Melanin-selenium nanoparticle@malassezia membrane complex system against malassezia and application thereof

Abstract

The application discloses a preparation method of a thearubigin-selenium nanoparticle@malassezia membrane composite system, and comprises the following steps: adding a sodium selenite solution and a vitamin C solution into a thearubigin solution and stirring; performing gradient centrifugation on the obtained mixed solution, and preparing a thearubigin-selenium nanoparticle suspension from the obtained precipitate; preparing a malassezia membrane by using a malassezia bacterial solution; mixing the thearubigin-selenium nanoparticle suspension and the malassezia membrane under ice bath ultrasonic mixing; then centrifuging to remove supernatant, adding deionized water to constant volume, and obtaining the thearubigin-selenium nanoparticle / malassezia membrane composite system. The application also simultaneously provides the use of the thearubigin-selenium nanoparticle@malassezia membrane composite system: for preparing an anti-malassezia drug or reagent.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine and cosmetics, and particularly relates to a theabrownin-selenium nanoparticle @ malassezia membrane composite system with anti-malassezia function and a preparation method and application thereof. BACKGROUND

[0002] Seborrheic dermatitis is a chronic, recurrent and inflammatory skin disease, and dandruff is a mild form of seborrheic dermatitis. The clinical manifestations are excessive fine grayish white dry or slightly oily bran-like dandruff on the scalp, which can be accompanied by itching, and the incidence rate in the population is high. The main pathogenesis of seborrheic dermatitis is that the excessive malassezia releases lipase and phospholipase and the hydrolysis of triglyceride to produce free unsaturated fatty acids, which causes significant damage to the epidermal barrier, leading to abnormal proliferation of keratinocytes, and the limiting malassezia is the main dominant species. Malassezia mainly grows on the surface of the scalp and the sebaceous gland area, so anchoring the hair follicle microstructure to develop anti-malassezia research is the key to treating seborrheic dermatitis. At present, antifungal drugs can effectively treat seborrheic dermatitis and dandruff by reducing the number of malassezia, but long-term use will have a certain negative impact on human health. Therefore, it is of great significance to develop safe and effective hair care products that can prevent and treat malassezia in the hair follicle, which can provide effective and convenient treatment for patients.

[0003] Compared with conventional therapy, nanomaterials can better penetrate into the hair follicle and skin layer through their special physical properties and surface characteristics. Studies have shown that particles with a medium particle size (about 640 nm) have the strongest ability to enter the hair follicle, and by changing the particle size, different parts of the hair follicle can be selectively targeted. Therefore, nanotechnology is expected to provide a new strategy for the development of anti-malassezia products for anchoring the hair follicle structure. This targeted antibacterial effect can effectively remove malassezia that is difficult to remove in the sebaceous gland.

[0004] Theabrownin is a high polymer with a benzene ring as the main body, complexing with polysaccharides and proteins, and rich in carboxyl, hydroxyl, methyl and other groups. Although they are oxidation products of tea polyphenols, they still maintain relatively high antioxidant activity. Studies have shown that theabrownin has various biological activities such as antioxidant, anti-obesity, anticancer and regulation of intestinal microorganisms. Theabrownin can destroy the cell membrane of Staphylococcus aureus and Escherichia coli and increase the level of intracellular reactive oxygen species, which affects the normal metabolic activity of cells through oxidative stress, thereby causing abnormal death of bacteria. However, the anti-malassezia effect of single theabrownin component is not good, and a composite system needs to be constructed.

[0005] Previous studies have shown that selenium nanoparticles have bactericidal, immune regulation, anti-inflammatory, anti-viral and other functions. Further studies have speculated that the possible bactericidal mechanism is that excessive nanoparticles of selenium replace sulfur without selection, so that the thiol required for microbial growth is converted into selenol, oxidative stress, and thus cell death. However, the exact mechanism of nanometer selenium against Malassezia still needs further study. SUMMARY

[0006] The problem to be solved by the present application is to provide a theabrownin-selenium nanoparticle-Malassezia membrane composite system with anti-Malassezia function and its application.

