| Guided by the anti-bacterial screening results against Acinetobacter baumannii,the ethyl acetate part of 80%ethanol ultrasonic extract of Sophora flavescens with antibacterial activity(MIC95 at about 512 μg/mL)were isolated and purified by various column chromatography including silica gel,reverse phase silica gel,polyamide,Sephadex LH-20 and high performance liquid chromatography.The structures of the compounds were identified by nuclear magnetic resonance(NMR)and mass spectrometry.A total of 24 compounds were identified,including two new compounds(-)-5-Methoxy-7-hydroxy-8-lavandulyl-chromone(13)and(-)-(2S,βS)-Sophobiflavonoid CE(19),one first isolated compound(2,5-Dihydroxybenzoic acid)(24),along with twenty-one known compounds,(-)-(6aR,11aR)-Maackiain(1),(-)-(2S)-8-Prenylnaringenin(2),(-)-(2S)-Exiguaflavanone K(3),(-)-(2S)-Sophoraflavanone G(4),(-)-(2S)-Leachianone A(5),(-)-(2S)-Kushenol E(6),(-)-(2S)-Leachianone G(7),Kushenol F(8),(-)-(2S)-Kurarinone(9),(-)-(2S)-Kurarinol(10),(-)-(2R,3R)-3,7,4’-Trihydroxy-5-methoxy-8-prenylflavanone(11),(-)-(2S)-Isoxanthohumol(12),(-)-(2S)-2’-Methoxykurarinon-e(14),(+)-(2R,3R)-Kushenol I(15),Calycosin(16),Kuraridin(17),(-)-(2S)-Kushenol A(18),Trifolirhizin(20),Kushenol D(21),(-)-(2R,3S)-Kushenol N(22)and Kushenol O(23).All the isolated compounds were assayed for their antibacterial activities against A.baumannii using broth dilution method.The results showed that compounds 1,2,5 and 15 exhibited dose-dependent antibacterial activity,with MIC95 value of 128-256μg/mL for 2 and 256-512 μg/mL for 1,5 and 15.Although the antibacterial activity of compound 4 was weak at 512 μg/mL,its activity reached 45.05 ± 1.77%at low dosage of 8 μg/mL,suggesting that these five flavonoids are the main active components of S.flavescens against A.baumannii.The structure-activity relationship analysis indicated the replacement of hydroxyl group by methoxy group at C5 could decrease the antibacterial activity of flavonone in S.flavescens,while the presence of hydroxyl group at C3 as in the case of flavononol could enhance the antibacterial activity as compared with flavonone.In addition,the antibacterial activity of flavononol against A.baumannii was higher than its chalcone analogue,and the glycosidation of the hydroxyl group of maackiain would make its antibacterial activity against A.baumannii completely disappear.Based on the above results,the HPLC method for the determination of 14 flavonoids in 80%ethanol extracts of S.flavescen and its fingerprint were established for the first time in this paper,and the contents of 14 flavonoids in 13 batches of S.flavescen extracts were determined.By analyzing the flavonoids content of extract S1which exhibited strong anti-bacterial activity,the results showed that the(-)-(2S)-Kurarinone(9)with very weak antibacterial activity had the highest content(7.29%),followed by(-)-(2S)-Sophoraflavanone G(4)(5.29%)prossessing weak antibacterial activity.Among the four flavonoids with strong antibacterial activity,the contents of(+)-(2R,3R)-Kushenol I(15)and(-)-(2S)-Leachianone A(5)were higher,which were 1.15%and 0.77%,respectively.The contents of(-)-(6aR,11aR)-Maackiain(1)and(-)-(2S)-8-Prenylnaringenin(2)were 0.37%and 0.12%,respectively.OSC-PLSR analysis was used to establish the quantity-effect relationship model between the contents of 14 flavonoids and the inhibition rate against A.baumannii at 512 μg/mL.It was found that the 5 flavonoids were closely correlated with the inhibition activity against A.baumannii,among which Trifolirhizin(20),(-)-(2S)-Isoxanthohumol(12)and(-)-(2S)-Sophoraflavanone G(4)were positively correlated,and(-)-(2R,3S)-Kushenol N(22)and(-)-(2S)-Kurarinone(9)were negatively correlated.The predicted values of calibration set and validation set samples of OSC-PLSR model were well correlated with the reference values(R2Y=0.868).Therefore,the content analysis of 14 flavonoids in 80%ethanol extract of S.flavescens could be used to predict its anti-Acinetobact baumannii activity. |
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