Cacumen biotae vesicle as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses a cacumen biotae vesicle as well as a preparation method and application thereof. The preparation method comprises the following steps: soaking crushed and sieved cacumen biotae in an ethanol solution for 10 days, stirring and filtering to obtain filtrate; carrying out segmented centrifugation for three times, and taking supernate; filtering through a filter membrane, performing ultrafiltration through an ultrafiltration tube, and collecting permeate; and performing ultrafiltration through an ultrafiltration tube, and retaining intercepted components to obtain the cacumen biotae vesicles. The invention discloses a cacumen biotae vesicle as well as a preparation method and application thereof, the cacumen biotae vesicle is used as a carrier, so that the transdermal efficiency of a medicine and the bioavailability of cacumen biotae are improved, and a better choice is provided for clinical treatment of diseases.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a biota leaf vesicle and a preparation method and application thereof. BACKGROUND

[0002] As a traditional Chinese medicinal material, biota leaf has important application value in the field of biological medicine, and its active ingredients show good potential in promoting hair growth, anti-inflammatory, anti-tumor and the like. However, the crude extract obtained by the traditional extraction process (such as decoction, alcohol extraction and the like) of biota leaf has many limitations, for example, poor stability of active ingredients, low transdermal absorption efficiency, low bioavailability and the like, which leads to the need for a high dose and a long course of treatment in clinical application, and seriously affects the treatment effect and patient compliance.

[0003] In recent years, extracellular vesicles of plant origin have attracted widespread attention due to their unique structure and function. Such vesicles usually have a lipid bilayer membrane structure, can encapsulate active ingredients of the plant itself, and when used as a drug carrier, can improve the stability and delivery efficiency of the drug, enhance the ability to cross biological barriers, and thus improve the bioavailability. In addition, plant vesicles also have the advantages of abundant source, relatively simple preparation process, good biocompatibility and low immunogenicity, and show great application prospects in the field of drug delivery and disease treatment.

[0004] In view of the application status of biota leaf, how to extract and prepare vesicles with high drug loading capacity and excellent biological activity from biota leaf by using modern biological technology to solve the defects of traditional extracts has become a research hotspot in the field of biological medicine. Developing a new type of biota leaf vesicle which can not only retain the medicinal activity of biota leaf itself, but also improve the transdermal efficiency and bioavailability of related drugs as a drug carrier, has important significance for promoting the application of biota leaf in clinical treatment. SUMMARY

[0005] The application aims to provide a biota leaf vesicle and a preparation method and application thereof, which improves the transdermal efficiency of the drug and the bioavailability of biota leaf as a carrier, and provides a better choice for the clinical treatment of androgenetic alopecia.

[0006] To solve the above technical problems, the technical scheme adopted by the application is as follows:

[0007] A preparation method of a biota leaf vesicle, comprising the following steps:

[0008] S1, washing and drying fresh biota leaves, crushing and sieving, soaking in an ethanol solution with a mass fraction of 50% for 3-60 days, stirring daily, filtering, and obtaining a filtrate;

[0009] S2, subjecting the filtrate obtained in S1 to three times of segmented centrifugation, and taking the supernatant;

[0010] S3, filtering the supernatant obtained in S2 through a filter membrane, and then performing ultrafiltration through a 100kD ultrafiltration tube, and collecting the permeate;

[0011] S4, performing ultrafiltration through a 10kD ultrafiltration tube on the permeate obtained in S3, and retaining the intercepted components to obtain the Platycladus orientalis vesicle.

[0012] Preferably, in S2, the three-stage centrifugation is specifically: sequentially performing centrifugation at 1000g for 20-60min, centrifugation at 4000g for 20-60min, and centrifugation at 10000g for 20-60min.

[0013] Preferably, in S3, after filtering the supernatant obtained in S2 through a 0.22-0.45μm filter membrane, performing ultrafiltration through a 100kD ultrafiltration tube by centrifugation at 4000g for 10-40min, and collecting the permeate.