[0007] To solve the above problems, the present application provides a theabrownin-selenium nanoparticle-Malassezia membrane composite system, comprising the following steps:

[0008] (1) Dissolve 25 mg of theabrownin in 24±2 mL of deionized water and stir uniformly;

[0009] (2) Add 90-110 mM (preferably 100 mM) of sodium selenite solution 9-11 mL (preferably 10 mL) to the theabrownin solution obtained in step (1), mix uniformly and stand (standing time is 10-20 min); then add 450-550 mM (preferably 500 mM) of vitamin C solution 15-17 mL (preferably 16 mL), stir for 0.8-1.2 hours (preferably 1 hour);

[0010] (3) Gradient centrifugation:

[0011] The mixture obtained in step (2) is first centrifuged at 4000±500 rpm for the first time, and the first centrifugation time is 15±3 min, and then the supernatant obtained by the first centrifugation is centrifuged at 6000±500 rpm for the second time, and the second centrifugation time is 15±3 min; the precipitate obtained by the second centrifugation is washed with deionized water (washed 2-4 times, and centrifuged after each washing is finished), and then deionized water is added to make up to 10 mL, to obtain a theabrownin-selenium nanoparticle (TB-Se) suspension;

[0012] Note: The present application adopts differential centrifugation, and the first centrifugation is to remove larger particles of TB-Se;

[0013] (4) Wash 50±5 ml of Malassezia liquid with PBS buffer and then perform crushing treatment, and then centrifuge (6000±500g centrifugation for 5±1 min) to collect the supernatant; after the supernatant is filtered, the permeate is centrifuged (12000±1000g centrifugation for 15±3 min) to collect the precipitate, and the precipitate is washed with sterile water (washed three times) to obtain Malassezia membrane;

[0014] (5) 5 mL of the suspension of the theabrownin-selenium nanoparticles (TB-Se) obtained in step (3) is mixed with the Malassezia film obtained in step (4) under ice-bath ultrasonication; then centrifugation (6000 ± 500 rpm for 5 ± 1 min) is performed to remove the supernatant (Malassezia film not completely coated with TB-Se), deionized water is added to make up to 5 mL, and a theabrownin-selenium nanoparticle / Malassezia film composite system (TB-Se@M containing Malassezia film-coated theabrownin selenium nanoparticles) is obtained.

[0015] As an improvement of the preparation method of the theabrownin-selenium nanoparticle@Malassezia film composite system of the present application: the bacterial content of the Malassezia bacterial solution is 10 10 individuals / ml.

[0016] As a further improvement of the preparation method of the theabrownin-selenium nanoparticle@Malassezia film composite system of the present application: the membrane in step (4) is a 0.45 μm water-based filter membrane.

[0017] As a further improvement of the preparation method of the theabrownin-selenium nanoparticle@Malassezia film composite system of the present application: in step 3), the precipitate obtained by secondary centrifugation is washed with deionized water 2-4 times (preferably 3 times) under ultrasonic conditions (40 KHz), with each washing time being 3 ± 1 min, and the precipitate is dispersed uniformly by blowing during the washing process. After each washing, centrifugation is performed. After the last washing (and centrifugation), deionized water is added to make up to 10 mL, and a suspension of theabrownin-selenium nanoparticles (TB-Se) is obtained.

[0018] As a further improvement of the preparation method of the theabrownin-selenium nanoparticle@Malassezia film composite system of the present application: in step 4), glass beads with a diameter of 425-600 μm (about 0.5 g) are added for crushing.

[0019] The present application also simultaneously provides an anti-Malassezia theabrownin-selenium nanoparticle@Malassezia film composite system prepared by any of the above methods.

[0020] The present application also simultaneously provides the use of the theabrownin-selenium nanoparticle@Malassezia film composite system prepared by any of the above methods: for preparing an anti-Malassezia drug or agent.

[0021] As an improvement of the use of the present application: for preparing a drug or agent for treating seborrheic dermatitis.

[0022] As a further improvement of the use of the present application: the anti-Malassezia agent can be used in cosmetics.

[0023] That is, the drug or agent for treating seborrheic dermatitis includes a pharmaceutical preparation, a cosmetic, a care product, and a beauty product.

[0024] As a further improvement of the use of the present application: the cosmetic is any of the following: shampoo, hair conditioner, hair serum, hair oil, scalp hair nourishing product, hair gel, hair mask, mascara / brow care liquid.

[0025] The theasinensin-selenium nanoparticle@malassezia membrane composite system with anti-malassezia function of the present application can be prepared into 100mg / mL aqueous solution for application on the scalp, and applied 1-3 times per day.

