Pharmacokinetics of nitrogen-containing metabolites R-gentiandiol and S-gentiandiol in rat plasma after oral administration of swertiamarin

Objective:A chiral resolution method for enantiomers of two chiral nitrogen-containing metabolites R-gentiandiol and S-gentiandiol of swertiamarin in plasma was developed,and the pharmacokinetics of the metabolites were studied.Methods:The metabolites of swertiamarin in vivo were detected by LC-MS/MS using Astec CyclobondⅡCyclodextrin column(4.6 mm×100 mm,5μm),gradient elution with acetonitrile-water as mobile phase,and monitored by multiple reaction monitoring(MRM)method in positive mode.The ion pairs for quantitative analysis are R-gentiandiol(m/z 210.04→192.06),S-gentiandiol(m/z 210.04→192.06)and gentianone(m/z 192.02→162.08).Results:The linear correlation coefficients of the method developed were greater than 0.999,the precision was less than 7.00%,the recovery was 99.57%-102.65%,and the matrix effect was 90.94%-91.34%.The peak t_(max)of R-gentiandiol and S-gentiandiol in rat plasma after oral administration of swertiamarin were(1.63±0.23)h and(1.58±0.21)h,t_(1/2)was(6.23±0.52)h and(5.46±0.38)h,C_(max)was(86.79±20.81)ng/mL and(60.72±18.95)ng/mL,and the AUC_(0-24)were(1094.58±86.37))(ng·h)/mL and(724.67±58.38)(ng·h)/mL,respectively.Conclusion:The method was highly sensitive with good accuracy and precision,and it was successfully applied for chiral resolution and pharmacokinetics study of R-gentiandiol and S-gentiandiol in plasma.The method developed and experimental results will provide scientific basis for the study of pharmacodynamics and pharmacodynamic material basis of swertiamarin,and lay a foundation for clinical application and resource development of TCM monomer.

Förster resonance energy transfer reveals phillygenin and swertiamarin concurrently target AKT on different binding domains to increase the anti-inflammatory effect

The clinical benefit of combination therapy is significant,but it is not easy to define the mechanism of complexity and diversity.Previous studies illustrate that phillygenin(Phi)binds in the allosteric inhibit pocket of protein kinase B(AKT),and swertiamarin(Swe)acts on the pleckstrin homology(PH)domain of AKT.However,the combined synergistic effect of relieving the inflammatory response has yet to be elucidated.Based on high sensitivity,specificity and fast-responsibility fluorescent sensors,the Förster resonance energy transfer(FRET)technique offers a route to provide clear insights into physiological and pathological processes.In the study,molecular docking,the fluorescent probes of Phi and Swe for FRET were designed and synthesized.FRET analysis shown that Swe and Phi concurrently acted on the PH domain and allosterically inhibited pocket of AKT,respectively.The combination of Swe and Phi significantly increased the heat stability of AKT and decreased protease-induced degeneration.In lipopolysaccharides(LPS)-induced mice and cells,the combination arrested AKT activation,nuclear factor kappa-B(NF-κB)phosphorylation,and the expression of tumor necrosis factor-α(TNF-α),interleukin(IL)-6 and IL-8.In conclusion,FRET revealed Phi and Swe concurrently targeted AKT on different domains and the combination of Phi and Swe enhanced the anti-inflammatory effect.

Anti-mutagenicity of Swertiamarin and Its Metabolite in Incubated System of Human Intestinal Flora

Objective To study the biotransformation regulation and pharmacological effect of swertiamarin and its metabolite in incubated system of human intestinal flora. Methods Incubated system of human intestinal flora was utilized to research the intestinal metabolism of swertiamarin. Furthermore, mutagenic test and anti-mutagenic test were carried out to research the activity relationship of swertiamarin and its metabolite. Results Gentianine was found in the metabolites of swertiamarin. The pharmacological experiment indicated that swertiamarin and its metabolite both had good anti-mutagenic effect. Conclusion Swertiamarin is partly metabolized to gentianine after oral administration. They show similar anti-mutagenicity effects.

