Anti-hair loss shampoo and preparation method thereof

Abstract

The application discloses a hair loss prevention shampoo targeting CD133 positive hair follicle dermal papilla cells and a preparation method thereof. The shampoo comprises a hair follicle targeting single-domain antibody-active peptide fusion, a liposome-encapsulated compound plant extract, a biological surfactant, a protein stability surfactant and an auxiliary material. The fusion is formed by fusing an anti-CD133 VHH single-domain antibody and a 5alpha-reductase inhibiting peptide DK-HP-7 through a flexible connecting peptide. Based on the discovery that CD133 positive dermal papilla cells are a key cell group driving hair follicle formation, the application uses single-domain antibodies to achieve precise targeting of active ingredients. The DK-HP-7 designed through molecular docking has an IC 50 of 4.28 μM. The rhamnolipid and Kolliphor HS 15 complex system can make the fusion protein activity retention rate higher than 89% after being stored at 40 ℃ for 3 months. The shampoo can significantly antagonize dihydrotestosterone-induced hair follicle growth inhibition, promote hair follicles to enter the growth phase and increase hair follicle density, and has good skin safety and stability.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of biomedicine and daily chemical care, and specifically relates to an anti-hair loss shampoo and its preparation method. Background Technology

[0002] Hair loss, a common clinical condition characterized by hair follicle miniaturization and shortened growth phase, exhibits a significant multifactorial synergistic effect in its pathogenesis. This involves complex biological processes such as genetic susceptibility, androgen metabolism disorders (e.g., abnormally elevated dihydrotestosterone levels), a micro-inflammatory environment around the hair follicle, local microcirculatory disturbances, and functional decline of hair follicle stem cells. Currently, commercially available anti-hair loss products mainly employ two technological approaches: one is the chemical drug intervention system, represented by minoxidil. While it can slow down the hair loss process to some extent, it has limitations such as significant skin irritation, high relapse rate after discontinuation, and a single target. The other is functional shampoos based on plant extracts. These have a relatively gentler action, but due to the barrier function of the scalp stratum corneum and the special anatomical structure of the hair follicles, active ingredients are difficult to effectively accumulate in the hair follicle area, resulting in low overall bioavailability.

[0003] Significant progress has been made in hair follicle biology research in recent years. Researchers have confirmed that dermal papillary cells (DP cells), as key mesenchymal cells regulating the cyclical regeneration and growth of hair follicles, have specific subsets whose functional status directly affects the morphogenesis and maintenance of hair follicles. Zhao et al. (2025, PMCID: PMC12045375) discovered through cell sorting technology that the CD133-positive (CD133+) DP cell subset can form more dense cell spheroids in a three-dimensional culture system, with significantly enhanced alkaline phosphatase activity and significantly upregulated expression levels of DP characteristic genes. Further in vivo skin remodeling experiments showed that mice transplanted with CD133+ DP cell spheroids had significantly more new hair growth in the wound area than the control group, suggesting that this cell subset plays a core role in driving hair follicle regeneration. At the mechanistic level, Zhou et al. (2016, https: / / doi.org / 10.1042 / BCJ20160466) revealed that CD133+ DP cells mainly affect the periodic activity of hair follicles by regulating the Wnt / β-catenin signaling pathway. The Wnt ligands secreted by these cells maintain the hair follicle induction potential of DP cells through autocrine and paracrine mechanisms and mediate the signaling dialogue between mesenchymal and epithelial cells. Activation of this pathway can effectively accelerate the transformation of hair follicles from the resting phase to the anagen phase, promote the proliferation and differentiation of matrix keratinocytes, and increase the number of DP cells in the anagen phase hair follicles.

[0004] The aforementioned research progress suggests that precisely delivering active ingredients to CD133+ DP cell subsets and activating their intrinsic hair growth signaling pathways may become a new strategy for treating hair loss. However, achieving selective enrichment of active ingredients in specific cell subsets remains a key technical challenge that urgently needs to be overcome in this field. Furthermore, the application of peptide and protein active ingredients in shampoos faces another significant challenge: commonly used anionic surfactants in traditional shampoos (such as sodium lauryl ether sulfate) can cause significant denaturation of protein structures; existing research data shows that conventional SLS / SLES systems can lead to up to 40% loss of protein activity. Therefore, developing mild surfactant systems that can maintain protein activity is a prerequisite for the effective application of protein active ingredients in anti-hair loss shampoos. Summary of the Invention

[0005] The purpose of this invention is to provide an anti-hair loss shampoo that targets CD133-positive dermal papillary cells of hair follicles, its preparation method, and its application. To achieve the above objective, this invention provides the following technical solution:

[0006] A hair loss prevention shampoo comprises the following ingredients by weight percentage: 0.5-2% hair follicle-targeting single-domain antibody-active peptide fusion, 5-10% liposome-encapsulated compound plant extract, 15-25% biosurfactant, 0.5-2% protein-stabilizing surfactant, and the remainder being excipients and deionized water. The hair follicle-targeting single-domain antibody-active peptide fusion is expressed by fusion of a single-domain antibody that specifically recognizes CD133-positive dermal papillary cells of hair follicles and a 5α-reductase inhibitory peptide via a flexible linker peptide.

[0007] Furthermore, the single-domain antibody is an anti-CD133 VHH antibody, and its variable region sequence is shown in SEQ ID NO:3. This antibody was obtained through alpaca immunization, phage display library construction, and affinity screening. Clone D5 exhibits highly selective binding ability to CD133+ DPC (selectivity index 24.8) and an affinity constant KD = 2.3 × 10⁻⁶. -9 M.

[0008] Furthermore, the amino acid sequence of the 5α-reductase inhibitory peptide DK-HP-7 is shown in SEQ ID NO:2. This peptide was obtained through molecular docking design based on the structure of human 5α-reductase type 2, forming four hydrogen bonds and strong hydrophobic interactions with the enzyme active site, with a docking score of -9.2 kcal / mol. In vitro enzyme activity assays showed that DK-HP-7 has an IC50 value for 5α-reductase. 50 =4.28±0.52 μM, enzyme kinetic analysis showed that it was a competitive inhibitor, Ki=3.2±0.4 μM.

[0009] Furthermore, the amino acid sequence of the flexible linker peptide is shown in SEQ ID NO:4, which is (GGGGS)3.

[0010] Furthermore, the amino acid sequence of the hair follicle-targeting single-domain antibody-active peptide fusion compound is shown in SEQ ID NO:5. This fusion protein was obtained through genetic engineering construction, prokaryotic expression, and purification, with a purity >95% and a yield of approximately 48 mg / L fermentation broth. Activity verification showed that the fusion protein binds to CD133 protein at EC50. 50 =0.13±0.02 μg / mL, comparable to parental VHH antibody; 5α-reductase inhibitory activity IC50 was 0.13±0.02 μg / mL. 50 =4.85±0.61 μM, comparable to free peptides; the selective binding fold to CD133+ DPC reached 26.5-fold.

[0011] Furthermore, the biosurfactant is selected from one or more of rhamnolipids and sophorolipids; the protein-stabilizing surfactant is selected from one or more of Kolliphor HS 15 and Poloxamer 188. Preferably, a compound system of 12% rhamnolipids + 6% sophorolipids + 1% Kolliphor HS 15 is used. This system retains 92.5% of the activity of the fusion protein, has a protein aggregation rate of only 2.8%, and maintains excellent foaming properties (foaming power 178 mm) and oil removal rate (91.5%).

[0012] Furthermore, the compound plant extract encapsulated in the liposomes was prepared from the following medicinal materials in parts by weight: 20 parts of Platycladus orientalis leaves, 15 parts of Ligustrum lucidum, 10 parts of Salvia miltiorrhiza, 10 parts of Ligusticum chuanxiong, and 5 parts of Glycyrrhiza uralensis. After extraction with ethanol and enrichment with AB-8 macroporous adsorption resin, nanoliposomes were prepared using a thin-film dispersion method. The resulting liposomes had an average particle size of 145±9 nm, a Zeta potential of -33.8±3.1 mV, an encapsulation efficiency of tanshinone IIA of 85.6±3.5%, and an encapsulation efficiency of ligustrolactone A of 78.9±4.2%.

[0013] Furthermore, the excipients include 0.1-0.5% adenosine, 1-3% red clover extract, 0.05-0.2% biotin, 0.5-1.5% thickener, 0.1-0.3% preservative, and an appropriate amount of pH adjuster. The thickener is selected from one or more of guar gum hydroxypropyltrimethylammonium chloride and carbomer; the preservative is phenoxyethanol; and the pH adjuster is citric acid.

[0014] The present invention also provides a method for preparing the aforementioned anti-hair loss shampoo, comprising the following steps: adding a biosurfactant and a protein-stabilizing surfactant to deionized water, heating to 45-50°C and stirring to dissolve, to obtain phase A; dispersing a thickener in glycerin, adding phase A, and homogenizing and stirring until completely dispersed; sequentially adding a hair follicle-targeting single-domain antibody-active peptide fusion, a liposome-encapsulated compound plant extract, and other excipients, and stirring evenly; adjusting the pH to 5.5-6.5, allowing it to stand to defoam, and thus obtaining the product.

[0015] The present invention also protects the hair follicle-targeting single-domain antibody-active peptide fusion itself, the amino acid sequence of which is shown in SEQ ID NO:5.

