Ginsenoside rg3-loaded bletilla striata polysaccharide microneedle, preparation method therefor, and use thereof

Abstract

A ginsenoside Rg3-loaded Bletilla striata polysaccharide microneedle, a preparation method therefor, and a use thereof, relating to the field of medicines. The ginsenoside Rg3-loaded Bletilla striata polysaccharide microneedle is prepared from the following raw materials in parts by weight: 1-6 parts of Bletilla striata polysaccharide, 5-15 parts of hyaluronic acid, 0.1-1 part of ginsenoside Rg3, and 15-20 parts of polyvinyl alcohol. The microneedle can realize transdermal administration to treat alopecia. The ginsenoside Rg3-loaded Bletilla striata polysaccharide microneedle has an excellent alopecia treatment effect. Compared with the use of ginsenoside Rg3 alone, Bletilla striata polysaccharide exerts the effect of an adjuvant in the treatment of alopecia, and enhances the hair growth effect of ginsenoside Rg3, thereby exerting, together with ginsenoside Rg3, a synergistic hair growth promotion effect. The prepared microneedle has good prospects for application in the treatment of alopecia.

Description

A bletilla striata polysaccharide microneedle loaded with ginsenoside Rg3 and its preparation method and use Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to a bletilla striata polysaccharide microneedle loaded with ginsenoside Rg3, a preparation method thereof, and uses thereof. Background Art

[0002] Hair loss has become one of the most troubling issues for the public today. Data shows that over 250 million people in my country are experiencing hair loss, of which approximately 163 million, or nearly 70%, are men, and nearly 70% are under the age of 30. Furthermore, hair loss is becoming more common among younger people, with 46.7% of those born after 1990 experiencing severe hair loss. Addressing this issue is urgent.

[0003] Microneedle technology is an emerging dermatological treatment for hair loss. Microneedle therapy utilizes a tiny needle-like device to puncture the skin, creating numerous micropores. This technique enhances transdermal drug penetration, promotes epidermal repair and regeneration, releases growth factors, and enhances dermal collagen and elastin formation. It is widely used for a variety of skin conditions. Furthermore, microneedle drug delivery induces mechanical stimulation of the superficial skin layers. This stimulation can upregulate the expression of pro-angiogenic factors in the skin, promote angiogenesis, activate hair follicle cells, and thus induce hair regeneration, further benefiting the treatment of hair loss disorders. With the advancement of micro- and nanofabrication technologies in the 1990s, microneedle technology has rapidly developed, resulting in the emergence of a variety of microneedle devices. Dissolvable microneedles, among others, have been extensively researched due to their biodegradability, excellent biocompatibility, and high safety. Made from a biodegradable polymer material and the drug, soluble microneedles offer advantages such as minimal invasiveness and improved penetration. During use, the needle body made of degradable material will gradually dissolve, releasing the drug synchronously and entering the human body through the subcutaneous tissue, which can effectively prevent wound infection. It is highly safe and no sharp waste remains after piercing the skin. The wound can also heal automatically after use.

[0004] Material selection is crucial when preparing soluble microneedles. Bletilla striata polysaccharide (BSP), a natural soluble polymer extracted from the traditional Chinese medicine Bletilla striata, is composed of α-mannose, β-mannose, and β-glucose. It plays an important role in the manufacture of adhesives, wound dressings, and other biomedical materials. Bletilla striata polysaccharide is also a key material for preparing soluble microneedles, demonstrating excellent mechanical properties, stability, and biocompatibility.

[0005] Ginsenoside Rg3 is one of the main active substances in ginseng, possessing a broad spectrum of pharmacological activities, including antioxidant, anti-tumor, and anti-aging properties. Recent literature reports indicate that ginseng and its main bioactive component, ginsenosides, enhance hair regeneration by regulating cell signaling pathways such as DKK1 and JAK2-STST3, modulating hair follicle-related genes and inflammatory cytokines such as interferon (IFN) and CD8+. Ginsenoside Rg3 significantly activates hair follicle stem cells and promotes hair regeneration by increasing the expression of human dermal papilla (DP) cells and vascular endothelial growth factor (VEGF) and CD34.

[0006] Currently, there are no reports of microneedles using Bletilla striata polysaccharide and ginsenoside Rg3 for the treatment of hair loss. Whether Bletilla striata polysaccharide / ginsenoside Rg3 microneedles can be successfully prepared, and how effective these microneedles are in treating hair loss, requires further research.

[0007] Summary of the Invention

[0008] The purpose of the present invention is to provide a bletilla striata polysaccharide microneedle loaded with ginsenoside Rg3 and a preparation method and use thereof.

[0009] The first aspect of the present invention provides a bletilla striata polysaccharide microneedle loaded with ginsenoside Rg3, wherein the microneedle is prepared from raw materials in the following weight ratio:

[0010] 1-6 parts of Bletilla striata polysaccharide, 5-15 parts of hyaluronic acid, 0.1-1 part of ginsenoside Rg3, and 15-20 parts of polyvinyl alcohol.

[0011] In another preferred embodiment, the aforementioned Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3 are prepared from the following raw materials in the following weight ratio:

[0012] 1-6 parts of Bletilla striata polysaccharide, 5-15 parts of hyaluronic acid, 0.6 parts of ginsenoside Rg3, and 20 parts of polyvinyl alcohol.

[0013] In another preferred embodiment, the aforementioned Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3 are prepared from the following raw materials in the following weight ratio:

[0014] 6 parts of Bletilla striata polysaccharide, 15 parts of hyaluronic acid, 0.6 parts of ginsenoside Rg3, and 20 parts of polyvinyl alcohol.

[0015] In another preferred embodiment, the height of a single microneedle in the microneedle is 900 μm, the width of the base is 400 μm, and the spacing between each microneedle is 700 μm.

[0016] In another preferred embodiment, the bletilla striata polysaccharide is composed of mannose and glucose in a molar mass ratio of 1.99 to 2.95:1, and the weight average molecular weight of the bletilla striata polysaccharide is 1.10×10 5~3.99×10 5 g / mol.

