DPPH-HPLC-QTOF-MS/MS快速筛选和鉴定杜仲黑茶中抗氧化活性成分

建立DPPH-HPLC-QTOF-MS/MS方法快速筛选、鉴定和测定杜仲黑茶中抗氧化活性成分。杜仲黑茶提取物与DPPH自由基反应30 min后,9个抗氧化活性成分的HPLC峰消失或峰面积明显减少,结合QTOF-MS/MS谱图及与标准品对照,确定抗氧化活性成分为:没食子酸,新绿原酸、原儿茶酸、绿原酸、咖啡酸、表没食子儿茶素没食子酸酯、槲皮素-3-O-桑布双糖苷、芦丁和异槲皮苷。并建立HPLC法同时测定9个抗氧化活性成分的含量。所建立的方法无须繁琐的分离纯化过程,可快速、灵敏、准确、高通量筛选复杂体系中的抗氧化活性成分,并为杜仲黑茶的质量控制提供科学依据。

DPPH-HPLC-QTOF-MS/MS快速筛选和鉴定臭牡丹中抗氧化活性成分

通过DPPH自由基清除实验对臭牡丹水提物的抗氧化活性进行评估,并建立DPPHHPLC-QTOF-MS/MS方法快速筛选和鉴定臭牡丹中抗氧化活性成分.臭牡丹水提物与DPPH自由基反应30min后,13个主要抗氧化活性成分的HPLC峰消失,根据抗氧化活性成分的保留时间、紫外吸收特征光谱、质谱裂解规律和诊断离子,以及与标准品对照,初步确定了13个抗氧化活性成分的结构:12个苯乙醇苷类化合物和咖啡酸,其中8个苯乙醇苷类物质为首次在大青属植物中发现,咖啡酸为首次报道存在于臭牡丹中.该方法无需繁琐的分离纯化过程,可对复杂组分中的抗氧化活性物质进行快速筛选和鉴定,为天然产物的质量控制和药效物质基础研究提供了简便、快捷、灵敏的方法.

HPLC-QTOF-MS分析六味地黄丸不同部位的化学成分

目的采用高效液相色谱—四级杆—飞行时间串联质谱(HPLC-QTOF-MS)法对六味地黄丸不同极性溶剂提取部位中的化学成分进行鉴定,并比较不同极性提取部位之间的成分差异。方法色谱分离采用WatersXBridge BEH C_(18)色谱柱(4.6 mm×250 mm,5μm),以乙腈(A)和0.1%甲酸溶液(B)为流动相梯度洗脱,流速为0.6 m L·min^(-1);质谱采用电喷雾离子源(ESI),在正负离子模式下扫描采集数据,根据准分子离子及二级碎片离子,并结合对照品及文献报道的质谱数据对六味地黄丸不同极性溶剂提取部位中的化学成分进行鉴定。结果从六味地黄丸的水、50%乙醇、75%乙醇、乙醇、石油醚和混合溶剂提取部位中分别鉴定出41、41、41、40、7和39个化合物。结论本研究首次对六味地黄丸的不同极性提取部位中的化学成分进行了鉴定和对比分析,丰富了六味地黄丸化学成分的结构信息,可为六味地黄丸的药效物质研究提供了科学参考。

HPLC-QTOF-MS法鉴别雷公藤多苷工艺残渣中的化学成分

建立高效液相色谱串联四级杆飞行时间质谱法(HPLC-QTOF-MS)快速鉴定雷公藤多苷柱色谱工艺残渣化学成分。采用Zorbax SB-C_(18)色谱柱(4.6mm×50 mm,1.8μm),流动相为乙腈-0.2%甲酸溶液,梯度洗脱,采用ESI-Q-TOF检测,正离子模式扫描。根据色谱峰精确相对分子质量、碎片离子信息及液相色谱保留时间比较鉴别化合物。结果表明,采用HPLC-QTOF-MS法鉴定了雷公藤多苷工艺残渣中30个成分,包括15个生物碱类化合物,10个二萜类化合物,4个三萜类化合物,1个不饱和脂肪酸。本研究工作为雷公藤多苷工艺残渣的综合开发利用提供定性分析依据。

