Alisol B 23-acetate-induced HepG2 hepatoma cell death through mTOR signaling-initiated G_1 cell cycle arrest and apoptosis: A quantitative proteomic study

Objective: The present study aimed to investigate the molecular events in alisol B 23-acetate(ABA) cytotoxic activity against a liver cancer cell line.Methods: First, we employed a quantitative proteomics approach based on stable isotope labeling by amino acids in cell culture(SILAC) to identify the different proteins expressed in HepG2 liver cancer cells upon exposure to ABA. Next, bioinformatics analyses through DAVID and STRING on-line tools were used to predict the pathways involved. Finally, we applied functional validation including cell cycle analysis and Western blotting for apoptosis and mTOR pathway-related proteins to confirm the bioinformatics predictions.Results: We identified 330 different proteins with the SILAC-based quantitative proteomics approach. The bioinformatics analysis and the functional validation revealed that the mTOR pathway, ribosome biogenesis, cell cycle, and apoptosis pathways were differentially regulated by ABA. G1 cell cycle arrest, apoptosis and mTOR inhibition were confirmed.Conclusions: ABA, a potential mTOR inhibitor, induces the disruption of ribosomal biogenesis. It also affects the mTOR-MRP axis to cause G1 cell cycle arrest and finally leads to cancer cell apoptosis.

A UFLC/MS/MS method for simultaneous quantitation of alisol A and alisol B 23-acetate from Alisma orientale (Sam.) Juz. in rat plasma

A sensitive and reliable ultra fast liquid chromatography tandem mass spectrometry(UFLC-MS/MS)method has been developed and validated for simultaneous quantitation of alisol A and alisol B 23-acetate from Alisma orientale(Sam.)Juz.in rat plasma using diazepam as an internal standard(IS).The plasma samples were extracted by liquideliquid extraction with methyl tert-butyl ether and separated on a Venusil MP C18 column(100 mm×2.1 mm,3.0 mm)(Venusil,China)using gradient elution with the mobile phase consisting of methanol and 0.1%acetic acid in water at a flow rate of 0.4 ml/min.The two analytes were monitored with positive electrospray ionization by multiple reaction monitoring mode(MRM).The lower limit of quantitation was 5.00 ng/ml for alisol A and 5.00 ng/ml for alisol B 23-acetate.The calibration curves were linear in the range of 5.00 e2500 ng/ml for alisol A and 5e2500 ng/ml for alisol B 23-acetate.The mean extraction recoveries were above 63.8%for alisol A and 68.0%for alisol B 23-acetate from biological matrixes.Both intra-day and inter-day precision and accuracy of analytes were well within acceptance criteria(15%).The validated method was successfully applied to the pharmacokinetic study of alisol A and alisol B 23-acetate in rat plasma after oral administration of alcohol extract of Alismatis Rhizoma.

泽泻中23-乙酰泽泻醇B的含量测定研究

目的 主要是确定泽泻的最佳采收时间、最佳炮制工艺、制定泽泻的定量标准 ,确保泽泻临床用药安全有效。方法 采用高效液相色谱法测定各样品中 2 3 -乙酰泽泻醇B的含量。结果 2 3 -乙酰泽泻醇B的平均回收率为 98.80 % ,符合分析要求。泽泻中 2 3 -乙酰泽泻醇B的含量以不低于 0 .0 5 0 %为宜。

泽泻炮制过程中23-乙酰泽泻醇B的转化

目的 分析泽泻中三萜类成分在炮制加工过程中的变化.方法 采用超临界流体色谱分离技术（SFC）及HPLC测定方法,对泽泻加工炮制前后的三萜类成分的变化进行研究.结果 在泽泻药材加工成生泽泻饮片的烘干（70℃）过程中,有少量23-乙酰泽泻醇B转化成了24-乙酰泽泻醇A和泽泻醇B；而在泽泻盐制（190～200℃）及麸制（160～170℃）过程中,23-乙酰泽泻醇B则大量转化为24-乙酰泽泻醇A和泽泻醇B,两者又进一步转化成了泽泻醇A.结论 在高温炮制过程中,泽泻药材中三萜类主成分23-乙酰泽泻醇B出现两条转变途径,一条是氧环开裂并重排生成24-乙酰泽泻醇A,进一步脱乙酰基转化成泽泻醇A；另一条是先脱乙酰基生成泽泻醇B,继而氧环开裂转化成泽泻醇A.

