Effects of Tribulus terrestris saponins on proliferation and invasion of A549 cells

Objective:Tribulus terrestris saponin is a traditional Chinese medicine in China.This experiment was designed to investigate the effects of tribulus terrestris saponin on the proliferation and invasion ability of non-small cell lung cancer A549 cells.Methods:A549 cells were divided into normal control and experimental groups(Tribulus terrestris saponin 250μg/mL group,Tribulus terrestris saponin 200μg/mL group,Tribulus terrestris saponin 150μg/mL group,Tribulus terrestris saponin 100μg/mL group,Tribulus terrestris saponin 50μg/mL group).The proliferation viability of the cells in each group was detected by CCK8,the invasion of tumor cells was detected by Transwell model.The mRNA expression of MMP9 and caspase-3 in each group of cells was detected by RT-PCR.Immunofluorescence staining was used to observe the fluorescence intensity of caspase-3 in each group of cells.Results:Compared with the normal control group,tribulus terrestris saponin significantly inhibited the proliferation activity and invasion ability of A549 cells,which was statistically significant(P<0.01).In the invasion assay,compared with the control group,MMP9 expression was significantly reduced and caspase-3 expression was significantly increased in the tribulus terrestris saponin group,and both were concentration-dependent,with statistically significant differences(P<0.01).By cellular immunofluorescence staining experiments,it was found that the fluorescence expression of caspase-3 was enhanced in the experimental group compared with the normal control group,in which the high concentration saponin group was significantly higher than the low concentration group.Conclusion:Tribulus terrestris saponin can inhibit the invasive ability of A549 cells by down-regulating the expression of MMP9,and induce irreversible apoptosis by up-regulating the activation of caspase-3 expression to form caspase-3.

Two New Furostanol Saponins from Tribulus Terrestris L.

Two new furostanol glucosides, named tribufurosides G and H, were isolated from Tribulus terrestris L. The structures of the two new furostanol glucosides were elucidated as 26-O-β-D-glucopyranosyl-（25R）-5α-furost12-one-3β,22α,26-tetraol-3-O-β-D-glucopyranosyl-（ 1→2 ）-β-D-glucopyranosyl-（ 1→4 ）-β-D-galactopyranoside（ 1 ） and 26-O-β-D-glucopyranosyl-（25S）-5 α- 12-one-2 α,3,8,22 α,26-tetraol-3 -O-β-D-glucopyranosyl-（ 1 → 2 ）-β-D-glucopyranosyl-（1→4）-β-D-galactopyranoside（2） by 1 D, 2D NMR techniques, ESI-MS analysis as well as chemical methods.

Steroidal saponins with anti-inflammatory activity from Tribulus terrestris L.

Objective:Tribulus terrestris L.(T.terrestris)is a highly valuable traditional Chinese medicine used to treat stroke,inflammation,pulmonary fibrosis,liver cancer,and urolithiasis.To identify the basic substance responsible for the anti-inflammatory effect of TST(total saponins of Tribulus),its chemical composition was systematically studied,and its effect of inhibiting nitric oxide generation and the expression of related inflammatory factors were determined.Methods:To separate chemical constituents from T.terrestris by column chromatography.Spectroscopic methods,including 1D and 2D nuclear magnetic resonance spectroscopy(NMR)and mass spectrometry(MS)techniques,were used to elucidate the isolated compounds.The anti-inflammatory activities of TST and several compounds were evaluated in vitro.Results:Fifteen steroidal saponins,including 9 furostanol steroidal saponins(1,2,3,4,5,6,7,8,and 15)and 6 isospirostanol steroidal saponins(9,10,11,12,13,and 14),were isolated from T.terrestris.TST significantly decreased the expression of tumor necrosis factor-a(TNF-a)and interleukin-6(IL-6)in RAW 264.7 cells stimulated by lipopolysaccharides.Compounds 13 and 15 evidently reduced TNF-a expression.Compounds 6,10,12,13,and 15 markedly reduced IL-6 expression.Conclusions:Compounds 1 was a novel furostanol steroidal saponin,named 26-O-b-D-glucopyranosyl-(25R)-5afurostan-12-carbonyl-20(22)-en-3b,26-diol-3-O-{b-D-xylopyranosyl-(1→2)-[b-D-xylopyranosyl-(1→3)]-b-D-glucopyranosyl-(1→4)-[a-L-rhamnopyranosyl-(1→2)]-b-D-galactopyranoside}.Compounds 2 was isolated from the family Zygophyllaceae for the first time,and 5 and 6 were isolated from the Tribulus genus.TST and compounds 6,10,12,13,and 15 exerts antiinflammatory activity.

