百合知母汤的高效液相色谱指纹图谱及其与组方药味的相关性

研究和建立了百合知母汤的高效液相色谱(HPLC)指纹图谱,为研究百合知母汤的药效物质基础及配伍变化提供了手段。采用Agela Venusil XBP-C18色谱柱(250 mm × 4.6 mm,5 μm),以乙腈和0.1%甲酸为流动相二元梯度洗脱,流速1 mL/min,检测波长315 nm,柱温25 ℃。以芒果苷为参照物,在相同的色谱条件下测定了10批不同产地的百合与知母制备的百合知母汤的指纹图谱,获得了16个共有指纹峰,通过与对照品的保留时间及紫外光谱比较,标定了5-羟甲基糠醛(5-HMF)、新芒果苷、芒果苷、异芒果苷、王百合苷B的出峰位置。该方法得到的百合知母汤的指纹图谱特征性和重现性较好,方法稳定、可靠,可以为百合知母汤的质量控制提供参考。通过实验归属了百合知母汤指纹图谱中的主要色谱峰,并确定了煎煮过程中的主要变化成分为5-HMF。     

基于超高效液相色谱-紫外检测定量指纹图谱结合化学模式识别的复方金钱草颗粒质量评价

复方金钱草颗粒具有利尿、抑制泌尿系结石形成、抗炎、抗氧化作用,且具有较大的市场需求。因此,采用超高效液相色谱-紫外检测(UPLC-UV)法建立定量指纹图谱,并结合化学模式识别技术对不同年份的复方金钱草颗粒进行质量评价,可为其质量控制提供依据。采用聚类分析(HCA)和主成分分析(PCA)等化学模式识别技术对35批复方金钱草颗粒样品的指纹图谱数据进行分析,筛选出质量差异标志物芒果苷和异芒果苷,并对二者进行含量测定。在复方金钱草颗粒指纹图谱中共指认出12个共有峰,且35批样品的相似度均在0.952以上。在HCA中,将35批样品分为了两类,其中2018年和2019年的样品为一类,2020年和2021年的样品为一类。此外,PCA结果显示了与聚类分析相同的聚类趋势。在此基础上,进一步通过正交偏最小二乘法分析 (OPLS-DA)筛选出了导致2018年、2019年与2020年、2021年的样品产生差异的差异标志物芒果苷和异芒果苷。以两个差异标志物芒果苷和异芒果苷为指标进行含量测定,结果显示色谱峰的分离度良好,线性关系良好,平均加标回收率分别为101.7%~105.6%和103.4%~105.5%,且相对标准偏差(RSD)均低于1.43%。在35批样品中,2020年、2021年的样品与2018年、2019年的样品相比,芒果苷与异芒果苷含量更高且波动范围更小。该研究建立了准确、可靠的复方金钱草颗粒质控方法,实现了对不同年份的复方金钱草颗粒样品合理、有效的质量评价,可为建立更系统、更全面的质量控制标准提供借鉴与参考。     

炮制方法对连翘主要化学成分连翘酯苷的影响

探讨炮制方法对连翘中化学成分的影响,为连翘的合理炮制提供依据.采用HPLC法分析比较清蒸炮制时间对连翘化学成分含量影响.连翘炮制品与生品比较化学成分有明显变化,尤以连翘酯苷的变化最明显.炮制对连翘中化学成分有较大影响,以连翘酯苷为考察指标评价炮制方法,蒸10～15 min连翘质量最佳.     

连翘鲜果粗酶对连翘酯苷A的酶解作用

探讨连翘鲜果粗酶对连翘酯苷A含量的影响,为连翘的炮制提供依据。采用高效液相色谱法(HPLC)分析不同浓度粗酶以及在不同孵育温度、孵育时间条件下,粗酶对连翘酯苷A酶解程度的影响,并利用水煮杀酶的方式测试了不同炮制时间对连翘果实中酶的灭活作用。结果表明,连翘果实中存在对连翘酯苷A有酶解作用的酶;用沸水煮的方法炮制连翘时,水煮数分钟即可达到杀酶保苷作用。此外,通过HPLC分析确认连翘酯苷A在粗酶液的作用下分解产物之一为咖啡酸。     

百合知母汤及其组方药味的高效液相色谱-电喷雾质谱研究

建立了百合知母汤的高效液相色谱-电喷雾质谱(HPLC-ESI-MS)分析方法,对百合知母汤及其组方药味中的主要成分进行了鉴定.根据正离子模式和负离子模式下的分子离子峰获得化合物分子量信息,通过与文献数据或部分对照品对照,确定化合物的可能结构.实验表明,百合知母汤中各主要化学成分在正离子模式中响应较好.在相同的条件下,通过对百合知母汤及其组方药味进行比较分析,归属并鉴定了百合知母汤中的38个成分,包括3个黄酮,4个酚酸糖苷和31个皂苷类成分.本实验为鉴别百合知母汤中的化学成分提供了一种简便、快速的方法.     

