Dioscin (CCRIS 4123)   中文名称: 薯蓣皂苷

DNA; apoptosis

在 HeLa 和 SiHa 细胞中，薯蓣皂苷（1.25–5 μg/mL；6–24 小时）会提高细胞内钙水平并引发细胞凋亡[4]。薯蓣皂苷（1.25–5 μg/mL；6–24 小时）：薯蓣皂苷下调 Bcl-2 和 Bcl-xl 水平蛋白，上调 Bak、Bax、Bid、p53、caspase-3 和 caspase-9 蛋白[ 4]。

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薯蓣皂苷是一种天然甾体皂苷，来源于多种植物，对多种肿瘤细胞系具有强大的抗癌作用。在本研究中，我们研究了薯蓣皂苷对人LNCaP细胞的抗癌活性，并评估了其抗肿瘤作用的可能机制。研究发现，薯蓣皂苷（1、2和4μmol/L）可以以时间和浓度依赖的方式显著抑制LNCaP细胞的存活率。流式细胞术显示，薯蓣皂苷处理LNCaP细胞24h后细胞凋亡率升高，说明凋亡是薯蓣皂苷抑制癌症的重要机制。Western blot检测LNCaP细胞中caspase-3、Bcl-2和Bax的表达。切割的半胱氨酸天冬氨酸蛋白酶-3的表达显著增加，同时前天冬氨酸酶-3的表达明显降低。抗凋亡蛋白Bcl-2的表达下调，而促凋亡蛋白Bax的表达上调。Bcl-2/Bax比值显著降低。这些结果表明，薯蓣皂苷通过凋亡途径在人LNCaP细胞中具有潜在的抗肿瘤活性，这可能与半胱氨酸天冬氨酸蛋白酶-3和Bcl-2蛋白家族有关。[1]

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在这项研究中，我们首次证明薯蓣皂苷在体外以剂量依赖的方式抑制RANKL介导的破骨细胞分化和骨吸收。薯蓣皂苷的抑制作用得到了破骨细胞特异性标志物表达减少的支持。进一步的分子分析表明，薯蓣皂苷消除了AKT磷酸化，从而损害了RANKL诱导的核因子κB（NF-κB）信号通路并抑制了NFATc1的转录活性。[2]

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确定薯蓣皂苷是否通过防止凋亡来保护心肌细胞免受缺血/再灌注（I/R）损伤。对心脏H9c2细胞进行模拟I/R。通过甲基噻唑四氮唑（MTT）比色法评估细胞存活率。用二氯二氢荧光素（DCF）检测活性氧（ROS）。用流式细胞术评估细胞凋亡。罗丹明123（Rho123）用于测量线粒体膜电位（ΔΨm）。ELISA用于检测细胞色素c（Cyt-c）从线粒体释放到细胞质中的情况。RT-PCR检测Bax和Bcl-2 mRNA的表达。薯蓣皂苷减少了I/R诱导的细胞凋亡和细胞色素c从线粒体释放到细胞质中的细胞死亡和乳酸脱氢酶（LDH）释放，而薯蓣皂甙可以阻止这一过程。作为支持，薯蓣皂苷降低了Bax，但增加了Bcl-2 mRNA的表达。薯蓣皂苷阻止了I/R诱导的ΔΨm的耗散。最后，薯蓣皂苷增加了超氧化物歧化酶（SOD）的表达，但降低了细胞内ROS和丙二醛（MDA）的水平。薯蓣皂苷通过减轻氧化应激调节线粒体凋亡途径，保护H9c2细胞免受H/R损伤。[3]

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Dioscin是一种天然产物，具有对抗多形性胶质母细胞瘤、肺癌和癌症的活性。本研究进一步证实了薯蓣皂苷对人宫颈癌HeLa和SiHa细胞的作用，并探讨了可能的机制。透射电子显微镜（TEM）分析和DAPI染色用于检测细胞形态。流式细胞术用于检测细胞凋亡、ROS和Ca（2+）水平。单细胞凝胶电泳和免疫荧光分析用于检测DNA损伤和细胞色素C释放。结果表明，薯蓣皂苷显著抑制HeLa和SiHa细胞的增殖，并导致DNA损伤。机制研究表明，薯蓣皂苷导致细胞色素C从线粒体释放到细胞质中。此外，薯蓣皂苷显著上调Bak、Bax、Bid、p53、caspase-3、caspase-9的蛋白水平，下调Bcl-2和Bcl-xl的蛋白水平。因此，我们的研究表明，薯蓣皂苷通过调节ROS介导的DNA损伤和线粒体信号通路，显著诱导HeLa和SiHa细胞凋亡[4]。