基于质量源于设计理念的小儿腹痛草标准汤剂的制备工艺及质量标准

目的运用质量源于设计理念(QbD),优化小儿腹痛草标准汤剂的制备工艺,并建立其质量标准。方法根据中药质量标志物理论预测分析关键质量属性;采用失效模型与影响分析筛选关键工艺参数(CPPs);建立关键质量属性的测定方法;根据单因素实验确定初步范围后使用Box-Behnken实验优化提取工艺;以熵值法进行综合评分;建立设计空间,选取较优操作空间进行工艺验证;以最佳工艺制备15批不同产地小儿腹痛草饮片的标准汤剂,最终建立其出膏率、浸出物、薄层色谱、指纹图谱、含量及含量转移率的质量标准。结果以獐牙菜苦苷含量、龙胆苦苷含量及出膏率为关键质量属性;以浸泡时间、加水量、煎煮时间为关键工艺参数;所建立的模型差异有统计学意义;筛选的最佳工艺为:浸泡时间90 min、加水量15倍、煎煮时间30 min(第二煎20 min);出膏率21.13%~30.73%;浸出物含量在82.00%~88.00%;薄层色谱中獐牙菜苦苷斑点清晰;15批样品与参照图谱的相似度均>0.85;獐牙菜苦苷含量250.64~385.21 mg·g^(-1),转移率43.76%~77.73%;龙胆苦苷含量0.69~2.70 mg·g^(-1),转移率56.02%~105.29%。结论基于以上方法及技术筛选出小儿腹痛草标准汤剂制备工艺,可为其配方颗粒的制剂开发及质量控制提供参考。

Lomatogonium rotatum extract alleviates diabetes mellitus induced by a high-fat,high-sugar diet and streptozotocin in rats

BACKGROUND Lomatogonium rotatum(LR)is traditionally used in Mongolian folk medicine as a hypoglycemic agent,but its evidence-based pharmacological effects and mechanisms of action have not been fully elucidated.AIM To emphasize the hypoglycemic action mechanism of LR in a type 2 diabetic rat model and examine potential biomarkers to obtain mechanistic understanding regarding serum metabolite modifications.METHODS A high-fat,high-sugar diet and streptozotocin injection-induced type 2 diabetic rat model was established.The chemical composition of the LR was identified by high performance liquid chromatography.LR extract administrated as oral gavage at 0.5 g/kg,2.5 g/kg,and 5 g/kg for 4 wk.Anti-diabetic effects of LR extract were evaluated based on histopathological examination as well as the measurement of blood glucose,insulin,glucagon-like peptide 1(GLP-1),and lipid levels.Serum metabolites were analyzed using an untargeted metabolomics approach.RESULTS According to a chemical analysis,swertiamarin,sweroside,hesperetin,coumarin,1.7-dihydroxy-3,8-dimethoxyl xanthone,and 1-hydroxy-2,3,5 trimethoxanone are the principal active ingredients in LR.An anti-diabetic experiment revealed that the LR treatment significantly increased plasma insulin and GLP-1 levels while effectively lowering blood glucose,total cholesterol,triglycerides,low-density lipoprotein cholesterol,and oral glucose tolerance test compared to the model group.Furthermore,untargeted metabolomic analysis of serum samples detected 236 metabolites,among which 86 were differentially expressed between the model and the LR group.It was also found that LR considerably altered the levels of metabolites such as vitamin B6,mevalonate-5P,Dproline,L-lysine,and taurine,which are involved in the regulation of the vitamin B6 metabolic pathway,selenium amino acid metabolic pathway,pyrimidine metabolic pathway,and arginine and proline metabolic pathways.CONCLUSION These findings indicated that LR may have a hypoglycemic impact and that its role may be related to changes in the serum metabolites and to facilitate the release of insulin and GLP-1,which lower blood glucose and lipid profiles.

