Trypsin inhibitor and application thereof

Abstract

The invention provides a trypsin inhibitor and application thereof, and relates to the technical field of molecular biology. According to the invention, it is found for the first time that zinc pyrithione, ketoconazole and / or selenium disulfide have an inhibition effect on trypsin activity, and new functional components which are convenient to obtain are provided for the trypsin inhibitor. The invention provides three compositions containing zinc hyaluronate, the zinc hyaluronate is respectively compounded with zinc pyrithione, ketoconazole or selenium disulfide according to a specific mass ratio, and it is found for the first time that the zinc hyaluronate and the zinc pyrithione, ketoconazole or selenium disulfide show the activity of synergistically inhibiting trypsin. The composition provided by the invention is simple to obtain, can be continuously compounded with other functional active components or auxiliary material components to obtain commercial products, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology, and particularly relates to a trypsin inhibitor and application thereof. BACKGROUND

[0002] Zinc hyaluronate is a zinc salt of hyaluronic acid, which is prepared by metal ion exchange between zinc ions and carboxyl groups of hyaluronate. In addition to the functions of both, it also significantly improves the biocompatibility and safety of zinc ions.

[0003] Zinc pyrithione (ZPT) is widely used as an antibacterial agent in shampoos and hair care ingredients. Its application on the scalp mainly includes dandruff control, control of seborrheic dermatitis, and maintenance of scalp health.

[0004] Ketoconazole is a widely used antifungal drug, which belongs to imidazole derivatives. This drug has inhibitory and bactericidal activity against a variety of fungi and is used to treat and prevent infections caused by various fungi. In the application of the scalp, ketoconazole is usually used as an external lotion or shampoo to treat seborrheic dermatitis, dandruff and other scalp problems.

[0005] Selenium disulfide is commonly used in shampoo products, which can regulate cell metabolism on the scalp, reduce the rate of cell shedding, and thus reduce the generation of dandruff. It is also an antifungal ingredient used to treat tinea versicolor.

[0006] Proteases mainly have two functions in the body: one is to degrade proteins as exogenous proteases, thereby affecting protein synthesis and accelerating protein degradation; the other is to interact with endogenous proteases to regulate various life activities of body cells. Trypsin, as an important active enzyme component in the protease system, has a key influence on protein decomposition and synthesis in the human body. In addition, the strength of trypsin activity also has an important influence on the extraction of insulin. When the activity of trypsin is high, it is difficult to extract insulin, and when the activity of trypsin is inhibited, a larger amount of insulin can be extracted.

[0007] In recent years, there have been continuous research and reports on protease inhibiting ingredients. Some results show that protease inhibiting ingredients can participate in many life activities and have various physiological functions. However, most trypsin inhibiting ingredients are polypeptide or protein products, which are difficult to obtain in practical applications and cannot be widely used. SUMMARY

[0008] To solve the above problems, the purpose of the present application is to provide a new component of trypsin inhibitor, trypsin inhibitor and application thereof, which is convenient to obtain and can be widely used.

[0009] In one aspect, the present application provides the use of zinc pyrithione, ketoconazole and / or selenium disulfide in the preparation of a trypsin inhibitor.

[0010] It is first discovered in the present application that pyrithioxin zinc, ketoconazole and / or selenium disulfide have inhibitory effect on trypsin activity, which provides new and convenient efficacy ingredients for trypsin inhibitors.

[0011] Preferably, the trypsin inhibition rate of pyrithioxin zinc is greater than or equal to 40%.

[0012] Preferably, the trypsin inhibition rate of ketoconazole is greater than or equal to 10%; preferably, greater than or equal to 13%.

[0013] Preferably, the trypsin inhibition rate of selenium disulfide is greater than or equal to 10%; preferably, greater than or equal to 12%.

[0014] In a preferred embodiment, the concentration of pyrithioxin zinc, ketoconazole and / or selenium disulfide in the trypsin inhibitor is 0.1%. The concentration can be adjusted by those skilled in the art according to actual needs, and therefore the present application does not make specific limitations on this content.

[0015] Further, the trypsin inhibitor further comprises hyaluronic acid or a salt thereof; preferably, the hyaluronic acid or a salt thereof comprises one or more of hyaluronic acid, zinc hyaluronate, sodium hyaluronate, potassium hyaluronate; more preferably, zinc hyaluronate.