[0017] In another preferred embodiment, the preparation method of the bletilla striata polysaccharide comprises the following steps:

[0018] Take Bletilla striata powder, first extract it with anhydrous ethanol at 60-80°C for 1-3 times, then extract the residue with petroleum ether at 60-90°C for 1-3 times, then extract the residue with water at 70-80°C for 1-3 times, concentrate the obtained extract and deproteinize it, add 90-95% ethanol aqueous solution by volume to precipitate, wash the precipitate and dry it to obtain Bletilla striata polysaccharide.

[0019] In another preferred embodiment, the ratio of the Bletilla striata powder to anhydrous ethanol is 1 g: 5-10 mL.

[0020] In another preferred embodiment, the ratio of the Bletilla striata powder to petroleum ether is 1 g: 5-10 mL.

[0021] In another preferred embodiment, the ratio of the Bletilla striata powder to water is 1 g: 40-50 mL.

[0022] In another preferred embodiment, when extracting with anhydrous ethanol, the extraction time is 2 to 5 hours each time.

[0023] In another preferred embodiment, when petroleum ether is used for extraction, the extraction time for each time is 2 to 5 hours.

[0024] In another preferred embodiment, when extracting with water, the extraction time is 2 to 5 hours each time.

[0025] In another preferred embodiment, the deproteinization is performed by the Sevage method.

[0026] In another preferred embodiment, the precipitate is washed with anhydrous ethanol, acetone and ether in sequence.

[0027] The second aspect of the present invention provides a method for preparing the aforementioned Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3, the method comprising the following steps:

[0028] (1) dispersing bletilla striata polysaccharide, hyaluronic acid, and ginsenoside Rg3 in water to obtain a mixed solution;

[0029] (2) filling the mixed solution prepared in step (1) into a mold and centrifuging;

[0030] (3) dissolving polyvinyl alcohol in water to obtain a polyvinyl alcohol solution;

[0031] (4) adding the polyvinyl alcohol solution obtained in step (3) to the mold of step (2) and centrifuging;

[0032] (5) Drying to obtain the Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3.

[0033] In another preferred embodiment, in step (1), the concentration of Bletilla striata polysaccharide in the mixed solution is 0.003-0.02 g / mL.

[0034] In another preferred embodiment, in step (2), the height of a single microneedle in the mold is 900 μm, the width of the base is 400 μm, and the spacing between each microneedle is 700 μm.

[0035] In another preferred embodiment, in step (2), the centrifugation is performed at 4000 rpm for 20 min.

[0036] In another preferred embodiment, in step (3), the concentration of polyvinyl alcohol in the polyvinyl alcohol solution is 20 wt%.

[0037] In another preferred embodiment, in step (3), the volume ratio of the polyvinyl alcohol solution to the mixed solution of step (1) is (3-5):1.

[0038] In another preferred embodiment, in step (4), the centrifugation is performed at 4000 rpm for 5 minutes.

[0039] In another preferred embodiment, in step (5), the drying is performed at 30-37°C.

[0040] In another preferred embodiment, in step (1), the concentration of Bletilla striata polysaccharide in the mixed solution is 0.02 g / mL.

[0041] In another preferred embodiment, in step (3), the volume ratio of the polyvinyl alcohol solution to the mixed solution of step (1) is 3:1.

[0042] The third aspect of the present invention provides the use of the aforementioned Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3 in the preparation of a drug for treating hair loss.

[0043] The fourth aspect of the present invention provides a method for treating hair loss, comprising the steps of administering the bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3 described in the first aspect of the present invention to a subject in need.

[0044] In another preferred embodiment, the subject includes rodents (such as mice and rats) and primates (such as humans).

[0045] The present invention provides Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3, which can be used for transdermal drug delivery to treat hair loss. The Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3 are highly effective in treating hair loss. Compared with using ginsenoside Rg3 alone, Bletilla striata polysaccharide acts as an adjuvant in treating hair loss, enhancing the hair growth effect of ginsenoside Rg3 and thereby exerting a synergistic, synergistic effect in promoting hair growth. The microneedles prepared by the present invention have promising application prospects in treating hair loss.

[0046] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 shows the mechanical properties of Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3 containing different concentrations of Bletilla striata polysaccharide.

[0048] Figure 2 shows the mechanical properties of Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3 and containing different concentrations of hyaluronic acid.

[0049] Figure 3 is a graph showing the mechanical properties of Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3 prepared with 0.06g Bletilla striata polysaccharide, 0.05g hyaluronic acid, and 0.006g ginsenoside Rg3.

[0050] FIG4 is a morphological diagram of the Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3 of the present invention; wherein A is an optical microscope image, B is a scanning electron microscope image, and C is a fluorescence microscope image.

[0051] FIG5 is a diagram showing the mechanical properties of the Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3 prepared in Preparation Example 1 of the present invention.

[0052] FIG6 is a HE staining result of the in vivo insertion performance study of the Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3 prepared in Preparation Example 1 of the present invention.

[0053] FIG7 is a graph showing the recovery performance of the mouse skin after insertion of the Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3 prepared in Preparation Example 1.

[0054] FIG8 is a graph showing the in vivo dissolution performance of the Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3 prepared in Preparation Example 1 of the present invention.

[0055] FIG9 is a graph showing the drug penetration results in vivo after the Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3 prepared in Preparation Example 1 of the present invention are inserted into the skin.

[0056] FIG10 is a graph showing the drug release results of ginsenoside Rg3 in vitro from the Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3 prepared in Preparation Example 1 of the present invention.

[0057] FIG11 is a graph showing the hair growth of mice in each group within 15 days of drug administration.

[0058] FIG12 is an H&E-stained section of hair follicle cells of mice in each group on day 8 of administration.

