Composition for improving recurrent dandruff, preparation method and application thereof

Abstract

The present invention provides a composition for improving recurrent dandruff, as well as its preparation method and application, relating to the technical field of shampoo products. The composition comprises tropone and hinokitiol, wherein the weight ratio of tropone and hinokitiol is 2 to 8:1. The composition has synergistic antibacterial and synergistic antioxidant effects; can effectively remove mature biofilms, reduce the ability of bacterial species to form biofilms, and avoid the drug resistance problem of antidandruff agents; has low cytotoxicity, and reduces the irritation problem of antidandruff agents; the composition effectively inhibits the occurrence of recurrent dandruff by taking a multi-pathway strategy of reducing drug resistance and repairing oxidative barrier damage, thereby achieving a long-lasting and highly effective effect, and solves the technical problem that long-term use of antidandruff agents can damage the scalp barrier and cause drug resistance in the resident scalp flora, thereby triggering an inflammatory response and exacerbating dandruff or causing a sensitive scalp, further deepening dandruff, and causing recurring dandruff.

Description

Technical Field

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

[0002] Currently, excessive and difficult-to-eliminate dandruff has become the second most common hair health issue plaguing consumers. Approximately 50% of adults worldwide, and 60% in my country, suffer from dandruff-related issues, severely impacting their daily lives.

[0003] Compared to facial skin, the scalp has abundant hair, and the number of sebaceous and sweat glands is large, with high secretion volumes. Therefore, mineral ions, lipids, and water provide a suitable growth environment and a rich source of nutrients for microorganisms, and microbial esterase activity is stronger. Most studies have shown that Malassezia is most closely associated with the development of dandruff. Most Malassezia species rely on sebum for growth. When the scalp secretes excessive sebum, Malassezia secretes lipase to break down triglycerides and saturated fatty acids in the sebum into unsaturated fatty acids, destroying 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 keratinocyte hyperkeratinization and increased itching, accelerating the formation of dandruff.

[0004] Drug resistance is one of the main reasons for the failure of current antifungal (Malassezia) treatments and persistent and recurrent dandruff. The biofilm formation process is closely related to the degree of drug resistance in Malassezia: the longer the formation period, the greater the tolerance to drugs. Malassezia in biofilms is hundreds of times more drug-resistant than planktonic Malassezia. The original inhibitory concentration for Malassezia in biofilm form is only sub-MIC, which not only fails to achieve inhibition but also induces the formation and growth of biofilms, thereby increasing drug resistance. Biofilms have been called the "protective suit" of microorganisms in extreme environments. Biofilms possess a strong resistance to drug-induced environments. They regulate the expression of a series of biofilm-forming genes through quorum sensing and nucleotide second messenger signal transduction, giving microorganisms the ability to withstand these environments. Biofilm formation and drug resistance play a crucial role in the persistence of microorganisms in the body. However, current research on antidandruff agents focuses on inhibiting planktonic bacteria, with limited research on the elimination of Malassezia populations and the biofilms they form. Therefore, the discovery and development of Malassezia biofilm inhibitors is particularly important for the prevention and control of recurrent dandruff.

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

[0006] Currently, commercially available chemical antidandruff agents are generally divided into three categories: microbial inhibitors, exfoliants, and cell growth inhibitors. Microbial inhibitors, such as zinc pyrithione (ZPT, banned in the EU), climbazole, and piroctone olamine (OCT), are potent fungicides. Most have poor solubility and are irritating to the skin. Furthermore, drug interactions can cause water pollution, pose a serious threat to humans and ecosystems, and limit their practical application. Exfoliants, such as salicylic acid, lack significant antifungal activity and are highly irritating, easily exfoliating and causing damage. Cell growth inhibitors, such as selenium disulfide, are highly irritating and can cause dryness after use. Long-term use can cause hair loss and odor. Most of these chemical antidandruff agents are highly irritating and will bring more oxidative stress to the scalp. Although they can achieve an immediate dandruff removal effect, long-term use of chemical antidandruff agents will increase oxidative damage to the scalp, damage the scalp barrier, trigger an inflammatory response, aggravate dandruff problems or cause sensitive scalp problems, further deepen dandruff, and make dandruff persistent and recurring.

