Composition for improving recurrent scurf as well as preparation method and application of composition

Abstract

The invention provides a composition for improving recurrent dandruff and a preparation method and application thereof, and relates to the technical field of shampoo supplies, the composition comprises tropolone and hinokiol, and the weight ratio of tropolone to hinokiol is (2-8): 1. The composition has the effects of synergistic bacteriostasis and synergistic antioxidation; mature biofilms can be effectively removed, the biofilm generation capability of strains is reduced, and the problem of drug resistance of the anti-dandruff agent is avoided; the anti-dandruff agent has small cytotoxicity, and the irritation problem of the anti-dandruff agent is reduced; the composition is used for effectively inhibiting recurrent dandruff from a multi-channel strategy of reducing drug resistance and repairing oxidation barrier injury, so that a lasting and efficient effect is achieved, and the problems that a dandruff removing agent is used for a long time, so that scalp barrier injury and drug resistance of scalp resident flora are caused, and the dandruff removing effect is poor are solved. Therefore, the technical problems that inflammatory reaction is caused, dandruff is aggravated or sensitive scalp is generated, the dandruff is further deepened, and the dandruff repeatedly exists are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of hair care products, and particularly to a composition for improving recurrent dandruff, a preparation method thereof and an application thereof. Background Art

[0002] At present, excessive dandruff that is difficult to eradicate has become the second major hair health problem troubling consumers. Approximately 50% of the world's population and even 60% of adults in China are troubled by dandruff-related problems, seriously affecting people's daily lives.

[0003] Compared with facial skin, the scalp environment has thick hair growth, a large number of sebaceous glands and sweat glands with high secretion levels. Therefore, mineral ions, lipids, moisture, etc. provide a suitable growth environment and rich nutrient sources for microorganisms, and the microbial-derived esterase activity is stronger. Most studies have shown that Malassezia is most closely related to dandruff production. Most Malassezia rely on sebum for growth. When sebum secretion on the scalp is excessive, Malassezia secretes lipase to break down triglycerides and saturated fatty acids in sebum into unsaturated fatty acids, damaging the stratum corneum barrier and causing inflammation and itching. In addition, unsaturated fatty acids promote the entry of calcium ions into cells, leading to excessive keratinization of keratinocytes and increased itching, accelerating dandruff formation.

[0004] Drug resistance is one of the main reasons for the failure of current antifungal (Malassezia) treatment and the persistence and recurrence of dandruff. The formation process of biofilms is closely related to the drug resistance level of Malassezia. The longer the formation time, the stronger the drug tolerance. Malassezia in biofilms has hundreds of times higher drug resistance than planktonic Malassezia. The original antibacterial concentration is only the sub-MIC concentration for Malassezia in the biofilm state, which not only fails to achieve the antibacterial result but instead induces the generation and increase of biofilms, increasing drug resistance. Biofilms are called the "protective suits" of microorganisms in extreme environments. Biofilms have strong resistance to the drug environment and regulate the expression of a series of biofilm formation genes through quorum sensing, signal transduction of nucleotide second messengers, etc., enabling microorganisms to have the ability to resist the environment. The formation and drug resistance of biofilms play an important role in the persistent existence of microorganisms in the in vivo environment. However, current research on antidandruff agents still focuses on the inhibition of planktonic bacteria, and there is less research on the clearance of Malassezia populations and the biofilms they form. Therefore, the discovery and development of Malassezia biofilm inhibitors are particularly important for the prevention and control of recurrent dandruff.

[0005] In addition, the antioxidant capacity of dandruff-prone scalps decreases, including reduced levels of superoxide dismutase and catalase, as well as a decrease in the content of factors that protect the skin against free radicals. In dandruff-prone scalps, the reduction of superoxide dismutase and hydrogen peroxide indicates that not only the innate immune defense system is affected, but also the antioxidant defense mechanism is affected. Additionally, Malassezia-derived lipoxygenase can peroxidize free unsaturated fatty acids, triglycerides, cholesterol, etc., and its products are cytotoxic and play a role in the pathogenesis of dandruff. Therefore, the repair of the oxidative barrier also cannot be underestimated in the prevention and treatment of dandruff.

[0006] Currently, commercially available chemical anti-dandruff agents are generally divided into three categories: microbial inhibitors, keratolytic agents, and cell growth inhibitors. Microbial inhibitors: such as zinc pyrithione (ZPT, which has been banned in the EU), clotrimazole, octopirox ethanolamine salt (OCT), etc., are strong fungicides. Most of them have poor solubility, are irritating to the skin, and have side effects of drug interactions, pollute water bodies, and pose serious threats to the human body and the ecosystem, limiting the practical application of the drugs. Keratolytic agents: such as salicylic acid, do not have obvious antifungal effects, are highly irritating, and easily exfoliate the skin causing damage; Cell growth inhibitors: such as selenium disulfide, are highly irritating, make the scalp feel dry and astringent after use, and long-term retention can cause hair loss and odors. Most of these chemical anti-dandruff agents are highly irritating and will bring more oxidative stress to the scalp. Although they can achieve the effect of immediate dandruff removal, long-term use of chemical anti-dandruff agents will increase oxidative damage to the scalp, damage the scalp barrier, trigger inflammatory reactions, exacerbate dandruff problems or cause sensitive scalp problems, further deepen dandruff, and make dandruff persistent and recurrent.

