Use of isorhamnetin-3-o neohesperidoside in the preparation of a medicament for treating hair loss

Abstract

The application belongs to the technical field of biological medicine, and provides a use of isorhamnetin-3-O neohesperidoside in preparation of a drug for treating alopecia, and finds that isorhamnetin-3-O neohesperidoside can promote hair growth and protect hair follicle stem cells, and as a natural extract, has many advantages such as low cost, few side effects, good patient compliance and easy to obtain, and provides a new idea for preparation of a drug for treating alopecia, in particular, a drug for treating androgenetic alopecia.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the use of isorhamnetin-3-O neohesperidin in the preparation of drugs for treating hair loss. Background Technology

[0002] Isorhamnetin-3-O-neohesperidoside (ISO) is a natural flavonoid glycoside compound. Its chemical name is 3-(((2S,3R,4S,5S,6R)-4,5-dihydroxy-6-(hydroxymethyl)-3-(((2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-5,7-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-4H-benzopyran-4-one, and its molecular formula is C2. 28 H 32 O 16 With a molecular weight of 624.54 g / mol, this compound is one of the main active ingredients of the traditional Chinese medicine Pollen Typhae. It can also be extracted from plants such as Acacia farnesiana. The wide range of plant sources and long history of application of these plants provide preliminary evidence for the safety of ISO and lay the material foundation for its development as a natural medicine. In addition, its extraction process is mature, its material basis is clear, and it has the feasibility of industrial production.

[0003] Existing research has confirmed that ISO has pharmacological activities in multiple fields: in the field of bone and teeth health, it can promote osteoclast formation and be used to treat delayed tooth eruption; in the field of the urinary system, it can improve benign prostatic hyperplasia through anti-angiogenic effects; in the field of gynecology, it can alleviate endometriosis fibrosis by regulating endometrial mesenchymal stem cells; and in the cardiovascular field, it can exert anti-atherosclerotic effects by improving endothelial dysfunction.

[0004] However, to date, there have been no research reports on ISO's work in preventing or treating hair loss, nor have any related patents been published.

[0005] Hair loss is a common condition characterized by decreased hair density or thinning hair, primarily due to hair follicle atrophy. Its prevalence has been steadily increasing in recent years. Hair loss not only affects appearance but also severely damages patients' mental health, leading to feelings of inferiority and anxiety. Based on its pathogenesis, hair loss can be divided into two main categories: cicatricial alopecia (such as lupus erythematosus alopecia, post-traumatic scalp alopecia) and non-cicatricial alopecia (such as androgenetic alopecia, telogen effluvium, and alopecia areata). Cicatricial alopecia is characterized by permanent damage to hair follicles and loss of hair regrowth ability; while non-cicatricial alopecia (such as androgenetic alopecia and alopecia areata) does not involve irreversible damage to hair follicles, delayed or improper treatment can easily lead to a prolonged condition. Non-cicatricial alopecia accounts for more than 80% of hair loss cases and is currently the primary target for hair loss treatment.

[0006] Currently, the main treatments for hair loss include: (1) Topical medications: Minoxidil, which works by dilating blood vessels and prolonging the hair growth cycle, but its effectiveness is only about 40%, and it needs to be used for a long time. It is easy to relapse after stopping the medication; (2) Oral medications: Finasteride, which reduces DHT production by inhibiting 5α-reductase, but it has side effects such as sexual dysfunction, and about 10% of patients cannot tolerate it; (3) Physical therapy: Scalp microneedling therapy and fractional radiofrequency therapy, which are expensive; (4) Surgical treatment: Hair transplantation, which is expensive and only applicable to specific types of hair loss.

[0007] In summary, existing hair loss treatments suffer from uneven efficacy, need for improved safety, and high costs, highlighting the urgent clinical need for safe, effective, and economical new treatment options. Natural extracts, with their advantages of low cost, fewer side effects, better patient compliance, and readily available raw materials, have become an important direction for the research and development of new hair loss treatments. Developing new, safe, and effective hair loss prevention and treatment methods has significant clinical importance and market value. Summary of the Invention

[0008] The purpose of this invention is to provide the use of isorhamnetin-3-O neohesperidin in the preparation of drugs for treating hair loss, in order to solve the problems mentioned in the background art.

[0009] The present invention is implemented as follows: the use of isorhamnetin-3-O neohesperidin in the preparation of a drug for treating hair loss, wherein the structural formula of isorhamnetin-3-O neohesperidin is shown below:

[0010]

[0011] Molecular weight: 624.54;

[0012] Molecular formula: C 28 H 32 O 16 ;

[0013] Chemical name: 3-(((2S,3R,4S,5S,6R)-4,5-dihydroxy-6-(hydroxymethyl)-3-(((2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-5,7-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-4H-benzopyran-4-one.

