| "Kushen" and "Shandougen" are the traditional Chinese medicines derived from Sophora plant that commonly used as heat-clearing and detoxicating."Kushen" is the dried roots of Sophora flavescens,"Shandougen" is the dried roots and rhizomes of S. tonkinensis. The two crude drugs are so similar in the chemical constituents that they were both stated to contain quinolizidine alkaloids, prenylated flavonoids and oleanene triterpenoids. But their clinical applications are quite different."Kushen" is mostly for external application which used to treat dermatosis and gynaecopathia diseases, while "Shandougen" is mainly for oral use that used to treat acute and chronic pharyngitis. We had compaired the fingerprints of alkaloid and flavonoid compositions of S. flavescens and S. tonkinensis, and we found some interesting facts: through the dendrogram of alkaloids, we saw that the two kinds of medicine were interlaced, we can hardly distinguish them; while the dendrogram of flavonoids had significant difference, the two kinds of medicine were specifically distinguished. Accordingly, if we investigate the flavonoids of S. flavescens and S. tonkinensis systemically, we may identify the active compounds which make them have different pharmacological actions and discover some new ideas for further researches. In this assay, the flavonoids of S. flavescens and S. tonkinensis were isolated systemically by methods of natural medicinal chemistry and analytical chemistry. The biological activities of the flavonoids such as cytotoxic, anticomplement and antibiosis were detected. The qualitative and quantitative analysis of flavonoids from S. flavescens and S. tonkinensis by LC/MS and HPLC were preformed respectively. And the stabilities of total flavonoids from S. flavescens and S. tonkinensis in artificial gastric and intestinal juice were also studied by HPLC.1. Flavonoids of S. flavescens and S. tonkinensis1.1Flavonoids from EtOAc fraction of S. flavescens:Twenty-nine compounds including twenty flavonoids were isolated from the EtOAc fraction of the ethanol extracts of the roots of S. flavescens by repeated column chromatography and preparative TLC method. Structures of the compounds were elucidated by spectroscopic methods as follows:Glabranin (SF-1), Sophoraflavanone B (SF-2), Isoxanthohumol (SF-3), Sophoraflavanone G (SF-4), Kurarinone (SF-5), Leachianone (SF-6), Kushenol T (SF-7),(-)-Trifolirhizin (SF-8),(-)-Maackiain (SF-9), Daidzein (SF-10), Genistein (SF-11), Formononetin (SF-12), Calycosin (SF-13), Pseudobaptigenin (SF-14), Pseudoindorin (SF-15), Xanthohumol (SF-16),7,9,2’,4’-tetrahydroxy-8-isopentenyl-5-methoxychalcone (SF-16), Kuraridine (SF-18), Noranhyoicaritin (SF-19),7,4’-Dihydroxyflavone (SF-20), Daphnetin (SF-21),7-Hydroxycoumarin (SF-22), Anisic acid (SF-23), Mono-stearin (SF-24), Pentadecanoic acid (SF-25), Pentacosanoic acid (SF-26), Triacontane (SF-27), Stigmasterol (SF-28) and β-sitosterol (SF-29). Compound SF-15and SF-21were isolated from this genus for the first time, and Compound SF-1was firstly found in S. flavescens.1.2Flavonoids from EtOAc fraction of S. tonkinensis:Twenty-seven compounds including fifteen flavonoids were isolated from the EtOAc fraction of the ethanol extracts of the roots and rhizomes of S. tonkinensis, by repeated column chromatography and preparative TLC method. Structures of the compounds were elucidated by spectroscopic methods as follows:Isobavachin (ST-1), Sophoranone (ST-2),2-(3-Hydroxy-2,2-dimethyl-8-prenyl-6-chromanyl)-7-hydroxy-8-prenyl-4-chromanone (ST-3), Sophoranochromene (ST-4),(-)-Trifolirhizin (ST-5),(-)-Maackiain (ST-6), Daidzein (ST-7), Genistein (ST-8), Formononetin (ST-9), Daidzein dimethy ether (ST-10),5-Hydroxypseudobaptigenin (ST-11), Quercetin (ST-12), Rutin (ST-13), Quercitrin (ST-14),7,4’-Dihydroxyflavone (ST-15), Esculetin (ST-16), lupeol (ST-17), p-methoxyl phenol (ST-18), p-hydroxybenzcic acid (ST-19), p-Coumaric acid ethyl ester (ST-20), Propylparaben (ST-21), docosyl caffeate (ST-22), Mono-stearin (ST-23), tricosanic acid (ST-24), isobutyl phthalate (ST-25), Stigmasterol (ST-26), β-sitosterol (ST-27). Compound ST-10and ST-11were isolated from this genus for the first time, and Compound ST-1and ST-14were firstly found in S. tonkinensis.1.3Flavonoids from n-BuOH fraction of S. tonkinensis:Six compounds including three flavonoids were isolated from the n-BuOH fraction of the ethanol extracts of the roots and rhizomes of S. tonkinensis that showed good anticomplement activity (CH50=0.44mg/mL), by repeated column chromatography and preparative TLC method. Structures of the compounds were elucidated by spectroscopic methods as follows:Quercitrin,7,4’-Dihydroxyflavone, rutin, p-hydroxybenzoic acid, mono-stearin and isobutyl phthalate. These six compounds have been isolated from the EtOAc parts of S. tonkinensis.2. Bioactivity evaluation of flavonoids2.1Anticomplementary activity test:The flavanoids isolated from S. flavescens and S. tonkinensis were screened for anticomplementary activity for the first time, result showed as follows:seven flavonoids including (-)-Maackiain, Noranhyoicaritin, Quercetin, Quercitrin, Rutin,7,4’-Dihydroxyflavone and Formononetin indicated anticomplementary activity with the CH50value from0.09to1.65mg/mL. Structure-activity relationship showed that flavonols and their glucosides were the main flavonoids for anticomplement effect. S. tonkinensis showed better activity than that of S. flavescens. Different flavonoids indicated various activation targets of complement system, it was great finding and worthy for further