An external preparation of anthelmintic Vernonia extract and preparation method thereof

Abstract

The present invention relates to an external preparation of an anthelmintic Vernonia extract and a preparation method thereof. The method uses the anthelmintic Vernonia extract as a raw material drug to prepare an external preparation of a gel and a microemulsion gel. The gel and microemulsion gel obtained by the method of the present invention have uniform appearance and stable physical and chemical properties, and the content of the main components, Vernonia oleracea and 3,5-dicaffeoylquinic acid, is stable as determined by HPLC; and the intradermal retention and cumulative transdermal amount of the main components of the external preparations such as the prepared gel and microemulsion gel are evaluated through a transdermal test, and it is determined that the microemulsion gel has a higher intradermal retention capacity and is very suitable for topical administration treatment. The present invention uses Vernonia oleracea and 3,5-O-dicaffeoylquinic acid as the main components of the preparation for the first time, and prepares an external preparation with uniform appearance and stable content.

Description

Technical Field

[0001] The invention relates to an external preparation of anthelmintic Vernonia extract and a preparation method thereof, wherein the external preparation of gel and microemulsion gel is prepared by taking the anthelmintic Vernonia extract as raw material medicine. Background Art

[0002] Vitiligo is a localized or generalized lesion caused by the progressive reduction or disappearance of melanosome production due to the decrease or disappearance of tyrosinase activity in melanocytes in the skin and hair follicles. Human skin is composed of the epidermis, dermis and subcutaneous tissue. The production of melanin is located in the deepest part of the epidermis - the basal layer. For vitiligo, the basal layer of the skin is the target site of the topical drug delivery system, and the stratum corneum covering the surface of the skin is the main barrier to the penetration of the drug delivery system. In order to restore the color of the vitiligo site, the active substance must pass through the stratum corneum to reach the basal layer to exert its pharmacological effect. Therefore, the most suitable dosage form for improving local vitiligo is an external preparation. Vernonia Anthelmintica is the core medicinal material used by 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 supplementing trace elements. The research group conducted chemical analysis on the main components of Vernonia anthelmintica and confirmed that the effective components that promote black coloration are sesquiterpenes, quinic acid and flavonoids. Then, Vernonia anthelmintica and 3,5-O-dicaffeoylquinic acid were used as enrichment target components to optimize the extraction and purification process of Vernonia anthelmintica. The prepared Vernonia anthelmintica extract contains 15-25% (w / w) Vernonia anthelmintica and 10-20% (w / w) 3,5-O-dicaffeoylquinic acid. HPLC detection showed that the commercially available Vernonia anthelmintica injection contained 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid and 4,5-O-dicaffeoylquinic acid, but no Vernonia anthelmintica. The Vernonia anthelmintica extract used in the present invention contains higher Vernonia anthelmintica and 3,5-O-dicaffeoylquinic acid. Summary of the invention

[0003] The present invention aims to provide an external preparation of an anthelmintic Vernonia extract and a preparation method thereof. The preparation is an external preparation of a gel and a microemulsion gel prepared from the anthelmintic Vernonia extract as a raw material drug. The gel and the microemulsion gel obtained by the method of the present invention have uniform appearance and stable physical and chemical properties, and the content of the main components, Vernonia serrata and 3,5-dicaffeoylquinic acid, is stable as determined by HPLC. The intradermal retention and cumulative transdermal amount of the main components of the external preparations such as the prepared gel and microemulsion gel are evaluated by transdermal test research, and it is determined that the microemulsion gel has a high intradermal retention capacity and is very suitable for local administration treatment. The present invention is the first to use Vernonia serrata and 3,5-O-dicaffeoylquinic acid as the main components of the preparation to prepare an external preparation with uniform appearance and stable content.