Optimization of Extraction of Saponins from Tribulus terrestris L.

To promote the recovery of saponins from Tribulus terrestris L.,the water dissolution and ethanol precipitation method was adopted to extract saponins from the powder prepared from T.terrestris fruit.The optimal process was to extract with 9 fold of the extracting water at the ethanol concentration of 85 for 3 times,each time 3.5 h.The effects of the factors ranked as the extracting water quantity > treatment time > treatment times > ethanol concentration.Under these optimal conditions,the recovery of saponins was 2.81%.After these crude saponins were purified by D101 macroporous adsorptive resin,the purity was found to be promoted through the UV scanning method,and the recovery of the refined saponins was up to 1.53%.Both of the recovery values were higher than that of the existing process.

Protection by the gross saponins of Tribulus terrestris against cerebral ischemic injury in rats involves the NF-κB pathway

The aim of this study was to investigate whether the gross saponins of Tribulus terrestris(GSTT),a traditional Chinese herbal medicine,have neuroprotective effects on rats subjected to middle cerebral artery occlusion(MCAO),through nuclear factor-κB(NF-κB)pathway and inflammatory mediators.Cerebral ischemia was produced by MCAO in either untreated(control)or GSTT-pretreated rats,and the animals were examined for infarct volume,cerebral edema,neuro-behavioral abnormality and pathological changes.Meanwhile,the expression of NF-kB protein in brain tissue was analyzed on Western blots and the serum levels of TNF-α and IL-1 were determined by ELISA.The experimental results demonstrated that,compared with the control MCAO group,GSTT-pretreated MCAO group had significantly reduced infarct volume,brain edema and neuro-behavioral abnormality,and lesser degree of pathologic changes in the brain,as well as had lower levels of serum TNF-α and IL-1β,and higher levels of brain NF-κB(Po0.05).Furthermore,treatment with an NF-κB inhibitor pyrrolidine dithiocarbamate(PDTC)abolished the protective effects of GSTT against MCAO-induced cerebral ischemic injury.These results indicated that GSTT’s ability to protect against cerebral ischemic injury was mediated through the NF-κB signaling pathway,and that GSTT may act through inhibition of the production of inflammatory mediators.

Antiglycation and antioxidant activities of the crude extract and saponin fraction of Tribulus terrestris before and after microcapsule release

Objective:The present study investigated antiglycation and antioxidant activities of crude dry extract and saponin fraction of Tribulus terrestris.It also developed a method of microencapsulation and evaluated antiglycation and antioxidant activities of crude dry extract and saponin fraction before and after microcapsule release.Methods:Antiglycation activity was determined by relative electrophoretic mobility(REM),free amino groups and inhibition of advanced glycation end-product(AGE)formation.Antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl(DPPH),ferric ion-reducing antioxidant power(FRAP),nitric oxide(NO)and thiobarbituric acid reactive species(TBARS)tests.Microcapsules were prepared using maltodextrin as wall material and freeze-drying as encapsulation technique.Morphological characterization of microcapsules was evaluated by scanning electron microscopy,and encapsulation efficiency and microcapsule release were determined by total saponins released.Antiglycation and antioxidant assays were performed using crude dry extract and saponin fraction of T.terrestris before and after release.Results:Saponin fraction showed an increase of 32.8%total saponins.High-performance liquid chromatography-mass spectrometry analysis showed the presence of saponins in the obtained fraction.Antiglycation evaluation by REM demonstrated that samples before and after release presented antiglycation activity similar to bovine serum albumin treated with aminoguanidine.Additionally,samples inhibited AGE formation,highlighting treatment with saponin fraction after release(89.89%).Antioxidant tests demonstrated antioxidant activity of the samples.Crude dry extract before encapsulation presented the highest activities in DPPH(92.00%)and TBARS(32.49%)assays.Saponin fraction before encapsulation in FRAP test(499μmol Trolox equivalent per gram of dry sample)and NO test(15.13μmol nitrite formed per gram of extract)presented the highest activities.Conclusion:This study presented antiglycation activity of crude dry extract and saponin fraction of T.terrestris,besides it demonstrated promising antioxidant properties.It also showed that the encapsulation method was efficient and maintained biological activity of bioactive compounds after microcapsule release.These results provide information for further studies on antidiabetic and antiaging potential,and data for new herbal medicine and food supplement formulations containing microcapsules with crude extract and/or saponin fraction of T.terrestris.