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

[0008] One of the purposes of the present invention is to provide a composition for improving recurrent dandruff, so as to solve the technical problem in the prior art that long-term use of antidandruff agents can damage the scalp barrier and cause drug resistance in the resident scalp flora, thereby triggering an inflammatory response and aggravating dandruff or causing a sensitive scalp, further aggravating dandruff and causing recurrent dandruff.

[0009] A second object of the present invention is to provide a method for preparing the above-mentioned composition.

[0010] A third object of the present invention is to provide use of the above-mentioned composition or the composition prepared by the above-mentioned preparation method in the preparation of a product for improving recurrent dandruff.

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

[0012] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0013] 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.

[0014] Furthermore, it also includes a plant extract, and the weight ratio of tropone, hinokitiol and the plant extract is 2:1:1~12.

[0015] Furthermore, the plant extract includes at least one of tea leaf extract, pomegranate peel extract, Camellia sinensis extract, Cynanchum wilfordii extract or Hibiscus mutabilis leaf extract.

[0016] Furthermore, the plant extract includes at least two of tea leaf extract, pomegranate peel extract or Camellia sinensis extract.

[0017] Furthermore, the plant extracts include Camellia sinensis extract, Cynanchum wilfordii extract and Hibiscus mutabilis leaf extract.

[0018] In a second aspect, the present invention provides a method for preparing the above-mentioned composition, which is prepared by mixing alcohol solutions or aqueous solutions of the components according to the formula.

[0019] In a third aspect, the present invention provides use of the above composition or the composition prepared by the above preparation method in preparing a product for improving recurrent dandruff.

[0020] Furthermore, the recurrent dandruff includes recurrent dandruff caused by scalp barrier damage and / or biofilm production by scalp flora.

[0021] Furthermore, the product includes a shampoo or a hair care product.

[0022] 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.

[0023] The present invention provides a composition for improving recurrent dandruff, which combines tropone and cypermethrin in a weight ratio of 2 to 8:1. The composition has synergistic antibacterial and antioxidant effects, thereby improving the antibacterial effect and antioxidant activity of the composition. The composition can effectively remove mature biofilms, reduce the ability of bacterial species to generate biofilms, and avoid the problem of drug resistance of antidandruff agents. The composition has relatively low cytotoxicity, reducing the damage and irritation of cells caused by antidandruff agents. The composition effectively inhibits the occurrence of recurrent dandruff by taking multi-pathway strategies such as reducing drug resistance and repairing oxidative barrier damage, thereby achieving a long-lasting and highly effective effect, and solves the technical problem that long-term use of antidandruff agents can damage the scalp barrier and cause drug resistance of the resident bacteria in the scalp, thereby triggering an inflammatory response and aggravating dandruff or producing a sensitive scalp, further deepening dandruff, and causing recurring dandruff. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 This is a graph showing the test results of the scavenging ability of OCT, tropolone, hinokitiol and tea extract provided by the present invention on DPPH free radicals when used alone;

[0026] Figure 2 A graph showing the test results of the DPPH free radical scavenging ability of different compositions for improving recurrent dandruff provided by the present invention;

[0027] Figure 3 A graph showing the biofilm removal test results of different compositions for improving recurrent dandruff provided by the present invention;

[0028] Figure 4 This is a graph showing the test results of cell viability of different compositions for improving recurrent dandruff provided by the present invention. DETAILED DESCRIPTION

[0029] 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 meaning and scope of the terms should be clear; however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, the use of "or" means "and / or" unless otherwise stated. In addition, the use of the term "including" and other forms is non-limiting.

[0030] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0031] In one aspect, the present invention provides a composition for improving recurrent dandruff, comprising tropolone and hinokitiol, wherein the weight ratio of tropolone to hinokitiol is 2-8:1.

[0032] Compared with piroctone olamine (OCT), the combination used in the above ratio has synergistic antibacterial and synergistic antioxidant effects, improves the antibacterial effect and antioxidant activity of the composition, and reduces the cytotoxicity of the composition; and can effectively remove mature biofilms, reduce the ability of bacteria to generate biofilms, and avoid the problem of drug resistance of antidandruff agents; the composition effectively inhibits the occurrence of recurrent dandruff by starting from a multi-pathway strategy of reducing drug resistance and repairing oxidative barrier damage, thereby achieving a long-lasting and highly effective effect, and solves the technical problem that long-term use of antidandruff agents will increase oxidative damage to the scalp and damage the scalp barrier; trigger inflammatory reactions and aggravate dandruff problems or produce sensitive scalp problems, further deepen dandruff, and cause dandruff to recur.