不同扭结方向秦艽中环烯醚萜苷类含量比较

目的:通过三种环烯醚萜苷含量比较,对不同基原和产地的秦艽品质进行评价。方法:分别以马钱苷酸、獐芽菜苦苷、龙胆苦苷的含量及三者之和,评价不同基原、不同产地左旋秦艽和右旋秦艽的品质。结果:对24批左旋秦艽和右旋秦艽的样品比较,以马钱苷酸含量计算,其中5批样品的含量左旋高于右旋;以獐牙菜苦苷含量计算,左旋和右旋各占一半;以龙胆苦苷含量计算,其中5批样品的含量左旋高于右旋;以三者总含量计算,9批样品中右旋均高于左旋,占比75%。左旋秦艽中三种环烯醚萜苷类总含量分别为8.08%、102.50%、14.47%,右旋秦艽含量分别为11.77%、108.80%、14.61%;左、右旋秦艽中总环烯醚萜苷类含量累计值分别为125.05%和135.18%。结论:以上结果对秦艽科学分析和品质评价提供参考,也为论证《本草纲目》中“但以左文者为良,分秦与艽为二名,谬矣”的观点提供一定依据。

大鼠灌胃当药苦苷后血浆中含氮代谢物R⁃gentiandiol和S⁃gentiandiol药代动力学研究

目的:建立血浆中当药苦苷的一对对映异构含氮代谢物R‑gentiandiol和S‑gentiandiol的手性拆分方法,并对这两个异构体的药代动力学进行研究。方法:采用LC‑MS/MS对当药苦苷的体内代谢物进行检测,使用Astec CyclobondⅡ环糊精手性色谱柱(4.6 mm×100 mm,5μm),选择乙腈‑水为流动相的梯度洗脱方式,且在正离子模式下采用多级反应监测展开分析。研究采用的定量分析反应离子对分别为R‑gentiandiol(m/z 210.04→192.06)、S‑gentiandiol(m/z 210.04→192.06),内标龙胆碱酮(m/z 192.02→162.08)。结果:两种含氮代谢物的大鼠血浆样品测定方法的线性相关系数均大于0.999,精密度小于7.00%,回收率为99.57%~102.65%,基质效应为90.94%~91.34%。当药苦苷灌胃大鼠后血浆中R‑gentiandiol和S‑gentiandiol达峰时间tmax分别为(1.63±0.23)、(1.58±0.21)h;半衰期t1/2分别为(6.23±0.52)、(5.46±0.38)h,最大血药浓度Cmax分别为(86.79±20.81)、(60.72±18.95)ng/mL,药时曲线下面积AUC0‑24分别为(1094.58±86.37)、(724.67±58.38)(ng·h)/mL。结论:该方法灵敏度高、精确度好,成功用于当药苦苷的体内代谢物R‑gentiandiol和S‑gentiandiol的手性拆分及其药代动力学研究,本研究方法及实验结果可为当药苦苷药效作用实体研究和药效物质基础研究提供科学依据,为中药单体的临床应用及资源开发奠定基础。

肋柱花的定性定量分析

目的:研究肋柱花TLC鉴别及多成分同时测定的方法。方法:利用TLC法对肋柱花中1,8-二羟基-4,6-二甲氧基双苯吡酮、1,3,5,8-四羟基-5,6,7,8-四氢双苯吡酮、乌苏酸和獐牙菜苦苷进行色谱鉴别。采用HPLC法对不同批次肋柱花进行含量测定,色谱柱为Topsil C_(18)柱(4.6 mm×250 mm,5μm);以乙腈(A)-水(B)为流动相,梯度洗脱(B;0~15 min,85%→75%;15~30 min,75%→55%)。流速为1.0 mL·min^(-1),柱温40℃,进样量10μL。獐牙菜苦苷为237 nm,而异牡荆苷、当药黄素和当药醇苷为275 nm。结果:1,8-二羟基-4,6-二甲氧基双苯吡酮和1,3,5,8-四羟基-5,6,7,8-四氢双苯吡酮以石油醚-丙酮-甲酸(5∶1∶0.2)为展开剂,而乌苏酸和獐牙菜苦苷分别以环己烷-乙酸乙酯-冰醋酸(10∶2∶0.3)和二氯甲烷-甲醇-甲酸(5∶1∶0.1)为展开剂时,斑点清晰,Rf值符合要求。在选用色谱条件下,被检测成分在较宽浓度范围内呈良好的线性关系(r>0.9994)。其平均回收率分别为98.47%、96.44%、97.80%和96.52%,RSD分别为0.49%、1.6%、1.4%和1.5%。结论:创建了肋柱花的定性定量分析方法,为综合评价该药材质量提供依据。