[0016] Preferably, the molecular weight of the hyaluronic acid or a salt thereof can be 1 kDa-2000 kDa, 1 kDa-1000 kDa, 1 kDa-100 kDa, 100 kDa-1000 kDa, 500 kDa-1000 kDa, 100 kDa-500 kDa, 200 kDa-800 kDa, and any interval thereof.

[0017] Alternatively, the molecular weight of the hyaluronic acid or a salt thereof can be any value in the range of 1 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, 600 kDa, 650 kDa, 700 kDa, 750 kDa, 800 kDa, 850 kDa, 900 kDa, 950 kDa, 1000 kDa, 1100 kDa, 1200 kDa, 1300 kDa, 1400 kDa, 1500 kDa, 1600 kDa, 1700 kDa, 1800 kDa, 1900 kDa, 2000 kDa.

[0018] The molecular weight can be adjusted by those skilled in the art according to actual needs, and therefore no specific limitation is made in this application.

[0019] It is first discovered in this application that pyrithione zinc, ketoconazole and / or selenium disulfide has a significant synergistic effect of inhibiting trypsin activity when compounded with hyaluronic acid or its salt, thus providing a new and convenient composition selection for trypsin inhibitors.

[0020] Preferably, the mass ratio of pyrithione zinc, ketoconazole and / or selenium disulfide to hyaluronic acid or its salt is (1-110):1.

[0021] Preferably, the mass ratio of hyaluronic acid or its salt to pyrithione zinc is (10-110):1; preferably (20-100):1; more preferably (20-50):1.

[0022] Preferably, the mass ratio of hyaluronic acid or its salt to ketoconazole is (10-100):1; preferably (10-61.5):1; more preferably (20-50):1.

[0023] Preferably, the mass ratio of hyaluronic acid or its salt to selenium disulfide is (1-110):1; preferably (10-100):1.

[0024] In a preferred embodiment, the hyaluronic acid or its salt and pyrithione zinc synergistically inhibit trypsin activity, and the trypsin inhibition rate is greater than or equal to 64%; preferably greater than or equal to 66%; preferably greater than or equal to 70%.

[0025] In a preferred embodiment, the hyaluronic acid or its salt and ketoconazole synergistically inhibit trypsin activity, and the trypsin inhibition rate is greater than or equal to 65%; preferably greater than or equal to 68%.

[0026] In a preferred embodiment, the hyaluronic acid or its salt and selenium disulfide synergistically inhibit trypsin activity, and the trypsin inhibition rate is greater than or equal to 65%; preferably greater than or equal to 67%.

[0027] In a preferred embodiment, the concentration of the composition compounded by pyrithione zinc, ketoconazole and / or selenium disulfide and hyaluronic acid or its salt is 0.1%. Those skilled in the art can adjust the concentration according to actual needs, and therefore no specific limitation is made in this application.

[0028] In another aspect, the application also provides a trypsin inhibitor, which at least includes one or more of pyrithione zinc, ketoconazole and selenium disulfide.

[0029] Further, the trypsin inhibitor further comprises hyaluronic acid or a salt thereof; preferably, the hyaluronic acid or a salt thereof at least comprises one or more of hyaluronic acid, zinc hyaluronate, sodium hyaluronate, potassium hyaluronate; more preferably, zinc hyaluronate.

[0030] Preferably, the hyaluronic acid or a salt thereof has a molecular weight of 1 kDa-1000 kDa, further preferably, a molecular weight of 100 kDa-1000 kDa, and yet further preferably, a molecular weight of 500 kDa-1000 kDa.

[0031] Preferably, the mass ratio of the zinc pyrithione, ketoconazole and / or selenium disulfide to the hyaluronic acid or a salt thereof is (1-110):1.

[0032] Alternatively, the mass ratio of the zinc pyrithione, ketoconazole and / or selenium disulfide to the hyaluronic acid or a salt thereof can be any one of 1:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 110:1.

[0033] In a preferred embodiment, the concentration of the composition of the zinc pyrithione, ketoconazole and / or selenium disulfide compounded with the hyaluronic acid or a salt thereof in the trypsin inhibitor is 0.1%.

[0034] Further, the mass ratio of the hyaluronic acid or a salt thereof to the zinc pyrithione is (10-110):1; preferably, (20-100):1; more preferably, (20-50):1.