[0016] The present invention also protects the compound plant extract itself encapsulated in the liposomes, which is prepared by extracting and enriching the medicinal materials containing 20 parts of Platycladus orientalis leaves, 15 parts of Ligustrum lucidum, 10 parts of Salvia miltiorrhiza, 10 parts of Ligusticum chuanxiong, and 5 parts of Glycyrrhiza uralensis using a thin film dispersion method after extraction and macroporous adsorption resin enrichment.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. This invention is the first to introduce a single-domain antibody targeting CD133 into an anti-hair loss product. Based on the recent biological discovery that CD133+ dermal papilla cells are the key cell population driving hair follicle formation, the single-domain antibody achieves precise enrichment of the active ingredient at its target site. Experimental results show that the anti-CD133 VHH-DK-HP-7 fusion protein selectively binds to CD133+ DPC by 26.5-fold, and competitive inhibition experiments confirm that this binding is antigen-specific recognition. Human ex vivo scalp tissue distribution experiments show that the fusion protein is specifically concentrated in the dermal papilla region of the hair follicle, while the free peptide is diffusely distributed. Anti-Versican targeting cannot achieve hair follicle-specific distribution, confirming the superiority of CD133 as a target and the necessity of targeted delivery.

[0019] 2. This invention utilizes the high-resolution structure of human 5α-reductase type 2 to obtain the derived peptide DK-HP-7 through molecular docking virtual screening. This peptide forms four stable hydrogen bonds and strong hydrophobic interactions with the enzyme's active site, achieving a docking score of -9.2 kcal / mol. In vitro enzyme activity assays show that DK-HP-7 has an IC50 value for 5α-reductase. 50 =4.28±0.52 μM, enzyme kinetic analysis showed it to be a competitive inhibitor, Ki=3.2±0.4 μM. After fusion expression, the active peptide retained its inhibitory function, and the fusion protein IC50 was 4.28±0.52 μM. 50 =4.85±0.61 μM, comparable to free peptides.

[0020] 3. Addressing the technical challenge of protein-based drugs being easily inactivated in traditional shampoo bases, this invention innovatively employs a compound system of biosurfactants (rhamnolipin + sophorolipid) and a protein stabilizer (Kolliphor HS 15). Experiments show that the conventional SLES system retains only 31.2% of the fusion protein's activity, with a protein aggregation rate as high as 42.5%; while the preferred system of this invention (12% rhamnolipin + 6% sophorolipid + 1% Kolliphor HS 15) achieves a protein activity retention rate of 92.5% and a protein aggregation rate of only 2.8%, while maintaining excellent foaming properties and oil removal efficiency. Accelerated stability testing shows that after storage at 40℃ for 3 months, the product of this invention retains 89.5% of the fusion protein's activity and ≥87% of the liposome encapsulation components, demonstrating good stability.

[0021] 4. To address the problem of unstable encapsulation of crude extracts of traditional Chinese medicine, this invention employs macroporous adsorption resin to enrich lipophilic active components (tanshinone IIA content 8.2%, ligustrazine lactone A content 5.6%), followed by liposome encapsulation. The resulting liposomes have an average particle size of 145±9 nm, uniform distribution (PDI=0.22), a Zeta potential of -33.8 mV, and encapsulation rates of tanshinone IIA (85.6±3.5%) and ligustrazine lactone A (78.9±4.2%). Stability tests show that the retention rate of the liposome-encapsulated components is ≥87% after storage at 40℃ for 3 months, significantly better than that of the unencapsulated concentrates.

[0022] 5. Human in vitro hair follicle culture experiments showed that Example 5.1 of this invention significantly antagonized DHT-induced hair follicle growth inhibition, with the growth length recovering to 89.6% of the blank control group after 7 days, and the hair follicle survival rate reaching 90%, significantly superior to all comparative groups. Cytokine detection showed that Example 5.1 significantly inhibited the pro-apoptotic factor TGF-β1 while promoting the angiogenic factor VEGF, mechanistically supporting its hair growth-promoting effect. C57BL / 6 mouse alopecia model experiments showed that the skin on the back of mice in the Example 5.1 group began to darken at 16.8 days, significantly earlier than the model group (>28 days) and all comparative groups; the hair follicle count reached 34.5 / mm², recovering to 88.9% of the blank control group, the proportion of hair follicles in the anagen phase was 78.5%, and the Ki67 positivity rate was significantly higher than that in the model group, indicating that it can effectively promote hair follicles to enter the anagen phase and proliferate. Factorial analysis showed that the fusion protein and liposome encapsulation had a significant synergistic effect (interaction p=0.008), rather than a simple additive effect, which confirmed the advanced nature and non-obviousness of the technical solution of the present invention.

[0023] 6. Good skin safety. Rabbit skin irritation tests showed that the shampoo in Example 5.1 had a PII of 0, indicating it was non-irritating; Comparative Example 3 (SLES system) showed mild erythema (PII = 0.8), confirming the mildness of the biosurfactant. Guinea pig skin sensitization tests showed that Example 5.1 had a sensitization rate of 0%, indicating it was non-sensitizing. Human skin patch tests showed that the incidence of skin reactions in Example 5.1 was only 3.3%, indicating good safety.

[0024] In summary, this invention has successfully developed a highly efficient, stable, and safe anti-hair loss shampoo by precisely targeting CD133+ dermal papillary cells of hair follicles, designing active peptides based on structural biology, using a protein-friendly surfactant system, and employing liposome technology to enrich components. This represents a technological leap from "passive nourishment" to "active targeted regulation" and has broad application prospects. Attached Figure Description

[0025] Figure 1 Immunofluorescence co-localization map of anti-CD133 VHH clone D5 and human scalp hair follicle tissue. A: DAPI staining (blue); B: Anti-CD133 VHH clone D5 staining (green); C: Mouse anti-human CD133 monoclonal antibody staining (red); D: Overlay map showing high overlap of green and red fluorescence in the dermal papilla region of the hair follicle.

[0026] Figure 2 Flow cytometry diagram illustrating the binding of FITC-labeled anti-CD133 VHH-DK-HP-7 fusion protein to different cell types. A: CD133+ DPC; B: CD133- DPC; C: HDF (human dermal fibroblasts); D: HEK (human epidermal keratinocytes). Gray-filled peaks represent negative controls, and solid peaks represent fusion protein staining.

[0027] Figure 3 Representative distribution of FITC-labeled samples in human ex vivo scalp tissue using confocal microscopy. A: Anti-CD133 VHH-DK-HP-7 fusion protein; B: Anti-Versican VHH-DK-HP-7 fusion protein. Green indicates FITC-labeled samples, blue indicates DAPI nuclear staining, and arrows indicate hair follicle structures.

[0028] Figure 4 Representative photograph of hair growth on the back skin of C57BL / 6 mice on day 28.

[0029] Figure 5 Representative photographs of rabbits on day 14 of the skin irritation test. A: Example 5.1 group, normal skin; B: Comparative Example 3 group (SLES system), slight erythema is visible on the skin (indicated by arrows). Detailed Implementation

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0031] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0032] Example 1: Screening and preparation of anti-CD133 VHH single-domain antibodies

[0033] 1. Immunogen

[0034] Recombinant CD133 protein (amino acid sequence as shown in SEQ ID NO:1) was purchased from ProteinTech, Cat no:Ag1579.

[0035] 2. Alpaca Immunization

[0036] Two healthy adult alpacas were selected. For the initial immunization of each alpaca, 1 mg of immunogen was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple points on the back (approximately 20 points). Booster immunizations were then administered on days 14, 28, and 42, with 0.5 mg of immunogen emulsified with an equal volume of Freund's incomplete adjuvant and injected subcutaneously each time. Seven days after the final immunization, 100 mL of peripheral anticoagulated blood was collected from the jugular vein (using heparin sodium anticoagulant tubes) for lymphocyte separation.

[0037] 3. Phage display library construction:

[0038] Anticoagulated blood was diluted with an equal volume of PBS and slowly added to an equal volume of lymphocyte separation medium. The mixture was centrifuged at 400×g for 30 minutes, and the white blood cell layer was aspirated and washed twice with PBS. RNA extraction and cDNA synthesis: Total RNA was extracted using TRIzol reagent, and the A260 / A280 ratio was measured to be >1.9. 5 μg of total RNA was used to synthesize cDNA using the SuperScript IV reverse transcription kit. VHH gene amplification: Nested PCR was used. First-round amplification conditions: 94℃ pre-denaturation for 3 minutes; 30 cycles of 94℃ for 30 seconds, 55℃ for 30 seconds, and 72℃ for 45 seconds; extension at 72℃ for 5 minutes. The amplified product was approximately 700 bp. The second round used the first-round PCR product as a template, with the same amplification conditions, yielding a product of approximately 400 bp.

[0039] The purified VHH gene fragment was double-digested with SfiI and NotI, ligated into the pMECS phage vector (preserved in the laboratory) digested with the same enzymes, and electrotransformed into *E. coli* TG1. The transformation product was plated on 2×YT plates containing 100 μg / mL ampicillin and 2% glucose and incubated overnight at 30°C. All colonies were scraped off, and glycerol was added to a final concentration of 20% and stored at -80°C. Library volume was determined by serially diluting 10 μL of bacterial culture and plating it onto plates, calculating the colony count, and obtaining a library volume of 2.3 × 10⁻⁶. 8 CFU. Twenty-four randomly selected clones were identified by PCR, with an insertion rate of 95% and good diversity.