[0033] The weight ratio of tropone to hinokitiol may be, but is not limited to, 2:1, 3:1, 1.74:1, 6:1 or 8:1, or any ratio between 2 and 8:1.

[0034] In some specific embodiments, a plant extract is further included, and the weight ratio of tropone, hinokitiol and the plant extract is 2:1:1-12.

[0035] Among them, the weight ratio of the tropone, hinokitiol and plant extract can be but 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, or any ratio between 2:1:1 and 12.

[0036] The addition of plant extracts can form a synergistic antibacterial effect with the natural active ingredients tropone and hinokitiol, and the antibacterial effect reaches the antibacterial effect of piroctone olamine (OCT); at the same time, it can enhance the antioxidant effect of the composition and maintain the antioxidant balance of the scalp; reduce the dosage of tropone and hinokitiol, reduce the irritation to keratinocytes, and enhance the overall repair effect of the composition.

[0037] In some specific embodiments, the plant extract comprises at least one of tea leaf extract, pomegranate peel extract, Isodon serrata extract, Cyperus rotundus extract, or Hibiscus mutabilis leaf extract.

[0038] The combination of different plant extracts has a synergistic antibacterial effect (synergistic antibacterial index CI <1), which is superior to the antibacterial effect of each extract alone. Furthermore, the combination can be combined with tropolone and hinokitiol to achieve a synergistic antibacterial effect equivalent to that of OCT. The tea can be green tea.

[0039] In some specific embodiments, the plant extract comprises at least two of tea leaf extract, pomegranate peel extract, or Isodon serrata extract.

[0040] In some specific embodiments, the plant extracts include Isodon serrata extract, Cynanchum wilfordii extract, and Hibiscus mutabilis leaf extract.

[0041] The preparation method of the plant extract comprises taking medicinal materials, adding 10 times the amount of extractant for soaking, ultrasonic extraction 1-2 times, filtering, concentrating the filtrate to 1g / mL, and centrifuging to obtain the supernatant to obtain the plant extract.

[0042] Specifically, the extractant can be water, 70% ethanol, or 70% butylene glycol. To fully extract the active ingredients in the plant, the soaking time is 0.8-1.2 hours. The ultrasonication time is 20-60 minutes. Concentrating the filtrate includes combining the two filtrates and then concentrating them.

[0043] According to another aspect of the present invention, a method for preparing the above-mentioned composition is also provided, which is prepared by measuring the alcohol solutions or aqueous solutions of the components according to the formula and mixing them.

[0044] For ease of preparation, each component can be dissolved in 70% ethanol, 70% butylene glycol, or water as a stock solution. Measuring and mixing appropriate amounts of the stock solution according to the formula can improve preparation efficiency.

[0045] The present invention studies the antibacterial effect and antioxidant activity of the above composition and proves that it can effectively remove mature biofilms, reduce the ability of bacteria to form biofilms, and reduce cytotoxicity (irritation).

[0046] According to another aspect of the present invention, there is also provided use of the above composition or the composition prepared by the above preparation method in preparing a product for improving recurrent dandruff.

[0047] In some specific embodiments, the recurrent dandruff includes recurrent dandruff caused by scalp barrier damage and / or scalp flora producing biofilm.

[0048] Among them, scalp barrier damage includes oxidative damage; scalp biofilm includes biofilm produced due to enhanced drug resistance of Malassezia, or other bacteria or fungi, such as Staphylococcus aureus, which have enhanced drug resistance and produce biofilm.

[0049] In some specific embodiments, the product comprises a shampoo or a hair care product.

[0050] According to another aspect of the present invention, there is also provided a product for improving recurrent dandruff, comprising the above composition or the composition prepared by the above preparation method.

[0051] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0052] 1. Composition for improving recurrent dandruff

[0053] The specific component weight ratios of the compositions for improving recurrent dandruff provided in various examples and comparative examples are shown in Tables 1-5.

[0054] Table 1

[0055]

[0056] Table 2

[0057]

[0058] Table 3

[0059]

[0060] Table 4

[0061]

[0062] Table 5

[0063]

[0064] Dissolve OCT, tropone, and hinokitiol in ethanol (Tween solubilization) to prepare a 50 mg / mL stock solution; dilute the plant extract with sterile enzyme-free water to prepare a 50 mg / mL stock solution; seal the stock solution tube with parafilm and store in the dark at 4°C until ready to use.

