Preparation of Rehmanniae Radix Juice for Baihe Dihuangtang Based on UPLC-Q-TOF-MS / 中国实验方剂学杂志

ObjectiveTo compare the four preparation methods of Rehmanniae Radix juice described in ancient literature and find the method that is most suitable for the preparation of Rehmanniae Radix juice used in Baihe Dihuangtang. MethodThe ancient medical books record four methods for preparing Rehmanniae Radix juice： crushing fresh Rehmanniae Radix for juice， steaming fresh Rehmanniae Radix for juice， boiling fresh Rehmanniae Radix for juice， and boiling dry Rehmanniae Radix for juice. Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry （UPLC-Q-TOF-MS） was employed to detect the compounds in the four juice samples， followed by principal component analysis （PCA）. Result① Totally 27 compounds were identified in the juice samples， including 10 iridoid glycosides， 14 phenylethanoid glycosides， 2 phenolic acids， and 1 irisone. Among them， 15 common compounds were shared by the four juice samples， including 7 iridoid glycosides， 7 phenylethanoid glycosides， and 1 phenolic acid. ② Five common compounds in the four juice samples can be matched with the reference standards， which were catalpol， aucubin， rehmannioside D， ajugol， and purpureaside C. ③ Verbascoside and isoacteoside were not detected in the juice prepared by crushing fresh Rehmanniae Radix， while it was detected in the other three juice samples， which indicated that the two components were produced after heating rather than being the original components in fresh Rehmanniae Radix. ④ The comparison of the ion fragments demonstrated that verbascoside was produced from purpureaside C after the cleavage of the glycosidic bond and removal of a molecule of mannose. ⑤ Isoacteoside could be isomerized from verbascoside， and its relative content increased with the extension of heating time. However， the relative content of verbascoside and purpureaside C did not decrease significantly. Therefore， it was hypothesized that purpureaside C was produced from its upstream component. ConclusionThe juice prepared by crushing fresh Rehmanniae Radix has the chemical composition significantly different from the juice samples prepared with the other 3 methods， while the latter 3 juice samples had similar chemical composition. Although all the four methods can be used， it is more suitable to prepare Rehmanniae Radix juice by steaming fresh Rehmanniae Radix， boiling fresh Rehmanniae Radix， and boiling dry Rehmanniae Radix.

Isoacteoside attenuates acute kidney injury induced by severe acute pancreatitis.

Severe acute pancreatitis (SAP) is a common acute abdominal disease accompanied by systemic inflammatory response syndrome, which may be complicated by acute kidney injury (AKI). Isoacteoside (ISO) is the active ingredient of Monochasma savatieri Franch. ex Maxim and has been reported to have anti­inflammatory activities. The present study detected the effects of ISO on AKI induced by SAP in rat models, and the underlying mechanism. The optimum dose of ISO for treatment of AKI induced by SAP was determined. The serum levels of TNF­α and IL­6 were estimated using an ELISA. Kidney injury was evaluated by histopathological examination, and the expression levels of nitric oxide were also detected. The expression levels of Toll­like receptor 4 (TLR4) and NF­κB p65 were measured by immunohistochemistry and western blotting. The results revealed that ISO may serve a critical role in ameliorating AKI induced by SAP. These effects may be associated with the TLR4/NF­κB signaling pathway.

Seasonal variation in the chemical composition of two chemotypes of Lippia alba.

Lippia alba is a popular Brazilian herb known as 'cidreira' that presents several chemotypes which exhibit different chemical profile and they are widely used as seasonings and traditional medicine. This work describes the seasonal variation of metabolites of polar extracts of carvone and linalool chemotypes, identified by GC-MS analyses of the essential oils. A methodology was elaborated in order to obtain a seasonal variation in the chemical composition of leaf employing HPLC-DAD. Acteoside, isoacteoside, geneposidic acid, 8-epi-loganin, mussaenoside, luteolin 7-O-glucoside, apigenin 7-O-glucuronide and tricin 7-O-diglucuronide have been isolated and identified for validation procedures and chromatographic analysis. Geneposidic acid was presented in all samples, in contrast to the 8-epi-loganin and, mussaenoside which were presented only in the carvone-chemotype. Acteoside was the major metabolite detected from July to November while tricin-7-O-diglucuronide was the major compound in all other months. Besides, phenylpropanoids are predominant in winter and flavonoids in summer season.

