Study On The Flavonoids From Ponka And Their Anti-inflammatory Activity

Ponka is commonly used in Tibetan medicine.It refers to the dried whole plant of Aconitum tanguticum or Aconitum naviculare of Ranunculaceae.It is cold in nature and bitter in taste,with the functions of clearing heat and detoxifying.In clinic,Ponka is often used to treat infectious fever,liver and bile fever,lung and intestinal fever,influenza and food poisoning.At present,due to the differences of origin and producing areas,the contents of different chemical constituents in Ponka have great fluctuation,whether there is difference in the efficacy of different Ponka is unclear.To date,a total of 182 compounds has been isolated and identified from Ponka,including 102 alkaloids,21 flavonoids,41 phenolic acids and 18 other compounds.Modern pharmacological studies have shown that flavonoids are a kind of natural compounds with 2-phenyltryptophenone as the basic skeleton,widely existing in edible plants and medicinal plants.These substances have strong antioxidant,antiinflammatory,anti-tumor,anti-viral,hypolipidemic and anti-osteoporosis biological activities,and has few side effects,so people have conducted extensive research on it.However,there are few flavonoids isolated and identified from Ponka,so further exploration is needed to clarify the pharmacodynamic material basis of Ponka and enrich the structural types of natural products.Objectives.To study on the less-studied flavonoids in Ponka and their anti-inflammatory activities,so as to elucidate the pharmacodynamic material basis of Ponka for exerting clearing heat and detoxifying effects.Methods.1.The anti-inflammatory activity of Ponka from different bases and habitats was investigated by carrying out the effect of 70%ethanol extract of 17 batches of Ponka on the release of NO from LPS-induced mouse macrophages RAW264.7 cells.2.The chemical ingredients in 40%ethanol extract of Aconitum tanguticum(Maxim.)Stapf,one of the source plants of Ponka,were separated and purified by macroporous adsorbent resin,ODS,MCI and Sephadex LH-20 gels,high pressure preparative liquid chromatography and recrystallization.Their structures were identified by modern spectroscopic techniques(UV,IR,MS,NMR,2D-NMR,CD,etc.).3.Based on the literature and previous laboratory work,21 flavonoid components of Ponka were obtained and imported into the prediction database in SMILES format to predict their effective targets,and inflammation-related targets were obtained from Gene Cards and OMIM databases,and Cytoscape 3.8.2 software was used to perform network analysis.The network topology analysis was performed by Cytoscape 3.8.2 software to construct the "Ponka flavonoids-disease-targe"network diagram.Protein interaction analysis was performed using String database,and GO analysis of biological processes and KEGG functional enrichment analysis were performed using the opensource bioinformatics software Bioconductor.4.The HPLC method was established for the determination of two flavonoids in Ponka.Kromasil 100 C8(4.6 mm×250 mm,5μm)column was used as the stationary phase,and acetonitrile-0.1%formic acid aqueous solution was used as the mobile phase,and the gradient elution was performed at a flow rate of 1 mL/min.The column temperature was 30℃.The detection wavelength was 320 nm,and the injection volume was 10 μL to determine the content of ponkaroside C and ponkaroside DResults.1.In the absence of cytotoxicity,6 batches of Aconitum tanguticum(numbered 2,4,6,8,10,12)and 2 batches of Aconitum naviculare(numbered 14 and 16)inhibited NO release from RAW264.7 cells induced by LPS,showing good anti-inflammatory activity.The medicinal