[0007] In view of this, the present invention is specifically proposed. Summary of the Invention

[0008] One of the objectives of the present invention is to provide a composition for improving recurrent dandruff, so as to solve the technical problems in the prior art that long-term use of anti-dandruff agents will damage the scalp barrier and cause drug resistance in the resident scalp flora, thereby triggering inflammatory reactions and exacerbating dandruff or causing sensitive scalp, further deepening dandruff, and making dandruff exist repeatedly.

[0009] Another objective of the present invention is to provide a preparation method for the above composition.

[0010] A third objective of the present invention is to provide the application of the above composition or the composition prepared by the above preparation method in the preparation of products for improving recurrent dandruff.

[0011] A fourth objective of the present invention is to provide a product for improving recurrent dandruff.

[0012] In order to achieve the above objectives of the present invention, the following technical solutions are specifically adopted: In a first aspect, the present invention provides a composition for improving recurrent dandruff, comprising tropolone and hinokitiol, wherein the weight ratio of tropolone to hinokitiol is 2 to 8:1.

[0013] Furthermore, it further comprises a plant extract, and the weight ratio of tropolone, hinokitiol and the plant extract is 2:1:1 to 12.

[0014] Furthermore, the plant extract comprises at least one of tea extract, pomegranate peel extract, isodonis amethystoides extract, tinospora sinensis extract or hibiscus mutabilis leaf extract.

[0015] Furthermore, the plant extract comprises at least two of tea extract, pomegranate peel extract or isodonis amethystoides extract.

[0016] Furthermore, the plant extract comprises isodonis amethystoides extract, tinospora sinensis extract and hibiscus mutabilis leaf extract.

[0017] In a second aspect, the present invention provides a preparation method of the above composition, which is prepared by mixing the alcohol solutions or aqueous solutions of each component according to the formula amount.

[0018] In a third aspect, the present invention provides the application of the above composition or the composition prepared by the above preparation method in the preparation of a product for improving recurrent dandruff.

[0019] Furthermore, the recurrent dandruff includes recurrent dandruff caused by scalp barrier damage and / or biofilm formation by scalp flora.

[0020] Furthermore, the product includes hair washing products or hair care products.

[0021] In a fourth aspect, the present invention provides a product for improving recurrent dandruff, comprising the above composition or the composition prepared by the above preparation method.

[0022] A composition for improving recurrent dandruff provided by the present invention combines tropolone and hinokitiol in a weight ratio of 2-8:1; it has a synergistic antibacterial and antioxidant effect, improving the antibacterial effect and antioxidant activity of the composition; and it can effectively remove mature biofilms, reduce the ability of bacteria to form biofilms, and avoid the problem of drug resistance of antidandruff agents; it has relatively low cytotoxicity, reducing the damage and irritation of antidandruff agents to cells; the composition effectively inhibits the occurrence of recurrent dandruff by starting from multi-channel strategies such as reducing drug resistance and repairing oxidative barrier damage, thereby achieving a lasting and efficient effect, and solving the technical problems that long-term use of antidandruff agents will damage the scalp barrier and cause drug resistance in scalp resident flora, thereby triggering inflammatory reactions and exacerbating dandruff or producing sensitive scalps, further deepening dandruff, and making dandruff exist repeatedly. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0024] Figure 1 It is a test result diagram of the ability of OCT, tropolone, hinokitiol and tea extract provided by the present invention to scavenge DPPH free radicals alone; Figure 2 It is a test result diagram of the ability of different compositions for improving recurrent dandruff provided by the present invention to scavenge DPPH free radicals; Figure 3 It is a test result diagram of the biofilm clearance of different compositions for improving recurrent dandruff provided by the present invention; Figure 4 It is a test result diagram of the cell viability of different compositions for improving recurrent dandruff provided by the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meanings and scopes of the terms should be clear. However, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or extrinsic definition. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "comprising" and other forms is non-restrictive.

[0026] Unless otherwise specified, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in various general and more specific references, which are cited and discussed throughout this specification.

[0027] On the one hand, the present invention provides a composition for improving recurrent dandruff, comprising tropolone and hinokitiol, and the weight ratio of tropolone to hinokitiol is 2 - 8:1.

