Application of fucoxanthin in preparation of oral anti-alopecia medicine

Abstract

The invention belongs to the technical field of natural products, and provides application of fucoxanthin in preparation of oral anti-alopecia drugs. Animal experiments find that long-term high-fat diet can lead to large-piece shedding of hair accompanied by skin inflammation, and fucoxanthin has a treatment effect on high-fat diet induced mouse back hair shedding and skin inflammation, can obviously relieve high-fat diet induced subcutaneous cholesterol crystal accumulation and sebaceous gland cell hypertrophy, and has the effects of preventing and treating the high-fat diet induced mouse back hair shedding and skin inflammation and preventing and treating the high-fat diet induced mouse back hair shedding and the high-fat diet induced sebaceous gland cell hypertrophy. And skin epidermal hyperplasia and cuticle thickening induced by high fat diet can be improved. Besides, cell experiments also find that fucoxanthin intervention can reduce oleic acid infiltration of the hair follicle stem cells and reduce oleic acid induced death and apoptosis of the hair follicle stem cells, and prove that fucoxanthin has a protection effect on the hair follicle stem cells, has low toxic and side effects on cells and animals as a natural algae extract, and has a good application prospect. Good clinical application prospects are realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of natural products and relates to the application of fucoxanthin in the preparation of an oral anti-hair loss drug. Background Art

[0002] The hair follicle is the basic unit of hair. Hair grows from stem cells within the follicle. Hair grows in cycles, divided into the anagen phase, the catagen phase, and the telogen phase. The anagen phase is the longest, lasting approximately 2-5 years, while the catagen phase is approximately 2-3 weeks, and the telogen phase lasts for several months. During the anagen phase, the follicular stem cells within the follicle actively grow and differentiate into hair roots, contributing to the hair's continued length. The catagen phase, approximately 2-3 weeks after the anagen phase, is characterized by stagnant growth of the follicle stem cells and gradual hair aging. Following the catagen phase, the telogen phase lasts 2-3 months, during which the follicle ceases development and the roots begin to loosen, making the hair extremely susceptible to shedding. Normally, approximately 90-95% of hair is in the anagen phase, less than 1% is in the catagen phase, and approximately 5-10% is in the telogen phase. The number of hair follicles is determined at birth and only decreases thereafter. Hair follicles are fragile and easily damaged by chemical and physical factors, leading to hair loss.

[0003] Hair loss has become a common concern for many people. There are many causes of hair loss, including genetics, stress, circadian rhythm disruption, illness, and medication. Furthermore, a high-fat diet and obesity have recently been identified as significant contributing factors. Hair loss symptoms are also becoming younger, with 60% of men experiencing hair loss experiencing it between the ages of 25 and 30. While hair loss itself does not cause illness, it can diminish a person's appearance, damage their confidence and self-esteem, and negatively impact their emotions, interpersonal relationships, and social connections. In severe cases, it can even lead to anxiety and depression.

[0004] With lifestyle changes, the number of people suffering from metabolic syndrome, characterized by the "three highs," is increasing. Recent studies have found that a high-fat diet can cause skin inflammation and hair loss. Another study has also demonstrated that a high-fat diet increases metabolic stress in hair follicle stem cells, accelerating their apoptosis and leading to irreversible hair loss. Furthermore, some studies have found that a high-fat diet can induce perfusion dysfunction in the skin's microvasculature, leading to hair loss. This phenomenon also occurs in diabetic skin complications, where high glucose levels induce thickening of the capillary basement membrane, leading to nutritional disorders in the dermis and hair follicles, and inducing hair loss and skin ulcers.

[0005] Although hair loss can be caused by a variety of factors, the only drugs currently used clinically for the treatment of androgenic alopecia are those targeting androgenic alopecia. Finasteride, dutasteride, and milodinil are the three drugs currently marketed globally for the treatment of androgenic alopecia. Finasteride and dutasteride are 5-α-reductase inhibitors that treat male pattern baldness by inhibiting the conversion of testosterone to dihydrotestosterone. However, finasteride can cause side effects such as decreased libido and erectile dysfunction. Milodinil is the only drug currently available for the treatment of androgenic alopecia that can be used by both men and women. It is a potassium channel opener that induces vasodilation in the skin, increasing blood supply to the skin, enhancing nutrition to hair follicles, and promoting hair growth. However, all of these drugs for androgenic alopecia carry the risk of recurrence and potential toxicity with long-term use. Other drugs for androgenic alopecia, such as furetinol and clapriston, are under development. Furetan is also an androgen receptor antagonist, the first topical medication of its kind, and has entered Phase III clinical trials. Clapriston, a topical androgen receptor inhibitor similar to Furetan, is also actively undergoing Phase III clinical trials. Other studies have reported that various Chinese herbal medicines, including Polygonum multiflorum, scallion extract, mulberry root extract, snow bean curd and jade soup, artemisinin, saponins, and Pueraria root, have some therapeutic effects on hair loss, but their effectiveness in preventing hair loss has been suboptimal.

