Topical preparation of vernonia anthelmintica extract and method for preparing same

Abstract

Provided are a gel and microemulsion gel prepared from a Vernonia anthelmintica extract, and a method for preparing same. The gel comprises Carbomer 2020, water, the Vernonia anthelmintica extract, polyoxyethylene 40 hydrogenated castor oil or polyoxyethylene 35 hydrogenated castor oil, diethylene glycol monoethyl ether, and a neutralizer, wherein the pH value of the gel is 4.3-5.5. The microemulsion gel comprises the Vernonia anthelmintica extract, diethylene glycol monoethyl ether, polyoxyethylene 40 hydrogenated castor oil or polyoxyethylene 35 hydrogenated castor oil, olive oil, almond oil, corn oil or soybean oil, water, and Carbomer 2020. The active ingredients of the Vernonia anthelmintica extract in the gel and the microemulsion gel are vernodalin and 3,5-O-dicaffeoylquinic acid. The major ingredients, vernodalin and 3,5-O-dicaffeoylquinic acid, in the gel and the microemulsion gel feature stable contents and high intradermal retention capability, thus providing the gel and the microemulsion gel with suitability for topical administration for treating vitiligo.

Description

An external preparation of anthelmintic Vernonia extract and its preparation method Technical Field

[0001] The invention relates to the technical field of anthelmintic Vernonia extract, which uses the anthelmintic Vernonia extract as a raw material medicine to prepare gel and microemulsion gel external preparations. Background Art

[0002] Vitiligo is a localized or widespread lesion caused by a decrease or disappearance of tyrosinase activity in melanocytes within the skin and hair follicles, leading to a progressive decrease or disappearance of melanosome production. Human skin is composed of the epidermis, dermis, and subcutaneous tissue. Melanin production occurs in the deepest part of the epidermis—the basal layer. For vitiligo, the basal layer is the target site for topical drug delivery systems, while the stratum corneum covering the skin's surface is the primary barrier to drug delivery system penetration. To restore color to the vitiligo affected area, active substances must penetrate the stratum corneum to reach the basal layer to exert their pharmacological effects. Therefore, the most suitable dosage form for improving localized vitiligo is a topical preparation. Vernonia Anthelmintica is a core medicinal herb used in Uyghur medicine in Xinjiang for the treatment of vitiligo. It has the effects of promoting melanin production, increasing skin photosensitivity, improving skin microcirculation in vitiligo lesions, regulating immunity, and replenishing trace elements. The applicant conducted a chemical analysis of the main components of Vernonia anthelmintica and confirmed that the active ingredients that promote melanin regeneration include sesquiterpenes, quinic acids, and flavonoids. Furthermore, the applicant optimized the extraction and purification process for Vernonia anthelmintica, focusing on Vernonia anthelminticum and 3,5-O-dicaffeoylquinic acid as target components for enrichment. The resulting Vernonia anthelminticum extract contains 15-25% (w / w) Vernonia anthelminticum and 10-20% (w / w) 3,5-O-dicaffeoylquinic acid. HPLC analysis revealed that commercially available Vernonia anthelminticum injections contain 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid, but no Vernonia anthelminticum. However, the Vernonia anthelminticum extract used in the present invention contains higher levels of Vernonia anthelminticum and 3,5-O-dicaffeoylquinic acid.

[0003] Summary of the Invention

[0004] The present invention aims to provide a method for preparing a topical preparation from an extract of Vernonia anthelmintica. The method uses the extract of Vernonia anthelmintica as a raw material to prepare a topical preparation of a gel or microemulsion gel. The gel and microemulsion gel obtained by the method have a uniform appearance, stable physicochemical properties, and stable contents of the main components, Vernonia truncatula and 3,5-O-dicaffeoylquinic acid, as determined by HPLC. Transdermal studies evaluated the intradermal retention and cumulative transdermal delivery of the main components of the prepared gel and microemulsion gel, confirming that the microemulsion gel has a high intradermal retention capacity, making it very suitable for topical therapeutic administration. This invention is the first to prepare a topical preparation with a uniform appearance and stable contents using Vernonia truncatula and 3,5-O-dicaffeoylquinic acid as the main components.

[0005] In one aspect of the present invention, there is provided an external preparation of an anthelmintic Vernonia extract, comprising Vernonia macrobitterin and 3,5-O-dicaffeoylquinic acid as active ingredients, in the form of a gel or microemulsion gel, wherein:

[0006] 1) The gelling agent comprises, per 100g, the following:

[0007] Carbomer 2020 1.0-1.5%;

[0008] Water 13.5-40.0%;

[0009] Vernonia anthelmintic extract 0.1-4.0%;

[0010] Polyoxyl 40 hydrogenated castor oil or polyoxyl 35 hydrogenated castor oil 5.0-15.0%;

[0011] Diethylene glycol monoethyl ether 1.0-15.0%; and

[0012] neutralizing agent;

[0013] The pH value of the gel is 4.3-5.5.

[0014] Provided that the sum of the contents of the components is 100% (for example, if less than 100%, water is used to make up the difference);

[0015] 2) The microemulsion gel comprises, per 100 g, the following:

[0016] Vernonia anthelmintic extract 0.1-4.0%;

[0017] Diethylene glycol monoethyl ether 1.0-15.0%;

[0018] Polyoxyl 40 hydrogenated castor oil or polyoxyl 35 hydrogenated castor oil 5.0-30.0%;

[0019] Olive oil, almond oil, corn oil or soybean oil 1.0-10.0%;

[0020] Water 40.0-84.0%; and

[0021] Carbomer 2020 1.0-1.5%,

[0022] The condition is that the total content of each component is 100% (for example, if it is less than 100%, it can be made up with water).

[0023] In a preferred embodiment of the present invention, the topical preparation of the anthelmintic Vernonia extract is a gel, wherein the neutralizing agent is a weak alkaline saline solution with a pH value of 8.0-10.0, preferably 9.0, such as a weak alkaline saline solution with a pH value of preferably 9.0 prepared from an alkali metal acid salt such as Na2HPO4, K2HPO4, NaHCO3 or a combination thereof.

[0024] In a preferred embodiment of the present invention, the gel is prepared by a method comprising the following steps:

[0025] 1) Carbomer 2020 was added to water to swell and obtain a blank gel matrix;

[0026] 2) adding polyoxyethylene 40 hydrogenated castor oil (or polyoxyethylene 35 hydrogenated castor oil) and diethylene glycol monoethyl ether to the Vernonia anthelmintica extract in sequence, and then adding the gel matrix obtained in step 1), stirring uniformly to obtain a mixture;

[0027] 3) Adding a neutralizing agent to the mixture obtained in step 2) and stirring uniformly to adjust the pH value of the total system to 4.3-5.5, thereby obtaining a gelling agent.