[0065] The composition for improving recurrent dandruff is prepared by mixing the corresponding stock solutions according to the formula ratio of the composition provided in the above examples.

[0066] 2. Testing the Inhibitory Effect of Malassezia

[0067] The minimum inhibitory concentration (MIC) of the compositions of Examples 1 to 20 and the drugs of Comparative Examples 1 to 11 was determined by the broth microdilution method to evaluate the inhibitory effect of each component drug on Malassezia.

[0068] 1. Test materials

[0069] Malassezia furfur (M. furfur, ATCC44344) was purchased from Shanghai Microorganism Collection Center; piroctone ethanolamine (OCT) was purchased from Tongmeiyuan (Guangdong) Biotechnology Co., Ltd.; tropone and cypermethrin were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; the plant extract is preferably alcohol-extracted. In this embodiment, the tea extract was purchased from Xi'an Tianben Bioengineering Co., Ltd. with the product number 20241111I5-1-1; pomegranate peel, chrysanthemum vulgare, truncatum ulmoides, and hibiscus mutabilis leaf medicinal materials were purchased from Qingping Market. 15 g of the medicinal materials were weighed, added with 10 times 70% ethanol and soaked for 1 hour, ultrasonically extracted twice (20 min each time), the two filtrates were combined and concentrated to 1 g / mL, and the supernatant was taken by centrifugation at 7000RPM for 15 min to obtain the stock solution of pomegranate peel, chrysanthemum vulgare, truncatum ulmoides, and hibiscus mutabilis leaf extracts.

[0070] 2. Test Method

[0071] (1) Preparation of modified Dxion (mDixon) medium and modified Dxion (mDixon) agar medium: According to the instructions, accurately weigh the medium granules, agar powder and deionized water and place them in a beaker. Use a glass rod to stir until the granules are completely dissolved. Adjust the pH value to 6.1±0.1, divide the granules into conical flasks, seal them, and sterilize them by high pressure at 121℃ for 30min. After cooling, store them in a refrigerator at 4℃ for later use.

[0072] (2) Preparation of bacterial suspension: Take out the glycerol tube of the test bacteria stored in the -20℃ refrigerator, use an inoculation loop to take 20μL of bacterial solution, inoculate it on the agar slant by streaking method, and place it in a constant temperature incubator at 32℃ for 2 days. Rinse the slant with physiological saline to obtain a high-concentration bacterial suspension. Take another test tube, pour in an appropriate amount of raw salt, take an appropriate amount of high-concentration bacterial suspension, and make its turbidity consistent with that of a No. 5 McFadden tube, that is, obtain a final concentration of 1×10 6 CFU / mL bacterial suspension.

[0073] (3) Determination of MIC value

[0074] ① Culture method: Add 100 μL of modified Dixon (mDixon) liquid medium to columns 2 to 11 of a 96-well plate, and add 200 μL of modified Dixon (mDixon) liquid medium to column 12. Add 200 μL of each test drug to column 1, and take 100 μL to dilute it horizontally to column 10 (discard the excess 100 μL of drug solution). Then take the bacterial suspension prepared in (2) ② and add it to columns 1 to 11, 100 μL per well. At this time, the final volume of each well is 200 μL. Column 11 is the growth control well, and column 12 is the blank control well. Place the 96-well plate incubated at 32°C for 1 to 2 days.

[0075] ②Evaluation standard: Observe with the naked eye. The concentration of the drug solution in the sterile growth hole is the minimum inhibitory concentration (MIC).

[0076] (4) Screening the ratio of tropone and cypermethrin by chessboard method

[0077] ① Checkerboard screening: Based on the MIC results of method (3), the optimal synergistic ratio of tropone and cypermethrin was screened. Based on the MIC results, each drug solution was prepared into a drug stock solution with a concentration of 4 MIC. 50 μL of modified Dixon (mDixon) liquid medium was added to rows A to G and columns 2 to 10 of a 96-well plate. 100 μL of the above-mentioned cypermethrin experimental test stock solution was added to columns A1 to G1, and then diluted horizontally by two times to column 6. At this time, the volume of each well was 50 μL. 50 μL of the corresponding series of tropone stock solutions were added to rows A to G. Growth control wells and blank control wells were also set up. 100 μL of bacterial suspension was added, and the final volume of all wells was 200 μL. The 96-well plate was placed in a constant temperature incubation at 32°C for 1 to 2 days, and the observation standards were the same as above.

