Application of semen nigellae extract in preparation of product for preventing and / or treating androgenetic alopecia

Abstract

The invention discloses application of a semen nigellae extract in preparation of a product for preventing and / or treating androgenetic alopecia. The total content of nigella alkaloid and nigella alkaloid 4-O-sulfite in the nigella seed extract is not less than 2%. The preparation process comprises the following steps: degreasing nigellum nigrum medicinal material powder with petroleum ether, extracting with an acidic aqueous solution, and enriching with macroporous adsorption resin and specifically purifying with anion exchange resin in sequence. Experiments show that the extract can most significantly reduce the levels of testosterone (T), dihydrotestosterone (DHT) and creatinine (Cr) in an androgenetic alopecia model mouse body, increase the level of estradiol (E2) and effectively promote back hair regeneration, and the effect of the extract is significantly superior to that of a water extract, an alcohol extract and a positive control drug. The extract is clear in component and controllable in quality, can be used for preparing medicines or wash supplies with multi-target anti-hair loss and hair growth functions, and provides a novel efficient natural raw material for treating androgenetic alopecia.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of a black cumin seed extract in the preparation of products for the prevention and / or treatment of androgenetic alopecia. Background Technology Androgenetic alopecia (AGA) is a common, progressive hair loss disorder with a complex pathogenesis, primarily related to genetic factors and abnormal androgen metabolism. Dihydrotestosterone (DHT), a potent androgen converted from testosterone (T) by 5α-reductase, binds to androgen receptors in hair follicles, leading to follicle miniaturization, shortened growth cycles, and ultimately, thinning and hair loss. Furthermore, studies show that elevated levels of stress hormones such as creatinine (Cr) and an imbalance in the estrogen-androgen ratio may also exacerbate the hair loss process.

[0002] Currently, the main medications for treating androgenetic alopecia include minoxidil (a topical vasodilator) and finasteride (an oral 5α-reductase inhibitor). However, these medications have certain limitations. For example, finasteride may cause sexual dysfunction as a side effect, while minoxidil may cause local irritation or hirsutism, and the efficacy is difficult to maintain after discontinuation. Therefore, developing a novel prevention and treatment product with multiple targets, fewer side effects, and natural sources has significant clinical importance and application value.

[0003] Nigella plants—especially *Nigella glandulari* (known as "Siyadan" in Uyghur medicine)—are among the most widely studied medicinal plants. Its seeds, designated as "nigella seeds" in the pharmacopoeia, are highly valued in Uyghur medicine for their broad-spectrum therapeutic applications. This versatility stems from its multi-functional pharmacological properties, including antiparasitic, anti-inflammatory, neuroprotective, hepatoprotective, and antitumor activities. Among the more than 20 identified taxa of *Nigella*, *Nigella glandulari* has become a key species in phytophagological research. They can be eaten directly in small quantities, pressed in large quantities for oil extraction, ground into powder for daily morning consumption, or made into sauces to enhance food flavor. Furthermore, *Nigella seeds* have been included in the *Pharmacopoeia of the People's Republic of China*, named *Nigella seed*. It possesses diuretic, emmenagogue, blood-activating, lactation-promoting, and anti-aging functions. It also exhibits a range of activities including antioxidant, anti-inflammatory, antitussive, expectorant, and immunomodulatory effects. Currently, there are no reports on the effects of its water extract on hair loss. Summary of the Invention

[0004] In a first aspect, the present invention provides the use of black cumin seed extract in the preparation of products for the prevention and / or treatment of androgenetic alopecia, wherein the black cumin seed extract contains a total content of not less than 2% of nigellidine and nigellidine-4-O-sulfite.

[0005] Preferably, the extract also contains flavonoids such as kaempferol and quercetin, coumarin compounds such as esculetin, amino acids such as L-(-)-tyrosine, L-(-)-phenylalanine, and L-tryptophan, and nucleoside compounds such as guanosine and adenosine.

[0006] Preferably, the extract is prepared by a method comprising the following steps: (1) The black cumin seed raw material was defatted with petroleum ether to obtain defatted medicinal residue; (2) The defatted medicinal residue was extracted with acidic aqueous solution to obtain extract; (3) The extract was purified by sequentially passing it through macroporous adsorption resin column chromatography and anion exchange resin column chromatography to obtain the extract.

