Application of Saposhnikovia divaricata in the preparation of a drug that promotes hair growth in the bald areas of mice

Abstract

This invention belongs to the field of traditional Chinese medicine technology and discloses the application of *Saposhnikovia divaricata* in the preparation of drugs for treating alopecia areata. The preparation method of *Saposhnikovia divaricata* extract is as follows: *Saposhnikovia divaricata* is dried in the shade, pulverized, and then soaked in ethanol for at least 24 hours. The supernatant is then collected. This invention's *Saposhnikovia divaricata* extract can significantly shorten the time for skin to darken and the time for new hair to grow, enabling skin and hair to enter the growth phase more quickly. It can significantly increase the length and weight of newly grown hair and increase the number of primary hair follicles in the skin, thus promoting hair growth in alopecia areata and providing ideas and theoretical basis for the research and development of new drugs.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to the application of Saposhnikovia divaricata in the preparation of drugs for treating alopecia areata. Background Technology

[0002] Alopecia areata (AA) is a common and complex autoimmune dermatological disease characterized by non-scarring hair loss. Statistics show that from 1990 to 2019, in just 30 years, the number of people diagnosed with AA worldwide increased by more than 10.68 million, an increase of over 49.41%. Currently, the etiology of AA is not fully understood. AA patients show no preference based on gender, race, or age, and there is no significant correlation between AA and disease duration, a history of atopic diseases, or histopathological characteristics.

[0003] Treatment options for alopecia areata are very limited, primarily based on suppressing or stimulating the immune response. Corticosteroids, immunomodulators, minoxidil, and contact immunotherapy remain the main treatment methods, but their efficacy is often limited, and there is no cure or effective way to prevent recurrence. Therefore, its exact etiology and effective treatment methods require further investigation.

[0004] Therefore, providing a drug that is effective in treating alopecia areata and can effectively prevent recurrence is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an application of *Saposhnikovia divaricata* in the preparation of drugs for treating alopecia areata.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The application of Saposhnikovia divaricata in the preparation of drugs for treating alopecia areata.

[0008] Saposhnikovia divaricata, also known as Fangfengcao or Tufangfeng, is the whole herb of Anisomeles indica (L.) Kuntze, belonging to the genus Anisomeles of the Lamiaceae family. It can be used to dispel wind and dampness, strengthen muscles and bones, darken hair, and improve eyesight. It is commonly used in the treatment of skin eczema, itching, scabies, and insect bites. It contains volatile oils, alkaloids, flavonoids, phenylethyl glycosides, and other compounds, exhibiting anti-inflammatory, antioxidant, analgesic, and antiviral pharmacological activities. However, its application in the treatment of alopecia areata has not been found in existing technologies. This invention discovers that its active ingredients can be used in the treatment of alopecia areata, significantly shortening the time for skin to darken and for new hair to grow, enabling hair to enter the growth phase more quickly, significantly increasing the length and weight of new hair, and increasing the number of primary hair follicles, thus promoting hair growth in alopecia areata-affected skin.

[0009] Preferably, the medicament for treating alopecia areata contains the active ingredient of Saposhnikovia divaricata and any one or more pharmaceutically acceptable carriers.

[0010] Preferably, the *Saposhnikovia divaricata* extract is used.

[0011] Preferably, the preparation method of the extract of Saposhnikovia divaricata is as follows: after drying and pulverizing Saposhnikovia divaricata, soak it in ethanol for at least 24 hours, and then take the supernatant.

[0012] Preferably, the concentration of the ethanol is not less than 30%.

[0013] Preferably, the mass-to-volume ratio of the Saposhnikovia divaricata and the ethanol is 1g:5ml.

[0014] Preferably, the drug is a topical application.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] This invention, *Guangfangfeng*, can significantly shorten the time for skin to darken and for new hair to grow, enabling skin and hair to enter the growth phase more quickly. It can significantly increase the length and weight of new hair, increase the number of primary hair follicles in the skin, and promote hair growth in alopecia areata. It provides ideas and theoretical basis for the research and development of new drugs. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0018] Figure 1 This is a diagram of the skin and hair condition in the hairless area of ​​mice in the control group of Example 1 of the present invention on day 1 (before drug administration);

[0019] Figure 2 This is a diagram of the skin and hair condition in the hairless area of ​​mice in the 15% minoxidil tincture group on day 1 (before drug administration) of this invention.