Extraction parameters and drying methods of Cistanche deserticola / 中草药

Objective: The optimum extraction process parameters of Cistanche deserticola were selected to study the effects of different drying methods on five phenylethanoid glycosides. Methods: Single factor screening combined with Box-Behnken response surface method was used to optimize the extraction process. After optimal conditions were extracted, HPLC method was used to detect the content of echinacoside, cistanche A, verbascoside, isoacteoside, and 2’-acetylacteoside in different drying methods, and one-way ANOVA, cluster analysis, principal component analysis and close value analysis were used to analyze the content of five phenylethanoid glycosides to choose the best drying method. Results: Optimal extraction process was as following: methanol volume fraction was 55.14%, liquid to material ratio was 46.39, extraction time was 38.50 min. Cluster analysis, principal component analysis, and close value analysis showed that the quality of C. deserticola obtained by freeze-drying method was the best, followed by drying at 80 ℃ and the lowest at 40 ℃. Conclusion: Using this process to extract C. deserticola, the five phenylethanoid glycosides are completely and fully extracted. Although the freeze-drying method of C. deserticola has the highest active ingredient retention, from the production point of view, the 80 ℃ drying method can achieve a balance of cost and efficiency.

Analysis of HPLC Fingerprint and Multi-component Chemical Pattern Recognition of Plantaginis Semen from Different Producing Areas in Jiangxi Province / 中国实验方剂学杂志

Objective:To establish a HPLC fingerprint detection method of Plantaginis Semen, and analyze the samples from different producing areas in Jiangxi province by combining with chemical pattern recognition method, and the contents of five ingredients in Plantaginis Semen were determined. Method:A total of 34 batches of Plantaginis Semen medicinal materials were detected by HPLC. The similarity evaluation was carried out by the 2012 edition of similarity evaluation system of chromatographic fingerprint of traditional Chinese medicine. The chromatographic peak information was used as the data source, and three chemical pattern recognition methods were used to comprehensively analyze the quality of this medicinal herb. Quantitative analysis was performed on the 5 active components, including geniposidic acid, plantamajoside, acteoside, galuteolin and isoacteoside. Result:The similarities between Plantaginis Semen samples from different producing areas in Jiangxi province were >0.86. Orthogonal partial least squares-discriminant analysis (OPLS-DA) could distinguish samples from different producing areas, and be used to determine the chemical components, which had strong correlation with the quality of Plantaginis Semen. The contents of 5 active components in samples from different producing areas were different to some degree, especially in the content of plantamajoside. Conclusion:The established HPLC fingerprint of Plantaginis Semen has strong characteristics, combined with chemical pattern recognition method, it can effectively evaluate the quality of Plantaginis Semen and distinguish its producing areas.

Cistanches Herba: An overview of its chemistry, pharmacology, and pharmacokinetics property.

ETHNOPHARMACOLOGICAL RELEVANCE: Cistanches Herba is an Orobanchaceae parasitic plant. As a commonly used Traditional Chinese Medicine (TCM), its traditional functions include treating kidney deficiency, impotence, female infertility and senile constipation. Chemical analysis of Cistanches Herba revealed that phenylethanoid glycosides, iridoids, lignans, oligosaccharides, and polysaccharides were the main constituents. Pharmacological studies demonstrated that Cistanches Herba exhibited neuroprotective, immunomodulatory, hormonal balancing, anti-fatigue, anti-inflammatory, hepatoprotection, anti-oxidative, anti-bacterial, anti-viral, and anti-tumor effects, etc. The aim of this review is to provide updated, comprehensive and categorized information on the phytochemistry, pharmacological research and pharmacokinetics studies of the major constituents of Cistanches Herba. MATERIALS AND METHODS: The literature search was conducted by systematic searching multiple electronic databases including SciFinder, ISI Web of Science, PubMed, Google Scholar and CNKI. Information was also collected from journals, local magazines, books, monographs. RESULTS: To date, more than 100 compounds have been isolated from this genus, include phenylethanoid glycosides, carbohydrates, lignans, iridoids, etc. The crude extracts and isolated compounds have exhibited a wide range of in vitro and in vivo pharmacologic effects, such as neuroprotective, immunomodulatory, anti-inflammatory, hepatoprotection, anti-oxidative, anti-bacterial, and anti-tumor effects. The phenylethanoid glycosides, echinacoside and acteoside have attracted the most attention for their significantly neuropharmacology effects. Pharmacokinetic studies of echinacoside and acteoside also have also been summarized. CONCLUSION: Phenylethanoid glycosides have demonstrated wide pharmacological actions and have great clinical value if challenges such as poor bioavailability, fast and extensive metabolism are addressed. Apart from phenylethanoid glycosides, other constituents of Cistanches Herba, their pharmacological activities and underlying mechanisms are also need to be studied further.