[0028] Compared with piroctone olamine (OCT), when used in combination according to the above ratio, it has a synergistic antibacterial and synergistic antioxidant effect, improves the antibacterial effect and antioxidant activity of the composition, reduces the cytotoxicity of the composition; and can effectively remove mature biofilms, reduce the ability of bacteria to form biofilms, and avoid the problem of drug resistance of antidandruff agents; starting from a multi - pathway strategy of reducing drug resistance and repairing oxidative barrier damage, the composition effectively inhibits the occurrence of recurrent dandruff, thereby achieving a lasting and efficient effect, and solving the technical problems that long - term use of antidandruff agents will increase scalp oxidative damage and damage the scalp barrier; trigger inflammatory reactions and exacerbate dandruff problems or cause sensitive scalp problems, further deepen dandruff, and make dandruff exist repeatedly.

[0029] Among them, the weight ratio of tropolone to hinokitiol can be, but is not limited to, 2:1, 3:1, 1.74:1, 6:1 or 8:1, and can also be any ratio between 2 - 8:1.

[0030] In some specific embodiments, it further comprises a plant extract, and the weight ratio of tropolone, hinokitiol and the plant extract is 2:1:1 - 12.

[0031] Among them, the weight ratio of tropolone, hinokitiol and the plant extract can be, but is not limited to, 2:1:1, 2:1:2, 2:1:3, 2:1:4, 2:1:5, 2:1:6, 2:1:7, 2:1:8, 2:1:9, 2:1:10, 2:1:11 or 2:1:12, and can also be any ratio between 2:1:1 - 12.

[0032] The addition of the plant extract can form a synergistic antibacterial effect with the natural active ingredients tropolone and hinokitiol, and the antibacterial effect reaches that of piroctone olamine (OCT); at the same time, it can enhance the antioxidant effect of the composition, maintain the antioxidant balance of the scalp; reduce the dosage of tropolone and hinokitiol, reduce the irritation to keratinocytes, and enhance the overall repair effect of the composition.

[0033] In some specific embodiments, the plant extract comprises at least one of tea extract, pomegranate peel extract, isodonis amethystoides extract, stephania extract or hibiscus mutabilis leaf extract.

[0034] Different plant extract compositions have a synergistic antibacterial effect (synergistic antibacterial index CI < 1). The antibacterial effect of the composition is better than that of each extract used alone, and it can be compounded with tropolone and hinokitiol to achieve a synergistic antibacterial effect, achieving an antibacterial effect equivalent to that of OCT. Among them, the tea can be selected as green tea.

[0035] In some specific embodiments, the plant extract includes at least two of tea extract, pomegranate peel extract or isodonis amethystoides extract.

[0036] In some specific embodiments, the plant extract includes isodonis amethystoides extract, tetrastigma hypoglaucum extract and hibiscus mutabilis leaf extract.

[0037] Among them, the preparation method of the plant extract includes taking medicinal materials, adding 10 times the amount of the extractant for soaking, ultrasonic extracting 1-2 times, filtering, concentrating the filtrate to 1 g / mL, and centrifuging to take the supernatant to obtain the plant extract.

[0038] Specifically, the extractant can be selected from water, 70% ethanol or 70% butanediol. In order to fully extract the active ingredients in the plant, the soaking time is 0.8-1.2 h. The ultrasonic time is 20-60 min. Concentrating the filtrate includes concentrating after combining the two filtrates.

[0039] According to another aspect of the present invention, there is also provided a preparation method of the above composition, which is prepared by mixing the alcohol solutions or aqueous solutions of each component according to the formula amount.

[0040] For the convenience of preparation, each component can be dissolved in 70% ethanol, 70% butanediol or water as a stock solution. Taking an appropriate amount of the stock solution according to the formula amount and mixing them can improve the preparation efficiency.

[0041] The present invention has studied the antibacterial effect and antioxidant activity of the above composition, and proved that it can effectively remove mature biofilms, reduce the ability of bacterial strains to form biofilms, and reduce cytotoxicity (irritation).

[0042] According to another aspect of the present invention, there is also provided an application of the above composition or the composition prepared by the above preparation method in the preparation of products for improving recurrent dandruff.

[0043] In some specific embodiments, the recurrent dandruff includes recurrent dandruff caused by scalp barrier damage and / or scalp flora forming biofilms Among them, scalp barrier damage includes oxidative damage; scalp biofilms include biofilms formed due to enhanced drug resistance of malassezia, or other bacteria or fungi, such as staphylococcus aureus, etc., with enhanced drug resistance.

[0044] In some specific embodiments, the product includes hair shampoo or hair care products.

[0045] According to another aspect of the present invention, there is also provided a product for improving recurrent dandruff, including the above-mentioned composition or the composition prepared by the above-mentioned preparation method.

[0046] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative work shall fall within the scope of protection of the present invention.

[0047] I. Composition for improving recurrent dandruff The specific component weight ratios of the compositions for improving recurrent dandruff provided in each example and comparative example are shown in Tables 1-5.

