一测多评法同步测定人参和三七药材中多种人参皂苷的含量

通过建立人参皂苷Rb₁与其他8种皂苷间的紫外相对校正因子(RCF)，实现只用一个对照品测定人参和三七药材中多个人参皂苷类成分的含量，以解决人参等药材质量控制中，对照品供应不足问题。结果表明，在一定的线性范围内，人参皂苷Rb₁与Rg₁、 Re、 Rf、 Rh₁、 Rc、 Rb₂、 Rb₃、 Rd间的RCF值分别为1.400， 1.215， 1.517， 1.801， 0.944， 1.012， 1.143， 1.135，且在不同实验条件下重现性良好(RSD=0.30%～3.9%)。本方法只需测定人参和三七药材中Rb₁的含量，其余人参皂苷含量由其RCF值计算得到，实现一测多评；并与常规外标法比较，两种药材中一测多评法与外标法所得结果均无显著性差异；所建立的校正因子可同时用于人参及三七药材及其相关产品的定量分析及质量评价。     

人参皂苷水溶液热稳定性研究

目的 研究加热时间对人参皂苷水溶液的影响及受热后人参皂苷含量的变化情况。方法 红参须浓缩液加热不同时间,采用高效液相色谱法测定其人参皂苷Rg1、Re、Rb1、Rc和Rd的含量。结果 5种人参皂苷在加热6 h内,发生不同程度的降解反应,二醇类人参皂苷Rb1、Rc和Rd在加热2-3 h时,含量呈明显下降趋势,人参皂苷Rd降解速率最慢。三醇类人参皂苷Rg1和Re在加热3 h内含量快速下降,3 h后趋于平缓。结论 在常压受热条件下,人参皂苷水溶液主要成分人参皂苷Rg1、Re、Rb1、Rc和Rd的含量,随加热时间延长而不断下降,3 h后下降速率减缓。三醇类人参皂苷较二醇类对热更为敏感。     

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.     

RETRACTED ARTICLE: The retention behavior of ginsenosides in HPLC and its application to quality assessment of Radix Ginseng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 octadecylsiloxanebonded 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. This article was retracted as it was printed by error in this issue of APR.     

Biomass and content of ginsenosides and polyacetylenes in American ginseng roots can be increased without affecting the profile of bioactive compounds

Fifty selected roots from a 7-year-old American ginseng (Panax quinquefolium L.) plant population grown in Denmark, with root weights varying from 191 to 490 g fresh weight (FW), were investigated for bioactive ginsenosides and polyacetylenes (PAs) in order to determine the correlation between the content of ginsenosides and PAs and root FW. PAs (falcarinol, panaxydol) and ginsenosides (Rb₁, Rb₂, Rb₃, Rc, Rd, Re, Rg₁) were extracted from roots by sequential extraction with ethyl acetate and 80% methanol, respectively, and quantified in extracts by reverse-phase high-performance liquid chromatography (HPLC) using photodiode array detection. Total concentrations of PAs and ginsenosides varied between 150 and 780 mg/kg FW and 5,920 and 15,660 mg/kg FW, respectively. No correlation existed between the content of ginsenosides and PAs and root FW or between the total concentration of ginsenosides and PAs. Strong significant correlation was found between total content of ginsenosides and ginsenoside Rb₁ (r = 0.8190, P < 0.0001) and between total content of PAs and falcarinol (r = 0.9904, P < 0.0001). Based on the results of this study, it was concluded that it is possible to select large American ginseng roots for increased biomass production and concentration of bioactive ginsenosides and PAs without affecting the profile of bioactive compounds. Ginsenoside Rb₁ and falcarinol were found to be important selection parameters for identifying superior genotypes with the highest content of bioactive compounds.     