[0040] 4. Affinity Screening

[0041] Round 1: Another recombinant human CD133 protein (Bipsys) was diluted to 10 μg / mL with coating buffer (0.1 M NaHCO3, pH 8.6), added to immunotubes, and incubated overnight at 4°C. The next day, the tubes were washed three times with PBST (PBS containing 0.1% Tween-20), and blocked for 2 hours with PBST containing 2% skim milk. A phage library (approximately 1 × 10⁻⁶) was then added. 12 PFU (prepared from bacterial culture stored at -80℃ and rescued using helper phage M13K07) was incubated at room temperature for 2 hours. After washing 10 times with PBST, the bound phage was eluted with 1 mL of 100 mM triethylamine, and immediately neutralized with 0.5 mL of 1 M Tris-HCl (pH 7.4). The elution product was used to infect logarithmically growing TG1 cells, and after amplification, the cells were used for the next round of selection.

[0042] Rounds two through four: The washing intensity was gradually increased in each round (PBST washing times increased from 10 to 20) while the coating antigen concentration was decreased (10 μg / mL → 5 μg / mL → 2 μg / mL → 1 μg / mL). After four rounds of screening, 96 single clones were randomly selected for phage ELISA identification: The plates were coated with recombinant CD133 protein (1 μg / mL), and the supernatant of each clone was added. HRP / anti-M13 antibody detection was performed, and OD... 450 Clones with a value >1.0 and more than 3 times that of the negative control were considered positive, and a total of 23 positive clones were obtained.

[0043] 5. Specificity screening and identification

[0044] Twenty-three positive clones were expressed as soluble VHH antibodies. The method involved inoculating each clone into 2×YT medium (containing 100 μg / mL ampicillin and 0.1% glucose) and incubating at 37°C until OD500. 600=0.6, add IPTG to a final concentration of 1 mM, and induce overnight at 30°C. Collect the supernatant by centrifugation, purify by Ni-NTA, and dialyze the eluent (250 mM imidazole) into PBS.

[0045] 5.1 Flow cytometry screening

[0046] Cell sources: Human dermal papillary cells (DPC) were isolated and cultured from hair follicles of healthy human scalp under a microscope, and CD133+ and CD133- subsets were obtained by sorting with anti-CD133 magnetic beads; human dermal fibroblasts (HDF); and human epidermal keratinocytes (HEK) were preserved in our laboratory.

[0047] Detection: Each FITC-labeled VHH antibody (using the FluoReporter FITC Labeling Kit) was mixed with 1×10⁻⁶ ions at a concentration of 10 μg / mL. 6 Cells were incubated at 4°C for 30 minutes, washed with PBS, and the mean fluorescence intensity (MFI) was detected by flow cytometry. The results showed that the MFI of clone D5 against CD133+ DPC was 441.6±36.5, and the MFI against CD133- DPC was 17.8±3.2, with a selectivity index (CD133+ / CD133-) of 24.8, which was significantly higher than that of other clones.

[0048] 5.2 Determination of affinity by surface plasmon resonance

[0049] Recombinant human CD133 protein was immobilized on a CM5 chip via amino-coupling using a Biacore T200 system, with a coupling amount of approximately 500 RU. HBS-EP+ was used as the running buffer at a flow rate of 30 μL / min. The purified clone D5 VHH was diluted to 0.625, 1.25, 2.5, 5, 10, 20, and 40 nM and injected sequentially, with binding time of 180 seconds and dissociation time of 600 seconds. A 1:1 binding model was used for fitting, and the KD value was determined to be 2.3 × 10⁻⁶. -9 M (ka = 4.6 × 10) 5 M -1 s -1 kd = 1.06 × 10 -3 s -1 ).

[0050] 5.3 Validation by tissue immunofluorescence

[0051] Scalp tissue from healthy individuals (derived from discarded skin from plastic surgery, with the consent of the volunteers) was embedded in OCT and frozen sectioned (8 μm thick). Sections were fixed with 4% paraformaldehyde for 15 minutes, blocked with 5% BSA for 1 hour, and incubated overnight at 4°C with clone D5 VHH (10 μg / mL) and mouse anti-human CD133 monoclonal antibody (MCE, HY-P80054, 1:200 dilution). After washing, Alexa Fluor 488-labeled anti-alpaca secondary antibody (prepared in the lab, 1:500) and Alexa Fluor 594-labeled anti-mouse secondary antibody (1:500) were added, incubated at room temperature for 1 hour, and counterstained with DAPI. Observation was performed using a confocal microscope. Figure 1 The green fluorescence of clone D5 and the red fluorescence of CD133 antibody highly overlapped in the dermal papilla region of the hair follicle, with a co-localization rate of 92.5±3.8%.

[0052] Example 2: Design and Validation of Active Peptide DK-HP-7

[0053] 1. Molecular docking design

[0054] Based on the high-resolution cryo-electron microscopy structure of human 5α-reductase type 2 (SRD5A2), this study used AutoDock Vina 1.2.0 to virtually screen a library of candidate peptides targeting the enzyme's active site pocket while preserving the physiological activity of the key cofactor NADPH. By setting up a 30Å×30Å×30Å docking box and employing a peptide flexible docking protocol (exhaustiveness=32), the derived peptide DK-HP-7 (sequence WKLVSIK, SEQ ID NO: 2) with the lowest binding energy (-9.2 kcal / mol) was successfully obtained. Molecular docking results showed that this peptide could form a stable network of four hydrogen bonds with the Tyr91, Phe194, Trp201, and Glu197 residues in the active site of SRD5A2, accompanied by strong hydrophobic interactions, confirming its potential biochemical activity as a 5α-reductase inhibitor from a structural biology perspective.

[0055] 2. Assay for 5α-reductase inhibitory activity

[0056] 2.1 Enzyme source preparation: Liver of male Sprague-Dawley rats (200-250g) was used to prepare microsomes. Protein concentration was determined by Lowry method and aliquoted and stored at -80℃.

[0057] 2.2 Inhibitory activity assay: The reaction system (500 μL) contained 100 mM Tris-HCl (pH 7.2), 1 mM DTT, 0.5 mM NADPH, and 1 μM [ 14[C]-Testosterone (PerkinElmer, 0.1 μCi), different concentrations of test samples (0.01-100 μM), and an appropriate amount of enzyme solution (approximately 50 μg protein). The mixture was incubated at 37°C for 30 minutes, and the reaction was terminated by adding 1 mL of ethyl acetate followed by vigorous shaking and extraction. The organic phase was transferred to a new tube, dried under nitrogen, and the residue was dissolved in 50 μL of methanol and spotted onto a silica gel thin-layer plate. The plate was developed with chloroform:acetone (9:1). The plate was scanned using a Typhoon FLA 9500 phosphorus screen imaging system, and the grayscale values ​​of the testosterone and dihydrotestosterone bands were analyzed using ImageQuant software to calculate the conversion and inhibition rates. Finasteride was used as the positive control, and KPVSL peptide (synthetic, purity >95%) was used as the negative control. The experiment was repeated three times independently, with three replicates per well.

[0058] 2.3 Results: The inhibition rate of DK-HP-7 peptide was concentration-dependent. After nonlinear fitting using GraphPad Prism 9.0 (four-parameter logistic model), the IC50 value was... 50 =4.28±0.52 μM. Positive control finasteride IC50 50 =0.15±0.03 μM, consistent with previous reports. The negative control KPVSL peptide showed an inhibition rate of <5% at a concentration of 100 μM.

[0059] 3. Enzyme kinetic analysis

[0060] With a fixed enzyme concentration, the initial reaction rates of different concentrations of testosterone (2.5, 5, 10, 20, 40 μM) in the presence of 0, 2, and 5 μM DK-HP-7 were determined. Lineweaver-Burk double reciprocal plots showed that as the inhibitor concentration increased, Vmax remained constant (approximately 2.8 nmol / min / mg), while the Km value increased from 8.5 μM to 21.3 μM, indicating competitive inhibition. A quadratic plot of the slope against the inhibitor concentration was performed, and Ki was calculated to be 3.2 ± 0.4 μM (n = 3).

[0061] Example 3: Construction and expression of anti-CD133 VHH-DK-HP-7 fusion protein

[0062] 1. Fusion gene design

[0063] The anti-CD133 VHH antibody sequence (SEQ ID NO:3, which, after comparison, was found to be quite similar to the human antibody, one of the reasons for its good efficacy and safety, will not be elaborated here) was linked to the DK-HP-7 active peptide sequence (SEQ ID NO:2) via a flexible linker peptide (GGGGS)3 (SEQ ID NO:4), and a 6×His tag was added to the C-terminus. NcoI and XhoI restriction sites were introduced at both ends of the gene. The whole gene synthesis was completed by Sangon Biotech (Shanghai) Co., Ltd., and the synthesized product was cloned into the pUC57 vector. Based on this, the amino acid sequence of the anti-CD133 VHH-DK-HP-7 fusion protein is shown in SEQ ID NO:5 (without the 6×His tag).

[0064] 2. Construction of expression vector

[0065] The plasmid and pET-28a(+) vector were synthesized by double digestion with NcoI and XhoI (NEB). The target fragment and vector fragment were recovered and ligated overnight at 16°C using T4 DNA ligase. The ligation product was transformed into E. coli DH5α competent cells and plated on LB agar plates containing 50 μg / mL kanamycin. Single clones were picked and identified by colony PCR (primers T7 and T7ter). Positive clones were sent for sequencing. The sequencing results were completely consistent with the designed sequence.

[0066] 3. Induced expression

[0067] Transform the correctly sequenced recombinant plasmid into competent E. coli BL21(DE3) cells. Pick single colonies and inoculate them into 5 mL LB medium (containing 50 μg / mL kanamycin) and incubate overnight at 37°C and 220 rpm. Then, inoculate 1 L LB medium (containing 50 μg / mL kanamycin) at a 1:100 ratio and incubate at 37°C until OD (outcome limit). 600 =0.6. Add IPTG to a final concentration of 0.5 mM and induce expression at 16℃ for 16 hours. Collect bacterial cells by centrifugation at 6000×g for 10 minutes at 4℃.