[0048] Table 1

[0049] Table 2

[0050] Table 3

[0051] Table 4

[0052] Table 5

[0053] Dissolve OCT, tropolone and hinokitiol in ethanol (Tween solubilization) to prepare a stock solution of 50 mg / mL; dilute the plant extract with sterile and enzyme-free water to prepare a stock solution of 50 mg / mL; seal the mouth of the stock solution tube with a sealing film and store it in the dark at 4 °C for later use.

[0054] Prepare by mixing the corresponding stock solutions according to the formulation ratios of the compositions for improving recurrent dandruff provided in the above examples.

[0055] II. Testing the inhibitory effect on Malassezia The minimum inhibitory concentration MIC of the compositions of Examples 1-20 and the drugs of Comparative Examples 1-11 was determined by the microbroth dilution method to evaluate the inhibitory effect of each component drug on Malassezia.

[0056] 1. Test materials Malassezia furfur (M. furfur, ATCC44344) was purchased from the Shanghai Center for Microbial Culture Collection; Octopirox ethanolamine salt (OCT) was purchased from Tongmeiyuan (Guangdong) Biotechnology Co., Ltd.; tropolone and hinokitiol were purchased from Shanghai Macklin Biochemical Co., Ltd.; the plant extract was preferably extracted with alcohol. In this example, the tea extract was purchased from Xi'an Tianben Biotechnology Co., Ltd., and the product number was 20241111I5-1-1; the medicinal materials of pomegranate peel, isodon amethystoides, tinomiscium petiolare, and hibiscus mutabilis leaf were purchased from Qingping Market. Weigh 15 g of the medicinal materials, add 10 times 70% ethanol, soak for 1 h, extract ultrasonically twice (20 min each time), combine the two filtrates and concentrate to 1 g / mL, centrifuge at 7000 RPM for 15 min, and take the supernatant to obtain the stock solution of pomegranate peel, isodon amethystoides, tinomiscium petiolare, and hibiscus mutabilis leaf extract.

[0057] 2. Test method (1) Preparation of modified Dxion (mDixon) medium and modified Dxion (mDixon) agar medium: Accurately weigh the medium granules, agar powder and deionized water according to the instructions, place them in a beaker, stir with a glass rod until the granules are completely dissolved, adjust the pH value to 6.1 ± 0.1, dispense into conical flasks, seal, sterilize at 121 °C under high pressure for 30 min, and store in a 4 °C refrigerator for later use after cooling.

[0058] (2) Preparation of bacterial suspension: Take out the stored test bacterial glycerol tube from the -20 °C refrigerator, use an inoculation loop to take 20 μL of the bacterial liquid, inoculate it on the agar slant by the streaking method, and incubate it in a 32 °C constant temperature incubator for 2 d. Rinse the slant with normal saline to obtain a high-concentration bacterial suspension. Take another test tube, pour in an appropriate amount of normal saline, take an appropriate amount of the high-concentration bacterial suspension, and make its turbidity consistent with that of the No. 5 McFarland turbidity standard tube to obtain a bacterial suspension with a final concentration of 1×10 6 CFU / mL.

[0059] (3) Determination of MIC value ① Culture method: Add 100 μL of modified Dxion (mDixon) liquid medium to each of the 2nd to 11th columns of the 96-well plate, and add 200 μL of modified Dxion (mDixon) liquid medium to the 12th column. Add 200 μL of each test drug to the 1st column, take 100 μL and dilute it two-fold horizontally to the 10th column (discard the excess 100 μL of the drug solution). Then add the bacterial suspension prepared in (2)② to the 1st to 11th columns, 100 μL for each well. At this time, the final volume of each well is 200 μL. The 11th column is the growth control well, and the 12th column is the blank control well. Incubate the 96-well plate in a 32 °C constant temperature incubator for 1 - 2 d.

[0060] ②Evaluation criteria: Visually observed, the concentration of the liquid medicine in the sterile growth well is the minimum inhibitory concentration (MIC).

[0061] (4)Screening the ratio of tropolone and hinokitiol by checkerboard method ①Checkerboard method screening: According to the MIC results of method (3), screen the optimal synergistic ratio of tropolone and hinokitiol. According to the MIC results, prepare the drug stock solutions with each liquid medicine concentration at 4MIC concentration. Add 50 μL of modified Dixon (mDixon) liquid medium to rows A - G and columns 2 - 10 of the 96-well plate. Take the above hinokitiol experimental test stock solution and add 100 μL to columns A1 - G1 respectively, and dilute it two-fold horizontally to the 6th column. At this time, the volume of each well is 50 μL. Add 50 μL of the corresponding series of concentrations of tropolone stock solution to rows A - G respectively. Additionally, set up growth control wells and blank control wells. Add 100 μL of the bacterial suspension, and the final volume of all wells is 200 μL. Place the 96-well plate in a constant temperature incubator at 32 °C for 1 - 2 days, and the observation criteria are the same as above.

