Herba ecliptae compound hair-blacking preparation and preparation method thereof

Abstract

The invention provides a herba ecliptae compound hair-blacking preparation and a preparation method thereof, and belongs to the technical field of medicines. The herba ecliptae compound hair-blacking preparation is prepared from the following raw materials: 1.5 parts of herba ecliptae, 1 part of pericarpium granati, 1.5 parts of radix rehmanniae recen, 1.5 parts of semen juglandis, 1.5 parts of semen vaccariae, 1.5 parts of flos caryophylli, 1.5 parts of fructus chebulae and 1.5 parts of fragrant ink. According to the invention, the raw materials and the ratio are adjusted, and meanwhile, the traditional complicated preparation modes of drying medicinal materials in the shade in a formula of hapalogeny, refining honey into pills and the like are eliminated, and the effective components of the eight medicinal materials in the formula are extracted by adopting an ethanol-water reflux method. The nutritional value of the Chinese herbal medicines can be fully utilized, the dosage is reduced, the operation convenience is improved, and the application range is expanded. The herba ecliptae compound hair blackening preparation provided by the invention has good effects of improving white hair and promoting hair blackening in a white hair model caused by acute pressure stress of a C57BL / 6J mouse.

Description

Technical Field

[0001] The present invention relates to the technical field of medicine, and in particular to a hair-blackening preparation of an Eclipta prostrata compound and a preparation method thereof. Background Art

[0002] Hair growth is a complex and dynamic cyclical process that consists of three main phases: anagen, catagen, and telogen. In addition, there is a shedding phase, which is usually considered part of the telogen phase.

[0003] At a macro level, the growth phase is the longest stage in the hair growth cycle, usually lasting several years. During the growth phase, hair follicles grow actively, and hair fibers continue to grow and gradually extend outward; the catagen phase is a short transitional phase, usually lasting only a few weeks, during which hair follicles begin to degenerate and atrophy, and hair fibers stop growing, marking the transition of hair follicles from the growth phase to the resting phase; the resting phase is a dormant phase in the hair growth cycle, usually lasting several months. Hair follicles enter a dormant state, hair fibers no longer grow, but are still attached to the hair follicles, and the superstructure of the hair follicles is still undergoing large-scale remodeling in preparation for the arrival of the next growth phase.

[0004] At the cellular level, hair growth is primarily regulated by two types of stem cells: hair follicle stem cells (HFSCs) and melanocyte stem cells (MeSCs). During the growth phase, HFSCs located in the bulge are activated to generate new hair follicles, while MeSCs located in the hair germ are activated to proliferate and differentiate, producing melanocytes that migrate downward. Melanocytes further divide to produce melanin, which then keratinizes, triggering the accumulation of keratinocytes, ultimately giving rise to hair.

[0005] Gray hair can occur when stress is too high. Nowadays, high-intensity work tasks, fast-paced lifestyles, and fiercely competitive environments put people under long-term stress, which gradually reduces their bodily functions and affects the nutritional supply and stress state of hair follicles. Hair follicles are the foundation of hair growth, and insufficient nutrition disrupts the hair growth cycle. Long-term mental stress and anxiety can also lead to endocrine disorders, affecting melanin production.

[0006] Stress, an acute stressor, activates the sympathetic nervous system, which in turn promotes the release of the neurotransmitter norepinephrine (NA). Stress-induced melanocyte stem cell depletion stems from the effects of norepinephrine produced by sympathetic nerve activation. Neither exogenous norepinephrine nor adrenal NA can cause hair graying; this process is directly caused by overactivation of the sympathetic nerves. The sympathetic nerves control the microenvironment where melanocyte stem cells (MeSCs) reside. Norepinephrine, a neurotransmitter and adrenergic receptor agonist, binds to adrenergic receptors on the surface of melanocyte stem cells, triggering a series of intracellular signaling pathways. These signals are transmitted to previously quiescent melanocyte stem cells, prompting them to rapidly proliferate, differentiate into mature melanocytes, and migrate out of their microenvironment. The rapid proliferation, differentiation, and migration of melanocyte stem cells leads to their rapid depletion, meaning that insufficient stem cells exist in the hair follicle to replenish new melanocytes, resulting in insufficient melanin production and the graying or graying of hair.

[0007] Traditional Chinese medicine (TCM) compound prescriptions play a key role in TCM treatment and represent the crystallization of TCM wisdom. Guided by TCM theory, they adhere to the principle of syndrome differentiation and treatment. For example, when treating a cold, TCM practitioners will formulate a prescription based on whether the patient has a wind-cold or wind-heat cold. Patients with wind-cold colds often experience chills, clear runny nose, and cough with thin sputum. In such cases, a compound prescription containing herbs such as ephedra and cinnamon twig is often used. Ephedra can induce sweating, relieve exterior symptoms, and clear the lungs and relieve asthma, while cinnamon twig can induce sweating, relax muscles, and warm the meridians. Together, these two herbs, along with other herbs, dispel wind-cold and harmonize the Ying and Wei systems, allowing the patient to sweat out and resolve the disease. This approach to compound prescriptions, which organically combines multiple herbs based on the patient's specific condition, fully demonstrates the essence of TCM's syndrome differentiation and treatment approach and is crucial to the continuation and development of TCM culture.