[0028] In a preferred embodiment of the present invention, the topical preparation of the anthelmintic Vernonia extract is an oil-in-water (O / W) microemulsion gel having the following characteristics:

[0029] 1) pH 4.0-6.5, such as 4.0-5.5, such as 4.5, 4.7 and 4.9;

[0030] 2) a viscosity within the range of 60-160 Pa.S, such as 30-90 Pa.S, 70-80 Pa.S, 60-80 Pa.S and 130-140 Pa.S; and / or

[0031] 3) The particle size is 10 nm to 100 nm and the PDI (polymer dispersibility index) value is less than 0.50 as determined by a dynamic light scattering particle size analyzer.

[0032] In a particularly preferred embodiment of the present invention, the optimal excipient formula of 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). The microemulsion prepared according to this ratio has a particle size of 20.09±1.45nm and a PDI value of 0.355±0.027.

[0033] In a preferred embodiment of the present invention, the microemulsion gel is prepared by a method comprising the following steps:

[0034] 1) adding the Vernonia anthelmintic extract to diethylene glycol monoethyl ether and dissolving it, then sequentially adding polyoxyethylene 40 hydrogenated castor oil (or polyoxyethylene 35 hydrogenated castor oil) and olive oil (or almond oil, corn oil, or soybean oil) and stirring evenly to obtain a mixture;

[0035] 2) After stirring and mixing the mixture in step 1), water is added dropwise, stirred evenly, and ultrasonicated to obtain a microemulsion, 1.0-1.5% of Carbomer 2020 is added, and the mixture is allowed to stand until the Carbomer is completely swollen, and then stirred evenly to obtain a microemulsion gel.

[0036] The identification method of the microemulsion prepared by the method of the present invention is as follows:

[0037] After centrifugation, the emulsion prepared according to the present invention remained clear and transparent, showing no stratification, and remained a brownish-yellow liquid. It could be diluted with distilled water to a certain extent, exhibiting a light blue opalescence. Laser irradiation produced a distinct light path (the Tyndall effect unique to microemulsions), indicating that the sample prepared according to the present invention was a microemulsion. The diffusion rate of methylene blue in both the drug-containing microemulsion and the blank microemulsion was significantly faster than that of Sudan red, indicating that the prepared microemulsion was an oil-in-water (O / W) type.

[0038] In a preferred embodiment of the present invention, after preparing the microemulsion, the following microemulsion gelation is performed:

[0039] Carbomer 2020 is directly dissolved in the prepared microemulsion at a ratio of 1:100 (w / w) to 1.5:100 (w / w), and after the microemulsion is fully swollen, it is stirred evenly to obtain the anthelmintic Vernonia microemulsion gel of the present invention.

[0040] In a preferred embodiment of the present invention, the anthelmintic Vernonia extract is prepared according to the method described in CN104825518A, and contains 15-25 wt % of Vernonia oleracea and 10-20 wt % of 3,5-O-dicaffeoylquinic acid.

[0041] In a preferred embodiment of the present invention, the topical preparation of the Vernonia anthelmintic extract of the present invention is prepared by preparing a gel or microemulsion gel from the Vernonia anthelmintic extract supplemented with pharmaceutically acceptable excipients per 100 g. The specific operation is carried out according to the following steps:

[0042] Prepare the gel:

[0043] a. Add 1.0-1.5% carbomer 2020 to 13.5-40.0% water at room temperature to swell the mixture, based on mass percentage, to obtain a blank gel matrix.

[0044] b. Adding 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 to 0.1-4.0% of Vernonia anthelmintica extract, and then adding the gel matrix obtained in step a, stirring uniformly to obtain a mixture;

[0045] c. A weak alkaline salt solution having a pH value of 9.0 is prepared in advance with Na2HPO4, K2HPO4 or NaHCO3 as a neutralizing agent, and the neutralizing agent is added to the mixture obtained in step b, and stirred evenly to make the pH value of the total system 4.3-5.5 to obtain a gelling agent;

[0046] Preparation of microemulsion gel:

[0047] a. Dissolve 0.1-4.0% of Vernonia anthelmintic extract in 1.0-15.0% of diethylene glycol monoethyl ether at room temperature, and then sequentially add 5.0-30.0% of polyoxyethylene 40 hydrogenated castor oil or polyoxyethylene 35 hydrogenated castor oil and 1.0-10.0% of olive oil (or almond oil, corn oil, or soybean oil) and stir well to obtain a mixture;

[0048] b. Stir the mixture in step a at a rate of 300-600 r / min with a magnetic stirrer, then add 40.0-84.0% water dropwise, balance stirring for 10-30 min, ultrasonicate for 5-20 min, add 1.0-1.5% carbomer 2020, let stand until the carbomer is completely swollen, and then stir evenly to obtain a microemulsion gel.

[0049] The present invention also provides use of the external preparation of the anthelmintic Vernonia extract obtained by the method in preparing drugs for treating skin pigment loss such as vitiligo.

[0050] The topical preparation of an anthelmintic Vernonia extract of the present invention is obtained from the Vernonia extract in Chinese patent CN201510288742.3 (publication number CN104825518A), "Preparation method and anti-inflammatory use of anthelmintic Vernonia phenolic acid fraction." The specific operation is carried out according to the following steps:

[0051] Dried Vernonia anthelmintica medicinal materials are crushed using a traditional Chinese medicine flattening machine, weighed, and subjected to reflux extraction at a material-to-liquid ratio of 1:12. The solvent is 80% ethanol, and the extraction temperature is 75-80°C. Each extraction lasts 3 hours, and the extraction is repeated twice. The filtrates are then filtered, and the combined filtrates are concentrated under reduced pressure at 50-60°C to a crude drug equivalent of 21g per 100mL of extract. Column chromatography is then used to purify the extract: the extract is dispersed in water and loaded onto a sample at a concentration of approximately 10g of crude drug equivalent per 100mL of sample solution. The extract is then washed with three volumes of water for impurities removal, eluted with three volumes of 60% ethanol, and the eluate is collected. The extract is then concentrated under reduced pressure at 60°C and dried under vacuum at 50°C. HPLC analysis reveals that the Vernonia anthelmintica extract contains 15-25% vernonia quinic acid and 10-20% 3,5-O-dicaffeoylquinic acid.

[0052] The present invention uses the anthelmintic Vernonia extract prepared by the applicant in the early stage as the raw material drug, and uses modern pharmaceutical preparation technology to prepare it into a gel and microemulsion gel external preparation. In terms of its innovation, compared with the Vernonia injection currently used in clinical practice, the main ingredient Vernonia quinata bittering agent with the highest content in this preparation is not found in the commercially available Vernonia injection (see Figure 4); and in terms of its advanced nature, this preparation can significantly increase the intradermal retention of the main ingredient and reduce skin irritation (see Figures 10 to 11); and in terms of its preparation method alone, the external preparation method involved in the present invention has simple steps, simple operation, easy control of parameters, low cost, and is suitable for large-scale industrial production. In addition, the external preparation is more convenient to use than the injection, has strong compliance, and is more suitable for widespread clinical application. At the same time, it is undoubtedly a very meaningful work for fully tapping and utilizing Xinjiang Uyghur medicine resources.