[0062] ②Evaluation criteria: Use FICI as the judgment basis for the combined drug sensitivity test: ; Among them, FICI ≤ 0.5 indicates a synergistic effect; 0.5 < FICI ≤ 1 indicates an additive effect; 1 < FICI ≤ 2 indicates an irrelevant effect; FICI > 2 indicates an antagonistic effect.

[0063] (5)Screening the ratio of tropolone, hinokitiol and plant extract by Chou - Talalay model The Chou - Talalay model is a commonly used pharmacological model for evaluating the synergistic and antagonistic effects of compound combinations during the onset process. Calculate the combination index (CI) according to the Chou - Talalay formula to judge whether the composition is synergistic. The combination method of the composition is as shown in the preparation examples, and the calculation formula is as follows: ; ②Evaluation criteria: Ca, Cb, Cc and Cd are the concentrations corresponding to the antibacterial effects when component A, component B, component C and component D act alone; CA, CB, CC, CD represent the inhibitory effects observed at the corresponding concentrations when each component is compounded into the composition, and the result of CA - D = the experimental data of the MIC of the composition × the proportion of the CA - D component in the composition. CI < 1 indicates the existence of a synergistic effect; CI = 1 indicates the absence of interaction; CI > 1 indicates the existence of an antagonistic effect.

[0064] 3. Test results (1)Antibacterial results of the comparative examples (single drug) are shown in Table 6.

[0065] Table 6 Antibacterial results / μg / mL

[0066] As can be seen from Table 6, tropolone and hinokitiol have good antibacterial effects, and their MIC values are both less than that of OCT. The antibacterial effect of the plant extract is inferior to that of OCT.

[0067] (2)Table 7 shows the results of the optimal synergistic antibacterial ratio of tropolone and hinokitiol screened by the checkerboard method: Table 7 Ratio of tropolone and hinokitiol screened by the checkerboard method

[0068] The optimal synergistic ratio of tropolone and hinokitiol screened by the checkerboard method is that when tropolone:hinokitiol = 2:1, FICI = 0.425 < 0.5, showing a relatively significant synergistic antibacterial effect.

[0069] (3)The Chou-Talalay model was used to evaluate the synergistic effect of each example and comparative example by the CI synergy index. The results are shown in Tables 8 to 11.

[0070] Table 8 Synergistic test results of tropolone and hinokitiol compositions

[0071] The results show that Examples 2 to 6 have a synergistic antibacterial effect, and their antibacterial effect is 1 / 2 of the MIC OCT , significantly better than that of OCT, and the synergy coefficient CI of Example 2 (tropolone:hinokitiol = 2:1) is the smallest. Therefore, when tropolone:hinokitiol = (2 - 8):1, it can be an ideal antibacterial substitute for OCT.

[0072] Table 9 Synergistic test results of tropolone, hinokitiol and tea extract compositions

[0073] As can be seen from Table 9, the compositions of Examples 7 to 13 all have a synergistic antibacterial effect, but the antibacterial effect of Example 13 is lower than that of OCT. Among them, the antibacterial effects of the compositions of Examples 7 to 10 are significantly better than that of OCT, and they are respectively the MIC OCT1 / 4, 1 / 2.4, 1 / 2, and 1 / 2; the antibacterial effects of Examples 11 to 12 are equivalent to that of OCT. Therefore, when the plant extract is tea extract, when the weight ratio of tropolone, hinokitiol, and tea extract is 2:1:(1 to 9), it can be used as a substitute for OCT antibacterial, and the effect is the best when tropolone:hinokitiol:tea extract = 2:1:(1 to 4).

[0074] Table 10 Synergistic test results of tropolone, hinokitiol, and pomegranate peel extract composition

[0075] As can be seen from Table 10, Examples 14 to 17 have a synergistic antibacterial effect, and the antibacterial effect is better than or equivalent to that of OCT. When the plant extract is pomegranate peel extract, when tropolone:hinokitiol:pomegranate peel extract = 2:1:(1 to 12), the composition has a synergistic antibacterial effect and can be used as a substitute for OCT antibacterial.

[0076] Table 11 Synergistic test results of the composition composed of tropolone, hinokitiol and two or more different plant extracts

[0077] As can be seen from Table 11, Comparative Example 9 does not have a synergistic antibacterial effect; each component of the compositions of Comparative Example 10 and Comparative Example 11 has a synergistic antibacterial effect, but its antibacterial effect is much lower than that of OCT. Examples 18 to 20 are combinations of tropolone, hinokitiol and the plant extracts described in Comparative Example 9 to Comparative Example 11. The compositions of Examples 18 to 20 have a synergistic antibacterial effect. Therefore, when the plant extract is a combination of two or more, when used in combination with tropolone and hinokitiol, it also has a synergistic antibacterial effect, and the antibacterial effect is equivalent to that of OCT.

