Novel dammarane-type saponins from Gynostemma pentaphyllum and their neuroprotective effect

Abstract

Three novel dammarane-type saponins, 2α,3β,12β,20(S),24(S)-pentahydroxydammar-25-ene-3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-20-O-β-D-glucopyranoside (1, namely gypenoside J1), 2α,3β,12β,20(S),25-pentahydroxydammar-23-ene-3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-20-O-β-D-glucopyranoside (2, namely gypenoside J2) and 2α,3β,12β,20(S)-tetrahydroxydammar-25-en-24-one-3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-20-O-β-D-xylopyranosyl-(1→6)-β-D-glucopyranoside (3, namely gypenoside J3) along with one known gypenoside (gypenoside LVII) were isolated from the aerial parts of G. pentaphyllum using various chromatographic methods. Their structures were elucidated on the basis of IR, 1D- (¹H and ¹³C), 2D-NMR spectroscopy (HSQC, HMBC and COSY), and mass spectrometry (ESI-MS/MS). Their activity was tested using CCK-8 assay. These four compounds showed little anti-cancer activity with IC₅₀ values more than 100?μM against four types of human cancer lines. The effects of them against H₂O₂-induced oxidative stress in human neuroblastoma SH-SY5Y cells were evaluated and they all showed potential neuroprotective effects with 3.64–18.16% higher cell viability than the H₂O₂-induced model group.     

Enzymatic Biotransformation of Gypenoside XLIX into Gylongiposide I and Their Antiviral Roles against Enterovirus 71 In Vitro

Biotransformation of specific saponins in the valuable medical plants to increase their bioavailability and pharmaceutical activities has attracted more and more attention. A gene encoding a thermophilic glycoside hydrolase from Fervidobaterium pennivorans DSM9078 was cloned and expressed in Escherichia coli. The purified recombinant enzyme, exhibiting endoglucanase cellulase activity, was used to transform gypenoside XLIX into gylongiposide I via highly selective and efficient hydrolysis of the glucose moiety linked to the C21 position in gypenoside XLIX. Under the optimal reaction conditions for large scale production of gylongiposide I, 35 g gypenoside XLIX was transformed by using 20 g crude enzyme at pH 6.0 and 80 °C for 4 h with a molar yield of 100%. Finally, 11.51 g of gylongiposide I was purified using a silica gel column with 91.84% chromatographic purity. Furthermore, inhibitory activities of gypenoside XLIX and gylongiposide I against Enterovirus 71 (EV71) were investigated. Importantly, the EC₅₀ of gypenoside XLIX and gylongiposide I calculated from viral titers in supernatants was 3.53 μM and 1.53 μM, respectively. Moreover, the transformed product gylongiposide I has better anti-EV71 activity than the glycosylated precursor. In conclusion, this enzymatic method would be useful in the large-scale production of gylongiposide I, which would be a novel potent anti-EV71 candidate.     

Development of a targeted method for quantification of gypenoside XLIX in rat plasma,using SPE and LC–MS/MS

A sensitive, selective and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the quantification of gypenoside XLIX, a naturally occurring gypenoside of Gynostemma pentaphyllum in rat plasma and then validated according to the US Food and Drug Administration's Guidance for Industry: Bioanalytical Method Validation . Plasma samples were prepared by a simple solid‐phase extraction. Separation was performed on a Waters XBridgeTM BEH C₁₈ chromatography column (4.6 × 50 mm, 2.5 μm) using a mobile phase of acetonitrile and water (62.5:37.5, v /v). Gypenoside XLIX and the internal standard gypenoside A were detected in the negative ion mode using selection reaction monitoring of the transitions at m/z 1045.6 → 913.5 and 897.5 → 765.4, respectively. The calibration curve was linear (R ² > 0.990) over a concentration range of 10–7500 ng/mL with the lower quantification limit of 10 ng/mL. Intra‐ and inter‐day precision was within 8.6% and accuracy was ≤10.2%. Stability results proved that gypenoside XLIX and the IS remained stable throughout the analytical procedure. The validated LC–MS/MS method was then applied to analyze the pharmacokinetics of gypenoside XLIX after intravenous administration to rats (1.0, 2.0 and 4.0 mg/kg).     

三七皂苷NMR研究Ⅱ——3个原人参二醇型双糖链配糖体的NMR信号全归属

运用2D NMR技术，如DQF ¹H-¹H COSY，HMQC，HMBC，TOCSY等，对3 个原人参二醇型双糖链配糖体人参皂甙-Rd,三七皂甙-E和七叶胆皂甙XVII的氢和碳的化学位移首次进行了全归属.     

超高效液相色谱-电雾式检测器测定福建产绞股蓝中绞股蓝皂苷XLVI和LVI北大核心CSCD

该研究利用超高效液相色谱-电雾式检测器(UHPLC-CAD)建立了福建产绞股蓝中绞股蓝皂苷XLVI和LVI含量的测定方法。首先利用超高效液相色谱-四极杆-飞行时间质谱(UHPLC-Q-TOF/MS)结合UHPLC-CAD鉴定了福建产绞股蓝的主要成分,其中绞股蓝皂苷XLVI、LVI以及二者相应的含丙二酰基酸性皂苷为其主成分,因此在含量测定时先进行碱水解预处理将酸性皂苷转化为对应的去丙二酰基中性皂苷,再利用UHPLC-CAD测定碱水解后绞股蓝皂苷XLVI和LVI的含量。将绞股蓝样品粉末在乙醇-水-氨水(50∶46∶4,v/v/v)和料液比1∶150(g∶mL)条件下超声提取30 min,静置24 h后,在Waters ACQUITY UPLC BEH C18色谱柱(100 mm×2.1 mm,1.7μm)上分离,采用0.1%(v/v)甲酸水溶液和乙腈作为流动相进行梯度洗脱,流速0.5 mL/min,柱温40℃,电雾式检测器检测。结果表明,绞股蓝皂苷XLVI和LVI分别在9.94~318.00μg/mL和12.78~409.00μg/mL范围内具有良好的线性关系,相关系数(r)分别为0.9993和0.9995。方法精密度、重复性和24 h稳定性试验的相对标准偏差(RSD)均小于2.0%(n=6),绞股蓝皂苷XLVI与LVI的加标回收率分别在100.2%~107.2%与97.9%~104.2%范围内,RSD值分别为2.4%与2.6%。16批绞股蓝样品含量测定结果显示:绞股蓝皂苷XLVI含量占0.57%~2.57%,绞股蓝皂苷LVI含量占0.66%~2.99%。该方法灵敏度高,重复性好,可用于福建产绞股蓝的质量研究和质量控制。     

绞股蓝皂苷与牛血清白蛋白相互作用的荧光光谱研究

用荧光光谱技术研究了绞股蓝皂苷与牛血清白蛋白(BSA)在pH=7.40的Tris-HCl缓冲溶液中的相互作用;通过计算确定了绞股蓝皂苷与BSA的结合位点数和结合常数,利用热力学分析探讨了绞股蓝皂苷与BSA之间的结合方式;同时采用同步荧光技术考察了绞股蓝皂苷对BSA构象的影响.结果表明,绞股蓝皂苷对牛血清白蛋白的荧光猝灭过程为静态猝灭;二者主要靠疏水作用和静电引力结合.