[0059] Figure 13 shows the results of characterization of new hair growth in each group of mice; A shows the number of days after hair removal when the skin darkened, B shows the coverage of regenerated hair, C shows the density of regenerated hair, and D shows the thickness of the skin. The data of each group were compared with those of the Rg3 Bletilla polysaccharide-loaded microneedle group, with #p<0.05, ##p<0.01, and ###p<0.001. ns p>0.05. DETAILED DESCRIPTION

[0060] After extensive and in-depth research, the inventors unexpectedly discovered for the first time that when Bletilla striata polysaccharide and ginsenoside Rg3 are used simultaneously to prepare microneedles for treating hair loss, the effect is far superior to the additive effect of the two, resulting in an excellent synergistic effect in promoting hair growth. Based on this, the present invention was completed.

[0061] Ginsenoside Rg3

[0062] Ginsenosides are important active ingredients of ginseng. They belong to the triterpenoid glycoside compounds and can be divided into protopanaxadiol saponins (PPD-type saponins), protopanaxatriol saponins (PPT-type saponins) and oleanane-type saponins. More than 40 types of ginsenosides have been isolated from ginseng roots.

[0063] In the present invention, ginsenoside Rg3 is one of the main active substances in ginseng. In a preferred embodiment of the present invention, the ginsenoside is (S-type) ginsenoside, CAS: 14197-60-5, and its chemical structure is shown below:

[0064] Bletilla striata polysaccharide and its preparation

[0065] Bletilla striata polysaccharide (BSP), also known as "Bletilla striata polysaccharide," is a natural soluble polymer extracted from the traditional Chinese medicine Bletilla striata. It is composed of α-mannose, β-mannose, and β-glucose. In a preferred embodiment, the Bletilla striata polysaccharide is prepared by extracting the Bletilla striata polysaccharide from the traditional Chinese medicine Bletilla striata with an organic phase to obtain a filter residue, which is then extracted with water. In a more preferred embodiment, the Bletilla striata polysaccharide is extracted as follows:

[0066] The powder of Bletilla striata is added to anhydrous ethanol, and the mixture is refluxed and extracted at 70-80° C. The residue is then refluxed and extracted with petroleum ether at 60-90° C. The residue is then refluxed and extracted with water.

[0067] The filtrate after water bath extraction is concentrated, deproteinized, and ethanol is added to refrigerate to precipitate. The precipitate is collected and purified to obtain Bletilla striata polysaccharide.

[0068] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercial products.

[0069] (S-type) ginsenoside Rg3 was purchased from Destelemi Pharmaceuticals, CAS: 14197-60-5, purity: HPLC ≥ 98%.

[0070] Preparation of Bletilla striata polysaccharide: Take 100g of Bletilla striata powder and extract it with 500mL of anhydrous ethanol at 70-80℃ twice, each extraction time is 2 hours. Take the filter residue and extract it with 500mL of petroleum ether at 60-90℃ for 2 hours. Take the filter residue and extract it with 4000ml of water again, repeat twice, each time for 2 hours. Then concentrate the filtrate after water bath extraction to 1 / 3 of the original volume and deproteinize it using the Sevage method. Add 95% ethanol and refrigerate to precipitate. Collect the precipitate, wash it with anhydrous ethanol, acetone, and ether in sequence, and dry it to obtain Bletilla striata polysaccharide.

[0071] The polysaccharide prepared by this method is composed of mannose and glucose in a molar mass ratio of 1.99 to 2.95:1, and the weight average molecular weight (Mw) of the polysaccharide is 1.10×10 5 ~3.99×10 5 g / mol.

[0072] Preparation Example 1: Preparation of Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3

[0073] 0.06g of Bletilla striata polysaccharide, 0.15g of hyaluronic acid, and 0.006g of ginsenoside Rg3 were dissolved and dispersed in 3mL of water to obtain a mixed solution. The mixed solution was placed in a mold and filled with water and centrifuged at 4000rpm for 20 minutes. After scraping off the excess liquid, 1mL of a 20% polyvinyl alcohol aqueous solution was added to the mold and centrifuged at 4000rpm for 5 minutes to form a microneedle base. After standing for a while, the excess liquid was scraped off and the mold was dried at low temperature (30-37°C) to obtain Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3. (Mold dimensions: 1.5cm diameter circular mold, individual microneedles 900μm high, base width 400μm, and spacing 700μm between microneedles).

[0074] Preparation Example 2: Preparation of Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3

[0075] 0.01g of Bletilla striata polysaccharide, 0.05g of hyaluronic acid, and 0.006g of ginsenoside Rg3 were dissolved and dispersed in 3mL of water to obtain a mixed solution. The mixed solution was placed in a mold and filled with water and centrifuged at 4000rpm for 20 minutes. After scraping off the excess liquid, 1mL of a 20% polyvinyl alcohol aqueous solution was added to the mold and centrifuged at 4000rpm for 5 minutes to form a microneedle base. After standing for a while, the excess liquid was scraped off and the mold was dried at low temperature (30-37°C) to obtain Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3. (Mold dimensions: 1.5cm diameter circular mold, individual microneedles 900μm high, base width 400μm, and spacing 700μm between microneedles).

[0076] The beneficial effects of the present invention are demonstrated below by specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0077] Example 1. Process screening of Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3

[0078] (1) Dosage of Bletilla striata polysaccharide

[0079] 0.01 g, 0.02 g, 0.04 g or 0.06 g of bletilla striata polysaccharide (BSP), 0.15 g of hyaluronic acid (HA), and 0.006 g of ginsenoside Rg3 were weighed respectively, dissolved and dispersed in 3 mL of water to obtain a mixed solution, which was placed in a mold and filled, and centrifuged at 4000 rpm for 20 min; after scraping off the excess liquid solution, 1 mL of 20% polyvinyl alcohol aqueous solution was added to the mold, and the mold was centrifuged at 4000 rpm for 5 min to form a microneedle base; after standing, the excess liquid solution was scraped off and the mold was dried at low temperature (30-37°C) to obtain bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3 with different bletilla striata polysaccharide concentrations (mold size: a circular mold with a diameter of 1.5 cm, a single microneedle height of 900 μm, a base width of 400 μm, and a spacing of 700 μm between each microneedle). The process of preparing Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3 at different Bletilla striata polysaccharide concentrations was investigated to observe the mechanical properties of each microneedle. The specific operation is as follows:

[0080] A microneedle patch containing a 5×5 array was secured with double-sided tape and placed in the center of a stainless steel plate, with the microneedle tips facing upward. The distance between the microneedle tips and the sensor was set at 0.5 cm. The sensor parameters were then adjusted to move vertically toward the stainless steel plate at a constant speed of 0.5 mm / min. The sensor displacement after contact with the microneedle tips and the corresponding resistance to the microneedles were recorded. The results are shown in Figure 1. As shown in Figure 1, the mechanical properties of the microneedles decreased with increasing Bletilla striata polysaccharide microneedle concentration.

