Extraction Process Optimization Of Volatile Oil From Zanthoxylum Armatum Dc.and Study On Analgesic And Anti-inflammatory Activity

Objective:In this study,the ultrasonic assisted steam distillation method was used to optimize the preparation process of Zanthoxylum armatum DC.volatile oil.The analgesic and anti-inflammatory effects of Zanthoxylum armatum DC.volatile oil were tested,and the analgesic and anti-inflammatory mechanisms were explored through network pharmacology and in vitro verification experiments.Method:This experiment through the reference to the relevant literature and combined with the 2015 edition of "Pharmacopoeia of the people’s Republic of China" general rule 2204 volatile oil determination method,with the extraction rate of volatile oil as the evaluation index,the optimal levels of ultrasonic time,liquid-solid ratio and distillation time were investigated by using complete random design and response surface methodology.Gas chromatography-mass spectrometry(GC-MS)was used to analyze and identify the chemical components in Zanthoxylum armatum DC.volatile oil by using Agilent gas quality database NIST08.LIB matching and literature data.Analgesia test:SPF mice were selected and randomly divided into 6 groups:Control group(1.5 % Tween-80);High,medium and low volatile oil dose groups(144 mg/kg、40 mg/kg、16 mg/kg);Positive control group(Aspirin Enteric-coated Tablets 60 mg/kg);Model control group(1.5 % Tween-80,Hot plate method does not have this group),10 mice in each group,Intragastric administration,1 times a day,for 4 d.The analgesic activity of volatile oil from Zanthoxylum armatum DC.was studied by hot plate test and acetic acid writhing test in mice.Anti-inflammatory test:SPF mice were selected and randomly divided into 6 groups : Control group(1.5 % Tween-80);High,medium and low volatile oil dose groups(144 mg/kg、48 mg/kg、16 mg/kg);Positive control group(Aspirin Enteric-coated Tablets 60mg/kg);Model control group(1.5 % Tween-80),10 mice in each group,Intragastric administration,1 times a day,for 7 d.The anti-inflammatory effect(swelling inhibition rate)of the Volatile oil of Zanthoxylum armatum DC.were observed in mice model of the xylene-induced ear swelling.The targets related to compounds were collected by traditional Chinese medicine systems pharmacology(TCMSP),Pubchem,Swiss Target Prediction and Uniprot.The targets related to inflammatory and pain were screened by Gis Ge NET and Online mendelian inheritance in man(OMIM)database,to establish “compound-target-disease network” with Cytoscape software.The interaction network and tissue distribution of protein targets were analyzed by Bio GPS and SRTRING database respectively.In addition,the gene ontology(GO)function and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis of targets carried out by DAVID.Finally,molecular docking method was applied to verifying the interaction between the main components of volatile oil in Zanthoxylum armatum DC.and relevant core targets.COX-2 enzyme inhibitor screening kit was used to verify its activity in vitro.Result:The optimum extraction conditions of fresh Zanthoxylum armatum DC.volatile oil were ultrasonic time of 40 min,liquid-solid ratio of 1:20 and distillation time of 120 min.44 chemical constituents were identified from Zanthoxylum armatum DC.volatile oil,including 23 alkenes,7 alcohols,2 esters,1 benzene and its derivatives,and 11 other types and unknown components.The results showed that the high,middle and low dose groups of volatile oil of Zanthoxylum armatum DC.could inhibit the hot plate induced pain,writhing and ear swelling in mice.In the high-dose group,the percentage of pain threshold was 69.97%,the inhibition rate of torsion response was 30.00%,and the inhibition rate of ear swelling was 44.19 %,and compared with model group,there were significant differences.78 related targets were predicted,including prostaglandin-endoperoxide synthase 2(PTGS2),muscarinic acetylcholine receptor M3(CHRM3),peroxisome proliferator activated receptor alpha(PPARA)and cannabinoid receptor 2(CNR2)etc.GO function enrichment analysis revealed that selected targets proteins mainly involved in positive regulation of transcription from RNA polymerase II promoter,plasma membrane,enzyme binding and other biological processes and 60 signal pathways were screened by KEGG pathway enrichment analysis,including neuroactive ligand-receptor interaction,serotonergic synapse and insulin resistance etc.Molecular docking indicated that sesamin,cholesterol myristate and β-chamigrenethe have a certain affinity with key targets.At the same time,Zanthoxylum armatum DC.volatile oil,sesamin and farnesol could inhibit the activity of COX-2.Conclusion:In conclusion,the ultrasonic assisted steam distillation method is simple,the distillation time is reduced and the yield is improved.It is found that the components of the extracted Zanthoxylum armatum DC.volatile oil are diverse and complex.In vivo experiments show that it has significant analgesic and anti-inflammatory activity.The Zanthoxylum armatum DC.volatile oil and sesamin,farnesol monomer and other components are the core target proteins White pigment has a good affinity,has a significant inhibitory effect on COX-2 target,and plays its anti-inflammatory and analgesic effect.