contains a food-scientifically acceptable auxiliary material, which has any one or more of the following characteristics: 1) contains a flavoring agent, the flavoring agent is selected from one or more of the following: oligofructose, lactose, stevioside, xylitol, honey, syrup, peppermint oil, instant tea powder, fruit juice powder, food flavor, citric acid; 2) contains a dosage form auxiliary material, the dosage form auxiliary material is selected from one or more of the following: malt dextrin, calcium stearate, magnesium stearate, hydroxypropyl methyl cellulose, microcrystalline cellulose, silicon dioxide.

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