[0004] The invention discloses an external preparation of an anthelmintic Vernonia extract and a preparation method thereof. The preparation is prepared from the anthelmintic Vernonia extract and a commonly used pharmaceutical auxiliary material to form a gel or microemulsion gel, with 100 g as the base. The specific operation is carried out according to the following steps:

[0005] Preparation of gel:

[0006] a. According to the mass percentage, at room temperature, 1.0-1.5% of Carbomer 2020 is added to 13.5-40.0% of water to swell and obtain a gel matrix;

[0007] b. Add 5.0-15.0% of polyoxyethylene 40 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;

[0008] 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 mixed solution 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;

[0009] Preparation of microemulsion gel:

[0010] a. According to the mass percentage, at room temperature, 0.1-4.0% of the anthelmintic 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 and 1.0-10.0% of olive oil are added in sequence and stirred evenly to obtain a mixture;

[0011] 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, balance stirring for 10-30 min, ultrasonicate for 5-20 min, add 1.0-1.5% carbomer 2020, let stand, stir evenly after carbomer 2020 is completely swollen, and obtain a microemulsion gel.

[0012] A method for preparing an external preparation of an anthelmintic Vernonia extract is carried out according to the following steps, based on 100 g:

[0013] Preparation of gel:

[0014] a. According to the mass percentage, at room temperature, 1.0-1.5% of Carbomer 2020 is added to 13.5-40.0% of water to swell and obtain a gel matrix;

[0015] b. Add 5.0-15.0% of polyoxyethylene 40 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;

[0016] 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 mixed solution 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;

[0017] Preparation of microemulsion gel:

[0018] a. According to the mass percentage, at room temperature, 0.1-4.0% of the anthelmintic 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 and 1.0-10.0% of olive oil are added in sequence and stirred evenly to obtain a mixture;

[0019] 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.

[0020] The external preparation of the anthelmintic Vernonia extract has main components, Vernonia quinic acid and 3,5-O-dicaffeoylquinic acid, whose mass percentages are 0.15-0.20% and 0.10-0.15% respectively.

[0021] The external preparation of the anthelmintic Vernonia extract obtained by the method is used in preparing drugs for treating vitiligo and skin pigment loss.

[0022] The anthelmintic Vernonia extract external preparation of the present invention, the anthelmintic Vernonia extract in the preparation is obtained from the preparation method and anti-inflammatory use of anthelmintic Vernonia phenolic acid fraction in Chinese patent CN201510288742.3, and the specific operation is carried out according to the following steps:

[0023] The dried anthelmintic Vernonia medicinal material is crushed by a Chinese medicine flattening machine, weighed, and subjected to reflux extraction at a material-liquid ratio of 1:12, with 80% ethanol as the solvent, extraction temperature of 75-80°C, extraction time of 3 hours each time, extraction twice, filtration, and filtrate combination. After decompression concentration at 50-60°C to 21g of crude drug equivalent in every 100mL of extract, purification is then performed by column chromatography: the extract is dispersed in water and then loaded, the loading concentration is about 10g of crude drug equivalent in every 100mL of loading solution, impurities are removed by 3BV water, eluted by 3BV 60% ethanol and the eluate is collected, and then concentrated by decompression at 60°C and vacuum dried at 50°C to obtain the anthelmintic Vernonia extract; and HPLC method is used to detect that the anthelmintic Vernonia extract contains 15-25% of Vernonia quinic acid and 10-20% of 3,5-O-dicaffeoylquinic acid.

[0024] The present invention uses the anthelmintic Vernonia extract prepared by the research group in the early stage as the raw material medicine, and uses modern pharmaceutical preparation technology to make 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 component Vernonia oleracea in the preparation with the highest content is Vernonia oleracea, which is not found in the commercially available Vernonia injection (see attached Figure 1 ); and in terms of its advanced properties, the preparation can significantly increase the intradermal retention of the main ingredient and reduce skin irritation (see attached Figure 7-8 ); and from the perspective of its preparation method alone, the preparation method of the external preparation involved in the present invention has simple steps, simple operation, easy control of parameters, and low cost, and is suitable for large-scale industrial production. Moreover, the external preparation is easier to use than the injection, has strong compliance, and is more suitable for extensive clinical application. At the same time, it is undoubtedly a very meaningful work to fully tap and utilize Xinjiang Uyghur medicine resources.

[0025] The difference between the present invention and the prior art is:

[0026] 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 without a neutralizer by adjusting the mass fraction of CMC-Na to prepare a gel with suitable viscosity, but it becomes a solution after being placed for 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 made into a gel or microemulsion gel with a conventional carbomer matrix such as carbomer 940, carbomer 971, carbomer 980, etc., 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 the viscosity gradually decreases after exceeding pH = 9. However, compared with commonly used models such as Carbomer 940, Carbomer 971, and Carbomer 980, it can also maintain a higher viscosity when the pH is lower than 5.