Simultaneous Determination of Geniposidic Acid, Caffeic Acid, Acteoside and Isoacteoside in Plantaginis Semen Formula Granules by HPLC / 中国药学杂志

OBJECTIVE: To establish an HPLC method to simultaneously determine the contents of geniposidic acid, caffeic acid, acteoside and isoacteoside in Plantaginis Semen formula granules, in order to provide basis for studying its quality standards. METHODS: An HPLC method was developed. Kinetex C18 column (2.1 mm×100 mm, 2.6 μm)was used and eluted with mobile phase of 0.5% acetic acid-acetonitrile at the flow rate of 0.3 mL·min-1. The wavelength was set at 239 nm for geniposidic acid, 325 nm for caffeic acid, and 330 nm for acteoside and isoacteoside. The column temperature was maintained at 30℃. RESULTS: The good linear relationships between the concentration and peak area were in the range of 0.292 1-2.92 1 μg for geniposidic acid, 0.003 4-0.033 6 μg for caffeic acid, 0.047 6-0.476 μg for acteoside and 0.102 7-1.027 μg for isoacteoside (r≥0.999 8), respectively. The average recoveries were 99.32%, 98.62%, 98.23% and 98.51% with RSDs of 1.47%, 1.36%, 1.62% and 1.53%, respectively. CONCLUSION: The method is simple, feasible and reproducible and can be used for the quality control of Plantaginis Semen formula granules.

Acteoside and Acyl-Migrated Acteoside, Compounds in Chinese Kudingcha Tea, Inhibit α-Amylase In Vitro.

Acteoside, the predominant polyphenol of small-leaved kudingcha, the Chinese tea, has various biological activities. In this study, we examined the acyl migration of acteoside to isoacteoside with high-temperature treatment of acteoside. The inhibitory effects of acyl-migrated acteoside and acteoside on α-amylase were investigated, as were their binding interaction with α-amylase. The binding of acteoside and isoacteoside to α-amylase was investigated by using the fluorescence spectra assay, circular dichroism, and protein-ligand docking studies. Acteoside was more effective than preheated acteoside and isoacteoside in inhibiting α-amylase activity. Acteoside and isoacteoside binding to α-amylase may induce conformational changes to α-amylase, and the binding site of acteoside and isoacteoside being near the active site pocket of α-amylase may explain the decreased activity of α-amylase. The different affinities and binding sites of acteoside and isoacteoside for α-amylase resulted in different inhibition rates, which may be due to structural differences between acteoside and isoacteoside.

Separation process of three phenolic glycosides from Monochasma savatieri / 中草药

Objective: The optimal simultaneous separation process of three phenylethanoid glycosides (acteoside, isoacteoside, and torenoside B) from Monochasmasavatieri were established using of macrophage resins and dynamic axial column (DAC). Methods: The adsorption/desorption experiments and dynamic separation experiments were performed on eight types of resins (AB-8, D 101, HPD 100, LSA-10, LX-11, LX-17, LX-38, and XDA-6) to find the optimal resin. Then the optimal separation parameters were investigated on the chosen resin. The total phenylethanoid glycosides obtained from the large-scale experiment were further seperated to get acteoside, isoacteoside, and torenoside B, respectively using of DAC system. Results: Among these candidate resins, LX-17 was chosen to further obtain the optimal parameters: The optimal feeding concentration of raw materials was 1.8 g/mL; The optimal adsorption time was 150 min; The optimal gradient elute conditions were ethanol/water (0/100, 4 BV; 15/85, 4 BV; 60/40, 5 BV; 90/10, 2 BV). The large-scale experiments were amplified to 10 folds on the basis of optimal parameters to obtain total phenylethanoid glycosides. Acteoside, isoacteoside, and torenoside B were simultaneously obtained from total phenylethanoidglycosides using DAC system. Conclusion: LX-17 and DAC system can be used for the purification of phenylethanoid glycosides, which will have a goodfuture for the application in industry.

