Total saponins of Panax ginseng effects on proliferation and differentiation of human embryonic neural stem cells and in a Parkinson's disease mouse model

BACKGROUND： Total saponins of Panax ginseng （TSPG） exhibits neuroprotection against Parkinson＇s disease in the substantia nigra. OBJECTIVE： To investigate the effects of TSPG on human embryonic neural stem cells （NSCs） proliferation and differentiation into dopaminergic neurons using in vitro studies, and to observe NSC differentiation in a mouse model of Parkinson＇s disease, as well as behavioral changes before and after transplantation. DESIGN, TIME AND SETTING： In vitro neural cell biology trial and in vivo randomized, controlled animal trial were performed at the Institute of Basic Medical Sciences, Chongqing Medical University between September 2004 and December 2007. MATERIALS： TSPG （purity 〉 95%） was isolated, extracted, and identified by Chongqing Academy of Chinese Materia Medica. Recombinant human basic fibroblast growth factor （bFGF） and recombinant human epidermal growth factor （EGF） were purchased from PeproTech, USA. A total of 25 C57/BL6J mice, aged 18-20 weeks were included. Twenty were used to establish a Parkinson＇s disease model with i.p. injection of MPTP （1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine） and TSPG alone or combined with interleukin-1 （IL-1）-treated NSCs prior to transplantation into the corpus striatum. The remaining five mice were pretreated for 3 days with TSPG prior to MPTP injection, serving as the TSPG prevention group. METHODS： Primary NSCs were isolated, cultured and purified from embryonic cerebral cortex. Immunocytochemistry was employed to detect specific antigen expression in the NSCs. In vitro experiment： （1） to induce proliferation, NSCs were treated with TSPG, EGF＋bFGF, or TSPG＋EGF＋bFGF, respectively; （2） to induce dopaminergic neuronal differentiation, NSCs were treated with TSPG, IL-1, or TSPG＋IL-1, respectively. MAIN OUTCOME MEASURES： In vitro experiment： the effects of TSPG on NSCs proliferation were evaluated with flow cytometry and MTT assay. Tyrosine hydroxylase expression was determined by immunocytochemistry assay to observe effects of TSPG on dopaminergic neuronal differentiation. In vivo experiment： differentiation of grafted NSCs in the mouse brain was determined by immunohistochemical staining. Behavioral changes were evaluated by spontaneous activity frequency, memory function, and score of paralysis agitans. RESULTS： （1） NSCs were cultured and passaged for more than three passages. Immunocytochemistry revealed positive nestin staining, as well as neurofilament protein and glial fibrillary acidic protein. （2） TSPG significantly increased NSC proliferation, in particular when combined with EGF and bFGF, which was twice as effective as FGF or bFGF alone. TSPG also induced dopaminergic differentiation in NSCs, in particular when TSPG was added together with IL-1, resulting in an effect five times greater than that of IL-1 alone. （3） At day 30 following transplantation, most NSCs in the TSPG prevention group differentiated into dopaminergic neurons, and the scores of paralysis agitans, spontaneous activity, and memory function were significantly increased compared with TSPG alone or TSPG＋IL-1 groups （P 〈 0.05）. CONCLUSION： TSPG stimulated NSC proliferation, in particular when combined with FGF and bFGF. TSPG significantly induced dopaminergic neuronal differentiation of NSCs, and the effect was greater when combined with IL-1. In addition, TSPG greatly improved behavior in the Parkinson＇s disease mouse model following NSC transplantation. Following NSC transplantation, TSPG pretreatment exhibited superior efficacy over either TSPG alone or TSPG in combination with IL-1, in terms of behavioral improvements in the Parkinson＇s disease mouse model.

Determination of the Content of Total Saponins in Paris daliensis H.Li et V. G. Soukoup and Paris dulongensis H. Li et S. kurita

[Objectives]To determine the content of total saponins in Paris daliensis H. Li et V. G. Soukoup and Paris dulongensis H. Li et S.kurita,fill the gap in the study of Paris daliensis H. Li et V. G. Soukoup and Paris dulongensis H. Li et S. kurita,and lay a foundation for searching new medicinal resources of Paris L. [Methods]The perchloric acid colorimetric method and UV spectrophotometer were used to determine the content of total saponins in Paris daliensis H. Li et V. G. Soukoup and Paris dulongensis H. Li et S. kurita at 406 nm. [Results]The lowest content of total saponins in Paris daliensis H. Li et V. G. Soukoup was 2. 637%,while the highest content reached 5. 474%; the lowest content of total saponins in Paris dulongensis was 1. 806%,while the highest content reached 3. 532%. Through the methodological examination,it was found that this method was stable and reliable. [Conclusions] The content of total saponins was high in Paris daliensis H.Li et V. G. Soukoup and Paris dulongensis H. Li et S. kurita,and it was suggested to further study and find the medicinal resources of Paris L.