[0078] ②Evaluation criteria: FICI is used as the basis for judging the combined drug sensitivity test:

[0079] ;

[0080] Among them, FICI≤0.5 indicates synergistic effect; 0.5<FICI≤1 indicates additive effect; 1<FICI≤2 indicates irrelevant effect; FICI>2 indicates antagonistic effect.

[0081] (5) Chou-Talalay model screening of the ratio of tropone, cypermethrin and plant extracts

[0082] The Chou-Talalay model is a commonly used pharmacological model used to evaluate the synergistic and antagonistic effects of compound combinations during the onset of action. The synergistic effect index (CI) is calculated according to the Chou-Talalay formula to determine whether the combination is synergistic or not. The combination method is shown in the preparation example, and the calculation formula is as follows:

[0083] ;

[0084] ② Evaluation Criteria: Ca, Cb, Cc, and Cd represent the concentrations corresponding to the antibacterial effects of components A, B, C, and D, respectively, when acting alone. CA, CB, CC, and CD represent the inhibitory effects observed when the components are combined into a composite. The CA-D result = the composite MIC data × the percentage of components CA-D in the composite. A CI < 1 indicates a synergistic effect; a CI = 1 indicates the absence of an interaction; and a CI > 1 indicates an antagonistic effect.

[0085] 3. Test results

[0086] (1) The antibacterial results of the comparative example (single drug) are shown in Table 6.

[0087] Table 6 Antibacterial results / μg / mL

[0088]

[0089] As shown in Table 6, tropone and hinokitiol have good antibacterial effects, and their MICs are both lower than OCT. The antibacterial effect of plant extracts is not as good as that of OCT.

[0090] (2) Table 7 shows the optimal synergistic antibacterial ratio of tropone and hinokitiol screened by the chessboard method:

[0091] Table 7 Screening ratio of tropone and hinokitiol by chessboard method

[0092]

[0093] The optimal synergistic ratio of tropone and hinokitiol was screened by the chessboard method. When tropone:hinokitiol = 2:1, FICI = 0.425 < 0.5, which has a more significant synergistic antibacterial effect.

[0094] (3) The synergistic effect of each embodiment and comparative example was evaluated by evaluating the CI synergistic index using the Chou-Talalay model. The results are shown in Tables 8 to 11.

[0095] Table 8 Synergistic test results of tropone and hinokitiol combination

[0096]

[0097] The results showed that Examples 2 to 6 had a synergistic antibacterial effect, and the antibacterial effect was MIC OCT The synergistic coefficient CI of Example 2 (tropone:hinokitiol = 2:1) was the smallest. Therefore, when tropone:hinokitiol = (2-8):1, it can be used as an ideal antibacterial substitute for OCT.

[0098] Table 9 Synergistic test results of tropolone, hinokitiol and tea extract composition

[0099]

[0100] As shown in Table 9, the compositions of Examples 7 to 13 all have synergistic antibacterial effects, 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 those of OCT, and are MIC OCT The antibacterial effects of Examples 11 and 12 were comparable to those of OCT. Therefore, when the plant extract is tea extract, a weight ratio of tropolone, hinokitiol, and tea extract of 2:1:(1-9) can serve as an antibacterial substitute for OCT, with the best effect achieved when the weight ratio of tropolone:hinokitiol:tea extract = 2:1:(1-4).

[0101] Table 10 Synergistic test results of tropone, hinokitiol and pomegranate peel extract composition

[0102]

[0103] Table 10 shows that Examples 14-17 exhibit synergistic antibacterial effects that are superior to or comparable to those of OCT. When the plant extract is pomegranate peel extract, and the ratio of tropolone:hinokitiol:pomegranate peel extract is 2:1:(1-12), the composition exhibits synergistic antibacterial effects and can be used as an alternative to OCT for antibacterial purposes.

[0104] Table 11 Synergistic test results of compositions consisting of tropone, hinokitiol and two or more different plant extracts

[0105]

[0106] As shown in Table 11, Comparative Example 9 lacks a synergistic antibacterial effect. The components of the compositions of Comparative Examples 10 and 11 exhibit synergistic antibacterial effects, but their antibacterial effects are far lower than those of OCT. Examples 18-20 combine tropolone and hinokitiol with the plant extracts described in Comparative Examples 9-11. The compositions of Examples 18-20 exhibit synergistic antibacterial effects. Therefore, when two or more plant extracts are used in combination with tropolone and hinokitiol, they also exhibit synergistic antibacterial effects, and their antibacterial effects are comparable to those of OCT.