[0035] Alternatively, the mass ratio of the hyaluronic acid or a salt thereof to the zinc pyrithione can be any one of 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 61.5:1, 70:1, 80:1, 90:1, 100:1, 110:1.

[0036] Alternatively, the mass ratio of the hyaluronic acid or a salt thereof to the zinc pyrithione is 20:1.

[0037] In a preferred embodiment, the trypsin inhibitor comprises, in terms of the mass percentage of the trypsin inhibitor, 0.09524%-0.09901% of the hyaluronic acid or a salt thereof and 0.00099%-0.00476% of the zinc pyrithione.

[0038] Further, the mass ratio of the hyaluronic acid or a salt thereof to the ketoconazole is (10-100):1; preferably, (10-61.5):1; more preferably, (20-50):1.

[0039] Optionally, the mass ratio of the hyaluronic acid or salt thereof to ketoconazole can be any of 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 61.5:1, 70:1, 80:1, 90:1, 100:1.

[0040] In a preferred embodiment, the trypsin inhibitor comprises, in mass percentage of the trypsin inhibitor, 0.09524%-0.0980% of hyaluronic acid or salt thereof, 0.00196%-0.00476% of ketoconazole.

[0041] Optionally, the mass ratio of the hyaluronic acid or salt thereof to selenium disulfide can be any of 1:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 110:1.

[0042] Optionally, the mass ratio of the hyaluronic acid or salt thereof to selenium disulfide can be any of 1:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 110:1.

[0043] Optionally, the mass ratio of the hyaluronic acid or salt thereof to selenium disulfide can be 50:1.

[0044] In a preferred embodiment, the trypsin inhibitor comprises, in mass percentage of the trypsin inhibitor, 0.09091%-0.09901% of hyaluronic acid or salt thereof, 0.00099%-0.00909% of selenium disulfide.

[0045] The trypsin inhibitor of the present application can also add adjuvants, which can be appropriate solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integration agents, penetration promoters, pH regulators, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickening agents, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filtration aids, release retardants, etc.

[0046] In a preferred embodiment, the solvent is water.

[0047] The trypsin inhibitor of the present application can be prepared by a general method, in which one or more diluents or carriers can be added to prepare different dosage forms, such as ointments, creams, milks, patches, tinctures, etc.

[0048] In a preferred embodiment, the preparation method of the trypsin inhibitor comprises: stirring zinc pyrithione, ketoconazole and / or selenium disulfide uniformly with hyaluronic acid or salt thereof, and dissolving in a solvent.

[0049] One aspect of the present application relates to the trypsin inhibitor, wherein each component includes but is not limited to simultaneous use or sequential use of each component. The "simultaneous use" includes use together in the same preparation or use separately in different preparations. The "sequential use" includes sequential use in different preparations, and there is no limitation on the order of sequential use.

[0050] In another aspect, the present application also provides a product comprising the trypsin inhibitor.

[0051] Preferably, the product can comprise conventional adjuvants.

[0052] The skilled person can compound the trypsin inhibitor with known efficacy products to obtain a product with composite functions according to the needs.

[0053] Further, the product includes medical products, daily use products.

[0054] In a preferred embodiment, the product can be a lotion, a hair conditioner or a shampoo.

[0055] Further, the daily use product includes a scalp care product, a hair cleansing product and / or a hair care product.

[0056] In the extracellular space, human stratum corneum trypsin (HSCTE) (hK5) and chymotrypsin (HSCCE) (hK7) are believed to promote desquamation by hydrolyzing proteins that make up the intercellular adhesive structure known as corneodesmosomes. Since hK5 is able to activate pro-hK7 and other pro-hKs, its role in desquamation is crucial.

[0057] In another aspect, the present application also provides the use of zinc pyrithione, ketoconazole, selenium disulfide, the trypsin inhibitor or the product in inhibiting trypsin activity and / or promoting protein synthesis for non-disease diagnosis and treatment purposes.

[0058] In another aspect, the present application also provides the use of zinc pyrithione, ketoconazole, selenium disulfide, the trypsin inhibitor or the product in extracting insulin, cleaning or caring for hair and / or preventing hair loss.

[0059] The present application has the following beneficial effects:

[0060] Zinc pyrithione, ketoconazole and / or selenium disulfide are found for the first time in the present application to have inhibitory effects on trypsin activity, providing new and readily available efficacy components for the trypsin inhibitor.