高效液相色谱法同时测定忍冬中7种成分

建立了在不同时间段内转换使用不同波长同时测定忍冬花和叶中7种化学成分(绿原酸、咖啡酸、芦丁、木犀草苷、异绿原酸A、异绿原酸B、异绿原酸C)的高效液相色谱分析方法,同时应用该方法分析了忍冬花、忍冬老叶和新叶中成分含量的差异。色谱柱为Agilent Eclipse Plus C18(250 mm×4.6 mm, 5 μm);流动相为0.3%甲酸水溶液(A)和乙腈(B),梯度洗脱,流速1 mL/min;采用VWD紫外检测器转换波长(330 nm、350 nm)检测。应用所建立的方法测定忍冬新叶中绿原酸、木犀草苷含量分别为2.572%、1.498‰,均比药典中规定的含量高,有必要进一步的研究和开发利用。该方法准确、简便、灵敏度高,适用于忍冬中7种化学成分含量的同时测定和忍冬的质量控制及综合评价。     

八种海参体内皂苷类成分的SPE-HPLC分析比较

采用SPE-HPLC方法研究8种海参的总皂苷特征图谱,比较不同种海参间皂苷的组成及含量差异,为海参的品种鉴定提供依据.使用zorbax SB-C<,18>(4.6 mm×250mm,5μm)色谱柱,以乙腈-1g/L的三氟乙酸溶液为流动相梯度洗脱,柱温30℃,流速1.0mL/min,检测波长205nm.对不同海参体内皂苷类成分进行HPLC分析,所得图谱特征性强,8种海参的各皂苷成分均得到很好的色谱分离.结果表明不同海参体内的皂苷组成及含量有明显差异,所建立的方法可用于海参的品种鉴定.     

超高效液相色谱-电雾式检测器测定福建产绞股蓝中绞股蓝皂苷XLVI和LVI北大核心CSCD

该研究利用超高效液相色谱-电雾式检测器(UHPLC-CAD)建立了福建产绞股蓝中绞股蓝皂苷XLVI和LVI含量的测定方法。首先利用超高效液相色谱-四极杆-飞行时间质谱(UHPLC-Q-TOF/MS)结合UHPLC-CAD鉴定了福建产绞股蓝的主要成分,其中绞股蓝皂苷XLVI、LVI以及二者相应的含丙二酰基酸性皂苷为其主成分,因此在含量测定时先进行碱水解预处理将酸性皂苷转化为对应的去丙二酰基中性皂苷,再利用UHPLC-CAD测定碱水解后绞股蓝皂苷XLVI和LVI的含量。将绞股蓝样品粉末在乙醇-水-氨水(50∶46∶4,v/v/v)和料液比1∶150(g∶mL)条件下超声提取30 min,静置24 h后,在Waters ACQUITY UPLC BEH C18色谱柱(100 mm×2.1 mm,1.7μm)上分离,采用0.1%(v/v)甲酸水溶液和乙腈作为流动相进行梯度洗脱,流速0.5 mL/min,柱温40℃,电雾式检测器检测。结果表明,绞股蓝皂苷XLVI和LVI分别在9.94~318.00μg/mL和12.78~409.00μg/mL范围内具有良好的线性关系,相关系数(r)分别为0.9993和0.9995。方法精密度、重复性和24 h稳定性试验的相对标准偏差(RSD)均小于2.0%(n=6),绞股蓝皂苷XLVI与LVI的加标回收率分别在100.2%~107.2%与97.9%~104.2%范围内,RSD值分别为2.4%与2.6%。16批绞股蓝样品含量测定结果显示:绞股蓝皂苷XLVI含量占0.57%~2.57%,绞股蓝皂苷LVI含量占0.66%~2.99%。该方法灵敏度高,重复性好,可用于福建产绞股蓝的质量研究和质量控制。     