[0107] 3. Determination of the DPPH free radical scavenging ability of the composition

[0108] This test uses the compositions of Examples 2 and 7 to 13 as examples to test the antioxidant capacity of the compositions. The antioxidant capacity of each test sample and composition is evaluated by measuring the scavenging ability of DPPH free radicals, and the IC 50 The synergistic antioxidant effect was evaluated by the synergistic effect index (CI).

[0109] 1. Test materials: 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0110] 2. Test Method

[0111] (1) Antioxidant test (DPPH free radical scavenging experiment)

[0112] Dilute the sample to be tested in a serial dilution series. Use a pipette to take 500 μL of each sample solution at different concentrations into a plastic centrifuge tube. Add 500 μL of DPPH (0.2 mmol / L, prepared in ethanol) solution. Shake well and protect from light for 30 minutes. Measure the absorbance at 517 nm. Perform three parallel experiments. The specific calculation formula is as follows:

[0113] ;

[0114] Where D1 is the absorbance of the test sample or positive control; D2 is the absorbance of the blank control.

[0115] (2) Drawing and IC 50 calculate

[0116] The index of each test drug concentration (Log drug concentration) was used as the horizontal axis, and the scavenging rate of each test drug on DPPH free radicals was used as the vertical axis (DPPH free radical scavenging rate / %). Graphpad Prism 9 was used to draw a graph and obtain the half inhibition rate IC 50 .

[0117] (3) Evaluation criteria

[0118] The Chou-Talalay model is a commonly used pharmacological model used to evaluate the synergistic and antagonistic effects of test drug combinations during the onset of action. The synergistic effect index (CI) is calculated according to the Chou-Talalay formula, which is as follows:

[0119]

[0120] Among them, C a 、C b 、C c 、C d The IC values ​​of components A, B, C, and D are respectively 50 ; C A 、C B 、C C 、C D Indicates the IC corresponding to the compound when the test drug is compounded 50 , C A-D Result = Composition IC 50 ×C A-D The proportion of a component in the composition. CI < 1 indicates a synergistic effect; CI = 1 indicates no interaction; CI > 1 indicates an antagonistic effect.

[0121] 3. Test results

[0122] (1) Test the IC of each component acting on DPPH free radicals 50 , the results are shown in Table 12 and Figure 1 As shown, Figure 1 A represents OCT; B represents tropone; C represents hinokitiol; and D represents tea extract.

[0123] Table 12 IC of single drug for DPPH free radical scavenging 50

[0124]

[0125] From Table 12 and Figure 1 It can be seen that the IC values ​​of tropone, hinokitiol and tea extract alone are 50 The antioxidant activities of tropone, hinokitiol, and tea extract were much better than those of OCT, which were 11.59%, 54.20%, and 0.02% of those of OCT, respectively.

[0126] (2) Test the IC of each composition on DPPH free radical 50 , the results are shown in Table 13 and Figure 2As shown, wherein 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.

[0127] Table 13 IC values ​​of DPPH free radical scavenging by different compositions 50

[0128]

[0129] As shown in Table 13, the antioxidant synergy coefficient CI of Example 2 (tropone: hinokitiol = 2:1) is less than 1, which shows a synergistic antioxidant effect and is significantly better than the IC of OCT. 50 , and can serve as an ideal antioxidant alternative to OCT. Combined with the antibacterial results, it can be seen that when the ratio of tropone ketone to hinokitiol is 2:1, the combination has both synergistic antibacterial and synergistic antioxidant effects, and both effects are significantly better than OCT, making it a more gentle alternative to OCT.

[0130] Antioxidant IC corresponding to Examples 7 to 13 50 Both are smaller than the IC of OCT 50 , its antioxidant capacity is significantly better than OCT, and it 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, and has a synergistic antioxidant effect. Combined 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 OCT). Although there is no synergistic antioxidant effect, its antioxidant capacity is far better than OCT. The compositions of Examples 8 to 10 have both synergistic antibacterial and synergistic antioxidant effects, and both effects are significantly better than OCT, and can replace the use of OCT. The compositions of Examples 11 and 12 have both synergistic antibacterial and synergistic antioxidant effects, and the antibacterial effect is comparable to that of OCT, and the antioxidant capacity is far better than OCT, so they can be used as a substitute for OCT. The composition of Example 13 has both synergistic antibacterial and synergistic antioxidant effects. The antibacterial effect is not as good as OCT, but the antioxidant capacity is far better than OCT.