不同等级川泽泻中23-乙酰泽泻醇B含量测定

目的:主要测定不同等级川泽泻中23-乙酰泽泻醇B含量,为饮片的分级标准提供参考。方法:通过HPLC法。结果:23-乙酰泽泻醇B的加样回收率为100.17%,RSD为1.24%符合分析要求。结论:川泽泻单以现代的分析方法测定其中的23-乙酰泽醇B含量很难区分开不同等级的药材,要区分开不同等级的川泽泻还要借鉴传统的分级标准。

泽泻不同采收期23-乙酰泽泻醇B含量变化

对福建龙海产泽泻不同采收期的23-乙酰泽泻醇B含量变化分析,结果表明:1月至3月含量差异不明显,4月份采收的样品含量明显增高。

HPLC法测定龙胆泻肝丸(大蜜丸)中23-乙酰泽泻醇B的含量

目的：建立HPLC法测定龙胆泻肝丸（大蜜丸）中23-乙酰泽泻醇B的含量。方法：色谱柱为Waters Symmetry C18（250 mm×4.6 mm,5μm）,流动相为乙腈-0.1%磷酸水溶液（62∶38）,流速为1.0 ml·min^-1,柱温为35℃,检测波长为208nm。结果：23-乙酰泽泻醇B在19.999 5-1 999.9500 ng范围内线性关系良好（r=0.999 9）,平均回收率为95.56%,RSD为0.7%（n=6）。结论：该方法简便、可靠、准确,具有良好的重复性和回收率,能用于控制龙胆泻肝丸（大蜜丸）的质量。

高效液相色谱法测定五苓滴丸中23-乙酰泽泻醇B的含量

目的采用高效液相色谱（HPLC）法测定五苓滴丸中23-乙酰泽泻醇B的含量。方法色谱柱：Agilent Eclipse XDB-C18柱（4.6 mm×250 mm,5μm）;流动相：乙腈-水（70∶30）;流速：1.0 mL/min;检测波长210 nm;柱温：30℃。结果 23-乙酰泽泻醇B在2.9-145.0μg/mL浓度范围内与峰面积具有良好的线性关系（r=0.999 9）,平均加样回收率为97.86%,RSD=1.29%。结论 HPLC法简便、准确、专属性高,可用于五苓滴丸的质量控制。

回流法与闪式提取法提取泽泻中23-乙酰泽泻醇B的工艺对比研究

为了对比研究回流提取法与闪式提取法提取泽泻中23-乙酰泽泻醇B的工艺,采用超快速液相色谱法测定泽泻中23-乙酰泽泻醇B的含量,回流法和闪式提取法均以乙醇浓度、提取时间、料液比为考察因素,以泽泻中23-乙酰泽泻醇B的含量为考察指标,采用L_9(3~4)正交试验优选出最佳提取工艺。结果回流提取法的最佳提取工艺:提取溶剂为95%乙醇,提取时间为2 h,料液比为1∶50;闪式提取法的最佳提取工艺:提取溶剂为95%乙醇,提取时间为1 min,料液比为1∶60。表明闪式提取法所得泽泻中23-乙酰泽泻醇B含量高于回流提取法。闪式提取法的操作简单、耗时短、提取率高、节省能源,可为泽泻药材的新药开发和工业化生产提供依据,同时为新兽药开发利用提供参考。

高效液相色谱法检测七味都气丸中活性成分五味子醇甲、马钱苷和23-乙酰泽泻醇B的含量

目的研究建立采用高效液相色谱法检测七味都气丸中活性成分五味子醇甲、马钱苷和23-乙酰泽泻醇B含量的分析方法。方法研究起止时间为2019年3月至2022年5月。七味都气丸样品经粉碎,90%甲醇水溶液超声提取后,采用Waters X-bridge C_(18)色谱柱分离,柱温27℃,检测波长228 nm,进样体积25µL,流动包括流动相A(乙腈),流动相B(0.3%磷酸水溶液),梯度洗脱,洗脱程序为0~7 min 8%A,7~18 min 8%~60%A,18~28 min 60%~91%A,28~30 min 91%~8%A,30~35min 8%A,外标法定量检测。结果马钱苷、五味子醇甲和23-乙酰泽泻醇B在质量浓度0.1~20.0 mg/L范围内具有良好的线性关系,相关系数(r)均大于0.995;重复性RSD(n=6)分别为2.7%、3.1%和3.4%;加样回收率分别为99.67%、100.11%和100.49%。结论该方法具有简便、快速、准确度好等优点,适用于七味都气丸品种的质量控制。