[0020] Figure 3 This is a diagram of the skin and hair condition in the hairless area of ​​mice in Group A of the Guangfangfeng medicinal liquid model in Example 1 of the present invention on day 1 (before drug administration);

[0021] Figure 4 This is a diagram of the skin and hair condition in the hairless area of ​​mice in Group B of the Guangfangfeng herbal liquid model in Example 1 of the present invention, on day 1 (before drug administration).

[0022] Figure 5This is a diagram showing the condition of the skin and hair in the hairless area of ​​mice on day 19 of the control group in Example 1 of this invention.

[0023] Figure 6 This is a diagram showing the condition of the skin and hair in the hairless area of ​​mice on day 19 after administration of 15% minoxidil solution in this embodiment of the invention.

[0024] Figure 7 This is a diagram showing the condition of the skin and hair in the hairless area of ​​mice on day 19 after administration of the Guangfangfeng herbal liquid in Group A of Example 1 of the present invention.

[0025] Figure 8 This is a diagram showing the condition of the skin and hair in the hairless area of ​​mice on day 19 after administration of the Guangfangfeng herbal liquid in Group B of Example 1 of the present invention.

[0026] Figure 9 This is a histological change diagram of the skin tissue in the hair-plucking area of ​​mice in the control group of Example 1 of the present invention;

[0027] Figure 10 This is a histological change image of the skin tissue in the hair-plucking area of ​​mice in the 15% minoxidil tincture group of this invention.

[0028] Figure 11 This is a histological change diagram of the skin tissue in the hair-plucking area of ​​mice in Group A of the Guangfangfeng medicinal liquid in Example 1 of the present invention;

[0029] Figure 12 This is a histological change diagram of the skin tissue in the hair-plucking area of ​​mice in Group B of the Guangfangfeng herbal solution according to Example 1 of the present invention;

[0030] Figure 13 This is the total ion chromatogram of the negative ion mode of the Guangfangfeng medicinal liquid A in Example 1 of the present invention;

[0031] Figure 14 This is the total ion chromatogram in positive ion mode for the Guangfangfeng medicinal liquid A in Example 1 of the present invention. Detailed Implementation

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Example 1

[0034] This invention provides a method for preparing an extract of Saposhnikovia divaricata, specifically including the following steps:

[0035] (1) Dry the whole herb of Saposhnikovia divaricata in the shade, grind it into fine powder, take 100g of fine powder, add an appropriate amount of 70% ethanol to fully soak it, then continue to add to a total volume of 500ml, soak at room temperature for 24h, filter and take the filtrate, centrifuge at 3500r / min for 10min, collect about 250ml of supernatant, which is Saposhnikovia divaricata solution A;

[0036] (2) Dilute the Guangfangfeng medicinal solution A with an equal volume of 70% ethanol to obtain Guangfangfeng medicinal solution B;

[0037] Verification of the efficacy of Saposhnikovia divaricata extract in treating alopecia areata

[0038] Drugs and dosage: Guangfangfeng liquid A, Guangfangfeng liquid B, and 5% minoxidil tincture, all at a dosage of 50 μl / time;

[0039] The method is as follows:

[0040] Six-week-old male C57BL / 6J mice (male, body weight 18-22g, SPF grade; provided by Changsha Tianqin Biotechnology Co., Ltd.) were selected. The hair on their backs was shaved under anesthesia, and then hair removal cream was applied. Mice with pink skin (hair in the resting phase), smooth skin, and no residual hair in the hair removal area were selected as qualified mice.

[0041] Qualified mice were randomly divided into four groups: control group (physiological saline), 5% minoxidil tincture group (90ml / bottle, batch number 20210830, Zhejiang Wansheng Pharmaceutical Co., Ltd.), Guangfangfeng liquid A group, and Guangfangfeng liquid B group, with 7-9 mice in each group. The medication was applied to the skin in the hair-removing area once daily for 20 consecutive days. Daily observations were made of skin color changes and hair growth in the hair-removing area, recording the time it took for the skin color to change from pink (resting phase) to black (anagen phase) and the time when new hair began to grow. Simultaneously, the overall recovery of hair in the hair-removing area was observed daily. Mice in each group were photographed before administration and on days 10, 16, and 19 after administration (results are shown in [link to results]). Figure 1-8 The recovery of hair in the hair-removed area was scored, and the scoring criteria are shown in Table 1. On the 20th day after drug administration, mice were anesthetized and sacrificed. Skin discs of the newly grown hair area were taken using a 13mm diameter trephine and weighed. The hair weight was calculated (hair weight = weight of skin disc with newly grown hair - weight of skin disc). Hair was plucked from the center of each skin disc and the length of 5 hairs was randomly measured. The average value was calculated, and the differences in hair length between the groups of mice were compared. Then, the skin of the hair-removed observation area of ​​the mice was fixed with 10% formaldehyde, prepared according to routine procedures, stained with hematoxylin and eosin (HE), and the number of primary and secondary hair follicles (converted to: number / cm) and their diameter (μm) were observed and counted under a light microscope.