UPLC法评价硫磺熏蒸对西洋参皂苷类成分的影响

目的：评价硫磺熏蒸对西洋参皂苷类成分的影响。方法：建立超高效液相色谱法,测定并比较分析硫磺熏蒸前后西洋参中人参皂苷Rg₁、Re和Rb₁的含量。结果：硫磺熏蒸后西洋参中人参皂苷Rg₁、Re和Rb₁总量仍符合《中国药典》规定;但当硫磺熏蒸致二氧化硫残留量大于400 mg·kg^-1时,人参皂苷Re、Rb₁的含量及人参皂苷Rg₁、Re和Rb₁三者的总量显著降低。当二氧化硫残留量不大于150 mg·kg^-1时,人参皂苷Rg₁、Re和Rb₁的含量及其总量基本不受影响。结论：《中国药典》规定的西洋参二氧化硫残留量不得大于150 mg·kg^-1有其科学合理性,硫磺过度熏蒸西洋参（二氧化硫残留量大于400mg·kg^-1）对西洋参中皂苷类成分的含量有显著影响。     

Drug-biomacromolecule interaction V

The binding properties of three ginsenosides, Rb₁, Rc and Re, to bovine and human serum albumins have been examined by fluorescence probe technique. 1-anilinonaphthalene-8-sulfonate (ANS) was used as the fluorescence probe. Protopanaxatriol glycoside, Re, did not quench the fluorescence of ANS to the bovine serum albumin. Competitive bindings between protopanaxadiol glycosides, Rb₁ and Rc, and ANS were observed. The numbers of binding sites of bovine serum albumin for Rb₁ and Rc are both 3.3. The binding constants for Rb₁ and Rc with bovine serum albumin were 1.91×10⁴M^?1 and 1.04×10⁴M^?1, respectively. The ginsenosides, Rb₁, Rc and Re did not quench the fluorescence of ANS bound to human serum albumin.     

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。     

基于近红外光谱分析技术的注射用益气复脉(冻干)红参醇提过程在线监测

目的 利用近红外光谱分析技术建立注射用益气复脉（冻干）主要原料红参醇提过程中3种单体皂苷——人参皂苷Rg₁、Re和Rb₁的定量模型,实现提取过程中关键指标的快速检测。方法 在线采集红参醇提过程的近红外光谱,以超高效液相色谱（UPLC）法测定提取过程药液中人参皂苷Rg₁、Re和Rb₁的量为参考值,采用偏最小二乘法建立光谱与测定值之间的定量校正模型,进而对提取过程进行在线分析。结果 人参皂苷Rg₁和Re的建模波段均为9 403.7～7 498.3 cm^-1和6 102～5 446.3 cm^-1组合波段;人参皂苷Rb₁的建模波段为5 774.1～5 446.3 cm^-1。人参皂苷Rg₁、Re、Rb₁定量模型的交叉验证决定系数（R²）分别为99.40、99.44、99.41,交叉验证均方根误差分别为5.18、2.77、11.00。结论 所建立的3种单体皂苷定量模型预测性能良好,能够有效测定红参醇提过程中人参皂苷Rg₁、Re和Rb₁的量。     

Simultaneous quantification of 19 ginsenosides in black ginseng developed from Panax ginseng by HPLC–ELSD

A high-performance liquid chromatographic method with evaporative light scattering detection (HPLC–ELSD) has been developed to identify and quantify 19 ginsenosides (Rg₁, Re, Rf, Rb₁, Rc, Rb₂, Rd, F₄, Rg₆, Rk₃, Rh₄, 20(S)-, 20(R)-Rg₃, 20(S)-, 20(R)-Rs₃, Rk₁, Rg₅, Rs₄, and Rs₅) in black ginseng (BG, Korean white ginseng that was subjected to nine cycles of steam treatment). Ultrasonication is employed for sample preparation, and the analysis is achieved on a Discovery C₁₈ column using gradient elution of CH₃CN–H₂O–CH₃COOH without buffer in 40 min. The method was validated by linearity (r² ≥ 0.9994), precision (92.0–107.5%), intra- and inter-day accuracy (R.S.D. < 3.21%), and limit of detection (LOD ≤ 93 ng). The quantification method was applied to analyze the composition of ginsenosides in Korean white, red, and black ginsengs. During the preparatory process of BG, ginsenosides transform into constituents of low polarity by hydrolysis, isomerization, and dehydration at C-20, and hydrolysis also occurs at C-3 or C-6. The validated HPLC method is expected to provide the basis for the quality assessment of ginseng products.     