[0006] As can be seen from the above, although there are currently reports of anti-hair loss drugs, they generally have significant limitations and are difficult to achieve ideal anti-hair loss effects. Moreover, these drugs mainly target hereditary hair loss and have not been reported for the growing number of hair loss induced by high-fat diets. Therefore, there is an urgent need to develop a drug with good anti-hair loss efficacy, especially for hair loss caused by high-fat diets. Summary of the Invention

[0007] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes the use of fucoxanthin in the preparation of an oral anti-hair loss drug. For hair loss caused by a high-fat diet, fucoxanthin can protect hair follicle stem cells from apoptosis and necrosis caused by high-fat damage, thereby improving local skin microcirculation, increasing blood supply and nutrition to hair follicles and dermis, and achieving excellent anti-hair loss effects. The present invention found through animal experiments that feeding fucoxanthin can improve hair loss and skin inflammation induced by a high-fat diet in mice, providing a treatment plan for the problems of hair loss and skin inflammation induced by a high-fat diet, but is not limited to hair loss induced by a high-fat diet.

[0008] The first aspect of the present invention provides the use of fucoxanthin in the preparation of an oral anti-hair loss drug.

[0009] Specifically, the use of fucoxanthin in the preparation of oral anti-hair loss drugs.

[0010] Fucoxanthin (Fx), a natural algae extract, has low toxicity to cells and animals. The present invention has found that oral administration of fucoxanthin has a significant therapeutic effect on hair loss and skin inflammation induced by a long-term high-fat diet.

[0011] Application of fucoxanthin in the preparation of oral drugs for preventing and treating hair loss caused by high-fat diet.

[0012] Fucoxanthin can be used to prevent and treat hair loss caused by a high-fat diet.

[0013] Application of fucoxanthin in the preparation of oral drugs for treating skin inflammation caused by high-fat diet.

[0014] The invention discloses an application of fucoxanthin in the preparation of an oral drug for preventing and treating hair loss caused by a high-fat diet and for treating skin inflammation caused by a high-fat diet.

[0015] A high-fat diet refers to: According to the "Guidelines for Dietary Regimens with Hyperlipidemia in Adults (2023 Edition)" issued by the National Health Commission, hereinafter referred to as the "Guidelines", fasting venous serum test indicators divide dyslipidemia into four types: hypercholesterolemia (total cholesterol, TC ≥ 5.2 mmol / L), hypertriglyceridemia (triglycerides, TG ≥ 1.7 mmol / L), high low-density lipoprotein cholesterol (low-density lipoprotein cholesterol, LDL-C ≥ 3.4 mmol / L), low high-density lipoprotein cholesterol (high-density lipoprotein cholesterol, HDL-C < 1.0 mmol / L). When one or more of the above blood lipid indicators are abnormal, dyslipidemia can be diagnosed. Given that low-density lipoprotein cholesterol and triglycerides are the main causes of atherosclerosis, as long as the daily diet causes the two to increase alone or simultaneously, it can be called a high-fat diet. The guidelines recommend that fat intake should account for 20-25% of total energy. People with hypertriglyceridemia should reduce their total daily fat intake as much as possible. Taking an adult's daily energy intake of 1800-2000kcal as an example, the equivalent fat intake from various food sources throughout the day (including cooking oil, animal foods, nuts and other foods) is between 40-55g. The daily cooking oil intake should not exceed 25g, so exceeding the above standards can also be considered a high-fat diet.

[0016] A second aspect of the present invention provides an oral medicine kit for preventing and treating hair loss caused by a high-fat diet and treating skin inflammation caused by a high-fat diet.

[0017] A medicine kit for preventing and treating hair loss caused by a high-fat diet and treating skin inflammation caused by a high-fat diet, the medicine kit comprising fucoxanthin, wherein the fucoxanthin content in the medicine kit is formulated according to a daily dosage standard of 0.001-0.05 mg / kg / d.

