Comparative Study On Anti-inflammatory And Anti-oxidant Effects Of Alpinia Oxyphylla From Different Producing Areas

Objective:The content of active compounds is an important index to evaluate the quality of Alpinia oxyphylla Miq.,which is the basis of pharmacological action.To study the difference of anti-inflammatory activity of A.oxyphylla from different producing areas,to investigate the therapeutic effect and mechanism of A.oxyphylla extract(AOE)on iodoacetamide(IA)induced chronic gastritis(CG)in rats,and to explore the possible difference of A.oxyphylla in the treatment of chronic gastritis in producing areas.To analyze the molecular mechanism efficacy in A.oxyphylla from different producing areas,and select important secondary metabolites,providing a theoretical basis for improving the quality of medicinal materials,breed selection and germplasm resources optimization.Methods: In this study,16 batches of A.oxyphylla samples from Hainan,a geo-authentic producing area,and 4 non-geo-authentic producing areas but belonging to the main cultivation areas(Guangdong,Guangxi,Fujian,and Yunnan)were selected as the research objects.(1)Determine the contents of nootkatone and tectochrysin by High performance liquid chromatography(HPLC),and to compare the content differences of these two components in 16 batches of A.oxyphylla samples.(2)Transcriptome sequencing(RNA-seq)was performed on A.oxyphylla from different regions through the high-throughput Illumina Hi Seq sequencing platform,and the metabolome analysis.To analyze and understand the functional information of the differentially expressed genes,GO functional annotation and KEGG pathway annotation were performed on the differentially expressed genes,and to find the metabolic pathways in which the differentially expressed metabolites were mainly enriched,and the differentially expressed metabolites related to the pharmacological effect was excavated in A.oxyphylla.(3)To investigate the difference of anti-inflammatory activity of AOE between different producing areas,we detected the content of NO and PGE2 in RAW264.7 macrophages cell induced by lipopolysaccharide(LPS).(4)The rat model of chronic gastritis(CG)was established by iodoacetamide(IA)induced to study the therapeutic effect and mechanism of AOE on iodoacetamide induced chronic gastritis in rats,and to explore the differences of AOE in the treatment of chronic gastritis in different producing areas.Results:(1)HPLC analysis of the content of nootkatone and tectochrysin.The results showed that the content of nootkatone and tectochrysin in the A.oxyphylla in different producing areas was significantly different(P < 0.05).The highest content of nootkatone in Danzhou,while Zhangpu and Nanning had the lowest content of nootkatone.The lowest content of tectochrysin was found in Baoting.The A.oxyphylla samples from Danzhou and Nanning were screened out for transcriptome sequencing analysis.(2)The results showed that there were 4797 differentially expressed genes between Danzhou and Nanning.We screened 30 differential metabolites which lead to differences in pharmacological effect of A.oxyphylla.(3)The CCK-8 assay results indicated that AOE concentration in the range of200～800 μg/m L did not affect RAW 264.7 cell viability.AOE treatment significantly inhibited NO secretion induced by LPS stimulation(P < 0.01),and AOE in Danzhou and Nanning had similar inhibitory effect on NO(P > 0.05);AOE significantly reduced the production of PGE2 in RAW 264.7 cells(P < 0.01).At lower concentration,the inhibitory effect of AOE in Nanning on PGE2 secretion was stronger than that of AOE in Danzhou(P < 0.05).The AOE in Danzhou showed a strong inhibition of PGE2 secretion,but the difference was not statistically significant when compared with Nanning(P > 0.05).(4)The results showed that AOE could significantly reduce the contents of IL-1β,IL-6,TNF-α and MPO in serum(P < 0.01),thereby alleviating the inflammatory response in rats of CG;AOE significantly increased SOD activity(P < 0.05,P < 0.01),significantly decreased MDA content(P < 0.05,P < 0.01),reduce the oxidative damage caused by IA;AOE can inhibit IA-induced CG by down-regulating the protein levels of p-NF-κB65,NF-κB65 and IKKβ,and up-regulating the protein expression level of IκBα.The anti-inflammatory activity and inhibition of NF-κB pathway related protein expression of AOE were similar between Danzhou and Nanning producing area.The antioxidant stress effect of AOE in Nanning producing area is stronger than Danzhou producing area.Conclusion: There were difference in the contents of nootkatone and tectochrysin of A.oxyphylla from different producing areas.The gene information and metabolite information of A.oxyphylla was obtained through transcriptome sequencing and metabolome analysis.was used to understand the functional information of differentially expressed genes by Bioinformatics analysis,and 30 differentially expressed metabolites related to pharmacological effect of A.oxyphylla were screened out.Bioactivity and pharmacological experimental results showed that AOE from different producing areas had similar anti-inflammatory activity and inhibitory effects on NF-κB pathway related protein expression.However,there were differences in the effect on anti-oxidative stress.The mechanism of AOE in the treatment of Ia-induced CG rats may be to reduce inflammatory response and oxidative stress response by inhibiting the activation of NF-κB pathway.