加压毛细管电色谱-微流蒸发光散射检测器联用系统的构建及其应用

构建微流蒸发光散射检测器(μELSD)与加压毛细管电色谱(pCEC)联用系统,测定中药提取物注射用血塞通(冻干)中三七皂苷R1、人参皂苷Rg1,Re,Rb1、Rd考察系统的实用性和稳定性。用C18毛细管色谱柱,通过对流动相体系、梯度洗脱条件、雾化载气流速、蒸发温度、施加电压等参数的优化,确定了注射用血塞通(冻干)5种成分含量测定的最佳测定参数。最佳测定参数如下,流动相A为15 mmol/L甲酸-三乙胺乙腈溶液(pH=7.0),流动相B为15 mmol/L甲酸-三乙胺溶液(pH=7.0);梯度洗脱条件:0~10 min,19%A;10~30 min,22%A;30~35 min,36%A;35~45 min,40%A。雾化载气流速2 L/min;蒸发温度120℃;施加电压+8 kV。5种成分线性范围为8.6~146.9 ng(三七皂苷R1)、6.9~189.7 ng(人参皂苷Rg1)、6.8~171.4 ng(人参皂苷Re)、9.4~156.1 ng(人参皂苷Rb1)、7.5~180.5 ng(人参皂苷Rd),5种成分回收率都在95%~105%之间。实验表明,构建的pCEC-μELSD联用系统能用于药物中有效成分的含量测定。μELSD的构建为毛细管液相色谱、毛细管电色谱和毛细管电泳分离技术提供了一种全新的检测手段。     

不同炮制工艺麦冬总皂苷含量与活性评价

根据《中国药典》和参考文献所述炮制方法,对麦冬进行抽心、轧扁(捣裂)、整粒和切片炮制处理,提取分离其总皂苷,并开展体外抑菌活性评价。结果表明:不同炮制工艺麦冬皂苷提取率从高到低的顺序为整粒>抽心>轧扁、切片;4种炮制品的麦冬皂苷类成分对大肠杆菌和金黄色葡萄球菌均有较强的抑菌效果,其中整粒的效果最佳。考察不同麦冬炮制品提取总皂苷含量与抑菌率的相关性研究,发现整粒炮制工艺具有提取率高、工艺稳定性、体外抑菌活性高和生产成本低的优点,可为麦冬炮制工艺的筛选提供参考。     

Identification of major xanthones and steroidal saponins in rat urine by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry technology following oral administration of Rhizoma Anemarrhenae decoction

Rhizoma Anemarrhenae (Zhimu in Chinese), the dried rhizome of Anemarrhena asphodeloides Bge. (Fam. Liliaceae), is a well-known traditional Chinese medicinal herb and has been used clinically in China for centuries to cure various diseases. However, like other traditional Chinese medicines, the effective constituents of this medicine, especially the assimilation and metabolites in vivo, which are very important to show their effects, have not been systematically studied. In this paper, solid-phase extraction and liquid chromatography-atmospheric pressure chemical ionization mass spectrometry technologies were used to study the constituents absorbed into rat urine and their metabolites after oral administration of Rhizoma Anemarrhenae decoction. A total of 11 compounds, including two xanthones, three of their metabolites and six steroidal saponins, were identified in rat urine sample. They were neomangiferin (1), glucuronide and monomethyl conjugate of mangiferin (2), mangiferin (3), monomethyl conjugate of mangiferin (4), dimethyl conjugate of mangiferin (5), timosaponin N or timosaponin E1 (6), timosaponin BII (7), timosaponin BIII (8), anemarrhenasaponin I or anemarrhenasaponin II (9), timosaponin AII (10) and timosaponin AIII (11). The results would efficaciously narrow the potentially active compounds range in Rhizoma Anemarrhenae decoction, and pave a helpful way for follow-up mechanism of action research.     

In Vivo Metabolism Study of Timosaponin BIII in Rat Using HPLC-QTOF-MS/MS

Timosaponin BIII, as one of the steroid saponins isolated from Anemarrhena asphodeloides Bge., was proved to have many pharmacological activities in recent years and became a natural active compound with good development prospect. In the present study, a simple and rapid method using high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry was developed for the determination of the structures of timosaponin BIII and its metabolites in rats after administrating intragastrically at 300 mg kg^?1. By comparing their changes in molecular masses (ΔM), retention times and spectral patterns with those of the parent compound, nine metabolites were detected and identified in urine, and eight in plasma as well as four in brain. It is also indicated that the deglycosylation and oxidation reactions were the main metabolic pathways in the biotransformation of timosaponin BIII in vivo and the structures of the nine metabolites were identified and proposed to be timosaponin BII(M1), the hydroxylated metabolite of TBII(M2), the hydroxylated metabolites of TBIII(M3 and M4), deglycosylation and monooxygenation product of TBIII(M5), the deglycosylation product of TBII(M6), timosaponin AIII(M8), the isomers of timosaponin AIII(M7 and M9).     