materials numbered 6,7,10,12 and 16 were dose-dependent.2.14 compounds were obtained from the 40%ethanol extract of whole herbs of Aconitum tanguticum,among which 13 compounds structures were identified by spectral analysis and comparison with literature values.They were vanillic acid(1)、caprolactam(2)、kaempferol-3-O-[β-D-glucopyranosyl-(1→3)-(4-O-trans-pcoumaroyl)-α-L-rhamnopyranosyl-(1→6)-β-D-galactopyranoside]-7-O-β-Dglucopyranosyl-(1→3)-α-L-rhamnopyranosyl(Ponkaroside D,3)、quercetin-3O-α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)-α-L-(4-O-trans-βcoumaroyl-rhamnopyranosyl)-(1→6)]-β-D-galactopyranoside-7-O-α-Lrhamnopyranoside(4)、kaempferol-3-O-[α-L-rhamnopyranosyl-(1→6)-β-Dgalactopyranoside]-7-O-α-L-rhamnopyranoside(5)、4-dihydroxyphenethoxy-8-O-βD-[6-O-(4-O-β-D-glucopyranosyl)-feruloyl]-glucopyranoside(6)、quercetin-3-O-[βD-glucopyranosyl-(1→3)-(4-O-trans-β-coumaroyl)-α-L-rhamnopyranosyl-(1→6)-βD-galactopyranoside]-7-O-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl(Ponkaroside C,7)、kaempferol-3-O-β-D-glucopyranosyl-(1→2)-O-[α-Lrhamnopyranosyl-(1→6)]-β-D-galactopyranoside-7-O-α-L-rhamnopyranoside(8)、quercetin-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-galactopyranoside-7-O-α-Lrhamnopyranoside(9)、quercetin-3-O-[β-D-glucopyranosyl-(1→3)-(4-O-trans-βcaffeoyl)-α-L-rhamnopyranosyl-(1-6)-β-D-galactopyranoside]-7-O-3-Dglucopyranosyl-(1→3)-α-L-rhamnopyranosyl(Ponkaroside A,10).2-methoxy-4carboxyl-phenyl-O-β-D-[6-O-(4-O-β-D-glucopyranosyl)-feruloyl]-glucopyranoside(11)、quercetin-3-O-[β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→6)-βD-galactopyranoside]-7-O-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl(Ponkaroside E,12)、quercetin-3-O-[β-D-glucopyranosyl-(1→3)-(4-O-trans-βcaffeoyl)-α-L-rhamnopyranosy-(1→6)-β-D-rhamnopyranosyl]-7-O-α-Lrhamnopyranosyl(Ponkaroside F,13)。Among them,compound 8 was isolated from Ponka plant for the first time and Compounds 11-13 are new compounds.Studies on monomers showed that compounds(3,4,7,8,10,12)had no significant anti-inflammatory effects.3.The analysis of potential active targets and anti-inflammatory mechanism of action in 21 flavonoid components of Ponka was analyzed by network pharmacology,and 128 anti-inflammatory targets of 21 flavonoids were found.The key antiinflammatory targets of Ponka flavonoids were TNF、ALB and VEGFA,respectively.GO and KEGG enrichment analysis showed that the anti-inflammatory effects of Ponka flavonoid components were mainly exerted through oxidative stress,inflammation and other responses.4.The contents of Ponkaroside C and Ponkaroside D in Ponka were determined by HPLC method,and the results showed that the two components showed good linearity(R2≥0.9994)in their respective concentration ranges,with mean recoveries of 96.3%and 100.8%,RSD values of 1.3%and 0.7%,and mass fractions of 0.08～12.58,0.01～10.33 mg/g-1,respectively.Conclusions.1.The anti-inflammatory activity of Ponka from different sources is very different.Different bases and habitats are the main reasons for this difference.2.The flavonoid components in Ponka were separated and purified by various modern chromatographic techniques,and the results enriched the chemical composition of Ponka,which laid the foundation for the elucidation of the material basis of Ponka’s medicinal effects.3.The anti-inflammatory mechanism of flavonoids in Ponka was preliminarily discussed,which provided a basis for the pharmacodynamic substance basis of Ponka.4.A preliminary HPLC method was developed for the determination of two flavonoid components of Ponka.The method is characterized by strong specificity and good separation,which can provide a basis for the quality control of Ponka.