[0042] SPSS 16.0 statistical software was used. Data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups when the variances were homogeneous, and Dunnett's T3 test was used when the variances were unequal. LSD test was used for comparisons between two groups. P < 0.05 was considered statistically significant.

[0043] The results are shown in Table 2-6;

[0044] Table 1 Scoring criteria for the recovery of new hair in the bald areas of mice.

[0045] score standard 0 The skin in the hair removal area is flesh-colored or pink. 1 The skin in the hair removal area appears light blue or light black. 2 The skin in the hair removal area is mostly blue or black. 3 The skin in the hair removal area is black with a few hairs growing back (visible or faintly visible on the skin). 4 The area where hair was removed is now covered with black hair (not visible on the skin), but there is a clear boundary between it and the surrounding hair. 5 The new hair in the hair removal area is the same length as the surrounding old hair, with no clear boundary.

[0046] Table 2. Effects of Guangfangfeng liquid on the time to skin darkening and new hair growth in the hair removal area (x±s)

[0047]

[0048] Note: Compared with the control group, *P<0.05, **P<0.01; compared with the minoxidil group, # P<0.05, ## P<0.01

[0049] The experimental results showed that, compared with the control group, Guangfangfeng liquid A could significantly shorten the time for the skin to turn black and the time for new hair growth in mice; compared with the minoxidil tincture group, it could also significantly shorten the time for new hair growth.

[0050] Table 3. Effects of Guangfangfeng liquid on the hair loss recovery score in mice.

[0051]

[0052] Note: Compared with the control group, *P<0.05, **P<0.01; compared with the minoxidil group, # P<0.05, ## P<0.01

[0053] The experimental results showed that, compared with the control group, Guangfangfeng liquid A significantly improved the hair recovery score of the bald area in mice on days 16 and 19 after administration; compared with the 5% minoxidil tincture group, it also significantly improved the hair recovery score of mice on day 19 after administration.

[0054] Table 4. Effects of Guangfangfeng liquid on the length and weight of new hair growth.

[0055]

[0056] Note: Compared with the control group, *P<0.05, **P<0.01; compared with the minoxidil group, # P<0.05, ##P<0.01

[0057] The experimental results showed that, compared with the control group, Guangfangfeng liquid A and 5% minoxidil tincture could significantly increase the length and weight of newly grown hair in mice; Guangfangfeng liquid B had no significant effect on the length and weight of newly grown hair in mice; compared with 5% minoxidil tincture, Guangfangfeng liquid A could also significantly increase the length of newly grown hair in mice.

[0058] Table 5. Effects of Guangfangfeng liquid on the number of primary and secondary hair follicles in mouse skin.

[0059]

[0060] Note: Compared with the control group, *P<0.05, **P<0.01.

[0061] Table 6. Effects of Guangfangfeng liquid on the diameter of primary and secondary hair follicles in mouse skin.

[0062]

[0063] Note: Compared with the control group, *P<0.05, **P<0.01.

[0064] As shown in Table 5, compared with the control group, Guangfangfeng extract A significantly increased the number of primary hair follicles per inch in the hairless skin of mice, but had no significant effect on the number of secondary hair follicles; Guangfangfeng extract B had no significant effect on either the number of primary or secondary hair follicles. As shown in Table 6, compared with the control group, 5% minoxidil tincture significantly increased the diameter of secondary hair follicles in the hairless skin of mice, but had no significant effect on the diameter of primary hair follicles. Guangfangfeng extract A and Guangfangfeng extract B had no significant effect on the diameter of either the primary or secondary hair follicles in the hairless skin of mice.

[0065] like Figure 1-4 The images, in order, show the skin and hair condition of mice in the hairless areas on day 1 (before drug administration) of the control group, the 5% minoxidil tincture group, the Guangfangfeng liquid A group, and the Guangfangfeng liquid B group. Figure 5-8 The images, in order, show the skin and hair condition of mice in the bald areas on day 19 after administration of the control group, the 5% minoxidil tincture group, the Guangfangfeng liquid A group, and the Guangfangfeng liquid B group. Figure 9-12The images show histological changes in the skin tissue of mice in the hair-plucked areas (HE staining, ×100) in the control group, 5% minoxidil tincture group, Guangfangfeng liquid A group, and Guangfangfeng liquid B group, respectively. Primary hair follicles are indicated by green arrows, and secondary hair follicles by red arrows. As shown in the figures, compared with the control group, Guangfangfeng liquid A significantly shortened the time for the skin to turn black and the time for new hair to grow (i.e., it enabled the skin and hair to enter the growth phase more quickly); it significantly improved the hair recovery score of the hair-plucked area on days 16 and 19 after administration; it significantly increased the length and weight of newly grown hair in mice; and it significantly increased the number of primary hair follicles per centimeter of skin in the hair-plucked area of ​​mice, thus promoting hair growth in the hair-plucked area (alopecia areata) of mice.