[0014] Another objective of this invention is to provide a medicament for treating hair loss, the medicament comprising isorhamnetin-3-O neohesperidin.

[0015] The present invention has shown that isorhamnetin-3-O-neohesperidin can promote hair growth and protect hair follicle stem cells. As a natural extract, it has many advantages, including low cost, few side effects, good patient compliance, and easy availability, providing a new approach for the preparation of drugs for treating hair loss, especially drugs for treating androgenetic alopecia. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of mouse hair morphology observation provided in Example 1 of the present invention;

[0017] Figure 2 The data results provided in Embodiment 1 of the present invention (A is body weight, B is hair length, C is hair growth score, D is hair weight) * p <0.05、 ** p <0.01、 **** p <0.001 vs control; # p <0.05、 ### p <0.001、 ##### p <0.0001 vs AGA; + p <0.05、 ++ p <0.01、 +++ p <0.001、 ++++ p <0.0001 vs ISO low);

[0018] Figure 3 The results of HE staining of mouse hair follicles provided in Example 1 of this invention;

[0019] Figure 4The data results provided in Embodiment 1 of the present invention (A represents the number of hair follicles in the growth phase, B represents the number of hair follicles in the growth / resting phase, * p <0.05、** p <0.01、**** p <0.001 vs control; # p <0.05、 #### p <0.001、 ##### p <0.0001 vs AGA; + p <0.05 vs ISO low);

[0020] Figure 5 Serum DHT level results provided in Example 2 of the present invention (**) p <0.01 vs control; # p <0.05、 ## p <0.01 vs AGA);

[0021] Figure 6 The results show the protein expression levels of Wnt5a and β-catenin in skin tissue provided in Example 3 of this invention;

[0022] Figure 7 The data results provided in Example 3 of this invention (A is a quantitative map of β-catenin protein expression level, B is a quantitative map of Wnt5a protein expression level, ** p <0.01 vs control; # p <0.05、 ### p <0.001 vs AGA; + p <0.05 vs ISO low);

[0023] Figure 8 This is an immunohistochemical image provided in Embodiment 3 of the present invention;

[0024] Figure 9 The experimental results of CCK8 with added DHT provided in Example 4 of this invention (A = 24h, B = 48h, C = 72h, ** p <0.01、*** p <0.001、**** p <0.0001 vs control);

[0025] Figure 10 The CCK8 experimental results of adding isorhamnetin-3-O-neohesperidin provided in Example 4 of the present invention (D is 24 h, E is 48 h, F is 72 h, ** p <0.01, *** p <0.001, **** p <0.0001 vs control);

[0026] Figure 11 The protein expression results of Wnt5a, β-catenin, and DKK1 in HDPC provided in Example 5 of the present invention;

[0027] Figure 12 The schematic diagram of the hair condition of mice provided in Example 6 of the present invention;

[0028] Figure 13 The schematic diagram of the hair condition of mice provided in Example 7 of the present invention. Detailed implementation manners

[0029] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.

[0030] The following describes the specific implementation of the present invention in detail with reference to specific embodiments.

[0031] Example 1. Experimental analysis of isorhamnetin-3-O-neohesperidin promoting hair regeneration in mice:

[0032] 1. Materials: 6-week-old SPF-grade male C57BL / 6 mice, weighing 18 ± 2 g, were purchased from Beijing Sparff Biotech Co., Ltd. (experimental animal production license: SCXK (Beijing) 2024-0001), and were raised under specific non-pathogenic conditions at a temperature of 22 ± 1 °C and a light / dark cycle of 12 hours. The feeding and operation of experimental animals followed the experimental procedures of the Animal Protection and Use Committee of Jilin University, and obtained the experimental animal ethics approval of the School of Public Health of Jilin University (KT: 2024-12-001). After 1 week of adaptive feeding, subsequent experiments were carried out;

[0033] Isorhamnetin-3-O-neohesperidin was purchased from Chengdu Dest Biotechnology Co., Ltd., testosterone was purchased from Solarbio (Beijing, China), and absolute ethanol was purchased from Tangshan Zhongxu Shengjin Trading Co., Ltd.