[0061] The application provides three hyaluronic acid zinc-containing compositions, hyaluronic acid zinc is respectively compounded with zinc pyrithione, ketoconazole or selenium disulfide in a specific mass ratio, and it is found for the first time that the two exhibit synergistic trypsin inhibiting activity.

[0062] The trypsin inhibitor provided by the application has good biocompatibility, high safety, simple obtaining method, and can be further compounded with other active ingredients or excipient ingredients to obtain commercial products, and has wide application prospect. DETAILED DESCRIPTION

[0063] In order to more clearly illustrate the overall concept of the application, the following will be described in the form of examples. In the following description, a large number of specific details are given in order to provide a more thorough understanding of the application. However, it is obvious to those skilled in the art that the application can be implemented without one or more of these details. In other examples, some technical features known in the art are not described in order not to obscure the application.

[0064] Before further describing the specific embodiments of the application, it should be understood that the scope of protection of the application is not limited to the following specific embodiments; it should also be understood that the terms used in the embodiments of the application are for the purpose of describing the specific embodiments, and are not intended to limit the scope of protection of the application. The test methods in the following examples are not specified, and are usually carried out under conventional conditions, or under the conditions recommended by the manufacturers.

[0065] It should be noted that the terms used herein are only for the purpose of describing the specific embodiments, and are not intended to limit the scope of protection according to the exemplary embodiments of the application. As used herein, the singular form is intended to include the plural form, unless the context clearly indicates otherwise, and it should also be understood that when the terms "comprise" and / or "include" are used in the specification, there is a feature, step, operation, device, component and / or combination thereof.

[0066] When the embodiments give numerical ranges, it should be understood that, unless otherwise stated by the application, each numerical range can be selected from both ends of the range and any numerical value between the two ends. Unless otherwise defined, all technical and scientific terms used in the application have the same meaning as generally understood by those skilled in the art. In addition to the specific methods, devices, materials used in the examples, any method, device and material of the prior art similar or equivalent to those described in the embodiments of the application can also be used to implement the application according to the master of the prior art and the description of the application by those skilled in the art.

[0067] In the following examples, reagents or instruments used are not specified by the manufacturer unless otherwise stated, and are all conventional products available on the market.

[0068] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present application all employ conventional techniques in microbiology, biochemistry, analytical chemistry, cell culture, and related fields.

[0069] In addition, the "water" described in the present application includes deionized water, distilled water, ion exchange water, double distilled water, high-purity water, purified water, and any other available water in the field.

[0070] In the following examples, "%" means "wt%" unless otherwise specified, i.e., weight percentage.

[0071] The reagents and instruments involved in the following examples are shown in Table 1.

[0072] Table 1 Reagents and instruments

[0073]

[0074] Example 1

[0075] This example provides a series of mixed solutions containing different mass ratios of zinc hyaluronate and zinc pyrithione (ZPT), and the specific preparation process is as follows:

[0076] (1) 0.09091 g of zinc hyaluronate and 0.00909 g of zinc pyrithione were weighed into deionized water, respectively, and diluted to 100 mL. After stirring uniformly until completely dissolved, a mixed solution containing zinc hyaluronate and zinc pyrithione was obtained, which was recorded as Leg-1.

[0077] (2) 0.09524 g of zinc hyaluronate and 0.00476 g of zinc pyrithione were weighed into deionized water, respectively, and diluted to 100 mL. After stirring uniformly until completely dissolved, a mixed solution containing zinc hyaluronate and zinc pyrithione was obtained, which was recorded as Leg-2.

[0078] (3) 0.09804 g of zinc hyaluronate and 0.00196 g of zinc pyrithione were weighed into deionized water, respectively, and diluted to 100 mL. After stirring uniformly until completely dissolved, a mixed solution containing zinc hyaluronate and zinc pyrithione was obtained, which was recorded as Leg-3.

[0079] (4) 0.09901 g of zinc hyaluronate and 0.00099 g of zinc pyrithione were weighed into deionized water, respectively, and diluted to 100 mL. After stirring uniformly until completely dissolved, a mixed solution containing zinc hyaluronate and zinc pyrithione was obtained, which was recorded as Leg-4.