蒺藜皂苷提取工艺及其药理活性研究进展

蒺藜皂苷是中药材蒺藜的主要成分,其药理活性显著,具有抗癌、抗氧化、抗炎、抗菌、抗抑郁、抗疲劳、降血糖、保护心脑血管和保护神经等功效.综述了蒺藜皂苷的提取方法和药理活性,以期为蒺藜皂苷的深加工综合利用提供理论参考.

HPLC-CAD法与ELSD法用于测定蒺藜皂苷提取物中蒺藜皂苷K的对比研究

目的:分别建立高效液相色谱-电雾式(CAD)和蒸发光散射(ELSD)2种通用型检测器测定蒺藜皂苷提取物中蒺藜皂苷K含量的方法。方法:采用高效液相色谱法,Waters Symmetry Shield RP18为色谱柱,以乙腈-水为流动相,梯度洗脱,体积流速为1.0 mL·min-1,柱温为25℃;使用CAD和ELSD检测器测定,并对样品实测结果进行比较。结果:2种方法线性关系均良好,精密度、重复性、稳定性及加样回收率皆符合方法学要求,CAD的检出限和定量限均比ELSD低。结论:2种检测方法都可用于蒺藜皂苷提取物中蒺藜皂苷K的含量测定,CAD检测器比ELSD检测器具有更高的灵敏度和精密度。

基于TLR4/NF-κB通路研究蒺藜总皂苷对周围神经病理性疼痛的镇痛作用

目的:基于TLR4/NF-κB通路研究蒺藜总皂苷(gross saponins from tribulus terrestris,GSTT)对大鼠慢性坐骨神经损伤(chronic constriction injury,CCI)所引起的周围神经病理性疼痛是否具有镇痛作用。方法:选取雄性SD大鼠40只,随机分为假手术组(SHAM组)、CCI组、GSTT 100 mg/kg+CCI组、GSTT 200 mg/kg+CCI组、普瑞巴林+CCI组(阳性对照)5组,每组8只。除SHAM组,其余各组均制备CCI大鼠模型,分别以药物处理后,检测各组大鼠机械缩足反射阈值(paw withdrawal mechanical threshold,PWMT)、热敏潜伏期;以HE染色检测各组大鼠脊髓及背根神经节组织病理形态变化;免疫组化检测脊髓及背根神经节组织中NF-κB的表达。结果:与SHAM组相比,CCI组大鼠PWMT、热敏潜伏期明显降低(P<0.05);损伤侧脊髓背角及背根神经节的神经元、胶质细胞染色较深,并出现肿胀、萎缩等损伤,细胞核浓缩、丢失;损伤侧脊髓背角及背根神经节中NF-κB表达水平明显增高(P<0.05)。与CCI组相比,GSTT 100 mg/kg+CCI组、200 mg/kg+CCI组,普瑞巴林+CCI组大鼠PWMT、热敏潜伏期升高(P<0.05),上述病理变化也明显改善,损伤侧脊髓背角及背根神经节中NF-κB水平降低。结论:GSTT对CCI大鼠引起的周围神经病理性疼痛具有镇痛作用,可能与TLR4/NF-κB通路有关。

蒺藜皂苷对A549细胞增殖及侵袭能力影响的研究

目的:蒺藜皂苷是我国的一种传统中药,本实验拟探讨蒺藜皂苷对非小细胞肺癌A549细胞增殖和侵袭能力的影响。方法:将A549细胞分为正常对照组和实验组(蒺藜皂苷250μg/mL组,蒺藜皂苷200μg/mL组,蒺藜皂苷150μg/mL组,蒺藜皂苷100μg/mL组,蒺藜皂苷50μg/mL组),采用CCK8法检测各组细胞的增殖活力,Transwell小室模型检测肿瘤细胞的侵袭能力,RT-PCR检测各组细胞中MMP9、caspase-3的mRNA表达差异。利用细胞免疫荧光染色观察各组细胞caspase-3荧光强度。结果:与正常对照组相比,蒺藜皂苷明显抑制A549细胞的增殖活性及侵袭能力,差异具有统计学意义(P<0.01)。在侵袭实验中,RT-PCR实验结果表明与对照组相比,蒺藜皂苷组MMP9表达明显降低,caspase-3表达明显增加,且都呈浓度依赖性,差异有统计学意义(P<0.01)。通过细胞免疫荧光染色实验发现,相比正常对照组,实验组caspase-3的荧光表达增强,其中高浓度皂苷组明显高于低浓度组。结论:蒺藜皂苷可以通过下调MMP9的表达以抑制A549细胞的侵袭能力,通过上调caspase-3表达来诱导细胞发生不可逆的凋亡。