[0066] Component analysis

[0067] LC-MS was used to analyze the components of Guangfangfeng liquid A.

[0068] Instruments and equipment

[0069] High-resolution liquid chromatography-mass spectrometry (Thermo QE FOCUS), ultra-high performance liquid chromatography (UltiMate 3000), chromatographic column (Thermo Hypersil GOLD 1.9μm 2.1mm×100mm);

[0070] Chromatographic conditions

[0071] The chromatographic column was (Thermo Hypersil GOLD 1.9μm 2.1mm×100mm), the column temperature was 30℃, the injection volume was 5μL, and the mobile phase was gradient elution.

[0072] Mass spectrometry conditions

[0073] Ion source: ESI; Scan mode: Full scan (FULL MS); Scan mode: Positive / Negative ion; Sheath gas flow rate: 40 (user unit); Assist gas flow rate: 10 (user unit); Assist gas temperature: 320℃; Capillary voltage: +3.2 / -3.5kV; Scan range: 120~1500 (m / z); Resolution: 70000; Secondary mass spectrometry collision energy (CE): 10, 30, 45 (auto-matched);

[0074] The results are shown in Table 7 and Figure 13-14 Based on molecular ion peaks, fragment ion peaks, and literature, the alcohol extract contains effective components such as verbascoside, isorhodoside, rosmarinic acid, and ursolic acid.

[0075] Table 7. LC-MS Component Analysis Results of Guangfangfeng Liquid

[0076]

[0077]

[0078] Comparative Example 1

[0079] This invention provides a method for preparing an extract of Saposhnikovia divaricata, specifically including the following steps:

[0080] The difference from Example 1 is that 30% ethanol was used to obtain the Guangfangfeng medicinal liquid C.

[0081] Comparative Example 2

[0082] A method for preparing an extract of Saposhnikovia divaricata is provided, specifically including the following steps:

[0083] The whole herb of Saposhnikovia divaricata was dried in the shade and ground into a fine powder. 100g of the powder was soaked in 10 times its weight of purified water. The mixture was heated to boiling over high heat and kept simmering over low heat for 2 hours. The mixture was then concentrated to a total volume of 500ml. The filtrate was filtered and centrifuged at 3000r / min for 15min. The supernatant was collected to obtain Saposhnikovia divaricata liquid D.

[0084] Effect verification

[0085] Mouse experiments were conducted using the drug solutions from Example 1 and Comparative Examples 1-2.

[0086] The method was the same as the efficacy verification experiment in Example 1, except that the administration time was 18 days. The differences in the length and weight of new hair in mice in each group were compared. The results are shown in Table 8.

[0087] Table 8. Effects of the drug solutions from Example 1 and Comparative Examples 1-2 on hair growth in mice. n=8)

[0088]

[0089]

[0090] Note: Compared with the control group, *P<0.05, **P<0.01

[0091] Table 8 shows that, compared with the control group, Example 1 and 5% minoxidil tincture significantly increased the length and weight of newly grown hair in mice, while Comparative Examples 1-2 had no significant effect on the length and weight of newly grown hair in mice.

[0092] The various embodiments are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. Similar or identical parts between the various embodiments can be referred to each other.

[0093] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The use of *Saposhnikovia divaricata* in the preparation of a drug that promotes hair growth in the hair-removing area of ​​mice; wherein the hair removal is caused by a hair removal cream; The aforementioned *Saposhnikovia divaricata* extract is used; The preparation method of the extract of Saposhnikovia divaricata is as follows: after drying and pulverizing Saposhnikovia divaricata, soak it in ethanol for at least 24 hours, and take the supernatant. The concentration of the ethanol is not less than 30%; The mass-to-volume ratio of the aforementioned Saposhnikovia divaricata and the ethanol is 1g:5ml.

2. Use according to claim 1, characterized in that, The drug is composed of Saposhnikovia divaricata extract and any one or more pharmaceutically acceptable carriers.

3. Use according to claim 1, characterized in that, The medication is a topical application.

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