[0080] (5) 0.09910 g of zinc hyaluronate and 0.00090 g of zinc pyrithione were weighed into deionized water, respectively, and made up to 100 mL, stirred uniformly until completely dissolved, to obtain a mixed solution including zinc hyaluronate and zinc pyrithione, recorded as Leg-5.

[0081] (6) 0.1 g of zinc hyaluronate was weighed into deionized water, made up to 100 mL, stirred uniformly until completely dissolved, to obtain a solution of zinc hyaluronate, recorded as Leg-6.

[0082] (7) 0.1 g of zinc pyrithione was weighed into deionized water, made up to 100 mL, stirred uniformly until completely dissolved, to obtain a solution of zinc pyrithione, recorded as Leg-7.

[0083] The above solutions were subjected to trypsin inhibition effect detection, and the results are shown in Table 2.

[0084] The experimental method of trypsin inhibition effect detection is as follows:

[0085] A reaction mixture containing trypsin (1.0 μg / mL), casein substrate (final concentration 5 μg / mL) and Tris-HCl (pH 7.8, 10 mmol / L) was placed in a 96-well plate, covered with foil and incubated at 37°C. Deionized water, Leg 1-7 was added to the reaction mixture, the amount added was 10 μL. After 1 hour of incubation, the fluorescence was measured at excitation / emission wavelength 485 / 530 nm. The increase in fluorescence is proportional to the activity of the protease, and the trypsin inhibition rate is calculated, the calculation formula is as follows:

[0086] Trypsin inhibition rate = (V 原始 -V 样品 ) / V 原始 * 100%;

[0087] Wherein V 原始 represents the fluorescence signal intensity of the deionized water group, V 样品 represents the fluorescence signal intensity of the sample group.

[0088] Table 2

[0089]

[0090]

[0091] Wherein the asterisk represents the significance of the zinc hyaluronate and ZPT composition relative to zinc hyaluronate (Leg-6),

[0092] * represents P≤0.05, ** represents P≤0.01, and *** represents P≤0.001;

[0093] wherein # represents the significance of the hyaluronate zinc and ZPT composition relative to ZPT (Leg-7),

[0094] # represents P < 0.05, ## represents P < 0.01, and ### represents P < 0.001.

[0095] As can be seen from the results in Table 2, both zinc pyrithione (ZPT) and hyaluronate zinc have trypsin inhibiting effects. And, comparing the hyaluronate zinc and ZPT composition of different proportions with the monomer hyaluronate zinc and the monomer ZPT, it is found that the hyaluronate zinc:ZPT in the range of 20:1-100:1 has a significant difference in statistical significance relative to each monomer, proving that the two can effectively synergistically inhibit trypsin activity in the proportion range.

[0096] Example 2

[0097] This example provides a series of mixed solutions containing different mass proportions of hyaluronate zinc and ketoconazole, and the specific preparation process is as follows:

[0098] (1) 0.09091 g of hyaluronate zinc and 0.00909 g of ketoconazole were weighed into deionized water, respectively, and made up to 100 mL, stirred uniformly until completely dissolved, to obtain a mixed solution containing hyaluronate zinc and ketoconazole, denoted as Leg-8.

[0099] (2) 0.09524 g of hyaluronate zinc and 0.00476 g of ketoconazole were weighed into deionized water, respectively, and made up to 100 mL, stirred uniformly until completely dissolved, to obtain a mixed solution containing hyaluronate zinc and ketoconazole, denoted as Leg-9.

[0100] (3) 0.09804 g of hyaluronate zinc and 0.00196 g of ketoconazole were weighed into deionized water, respectively, and made up to 100 mL, stirred uniformly until completely dissolved, to obtain a mixed solution containing hyaluronate zinc and ketoconazole, denoted as Leg-10.

[0101] (4) 0.0984 g of hyaluronate zinc and 0.0016 g of ketoconazole were weighed into deionized water, respectively, and made up to 100 mL, stirred uniformly until completely dissolved, to obtain a mixed solution containing hyaluronate zinc and ketoconazole, denoted as Leg-11.

[0102] (5) 0.1 g of hyaluronate zinc was weighed into deionized water, and made up to 100 mL, stirred uniformly until completely dissolved, to obtain a solution of hyaluronate zinc, denoted as Leg-12.

[0103] (6) 0.1 g of ketoconazole was weighed into deionized water, and made up to 100 mL, stirred uniformly until completely dissolved, to obtain a solution of ketoconazole, denoted as Leg-13.