[0018] Preferably, the content of fucoxanthin in the medicine kit is formulated according to a daily dosage standard of 0.009-0.018 mg / kg / d.

[0019] The third aspect of the present invention provides the use of fucoxanthin in the preparation of an oral drug for protecting hair follicle stem cells.

[0020] Application of fucoxanthin in the preparation of oral drugs for protecting hair follicle stem cells.

[0021] Preferably, the fucoxanthin protects hair follicle stem cells by reducing hair follicle stem cell damage and stably maintaining the number of hair follicle stem cells.

[0022] Use of fucoxanthin in the preparation of a drug for preventing hair loss by protecting hair follicle stem cells.

[0023] Preferably, the concentration of fucoxanthin in the drug is 0.01-10 μmol / L.

[0024] Further preferably, the concentration of fucoxanthin in the drug is 0.25-5 μmol / L.

[0025] More preferably, the concentration of fucoxanthin in the drug is 0.25-1 μmol / L.

[0026] A fourth aspect of the present invention provides an oral anti-hair loss drug.

[0027] An oral anti-hair loss medicine comprising fucoxanthin.

[0028] Preferably, the oral anti-hair loss medicine further contains pharmaceutically acceptable excipients.

[0029] Preferably, the oral anti-hair loss drug is in the form of tablets, capsules, oral liquids, and granules.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The present invention provides the use of fucoxanthin in the preparation of oral anti-hair loss drugs. The present invention found through animal experiments that mice that had been fed a high-fat diet for 20 weeks had large patches of hair loss on their backs, accompanied by skin inflammation. After being treated with fucoxanthin, the mice's hair became shiny and smooth, and no hair loss or skin inflammation occurred. This proves that fucoxanthin has a therapeutic effect on hair loss and skin inflammation on the backs of mice induced by a high-fat diet, and can significantly reduce the accumulation of subcutaneous cholesterol crystals and sebaceous gland cell hypertrophy induced by a high-fat diet, and significantly improve the skin epidermal hyperplasia and stratum corneum thickening induced by a high-fat diet. In addition, the present invention also found through cell experiments that fucoxanthin intervention can reduce the infiltration of hair follicle stem cells by oleic acid, and reduce the death and apoptosis of hair follicle stem cells induced by oleic acid, proving that fucoxanthin has a protective effect on hair follicle stem cells. Fucoxanthin can protect hair follicle stem cells from apoptosis and necrosis caused by high-fat damage, thereby improving local skin microcirculation, increasing blood supply and nutrition to hair follicles and dermis, and promoting hair growth.

[0032] (2) Oral administration of fucoxanthin has a very significant therapeutic effect on hair loss and skin inflammation induced by a long-term high-fat diet. Moreover, as a natural carotenoid derived from brown algae, fucoxanthin has low toxicity and side effects on cells and animals, and its oral safety has been supported by multiple studies. Animal experiments and initial clinical trials have shown that within the recommended dose range, fucoxanthin does not show significant toxicity or serious adverse reactions. Its metabolites are safely cleared by the liver and kidneys, and the safety window is relatively wide. Therefore, fucoxanthin has good prospects for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 These are photos of hair loss and skin inflammation in each group of mice in the animal experiment;

[0034] Figure 2 Figures of hematoxylin-eosin staining (HE staining) of skin tissues of mice in each group in the animal experiment and the statistical results of cholesterol crystals and sebaceous gland cells (n=4, **** indicates p<0.0001);

[0035] Figure 3 Masson staining of skin tissue of mice in each group in the animal experiment and statistical results of epidermal thickness and stratum corneum thickness (n=4, ** indicates p<0.01; *** indicates p<0.001; **** indicates p<0.0001);

[0036] Figure 4 The immunofluorescence staining results of hair follicle stem cell markers K15 and CD34 and the statistical results of relative fluorescence intensity of the skin tissues of mice in each group in the animal experiment;

[0037] Figure 5The results of in situ terminal transferase-labeling (TUNEL) staining of hair follicle stem cells in the skin tissue of each group of mice in the animal experiment and the statistical results of the relative fluorescence intensity of TUNEL staining;

[0038] Figure 6 Figure 3 shows the oil red staining and cell count of hair follicle stem cells in each group in the cell experiment, as well as the statistical results of the positive rate of oil red staining (n=4, * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001; **** indicates p<0.0001; NS (nosignificance) indicates no significance);