[0078] III. Determination of the scavenging ability of the composition on DPPH free radicals In this test, the compositions of Example 2 and Examples 7 to 13 were selected as examples to test the antioxidant capacity of the compositions. The antioxidant capacity of each test sample and composition was evaluated by measuring the scavenging ability on DPPH free radicals, and IC 50 was obtained by plotting; the synergistic antioxidant effect was evaluated by the synergistic effect index (CI).

[0079] 1. Test materials; 1,1-diphenyl-2-trinitrophenylhydrazine (abbreviation DPPH) was purchased from Shanghai Macklin Biochemical Co., Ltd.

[0080] 2. Test methods (1) Antioxidant test (DPPH free radical scavenging experiment) Dilute the sample to be tested in a serial dilution gradient. Use a pipette to take 500 μL of sample solutions with different concentrations into plastic centrifuge tubes respectively. Add 500 μL of DPPH (0.2 mmol / L, prepared with ethanol) solution. After shaking well and keeping in the dark for 30 min, measure the absorbance at 517 nm, and conduct 3 parallel experiments. The specific calculation formula is as follows: ; Among them, D1 is the absorbance of the test sample or positive control; D2 is the absorbance of the blank control.

[0081] (2)Plotting and IC 50 Calculation Taking the logarithm (Log drug concentration) of each test drug concentration as the abscissa and the scavenging rate of each test drug on DPPH free radicals as the ordinate (DPPH free radical scavenging rate / %), use Graphpad prism 9 to plot a curve graph and obtain the half inhibitory concentration IC 50 .

[0082] (3)Evaluation criteria The Chou-Talalay model is a commonly used pharmacological model for evaluating the synergistic and antagonistic effects of test drug combinations during the onset process. Calculate the synergistic effect index (CI) according to the Chou-Talalay formula, and the formula is as follows:

[0083] Among them, C a , C b , C c , C d are the IC 50 when components A, B, C, and D act alone respectively; C A , C B , C C , C D represent the corresponding IC 50 when the test drugs are compounded into a composition, and the result of C A-D = composition IC 50 × C A-D The proportion of the component in the composition. CI < 1 indicates the existence of a synergistic effect; CI = 1 indicates the absence of interaction; CI > 1 indicates the existence of an antagonistic effect.

[0084] 3. Test results (1)Test the IC 50 of each component acting on DPPH free radicals alone, and the results are shown in Table 12 and Figure 1 as follows, Figure 1 where A represents OCT; B represents tropolone; C represents hinokitiol; D represents tea extract.

[0085] Table 12 IC of single drug against DPPH free radical scavenging 50

[0086] From Table 12 and Figure 1 it can be seen that the IC of tropolone, hinokitiol and tea extract acting alone 50 is much smaller than that of OCT, which are 11.59%, 54.20% and 0.02% of OCT respectively. It can be seen that the antioxidant activities of tropolone, hinokitiol and tea extract are far superior to that of OCT.

[0087] (2)Test the IC of each composition against DPPH free radical 50 , and the results are shown in Table 13 and Figure 2 as follows, where A represents Example 2, B represents Example 7, C represents Example 8, D represents Example 9, E represents Example 10, F represents Example 11, G represents Example 12, and H represents Example 13.

[0088] Table 13 IC of different compositions against DPPH free radical scavenging 50

[0089] From Table 13, it can be seen that the antioxidant synergy coefficient CI of Example 2 (tropolone: hinokitiol = 2:1) < 1, having a synergistic antioxidant effect, and is significantly superior to the IC of OCT 50 , and can be used as an ideal antioxidant substitute for OCT. Combining the antibacterial results, it can be known that when tropolone: hinokitiol = 2:1, the composition has both synergistic antibacterial and synergistic antioxidant effects, and both effects are significantly superior to those of OCT, and can replace the use of OCT, being more mild.

[0090] The antioxidant IC corresponding to Examples 7 - 13 50 are all smaller than the IC of OCT 50, its antioxidant ability is significantly better than that of OCT and can be used as an ideal antioxidant substitute for OCT. Among them, the antioxidant synergy coefficient CI of Examples 8 to 13 is less than 1, showing a synergistic antioxidant effect. Combining with the antibacterial results, it can be seen that the composition of Example 7 has a synergistic antibacterial effect (the antibacterial effect is better than that of OCT). Although there is no synergistic effect in the antioxidant effect, its antioxidant ability is much better than that of OCT. The compositions of Examples 8 to 10 have both synergistic antibacterial and synergistic antioxidant effects, and both effects are significantly better than those of OCT, and can replace the use of OCT. The compositions of Examples 11 to 12 have both synergistic antibacterial and synergistic antioxidant effects, the antibacterial effect is comparable to that of OCT, and the antioxidant ability is much better than that of OCT, and can replace the use of OCT. The composition of Example 13 has both synergistic antibacterial and synergistic antioxidant effects, the antibacterial effect is inferior to that of OCT, but the antioxidant ability is much better than that of OCT.