[0014] Preferably, in S4, performing ultrafiltration through a 10kD ultrafiltration tube on the permeate obtained in S3 by centrifugation at 4000g for 10-40min, and retaining the intercepted components to obtain the Platycladus orientalis vesicle.

[0015] The application further provides a Platycladus orientalis vesicle prepared by the preparation method.

[0016] The application further provides an application of the Platycladus orientalis vesicle in the preparation of a hair growth promoting preparation.

[0017] The application further provides a hair growth promoting preparation, which comprises the Platycladus orientalis vesicle.

[0018] The application further provides a preparation method of the hair growth promoting preparation, which comprises the following steps:

[0019] dissolving the modulator in an anhydrous ethanol solution, mixing the modulator with the Platycladus orientalis vesicle of claim 5, incubating, and then washing 1-5 times through a 10kD ultrafiltration tube to obtain modulator-Platycladus orientalis vesicle.

[0020] Preferably, dissolving the modulator in an anhydrous ethanol solution, mixing the modulator with the Platycladus orientalis vesicle of claim 5, incubating at 37℃ under the condition of 200rpm for 10-60min, performing centrifugation at 4000g for 10-30min through a 10kD ultrafiltration tube, and washing 1-5 times to obtain modulator-Platycladus orientalis vesicle.

[0021] Preferably, the modulator comprises one of the following: 5α-reductase inhibitor, external antihypertensive agent / potassium channel opener, aldosterone receptor antagonist, non-steroidal pure antiandrogen, antifungal / steroid synthesis inhibitor, prostaglandin analogue, calcineurin inhibitor (macrolide immunosuppressant), JAK inhibitor, and glucocorticoid.

[0022] Preferably, the modulator comprises one of finasteride, dutasteride, minoxidil, spironolactone, bicalutamide, ketoconazole, bimatoprost, latanoprost, tacrolimus, tofacitinib, baricitinib, prednisone, methylprednisolone, halometasone, mometasone furoate, ruxolitinib.

[0023] The application also provides application of the Platycladus orientalis vesicle in preparation of an anti-tumor drug.

[0024] The application also provides an anti-tumor drug composition, wherein the drug composition comprises the Platycladus orientalis vesicle as a drug carrier.

[0025] Compared with the prior art, the application has the following advantages and technical effects:

[0026] The application discloses a Platycladus orientalis vesicle and a preparation method and application thereof. The Platycladus orientalis vesicle (PcNs) can remove excess active oxygen induced by H2O2 in HaCaT cells in a concentration-dependent manner, significantly reduces the cell oxidative stress state, can inhibit the release of inflammatory factor TNF-α by the cells, and can up-regulate the expression of anti-inflammatory factor IL-10, effectively improves the inflammation and oxidative damage of the hair follicle microenvironment, and in-vivo experiments further prove that the PcNs can promote the transdermal absorption of drugs, and the fluorescence signal can still be detected 48-60 hours after local administration in the high-concentration group, indicating that the PcNs can not only break through the skin barrier, but also can be continuously released in the local skin, thereby prolonging the drug action time and the local concentration. The Platycladus orientalis vesicle as the carrier improves the transdermal efficiency of the drug and the bioavailability of the Platycladus orientalis, and provides a better choice for the clinical treatment of androgenetic alopecia.

[0027] The technical scheme of the application is further described in detail below with reference to the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The scale is 100 microns for the ROS detection results of the HaCaT cells in Example 1;

[0029] Figure 2 The PcNs are Platycladus orientalis vesicles, and the ROS is reactive oxygen species. Figure 2 A in the figure is a histogram detected by a flow cytometer, Figure 2 B in the figure is the mean fluorescence intensity.

[0030] Figure 3 The PcNs are Platycladus orientalis vesicles, and the ROS is reactive oxygen species. Figure 3 A in the figure is a histogram detected by a flow cytometer, Figure 3 B in the figure is the mean fluorescence intensity.

