The retention behavior of ginsenosides in HPLC and its application to quality assessment of radix ginseng

This study systematically investigated the retention behavior of seven neutral ginsenosides Rg₁, Re, Rf, Rb₁, Rb₂, Rc, Rd, and an acidic ginsenoside R₀, the major pharmacologically active components of Radix Ginseng with RP-HPLC. The effects of solvent, pH value, ionic strength of the mobile phase, and column temperature were investigated using an octadecylsiloxane-bonded silica gel column. Based on the ginsenosides’ retention characteristics, the concentration of acetonitrile and the gradient of the mobile phase needed to maintain the baseline separation of the major neutral ginsenosides in Radix Ginseng were theoretically predicted. Furthermore, the ionic strength of mobile-phase necessary to achieve good resolution of the neutral ginsenosides and acidic ginsenosides was carefully investigated. According to the results of the quantitative analysis of ginsenosides in eight batches of ginseng samples from different sources, the developed HPLC technique may be a valuable tool for the quality assessment of Radix Ginseng.     

HPLC法测定石柱参药材及其片剂中5种人参皂苷的含量

目的建立测定石柱参药材及其片剂中5种人参皂苷含量的方法,为石柱参的质量控制提供科学依据。方法色谱柱为Kromasil C18柱;流动相为乙腈-水,梯度洗脱;柱温为25℃;流速为1.0 mL.min-1;检测波长为203 nm。结果5种人参皂苷的色谱峰与相邻色谱峰分离良好。人参皂苷Rg1质量浓度在19.8~198 mg.L-1内、人参皂苷Re质量浓度在20.6~206 mg.L-1内、人参皂苷Rb1质量浓度在33.0~330 mg.L-1内、人参皂苷Rc质量浓度在18.0~180 mg.L-1内、人参皂苷Rb2质量浓度在13.0~130 mg.L-1内与峰面积呈良好的线性关系。石柱参药材中人参皂苷Rg1、Re、Rb1、Rc、Rb2的平均回收率分别为99.8%、98.3%、99.2%、95.4%、96.8%,RSD分别为3.0%、3.0%、2.6%、2.2%、2.8%(n=6);石柱参片剂中人参皂苷Rg1、Re、Rb1、Rc、Rb2的平均回收率分别为100.2%、99.0%、99.7%、96.4%、98.4%,RSD分别为2.2%、3.1%、3.6%、2.6%、2.8%(n=6)。结论本实验中所建立的方法可用于石柱参药材及其片剂的质量控制。     

红参高效液相色谱指纹图谱研究

目的 建立红参药材高效液相色谱(HPLC)指纹图谱,为其质量评价提供依据。方法 Sharpsil-T C₁₈色谱柱(4.6 mm×250 mm,5 μm);以乙腈-水为流动相梯度洗脱;柱温为30℃;流速为1.0 ml/min;检测波长为203 nm。结果 以人参皂苷Rb1为参照峰,建立10批红参的对照指纹图谱,识别出24个共有峰,其相似度均大于0.940。通过质谱推断、对照品比对确证了其中11个共有峰。结论 该方法操作简便、准确可靠、重复性较好,为红参药材的质量控制提供了有效手段。     

HPLC测定地黄及其制剂中梓醇的含量

目的　采用HPLC测定地黄及其制品中梓醇的含量。方法　以InertsilODS - 2为固定相 ,水 -乙腈 (99.5∶0 .5 )为流动相 ,检测波长 2 10nm。结果　梓醇与其他峰均能达到基线分离 ,其色谱峰形好。梓醇的进样量在 0 .4 84～ 4 .84 μg时与峰面积呈良好的线性关系 (Y =7.4 91× 10 4X - 3.5 35× 10 2 ,r=0 .9999) ,平均回收率 99.0 % ,方法精密度RSD =1.3% (n =6 )。结论　所用方法简便 ,准确 ,重复性好 ,可作为质控方法     