HPLC-QTOF-MS/MS结合NMR分析甘草酸单铵盐原料及其高温强降解杂质

目的:分析鉴定甘草酸单铵盐原料及其高温强降解有关杂质成分。方法:采用Shim-pack GIST C18色谱柱(150 mm×4.6 mm,5μm),以乙腈-1%乙酸水溶液(34.4:65.6)为流动相,对甘草酸单铵盐及高温强降解原料中的有关物质进行分离,采用四级杆飞行时间质谱进行检测,结合其分子离子峰、碎片离子及裂解规律进行分析,鉴定其化学结构;进一步采用C18中压柱(800 g,25~50μm,6.5 cm×50 cm),以甲醇-水-乙酸(40:60:1、50:50:1、60:40:1)洗脱,分离制备有关物质,并用核磁共振波谱(^(1)H-NMR、^(13)C-NMR、HSQC、HMBC)对其中2个未知杂质成分进行结构确证。结果:通过对照品对照及HPLC-QTOF-MS/MS分析,鉴定了甘草酸单铵盐及其高温强降解原料中13个三萜皂苷类杂质成分,其中2个未知杂质成分经鉴定分析,结构确证为6’-甲基甘草酸及6″-甲基甘草酸。结论:HPLC-QTOF-MS/MS法可以快速分析鉴定甘草酸单铵盐中的未知杂质,可为其原料及制剂杂质限度的制定奠定基础。

桂枝茯苓胶囊HPLC-ESI-QTOF/MS化学成分分析过程中提取溶剂的优化

采用高效液相色谱电喷雾四极杆飞行时间串联质谱(HPLC-ESI-QTOF/MS)对桂枝茯苓胶囊中的化学成分进行分离和鉴别。运用Agilent Eclipse XDB-C18(4.6 mm×250 mm,5μm)色谱柱,以0.1%甲酸水溶液-乙腈为流动相梯度洗脱;使用ESI离子源,正离子与负离子模式下采集数据。根据Q-TOF/MS正、负离子质谱信息、通过元素组成分析、化合物同位素分布验证和对质谱裂解碎片解析并与相关文献对照,鉴别了桂枝茯苓胶囊中18个化合物。本法为桂枝茯苓胶囊中的化学成分分析提供了一种快速、有效的方法。

Comprehensive metabolic profiling of Alismatis Rhizoma triterpenes in rats based on characteristic ions and a triterpene database

Alismatis Rhizoma(AR)is widely used in Chinese medicine,and its major bioactive components,triterpenes,reportedly possess various pharmacological activities.Therefore,it is very important to study the metabolism of triterpenes in vivo.However,the metabolism of AR triterpene extract has not been comprehensively elucidated due to its complex chemical components and metabolic pathways.In this study,an ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry method,which was based on the characteristic ions from an established database of known triterpenes,was used to analyze the major metabolites in rats following the oral administration of Alismatis Rhizoma extracts(ARE).As a result,a total of 233 constituents,with 85 prototype compounds and 148 metabolites,were identified for the first time.Hydrogenation,oxidation,sulfate and glucuronidation conjugation were the major metabolic pathways for triterpenes in AR.In addition,the mutual in vivo transformation of known ARE triterpenes was discovered and confirmed for the first time.Those results provide comprehensive insights into the metabolism of AR in vivo,which will be useful for future studies on its pharmacodynamics and pharmacokinetics.Moreover,this established strategy may be useful in metabolic studies of similar compounds.