In Vivo Metabolism Study of Timosaponin BIII in Rat Using HPLC-QTOF-MS/MS

Timosaponin BIII, as one of the steroid saponins isolated from Anemarrhena asphodeloides Bge., was proved to have many pharmacological activities in recent years and became a natural active compound with good development prospect. In the present study, a simple and rapid method using high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry was developed for the determination of the structures of timosaponin BIII and its metabolites in rats after administrating intragastrically at 300 mg kg⁻¹. By comparing their changes in molecular masses (ΔM), retention times and spectral patterns with those of the parent compound, nine metabolites were detected and identified in urine, and eight in plasma as well as four in brain. It is also indicated that the deglycosylation and oxidation reactions were the main metabolic pathways in the biotransformation of timosaponin BIII in vivo and the structures of the nine metabolites were identified and proposed to be timosaponin BII(M1), the hydroxylated metabolite of TBII(M2), the hydroxylated metabolites of TBIII(M3 and M4), deglycosylation and monooxygenation product of TBIII(M5), the deglycosylation product of TBII(M6), timosaponin AIII(M8), the isomers of timosaponin AIII(M7 and M9).

     

知母及制剂中菝葜皂甙元的反相高效液相色谱测定

用反相高效液相色谱法测定了知母及其制剂中的菝葜皂甙元，建立了中药及制剂中菝葜皂甙元测定的色谱方法。为知母及其制剂的质量评价提供了一种新的分析方法。     

Identification of major alkaloids and steroidal saponins in rat serum by HPLC-diode array detection-MS/MS following oral administration of Huangbai-Zhimu herb-pair Extract

Huangbai-Zhimu herb-pair (HBZMHP) is a widely used Chinese traditional medicine formula in treating various diseases; however, its active components have remained unknown. In this paper, serum chemistry and combined high-performance liquid chromatography (HPLC), diode-array detection and mass-spectrometry (MS) techniques were used to study the constituents of HBZMHP extract absorbed into rat serum after oral administration. A total of nine characteristic HPLC peaks in the TIC chromatograms were identified as magnoflorine (1), menisperine (2), palmatine (3), berberine (4), timosaponin N or timosaponin E1 (5), timosaponin D (6), timosaponin BIII, anemarsaponin C or xilingsaponin B (7) timosaponin BII (8) and timosaponin AIII (9). All of the identified peaks were constituents of HBZMHP extract. The results narrow the range of active compounds to be found in HBZMHP extract, and pave the way for the follow-up action mechanism research.     

A novel ultra high‐performance liquid chromatography‐tandem mass spectrometry method for the simultaneous determination of xanthones and steroidal saponins in crude and salt‐processed Anemarrhenae Rhizoma aqueous extracts

We established a rapid and sensitive ultra high‐performance liquid chromatography tandem mass spectrometry method for the simultaneous quantification of xanthones and steroidal saponins in rat plasma. Chromatographic separation was achieved on a C₁₈ column with a mobile phase comprising acetonitrile and 0.1% formic acid. The detection was performed by negative electrospray ionization in multiple reaction monitoring mode. The validated method showed good linearity within the tested range (r > 0.9945). The intra‐ and interday precision at high, medium, and low concentrations was less than 7.96%. The bias of accuracies ranged from −1.92 to 9.62%. The extraction recoveries of the compounds ranged from 84.78 to 88.69%, and the matrix effects ranged from 96.76 to 108.59%. This method was successfully applied to a pharmacokinetic comparison of crude and salt‐processed Anemarrhenae Rhizoma aqueous extracts after oral administration in rats. The maximum plasma concentration and area under concentration–time curve of timosaponin BIII and timosaponin AIII increased significantly (P < 0.05 or 0.01) and those of timosaponin BII decreased significantly (P < 0.05) after processing. These results could contribute to the clinical application of crude and salt‐processed Anemarrhenae Rhizoma and reveal the processing mechanism.     