[0026] The present application first considers preparing the theasinensin-selenium nanoparticle, then separating and extracting the malassezia membrane, and constructing the theasinensin-selenium nanoparticle@malassezia membrane composite system, which utilizes the synergistic effect of theasinensin and selenium nanoparticles in killing malassezia in the skin and repairing the skin barrier, improves seborrheic dermatitis, provides a new method for solving the problem of anti-malassezia, and provides a theoretical basis for related research on the regulation of scalp microecology by nanoparticles.

[0027] The present application has the beneficial effects that: the composite nanomaterial based on theasinensin-selenium nanoparticles is successfully prepared, which expands the application field of theasinensin-selenium nanoparticles. The nanoscale composite system prepared by the present application can effectively penetrate into the hair follicle, kill malassezia, and improve seborrheic dermatitis, which can be developed into a natural shampoo for removing dandruff and improving seborrheic dermatitis, replacing artificially synthesized shampoo and anti-malassezia products; the present application is designed scientifically and reasonably, and has good practical application value.

[0028] In summary, the present application prepares theasinensin-selenium nanoparticles, and combines with the malassezia membrane, and after centrifugal washing, obtains the theasinensin-selenium nanoparticle@malassezia membrane composite system. The composite system has significant effect in killing malassezia and repairing the skin barrier. BRIEF DESCRIPTION OF DRAWINGS

[0029] The specific embodiments of the present application will be further described in detail below in combination with the drawings.

[0030] Figure 1 The preparation process diagram of theasinensin-selenium nanoparticles and theasinensin-selenium nanoparticle@malassezia membrane composite system;

[0031] Figure 2 The SEM (up) and TEM (down) pictures of theasinensin-selenium nanoparticles and theasinensin-selenium nanoparticle@malassezia membrane composite system;

[0032] Figure 3 In the drawings, a is a nanoparticle size analysis experiment diagram; b is a Zeta potential analysis experiment diagram; c is an ultraviolet-visible spectrum experiment diagram; d is an infrared spectrum experiment diagram;

[0033] Figure 4The in vitro sterilization effect of different nano-system treatment groups on Malassezia;

[0034] Figure 5 The ear scaling of mice in different administration groups on the 8th day;

[0035] Figure 6 The H&E staining section of the ear tissue of mice in different administration groups on the 8th day;

[0036] Figure 7 The stratum corneum thickness of the ear tissue of mice in different administration groups

[0037] Figure 8 The SEM picture of TB-Se synthesized by using sodium selenite and zinc selenite as raw materials in Comparative Example 1;

[0038] Figure 9 The anti-Malassezia effect of TB-Se synthesized by using sodium selenite and zinc selenite as raw materials;

[0039] Figure 10 The anti-Malassezia effect of TB-Se synthesized by using different stirring times;

[0040] Figure 11 The SEM picture of TB-Se obtained by using different centrifugal methods in Comparative Example 3;

[0041] Figure 12 The anti-Malassezia effect of TB-Se obtained by using different centrifugal methods. DETAILED DESCRIPTION

[0042] The present application will be further described in conjunction with specific examples, but the protection scope of the present application is not limited to the following:

[0043] In the following examples, the experimental methods are conventional methods unless otherwise specified; and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0044] The theabromine can be purchased from Zhejiang Zhonglu Songbo Biological Engineering Co., Ltd. (the content of the effective component theabromine is >99%).

[0045] The Malassezia is the freeze-dried powder of Malassezia restricta with the number 185492 of BNCC, and then the conventional activation culture is carried out according to the culture conditions informed by it: 30℃, aerobic, YM culture medium, 24-48h. YM culture medium: yeast extract 3.0g, malt extract 3.0g, glucose 10.0g, peptone 5.0g, agar 20.0g (liquid culture medium does not contain), distilled water 1.0L, pH 6.2±0.2. 121℃, 15min sterilization.

[0046] Thus, a Malassezia liquid containing 10 10 cells / ml was obtained.