[0034] 2. Experimental method process: After 7 days of adaptive feeding with ordinary feed, the experimental animals were randomly divided into 4 groups, and the specific grouping is shown in Table 1:

[0035] Table 1

[0036]

[0037] The method for preparing the AGA androgenetic alopecia model includes the following steps:

[0038] (1) Mice in each group were anesthetized by intraperitoneal injection of 1% pentobarbital at a dose of 0.1 ml / 20 g;

[0039] (2) After anesthesia, the mice were fixed in a prone position on the operating table, and 2×3cm hairs were removed with a shaver along the spine. 2 After removing the hair, apply hair removal cream evenly to the area. After 2-3 minutes, gently wipe off the hair removal cream with gauze. The mouse's back is smooth and hairless. This day is recorded as DAY 0.

[0040] Administration method: Starting from DAY 1, mice in the model group were given 0.1 ml of 0.4% (w / v) testosterone on their backs daily. The testosterone was dissolved in 50% ethanol solution. In the ISO low-dose group, after applying testosterone to the back, mice were given 100 mg / kg of isorhamnetin-3-O-neohesperidin solution. In the ISO high-dose group, mice were given 200 mg / kg of isorhamnetin-3-O-neohesperidin solution. The feeding continued for 21 days.

[0041] During the administration period, the mice were weighed once a week to observe their growth and to change their bedding in a timely manner.

[0042] Starting from DAY12, the mice were photographed every 3 days to record their hair growth.

[0043] Starting from DAY12, hair growth will be scored. The scoring criteria are as follows: 0 for no growth; 1 for ≤20% growth; 2 for 20-40% growth; 3 for 40-60% growth; 4 for 60-80% growth; and 5 for 80% to complete growth.

[0044] Starting from DAY 15, hair length will be measured every 3 days;

[0045] At 21 days, the mice were shaved and weighed.

[0046] At day 21, blood was collected from the eyeballs in blood collection tubes and centrifuged at 4°C and 3000 g for 10 minutes to obtain serum samples for further analysis.

[0047] Subcutaneous fascia was separated to obtain skin tissue from the back of mice. The collected tissue was preserved in 4% paraformaldehyde fixative for subsequent tissue sectioning, H&E staining, and immunohistochemical staining. The skin tissue was required to be laid flat on filter paper before being immersed in the fixative to prevent the skin tissue from curling and affecting the experimental results. The remaining tissue was preserved in an ultra-low temperature freezer at -80℃ for subsequent protein and biochemical detection.

[0048] 3. Results and Conclusions:

[0049] The schematic diagram of mouse hair morphology observation is shown below. Figure 1 As shown; the statistical results of the data are as follows: Figure 2 As shown in the figure, the analysis revealed differences in body weight among the groups ( Figure 2 There was no significant difference in hair length (A) compared to the previous year. Figure 2 (B) From day 15, the model group showed significantly lower hair length than the control group, while the treatment group had longer hair than the model group. Furthermore, among the treatment groups, the higher dose was superior to the lower dose. Hair growth score ( Figure 2 In the middle C), the model group scored lower than the control group at all time points, while the treatment group scored higher than the model group. On day 12, the high-dose treatment group scored higher than the model group, and hair weight ( Figure 2 The hair weight in the model group (D) was significantly lower than that in the control group, while the hair weight in the treatment group was better than that in the model group, and the high-dose group was better than the low-dose group; the HE staining results of mouse hair follicles were as follows. Figure 3 As shown, the statistical results of the data are as follows: Figure 4 As shown, the number of hair follicles in the growth phase ( Figure 4 (A) and the number of hair follicles in the growth / resting phase ( Figure 4 The proportion of B in the model group was significantly lower than that in the control group, while the treatment group was superior to the model group.

[0050] Example 2: Experimental analysis of the inhibition of serum dihydrotestosterone levels in mice by isorhamnetin-3-O-neohesperidin:

[0051] 1. Materials: The mouse dihydrotestosterone (DHT) ELISA research kit was purchased from Jiangsu Jingmei Biotechnology Co., Ltd., and the cell imaging microplate detection system (Cytation 5) was purchased from BioTek.

[0052] 2. Method: The collected mouse serum was measured at 450 nm using an ELISA reader, following the instructions of the ELISA kit.

[0053] 3. Results and Conclusions: High levels of dihydrotestosterone (DHT) are a major trigger for the initiation and development of atrophic juvenile hormone (AGA). Therefore, serum DHT levels were measured, and the results are as follows: Figure 5 As shown, the DHT level in the model group was higher than that in the control group, while the DHT level in the treatment group was lower than that in the model group. The treatment of isorhamnetin-3-O-neohesperidin reduced the serum dihydrotestosterone content in mice in a dose-dependent manner.