[0027] When Carbomer 2020 was used as the matrix material, the effects of adjusting pH value and viscosity of the preparation with different neutralizers were investigated.

[0028] ① When NaOH was used as a neutralizing agent and the pH was adjusted to 5.5-7.0, a gel with suitable viscosity could be obtained, but the content of Vernonia oleracea in the product decreased by about 20% after 10 days;

[0029] 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 Vernonia oleracea in the product decreased by about 40% after 10 days.

[0030] 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 Vernonia oleracea in the product decreased by about 45% after 10 days.

[0031] 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.

[0032] ⑤ After the microemulsion gel system is made into a microemulsion, Carbomer 2020 is directly added into the microemulsion. By adjusting the dosage 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

[0033] Figure 1 The invention is to distinguish the components of the commercial Vernonia injection and the anthelmintic Vernonia extract of the present invention; through HPLC detection, the commercial Vernonia injection contains 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid and 4,5-O-dicaffeoylquinic acid, but does not contain Vernonia quinine, while the anthelmintic Vernonia extract of the present invention contains a large amount of 3,5-O-dicaffeoylquinic acid and Vernonia quinine;

[0034] Figure 2 The microemulsion gel and common gel of the present invention under transmission electron microscope, wherein the left: 1% anthelmintic Vernonia microemulsion gel; the right: 1% anthelmintic Vernonia gel; the particle size of the present invention is much smaller than that of the common gel, and the distribution is uniform, without adhesion and aggregation, while the particle size of the common gel is about 500nm, and some particles are aggregated in blocks;

[0035] Figure 3 This is the observation of the therapeutic effect of the 1% gel of the present invention on the vitiligo model of mice induced by hydroquinone;

[0036] Figure 4 This is the progression change of the vitiligo model used in the present invention without drug intervention;

[0037] Figure 5 The efficacy of the anthelmintic Vernonia microemulsion gel in the present invention on the vitiligo mouse model induced by monobenzone is observed;

[0038] Figure 6 The present invention shows the effect of the anthelmintic Vernonia microemulsion gel on the melanin-containing hair follicles in the skin of mice with vitiligo induced by monobenzone.

[0039] Figure 7 The cumulative transdermal amounts and intradermal retention amounts of the main component Vernonia oleracea in the anthelmintic Vernonia 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.

[0040] Figure 8 The scanning electron microscope pictures of skin tissue after the transdermal test of the present invention are shown, wherein the scanning electron microscope is 500 times; A: anthelmintic Vernonia extract solution group; B: anthelmintic Vernonia microemulsion gel group; C: anthelmintic Vernonia gel group. DETAILED DESCRIPTION

[0041] The present invention is not limited by the following embodiments. Unless otherwise specified, the various chemical reagents and chemicals mentioned are all chromatographically pure or analytically pure products known in the prior art; the various pharmaceutical excipients mentioned are all pharmaceutical grade products known in the prior art unless otherwise specified.

[0042] The present invention will be further described below in conjunction with embodiments:

[0043] Example 1

[0044] Preparation of gel (mass percentage, based on 100g):

[0045] a. At room temperature, add 1.0% Carbomer 2020 to 14.0% water for pre-swelling to obtain a blank gel matrix;

[0046] b. Add 10.0% of polyoxyethylene 40 hydrogenated castor oil and 10.0% of diethylene glycol monoethyl ether to 1.0% of the anthelmintic Vernonia extract in sequence, and then add the gel matrix obtained in step a, stir evenly, and obtain a mixed solution;

[0047] c. Prepare a weak alkaline salt solution with a pH value of 9.0 using Na2HPO4 as a neutralizer in advance, add 64.0% of the neutralizer to the mixed solution obtained in step b, and stir evenly to obtain an anthelmintic Vernonia gel with a pH value of 4.8 and a drug loading of 1.0%.