响应面法优选蒺藜炮制工艺

响应面法优选蒺藜最佳炮制工艺。采用紫外可见分光光度法,以呋甾皂苷含量为考察指标,选取影响总呋甾皂苷含量的主要因素烘制温度、烘制时间,进行2因素5水平的中心组合试验设计,优选蒺藜最佳炮制工艺。通过Design-Expert 12.0.3.0软件分析预测得出蒺藜最佳炮制工艺为烘制温度190.1℃、烘制时间10.51 min,在此条件下得到的呋甾皂苷含量为1.85%。优选的蒺藜炮制工艺稳定可行。

蒺藜及蒺藜皂苷治疗勃起功能障碍的研究进展

目的探讨蒺藜及蒺藜皂苷对勃起功能障碍的作用研究,为临床应用提供理论依据。方法通过分析中医药传统医书记载和现代国内外文献,对蒺藜及蒺藜皂苷改善勃起功能进行综述阐释。结果研究证实,蒺藜及蒺藜皂苷通过提高睾丸激素水平、抗氧化、控制血糖、调理血脂及发挥抗动脉硬化等作用改善勃起功能。结论蒺藜及蒺藜皂苷在改善勃起功能的临床应用及其基础研究中具有重要价值。

蒺藜皂苷对小鼠急性酒精性肝损伤的保护作用研究

目的寻找小鼠急性酒精性肝损伤相关的潜在代谢标志物,评价蒺藜皂苷(GSTT)对小鼠急性酒精性肝损伤的保护作用。方法小鼠单次大剂量酒精灌胃建立急性酒精性肝损伤模型,并以GSTT进行干预,测定肝脏和血清生化指标,同时采用核磁共振氢谱(^(1)H-NMR)技术结合数据的多元统计分析方法,研究小鼠急性酒精性肝损伤所致肝脏代谢物谱的变化并鉴定相关代谢标志物。结果与Con组相比,Model组小鼠血清ALT、AST、MDA活力显著升高(P<0.01),SOD、GSH活力显著降低(P<0.01);与Model组相比,蒺藜皂苷给药组出现剂量依赖性差异,小鼠血清ALT、AST、MDA活力显著降低(P<0.01),SOD、GSH活力显著升高(P<0.01);GSTT能明显回调琥珀酸、延胡索酸、乳酸等13种肝脏代谢标志物,纠正代谢紊乱。结论GSTT可以使急性酒精性肝损伤小鼠肝脏中代谢物水平得到不同程度的恢复,其保护作用可能与降低氧化应激,改善能量代谢、氨基酸代谢、脂质代谢、嘌呤代谢等方面相关。

蒺藜皂苷对人肝癌细胞BEL7402生长抑制和诱导凋亡作用的研究

目的 :探讨蒺藜皂苷 (STT)对体外培养的人肝癌细胞BEL 74 0 2的增殖抑制作用及诱导凋亡作用。方法 :应用MTT与SRB法检测蒺藜皂苷对人肝癌细胞BEL 74 0 2的抑制作用 ,采用形态学观察以及流式细胞术 (FCM)检测了STT诱导人肝癌细胞BEL 74 0 2凋亡的效应 ,采用流式细胞术检测BEL 74 0 2细胞Bcl 2蛋白表达。结果 :①STT能够剂量依赖地抑制人肝癌细胞BEL 74 0 2的增殖 ;②细胞显现典型的凋亡形态学改变 ,FCM出现凋亡峰 ;③STT能降低BEL 74 0 2细胞的Bcl 2蛋白表达。结论 :STT在体外能抑制BEL 74 0 2细胞的增殖并能诱导细胞凋亡 ,其诱导凋亡作用的途径之一是下调Bcl 2蛋白表达。