[0008] Numerous TCM texts mention the blackening effect of Eclipta prostrata. Volume 49 of Puji Fang states, "There's a recipe for instantly blackening beards and temples (from Shenghui Fang). Rehmannia root flower, purple yew flower, nightshade flower, green walnut pulp, purple hollyhock flower, wheat indigo seeds (those grown in wheat fields), lotus seed grass, and a large sour pomegranate. Wrap the above ingredients in cotton and dry in the shade. One ounce each of mother clove, holly bark, and fragrant ink. Mix the above with honey and make into pills the size of marbles. Take one pill each time, grinding it with one cup of wine on an empty stomach. Take slowly, letting the wine run dry. Take three pills after three cups of wine, five pills after five cups, and see results when you're drunk. Avoid radish and the five pungent spices." Eclipta prostrata, also known as lotus seed grass, nourishes the kidneys and yin, promoting black hair. It is recorded in Compendium of Materia Medica: "Enchantment intestine, Tang Materia Medica. Explanation of the name, lotus seed grass; Tang version, Houttuynia cordata; Illustrated, Jinling grass; Illustrated, ink tobacco; Gangmu, ink head grass; Gangmu, ink vegetable; Gangmu, monkey head; Must use, pig tooth grass. Shizhen said, it is eel Aconitum, its intestine is also black, this grass has ink when the soft stem is broken, hence the name, commonly called ink vegetable, the thin fruit is quite like a lotus pod, hence the name lotus", "the grass has a sweet smell, sour and flat, non-toxic, mainly used to treat bloody dysentery, acupuncture and moxibustion, sores with unstoppable bleeding. Fu Zhili, has applied the juice to the eyebrows, and the hair grows fast and profusely. Tang version, black beard hair, benefits kidney yin". Eclipta prostrata flavonoids have antioxidant effects and can delay aging; the water extract and alcohol extract of Eclipta prostrata can significantly activate tyrosinase activity, playing a role in blackening hair.

[0009] However, the modern names of some of the ingredients in the ancient recipe "Black Beard, Instant Proof of a Black Prescription" are difficult to determine, and therefore their efficacy remains uncertain. The recipe's preparation is complex, involving drying the ingredients in the shade and making them into pills. The method of administration involves mixing the ingredients with alcohol, sometimes even requiring intoxication. The "refined honey pills" method also has the disadvantage of being high in sugar, which limits the effective ingredients and makes them difficult to transport and consume. Summary of the Invention

[0010] In response to the deficiencies of the above-mentioned prior art, the present invention provides a compound hair-blackening preparation containing Eclipta prostrata and a preparation method thereof based on the "Black Beard and Bind Blackening Prescription". The compound hair-blackening preparation can reduce hair whitening induced by acute stress, promote hair blackening and prevent gray hair.

[0011] To achieve the above purpose, the specific technical solutions of the present invention are as follows:

[0012] In a first aspect, the present invention provides a hair-blackening preparation of a Herba Ecliptae compound, the raw materials of which include: 0.9-1.1 parts of pomegranate peel, 1.5-1.7 parts of Rehmannia root, 1.3-1.7 parts of green walnut kernel, 1.5-1.7 parts of Vaccaria segetalis, 1.3-1.7 parts of Herba Ecliptae, 1.3-1.7 parts of Clove, 1.3-1.7 parts of Terminalia chebula, and 1.3-1.7 parts of fragrant ink.

[0013] The present invention addresses the shortcomings of the prior art in honey pills containing Ecliptae chinensis, such as uncertain efficacy and complex preparation methods. By adjusting the raw materials, proportions, and preparation methods, the present invention prepares a compound hair-blackening preparation containing Ecliptae chinensis. In the formula, pomegranate peel has the effects of astringing the intestines, stopping diarrhea, stopping bleeding, and expelling parasites; raw rehmannia root has the effects of clearing heat and cooling blood, nourishing yin and promoting fluid production; green walnut kernel has the effects of tonifying the lungs and kidneys, promoting qi and relieving asthma, and moistening the intestines and promoting bowel movements; fenugreek has the effects of promoting blood circulation and menstruation, promoting lactation and reducing swelling, and promoting diuresis and relieving stranguria; Ecliptae chinensis has the effects of tonifying the kidneys and replenishing yin, and blackening hair; clove has the effects of warming the middle and relieving adverse reactions, tonifying the kidneys and supporting yang; terminalia chebula has the effects of astringing the intestines, stopping diarrhea, astringing the lungs and relieving cough, and reducing heat and relieving sore throat; and fragrant moxa has the effects of stopping bleeding and reducing swelling, clearing heat and detoxifying, and astringing and promoting tissue regeneration. This formula is mainly based on Eclipta prostrata, which has been clearly documented to have the effect of blackening hair, and is supplemented with medicinal materials that have the effects of clearing heat and activating blood circulation. They synergistically exert a significant effect of blackening hair, effectively promoting hair blackening and preventing gray hair.

[0014] Furthermore, the raw materials of the hair-blackening preparation of the Eclipta compound include: 1 part of pomegranate peel, 1.5 parts of raw rehmannia root, 1.5 parts of green walnut kernel, 1.5 parts of fenugreek, 1.5 parts of Eclipta, 1.5 parts of cloves, 1.5 parts of Terminalia chebula, and 1.5 parts of fragrant ink.

[0015] In a second aspect, the present invention provides a method for preparing the black hair preparation of the Eclalaria compound, comprising the following steps:

[0016] The raw materials are weighed respectively, mixed and soaked in ethanol water, then extracted under reflux conditions, filtered, concentrated and dried to obtain the hair-blackening preparation of the Eclipta prostrata compound.

[0017] Preferably, the drying is freeze-drying.

[0018] Preferably, the concentration of the ethanol water is 60% to 80%.

[0019] Preferably, the amount of ethanol water used is 5 to 15 times the total weight of the raw materials.

[0020] Preferably, the raw material is soaked in ethanol water for 30 to 60 minutes.

[0021] Preferably, the extraction is repeated multiple times, specifically 2 to 3 times. After each reflux extraction, the mixture is filtered while hot, the filtrates are combined, concentrated under reduced pressure, and dried to obtain the black hair preparation of the Eclipta prostrata compound.

[0022] Preferably, the temperature of the reduced pressure concentration is 55-60°C.

