Vernonia anthelimenticas extract preparation for external use and its preparation method

Abstract

The present invention relates to the technical field of Vernonia anthelimenticas topical preparations, specifically gels and microemulsion gels prepared using Vernonia anthelimenticas extract as a drug substance, and methods for preparing the same. The gels and microemulsion gels prepared according to the present invention have a uniform appearance, good absorption, stable physicochemical properties, and stable contents of the main components of the preparations, vernodaline and 3,5-O-dicaffeoylquinic acid, as measured by HPLC. Furthermore, skin permeation studies were conducted to evaluate the intradermal retention of the main components of each topical preparation, confirming that Vernonia anthelimenticas microemulsion gel has high intradermal retention capacity and is highly suitable for topical administration to treat vitiligo.

Description

[Technical Field]

[0001] The present invention relates to the technical field of Vernonia anthelimentiica extract, and to topical preparations of gel and microemulsion gel prepared using Vernonia anthelimentiica extract as a drug substance. [Background technology]

[0002] Vitiligo is a localized or systemic lesion caused by a progressive decrease or loss of melanosome production due to a decline or loss of tyrosinase activity in melanocytes within the skin and hair follicles. Human skin is composed of the epidermis, dermis, and subcutaneous tissue. Melanin production occurs in the basal layer, the deepest part of the epidermis. For vitiligo, the basal layer of the skin is the target site of action for drug delivery systems, while the stratum corneum covering the skin surface is the main barrier to drug delivery system penetration. To repigment vitiligo areas, active substances must pass through the stratum corneum to reach the basal layer and thereby exert their therapeutic effects. Therefore, topical formulations are the optimal formulation for localized vitiligo treatment. Vernonia anthelmintica is an important medicinal herb used in the treatment of vitiligo in the Xinjiang Uyghur Autonomous Region. Its effects include promoting melanin production, increasing skin photosensitivity, improving microcirculation in vitiligo-affected skin, regulating the immune system, and providing trace element supplementation. The applicant conducted a chemical analysis of the main components of Vernonia anthelimentiica and found that the active ingredients that promote blackening and re-pigmentation are sesquiterpenes, quinic acids, and flavonoids. Based on this, the applicant targeted vernodalin and 3,5-O-dicaffeoylquinic acid for enrichment and optimized the Vernonia anthelimentiica extraction and purification process. The resulting Vernonia anthelimentiica extract contains 15-25% (w / w) vernodalin and 10-20% (w / w) 3,5-O-dicaffeoylquinic acid. HPLC analysis showed that commercially available Vernonia injection contains 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid, but does not contain vernodaline, whereas the Vernonia antherimentiica extract used in this invention contains a large amount of vernodaline and 3,5-O-dicaffeoylquinic acid. Summary of the Invention [Problem to be solved by the invention]

[0003] DISCLOSURE OF THE INVENTION The present invention provides a method for preparing topical formulations from Vernonia anthelimentiica extract. Specifically, it provides a method for preparing topical gel and microemulsion gel formulations using Vernonia anthelimentiica extract as a drug substance. The gel and microemulsion gel obtained by the method of the present invention have a uniform appearance, stable physicochemical properties, and stable contents of the main components, vernodaline and 3,5-O-dicaffeoylquinic acid, as measured by HPLC. Furthermore, skin permeation studies have evaluated the intradermal retention and cumulative permeation of the main components of the prepared topical formulations, such as gels and microemulsion gels. The results show that microemulsion gels have a high intradermal retention capacity and are highly suitable for topical administration. For the first time, the present invention has successfully prepared topical formulations with a uniform appearance and stable contents, using vernodaline and 3,5-O-dicaffeoylquinic acid as the main components. [Means for solving the problem]

[0004] In a first aspect of the present invention, there is provided an external preparation of Vernonia anthelimentiica extract, the external preparation of Vernonia anthelimentiica extract comprising vernodalin and 3,5-O-dicaffeoylquinic acid as active ingredients, in the form of a gel or microemulsion gel.

[0005] 1) The above gel formulation is per 100g, Carbomer 2020 1.0-1.5% and 13.5 to 40.0% water, Vernonia antherimentica extract 0.1-4.0% 5.0 to 15.0% of polyoxyethylene (40) hydrogenated castor oil or polyoxyethylene (35) hydrogenated castor oil, Diethylene glycol monoethyl ether 1.0 to 15.0% a neutralizing agent, The pH value of the gel is 4.3 to 5.5, However, the total content of each of the above ingredients must be 100% (for example, if it is less than 100%, add water to make up for it).

[0006] 2) The amount of the above microemulsion gel is, based on 100g, Vernonia antherimentica extract 0.1-4.0% Diethylene glycol monoethyl ether 1.0 to 15.0% 5.0 to 30.0% of polyoxyethylene (40) hydrogenated castor oil or polyoxyethylene (35) hydrogenated castor oil, 1.0 to 10.0% olive oil, almond oil, corn oil or soybean oil, 40.0 to 84.0% water, Carbomer 2020 1.0-1.5% However, the total content of each of the above ingredients must be 100% (for example, if it is less than 100%, add water to make up for it).

[0007] In a preferred embodiment of the present invention, the external preparation of the Vernonia anthelimntheica extract is a gel, and the neutralizing agent is a weakly alkaline salt solution having a pH value of 8.0 to 10.0, preferably a weakly alkaline salt solution having a pH value of 9.0, such as a weakly alkaline salt solution prepared from an alkali metal acid salt such as Na2HPO4, K2HPO4, NaHCO3, or a combination thereof, and having a pH value of preferably 9.0.

[0008] In a preferred embodiment of the present invention, the gel is prepared by a method comprising the following steps: 1) Add water to Carbomer 2020 to swell it and obtain a blank gel matrix. 2) Add polyoxyethylene (40) hydrogenated castor oil (or polyoxyethylene (35) hydrogenated castor oil) and diethylene glycol monoethyl ether to the Vernonia antherimentiica extract in this order, then add the gel matrix obtained in step 1), and stir to obtain a homogeneous mixture. 3) Adding a neutralizer to the mixture obtained in step 2) and stirring uniformly to adjust the pH value of the whole system to 4.3 to 5.5 to obtain a gel.

[0009] In a preferred embodiment of the present invention, the topical preparation of the Vernonia anthelimentiica extract is an oil-in-water (O / W) microemulsion gel, 1) pH is 4.0 to 6.5, for example 4.0 to 5.5, for example 4.5, 4.7 and 4.9; 2) viscosity in the range of 60 to 160 Pa.S, for example, in the range of 30 to 90 Pa.S, in the range of 70 to 80 Pa.S, in the range of 60 to 80 Pa.S, and in the range of 130 to 140 Pa.S; and / or 3) The particle size is 10nm to 100nm and the PDI (polymer dispersibility index) value is less than 0.50 as detected by a dynamic light scattering particle size analyzer. It has.