[0007] Preferably, in step (2), the pH of the acidic aqueous solution is 4.0-5.5. This acidic condition is beneficial for stabilizing the sulfate bonds in Nigellidine-like components and promoting their dissolution from the plant matrix. The extraction method is hot reflux extraction.

[0008] Preferably, in step (3), the macroporous adsorption resin used in the macroporous adsorption resin column chromatography is HPD-400, and the elution method is: gradient elution with 0-35% ethanol aqueous solution; more preferably, 4-5 column volumes are eluted with deionized water first, then 3-4 column volumes are eluted with 15% ethanol solution to remove strongly polar impurities; finally, 4-5 column volumes are eluted with 35% ethanol solution and this portion is collected.

[0009] Preferably, the anion exchange resin used in the column chromatography is type D-301, and the elution method is gradient elution with a 0.1-0.5 M alkaline salt solution (such as 0.1-0.5 M ammonium bicarbonate). This selectively adsorbs and elutes negatively charged Nigellidine-4-O-sulfite and other acidic active components. During elution, the eluent rich in Nigellidine-4-O-sulfite is monitored by TLC (with Nigellidine-4-O-sulfite as a control) or HPLC, and the eluent fraction is collected.

[0010] Preferably, step (3) is followed by a desalination and drying step. More preferably, the desalination is performed using Seplite® LSD-1 type desalination resin column, and the drying method is freeze drying.

[0011] Preferably, the product can reduce the levels of testosterone (T), dihydrotestosterone (DHT), and creatinine (Cr) in the body or target tissues, and / or can increase the level of estradiol (E2). This indicates that the product of the present invention can intervene in androgenetic alopecia from multiple dimensions, such as regulating androgen levels, reducing stress response, and balancing sex hormones.

[0012] Preferably, the product is a medicine or a personal care product.

[0013] Preferably, when the product is a drug, it also includes pharmaceutically acceptable excipients and can be formulated into various dosage forms, preferably topical preparations, including but not limited to gels, ointments, sprays or liniments.

[0014] More preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of the following: sustained-release agents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, and lubricants. The combination of at least two is, for example, a combination of binders and excipients, a combination of binders and flavoring agents, a combination of binders and fillers, etc. Other combinations are also possible and will not be described in detail here.

[0015] Preferably, when the product is a hair care product, it can be selected from the form of shampoo, hair serum, scalp care liquid, hair growth tincture, etc., which is convenient for local external use and can act directly on the scalp and hair follicles.

[0016] In a second aspect, the present invention provides the use of black cumin seed extract in the preparation of testosterone (T), dihydrotestosterone (DHT), creatinine (Cr) inhibitors or estradiol (E2) activators, wherein the total content of black cumin alkaloid and black cumin alkaloid-4-O-sulfite in the black cumin seed extract is not less than 2%.

[0017] The extract also exhibits activity in regulating these key biomarkers in in vitro models and can be used as a reagent for related research.

[0018] Compared with the prior art, the present invention has at least the following beneficial effects: 1. This invention discloses for the first time the clear application of black cumin seed extract, especially an extract containing a total content of not less than 2% of Nigellidine and Nigellidine-4-O-sulfite, in the prevention and / or treatment of androgenetic alopecia, providing new natural source candidates and clear indicators of effective ingredients for the development of AGA therapeutic drugs or products.

[0019] 2. The extract not only significantly reduces the levels of key androgens closely related to hair loss—testosterone (T) and dihydrotestosterone (DHT)—but also lowers the stress-related marker creatinine (Cr) and increases the protective level of estradiol (E2). This indicates that the extract can intervene in AGA through multiple synergistic mechanisms, including inhibiting pathogenic androgens, reducing tissue stress, and balancing the sex hormone microenvironment, potentially offering more comprehensive therapeutic effects and potential than single-target drugs.

[0020] 3. The extraction and purification process of the extract is scientific, stable, and highly targeted: Hot reflux extraction was performed using an acidic aqueous solution with a pH of 4.0-5.5. This condition effectively stabilized the key sulfate ester bond in Nigellidine compounds (the characteristic structure of Nigellidine-4-O-sulfite) and promoted its efficient dissolution from the plant matrix, laying a foundation for highly active materials in subsequent purification. First, macroporous resin was used to remove most impurities and preliminarily enrich the extract. Then, anion exchange resin was used to specifically adsorb negatively charged acidic components such as Nigellidine-4-O-sulfite, achieving highly selective separation and effective enrichment of active ingredients. The extract prepared by this process showed significantly higher activity than the water extract, ethanol extract, and petroleum ether extract.

