Diosgenin revealed potential effect against cerebral ischemia reperfusion through HIKESHI/HSP70/NF-κB anti-inflammatory axis.

BACKGROUND: It is a research hotspot to use natural compounds in treatment of cerebral ischemia reperfusion (I/R) for a refractory disease throughout the worldwide without available drugs or treatments at present. Our previous study has demonstrated that diosgenin (DIO), a starting material to synthesize various steroid anti-inflammatory drugs in medical industry, showed medicinal effect against I/R via inhibiting aberrant inflammatory reaction induced by I/R. However, the detailed anti-inflammatory network of DIO in treatment of I/R still remains to be further explored. PURPOSE: HIKESHI was firstly identified as a novel target of DIO used for I/R by rat brain proteomic analysis, and mechanistic efforts were focused based on this gene. Hopefully, extensive detailed molecular mechanisms of DIO against I/R was established and confirmed. METHODS: The effect of DIO against I/R was examined in vitro and in vivo, which cells (SH-SY5Y and PC12) and rats were experienced to ORD/RP and MCAO exposures, respectively, to establish I/R modes. Staining was used to evaluate the pathological procedure of DIO used for I/R. Protein changes including expression, interaction, and activity during DIO's anti-I/R effect were assessed with real time PCR, western blot, Co-IP, luciferase reporter assay. RESULTS: In the current study, HIKESHI and HSP70 were both upregulated, when I/R cells and rats were treated with DIO in vitro and in vivo. Mechanistically, DIO stimulated the binding of HIKESHI to HSP70 and facilitated the translocation of HSP70 into nucleus. Subsequently, HSP70 blunted the transcription activity of NF-κB after physical interaction with this transcription factor, and therefore led to the suppression of its downstream pro-inflammatory cytokine (TNF-α, IL-1ß, and IL-6) release into surrounding I/R lesion area. Conversely, HIKESHI or HSP70 knockdowns attenuated the nuclear translocation and restraint on NF-κB-mediated inflammation, finally resulting in the abolishment of DIO-induced anti-I/R effect. NF-κB activation also relieved the inhibitory inflammation and reversed DIO's effect against I/R, suggesting that NF-κB was the downstream target of HIKESHI and HSP70 in I/R treatment with DIO. CONCLUSIONS: These findings established a novel HIKESHI/HSP70/NF-κB signaling pathway associated with DIO-treated I/R, which might be as therapeutic targets or drugs with potential implications for the therapeutic use of I/R in clinic.

New steroid saponins from Dioscorea zingiberensis yam and their medicinal use against I/R via anti-inflammatory effect.

Steroid saponins are the medicinal compounds and nutrition ingredients of medicine food  homology (MFH) Dioscorea zingiberensis C. H. Wright (D. zingiberensis) yam. Our phytochemical investigation of the edible rhizomes resulted in 9 new furostanol steroid saponins named dioscins A-I (1-9), together with 11 known steroid saponins. Their chemical structures were elucidated based on spectroscopic and chemical analyses. The new dioscins were evaluated for their anti-inflammatory and beneficial effects against cerebral ischemia reperfusion (I/R) injury on RAW264.7 and PC12 cells in vitro, respectively. Dioscins A, B, and G revealed considerable anti-I/R effect through an anti-inflammatory mechanism based on the decreasing concentration of pro-inflammatory (TNF-α and IL-6) and down-regulating the NF-κB expression. The present research demonstrated that daily consumption of this yam plant probably prevented the I/R occurrence via the anti-inflammatory property of steroid saponins, and it also enriched the steroid saponin library, providing the possibility to develop MFH-containing steroid saponins into functional foods for maintenance of human health or drugs for the treatment of I/R disease.

Prevention properties on cerebral ischemia reperfusion of medicine food homologous Dioscorea yam-derived diosgenin based on mediation of potential targets.