[0053] Through research, the inventors unexpectedly discovered that, in the gel of the present invention, commonly used moisturizers for gels, such as polyols and fatty acid esters, should not be added, as their addition would cause degradation and other transformations of the main component of the extract, the vernonia quinone; commonly used alkaline components, such as triethanolamine and sodium hydroxide, should not be added, as their addition would cause alkaline hydrolysis and ring opening of the main component of the extract, the vernonia quinone; and the use of an ion-resistant gel matrix, Carbomer 2020, and pre-adjustment of the pH of the water used in the gel with Na2HPO4 or the like, thereby stabilizing the content of the sesquiterpenoid component vernonia quinone in the extract (its percentage in the extract is not less than 20% as determined by HPLC).

[0054] The inventors also unexpectedly found through research that in the preparation of the microemulsion gel of the present invention, the preparation method is to directly disperse carbomer 2020 into the microemulsion liquid prepared by the method of the present invention, wait for it to fully swell, and stir evenly to obtain the result; it is characterized in that the microemulsion gel prepared by this method does not require the use of organic or inorganic bases to adjust the pH, for example, the pH value can be obtained in the range of 4.0-6.5 (such as 5.0-6.5) and the viscosity can be obtained in the range of 20-160 Pa.S (such as 30-90 Pa.S), which meets the pH and viscosity requirements of the external gel.

[0055] The microemulsion and microemulsion gel of the present invention have uniform and delicate appearance, stable physical and chemical properties, and the content of the main components, vernonia quinata and isochlorogenic acid A, is stable within 30 days.

[0056] The difference between the present invention and the prior art is:

[0057] When selecting the gel matrix material of the present invention, in addition to the conventional system viscosity, the content stability of the main component of the preparation needs to be fully considered. Studies have shown that a neutral gel matrix such as CMC-Na can be used to prepare a gel with suitable viscosity by adjusting the mass fraction of CMC-Na without a neutralizer, but it becomes a solution after 48 hours and loses its semi-solid form. If the amount of CMC-Na is further increased, the initial viscosity will be too high and the gel will lose its proper fluidity. When further screening conventional matrix materials, when the anthelmintic Vernonia extract is prepared into a gel or microemulsion gel with a conventional carbomer matrix such as carbomer 940, carbomer 971, carbomer 980, the content of the main component Vernonia oleracea is unstable, and its content decreases by more than 20%. Only carbomer 2020 can meet the requirements of viscosity and content at the same time. Carbomer 2020 has long rheology, ion resistance, high transparency, excellent thickening and suspension properties, and is suitable for surfactant systems and transparent gel systems. Carbomer 2020 has a strong and long pH viscosity range. The viscosity begins to increase at pH = 4 and gradually decreases after exceeding pH = 9. However, compared with commonly used models such as Carbomer 940, Carbomer 971, and Carbomer 980, it can maintain a higher viscosity even when the pH is below 5.

[0058] When Carbomer 2020 was used as the matrix material, the effects of adjusting pH and viscosity of the formulation with different neutralizers were investigated.

[0059] ① Using NaOH as a neutralizing agent, a gel with suitable viscosity can be obtained when the pH is adjusted to 5.5-7.0, but the content of Vernonia serrata in the product decreases by about 20% after 10 days;

[0060] ② When triethanolamine was used to adjust the pH to 5.5-7.0, a gel with suitable viscosity could be obtained, but the content of scutellaria baicalensis in the product decreased by about 40% after 10 days.

[0061] ③ When tromethamine was used to adjust the pH to 5.5-7.0, a gel with suitable viscosity could be obtained, but the content of euphorbia pulegone in the product decreased by about 45% after 10 days.

[0062] ④ When the pH value of the system is adjusted to 4.3-5.5 using Na2HPO4, K2HPO4, and NaHCO3 as neutralizers, an anthelmintic Vernonia gel with uniform appearance, stable properties, and controllable quality can be obtained. When the amount of the neutralizer is further increased (i.e., pH>5.5), the viscosity of the gel continues to decrease, and when the pH value is>6.0, it is in a solution state.

[0063] ⑤ After the microemulsion is prepared, carbomer 2020 is directly added into the microemulsion. By adjusting the amount of carbomer, an anthelmintic Vernonia microemulsion gel with suitable viscosity and stable main components can be obtained without a neutralizer. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] FIG1 shows the identification and morphology of the microemulsion prepared by the present invention;

[0065] FIG2 shows the microscopic morphology and particle size examination of the microemulsion prepared in the present invention;

[0066] FIG3 shows transmission electron microscopic images of a microemulsion gel prepared in the present invention and a conventional gel (left: 1% Vernonia anthelmintic microemulsion gel; right: 1% Vernonia anthelmintic gel; the particle size of the present invention is much smaller than that of the conventional gel, and the distribution is uniform, without adhesion or aggregation. The conventional gel has a particle size of approximately 500 nm, and some particles are aggregated in a lumpy manner);

[0067] Figure 4 shows the difference in composition between the commercially available Vernonia injection (Wuhu Yangyan Pharmaceutical Co., Ltd., Le Manxin, National Medicine Standard Z20063652, Batch No. 190407) and the anthelmintic Vernonia extract of the present invention; HPLC analysis revealed that the commercially available Vernonia injection contained 3,4-O-dicaffeoylquinic acid (Shanghai Chunyou Biotechnology Co., Ltd., Batch No. 17121102), 3,5-O-dicaffeoylquinic acid (Beijing Hengyuan Qitian Chemical Technology Research Institute, Batch No. 16031611), and 4,5-O-dicaffeoylquinic acid (China Food and Drug Control Institutes, Batch No. 111894-201102), but did not contain Vernonia quinacridin, whereas the anthelmintic Vernonia extract of the present invention contained a large amount of 3,5-O-dicaffeoylquinic acid and Vernonia quinacridin.

[0068] FIG5 is an observation of the therapeutic effect of 1% gel of the present invention on a hydroquinone-induced vitiligo mouse model;

[0069] FIG6 shows the progression of the vitiligo model used in the present invention without drug intervention;

[0070] FIG7 is an observation of the therapeutic effect of the anthelmintic Vernonia microemulsion gel of the present invention on a vitiligo mouse model induced by monobenzone;

[0071] FIG8 shows the effect of the anthelmintic Vernonia microemulsion gel of the present invention on melanin-containing hair follicles in the skin of mice with vitiligo induced by monobenzone.

[0072] FIG9 shows the cumulative transdermal amounts and intradermal retention amounts of the main component Vernonia quinata extract solution, the anthelmintic Vernonia microemulsion gel, and the anthelmintic Vernonia gel in the transdermal test of the present invention; left: cumulative transdermal amount; right: intradermal retention amount.