[0047] Example 1, a preparation method of a theabrownin-selenium nanoparticle / Malassezia membrane composite system, in turn comprising the following steps:

[0048] (1) 25 mg of theabrownin was weighed into a 50 mL centrifuge tube, 24 mL of deionized water was added, and the mixture was stirred until uniform;

[0049] (2) 10 mL of 100 mM sodium selenite solution was added to the theabrownin solution obtained in step (1), the mixture was stirred until uniform, and then allowed to stand for 15 min; then 16 mL of 500 mM vitamin C solution was added, and the mixture was stirred at 25°C for 1 h to mix;

[0050] (3) Gradient centrifugation:

[0051] The mixture obtained in step (2) was first centrifuged at 4000 rpm for 15 min, and then the supernatant obtained from the first centrifugation was centrifuged at 6000 rpm for 15 min; the precipitate obtained from the second centrifugation was washed with deionized water under ultrasonic conditions at 40 KHz, and the precipitate was dispersed uniformly by blowing during the washing process; after each washing, the water was removed by centrifugation; the washing with deionized water was repeated for a total of 3 times, and each washing time was 3 min; after the last washing and centrifugation, the precipitate was resuspended in 10 mL of deionized water, and the theabrownin-selenium nanoparticle (TB-Se) suspension was obtained, which was stored at 4°C for subsequent experiments.

[0052] Note: The purpose of the first centrifugation is to remove larger particles (waste disposal), and the theabrownin-selenium nanoparticles obtained in this step have a particle size of about 60-80 nm.

[0053] (4) 50 mL of Malassezia liquid (containing 10 10 cells / ml) was placed in a 2 mL round-bottom centrifuge tube, washed three times with PBS buffer (0.01 M, pH 7.2-7.4), and then broken by adding 0.5 g of grinding beads (diameter 425-600 μm) (broken for 3 min at a frequency of 60 Hz), centrifuged at 6000 g for 5 min to collect the supernatant, and the precipitate (which was the cell wall and organelles of Malassezia) was discarded. The supernatant was filtered (through a 13 mm diameter x 0.45 μm water filter membrane), and the permeate was centrifuged at 12000 g for 15 min to collect the precipitate (mainly the cell membrane of Malassezia), which was washed three times with sterile water to obtain the Malassezia membrane (M).

[0054] (5) 5 mL of the suspension of the theabrownin-selenium nanoparticles (TB-Se) obtained in step (3) was mixed with the Malassezia film obtained in step (4) under ice-bath ultrasonication (so as to load the Malassezia film onto the theabrownin-selenium nanoparticles). Subsequently, centrifugation was performed at 6000 rpm for 5 min to remove the supernatant (Malassezia film not completely coated with TB-Se), deionized water was added to make up to 5 mL, and a theabrownin-selenium nanoparticle / Malassezia film composite system (i.e., Malassezia film-coated theabrownin-selenium nanoparticles, TB-Se@M) was obtained.

[0055] Description: The selenium nanoparticles were also prepared according to the following method: 10 mL of a 100 mM sodium selenite solution was mixed with 16 mL of a 500 mM vitamin C solution under stirring at 25 °C for 1 h so as to mix uniformly. The obtained mixed solution was first centrifuged at 4000 rpm for 15 min, and then the supernatant obtained after the first centrifugation was centrifuged at 6000 rpm for 15 min. The precipitate obtained after the second centrifugation was washed with deionized water (washed with deionized water for 3 times, each time for 3 min, and centrifuged after each washing); after the last centrifugation, water was added to make up to 10 mL, and a suspension of selenium nanoparticles (Se) was obtained, which was stored at 4 °C for subsequent experiments.

[0056] Experiment 1, characterization of the theabrownin-selenium nanoparticle / Malassezia film composite system

[0057] The theabrownin-selenium nanoparticle / Malassezia film composite system prepared in Example 1 was characterized by scanning electron microscopy and transmission electron microscopy, ultraviolet absorption spectrometry, Zeta potential analysis, nanoparticle size analysis, infrared spectroscopy, and the like.

[0058] Scanning electron microscopy and transmission electron microscopy were used to characterize the morphology of the nanoparticles. The theabrownin-selenium nanoparticles (the suspension of the theabrownin-selenium nanoparticles (TB-Se) obtained in step (3)) and the theabrownin-selenium nanoparticle / Malassezia film composite system (TB-Se@M) prepared in Example 1 were dropped onto a silicon wafer, gold was sprayed after natural air-drying, and the size and morphology were observed by scanning electron microscopy; the theabrownin-selenium nanoparticles and the theabrownin-selenium nanoparticle / Malassezia film composite system prepared in Example 1 were dropped onto a copper mesh, gold was sprayed after natural air-drying, and the size and morphology were observed under a transmission electron microscope. It was found that the particle size of the theabrownin-selenium nanoparticles was 60-80 nm, and the particle size of the theabrownin-selenium nanoparticle / Malassezia film composite system was 100-120 nm.