[0031] Figure 4 Figure 6 is the cumulative penetration curve of the cypress leaf vesicles PcNs in Example 1, wherein A is the cumulative penetration curve, Figure 4 Figure 4 Figure 7 is the skin retention of the cypress leaf vesicles PcNs in Example 1, wherein B is the skin retention;

[0032] Figure 5 Figure 8 is the dynamic change of the DiD-PcN transdermal penetration in Example 1 detected by small animal live imaging;

[0033] Figure 6 Figure 9 is the DiD fluorescence signal analysis result in Example 1;

[0034] Figure 7 Figure 7 Figure 10 is the in vivo skin penetration test result of the cypress leaf vesicles PcNs in Example 1, wherein A is the fluorescence map of DiD in the skin, and the scale is 100 μm, Figure 7

[0035] Figure 8 Figure 11 is the hair growth of mice;

[0036] Figure 9 Figure 12 is the hair growth score and the new hair coverage rate after hair loss of mice in each group, wherein A is the hair growth score of mice in each group, Figure 9 Figure 9

[0037] Figure 10 Figure 13 is the weight of the regrown hair of mice in each group;

[0038] Figure 11 Figure 14 is the H&E staining of the back skin of mice in each group, and the red arrow indicates the hair follicle, and the scale is 100 μm;

[0039] Figure 12 Figure 15 is the quantitative analysis result of the skin thickness and hair bulb diameter of mice in each group, wherein A is the skin thickness statistical result, Figure 12 Figure 12

[0040] Figure 13 Figure 16 is the Ki67 immunofluorescence staining of the skin tissue of different treatment groups, and the scale is 50 μm;

[0041] Figure 14 Figure 17 is the Ki67 fluorescence intensity of the skin tissue of different treatment groups. DETAILED DESCRIPTION

[0042] The technical solutions of the present application are further illustrated by the accompanying drawings and examples.​​​​​​​

[0043] 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 application belongs.

[0044] In the present application, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in the art, which can be purchased through commercial channels.

[0045] Example 1

[0046] A preparation method of a Platycladus orientalis L. capsule, comprising the following steps:

[0047] S1, wash and dry fresh Platycladus orientalis L. leaves, crush and sieve, soak in a 50% ethanol solution for 10 days, stir daily, filter to obtain a filtrate;

[0048] S2, centrifuge the filtrate obtained in S1 at 1000g for 40 min, at 4000g for 40 min, and at 10000g for 40 min, and take the supernatant;

[0049] S3, filter the supernatant obtained in S2 through a 0.22μm filter membrane, and then perform ultrafiltration on the filtrate by using a 100kD ultrafiltration tube at 4000g for 30 min, and collect the permeate;

[0050] S4, centrifuge the permeate obtained in S3 by using a 10kD ultrafiltration tube at 4000g for 30 min, and retain the cut-off component to obtain the Platycladus orientalis L. capsule PcNs.

[0051] Example 2

[0052] A preparation method of a finasteride-Platycladus orientalis L. capsule, comprising the following steps:

[0053] Dissolve finasteride FIN in anhydrous ethanol solution, mix with the Platycladus orientalis L. capsule PcNs obtained in Example 1, incubate at 37℃ under the condition of 200rpm for 60 min, centrifuge at 10kD ultrafiltration tube at 4000g for 20 min, and wash 3 times to obtain the finasteride-Platycladus orientalis L. capsule FIN-PcNs.

[0054] The Platycladus orientalis L. capsule PcNs provided in Example 1 and the finasteride-Platycladus orientalis L. capsule FIN-PcNs provided in Example 2 are verified for effects through the following tests.

[0055] 1. Detection of the antioxidant activity of the Platycladus orientalis L. capsule PcNs

[0056] (1) HaCaT cells are seeded in a 96-well plate at a density of 5×10 4The cells were inoculated in 48-well plates, and when the cells grew to about 60%, different concentrations of PcNs (50 μg / mL, 150 μg / mL, 250 μg / mL) were added for 12 h, and then H2O2 was added for 24 h to induce ROS production.