[0068] 4. Fusion protein purification

[0069] Bacterial cells were resuspended in lysis buffer (20 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole, pH 8.0) and sonicated (300 W, 3 seconds on, 5 seconds off, for a total of 30 minutes). The cells were centrifuged at 12000 × g for 30 minutes at 4°C, and the supernatant was filtered through a 0.45 μm filter. The supernatant was loaded onto a Ni-NTA affinity chromatography column and eluted with a gradient of elution buffers containing 20 mM, 50 mM, 100 mM, and 250 mM imidazole. The 250 mM imidazole elution peak was collected. The eluent was concentrated to 2 mL using an ultrafiltration tube and loaded onto a Superdex 75 10 / 300 GL size exclusion chromatography column. Elution was performed with PBS (pH 7.4), and the monomer peaks were collected.

[0070] 5. Product Identification

[0071] 5.1 SDS-PAGE: The purified product was subjected to 15% SDS-PAGE and stained with Coomassie Brilliant Blue. The results showed a single band at approximately 18 kDa, and the purity was >95% according to gel scanning quantification.

[0072] 5.2 Yield: Protein concentration was determined by BCA method, and approximately 48 mg of purified fusion protein was obtained per liter of fermentation broth.

[0073] 6. Validation of fusion protein activity

[0074] 6.1 ELISA Conjugation Assay: Recombinant human CD133 protein (2 μg / mL) was coated onto a 96-well microplate and incubated overnight at 4°C. After blocking with 5% skim milk powder for 1 hour, serially diluted fusion protein or parental VHH antibody (0.001-10 μg / mL) was added, and the plate was incubated at room temperature for 2 hours. HRP-labeled anti-His tag antibody (1:5000) was added, and the plate was incubated at room temperature for 1 hour. TMB was developed, and OD was measured. 450 Fitting dose-response curves for the fusion protein EC 50 =0.13±0.02 μg / mL, parental VHH EC 50 =0.11±0.02 μg / mL (n=3), no significant difference (p>0.05).

[0075] 6.2 5α-Reductase Inhibitory Activity: The IC50 of the fusion protein was determined according to the method in Example 2. 50 =4.85±0.61 μM, comparable to the free peptide (4.28±0.52 μM) (p>0.05). Enzyme kinetics showed competitive inhibition, Ki=3.2±0.4 μM.

[0076] 6.3 Cell binding specificity: After incubation with the FITC-labeled fusion protein (10 μg / mL) in various cells, flow cytometry analysis showed that the MFI of CD133+ DPC was 432.5±35.8, and the MFI of CD133- DPC was 16.3±3.2, with a selectivity fold of 26.5-fold. Competitive inhibition assays (pre-addition of 10-fold excess of unlabeled VHH) reduced the MFI to 28.5±5.2, achieving an inhibition rate of 93.5%.

[0077] Example 4: Preparation of compound plant extracts encapsulated in liposomes

[0078] 1. Extraction of medicinal materials

[0079] Weigh the following medicinal materials according to the specified proportions: 20 parts of Platycladus orientalis leaves, 15 parts of Ligustrum lucidum fruit, 10 parts of Salvia miltiorrhiza root, 10 parts of Ligusticum chuanxiong rhizome, and 5 parts of Glycyrrhiza uralensis root. After pulverizing the medicinal materials, pass them through a 40-mesh sieve. Add 10 times the amount of 60% ethanol and reflux at 80°C for 2 hours, then filter. Add 8 times the amount of 60% ethanol to the residue and reflux at 80°C for 2 hours, then filter again. Combine the two filtrates and concentrate under reduced pressure at 50°C (using a rotary evaporator) until no alcohol odor remains, obtaining a crude extract.

[0080] 2. Enrichment with macroporous adsorption resins

[0081] The crude extract was dispersed in an appropriate amount of water and loaded onto an AB-8 macroporous adsorption resin column (2.5 cm diameter, 25 cm height, approximately 120 mL volume). The loading flow rate was 2 BV / h, and the loading amount was equivalent to 100 g of crude drug. Elution was performed sequentially with water (3 BV), 30% ethanol (3 BV), 70% ethanol (5 BV), and 95% ethanol (3 BV), collecting the 70% ethanol eluent. The eluent was concentrated to dryness under reduced pressure at 50°C and then vacuum dried (40°C, 24 hours) to obtain the enriched powder. The enrichment yield was approximately 3.5% of the crude drug weight.

[0082] 3. HPLC fingerprint analysis

[0083] An Agilent 1260 HPLC system was used with an Agilent ZORBAX SB-C18 column (4.6 × 250 mm, 5 μm). Mobile phase A was acetonitrile, and mobile phase B was 0.1% phosphoric acid aqueous solution. Gradient elution was used: 0–10 min 30% A, 10–20 min 30–70% A, 20–30 min 70% A, 30–35 min 70–30% A, and 35–40 min 30% A. The flow rate was 1.0 mL / min, and the detection wavelengths were 270 nm (tanshinone IIA) and 320 nm (ligustrazine A). The column temperature was 30 °C. Standard curves were established using tanshinone IIA (China National Institutes for Food and Drug Control) and ligustrazine A (Sigma) as reference standards. The concentration of tanshinone IIA in the concentrate was 8.2%, and the concentration of ligustrazine A was 5.6%.

[0084] 4. Liposome preparation

[0085] Nanoliposomes were prepared using a thin-film dispersion method: 4.0 g of soybean lecithin, 0.5 g of cholesterol, and 0.5 g of enrichment were weighed and dissolved in 50 mL of anhydrous ethanol. The solution was transferred to a 500 mL round-bottom flask, and the ethanol was removed by rotary evaporation at 40 °C, forming a uniform thin film on the flask wall. 100 mL of pH 7.4 phosphate buffer (containing 10 mM PBS) was added, and the mixture was hydrated and shaken at 40 °C for 30 minutes. The liposome suspension was transferred to a 50 mL centrifuge tube and sonicated under ice bath conditions (200 W, 3 seconds on, 3 seconds off, for a total of 10 minutes). After sonication, the liposomes were filtered through a 0.45 μm filter membrane to obtain the nanoliposome suspension, which was stored at 4 °C.

[0086] 5. Liposome Characterization

[0087] 5.1 Particle size and Zeta potential: An appropriate amount of liposome suspension was diluted with PBS to a suitable concentration before measurement. The average particle size was 145±9 nm, and the polydispersity index (PDI) was 0.22±0.03 (n=3). The measured Zeta potential was -33.8±3.1 mV.

[0088] 5.2 Encapsulation efficiency determination: Take 1 mL of liposome suspension, place it in an ultrafiltration centrifuge tube (MWCO 10 kDa), centrifuge at 10000×g for 30 minutes at 4℃, and collect the filtrate (free drug). Take another equal volume of liposome suspension, add 9 volumes of methanol to demulsify, sonicate for 5 minutes, and determine the total drug content by HPLC. Encapsulation efficiency (%) = (total drug content - free drug content) / total drug content × 100%. Initial encapsulation efficiency of tanshinone IIA = 85.6 ± 3.5%, initial encapsulation efficiency of ligustrazine lactone A = 78.9 ± 4.2% (n=3).

[0089] Example 5: Preparation of anti-hair loss shampoo

[0090] Example 5.1: Basic formulation (per 1000 g)

[0091] Table 1 Basic Formula (per 1000 g)

[0092]

[0093] Preparation process:

[0094] Phase A preparation: Take about 500 g of deionized water, heat to 45°C, add rhamnolipin, sophorolipolipin and Kolliphor HS15, stir to dissolve evenly (magnetic stirrer, 500 rpm), and keep warm at 45°C.

[0095] Phase B preparation: Take glycerol (20 g), add guar hydroxypropyltrimethylammonium chloride, and stir to disperse evenly.

[0096] Mixing: Slowly add phase B to phase A, stir homogenously (8000 rpm) for 10 minutes until fully dispersed, and continue to keep warm at 45°C.

[0097] Add active ingredients: Add the anti-CD133 VHH-DK-HP-7 fusion protein (pre-dissolved in a small amount of PBS), liposome-encapsulated compound plant extract, adenosine, red clover extract, and biotin in sequence. Stir for 5 minutes after each addition until homogeneous.

[0098] Adjust pH: Add phenoxyethanol and adjust the pH to 5.5-6.5 with citric acid (10% aqueous solution) (target pH 6.0, monitored with a pH meter), and continue stirring for 30 minutes.

[0099] Make up to 1000 g of deionized water and stir well.

[0100] Defoaming after standing: Let stand for 24 hours until the bubbles disappear, then dispense into plastic bottles and store at room temperature.

[0101] Example 5.2: High-concentration formula (suitable for severe hair loss)

[0102] The difference from Example 5.1 is that the fusion protein is increased to 2.0%, the liposome encapsulation is increased to 10.0%, rhamnolipin is 15% + sophorolipid is 10% (total surfactant is 25%), and the rest are the same.

[0103] Example 5.3: Gentle Formula (Suitable for Sensitive Scalps)

[0104] The difference from Example 5.1 is that the fusion protein was reduced to 0.5%, the liposome encapsulation was reduced to 5.0%, and all biosurfactants were sophorolipids (15%), while the rest were the same.