[0081] (2) Dosage of hyaluronic acid

[0082] 0.05g, 0.1g, or 0.15g of hyaluronic acid (HA), 0.06g of Bletilla striata polysaccharide, and 0.006g of ginsenoside Rg3 were weighed, dispersed, and dissolved in 3mL of water to obtain a mixed solution. The mixed solution was then placed in a mold and centrifuged at 4000rpm for 20min. After scraping off the excess liquid, 1mL of a 20% polyvinyl alcohol aqueous solution was added to the mold and centrifuged at 4000rpm for 5min to form a microneedle base. After standing for a while, the excess liquid was scraped off and the mold was dried at low temperature (30-37°C). This yielded Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3 at varying HA concentrations. (Mold dimensions: 1.5cm diameter circular mold, individual microneedles 900μm high, base 400μm wide, 700μm spacing between microneedles). The process of preparing Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3 and different hyaluronic acid concentrations was investigated to observe the mechanical properties of each microneedle. The specific operation is as follows:

[0083] A microneedle patch containing a 5×5 array was secured with double-sided tape and placed in the center of a stainless steel plate, with the microneedle tips facing upward. The distance between the microneedle tips and the sensor was set at 0.5 cm. The sensor parameters were then adjusted to move vertically toward the stainless steel plate at a constant speed of 0.5 mm / min. The sensor displacement after contact with the microneedle tips and the corresponding resistance to the microneedles were recorded. The results are shown in Figure 2. Figure 2 shows that the mechanical properties of the microneedles significantly increase with increasing hyaluronic acid concentration.

[0084] Based on the results in Figures 1-2, 0.06g of Bletilla striata polysaccharide, 0.05g of hyaluronic acid, and 0.006g of ginsenoside Rg3 were weighed and dissolved in 3mL of water to obtain a mixed solution. The mixed solution was then placed in a mold and centrifuged at 4000rpm for 20min. After scraping off the excess liquid, 1mL of a 20% polyvinyl alcohol aqueous solution was added to the mold and centrifuged at 4000rpm for 5min to form a microneedle base. After standing for a while, the excess liquid was scraped off and the mold was dried at low temperature (30-37°C) to obtain the microneedles. (Mold dimensions: 1.5cm diameter circular mold, individual microneedles 900μm high, base width 400μm, spacing 700μm between microneedles). The microneedles were then placed on the center of a stainless steel plate using double-sided tape, with the microneedle tips facing upwards. The distance between the microneedle tips and the sensor was set at 0.5cm. Subsequently, the sensor parameters were adjusted so that it moved vertically toward the stainless steel plate at a constant speed of 0.5 mm / min. The sensor displacement after contact with the microneedle tip and the corresponding resistance to the microneedle were recorded. The mechanical properties of the microneedle are shown in Figure 3. As shown in Figure 3, no turning points were observed in the force-displacement curve. Furthermore, the compressive force of the microneedle increased with displacement. When the displacement increased to 0.34 mm, the compressive force of a single needle exceeded 0.6 N. This demonstrates that the microneedle has sufficient mechanical properties to penetrate the skin.

[0085] Summary: According to Figures 1-2, as the concentration of Bletilla striata polysaccharide increases, the mechanical properties of the microneedles decrease; while as the concentration of hyaluronic acid increases, the mechanical properties of the microneedles increase. Microneedles prepared with high concentrations of Bletilla striata polysaccharide and low concentrations of hyaluronic acid also show sufficient mechanical properties to pierce the skin to deliver drugs. Therefore, any ratio of Bletilla striata polysaccharide to hyaluronic acid within the range of 0.01-0.06g Bletilla striata polysaccharide and 0.05-0.15g hyaluronic acid should pierce the skin and deliver drugs. The raw materials and dosages used to successfully prepare microneedles in the present invention are: 0.01-0.06g Bletilla striata polysaccharide, 0.05-0.15g hyaluronic acid, and 0.006g ginsenoside Rg3.

[0086] Example 2: Characterization of the physical and chemical properties of Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3

[0087] 1. Optical morphology characterization

[0088] The surface morphology and dimensions of the microneedles prepared in Preparation Example 1 were examined using an optical microscope. Heavy metal atoms were spray-coated onto the surface of the microneedles prepared in Preparation Example 1 under vacuum conditions, and the three-dimensional morphology of the microneedles was observed using a scanning electron microscope. To clarify the distribution of the drug within the microneedles and their three-dimensional structure, 1 mg of fluorescein isothiocyanate (FITC) was dissolved in 15 ml of water as a fluorescent probe. 3 ml of this solution was then used as a solvent to disperse bletilla striata polysaccharide, hyaluronic acid, and ginsenoside Rg3. Ginsenoside Rg3-loaded bletilla striata polysaccharide microneedles (FITC-Rg3-MNs) were prepared according to the method described in Preparation Example 1, and the microneedle morphology was imaged using a confocal laser scanning microscope (CLSM).

[0089] Figure 4 shows the microneedle morphology, which, from left to right, is captured using optical microscopy, scanning electron microscopy, and fluorescence microscopy. Figure 4 shows that the microneedles prepared in this invention are approximately 900 μm tall, with a base width of 400 μm and a spacing of 700 μm between each microneedle.