[0057] (2) Discard the original culture medium, wash 3 times with PBS. Dilute the DCFH-DA mother liquor to 10 μM with serum-free DMEM medium, add 200 μL per well, and incubate at 37°C in the dark for 45 min.

[0058] (3) Discard the supernatant, wash 3 times with PBS, observe under a fluorescence inverted microscope and take pictures, and the results are shown in Figure 1

[0059] (4) After washing the cells with PBS, add trypsin for digestion for 9 min, add an equal volume of DMEM complete medium to terminate digestion, then blow the cells until all the cells are detached from the bottom of the culture dish. Transfer the cell suspension blown off to a 1.5 mL centrifuge tube, centrifuge at 1800 rpm for 5 min. Take out the cells from the centrifuge, discard the supernatant, and wash twice with pre-cooled PBS. Finally, resuspend the cells in 250 μL PBS, filter through a 200-mesh gauze, and transfer to a flow tube, and detect and analyze by flow cytometry, and the results are shown in Figure 2

[0060] As can be seen from Figure 1 , compared with the PBS group, the DCF fluorescence in the H2O2 group was significantly enhanced, indicating that the HaCaT cells were in an oxidative stress state due to excessive ROS production, and the fluorescence intensity of the cells pretreated with the PcNs in Example 1 decreased in a concentration-dependent manner.

[0061] As can be seen from Figure 2 , compared with the PBS group, the H2O2 group was significantly right-shifted, indicating that a large amount of ROS was produced in the cells after H2O2 induced HaCaT cells, and the cells pretreated with 250 μg / mL PcNs were significantly left-shifted compared with the H2O2 group, indicating that most of the ROS produced by oxidation in the cells was removed. Statistical analysis of the average fluorescence intensity showed that PcNs could significantly remove excess ROS in the cells, and there was no significant difference compared with the PBS group. It was shown that PcNs could improve the microenvironment of hair follicles by removing ROS and reducing oxidative damage.

[0062] 2. Anti-inflammatory activity detection:

[0063] (1) RAW264.7 cells were inoculated in 96-well plates, and when the cells grew to the logarithmic growth phase, the culture medium was discarded, and different concentrations of the sample to be tested were added according to the experimental design for 6 h.

[0064] ​​(2) Take 10 μL LPS and dilute to 1 mL, then take 100 μL of the 0.1 mg / mL LPS solution after dilution and continue to dilute to 1 mL to obtain a 10 μg / mL LPS solution. Add 2 μL LPS to the well plate and continue to incubate for 12 h.

[0065] (3) Collect the cell supernatant and detect TNF-α and IL-10 according to the product manual of the ELISA kit. Perform double-wavelength measurement and reading in the enzyme label instrument. Subtract the wavelength 570 nm absorbance from the wavelength 450 nm absorbance, and calculate the concentration of each well according to the standard curve. The results are shown in Figure 3

[0066] As can be seen from Figure 3 , after 12 h of LPS stimulation, the TNF-α level in the cell supernatant of Raw264.7 cells was significantly increased, while the PcNs pretreatment effectively inhibited the expression of TNF-α and up-regulated the level of anti-inflammatory factor IL-10, reducing the release of inflammatory factors. The above data prove that PcNs can significantly regulate the inflammatory cytokines produced by LPS-stimulated Raw264.7 cells.

[0067] 3. In vitro skin penetration of Platycladus orientalis vesicles PcNs

[0068] (1) In order to label PcNs, lipophilic fluorescent dye DiD was used for staining treatment. DiD (400X) was mixed with staining enhancer (400X) uniformly, added to PcNs, and incubated at 37°C for 30 min in the dark, and free DiD was removed by ultrafiltration to obtain labeled PcNs solution (DiD-PcN).

[0069] Preparation of control solution DiD-PBS solution: Take an appropriate amount of DiD (400X) and mix it uniformly with staining enhancer (400X), add it to PBS solution and mix well, incubate at 37°C for 30 min in the dark, and store at 4°C for standby.