Metabolic enzyme inhibitory abilities, in vivo hypoglycemic ability of palmleaf raspberry fruits extracts and identification of hypoglycemic compounds

The unripe palmleaf raspberry,namely Fupenzi(FPZ),is an important medicinal and edible food.This study aims to evaluate the potential of FPZ extracts prepared with different approaches in attenuating hyperglycemia,gout,Alzheimer’s disease,and pigmentation,to obtain the enriching fraction and to identify the major active compounds.Results indicated that FPZ extracts showed weak activity against acetylcholinesterase,considerable ability against tyrosinase and xanthine oxidase,but excellent inhibition onα-glucosidase.Ultrasound-assisted 40%ethanol extract(40EUS)gave the highest phenolics content,and the bestα-glucosidase inhibition(IC_(50)=0.08μg/mL),which is 877-fold higher than that of positive control acarbose.The 40%ethanol eluting fraction of 40EUS showed the strongestα-glucosidase inhibition with the IC_(50) value of 37.79 ng/mL,it could also effectively attenuate the fasting blood glucose level and oral glucose tolerance of C57BL/6 mice.Twenty-six compounds were identified from 40%ethanol fraction by using HPLC-QTOF-MS/MS,hydrolysable tannins(including 11 ellagitannins and 4 gallotannins)were the major compounds,phenolic acids came to the second.Above results could provide important technical supporting for the further application and research of FPZ in health foods and drugs against diabetes.

基于UPLC-QTOF-MS和HPLC-QQQ-MS技术的鹰嘴豆酚类化合物定性、定量分析及其抗氧化能力研究

鹰嘴豆为一种高营养豆类植物。为深入了解其营养价值,用80%甲醇溶液提取酚类物质,对提取物中不同存在形态的酚类化合物及其抗氧化活性进行测定,并对其提取物进行酚类的定性及定量分析。结果表明,酚酸含量最高部分为糖苷键合态酚类物质(0.81 mg/g),黄酮含量最高部分为游离态酚类物质(0.26 mg/g),不同存在形式酚类物质含量及抗氧化能力不同;80%甲醇提取物通过UPLC-QTOF-MS定性分析,对其24种化合物进行鉴定,包括12种黄酮、4种酚酸;HPLC-QQQ-MS定量分析表明:对羟基苯甲酸、香豆酸、原儿茶酸类化合物含量丰富。

体外模拟消化过程中余甘子果实及醇提物的活性成分和抗氧化能力的变化

为探究余甘子冻干粉及其乙醇提取物在体外消化(口腔、胃、小肠和大肠)过程中的差异,本研究对余甘子的主要成分进行了鉴定,然后比较了两者消化前后的总酚和总黄酮含量以及抗氧化能力。结果表明,余甘子富含多酚和黄酮类物质,其中,冻干粉结合型多酚含量更高,而醇提物游离型多酚含量更高。体外模拟消化前,冻干粉的总酚和总黄酮的含量分别为5.40±0.07 mg GAE/mL和1.48±0.15 mg RT/mL,醇提物的总酚和总黄酮含量分别为3.65±0.05 mg GAE/mL和14.80±0.74 mg RT/mL。和消化前相比,在消化过程中,两者的总酚含量均显著降低(P<0.05),但两者的总黄酮含量在口腔消化液中升高。此外,消化前的余甘子醇提物对ABTS^(+)、DPPH自由基的清除能力和Fe^(3+)还原能力都显著低于冻干粉(P<0.05),但消化后余甘子醇提物的抗氧化能力显著增强(P<0.05)。对总酚、总黄酮含量与抗氧化能力的相关性分析表明,总酚含量与抗氧化能力之间存在显著的正相关性(P<0.05)。综上,消化后余甘子果实冻干粉及醇提物的活性成分和抗氧化能力发生明显的变化,且和余甘子冻干粉相比,醇提物的抗氧化能力更强。表明经提取及消化处理加工后的余甘子产品可能会有更高的抗氧化健康效益。

Identification and characterization of phenolics and terpenoids from ethanolic extracts of Phyllanthus species by HPLC-ESI-QTOF-MS/MS

Phyllanthus species plants are a rich source of phenolics and widely used due to their medicinal properties. A liquid chromatography–tandem mass spectrometry(LC–MS/MS) method was developed using high-pressure liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry(HPLC-ESI-QTOFMS/MS) for the identification and characterization of quercetin, kaempferol, ellagic acid and their derivatives in ethanolic extracts of Phyllanthus species. The chromatographic separation was carried out on Thermo Betasil C_8 column(250 mm×4.5 mm, 5 μm) using 0.1% formic acid in water and 0.1% formic acid in methanol as the mobile phase. The identification of diagnostic fragment ions and optimization of collision energies were carried out using 21 reference standards. Totally 51 compounds were identified which include 21 compounds identified and characterized unambiguously by comparison with their authentic standards and the remaining 30 were tentatively identified and characterized in ethanolic extracts of P. emblica, P. fraternus, P. amarus and P.niruri.