滇重楼地上部分与地下部分总皂苷止血、镇痛、抗炎作用比较

以宫血宁胶囊为阳性对照，观察滇重楼茎叶、果实与根茎总皂苷对小鼠尾尖出血时间的影响、对醋酸致小鼠疼痛扭体反应的影响以及对二甲苯致小鼠耳廓肿胀的影响，结果表明滇重楼根茎总皂苷的止血和抗炎作用均强于茎叶和果实的总皂苷作用；根茎、茎叶、果实的总皂苷镇痛作用相当。因此滇重楼茎叶、果实与根茎总皂苷均具有止血、抗炎和镇痛作用。

紫外分光法测定无患子果皮中总皂苷的含量

以甲醇作溶剂，提取料液比为1：10，超声提取2次（每次30rain）；以香草醛冰醋酸-高氯酸为显色剂，（显色温度70℃，显色时间15min）在541nm下，用紫外分光光度法测定总皂苷的含量。结果表明，无患子总皂苷含量在28～126μg之间与吸光度线性关系良好，相关系数r=0．9997，方法平均回收率为98．68％，RSD（n=5）为2．06％。本方法重现性好，结果稳定可靠，可为无患子果皮中总皂苷含量的测定提供参考。

大孔树脂纯化无患子总皂苷工艺研究

本文研究了D-101大孔树脂纯化无患子总皂苷的工艺条件。通过考察上柱样品量、吸附时间、洗脱剂浓度、洗脱剂用量、洗脱速度等因素的影响，确定了较优的工艺方案：30g大孔树脂柱（2cm×40cm）上样20mL，吸附30min，200mL水洗除杂，300mL50％乙醇以3BV／h的速度洗脱。经过大孔吸附树脂富集、纯化后，无患子总皂苷纯度可达64％以上，说明采用本法富集、纯化无患子总皂苷可行。

连续泡沫分离法纯化无患子总皂苷的研究

目的采用连续泡沫分离法纯化无患子总皂苷,确定泡沫分离最佳工艺条件。方法采用正交试验法与分光光度法,以无患子总皂苷富集比、收率和质量分数为评价指标,确定泡沫分离的最佳工艺条件。结果连续泡沫法分离无患子总皂苷的最佳工艺条件为:进料液中无患子总皂苷质量浓度为20 mg/L,pH值为4.40,进料口高度为40 cm,气体体积流量为0.6 L/min,进料液体积流量为35 mL/min;通过连续泡沫分离法纯化得到无患子总皂苷,收率为23.64%、质量分数为90.27%。结论连续式泡沫分离法纯化无患子总皂苷具有操作简单、能耗低等优点,可工业化应用。

不同蒸制法对三七主根中皂苷的影响

目的研究不同蒸制法对三七主根中皂苷的影响。方法采用比色法测定主根中总皂苷的量,采用HPLC法测定主根中单体皂苷的量。结果蒸制后主根中总皂苷及单体皂苷三七皂苷R1和人参皂苷Rg1、Re、Rb1、Rd的量均有不同程度的降低,而人参皂苷Rh1、Rh4、Rk3和20(S)-、20(R)-人参皂苷Rg3这5种单体皂苷的量均有不同程度的增加。结论不同蒸制法对三七中皂苷成分的影响不同,总皂苷和5种主要皂苷成分的量下降程度和新产生成分的量增加程度与蒸制时间和温度相关。该方法可用于三七炮制品中皂苷类成分的定量测定和质量控制,为三七"生打熟补"与物质变化之间的相关性研究提供依据,同时为研究稀有皂苷的积累规律提供了一定的理论基础。

三七总皂苷对小鼠肝脏谷胱甘肽-S-转移酶的抑制作用及其动力学分析

目的:研究三七总皂苷(total saponins of Panax notoginseng,PNS)对小鼠肝脏谷胱甘肽-S-转移酶(glutathione-S-transferase,GST)的抑制作用及其酶动力学。方法:采用差速离心法制备小鼠胞质液酶,用半数效量概率单位法及Lineweaver-Burk双倒数作图法求得小鼠肝脏GST的米氏常数(Km)、最大反应速度(Vmax)和三七总皂苷对小鼠肝脏GST的半数抑制浓度(IC50)以及判断三七总皂苷对GST的抑制类型和抑制常数(KI,KIS)。结果:三七总皂苷对小鼠肝脏GST有抑制作用,其IC50是189.54 mg.L-1;对底物还原型谷胱甘肽(GSH),GST的Km是0.456 3 mmol.L-1,Vmax是476.19 U.mg-1;对底物1-氯-2,4-二硝基苯(CDNB),GST的Km是0.109 7 mmol.L-1,Vmax是400.00 U.mg-1。对底物GSH,三七总皂苷对小鼠肝脏GST属于混合性抑制,其抑制常数KI是0.27 mg.L-1,KIS是0.68 mg.L-1;对底物CDNB,三七总皂苷对小鼠肝脏GST属于混合性抑制,其抑制常数KI是0.21 mg.L-1,KIS是0.66 mg.L-1。结论:三七总皂苷对小鼠肝脏GST的活性有明显抑制作用。