[0010] In a particularly preferred embodiment of the present invention, the optimal excipient formulation for the microemulsion gel is olive oil (1.0%, w / w), polyoxyethylene (40) hydrogenated castor oil (RH40) (22.0%, w / w), diethylene glycol monoethyl ether (Transcutol P) (11.0%, w / w), and water (66.0%, w / w), and the particle size of the microemulsion prepared with this formulation is 20.09±1.45 nm and the PDI value is 0.355±0.027.

[0011] In a preferred embodiment of the present invention, the microemulsion gel is prepared by a method comprising the following steps: 1) Adding and dissolving Vernonia antherimentiica extract to diethylene glycol monoethyl ether, adding polyoxyethylene (40) hydrogenated castor oil (or polyoxyethylene (35) hydrogenated castor oil) and olive oil (or almond oil, corn oil, or soybean oil) in that order, and then stirring to obtain a uniform mixture. 2) After the mixture obtained in step 1) is uniformly stirred, water is added drop by drop, the mixture is uniformly stirred, and ultrasonically treated to obtain a microemulsion. Carbomer 2020 is added at 1.0 to 1.5%, and the mixture is allowed to stand until the carbomer is completely swollen, and then the mixture is uniformly stirred to obtain a microemulsion gel.

[0012] The method for identifying the microemulsion prepared by the method of the present invention is as follows.

[0013] The emulsion prepared by this invention, even after centrifugation, remains clear and transparent with no observed layer separation phenomenon, remaining a yellowish-brown liquid. It can be diluted with distilled water; upon dilution to a certain extent, it exhibits a pale blue opalescence, and when irradiated with a laser pointer, a clear light path (the Tyndall effect specific to microemulsions) is generated, proving that the sample prepared by this invention is a microemulsion. The diffusion rates of methylene blue in the drug-containing microemulsion and the blank microemulsion are both significantly faster than that of Sudan Red, proving that the prepared microemulsion is an oil-in-water (O / W) type.

[0014] In one preferred embodiment of the present invention, after the microemulsion is prepared, microemulsion gelation is carried out by the following steps: A step of directly swelling Carbomer 2020 in the prepared microemulsion at a mixing ratio of Carbomer 2020:microemulsion of 1:100 (w / w) to 1.5:100 (w / w), and after sufficient swelling, uniformly stirring to obtain the Vernonia anthelimentiica microemulsion gel of the present invention.

[0015] In a preferred embodiment of the present invention, the Vernonia anthelimntheica extract is prepared by the method described in CN104825518A and contains 15-25 wt% vernodalin and 10-20 wt% 3,5-O-dicaffeoylquinic acid.

[0016] In a preferred embodiment of the present invention, the topical formulation of Vernonia anthelimentiica extract according to the present invention is prepared as a gel or microemulsion gel from Vernonia anthelimentiica extract and pharmaceutically acceptable excipients according to the following specific steps, based on a 100g basis: For preparing gels a. Add 1.0-1.5% Carbomer 2020 to 13.5-40.0% water at room temperature to swell the mixture to obtain a blank gel matrix. b. Add 5.0-15.0% of polyoxyethylene (40) hydrogenated castor oil or polyoxyethylene (35) hydrogenated castor oil and 1.0-15.0% of diethylene glycol monoethyl ether in this order to 0.1-4.0% of Vernonia antherimentica extract, then add the gel matrix obtained in step a, and stir to obtain a uniform mixture. c. A weakly alkaline salt solution having a pH value of 9.0 is prepared in advance using Na2HPO4, K2HPO4 or NaHCO3 as a neutralizing agent, and the obtained neutralizing agent is added to the mixture obtained in step b, and the mixture is stirred uniformly to adjust the pH value of the whole system to 4.3 to 5.5, thereby obtaining a gel. For preparation of microemulsion gel a. Add 0.1 to 4.0% by mass of Vernonia antherimentiica extract to 1.0 to 15.0% diethylene glycol monoethyl ether and dissolve it at room temperature, then add 5.0 to 30.0% polyoxyethylene (40) hydrogenated castor oil or polyoxyethylene (35) hydrogenated castor oil and 1.0 to 10.0% olive oil (or almond oil, corn oil, or soybean oil) in that order and stir to obtain a uniform mixture. b. The mixture obtained in step a is uniformly stirred with a magnetic stirrer at a speed of 300-600 r / min, and then 40.0-84.0% water is added dropwise, followed by equilibrium stirring for 10-30 minutes, ultrasonication for 5-20 minutes, addition of 1.0-1.5% carbomer 2020, and the mixture is allowed to stand until the carbomer is completely swollen, followed by uniform stirring to obtain a microemulsion gel.

[0017] The present invention further provides the use of an external preparation of Vernonia anthelimnteica extract obtained by the above method in the preparation of a medicament for treating skin depigmentation disorders such as vitiligo.

[0018] In the external preparation of Vernonia anthelimentiica extract of the present invention, the Vernonia anthelimentiica extract in this preparation is obtained by Chinese patent CN201510288742.3 (publication number CN104825518A) "Preparation method of phenolic acid moiety of Vernonia anthelimentiica and its anti-inflammatory use", and the specific operation is carried out according to the following steps:

[0019] Dried Vernonia antherimenteica herbal material was crushed in a traditional Chinese medicine rolling mill, weighed, and then reflux-extracted at a material-to-liquid ratio of 1:12. The extract was extracted twice using 80% ethanol at 75-80°C for 3 hours each time. The extract was then filtered and the combined filtrate was concentrated under reduced pressure at 50-60°C until the extract reached 21 g of herbal equivalent per 100 mL. The extract was then purified by column chromatography. The extraction was carried out as follows: the extract was dispersed in water and loaded (sample loading concentration was approximately 10 g of herbal equivalent per 100 mL of sample loading solution). Impurities were removed with three volumes of water, followed by elution with three volumes of 60% ethanol. The eluate was collected, concentrated under reduced pressure at 60°C, and then vacuum-dried at 50°C to obtain the Vernonia antherimenteica extract. HPLC analysis showed that Vernonia antherimentiica extract contained 15-25% vernodalin and 10-20% 3,5-O-dicaffeoylquinic acid.

[0020] In this invention, the applicant has used a Vernonia antheriminteica extract prepared in advance by the applicant as a drug substance to prepare topical gel and microemulsion gel formulations using modern pharmaceutical formulation technology. In terms of innovation, compared to currently clinically used Vernonia injections, vernodaline, the main ingredient with the highest content in the formulations of this invention, is not present in commercially available Vernonia injections (Figure 4). In terms of cutting-edge technology, the formulations of this invention significantly increase the intradermal retention of the main ingredient and reduce skin irritation (Figures 10-11). In terms of manufacturing method, the manufacturing method for the topical formulation of this invention is simple, easy to operate, easy to control parameters, low cost, and suitable for large-scale industrial production. Furthermore, topical formulations are easier to use than injections, have better compliance, and are suitable for a wider range of clinical applications. Furthermore, this work is highly meaningful for maximizing the development and utilization of medicinal resources in the Xinjiang Uygur Autonomous Region.