给予薯蓣皂苷/Dioscin（75-300mg/kg，10mL/kg；口服灌胃；90天）后，雌性大鼠无亚慢性毒性，雄性大鼠有轻度亚慢性毒性。在雄性大鼠中，多利辛使胃肠道适度扩张，并在血液学检查中显示出溶血性贫血[5]。薯蓣皂苷可以通过抑制氧化硝化应激、细胞凋亡和炎症来减轻大鼠肝脏缺血再灌注损伤[6]。

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薯蓣皂苷是许多中药中的主要活性化合物，但这种天然产物的安全性评估尚未得到研究。因此，本研究旨在评估Dioscin/薯蓣皂苷 对大鼠的90天亚慢性毒性。将大鼠分为四组，分别以0、75、150和300mg/kg/天的剂量口服<强>薯蓣皂苷。根据临床观察、眼科检查、体重、食物和饮水量、尿液分析、血液学、临床生化和病理学评估薯蓣皂苷的毒性。结果表明，薯蓣皂苷对雌性大鼠没有亚慢性毒性，对雄性大鼠有轻微的亚慢性毒性。然而，300mg/kg/天组的雄性大鼠在治疗期间表现出轻微的胃肠道扩张，在血液学评估中表现出溶血性贫血。与对照组相比，雄性大鼠的体重增加显著减少。在雄性和雌性组中，其他显著变化与薯蓣皂苷无关。总之，雌性和雄性大鼠的薯蓣皂苷无观测不良反应水平（NOAEL）和最低观测不良反应程度（LOAEL）估计分别为300mg/kg/天。我们的工作为薯蓣皂苷的进一步研究和新药开发提供了有用的数据。[5]

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研究发现，薯蓣皂苷显著降低了血清丙氨酸氨基转移酶和天冬氨酸氨基转移酶的活性，提高了大鼠的存活率，改善了I/R诱导的肝细胞异常。此外，薯蓣皂苷明显提高了SOD、CAT、GSH-Px、GSH的水平，降低了MDA、TNOS、iNOS、NO的水平，并防止了I/R损伤引起的DNA断裂。进一步的研究表明，Dioscin/薯蓣皂苷显著降低了白介素-1β、白介素-6、肿瘤坏死因子-α、细胞间粘附分子-1、MIP-1α、MIP-2、Fas、FasL的基因表达，降低了NF-κB、AP-1、COX-2、HMGB-1、CYP2E1、Bak、caspase-3、p53、PARP、caspase-9的蛋白表达，降低JNK、ERK和p38 MAPK磷酸化水平，上调Bcl-2和Bcl-x水平。 结论：上述结果表明，薯蓣皂苷通过抑制炎症、氧化硝化应激和细胞凋亡对肝脏I/R损伤具有强效作用，应作为未来治疗肝脏I/R伤害的新药开发[6]。

透射电子显微镜（TEM）分析[4]
将HeLa和SiHa细胞（2×105个细胞/mL）铺在6孔板中，用Dioscin/薯蓣皂苷处理，收获，并在4%戊二醛中于4°C下固定过夜。如前所述，对样品进行处理。然后对获得的切片进行染色，并使用透射电子显微镜进行观察。

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DAPI染色[4]
将HeLa和SiHa细胞接种在六孔板中并培养过夜，然后分别用Dioscin/薯蓣皂苷（1.25、2.5和5.0μg/mL）处理12小时和24小时。对于DAPI染色，如上所述处理细胞，然后用DAPI（1.0μg/mL）溶液染色。最后，用荧光显微镜拍摄图像。

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细胞内ROS积累的检测[4]
HeLa和SiHa细胞以1×105个细胞/孔的密度铺在6孔板上，并用Dioscin/薯蓣皂苷（1.25、2.5和5.0μg/mL）处理。收集细胞并重新悬浮在500μL DCFH-DA（10.0μM）中，所有细胞均通过流式细胞术进行分析。

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细胞凋亡和细胞内Ca2+释放的检测[4]
HeLa和SiHa细胞以1×105个细胞/孔的密度铺在6孔板上，用Dioscin/薯蓣皂苷（1.25、2.5和5.0μg/mL）处理，然后收集并重新悬浮在500μL Fluo-3/AM（2.5μM）中，所有细胞均通过流式细胞术进行分析。