Drying of steamed Asian ginseng (Panax ginseng) roots by microwave-hot air combination

Steamed Asian ginseng (Panax ginseng) roots were dried by a combined microwave-hot air method in a modified experimental microwave oven. Hot air drying was used as a reference method. The drying time to achieve the desired moisture level (10%) as well as the ginsenoside contents and the color of the final product were determined. The ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, Rg1 and Ro were analyzed by HPLC. Compared with hot air drying, the combined microwave-hot air drying method resulted in a substantial decrease (approximately 30-40%) in drying time and had little influence on the ginsenoside contents and the color of the final product.     

Antinociceptive effects of ginsenosides injected intracerebroventricularly or intrathecally in substance P-induced pain model

We have examined the effects of several ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1 and Rg3) administered intracerebroventricularly (i.c.v.) or intrathecally (i.t.) on the nociceptive behavior induced by substance P (0.7 microg) injected i.t. Among the several ginsenosides studied, Rb2, Rc, Rd, and Re, but not Rb1, Rf, Rg1 and Rg3, treated i.c.v. (50 microg) attenuated the nociceptive behavior induced by substance P injected i.t. On the other hand, we found that i.t. treatment with 50 microg of Rb1, Rb2, Rd, or Rf effectively attenuated the nociceptive behavior induced by i.t. injected substance P. However, the i.t. treatment with the same doses of Rc, Re, Rg1 or Rg3 was not effective for antagonizing i.t. injected substance P-induced nociceptive behavior. Our results show that ginsenosides Rb2, Rc, Rd, or R2 injected supraspinally exert a antinociceptive effect in the substance P-induced pain model. Furthermore, Rb1, Rb2, Rd, or Rf treated spinally produce antinociception in the substance P-induced pain model.     

Ginsenosides that produce differential antinociception in mice.

Ginsenoside Rc, Rd, and Re induced antinociception in writhing and formalin tests among five representative ginsenosides: Rb1, Rc, Rd, Re, and Rg1. However, these ginsenosides had no effect in the tail-flick test. The antinociceptive effects induced by three ginsenosides were dose dependent. ED50 was 20.5 (7.3-57.4 mg/kg) for Rc, 17 (11.0-27.6 mg/kg) for Rd, and 3.5 (1-12 mg/ kg) for Re in the writhing test and 62 (42-90 mg/kg) for Rc, 45 (20.5-99.0 mg/kg) for Rd, and 82 (48-139 mg/kg) for Re in the second phase of the formalin test. The antinociceptive effects were not blocked by the opioid receptor antagonist naloxone in the writhing and formalin tests. These three ginsenosides did not affect motor function. Ginsenoside Rc and Rd induced hypothermia for 30 to 60 min, and ginsenoside Rc induced hyperthemia after 150 min of treatment at doses of 100 mg/kg. These results suggest that ginsenosides such as Rc, Rd, or Re inhibit mainly chemogenic pain rather than thermal pain by the nonopioid system in mice.     

Molecular mechanisms governing different pharmacokinetics of ginsenosides and potential for ginsenoside-perpetrated herb–drug interactions on OATP1B3

Background and Purpose

Ginsenosides are bioactive saponins derived from Panax notoginseng roots (Sanqi) and ginseng. Here, the molecular mechanisms governing differential pharmacokinetics of 20(S)-protopanaxatriol-type ginsenoside Rg₁, ginsenoside Re and notoginsenoside R₁ and 20(S)-protopanaxadiol-type ginsenosides Rb₁, Rc and Rd were elucidated.

Experimental Approach

Interactions of ginsenosides with human and rat hepatobiliary transporters were characterized at the cellular and vesicular levels. A rifampin-based inhibition study in rats evaluated the in vivo role of organic anion-transporting polypeptide (Oatp)1b2. Plasma protein binding was assessed by equilibrium dialysis. Drug–drug interaction indices were calculated to estimate potential for clinically relevant ginsenoside-mediated interactions due to inhibition of human OATP1Bs.

Key Results

All the ginsenosides were bound to human OATP1B3 and rat Oatp1b2 but only the 20(S)-protopanaxatriol-type ginsenosides were transported. Human multidrug resistance-associated protein (MRP)2/breast cancer resistance protein (BCRP)/bile salt export pump (BSEP)/multidrug resistance protein-1 and rat Mrp2/Bcrp/Bsep also mediated the transport of the 20(S)-protopanaxatriol-type ginsenosides. Glomerular-filtration-based renal excretion of the 20(S)-protopanaxatriol-type ginsenosides was greater than that of the 20(S)-protopanaxadiol-type counterparts due to differences in plasma protein binding. Rifampin-impaired hepatobiliary excretion of the 20(S)-protopanaxatriol-type ginsenosides was effectively compensated by the renal excretion in rats. The 20(S)-protopanaxadiol-type ginsenosides were potent inhibitors of OATP1B3.