[0021] As a result of research, the inventor surprisingly discovered that the gel of the present invention cannot contain moisturizing agents commonly used in gels, such as polyols and fatty acid esters, which cause decomposition or other conversion of the main component in the extract, vernodaline, and that it cannot contain commonly used alkaline components, such as triethanolamine and sodium hydroxide, which cause alkaline hydrolysis and ring-opening of the main component in the extract, vernodaline. Furthermore, by using the ion-resistant gel matrix Carbomer 2020 and adjusting the pH of the water used in the gel in advance with Na2HPO4 or the like, the content of vernodaline, a sesquiterpene component in the extract (measured by HPLC, the content in the extract is 20% or more) can be stabilized.

[0022] As a result of research, the present inventors have surprisingly discovered that the microemulsion gel of the present invention can be prepared by directly dispersing carbomer 2020 in the microemulsion liquid prepared by the method of the present invention, allowing it to swell sufficiently, and then stirring uniformly. That is, this preparation method makes it possible to obtain a microemulsion gel that satisfies the pH and viscosity requirements for external use gels, for example, a pH value within the range of 4.0 to 6.5 (e.g., 5.0 to 6.5) and a viscosity within the range of 20 to 160 Pa.S (e.g., 30 to 90 Pa.S), without adjusting the pH with an organic or inorganic alkali.

[0023] The microemulsion and microemulsion gel of the present invention have a uniform and silky appearance and stable physicochemical properties, and the contents of its main components, vernodaline and isochlorogenic acid A, are stable within 30 days.

[0024] The differences between the present invention and the prior art are as follows.

[0025] In the present invention, when selecting a gel matrix material, consideration must be given to the stability of the formulation's main ingredient content, in addition to the usual system viscosity. Research has revealed the following: When using a neutral gel matrix such as CMC-Na, a gel with an appropriate viscosity can be prepared by adjusting the mass fraction of CMC-Na without the need for a neutralizer. However, after 48 hours, the gel becomes liquid and loses its semi-solid form. Further increasing the amount of CMC-Na causes the initial viscosity to become too high, resulting in loss of gel fluidity. Further screening of conventional matrix materials revealed that when Vernonia anthelimenticas extract is formulated into a gel or microemulsion gel using conventional carbomer matrices, such as Carbomer 940, Carbomer 971, or Carbomer 980, the content of the main ingredient, vernodaline, becomes unstable, with a content loss of more than 20%. Only Carbomer 2020 can simultaneously meet the viscosity and content requirements. Carbomer 2020 has long rheology, ion resistance, high transparency, and excellent thickening and suspending properties, making it suitable for surfactant and transparent gel systems. Carbomer 2020 has a strong and long pH viscosity range, with viscosity increasing at pH 4 and gradually decreasing above pH 9. However, compared to commonly used products such as Carbomer 940, Carbomer 971, and Carbomer 980, it maintains a high viscosity even at pH below 5.

[0026] When Carbomer 2020 was used as the matrix material, different neutralizing agents were considered to adjust the pH value and formulation viscosity.

[0027] (1) When NaOH was used as a neutralizing agent and the pH was adjusted to 5.5-7.0, a gel with appropriate viscosity was obtained, but the vernodaline content in the product decreased by about 20% within 10 days.

[0028] (2) When the pH was adjusted to 5.5-7.0 with triethanolamine, a gel with appropriate viscosity was obtained, but the vernodaline content in the product decreased by about 40% within 10 days.

[0029] (3) When the pH was adjusted to 5.5-7.0 with tromethamine, a gel with appropriate viscosity was obtained, but the vernodaline content in the product decreased by approximately 45% within 10 days.

[0030] (4) When the pH value of the system was adjusted to 4.3-5.5 using Na2HPO4, K2HPO4, or NaHCO3 as a neutralizing agent, a Vernonia anthelimina gel with a uniform appearance, stable properties, and controllable mass was obtained. When the dosage of the neutralizing agent was further increased (i.e., when the pH was greater than 5.5), the gel viscosity continued to decrease, and when the pH was greater than 6.0, it became a solution.

[0031] (5) After preparing a microemulsion using a microemulsion gel system, Carbomer 2020 was added directly to the microemulsion. By adjusting the amount of Carbomer used, a Vernonia anthelimentiica microemulsion gel was obtained that did not require a neutralizer, had an appropriate viscosity, and had a stable main component. [Brief explanation of the drawings]

[0032] [Figure 1] FIG. 1 shows the identity and morphology of the microemulsions prepared in this invention. [Figure 2] FIG. 2 shows the microscopic morphology and particle size examination of the microemulsion prepared in this invention. [Figure 3] Figure 3 shows the microemulsion gel prepared in accordance with the present invention and a conventional gel, as observed under a transmission electron microscope (left: 1% Vernonia anthelimentiae microemulsion gel; right: 1% Vernonia anthelimentiae gel. In the present invention, the particle size is much smaller than that of the conventional gel, the distribution is uniform, and no blocking or aggregation occurs; in contrast, the particle size of the conventional gel is approximately 500 nm, and some particles aggregate into clumps). [Figure 4]Figure 4 shows the differences in components between a commercially available Vernonia injection (Wuhu Yangyan Pharmaceutical Co., Ltd., Le Manxin, National Drug Approval Number Z20063652, Batch Number 190407) and the Vernonia anthelimentiica extract of the present invention. HPLC analysis revealed that the commercially available Vernonia injection contained 3,4-O-dicaffeoylquinic acid (Shanghai Junyou Biotechnology Co., Ltd., Batch Number 17121102), 3,5-O-dicaffeoylquinic acid (Beijing Hengyuan Qitian Chemical Technology Research Institute, Batch Number 16031611), and 4,5-O-dicaffeoylquinic acid (China Food and Drug Testing Institute, Batch Number 111894-201102), but no vernodaline. The Vernonia anthelimentiica extract of the present invention contained significant amounts of 3,5-O-dicaffeoylquinic acid and vernodaline. [Figure 5] FIG. 5 shows the results of observing the therapeutic effect of a 1% gel of the present invention on a mouse vitiligo model caused by hydroquinone. [Figure 6] FIG. 6 shows the evolution of the vitiligo model used in the present invention without drug intervention. [Figure 7] FIG. 7 shows the observation results of the therapeutic effect of Vernonia anthelimina microemulsion gel on a mouse vitiligo model induced by monobenzone in the present invention. [Figure 8] FIG. 8 shows the effect of Vernonia anthelimina microemulsion gel on the melanin content of hair follicles in the skin of monobenzone-induced vitiligo mice in the present invention. [Figure 9] Figure 9 shows the cumulative permeation amount and intradermal retention amount of the main ingredient vernodaline in the Vernonia anthelimentiica extract solution, the Vernonia anthelimentiica microemulsion gel of the present invention, and the Vernonia anthelimentiica gel in the skin permeation test of the present invention. Left: cumulative permeation amount, right: intradermal retention amount. [Figure 10]Figure 10 shows scanning electron microscope photographs of skin tissues after the skin permeation test of the present invention, where scanning electron microscope: 500x magnification, A: Vernonia anthelimentiica extract solution group, B: Vernonia anthelimentiica microemulsion gel group, C: Vernonia anthelimentiica gel group. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention is not limited by the following examples, in which all chemical reagents and chemicals referred to are of chromatographic or analytical purity grades known in the art unless otherwise specified. All pharmaceutical excipients referred to are of pharmaceutical grades known in the art unless otherwise specified. All percentages are by weight unless otherwise specified or clearly contradicted by context.