I/R (cerebral ischemia reperfusion injury) is the secondary complication of ischemic stroke patients that are immediately treated with drug thrombolysis or vascular recanalization in clinic. Diosgenin (DIO) purified from medicine food homologous (MFH) Dioscorea yam source is served as a fatal starting material to synthesize multifarious steroidal anti-inflammatory drugs in medicinal field, and has previously been demonstrated the potential prevention of I/R. However, the detailed mechanisms of neuroprotective effects against I/R remain elusively understood. Here, a global proteomic dynamics of rat right hemisphere brains was executed to investigate the protein expression patterns with a quantitative LC-MSn. In total, 5043 proteins were identified and 418 ones were determined to be significantly dysregulated DEPs (differentially expressed proteins) in comparison of Sham verse I/R and I/R verse DIO after onset stage of I/R, among which 5 DEPs namely BICD2, HNRNPK, CEP41, PPM1K, and ARL2BP, whose biological functions were mainly clustered into the mediation of nervous system, were selected for further validation in vitro and in vivo, and the change tendency expectedly supported the proteomic findings. Additionally, the AUC value of the combined ROC of these 5 DEPs was 0.988 with P < 0.0001, higher than every single one. Collectively, these scientific findings attributed to a typical investigation of dietary Dioscorea-enriched diosgenin in MFH research, suggesting that diosgenin or its derivatives were potential to be developed into food supplements or healthy food products to reveal healthy benefits in natural prevention and reduction risk of I/R. This work also promoted reasonable consumption of Dioscorea yams and contributed to the function of diosgenin-derived products and their applications in food industry.

Botany, traditional use, phytochemistry, pharmacology, quality control, and authentication of Radix Gentianae Macrophyllae-A traditional medicine: A review.

BACKGROUND: Radix Gentianae Macrophyllae, commonly known as Qinjiao (in Chinese), is dried roots of medicinal plants that belong to Gentianaceae family and Gentiana genus. It has medically been used for the treatment of various diseases such as arthritis, stroke, facial paralysis, and scapulohumeral periarthritis in China since ancient times. PURPOSE: The aim of this paper is to provide a comprehensive and constructive overview of Qinjiao mainly containing Gentiana macrophylla Pall., Gentiana straminea Maxim., Gentiana crasicaulis Duthie ex Burk., and Gentiana daurica Fisch. in botany, traditional use, phytochemicals, pharmacology (biological activities and pharmacokinetics), quality control, and authentication according to the up-to-date data of available scientific literatures. MATERIALS AND METHODS: All information regarding these four plants was collected from various academic search engines for example Google, Google Scholar, Web of Science, SciFinder, Pubmed, CNKI, and Wanfang. Additional information was obtained from botanical books, Chinese classic texts, and medical monographs. RESULTS: So far 166 compounds have been isolated and identified from Qinjiao plants together with Gentiana tibetica King ex Hook. f., Gentiana siphonantha Maxim., Gentiana officinalis H. Smith, and Gentiana waltonii Burk. Their constituents are mainly classified into iridoid glycosides, triterpenes, flavones, sterols, benzene derivatives, etc. The pharmacological studies demonstrate that Qinjiao plants display a wide range of bioactivities e.g. anti-inflammatory, anti-oxidant, hepato-protective, cardio- and neuro-protective, insecticidal, and anti-influenza effects. The conventional quality control is performed by determination of the concentration of some compounds, e.g., gentiopicroside, or establishment of the fingerprint. In addition to seed propagation, tissue culture technology has been used to address the limited supplies and guarantee the sustainable development of Qinjiao in the experimental scale. CONCLUSIONS: Although the identification of compounds from Qinjiao and demonstration of medicinal uses in vitro and in vivo have been carried out, various other studies on these plants should deserve our more attention. More efforts should be concentrated on the underlying mechanisms of their beneficial bioactivities. The proper toxic evaluation is indispensable to guarantee the safety, efficacy, and eligibility for medical use. To sum up, the summarized achievements could highlight the importance of Qinjiao and provide a solid foundation for scientists not only to further exploit the therapeutic potentials, but also possibly develop novel drugs in the subsequent research.

Purification of R-phycoerythrin from Gracilaria lemaneiformis by centrifugal precipitation chromatography.

Centrifugal precipitation chromatography (CpC) is a powerful chromatographic technique invented in the year 2000 but so far very little applied. The method combines dialysis, counter-current and salting out processes. The separation rotor consists of two identical spiral channels separated by a dialysis membrane (6-8 K MW cut-off) in which the upper channel is eluted with an ammonium sulfate gradient and the lower channel with water, and the mixtures are separated according to their solubility in ammonium sulfate as a chromatographic technique. In the present study, the method was successfully applied for separation and purification of R-phycoerythrin (R-PE), a protein widely used as a fluorescent probe, from the red alga Gracilaria lemaneiformis. The separation was performed with the elution of ammonium sulfate from 50% to 0% in 21.5 h at a flow rate of 0.5 ml/min, while the lower channel was eluted with water at a flow rate of 0.05 ml/min after sample charge, and the column was rotated at 200 rpm. After a single run, the absorbance ratio A565/A280 (a criterion for the purity of R-PE) was increased from 0.5 of the crude to 6.5. The purified R-PE exhibited a typical "three peaks" spectrum with absorbance maximum at 497, 538 and 565 nm. The Native-PAGE showed one single protein band and 20 kDa (subunits α and ß) and 30 kDa (subunit γ) can be observed in SDS-PAGE analysis which were consistent with the (αß)6γ subunit composition of R-PE. The results indicated that CpC is an efficient method to obtain protein with the high purity from a complex source.

