Study On The Anti-Hbv Primary Screening Of The Lipid-Soluble Components Of Arnebia Euchroma(Royle) Johnst. And Its Liposome Preparation Process

Object:The object of the study were to extract and isolate the chemical constituents from the roots of Arnebia euchroma（Royle）Johnst.（AERJ）.The structure of the chemical components is identified by nuclear magnetic resonance spectroscopy（NMR）and literature comparison.The network pharmacology is used to predict the action target and signal pathway of AERJ effective components in the treatment of hepatitis B virus（HBV）.Based on the predicted results,the active ingredients with the most significant anti-HBV effects were screened by in vitro cell MTT assay,and the liposome preparation process,preparation process optimization and characterization experiments were carried out to provide a basis for the clinical formulation of AERJ.Methods:Extraction and separation experimental method:The dried AERJ roots were crushed,soaked in petroleum ether overnight at room temperature,extracted the next day and concentrated to obtain an infusion.The petroleum ether extracted AERJ was further extracted with 60%ethanol and the concentrated infusion was mixed with pure water and extracted with different polar solvents ethyl acetate and n-butanol in turn to obtain the infusion of each constituent part.Further separation of the petroleum ether extracted parts and ethyl acetate extracted parts was carried out by various chromatographic methods such as normal phase silica gel column,C-18 reverse silica gel column and normal phase preparative thin layer chromatography,and the monomeric compounds were purified using a series of separation methods.Nuclear magnetic resonance spectrometry（NMR）and literature comparisons were used to determine the structure in plan and stereo structure of the compounds to determine their nomenclature.Network pharmacology experimental method:Pub Chem,Swiss Target Prediction and HERB databases were used to collect the targets of the isolated active ingredients,the intersecting targets were entered into STRING database to retrieve their protein interaction information and Cytoscape software was used to draw the PPI network relationship map.The intersecting targets were entered into the STRING database to retrieve their protein interaction information and Cytoscape software was used to map the PPI network,followed by GO and KEGG enrichment analysis using the Microbiology online mapping platform to obtain the key pathways.MTT assays were used to investigate the cytotoxic effects of Isovaleryl-shikonin（Iso SHK）,Acetylshikonin（Ace SHK）,2,3-dimethylpentenyl shikonin（23DSHK）and shikonin（SHK）on Hep G2.2.15cells.Isovalerylshikonin-liposome（Iso SHK-lip）preparation method:Iso SHK-lip was prepared by the thin film dispersion method,and the optimal Iso SHK-lip was characterized by the response surface optimization method,using A:Lecithin-Cholesterol mass ratio,B:Lecithin-Isovalerylshikonin mass ratio and C:Volume of Hydrated Solvent as 3 factors and 3 levels of preparation protocols,and the particle size,PDI index,Zeta potential and morphology were characterized.Results:The results of extraction and separation experiments:After 10 kg of dried AERJ roots were extracted in constant temperature waters to obtain petroleum ether part extract（120.0 g）,the petroleum ether extracted herbs were continued to be extracted with 60%ethanol to obtain ethanol extract,and different solvents were used to extract the ethanol extract to obtain ethyl acetate extract part（54.69 g）,n-butanol extract part（60.21 g）,and water part extract（225.93 g）.After the separation and purification of the petroleum ether part,six compounds were obtained,which were structurally identified as six naphthoquinones by NMR detection,literature comparison and physicochemical property analysis.One compound was isolated and purified from the ethyl acetate extraction site and structurally identified as a naphthoquinone by NMR detection,literature comparison and physicochemical analysis.The known compounds were identified as:Propionylshikonin（WR2-34-1）,Isovaleryl shikonin（WR2-34-2）,Acetylshikonin（WR2-34-4）,2,3-Dimethylpentenoyl-shikonin（WR2-35-3）,Shikonin（WR2-35-4）,β,β-Dimethylacrylshikonin（WR2-35-6）,andβ-Acetoxyisovalerylshikonin（MLY-1-4-4）.Network pharmacology experimental results:7 naphthoquinones extracted and isolated as active ingredients,Swiss ADME database analysis shows that all 7 naphthoquinones have good GI absorption properties as well as druglikeness properties,302 potential targets of 7 active ingredients and 2655 HBV disease-related targets were collected through the database,162 active ingredient-disease intersection targets were collected The 10 core key targets obtained from the intersection of C-T-P network and PPI network analysis were AKT1,PIK3R1,PIK3CA,MAPK3,MAPK1,NFKB1,EGFR,MAPK14,CCND1,MTOR.GO functional enrichment yielded a total of 5362 functional messages,and KEGG signaling pathway enrichment yielded 256 signaling pathways,among which AERJ can treat HBV through PI3K-Akt,TNF,Erb B and other signaling pathways.Molecular docking analysis revealed that the combinations of active ingredients with the lowest binding capacity to the 10 core key targets were:23DSHK-AKT1,Iso SHK-AKT1,ββDSHK-PIK3CA,SHK-MTOR,Pro SHK-PIK3CA,βAc SHK-PIK3R1 and Ace SHK-PIK3CA.23DSHK-AKT1 and Iso SHK-AKT1 bound best.MTT assay showed that all four naphthoquinones had good inhibitory effect on Hep G2.2.15 cells.The results of Iso SHK-lip preparation experiments:The detection wavelength of Iso SHK was 518.5 nm by UV spectrophotometer,and the labeling curve of Iso SHK was Y=0.01571X+0.003056,R²=0.9993（n=3）,indicating that Iso SHK has good linearity in the range of 8～40μg-m L^-1.Iso SHK stability,precision,and recovery experiments showed that Iso SHK could be stored stably for 12 h,and the precision,and recovery were good.The response surface optimization experiments were conducted by single-factor experiments with a lecithin-cholesterol-isovalerylshikonin ratio of 90:10:3（lecithin:90 mg,cholesterol:10 mg,isovaleryl-shikonin:3 mg）and a hydration medium volume of 30 m L.The optimal preparation conditions of Iso SHK-lip were predicted to be 8.82:1 lecithin-cholesterol mass ratio,30.65:1 lecithin-isovaleryl shikonin mass ratio,and 29.22 m L of water and medium volume.The average encapsulation rate of Iso SHK-lip was 90.03%,the mean particle size was 117.48 nm,the mean PDI index was 0.246 and the mean Zeta potential was-13.59 m V.The stability experiments showed that the particle size,PDI index and Zeta potential of Iso SHK-lip did not change significantly.Transmission electron microscopy results showed that Iso SHK-lip was prepared in the range of 100-200 nm with subspherical particle size.Conclusion:Conclusion of extraction and isolation experiments:Seven compounds were extracted and purified,of which all seven were naphthoquinones,and the main chemical component in AERJ was identified as isovalerylshikonin.From the chemical taxonomic point of view,the affinity between AERJ and other species of Lithospermum L.was verified,which provided the material basis for the subsequent network pharmacological research.The combination of network pharmacology and in vitro cellular assays verified that Xinjiang soft comfrey can treat HBV through 23DSHK-AKT1,Iso SHK-AKT1 active ingredient-core key target and PI3K-Akt,TNF,Erb B signaling pathways,and Iso SHK has better anti-HBV effect,so Iso SHK was used for subsequent dosage form experiments.Conclusion of Iso SHK-lip preparation experiments:Iso SHK-lip was prepared after three repetitions of the optimal results obtained by the response surface method optimization,and nearly spherical,smaller particle size,uniform particle size distribution and better stability of Iso SHK-lip were prepared,which provided the basis for the subsequent pharmacological studies of the preformed dosage forms.