[0070] (2) Fresh pig abdominal skin was selected, washed clean with normal saline, and surgical knife was used to remove residual subcutaneous fat and impurities, and then washed clean with normal saline. The pig skin was cut into round pieces of appropriate size for diffusion pool with scissors, and the surface moisture of the pig skin was absorbed with kitchen paper.

[0071] ​(3) The treated pigskin was fixed in the Franz diffusion cell with the stratum corneum facing up and the dermis facing down, ensuring the tightness of the skin to the donor and receiver cells. About 3 mL of normal saline was added to the receiver cell as the receiving medium, which could completely immerse the skin. The diffusion cell was placed in a constant temperature water bath, and the temperature was set to 32 ± 1 ℃ to simulate the temperature of human skin. 0.5 mL of DiD-PcN and DiD-PBS solution was added to the donor cell, respectively, and the contact area was 0.785 cm 2 , and the rotation speed was set to 280 rpm.

[0072] (4) The constant temperature water bath and magnetic stirrer were started, and 100 μL of sample was taken at 5, 10, 20 min, 1, 2, 3, 6, 8, 10, 12 h after drug administration, and immediately replenished with the same volume of fresh receiving medium to maintain the constant volume of the receiving cell.

[0073] (5) DiD dye was diluted with normal saline solution to prepare DiD standard solutions with concentrations of 0.313, 0.625, 1.250, 2.500, and 5.000 μM, respectively. 50 μL of the collected sample was taken into a black opaque 96-well plate, and the multifunctional enzyme labeler was used to measure the sample absorbance at 680 nm. The standard curve was drawn and the sample concentration was calculated. The cumulative transdermal amount of drug per unit area (Q) was calculated according to the following formula.

[0074]

[0075] , wherein Q i represents the cumulative transdermal amount of drug per unit area at the i th time; C i represents the drug concentration of the receiving medium at the i th time; V represents the volume of the receiving cell; V i represents the sampling volume; and A represents the contact area of drug administration.

[0076] (6) After the in vitro percutaneous penetration experiment, the pigskin was taken out of the diffusion cell, the skin surface was washed with normal saline, and the surface moisture was absorbed with kitchen paper. Then the pigskin was cut into pieces with scissors, 1 mL of methanol solution was added, and ultrasonic was performed for 15 min. After centrifugation at 2000 rpm for 5 min, the supernatant was taken to determine the drug content.

[0077] The standard solution was prepared by diluting DiD dye with methanol solution to prepare DiD standard solutions with concentrations of 0.313, 0.625, 1.250, 2.500, and 5.000 μM, respectively. 50 μL of the standard solution and the supernatant were taken into a black opaque 96-well plate, and the multifunctional enzyme labeler was used to measure the sample absorbance at 680 nm. The standard curve was drawn and the drug skin retention was calculated, and the results are shown in Figure 4

[0078] ​Depend on Figure 4 The results showed that the cumulative permeation of DiD-PBS solution into the skin was very low, only about one-third that of the DiD-PcN group. This indicates that DiD has difficulty penetrating the skin barrier in PBS solution, resulting in only trace amounts of DiD entering the skin. DiD-PcN exhibited a higher cumulative permeation, possibly because the PcN solution can utilize the ethanol component in the solution to dissolve lipid components in the stratum corneum, enhancing skin lipid fluidity and disrupting the intercellular lipid arrangement to create channels for easy drug penetration. It can also penetrate the skin through the unique properties of vesicles via skin appendages such as hair follicles and sweat glands. Furthermore, the skin retention of DiD in PBS solution was very low, while PcNs could carry DiD and remain in the skin, with an accumulation approximately six times that of the DiD-PBS group. These results demonstrate that PcNs can not only penetrate the skin barrier to enter the skin but also remain locally within the skin and continuously release it.