[0021] 4. A wide range of products can be developed based on this extract. It can be prepared into topical drug dosage forms such as gels, ointments, and sprays, which are convenient for local administration and direct action on the hair follicles of the scalp. It can also be incorporated into daily washing and care products such as shampoos and hair serums to achieve a combination of prevention and treatment, improve patient compliance, and has broad market application prospects. Attached Figure Description

[0022] Figure 1 The results of component analysis of the black cumin seed extract prepared in Example 1; Figure 2 Photos of mice in each group on day 1 (after shaving), day 7, day 14, and day 21 after the start of treatment in Experiment Example 1; Figure 3 The mean length and mean weight of new hair in the shaved area on the back of each group of mice in Experiment 1; Figure 4 Serum testosterone levels in mice of each group in Experiment 1; Figure 5 The serum dihydrotestosterone levels in mice in each group of Experiment 1; Figure 6 Serum creatinine levels in the serum of mice in each group of Experiment 1; Figure 7 The estradiol levels in the serum of mice in each group of Experiment 1; Figure 8The results of He staining of skin tissue in the drug-treated area on the back of mice in each group of Experiment 1. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0024] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0025] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of any conflict, the specification containing the definitions shall prevail.

[0026] The following are the main sources of raw materials in the examples: Seeds of *Nigeria nigra* (Ranunculaceae family) were collected from Xishuangbanna, Yunnan Province, China (altitude approximately 750 meters). The plant material was identified by Professor Tan Wenhong of Yunnan University of Traditional Chinese Medicine, and the voucher specimens are preserved in the university's herbarium. One-step testosterone ELISA kit (catalog number: FY-EU10175S) and dihydrotestosterone ELISA kit (catalog number: FY-EU10176S) were purchased from Wuhan Feiyue Biotechnology Co., LTD, East Lake High-tech Development Zone, Wuhan, China; estradiol (E2) ELISA kit (purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd.); and serum creatinine (Cr) biochemical assay reagent (purchased from Nanjing Jiancheng Bioengineering Institute).

[0027] Example 1: Preparation of Niger Seed Extract (1) Raw material pretreatment and defatting: Take 1 kg of dried black cumin seeds, wash and dry at 50℃, then pulverize and pass through an 80-mesh sieve. Mix the medicinal powder with 15 L of petroleum ether (boiling range 30-60℃), reflux extract at 60℃ for 5 hours, and filter. After defatting, evaporate the solvent from the residue at room temperature.

[0028] (2) Acidic hot water directional extraction: Mix defatted drug residue with 12L of deionized water (solid-to-liquid ratio 1:12), and adjust the pH to 4.5 with dilute acetic acid. Reflux extraction twice at 85℃, 1.5 hours each time, followed by filtration. Combine the extracts, immediately cool in an ice bath, and then centrifuge at 8000 r / min for 20 minutes, collecting the supernatant.

[0029] (3) Macroporous resin enrichment: Load the supernatant into a pretreated HPD-400 macroporous adsorption resin column (column volume 2L, diameter-to-height ratio 1:8). Use the following sequentially: Elute with 5 BV (column volume) of deionized water (pH 5.0) and discard. 3 BV eluted with 15% ethanol solution and discarded; Elute with 5 BV 35% ethanol solution and collect this fraction (target fraction).

[0030] The collected liquid is concentrated under reduced pressure (≤50℃) until no alcohol odor remains, yielding a concentrated aqueous solution.

[0031] (4) Anion exchange-specific purification: The concentrated solution was loaded onto a pretreated D-301 weak anion exchange resin column (column volume 1L). After washing with deionized water until the eluent was colorless, gradient elution was performed with 0.1 M, 0.3 M, and 0.5 M ammonium bicarbonate aqueous solutions. The eluent fractions rich in the target components (usually the 0.3 M and 0.5 M fractions) were collected by TLC (with Nigellidine-4-O-sulfite as a control) or HPLC monitoring.

[0032] (5) Desalting, purification and content determination: The target fractions were combined and desalted by Seplite® LSD-1 desalting resin column. The desalted solution was freeze-dried to obtain a light brown powder of black cumin seed extract (about 18.5 g).