[0034] The present invention will now be further described with reference to the following examples.

[0035] Selection of inert solvent Vernodalin, the most abundant component in the active ingredient of Vernonia anthelimentiica, is a sesquiterpene compound with a lactone ring structure that is susceptible to hydrolysis in alkaline environments. Therefore, it was necessary to first identify an inert solvent that could stabilize vernodalin and completely dissolve the active ingredient. Furthermore, the compatibility of commonly used excipients with the raw material was examined. Several solvents (diethylene glycol monoethyl ether, Transcutol P, 60% methanol, 60% ethanol, polyethylene glycol, and acetonitrile) were selected, and the change in vernodalin content in each solvent was measured at room temperature using HPLC. Next, appropriate amounts of Vernonia anthelimentiica extract were dissolved in the corresponding solvents, and the content of total organic acids (isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C) was measured at 0, 10, and 30 days (days), and the percentage change was calculated. The results are shown in Table A.

[0036] [Table A]

[0037] Thus, the inert solvent diethylene glycol monoethyl ether Transcutol P can be used as an inert solvent in the gel or microemulsion gel of the present invention.

[0038] Excipient compatibility testing Transcutol P was selected as the inert solvent, capable of stabilizing vernodalin and completely dissolving Vernonia anthelimentiica extract. Commonly used excipients for microemulsions and emulsions were mixed with the Vernonia anthelimentiica extract dissolved in Transcutol P. The changes in the content of the main component vernodalin and total organic acids (the combined content of isochlorogenic acids A, B, and C) were monitored at 0, 10, and 30 days after mixing. The evaluation criteria were a change in the content of the main component of less than 5.0% between 10 and 30 days, with the formula: change rate = 100% x (content at 0 days - content at 30 days) / content at 0 days. Excipients with good compatibility with microemulsions were selected, and further formulation and preparation process testing was conducted. The results are shown in Table B.

[0039] [Table B]

[0040] Through screening, the excipients that are compatible with Vernonia antherimentiica extract are polyoxyethylene (40) hydrogenated castor oil (RH40), polyoxyethylene (35) hydrogenated castor oil (ELP), glyceryl monostearate (GMS) and lecithin.

[0041] Optimization and Selection of Preparation Process for Microemulsion / Microemulsion Gel In the topical preparation of Vernonia anthelimntaica extract, the microemulsion in the microemulsion gel is prepared by a method comprising the following steps: 1) Adding and dissolving Vernonia antherimentiica extract to diethylene glycol monoethyl ether, adding polyoxyethylene (40) hydrogenated castor oil (or polyoxyethylene (35) hydrogenated castor oil) and olive oil (or almond oil, corn oil, or soybean oil) in that order, and then stirring to obtain a mixture (oil phase). 2) After uniformly stirring the mixture obtained in step 1), water (aqueous phase) is added dropwise, stirred uniformly, and treated with ultrasound.

[0042] If it is necessary to further form a microemulsion gel, add 1.0 to 1.5% of Carbomer 2020 to the microemulsion prepared above, leave it to stand until the Carbomer is completely swollen, and then stir evenly to obtain a microemulsion gel.

[0043] A) Selection of preparation method A microemulsion with a drug (i.e., Vernonia antherimentiica extract) loading of 0.1% was prepared using the formula in Example 7 below. It was then subjected to equilibrium stirring for 10 minutes at maximum rotation speed using a high-speed emulsifier, a vortex mixer, and a magnetic stirrer. After 10 minutes of ultrasonication, the particle size and PDI of the prepared microemulsions were examined, and the results are shown in the table below. Because the particle size and PDI values ​​of the microemulsion prepared using a magnetic stirrer were lowest, the magnetic stirrer was used to prepare the microemulsion.

[0044] [Table C]

[0045] B) Consideration of drug addition order Microemulsions with drug (i.e., Vernonia antherimentica extract) loadings of 0.1%, 0.5%, and 1.0% were prepared using the formulation in Example 7 below. The drug was either dissolved in Transcutol P before addition, or added directly to a blank microemulsion. After 10 minutes of equilibrium stirring using a magnetic stirrer and 10 minutes of ultrasonication, the effects of the two drug addition orders on the particle size, PDI, and drug loading capacity of the microemulsions were examined. The results are shown in the table below. As the drug loading increased, the particle size and PDI values ​​of the microemulsions prepared after dissolving the drug were lower; therefore, the drug was dissolved before preparing the microemulsion.

[0046] [Table D]

[0047] C) Microemulsion Identification and Morphology The emulsion prepared according to the above formula remains clear and transparent after centrifugation, exhibiting no layer separation and remaining a yellowish-brown liquid. The emulsion prepared according to the present invention can be diluted with distilled water. Upon dilution to a certain extent, it exhibits a pale blue opalescence and exhibits the Tyndall phenomenon, proving that the sample prepared in this study is a microemulsion. The diffusion rates of methylene blue in both the drug-containing microemulsion and the blank microemulsion are significantly faster than those of Sudan Red, proving that the prepared microemulsion is an oil-in-water (O / W) type. The results are shown in Figure 1.

[0048] D) Microscopic morphology and particle size examination of microemulsions The drug-containing microemulsion prepared according to the above formula was diluted with purified water and shaken uniformly. One drop of the microemulsion was placed on a copper mesh designed for transmission electron microscopy. After air-drying, one drop of 2% phosphotungstic acid was added and stained for 2-3 minutes. After air-drying, the microemulsion morphology was observed using an H-600 transmission electron microscope. Transmission electron microscopy revealed that the microemulsion particles were round and uniformly spherical, with smooth surfaces, no aggregation, and good dispersion. As shown in Figure 2, the particle size of the microemulsion was observed to be approximately 10-100 nm, which is essentially consistent with the results of dynamic light scattering (DLS) measurements.