[0073] FIG10 is a scanning electron micrograph of skin tissue after the transdermal test of the present invention, wherein the scanning electron microscope is at 500 times magnification; A: anthelmintic Vernonia extract solution group; B: anthelmintic Vernonia microemulsion gel group; C: anthelmintic Vernonia gel group. DETAILED DESCRIPTION

[0074] The present invention is not limited to the following examples. Unless otherwise specified, all chemical reagents and chemicals mentioned are of chromatographic or analytical grade, as known and used in the art. All pharmaceutical excipients mentioned are of pharmaceutical grade, as known and used in the art, as known and used in the art. Percentages are by weight unless otherwise specified or clearly contradicted by the context.

[0075] The present invention will be further described below in conjunction with the embodiments:

[0076] Selection of inert solvents

[0077] Considering that Vernonia serrata, the most abundant component in the active fraction of the anthelmintic Vernonia plant, is a sesquiterpenoid with a lactone ring in its structure and is susceptible to hydrolysis in alkaline environments, the first step was to identify an inert solvent that would both stabilize Vernonia serrata and completely dissolve the active fraction. Compatibility testing of commonly used excipients was then conducted. Several solvents were selected (Transcutol P, 60% methanol, 60% ethanol, polyethylene glycol, and acetonitrile as references). The HPLC method was used to measure the changes in Vernonia serrata content in each solvent at room temperature. Appropriate amounts of Vernonia serrata extract were then dissolved in the corresponding solvents. The total organic acid (isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C) content was measured at 0, 10, and 30 days (d), and the rate of change was calculated. The results are shown in Table A.

[0078] Table A. Inert solvent selection criteria

[0079] Therefore, 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.

[0080] Excipient compatibility test

[0081] Transcutol P, which stabilizes Vernonia serrata and fully dissolves the anthelmintic Vernonia extract, was selected as an inert solvent. Commonly used excipients for microemulsions / emulsions were mixed with the anthelmintic Vernonia extract dissolved in Transcutol P. Changes in the content of the main component, Vernonia serrata, and total organic acids (the sum of isochlorogenic acids A, B, and C), were measured at 0, 10, and 30 days after mixing. The evaluation criteria for the main component content change within 10 and 30 days was less than 5.0%, with the change rate = 100% × (0 day content - 30 day content) / 0 day content. Microemulsion excipients with good compatibility were selected for formulation screening and preparation process testing. The results are shown in Table B.

[0082] Table B. Compatibility test of raw materials and excipients of the active part of Vernonia anthelmintica

[0083] After screening, the excipients with good compatibility with the anthelmintic Vernonia extract are: polyoxyethylene 40 hydrogenated castor oil (RH40), polyoxyethylene 35 hydrogenated castor oil (ELP), glyceryl monostearate (GMS) and lecithin.

[0084] Optimization and screening of preparation technology of microemulsion / microemulsion gel

[0085] An external preparation of an anthelmintic Vernonia extract, wherein the microemulsion in the microemulsion gel is prepared by a method comprising the following steps: 1) dissolving the Vernonia extract in diethylene glycol monoethyl ether, and then sequentially adding polyoxyethylene 40 hydrogenated castor oil (or polyoxyethylene 35 hydrogenated castor oil) and olive oil (or almond oil, corn oil or soybean oil) and stirring the mixture to obtain a mixture (oil phase); and

[0086] 2) The mixture in step 1) was stirred and mixed, and then water (aqueous phase) was added dropwise, stirred evenly, and ultrasonicated.

[0087] If a microemulsion gel is to be further formed, 1.0-1.5% of Carbomer 2020 is added to the microemulsion prepared above, and the mixture is allowed to stand until the Carbomer is completely swollen and then stirred evenly to obtain the microemulsion gel.

[0088] A) Selection of preparation method

[0089] A microemulsion with a drug loading of 0.1% (i.e., Vernonia anthelmintica extract) was prepared according to the formulation of Example 7 below. The particle size and PDI of the microemulsions prepared using a high-speed emulsifier, a vortex mixer, and a magnetic stirrer were investigated after balanced stirring for 10 minutes at each maximum speed and ultrasonication for 10 minutes. The results are shown in the table below. The microemulsion prepared using a magnetic stirrer had the lowest particle size and PDI values, so the magnetic stirrer was used to prepare the microemulsions.

[0090] B) Investigation of the order of drug addition

[0091] Microemulsions with drug loadings (i.e., Vernonia anthelmintica extract) of 0.1%, 0.5%, and 1.0% were prepared according to the formulation of Example 7 below. The drug was first dissolved in Transcutol P and then added, and the drug was directly added to a blank microemulsion. The mixture was balanced stirred for 10 minutes with a magnetic stirrer and ultrasonicated for 10 minutes. The effects of the two drug addition sequences on the particle size, PDI, and drug loading capacity of the microemulsion were investigated. The results are shown in the table below. As the drug loading increased, the particle size and PDI values ​​of the microemulsion prepared when the drug was dissolved first were lower. Therefore, it was recommended that the drug be dissolved first before preparing the microemulsion.

[0092] C) Identification and morphology of microemulsions

[0093] After centrifugation, the emulsion prepared according to the above recipe remained clear and transparent, with no visible stratification. It remained a brownish-yellow liquid and could be diluted with distilled water. Upon dilution to a certain extent, it exhibited a light blue opalescence, demonstrating the Tyndall phenomenon, indicating that the sample prepared in this study was a microemulsion. The diffusion rate of methylene blue in both the drug-containing microemulsion and the blank microemulsion was significantly faster than that of Sudan red, indicating that the prepared microemulsion was an oil-in-water (O / W) type. The results are shown in Figure 1.

[0094] D) Microscopic morphology and particle size examination of microemulsion

[0095] The drug-containing microemulsion prepared with the above formula was diluted with purified water and shaken well. One drop of the microemulsion was placed on a copper grid for transmission electron microscopy. After air drying, one drop of 2% phosphotungstic acid was added. The grid was stained for 2-3 minutes and air dried. The microemulsion was then observed under an H-600 transmission electron microscope for morphology. Transmission electron microscopy revealed that the microemulsions were round, uniform, spherical particles with smooth surfaces. There was no aggregation between the microemulsions, indicating good dispersion. As shown in Figure 2, the particle size of the microemulsions was observed to be approximately 10-100 nm, which is consistent with the results of dynamic light scattering (DLS) measurements.

[0096] E) Gelation of microemulsion

[0097] After preparing the microemulsion according to the above microemulsion formula and process, 1.0% Carbomer 2020 was immediately evenly applied to the surface of the microemulsion. The mixture was allowed to stand for complete swelling of the Carbomer 2020, then stirred evenly. The pH value was determined to be 4.8-5.0, and the viscosity was within the range of 50-90 Pa.s. A Vernonia microemulsion gel and a standard Vernonia gel were prepared, both with a drug loading of 1%. Transmission electron microscopy revealed that the microemulsion gel had a much smaller particle size than the standard gel, exhibiting a uniform distribution and lacking adhesion or aggregation. The standard gel had a particle size of approximately 500 nm, with some particles exhibiting clumping. Transmission electron microscopic images of the microemulsion and standard gel are shown in Figure 3: Left: 1% Vernonia microemulsion gel; Right: 1% Vernonia gel.

