Optimization Of The Extraction And Purification Process Of Total Flavonoids From Vernonia Anthelmintica (L.) Willd.and Research Of Anti-vitiligo Preparations

Object:Vernonia anthelmintica（L.）Willd.is used in Uyghur medicine for the treatment of vitiligo.Modern drug analysis and pharmacological studies have confirmed that flavonoids are one of the main components in Vernonia anthelmintica（L.）Willd.and play a role in the treatment of vitiligo.In order to improve the utilisation of the total flavonoids active ingredient,it is particularly important to use a suitable extraction and purification method.Vitiligo is a skin disease in which white patches appear due to the destruction of melanocytes in the skin.To improve the efficacy of the drug,the development of a transdermal delivery formulation has many advantages,such as being portable and conducive to improved patient compliance.Based on this,the optimized process conditions for the extraction and purification of the total flavonoids from Vernonia anthelmintica（L.）Willd.were constructed in this study,the ointment formulation and the ethosome gel of the total flavonoids from Vernonia anthelmintica（L.）Willd.were prepared,which provided a theoretical basis for the extraction and purification of the total flavonoids of Vernonia anthelmintica（L.）Willd.and the development of the transdermal formulation.Methods:The Na NO₂-Al（NO₃）₃-Na OH colorimetric method was used to determine the total flavonoids content,and the response surface method was used to optimize the total flavonoids yield in the herb on the basis of the single-factor tests,and the total flavonoids with higher yield were obtained.Then,the purification process of total flavonoids from crude extracts were investigated by combining single-factor and uniform design,using macroporous resin as filler,and the total flavonoids with higher purity were prepared.The validity of the purification method established was confirmed by comparing the antioxidant properties and the effect on tyrosinase activity of crude extracts with purified ones,which provided the material basis for the later formulation studies.Subsequently,an ointment of the crude extract was prepared and the anti-vitiligo effect of the total flavonoids from Vernonia anthelmintica（L.）Willd.was initially confirmed by in vivo experiments.Further,the preparation of ethosomes were explored by single-factor tests on the preparation method,alcohol phase selection,cholesterol to lecithin ratio,alcohol phase percentage,ethanol to 1,2-propanediol ratio and soybean lecithin content to obtain drug-loaded ethosomes.In combination with carbomer gels,the gel matrix swelling solution,concentration of gel matrix,p H,and drug matrix ratio were investigated to prepare a drug-loaded ethosome gel system for transdermal drug delivery.Finally,the efficacy of the nano-gel formulation was verified by inter-group comparison with the traditional preparation ointment prepared by the extraction.The effectiveness of the ethosome gel was confirmed by using a solution containing the same concentration of drug as the gel system as an in-group reference,observing the melanin status by HE and Masson-Fontana staining of skin issue,measuring the TYR,MAO,CHE,SOD and MDA indexes in mice.Results:（1）Optimal processes for the extraction and purification of total flavonoids were established.The optimum extraction process was established as follows:ethanol concentration of 50%,material-to-liquid ratio of 1:14,extraction time of 3.5 h,extraction frequency of 3 times,and the average yield of total flavonoids were 103.65 mg·g^-1 at an extraction temperature of 80℃,and the content was determined to be 40.35%±0.04%（n=3）.（2）The optimum purification process was established as follows:the concentration of the loading solution was 1.0 mg·m L^-1,the p H was 0.5,the maximum loading volume was 2.3 BV,the p H of the removal solvent was adjusted to≤2,the concentration of the eluent was 75%alcohol（v/v）,the volume of the eluent was 6 BV,and the elution flow rate was 1 m L·min^-1.The average recovery of total flavonoids were 94.37%,and the content was determined as 81.02%±0.35%.The results of in vitro antioxidant assay and in vitro tyrosinase activity assay showed that the total flavonoids from Vernonia anthelmintica（L.）Willd.had good antioxidant and tyrosinase activating effects.（3）The preparation of extraction ointment and animal experiments showed that the water-soluble matrix ointment had good in vitro drug release performance,and the preparation of 5%total flavonoids extraction ointment of Vernonia anthelmintica（L.）Willd.had certain effect on the treatment of vitiligo.（4）The ethosomes of total flavonoids from Vernonia anthelmintica（L.）Willd.were prepared with a particle size of 98.84±0.3 nm,PDI of 0.239±0.014,Zeta potential of-22.3±0.4 m V and encapsulation efficiency of 72.85±0.42%.A more stable ethosome gel transdermal drug delivery system was obtained by combining it with an aqueous solution of glycerol-propanediol（10%glycerol,5%propylene glycol）in the swelling solution and carbomer 934 at a concentration of 0.7%and p H 7.0 in a drug matrix ratio of 1:1.The system was used to treat a mouse model of vitiligo induced by hydroquinone by up-regulating TYR,SOD and down-regulating MAO,CHE,and MDA.The system has a great potential to be used in the treatment of mouse models of vitiligo induced by hydroquinone.Conclusions:In this study,optimal processes for the extraction and purification of the total flavonoids from Vernonia anthelmintica（L.）Willd.were successfully constructed,providing a theoretical basis for their further development and utilization.The preparation of the traditional form of ointment and the nano-form of ethosome gel and their therapeutic effects on mice with hydroquinone-induced vitiligo were investigated,indicating that the transdermal delivery form had a broader application in the treatment of skin diseases.