[0079] 4. In vivo and skin penetration of Platycladus orientalis leaf vesicle PcNs

[0080] (1) After anesthetizing the mouse, use a small animal hair removal tool to shave the hair off the back of the mouse. Then, apply hair removal cream evenly with a cotton swab in the direction of hair growth, completely cover the hair removal area and leave it for 4 minutes. Then, wipe off the hair against the direction of hair growth.

[0081] (2) One day after the hair loss in mice, 0.2 mL of DiD-PBS and DiD-PcN (PcN concentrations of 0.75, 1.5, and 3.0 mg / mL) solutions were applied to the hair-removed areas on the back. In vivo imaging was then performed at 6, 12, 24, 36, 48, and 60 hours. The results are shown in the figure. Figures 5-6 .

[0082] (3) The retention behavior of DiD-PcN on the skin was observed using confocal microscopy. DiD-PcN and 0.2 mL of DiD-PBS solution were applied to the back of mice. Twelve hours after administration, the mice were euthanized by cervical dislocation. The skin was rinsed with physiological saline and dried with kitchen paper. The skin of the experimental area was then gently separated with scissors, laid flat in aluminum foil to protect it from light, and fixed in paraformaldehyde fixative for at least 24 hours. Frozen sections were prepared, observed, and photographed under a confocal microscope. The results are shown below. Figure 7 .

[0083] Depend on Figure 5 It was found that in the DiD-PBS group, only a small amount of DiD was visible on the backs of mice 6 hours after application; it not only failed to penetrate the skin but was also rapidly metabolized and cleared. In contrast, all concentrations of PcNs solution in the experimental groups effectively promoted the absorption of DiD on the mouse skin, increasing absorption by more than three times. Figure 6It can be seen that the high concentration group of PcNs can still be detected for DiD fluorescence signal 2 days after local administration, indicating that the PcNs solution can significantly prolong the retention of drugs in the local skin and slowly release to play a role in local long-acting treatment.

[0084] By Figure 7 It can be seen that DiD can only stay in the epidermis in a small amount in PBS solution, and the skin fluorescence intensity is low, about 21 AU. In comparison, DiD can penetrate into the whole layer of skin and subcutaneous tissue in the PcNs solution, and its fluorescence intensity is also significantly higher than that of the control group, about 4.8 times that of the DiD-PBS group, indicating that the nanovesicle exhibits superior skin penetration capacity. This is consistent with the above-mentioned in vitro percutaneous penetration research results of DiD-PcN, which once again proves that the percutaneous delivery system based on biota capsule has great potential for local treatment of transdermal drug delivery.

[0085] 5. Evaluation of anti-androgen alopecia effect of finasteride-biota vesicle FIN-PcNs

[0086] (1) Preparation of 0.5% testosterone solution (TTE): weigh 1.0 g of testosterone raw material powder, add 108 mL of anhydrous ethanol, stir to dissolve the powder, then add 52 mL of propylene glycol and 40 mL of ultrapure water, mix well to obtain 0.5% testosterone solution; preparation of minoxidil solution (MNX): weigh 0.25 g of minoxidil raw material powder, add 27 mL of anhydrous ethanol, vortex to dissolve the powder, then add 13 mL of propylene glycol and 10 mL of ultrapure water, mix well to obtain 0.5% minoxidil solution; preparation of finasteride solution (FIN): take an appropriate amount of finasteride powder, dissolve in 50% ethanol solution, and the finasteride content is the same as that of FIN-PcNs, i.e. FIN solution.

[0087] (2) Anesthetize 6-week-old C57BL / 6 mice with isoflurane, remove the hair on their backs with a hair remover, and expose the pink skin by removing the remaining hair with depilatory cream. Randomly divide them into 6 groups, 3 in each group, and start giving them the drugs according to the grouping shown in Table 1 every day from the next day.

[0088] Table 1: Mouse grouping and drug administration method

[0089]

[0090]

[0091] (3) During the experiment, the skin color and hair growth of the experimental area of the mice were observed every day, and photographs were taken before administration after the mice were depilated (day 1) and during administration (days 8, 12, 16, and 21), and the results are shown in Figure 8 .