[0090] 2. Mechanical properties test

[0091] The mechanical properties of the microneedles prepared in Preparation Example 1 were characterized using a universal testing machine. A microneedle patch containing a 5×5 array was placed in the center of a stainless steel plate with double-sided tape, with the microneedle tips facing upward. The distance between the microneedle tips and the sensor was set at 0.5 cm. The sensor parameters were then adjusted to move perpendicularly toward the stainless steel plate at a constant speed of 0.5 mm / min. The sensor displacement after contact with the microneedle tips and the corresponding resistance to the microneedles were recorded.

[0092] Figure 5 shows the mechanical properties of the microneedles. As shown in Figure 5, no turning points are observed in the force-displacement curves, and the compressive force of the microneedles increases with displacement. When the displacement increases to 0.3 mm, the compressive force of a single needle exceeds 0.6 N. This demonstrates that the microneedles of the present invention have sufficient mechanical properties to penetrate the skin and deliver drugs to hair follicles.

[0093] 3. In vivo insertion performance test

[0094] The microneedle's ability to penetrate the skin was tested on C57BL / 6 mice. Prior to the experiment, the mouse's back hair was removed and the skin disinfected with alcohol. After the skin recovered for 5 minutes, the microneedles prepared in Preparation Example 1 were inserted into the mouse skin. After 5 minutes, the penetrated skin was excised, fixed with 4% paraformaldehyde for 48 hours, embedded in paraffin, and cut into 5μm slices. The slices were then stained with hematoxylin and eosin (H&E). Finally, the sections were observed under a light microscope.

[0095] HE staining results, shown in Figure 6, demonstrate that the microneedles of the present invention successfully penetrated the skin, a crucial requirement for in vivo pharmacodynamic studies. Notably, due to the inherent elasticity of the skin, the microneedles of the present invention penetrated to a depth of approximately 300 μm, confirming that the microneedles penetrated the stratum corneum and epidermis, achieving successful drug delivery.

[0096] 4. Skin recovery test

[0097] To evaluate skin irritation caused by microneedle insertion, the backs of healthy C57BL / 6 mice were shaved and the skin disinfected with 75% alcohol. After the skin recovered for 5 minutes, a microneedle prepared according to the method described in Preparation Example 1 was inserted into the back skin of the C57BL / 6 mice and held for 5 minutes before removal. The treated area was photographed within 1 hour after microneedle removal to observe skin recovery.

[0098] The ideal microneedle loaded with ginsenoside Rg3 should have excellent biocompatibility. The treated skin was photographed and recorded at 0min, 20min, 40min, and 60min after the microneedles were removed. As shown in Figure 7, after the microneedles were removed, the microneedle holes on the skin were more obvious, and the skin was slightly red. As time went on, the microneedle holes gradually disappeared, and the microneedle holes on the skin basically disappeared completely after 1 hour. During the entire observation period, there was no obvious irritation reaction on the skin (such as swelling and erythema, etc.). The results of the skin recovery test showed that the bletilla polysaccharide microneedle loaded with ginsenoside Rg3 of the present invention can penetrate the skin, and has good biocompatibility and fast recovery, which can increase the safety of frequent administration and the acceptability of patients.

[0099] 5. In vivo solubility test

[0100] Healthy C57BL / 6 mice were anesthetized with chloral hydrate, and most of the hair on their backs was removed. Depilatory cream was then used to remove the hair. After a 1-hour recovery period, microneedles prepared in Preparation Example 1 were inserted into the skin and removed at intervals of 15 and 25 minutes. The resulting microneedles were imaged using an optical microscope to determine the remaining tip length.

[0101] Ideally, ginsenoside Rg3-loaded microneedles should dissolve rapidly upon insertion into the skin, subsequently releasing the drug. As shown in Figure 8 , the tips of the microneedles of the present invention become short and blunt upon insertion into the skin. The microneedles dissolve relatively quickly after insertion into the skin, almost completely within 25 minutes. Rapid dissolution of the microneedles can help reduce administration time, facilitate practical use, and lead to greater patient convenience and compliance.

[0102] 6. Determination of drug loading capacity of Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3

[0103] The tips of the microneedles prepared in Preparation Example 1 were scraped off with a scalpel and completely dissolved in a small amount of deionized water. An appropriate amount of methanol was added to break the emulsion, followed by filtration and evaluation of the drug loading of ginsenoside Rg3 using high-performance liquid chromatography (HPLC) using a C18 column. The mobile phase consisted of acetonitrile and 0.1% phosphoric acid (50:50, v / v) at a flow rate of 1 mL / min. Ginsenoside Rg3 was detected at 203 nm.

[0104] The drug content of ginsenoside Rg3 per microneedle is 127.13 ± 0.67 μg / microneedle. Each microneedle is 900 μm tall, 400 μm wide, and 700 μm apart. The entire microneedle patch is a circular patch with a diameter of 1.5 cm.

[0105] 7. In vivo skin penetration test

[0106] C57BL / 6 mice were anesthetized and shaved, and a microneedle patch loaded with fluorescent FITC was inserted into the mouse skin, pressed for 5 minutes, and covered with tape for 30 minutes. The treated mice were then euthanized, and the treated skin was removed. The skin was scanned at different Z-axis heights using a confocal laser scanning microscope (CLSM) at an excitation wavelength of 488nm to visualize the insertion depth of the microneedles. Scans were performed every 25μm until no fluorescent signal was detected. Finally, after determining the maximum and minimum Z-axis heights scanned by the CLSM, images were obtained from the XY plane as the Z-axis height gradually increased. 3D reconstructed images were obtained by overlapping the XY plane images at different Z-axis heights.

[0107] The preparation method of the microneedle patch loaded with fluorescent FITC is the same as the method described in "1. Optical morphology characterization": 1 mg of fluorescein isothiocyanate (FITC) was dissolved in 15 ml of water as a fluorescent probe, and 3 ml of the solution was aspirated as a solvent to disperse the bletilla striata polysaccharide, hyaluronic acid and ginsenoside Rg3. The bletilla striata polysaccharide microneedles (FITC-Rg3-MNs) loaded with ginsenoside Rg3 were prepared according to the method described in Preparation Example 1.