[0039] Figure 7 The results of in situ terminal transferase labeling (TUNEL staining) of hair follicle stem cells in each group in the cell experiment and the statistical results of relative fluorescence intensity of TUNEL staining (n=4, ** indicates p<0.01; **** indicates p<0.0001);

[0040] Figure 8 This is the Oil Red staining image of hair follicle stem cells after being stimulated by oleic acid and treated with astaxanthin at different concentrations;

[0041] Figure 9 This is the oil red staining image of hair follicle stem cells after being stimulated by oleic acid and treated with different concentrations of lutein and carotene;

[0042] Figure 10 This is the Oil Red staining image of hair follicle stem cells after being stimulated by oleic acid and treated with fucoxanthin at different concentrations;

[0043] Figure 11 This is a comparison of the results of oil red staining of hair follicle stem cells stimulated by oleic acid after intervention with different concentrations of fucoxanthin, astaxanthin, lutein, and carotene;

[0044] Figure 12 This is the TUNEL staining diagram of hair follicle stem cells stimulated by oleic acid after intervention with different concentrations of fucoxanthin, astaxanthin, lutein and carotene;

[0045] Figure 13 This is a comparison of the TUNEL staining results of hair follicle stem cells stimulated by oleic acid after intervention with different concentrations of fucoxanthin, astaxanthin, lutein and carotene. DETAILED DESCRIPTION

[0046] In order to make the technical solution of the present invention more clearly understood by those skilled in the art, the following examples are given for illustration. It should be noted that the following examples do not limit the scope of protection claimed by the present invention.

[0047] Unless otherwise specified, the raw materials, reagents, or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0048] Application effect verification

[0049] 1. Effects of fucoxanthin on the hair and skin of mice fed a high-fat diet (animal experiment)

[0050] 1.1 Experimental animals and groups

[0051] Twenty-four ApoE- / - mice were purchased and randomly divided into four groups, with six mice in each group, respectively marked as: (1) normal diet group (Normal Feed, NF); (2) high-fat feed plus normal saline group (High-fat Feed plus Normal Saline, HF+NS); (3) high-fat feed plus low dose of fucoxanthin intervention group (High-fat Feed plus Low Dose of Fucoxanthin, HF+LFx, where the fucoxanthin feeding dose was 0.009 mg / kg / d); (4) high-fat feed plus high dose of fucoxanthin intervention group (High-fat Feed plus High Dose of Fucoxanthin, HF+HFx, where the fucoxanthin feeding dose was 0.018 mg / kg / d).

[0052] 1.2 Animal feeding regimen

[0053] All mice were adaptively fed for 1 week after entering the barrier system. Thereafter, the normal diet group (NF) maintained a normal diet without any intervention. The other groups were first fed a high-fat diet for 8 weeks without any intervention. After the end of the period, they continued to be fed a high-fat diet and received different interventions for each group: the high-fat diet plus normal saline group (HF+NS) continued to be fed a high-fat diet and received normal saline by gavage for 12 weeks; the high-fat diet plus low-dose fucoxanthin intervention group (HF+LFx) continued to be fed a high-fat diet and received a low-dose fucoxanthin (0.009 mg / kg / d) by gavage for 12 weeks; and the high-fat diet plus high-dose fucoxanthin intervention group (HF+HFx) continued to be fed a high-fat diet and received a high-dose fucoxanthin (0.018 mg / kg / d) by gavage for 12 weeks.

[0054] The high-fat diet feeding mentioned above refers to: feeding with Jiangsu Collaborative Biological Company's XT108C high-fat model feed, which contains 20% fat and 1.25% cholesterol; the initial weight of the mice is about 20g; the daily feed intake of each mouse is about 5g; and the feeding method is ad libitum.

[0055] 1.3 Sampling and index testing

[0056] At week 21 of the experiment, mice were harvested and hair loss was observed and recorded in each group. Skin tissue was collected and paraffin sections were prepared. Hematoxylin and eosin (HE) and Masson staining were performed to observe the structure and morphology of hair follicles. Perifollicular fat infiltration and the degree of perifollicular fibrosis were assessed. Hair follicle stem cell density was assessed in the hair follicles of each group by double staining with keratin K15 and glycoprotein CD34. TUNEL staining was used to assess the degree of hair follicle cell apoptosis.