獐芽菜苦苷结构修饰产物对柞蚕丝绸的染色性能

选取天然环烯醚萜类化合物獐芽菜苦苷进行结构修饰,研究了修饰产物对柞蚕丝绸的染色性能。利用UVvis分析了整个呈色反应过程,确定了最佳染色工艺条件,测试了染色柞蚕丝绸的色牢度,初步探讨了獐芽菜苦苷结构修饰的原理和染色机理。结果表明:獐芽菜苦苷可通过“苷交换”结构修饰方法获得与母体结构相似的目标产物甲基獐芽菜苦苷苷元,在其质量分数为5%(omf)(以干柞蚕丝绸质量为计),pH为7.5~8.0,染色温度50℃,染色时间2 h的条件下,染色柞蚕丝绸的K/S值最高,色牢度达到4~5级及以上。

大花肋柱花的质量标准研究

目的建立大花肋柱花的质量标准。方法用显微及薄层色谱法对大花肋柱花进行定性分析;参考《中华人民共和国药典》方法进行该药材浸出物的研究:以高效液相色谱法测定獐牙菜苦苷和当药苷2种成分的含量。结果大花肋柱花粉末显微特征清晰,含有色素块、纤维、花粉粒、螺纹导管、表皮细胞、木栓细胞。以三氯甲烷-甲醇-水(30∶10∶1)溶液为展开剂,以100 g·L^(-1)硫酸乙醇溶液为显色剂,加热至斑点显色清晰,在紫外光灯365 nm处检视,所得的薄层图谱分离度好,斑点清晰。该药材浸出物的含量均值为31.95%;药材中獐牙菜苦苷、当药苷的含量测定线性范围分别为0.1213~0.8008 mg·mL^(-1)(r=0.9996)、0.0102~0.1101 mg·mL^(-1)(r=0.9994),平均回收率分别为97.43%、98.02%,RSD值分别为1.87%、2.95%。结论所建立的大花肋柱花定性定量测定方法简单、准确,能够为大花肋柱花的质量控制提供有效数据。

响应面法优化藏茵陈中獐牙菜苦苷的提取工艺研究

本文以甲醇体积分数、提取时间、液料比为考察因素,獐芽菜苦苷的提取率为考察指标,用高效液相色谱法测定獐芽菜苦苷的含量,通过单因素试验及响应面实验优化藏茵陈中獐芽菜苦苷的提取工艺。结果表明,藏茵陈中獐芽菜苦苷的最佳提取工艺条件为甲醇体积分数70%,提取2.0 h,液料比25∶1,在此提取条件下獐芽菜苦苷的平均提取率为2.09%,优化的工艺模型拟合度良好。优化后的方法可用于藏茵陈中獐芽菜苦苷的提取,工艺简单且稳定。

HPLC法测定獐牙菜及其近缘植物中4种有效成分的含量

目的:建立反相高效液相色谱法同时测定獐牙菜及其近缘植物中番木鳖酸、獐牙菜苦苷、龙胆苦苷、芒果苷的含量。方法:采用ZORBAX SB—C18(250 mm×4.6mm,5μm)色谱柱,以流动相甲醇和水(含0.04%磷酸)的比例在0-24 min内由22:78至38:62线性梯度洗脱,流速1 mL·min～1,检测波长254 nm,柱温30℃。结果:4种成分均达到基线分离,番木鳖酸、獐牙菜苦苷、龙胆苦苷、芒果苷的线性范围分别为0.05—6.25μg(r=0.9999),0.0095—2.9 μg(r=0.9998),0.0486—2.56μg(r=0.9999),0.0056—2.8μg(r=0.9998);回收率为102%(RSD=4.4%),97.7%(RSD=4.3%),99.5%(RSD=3.5%),103%(RSD=1.1%)。结论:方法测定快速,结果准确、可靠。