[0048] Example 2

[0049] Preparation of gel (mass percentage, based on 100g):

[0050] a. At room temperature, add 1.5% Carbomer 2020 to 21.0% water for pre-swelling to obtain a blank gel matrix;

[0051] b. Add 5.0% of polyoxyethylene 40 hydrogenated castor oil and 1.0% of diethylene glycol monoethyl ether to 0.1% of the anthelmintic Vernonia extract in sequence, and then add the blank gel matrix obtained in step a, stir evenly, and obtain a mixed solution;

[0052] c. Use K2HPO4 to prepare a weak alkaline salt solution with a pH value of 9.0 as a neutralizer in advance, add 71.4% of the neutralizer to the mixed solution obtained in step b, and stir evenly to obtain an anthelmintic Vernonia gel with a pH value of 4.5 and a drug loading of 0.1%.

[0053] Example 3

[0054] Preparation of gel (mass percentage, based on 100g):

[0055] a. At room temperature, add 1.2% Carbomer 2020 to 16.8% water for pre-swelling to obtain a blank gel matrix;

[0056] b. Add 11.0% of polyoxyethylene 40 hydrogenated castor oil and 15.0% of diethylene glycol monoethyl ether to 4.0% of the anthelmintic Vernonia extract in sequence, and then add the blank gel matrix obtained in step a, stir evenly, and obtain a mixed solution;

[0057] c. Prepare a weak alkaline salt solution with a pH value of 9.0 using NaHCO3 as a neutralizer in advance, add 52% of the neutralizer to the mixed solution obtained in step b, and stir evenly to obtain an anthelmintic Vernonia gel with a pH value of 4.6 and a drug loading of 4.0%.

[0058] Example 4

[0059] Preparation of gel (mass percentage, based on 100g):

[0060] a. At room temperature, add 1.0% Carbomer 2020 to 14.0% water for pre-swelling to obtain a blank gel matrix;

[0061] b. Add 15.0% of polyoxyethylene 40 hydrogenated castor oil and 1.0% of diethylene glycol monoethyl ether to 2.0% of the anthelmintic Vernonia extract in sequence, and then add the gel matrix obtained in step a, stir evenly, and obtain a mixed solution;

[0062] c. Prepare a weak alkaline salt solution with a pH value of 9.0 using Na2HPO4 as a neutralizer in advance, add 67.0% of the neutralizer to the mixed solution obtained in step b, and stir evenly to obtain an anthelmintic Vernonia gel with a pH value of 4.9 and a drug loading of 2.0%.

[0063] Example 5

[0064] Preparation of microemulsion gel (mass percentage, based on 100g):

[0065] a. At room temperature, 0.1% of Vernonia extract was added to 1.0% of diethylene glycol monoethyl ether (Transcutol P) to dissolve, and then 30.0% of polyoxyethylene 40 hydrogenated castor oil and 5.0% of olive oil were added in sequence and stirred evenly to obtain a mixture;

[0066] b. The mixture in step a was stirred and mixed at a rate of 300 r / min by a magnetic stirrer, and then 62.4% of water was added dropwise. The mixture was balanced and stirred for 10 min, and ultrasonicated for 20 min. 1.5% of carbomer 2020 was added, and the mixture was allowed to stand. After the carbomer was completely swollen, the mixture was stirred evenly to obtain an anthelmintic Vernonia microemulsion gel with a drug loading of 0.1%. The pH value thereof was 4.5, and the viscosity was within the range of 130-140 Pa.S.

[0067] Example 6

[0068] Preparation of microemulsion gel (mass percentage, based on 100g):

[0069] a. At room temperature, 4.0% of Vernonia anthelmintic extract was added to 15.0% of diethylene glycol monoethyl ether (Transcutol P) to dissolve, and then 5.0% of polyoxyethylene 40 hydrogenated castor oil and 10.0% of olive oil were added in sequence and stirred evenly to obtain a mixture;

[0070] b. The mixture in step a was stirred and mixed at a rate of 600 r / min by a magnetic stirrer, and then 65.0% of water was added dropwise, and balanced stirring was performed for 30 min. Ultrasound was performed for 15 min, and 1.0% of carbomer 2020 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 thereof was 4.7, and the viscosity was within the range of 70-80 Pa.S.