[0098] Example 1

[0099] Preparation of gel (mass percentage, per 100g):

[0100] a. At room temperature, Carbomer 2020 (Lubrizol ETD 2020 polymer) 1.0% was added to 14.0% water for pre-swelling to obtain a blank gel matrix;

[0101] b. 10.0% of polyoxyethylene 40 hydrogenated castor oil (Beijing Fengli Jingqiu Pharmaceutical Co., Ltd.) and 10.0% of diethylene glycol monoethyl ether (Shanghai MacLean Biochemical Technology Co., Ltd., Transcutol P) were sequentially added to 1.0% of Vernonia anthelmintica extract (prepared by the method described in the Summary of the Invention), and then the gel matrix obtained in step a was added, and the mixture was stirred to obtain a mixed solution.

[0102] c. A weak alkaline salt solution with a pH value of 9.0 was prepared in advance with Na2HPO4 as a neutralizing agent, and 64.0% of the neutralizing agent was added to the mixed solution obtained in step b, and stirred evenly to obtain an anthelmintic Vernonia gel with a pH value of 4.8 and a drug loading of 1.0%.

[0103] Example 2

[0104] Preparation of gel (mass percentage, per 100g):

[0105] a. At room temperature, 1.5% Carbomer 2020 was added to 21.0% water to pre-swell to obtain a blank gel matrix;

[0106] b. 5.0% of polyoxyethylene 40 hydrogenated castor oil and 1.0% of diethylene glycol monoethyl ether were sequentially added to the above-mentioned 0.1% of the anthelmintic Vernonia extract, and then the blank gel matrix obtained in step a was added and stirred to obtain a mixed solution;

[0107] c. A weak alkaline salt solution with a pH value of 9.0 was prepared in advance with K2HPO4 as a neutralizing agent, and 71.4% of the neutralizing agent was added to the mixed solution obtained in step b, and stirred evenly to obtain an anthelmintic Vernonia gel with a pH value of 4.5 and a drug loading of 0.1%.

[0108] Example 3

[0109] Preparation of gel (mass percentage, per 100g):

[0110] a. At room temperature, 1.2% Carbomer 2020 was added to 16.8% water to pre-swell to obtain a blank gel matrix;

[0111] b. 11.0% of polyoxyethylene 40 hydrogenated castor oil and 15.0% of diethylene glycol monoethyl ether were sequentially added to the 4.0% of the above-mentioned Vernonia anthelmintic extract, and then the blank gel matrix obtained in step a was added and stirred to obtain a mixed solution;

[0112] c. A weak alkaline salt solution with a pH value of 9.0 was prepared in advance with NaHCO3 as a neutralizing agent, and 52% of the neutralizing agent was added to the mixed solution obtained in step b, and stirred evenly to obtain an anthelmintic Vernonia gel with a pH value of 4.6 and a drug loading of 4.0%.

[0113] Example 4

[0114] Preparation of gel (mass percentage, per 100g):

[0115] a. At room temperature, add 1.0% Carbomer 2020 to 14.0% water to pre-swell to obtain a gel matrix;

[0116] b. 15.0% of polyoxyethylene 40 hydrogenated castor oil and 1.0% of diethylene glycol monoethyl ether were sequentially added to the 2.0% of the above-mentioned Vernonia anthelmintic extract, and then the gel matrix obtained in step a was added and stirred to obtain a mixed solution;

[0117] c. A weak alkaline salt solution with a pH value of 9.0 was prepared in advance using Na2HPO4 as a neutralizing agent, and 67.0% of the neutralizing agent was added to the mixed solution obtained in step b, and stirred evenly to obtain an anthelmintic Vernonia gel with a pH value of 4.9 and a drug loading of 2.0%.

[0118] Example 5

[0119] Preparation of microemulsion gel (mass percentage, per 100g):

[0120] a. At room temperature, 0.1% of the above-mentioned Vernonia extract was dissolved in 1.0% of diethylene glycol monoethyl ether (Transcutol P), and then 30.0% of polyoxyethylene 40 hydrogenated castor oil and 5.0% of olive oil were added in sequence and stirred to obtain a mixture;

[0121] b. The mixture in step a was stirred and mixed at a rate of 300 r / min using a magnetic stirrer, and then 62.4% water was added dropwise. The mixture was balanced and stirred for 10 min, and ultrasonicated (ultrasonic frequency 40 kHz, amplitude 60%) for 20 min. Carbomer 2020 1.5% was added, and the mixture was allowed to stand until the carbomer was completely swollen, and then stirred evenly to obtain an anthelmintic Vernonia microemulsion gel with a drug loading of 0.1%. The pH value was 4.5 and the viscosity was within the range of 130-140 Pa.S (Shanghai Lichen Instrument Technology Co., Ltd., NDJ-5S digital display viscometer, referring to the first method of viscosity determination in Part IV of the 2020 edition of the Chinese Pharmacopoeia, at 60 r.min -1 The rotation speed is measured).

[0122] Example 6

[0123] Preparation of microemulsion gel (mass percentage, per 100g):

[0124] a. At room temperature, 4.0% of Vernonia anthelmintic extract was dissolved in 15.0% of diethylene glycol monoethyl ether (Transcutol P), and then 5.0% of polyoxyethylene 40 hydrogenated castor oil and 10.0% of olive oil were added in sequence and stirred to obtain a mixture;

[0125] b. The mixture in step a was stirred and mixed at a rate of 600 r / min using a magnetic stirrer, and then 65.0% water was added dropwise. The mixture was balanced and stirred for 30 min, and ultrasonicated for 15 min. Carbomer 2020 1.0% was added. The mixture was allowed to stand until the carbomer was completely swollen, and then stirred evenly to obtain an anthelmintic Vernonia microemulsion gel with a drug loading of 4%. The pH value was 4.7 and the viscosity was within the range of 70-80 Pa.S.

[0126] Example 7

[0127] Preparation of microemulsion gel (mass percentage, per 100g):

[0128] a. At room temperature, 1.0% of Vernonia anthelmintic extract was dissolved in 11.0% of diethylene glycol monoethyl ether (Transcutol P), and then 22.0% of polyoxyethylene 40 hydrogenated castor oil (RH40) and 1.0% of olive oil were added in sequence and stirred to obtain a mixture;

[0129] b. The mixture in step a was stirred and mixed at a rate of 300 r / min using a magnetic stirrer, and then 66% water was added dropwise. The mixture was balanced and stirred for 30 min, and ultrasonicated for 5 min. Carbomer 2020 1.0% was added. The mixture was allowed to stand until the carbomer was completely swollen, and then stirred evenly to obtain an anthelmintic Vernonia microemulsion gel with a drug loading of 1.0%. The pH value was 4.9, the viscosity was within the range of 60-80 Pa.S, the particle size of the microemulsion prepared according to this ratio was 20.09±1.45 nm, and the PDI value was 0.355±0.027.