Simultaneous quantitative determination of 13 active components in the traditional Chinese medicinal preparation Suanzaoren oral liquid by HPLC coupled with diode array detection and evaporative light scattering detection

To control the quality of different forms of Suanzaoren decoction, an effective and reliable method for the simultaneous determination of 13 major components (neomangiferin, mangiferin, spinosin, liquiritin apioside, liquiritin, 6′′′‐feruloylspinosin, senkyunolide I, timosaponin BII, isoliquiritoside, timosaponin C, jujuboside A, jujuboside B, and timosaponin AIII) was developed and validated for the first time in this study using high‐performance liquid chromatography with diode array detection and evaporative light scattering detection. The chromatographic separation was performed on a Venusil MP C₁₈ column (250 mm × 4.6 mm, 5 μm) at 30°C with a gradient of acetonitrile/redistilled water as the mobile phase. Diode array detection was carried out at a wavelength of 275 nm. The drift tube temperature and the nitrogen gas flow rate of the evaporative light scattering detection were set at 50°C and 1.6 L/min, respectively. The newly developed method was successfully applied to the determination of 13 components in lab‐prepared Suanzaoren oral liquid, Suanzaoren mixture, and clinical Suanzaoren granules, and this study showed that this was a useful way to comprehensively evaluate the quality of Suanzaoren decoction in different forms of the preparation.     

Pharmacokinetic study of free mangiferin in rats by microdialysis coupled with microbore high-performance liquid chromatography and tandem mass spectrometry

Mangiferin (2-beta-D-glucopyranosyl-1,3,6,7-tetrahydroxyxanthen-9-one) has been isolated from the herbal root of Anemarrhena asphodeloides Bung showing antioxidative, antiviral, and anticancer effect. An in vivo microdialysis sampling method coupled to microbore high-performance liquid chromatography (HPLC) was employed for continuous monitoring of free mangiferin in rat blood. Microdialysis probes were inserted into the jugular vein/right atrium and brain striatum of Sprague-Dawley rats, and mangiferin at doses of 10, 30 or 100 mg/kg were then administered via the femoral vein. Dialysates were collected every 10 min and injected directly into a microbore HPLC system. Mangiferin was separated by a reversed-phase C18 microbore column (150 x 1 mm) from dialysate within 10 min. The mobile phase consisted of acetonitrile-0.05% phosphoric acid-tetrahydrofuran (10:75:15, v/v/v) with a flow-rate of 0.05 ml/min. The wavelength of the UV detector was set at 257 nm. The limit of quantification for mangiferin was 0.05 microg/ml and in vivo recovery of mangiferin at concentrations of 1, 5 and 10 microg/ml was in range of 37.7-39.8%. The results indicate that the pharmacokinetics of mangiferin at doses of 10-30 mg/kg reveals a linear relation, while doses of 30-100 mg/kg show a nonlinear pharmacokinetic phenomenon. Mangiferin was undetectable in brain dialysate. The proposed method provides a technique for rapid and sensitive analysis of free mangiferin in rat blood and further application in pharmacokinetic study. Furthermore, the metabolites of mangiferin in the rat bile were confirmed by LC electrospray ionization (ESI) tandem mass spectrometry (MS-MS).     

Biotransformation of Timosaponin BII into Seven Characteristic Metabolites by the Gut Microbiota

Timosaponin BII is one of the most abundant Anemarrhena saponins and is in a phase II clinical trial for the treatment of dementia. However, the pharmacological activity of timosaponin BII does not match its low bioavailability. In this study, we aimed to determine the effects of gut microbiota on timosaponin BII metabolism. We found that intestinal flora had a strong metabolic effect on timosaponin BII by HPLC-MS/MS. At the same time, seven potential metabolites (M1–M7) produced by rat intestinal flora were identified using HPLC/MS-Q-TOF. Among them, three structures identified are reported in gut microbiota for the first time. A comparison of rat liver homogenate and a rat liver microsome incubation system revealed that the metabolic behavior of timosaponin BII was unique to the gut microbiota system. Finally, a quantitative method for the three representative metabolites was established by HPLC-MS/MS, and the temporal relationship among the metabolites was initially clarified. In summary, it is suggested that the metabolic characteristics of gut microbiota may be an important indicator of the pharmacological activity of timosaponin BII, which can be applied to guide its application and clinical use in the future.     

The structural elucidation of two new artificial steroidal saponins

Two novel steroidal saponins(timosaponin BⅢ-a,timosaponin BⅢ-b) together with a known steroidal saponin(timosaponin BⅡ-b) were prepared by the dilute acid hydrolysis of timosaponin BⅢ.Their structures were elucidated by means of 1D,2D NMR and TOF-MS spectral analysis.