Structural characterization of monoterpene indole alkaloids in ethanolic extracts of Rauwolfia species by liquid chromatography with quadrupole time-of-flight mass spectrometry

Rauwolfia species(Apocynaceae) are medicinal plants well known worldwide due to its potent bioactive monoterpene indole alkaloids(MIAs) such as reserpine,ajmalicine,ajmaline,serpentine and yohimbine.Reserpine,ajmalicine and ajmaline are powerful antihypertensive,tranquilizing agents used in hypertension.Yohimbine is an aphrodisiac used in dietary supplements.As there is no report on the comparative and comprehensive phytochemical investigation of the roots of Rauwolfia species,we have developed an efficient and reliable liquid chromatography-tandem mass spectrometry(LC–MS/MS) method for ethanolic root extract of Rauwolfia species to elucidate the fragmentation pathways for dereplication of bioactive MIAs using highperformance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry(HPLC–ESI–QTOF–MS/MS) in positive ion mode.We identified and established diagnostic fragment ions and fragmentation pathways using reserpine,ajmalicine,ajmaline,serpentine and yohimbine.The MS/MS spectra of reserpine,ajmalicine,and ajmaline showed C-ring-cleavage whereas E-ring cleavage was observed in serpentine via Retro Diels Alder(RDA).A total of 47 bioactive MIAs were identified and characterized on the basis of their molecular formula,exact mass measurements and MS/MS analysis.Reserpine,ajmalicine,ajmaline,serpentine and yohimbine were unambiguously identified by comparison with their authentic standards and other 42 MIAs were tentatively identified and characterized from the roots of Rauwolfia hookeri,Rauwolfia micrantha,Rauwolfia serpentina,Rauwolfia verticillata,Rauwolfia tetraphylla and Rauwolfia vomitoria.Application of LC–MS followed by principal component analysis(PCA) has been successfully used to discriminate among six Rauwolfia species.

毛蕊花糖苷在常氧、缺氧大鼠体内药动学的比较

目的比较毛蕊花糖苷在常氧、缺氧大鼠体内药动学。方法采用低压低氧动物实验舱模拟高原环境制备急性缺氧大鼠模型,常氧、缺氧大鼠灌胃给予毛蕊花糖苷(300 mg/kg),于5、10、15、30、45 min及1、1.5、2、4、6、8、12、24 h采血,HPLC-QTOF-MS法检测毛蕊花糖苷、羟基酪醇、咖啡酸血药浓度,计算主要药动学参数。结果常氧、缺氧大鼠血浆中均未检测到羟基酪醇。与常氧大鼠血浆比较,缺氧大鼠血浆中毛蕊花糖苷AUC_(0~)_(t)、AUC_(0~∞)、C_(max)降低(P<0.05),T_(max)、V_(z)/F升高(P<0.05);咖啡酸AUC_(0~)_(t)、AUC_(0~∞)、C_(max)降低(P<0.05),MRT_(0~∞)、T_(max)、V_(z)/F升高(P<0.05)。结论在缺氧条件下,毛蕊花糖苷及其活性代谢产物咖啡酸的药动学参数发生明显变化。

Anti-asthmatic mechanism of the Huashanshen dripping pill via suppressing contraction of the airway smooth muscle