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单细胞凝胶电泳分析[4]
癌症细胞用Dioscin（1.25、2.5和5.0μg/mL）处理后，用单细胞凝胶电泳（SCGE）法检测Dioscin诱导的DNA损伤。根据制造商的说明，通过荧光显微镜（奥林巴斯）获得细胞的图像。最终，从三个重复的孔中随机选择50多个细胞，并通过彗星分析软件项目（CASP）1.2.2进行分析。

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细胞色素C释放的检测[4]
将HeLa和SiHa细胞接种在六孔板中，用Dioscin/薯蓣皂苷（1.25、2.5和5.0μg/mL）处理，然后与第一抗体一起孵育过夜。之后，将平板与第二抗体在37°C下孵育1小时，并用DAPI（5.0μg/mL）染色5分钟。通过激光扫描共聚焦显微镜获得细胞图像。

MTT法检测细胞存活率[1]
根据文献记载的方法，通过MTT法测定薯蓣皂苷对LNCaP细胞的细胞毒性作用。简而言之，将8×103个细胞接种到96孔平底板中，每个孔有200μL完全生长培养基。24小时后，用不同浓度的薯蓣皂苷（0-10μmol/L）处理LNCaP细胞12、24和48小时。此后，向每个孔中加入10μL 5mg/mL MTT，并将平板再孵育4小时。实验结束时，从每个孔中取出培养基，加入150μL DMSO终止反应。使用自动ELISA酶标仪 测量570nm波长下的吸光度（A）。抑制率（%）根据以下方程式计算：抑制率（百分比）=[（对照-Atreated）/Acontrol]×100%，其中Acontrol是载体处理孔的A值，Atreated是薯蓣皂苷处理孔的B值。薯蓣皂苷/Dioscin对LNCaP细胞的细胞毒性以IC50值表示（相对于0.1%DMSO处理的细胞，用受试药物处理的细胞对细胞存活率的抑制率为50%，并通过Probit正常法计算）。

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细胞凋亡的流式细胞术检测[1]
按照以下说明，使用AnnexinⅤ-FITC/PI凋亡检测试剂盒通过流式细胞术测定细胞凋亡。简而言之，收获2×105个细胞，用冰冷的PBS（pH 7.4）洗涤两次。然后，将细胞悬浮在500μL结合缓冲液中，用5μL膜联蛋白Ⅴ-FITC和5μL碘化丙啶（PI）双重标记后，在室温下黑暗中孵育15分钟。然后，对约1×104个染色的凋亡细胞进行流式细胞术计数，结果如下：左下象限代表活细胞（Annexin V-/PI-）；右下象限代表早期凋亡细胞（膜联蛋白V+/PI-）；右上象限代表晚期凋亡细胞（膜联蛋白V+/PI+）；左上象限代表原发性坏死细胞（膜联蛋白V–/PI+）。

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蛋白质印迹[1]
Western blot检测caspase-3、Bcl-2和Bax的表达。简而言之，用薯蓣皂苷处理24小时后，收获细胞，置于冰上，用冰冷的PBS洗涤，然后在冰上裂解缓冲液中裂解30分钟。将缓冲液在4°C下以15000 g离心10分钟后，收集所得上清液作为总细胞蛋白。使用BCA蛋白测定试剂盒 检测蛋白质浓度。蛋白质提取物（20μg，20μL）通过10%十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）分离，然后通过电印迹转移到聚二氟乙烯（PVDF）膜上。转移的膜在室温下用TBST（1 mol/L Tris缓冲盐水，pH 7.4；0.1%吐温-20）中的5%脱脂奶粉封闭1小时，然后在4°C下用一抗（cas-pase-3、Bcl-2和Bax，1:1000稀释）培养过夜。将膜在TBST缓冲液中洗涤三次，然后在室温下用与辣根过氧化物酶连接的适当二抗（1:2000稀释）孵育90分钟。用TBST洗涤膜4次后，使用增强化学发光试剂在膜上显影印迹中的蛋白质。使用Bio-Rad Quantity One V4.62对上述蛋白质的表达水平进行密度测定。β-actin以与上述相同的方式检测，并用作内部负荷参考。