[0033] Content determination: (1) Chromatographic conditions: Instrument: Agilent 1260 high performance liquid chromatograph with diode array detector (DAD). Column: Agilent ZORBAX SB-C18 column (4.6 × 150 mm, 5 μm). Mobile phase: Acetonitrile (A) - 0.1% formic acid aqueous solution (B), gradient elution (0-20 min, 15%→40% A). Flow rate: 1.0 mL / min. Column temperature: 30℃. Detection wavelength: 280 nm. Injection volume: 10 μL. (2) Solution preparation: Accurately weigh Nigellidine reference standard and Nigellidine-4-O-sulfite reference standard, add methanol to prepare a mixed reference standard stock solution. Accurately weigh 10 mg of black cumin seed extract (NW-Enr) sample, and prepare the test solution in the same way.

[0034] (3) Determination method: Accurately pipette the reference solution and the test solution separately and inject them into the liquid chromatograph, and record the chromatogram. Calculate the content according to the external standard method.

[0035] (4) Results: The contents of Nigellidine-4-O-sulfite in the black cumin seed extract (NW-Enr) prepared in this example were determined to be 1.65% (w / w) and 0.38% (w / w) of Nigellidine. The total contents of both were 2.03% (w / w).

[0036] Chemical composition analysis was performed using an ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry system, and the results are shown in [Figure number missing]. Figure 1 The results showed that the black cumin seed extract (NW-Enr) prepared in the examples contained Kaempferol, Quercetin, 3aS)-2,2,3a,6,6,8,9-heptamethyl-4H-pyrrolo[2,3-g]quinolin-3-one, Nigellidine, L-(-)-Tyrosine, L-(-)-Phenylalanine, L-Tryptophan, Guanosine, Adenosine, Esculetin and other components.

[0037] Comparative Example 1: Preparation of Ethanol Extract from Black Seeds (1) Raw material processing: Take 1 kg of dried black cumin seeds, wash and dry them, crush them, and pass them through a 100-mesh sieve to obtain coarse powder of the medicinal material.

[0038] (2) Degreasing treatment: Mix the above-mentioned crude powder of medicinal materials with 15L of petroleum ether (material-liquid ratio 1:15, g / mL), place it in a reflux extraction device, and reflux extract at 60℃ for 5 hours. Filter, collect the filter residue (medicinal material residue after degreasing), drain and evaporate the residual solvent at room temperature.

[0039] (3) Alcohol extraction: Mix the defatted medicinal residue with 8L of 60% ethanol aqueous solution (material-liquid ratio of about 1:8, g / mL), and extract by hot reflux at 80℃. Filter, collect the filtrate, extract twice, 1 hour each time, and combine the filtrates.

[0040] (4) Post-processing: Cool the filtrate to room temperature, centrifuge at 5000 r / min for 15 minutes, and collect the supernatant. Concentrate the supernatant under reduced pressure at 60℃ to a thick paste, then transfer it to a vacuum drying oven and dry it at 50℃ to constant weight. Pulverize to obtain 265.8g of black cumin seed alcohol extract.

[0041] The niger seed alcohol extract was tested according to the method in Example 1. The content of Nigellidine-4-O-sulfite in NE was 0.05% (trace), the content of Nigellidine was 0.55%, and the total content of the two was 0.60%.

[0042] Comparative Example 2: Enrichment of Nigella seed ethanol extract Take the ethanol extract of Comparative Example 1 and enrich and purify it using macroporous resin and anion exchange resin, in the same way as in Example 1.

[0043] The ethanol extract of black cumin seeds was tested according to the method in Example 1. In NE-Enr, the content of Nigellidine-4-O-sulfite was 0.08%, the content of Nigellidine was 1.05%, and the total content of the two was 1.13%.

[0044] Comparative Example 3: Preparation of Niger Seed Aqueous Extract (1) Raw material processing: Take 1 kg of dried black cumin seeds, wash and dry them, crush them, and pass them through a 100-mesh sieve to obtain coarse powder of the medicinal material.

[0045] (2) Degreasing treatment: Mix the above-mentioned crude powder of medicinal materials with petroleum ether (material-liquid ratio 1:15, g / mL), place it in a reflux extraction device, and reflux extract at 60°C for 5 hours. Filter, collect the filter residue (medicinal material residue after degreasing), drain and evaporate the residual solvent at room temperature.

[0046] (3) Water extraction: The defatted medicinal residue was mixed with 12L of deionized water (solid-to-liquid ratio 1:12), and the pH was adjusted to 4.5 with dilute acetic acid. The mixture was refluxed twice at 85℃ for 1.5 hours each time, and then filtered. The extracts were combined, cooled immediately in an ice bath, and then centrifuged at 8000r / min for 20 minutes. The supernatant was collected, concentrated, and dried to obtain 265.8g of black cumin seed water extract.