Dioscorea zingiberensis C. H. Wright: An overview on its traditional use, phytochemistry, pharmacology, clinical applications, quality control, and toxicity.

ETHNOPHARMACOLOGICAL RELEVANCE: Dioscorea zingiberensis C. H. Wright (D. zingiberensis), Dioscoreaceae, is used extensively in traditional Chinese medicines. The aim of the current review paper is to give a comprehensive overview of the traditional usage and phytochemistry of the plant. Clinical studies performed and products prepared from the plant and active principles will be mentioned. In addition a review of the taxonomy of the genus Dioscorea is given. MATERIALS AND METHODS: A systematic search on literature has been performed in databases like Google Scholar, Science Direct, Scifinder, Web of Science, and in Chinese databases (China Knowledge Resource Integrated, i.e., CNKI and Wanfang) including Ph.D. and M.Sc. dissertations. These from the period 1983-2016 have been searched. RESULTS: D. zingiberensis is an endemic plant in China widely distributed in some provinces, especially Hubei and Shaanxi. The rhizomes are the medicinal part in Dun-Ye-Guan-Xin-Ning tablets and are used for isolation of diosgenin. Steroidal saponins are believed to be the active principles. More than 70 compounds have been identified. Several of these have been tested in preclinical assays and clinical trials. A wide spectrum of biological effects including cardiovascular, anti-thrombosis, hyperlipidemia, neuroprotection, anti-inflammatory, and anthelmintic effect has been verified. CONCLUSIONS: Because of the promising results from the investigations on the plant material of D. zingiberensis, further in depth analyses ought to be performed to evaluate its potential as either a traditional drug or a source of bioactive principle. The presently performed studies do not explain mechanism of action, pharmacokinetics (ADME properties), or toxicity. All of these topics need more elaborate investigations.

Safety investigation on total steroid saponins extracts from Dioscorea zingiberensis C.H. Wright: Sub-acute and chronic toxicity studies on dogs.

Sub-acute and chronic toxic effects of total steroidal saponins (TSSN) extracts from Dioscorea zingiberensis C.H. Wright on various internal organs and biochemical indicators have never been studied before and this study is the first of its kind to demonstrate sub-acute and chronic toxicities of TSSN on dogs. Administration of TSSN extracts at doses up to 3000 mg/Kg daily for 14 days, no biochemical and organ changes were observed on the experimental groups of dogs. Further, chronic toxicity study through oral administration of TSSN extracts at the gradual doses of 50, 250 and 500 mg/Kg for 90 days followed by a 2-week recovery assay revealed absence of significant architectural and morphological changes in internal organs which were confirmed through histopathological examination and merely no significant alteration in the biochemical indicators including hematologic and urine analysis and electrocardiogram compared to the control dogs. This toxicological evaluation came across with the finding that the herbal preparation can be considered as nontoxic and animals could tolerate the extracts at doses up to 500 mg/Kg with LD50 greater than 3000 mg/Kg. It may serve as a preliminary scientific evidence for further therapeutic investigations.

SEPARATION AND PURIFICATION OF TWO MINOR COMPOUNDS FROM RADIX ISATIDIS BY INTEGRATIVE MPLC AND HSCCC WITH PREPARATIVE HPLC.

Radix isatidis has been widely used as a Chinese traditional medicine for its anti-virus and anticancer activities where the minor components may contribute to these beneficial pharmaceutical effects. In order to enrich the target minor compounds effectively and rapidly, extraction, medium-pressure liquid chromatography (MPLC), high-speed countercurrent chromatography (HSCCC) and preparative high-performance liquid chromatography (pre-HPLC) were integratively used for separation and purification of two target minor compounds indole-3-acetonitrile-6-O-ß-D-glucopyranoside (target 1) and clemastanin B (target 2) in the present study. Radix isatidis was dried, pulverized and extracted with 50% methanol at room temperature, then concentrated and subjected to pretreatment with D-101 macroporous resin chromatography and extraction by MPLC. The first target compound was separated by MPLC at the purity raised to 70-80%, but without the second minor compounds which were irreversibly adsorbed by C18 solid support. Therefore, the second target compound in the crude extract was directly separated by HSCCC at purity of 80-90%. Finally these refined samples were further separated by pre-HPLC to obtain a high purity at 98-99%. The chemical structure identification of each target compound was carried out by IR, ESI-MS and 1H NMR.