[0023] Specifically, the preparation method of the black hair preparation of the Ecliptae compound comprises the following steps:

[0024] Respectively weigh raw rehmannia root, green walnut kernel, vaccaria segetalis, Eclampsia chinensis, clove, Terminalia chebula, fragrant black loquat and pomegranate peel, and crush the raw rehmannia root, green walnut kernel, vaccaria segetalis, Eclampsia chinensis, clove, Terminalia chebula, fragrant black loquat and pomegranate peel into powder. Mix the raw materials, soak them in 60%-80% ethanol water, and then extract them 2-3 times under reflux conditions. After each reflux extraction, filter while hot, concentrate the filtrate under reduced pressure at 55-60°C, and freeze-dry to obtain the black hair preparation of the Eclampsia chinensis compound.

[0025] In a third aspect, the present invention provides the use of the Eclalaria compound hair-blackening preparation in the preparation of a product for improving hair graying caused by acute stress.

[0026] Specifically, the Ecliptaecarpa compound hair-blackening preparation of the present invention helps increase the proportion of black hair on the back of mice; improves hair graying induced by acute stress; increases the amount of melanin in hair follicles in the back tissue of mice, promoting melanin synthesis; promotes melanin synthesis by increasing the expression of tyrosinase (TYR) and the transcription factor MITF; inhibits the expression of Wnt5a, thereby increasing the expression of p-β-catenin, inhibiting the non-canonical Wnt pathway, increasing the stability of β-catenin, and enhancing melanocyte proliferation and melanin synthesis. It regulates the proliferation and differentiation of melanocytes to maintain normal levels by regulating the expression levels of CDK2 and KIT mRNA; increases the melanin synthesis capacity of melanocytes by enhancing the expression levels of TYR, tyrosine-related protein-1 (TYRP-1), and TYRP-2 mRNA; and regulates the normal synthesis and transport of melanosomes by enhancing the expression levels of PMEL and OCA2 mRNA, stabilizing the structure and internal environment of melanosomes, thereby promoting melanin synthesis.

[0027] In a fourth aspect, the present invention provides a product for improving hair graying caused by acute stress, the active ingredient of which includes the hair-blackening preparation of the Eclalaria compound.

[0028] Preferably, the dosage form of the product for improving hair graying caused by acute stress includes an oral preparation.

[0029] Preferably, the oral preparations include but are not limited to granules, compressed tablets, and oral liquids.

[0030] Compared with the prior art, the present invention is beneficial in that:

[0031] 1. The present invention adjusts the prescription ratio and preparation method of the traditional "Hei Ru Tai Li Yan Yi Wu Fang" to eliminate the complicated production method, and adopts the ethanol-water reflux method to extract the effective ingredients of medicinal materials such as raw rehmannia root, green walnut kernel, fenugreek, Eclipta prostrata, clove, Terminalia chebula, fragrant ink and pomegranate peel. The present invention can fully utilize the nutritional value of Chinese herbal medicine, reduce the dosage, improve the convenience of operation, and expand the scope of application. The black hair preparation of the Eclipta prostrata compound provided by the present invention has a good effect of improving gray hair and promoting hair blackening in the gray hair model caused by acute stress in C57BL / 6J mice.

[0032] 2. The preparation method of the hair-blackening preparation of the present invention has simple process and low raw material cost, which is conducive to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The effect of the preparation of the present invention on the RTX-induced gray hair mouse model;

[0034] Figure 2 This is a graph showing the percentage of white hair on the back of mice in each experimental group;

[0035] Figure 3 Figure 2 is a graph showing the weight growth rate of mice in each experimental group;

[0036] Figure 4 The results of H&E-stained longitudinal sections of the back skin of mice in each experimental group;

[0037] Figure 5 The results of H&E-stained cross-sections of the dorsal skin of mice in each experimental group;

[0038] Figure 6 Figure 2 is the number of melanin-containing hair follicles on the back of mice in each experimental group;

[0039] Figure 7 The results of the CDK2 and CD117 gene mRNA expression levels in mice of each experimental group are shown;

[0040] Figure 8 The results of TYRP-1 and DCT gene mRNA expression levels in mice of each experimental group;

[0041] Figure 9 The results of PMEL and OCA2 gene mRNA expression levels in mice of each experimental group are shown;

[0042] Figure 10 The results of the mRNA expression levels of CTNNB1, Mc1R and MITF genes in mice of each experimental group are shown;

[0043] Figure 11The results of the Wnt 5a and p-β-catenin protein expression levels in mice of each experimental group are shown;

[0044] Figure 12 These are the results of TYR and MITF protein expression levels in mice in each experimental group. DETAILED DESCRIPTION

[0045] The technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0046] The invention provides a hair-blackening preparation of an Eclipta multiflora compound. The raw materials of the preparation comprise: 0.9-1.1 parts of pomegranate peel, 1.5-1.7 parts of raw rehmannia root, 1.3-1.7 parts of green walnut kernel, 1.5-1.7 parts of fenugreek, 1.3-1.7 parts of Eclipta multiflora, 1.3-1.7 parts of clove, 1.3-1.7 parts of terminalia chebula and 1.3-1.7 parts of fragrant ink.

[0047] The preparation method of the Ecliptaecarpa compound hair-blackening preparation comprises the following steps:

[0048] The raw materials were weighed separately, mixed and soaked in 60% to 80% ethanol water (5 to 15 times the total weight of the raw materials) for 30 to 60 minutes, and then extracted 2 to 3 times under reflux conditions. After each reflux extraction, the mixture was filtered while hot, the filtrates were combined, concentrated under reduced pressure at 55 to 60°C, and dried to obtain a hair-blackening preparation of the Eclipta prostrata compound.

[0049] Example 1

[0050] A hair-blackening preparation of Ecliptaecarpa compound, the preparation method comprising the following steps:

[0051] (1) Weigh 37.37 g of Radix Rehmanniae, 37.36 g of green walnut kernel, 37.35 g of Vaccaria segetalis, 37.48 g of Eclipta prostrata, 37.33 g of Clove, 37.39 g of Terminalia chebula, 37.38 g of Rhizoma Cimicifugae, and 25.23 g of Pomegranate peel.