Background:The Huashanshen(HSS)dripping pill has been widely used in asthma for a long time in China.However,the relaxant mechanism of HSS is not well understood.Methods:In this report,high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was used to identify the constituents in rat plasma after oral administration of HSS.Ovalbumin-sensitized allergic asthma and isolated trachea were studied for the anti-asthmatic mechanism of HSS.Results:D-anisodamine,L-anisodamine,scopolamine and atropine were detected in the rat plasma containing HSS.It was clear that the HSS inhibited the release of inflammatory mediators,regulated the balance of T-helper 1 and T-helper 2 to reduce the airway inflammation,and relaxed the tracheal smooth muscle by controlling the KCa channel,Ca^(2+)influx and release to reduce the airway hyperresponsiveness.Conclusion:Atropine,anisodamine and scopolamine might be active compounds of HSS which inhibited the release of inflammatory mediators,regulated the balance of Th1/Th2,and relaxed the tracheal smooth muscle to reduce airway hyperresponsiveness.

Separation, Identification, Isolation and Characterization of Degradation Product of Osimertinib Tablets by UPLC-QTOF-MS/MS and NMR: Evaluation of <i>In-Silico</i>Safety Assessment for Osimertinib (OSM) and Degradation Product (DP)

The present work encompasses identification and characterization of major degradation product (DP) of OSM observed in base hydrolytic stress study. The separation of DP was carried out on a non-polar stationary phase by using high-performance liquid chromatography system (HPLC). Using waters X-bridge (250 mm × 4.6 mm, 5 μm) C18 column with gradient elution program. For the characterization study, stress samples were subjected to HPLC and UPLC-QTOF-MS/MS and based on mass fragmentation pattern, plausible structure was deduced. Further, the DP was isolated using semi-prepara- tive liquid chromatography and concentrated the fractions using lyophilization. The isolated DP was subjected to extensive 1D (1H, 13C, and DEPT-135) and 2D (COSY, HSQC and HMBC) nuclear magnetic resonance (NMR) studies to authenticate the structure. The impurity was unambiguously named as N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-metho-xy-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)phenyl)-3-methoxypropanamide. Add- itionally, the In-Silico structure activity relation (QSAR) assessed through statistical based software’s DEREK NexusTM, and MultiCASE, Case UltraTM widely accepted and respected software’s for DP and OSM.

Integrating qualitative and quantitative characterization of Prunellae Spica by HPLC-QTOF/MS and HPLC-ELSD

The present study was designed to analyze the major constituents in Prunellae Spica and establish a method for simultaneous determination of two constituents contained in Prunellae Spica. High performance liquid chromatography coupled with time-of-flight mass spectrometry(HPLC-QTOF-MS/MS) technique was used to identify the constituents in the extractive of Prunellae Spica. High performance liquid chromatography coupled with evaporative light scattering detection(HPLC-ELSD) was used to simultaneously quantify two kinds of constituents contained in Prunellae Spica. Principal component analysis(PCA) was applied to compare the similarity and difference among samples from different regions of China. In the present study, 22 compounds were identified and some new fragmental pathways of triterpenic acids were discovered. An accurate and reliable HPLC-ELSD method was developed and validated for the first time to simultaneously quantify multiple constituents, including rosmarinic acid, maslinic acid, corosolic acid, betulin, oleanolic acid, and ursolic acid in the extract of Prunellae Spica.(PCA) revealed some similarities and differences among different samples from different regions of China. In conclusion, our results from this study would be helpful in establishing a scientific and rational quality control method for Prunellae Spica.

高效液相色谱-四极杆飞行时间质谱法推定奥替拉西钾中主要杂质

目的:利用高效液相色谱-四极杆飞行时间质谱法(HPLC-QTOF/MS)对奥替拉西钾中主要杂质进行结构推定,并且分析其来源。方法:采用Waters Sunfire C_(18)(250mm×4.6 mm,5μm)色谱柱,柱温30℃,以甲醇-10 mmol·L^(-1)乙酸铵溶液(用乙酸调节pH至4.3)(5∶95)为流动相,流速0.8 mL·min^(-1),检测波长220 nm;采用ESI离子源,正离子扫描模式。结果:通过对奥替拉西钾及其主要杂质的一级与二级高分辨质谱图进行分析,推定主要杂质的分子结构为1,3,5-三嗪-2,4-(1氢,3氢)-二酮和2-(3-氨基甲酰脲基)-2-羰基乙酸。2个主要杂质均由奥替拉西钾降解得到。结论:所建立的方法能够有效地将奥替拉西钾中的主要杂质分离检测,杂质结构的确定为药品的质量控制提供了依据。