[0047] The water extract of black cumin seeds was tested according to the method in Example 1. The content of Nigellidine-4-O-sulfite in NW was 0.35%, the content of Nigellidine was 0.15%, and the total content of the two was 0.50%.

[0048] Comparative Example 4: Preparation of Petroleum Ether Extract of Niger Seeds (1) Raw material processing: Take 1 kg of dried black cumin seeds, wash and dry them, crush them, and pass them through a 100-mesh sieve to obtain coarse powder of the medicinal material.

[0049] (2) Degreasing treatment: Mix the above-mentioned crude powder of medicinal materials with 12L of petroleum ether (material-liquid ratio 1:12, g / mL), place it in a reflux extraction device, and extract by hot reflux at 60℃ for 6 hours. Filter, collect the filtrate, concentrate and dry to obtain petroleum ether extract.

[0050] The petroleum ether extract of black cumin seeds was tested according to the method in Example 1. Nigellidine-4-O-sulfite and Nigellidine were not detected in PL (low-dose petroleum ether extract) and PH (high-dose petroleum ether extract).

[0051] Experimental Example 1: Effects of Nigella seed extract on hormone levels in a mouse model of androgenetic alopecia 1. Animal grouping, model establishment, and drug administration: Fifty-four healthy male C57BL / 6 mice (weighing 20±2 g) were randomly divided into 9 groups, with 6 mice in each group: Normal control group (NC): No treatment was given, and the hair on the back was shaved.

[0052] Model control group (MC): 200 μl of 0.2% testosterone solution was applied to the shaved back skin daily.

[0053] Positive control group (PC): The modeling was the same as in the MC group, and 200 μl of 5% minoxidil tincture was applied daily.

[0054] Treatment group (6 groups in total): The model was established in the same way as the MC group, but different extracts were administered: Basic water extract group (NW): Apply 200 μl of 0.5% conventional water extract (sample prepared in Comparative Example 3, dissolved in water) solution.

[0055] Enriched water extract group (NW-Enr): Apply 200 μl of 0.5% of the enriched water extract of the present invention (sample prepared in Example 1, dissolved in water).

[0056] Basic alcohol extract group (NE): Apply 200 μl of 0.5% conventional alcohol extract (sample prepared in Comparative Example 1, dissolved in 50% ethanol) solution.

[0057] Enriched alcohol extract group (NE-Enr): Apply 200 μl of 0.5% enriched alcohol extract (sample prepared in Comparative Example 2, dissolved in 50% ethanol) solution.

[0058] Low-dose petroleum ether extract group (PL): 200 μl of 0.3% black cumin seed petroleum ether extract (sample prepared in Comparative Example 4, dissolved in physiological saline containing Tween 80) was applied.

[0059] High-dose petroleum ether extract group (PH): Apply 200 μl of 0.5% black cumin seed petroleum ether extract (sample prepared in Comparative Example 4, dissolved in physiological saline containing Tween 80).

[0060] All topical interventions were applied once daily for 21 consecutive days. Modeling (applying testosterone) continued throughout the intervention period.

[0061] 2. Observation and evaluation of hair regrowth: On days 1 (after shaving), 7, 14, and 21 after the start of treatment, photographs were taken of the shaved areas on the backs of mice in each group to visually record and compare the progress of hair regrowth. At the end of the experiment on day 21, the average length of newly grown hair on the backs of each mouse in each group was measured using precision electronic calipers (five points were randomly selected), and all newly grown hair in the area was gently shaved off. The average weight of the hair was recorded using a precision electronic balance.

[0062] 3. Biochemical indicator testing Following the last administration (day 21), mice were fasted for 12 hours (water was permitted), anesthetized, and blood was collected via the heart. Serum was separated by centrifugation after standing at room temperature. Simultaneously, full-thickness skin tissue from the dorsal administration area was collected, washed with physiological saline, and prepared into a 10% tissue homogenate. The supernatant was collected by centrifugation. Following strict adherence to the instructions of each kit, the levels of testosterone (T), dihydrotestosterone (DHT), estradiol (E2), and creatinine (Cr) in the serum and skin tissue homogenate supernatants were measured.