[0052] (2) The weighed raw Rehmannia root, green walnut kernel, Vaccaria segetalis, Ecliptae macrophylla, clove, Terminalia chebula, Fragrant ink and pomegranate peel were crushed into powder, added with 60% ethanol water (10 times the total mass of the total mass of the raw Rehmannia root, green walnut kernel, Vaccaria segetalis, Ecliptae macrophylla, clove, Terminalia chebula, Fragrant ink and pomegranate peel) and soaked for 30 min. The mixture was extracted under reflux for 2 h. The extraction was repeated twice. After each reflux extraction, the mixture was filtered with gauze while hot. The filtrates were combined and concentrated under reduced pressure at 60 °C to obtain a mixture.

[0053] (3) The mixture was freeze-dried in a freeze dryer to obtain 83.01 g of freeze-dried powder, which was stored at -20 °C for future use.

[0054] Comparative Example 1

[0055] Weigh 100 g, 100 g, 100 g, 100 g, 100 g, 100 g, 100 g, 100 g, 100 g, and 67 g of Rehmannia root, green walnut kernel, Vaccaria segetalis, Ecliptae macrophylla, clove, Terminalia chebula, Rhizoma Cimicifugae, and pomegranate peel, respectively. Soak in 10 times the total weight of purified water for 2 h, then boil and simmer for 30 min. The liquid was then decanted and collected, and 10 times the total weight of purified water was added again, followed by boiling and simmering for 30 min. The resulting decoctions were combined, concentrated under reduced pressure at 60°C, and lyophilized in a freeze dryer to yield 123 g of lyophilized powder. Store at -20°C until use.

[0056] Application Examples

[0057] Effect and mechanism of action of the hair-blackening preparation of Ecliptaecarpa compound on improving gray hair in a mouse model

[0058] 1. Experimental solution and its preparation method

[0059] The medicinal solutions used in this example and their preparation methods are shown in Table 1. The medicinal solutions in Table 1 were stored at 4 ° C and used within 3 days.

[0060] Table 1: Drug solutions and their preparation methods

[0061]

[0062] 2. Experimental Animals

[0063] This application example used 60 4-week-old, SPF-grade female Kunming C57BL / 6J mice weighing 15-17 g, provided by the Hubei Provincial Center for Disease Control and Prevention. Animal experiments were conducted in accordance with internationally recognized standards and in strict compliance with the "Guide for the Care and Use of Laboratory Animals" and relevant regulations. Animals were housed in polyethylene cages at room temperature (25 ± 2°C), natural humidity, and a 12-h light / dark cycle. Standardized diet and distilled water were used. Each group of mice was housed in separate cages and allowed to acclimate for several days. Experiments were performed after the mice's hair growth entered its telogen phase.

[0064] 3. Experimental methods

[0065] 3.1 Animal grouping and drug administration

[0066] After several days of adaptive feeding, all mice were fed daily in the morning and had free access to water. They were randomly divided into five groups: a blank group, a model group, a propranolol group, an Example 1 group, and a comparative example 1 group. Each group consisted of 12 mice, which were randomly divided into three cages for feeding, weighing, and labeling. The groups and dosages are shown in Table 2. Since there is no effective treatment for gray hair, research literature has documented that topical application of propranolol has a certain effect on the treatment of gray hair. Therefore, propranolol was selected as a positive control and formulated into a gel formulation for topical administration.

[0067] 3.2 Establishment of stress-induced gray hair model in mice

[0068] When 4-week-old female C57BL / 6J mice entered the telogen phase (the skin was pink), a 3 × 4 cm 2 The area was first shaved with an electric shaver, followed by a 5-minute application of depilatory cream until the shaved area was smooth. Following depilation, mice were injected with RTX solution (prepared at a concentration of 20 μg / mL in PBS containing 2% DMSO and 0.15% Tween-80) the following day. Dosing began once daily for 30 days, and the experiment ended. RTX solution was administered subcutaneously into the flank of mice at doses of 30 μg / kg (on the first day), 70 μg / kg (on the second day), and 100 μg / kg (on the third day) once daily for 3 consecutive days. A blank control group received subcutaneous saline (see Table 2 for details) and was returned to their cages for free movement except for injections.

[0069] Resiniferatoxin (RTX) is a natural compound extracted from the Moroccan resin euphorbia (Euphorbiaresinifera). With a pungent intensity of 16 billion SHU (SHU), it is the strongest known capsaicin receptor TRPV1 agonist and the hottest substance known. Studies have shown that subcutaneous injection of RTX into mice causes significant pain, resulting in intense mental stress, and can be used to induce hair graying in mice.

[0070] Table 2: Animal experiment groups, modeling and drug administration

[0071]

[0072] 3.3 Scoring of white hair ratio in mice

[0073] After 30 days, when the hair on the backs of the mice had fully grown, all mice were sacrificed using a carbon dioxide anesthesia machine. Six mice were randomly selected from each group and placed on white A4 paper. The LED lights were adjusted and the backs, flanks, and bellies of the mice were photographed with a camera to observe the proportion of white hair in each area. Fiji-ImageJ software was used to effectively determine the color ratio in the captured images. The software usage process is as follows:

[0074] The mouse's head, paws, and feet were cut off from the image, leaving only the back, flank, and belly. The outline of the mouse was then outlined using a mobile phone image processing software to remove the interference of the A4 paper.

[0075] Start Fiji-ImageJ software and import the image to be analyzed by selecting File–open;

[0076] Go to Image-Type and convert the image to 8-bit format and grayscale;

[0077] Go to Image > Adjust > Threshold, set a threshold to isolate the color of interest, and adjust the image threshold slider to include only the desired color range to separate the black and white hair areas. Note that the threshold must be consistent across all images during analysis.

[0078] Go to Analyze-Set measurements and select the required measurement indicators. Select the three indicators: Area, Areafraction, and Limit to threshold.