[0091] IV. Determination of the clearance effect of the composition on biofilms In this test, the compositions of Example 2, Example 9 and Example 12 were selected as examples to test the ability of each composition to clear biofilms. Malassezia biofilms (drug-resistant Malassezia) were constructed through microplates, and the clearance effect of the test samples on mature biofilms was tested by the crystal violet method.

[0092] 1. Test materials: Crystal violet was purchased from Shanghai Macklin Biochemical Co., Ltd.

[0093] 2. Test method: Add 200 μL of modified Dixon (mDixon) liquid medium containing bacterial liquid to each well of a 96-well plate, incubate in a constant temperature incubator at 32 °C for 5 days, and change the medium every two days. After the bacterial biofilm is formed on the tube wall, discard the bacterial liquid. Add the medium with the final drug concentrations of MIC, 2MIC, 4MIC and 8MIC, incubate in a constant temperature incubator at 32 °C for 24 h, and discard the bacterial liquid. The crystal violet staining method was used to determine the biofilm content. That is, the planktonic bacteria were removed by rinsing with physiological saline, dehydrated with methanol, then stained with 200 μL of 0.1% crystal violet staining solution for 15 min. After discarding, the excess staining solution was washed off with physiological saline, 200 μL of 95% ethanol was added to dissolve for 10 min, and the OD 570 was measured with an enzyme-linked immunosorbent assay (ELISA) reader, and the biofilm inhibition rate was calculated by the following formula: ; Among them, OD y represents the absorbance value of the test sample well, OD c represents the absorbance value of the growth control well, and OD0 represents the absorbance value of the blank control well.

[0094] 3. Test results: (1) The drug resistance test of different compositions and OCT, and the results are as Figure 3As shown in the figure, where Control represents the growth control group; Example 2 represents tropolone: hinokitiol = 2:1, Example 9 represents tropolone: hinokitiol: tea extract = 2:1:3, and Example 12 represents tropolone: hinokitiol: tea extract = 2:1:9; * indicates P < 0.05 compared with the growth control group, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001, and ns indicates P ≥ 0.05; # indicates P < 0.05 compared with OCT, ## indicates P < 0.01, and NS indicates P ≥ 0.05.

[0095] The clearance effects of each test sample on mature biofilms at MIC concentration and 2MIC concentration were poor, showing no significant difference from the growth control.

[0096] At 4MIC concentration: Compared with the growth control group, Example 2 (tropolone: hinokitiol = 2:1), Example 9 (tropolone: hinokitiol: tea extract = 2:1:3), and Example 12 (tropolone: hinokitiol: tea extract = 2:1:9) could significantly reduce the total amount of biofilms (Example 2, P < 0.001, with 29.40% clearance; Example 9 and Example 12, P < 0.05, with 15.43% and 15.67% clearance respectively), while there was no significant difference between OCT and the growth control group (P > 0.05). There was a significant difference between Example 2 and OCT (P < 0.05), and there was no significant difference between Example 9, Example 12 and OCT (P > 0.05).

[0097] At 8MIC concentration: Compared with the growth control group, all test samples had a highly significant clearance effect on mature biofilms (P < 0.0001), with clearances of 33.55%, 47.78%, 34.40%, and 33.02% respectively. There was a highly significant difference in the clearance effect between Example 2 and OCT (P < 0.01), while Example 9 and Example 12 were comparable to OCT.

[0098] V. Determination of the irritation of the composition In this test, the compositions of Example 2, Example 9, and Example 12 were selected as examples to test the irritation of the compositions through a cytotoxicity experiment.

[0099] 1. Test method: Take HaCaT cells in the logarithmic growth phase and adjust them to 1×10 with DMEM complete medium 5Cells were inoculated into a 96-well plate at a density of [[ID=]], with 100 μL per well, and cultured in a CO2 incubator at 37 °C with a volume fraction of 5% CO2 for 24 h. The culture medium was discarded, and the composition was diluted to the experimental concentration (0.01%, 0.1%) with complete DMEM medium. Then, 100 μL of the diluted composition was added to each well, and 5 replicate wells were set for each drug group. Meanwhile, a blank group and a zero-adjustment group were also set. After culturing for 24 h, the supernatant was discarded. Then, 100 μL of MTT at a concentration of 0.5 mg / mL was added to each well, and the cells were cultured for another 4 h. After that, the supernatant was discarded, and 150 μL of DMSO was added to each well. The OD value at 490 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0100] 。

[0101] 2. Test results: Table 14 Cell viability of different compositions (%)

[0102] Figure 4 In the table, A represents the cell viability corresponding to a sample concentration of 0.01%, B represents the cell viability corresponding to a sample concentration of 0.1%. **** indicates that compared with the control example (OCT), P < 0.0001; # indicates that compared with the Control (blank group), P < 0.05, ## indicates P < 0.01, indicates P < 0.001, indicates P < 0.0001, and ns indicates P ≥ 0.05.