Conclusion and Implications

Differences in hepatobiliary and in renal excretory clearances caused markedly different systemic exposure and different elimination kinetics between the two types of ginsenosides. Caution should be exercised with the long-circulating 20(S)-protopanaxadiol-type ginsenosides as they could induce hepatobiliary herb–drug interactions, particularly when patients receive long-term therapies with high-dose i.v. Sanqi or ginseng extracts.     

注射用血栓通(冻干)透过血脑屏障研究

目的 研究大鼠脑缺血再灌注后,血栓通主要成分人参皂苷Rb₁、Rg₁、Re、Rd及三七皂苷R₁在脑内的分布状况。方法 采用超高效液相色谱质谱联用法(UPLC-MS/MS)测定给予不同浓度的血栓通后,不同时间点血栓通主要成分在正常脑组织和缺血脑组织中的含量。结果 结果表明,血栓通活性成分在脑缺血大鼠脑中的分布远高于正常大鼠,并且其在脑内的含量随再灌注时间的延长降低。结论 脑缺血再灌注后,血脑屏障开放,注射用血栓通(冻干)可以透过血脑屏障发挥作用。     

Effects of ginsenosides on GABAA receptor channels expressed in xenopus oocytes

Ginsenosides, major active ingredients of Panax ginseng, are known to regulate excitatory ligand-gated ion channel activity such as nicotinic acetylcholine and NMDA receptor channel activity. However, it is not known whether ginsenosides affect inhibitory ligand-gated ion channel activity. We investigated the effect of ginsenosides on human recombinant GABA(A) receptor (alpha1beta1gamma2S) channel activity expressed in Xenopus oocytes using a two-electrode voltage-clamp technique. Among the eight individual ginsenosides examined, namely, Rb1, Rb2, Rc, Rd, Re, Rf, Rg1 and Rg2, we found that Rc most potently enhanced the GABA-induced inward peak current (I(GABA)). Ginsenoside Rc alone induced an inward membrane current in certain batches of oocytes expressing the GABA(A) receptor. The effect of ginsenoside Rc on I(GABA) was both dose-dependent and reversible. The half-stimulatory concentration (EC50) of ginsenoside Rc was 53.2 +/- 12.3 microM. Both bicuculline, a GABA(A) receptor antagonist, and picrotoxin, a GABA(A) channel blocker, blocked the stimulatory effect of ginsenoside Rc on I(GABA). Niflumic acid (NFA) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), both Cl- channel blockers, attenuated the effect of ginsenoside Rc on I(GABA). This study suggests that ginsenosides regulated GABA(A) receptor expressed in Xenopus oocytes and implies that this regulation might be one of the pharmacological actions of Panax ginseng.     

Comparative studies of saponins in 1–3-year-old main roots, fibrous roots, and rhizomes of Panax notoginseng, and identification of different parts and growth-year samples

Notoginsenosides R₁, R₄, Fa, and K (N-R₁, N-R₄, N-Fa, and N-K), as well as ginsenosides Rg₁, Rb₁, Rd, Re, Rf, Rg₂ and Rh₁ (G-Rg₁, G-Rb₁, G-Rd, G-Re, G-Rf, G-Rg₂ and G-Rh₁) in 47 Notoginseng samples including 1-, 2- and 3-year-old main roots, rhizomes and fibrous roots of Panax notoginseng were determined by high-performance liquid chromatography-diode array detection method. Total contents (%) of the 11 saponins were 9.82–14.57 for 2-year old and 14.20–16.00 for 3-year-old rhizomes; 2.72–4.50 for 2-year-old and 1.98–4.92 for 3-year-old fibrous roots; 1.75–3.05 for 1-year-old whole roots; and 3.71–8.98 for 2-year-old and 7.03–11.23 for 3-year-old main roots. Contents of most saponins and total content of 11 saponins were in the order 3- >2- >1-year-old main root samples. G-Rf content, sum of G-Rf and G-Rh₁ were, respectively, 0.08–0.18 and 0.14–0.32 for 2- or 3-year-old rhizomes, and 0.01–0.07 and 0.03–0.10 for 2- or 3-year-old main roots. Combined contents of N-R₁, G-Rg₁ and G-Rb₁ were 5.78–9.37 in 3-year-old main roots, and 2.99–7.13 in 2-year-old main roots, of which nearly one-third of samples were lower than the limit (5 %) in the Chinese Pharmacopoeia. Those of 2- or 3-year-old fibrous roots (1.47–3.83) and 1-year-old whole roots (1.41–2.44) were much lower than the limit, and were considered not suitable for use as Notoginseng. Two-year-old main roots are not appropriate for collection as Notoginseng. Different parts and growth years of P. notoginseng can be identified from each another according to differences in saponin content.     