[0079] Select Analyze - Measure, and the software will automatically calculate the area and area ratio of the target area;

[0080] The results will be displayed in the Results window. Click File-Save As to save the results as an Excel table or other format. If you need to further analyze multiple images, you can use the batch processing function.

[0081] 3.4 Skin tissue pathological staining

[0082] Paraffin embedding of tissue: Take the skin of the same position in the center of the mouse back, about 0.5 × 0.5 cm 2 When sampling, avoid pulling the skin to cause deformation. After sampling, immediately immerse the tissue in 4% paraformaldehyde solution for tissue fixation at 4°C for 48 h, and use conventional methods to dehydrate, embed and slice.

[0083] (1) Section staining

[0084] Cut skin tissue sections need to be dewaxed before histopathological staining. Paraffin embedding is performed in xylene I for 20 minutes, xylene II for 20 minutes, ethanol I for 5 minutes, ethanol II for 5 minutes, and 75% ethanol for 5 minutes. Dewaxing is performed in a gradient manner and washed with water. H&E staining is performed.

[0085] (2) Microscope photography

[0086] H&E-stained longitudinal sections of skin were obtained from both the control and model groups and observed under microscopes at 4x, 10x, and 20x magnifications. The number of mature hair follicles in three random locations on each section was determined. The primary focus was on the number of mature hair follicles (which resemble tadpole-like structures) in the skin tissue. The results were analyzed using a t-test.

[0087] H&E-stained transverse sections were obtained from each group and examined under a microscope at 4x, 10x, and 20x magnification. Epidermal thickness and the number of mature hair follicles were measured at three random locations on each section. The thickness of the epidermis and dermis (perpendicular to the subcutaneous fat layer) and the number and morphology of hair follicles were primarily observed. The results were analyzed using t-tests.

[0088] 3.5 Real-time fluorescence quantitative RT-PCR

[0089] (1) Extract RNA from skin tissue using a tissue disruptor and RNA extraction kit

[0090] Because skin contains a large amount of RNases and is particularly susceptible to degradation, RNA extraction is challenging. Therefore, we used a tissue disruptor and an RNA extraction kit (RK30120, Abiotech) to extract total RNA from skin tissue. RNA concentration and quality were measured using a NanoDrop ultraviolet-visible spectrophotometer, followed by reverse transcription. The RNA reverse transcription reaction system is shown in Table 3. Real-time quantitative PCR (RT-qPCR) was used to measure the transcription levels of genes such as CDK2, CD117, and TYRP1 in mouse skin. The qPCR system and amplification procedure are shown in Table 4.

[0091] Table 3: Reverse transcription reaction system

[0092]

[0093] Prepare the reaction mixture according to Table 3. Gently flick the bottom of the tube to mix thoroughly, then centrifuge for 1 minute. Place the reaction mixture in a PCR instrument and incubate at 42°C for 60 minutes. Then, heat to 72°C for 5 minutes to terminate the reaction. Store the cDNA at -20°C until ready for use.

[0094] Primers were designed based on the cDNA sequence in Genbank. The primer specificity was verified by nucleic acid gel electrophoresis after PCR amplification, and the primer amplification efficiency was verified by real-time fluorescence quantitative RT-PCR. The appropriate dilution factor of cDNA was first determined through preliminary experiments. An appropriate amount of cDNA stock solution was diluted to 50 ng / μL with high-purity water without nuclease, and eight consecutive PCR tubes were placed on an ice box. Each component was added, mixed, and centrifuged for 1 min. The PCR tube was then placed in a real-time fluorescence quantitative PCR instrument. The qPCR system and amplification program are shown in Tables 4 and 5. GAPDH was used as an internal reference in the experiment to obtain the number of cycles Ct experienced when each group of cDNA amplification reached the threshold, and 2 -ΔΔCt The calculation method was used to analyze the data.

[0095] Table 4: qPCR system

[0096]

[0097] Table 5: qPCR amplification procedure

[0098]

[0099] 3.6 Western-blot analysis of skin tissue

[0100] 3.6.1 Skin tissue pretreatment

[0101] Pre-cool the grinder and high-speed centrifuge. Remove the skin tissue from a -80°C freezer and weigh approximately 35 mg of dorsal skin tissue into a grinding tube. Place on ice. Add 400 µL of the lysis buffer mixture to each tube and homogenize using a tissue grinder until no tissue fragments are visible. Grind at -40°C, 7 m / s, for 15 seconds on, 30 seconds off, and repeat six times. After grinding to allow the foam to settle, centrifuge at 13,000 rpm for 10 minutes at 4°C. Transfer 350 µL of the supernatant to a labeled EP tube.

[0102] 3.6.2 Protein quantification

[0103] The extracted protein was quantified by the Coomassie Brilliant Blue method, and the protein concentration was calculated using the Coomassie Brilliant Blue standard curve so that equal amounts of protein samples could be accurately added in subsequent experiments.

[0104] 3.6.3 Protein denaturation

[0105] Mix the protein loading buffer and protein sample in a ratio of 1:4. Use a syringe needle to poke a small hole in the EP tube cap. Place the tube in a tube rack and heat in a 100°C water bath. The cooked protein should be frozen at -80°C. Before loading, the protein sample must be heated again in a water bath.

[0106] 3.6.4 SDS-PAGE electrophoresis

[0107] Electrophoresis: Add protein samples and protein markers to the sample wells of polyacrylamide gel, with a starting current of 20 mA and a starting voltage of 80 V. When the bromophenol blue front enters the separation gel, the current is changed to 30 mA and the voltage is fixed at 120 V. Electrophoresis is performed for approximately 80 min.

[0108] Transfer: The current was fixed at 350 mA, and the membrane was transferred for 1.5 h. The proteins in the gel were transferred to the membrane through the action of the electric field.