GTS40, an active fraction of Gou Teng-San (GTS), protects PC12 from H2O2-induced cell injury through antioxidative properties

Oxidative stress, a predominant cause of apoptosis cascades triggered in neurodegenerative disorders, has been regarded as a critical inducement in the pathogenesis of Alzheimer's disease(AD). Gou Teng-San(GTS) is a traditional Chinese herbs preparation commonly utilized to alleviate cognitive dysfunction and psychological symptoms of patients with dementia. The present study aimed to investigate the protective effects of GTS40, an active fraction of GTS, on H_2O_2-induced oxidative damage and identify the potential active ingredients. Our results revealed that GTS40 exhibited radical scavenging activity, elevated cell viability, decreased the levels of intracellular reactive oxygen species(ROS), and stabilized mitochondrial transmembrane potential(MMP) in H_2O_2-treated PC12 cells. In addition, GTS40 blocked the apoptotic cascade by reversing the imbalance of Bcl-2/Bax and inhibiting the activity of caspase-3. Furthermore, an HPLC-QTOFMS method was developed to characterize major chemical constituents in GTS40. Our results revealed twenty-seven identified or tentatively characterized compounds through comparing their retention time(t_R) and MS spectra with reference standards. These results suggested that GTS40 was a promising active fraction that may be beneficial in the prevention and treatment of oxidative stress-mediated neurodegenerative disorders.

Phytochemicals of six selected herbal plants and their inhibitory activities towards free radicals and glycation

In type 2 diabetes mellitus patients,hyperglycaemia speed up the production of advanced glycation end products(AGEs),leading to oxidative stress and various diabetic complications.In order to identify natural products with potential antioxidant and antiglycative properties,six herbal plants,namely Musa acuminata(flower buds),Bletilla striata(rhizomes),Amomum villosum(dried nuts),Moghania philippinensis(roots),Millettia speciosa(rhizomes),and Spatholobus suberectus(stems)were assessed for their antioxidant and antiglycative properties.Among all the selected samples,both water and ethanolic extracts of S.suberectus possessed the highest phenolic,flavonoid and triterpenoid contents,ranging from three to six-fold higher than other plant extracts.In consistent with its phytochemical content,S.suberectus also showed the highest antioxidant activities which are comparable,if not higher than the known antioxidants,butylated hydroxytoluene,gallic acid,rutin,quercetin and ursolic acid.Similarly,S.suberectus also displayed the highest antiglycative activities among the six herbal plants measured by two glycation assays using bovine serum albumin-glucose and bovine serum albumin-methylglyoxal as model systems.HPLC-QToF-MS/MS analysis identified 48 polyphenols and 76 terpenoids,from the water and ethanolic extracts of S.suberectus,where 12 polyphenols and 9 terpenoids are common in both extracts.The results also suggested that S.suberectus contains compounds such as quercetin,luteolin,apigenin and saponin derivatives,which were reported to exert high antioxidant and antiglycative properties.The high antioxidant and antiglycative properties suggest the great potential of S.suberectus in preventing the formation of AGEs and oxidative stress seen in type 2 diabetes.

HPLC测定野烟叶中的1,5-二咖啡酰奎宁酸

目的采用串联四极杆飞行时间质谱鉴别苗药野烟叶的指标成分,并用HPLC法测定野烟叶中指标成分1,5-二咖啡酰奎宁酸(CQA)的含量。方法采用Inertsil ODS-SP色谱柱(150 mm×4.6 mm,5μm),流动相为乙腈-0.4%甲酸(14:86),检测波长325 nm,流速0.7 mL·min^(-1),柱温30℃。结果CQA的线性范围为0.061~3.882μg,加样回收率为93.14%,RSD=1.74%。结论所用方法简便、准确、可靠、可用于野烟叶的质量控制。