[0130] The following comparative examples were prepared using the anthelmintic Vernonia gel of Example 1 and the anthelmintic Vernonia microemulsion gel of Example 7 as representatives, respectively, to compare the stability of the main component content of the preparations of the present invention.

[0131] Comparative Example 8

[0132] Preparation of gel (mass percentage, per 100g):

[0133] a. At room temperature, add 1.0% Carbomer 980 to 14.0% water to pre-swell to obtain a gel matrix;

[0134] b. Add 10.0% of polyoxyethylene 40 hydrogenated castor oil and 10.0% of diethylene glycol monoethyl ether to 1.0% of Vernonia anthelmintica extract, then add the gel matrix obtained in step a, stir well, and obtain a mixed solution;

[0135] c. Add 1.3% triethanolamine to 62.7% water, stir evenly, then add to the mixed solution obtained in step b, stir evenly, to obtain an anthelmintic Vernonia gel with a pH value of 6.5 and a drug loading of 1.0%.

[0136] Comparative Example 9

[0137] Preparation of gel (mass percentage, per 100g):

[0138] a. At room temperature, add 1.0% Carbomer 2020 to 14.0% water to pre-swell to obtain a gel matrix;

[0139] b. Add 10.0% of polyoxyethylene 40 hydrogenated castor oil and 10.0% of diethylene glycol monoethyl ether to 1.0% of Vernonia anthelmintica extract, then add the gel matrix obtained in step a, stir well, and obtain a mixed solution;

[0140] c. A solution with a pH value of 9.0 was prepared in advance with NaOH as a neutralizing agent, and 64.0% of the neutralizing agent was added to the mixed solution obtained in step b, and stirred evenly to obtain an anthelmintic Vernonia gel with a pH value of 5.0 and a drug loading of 1.0%.

[0141] Comparative Example 10

[0142] Preparation of microemulsion gel (mass percentage, per 100g):

[0143] a. At room temperature, 1.0% of Vernonia anthelmintic extract was dissolved in 11.0% of diethylene glycol monoethyl ether (Transcutol P), and then 22.0% of polyoxyethylene 40 hydrogenated castor oil and 1.0% of olive oil were added in sequence and stirred to obtain a mixture;

[0144] b. The mixture in step a was stirred and evenly mixed at a rate of 300 r / min using a magnetic stirrer, and 63% water was added dropwise. The mixture was balanced and stirred for 30 min, and ultrasonicated for 20 min. Carbomer 980 1.0% was added, and the mixture was allowed to stand until the carbomer was completely swollen and then stirred evenly. When the mixture was still in a solution state, 1% triethanolamine was added and stirred evenly to obtain a Vernonia anthelmintic microemulsion gel with a drug loading of 1.0%. The pH value thereof was 6.0 and the viscosity was within the range of 80-90 Pa.s.

[0145] Example 11

[0146] Stability of the main component of the external preparation of the anthelmintic Vernonia extract of the present invention:

[0147] The HPLC method was referenced to the method in the literature (Zulipiya Maimaiti. Optimization of the preparation process of the effective component of Vernonia anthelminticum and its quality standard research [D]. Beijing: University of Chinese Academy of Sciences, 2019.), and methodological verification was performed. The HPLC method was used to detect the changes in the contents of 3,5-O-dicaffeoylquinic acid and Vernonia anthelminticum in the gel Example 1, and the comparison of Example 8 and Example 9, as well as the microemulsion gel Example 7 and Example 10 within 90 days; the results are shown in Tables 1 and 2. The results show that the main component content change rate of the Vernonia anthelminticum gel and the Vernonia anthelminticum microemulsion gel prepared by the method of the present invention is less than 5%, indicating that the main component content of the external preparation is stable, while the preparations prepared by conventional methods in Examples 8-10 cannot obtain preparations with stable main component content.

[0148] Example 12

[0149] Transdermal properties of the topical preparation of the anthelmintic Vernonia extract of the present invention:

[0150] The transdermal rate was studied by diffusion cell. Healthy rats with qualified body weight (SD rats, purchased from the Animal Experiment Center of Xinjiang Medical University, production license number: SCXK (new) 2018-0003) were taken, killed by cervical dislocation, and the hair on the abdomen was pushed; the abdominal skin was cut off, and the short hair, subcutaneous tissue and fascia on the skin were removed, and the integrity of the skin stratum corneum was ensured by observation under a magnifying glass; it was rinsed clean with normal saline and set aside. The skin was clamped in a double-chamber diffusion cell, with the stratum corneum facing the supply cell and the dermis facing the receiving solution; 1 ml of sample was added to the supply cell to contact the skin stratum corneum; the samples were 1% anthelmintic Vernonia extract solution, 1% anthelmintic Vernonia microemulsion gel, and 1% anthelmintic Vernonia gel dissolved in the receiving solution; a normal saline solution containing 20% ​​ethanol was used as the receiving medium, kept warm in a 37°C water bath, and stirred at a speed of 400r / min; 1 mL of sample was taken at 0, 2, 4, and 6 h, respectively, and the same solution kept warm at 37°C was supplemented at the same time. A blank receiving solution was measured, and the active ingredient content in the receiving solution was determined according to the content determination method; after 6 hours, the skin was removed and the test substance remaining on the skin surface was carefully removed. The test substance was then chopped, homogenized, and filtered in 1 mL of a normal saline solution containing 20% ​​ethanol, and then injected and tested according to the above-mentioned HPLC method. The cumulative transdermal amount and the intradermal retention amount after 6 hours were calculated. The results are shown in Figure 9, which shows the left side: the cumulative transdermal amount curves of the Vernonia anthelmintica extract solution and the three preparations; the right side: the intradermal retention amount after 6 hours of exposure to the Vernonia anthelmintica extract solution and each preparation;

[0151] Results and conclusion: As shown in Figure 9, the main component in the Vernonia anthelmintic extract solution penetrates the skin in large quantities in a very short period of time, and the intradermal retention amount is extremely low. Compared with the extract solution, both the Vernonia anthelmintic microemulsion gel and the Vernonia anthelmintic gel can significantly increase the intradermal retention amount of the main component, indicating that the preparation product prepared by the present invention can increase the intradermal retention amount of the main component, which is beneficial to the exertion of the drug efficacy.