高通量测序分析附子、红参不同比例配伍对大鼠肠道菌群的影响

目的 初步研究附子、红参不同比例配伍对大鼠肠道菌群的影响。方法 红参、附子分别采用8倍体积的70%乙醇加热回流提取2次,每次1 h,制备醇提物。将15只SD大鼠随机分成A、B、C 3组,每组5只,A组为附子醇提物单独给药组,给药量为0.415 g/kg,B、C组分别为附子、红参配伍比例1∶1（红参醇提物1.17 g/kg）和1∶2（红参醇提物2.34 g/kg）给药组,每天ig给药1次,连续给药7 d。给药结束后,于第8天收集大鼠粪便,提取粪便中细菌的DNA,对粪便菌群的16Sr-RNA的V4区进行扩增,利用Miseq高通量测序平台进行基因序列的测定,利用Uparse software对序列进行分析,将相似性在97%以上的序列进行归并,生成分类操作单元（OTU）;利用QⅡME软件计算样品的菌群丰度指数（Ace、Chao1）、菌群多样性指数（Simpson、Shannon）。结果 A组和B组的OTU数量较C组显著增多（P<0.05）;A组和B组Ace、Chao1指数为1 393、1 368和1 085、1 057,C组Ace和Chao1指数为889和884,A组和B组较C组显著增多（P<0.05、0.01）;厚壁菌门、拟杆菌们和变形菌门均为3组样品中的优势菌门;在属的水平上,C组中乳酸杆菌属和拟杆菌属含量较A组和B组增加。结论 红参与附子不同比例配伍可对大鼠肠道菌群产生显著的影响,随红参比例的增加,可能在一定程度上改善肠道的微生态环境。     

Simultaneous determination of ginsenosides Rg1, Re and Rb1 in rat plasma by HPLC and its application to pharmacokinetic study of SHENMAI injection

In the present study, we developed a sensitive and efficient high performance liquid chromatography (HPLC) method for the simultaneous determination of three ginsenosides (Rg₁, Re, Rb₁) in rat plasma. Chromatographic separation was performed on a C₁₈ (150 mm×4.6 mm) column utilizing gradient elution profile and a mobile phase consisting of (A) water and (B) acetonitrile. The calibration curve, with a great correlation coefficient greater than 0.998, was linear over the range of 1.0–30.0 μg/mL for ginsenoside Rg₁, 0.5–15.0 μg/mL for ginsenoside Re, and 0.5–200.0 μg/mL for ginsenoside Rb₁. The intra- and inter-day precisions for three ginsenosides (Rg₁, Re, Rb₁) were all less than 6.0%, and average recovery, examined at three concentration levels, ranged from 96.1% to 118.6%. The samples was stable within 24 h at 4 ºC storage, and 30 d at –20 ºC storage with three freeze-thaw-assay cycles. The low limits of quantification (LOQ) were 1.0, 0.5 and 0.5 μg/mL for Rg₁,Re and Rb₁, respectively. Taken together, the newly developed method was successfully applied to study the pharmacokinetics of ginsenoside Rg₁, Re and Rb₁ in rat plasma after intravenous administration of SHENMAI injection (SMI).     

Quality assessment of Rhizoma et Radix Notopterygii by HPTLC and HPLC fingerprinting and HPLC quantitative analysis

This paper describes an improved quality assessment method for Rhizoma et Radix Notopterygii (the rhizome and root of Notopterygium incisum Ting ex H.T. Chang or Notopterygium forbesii Boiss). The method was established by using fingerprinting and quantitation of marker compounds (isoimperatorin, notopterol and bergapten) in this herbal medicine. The authentication of Rhizoma et Radix Notopterygii using high performance thin-layer chromatography (HPTLC) fingerprinting was achieved by comparing the colors and Rf values of the bands in TLC fingerprints with those of the marker compounds. The HPLC fingerprints of 16 batches of herbal samples from different regions of China showed similar chromatographic patterns. Five peaks were selected as characteristic peaks, and three of these were identified by using LC-MS-MS techniques. The relative retention times of these characteristic peaks in the HPLC fingerprint were established as an important parameter for identification of Rhizoma et Radix Notopterygii. Finally, the pharmacologically active marker compounds isoimperatorin, notopterol and bergapten in this herb were quantitatively determined using a validated reverse-phase HPLC method.     

HPLC法测定人参毛状根及不同人参样品中9种人参皂苷的含量

目的采用HPLC法测定不同人参样品中9种人参皂苷(Rc、Rb1、Rb2、Re、Rd、Rg1、Rg2、Rg3和Rh2)的含量。方法色谱条件:Zorbax SB C18柱(4.6mm×250mm,5μm),保护柱Extend-C18柱(4.6mm×12.5mm,5μm);以乙腈-水为流动相,梯度洗脱;流速:1.0ml/min;检测波长:203nm;柱温:35℃。结果 9种人参皂苷Rg1、Rb1、Re、Rc、Rg2、Rh2、Rg3、Rb2和Rd在120min内基线分离。方法学表明其线性关系良好,精密度、稳定性和重复性RSD均小于2.0%,加样回收率在98.3%~102%之间。测得人参叶和人参须根中的总皂苷含量最高,分别为48.9、23.6mg/g;人参毛状根中的总皂苷与人参主根和人参果的总皂苷含量差别不大,为7.47mg/g。结论该法准确性高,操作简便、快速,重复性好,精密度高,可用于不同人参样品中9种人参皂苷的含量测定。     

