Study On Anti-infective Active Constituents Of Pulsatilla Cernua(Thunb.) Bercht.ex J. Presl

Pulsatilla cernua（Thunb.）Bercht.ex J.Presl,its rhizome is used for medicinal purposes.The plant is mainly distributed in the south of Liaoning and east of Jilin in China,and it is mainly distributed in North Korea,Japan and the Russian Far East abroad.Its main functions are clearing away heat and detoxifying.Studies have shown that triterpene saponins are the main chemical components of P.cernua and have a variety of pharmacological activities,such as anti-tumor,anti-inflammation,anti-virus,anti-bacterial,anti-trichomonas,liver protection and immune function enhancement.In order to study the anti-coronavirus and anti-pathogenic bacteria（bacteria and fungi）role of P.cernua,this project selected P.cernua in Dandong City,Liaoning Province for the process of separating and extracting active components,structural identification,and research on anti-infective activity.Using traditional natural medicinal chemistry methods to gradually extract and separate the chemical components of P.cernua,obtain monomeric compounds,and combine NMR to analyze the structure of the substances.Finally,as a result of the 70%water-ethanol extract of P.cernua,22 compounds were isolated,and the structures of 22compounds were identified.They are:1-phenyl-1,2-ethanediol（1）,1-（4-ethylphenyl）-1,2-ethanediol（2）,Pulsatilla saponin D（3）,Beesioside Q（4）,3-O-β-D-ribopyranosyl-（1→3）-α-L-rhamnopyranosyl-（1→2）-[β-D-glucopyranosyl-（1→4）]-α-L-arabinopyranosyl oleanolic acid 28-O-β-D-glucopyranoside（5）,squarroside II（6）,Oleanolic acid 3-O-β-D-glucopyranosyl-（1→3）-α-L-rhamnopyranosyl-（1→2）-α-L-arabinopyranoside（7）,oleanolic acid-3-O-α-L-rhamno-pyranosyl-（1→2）-[β-D-glucopyranosyl-（1→4）-]-α-L-arabinopyranoside（8）,α-hedera（9）,Ciwujianoside C3（10）,3-O-β-D-glucopyranosyl-（1→3）-α-L-rhamnopyranosyl-（1→2）-α-L-arabinopyranosyl oleanolic acid 28-β-D-glucopyranoside（11）,hederagenin 28-O-α-L-rhamnopyranosyl-（1→4）-β-D-glucopyranosyl-（1→6）-β-D-glucopyranoside（12）,3-O-α-L-rhamnopyranosyl-（1→2）-α-L-arabinopyranosyl oleanolic acid 28-β-D-glucopyranoside（13）,3-O-α-L-arabinopyranosyl oleanolic acid 28-β-D-glucopyranoside（14）,oleanolic acid 3-O-β-D-glucopyranosyl-（1→4）-β-D-glucopyranosyl-（1→3）-α-L-rhamnopyranosyl-（1→2）-α-L-arabinopyranoside（15）,Oleanolic acid 3-O-β-D-glucopyranosyl-（1→3）-α-L-rhamnopyranosyl-（1→2）[α-L-rhamnopyranosyl-（1→6）-β-D-glucopyranosyl-（1→4）]-α-L-arabinopyranoside（16）,hedera saponin E（17）,3-O-β-D-Ribopyranosyl-（1→3）-α-L-rhamnopyranosyl-（1→2）-[β-D-glucopyranosyl-（1→4）]-α-L-arabinopyranosyl oleanolic acid 28-O-α-L-rhamnopyranosyl-（1→4）-β-D-glucopyranosyl-（1→6）-β-D-glucopyranoside（18）,3-O-β-D-ribopyranosyl-（1→3）-α-L-rhamnopyranosyl-（1→2）-[β-D-glucopyranosyl-（1→4）]-α-L-arabinopyranosyl oleanolic acid 28-O-β-D-glucopyranosyl-（1→3）-α-L-rhamnopyranosyl-（1→4）-β-D-glucopyranosyl-（1→6）-β-D-glucopyranoside（19）,3-O-β-D-glucopyranosyl-（1→4）-β-D-xylopyranosyl-（1→3）-α-L-rhamnopyranosyl-（1→2）-[β-D-glucopyranosyl-（1→4）]-α-L-arabinopyranosyl oleanolic acid 28-O-β-D-glucopyranosyl-（1→6）-β-D-glucopyranoside（20）,indole-3-carboxamide（21）,3-O-β-D-glucopyranosyl-（1→4）-α-L-arabinopyranosyl hederagenin 28-O-α-L-rhamnopyranosyl-（1→4）-β-D-glucopyranosyl-（1→6）-β-D-glucopyranoside（22）.Among them,compounds 1-2 are alcohols,compound21 is indole,compounds 3-20 and 22 are triterpene saponins,compounds 1,2,5,11,18,19,20 and 21 were isolated from this genus for the first time.The anti-coronavirus activity of 22 compounds was virtual screened using molecular docking technology,and molecular docking was carried out with the two proteins 3CLpro and PLpro of SARS-Co V-2.The results showed that all compounds had binding ability for both target proteins.Among them,triterpene saponins have strong binding energy,and the more sugars connected to saponin,the stronger the binding ability.The binding energy of compounds 18,19,and 20 to the two target proteins are all less than-10 Kcal·mol^-1.It can be seen from the results of virtual screening that triterpenoid saponins may be potential anti-new coronavirus drugs.However,through the in vitro anti-SARS-Co V-2 activity test,it was found that among compounds 1-20 and 22,only compounds 2 and 14 had an inhibition rate of more than 50%on the main protease at a concentration of 200μM,and the inhibition rates were58.3%and 71.9%,respectively.Afterwards,the IC₅₀ value of compound 14 against the main protease of SARS-Co V-2 was determined to be 84.47μM.Therefore,compound 14,namely3-O-α-L-arabinopyranosyl oleanolic acid 28-β-D-glucopyranoside,may be the lead compound for the treatment of novel coronavirus.The bacteriostatic effects of compounds 1-20 and 22 on Escherichia coli,Staphylococcus aureus and Saccharomyces were tested by turbidimetric method.Only compound 1 had a good inhibitory effect on S.aureus,and the other compounds had no obvious antibacterial effect.The MIC value of compound 1 against S.aureus was determined to be 32μg/m L by the mini broth dilution susceptibility test.