[0108] FITC-loaded ginsenoside Rg3 microneedles were used to study the skin penetration ability of drugs in the microneedles. In order to determine the penetration distribution of microneedles in mouse skin, the present invention captured confocal images of fluorescence at different skin depths after the application of microneedles, from the appearance of fluorescence until the disappearance of fluorescence. As shown in Figure 9: The FITC green fluorescence depth in the microneedle is about 300 microns, which indicates that the microneedle penetrates the stratum corneum and epidermis. However, this result is smaller than the geometric height of the microneedle (~900 microns), which may be due to the deformation of the skin when the microneedle is forced to hit the skin surface. The reconstructed three-dimensional fluorescence image shows an inverted triangle shape from the stratum corneum to the epidermis, which indicates the drug release characteristics of the prepared microneedle in vivo and shows that the drug diffuses widely in the skin.

[0109] 8. In vitro drug permeability test

[0110] The microneedles prepared in Preparation Example 1 were subjected to in vitro transdermal drug delivery studies using a Franz diffusion cell. The back hair of healthy C57BL / 6 mice was shaved and the skin was disinfected with 75% alcohol. After the skin recovered for 1 hour, the mice were killed. After cleaning the depilated area, the moisture was absorbed with filter paper, and the microneedle patch was inserted into the skin for 5 minutes and then fixed with tape. The treated skin sample was then installed between the receptor chamber and the donor chamber of the Franz diffusion cell. PBS buffer (pH = 7.4) was filled into the receptor chamber as the receiving medium to eliminate all bubbles under the skin. Continuously stir with a magnetic stirrer at 300 rpm and maintain at 37±0.5°C in a circulating water bath. At specified time intervals, an appropriate amount of receiving fluid was removed from the receptor chamber, and an isothermal and equal volume of PBS buffer solution was added at the same time. Three parallel experiments were performed.

[0111] The in vitro transdermal diffusion rate of Rg3 in the microneedles was measured using a Franz diffusion cell. As shown in Figure 10, the Rg3 in the microneedles released the drug through the skin, reaching a drug penetration rate of 37.63% within 4 hours and 83.87% within 72 hours, indicating nearly complete drug release. These results demonstrate that the Rg3 microneedle tips can penetrate the skin's stratum corneum, facilitating transdermal drug delivery.

[0112] Example 3: Efficacy test of Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3

[0113] 1. Experimental methods

[0114] In vivo hair regeneration experiments were conducted using C57BL / 6 male mice. At 7 weeks of age, the mice had their back hair completely removed using animal clippers and depilatory cream. On the 1st, 4th, and 7th days after depilation, the mice were topically treated with the ginsenoside Rg3-loaded Bletilla striata polysaccharide microneedles (Bletilla striata polysaccharide microneedles loaded with Rg3), the ginsenoside Rg3-loaded Bletilla striata polysaccharide microneedles without Bletilla striata polysaccharide (Bletilla striata polysaccharide microneedles loaded with Rg3), the Bletilla striata polysaccharide microneedles without ginsenoside Rg3 (Bletilla striata polysaccharide microneedles), the blank microneedles without ginsenoside Rg3 and without Bletilla striata polysaccharide (blank microneedles), and the Rg3 solution. Within the first 10 seconds, the microneedle patch was pressed firmly to penetrate the epidermis, and gently pressed for 5 minutes to allow the patch to absorb the liquid. The patch base was removed 4 hours after insertion into the skin, leaving the microneedles to settle in the skin for further sustained drug release. Shaved mice without any treatment were used as blank control group (blank).

[0115] Hair growth in each group of mice was observed starting on day 1, and continuing for 1, 5, 10, and 15 days. On day 8, H&E-stained sections were used to observe hair follicle activation in each group. Newly generated hair was also characterized on day 15.

[0116] Preparation of Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3 (Bletilla striata polysaccharide microneedles loaded with Rg3): 0.06g of Bletilla striata polysaccharide, 0.15g of hyaluronic acid, and 0.006g of ginsenoside Rg3 were dissolved and dispersed in 3mL of water to obtain a mixed solution. The mixed solution was placed in a mold and filled with the solution and centrifuged at 4000rpm for 20min. After scraping off the excess liquid, 1mL of a 20% polyvinyl alcohol aqueous solution was added to the mold and centrifuged at 4000rpm for 5min to form a microneedle base. After standing, the excess liquid was scraped off and the mold was dried at 30-37°C to obtain Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3. Mold dimensions: 1.5cm diameter circular mold, individual microneedles 900μm high, base 400μm wide, and 700μm spacing between microneedles.

[0117] Bletilla striata polysaccharide microneedles without ginsenoside Rg3 (Bletilla striata polysaccharide microneedles): Dissolve 0.06g of Bletilla striata polysaccharide and 0.15g of hyaluronic acid in 3mL of water to obtain a mixed solution. Place the mixed solution in a mold and centrifuge at 4000rpm for 20min. After scraping off the excess liquid, add 1mL of 20% polyvinyl alcohol aqueous solution to the mold and centrifuge at 4000rpm for 5min to form a microneedle base. After standing, scrape off the excess liquid and dry at 30-37°C to obtain Bletilla striata polysaccharide microneedles without ginsenoside Rg3. Mold dimensions: 1.5cm diameter circular mold, individual microneedles are 900μm tall, base width is 400μm, and spacing between microneedles is 700μm.

[0118] Blank microneedles without ginsenoside Rg3 and Bletilla striata polysaccharide (blank microneedles): Dissolve 0.15g of hyaluronic acid in 3mL of water to obtain a solution. Place the solution in a mold and centrifuge at 4000rpm for 20min. After scraping off the excess liquid, add 1mL of 20% polyvinyl alcohol aqueous solution to the mold and centrifuge at 4000rpm for 5min to form a microneedle base. After standing, scrape off the excess liquid, and dry at 30-37°C to obtain blank microneedles. Mold dimensions: 1.5cm diameter circular mold, individual microneedles are 900μm tall, 400μm wide, and 700μm between each microneedle.