[0105] Example 5.4: Simplified Formulation (Verifying the Necessity of Core Components)

[0106] The difference from Example 5.1 is that it does not contain red clover extract and biotin, but is otherwise the same.

[0107] Example 5.5: Poloxamer Alternative Formulation

[0108] The difference from Example 5.1 is that Kolliphor HS 15 is replaced with an equal amount of Poloxamer 188, otherwise the same.

[0109] Example 6: Scale Setting

[0110] Comparative Example 1 (without fusion protein): The formulation was the same as in Example 5.1, but without the anti-CD133 VHH-DK-HP-7 fusion protein, and was replaced with an equal amount of PBS.

[0111] Comparative Example 2 (without liposome encapsulation): The formulation is the same as in Example 5.1, but without liposome-encapsulated plant extracts. Instead, an equal amount of unencapsulated plant extracts is used (i.e., extract powder is added directly).

[0112] Comparative Example 3 (Conventional Surfactant System): The formulation is the same as in Example 5.1, but the surfactant system is replaced with: sodium lauryl ether sulfate (SLES, 70% solution) 12% (dry weight) and cocamidopropyl betaine (CAB, 30% solution) 6% (dry weight), without Kolliphor HS 15.

[0113] Comparative Example 4 (Anti-Versican Targeting): Same formulation as Example 5.1, but the targeting antibody in the fusion protein was replaced with anti-Versican VHH (obtained by screening in a similar manner, clone V1, KD=1.5×10⁻⁶). -7 M (sequence not listed), the active peptide portion remains DK-HP-7.

[0114] Comparative Example 5 (commercially available product): A well-known commercially available anti-hair loss shampoo (containing Polygonum multiflorum and Platycladus orientalis leaf extracts, with SLES as the main surfactant).

[0115] Comparative Example 6 (Non-targeted fusion protein): The formulation is the same as in Example 5.1, but the targeting antibody in the fusion protein is replaced with an irrelevant VHH (anti-GFP VHH, laboratory-preserved, KD does not bind to CD133), and the active peptide portion remains DK-HP-7.

[0116] Comparative Example 7 (Unoptimized Active Peptide): The formulation is the same as in Example 5.1, but the active peptide in the fusion protein is replaced with the KPVSL peptide of the original application sequence (sequence: KPVSL), and the targeting antibody is still anti-CD133 VHH.

[0117] Comparative Example 8 (Physical Mixture): The formulation was the same as in Example 5.1, but the anti-CD133 VHH antibody (equimolar amount with the fusion protein, about 6 g) and the DK-HP-7 active peptide (about 6 g) were added in a physical mixture form instead of fusion expression.

[0118] Experiment 1: Validation of fusion protein targeting specificity (flow cytometry)

[0119] 1. Experimental Methods

[0120] Cells: Human hair follicle dermal papilla cells (CD133+ and CD133- subsets), HDF, HEK, cultured to logarithmic growth phase, trypsin digested, washed with PBS, and cell density adjusted to 1×10⁻⁶. 6 / mL.

[0121] Staining: Add 100 μL of cell suspension (1×10⁻⁶) to each tube. 5 Cells were incubated with FITC-labeled anti-CD133 VHH-DK-HP-7 fusion protein (final concentration 10 μg / mL) at 4°C in the dark for 30 minutes. Cells were washed twice with PBS, resuspended in 300 μL PBS, and analyzed by flow cytometry. 10,000 cells were collected for each sample, and mean fluorescence intensity (MFI) was analyzed using FlowJo 10.0.

[0122] Competitive inhibition group: Cells were incubated with 10 times molar excess of unlabeled anti-CD133 VHH antibody at 4°C for 30 minutes, and then FITC-labeled fusion protein was added and detected as above.

[0123] 2. The results are shown in Table 2 and Figure 2 As shown, the fusion protein exhibits highly selective binding ability to CD133+ DPC (selectivity fold > 25-fold). Competitive inhibition experiments confirmed that this binding is antigen-specific recognition, with virtually no binding to CD133- cells and other skin cells, indicating that it can achieve precise targeted delivery.

[0124] Table 2 Experimental results (mean±SD, n=3)

[0125]

[0126] Note: Compared with the CD133+ DPC group, **p<0.01 (t-test). Relative binding fold = MFI of each cell type / MFI of CD133+ DPC, used to characterize the binding specificity of the fusion protein.

[0127] Experimental Example 2: Tissue Distribution Study of Fusion Proteins (Human Ex vivo Scalp Tissue)

[0128] 1. Experimental Methods

[0129] Tissue Acquisition: Scalp tissue was taken from healthy individuals (derived from discarded skin from plastic surgery, with the consent of the volunteers), subcutaneous fat was trimmed, and the tissue was cut into pieces approximately 5 mm × 5 mm in size and approximately 1 mm thick.

[0130] Culture: Tissues were placed in Transwell chambers (Corning, 0.4 μm pore size) and cultured at the gas-liquid interface (600 μL Williams E medium containing 2 mM L-glutamine, 10 ng / mL hydrocortisone, 10 μg / mL insulin, and 100 U / mL penicillin / streptomycin were added to the lower layer).

[0131] Treatment: Add the following FITC-labeled samples (10 μg / mL each): (1) anti-CD133 VHH-DK-HP-7 fusion protein; (2) anti-Versican VHH-DK-HP-7 fusion protein; (3) unrelated VHH-DK-HP-7 fusion protein; (4) FITC-labeled DK-HP-7 free peptide. Three tissue blocks were prepared for each group. Incubate at 37°C for 24 hours.

[0132] Slide preparation: Tissue was removed, washed with PBS, fixed with 4% paraformaldehyde for 4 hours, dehydrated with 30% sucrose overnight, embedded with OCT, frozen sectioned (10 μm thick), and mounted with DAPI.

[0133] Observation: Confocal microscopy was used for observation, with excitation wavelengths of 488 nm (FITC) and 405 nm (DAPI).

[0134] 2. Experimental results are as follows Figure 3 As shown

[0135] Anti-CD133 VHH fusion protein group: FITC fluorescence is specifically concentrated in the dermal papilla region of the hair follicle, showing a bright punctate or ring-like distribution; the fluorescence signal in the epidermis and dermis surrounding the hair follicle is weak. Anti-Versican VHH fusion protein group: Fluorescent signals are widely distributed in the dermis and connective tissue surrounding the hair follicle, without obvious region specificity. Irrelevant VHH fusion protein group: No obvious fluorescence signal was observed in the entire tissue layer. Free DK-HP-7 peptide group: Fluorescent signals are diffusely distributed throughout the entire tissue section, without region specificity.

[0136] Therefore, the anti-CD133 VHH fusion protein can specifically target the dermal papilla region of the hair follicle, achieving precise enrichment of the active ingredient at the site of action. In contrast, the anti-Versican targeting cannot achieve follicle-specific distribution, while the free peptides exhibit diffuse distribution, confirming the necessity of targeted delivery.

[0137] Experimental Example 3: Human Ex vivo Hair Follicle Culture Experiment

[0138] 1. Experimental Materials and Methods

[0139] 1.1 Hair follicle separation: Take scalp tissue from healthy individuals (source as above), and separate hair follicles in the growth phase under a stereomicroscope using micro-forceps and micro-scissors. Select hair follicles that are morphologically intact, have a full hair bulb, an intact hair shaft, and a length of about 2.5-3.0 mm.

[0140] 1.2 Culture: Place a single hair follicle in a 24-well plate, add 500 μL of Williams E medium (same as in Experiment 2) to each well, and culture at 37°C with 5% CO2.

[0141] 1.3 Grouping: Hair follicles were randomly divided into 12 groups, with 20 follicles in each group. A model of androgen damage was established by adding 10 μM dihydrotestosterone (DHT) to the culture medium in each group, and corresponding samples (the active ingredient mixture was prepared according to the proportions in Example 5, without surfactants, and sterilized by filtration through a 0.22 μm filter membrane) were added. Specific groups were: blank control group (without DHT), model group (DHT), Example 5.1 group, Example 5.2 group, Example 5.3 group, Example 5.4 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 4 group, Comparative Example 6 group, Comparative Example 7 group, and Comparative Example 8 group. The culture medium was replaced with fresh medium every 2 days.

[0142] 1.4 Detection indicators:

[0143] (1) Hair follicle length measurement: On days 0, 1, 3, 5 and 7, take pictures under an inverted microscope and use ImageJ to measure the hair follicle length (from the bottom of the hair bulb to the top of the hair shaft) and calculate the growth length.

[0144] (2) Hair follicle survival rate: On the 7th day, the survival standard is that the hair bulb is intact and there are no degenerative changes (such as hair bulb atrophy or melanin diffusion). The number of surviving hair follicles is counted.

[0145] (3) Histological analysis: On day 7, 10 hair follicles were taken from each group, fixed with 4% paraformaldehyde, embedded in paraffin, longitudinally sectioned (5 μm thick), stained with HE, and the diameter of the hair bulb and the thickness of the hair matrix were measured under a microscope.

[0146] (4) Cytokine detection: On day 7, 10 hair follicles from each group were taken, washed with PBS, and 100 μL of RIPA lysis buffer (containing protease inhibitor) was added. After homogenization, the mixture was centrifuged at 12000×g for 10 minutes at 4℃, and the supernatant was collected. The contents of TGF-β1 and VEGF were detected by ELISA according to the kit instructions. The results are expressed as pg / mg total protein (total protein was measured by BCA method).

[0147] 2. The experimental results are shown in Tables 3 to 5.