高效液相色谱法同时测定黄芩及其制剂中4种黄酮的含量

目的建立反相高效液相色谱法同时测定黄芩药材及其制剂中黄芩苷（1）、汉黄芩苷（2）、黄芩素（3）和汉黄芩素（4）的含量。方法采用Agilent Zorbax SB-C18色谱柱（4.6 mm×250 mm,5μm）;以乙腈-0.15%三氟乙酸水为流动相,梯度洗脱;流速：1.0 ml·min^-1;检测波长：280 nm;柱温：室温。结果已测定的4种黄酮在以下浓度范围内呈现良好的线性：（1）0.51～164 mg·L^-1（r=0.9999）,（2）0.33～104 mg·L^-1（r=0.9998）,（3）0.24～76 mg·L^-1（r=0.999 8）,（4）0.31～100mg·L^-1（r=0.9999）。平均回收率（n=3）分别为：（1）98.47%（RSD=1.35%）,（2）100.73%（RSD=0.57%）,（3）101%（RSD=2.63%）,（4）100.77%（RSD=0.27%）。结论该方法简便,准确,灵敏,结果可靠,可用于黄芩药材及其制剂的质量控制。     

Survey of Glycyrrhizae Radix resources in Mongolia: chemical assessment of the underground part of Glycyrrhiza uralensis and comparison with Chinese Glycyrrhizea Radix

In order to reveal the chemical characteristics of Glycyrrhiza uralensis growing in Mongolia and to clarify whether it can be the source of Glycyrrhizae Radix used in Japan, eight major bioactive constituents in the underground parts of G. uralensis collected in Mongolia were quantitatively analyzed and compared with Glycyrrhizae Radix produced in China. Most of the 15 samples from eastern, southern and western parts of Mongolia contained 26.95–58.55 mg/g of glycyrrhizin, exceeding the criterion (25 mg/g) assigned in the Japanese Pharmacopoeia. The sample collected in Tamsagiyn hooly, Dornod province, in eastern Mongolia was of the highest content 58.55 mg/g. The contents of three flavanone constituents (liquiritin apioside, liquiritin and liquiritigenin) and three chalcones (isoliquiritin apioside, isoliquiritin and isoliquiritigenin) varied significantly according to collection places; the subtotal of the three flavanones ranged from 3.00 to 26.35 mg/g, and the subtotal of the three chalcones ranged from 1.13 to 10.50 mg/g. The content of glycyrrhizin and subtotal contents of flavanones and chalcones in the underground parts of G. uralensis from Mongolia were obviously lower than wild samples, but higher than cultivated samples derived from the same species produced in China. Glycycoumarin, a species-specific constituent of G. uralensis, was detected in all Mongolian samples. Its contents in samples from eastern Mongolia, Sergelen and Tamsagiyn hooly of Dornod province were very high and were compatible with Tohoku-kanzo derived from wild Chinese G. uralensis. The present study suggested that Mongolian G. uralensis could be a source of Glycyrrhizae Radix, mostly of Japanese Pharmacopoeia grade. However, the producing area should be taken into consideration to ensure relatively high quality. In addition, planned use and promotion of cultivation must be advocated to avoid confronting Mongolian Glycyrrhiza with the same threat as its congener in China. Our study sheds some light on selecting cultivation areas and superior strains, which are important tasks to promote cultivation.     

Simultaneous quantification of 14 ginsenosides in Panax ginseng C.A. Meyer (Korean red ginseng) by HPLC-ELSD and its application to quality control

A new method of high-performance liquid chromatography coupled with evaporative light scattering detection (HPLC-ELSD) was developed for the simultaneous quantification of 14 major ginsenosides, which are the marker compounds of Panax ginseng C.A. Meyer (Korean red ginseng). Various types of ginseng samples were extracted, and the amounts of the 14 ginsenosides (Rg1, Re, Rf, Rh1, Rg2, Rb1, Rc, Rb2, Rb3, Rd, Rg3, Rk1, Rg5, and Rh2) were determined by reverse-phase HPLC-ELSD using digoxin as an internal standard. The mobile phase consisted of a programmed gradient of aqueous acetonitrile. Calibration curves for each ginsenoside were determined for the quantification. The method was validated for linearity, precision, accuracy, limit of detection, and limit of quantification. This quantification method was applied to several finished ginseng products including white ginseng, red ginseng powder, and red ginseng concentrate. The amounts of the 14 ginsenosides in the various ginseng samples could be analyzed simultaneously. This validated HPLC method is expected to provide a new basis for the quality assessment of ginseng products.     