[0092] By Figure 8It was found that, compared with the control group, the hair regeneration rate of AGA mice induced by testosterone was significantly delayed, with almost no pigmentation observed, hair follicles in the resting phase, and minimal hair growth. These results indicate that the AGA model was successfully established. During the treatment period, mice in each experimental group showed varying degrees of hair growth, with the FIN-PcN group showing the fastest hair regeneration and a slightly better growth-promoting effect than minoxidil. In the finasteride free solution group, only a small amount of pigmentation appeared on the 16th day, and the hair growth recovery effect was not ideal. This may be due to the thicker scalp keratin and the larger molecular weight of the drug, resulting in low effective transdermal absorption of free finasteride and easy metabolism, making it difficult to reach the hair follicles and accumulate sufficient local concentration to exert its effect. On day 12, all groups except the model group and the FIN group showed varying degrees of gray-black areas. On day 16, a large amount of melanin deposition was visible on the back skin of mice in the FIN-PcN group, with the color changing from gray-black to black and hair shafts growing densely. By day 21, the regenerated hair of mice in the FIN-PcN treatment group almost completely and densely covered the hair loss area, which was close to the hair growth rate and coverage of mice in the blank group, indicating that local application of FIN-PcN can significantly improve hair growth in AGA mice.

[0093] Hair growth was assessed according to the scoring criteria shown in Table 2, and the coverage rate of new hair growth was calculated using ImageJ software. The results are shown below. Figure 9 .

[0094] Table 2 Hair Scoring

[0095] Score Growth 1 Pink skin in depilated area 2 Grey skin in depilated area 3 Black skin in depilated area 4 Black skin in depilated area with little hair growth 5 Black skin in depilated area with partial hair growth 6 Almost complete hair growth in depilated area

[0096] Depend on Figure 9 Based on the hair score and hair area, it can be seen that the FIN-PcN group of mice had the fastest hair regeneration progress, which was better than the Model group and the FIN free solution group, showing a statistically significant difference.

[0097] At the end of the 21st day of the experiment, newly grown hair in the experimental area was shaved off from the root using a razor and collected. The hair was weighed using an analytical balance and the results were recorded. (See attached table). Figure 10 .

[0098] Depend on Figure 10It can be seen that the hair of the Model group is thin, with a hair weight of 15.1 mg, and the hair of the non-FIN free solution group is slightly better than that of the Model group, but the hair is thin and not long. The hair weight of the mice in the blank group is 110.2 mg, and the hair is hard and grows densely. The hair weight of the mice in the PcN group, the FIN-PcN group and the minoxidil group is 84.4, 100.4 and 84.7 mg respectively, and the hair regrowth amount is significantly higher than that of the Model group, the hair is hard and dense, and is close to the hair weight of the blank group. Among all the treatments, FIN-PcNs promote the hair regrowth of AGA mice most obviously. Notably, the hair weight of the FIN-PcNs group is increased by about 1.1 times compared with the free FIN group, and is slightly higher than the effect of the minoxidil group.

[0099] On the 21st day, the skin of the experimental area on the back of the mice was carefully separated with scissors, flattened and wrapped in tin foil paper, and then placed in a paraformaldehyde fixing solution for fixation. After paraffin sectioning, hematoxylin and eosin staining was performed, and then the results were photographed and analyzed under an optical microscope, as shown in Figure 11 and Figure 12 .

[0100] As can be seen from Figure 11 , the skin of the mice in the blank group has complete hair follicle structure, including hair papilla, sebaceous gland and other structures, the hair bulb part is obviously swollen and extends to the deep subcutaneous part, the number of hair follicles is large and the hair shaft is dark, and most of the hair follicles are in the hair growth phase. The skin of the mice in the Model group and the non-steroidal free solution group has almost no complete hair follicle structure, the hair follicle atrophies and becomes small and is located in the dermis, there is lymphocyte, neutrophil and multinucleated macrophage infiltration in the dermis, fibroblasts surround the hair bulb, and the number of hair follicles is small and most of them are still in the resting phase and have not entered the growth phase. Compared with the Model group, the number and density of hair follicles in the skin of the mice in the PcN group, the FIN-PcN group and the minoxidil group are significantly increased, the hair shaft is dark, and there is no obvious inflammatory cell infiltration in the dermis, which eliminates the inhibitory effect of androgens on hair growth and enters the growth phase.