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细胞活力测定[2]
使用细胞计数试剂盒-8评估薯蓣皂苷对BMMs细胞的抗增殖作用。简而言之，处理后，向每个孔中加入10μl CCK-8溶液；孵育4小时后，使用酶标仪在450nm处测量吸光度。Dioscin薯蓣皂苷对细胞存活率的影响以细胞存活率百分比表示，载体处理的对照细胞设置为100%。

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体外破骨细胞生成试验[2]
进行体外破骨细胞生成试验，以检查薯蓣皂苷对破骨细胞分化的影响。如前所述制备骨髓巨噬细胞（BMM）细胞。简而言之，从6周龄C57/BL6小鼠的股骨和胫骨中提取的细胞在T-75 cm2烧瓶中的全细胞培养基和30 ng/mL M-CSF中孵育以增殖。更换培养基时，清洗细胞以耗尽残留的基质细胞。达到90%融合后，用磷酸缓冲盐水（PBS）洗涤细胞三次，并用胰蛋白酶处理30分钟以收获BMM。培养皿底部的粘附细胞被归类为BMM；将这些BMM以8×103个细胞/孔的密度铺在96孔板上，一式三份，在37°C下含5%CO2的加湿培养箱中孵育24小时。然后用不同浓度的Dioscin/薯蓣皂苷（0、1或4μM）加M-CSF（30 ng/mL）和RANKL（50 ng/mL）处理细胞。五天后，固定细胞并对TRAP活性进行染色。TRAP+具有五个以上核的多核细胞被计数为破骨细胞。

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吸收坑分析[2]
为了进行如上所述的骨吸收试验，将BMM以8×103个细胞/孔的密度接种在96孔板的骨切片上，进行三次重复，并用M-CSF（30 ng/mL）加RANKL（50 ng/mL）刺激。三天后，细胞在培养后用指定浓度的Dioscin/薯蓣皂苷处理48小时。然后用2.5%戊二醛固定细胞。使用放大倍数为200倍、电压为10 kV的扫描电子显微镜 对骨切片进行成像。为每个骨切片随机选择三个视场进行进一步分析。使用Image J软件对凹坑区域进行量化。类似的独立实验重复了至少三次。

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蛋白质印迹分析[2]
将BMMS细胞以5×105个细胞/孔的速度接种到6孔板中，在RANKL刺激（50 ng/mL）之前，用或不用Dioscin/薯蓣皂苷 4μM）预处理4小时，持续指定时间（0、5、15或30分钟）。将BMMs以5×105个细胞/孔的速度接种到6孔板中，并用或不用薯蓣皂苷（4μM）和RANKL（50 ng/mL）处理指定时间。细胞在含有50 mM Tris-HCl、150 mM NaCl、5 mM EDTA、1%Triton X-100、1 mM氟化钠、1 mM钒酸钠、1%脱氧胆酸盐和蛋白酶抑制剂混合物的RIPA裂解缓冲液中裂解。将裂解物在12000rcf下离心10分钟，收集上清液中的蛋白质。通过BCA测定法测量蛋白质浓度。使用8-10%的凝胶通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离每种蛋白质裂解物的30微克，然后将蛋白质转移到聚偏二氟乙烯膜上。用5%脱脂乳阻断非特异性相互作用1小时，然后在4°C下用指定的一抗探测膜过夜。用与IRDye 800CW（分子量，1166 Da）偶联的适当二抗孵育膜，并通过在Odyssey红外成像系统（Li-COR）中暴露来检测抗体反应性。

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萤光素酶报告基因活性测定[2]
如前所述，使用用NF-κB荧光素酶报告构建体稳定转染的RAW264.7细胞测量了薯蓣皂苷对RANKL诱导的NF-κB活化的影响。简而言之，将细胞接种到48孔板中，并在细胞培养基中保持24小时。然后用或不用指定浓度的Dioscin/薯蓣皂苷预处理细胞1小时，然后加入RANKL（50 ng/mL）8小时。使用Promega萤光素酶测定系统测量萤光素酶活性，并使其标准化为载体对照。同样，薯蓣皂苷对RANKL诱导的AP-1或NFATc1依赖性萤光素酶报告基因检测的影响如前所述进行了测定。