高效液相色谱法测定金泽冠心胶囊中泽泻醇B-23-乙酸酯含量

目的应用高效液相色谱法测定金泽冠心胶囊中泽泻醇B-23-乙酸酯的含量。方法采用DiamonsilTM(钻石)C18色谱柱(250mm×4.6mm,5μm),流动相为乙腈-水(70∶30),流速为1.0mL/min,检测波长为208nm。结果泽泻醇B-23-乙酸酯进样量在0.7312～2.9248μg范围内与峰面积线性关系良好(r=0.9999),平均加样回收率为99.15%,RSD为0.96%(n=6)。结论该法操作简便、准确,灵敏度高,重现性好,可用于金泽冠心胶囊中泽泻醇B-23-乙酸酯的含量测定。

建泽泻中泽泻醇B-23-乙酸酯含量的HPLC测定

采用高效液相色谱法测定不同商品规格的福建泽泻 (建泽泻 )中泽泻醇 B-2 3 -乙酸酯的含量 ,并对测定方法进行优化。结果表明不同等级的建泽泻中泽泻醇 B-2 3 -乙酸酯含量未见显著差异 ,优化建立的高效液相色谱方法准确、稳定、简便、可行。

反相高效液相色谱法测定泽泻提取物中23-乙酰泽泻醇B含量

目的利用反相高效液相色谱法(RP-HPLC)测定泽泻提取物中23-乙酰泽泻醇B含量。方法利用RP-HPLC检测,选用Kromasil 100-5C18(250 mm×4.6 mm)色谱柱,以乙腈-水为流动相等梯度洗脱,流速1 ml.min-1;检测波长208 nm。结果 23-乙酰泽泻醇B在进样量范围内与峰面积线性关系良好,仪器精密度和稳定性RSD均小于2%,平均回收率在99%～101%(n=3)。测定了5批提取物中23-乙酰泽泻酐B,平均的含量为2.550 mg/g。结论该方法快速、准确、重复性好,可对泽泻指标性成分23-乙酰泽泻醇B进行定量,从而控制提取物质量。

不同诱导子对泽泻中23-乙酰泽泻醇B积累的影响

采用不同浓度的水杨酸、赤霉素和脱落酸处理泽泻植株,研究不同诱导子对泽泻中23-乙酰泽泻醇B积累的影响。结果表明,水杨酸、赤霉素和脱落酸在低浓度下都能促进泽泻中23-乙酰泽泻醇B的积累,在高浓度下则表现出一定的抑制作用。150μg/m L的水杨酸处理第8天时,23-乙酰泽泻醇B的含量为715.4μg/g,为所有处理中的最大值。赤霉素和脱落酸的处理效果与水杨酸相比较差。

HPLC法测定复方泽泻滴丸中23-乙酰泽泻醇B的含量

目的建立HPLC法测定复方泽泻滴丸中23-乙酰泽泻醇B含量的方法。方法采用岛津LC-10AT依利特Hypersil C18色谱柱(5μm,4.6 mm×250 mm);以乙腈∶水=90∶10为流动相;流速为0.8 mL.min-1;检测波长为208 nm;柱温:30℃。结果 23-乙酰泽泻醇B在0.012～0.12mg/mL范围与峰面积呈良好的线性关系。平均加样回收率为100.24%,RSD%为1.21%(n=9)。结论该方法灵敏简便,快速可靠,可用于复方泽泻滴丸的质量控制。

MPLC合p-HPLC法分离制备泽泻中23-乙酰泽泻醇B

目的建立快速制备泽泻药材中23-乙酰泽泻醇B对照品的方法。方法采用中低压正相硅胶柱色谱(MPLC)合高压反相色谱法(p-HPLC),MPLC:硅胶柱:3 cm×13 cm,40 g;流动相:石油醚-乙酸乙酯梯度洗脱;流速:50 ml/min;p-HPLC:Ultimate XB-C18:21.2 mm×250 mm,10μm;流动相:乙腈-水(65:35);流速:15 mL/min;检测波长:208 nm。结果 MPLC合p-HPLC法一次可以从2 g泽泻乙酸乙酯粗提物中分离制备得到23-乙酰泽泻醇B 40.2 mg。结论建立的MPLC合p-HPLC法简便、快捷、高效地获得泽泻中23-乙酰泽泻醇B对照品。