[0103] From Table 14 and Figure 4 it can be seen that compared with Comparative Example 1 (OCT): when the sample concentration is 0.01%, the cytotoxicities of the compositions of Example 2, Example 9, and Example 12 are all relatively small. Among them, when the sample concentration is 0.01%, the cytotoxicity of Example 12 has no significant difference from that of the blank group, P ≥ 0.05. The cell viabilities of Example 2, Example 9, and Example 12 are 33.33% - 34.89% higher than that of Comparative Example 1 (OCT) (P < 0.0001, with significant differences). When the sample concentration is 0.1%, the cytotoxicities of Example 2, Example 9, and Example 12 are much smaller than that of OCT (P < 0.0001, with significant differences). The cell viabilities of Example 2, Example 9, and Example 12 are 28.79% - 31.53% higher than that of Comparative Example 1 (OCT).

[0104] VI. Application of different compositions in shampoo products In this application, the compositions of Example 2, Example 9, and Example 12 were selected as examples to test the application effects of the compositions in shampoo products. The weight parts of each component of the shampoo for improving recurrent dandruff are shown in Table 15. Its preparation method includes the following steps: S1. Add the A1 phase water into the main pot, add the remaining ingredients of the A1 phase under homogenization, disperse completely, add the A2 phase ingredients, stir well, heat to 80-85°C, add the A3 phase ingredients and stir to dissolve completely; S2. Add phase B1, stir thoroughly until completely dissolved, then add phase B2 and stir until completely dissolved; S3. Cool down to 60~65℃ and add C1 phase raw materials, stirring evenly; S4. Cool down to 45`50 ℃, add C2 phase raw materials in sequence and stir evenly; S5. Add the raw materials of phase D in sequence, stir and cool to room temperature.

[0105] Table 15 Shampoo formula

[0106] (2) Consumer testing 120 male and female volunteers aged 18 to 50 were recruited (the inclusion criteria were healthy volunteers with oily hair, dandruff, and itchy scalp), 30 people in each group, and used shampoo once every other day (once every 2 days) for 4 consecutive weeks; they were scored and evaluated in three aspects: hair fluffy, dandruff removal, and itching. The scoring range is 0 to 7 points (7 points for extremely satisfied, 6 points for very satisfied, 5 points for relatively satisfied, 4 points for average feeling, 3 points for not very satisfied, 2 points for very dissatisfied, and 1 point for extremely dissatisfied). The higher the sense of identity, the higher the score; overall satisfaction refers to the proportion of people who scored 5 to 7 points. The results of the consumer test are shown in Table 16.

[0107] Table 16 Consumer test results

[0108] As shown in Table 16, the hair fluffiness recognition of Examples 2, 9 and 12 is similar to that of OCT, but the anti-dandruff and anti-itching recognition is much better than that of OCT. Therefore, the composition provided by the present invention can replace OCT.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composition for improving recurrent dandruff, characterized in that, It includes tropolone and hinokitiol, and the weight ratio of tropolone to hinokitiol is 2 to 8:

1.

2. The composition according to claim 1, characterized in that, It further includes a plant extract, and the weight ratio of tropolone, hinokitiol and the plant extract is 2:1:1 to 12.

3. The composition according to claim 2, characterized in that, The plant extract includes at least one of tea extract, pomegranate peel extract, isodon extract, tinospora extract or hibiscus mutabilis leaf extract.

4. The composition according to claim 3, characterized in that, The plant extract includes at least two of tea extract, pomegranate peel extract or isodon extract.

5. The composition according to claim 3, characterized in that, The plant extract includes isodon extract, tinospora extract and hibiscus mutabilis leaf extract.

6. A method for preparing the composition according to any one of claims 1 to 5, characterized in that, It is prepared by mixing the alcohol solutions or aqueous solutions of each component according to the formula amount.

7. Use of the composition according to any one of claims 1 to 5 or the composition prepared by the preparation method according to claim 6 in the preparation of a product for improving recurrent dandruff.

8. The use according to claim 7, characterized in that, The recurrent dandruff includes recurrent dandruff caused by scalp barrier damage and / or the formation of biofilms by scalp flora.

9. The use according to claim 7, characterized in that, The product includes hair washing products or hair care products.

10. A product for improving recurrent dandruff, characterized in that, It includes the composition according to any one of claims 1 to 5 or the composition prepared by the preparation method according to claim 6.

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