[0071] Example 7

[0072] Preparation of microemulsion gel (mass percentage, based on 100g):

[0073] a. At room temperature, 1.0% of Vernonia anthelmintic extract was added to 11.0% of diethylene glycol monoethyl ether (Transcutol P) to dissolve, and then 22.0% of polyoxyethylene 40 hydrogenated castor oil and 1.0% of olive oil were added in sequence and stirred evenly to obtain a mixture;

[0074] b. The mixture in step a was stirred and mixed at a rate of 300 r / min with a magnetic stirrer, and then 64% water was added dropwise. The mixture was balanced and stirred for 30 min, and ultrasonicated for 5 min. 1.0% of carbomer 2020 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 and the viscosity was within the range of 60-80 Pa.S.

[0075] The following comparative examples were prepared by conventional methods 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 content of the main components of the preparations of the present invention.

[0076] Example 8 Comparison

[0077] Preparation of gel (mass percentage, based on 100g):

[0078] a. At room temperature, add 1.0% Carbomer 980 to 14.0% water to pre-swell to obtain a gel matrix;

[0079] b. Add 10.0% of polyoxyethylene 40 hydrogenated castor oil and 10.0% of diethylene glycol monoethyl ether to 1.0% of the anthelmintic Vernonia extract in sequence, and then add the gel matrix obtained in step a, stir evenly, and obtain a mixed solution;

[0080] 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%.

[0081] Example 9 Comparison

[0082] Preparation of gel (mass percentage, based on 100g):

[0083] a. At room temperature, add 1.0% Carbomer 2020 to 14.0% water to pre-swell to obtain a gel matrix;

[0084] b. Add 10.0% of polyoxyethylene 40 hydrogenated castor oil and 10.0% of diethylene glycol monoethyl ether to 1.0% of the anthelmintic Vernonia extract in sequence, and then add the gel matrix obtained in step a, stir evenly, and obtain a mixed solution;

[0085] c. Prepare a solution with a pH value of 9.0 using NaOH as a neutralizer in advance, add 64.0% of the neutralizer to the mixed solution obtained in step b, and stir evenly to obtain an anthelmintic Vernonia gel with a pH value of 5.0 and a drug loading of 1.0%.

[0086] Example 10 Comparison

[0087] Preparation of microemulsion gel (mass percentage, based on 100g):

[0088] a. At room temperature, 1.0% of Vernonia anthelmintic extract was added to 11.0% of diethylene glycol monoethyl ether (Transcutol P) to dissolve, and then 22.0% of polyoxyethylene 40 hydrogenated castor oil and 1.0% of olive oil were added in sequence and stirred evenly to obtain a mixture;

[0089] b. The mixture in step a was stirred and mixed at a rate of 300 r / min by a magnetic stirrer, and then 63% of water was added dropwise, balanced stirring was performed for 30 min, and ultrasonication was performed for 20 min. 1.0% of carbomer 980 was added, and the mixture was allowed to stand until the carbomer was completely swollen, and then stirred evenly. At this time, it was still in a solution state, and 1% of triethanolamine was added and stirred evenly to obtain an anthelmintic Vernonia 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 after testing.

[0090] Embodiment 11

[0091] Stability of main components of the external preparation of the anthelmintic Vernonia extract of the present invention:

[0092] The HPLC method was based on the method in the reference (Zulipiya Maimaiti. Optimization of the preparation process of the effective components of Vernonia anthelminticum and its quality standard research [D]. Beijing: University of Chinese Academy of Sciences, 2019.), and the 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 of the present invention, 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 <5%, indicating that the main component content of the external preparation is stable, while the comparison of Examples 8-10 prepared by the conventional method cannot obtain a preparation with a stable main component content;

[0093] Table 1 Changes in the content of main components of the anthelmintic Vernonia gel in 90 days

[0094]

[0095] Table 2 Changes in the main component content of the anthelmintic Vernonia microemulsion gel within 90 days

[0096]

[0097] Example 12

[0098] The transdermal properties of the topical preparation of the anthelmintic Vernonia extract of the present invention are as follows:

[0099] The permeation rate was studied by diffusion cell. Healthy rats with qualified body weight were taken and killed by cervical dislocation. The hair on the abdomen was removed. The skin on the abdomen was cut, and the short hair, subcutaneous tissue and fascia on the skin were removed. The integrity of the stratum corneum of the skin was ensured by observation under a magnifying glass. The rats were rinsed with physiological 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 stratum corneum of the skin; the samples were 1% anthelmintic Vernonia extract solution dissolved in the receiving solution, 1% anthelmintic Vernonia microemulsion gel, and 1% anthelmintic Vernonia gel; a physiological saline solution containing 20% ​​ethanol was used as the receiving medium, kept warm in a water bath at 37°C, and stirred at a speed of 400 r / min; 1 mL of sample was taken at 0, 2, 4, and 6 h, and an equal amount of blank receiving solution kept warm at 37°C was added at the same time, and the content of the active ingredient in the receiving solution was determined according to the content determination method; the skin was removed at 6 h and the test substance remaining on the skin surface was carefully removed, and it was cut into pieces, homogenized, and filtered in 1 mL of physiological saline solution containing 20% ​​ethanol in sequence, and then sampled and tested according to the above-mentioned HPLC method, and the cumulative transdermal amount and the intradermal retention at 6 h were calculated. The results are shown in Figure 7 , where left: cumulative permeation curves of the anthelmintic Vernonia extract solution and three preparations; right: intradermal retention of the anthelmintic Vernonia extract solution and each preparation after 6 hours of action;

[0100] Results and conclusions: Figure 7 It can be seen that the main component in the anthelmintic Vernonia extract solution penetrates the skin in large quantities in a very short time, and the intradermal retention amount is extremely low. Compared with the extract solution, the anthelmintic Vernonia microemulsion gel and the anthelmintic Vernonia 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.

[0101] Carefully remove each skin sample after transdermal penetration 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 pretreated skin was fixed in 2.5% glutaraldehyde fixative overnight and fixed with 0.1 mol·L -1 After rinsing with phosphate buffer for 3 times, the skin samples were dehydrated with ethanol-water solutions of different volume fractions (10%, 30%, 50%, 75%, 100%), with each gradient dehydration lasting 15-20 min, and dehydrated twice with pure ethanol, and then gradiently transitioned to acetone, freeze-dried, and prepared for skin sample coating by ion sputtering and gold coating, and observed and photographed under a scanning electron microscope. The skin surface morphology under the electron microscope is shown in Figure 8 In the transdermal experimental group of the Vernonia microemulsion gel group, slightly regular concave structures were observed, and the concave walls were wrinkled; in the transdermal experimental group of the gel group, concave structures were observed, but the degree of concave was lower, and the concave walls were not obviously wrinkled; while in the skin samples of the transdermal experimental group of the Vernonia extract solution, the concave structures had basically disappeared, the stratum corneum expanded outward, and the penetration surface increased, indicating that the Vernonia extract solution has certain skin irritation, and making the Vernonia extract into microemulsion gel and gel can increase the intradermal retention amount and reduce skin irritation.

[0102] Embodiment 13

[0103] The effectiveness of the external preparation gel of the anthelmintic Vernonia extract of the present invention:

[0104] Study on the effect of anthelmintic Vernonia gel on hydroquinone-induced vitiligo model in mice:

[0105] 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. The effectiveness of the Vernonia anthelmintica gel was preliminarily investigated using a hydroquinone-induced vitiligo mouse model (the preparation method of the model is shown in reference: Gao Li, Huo Shixia, Peng Xiaoming, et al. Effect of galangin on hydroquinone-induced vitiligo mouse model [J]. Chinese Herbal Medicine, 2014, 45(16): 2358-2363.);

[0106] Starting from the 21st day of modeling, each group started to apply medication 1 hour after applying 5% hydroquinone: the blank control group and 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, twice a day, for 21 consecutive days; after the 21st day of medication, the efficacy of each group was evaluated, and the 3cm center of the medication site was selected 2 The results were divided into two groups: one observation unit, excellent means that the pigmentation of the test area basically returned to normal; good means that the pigmentation area of ​​the test area was >50%; fair means that the pigmentation area of ​​the test area was <50%; poor means that the skin of the test area was pale or white. The total effective rate was calculated as excellent plus good. The effective rate is shown in Table 3.

[0107] Table 3 Statistics on the efficacy of Vernonia gel for anthelmintic

[0108]

[0109] Results and conclusions: Figure 3 As shown, 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 effective part gel of the anthelmintic Vernonia scabra with a drug loading of 1% had an efficacy of about 60% on vitiligo model mice, which was better than the therapeutic effect of the compound Kali cumin tincture during the same period.