[0148] Example 5.1 significantly antagonized DHT-induced hair follicle growth inhibition, restoring hair length to 89.6% of the blank control group after 7 days, with a hair follicle survival rate of 90% and morphological parameters significantly superior to the model group. Comparative Example 1 (without fusion protein) and Comparative Example 2 (without liposome encapsulation) showed significantly lower effects than Example 5.1, confirming the necessity of fusion protein and liposome encapsulation technology. Comparative Example 4 (anti-Versican targeting) showed better effects than Comparative Example 1 but lower than Example 5.1, indicating that CD133 is a better target. Comparative Example 8 (physical mixture) showed lower effects than the fusion protein, indicating that fusion expression is beneficial for synergistic effects. Cytokine assays showed that Example 5.1 significantly inhibited the pro-apoptotic factor TGF-β1 while promoting the angiogenic factor VEGF, mechanistically supporting its hair growth-promoting effect.

[0149] Table 3. Dynamic changes in hair follicle growth length (mm, mean ± SD, n=20)

[0150]

[0151] Note: Compared with the blank control group, **p<0.01; compared with the model group, #p<0.05, ##p<0.01; compared with the Example 5.1 group, *p<0.05, ANOVA + Dunnett test.

[0152] Table 4. Hair follicle survival rate and morphological parameters (day 7, mean ± SD, n=20)

[0153]

[0154] Table 5 Cytokine levels (day 7, pg / mg total protein, mean ± SD, n=10)

[0155]

[0156] Experiment Example 4: Factorial Analysis of Synergistic Effects

[0157] A 2×2 factorial design was used to investigate two factors: fusion protein (present / absent) and liposome encapsulation (present / absent). There were four groups, each with 20 hair follicles, treated as in Experiment 3, using DHT to induce the model. The dependent variable was the hair follicle growth length on day 7.

[0158] Table 6 Experimental results (mean±SD, n=20)

[0159]

[0160] Statistical analysis: Two-way ANOVA (SPSS 26.0): Main effect of fusion protein: F=92.5, p<0.001; Main effect of liposome encapsulation: F=45.8, p<0.001; Interaction: F=8.6, p=0.008.

[0161] Therefore, the statistically significant interaction (p=0.008) indicates that the fusion protein and liposome encapsulation have a synergistic effect rather than a simple additive effect, which confirms the advanced nature and non-obviousness of the technical solution of the present invention.

[0162] Experimental Example 5: C57BL / 6 Mouse Hair Loss Model Experiment

[0163] 1. Experimental Materials and Methods

[0164] 1.1 Animals: Eight-week-old male C57BL / 6 mice, weighing 20-25g, SPF grade, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. They were housed in an IVC system at a temperature of 22±2℃ and humidity of 50±10%, with 12 hours of light / 12 hours of darkness, free access to water and food, and were acclimatized for one week.

[0165] 1.2 Model Establishment and Grouping: Mice were shaved on the back (2 cm × 3 cm area). Except for the blank control group, the other groups were injected subcutaneously with testosterone propionate (1 mg / kg, dissolved in sterile olive oil) daily to establish an androgenic alopecia model. They were randomly divided into 12 groups, with 10 mice in each group: (1) Blank control group: physiological saline applied and rinsed; (2) Model group: physiological saline applied and rinsed; (3) Example 5.1 group: 5% Example 5.1 shampoo solution applied and rinsed; (4) Example 5.2 group: 5% Example 5.2 shampoo solution applied and rinsed; (5) Example 5.3 group: 5% Example 5.3 shampoo solution applied and rinsed; (6) Example 5.4 group: 5% Example 5.4 shampoo solution applied and rinsed. (7) Comparative Example 1: 5% Comparative Example 1 shampoo solution applied and rinsed; (8) Comparative Example 2: 5% Comparative Example 2 shampoo solution applied and rinsed; (9) Comparative Example 3: 5% Comparative Example 3 shampoo solution applied and rinsed; (10) Comparative Example 4: 5% Comparative Example 4 shampoo solution applied and rinsed; (11) Comparative Example 5: 5% commercially available shampoo solution applied and rinsed; (12) Comparative Example 8: 5% Comparative Example 8 shampoo solution applied and rinsed.

[0166] 1.3 Treatment method: Apply the corresponding sample (0.2 mL) to the back skin daily, leave it on for 10 minutes, and then rinse it off with warm water (about 37℃). Repeat once a day for 28 consecutive days.

[0167] 1.4 Observation indicators:

[0168] (1) Skin color change: Observe and record the time when the skin color on the back changes from pink to black every day (indicating that the hair follicles have entered the growth phase).

[0169] (2) Hair growth score: Back photos were taken on days 7, 14, 21 and 28 (same angle and same lighting). The hair growth score was scored independently by two people using a double-blind method: 0 points = no hair growth; 1 point = coverage area <25%; 2 points = coverage area 25-50%; 3 points = coverage area 50-75%; 4 points = coverage area >75%.

[0170] (3) Hair follicle counting: Mice were sacrificed on day 28, and skin from the central back (1 cm × 1 cm) was taken, fixed in 10% neutral formalin, embedded in paraffin, and transversely serially sectioned (5 μm thick). One section was taken every 100 μm, stained with hematoxylin and eosin (HE), and the number of hair follicles per unit area (number / mm²) was counted under a microscope. 2 Take 3 slices and average them.

[0171] (4) Histological analysis: Measure hair follicle density, hair follicle diameter, hair follicle depth and the ratio of growth phase to resting phase (judged according to hair follicle morphology).

[0172] (5) Immunohistochemistry: Sections were dewaxed and hydrated, antigens were retrieved, 3% H2O2 was used for blocking, 5% BSA was used for blocking, rabbit anti-Ki67 antibody (1:200) was added and incubated at 4°C overnight, secondary antibody was incubated, DAB staining was performed, and hematoxylin was used for counterstaining. The proportion of Ki67 positive cells was counted under a microscope (5 fields of view were randomly selected from each section).

[0173] 2. The experimental results are shown in Tables 7-9. Figure 4 As shown.

[0174] In Example 5.1, the skin on the back of mice began to darken at 16.8 days, significantly earlier than the model group (>28 days) and all comparative groups, indicating that it can effectively promote hair follicles to enter the growth phase. The hair follicle count in Example 5.1 reached 34.5 follicles / mm². 2 The hair follicle recovery rate was 88.9% of the control group, with 78.5% in the growth phase. The Ki67 positivity rate was significantly higher than the model group, indicating that it promotes hair follicle proliferation. Comparative Example 3 (conventional surfactant system) showed the worst effect, with a hair follicle recovery rate of only 43.3%, which was not significantly different from the model group, confirming the necessity of the protein-friendly surfactant system. Comparative Example 4 (anti-Versican targeted) showed better results (hair follicle recovery rate of 60.6%) than Comparative Example 1 but lower than Example 5.1, consistent with the results of the in vitro experiment. Comparative Example 5 (commercially available product) showed limited effect, with a hair follicle recovery rate of only 46.9%.

[0175] Table 7. Skin darkening time and hair follicle count (mean±SD, n=10)

[0176]

[0177] Note: Compared with the blank control group, **p<0.01; compared with the model group, #p<0.05, ##p<0.01; compared with the group in Example 5.1, *p<0.05)

[0178] Table 8. Dynamic changes in hair growth scores (mean±SD, n=10)

[0179]

[0180] Table 9. Histological and immunohistochemical results (day 28, mean ± SD, n=10)

[0181]

[0182] Experiment Example 6: Optimization and Verification of Surfactant System

[0183] 1. Experimental Methods

[0184] The anti-CD133 VHH-DK-HP-7 fusion protein (100 μg / mL) was co-incubated with different surfactant systems (formulations shown in the table) (37℃, 7 days), and the following indicators were detected:

[0185] Protein activity retention rate: After incubation, the surfactant was removed by hydrophobic interaction chromatography, the protein components were collected, and the 5α-reductase inhibitory activity was determined according to the method in Example 2. The retention rate was calculated with the initial activity as 100%.

[0186] Protein aggregation rate: The monomer peak area was analyzed by SEC-HPLC (Agilent 1260, TSKgel G2000SWXL column). The proportion of monomer reduction due to aggregation was calculated with the initial monomer peak area as 100%.

[0187] Foaming power: Ross-Miles method (GB / T 7462-94), take 200 mL of sample solution (5%, w / v) and let it flow down from a height of 90 cm, and measure the initial foam height (mm).

[0188] Oil removal rate: Artificial sebum method (refer to the "Cosmetic Hygiene Standard"), weigh artificial sebum (formula: stearic acid: palmitic acid: paraffin oil: squalane: cholesterol = 20:15:25:30:10) and spread it on a glass slide. After weighing, immerse it in the sample solution (5%, w / v) and shake for 10 minutes. After drying, weigh it and calculate the oil removal rate.

[0189] 2. Surfactant system design

[0190] Table 10 Surfactant Systems

[0191]

[0192] 3. The experimental results are shown in Table 11.

[0193] The conventional SLES system (S1) exhibited a protein activity retention rate of only 31.2% and a protein aggregation rate as high as 42.5%, confirming its severe destructive effect on proteins. The single-system biosurfactant system (S2, S3) showed a protein activity retention rate of approximately 75-78%, significantly better than the conventional system, but still suffered about 20% activity loss. Adding Kolliphor HS15 or Poloxamer 188 (S5-S8) increased the protein activity retention rate to 88-92% and reduced the protein aggregation rate to below 3%, indicating that protein stabilizers can effectively protect protein structure. The preferred system of this invention (S7: rhamnolipid + sophorolipid + Kolliphor HS15) achieved the highest protein activity retention rate (92.5%) and the lowest protein aggregation rate (2.8%), while maintaining excellent foaming properties and oil removal efficiency.