高效液相色谱法测定苦参药材及其黄酮部位中降苦参酮、黄腐醇和苦参啶的含量

目的:建立测定苦参药材及其黄酮部位中3种黄酮类成分降苦参酮、黄腐醇和苦参啶含量的高效液相色谱方法。方法:采用 Phenomenex Luna C_(18)柱(250 mm×4.6 mm,5 μm),以乙睛-水为流动相,梯度洗脱(0～35 min,45:55→69∶31),流速1.0 mL·min~(-1),柱温35℃,检测波长 294 nm(降苦参酮)和365 nm(黄腐醇、苦参啶)。结果:降苦参酮、黄腐醇、苦参啶的线性范围分别为0.12～0.60μg(r=0.9999),0.004～0.200μg(r=0.9996),0.20～1.02μg(r=0.9997);苦参黄酮部位平均加样回收率(n=6)分别为102.5%,101.5%,99.8%;苦参药材平均加样回收率(n=6)分别为101.8%,103.5%,99.9%。结论:3批样品测定结果表明,该方法简便准确,可用于苦参药材及其黄酮部位中降苦参酮、黄腐醇和苦参啶3种黄酮类成分的含量测定。     

Development of an in-line HPLC fingerprint ion-trap mass spectrometric method for identification and quality control of Radix Scrophulariae

Chromatographic fingerprinting has been widely accepted as a crucial method for qualitative and quantitative analyses of bioactives within traditional Chinese medicine. A fingerprint provides detailed information, specific for any given herb, thus facilitating the quality control measures of a given traditional Chinese medicine. In this article, quality assessment of Radix Scrophulariae was achieved by using high performance liquid chromatography combining diode-array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI/MS). Eight batches of sample obtained from different origins in China were used to establish the fingerprint and quantitative analyses. By comparing the retention times, UV and MS spectral data with reference standards, four characteristic peaks in the chromatograms were confirmed as corresponding to acetoside, angoroside C, cinnamic acid, and harpagoside. In addition, other two characteristic peaks were tentatively identified, following the literature interpretation of HPLC-ESI-MS and LC-MS/MS (affording structural information) to be sibirioside A and scrophuloside B₄, respectively. The results indicated that the newly developed HPLC-DAD-MS fingerprint method would be suitable for quality control of Radix Scrophulariae.     

中药多组分提取物七叶皂苷钠的指纹图谱研究及其在质量控制中的应用

目的：建立中药多组分提取物七叶皂苷钠的指纹图谱检测方法,为七叶皂苷钠及其制剂的质量控制提供科学依据。方法：利用Kromasil 100-5C₁₈色谱柱（250 mm×4.6 mm,5 μm）,以乙腈-0.2%磷酸为流动相梯度洗脱,检测波长220 nm,共收集了11批七叶皂苷钠进行测定,运用"中药色谱指纹图谱相似度评价软件（2004 A）"进行指纹图谱相似度评价,运用SPSS 19.0对七叶皂苷钠进行主成分分析。结果：获得了较好七叶皂苷钠的HPLC指纹图谱,共有15个主要共有峰;11批样品指纹图谱的相似度均>0.995,4个主成分的累积方差贡献率为93.52%。结论：本研究建立了七叶皂苷钠的指纹图谱分析方法,为其质量控制提供依据。     

Hashish extract impairs retention of defeat-induced submissive behavior in mice

The effects of hashish extract on adaptive behavior of male mice were studied in a paradigm which allows the investigation of learning mechanisms in a social context. Mice of the C3H strain, which were not submissive in a confrontation with a nonaggressive DBA mouse on day 1, were defeated on day 2 over 3 min by aggressive, isolated DBA mice, and showed conditioned submissive behavior upon mere contact with a nonaggressive DBA mouse on day 3. A hashish extract containing 38.6–39.4% ⁹-tetrahydrocannabinol ( ⁹-THC), 11.6–12.0% cannabinol and 47.7–48.5% cannabidiol was administered orally in all experiments. Hashish extract given 90 min before defeat on day 2, in dosages corresponding to 1, 5, and 10 mg ⁹-THC/kg, impaired retention of defensive upright, defensive sideways and immobility on day 3 (experiment 1). Experiment 2 showed that the drug (5, and 10 mg ⁹-THC/kg) had no antinociceptive potency in mice and did not modify defeat-induced analgesia. Experiment 3, with drug (5 mg ⁹-THC/kg) or solvent administration on day 2 and day 3, showed that the retention deficit was neither due to state-dependent learning, nor to impaired retrieval. It is suggested that hashish extract administered before learning may interfere with memory processing.     