[0049] E) Gelation of microemulsions According to the above microemulsion formulation and process, immediately after preparing the microemulsion, 1.0% Carbomer 2020 was evenly spread on the surface of the microemulsion. The microemulsion was allowed to stand until the Carbomer 2020 was fully swollen, and then stirred uniformly to obtain a gel. The pH was determined to be between 4.8 and 5.0, with a viscosity ranging from 50 to 90 Pa.S. Vernonia anthelimenticas microemulsion gels with 1% drug loading and standard Vernonia anthelimenticas gels were prepared, and observations under a transmission electron microscope revealed that the particle size of the microemulsion gel was significantly smaller than that of the standard gel, was uniformly distributed, and no blocking or aggregation occurred. The particle size of the standard gel was approximately 500 nm, with some particles forming clumps and agglomerating. The microemulsion gel and standard gel observed under a transmission electron microscope are shown in Figure 3. Left: 1% Vernonia anthelimentiae microemulsion gel, Right: 1% Vernonia anthelimentiae gel. [Example]

[0050] Example 1 Preparation of gel (mass percentage, based on 100g) a. At room temperature, Carbomer 2020 (Lubrizol Carbopol (R)A blank gel matrix was prepared by adding 1.0% of ETD 2020 polymer to 14.0% of water to swell the matrix in advance.

[0051] b. 10.0% polyoxyethylene (40) hydrogenated castor oil (Beijing Fengli Jingjiu Pharmaceutical Co., Ltd.) and 10.0% diethylene glycol monoethyl ether (Shanghai McLin Biochemical Technology Co., Ltd., Transcutol P) were added in sequence to 1.0% Vernonia antherimentica extract (prepared by the method described in the "Disclosure of the Invention"). The gel matrix obtained in step a was then added and stirred uniformly to obtain a mixed solution.

[0052] c. A weakly alkaline salt solution was prepared in advance with Na2HPO4 to adjust the pH value to 9.0, and used as a neutralizer. 64.0% of this neutralizer was added to the mixed solution obtained in step b and stirred uniformly to obtain a Vernonia anthelimina gel with a pH value of 4.8 and a drug loading of 1.0%.

[0053] Example 2 Preparation of gel (mass percentage, based on 100g) a. A blank gel matrix was obtained by pre-swelling 1.5% Carbomer 2020 in 21.0% water at room temperature.

[0054] b. 5.0% polyoxyethylene (40) hydrogenated castor oil and 1.0% diethylene glycol monoethyl ether were added in that order to the above Vernonia antherimentica extract (0.1%), and then the blank gel matrix obtained in step a was added and stirred uniformly to obtain a mixed solution.

[0055] c. A weakly alkaline salt solution was prepared in advance with K2HPO4 to adjust the pH value to 9.0, and used as a neutralizer. 71.4% of this neutralizer was added to the mixed solution obtained in step b and stirred uniformly to obtain a Vernonia anthelimina gel with a pH value of 4.5 and a drug loading of 0.1%.

[0056] Example 3 Preparation of gel (mass percentage, based on 100g) a. A blank gel matrix was obtained by pre-swelling 1.2% Carbomer 2020 in 16.8% water at room temperature.

[0057] b. 11.0% polyoxyethylene (40) hydrogenated castor oil and 15.0% diethylene glycol monoethyl ether were added in that order to the above 4.0% Vernonia antherimentiica extract, and then the blank gel matrix obtained in step a was added and stirred uniformly to obtain a mixed solution.

[0058] c. A weakly alkaline salt solution was prepared in advance with NaHCO3 to adjust the pH value to 9.0 as a neutralizer, and 52% of this neutralizer was added to the mixed solution obtained in step b and stirred uniformly to obtain Vernonia anthelimentiga gel with a pH value of 4.6 and a drug loading of 4.0%.

[0059] Example 4 Preparation of gel (mass percentage, based on 100g) a. 1.0% Carbomer 2020 was pre-swollen in 14.0% water at room temperature to obtain a gel matrix.

[0060] b. 15.0% polyoxyethylene (40) hydrogenated castor oil and 1.0% diethylene glycol monoethyl ether were added in that order to the above 2.0% Vernonia antherimentiica extract, and then the gel matrix obtained in step a was added and stirred uniformly to obtain a mixed solution.

[0061] c. A weakly alkaline salt solution was prepared in advance with Na2HPO4 to adjust the pH value to 9.0, and used as a neutralizer. 67.0% of this neutralizer was added to the mixed solution obtained in step b and stirred uniformly to obtain a Vernonia anthelimina gel with a pH value of 4.9 and a drug loading of 2.0%.

[0062] Example 5 Preparation of microemulsion gel (mass percentage, based on 100g) a. At room temperature, 0.1% of the above Vernonia antherimentiica extract was dissolved in 1.0% diethylene glycol monoethyl ether (Transcutol P), and then 30.0% polyoxyethylene (40) hydrogenated castor oil and 5.0% olive oil were added in that order and stirred uniformly to obtain a mixture.

[0063] b. The mixture obtained in step a was stirred uniformly using a magnetic stirrer at a speed of 300 r / min, followed by adding 62.4% water dropwise and stirring at equilibrium for 10 minutes. Ultrasonication was then performed for 20 minutes (ultrasonic frequency 40 kHz, amplitude 60%), followed by adding 1.5% Carbomer 2020, allowing the mixture to stand until the carbomer was completely swollen, and then stirring uniformly to obtain a Vernonia anthelimentiae microemulsion gel with a drug loading of 0.1%. The pH was found to be 4.5 and the viscosity was in the range of 130-140 Pa.S (Shanghai Lichen Instrument Technology Co., Ltd., NDJ-5S digital viscometer, according to Method 1 of Part 4 of the 2020 edition of the Chinese Pharmacopoeia, 60 r / min). -1 (Measured at rpm).

[0064] Example 6 Preparation of microemulsion gel (mass percentage, based on 100g) a. At room temperature, 4.0% of Vernonia antherimentiica extract was dissolved in 15.0% diethylene glycol monoethyl ether (Transcutol P), and then 5.0% polyoxyethylene (40) hydrogenated castor oil and 10.0% olive oil were added in that order and stirred uniformly to obtain a mixture.

[0065] b. The mixture obtained in step a was stirred uniformly at 600 r / min using a magnetic stirrer, followed by adding 65.0% water dropwise, 30 minutes of equilibrium stirring, 15 minutes of ultrasonication, 1.0% Carbomer 2020, and the mixture was allowed to stand until the carbomer was completely swollen, after which it was stirred uniformly to obtain a Vernonia anthelimenticas microemulsion gel with a drug loading of 4%. The pH was 4.7 and the viscosity was in the range of 70-80 Pa.S.

[0066] Example 7 Preparation of microemulsion gel (mass percentage, based on 100g) a. At room temperature, 1.0% of Vernonia antherimentiica extract was dissolved in 11.0% diethylene glycol monoethyl ether (Transcutol P), and then 22.0% polyoxyethylene (40) hydrogenated castor oil (RH40) and 1.0% olive oil were added in that order and stirred uniformly to obtain a mixture.