[0110] Embodiment 14

[0111] The effectiveness of the external preparation microemulsion gel of the anthelmintic Vernonia extract of the present invention:

[0112] Study on the effect of anthelmintic Vernonia microemulsion gel on the vitiligo model of mice induced by monobenzone (the preparation method of the model is shown in the 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.)

[0113] Grouping and drug intervention:

[0114] Starting from the 21st day of modeling, each group started to receive medication according to the set dose and frequency in Table 4 4 ​​hours after applying monobenzone cream every day (wipe it off with a cotton swab soaked in water before medication): 30 days of medication;

[0115] Table 4: Dosage method for each group

[0116]

[0117] Note: VAF microemulsion gel - anthelmintic Vernonia microemulsion gel;

[0118] Results and conclusion: From the 10th day of modeling, local whitening of the skin in the test area of ​​the mice was observed, and small-scale white spots appeared. The hair growth rate was significantly slower than that of the blank group. With the extension of modeling time, white spots appeared in the test area of ​​each modeling animal to varying degrees, and white hair grew. After the 40th day, the animals in the model group gradually developed hair decolorization in non-modeling areas, and pinpoint decolorization of the tail and ears. Figure 4 ;

[0119] From the 10th day after the 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. 2 The results of the observation were 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; the total effective rate was calculated as excellent plus good; the visual observation score results showed that the efficacy of the medium and high doses of VAF microemulsion gel was better than that of the positive control group, as shown in Table 5. Figure 5 ;

[0120] Melanin staining:

[0121] Skin tissue was embedded in paraffin and stained with melanin. Hair follicles were observed under a 200x light microscope, and the number of hair follicles containing melanin in every 50 hair follicles was counted. The data were analyzed and processed using GraphPad prism6.0 statistical software, and the mean comparison among multiple groups was performed using one-way analysis of variance. P<0.05 was considered significant.

[0122] The blank control group had a large number of hair follicles in the epidermis, high pigment content and dark color; no obvious pigmentation was found in the hair follicles of most modeling group animals, and the number of melanin-containing hair follicles was significantly reduced, with a statistically significant difference (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, with a statistically significant difference (P<0.05). The increase in the number of melanin-containing hair follicles in the back skin of each dosed mouse had a dose-effect relationship, as shown in the results. Figure 6 , Table 5;

[0123] Table 5 Observation on the efficacy of VAF microemulsion gel on the vitiligo mouse model induced by monobenzone

[0124]

[0125] 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, characterized in that The preparation is made of anthelmintic Vernonia extract and adjuvants commonly used in pharmacy to form a gel, with 100g as the base. The specific operation is carried out according to the following steps: Extract preparation: The dried Vernonia anthelminticum medicinal material is crushed by a Chinese medicine flattening machine, weighed, and refluxed for extraction at a material-liquid ratio of 1:12, the solvent is 80% ethanol, the extraction temperature is 75-80°C, the extraction time is 3h each time, the extraction is performed twice, the extraction is filtered, the filtrate is combined and concentrated under reduced pressure at 50-60°C to 21g of the crude drug equivalent in every 100mL of the extract, and then purified by column chromatography: the extract is dispersed in water and loaded, the loading concentration is about 10g of the crude drug equivalent in every 100mL of the loading solution, 3BV of water is used for impurity removal, 3BV of 60% ethanol is used for elution and the eluate is collected, and then the extract is concentrated under reduced pressure at 60°C and vacuum dried at 50°C to obtain the extract; HPLC method is used to detect that the Vernonia anthelminticum extract contains 15-25% of Vernonia anthelminticum and 10-20% of 3,5-O-dicaffeoylquinic acid; Preparation of gel: a. According to the mass percentage, at room temperature, 1.0-1.5% of Carbomer 2020 is added to 13.5-40.0% of water to swell and obtain a gel matrix; b. Add 5.0-15.0% of polyoxyethylene 40 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 mixed solution obtained in step b, stir evenly, and make the pH value of the total system 4.3-5.5 to obtain a gelling agent.