[0057] Statistical analysis: SPSS software (statistical products and service solutions software) was used for statistical analysis, and the measurement data were expressed as mean ± standard deviation. express.

[0058] 1.4 Experimental Results

[0059] 1.4.1 Effects of fucoxanthin on high-fat diet-induced back hair loss and skin inflammation in mice

[0060] In the record of skin and hair loss on the back of mice at week 21, Figure 1 The hair and skin of mice in the NF group were completely normal. However, mice in the HF+NS group experienced large-scale hair loss on their backs after being fed a high-fat diet, and the exposed skin tissue also showed obvious erythema and scaling. However, mice in the HF+LFx and HF+HFx groups did not experience significant hair loss, nor did their skin tissue show obvious erythema and scaling.

[0061] The above observations on hair and skin indicate that a continuous high-fat diet can induce hair loss and skin inflammation on the back of mice, while treatment with fucoxanthin can significantly improve the above skin pathological conditions.

[0062] 1.4.2 Effects of fucoxanthin on subcutaneous cholesterol crystallization and sebaceous gland hypertrophy in mice induced by a high-fat diet

[0063] The skin tissues of mice in each group were stained with HE to observe the pathological changes of the skin tissues of mice in each group. Figure 2As shown, through HE sections, it can be observed that compared with the HE sections of the skin of the mice in the NF group, a large amount of cholesterol crystals accumulated in the subcutaneous soft tissue of the mice in the HF+NS group, while almost no cholesterol crystals were found in the subcutaneous soft tissue of the mice in the HF+LFx group and the HF+HFx group after fucoxanthin treatment. In addition, it was observed that the sebaceous glands of the mice in the HF+NS group showed obvious hypertrophy, and the area of sebaceous gland cells also increased significantly, while no obvious sebaceous gland hypertrophy and sebaceous gland cell hypertrophy were observed in the skin tissue sections of the mice in the fucoxanthin-treated groups (HF+LFx group and HF+HFx group). Further quantitative analysis of the cholesterol crystal area and sebaceous gland cell area in the skin sections showed that the cholesterol crystal area and sebaceous gland cells in the HF+NS group were significantly increased compared with the NF group; after fucoxanthin intervention, no cholesterol crystals were found in the HF+LFx group, and very small amounts of cholesterol crystals were seen in the HF+HFx group. The sebaceous gland cell area of the two groups also returned to a level close to that of the NF group. Among them Figure 2 In the figure, Area of CholesterolCrystal is the cholesterol crystal area, and Area of Sebocytes is the sebocyte area.

[0064] The above HE staining results showed that fucoxanthin intervention can significantly reduce the subcutaneous cholesterol crystal accumulation and sebaceous gland cell hypertrophy induced by a high-fat diet.

[0065] 1.4.3 Effects of fucoxanthin on epidermal hyperplasia and stratum corneum thickening induced by high-fat diet in mice

[0066] From the above experiments, we can see that the results of HE sections showed that the epidermis and stratum corneum of the mice in the HF+NS group were significantly thickened, and the skin tissue showed pathological hyperplasia ( Figure 2 ), which manifests itself as rough, hardened skin with scaling ( Figure 1 In order to more clearly compare the differences in epidermal thickness and stratum corneum thickness between the groups, the skin tissues of each group were stained with Masson staining, and the epidermal thickness and stratum corneum thickness of each group of mice were quantitatively analyzed. The results are shown in Figure 2. Figure 3 The results of Masson staining, as well as the statistical results of epidermal thickness and stratum corneum thickness, showed that the epidermis and stratum corneum of the mice in the HF+NS group were significantly thickened. In addition, large pieces of epidermal tissue were found to be peeled off from the subcutaneous tissue. However, the epidermis and stratum corneum of the mice in the HF+LFx and HF+HFx groups did not show the above pathological conditions, and there was no significant difference from the NF group. Figure 3 Cuticle thickness refers to the thickness of the stratum corneum, and Epidermal thickness refers to the thickness of the epidermis.

[0067] The above Masson staining results show that fucoxanthin intervention can significantly improve the pathological conditions of skin epidermal hyperplasia and stratum corneum thickening induced by high-fat diet in mice.