researches.2.2Cytotoxic test:The flavanoids isolated from S. flavescens and S. tonkinensis were screened for cytotoxic activity, result showed as follows:twelve flavonoids including Sophoranochromene, Glabranin, Noranhyoicaritin, Isoxanthohumol, Daidzein dimethy ether, Genistein, Sophoraflavanone G, Kushenol T, Quercetin, Sophoranone and7,9,2’,4’-tetrahydroxy-8-isopentenyl-5-methoxychalcone had inhibition to several kinds of people’s tumour cell strain, the GI50value varies from2.94to16.37mg/mL. Structure-activity relationship showed that flavanones and flanonols were the main flavonoids for antitumor activity. And S. flavescens showed better effect than that of S. tonkinensis.2.3Antibacterial Activity Test:The flavanoids isolated from S. flavescens and S. tonkinensis were screened for antibacterial activity, two flavonoids showed favourable antibacterial activity.3. Qualitative analysis of flavonoids in S. flavescens and S. tonkinensis by LC/MS3.1Qualitative analysis of flavonoids in S. flavescens:Using LC/MS technology for detection, a total of24flavonoids was identified in S. flavescens by comparing the reference substance, analyzing fragment ions and other informations, including11flavanones,3chalcones,3isoflavones,3pterocarpans,1isoflavanone,1flavanonol and1flavonol. 3.2Qualitative analysis of flavonoids in S. tonkinensis:Using LC/MS technology for detection, a total of17flavonoids was identified in S. tonkinensis by comparing the reference substance, analyzing fragment ions and other informations, including6isoflavones,4pterocarpans,4flavanones,2chalcones and1flavonol.4. Quantitative analysis of flavonoids in S. flavescens and S. tonkinensis by HPLC4.1Quantitative analysis of S. flavescens:the assay was successfully applied to the quantification of seven flavonoids in twenty-one samples of S. flavescens. The major constituent of S. flavescens is Kurarinone (10.47mg/g, flavanone), and the contents of the other six flavonoids are as fllows:Sophoraflavanone G (2.73mg/g, flavanone), Trifolirhizin (2.05mg/g, petrocarpan), Sophoraflavanone B (0.59mg/g, flavanone), Maackiain (0.56mg/g, petrocarpan), Isoxanthohumol (0.14mg/g, flavanone) and Kuraridine (0.34mg/g, chalcone). There was a significant variability in the content of flavonoids of the twenty-one S. flavescens samples. The total amounts of seven flavonoids varied from9.69to36.28mg/g and the average level is16.89mg/g.4.2Quantitative analysis of S. tonkinensis:the assay was successfully applied to the quantification of five flavonoids in seventeen samples of S. tonkinensis. The major constituent of S. tonkinensis is Sophoranone (2.53mg/g, flavanone), and the contents of the other four flavonoids are as follows:Maackiain (1.13mg/g, petrocarpan), Trifolirhizin (0.86mg/g, petrocarpan), Sophoranochromene (0.35mg/g, flavanone) and Quercetin (0.16mg/g, flavonol). There was variability in the content of flavonoids of the seventeen S. tonkinensis samples. The total amounts of five flavonoids varied from3.18to8.25mg/g and the average level is5.05mg/g.5. Stability test of total flavonoids from S. flavescens and S. tonkinensis in artificial gastric and intestinal juice by HPLCIn this assay, the HPLC peak areas of total flavonoids from S. flavescens and S. tonkinensis in artificial gastric and intestinal juice were detected for six hours, results displayed that:flavonoids were stabile in six hours, neither degradation nor transformation. It was thus evident that the flavonoids didn’t influenced by gastric and intestinal juice.Our research was different from the traditional jobs which always focused on alkaloids of S. flavescens and S. tonkinensis. The comparative studies on flavonoids and their bioactivities of these two herbs were developed. Due to the systematically chemical researches and LC/MS identity results of the flavonoids, we had made clear the different types of flavonoids in these two herbs:the main flavonoids in S. flavescens were flavanones along with isoflavones, chalcones and petrocarpans, while the main flavonoids of S. tonkinensis were flavonones, isoflavones and petrocarpans along with chalcones, flavones and flavonols. The HPLC assays were performed to make clear the different contents of main active flavonoids in S. flavescens and S. tonkinensis. The major flavonoid of S. flavescens was Kurarionoe, and the characteristic flavonoids different from S. tonkinensis were SFQ SFB, Isoxanthohumol and Kuraridine. The major flavonoid of S. tonkinensis was Sophoranoe, and the characteristic flavonoids different from S. flavescens were Sophoranochromene and Quercetin. And it had significant meaning of consummating the quality control methods and carrying further researches for S. flavescens and S. tonkinensis. Bioactivity tests were also applied to investigate the various pharmacological effects of these two herbs. Flavonoids isolated from S. tonkinensis showed better anticomplementary activities, while that of S. flavescens indicated better cytotoxic and antibacterial effects. Flavanones with prenyl or lavandulyl chains from S. flavescens had significant antibacterial activities, and this was one of the important reasons that S. flavescens was always in external application for the treatments of dermatological and gynecological diseases clinically. In addition, the flavonoids that manifestated good biological aptivities such as anticomplement, cytotoxic and antibacterial activities, offered the ideas for the development of new drugs and the further exploitation of Sophora medicinal plants resources. |
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