Based on a previously published study on the acute toxic effects of Dioscin (Liang et al., 2010), the rats received Dioscin at doses of 0 (control), 75, 150, and 300 mg/kg/day, respectively. Dioscin was mixed with a solution of 0.5% carboxymethylcellulose sodium in distilled water. An appropriate amount of Dioscin or control (vehicle) was administered daily (7 days/week) to each rat for 90 consecutive days. Each animal was dosed by oral intubation using a ball-tipped gavage needle attached to an appropriate syringe. Dosing was at the same time each day ±1 h. Individual doses were calculated based on body weight and were adjusted each day to maintain the target dose level in all rats. All doses were administered volumetrically after correcting for dilution. Doses were administered to all groups at a constant dose volume of 10 mL/kg. The control group received vehicle only at the same volume as the test animals. The present study was conducted based on the US Food and Drug Administration principles (FDA, 2000) and OECD Test Guideline 408 for ‘Repeated dose 90-day oral toxicity study in rodents’. [5]

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Ophthalmological examination : An ophthalmological examination was conducted on all rats (40 males; 40 females) on the first day of the experiment prior to the administration of Dioscin and on the 90th day at the end of the study. The peripheral and internal structure of both eyes in each rat was examined by the naked eye and indirect ophthalmoscopy, respectively.

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The animals were allowed to adapt to the environment for 1 week before the experiments. As shown in Figure 5(B), in the prophylactic test, Dioscin was administered intragastrically to the animals at the doses of 20, 40, and 60 mg/kg once daily for seven consecutive days. The rats in the sham operation and model groups were administered with vehicle. On the eighth day, the murine model of 70% partial hepatic ischemia was established as described previously. Briefly, the rats were anesthetized, and the livers were exposed by midline laparotomy, then the inflow of the left lateral and median lobes of the livers was choked by placing a bulldog clamp, whereas the right lobes were remainedly perfused to prevent intestinal congestion occlusion. After 60 min of hepatic ischemic, the bulldog clamp was removed, and the liver was reperfused for 6 hr. At the end of the surgery, the animals were anesthetized for collecting the blood and then killed to collect the left lateral and middle lobes of the livers. As shown in Figure 5(C), in the therapeutic test, the rats were randomized into three groups, of which the animals in sham and model groups were given vehicle, and the rats in Dioscin group received Dioscin intragastrically at a dose 60 mg/kg once 2 hr before I/R. After 60 min of hepatic ischemic, the bulldog clamp was removed, and the orbital blood samples were collected at 0.5, 1, 2, 4, 6, 12, 24, 48, and 96 hr after reperfusion.. In addition, the survival rate of the animals was assayed. [6]

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Background: Dioscin shows potent effects against liver damage in our previous studies; however, the action of it on hepatic ischemia-reperfusion (I/R) injury is still unknown. In the present article, the effects and possible mechanisms of dioscin against hepatic I/R injury were investigated. Methods: Seventy percent partial hepatic warm ischemia was induced in Wistar rats for 60 min followed by succedent reperfusion. In the prophylactic test, dioscin was administered intragastrically to the rats at doses of 20, 40, and 60 mg/kg once daily for seven consecutive days before I/R. In the therapeutic test, the rats received dioscin intragastrically at a dose of 60 mg/kg once 2 hr before I/R. [6]

mouse LD50 subcutaneous >300 mg/kg Japanese Kokai Tokyo Koho Patents., #91-271224

[1]. Dioscin-induced apoptosis of human LNCaP prostate carcinoma cells through activation of caspase-3 and modulation of Bcl-2 protein family. J Huazhong Univ Sci Technolog Med Sci. 2014 Feb;34(1):125-30.

[2]. Dioscin inhibits osteoclast differentiation and bone resorption though down-regulating the Akt signaling cascades. Biochem Biophys Res Commun. 2014 Jan 10;443(2):658-65.

[3]. Dioscin prevents the mitochondrial apoptosis and attenuates oxidative stress in cardiac H9c2 cells. Drug Res (Stuttg). 2014 Jan;64(1):47-52.

[4]. Dioscin Induces Apoptosis in Human Cervical Carcinoma HeLa and SiHa Cells through ROS-Mediated DNA Damage and the Mitochondrial Signaling Pathway. Molecules. 2016 Jun 4;21(6):730.

[5]. A 90-day subchronic toxicological assessment of dioscin, a natural steroid saponin, in Sprague-Dawley rats. Food Chem Toxicol. 2012 May;50(5):1279-87.

[6]. Dioscin attenuates hepatic ischemia-reperfusion injury in rats through inhibition of oxidative-nitrative stress, inflammation and apoptosis. Transplantation. 2014 Sep 27;98(6):604-11.