[0109] Blocking: After transfer, wash with TBST and block with 5% BSA solution for 1 h to block the sites of unbound nonspecific proteins on the membrane, prevent nonspecific binding, and reduce background interference.

[0110] Antibody incubation: First, incubate the membrane with the primary antibody. After incubation, wash the membrane with TBST to remove unbound primary antibody, and then incubate the membrane with a fluorescent secondary antibody.

[0111] Detection: Detection is performed using a fluorescence imaging system, and the results are then analyzed using analysis software to determine the expression of the target protein.

[0112] 3.7 Statistical analysis

[0113] The experiments were repeated three times, and the results were analyzed using Graphpad Prism 9 statistical software. The mean and standard deviation were calculated, and the experimental data were expressed as mean ± SD. The t-test and one-way analysis of variance were used to compare the data between two groups and / or within two groups.

[0114] 4. Experimental results

[0115] 4.1 Effects of the Preparation of the Present Invention on RTX-Induced White Hair Mice

[0116] Figure 1 The effects of the preparation of the present invention on an RTX-induced gray hair mouse model are demonstrated. As shown in the figure, the area of ​​gray hair in the model group mice increased significantly compared to the blank group, indicating that the RTX gray hair model was successfully established. Photographs of the backs, flanks, and abdomens of the mice revealed a significant increase in the proportion of black hair in the propranolol group, the comparative example 1 group, and the example 1 group compared to the model group. This suggests that propranolol and the hair-blackening preparations prepared in comparative example 1 and example 1 all have a certain effect on gray hair, with the Example 1 group showing the most significant effect.

[0117] 4.2 Scoring results of white hair ratio on the back of mice

[0118] Figure 2 The figure shows the percentage of white hair on the back of mice in each experimental group. As shown in the figure, after 30 days of continuous administration, the percentage of white hair in the model group increased significantly compared to the blank group, indicating that the RTX white hair model was successfully established. Compared to the model group, the percentage of white hair in the propranolol group, Example 1 group, and Comparative Example 1 group all decreased significantly, with the Example 1 group showing the most significant effect. Figure 3 The figure shows the weight gain rate of mice in each test group. As can be seen from the figure, although the pain and discomfort caused by the injection of RTX solution affected the mice's eating and weight gain, by the end of the second week, the weight of mice in all test groups had increased, indicating that the hair blackening preparation of the present invention has no obvious toxicity.

[0119] 4.3 Histological examination results

[0120] Figure 4 The image shows the results of H&E-stained longitudinal sections of mouse dorsal skin after drug administration. As can be seen in the blank group, the hair follicles in the mice exhibited normal mature morphology, with relatively dense, intact, and regular epidermal tissue, mature and standard hair follicles, and a high follicle density. In contrast, the skin layer of the mice in the model group was thin, and the epidermal tissue was atrophied, indicating that RTX modeling caused some damage to the epidermis. Compared with the blank group, the hair follicles in the model group were severely damaged, with almost no mature follicles, demonstrating the success of the RTX gray hair model.

[0121] Figure 5 The image shows H&E-stained cross-sections of mouse dorsal skin after drug administration. As can be seen, the epidermal tissue in the blank group was relatively dense, intact, and morphologically regular. The hair follicles in the blank group exhibited normal, mature morphology, with the highest number of melanin-containing follicles.

[0122] Mature hair follicles containing melanin are an important sign of black hair. They are quantified by counting the number of hair follicles containing melanin in cross-sections of skin tissue. Figure 6 (* P < 0.05, *** P < 0.001, **** P < 0.0001, other groups compared with the model group). As shown in the figure, the number of melanin-containing hair follicles in the model group and the comparative example 1 group was almost zero, while the hair follicles in the propranolol group and the example 1 group were relatively mature in morphology, and the number of melanin-containing hair follicles was significantly greater than that in the model group, indicating that the hair-blackening preparation of the present invention has a significant effect in improving stress-induced gray hair.

[0123] 4.4 Effects of black hair preparations on the transcriptional levels of cell proliferation factors in mouse skin tissue

[0124] Cyclin-dependent kinase 2 (CDK2) and the CD117 gene play important roles in both the physiological and pathological processes of melanocytes. CDK2 is a cell cycle-dependent kinase that regulates cell cycle progression primarily by binding to the cyclins CyclinA and CyclinE. CDK2 plays a key role in the G1-to-S phase transition of melanocytes, a regulation crucial for normal melanocyte proliferation and differentiation. CDK2 promotes cell entry into the S phase by phosphorylating substrates such as retinoblastoma protein. Although CDK2 is not directly involved in melanin synthesis, it indirectly influences the metabolic state of melanocytes by regulating the cell cycle. Increased metabolic activity during the S phase of the cell cycle promotes the expression of genes involved in melanin synthesis. c-Kit, encoded by CD117, is a type III transmembrane receptor tyrosine kinase with TYR activity that promotes melanocyte proliferation. Its ligand is the stem cell factor (SCF), which is highly expressed in dark skin. The c-Kit / SCF signaling pathway plays a key role in melanocyte proliferation, differentiation, development, and survival. Under normal circumstances, c-Kit activity is tightly regulated. However, in melanoma, mutations or abnormal expression of the c-Kit gene can lead to its persistent activation. The c-Kit / SCF signaling pathway can also promote melanin synthesis by activating MITF. As a key transcription factor for melanin synthesis, MITF regulates the expression of TYR, TYRP-1, and TYRP-2, thereby promoting melanin production.

[0125] Figure 7 The results of the CDK2 and CD117 gene mRNA expression levels in mice of each experimental group are shown in the figure. As can be seen from the figure, compared with the model group, the CDK2 gene expression levels in the blank group, propranolol group and Example 1 group were significantly increased, indicating that RTX modeling promotes the proliferation and differentiation of MeSCs and accelerates the depletion of MeSCs. The high expression level of CDK2 in Example 1 group verifies that the MeSCs of the mice are in a normal proliferation and differentiation cycle, maintaining the MeSC depletion rate at a normal speed. Compared with the model group, the CD117 expression levels in the blank group, propranolol group and Example 1 group were significantly increased, indicating that RTX modeling affects the proliferation of melanocytes, and the high expression level of CD117 in Example 1 group verifies that its TYR activity is strong and its ability to synthesize melanocytes is strong.