[0101] As can be seen from Figure 12 , the skin of the mice in the Model group is thin and the hair follicles are obviously miniaturized, and the skin thickness and hair bulb diameter increase after treatment, which has a significant difference from the Model group, indicating that FIN-PcNs treatment can reverse the damage of androgens to hair follicles and promote hair follicles to enter the growth phase.

[0102] Paraffin sections were subjected to immunofluorescence staining to detect the expression of Ki67 in the skin tissue, and the results are shown in Figures 13-14 .

[0103] Figures 13-14It is known that Ki67 is mainly expressed in cell proliferation, located in the nucleus, and is a marker for evaluating cell proliferation activity. Compared with the blank group, the expression of Ki67 in the hair follicle cells of the model group was significantly reduced, indicating that androgens inhibited the hair into the growth phase and stagnated in the prolonged resting phase. Notably, the expression of the cell proliferation marker Ki67 at the skin hair follicle of the FIN-PcN group of mice was the highest among the various treatment groups. This result further demonstrates that the hair growth of the FIN-PcN group of mice is vigorous, and the FIN-PcN group of mice has the best effect of promoting hair growth in AGA mice.

[0104] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application rather than limit them, although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that: it can still modify or equivalently replace the technical solutions of the present application, and these modifications or equivalent replacements also cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. A method for preparing a Plumbago zeylanica capsule, characterized by, Comprising the following steps: S1, washing and drying fresh Platycladus orientalis leaves, crushing and sieving, soaking in 50% ethanol solution for 3-60 days, stirring daily, filtering to obtain filtrate; S2, centrifuging the filtrate obtained in S1 three times, taking the supernatant; S3, filtering the supernatant obtained in S2 through a filter membrane, then ultrafiltering through a 100kD ultrafilter tube, collecting the permeate; S4, ultrafiltering the permeate obtained in S3 through a 10kD ultrafilter tube, retaining the cut-off component to obtain Platycladus orientalis vesicles.

2. The method of claim 1, wherein, In S2, the three times of segmental centrifugation are as follows: 1000g centrifugation for 20-60min, 4000g centrifugation for 20-60min, and 10000g centrifugation for 20-60min.

3. The preparation method according to claim 1, characterized in that, In S3, after filtering the supernatant obtained in S2 through a 0.22-0.45μm filter membrane, ultrafiltering through a 100kD ultrafilter tube at 4000g for 10-40min, the permeate is collected.

4. The preparation method according to claim 1, characterized in that, In S4, the permeate obtained in S3 is centrifuged at 4000g for 10-40min through a 10kD ultrafilter tube, and the cut-off component is retained to obtain Platycladus orientalis vesicles.

5. The Platycladus orientalis vesicles prepared by the preparation method of any one of claims 1-4.

6. The use of the Platycladus orientalis vesicles of claim 5 in the preparation of a preparation for promoting hair growth.

7. A hair growth promoting preparation, characterized by comprising The preparation comprises the Platycladus orientalis vesicles of claim 5.

8. A method of preparing the hair growth promoting preparation according to claim 7, characterized by, Comprising the following steps: The regulator is dissolved in anhydrous ethanol solution, mixed with the Platycladus orientalis vesicles of claim 5, incubated, washed 1-5 times through a 10kD ultrafilter tube to obtain regulator-Platycladus orientalis vesicles.

9. The use of the Platycladus orientalis vesicles of claim 5 in the preparation of an antitumor drug.

10. An antitumor drug composition, characterized in that, The pharmaceutical composition comprises the Platycladus orientalis vesicles of claim 5 as a drug carrier.

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