[0068] 1.4.4 Protective effect of fucoxanthin on apoptosis of epidermal hair follicle stem cells in mice induced by a high-fat diet

[0069] The hair follicles of mice in each group were completely sectioned, and the hair follicle stem cells of mice were co-labeled with K15 and CD34 dual fluorescent proteins to detect the protective effect of fucoxanthin feeding on hair follicle stem cells. Figure 4 As shown, the fluorescence intensity of K15 and CD34 in hair follicle sections of mice in the HF+NS group was significantly reduced compared with the NF group, while the fluorescence intensity of K15 and CD34 in the HF+LFx group was significantly increased compared with the HF group. A similar situation occurred in the HF+HFx group, where the fluorescence intensity of K15 and CD34 in the HF+HFx group was also significantly increased compared with the HF group. To ensure the statistical accuracy of the results, the fluorescence intensity of K15 and CD34 in each group was normalized to the basal nuclear DAPI fluorescence intensity using ImageJ software. The statistical results showed that the relative fluorescence intensity of K15 and CD34 in the HF+NS group was significantly reduced compared with the HF group, while the relative fluorescence intensity of K15 and CD34 in the HF+LFx and HF+HFx groups was significantly increased compared with the HF group.

[0070] The above results indicate that a high-fat diet induces a significant decrease in the number of hair follicle stem cells in the hair follicles of mice, and after feeding with fucoxanthin, the number of hair follicle stem cells increased significantly. In addition, it was observed that the hair follicle stem cells marked by K15 and CD34 in the HF group migrated, that is, the fluorescence intensity of K15 and CD34 in the hair follicle shaft and tail was significantly enhanced compared with the hair follicle head, which may be the reason why a high-fat diet leads to a decrease in hair follicle stem cells. However, in mice fed with fucoxanthin, there was no significant migration of hair follicle stem cells. In order to further confirm the deep mechanism of the protective effect of fucoxanthin on hair follicle stem cells, the TUNEL method was used to detect the level of cell apoptosis around the hair follicles. Figure 5 As shown, the TUNEL fluorescence intensity at the base of the hair follicles in the HF group fed a high-fat diet was significantly higher than that in the NF group, while the TUNEL fluorescence intensity in the HF+LFx and HF+HFx groups was significantly lower than that in the HF group. To ensure the accuracy of the experimental results, the TUNEL fluorescence intensity of each group was normalized by DAPI nuclear staining. The statistical results showed that the relative TUNEL fluorescence intensity in the HF group was significantly higher than that in the NF group, while the relative TUNEL fluorescence intensity in the HF+LFx and HF+HFx groups was significantly lower than that in the HF group.

[0071] The above hair follicle stem cell test results show that a high-fat diet induces a significant decrease in the number of hair follicle stem cells and a significant apoptosis in the hair follicle region. However, after feeding with fucoxanthin, the number of hair follicle stem cells rebounded significantly, and the level of apoptosis in the hair follicle region was significantly reduced. This shows that the underlying mechanism by which fucoxanthin improves hair loss in mice is closely related to reducing hair follicle stem cell damage and stabilizing the number of hair follicle stem cells. Fucoxanthin can significantly improve the reduction in hair follicle stem cell numbers in the skin induced by a high-fat diet and alleviate hair follicle cell apoptosis induced by a high-fat diet.

[0072] 2. Effect of fucoxanthin on hair follicle stem cells stimulated by oleic acid (cell experiment)

[0073] 2.1 Experimental Grouping

[0074] Hair follicle stem cells were extracted from mouse hair follicles and divided into 6 groups: (1) normal group (CTL); (2) oleic acid stimulation group (OA); (3) oleic acid stimulation + 0.25 μmol / L fucoxanthin intervention group (OA+0.25Fx); (4) oleic acid stimulation + 1 μmol / L fucoxanthin intervention group (OA+1Fx); (5) oleic acid stimulation + 2.5 μmol / L fucoxanthin intervention group (OA+2.5Fx); (6) oleic acid stimulation + 5 μmol / L fucoxanthin intervention group (OA+5Fx).

[0075] 2.2 Experimental plan

[0076] The extracted hair follicle stem cells were randomly and evenly divided into the six groups described above. Except for the CTL and OA groups, the remaining four groups were pre-treated with corresponding concentrations of fucoxanthin for 24 hours. Following treatment, cells in each group were stimulated with 200 μM oleic acid for 24 hours. After model establishment, lipid infiltration of hair follicle stem cells was assessed by Oil Red staining, and apoptosis was assessed by TUNEL staining.

