Preparation Of Zhachong Thirteen Nanoemulsion And Preliminary Evaluation Of Its Anti-inflammatory Effect In Vivo

Objective:Zhachong Shisanwei Wan is a classic prescription of Mongolian medicine.Its composition is complex,and it consists of 13 herbs,including 8 herbs,3 minerals and 2 animals.In this study,the dosage form of Zhachong Shisanwei Wan was reformed by nanoemulsion technology to prepare Zhachong thirteen nanoemulsion with stable properties and enhanced anti-inflammatory effect,in order to provide reference for the research and development of new oral dosage forms of compound Chinese and Mongolian medicines.Methods:1.GC-MS analysis of volatile oil of Zhachong thirteen:Eight kinds of volatile oils（volatile oil-1）and four kinds of volatile oils（volatile oil-2）from Zhachong thirteen prescriptions were extracted respectively,and the components were analyzed by gas chromatography-mass spectrometry（GC-MS）.The relative percentage content of each chemical component was calculated by peak area normalization method,and the chemical composition differences between volatile oil-1 and volatile oil-2 were compared.2.Study on the preparation technology and quality evaluation of volatile oil nanoemulsion and Zhachong thirteen nanoemulsion:With volatile oil-2 as the oil phase and ultrapure water as the water phase,the auxiliary materials were screened by pseudo ternary phase diagram method,and the significant factors were screened by single factor experiment.With particle size and polydispersity index（PDI）as indicators,the central composite design（CCD）method was used to optimize the formulation of volatile oil nanoemulsion,and the preliminary quality evaluation of particle size distribution and drug loading was carried out.On the basis of the prescription of volatile oil nanoemulsion,other components in Zhachong Shisanwei Pill were added to prepare Zhachong thirteen nanoemulsion,and its appearance,particle size distribution,drug loading and stability were evaluated.3.Exploring the anti-inflammatory mechanism of Zhachong thirteen based on network pharmacology:The anti-inflammatory target of Zhachong thirteen was predicted by using the network pharmacology database,and the anti-inflammatory related pathways were enriched and analyzed to explore its potential mechanism.4.Preliminary evaluation of anti-inflammatory effect of Zhachong thirteen nanoemulsion in vivo:The models of mouse auricle swelling induced by xylene and paw swelling induced by Carr were established,and the ear swelling and paw swelling of mice were measured and calculated.Enzyme-linked immunosorbent assay（ELISA）was used to determine the level of inflammatory factors in Carr-induced mice.Pathological changes were observed in inflammatory tissue sections,and the anti-inflammatory effects of Zhachong thirteen nanoemulsion and volatile oil nanoemulsion containing volatile oil-2 were preliminarily evaluated.Results:1.GC-MS analysis of volatile oil of Zhachong thirteen:44 and 43 chemical compon-ents were identified from volatile oil-1 and volatile oil-2,and the maximum relative percent-age of eugenol was 10.97%and 16.50%,respectively.The volatile components identified by both were almost the same,but the relative percentage was slightly different.2.Study on the preparation technology and quality evaluation of volatile oil nanoemulsion and Zhachong Thirteen nanoemulsion:Polyoxyethylene castor oil（CO-40）and anhydrous ethanol were screened by pseudo-ternary phase diagram method.Three significant factors,namely Km value,volatile oil content（%）and mixed emulsifier content（%）,were screened by single factor experiment.The optimal formula was optimized by CCD method,which contained CO-4017.33%,anhydrous ethanol 8.67%,volatile oil-2 12%and ultrapure water 62%.The particle size of the volatile oil nanoemulsion was（19.9±0.1）nm,the PDI was 0.180±0.020,the Zeta potential was（-41.2±3.3）m V,the drug loading was（39.77±0.04）mg/m L,and the storage stability at 4℃was good.Based on the optimal formula of volatile oil nanoemulsion,Zhachong thirteen nanoemulsion was prepared with water extraction and alcohol precipitation as water phase and artificial musk ethanol extraction as co-emulsifier.The formula consisted of CO-4017.33%,absolute ethanol 8.24%,artificial musk 0.43%,volatile oil-2 12%and 90%water extraction and alcohol precipitation 62%.The particle size of Zhachong thirteen nano emulsion is（21.8±0.4）nm,the PDI is 0.126±0.019,the Zeta potential is（-30.0±1.5）m V,the viscosity is（2.03±0.04）Pa·s,the p H is 5.62±0.01,and the drug loading is（44.61±0.03）mg/m L.Under the condition of high humidity,the stability of nanoemulsion is good within 10 days,and the particle size becomes larger and the color becomes darker when irradiated by high temperature and strong light,so it should be avoided to store it.3.Exploring the anti-inflammatory mechanism of Zhachong thirteen based on network pharmacology:Network pharmacology predicted 106 anti-inflammatory related active ingredients such as quercetin,kaempferol,eugenol andβ-asarether,and screened out 20 key targets such as tumor necrosis factor（TNF）,prostaglandin peroxidase synthetase 2（PTGS2）and interleukin-6（IL-6）.It involves HIF-1,IL-17,TNF and other inflammatory signaling pathways.4.Preliminary evaluation of anti-inflammatory effect of Zhachong thirteen nanometer emulsion in vivo:Zhachong thirteen nanoemulsion,volatile oil nanoemulsion containing volatile oil-2 and Zhachong Shisanwei Wan can all reduce the ear swelling and plantar swelling of mice to varying degrees,and the inhibition rates of auricular swelling and plantar swelling of Zhachong thirteen nanoemulsion at 5 h are the highest,41.94%and 37.68%respectively.Except for the volatile oil nanoemulsion group,the levels of interleukin-6（IL-6）in serum of Carr-induced inflammatory mice were significantly decreased in all groups.The levels of tumor necrosis factor-α（TNF-α）in serum and prostaglandin E2（PGE₂）in inflammatory tissues were significantly decreased in all groups.All groups can reduce the infiltration of inflammatory cells in inflammatory feet.Among them,Zhachong thirteen nanoemulsion group has the strongest inhibitory effect on TNF-α,IL-6 and PGE₂ levels in Carr-induced inflammatory mice,and Zhachong thirteen nanoemulsion has a good anti-inflammatory effect.Conclusion:The Zhachong thirteen nanoemulsion prepared in this paper is uniform,clear and transparent,and its properties are stable,which shows that it is feasible to reform the Zhachong Shisanwei Wan form into Zhachong thirteen nanoemulsion,and the volatile oil nanoemulsion and Zhachong thirteen nanoemulsion have certain anti-inflammatory effects.This study provides a new idea for the development of new dosage forms of Zhachong thirteen and Mongolian medicine compound,and provides important theoretical significance and reference value for the follow-up research.