[0067] b. After the mixture obtained in step a was stirred uniformly using a magnetic stirrer at a speed of 300 r / min, 66% water was added dropwise, followed by 30 minutes of equilibrium stirring and 5 minutes of ultrasonication. 1.0% Carbomer 2020 was added, and the mixture was allowed to stand until the carbomer was completely swollen. After further stirring, a Vernonia anthelimenticas microemulsion gel with a drug loading of 1.0% was obtained. The pH was 4.9, the viscosity was in the range of 60-80 Pa.S, and the microemulsion prepared at this ratio had a particle size of 20.09±1.45 nm and a PDI of 0.355±0.027.

[0068] Using the Vernonia anthelimentiica gel of Example 1 and the Vernonia anthelimentiica microemulsion gel of Example 7 as representatives, the following corresponding comparative examples were prepared using conventional methods to compare the stability of the main ingredient content of the preparations of the present invention.

[0069] Comparative Example 8 Preparation of gel (mass percentage, based on 100g) a. 1.0% Carbomer 980 was pre-swollen in 14.0% water at room temperature to obtain a gel matrix.

[0070] b. 10.0% polyoxyethylene (40) hydrogenated castor oil and 10.0% diethylene glycol monoethyl ether were added in that order to 1.0% Vernonia antherimentiica extract, and then the gel matrix obtained in step a was added and stirred uniformly to obtain a mixed solution.

[0071] c. 1.3% triethanolamine was added to 62.7% water, stirred evenly, and then added to the mixed solution obtained in step b, and stirred evenly to obtain Vernonia anthelimina gel with a pH value of 6.5 and a drug loading of 1.0%.

[0072] Comparative Example 9 Preparation of gel (mass percentage, based on 100g) a. 1.0% Carbomer 2020 was pre-swollen in 14.0% water at room temperature to obtain a gel matrix.

[0073] b. 10.0% polyoxyethylene (40) hydrogenated castor oil and 10.0% diethylene glycol monoethyl ether were added in that order to 1.0% Vernonia antherimentiica extract, and then the gel matrix obtained in step a was added and stirred uniformly to obtain a mixed solution.

[0074] c. A solution adjusted to pH 9.0 with NaOH was prepared as a neutralizer, and 64.0% of this neutralizer was added to the mixed solution obtained in step b and stirred uniformly to obtain a Vernonia anthelimina gel with a pH of 5.0 and a drug loading of 1.0%.

[0075] Comparative Example 10 Preparation of microemulsion gel (mass percentage, based on 100g) a. At room temperature, 1.0% of Vernonia antherimentiica extract was dissolved in 11.0% diethylene glycol monoethyl ether (Transcutol P), and then 22.0% polyoxyethylene (40) hydrogenated castor oil and 1.0% olive oil were added in that order and stirred uniformly to obtain a mixture.

[0076] b. After stirring the mixture obtained in step a uniformly at 300 rpm using a magnetic stirrer, add 63% water dropwise, stir for 30 minutes, and then sonicate for 20 minutes. Add 1.0% Carbomer 980, and allow to stand until the carbomer is completely swollen. After stirring uniformly, the mixture remains in a solution state. Add 1% triethanolamine and stir uniformly to obtain a Vernonia anthelimenticas microemulsion gel with a drug loading of 1.0%. The pH was found to be 6.0 and the viscosity was in the range of 80-90 Pa.S.

[0077] Example 11 Stability of the main component of the topical preparation of Vernonia antherimentica extract according to the present invention The HPLC method is

number

[0078] [Table E]

[0079] [Table F]

[0080] Example 12 Transdermal properties of topical preparations of Vernonia anthelimentiae extract according to the present invention Transdermal rate was studied using a diffusion cell. Healthy rats (SD rats, purchased from the Animal Experiment Center of Xinjiang Medical University, production permit number: SCXK(New)2018-0003) of acceptable weight were harvested and sacrificed by cervical dislocation, and their abdomens were depilated. The abdominal skin was then excised, and short hairs, subcutaneous tissue, and fascia were removed. The integrity of the stratum corneum was confirmed under magnification. The skin was then thoroughly washed with saline. The skin was sandwiched between a double-chamber diffusion cell, with the stratum corneum facing the donor cell and the dermis facing the receptor fluid. A 1ml sample was placed in the donor cell and contacted with the stratum corneum. The samples were 1% Vernonia anthelimentiica extract solution, 1% Vernonia anthelimentiica microemulsion gel, and 1% Vernonia anthelimentiica gel, dissolved in receptor fluid. The receptor medium was a saline solution containing 20% ​​ethanol, which was kept in a 37°C water bath and stirred at 400 rpm. 1 mL samples were taken at regular intervals at 0, 2, 4, and 6 h. Equal volumes of blank receptor solution, also kept at 37°C, were simultaneously added. The active ingredient content in the receptor solution was measured using the content determination method. At 6 h, the skin was removed, and any remaining test substance on the skin surface was carefully removed. After being finely chopped, the test substance was placed in 1 mL of saline solution containing 20% ​​ethanol, homogenized, and filtered. The sample was then injected and detected using the HPLC method described above. The cumulative permeation volume and intradermal retention volume at 6 h were calculated. The results are shown in Figure 9. Left: Cumulative permeation curves for the Vernonia anthelimentiica extract solution and the three formulations. Right: Intradermal retention volume of the Vernonia anthelimentiica extract solution after 6 h of exposure to each formulation.

[0081] Results and conclusion: Figure 9 shows that the main component of the Vernonia anthelimentiica extract solution penetrates the skin in large amounts in a short period of time, but the amount retained within the skin is extremely low. Compared to the extract solution, both the Vernonia anthelimentiica microemulsion gel and Vernonia anthelimentiica can significantly improve the amount of the main component retained within the skin. This indicates that the formulation prepared by the present invention can increase the amount of the main component retained within the skin, which is advantageous for exerting medicinal effects.

[0082] After percutaneous application, each skin sample was carefully removed and the skin surface was repeatedly wiped with a water-alcohol cotton ball to ensure that all drug was removed from the skin surface. Careful handling was required to avoid damaging the skin surface during manual handling. The pretreated skin was fixed overnight in 2.5% glutaraldehyde fixative at 0.1 mol L. -1 After washing three times with phosphate buffer, the skin samples were dehydrated in various volume fractions of ethanol-water solutions (10%, 30%, 50%, 75%, and 100%) for 15–20 min at each gradient. Then, they were dehydrated twice in pure ethanol, gradient-shifted to acetone, freeze-dried, and gold-plated by ion sputtering to prepare the skin samples. They were then observed and photographed under a scanning electron microscope. The morphology of the skin surface observed under an electron microscope is shown in Figure 10. The skin in the Vernonia anthelimntaica microemulsion gel transdermal experimental group exhibited a fairly regular depression structure, with the depression walls exhibiting wrinkled appearance. The skin in the gel transdermal experimental group exhibited depression structure, but the depressions were less pronounced and the depression walls were less pronounced and more wrinkled. The depressions in the skin samples from the transdermal test group for the Vernonia anthelimentiica extract solution almost disappeared, the stratum corneum expanded outward, and the penetration surface increased to a certain extent. Based on the above, it was suggested that the Vernonia anthelimentiica extract solution has a certain degree of skin irritation, and that by preparing the Vernonia anthelimentiica extract into a microemulsion gel or gel, it is possible to increase the intradermal retention amount and reduce skin irritation.