[0077] 2.3 Experimental Results

[0078] 2.3.1 Effects of fucoxanthin on oleic acid-infiltrated hair follicle stem cells and oleic acid-induced hair follicle stem cell death

[0079] The protective effect of fucoxanthin intervention on hair follicle stem cells was further explored through in vitro experiments on hair follicle stem cells. Different doses of fucoxanthin were used to intervene in hair follicle stem cells for 24 hours before stimulating hair follicle stem cells with 200 μM oleic acid to construct a high-fat stimulated hair follicle stem cell model. The cells were stained with oil red and the number of surviving hair follicle stem cells and the proportion of oil red-stained positive cells under random high-power microscope fields were observed, calculated and statistically analyzed. The results are as follows: Figure 6As shown in the results of cell counting, the number of live cells under random high-power microscope fields was significantly reduced compared with the CTL group, and a large number of hair follicle stem cells in the OA group died. After fucoxanthin treatment, the number of surviving hair follicle stem cells in the OA+0.25Fx, OA+1Fx, and OA+2.5Fx groups was significantly higher. However, the survival of hair follicle stem cells in the OA+5Fx group was lower, with no significant difference from the OA group. Judging from the results of oil red staining, the oil red staining positivity rate of hair follicle stem cells in the OA group was significantly higher than that in the CTL group, the cell morphology was wrinkled, the volume was reduced, and the intracellular positive lipids filled the entire cytoplasm; the proportion of oil red staining positive cells in the OA+0.25Fx group decreased most significantly, and the cell wrinkling was also significantly improved. In a small number of oil red staining positive cells, the positive lipids were located on one side of the cell; the OA+1Fx group and the OA+2.5Fx group also showed significant improvement compared with the OA group, and were slightly inferior to the OA+0.25Fx group in terms of cell morphology; the proportion of oil red staining positive cells of hair follicle stem cells in the OA+5Fx group increased compared with the OA+0.25Fx, OA+1Fx and OA+2.5Fx groups, but was still significantly lower than the OA group. Figure 6 Cellcount refers to cell count, and Oilred staining positive refers to oil red staining positive.

[0080] The above cell oil red staining results show that oleic acid stimulation can increase the infiltration of hair follicle stem cells, change their morphology, and accelerate their death. Fucoxanthin intervention can significantly reduce the oleic acid infiltration of hair follicle stem cells, alleviate cell morphological changes, and reduce cell death, indicating that fucoxanthin has a protective effect on hair follicle stem cells, with 0.25μmol / L fucoxanthin having the best protective effect.

[0081] 2.3.2 Effect of fucoxanthin on oleic acid-induced apoptosis of hair follicle stem cells

[0082] In order to further detect the apoptosis level of hair follicle stem cells induced by oleic acid, TUNEL staining was performed on the hair follicle stem cells of each treatment group and the relative fluorescence intensity was statistically analyzed. Figure 7 As shown in the figure, the statistical results showed that compared with the CTL group, the relative fluorescence intensity of hair follicle stem cells in the OA group increased significantly, while after fucoxanthin intervention, the relative fluorescence intensity decreased significantly. Among the fucoxanthin intervention groups, the relative fluorescence intensity of the OA+1Fx and OA+2.5Fx groups decreased significantly, but there was no significant difference. The relative fluorescence intensity of OA+5Fx was the highest. Figure 7 Relative fluorescence intensity is the relative fluorescence intensity.

[0083] The above TUNEL staining results showed that fucoxanthin could significantly reduce the apoptosis of hair follicle stem cells induced by oleic acid, among which 1 μmol / L fucoxanthin had the best effect.

[0084] In summary, the present invention induces hair loss by giving ApoE- / - mice a high-fat diet for 20 consecutive weeks. In the 8th week, the treatment group was given different doses of fucoxanthin intervention (0.009 mg / kg / d and 0.018 mg / kg / d). The skin of each group of mice was collected, skin tissue sections were prepared, and the cholesterol deposition area, sebaceous gland cell area, epidermal thickness and stratum corneum thickness of each group were statistically analyzed. The above animal experimental results show that continuous high-fat diet can lead to skin inflammation and hair loss, increase cholesterol deposition in subcutaneous soft tissue and hypertrophy of sebaceous gland cells, and the epidermal thickness and stratum corneum thickness are also significantly increased. After fucoxanthin treatment, the above pathological conditions are significantly improved. In addition, in the cell experiment, fucoxanthin intervention was given in advance, and then a cell model was constructed by 200 μM oleic acid stimulation and the degree of oleic acid infiltration of hair follicle stem cells and the level of cell apoptosis were assessed by oil red staining and TUNEL staining. Finally, the cell counts, oil red staining positive rate and TUNEL relative fluorescence intensity under random high-power microscope fields of each group were statistically analyzed. The above cell experiment results show that oleic acid stimulation can significantly increase the degree of oleic acid infiltration of hair follicle stem cells, significantly reduce the number of hair follicle stem cells and induce their apoptosis; while the degree of oleic acid infiltration of hair follicle stem cells treated with fucoxanthin is significantly reduced, the number of stem cells under random high-power microscope field of view is significantly larger, and the relative fluorescence intensity of TUNEL staining is also significantly reduced; the present invention publicly proves at the cellular level that fucoxanthin can significantly reduce oleic acid-induced oleic acid infiltration and reduce hair follicle stem cell apoptosis and death, and fucoxanthin has a protective effect on hair follicle stem cells.