The Osmanthus fragrans flower phenylethanoid glycoside-rich extract: Acute and subchronic toxicity studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Osmanthus fragrans var. thunbergii (O. fragrans) flower has been consumed as folk medicine for thousands of years. O. fragrans flower extract is a well-characterized phenylethanoid glycoside-rich extract, which has been used as a natural anti-oxidant. The aim of this study was to evaluate the safety of O. fragrans flower phenylethanoid glycoside-rich extract (OFFE). MATERIALS AND METHODS: The OFFE was extracted by 80% (v/v) aqueous ethanol with 0.01% sodium isoascorbate (w/v) from the O. fragrans flower and purified on HPD300 resins. The total phenylethanoid glycosides content and individual phenylethanoid glycosides was determined by photocolorimetric method and reversed phase UPLC respectively. An acute oral toxicity study, reverse mutation test, bone marrow cell micronucleus test, and sperm abnormality test as well as a 90-day oral toxicity study were performed on experimental animals. RESULTS: The total content of phenylethanoid glycosides in OFFE was 73.4g acteoside equivalent per 100g of extract, include acteoside (52.5g per 100g of extract), salidroside (13.8g per 100g of extract), and isoacteoside (2.6g per 100g of extract) and so on. No acute lethal effect at the maximal tested OFFE dose of 10g/kg body weight (bw) in either rats or mice was observed, suggesting that OFFE can be considered nontoxic. No evidence for mutagenicity was detected in any of the three mutagenic tests. Administration at levels of 0.50, 1.00, and 2.00g/kg bw to rats for 90 days failed to induce any significant hematological, clinical, chemical, or histopathological changes. The no-observed adverse-effect-level for OFFE was >2.00g/kg bw for the study on subchronic toxicity. CONCLUSION: The results showed that consuming OFFE has no adverse effects and poses no health risk in the acute oral toxicity study, subchronic oral toxicity study, and in the micronucleus test, which may provide supportive evidence for the safety of OFFE powder that has been used in medicine as well as in functional foods, and dietary supplements.

Rational quality assessment procedure for less-investigated herbal medicines: Case of a Congolese antimalarial drug with an analytical report.

Herbal medicines are the most globally used type of medical drugs. Their high cultural acceptability is due to the experienced safety and efficiency over centuries of use. Many of them are still phytochemically less-investigated, and are used without standardization or quality control. Choosing SIROP KILMA, an authorized Congolese antimalarial phytomedicine, as a model case, our study describes an interdisciplinary approach for a rational quality assessment of herbal drugs in general. It combines an authentication step of the herbal remedy prior to any fingerprinting, the isolation of the major constituents, the development and validation of an HPLC-DAD analytical method with internal markers, and the application of the method to several batches of the herbal medicine (here KILMA) thus permitting the establishment of a quantitative fingerprint. From the constitutive plants of KILMA, acteoside, isoacteoside, stachannin A, and pectolinarigenin-7-O-glucoside were isolated, and acteoside was used as the prime marker for the validation of an analytical method. This study contributes to the efforts of the WHO for the establishment of standards enabling the analytical evaluation of herbal materials. Moreover, the paper describes the first phytochemical and analytical report on a marketed Congolese phytomedicine.

Screening and identification of three typical phenylethanoid glycosides metabolites from Cistanches Herba by human intestinal bacteria using UPLC/Q-TOF-MS.

Acteoside, isoacteoside, and 2'-acetylacteoside are three representative phenylethanoid glycosides (PhGs), which are widely distributed in many plants and also known as the active components of Cistanches Herba. However, the extremely low oral bioavailability of acteoside in rats implies that these structural similar components may go through multiple sequential routes of hydrolysis in gastrointestinal tract before they are absorbed into blood. Therefore, the metabolites of these three components and other PhGs from gastrointestinal tract such as echinacoside, are supposed to be the bioactive elements. In this study, we established an approach combining ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS) with MS(E) technology and MetaboLynx™ software for the rapid metabolic profiling of acteoside, isoacteoside, and 2'-acetylacteoside by human intestinal bacteria. As a result, 11 metabolites of acteoside, 7 metabolites of isoacteoside, and 11 metabolites of 2'-acetylacteoside were identified respectively. 8 metabolic pathways including deglycosylation, de-rhamnose, de-hydroxytyrosol, de-caffeoyl, deacetylation, reduction, acetylation, and sulfate conjugation were proposed to involve in the generation of these metabolites. Furthermore, we found that the degraded metabolites hydroxytyrosol (HT) and 3-hydroxyphenylpropionic (3-HPP) were transformed from acteoside, isoacteoside, and 2'-acetylacteoside by human intestinal bacteria and demonstrated similar bioactivities to their precursors. These findings are significant for our understanding of the metabolism of PhGs and the proposed metabolic pathways of bioactive components might be crucial for further pharmacokinetic evaluations of Cistanches Herba.