小儿化痰止咳颗粒特征图谱研究与质量评价

目的 建立小儿化痰止咳颗粒的HPLC特征图谱,考察其主要组成原料吐根酊的投料情况,评价其质量。方法 采用高效液相色谱法,色谱柱为Inertsil ODS-SP（250 mm×4.6 mm,5 μm）,乙腈-0.4%磷酸溶液为流动相,梯度洗脱,体积流量为1 mL/min,检测波长210 nm,柱温35℃。结果 建立了41个厂家181批样品的HPLC特征图谱,确定其中来自原料盐酸麻黄碱、吐根酊和桑白皮流浸膏的6个特征色谱峰;利用该特征图谱,发现部分厂家存在投入不合格吐根酊原料的情况。结论 该方法建立的小儿化痰止咳颗粒HPLC特征图谱专属性强,重复性好,可有效的控制该产品的质量。     

高效液相色谱法测定体外大鼠肠道菌液中大豆苷及其代谢物

目的 建立HPLC法测定体外大鼠肠道菌液中的大豆苷及其代谢物,研究大鼠肠道菌群对大豆苷的代谢情况。方法 采用大鼠离体粪便温孵法,将大豆苷与大鼠肠道菌群培养液进行厌氧温孵。温孵样品采用甲醇沉淀蛋白处理后进行HPLC法分析,色谱条件：色谱柱为Diamonsil C₁₈柱;流动相为0.2%乙酸水溶液与甲醇,梯度洗脱;检测波长为260 nm;柱温为25℃;进样量为10 μl。结果 大豆苷及其代谢物大豆苷元分别在98.0~490、57.6~288 μg/ml浓度范围内线性良好,高、中、低3个浓度的相对回收率在96.6%~114.0%之间,相对标准偏差小于9.9%,表明本法的准确度和精密度符合要求。大豆苷在肠道菌液中代谢生成大豆苷元,其消除半衰期为37.6 min。结论 所建立的HPLC法适合同时测定体外大鼠肠道菌液中的大豆苷及其代谢物,大豆苷在体外代谢的主要途径为糖苷键断裂转化成对应苷元。     

Authentication of Stephania tetrandra S. Moore (Fang Ji) and differentiation of its common adulterants using microscopy and HPLC analysis

Stephania tetrandra S. Moore (Hang Fang Ji) is used in traditional Chinese medicine as a diuretic, an antiphlogistic, and an antirheumatic. The name “fang ji” is applied to at least four different genera of plants, including Aristolochia fangchi Y. C. Wu ex L. D. Chow and S. M. Hwang, Cocculus orbiculatus (L.) DC., Stephania tetrandra S. Moore, and Sinomenium acutum Rehder and E. H. Wilson. Due to similarity in the use of their common names, Stephania tetrandra S. Moore is often confused with Aristolochia fangchi Y. C. Wu ex L. D. Chow and S. M. Hwang, which has potentially dangerous consequences. To aid rapid and easy differentiation between the roots of these four species, so as to avoid possible contamination, detailed macroscopic and microscopic observations were made using stereo-and light-microscopy. The powdered samples were further analyzed using HPLC.     

醋柴胡不同极性洗脱部位对黄芩苷在小鼠体内分布的影响以及成分分析

目的 考察醋柴胡不同极性洗脱部位对黄芩苷在小鼠体内分布的影响,并运用相关性分析初步探讨醋柴胡配伍黄芩苷在不同脏器中的增效活性成分.方法 通过多次给药,LC-MS/MS测定小鼠各组织中黄芩苷的含量,考察醋柴胡不同极性洗脱部位对黄芩苷在小鼠体内分布的影响;Q-Exactive Plus对醋柴胡不同极性部位的成分进行分析,S...     

HPLC同时测定穿心莲药材及其注射液中3种内酯类成分的含量

目的 利用HPLC同时测定穿心莲药材及其注射液中穿心莲内酯、脱水穿心莲内酯和新穿心莲内酯的含量。方法 色谱柱为Shimadzu Shim-pack VP-ODS柱(4.6 mm×150 mm,5 μm),以甲醇-水(53∶47)洗脱,流速：1 mL·min^-1,柱温：25 ℃,检测波长225 nm处检测穿心莲内酯,检测波长205 nm处检测脱水穿心莲内酯与新穿心莲内酯。结果 穿心莲药材及其注射液中3种内酯类成分能达到很好分离,线性关系良好(r≥0.999 5);平均加样回收率为99.6%~101.2%,RSD<3%。结论 本方法灵敏、准确、可靠、重复性好,可用于穿心莲药材及其注射液的质量评价与3种内酯类成分的快速测定。