[0085] 3. Comparison of the effects of different types of carotenoids

[0086] The present invention also explores the effects of different types of carotenoids in preventing and treating hair loss and skin inflammation induced by high fat. The test method is as follows: To explore the protective effect of fucoxanthin compared with other carotenoids on hair follicle stem cells, hair follicle stem cells were pre-treated with different concentrations of fucoxanthin (0.1μmol / L, 1μmol / L, 10μmol / L), astaxanthin (0.1μmol / L, 1μmol / L, 10μmol / L), lutein (0.1μmol / L, 1μmol / L, 10μmol / L), and carotene (0.1μmol / L, 1μmol / L, 10μmol / L) for 24 hours. The hair follicle stem cells were then stimulated with 200μM oleic acid to construct a high fat-stimulated hair follicle stem cell model. The CTL group was used as the control group and did not receive any treatment during the experiment. The OA group was used as the oleic acid stimulation group and did not receive any drug intervention but only oleic acid stimulation. After the experiment, the cells were uniformly stained with oil red, and the number of surviving hair follicle stem cells and the proportion of oil red positive cells under random high-power microscope fields were calculated and counted. Figure 8-11 As shown in the figure, the cell counting results showed that at the same dose, fucoxanthin had the most significant effect in improving the lipid infiltration of hair follicle stem cells and had the best protective effect on hair follicle stem cells stimulated by oleic acid. Moreover, as the concentration of fucoxanthin increased, the protective effect became more significant. Figure 11 Where OilRedAreaFraction is the oil red area fraction.

[0087] In order to further verify the protective effect of fucoxanthin compared with other carotenoids, TUNEL staining was performed on the hair follicle stem cells of each treatment group and the relative fluorescence intensity was statistically analyzed to further verify the protective effect of fucoxanthin compared with other carotenoids. Figure 12-13 As shown, the results show that at the same dose, fucoxanthin has the most significant effect on reducing oleic acid-induced hair follicle stem cell apoptosis and has the best protective effect on hair follicle stem cells. Figure 13 Relativefluorescenceintensity is the relative fluorescence intensity.

Claims

1. Application of fucoxanthin in the preparation of oral anti-hair loss drugs.

2. Application of fucoxanthin in the preparation of oral drugs for preventing and treating hair loss caused by high-fat diet.

3. Application of fucoxanthin in the preparation of oral drugs for treating skin inflammation caused by high-fat diet.

4. An oral medicine kit for preventing and treating hair loss caused by a high-fat diet and treating skin inflammation caused by a high-fat diet, characterized in that: The medicine kit includes fucoxanthin, and the content of fucoxanthin in the medicine kit is prepared according to the following daily dosage standard: Fucoxanthin: 0.001-0.05mg / kg / d.

5. Application of fucoxanthin in the preparation of oral drugs for protecting hair follicle stem cells.

6. The use according to claim 5, characterized in that The fucoxanthin protects the hair follicle stem cells by reducing hair follicle stem cell damage and stably maintaining the number of hair follicle stem cells.

7. Use of fucoxanthin in the preparation of a drug for preventing hair loss by protecting hair follicle stem cells.

8. The application according to claim 7, characterized in that: The concentration of fucoxanthin in the drug is 0.01-10 μmol / L.

9. An oral anti-hair loss drug, characterized in that: Contains fucoxanthin.

10. The oral anti-hair loss drug according to claim 9, characterized in that: The dosage form of the oral anti-hair loss medicine is one of tablets, capsules, oral liquids and granules.

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