Anti-inflammatory effects of isoacteoside from Abeliophyllum distichum.

Isoacteoside, a dihydroxypheynylethyl glycoside, is a major bioactive component of Abeliophyllum distichum (White Forsythia) which is a deciduous shrub native to the south and central areas of Korea. The present study is designed to evaluate the anti-inflammatory activities and underlying mechanisms of isoacteoside in human mast cell line, HMC-1 cells. We isolated isoacteoside from A. distichum. The anti-inflammatory effect of isoacteoside was investigated in HMC-1 cells by studying the following markers: phorbol 12-myristate 13-acetate and calcium ionophore A23187 (PMACI)-induced interleukin (IL)-1ß, IL-6, IL-8, and tumor necrosis factor alpha (TNF-α) secretion and mRNA expression by ELISA and RT-PCR, respectively. In addition, mechanism related to anti-inflammatory was investigated by Western blotting. Isoacteoside significantly suppressed the production and mRNA expression of proinflammatory cytokines including IL-1ß, IL-6, IL-8 and TNF-α in PMACI-stimulated HMC-1 cells without cytotoxicity. It was found that anti-inflammatory effects of isoacteoside are mediated by action on caspase-1, mitogen-activated protein kinases (c-Jun N-terminal kinase, p38, extracellular signal-regulated protein kinase) and nuclear factor-kappa B pathways. Taken together, the present findings provide new insights that isoacteoside may be a promising anti-inflammatory agent for inflammatory disorders.

Chemical constituents from Goldfussia pentstemonoides / 中草药

Objective: To study the chemical constituents from the whole plant of Goldfussia pentstemonoides. Methods: The compounds were separated and purified by chromatographic methods. The structures were identified by spectroscopic analyses. Results: Ten compounds were isolated from the n-butanol fraction of 95% ethanol extract in the whole plants of G. pentstemonoides and identified as salidroside (1), 1-(α-L-rhamnosyl-(1→6)-O-β-D-glucopyranosyloxy)-3,4,5-trimethoxybenzene (2), isoacteoside (3), isonuomioside A (4), benzyl-O-β-D-glucopyranoside (5), lcariside F2 (6), (-)-lyoniresinol 3α-O-β-D-glucopyranoside (7), (+)-isolariciresinol-9-O-β-D-glucopyranoside (8), decaffeoylverbascoside (9), and dehydrodiconiferyl alcohol 4-O-β-D-glucopyranoside (10). Conclusion: All compounds are isolated from G. pentstemonoides for the first time.

Simultaneous determination of three phenolic glycosides in Callicarpa nudiflora by UHPLC-MS methods and analysis of their pharmacokinetics in plasma of rats / 中草药

Objective: An UHPLC-MS/MS method was developed for the simultaneous determination of acteoside, isoacteoside, and forsythoside B in plasma of rats and the pharmacokinetic parameters for three phenolic glycosides were calculated as well. Methods: Samples of plasma of rats were obtained at different time after rats were administrated with Callicarpa nudiflora extract (5 g/kg). After the addition of acidification (hydrochloric acid, 0.25 mol/L) and deproteinization by acetonitrile, plasma samples were separated on a Phenomenex® Kinetex C18 column (50 mm × 2.1 mm, 1.7 μm) with gradient elution using acetonitrile-0.005% formic acid as mobile phase. Mass spectrometric detection was carried out by multiple reaction monitoring (MRM) using electrospray ionization in negative ion mode. Results: A good linearity of acteoside, isoacteoside, and forsythoside B was shown in the ranges of 7.77 - 3 880.00 ng/mL (r2 = 0.995 5), 5.04 - 2 520.00 ng/mL (r2 = 0.994 9), and 1.78 - 890.00 ng/mL (r2 = 0.995 1), respectively. The mean extraction recoveries of analytes were in the range of 75.2% - 89.9%, and the intra- and inter-day RSD values were less than 8.8%. The tmax of acteoside, isoacteoside, and forsythoside B was about 30 min, AUC0~t were (93 881.65 ± 18 326.65), (29 204.97 ± 8 499.88), and (15 027.05 ± 3763.82) ng∙min/mL, Cmax were (2 179.00 ± 355.60), (737.57 ± 210.31), and (227.30 ± 48.38) ng/mL, t1/2z were (235.41 ± 117.90), (151.56 ± 49.23), and (161.68 ± 63.92) min, respectively. Conclusion: The method is proved to be simple, rapid, and specific, and to be suitable for the simultaneous determination of acteoside, isoacteoside, and forsythoside B in plasma of rats and the pharmacokinetic study.