[0083] Example 13 Effectiveness of the topical gel formulation of Vernonia anthelimentiica extract according to the present invention Study on the effect of Vernonia anthelimintica gel on a mouse model of hydroquinone-induced vitiligo Using Vernonia anthelimentiica extract as the drug substance, a gel with a drug loading of 1% was prepared according to the method described in the present invention (see Example 1). A hydroquinone-induced vitiligo mouse model was used (for the model preparation method, see the following literature: Gao Li, Huo Shixia, Peng Xiaoming et al., Effects of flavones on a hydroquinone-induced vitiligo mouse model [J], Chinese herbal medicine, 2014, 45(16): 2358-2363.) to initially investigate the efficacy of the Vernonia anthelimentiica gel.

[0084] From the 21st day after model establishment, 5% hydroquinone was applied to each group, and the drug was applied one hour later. The blank control group and the vitiligo model group were given an equal volume of distilled water. The treatment group was given Vernonia anthelimintica gel, and the positive control group was given an equal volume of Compound Kaliziran tincture.

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[0085] The overall effectiveness rate was calculated as the sum of excellent and good, and the effectiveness rates are shown in Table 3.

[0086] [Table F]

[0087] Results and Conclusions: As shown in Figure 5, during the model establishment process, the skin in the test areas of mice in each group was pale and hair growth was slow. Over time, white spots appeared and newly grown hair turned white. After treatment, the skin color in the test areas of the vitiligo mice in the positive control group and the 1% gel group recovered to different degrees, and newly grown hair also appeared to have returned to black. After 21 days of administration, the efficacy rate of 1% Vernonia anthelimentiga gel in the vitiligo model mice was approximately 60%, superior to the therapeutic effect of Compound Kaliziran tincture over the same period.

[0088] Example 14 Efficacy of the topical preparation microemulsion gel of Vernonia anthelimntaica extract according to the present invention A mouse vitiligo model (for the preparation method, see the following literature: Zhu Yiping, et al.) using Vernonia anthelimentiae microemulsion gel (see Example 7 for the preparation method) and monobenzone was used.

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[0089] Grouping and drug intervention From the 21st day after model establishment, each group was given monobenzone cream (self-administered, the mass fraction of monobenzone in the cream was 40%, monobenzone was purchased from Sigma-Aldrich, product number 158348-100g, batch number STBK2160) every day (wiped off with a cotton swab soaked in water before administration), and 4 hours later, administration began at the dose and frequency set in Table 4, for 30 days.

[0090] [Table G]

[0091] Results and Conclusions: From day 10 after model establishment, the skin of the mice in the test areas became partially white, with small white spots appearing. Hair growth was significantly slower than in the blank group. Over time, varying degrees of white spots appeared on the skin in the test areas of each model-established animal, and white hair grew. From day 40 onward, the hair in the non-model-established areas of the model group gradually became discolored, and the tails and ears became pinpoint-like. The results are shown in Figure 6.

[0092] From the 10th day after the microemulsion gel treatment, the formation of black spots was observed on the skin of the test area of ​​the animals in each microemulsion gel dose group. As the drug treatment time progressed, the skin color gradually recovered, the color of the newly grown hair also gradually darkened, and the hair growth rate was significantly faster than that of the model group. On the 50th day of the experiment, 2 was selected as one observation unit. Excellent: Pigment in the test area returns to near normal Good: Pigment appears in >50% of the test area Medium: Pigment appears in less than 50% of the test area Bad: Pale or white patches of skin in the test area

[0093] The overall efficacy rate was calculated by adding up the excellent and good ratings. Visual observation and evaluation showed that the therapeutic effects of the medium and high dose groups of VAF microemulsion gel were superior to those of the positive control group. The results are shown in Table 5 and Figure 7.

[0094] Melanin staining Skin tissue was embedded in paraffin and stained for melanin. Hair follicles were observed under a 200x optical microscope and the number of melanin-containing follicles was counted for every 50 follicles. Data were analyzed using GraphPad Prism 6.0 statistical software. Means between groups were compared by single-factor analysis of variance, with P<0.05 indicating a significant difference.

[0095] The epidermal tissue of the blank control group had a higher number of hair follicles, higher pigment content, and deeper color. In the model group, no obvious pigmentation was observed within the hair follicles of many animals, and the number of melanin-containing hair follicles was significantly reduced, with statistically significant differences (P<0.05). Compared with the vitiligo model group and the blank gel group, the number of melanin-containing hair follicles in the skin of the low, medium, and high dose groups after treatment with VAF microemulsion gel was significantly increased, with statistically significant differences (P<0.05). The increase in the number of melanin-containing hair follicles in the back skin of each treated mouse showed a dose-response relationship. The results are shown in Figure 8 and Table 5.

[0096] [Table H]

Claims

1. An external preparation of Vernonia anthelimntheica extract, which contains vernodaline and 3,5-O-dicaffeoylquinic acid as active ingredients, and is in the form of a gel or microemulsion gel, 1) The gel contains, per 100 g, Carbomer 2020 1.0-1.5% 13.5 to 40.0% water, Vernonia antherimentica extract 0.1 to 4.0%, 5.0 to 15.0% of polyoxyethylene (40) hydrogenated castor oil or polyoxyethylene (35) hydrogenated castor oil, 1.0 to 15.0% diethylene glycol monoethyl ether; a neutralizing agent, and the pH value of the gel is 4.3 to 5.5; However, the total content of each component is 100%. 2) The microemulsion gel contains, per 100 g, Vernonia antherimentica extract 0.1 to 4.0%, 1.0 to 15.0% diethylene glycol monoethyl ether; 5.0 to 30.0% of polyoxyethylene (40) hydrogenated castor oil or polyoxyethylene (35) hydrogenated castor oil, 1.0 to 10.0% of olive oil, almond oil, corn oil or soybean oil; 40.0 to 84.0% water, Carbomer 2020 1.0 to 1.5%; However, this is an external preparation of Vernonia anthelimntaica extract in which the total content of each ingredient is 100%.

2. An external preparation of Vernonia anthelimntaica extract as a gel, wherein the neutralizing agent is a weakly alkaline salt solution having a pH value of 8.0 to 10.0, preferably a weakly alkaline salt solution having a pH value of 9.0, for example, Na ２ HPO ４ , K. ２ HPO ４ , NaHCO ３ 2. The topical formulation of claim 1, wherein the Vernonia anthelimntheica extract is a weakly alkaline aqueous salt solution prepared from an alkali metal acid salt such as benzoyl perfluorooctanoate, ...