[0152] Carefully remove each skin sample after transdermal treatment and wipe the skin surface repeatedly with a water-alcohol cotton ball to ensure that the drug on the skin surface is completely removed. Be careful to avoid damage to the skin surface caused by human manipulation. The pre-treated skin was fixed in 2.5% glutaraldehyde fixative overnight and fixed with 0.1 mol·L -1After rinsing three times with phosphate buffer, the skin was dehydrated with ethanol-water solutions of varying volume fractions (10%, 30%, 50%, 75%, and 100%), each dehydration step lasting 15-20 minutes. Dehydration was then repeated twice with pure ethanol, followed by a gradient transition to acetone. The skin samples were freeze-dried and gold-coated using ion sputtering. The skin samples were observed and photographed under a scanning electron microscope. The surface morphology of the skin under an electron microscope is shown in Figure 10. In the transdermal group treated with the Vernonia microemulsion gel, slightly regular depressions with wrinkled walls were observed. In the transdermal group treated with the gel, depressions were observed, but to a lesser degree, with less pronounced wrinkles. In the skin samples treated with the Vernonia extract solution, the depressions were essentially eliminated, the stratum corneum expanded outward, and the permeability increased. This suggests that the Vernonia extract solution exhibits some skin irritation. Formulating the Vernonia extract into a microemulsion gel or gel can increase intradermal retention and reduce skin irritation.

[0153] Example 13

[0154] The effectiveness of the external preparation gel of the anthelmintic Vernonia extract of the present invention:

[0155] Study on the effect of Vernonia anthelmintic gel on hydroquinone-induced vitiligo model in mice:

[0156] The extract of Vernonia anthelmintica was used as the raw material drug, and a gel with a drug loading of 1% was prepared according to the method of the present invention (see Example 1). The effectiveness of the Vernonia anthelmintica gel was preliminarily investigated using a hydroquinone-induced vitiligo mouse model (the preparation method of the model is described in reference: Gao Li, Huo Shixia, Peng Xiaoming, et al. Effects of galangin on hydroquinone-induced vitiligo mouse model [J]. Chinese Herbal Medicine, 2014, 45(16): 2358-2363.);

[0157] Starting from the 21st day of modeling, each group started to apply medication 1 hour after applying 5% hydroquinone: the blank control group and the vitiligo model group were given an equal volume of distilled water; the medication group was given anthelmintic Vernonia gel, and the positive control group was given an equal volume of compound Kali Cumin tincture (Xinjiang Wei'atang Pharmaceutical Co., Ltd., national medicine standard Z65020003), twice a day for 21 consecutive days; after the 21st day of medication, the efficacy of each group was evaluated, and the area 3 cm from the center of the medication site was selected for evaluation. 2 The results were divided into two groups: excellent (1) when the pigmentation of the test area basically returned to normal; good (2) when the pigmentation area of ​​the test area was >50%; fair (3) when the pigmentation area of ​​the test area was <50%; and poor (4) when the skin of the test area was pale or white. The total effective rate was calculated as excellent plus good. The effective rates are shown in Table 3.

[0158] Table 3 Statistics on the efficacy of Vernonia gel

[0159] Results and conclusion: As shown in Figure 5, during the modeling process, the skin color of the test area of ​​the modeling mice in each group was pale and the hair grew slowly. As the modeling time prolonged, white spots appeared and the new hair turned white. After treatment, the skin color of the test area of ​​the vitiligo mice in the positive control group and the 1% gel group recovered to varying degrees, and the new hair also recovered to black. After 21 days of administration, the anthelmintic Vernonia gel with a drug loading of 1% had an efficacy of about 60% for vitiligo model mice, which was better than the therapeutic effect of the compound Kali cumin tincture during the same period.

[0160] Example 14

[0161] The effectiveness of the microemulsion gel of the external preparation of the anthelmintic Vernonia extract of the present invention:

[0162] Study on the effect of Vernonia anthelmintic microemulsion gel (preparation method see Example 7) on the vitiligo model of mice induced by Monobenzone (preparation method of the model see reference: Zhu Yiping, Jin Rong, Wang Suiquan et al. Study on the mechanism of action of baicalin on vitiligo mouse model [J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2017, 22(01): 27-32.)

[0163] Grouping and drug intervention:

[0164] Starting from the 21st day of modeling, each group was treated with monobenzone cream (homemade, with a mass fraction of 40% monobenzone in the cream; monobenzone was purchased from Sigma-Aldrich, product number 158348-100g, batch number STBK2160) daily (wipe it off with a cotton swab soaked in water before administration). 4 hours later, the drug was administered according to the set dose and frequency in Table 4: administration for 30 days;

[0165] Table 4: Dosage methods for each group

[0166] Note: VAF microemulsion gel - anthelmintic Vernonia microemulsion gel

[0167] Results and Conclusion: Starting from the 10th day of modeling, local whitening of the skin in the test area of ​​the mice was observed, with small areas of white spots appearing. The hair growth rate was significantly slower than that of the blank group. As the modeling time prolonged, white spots appeared in the test area of ​​the model animals to varying degrees, and white hair grew. After the 40th day, the animals in the model group began to lose color in the hair of non-modeling areas, and the tail and ears showed pinpoint discoloration. The results are shown in Figure 6.

[0168] From the 10th day after microemulsion gel intervention, black spots were observed on the skin of the animals in the microemulsion gel groups. As the drug intervention time prolonged, the skin color gradually recovered, the color of the new hair gradually turned black, and the hair growth rate was significantly faster than that of the model group. On the 50th day of the experiment, a 3cm center of the drug site was selected. 2The results were summarized as follows: excellent: the pigmentation of the test area basically returned to normal; good: the pigmentation area of ​​the test area was >50%; fair: the pigmentation area of ​​the test area was <50%; poor: the skin of the test area was pale or white-spotted; the total effective rate was calculated as excellent plus good; the visual observation scoring results showed that the efficacy of the medium-dose and high-dose VAF microemulsion gel was better than that of the positive control group, as shown in Table 5 and Figure 7.

[0169] Melanin staining:

[0170] Skin tissue was embedded in paraffin and stained for melanin. Hair follicles were observed under a 200x microscope, and the number of melanin-containing follicles per 50 follicles was counted. Data were analyzed using GraphPad Prism 6.0 statistical software. Means between groups were compared using one-way analysis of variance. P < 0.05 was considered significant.

[0171] The blank control group had a large number of hair follicles in the epidermal tissue, high pigment content and dark color; no obvious pigmentation was found in the hair follicles of most model group animals, and the number of melanin-containing hair follicles was significantly reduced, and the difference was statistically significant (P<0.05); compared with the vitiligo model group and the blank gel group, after treatment with VAF microemulsion gel, the number of melanin-containing hair follicles in the skin of the low, medium and high dose groups was significantly increased, and the difference was statistically significant (P<0.05). The increase in the number of melanin-containing hair follicles in the back skin of the treated mice had a dose-effect relationship, as shown in Figure 8 and Table 5.

[0172] Table 5 Observation on the therapeutic effect of VAF microemulsion gel on the vitiligo mouse model induced by monobenzone

[0173] Note: Compared with the blank control group, # P<0.05; compared with the model group, * P<0.05, ## P<0.01.

Claims

1. An external preparation of an anthelmintic Vernonia extract, comprising Vernonia oleracea and 3,5-O-dicaffeoylquinic acid as active ingredients, in the form of a gel or microemulsion gel, wherein: 1) The gelling agent comprises, based on 100 g: Carbomer 2020 1.0-1.5%; Water 13.5-40.0%; Vernonia anthelmintic extract 0.1-4.0%; Polyoxyl 40 hydrogenated castor oil or polyoxyl 35 hydrogenated castor oil 5.0-15.0%; Diethylene glycol monoethyl ether 1.0-15.0%; and Neutralizer; The pH value of the gel is 4.3-5.