Simultaneous Determination of Echinacoside, Acteoside and Isoacteoside in Total Cistanchis glycosides Capsules by HPLC / 世界科学技术-中医药现代化

This study was aimed to establish an HPLC method for simultaneous determination of the content of echinacoside, acteoside and isoacteoside in Total Cistanchis glycosides Capsules. Waters C18 column (4.6 mm ×150 mm, 5 μm) was used with the mobile phase of methanol-0.1% formic acid at a flow rate of 1.0 mL·min-1. The detection wavelength was set at 330 nm. The column temperature was maintained at 30℃ . The results showed that the linear ranges of echinacoside, acteoside and isoacteoside were in the range of 27.792-277.92 μg·mL-1 (r = 0.9996,n = 6), 2.4184-24.184 μg·mL-1 (r = 0.9996, n = 6), 5.106-51.06 μg·mL-1 (r = 0.9998, n = 6). The average recoveries of three components were in accordance with the determination requirement. It was concluded that the method was simple and accurate, which can be used in the content determination of echinacoside, acteoside and isoacteoside in Total Cistanchis glycosides Capsules.

Inhibitory activities of acteoside, isoacteoside, and its structural constituents against protein glycation in vitro.

BACKGROUND: Advanced glycation end products (AGE) are substances that can induce insulin resistance in adipocyte, hepatocyte and muscle cells. This resistance correlates highly with cardiovascular disease and diabetic complications. Acteoside (A), a phenylethanoid glycoside, is an active compound in several plants and traditional herbal medicines. Acteoside, its structural isomer, isoacteoside (I), and their constituents, caffeic acid (C) and 3,4-dihydroxyphenylethanol (D), were used in the study to investigate the inhibitory activity against AGE formations in vitro. RESULTS: AGE formations were detected by anti-(Nϵ-(carboxymethyl)lysine (anti-CML), using bovine serum albumin (BSA)/glucose (glc) and BSA/galactose (gal) as models, or by anti-argpyrimidine (anti-AP), using BSA/methylglyoxal (MGO) as models. It was found that A, I, C, or D, each at 5 mM, could attenuate the CML formations detected by ELISA in the BSA/gal model of a 3-day or 5-day reaction, and showed significant differences (P < 0.01 or P < 0.001) compared to the control. However, these compounds showed a minor effect after a 7-day incubation. It was also found that C or D could lower the CML formations in the BSA/glc model and showed significant differences (P < 0.05 or P < 0.01) compared to the control after a 3-day, 5-day and 7-day reaction. It was found that A, I, C, or D, each at 0.5 mM or 5 mM, could attenuate the AP formations in the BSA/MGO model of a 3-day reaction and showed significant differences (P < 0.001) compared to the control. CONCLUSIONS: The results suggest the potential anti-glycation activities of A and I in vitro may apply to cell models at higher glucose concentrations or to diabetic animal models, and need further investigation.

Isolation and purification of phenylethanoid glycosides from Cistanche deserticola by high-speed counter-current chromatography.

Five phenylethanoid glycosides (PhGs), echinacoside, cistanoside A, acteoside, isoacteoside and 2'-acetylacteoside, were isolated and purified from Cistanche deserticola for the first time by high-speed counter-current chromatography (HSCCC) using two biphasic systems, one consisting of ethyl acetate-ethanol-water (5:0.5:4.5, v/v/v) and another of ethyl acetate-n-butanol-ethanol-water (0.5:0.5:0.1:1, v/v/v/v). A total of 28.5mg of echinacoside, 18.4mg of cistanoside A, 14.6mg of acteoside, 30.1mg of isoacteoside and 25.2mg of 2'-acetylacteoside were purified from 1412mg of the n-butanol extract of C. deserticola, each at over 92.5% purity as determined by HPLC. The structures were identified by their retention time, UV, LC-ESI-MS in the negative ion mode, and confirmed by NMR experiments. The characteristic LC-ESI-MS(n) fragmentation pattern of the five compounds is discussed, and found to be a very specific and useful tool for the structural identification of PhGs from this important medicinal plant.