[0054] Example 3: Experimental analysis of isorhamnetin-3-O-neohesperidin promoting the expression of hair growth protein in mice:

[0055] 1. Materials: High-speed low-temperature tissue homogenizer (KZ-III-FP) was purchased from Wuhan Saiwei Biotechnology, high-speed refrigerated centrifuge (V18R) was purchased from Dynamica, RIPA lysis buffer was purchased from Beijing Beyotime Biotechnology, 5X instant SDS-PAGE protein loading buffer was purchased from Affinity Biotechnology, and 4% paraformaldehyde was purchased from Wuhan Saiwei Biotechnology.

[0056] 2. Methods: 10 mg of mouse skin tissue was added to 100 μL of prepared RIPA lysis buffer and homogenized in a grinder to form a tissue homogenate. Then, it was centrifuged at 12,000 rpm and 4℃ for 10 min using a high-speed refrigerated centrifuge and the supernatant was collected. Protein loading buffer was added to prepare a protein loading solution for protein detection. Immunohistochemical detection was performed by embedding the skin tissue in paraffin after dehydration, sectioning and fixing it on a glass slide, dehydrating it, staining it with hematoxylin and eosin, mounting it and observing it under a microscope.

[0057] 3. Results and Conclusions: The protein expression levels of Wnt5a and β-catenin in skin tissue were obtained as follows: Figure 6 As shown, the statistical results of the data are as follows: Figure 7 As shown, the expression levels of Wnt / β-catenin pathway proteins, β-catenin, and Wnt5a in the model group were lower than those in the control group, while isorhamnetin-3-O-neohesperidin treatment promoted an increase in protein levels; immunohistochemical images are shown below. Figure 8 As shown in the figure, the model histone expression level was lower than that of the control, and isorhamnetin-3-O-neohesperidin treatment could restore the decrease in histone expression level.

[0058] Example 4: Experimental analysis of isorhamnetin-3-O-neohesperidin promoting HDPC cell proliferation:

[0059] 1. Materials: Human dermal papilla cells (HDPC) were purchased from Shanghai Xuanya Biotechnology Co., Ltd., dihydrotestosterone solution was purchased from Merck, cell imaging microplate detection system (Cytation 5) was purchased from BioTek, and CCK8 solution was purchased from GlpBio.

[0060] 2. Method: HDPC is divided into 4 × 10 3 Cells were seeded at a density of 0 / 12.5 / 25 / 50 / 100 / 200 μM DHT and 0 / 5 / 10 / 20 / 40 / 80 / 160 μg / mL isorhamnetin-3-O-neohesperidin, respectively, and incubated for 24 / 48 / 72 h. After adding CCK8 solution, the OD values ​​of each group were measured.

[0061] 3. Results and Conclusions: The results are as follows Figure 9 , Figure 10As shown, it can be seen that when adding different concentrations of DHT, the effect is considered comprehensively within 24 hours. Figure 9 (A), 48h Figure 9 (Chinese B), 72h ( Figure 9 The effect of C) on cell viability, 50 μM is a suitable concentration, when different concentrations of isorhamnetin-3-O-neohesperidin are added, 24h ( Figure 10 (middle D), 48h ( Figure 10 (E), 72h ( Figure 10 At concentrations of 10 / 20 μg / mL, isorhamnetin-3-O-neohesperidin showed a significant effect on promoting cell survival, while 80 / 160 μg / mL caused cytotoxicity. In subsequent experiments, DHT 50 μM and isorhamnetin-3-O-neohesperidin 10 / 20 μg / mL were used for the experiments.

[0062] Example 5: Experimental analysis of the effect of isorhamnetin-3-O-neohesperidin on the expression of HDPC hair regeneration protein:

[0063] 1. Materials: Ultrasonic cell disruptor (JY92IIN) was purchased from Ningbo Xinzhi Biotechnology, high-speed refrigerated centrifuge (V18R) was purchased from Dynamica, RIPA lysis buffer was purchased from Beijing Beyotime Biotechnology, and 5X instant SDS-PAGE protein loading buffer was purchased from Affinity Biotechnology.

[0064] 2. Method: HDPCs were transferred to four 6cm dishes and divided into four groups: control, DHT, DHT+ISO10, and DHT+ISO20. When the cell density was 70%-80%, 50μM DHT was added, and the ISO was 10 / 20μg / mL, respectively. After 24 hours, the cells were collected, and pre-prepared solution A was added. After centrifugation, the supernatant was separated to obtain cytoplasmic proteins. Solution B was added, and after incubation, the supernatant was centrifuged to obtain nuclear proteins. After separating the proteins, they were added to the loading buffer to prepare protein samples.