[0059] UV-Vis spectroscopy was used to characterize the optical properties of the nanoparticles. UV-Vis spectroscopy was measured (200-800 nm) by quartz microplate using a UV-Vis spectrophotometer. The UV-Vis spectra of the theabrownin-selenium nanoparticles and the theabrownin-selenium nanoparticles / malassezia membrane composite system were measured three times, respectively.

[0060] Nanoparticle size analysis and Zeta potential analysis were used to characterize the particle size distribution and Zeta potential of the nanoparticles. The particle size and Zeta potential of the theabrownin-selenium nanoparticles and the theabrownin-selenium nanoparticles / malassezia membrane composite system were measured by a nanoparticle size analyzer. After ultrasonic treatment for 3 min, they were dispersed uniformly and measured three times, respectively.

[0061] Fourier transform infrared spectroscopy was used to explore the surface chemical groups of different samples. The Fourier transform infrared spectra of the malassezia membrane (M), theabrownin (TB), theabrownin-selenium nanoparticle (TB-Se) suspension and theabrownin-selenium nanoparticle / malassezia membrane composite system (TB-Se@M) solution were measured after freeze-drying, and the wavelength range was set to 400-4000 cm -1 .

[0062] According to Figure 2 and Figure 3 (a-d), the addition of malassezia membrane changed the UV spectra, particle size and potential value of theabrownin-selenium nanoparticles, which proved the successful preparation of the theabrownin-selenium nanoparticles / malassezia membrane composite system.

[0063] Experiment two, the bactericidal effect of theabrownin-selenium nanoparticles / malassezia membrane composite system on malassezia

[0064] The antibacterial activity of the theabrownin group, the selenium group, the theabrownin-selenium nanoparticle group and the theabrownin-selenium nanoparticle / malassezia membrane group (the selenium concentration remained the same) was determined by plate counting method.

[0065] 1 mL of malassezia bacterial solution (1 x 10 5 CFU / mL) was taken from each group, centrifuged at 5500 rpm / min for 10 min to retain the precipitate, washed twice with sterile water, and 1.5 mL of theabrownin (250 mg / L), 1.5 mL of the selenium suspension obtained above, 1.5 mL of the theabrownin-selenium nanoparticle suspension obtained in step (3) of Example 1 and the theabrownin-selenium nanoparticle / malassezia membrane composite system obtained in step (5) of Example 1 were added as the corresponding experimental groups, and a control group (1.5 mL of sterile water was added) was set up. All treatment groups were placed in a 30°C shaking bed, and samples were taken after 24 hours. Sterile water was used to dilute by ten times, and the number of colonies was recorded after plate coating and incubation at 30°C for 24 hours. All experimental groups were counted in triplicate.

[0066] according to Figure 4 It can be seen that, compared with the control group, the single treatment with theaflavins (TB) and selenium (Se) was not effective against Malassezia, while theaflavins-selenium nanoparticles (TB-Se) and theaflavins-selenium nanoparticle / Malassezia membrane composite system (TB-Se@M) were both effective in killing Malassezia, and theaflavins-selenium nanoparticle / Malassezia membrane composite system (TB-Se@M) had the best anti-Malassezia effect, which confirms the synergistic anti-Malassezia effect of theaflavins and selenium.

[0067] Experiment 3: Effects of the theabrownin-selenium nanoparticle / Malassezia membrane composite system on the inflammation of seborrheic dermatitis