[0194] Table 11 Summary of experimental results (mean±SD, n=3)

[0195]

[0196] Note: Compared with group S4, *p<0.05, **p<0.01.

[0197] Experiment Example 7: Stability Test

[0198] 1. Experimental Methods

[0199] 1.1 The shampoo samples from Examples 5.1-5.5 and Comparative Example 3 were placed under the following conditions:

[0200] Accelerated stability: 40℃±2℃ / 75%±5% RH (constant temperature and humidity chamber, Binder KBF 240), stored for 6 months.

[0201] Long-term stability: 25℃±2℃ / 60%±5% RH, stored for 12 months.

[0202] Low temperature stability: 4℃±2℃ (refrigerator), stored for 12 months.

[0203] 1.2 Samples were taken and tested at 0, 1, 2, 3, 6, and 12 months (accelerated testing up to 6 months, low-temperature testing up to 12 months):

[0204] Appearance: Visually inspect for any layering, sedimentation, discoloration, or odor.

[0205] pH value: Take 10 g of sample, add deionized water to 100 g, stir well, and measure with a pH meter.

[0206] Fusion protein content: Take 1 mL of sample, remove surfactant with HiTrap Butyl HP column, collect protein components, determine fusion protein concentration by ELISA (same as Example 3.6.1), and calculate content retention rate (compared to 0 month).

[0207] Fusion protein activity: The 5α-reductase inhibitory activity was determined according to the method in Example 2, and the activity retention rate was calculated.

[0208] Liposome particle size: Take 1 mL of sample and determine the average particle size using dynamic light scattering method.

[0209] Liposome encapsulation efficiency: Take 1 mL of sample, centrifuge at ultraspeed (100,000×g, 4℃, 1 hour) to separate liposomes, resuspend the precipitate with PBS, determine the content of tanshinone IIA and ligustrazine A by HPLC, and calculate the retention rate (compared to 0 month).

[0210] Microbiological testing: The total number of colonies, molds and yeasts were tested according to the methods in the "Cosmetic Safety Technical Specifications".

[0211] 2. Accelerated stability test results

[0212] Comparative Example 3: Accelerated stability (40℃, 3 months): Protein content retention rate 42.5±5.8%, protein activity retention rate 28.6±4.5%, slight stratification in appearance.

[0213] Table 12 Accelerated stability test results (40℃, mean±SD, n=3)

[0214]

[0215] 3. Results of long-term stability tests

[0216] Low-temperature stability (4℃, 12 months): All indicators showed minimal change, protein content retention >95%, protein activity retention >94%, and no significant increase in liposome particle size. Microbiological testing: All samples had a total bacterial count <10 CFU / g and a total mold and yeast count <10 CFU / g at all time points, meeting the limits for cosmetics.

[0217] Table 13 Results of long-term stability test (25℃, mean±SD, n=3)

[0218]

[0219] 4. Summary

[0220] The product of this invention (Example 5.1), after being stored under accelerated conditions (40°C) for 3 months, retained 89.5% of the fusion protein activity and ≥87% of the liposome encapsulation components. Even after 6 months, it still maintained over 80% activity, demonstrating good stability. Long-term stability (25°C) for 12 months showed a protein activity retention rate of 91.2% and a liposome encapsulation component retention rate of ≥89%, meeting cosmetic requirements. Comparative Example 3 (conventional SLES system) showed rapid protein inactivation under accelerated conditions, with only 28.6% activity retention after 3 months, confirming the necessity of a protein-friendly surfactant system. Low-temperature conditions are more conducive to product stability; it is recommended to store the product in a cool place.

[0221] Experimental Example 8: Skin Safety Evaluation

[0222] 1. Experimental Methods 1.1 Rabbit Skin Irritation Test (Refer to the 2015 edition of the "Cosmetic Safety Technical Specifications")

[0223] (1) Animals: 6 New Zealand white rabbits (half male and half female, weighing 2.0-2.5kg, purchased from Beijing Vital River), kept in individual cages, and acclimatized for 3 days.

[0224] (2) Procedure: Hair was removed from the back (8 cm × 10 cm) 24 hours before the experiment. Shampoo (0.5 g) from Example 5.1 and physiological saline (control) were applied to both sides respectively. The areas were covered with gauze and secured with non-irritating adhesive tape. This was done once daily for 14 consecutive days. Erythema and edema were observed and recorded 24 hours after each application and scored according to standards.

[0225] Erythema: No erythema = 0, mild erythema = 1, moderate erythema = 2, severe erythema = 3, purplish-red erythema to mild eschar = 4;

[0226] Edema: No edema = 0, mild edema = 1, moderate edema = 2, severe edema = 3, extreme edema = 4;

[0227] Calculate the Primary Stimulation Index (PII) = (Total Erythema Score + Total Edema Score) / Number of Observations.

[0228] The same test was also performed on ratio 3 (the conventional SLES system).

[0229] 1.2 Skin sensitization test (guinea pig maximal test, refer to OECD 406)

[0230] Animals: 30 Hartley guinea pigs (half male and half female, weighing 250-300g) were randomly divided into 3 groups (10 in each group): Example 5.1 group, positive control group (2,4-dinitrochlorobenzene, DNCB), and negative control group (physiological saline).

[0231] Induction phase: On day 0, inject 0.1 mL of sample intradermally (5% shampoo solution for example 5.1 group, 0.1% DNCB for positive control group, and physiological saline for negative control group), apply sample topically on day 7 (0.2 mL, applied to the induction site), and apply again on day 14.

[0232] Triggering phase: On day 21, apply 0.2 mL of the sample to untreated areas and observe erythema and edema at 24 and 48 hours later, scoring according to the Magnusson grading system (0-4).

[0233] 1.3 Human skin patch test (refer to the "Cosmetic Safety Technical Specifications")

[0234] Subjects: 30 healthy volunteers (aged 18-60, half male and half female, with no history of allergies) were recruited and signed an informed consent form.

[0235] Procedure: Shampoo (5% aqueous solution) and physiological saline from Example 5.1 were placed in a Finn Chamber spot tester (8 mm) and applied to the back of the subject. The spot tester was removed after 48 hours, and the skin reaction was observed at 0.5, 24, and 48 hours and scored according to the standard.

[0236] 2. Experimental Results

[0237] 2.1 Skin irritation test on rabbits, as follows Figure 5 As shown

[0238] Example 5.1: No erythema or edema was observed in any of the rabbits during the 14-day observation period, and the PII was 0, indicating that the rabbits were non-irritating.

[0239] Comparative group 3: Mild erythema appeared on day 1 (0.3±0.5), and the erythema score was 0.8±0.4 on day 14, with PII=0.8, which is considered mild irritation.

[0240] 2.2 Skin sensitization test

[0241] Example 5.1: No skin reaction was observed in any guinea pigs 24 and 48 hours after stimulation, with a sensitization rate of 0%, indicating no sensitization.

[0242] Positive control group: All guinea pigs showed varying degrees of erythema after challenge, with a sensitization rate of 100%.

[0243] Negative control group: No skin reaction was observed, and the sensitization rate was 0%.

[0244] 2.3 Human skin patch test

[0245] Of the 30 volunteers, 29 had no skin reaction, and one developed a mild erythema (about 3 mm in diameter) after 48 hours, which subsided spontaneously within 24 hours. The incidence of skin reaction was 3.3%, which is considered low irritation.

[0246] 3. Summary

[0247] Example 5.1 showed a PII of 0 in the rabbit skin irritation test, indicating it was non-irritating; Comparative Example 3 showed mild erythema, confirming the mildness of the biosurfactant. The guinea pig skin sensitization test showed a sensitization rate of 0% for Example 5.1, indicating it was non-sensitizing. The human skin patch test showed a skin reaction rate of only 3.3% for Example 5.1, indicating good safety. The overall results demonstrate that the product of this invention has good skin safety and is suitable for daily use.

[0248] Experimental Example 9: Study on Mechanism of Action

[0249] 1. Experimental Methods

[0250] 1.1 Cell Culture and Processing: Human dermal papillary cells (DPCs) were cultured and CD133+ and CD133- subsets were obtained by flow cytometry sorting. CD133+ DPCs were seeded in 6-well plates (2 × 10⁻⁶). 5 Cells / well), cultured for 24 hours, then replaced with serum-free medium and treated separately:

[0251] (1) Blank control (no treatment);

[0252] (2) DHT group: 10 μM DHT;

[0253] (3) DHT + Active ingredient group of Example 5.1: DHT + 0.5 μM fusion protein (based on fusion protein concentration, derived from the active ingredient mixture of Example 5.1, after dialysis to remove surfactant);

[0254] (4) DHT+ Comparative Example 1 active ingredient group: DHT+ active ingredient mixture without fusion protein (equal volume);

[0255] (5) DHT+ Comparative Example 4 Active Ingredient Group: Cells were collected after being treated with a mixture of DHT+ active ingredients containing anti-Versican fusion protein (at equal concentration) for 48 hours.