The transformation of ginsenosides by acid catalysis in gastric pH

1. The ginsenosides of Korean ginseng decomposed profoundly to produce artifact products of prosapogenin A₁, A₂ and A₃ from ginsenoside Rg₁, prosapogenin C₁, C₂ and C₃ from ginsenoside Re, and prosapogenin E₁, E₂ and E₃ from ginsenoside Rb₁ by the acid treatment under physiological condition such as 37°C incubation in 0.1 N HCl. 2. The chemical structures of the artifact substances were determined by the analysis of CMR and mass spectra of TMS-derivatives as following;     

Three new dammarane-type triterpene saponins from the leaves of Panax ginseng C.A. Meyer

Three new dammarane-type triterpene ginsenosides, together with six known ginsenosides, were isolated from the leaves of Panax ginseng C.A. Meyer. The new saponins were named as ginsenoside Rh₁₁, ginsenoside Rh₁₂, and ginsenoside Rh₁₃. Their structures were elucidated as (20S)-3β,6α,12β,20-tetrahydroxydammara-25-ene-24-one 20-O-β-d-glucopyranoside (1), (20S)-3β,12β,20,24,25-pentahydroxydammarane 20-O-β-d-glucopyranoside (2), and (20S,23E)-3β,12β,20,25-tetrahydroxydammara-23-ene 20-O-β-d-glucopyranoside (3) on the basis of 1D and 2D NMR experiments and mass spectra. The known ginsenosides were identified as ginsenoside M_7cd, ginsenoside Rg₆, ginsenoside Rb₃, gypenoside XVII, gypenoside IX, and 20-(E)-ginsenoside F₄.     

Drug Metabolism: Intestinal Bacterial Hydrolysis is Required for the Appearance of Compound K in Rat Plasma after Oral Administration of Ginsenoside Rb1 from Panax ginseng

Ginsenoside Rb₁ from Panax ginseng root is transformed into compound K via ginsenosides Rd and F₂ by intestinal bacterial flora. Among 31 defined intestinal strains from man, only Eubacterium sp. A-44 transformed ginsenoside Rb₁ into compound K via ginsenoside Rd. The ginsenoside Rb₁-hydrolysing enzyme isolated from Eubacterium sp. A-44 was identical to a previously purified geniposide-hydrolysing β-D-glucosidase. When ginsenoside Rb₁ (200 mg kg^?1) was administered orally to germ-free rats, neither compound K nor any other metabolite was detected in the plasma, intestinal tract or cumulative faeces 7 or 15 h after administration. Most of the ginsenoside Rb₁ administered was recovered from the intestinal tract, especially the caeca, and cumulative faeces indicating poor absorption of ginsenoside Rb₁. When ginsenoside Rb₁ was administered orally to gnotobiote rats mono-associated with Eubacterium sp. A-44, a significant amount of compound K was detected in the plasma and considerable amounts were found in the caecal contents and cumulative faeces 7 and 15 h after administration. A small amount of ginsenoside Rb₁ was detected in the caecal contents only 7 h after administration. These results indicate that orally administered ginsenoside Rb₁ is poorly absorbed from the gut but that its metabolite compound K, produced by ginsenoside Rb₁-hydrolysing bacteria such as Eubacterium sp. A-44 in the lower part of intestine, is absorbed.