Dioscin is a spirostanyl glycoside that consists of the trisaccharide alpha-L-Rha-(1->4)-[alpha-L-Rha-(1->2)]-beta-D-Glc attached to position 3 of diosgenin via a glycosidic linkage. It has a role as a metabolite, an antifungal agent, an antiviral agent, an antineoplastic agent, an anti-inflammatory agent, a hepatoprotective agent, an apoptosis inducer and an EC 1.14.18.1 (tyrosinase) inhibitor. It is a spirostanyl glycoside, a spiroketal, a hexacyclic triterpenoid and a trisaccharide derivative. It is functionally related to a diosgenin. It derives from a hydride of a spirostan.
Dioscin has been reported in Dioscorea collettii, Dioscorea deltoidea, and other organisms with data available.
See also: Dioscorea polystachya tuber (part of).

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In this study, we have verified for the first time that natural compound Dioscin inhibited osteoclast differentiation and bone resorption, suggesting an additional protective effect of dioscin on osteoclast-related diseases. In addition, we revealed the molecular mechanisms of dioscin on osteoclasts are through suppressing Akt/NF-κB and Akt/NFATc1 signaling pathways.
In osteoclasts, the Akt signaling cascades is a critical downstream of three osteoclast surface receptors including c-fms, αvβ3 integrin and RANK. Previous studies demonstrated Akt phosphorylation is activated upon both M-CSF and RANKL stimulation and play critical roles in osteoclastogenesis by affecting both NF-κB and NFATc1 activation. Moon et al. demonstrated that overexpression of Akt in BMMs strongly induced NFATc1 expression and lead to enhanced osteoclastogenesis. Besides, activation of NF-κB can be initiated by several different kinases such as Akt and NF-κB inducing kinase. Gingery et al. demonstrated that AKT/NF-κB axis is critical in osteoclastogenesis and maintaining mature osteoclast survival. In consistent with these studies, we demonstrated that dioscin inhibited Akt phosphorylation and thus suppressed the RANKL-induced NF-κB activity and NFATc1 activity, both of which are critical for osteoclast differentiation. Interestingly, in the process of detecting the function of dioscin on MAPKs pathway, no significantly inhibitory impact was witnessed.
In summary, dioscin is capable of inhibiting osteoclast formation and function, indicating additional therapeutic benefits of dioscin for osteoclast-related diseases. In addition, this study also clearly revealed the molecular mechanisms of dioscin on osteoclasts are via impairing Akt/NF-κB and Akt/NFATc1 signaling pathways in vitro. In addition, our in vitro results further verified the bone protective role of dioscin on LPS-induced osteolysis model. However, further investigation of dioscin on other cells within bone is still required. [1]

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Dioscin induced apoptosis of human cervical cancer HeLa and SiHa cells through inducing ROS-mediated DNA damage and activating the mitochondrial signaling pathway. However, based on the in vitro results, the molecular mechanism of action and the drug targets of dioscin need further investigation.[4]