11种獐牙菜及近缘植物中有效成分的高效液相色谱测定

建立了獐牙菜中３种苦味甙，即獐牙菜苦甙（ｓｗｅｒｔｉａｍａｒｉｎ）、獐牙菜甙（ｓｗｅｒｏｓｉｄｅ）、龙胆苦甙（ｇｅｎｔｉｏｐｉｃｒｏｓｉｄｅ）及一种 酮甙──獐牙菜 酮甙（ｓｗｅｒｔｉａｎｏｌｉｎ）的高效液相色谱定量分析方法（μ－ＢｏｎｄｐａｋＣ１８柱，水—甲醇—异丙醇—四氢呋喃＝６５：３０：５：１作流动相）；对１１种獐牙菜及近缘植物椭圆叶花锚、坚龙胆进行了含量测定，并对他们之间的异同进行了比较。

藏药麻花艽中四种苦苷类化学成分的HPLC测定

建立了藏药麻花艽中龙胆苦苷（gentiopicroside）、落干酸（loganic acid）、獐牙菜苦苷（swertiamarin）和獐牙菜苷（sweroside） 4种苦苷类成分的HPLC分析方法. 色谱柱为Eclipse XDB-C8 （4.6 mm i.d. ×150 mm, 5 μm）, 流动相为体积分数5%乙腈＋10 mmoL甲酸～95%乙腈水溶液, 检测波长为240 nm. 结果表明, 该方法具有良好的线性关系和回收率, 为麻花艽中苦苷类成分的进一步开发利用提供了科学依据.

獐牙菜属及近缘植物中4种有效成分的HPLC定量分析

目的:比较獐牙菜属及近缘植物中獐牙菜苦苷、龙胆苦苷、芒果苷、齐墩果酸的含量,为资源利用提供依据。方法:獐牙菜苦苷、龙胆苦苷、芒果苷色谱条件:ZORBAX SB-C_(18)色谱柱(4.6 mm×150 mm,5μm),流动相甲醇-水(含0.1%磷酸)的比例在0→30 min 内由20:80线性变化至30:70,检测波长240 nm,流速0.60 mL·min^(-1),柱温30℃。齐墩果酸色谱条件:ZOR-BAX SB-C_(18)色谱柱(4.6 mm×150 mm,5μm);流动相乙腈-水(含0.1%磷酸)(75:25),检测波长210 nm;流速1.0 mL·min^(-1);柱温40℃。结果:獐牙菜苦苷、龙胆苦苷、芒果苷及齐墩果酸的线性范围为0.155～19.0μg(r=0.9999),0.0186～1.24μg(r=0.9999),0.00680～1.36μg(r=0.9998),0.150～3.90μg(r=0.9996),回收率分别为101.2%(RSD=1.6%),99.4%(RSD=1.2%),102.6%(RSD=1.5%),99.3%(RSD=1.8%)。结论:本实验说明在民间作为獐牙菜使用的植物中都含有4种有效成分,但种间差异较大。

高效液相色谱法测定川东獐牙菜中獐牙菜苦苷和去甲当药苷的含量

目的 建立测定川东獐牙菜中獐牙菜苦苷和去甲当药苷含量的高效液相色谱方法。方法 HYPERSILBDSC18(4.6 mm×200 mm,5μm)色谱柱,流动相为乙腈-磷酸二氢钠(0.03 mol·L-1,pH=3.0),梯度洗脱(ACN%;0 min 5％,20 min 15％),流速为0.8 mL·min-1,检测波长为250 nm,柱温为25℃。结果 獐牙菜苦苷和去甲当药苷分别在1.528-11.466μg,85.4-854 ng范围内线性良好,相关系数r分别为0.99992、0.99988, 平均回收率分别为99.1％和101.8％,RSD均<1％(n=5)。结论 本方法操作简便,分离效果好,灵敏度高,重 现性好,为控制川东獐牙菜质量提供参考。