3. 3. The topical preparation of Vernonia anthelimntheica extract according to claim 2, wherein the gel is prepared by a method comprising the steps of: 1) Add Carbomer 2020 to water to swell it and obtain a blank gel matrix; 2) Add polyoxyethylene (40) hydrogenated castor oil (or polyoxyethylene (35) hydrogenated castor oil) and diethylene glycol monoethyl ether in this order to the Vernonia antherimentiica extract, then add the gel matrix obtained in step 1), and stir uniformly to obtain a mixture; 3) Add the neutralizer to the mixture obtained in step 2) and stir evenly to make the pH value of the whole system 4.3-5.5 to obtain a gel.

4. An external preparation of Vernonia anthelimntaica extract, which is an oil-in-water (O / W) microemulsion gel, 1) the pH is 4.0 to 6.5, for example, 4.0 to 5.5, for example, 4.5, 4.7, and 4.9; 2) the viscosity is in the range of 20 to 160 Pa.S, for example, in the range of 30 to 90 Pa.S, in the range of 70 to 80 Pa.S, in the range of 60 to 80 Pa.S, and in the range of 130 to 140 Pa.S; 3) As a result of detection by a dynamic light scattering particle size analyzer, the particle diameter is 10 nm to 100 nm and the PDI (polymer dispersibility index) value is less than 0.

50. An external preparation of the Vernonia anthelimntheica extract according to claim 1.

5. 5. The topical preparation of Vernonia anthelimntheica extract according to claim 4, wherein the microemulsion gel is prepared by a method comprising the steps of: 1) Add and dissolve Vernonia antherimentiica extract in diethylene glycol monoethyl ether, add polyoxyethylene (40) hydrogenated castor oil or polyoxyethylene (35) hydrogenated castor oil and olive oil (or almond oil, corn oil, or soybean oil) in that order, and then stir to obtain a uniform mixture; 2) After the mixture obtained in step 1) is uniformly stirred, water is added dropwise, the mixture is uniformly stirred, and the mixture is treated with ultrasound. 1.0 to 1.5% of Carbomer 2020 is added, the mixture is allowed to stand until the Carbomer is completely swollen, and the mixture is uniformly stirred to obtain a microemulsion gel.

6. The Vernonia anthelimentiica extract according to any one of claims 1 to 5, wherein the Vernonia anthelimentiica extract is prepared by the method described in CN104825518A and contains 15 to 25 wt% of vernodalin and 10 to 20 wt% of 3,5-O-dicaffeoylquinic acid.

7. 1. An external preparation of Vernonia antheliminteica extract, characterized in that the external preparation is prepared as a gel or microemulsion gel from Vernonia antheliminteica extract and a pharmaceutically acceptable excipient based on the following steps, based on a 100 g basis: For the preparation of gels, a. Add 1.0 to 1.5% Carbomer 2020 to 13.5 to 40.0% water by mass at room temperature to swell the mixture to obtain a blank gel matrix; b. Add 5.0-15.0% of polyoxyethylene (40) hydrogenated castor oil (or polyoxyethylene (35) hydrogenated castor oil) and 1.0-15.0% of diethylene glycol monoethyl ether in this order to 0.1-4.0% of Vernonia antherimentiica extract, then add the gel matrix obtained in step a, and stir uniformly to obtain a mixture; c. As a neutralizer, Na ２ HPO ４ , K. ２ HPO ４ or NaHCO ３ a weakly alkaline salt solution having a pH value of 9.0 is prepared by adding the obtained neutralizing agent to the mixture obtained in step b, and stirring uniformly to make the pH value of the whole system 4.3-5.5, thereby obtaining a gel; For the preparation of microemulsion gels, a. Add 0.1 to 4.0% by mass of Vernonia antherimentiica extract to 1.0 to 15.0% diethylene glycol monoethyl ether at room temperature and dissolve, then add 5.0 to 30.0% polyoxyethylene (40) hydrogenated castor oil (or polyoxyethylene (35) hydrogenated castor oil) and 1.0 to 10.0% olive oil (or almond oil, corn oil, or soybean oil) in that order and stir uniformly to obtain a mixture; b. The mixture obtained in step a is stirred uniformly with a magnetic stirrer at a speed of 300-600 r / min, and then 40.0-84.0% water is added dropwise, followed by equilibrium stirring for 10-30 minutes, ultrasonic waves are applied for 5-20 minutes, 1.0-1.5% Carbomer 2020 is added, and the mixture is allowed to stand until the carbomer is completely swollen, followed by uniform stirring to obtain a microemulsion gel.

8. A method for preparing an external preparation of Vernonia anthelimntheica extract, characterized in that it is carried out by the following steps: For the preparation of gels, a. Add 1.0 to 1.5% Carbomer 2020 to 13.5 to 40.0% water by mass at room temperature to swell the mixture to obtain a blank gel matrix; b. Add 5.0-15.0% of polyoxyethylene (40) hydrogenated castor oil (or polyoxyethylene (35) hydrogenated castor oil) and 1.0-15.0% of diethylene glycol monoethyl ether in this order to 0.1-4.0% of Vernonia antherimentiica extract, then add the gel matrix obtained in step a, and stir uniformly to obtain a mixture; c. As a neutralizer, Na ２ HPO ４ , K. ２ HPO ４ or NaHCO ３ a weakly alkaline salt solution having a pH value of 9.0 is prepared by adding the obtained neutralizing agent to the mixture obtained in step b, and stirring uniformly to make the pH value of the whole system 4.3-5.5, thereby obtaining a gel; For the preparation of microemulsion gels, a. Add 0.1 to 4.0% by mass of Vernonia antherimentiica extract to 1.0 to 15.0% diethylene glycol monoethyl ether at room temperature and dissolve, then add 5.0 to 30.0% polyoxyethylene (40) hydrogenated castor oil (or polyoxyethylene (35) hydrogenated castor oil) and 1.0 to 10.0% olive oil (or almond oil, corn oil, or soybean oil) in that order and stir uniformly to obtain a mixture; b. The mixture obtained in step a is stirred uniformly with a magnetic stirrer at a speed of 300-600 r / min, and then 40.0-84.0% water is added dropwise, followed by equilibrium stirring for 10-30 minutes, ultrasonic waves are applied for 5-20 minutes, 1.0-1.5% Carbomer 2020 is added, and the mixture is allowed to stand until the carbomer is completely swollen, followed by uniform stirring to obtain a microemulsion gel.

9. The Vernonia antherimintei gel according to claim 1 or 7, characterized in that the contents of the main ingredients, vernodaline and 3,5-O-dicaffeoylquinic acid, are 0.15 to 0.20% and 0.10 to 0.15%, respectively, in percentages.

10. Use of an external preparation of Vernonia anthelimentiica extract according to any one of claims 1 to 7 or an external preparation of Vernonia anthelimentiica extract obtained by the method according to claim 8 in the preparation of a therapeutic agent for skin depigmentation disorders such as vitiligo.

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