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Due to the medicinal value and widespread use of Dioscin, this natural compound has great potential in future application and research in the pharmaceutical field. Thus, it is critically important to evaluate the toxicity of dioscin. The results from our 90-day subchronic toxicity study showed changing trends in dose dependency on individual body weight. Compared with the control group, body weight was significantly decreased in all male groups, especially in the male 300 mg/kg treatment group. This change was considered to be related to the properties of saponins such as reduced feeding caused by gastro-intestinal tract distension (Shen et al., 2008), decreased intestinal motility (Klita et al., 1996), and protein digestibility (Potter et al., 1993). Due to reduced feeding, food consumption was decreased and therefore body weight was lost. In the urinalysis, changes in urine pH, urine specific gravity and urine protein were noted in the male 75 mg/kg treatment group. Similar changes in the 300 mg/kg treatment group were not found. These changes were incidental without apparent dose dependence.
In the hematology assessment, changes in RBC and HCT were recorded in the male 300 mg/kg treatment group, suggesting hemolytic anemia. The bioactive compound of steroidal saponin has hemolytic activity (Santos et al., 1997, Zhang et al., 1999) and saponin is known to have a lytic action on erythrocyte membranes (Howard and Wallace, 1953), which was reported to result from the affinity of aglycone moieties for membrane sterols, particularly cholesterol, forming insoluble complexes in open pores in the membranes (Bangham et al., 1962, Cho et al., 2009). Membrane rupture of erythrocytes could release into the blood and cause slight nephrotoxicity. There were significant changes in BUN of males in the clinical biochemistry assessment, but no histopathological lesions were observed in the kidneys. Other changes in hematology were incidental as a dose–response relationship was not observed. In the clinical biochemistry assessment, increases in ALT observed in the female 300 and male 300 mg/kg treatment groups were judged to be related to Dioscin treatment, however, the changes in AST, AKP and T-BIL in the groups showed no apparent dose dependence. Although the changes in ALT in the groups were suggestive of hepatic damage, no changes in liver weight and no abnormalities on histopathological examination were observed. The significant changes in BUN in male and female rats indicated mild nephrotoxic effects, however, no histopathological lesions were recorded in the kidneys. While both males and females showed significant fluctuations in Cr, TG, γ-GT and glucose, the changes were incidental without apparent dose dependence or toxicological significance.
In the evaluation of organ weight and histopathology, although significant changes in absolute and relative weights were observed in organs such as the liver, kidneys, heart, and brain, no histopathological changes were observed and these changes could be considered to be due to decreased body weight. Body weight loss and relative organ weight increases are shown in Table 5, and these changes are considered to be incidental due to additional fat.
Conclusion: The development potential of the medicinal constituent, Dioscin, is significant. However, it is also necessary to focus on the toxicity of Dioscin based on the results obtained in this study. Our findings provide some evidence on the safety of dioscin for potential clinical application and the widespread use of dioscin, however, prior to the clinical application of dioscin further evaluation is required. In conclusion, dioscin at a dose of 300 mg/kg/day in female rats was identified as the NOAEL (no-observed-adverse-effect-level) and a dose of 300 mg/kg/day in male rats was identified as the LOAEL (lowest-observed-adverse-effect level) in this study. [5]

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In conclusion, dDioscin has a good protective effect against hepatic I/R injury in rats through attenuating oxidative-nitrative stress, inflammation, and apoptosis, which should be developed as a new and potent candidate for treatment of I/R-induced liver injury in the future. Of course, mechanisms, drug-target, and clinical applications of Dioscin are needed further investigations. [6]

C45H72O16

869.05

868.482

C, 62.19; H, 8.35; O, 29.46

19057-60-4

119245

White to off-white solid powder

1.4±0.1 g/cm3

294-296 ℃

1.614

7.24

235.68

8

16

7

61

1600

26

C[C@@H]1CC[C@@]2([C@H]([C@H]3[C@@H](O2)C[C@@H]4[C@@]3(CC[C@H]5[C@H]4CC=C6[C@@]5(CC[C@@H](C6)O[C@H]7[C@@H]([C@H]([C@@H]([C@H](O7)CO)O[C@H]8[C@@H]([C@@H]([C@H]([C@@H](O8)C)O)O)O)O)O[C@H]9[C@@H]([C@@H]([C@H]([C@@H](O9)C)O)O)O)C)C)C)OC1

VNONINPVFQTJOC-ZGXDEBHDSA-N

InChI=1S/C45H72O16/c1-19-9-14-45(54-18-19)20(2)30-28(61-45)16-27-25-8-7-23-15-24(10-12-43(23,5)26(25)11-13-44(27,30)6)57-42-39(60-41-36(52)34(50)32(48)22(4)56-41)37(53)38(29(17-46)58-42)59-40-35(51)33(49)31(47)21(3)55-40/h7,19-22,24-42,46-53H,8-18H2,1-6H3/t19-,20+,21+,22+,24+,25-,26+,27+,28+,29-,30+,31+,32+,33-,34-,35-,36-,37+,38-,39-,40+,41+,42-,43+,44+,45-/m1/s1

(2S,3R,4R,5R,6S)-2-[(2R,3S,4S,5R,6R)-4-hydroxy-2-(hydroxymethyl)-6-[(1S,2S,4S,5'R,6R,7S,8R,9S,12S,13R,16S)-5',7,9,13-tetramethylspiro[5-oxapentacyclo[10.8.0.02,9.04,8.013,18]icos-18-ene-6,2'-oxane]-16-yl]oxy-5-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxyoxan-3-yl]oxy-6-methyloxane-3,4,5-triol

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: ≥ 2.25 mg/mL (2.59 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 22.5 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.25 mg/mL (2.59 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 22.5 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.25 mg/mL (2.59 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 22.5 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。