HPLC法检测建泽泻中23-乙酰泽泻醇B含量

也目的页建立中药材建泽泻中23-乙酰泽泻醇B的提取及检测体系。也方法页采用石油醚超声处理，辅助旋转蒸发提取23-乙酰泽泻醇B，利用高效液相色谱法检测；色谱柱：Waters C18柱（4.6 mm＆#215;250 mm，5μm）；柱温：室温；流动相：乙腈-水（80：20）；流速：0.8 mL/min，检测波长：208 nm。也结果页建泽泻中23-乙酰泽泻醇B在25~500μg/mL的线性关系良好；平均回收率为98.1%，RSD为3.10%。也结论页该方法简便、准确、重复性好，适用于微量建泽泻的检测。

正交试验优选泽泻中23-乙酰泽泻醇B的提取工艺

目的优选泽泻中23-乙酰泽泻醇B的超声提取工艺。方法采用超快速液相色谱法测定泽泻中23-乙酰泽泻醇B的含量,以乙醇浓度、提取时间、超声功率、料液比为考察因素,以泽泻中23-乙酰泽泻醇B的含量为考察指标,采用L9(34)正交试验优选超声提取工艺条件。结果最佳提取工艺为提取溶剂95%乙醇,提取时间45 min,超声功率250 W,料液比1∶50。结论该提取工艺具有效率高、时间短、能耗低、环保等优点,可为泽泻中23-乙酰泽泻醇B的工业化生产和新药开发提供依据。

泽泻对照品23-乙酰泽泻醇B克级制备工艺研究

目的探索制备克级23-乙酰泽泻醇B化学对照品的工艺。方法通过高效液相色谱法比较提取溶剂、提取次数、提取时间和溶剂量等因素对23-乙酰泽泻醇B提取率的影响,选择最佳提取工艺放大到中试设备。结果采用95%乙醇,料液比10∶1回流提取药材2次,每次1 h,提取物经石油醚-醋酸乙酯混合液萃取后,经常规硅胶柱层析、精制,获得9.8 g对照品。结论该工艺高效、简便、廉价,适用于23-乙酰泽泻醇B对照品的克级制备。

Alisol B 23-acetate promotes white adipose tissue browning to mitigate high-fat diet-induced obesity by regulating mTOR-SREBP1 signaling

Objective Obesity is a global health concern with management strategies encompassing bariatric surgery and anti-obesity drugs;however,concerns regarding complexities and side effects persist,driving research for more effective,low-risk strategies.The promotion of white adipose tissue(WAT)browning has emerged as a promising approach.Moreover,alisol B 23-acetate(AB23A)has demonstrated efficacy in addressing metabolic disorders,suggesting its potential as a therapeutic agent in obesity management.Therefore,in this study,we aimed to investigate the therapeutic potential of AB23A for mitigating obesity by regulating metabolic phenotypes and lipid distribution in mice fed a high-fat diet(HFD).Methods An obesity mouse model was established by administration of an HFD.Glucose and insulin metabolism were assessed via glucose and insulin tolerance tests.Adipocyte size was determined using hematoxylin and eosin staining.The expression of browning markers in WAT was evaluated using Western blotting and quantitative real-time polymerase chain reaction.Metabolic cage monitoring involved the assessment of various parameters,including food and water intake,energy metabolism,respiratory exchange rates,and physical activity.Moreover,oil red O staining was used to evaluate intracellular lipid accumulation.A bioinformatic analysis tool for identifying the molecular mechanisms of traditional Chinese medicine was used to examine AB23A targets and associated signaling pathways.Results AB23A administration significantly reduced the weight of obese mice,decreased the mass of inguinal WAT,epididymal WAT,and perirenal adipose tissue,improved glucose and insulin metabolism,and reduced adipocyte size.Moreover,treatment with AB23A promoted the expression of browning markers in WAT,enhanced overall energy metabolism in mice,and had no discernible effect on food intake,water consumption,or physical activity.In 3T3-L1 cells,AB23A inhibited lipid accumulation,and both AB23A and rapamycin inhibited the mammalian target of rapamycin-sterol regulatory element-binding protein-1(mTOR-SREBP1)signaling pathway.Furthermore,3-isobutyl-1-methylxanthine,dexamethasone and insulin,at concentrations of 0.25 mmol/L,0.25μmol/L and 1μg/mL,respectively,induced activation of the mTOR-SREBP1 signaling pathway,which was further strengthened by an mTOR activator MHY1485.Notably,MHY1485 reversed the beneficial effects of AB23A in 3T3-L1 cells.Conclusion AB23A promoted WAT browning by inhibiting the mTOR-SREBP1 signaling pathway,offering a potential strategy to prevent obesity.