[0126] 4.5 Effects of black hair preparations on the transcriptional levels of factors directly involved in melanin synthesis in mouse skin tissue

[0127] Melanin synthesis is a complex biochemical process directly catalyzed by TYR and its related proteins, TYRP-1 and TYRP-2. These enzymes play a key role in melanin synthesis in melanocytes. TYR is the rate-limiting enzyme in melanin synthesis, catalyzing the conversion of tyrosine to dopaquinone in the initial step of melanin synthesis. TYRP1, also known as DHICA oxidase, catalyzes the oxidation of dihydroxyindolecarboxylic acid (DHICA) to produce eumelanin during melanin synthesis. Eumelanin is the predominant melanin type in skin and hair, and TYRP-1 activity is crucial for its synthesis. TYRP-1 also has antioxidant properties, protecting melanocytes from oxidative stress. TYRP2, also known as dopachrome isomerase, encoded by the DCT gene, catalyzes the conversion of dopachrome to dihydroxyindole (DHI) and DHICA during melanin synthesis. These products are precursors for eumelanin synthesis. The expression and activity of TYR, TYRP-1 and TYRP-2 are regulated by multiple signaling pathways, including the cAMP-PKA pathway, the Wnt / β-catenin pathway and the ERK signaling pathway. These pathways affect the gene expression of TYR, TYRP-1 and TYRP-2 by regulating the activity of the transcription factor MITF.

[0128] Figure 8 The results of TYRP-1 and DCT gene mRNA expression levels in mice from each experimental group are shown. As shown in the figure, compared with the model group, the blank group, the propranolol group, and the Example 1 group, TYRP1 and DCT expression levels were significantly increased, indicating that RTX modeling reduced the expression of key enzymes in melanin synthesis. In contrast, the expression levels of both genes in the Example 1 group were significantly increased compared with the model group, confirming that the mice in the Example 1 group had a strong ability to synthesize melanin.

[0129] 4.6 Effects on the transcriptional levels of melanin synthesis and transport-related factors in mouse skin tissue

[0130] Premelanosome protein (PMEL) is primarily involved in melanosome formation and melanin synthesis, playing a crucial role in the structural organization of melanosomes. The repetitive domains of PMEL are crucial for forming the fibrillar structure within melanosomes, which provides a scaffold for melanin deposition. During melanosome maturation, PMEL maintains the pH within the melanosome, providing a suitable environment for melanin synthases such as TYR, thereby promoting melanin synthesis. Furthermore, abnormal expression or functional loss of PMEL can alter melanosome morphology and structure, thereby impairing melanin production. Oculocutaneous Albinism Type 2 (OCA2) is a transmembrane protein widely present in melanocytes and retinal pigment epithelial cells. It participates in melanosome transport and melanin synthesis, primarily by influencing melanosome maturation, regulating melanosome pH, and participating in the first step of the melanin synthesis pathway. The OCA2 protein functions like an ion pump, regulating the pH within melanosomes. Since TYR is most active at neutral pH, maintaining an appropriate pH is crucial for its activity. PMEL and OCA2 collaborate during melanin synthesis, jointly maintaining the normal function of melanosomes and influencing melanin production and deposition. PMEL promotes melanin deposition primarily by forming melanosome fibers and regulating the internal environment of melanosomes, while OCA2 regulates the pH within melanosomes, providing optimal conditions for melanin synthesis.

[0131] Figure 9 The results of measuring PMEL and OCA2 gene mRNA expression levels in mice from each experimental group are shown. As shown, compared to the model group, PMEL and OCA2 expression levels were significantly increased in the blank group, propranolol group, and Example 1 group, indicating that RTX modeling reduced the expression of genes involved in melanin synthesis transporters. This suggests that the hair-blackening preparation of the present invention can enhance melanin transport capacity by increasing the expression levels of these two genes.

[0132] 4.7 Effects of black hair preparations on the transcriptional levels of other important factors related to melanin synthesis in mouse skin tissue

[0133] Wnt Family Member 5A (Wnt5a), a member of the Wnt family, inhibits melanocyte proliferation and melanin synthesis primarily by inhibiting the non-canonical Wnt / β-catenin pathway. Wnt5a competitively binds to the Frizzled receptor, preventing the binding of canonical Wnt ligands such as Wnt3a to the Frizzled receptor. This competitive binding prevents the Frizzled receptor from activating the downstream β-catenin signaling pathway, thereby suppressing melanocyte proliferation and melanin synthesis. Wnt5a promotes the phosphorylation and degradation of β-catenin by activating GSK-3β. Phosphorylated β-catenin (p-β-catenin), encoded by the gene CTNNB1, is a key protein in the Wnt / β-catenin signaling pathway, and its phosphorylation status determines the activity and stability of β-catenin. Because β-catenin cannot accumulate in the cytoplasm, its nuclear entry is significantly reduced, preventing it from activating downstream transcription factors TCF / LEF, thereby inhibiting the expression of genes involved in melanin synthesis, such as MITF. Mc1R belongs to the G protein-coupled receptor family and influences skin and hair color by regulating the type and amount of melanin. Its primary ligand is α-melanocyte-stimulating hormone (α-MSH). When α-MSH binds to Mc1R, it activates adenylate cyclase, leading to an increase in intracellular cAMP levels. This increase in cAMP activates protein kinase A, which in turn phosphorylates and activates the transcription factor CREB. Activated CREB promotes the expression of MITF, a key transcription factor in melanin synthesis and involved in multiple melanin synthesis pathways.