[0131] 4. Determination of the biofilm removal effect of the composition

[0132] This test used the compositions of Examples 2, 9, and 12 as examples to examine their biofilm-clearing abilities. Malassezia biofilms (drug-resistant Malassezia) were established using microplates, and the crystal violet method was used to test the effectiveness of the test samples in clearing mature biofilms.

[0133] 1. Test materials: Crystal violet was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0134] 2. Test method: Add 200 μL of modified Dixon (mDixon) liquid medium containing bacterial solution to each well of a 96-well plate, place in a 32°C constant temperature incubator and incubate for 5 days, and replace the medium every two days. When bacterial biofilm is formed on the tube wall, discard the bacterial solution. Add culture medium with final drug concentrations of MIC, 2MIC, 4MIC and 8MIC, place in a 32°C constant temperature incubator and incubate for 24 hours, then discard the bacterial solution. The crystal violet staining method is used to determine the biofilm content. That is, rinse with normal saline to remove planktonic bacteria, dehydrate with methanol, add 200 μL of 0.1% crystal violet staining solution and stain for 15 minutes, discard it, wash off the excess stain with normal saline, add 200 μL of 95% ethanol to dissolve for 10 minutes, and measure the OD with an enzyme reader. 570 The biofilm inhibition rate was calculated using the following formula:

[0135] ;

[0136] Among them, OD y Indicates the absorbance value of the sample well to be tested, OD c OD represents the absorbance value of the growth control well, and OD0 represents the absorbance value of the blank control well.

[0137] 3. Test results:

[0138] (1) Different compositions and OCT resistance test, the results are as follows Figure 3 As shown, wherein Control represents the growth control group; Example 2 represents tropone: cypress = 2:1, Example 9 represents tropone: cypress phenol: tea extract = 2:1:3, and Example 12 represents tropone: cypress phenol: tea extract = 2:1:9; * represents P < 0.05 compared with the growth control group, ** represents P < 0.01, *** represents P < 0.001, **** represents P < 0.0001, and ns represents P ≥ 0.05; # represents P < 0.05 compared with OCT, ## represents P < 0.01, and NS represents P ≥ 0.05.

[0139] The clearance effects of the tested samples on mature biofilm at MIC and 2MIC concentrations were poor, with no significant difference compared with the growth control.

[0140] At a concentration of 4 MIC, compared with the growth control group, Example 2 (tropone: hinokitiol = 2:1), Example 9 (tropone: hinokitiol: tea extract = 2:1:3), and Example 12 (tropone: hinokitiol: tea extract = 2:1:9) significantly reduced the total amount of biofilm (Example 2, P < 0.001, eliminated 29.40%; Example 9 and Example 12, P < 0.05, eliminated 15.43% and 15.67%, respectively), while OCT showed no significant difference compared with the growth control group (P > 0.05). Example 2 showed a significant difference compared with OCT (P < 0.05), while Example 9 and Example 12 showed no significant difference compared with OCT (P > 0.05).

[0141] At a concentration of 8 MIC: Compared with the growth control group, each test sample had a very significant clearing effect on mature biofilm (P < 0.0001), clearing 33.55%, 47.78%, 34.40%, and 33.02%, respectively. There was a very significant difference in the clearing effect between Example 2 and OCT (P < 0.01), while Examples 9 and 12 were equivalent to OCT.

[0142] 5. Determination of the irritation of the composition

[0143] This test uses the compositions of Example 2, Example 9 and Example 12 as examples, and tests the irritation of the compositions through a cytotoxicity experiment.

[0144] 1. Test method: HaCaT cells in the logarithmic growth phase were taken and adjusted to 1×10 5 100 µL of the combination was plated per well in a 96-well plate and incubated in a 37°C, 5% CO2 incubator for 24 hours. The culture medium was discarded, and the combination was diluted to the experimental concentration (0.01% or 0.1%) with complete DMEM medium. 100 µL was added to each well. Five replicates were set for each drug group, along with a blank control and a zero-adjustment control. After 24 hours of incubation, the supernatant was discarded, and 100 µL of 0.5 mg / mL MTT was added to each well. After an additional 4 hours of incubation, the supernatant was discarded, and 150 µL of DMSO was added to each well. The OD value at 490 nm was measured using a microplate reader.