蒺藜化学成分的研究

目的：研究蒺藜（ Ｔｒｉｂｕｌｕｓｔｅｒｒｅｓｔｒｉｓ Ｌ．） 中生物活性物质。方法：用硅胶柱色谱、ＨＰＬＣ进行分离，根据光谱数据鉴定其结构。结果：分离纯化得到两种呋甾皂苷，确定其结构为２６ＯβＤ吡喃葡糖基（２５ Ｒ，Ｓ）５α呋甾１２羰基２０（２２）烯基３β，２６二醇３ＯβＤ吡喃葡糖基（１ →４）βＤ吡喃半乳糖苷（Ｉ） ；２６ＯβＤ吡喃葡糖基（２５ Ｒ）５α呋甾１２羰基３β，２２α，２６三醇３ＯβＤ吡喃葡糖基（１→２）βＤ吡喃半乳糖苷（ＩＩ）。结论：Ｉ。

注射用蒺藜皂苷的抗血栓形成作用

目的 :研究注射用蒺藜皂苷 (GSTT)对体内外血栓形成的影响。方法 :1采用动 -静脉旁路血栓形成法、电刺激动脉血栓形成法和体外血栓法 ,观察 GSTT对体内血栓湿重、血栓形成时间以及体外血栓长度和干、湿重的影响 ;2建立血瘀模型 ,观察 GSTT对血瘀大鼠全血高切、低切粘度的影响。结果 :1GSTT2 0 .8和31.2 mg· kg- 1可降低血栓湿重 (P<0 .0 5或 P<0 .0 1) ,延长血栓形成时间 (P<0 .0 1) ,缩短体外血栓长度(P<0 .0 5 ) ,降低体外血栓湿重和干重 (P<0 .0 1或 P<0 .0 5 ) ;2 GSTT2 0 .8和 31.2 m g· kg- 1可明显降低血液黏度 (P<0 .0 1)。结论 :GSTT具有抗血栓形成作用 ,其机制与降低血液黏度、改善血液流变性有关。

蒺藜皂苷对乳腺癌细胞Bcap-37的体外抑制作用

采用生长曲线法、MTT法、蛋白质含量测定以及形态学方法观察了蒺藜皂苷(STT)对于体外培养Bcap-37细胞抑制作用,结果显示:STT对于Bcap-37细胞增殖有较强的抑制作用,且呈明显的剂量与时间依赖关系,经STT作用后,细胞形态学表现为细胞变圆、皱缩,核固缩、浓聚。

蒺藜皂苷对大鼠小肠α-葡萄糖苷酶的抑制作用

目的:研究蒺藜皂苷(STT)对大鼠小肠α-葡萄糖苷酶的抑制作用及对餐后血糖水平的影响。方法:提取大鼠小肠α-葡萄糖苷酶,测定STT对其活性的抑制作用;一次性灌胃蔗糖(2 g.kg-1)或葡萄糖(2 g.kg-1),同时灌胃STT(100 mg.kg-1),测定60 min后血糖水平的变化;每天一次性灌胃STT(100 mg.kg-1),连续14 d,测定小肠α-葡萄糖苷酶活性以及灌胃蔗糖(2 g.kg-1)60 min后血糖水平的变化。结果:0.1,1,10 mg.mL-1的STT能够显著抑制α-葡萄糖苷酶的体外活性(P<0.001),抑制率分别为(20.83±1.66)%,(43.73±2.39)%和(52.62±2.69)%;STT显著抑制大鼠一次性灌胃蔗糖60 min后血糖水平的升高(P<0.01),变化幅度为对照的(52.61±6.24)%,但对灌胃葡萄糖没有作用;连续灌胃STT 14 d后,α-葡萄糖苷酶活性显著低于对照(P<0.05),为对照的(58.17±3.24)%,灌胃蔗糖60 min后血糖变化幅度为对照的(69.50±4.28)%(P<0.05)。结论:STT抑制大鼠餐后血糖水平的升高,是由其对小肠α-葡萄糖苷酶的抑制作用实现的。

天然产物 T12抗真菌作用及其机制研究

目的:研究从刺蒺藜(Tribulus terrestris L.)分离的一种皂苷类化合物T12的抗真菌作用及机制。方法:采用微量液基稀释法测定T12体外MIC_(80)值;透射电镜观察T12对白色念珠菌超微结构的影响;薄层色谱法、气质联用色谱法分别研究T12对细胞膜麦角甾醇生物合成通路的影响。结果:T12具有强的抗真菌活性,对白色念珠菌的体卟MIC_(80)值为0.87μmol·L^(-1);透射电镜观察,可见T12作用后,细胞膜多处破裂,细胞壁未见明显改变;T12对细抱膜麦角甾醇生物合成通路有明显影响。结论:结构新型、具有强抗菌活性的T12,通过影响白色念珠菌细胞膜麦角甾醇的生物合成发挥抗真菌作用。