[0134] Figure 10 The results of measuring the mRNA expression levels of CTNNB1, Mc1R, and MITF genes in mice from each experimental group are shown. As can be seen from the figure, compared with the model group, the blank group, the propranolol group, and the Example 1 group all showed significantly increased expression of CTNNB1, Mc1R, and MITF, indicating that after RTX modeling, the expression of genes indirectly involved in melanin synthesis was also significantly suppressed, and the hair darkening preparation of the present invention can significantly relieve this suppression.

[0135] 4.8 Effects on the expression levels of Wnt5a / β-catenin pathway-related proteins in back skin tissue

[0136] As a ligand of the Wnt signaling pathway, Wnt5a upregulation may inhibit canonical Wnt / β-catenin signaling, forming a negative feedback loop. Activation of the non-canonical Wnt pathway disrupts β-catenin stability, whose stability and activity enhance melanocyte proliferation and melanin synthesis. Antagonism between Wnt5a and β-catenin further exacerbates the functional loss of the MITF-TYR axis.

[0137] Figure 11 The Wnt 5a and p-β-catenin protein expression levels of the experimental group mice are shown. As shown in the figure, the relative expression of Wnt5a protein in the propranolol group and the Example 1 group was significantly lower than that in the model group, indicating that RTX modeling triggered Wnt5a expression. Melanin synthesis in the RTX model group mice was lower than that in the other groups, resulting in gray hair. Compared to the model group, the expression of p-β-catenin protein in the Example 1 group mice was significantly higher, indicating that the hair-blackening preparation of the present invention can promote melanin synthesis by maintaining the stability of p-β-catenin.

[0138] 4.9 Effects on MITF and TYR protein expression levels in back skin tissue

[0139] MITF is one of the most important transcription factors in melanocytes, regulating their growth, survival, proliferation, and differentiation. TYR is a key enzyme in melanin synthesis, catalyzing the conversion of tyrosine to DOPA, which in turn synthesizes melanin. Reduced TYR activity leads to decreased melanin synthesis, which can cause gray hair. MITF promotes the expression of genes related to melanin synthesis, such as TYR, thereby influencing melanin production.

[0140] Figure 12 The expression levels of TYR and MITF proteins in mice from each experimental group are shown. As shown, the relative expression of TYR protein was significantly increased in the propranolol and Example 1 groups compared to the model group, indicating that the hair-blackening preparation of the present invention promotes the synthesis of key enzymes in melanin synthesis, thereby promoting melanin synthesis. MITF, a key factor in melanin synthesis, was significantly higher in the blank, propranolol, and Example 1 groups than in the model group. These results further support the effectiveness of the hair-blackening preparation of the present invention in improving the treatment of stress-induced graying hair.

[0141] In summary, the present invention is based on the traditional ancient recipe "Hei Xi Ting Li Yan Yi Wu Fang", and adjusts the formula and ratio to solve the difficulty of raw material ratio caused by the difference in dosage units between ancient and modern times; and adjusts the preparation method to eliminate the complex production methods of the ancient recipe "Hei Xi Ting Li Yan Yi Wu Fang" such as drying medicinal materials in the shade and refining honey into pills, and adopts the ethanol-water reflux method to extract the effective ingredients of the medicinal materials. The present invention can make full use of the nutritional value of Chinese herbal medicines, reduce the dosage, improve the convenience of operation, and expand the scope of application. The Eclipta Prostrata compound hair-blackening preparation provided by the present invention has a good effect in preventing and treating gray hair in the gray hair model caused by stress in C57BL / 6J mice.

[0142] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details of the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. A hair-blackening preparation of Ecliptae Polygoni Multiflori compound, characterized in that: The raw materials include: 0.9-1.1 parts of pomegranate peel, 1.5-1.7 parts of raw rehmannia root, 1.3-1.7 parts of green walnut kernel, 1.5-1.7 parts of fenugreek, 1.3-1.7 parts of Eclipta prostrata, 1.3-1.7 parts of clove, 1.3-1.7 parts of Terminalia chebula, and 1.3-1.7 parts of fragrant ink.

2. The method for preparing the hair-blackening preparation of the Ecliptae compound according to claim 1, characterized in that: The following steps are involved: The raw materials are weighed respectively, mixed and soaked in ethanol water, then extracted under reflux conditions, filtered, concentrated and dried to obtain the hair-blackening preparation of the Eclipta prostrata compound.

3. The method for preparing the hair-blackening preparation of the Ecliptae compound according to claim 2, characterized in that: The drying is freeze-drying.

4. The method for preparing the hair-blackening preparation of the Ecliptae compound according to claim 2, characterized in that: The amount of the ethanol water is 5 to 15 times the total weight of the raw materials.

5. The method for preparing the hair-blackening preparation of the Ecliptae compound according to claim 2, characterized in that: The raw materials are soaked in ethanol water for 30 to 60 minutes.

6. The method for preparing the hair-blackening preparation of the Ecliptae compound according to claim 2, characterized in that: The extraction is repeated for multiple times, specifically 2 to 3 times. After each reflux extraction, the mixture is filtered while hot, and the filtrates are combined, concentrated under reduced pressure, and dried to obtain the hair-blackening preparation of the Eclipta prostrata compound.

7. The method for preparing the hair-blackening preparation of the Ecliptae compound according to claim 6, characterized in that: The temperature of the reduced pressure concentration is 55-60°C.

8. Use of the hair-blackening preparation of the Eclaliya compound according to claim 1 or the hair-blackening preparation of the Eclaliya compound prepared by the method according to any one of claims 2 to 7 in the preparation of products for improving hair graying.

9. A product for improving hair graying caused by acute stress, characterized in that: The active ingredients include the hair-blackening preparation of the Eclaliya compound according to claim 1 or the hair-blackening preparation of the Eclaliya compound prepared by the method according to any one of claims 2-7.

10. The product for improving hair graying caused by acute stress according to claim 9, characterized in that: The dosage form of the product includes oral preparations.

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