[0119] Microneedles loaded with ginsenoside Rg3 and free of Bletilla striata polysaccharide (Rg3-loaded microneedles without Bletilla striata polysaccharide): 0.15g of hyaluronic acid and 0.006g of ginsenoside Rg3 were dispersed in 3mL of water to obtain a mixed solution. The mixed solution was placed in a mold and filled with the solution and centrifuged at 4000rpm for 20min. After scraping off the excess liquid, 1mL of a 20% polyvinyl alcohol aqueous solution was added to the mold and centrifuged at 4000rpm for 5min to form a microneedle base. After standing, the excess liquid was scraped off and the mold was dried at 30-37°C to obtain Rg3-loaded microneedles without Bletilla striata polysaccharide. Mold dimensions: 1.5cm diameter circular mold, individual microneedles 900μm high, base 400μm wide, and 700μm spacing between microneedles.

[0120] Rg3 solution: Take an appropriate amount of Rg3 and dilute it with pure water to 130μg / ml.

[0121] For the Rg3 solution group, 1 ml of solvent was applied for each administration. For each microneedle group, one microneedle was applied for each administration. The microneedle dimensions were approximately 900 μm in height, 400 μm in base width, and 700 μm between each microneedle.

[0122] 2. Experimental results

[0123] The growth of new hair in mice after different drug administration methods over 15 days is shown in Figure 11. The activation of hair follicles in mice on day 8 is shown in the H&E sections of Figure 12. The growth of new hair in mice in each group was characterized, and the results are shown in Figure 13. Figure 13 shows, from left to right: time to skin darkening after hair removal (days); coverage of regenerated hair at day 15 (%); and density of regenerated hair at day 15 (roots / cm). 2 ); Skin thickening value (micrometer) at 15 days. From the results of Figures 11 to 13, we can see that:

[0124] As the number of doses increased, the hair follicles of mice in the Rg3 Bletilla polysaccharide microneedle group began to shift from the resting phase to the growth phase on average 5.5 days after administration. H&E sections confirmed that on the 8th day, most hair follicle cells had been activated and were in different growth phases. On the 10th day, the shaved area almost completely turned black. By the 15th day, the new hair was black and shiny, with a new hair coverage rate of 89.7%, covering most of the shaved area, and the new hair density could reach 1502.1±398.9 hairs / cm 2 , the skin thickness increased by 153.3 microns.

[0125] The average number of days for the skin to darken in the Rg3 microneedle group without Bletilla polysaccharide was 6.7 days. HE sections showed that on the 8th day, the hair follicle cells transitioned from the resting phase to the growth phase, with a very small number of hair follicle cells entering the growth phase. On the 10th day, some hair follicle cells entered the growth phase, and the skin showed regional darkening. Within 10-15 days, the vast majority of hair follicle cells entered the growth phase. On the 15th day, hair grew on some skin, with a new hair coverage rate of 83.9% and a new hair density of 1097.2±313.0 roots / cm 2 , the skin thickness increased by 108 microns.

[0126] The average duration of skin darkening in the Bletilla polysaccharide microneedle group was 8.9 days. HE sections showed that on the 8th day, hair follicle cells transitioned from the resting phase to the growing phase. On the 10th day, some hair follicle cells entered the growing phase, and the skin showed regional darkening. Within 10-15 days, most hair follicle cells entered the growing phase. On the 15th day, some short hairs grew on some skin, with a new hair coverage rate of 79.2% and a new hair density of 868.4±270.9 hairs / cm 2 , the skin thickness increased by 80 microns.

[0127] The average time it took for the skin in the blank microneedle group to darken was 11.2 days. Most hair follicle cells began to grow between 10 and 15 days. On the 15th day, some areas of the skin became darker and a few short hairs grew. The density of new hair was 825.5 ± 89.9 hairs / cm 2 The skin thickness increased by 80 microns. Blank microneedles and Bletilla striata polysaccharide microneedles had similar effects on the density of new hair and the increase in skin thickness in mice.

[0128] In the Rg3 solution group, some hair follicle cells were activated on average at 9 days. On the 10th day, only a small part of the skin darkened. On the 15th day, only short, downy hair grew. The new hair coverage rate was 62.9%, and the new hair density was 741.2 ± 256.2 roots / cm 2 , the skin thickness increased by 76 microns.

[0129] The skin of the blank group darkened on average on day 11.3, and only a small amount of downy hair grew on day 15. The new hair coverage rate was 54.2%, and the new hair density was 565.0 ± 400.6 hairs / cm 2 , the skin thickness increased by 33 microns.

[0130] By comparing the growth of new hair, the activation of hair follicles in HE sections and the related characteristics of new hair, it can be seen that the bletilla polysaccharide microneedles loaded with ginsenoside Rg3 can significantly accelerate the process of hair follicles transitioning from the resting phase to the growth phase, and can significantly increase the density of new hair and skin thickness. However, the effects of the bletilla polysaccharide-free microneedle group, the bletilla polysaccharide microneedle group, the blank microneedle group and the Rg3 solution group loaded with ginsenoside Rg3 on promoting hair growth are weaker.

[0131] Combining the above results with Figure 13C, the density of new hair in the Bletilla striata polysaccharide microneedle group increased by about 42.9 hairs / cm compared with the blank microneedle group. 2 The density of new hair in the Rg3 microneedle group without Bletilla striata polysaccharide increased by about 271.7 hairs / cm 2 Unexpectedly, the density of new hair in the Rg3 Bletilla striata polysaccharide microneedle group increased by 676.6 hairs / cm 2 , which was significantly higher than the sum of the Bletilla striata polysaccharide microneedle group and the Rg3 microneedle group without Bletilla striata polysaccharide (676.6>42.9+271.7).