[0104] The above solutions were subjected to trypsin inhibition effect detection, and the results are shown in Table 3.

[0105] The experimental method for trypsin inhibition effect detection is as follows:

[0106] A reaction mixture containing trypsin (1.0 μg / mL), Casein substrate (final concentration 5 μg / mL) and Tris-HCl (pH 7.8, 10 mmol / L) was placed in a 96-well plate, covered with foil and incubated at 37°C. Deionized water, Leg 8-13 was added to the reaction mixture, and the amount added was 10 μL. After incubation for 1 hour, the fluorescence was measured at an excitation / emission wavelength of 485 / 530 nm. The increase in fluorescence was proportional to the protease activity, and the trypsin inhibition rate was calculated, and the calculation formula is as follows:

[0107] Trypsin inhibition rate = (V 原始 -V 样品 ) / V 原始 * 100%;

[0108] Wherein V 原始 represents the fluorescence signal intensity of the deionized water group, and V 样品 represents the fluorescence signal intensity of the sample group.

[0109] Table 3

[0110]

[0111] Wherein the asterisk represents the significance of the zinc hyaluronate and ketoconazole composition relative to zinc hyaluronate (Leg-12),

[0112] * represents P≤0.05, ** represents P≤0.01, and *** represents P≤0.001;

[0113] Wherein # represents the significance of the zinc hyaluronate and ketoconazole composition relative to ketoconazole (Leg-13),

[0114] # represents P≤0.05, ## represents P≤0.01, and ### represents P≤0.001.

[0115] From the results in Table 3, it can be seen that both ketoconazole and zinc hyaluronate have trypsin inhibition effect. And by comparing the zinc hyaluronate and ketoconazole composition with the monomers zinc hyaluronate and ketoconazole, it is found that zinc hyaluronate: ketoconazole in the range of 20:1-50:1 has significant difference in statistical significance relative to each monomer, proving that the two can effectively synergistically inhibit trypsin activity in the above ratio range.

[0116] Example 3

[0117] The present embodiment provides a series of mixed solutions containing different mass ratios of zinc hyaluronate and selenium disulfide, and the specific preparation process is as follows:

[0118] (1) 0.05 g of zinc hyaluronate and 0.05 g of selenium disulfide were weighed and added to deionized water, respectively, and then diluted to 100 mL. After stirring uniformly until completely dissolved, a mixed solution containing zinc hyaluronate and selenium disulfide was obtained, which was recorded as Leg-14.

[0119] (2) 0.09091 g of zinc hyaluronate and 0.00909 g of selenium disulfide were weighed and added to deionized water, respectively, and then diluted to 100 mL. After stirring uniformly until completely dissolved, a mixed solution containing zinc hyaluronate and selenium disulfide was obtained, which was recorded as Leg-15.

[0120] (3) 0.09524 g of zinc hyaluronate and 0.00476 g of selenium disulfide were weighed and added to deionized water, respectively, and then diluted to 100 mL. After stirring uniformly until completely dissolved, a mixed solution containing zinc hyaluronate and selenium disulfide was obtained, which was recorded as Leg-16.

[0121] (4) 0.09804 g of zinc hyaluronate and 0.00196 g of selenium disulfide were weighed and added to deionized water, respectively, and then diluted to 100 mL. After stirring uniformly until completely dissolved, a mixed solution containing zinc hyaluronate and selenium disulfide was obtained, which was recorded as Leg-17.

[0122] (5) 0.09901 g of zinc hyaluronate and 0.00099 g of selenium disulfide were weighed and added to deionized water, respectively, and then diluted to 100 mL. After stirring uniformly until completely dissolved, a mixed solution containing zinc hyaluronate and selenium disulfide was obtained, which was recorded as Leg-18.

[0123] (6) 0.09910 g of zinc hyaluronate and 0.00090 g of selenium disulfide were weighed and added to deionized water, respectively, and then diluted to 100 mL. After stirring uniformly until completely dissolved, a mixed solution containing zinc hyaluronate and selenium disulfide was obtained, which was recorded as Leg-19.

[0124] (7) 0.1 g of zinc hyaluronate was weighed and added to deionized water, and then diluted to 100 mL. After stirring uniformly until completely dissolved, a solution of zinc hyaluronate was obtained, which was recorded as Leg-20.