[0063] 3. HE staining Full-thickness skin tissue from the dorsal drug administration area was collected for HE staining. The tissue was first fixed and processed. Skin tissue samples from the mouse back (approximately 1 cm × 1 cm) were collected on day 21 after hair removal and immediately immersed in 4% paraformaldehyde phosphate buffer (4% PFA in PBS) for 24-48 hours at 4°C to ensure complete fixation. After fixation, the tissue was rinsed with running water, followed by gradient ethanol dehydration (70%, 80%, 95%, 100% ethanol, 1 hour each), xylene clearing, and finally embedding in melted paraffin. Sectioning and mounting: The embedded tissue blocks were serially sectioned on a paraffin microtome to a thickness of 4-5 μm. The sections were then flattened by floating in a 40°C water bath, then lifted with a glass slide and dried in a 60°C oven for 2 hours to ensure tight tissue adhesion. The hematoxylin-eosin (H&E) staining procedure strictly follows the standard process, as follows: Dewaxing and hydration: Immerse sections sequentially in xylene I and II for 10 minutes each to completely dewax, followed by hydration with a gradient of ethanol (100%, 95%, 80%, 70%) for 2 minutes each, and finally rinse with distilled water. Hematoxylin staining: Immerse sections in Harris hematoxylin staining solution for 5-8 minutes to stain the cell nuclei. Differentiation and blueing: Differentiate with 1% hydrochloric acid in 70% ethanol for a few seconds, rinse with tap water; then blue with 0.6% ammonia or saturated lithium carbonate solution, rinse with running water for 10-15 minutes. Eosin staining: Immerse sections in 0.5% eosin staining solution (aqueous or alcoholic solution) for 1-3 minutes to stain the cytoplasm and collagen fibers. Dehydration and Clearing: Rapidly dehydrated sections sequentially with gradient ethanol (70%, 80%, 95%, 100%), each step lasting from a few seconds to 30 seconds, followed by clearing twice with xylene, each time for 2 minutes. Mounting and Observation: Add neutral resin and mount with a coverslip. Observe and photograph the stained sections under an optical microscope. Images used for quantitative analysis (such as hair follicle density and diameter) should be systematically acquired at the same magnification (e.g., 100x or 200x).

[0064] 4. Experimental Results Hair observation photos (see) Figure 2 The results showed that hair regrowth was slow and sparse in the model group (MC) mice throughout the experiment. On day 21, both the positive control group (minoxidil, PC) and the various extract treatment groups showed varying degrees of improvement in hair regrowth. Quantitative analysis of newly grown hair (see...) Figure 3The results showed that the enriched water extract group (NW-Enr) performed best in promoting physical hair growth, with significantly better average length and weight of newly grown hair compared to all other extract treatment groups (including the basic water extract group NW, the basic alcohol extract group NE, the enriched alcohol extract group NE-Enr, and the petroleum ether extract group) and the positive control group (PC). This indicates that the enriched water extract prepared by the specific purification process of this invention is rich in highly active water-soluble components (such as Nigellidine alkaloids and their sulfates) that can directly stimulate hair follicles to enter the growth phase and accelerate hair shaft synthesis, resulting in a clear and powerful topical hair growth effect.

[0065] Regulation of androgen (T and DHT) levels (see) Figure 4-5 In the model group (MC) mice, serum and skin tissue levels of T and DHT were significantly elevated, indicating the successful establishment of the AGA model. Minoxidil (PC), a positive control drug, also had a regulatory effect on T and DHT, consistent with its main mechanism of directly stimulating hair follicle growth through opening potassium channels and dilating local blood vessels. Its hormonal changes may be an indirect, functional metabolic adjustment resulting from widespread activation of hair follicles. The preferred enriched water extract group (NW-Enr) exhibited the strongest androgen-suppressing effect, with the largest decrease in T and DHT levels, closest to normal levels, and significantly better than the basic water extract group (NW), basic alcohol extract group (NE), enriched alcohol extract group (NE-Enr), and petroleum ether extract group. This indicates that the present invention, through acidic water extraction combined with multi-step resin purification, can efficiently enrich and retain water-soluble Nigellidine-like active ingredients that can inhibit 5α-reductase activity or antagonize androgen receptor signaling, thereby addressing the core pathology of AGA at both the source and effector levels.