不同生长季节下藏药麻花秦艽活性成分含量研究

采用高效液相色谱法测定了野生与栽培藏药麻花秦艽(Gentianastraminea)根中龙胆苦苷、落干酸、獐牙菜苦苷和獐牙菜苷四种环烯醚萜苷类化学成分的含量及其在不同生长季节的变化趋势。结果表明,4种环烯醚萜苷类成分的含量随植物的生长季节而波动,其活性成分含量在野生种与栽培种之间发生了一定的差异。但是,栽培根中的龙胆苦甙已达到药典的标准,可供药用。

金银花中抗炎活性成分獐牙菜苷和獐牙菜苦苷定量方法的研究

目的：研究建立金银花中抗炎活性成分獐芽菜苷、獐牙菜苦苷同时定量的方法。方法：通过超高效液相色谱法对獐芽菜苷、獐牙菜苦苷进行含量测定。结果：结果表明獐芽菜苷进样量在6.85~548.00 ng、獐牙菜苦苷进样量在64.50~1 032.00 ng与峰面积呈良好线性关系,标准曲线分别为Y=495 534 7X-213 11（r=0.999 4）;Y=1 596 561X-24 132（r=0.999 9）。结论：所建立的定量方法快速简单,方法可靠,可作为金银花中抗炎活性成分的质量控制方法。

HPLC波长切换法同时测定粗茎秦艽中6个成分的含量

本实验建立了HPLC波长切换法同时测定云南丽江粗茎秦艽中马钱苷酸、獐牙菜苦苷、龙胆苦苷、獐牙菜苷、异荭草苷、异牡荆苷6种成分的含量,并对各成分含量间的相关性进行分析,为粗茎秦艽药材质量的全面评价提供了科学依据。采用Sepax Gp-C18（150 mm×4.6 mm,5μm）色谱柱,甲醇-0.1%磷酸为流动相体系,梯度洗脱,流速1.0 mL·min-1,柱温25℃,检测波长235 nm（0～51 min）、270 nm（51～57 min）、243 nm（57～65 min）、270 nm（65～80 min）。马钱苷酸、獐牙菜苦苷、龙胆苦苷、獐牙菜苷、异荭草苷、异牡荆苷的量分别在0.01000～10.0000、0.00216～2.1600、0.0480～48.0000、0.0010～1.0000、0.00034～0.3400、0.00026～0.2600μg范围内与色谱峰峰面积呈良好的线性关系,平均回收率为98.52%～99.31%,RSD均小于3.5%（n=6）;对各成分含量间相关性分析发现环稀醚萜苷类与黄酮类组分含量间无相关性,四种环烯醚萜苷类成分的含量之间存在着显著相关性,两种黄酮类成分的含量之间也存在相关性。该方法简便、快速、准确,重现性好,更符合中药材复杂体系的多指标质量控制发展趋势,为粗茎秦艽药材的质量控制找到了简便易行的方法。

西南獐牙菜的化学成分研究(I)

西南獐芽菜(Swertia cincta Burk)为一种龙胆科獐牙菜属的民间植物药.对采自云南省新平县的西南獐牙菜进行了化学成分研究.采用现代分离分析技术,从该植物80%甲醇提取物的正丁醇萃取部分分离出了7个环烯醚萜类化合物,运用现代波谱技术鉴定它们的结构为:獐芽菜苦苷(1)、龙胆苦苷(2)、獐芽菜苷(3)、6'-O-葡萄糖基-獐牙菜苷(4)、septemfidoside(5)、马钱苷酸(6)、6'-O-葡萄糖基-獐牙菜苦苷(7),其中化合物4～7是首次从该属植物中分离得到.