[0125] (8) 0.1 g of selenium disulfide was weighed and added to deionized water, and then diluted to 100 mL. After stirring uniformly until completely dissolved, a solution of selenium disulfide was obtained, which was recorded as Leg-21.

[0126] The above solutions were subjected to trypsin inhibition effect detection, and the results are shown in Table 4.

[0127] The experimental method for detecting the trypsin inhibitory effect is as follows:

[0128] A reaction mixture containing trypsin (1.0 μg / mL), casein substrate (final concentration 5 μg / mL) and Tris-HCl (pH 7.8, 10 mmol / L) was placed in a 96-well plate, covered with foil and incubated at 37°C. Deionized water, Leg 14-21, was added to the reaction mixture, respectively, and the amount of addition was 10 μL. After incubation for 1 hour, the fluorescence was measured at an excitation / emission wavelength of 485 / 530 nm. The increase in fluorescence was proportional to the protease activity, and the trypsin inhibition rate was calculated, and the calculation formula was as follows:

[0129] Trypsin inhibition rate = (V 原始 -V 样品 ) / V 原始 * 100%;

[0130] Wherein V 原始 represents the fluorescence signal intensity of the deionized water group, and V 样品 represents the fluorescence signal intensity of the sample group.

[0131] Table 4

[0132]

[0133] Wherein the asterisk represents the significance of the zinc hyaluronate and selenium disulfide composition relative to zinc hyaluronate (Leg-20), and the asterisk represents P≤0.05;

[0134] Wherein # represents the significance of the zinc hyaluronate and selenium disulfide composition relative to selenium disulfide (Leg-21), and ### represents P≤0.001.

[0135] From the results in Table 4, it can be seen that both selenium disulfide and zinc hyaluronate have trypsin inhibitory effect. And by comparing the zinc hyaluronate and selenium disulfide composition with the monomers of zinc hyaluronate and selenium disulfide, it is found that the zinc hyaluronate: selenium disulfide in the range of 10:1-100:1 has a significant difference in statistical significance relative to each monomer, proving that both can effectively inhibit trypsin activity in the above ratio range.

[0136] The above only describes the embodiments of the present application and is not intended to limit the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the scope of the claims of the present application.

Claims

1. Application of zinc pyrithione, ketoconazole and / or selenium disulfide in the preparation of trypsin inhibitors.

2. The application according to claim 1, characterized in that, The trypsin inhibitor further includes hyaluronic acid or its salt; preferably, the hyaluronic acid or its salt includes one or more of hyaluronic acid, zinc hyaluronic acid, sodium hyaluronic acid, and potassium hyaluronic acid; more preferably, zinc hyaluronic acid.

3. A trypsin inhibitor, characterized in that, The trypsin inhibitors include at least one or more of zinc pyrithione, ketoconazole, and selenium disulfide, and hyaluronic acid or its salts.

4. The trypsin inhibitor according to claim 3, characterized in that, The hyaluronic acid or its salts include at least one or more of hyaluronic acid, zinc hyaluronic acid, sodium hyaluronic acid, and potassium hyaluronic acid; more preferably, zinc hyaluronic acid.

5. The trypsin inhibitor according to claim 3 or 4, characterized in that, The mass ratio of the hyaluronic acid or its salt to zinc pyrithione is (10-110):1; preferably, (20-100):

1.

6. The trypsin inhibitor according to claim 3 or 4, characterized in that, The mass ratio of the hyaluronic acid or its salt to ketoconazole is (10-100):1; preferably, (20-50):

1.

7. The trypsin inhibitor according to claim 3 or 4, characterized in that, The mass ratio of the hyaluronic acid or its salt to selenium disulfide is (1-110):1; preferably, (10-100):

1.

8. A product comprising any of the trypsin inhibitors described in claims 3-7.

9. The use of zinc pyrithione, ketoconazole, selenium disulfide, any of the trypsin inhibitors described in claims 3-7, or the product described in claim 8, in the inhibition of trypsin activity and / or promotion of protein synthesis for non-disease diagnostic and therapeutic purposes.

10. The use of zinc pyrithione, ketoconazole, selenium disulfide, trypsin inhibitors as described in any of claims 3-7, or products as described in claim 8, in the extraction of insulin, cleaning or conditioning hair, and / or prevention of hair loss.

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