[0066] Regulation of metabolic stress markers (Cr) (see Figure 6 The elevated Cr levels in the model group suggest that modeling may cause metabolic burden or mild stress. Minoxidil and petroleum ether extracts had minimal effect on Cr levels. Notably, the Cr levels in the enriched water extract group (NW-Enr) were significantly reduced, and the effect was significantly better than that in the basic water extract group (NW). This unique effect strongly suggests that water-soluble components enriched by the process of this invention, such as (Nigellidine-like components), may have a systemic regulatory effect on improving systemic energy metabolism and reducing oxidative stress, demonstrating its holistic therapeutic concept of "systemic regulation - local onset".

[0067] Regulation of protective hormone (E2) levels (see) Figure 7The model group showed a significant decrease in E2 levels, reflecting hormonal imbalance in the AGA state. Among the treatment groups, the enriched water extract group (NW-Enr) exhibited the most significant E2-boosting effect, with E2 levels returning to the normal range. This boosting effect was significantly stronger than that of the basic water extract group (NW), the basic alcohol extract group (NE), and the petroleum ether extract group. This suggests that the active ingredients in the enriched extracts of this invention (such as Nigellidine compounds) may effectively correct the hormonal imbalance in AGA through phytoestrogens-like effects or by regulating aromatase, creating a favorable growth microenvironment for hair follicles.

[0068] HE staining results of full-thickness skin tissue from the drug administration area on the back of mice are shown in the figure. Figure 8 The results showed that, compared with the model control group, the NW-Enr group exhibited significantly increased hair follicle density, larger hair follicle diameter, and reduced interfollicle spacing, with a significantly better degree of improvement than the basic water extract group (NW), confirming that the extract of the present invention can effectively promote active hair follicle regeneration and development.

[0069] The above results demonstrate that the *Nigrum nigrum* seed extract prepared using the specific water extraction combined with multi-step resin purification process of this invention can most effectively reduce the levels of pathogenic factors T and DHT, reduce the level of metabolic stress marker Cr, and significantly increase the level of protective hormone E2. This multi-target, systemic regulation of hormones and metabolic homeostasis is the core advantage of the enriched water extract (NW-Enr) of this invention compared to the basic water extract (NW), basic alcohol extract (NE), enriched alcohol extract (NE-Enr), petroleum ether extract, and the traditional positive control drug minoxidil, providing a solid pharmacodynamic basis for its use in the prevention and treatment of androgenetic alopecia.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of Nigella sativa extract in the preparation of a product for the prevention and / or treatment of androgenetic alopecia, characterized in that, The total content of alkaloid nigelline and nigelline-4-O-sulfite in the extract of black seed is not less than 2%.

2. Use according to claim 1, characterized in that, The preparation method of the extract of black seed comprises the following steps: (1) defatting black seed raw material with petroleum ether to obtain defatted residue; (2) extracting the defatted residue with an acidic aqueous solution to obtain an extract; (3) separating and purifying the extract by macroporous adsorption resin column chromatography and anion exchange resin column chromatography in sequence to obtain the extract.

3. Use according to claim 2, characterized in that, In step (2), the pH value of the acidic aqueous solution is 4.0-5.5, and the extraction method is hot reflux extraction.

4. Use according to claim 2, characterized in that, In step (3), the macroporous adsorption resin used in the macroporous adsorption resin column chromatography is HPD-400, and the elution mode is gradient elution with 0-35% ethanol aqueous solution; the anion exchange resin used in the anion exchange resin column chromatography is D-301, and the elution mode is gradient elution with an alkaline salt solution with a concentration of 0.1-0.5 M.

5. Use according to claim 2, characterized in that, After step (3), the process further comprises desalination and drying steps.

6. Use according to any one of claims 1 to 5, characterized in that, The product can reduce the levels of testosterone (T), dihydrotestosterone (DHT) and creatinine (Cr) in the body or local target tissue, and / or can increase the level of estradiol (E2).

7. The use according to any one of claims 1 to 6, characterized in that, The product is a drug or a washing and caring product.

8. Use according to claim 7, characterized in that, When the product is a drug, the dosage form is a topical external preparation, including a gel, an ointment, a spray, a liniment or a tincture.

9. Use according to claim 7, characterized in that, When the product is a washing and caring product, it is selected from a shampoo, a hair care serum, a scalp care liquid or a hair growth tincture.

10. Use of Nigella sativa extract in the preparation of testosterone, dihydrotestosterone, creatinine inhibitors or estradiol activators, characterized in that, The total content of nigelline and nigelline-4-O-sulfite in the extract of black seed is not less than 2%.

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