[0068] Eight-week-old male C57BL / 6 mice were used in the experiment. After routine acclimatization culture (7 days), the mice were divided into a blank control group, a model group, a theabrownin group (treated with 250 mg / L theabrownin), a selenium group (treated with pure nano-selenium obtained above), a theabrownin-selenium nanoparticle group (treated with theabrownin-selenium nanoparticles obtained in Example 1), and a theabrownin-selenium nanoparticle / Malassezia membrane group (treated with the theabrownin-selenium nanoparticle / Malassezia membrane composite system obtained in Example 1), with 10 mice in each group. Before the experiment, Malassezia was resuspended in 100 μL of olive oil (10 9 A bacterial suspension was prepared using CFU / mL. For each group of mice, the skin barrier was disrupted by applying medical tape to the ears (5 sites per mouse, maintaining consistent treatment intensity). Except for the control group, each mouse in each group received 100 μL of the bacterial suspension daily for 7 consecutive days. Treatment was administered 2 hours after each modeling session. Defatted cotton soaked in theaflavins, selenium, or theaflavins-selenium nanoparticles was applied thoroughly to the mouse's ear for 2 minutes. The control and model groups received the same application using distilled water. Treatment ended on day 8. After the experiment, all mice were euthanized, and ear tissue was collected. The tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Pathological changes were observed under a microscope. During the experiment, the ear flaking was recorded by photograph every other day. Mice had free access to food and water during the experiment.

[0069] according to Figure 5 It was observed that large flakes of dandruff were produced in the model group from day 5 to day 7. Compared with the model group, the theabrownin group, selenium group, theabrownin-selenium nanoparticle group, and the theabrownin-selenium nanoparticle / Malassezi membrane group only had a small amount of dandruff. This indicates that compared with the model group, all material treatment groups could improve seborrheic dermatitis in mice and reduce dandruff production, but the theabrownin-selenium nanoparticle / Malassezi membrane group showed the best effect.

[0070] Experiment four, the influence of the theabrownin-selenium nanoparticle / malassezia film composite system on the ear tissue of seborrheic dermatitis mice

[0071] The blank control group (Control), the model group (Model), the theabrownin group (TB), the selenium group (Se), the theabrownin-selenium nanoparticle group (TB-Se), and the theabrownin-selenium nanoparticle / malassezia film group (TB-Se@M) were set in the same way as in Experiment three. After the experiment, all the mice were euthanized, and the ear tissues were collected. The tissues were fixed with 4% paraformaldehyde, embedded in paraffin, sliced, and stained with hematoxylin and eosin (H&E). The pathological changes of the tissues were observed under a microscope.

[0072] According to Figure 6 and Figure 7 , compared with the control group, the stratum corneum of the model group was significantly thickened. After treatment with theabrownin, selenium, theabrownin-selenium nanoparticle, and theabrownin-selenium nanoparticle / malassezia film composite system, the thickness of the stratum corneum became thinner, and the theabrownin-selenium nanoparticle / malassezia film composite system had the best improvement effect, proving the synergistic effect of theabrownin and selenium nanoparticles.

[0073] Comparative Example 1: In Example 1, sodium selenite was replaced with zinc selenite, and the selenium concentration and amount were kept unchanged, i.e., "100 mM sodium selenite solution 10 mL" was replaced with "100 mM zinc selenite solution 10 mL", and the rest was the same as Example 1. The SEM image of this comparative example 1 is compared with Example 1 as shown in Figure 8 According to Figure 8 , it can be known that the yield of TB-Se synthesized from sodium selenite is higher.

[0074] The product obtained in Comparative Example 1 was detected according to the method described in Experiment two, and the results are shown in Figure 9 The sterilization effect of this Comparative Example 1 was 80% (bacterial colony number 2×10 4 CFU), while the sterilization effect of the present application was 98% (bacterial colony number 2×10 3 CFU).

[0075] Comparative Example 2: In step (2) of Example 1, "stirring for 1 h" was replaced with 0.5 h and 2 h, respectively, and the concentrations and amounts of the solutions were kept unchanged, and the rest was the same as Example 1. Stirring for too short a time cannot obtain the relatively uniform nanoparticles described in Example 1.

[0076] The product obtained in Comparative Example 2 was detected according to the method described in Experiment two, and the results are shown in Figure 10 The sterilization effect corresponding to 0.5 h was 86% (bacterial colony number 1.4×10 4 CFU), and the sterilization effect corresponding to 2 h was 8% (bacterial colony number 9.2×10 4The sterilization effect of the product obtained by the method of Example 1 is 98%, while the sterilization effect of the product obtained by the method of Comparative Example 3 is 14%.