2. An external preparation of an anthelmintic Vernonia extract, characterized in that The preparation is made of anthelmintic Vernonia extract and supplemented with pharmaceutically common auxiliary materials to form a microemulsion gel, with each 100g as the base. The specific operation is carried out according to the following steps: Extract preparation: The dried Vernonia anthelminticum medicinal material is crushed by a Chinese medicine flattening machine, weighed, and refluxed for extraction at a material-liquid ratio of 1:12, the solvent is 80% ethanol, the extraction temperature is 75-80°C, the extraction time is 3h each time, the extraction is performed twice, the extraction is filtered, the filtrate is combined and concentrated under reduced pressure at 50-60°C to 21g of the crude drug equivalent in every 100mL of the extract, and then purified by column chromatography: the extract is dispersed in water and loaded, the loading concentration is about 10g of the crude drug equivalent in every 100mL of the loading solution, 3BV of water is used for impurity removal, 3BV of 60% ethanol is used for elution and the eluate is collected, and then the extract is concentrated under reduced pressure at 60°C and vacuum dried at 50°C to obtain the extract; HPLC method is used to detect that the Vernonia anthelminticum extract contains 15-25% of Vernonia anthelminticum and 10-20% of 3,5-O-dicaffeoylquinic acid; 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 and 1.0-10.0% of olive 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, balance stirring for 10-30 min, ultrasonicate for 5-20 min, add 1.0-1.5% carbomer 2020, let stand, stir evenly after carbomer 2020 is completely swollen, and obtain a microemulsion gel.

3. A method for preparing an external preparation of an anthelmintic Vernonia extract, characterized in that Based on 100g, follow the steps below: Extract preparation: The dried Vernonia anthelminticum medicinal material is crushed by a Chinese medicine flattening machine, weighed, and refluxed for extraction at a material-liquid ratio of 1:12, the solvent is 80% ethanol, the extraction temperature is 75-80°C, the extraction time is 3h each time, the extraction is performed twice, the extraction is filtered, the filtrate is combined and concentrated under reduced pressure at 50-60°C to 21g of the crude drug equivalent in every 100mL of the extract, and then purified by column chromatography: the extract is dispersed in water and loaded, the loading concentration is about 10g of the crude drug equivalent in every 100mL of the loading solution, 3BV of water is used for impurity removal, 3BV of 60% ethanol is used for elution and the eluate is collected, and then the extract is concentrated under reduced pressure at 60°C and vacuum dried at 50°C to obtain the extract; HPLC method is used to detect that the Vernonia anthelminticum extract contains 15-25% of Vernonia anthelminticum and 10-20% of 3,5-O-dicaffeoylquinic acid; Preparation of gel: a. According to the mass percentage, at room temperature, 1.0-1.5% of Carbomer 2020 is added to 13.5-40.0% of water to swell and obtain a gel matrix; b. Add 5.0-15.0% of polyoxyethylene 40 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 mixed solution obtained in step b, stir evenly, and make the pH value of the total system 4.3-5.5 to obtain a gelling agent.

4. A method for preparing an external preparation of an anthelmintic Vernonia extract, characterized in that Based on 100g, follow the steps below: Extract preparation: The dried Vernonia anthelminticum medicinal material is crushed by a Chinese medicine flattening machine, weighed, and refluxed for extraction at a material-liquid ratio of 1:12, the solvent is 80% ethanol, the extraction temperature is 75-80°C, the extraction time is 3h each time, the extraction is performed twice, the extraction is filtered, the filtrate is combined and concentrated under reduced pressure at 50-60°C to 21g of the crude drug equivalent in every 100mL of the extract, and then purified by column chromatography: the extract is dispersed in water and loaded, the loading concentration is about 10g of the crude drug equivalent in every 100mL of the loading solution, 3BV of water is used for impurity removal, 3BV of 60% ethanol is used for elution and the eluate is collected, and then the extract is concentrated under reduced pressure at 60°C and vacuum dried at 50°C to obtain the extract; HPLC method is used to detect that the Vernonia anthelminticum extract contains 15-25% of Vernonia anthelminticum and 10-20% of 3,5-O-dicaffeoylquinic acid; 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 and 1.0-10.0% of olive 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, balance stirring for 10-30 min, ultrasonicate for 5-20 min, add 1.0-1.5% carbomer 2020, let stand, and stir evenly after the carbomer is completely swollen to obtain a microemulsion gel.

5. Use of the external preparation of the anthelmintic Vernonia extract according to any one of claims 1 to 2 in the preparation of a drug for treating vitiligo.

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