[0256] 1.2 Western blot: Total protein extraction: RIPA lysis buffer (containing protease and phosphatase inhibitors), lyse on ice for 30 minutes, centrifuge at 12000×g for 10 minutes at 4℃, collect the supernatant, and determine protein concentration using the BCA method. Nucleoprotein extraction: Use the NE-PER nucleoprotein extraction kit. SDS-PAGE: Load 30 μg of protein per well, separate using 10% SDS-PAGE, and transfer to a PVDF membrane. Blocking: 5% skim milk powder (dissolved in TBST), incubate at room temperature for 1 hour. Primary antibodies: rabbit anti-β-catenin (1:1000), rabbit anti-p-GSK-3β (Ser9) (1:1000), rabbit anti-Axin2 (1:1000), rabbit anti-LEF1 (1:1000), rabbit anti-Cyclin D1 (1:1000), rabbit anti-Bcl-2 (1:1000), rabbit anti-Bax (1:1000), rabbit anti-Histone H3 (1:2000, nuclear internal control), rabbit anti-GAPDH (1:2000, total protein internal control), incubated overnight at 4°C. Secondary antibody: HRP-labeled goat anti-rabbit (1:3000), incubated at room temperature for 1 hour. Development: ECL chemiluminescence solution, detected by chemiluminescence imaging system, and band grayscale analyzed by ImageJ.

[0257] 1.3 Immunofluorescence: CD133+ DPCs were seeded into confocal dishes (2×10⁻⁶). 4 Cells / plate), treated as above. Fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, blocked with 5% BSA, added rabbit anti-β-catenin antibody (1:200) and incubated overnight at 4°C, labeled with goat anti-rabbit secondary antibody (1:500) with Alexa Fluor 488 and incubated at room temperature for 1 hour, nuclei stained with DAPI, and observed under a confocal microscope.

[0258] 1.4 qRT-PCR: Total RNA was extracted with TRIzol and reverse transcribed into cDNA. qPCR was performed using SYBR Premix Ex Taq on an ABI 7500. GAPDH was used as an internal control. - The relative expression level was calculated using the ΔΔCt method.

[0259] 1.5 Cell proliferation and apoptosis detection: CCK-8: Cells were seeded in 96-well plates (5 × 10⁻⁶ cells / well). 3 After 48 hours of treatment (cells / well), 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C for 2 hours. OD was then measured. 450 EdU incorporation: Using the EdU kit, follow the instructions and count the proportion of positive cells using a fluorescence microscope. Apoptosis detection: The apoptosis rate was detected by flow cytometry using the Annexin V-FITC / PI apoptosis detection kit.

[0260] 2. Experimental Results

[0261] 2.1 Western blot results

[0262] Table 14 Western blot results (relative gray values, mean ± SD, n=3)

[0263]

[0264] 2.2 Immunofluorescence results

[0265] Blank control group: β-catenin was distributed in both the cytoplasm and nucleus, and some cells showed nuclear positivity.

[0266] DHT group: β-catenin is mainly distributed in the cytoplasm, with very few nuclear positive cells (<5%).

[0267] DHT+ Example 5.1 group: β-catenin nuclear translocation was obvious, and the proportion of nuclear positive cells was about 40%, which is close to the blank control.

[0268] DHT+ control group 1: partial recovery of nuclear translocation, approximately 15% of cells were nuclear-positive.

[0269] DHT+ control group 4: approximately 25% of cells were nuclear positive.

[0270] 2.3 qRT-PCR results (relative mRNA expression level, mean ± SD, n=3)

[0271] Table 15 qRT-PCR results (relative mRNA expression levels, mean ± SD, n=3)

[0272]

[0273] 2.4 Cell proliferation and apoptosis results (mean±SD, n=3)

[0274] Table 16. Cell proliferation and apoptosis results (mean ± SD, n=3)

[0275]

[0276] 3. Summary

[0277] DHT treatment significantly inhibited the Wnt / β-catenin signaling pathway in CD133+ DPCs, manifested as decreased total and nuclear β-catenin protein, downregulated expression of downstream target genes, inhibited cell proliferation, and increased apoptosis. Example 5.1 showed that the active ingredient significantly activated the Wnt / β-catenin signaling pathway, restored β-catenin nuclear translocation, upregulated downstream target gene expression, promoted cell proliferation, and inhibited apoptosis. Comparative Example 1 (without fusion protein) showed significantly lower efficacy than Example 5.1, indicating that targeted delivery of the fusion protein is crucial for signaling pathway activation. Comparative Example 4 (anti-Versican targeting) showed efficacy between Comparative Example 1 and Example 5.1, indicating that non-specific targeting can partially deliver the active ingredient, but the effect is far less than CD133-specific targeting. This study confirms that the present invention activates the Wnt / β-catenin signaling pathway in CD133+ DPCs through targeted delivery, thereby antagonizing DHT-induced hair follicle damage and promoting hair growth.

[0278] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A single-domain antibody clone D5 against CD133 VHH, wherein the amino acid sequence of the single-domain antibody against CD133 VHH is shown in SEQ ID NO:

3.

2. A hair follicle-targeting single-domain antibody-active peptide fusion protein, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO:

5.

3. A hair loss prevention shampoo, characterized in that, Based on 1000 g, it comprises the following raw materials: 12 g of the fusion protein as described in claim 2, 80 g of liposome-encapsulated compound plant extract, 2 g of adenosine, 20 g of red clover extract, 1 g of biotin, 120 g of rhamnolipin, 60 g of sophorolipid, 10 g of Kolliphor HS 15, 8 g of guar hydroxypropyltrimethylammonium chloride, 2 g of phenoxyethanol, an appropriate amount of citric acid, and deionized water to make up to 1000 g, wherein the pH value is 5.5 to 6.

5.

4. A hair loss prevention shampoo, characterized in that, Based on 1000 g, it comprises the following raw materials: 20 g of the fusion protein as described in claim 2, 100 g of liposome-encapsulated compound plant extract, 2 g of adenosine, 20 g of red clover extract, 1 g of biotin, 150 g of rhamnolipin, 100 g of sophoroliposide, 10 g of Kolliphor HS 15, 8 g of guar hydroxypropyltrimethylammonium chloride, 2 g of phenoxyethanol, an appropriate amount of citric acid, and deionized water to make up to 1000 g, wherein the pH value is 5.5 to 6.

5.

5. A hair loss prevention shampoo, characterized in that, Based on 1000 g, it comprises the following raw materials: 5 g of the fusion protein as described in claim 2, 50 g of liposome-encapsulated compound plant extract, 2 g of adenosine, 20 g of red clover extract, 1 g of biotin, 150 g of sophorolipid, 10 g of Kolliphor HS 15, 8 g of guar hydroxypropyltrimethylammonium chloride, 2 g of phenoxyethanol, an appropriate amount of citric acid, and deionized water to make up to 1000 g, wherein the pH value is 5.5 to 6.

5.

6. A hair loss prevention shampoo, characterized in that, Based on 1000 g, it comprises the following ingredients: 12 g of the fusion protein as described in claim 2, 80 g of liposome-encapsulated compound plant extract, 2 g of adenosine, 1 g of biotin, 120 g of rhamnolipin, 60 g of sophorolipolipin, 10 g of Kolliphor HS 15, 8 g of guar hydroxypropyltrimethylammonium chloride, 2 g of phenoxyethanol, an appropriate amount of citric acid, and deionized water to make up to 1000 g, wherein the pH value is 5.5 to 6.5, and it does not contain red clover extract.

7. A hair loss prevention shampoo, characterized in that, Based on 1000 g, it comprises the following raw materials: 12 g of the fusion protein as described in claim 2, 80 g of liposome-encapsulated compound plant extract, 2 g of adenosine, 20 g of red clover extract, 1 g of biotin, 120 g of rhamnolipin, 60 g of sophorolipid, 10 g of poloxamer 188, 8 g of guar hydroxypropyltrimethylammonium chloride, 2 g of phenoxyethanol, an appropriate amount of citric acid, and deionized water to make up to 1000 g, wherein the pH value is 5.5 to 6.

5.

8. The shampoo according to any one of claims 3 to 7, characterized in that, The liposome-encapsulated compound plant extract was prepared by extracting 20 g of Platycladus orientalis leaves, 15 g of Ligustrum lucidum, 10 g of Salvia miltiorrhiza, 10 g of Ligusticum chuanxiong, and 5 g of Glycyrrhiza uralensis with 10 times the volume of 60% ethanol under reflux at 80°C for 2 hours each time. The filtrates were combined, concentrated, and loaded onto an AB-8 macroporous adsorption resin column. The column was eluted sequentially with water, 30% ethanol, 70% ethanol, and 95% ethanol. The 70% ethanol eluent was collected, concentrated, and dried to obtain the enriched product. 4.0 g of soybean lecithin, 0.5 g of cholesterol, and 0.5 g of the enriched product were dissolved in 50 mL of anhydrous ethanol. The ethanol was removed by rotary evaporation at 40°C to form a thin film. 100 mL of pH 7.4 phosphate buffer was added, and the mixture was hydrated at 40°C with shaking for 30 minutes. The mixture was then sonicated for 10 minutes with a 200 W probe for 10 seconds of continuous operation followed by 3 seconds of rest. The final product was filtered through a 0.45 μm filter membrane.

9. The shampoo according to claim 8, characterized in that, The liposome-encapsulated compound plant extract contained 8.2% tanshinone IIA and 5.6% ligustrazine lactone A. The average particle size of the liposome-encapsulated compound plant extract was 145±9 nm, the polydispersity index was 0.22±0.03, and the zeta potential was -33.8±3.1 mV. The encapsulation efficiency of tanshinone IIA was 85.6±3.5%, and the encapsulation efficiency of ligustrazine lactone A was 78.9±4.2%.

10. The use of the anti-hair loss shampoo according to any one of claims 3 to 7 in the preparation of a product for the prevention or treatment of androgenetic alopecia.

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