[0077] In Comparative Example 3, the gradient centrifugation of step (3) of Example 1 is changed to "4000 rpm centrifugation for 15 min to obtain the precipitate", or changed to "first centrifugation at 6000 rpm for 15 min to obtain the supernatant, and then second centrifugation at 12000 rpm for 15 min to obtain the precipitate", and the rest is the same as Example 1. The SEM image of the product is shown in Figure 11 Figure 11 It can be seen that the yield of TB-Se obtained by 4000 rpm centrifugation is low, the TB-Se obtained by first centrifugation at 4000 rpm to obtain the supernatant and then second centrifugation at 6000 rpm to obtain the precipitate has uniform size and high yield, and the yield of TB-Se obtained by first centrifugation at 6000 rpm to obtain the supernatant and then second centrifugation at 12000 rpm to obtain the precipitate is too low, which is not recommended.

[0078] The product obtained by "4000 rpm centrifugation for 15 min" of Comparative Example 3 is detected according to the method of Experiment 2, and the results are shown in Figure 12 4 The sterilization effect of the product obtained by "4000 rpm centrifugation to obtain the precipitate" is 14% (bacterial colony number 8.6 x 10 4 CFU), while the sterilization effect of the product of the present application is 98%.

[0079] In Comparative Example 4, the amount of Malassezia liquid in Example 1 is changed to 25 ml and 12.5 ml, respectively, and the rest is the same as Example 1. Only Example 1 can obtain TB-Se@M with relatively uniform size, while the two products obtained by Comparative Example 4 cannot achieve uniform size, which leads to poor sterilization effect.

[0080] Finally, it should be noted that the above only lists several specific embodiments of the present application. Obviously, the present application is not limited to the above embodiments, and there can be many variations. All variations that can be directly derived or inferred by those skilled in the art from the disclosure of the present application should be considered as falling within the scope of the present application.

Claims

1. A method for preparing a theabrownin-selenium nanoparticle@Malassezia membrane composite system, characterized in that... Includes the following steps: (1) Dissolve 25 mg of theabrownin in 24 ± 2 mL of deionized water and stir well; (2) Add 9-11 ml of 90-110 mM sodium selenite solution to the theabrownin solution obtained in step (1), mix well and let stand; then add 15-17 ml of 450-550 mM vitamin C solution and stir for 1 hour. (3) Gradient centrifugation: The mixture obtained in step (2) was first centrifuged at 4000±500 rpm for 15±3 min, and then the supernatant obtained from the first centrifugation was centrifuged at 6000±500 rpm for 15±3 min. The precipitate obtained from the second centrifugation was washed with deionized water and then deionized water was added to make up to 10 mL to obtain a suspension of tea brown pigment-selenium nanoparticles. (4) Wash 50±5ml of Malassezia bacterial culture with PBS buffer, add glass beads with a diameter of 425~600μm for disruption, then centrifuge and collect the supernatant; after passing the supernatant through the membrane, take the permeate, centrifuge and collect the precipitate, and wash with sterile water to obtain Malassezia membrane; The bacterial count of Malassezia culture was 10. 10 The membrane is a 0.45 μm aqueous filtration membrane; (5) Mix 5 mL of the suspension of theabrownin-selenium nanoparticles obtained in step (3) with the Malassezia membrane obtained in step (4) under an ice bath; then centrifuge to remove the supernatant, add deionized water to make up to 5 mL, and obtain the theabrownin-selenium nanoparticle / Malassezi membrane composite system.

2. The preparation method of the theabrownin-selenium nanoparticle@Malassezia membrane composite system according to claim 1, characterized in that... In step (3): the precipitate obtained by the second centrifugation is washed with deionized water 2 to 4 times under ultrasonic conditions. After the last wash, deionized water is added to make up to 10 mL to obtain a suspension of tea brown pigment-selenium nanoparticles.

3. The theabrownin-selenium nanoparticle@Malassezi membrane composite system prepared by any one of claims 1 or 2.

4. The use of the theaflavin-selenium nanoparticle@Malassezi membrane composite system as described in claim 3, characterized in that: Drugs or reagents used to prepare anti-Malassezia drugs.

5. The use according to claim 4, characterized in that: Used to prepare drugs or reagents for treating seborrheic dermatitis.

6. The use according to claim 4 or 5, characterized in that: The reagent for treating Malassezia can be used in cosmetics.

7. The use according to claim 6, characterized in that: Cosmetics include any of the following: shampoo, conditioner, hair serum, hair oil, hair gel, hair mask, and eyelash / eyebrow treatment.

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