5. The condition is that the sum of the contents of each component is 100%; 2) The microemulsion gel comprises, per 100 g, the following: Vernonia anthelmintic extract 0.1-4.0%; Diethylene glycol monoethyl ether 1.0-15.0%; Polyoxyl 40 hydrogenated castor oil or polyoxyl 35 hydrogenated castor oil 5.0-30.0%; Olive oil, almond oil, corn oil or soybean oil 1.0-10.0%; Water 40.0-84.0%; and Carbomer 2020 1.0-1.5%, The condition is that the sum of the contents of each component is 100%.

2. The external preparation of the anthelmintic Vernonia extract according to claim 1, which is a gel, wherein the neutralizer is a weakly alkaline saline solution with a pH value of 8.0-10.0, preferably 9.0, such as a weakly alkaline saline solution with a pH value of preferably 9.0 prepared from an alkali metal acid salt such as Na2HPO4, K2HPO4, NaHCO3 or a combination thereof.

3. The external preparation of the anthelmintic Vernonia extract according to claim 2, wherein the gel is prepared by a method comprising the following steps: 1) Carbomer 2020 was added into water for swelling to obtain a blank gel matrix; 2) adding polyoxyethylene 40 hydrogenated castor oil (or polyoxyethylene 35 hydrogenated castor oil) and diethylene glycol monoethyl ether to the anthelmintic Vernonia extract in sequence, and then adding the gel matrix obtained in step 1), stirring evenly to obtain a mixture; 3) Adding a neutralizing agent to the mixture obtained in step 2), stirring the mixture evenly to make the pH value of the total system be 4.3-5.5, thereby obtaining a gelling agent.

4. The external preparation of the anthelmintic Vernonia extract according to claim 1, which is an oil-in-water (O / W) microemulsion gel, having the following characteristics: 1) pH 4.0-6.5, such as 4.0-5.5, such as 4.5, 4.7 and 4.9; 2) Viscosity is in the range of 20-160Pa.S, such as 30-90Pa.S, 70-80Pa.S, 60-80Pa.S and 130-140Pa.S; 3) The particle size is 10nm-100nm and the PDI (polymer dispersibility index) value is less than 0.50 as measured by a dynamic light scattering particle size analyzer.

5. The external preparation of the anthelmintic Vernonia extract according to claim 4, wherein the microemulsion gel is prepared by a method comprising the following steps: 1) adding the anthelmintic Vernonia extract into diethylene glycol monoethyl ether to dissolve, and then adding polyoxyethylene 40 hydrogenated castor oil (or polyoxyethylene 35 hydrogenated castor oil) and olive oil (or almond oil, corn oil or soybean oil) in sequence and stirring evenly to obtain a mixture; 2) After stirring and mixing the mixture in step 1), water is added dropwise, stirred evenly, subjected to ultrasonic treatment, and 1.0-1.5% of Carbomer 2020 is added. The mixture is allowed to stand until the Carbomer is completely swollen and then stirred evenly to obtain a microemulsion gel.

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

7. An external preparation of an anthelmintic Vernonia extract, characterized in that The preparation is made of the extract of Vernonia anthelmintica and pharmaceutically acceptable excipients to form a gel or microemulsion gel, with 100g as the base. The specific operation is carried out according to the following steps: Preparation of gel: a. According to the mass percentage, at room temperature, add 1.0-1.5% of Carbomer 2020 to 13.5-40.0% of water to swell and 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 to 0.1-4.0% of the anthelmintic Vernonia extract, and then add the gel matrix obtained in step a, stir evenly, and obtain a mixture; c. Prepare a weak alkaline salt solution with a pH value of 9.0 using Na2HPO4, K2HPO4 or NaHCO3 as a neutralizer in advance, add the neutralizer to the mixture obtained in step b, stir evenly, and make the pH value of the total system 4.3-5.5, so as to obtain a gelling agent; Preparation of microemulsion gel: a. According to mass percentage, at room temperature, 0.1-4.0% of the Vernonia extract is added to 1.0-15.0% of diethylene glycol monoethyl ether to dissolve, and then 5.0-30.0% of polyoxyethylene 40 hydrogenated castor oil (or polyoxyethylene 35 hydrogenated castor oil) and 1.0-10.0% of olive oil (or almond oil, corn oil or soybean oil) are added in sequence and stirred evenly to obtain a mixture; b. Stir the mixture in step a at a rate of 300-600 r / min with a magnetic stirrer, add 40.0-84.0% water dropwise, stir evenly for 10-30 min, ultrasonicate for 5-20 min, add 1.0-1.5% carbomer 2020, let stand until the carbomer is completely swollen, and then stir evenly to obtain a microemulsion gel.

8. A method for preparing an external preparation of an anthelmintic Vernonia extract, characterized in that Follow these steps: Preparation of gel: a. According to the mass percentage, at room temperature, add 1.0-1.5% of Carbomer 2020 to 13.5-40.0% of water to swell and 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 to 0.1-4.0% of the anthelmintic Vernonia extract, and then add the gel matrix obtained in step a, stir evenly, and obtain a mixture; c. Prepare a weak alkaline salt solution with a pH value of 9.0 using Na2HPO4, K2HPO4 or NaHCO3 as a neutralizer in advance, add the neutralizer to the mixture obtained in step b, stir evenly, and make the pH value of the total system 4.3-5.5, so as to obtain a gelling agent; Preparation of microemulsion gel: a. According to mass percentage, at room temperature, 0.1-4.0% of the Vernonia extract is added to 1.0-15.0% of diethylene glycol monoethyl ether to dissolve, and then 5.0-30.0% of polyoxyethylene 40 hydrogenated castor oil (or polyoxyethylene 35 hydrogenated castor oil) and 1.0-10.0% of olive oil (or almond oil, corn oil or soybean oil) are added in sequence and stirred evenly to obtain a mixture; b. Stir the mixture in step a at a rate of 300-600 r / min with a magnetic stirrer, add 40.0-84.0% water dropwise, stir evenly for 10-30 min, perform ultrasound for 5-20 min, add 1.0-1.5% Carbomer 2020, and let stand. After the carbomer is completely swollen, stir evenly to obtain a microemulsion gel.

9. The insect repellent Vernonia gel according to claim 1 or 7, characterized in that: The main ingredients of the preparation, vernonia quinine and 3,5-O-dicaffeoylquinic acid, have percentages of 0.15-0.20% and 0.10-0.15% respectively.

10. Use of the external preparation of the anthelmintic Vernonia extract according to any one of claims 1 to 7 or the external preparation of the anthelmintic Vernonia extract obtained according to the method of claim 8 in the preparation of drugs for treating skin pigment loss such as vitiligo.

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