[0065] 3. Results and Conclusions: β-catenin is a core molecule in the Wnt / β-catenin signaling pathway. Its nuclear transfer can activate downstream target genes (such as the LEF / TCF family), regulating hair follicle stem cell proliferation and the hair follicle cycle (from the resting phase to the anagen phase). The nuclear transfer effect of β-catenin was detected by Western blotting, and the results are as follows: Figure 11 As shown, ISO treatment can promote β-catenin nuclear metastasis, restore Wnt5a protein expression, and inhibit the decrease in DKK1 expression in the Wnt5a pathway.

[0066] Example 6: Experimental analysis of isorhamnetin-3-O-neohesperidin promoting hair regeneration in radiation-damaged mice (topical application):

[0067] 1. Materials: Six-week-old SPF-grade female BALB / c mice, weighing 18±2 g, were purchased from Spiford (Beijing) Biotechnology Co., Ltd. They were housed under specific non-pathogenic conditions, with a temperature of 22±1℃ and a 12-hour light / dark cycle. The feeding and handling of the experimental animals followed the experimental procedures of the Animal Protection and Use Committee of Jilin University. After one week of acclimatization, subsequent experiments were conducted. Isorhamnoside-3-O-neohesperidin was purchased from Chengdu Dester Biotechnology Co., Ltd. Anhydrous ethanol was purchased from Tangshan Zhongxushengjin Trading Co., Ltd.

[0068] 2. Methods: After acclimatization to a normal diet for 7 days, the experimental animals were randomly divided into 3 groups of 8 mice each. In the radiation group, the hair on the back of the mice was removed, and 1 cm × 1 cm of skin was cut off and irradiated. In the treatment group, isorhamnetin-3-O-neohesperidin solution 90 mg / kg was applied daily after modeling. Starting from day 6, the mice were photographed every 3 days.

[0069] 3. Results: such as Figure 12 As shown, at 21 days, the hair coverage in the treatment group was better than that in the model group.

[0070] Example 7: Experimental analysis of isorhamnetin-3-O-neohesperidin promoting hair regeneration in radiation-damaged mice (by gavage):

[0071] 1. Materials: Six-week-old SPF-grade female BALB / c mice, weighing 18 ± 2 g, were purchased from Spiford (Beijing) Biotechnology Co., Ltd. They were housed under specific non-pathogenic conditions, with a temperature of 22 ± 1 ℃ and a 12-hour light / dark cycle. The feeding and handling of the experimental animals followed the experimental procedures of the Animal Protection and Use Committee of Jilin University. After one week of acclimatization, subsequent experiments were conducted. Isorhamnoside-3-O-neohesperidin was purchased from Chengdu Dester Biotechnology Co., Ltd. Anhydrous ethanol was purchased from Tangshan Zhongxushengjin Trading Co., Ltd.

[0072] 2. Methods: After 7 days of acclimatization with normal feed, the experimental animals were randomly divided into 3 groups of 8 mice each. In the radiation group, the hair on the back of the mice was removed, and 1 cm × 1 cm of skin was cut off and irradiated. In the treatment group, 60 mg / kg of isorhamnetin-3-O-neohesperidin solution was administered by gavage daily after modeling. Starting from day 1, the mice were photographed every 2 days.

[0073] 3. Results: such as Figure 13 As shown, at 17 days, the hair coverage in the treatment group was better than that in the radiation group.

[0074] In summary, isorhamnetin-3-O-neohesperidin can be used in the preparation of drugs for the treatment of hair loss. These drugs may include pharmaceutically acceptable carriers or excipients, including but not limited to excipients, solubilizers, preservatives, stabilizers, wetting agents, emulsifiers, salts that regulate osmotic pressure, buffers, etc.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of isorhamnetin-3-O neohesperidin in the preparation of drugs for treating hair loss.

2. The use of isorhamnetin-3-O neohesperidin according to claim 1 in the preparation of a drug for treating hair loss, characterized in that, The hair loss mentioned is one of the following: androgenetic alopecia, alopecia areata, telogen effluvium, anagen effluvium, cicatricial alopecia, or chemotherapy-induced hair loss.

3. The use of isorhamnetin-3-O neohesperidin according to claim 1 in the preparation of a drug for treating hair loss, characterized in that, The isorhamnetin-3-O neohesperidin promotes hair growth by reducing dihydrotestosterone levels and promoting the expression of hair growth protein.

Images