[0132] In addition, as shown in Figure D in Figure 13, compared with the blank microneedle group, the skin thickness of the Bletilla striata polysaccharide microneedle group increased by about 0, and the skin thickness of the Rg3 microneedle group without Bletilla striata polysaccharide increased by 28 microns. Unexpectedly, the skin thickness of the Rg3 Bletilla striata polysaccharide microneedle group increased significantly by 73.3 microns, which was significantly higher than the sum of the Bletilla striata polysaccharide microneedle group and the Rg3 microneedle group without Bletilla striata polysaccharide (73.3>0+28).

[0133] Conclusion: When Bletilla striata polysaccharide and ginsenoside Rg3 are prepared into microneedles together for use, Bletilla striata polysaccharide can play an adjuvant-like role, enhancing the hair growth effect of ginsenoside Rg3 and exerting a synergistic effect in promoting hair growth.

[0134] In summary, the present invention provides a Bletilla striata polysaccharide microneedle loaded with ginsenoside Rg3, which can be used to achieve transdermal drug delivery for the treatment of hair loss. The Bletilla striata polysaccharide microneedle loaded with ginsenoside Rg3 is highly effective in treating hair loss. Compared with using ginsenoside Rg3 alone, Bletilla striata polysaccharide acts as an adjuvant in the treatment of hair loss, enhancing the hair growth effect of ginsenoside Rg3 and thereby exerting a synergistic and synergistic effect in promoting hair growth. The microneedles prepared by the present invention have promising application prospects in the treatment of hair loss.

[0135] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A bletilla striata polysaccharide microneedle loaded with ginsenoside Rg3, characterized in that: The microneedle is prepared from raw materials in the following weight ratio: 1-6 parts of Bletilla striata polysaccharide, 5-15 parts of hyaluronic acid, 0.1-1 parts of ginsenoside Rg3, and 15-20 parts of polyvinyl alcohol.

2. The bletilla striata polysaccharide microneedle loaded with ginsenoside Rg3 according to claim 1, characterized in that: The microneedle is prepared from raw materials in the following weight ratio: 1-6 parts of Bletilla striata polysaccharide, 5-15 parts of hyaluronic acid, 0.6 parts of ginsenoside Rg3, and 20 parts of polyvinyl alcohol.

3. The bletilla striata polysaccharide microneedle loaded with ginsenoside Rg3 according to claim 2, characterized in that: The microneedle is prepared from raw materials in the following weight ratio: 6 parts of Bletilla striata polysaccharide, 15 parts of hyaluronic acid, 0.6 parts of ginsenoside Rg3, and 20 parts of polyvinyl alcohol.

4. The bletilla striata polysaccharide microneedle loaded with ginsenoside Rg3 according to any one of claims 1 to 3, characterized in that: The height of a single microneedle in the microneedle is 900 μm, the width of the base is 400 μm, and the spacing between each microneedle is 700 μm.

5. The bletilla striata polysaccharide microneedle loaded with ginsenoside Rg3 according to any one of claims 1 to 3, characterized in that: The bletilla striata polysaccharide is composed of mannose and glucose in a molar mass ratio of 1.99 to 2.95:1, and the weight average molecular weight of the bletilla striata polysaccharide is 1.10×10 5 ~3.99×10 5 g / mol.

6. The bletilla striata polysaccharide microneedle loaded with ginsenoside Rg3 according to claim 5, characterized in that: The preparation method of the bletilla striata polysaccharide comprises the following steps: Take Bletilla striata powder, first extract it with anhydrous ethanol at 60-80°C for 1-3 times, then extract the residue with petroleum ether at 60-90°C for 1-3 times, then extract the residue with water at 70-80°C for 1-3 times, concentrate the obtained extract and deproteinize it, add 90-95% ethanol aqueous solution by volume to precipitate, wash the precipitate and dry it to obtain Bletilla striata polysaccharide.

7. The bletilla striata polysaccharide microneedle loaded with ginsenoside Rg3 according to claim 6, characterized in that: The ratio of the Bletilla striata powder to anhydrous ethanol is 1 g: 5-10 mL; And / or, the ratio of the bletilla striata powder to petroleum ether is 1 g: 5-10 mL; And / or, the ratio of the Bletilla striata powder to water is 1 g: 40-50 mL; and / or, when extracting with anhydrous ethanol, each extraction time is 2 to 5 hours; and / or, when petroleum ether is used for extraction, each extraction time is 2 to 5 hours; and / or, when extracting with water, each extraction time is 2 to 5 hours; And / or, the deproteinization is carried out by using the Sevage method; And / or, the precipitate is washed with anhydrous ethanol, acetone and ether in sequence.

8. A method for preparing the bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3 according to any one of claims 1 to 7, characterized in that: The method comprises the following steps: (1) dispersing bletilla striata polysaccharide, hyaluronic acid, and ginsenoside Rg3 in water to obtain a mixed solution; (2) filling the mixed solution prepared in step (1) into a mold and centrifuging; (3) dissolving polyvinyl alcohol in water to obtain a polyvinyl alcohol solution; (4) adding the polyvinyl alcohol solution obtained in step (3) into the mold of step (2) and centrifuging; (5) Drying to obtain the Bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3.

9. The method according to claim 8, characterized in that: In step (1), the concentration of Bletilla striata polysaccharide in the mixed solution is 0.003-0.02 g / mL; And / or, in step (2), the height of a single microneedle in the mold is 900 μm, the width of the base is 400 μm, and the spacing between each microneedle is 700 μm; And / or, in step (2), the centrifugation is performed at 4000 rpm for 20 min; And / or, in step (3), the mass percentage concentration of polyvinyl alcohol in the polyvinyl alcohol solution is 20%; And / or, in step (3), the volume ratio of the polyvinyl alcohol solution to the mixed solution of step (1) is (3-5):1; And / or, in step (4), the centrifugation is performed at 4000 rpm for 5 min; And / or, in step (5), the drying is performed at 30-37°C.

10. Use of the bletilla striata polysaccharide microneedles loaded with ginsenoside Rg3 according to any one of claims 1 to 7 in the preparation of a medicament for treating hair loss.

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