[0145] .

[0146] 2. Test results:

[0147] Table 14 Cell viability of different compositions (%)

[0148]

[0149] Figure 4A indicates the cell viability corresponding to a sample concentration of 0.01%, B indicates the cell viability corresponding to a sample concentration of 0.1%, **** indicates P < 0.0001 compared with the control (OCT); # indicates P < 0.05 compared with the control (blank group), ## indicates P < 0.01, ### indicates P < 0.001, ### indicates P < 0.0001, and ns indicates P ≥ 0.05.

[0150] From Table 14 and Figure 4 It can be seen that compared with Comparative Example 1 (OCT), when the sample concentration was 0.01%, the cytotoxicity of the compositions of Examples 2, 9, and 12 was relatively low. Specifically, when the sample concentration was 0.01%, the cytotoxicity of Example 12 was not significantly different from that of the blank group (P ≥ 0.05). The cell viability of Examples 2, 9, and 12 was 33.33% to 34.89% greater than that of Comparative Example 1 (OCT) (P < 0.0001, significant difference). When the sample concentration was 0.1%, the cytotoxicity of Examples 2, 9, and 12 was much lower than that of OCT (P < 0.0001, significant difference). The cell viability of Examples 2, 9, and 12 was 28.79% to 31.53% greater than that of Comparative Example 1 (OCT).

[0151] 6. Application of different compositions in shampoo products

[0152] This application uses the compositions of Examples 2, 9, and 12 as examples to test the effects of the compositions in shampoo products. The weight percentages of the components of the shampoo for improving recurrent dandruff are shown in Table 15. The preparation method includes the following steps:

[0153] S1. Add the water of phase A1 to the main pot, add the remaining ingredients of phase A1 while homogenizing, disperse thoroughly, add the ingredients of phase A2, stir thoroughly, heat to 80-85°C, add the ingredients of phase A3, and stir to dissolve completely;

[0154] S2. Add phase B1, stir thoroughly until completely dissolved, then add phase B2 and stir to dissolve completely;

[0155] S3. Cool to 60~65℃ and add the C1 phase raw materials and stir evenly;

[0156] S4 cooled to 45`50 ℃, followed by adding C2 phase raw materials, stirring evenly;

[0157] S5. Add the ingredients of phase D in sequence, stir and cool to room temperature.

[0158] Table 15 Shampoo formula

[0159]

[0160] (2) Consumer testing

[0161] 120 male and female volunteers aged 18 to 50 were recruited (eligible subjects were healthy volunteers with oily hair, dandruff, or itchy scalp). Each group had 30 participants use the shampoo every other day (once every two days) for four weeks. They were evaluated on three aspects: hair volume, dandruff removal, and itching relief. The scores ranged from 0 to 7 (7 = extremely satisfied, 6 = very satisfied, 5 = somewhat satisfied, 4 = average, 3 = somewhat satisfied, 2 = very dissatisfied, and 1 = extremely dissatisfied). The higher the level of approval, the higher the score. Overall satisfaction refers to the percentage of participants who rated 5 to 7. The results of the consumer testing are shown in Table 16.

[0162] Table 16 Consumer test results

[0163]

[0164] As shown in Table 16, the hair volume recognition of Examples 2, 9, and 12 is similar to that of OCT, but the anti-dandruff and anti-itching recognition are much better than OCT. Therefore, the composition provided by the present invention can replace OCT.

[0165] 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 above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above 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: Includes tropolone, hinokitiol, and plant extracts; The weight ratio of tropolone, hinokitiol and plant extract is 2:1:2-12; The plant extract is tea extract.

2. The method for preparing the composition according to claim 1, wherein The alcohol solution or aqueous solution of each component is measured and mixed according to the formula to prepare the product.

3. Use of the composition according to claim 1 or the composition prepared by the preparation method according to claim 2 in preparing a product for improving recurrent dandruff.

4. The use according to claim 3, characterized in that The recurrent dandruff includes recurrent dandruff caused by scalp barrier damage and / or biofilm production by scalp flora.

5. The use according to claim 3, characterized in that The products include hair shampoo or hair care products.

6. A product for improving recurrent dandruff, characterized in that: The invention relates to a composition according to claim 1 or a composition prepared by the preparation method according to claim 2.

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