Separation of catalpol from Rehmannia glutinosa Libosch. by high-speed countercurrent chromatography.

The bioactive iridoid component catalpol was successfully separated by high-speed countercurrent chromatography with high purity from the partially purified crude extract of Rehmannia glutinosa. A polar two-phase solvent system composed of ethyl acetate-n-butanol-water (2:1:3, v/v/v) was selected by thin-layer chromatography and run on a preparative scale where the lower aqueous phase was used as the mobile phase with a head-to-tail elution mode. A 105 mg quantity of the partially purified sample containing 39.2% catalpol was loaded on a 270-mL capacity high-speed countercurrent separation column, yielding 35 mg of catalpol at 95.6% purity. The chemical structure of catalpol was determined by comparison with the high-performance liquid chromatography retention time of standard substance as well as the (1)H NMR spectrum.

Simultaneous quantification of five steroid saponins from Dioscorea zingiberensis C.H. Wright in rat plasma by HPLC-MS/MS and its application to the pharmacokinetic studies.

A simple, reliable and sensitive high-performance liquid chromatography tandem mass spectrometry method (HPLC-MS/MS) was established for simultaneous analyses of the following 5 steroid saponins in rat plasma after the single dose administration of total steroid saponins extracted from the rhizome of Dioscorea zingiberensis C.H. Wright for the first time. Protodioscin, huangjiangsu A, zingiberensis new saponin, dioscin, and gracillin were quantified using ginsenoside Rb1 as the internal standard (IS). The plasma samples were pretreated by a single step acetonitrile-mediated protein precipitation. The chromatographic separation was performed on an Inersil ODS-3 C18 column (250mm×4.6mm, 5µm) with the mobile phase composed of acetonitrile and water containing 0.1% formic acid under a gradient elution mode at 0.2mLmin(-1) using a microsplit after the eluent from the HPLC apparatus. The quantification was accomplished on a triple quadrupole tandem mass spectrometer using the multiple reaction monitoring (MRM) in the positive ionization mode. The above five analytes were stable under sample storage and preparation conditions applied in the present study. The linearity, precision, accuracy, and recoveries of the analysis confirmed the requirements for quality-control purposes. After validation, this proposed method was successfully adopted to investigate the pharmacokinetic parameters of these five analytes.

Purification of active myrosinase from plants by aqueous two-phase counter-current chromatography.

INTRODUCTION: Myrosinase (thioglucoside glucohydrolase; E.C. 3.2.1.147), is a plant enzyme of increasing interest and importance to the biomedical community. Myrosinase catalyses the formation of isothiocyanates such as sulforaphane (from broccoli) and 4-(α-l-rhamnopyranosyloxy)benzyl isothiocyanate (from moringa), which are potent inducers of the cytoprotective phase-2 response in humans, by hydrolysis of their abundant glucosinolate (ß-thioglucoside N-hydroxysulphate) precursors. OBJECTIVE: To develop an aqueous two-phase counter-current chromatography (CCC) system for the rapid, three-step purification of catalytically active myrosinase. METHODS: A high-concentration potassium phosphate and polyethylene glycol biphasic aqueous two-phase system (ATPS) is used with a newly developed CCC configuration that utilises spiral-wound, flat-twisted tubing (with an ovoid cross-section). RESULTS: Making the initial crude plant extract directly in the ATPS and injecting only the lower phase permitted highly selective partitioning of the myrosinase complex before a short chromatography on a spiral disk CCC. Optimum phase retention and separation of myrosinase from other plant proteins afforded a 60-fold purification. CONCLUSION: Catalytically active myrosinase is purified from 3-day broccoli sprouts, 7-day daikon sprouts, mustard seeds and the leaves of field-grown moringa trees, in a CCC system that is predictably scalable.

Preparative separation of quaternary ammonium alkaloids from Coptis chinensis Franch by pH-zone-refining counter-current chromatography.

pH-zone-refining counter-current chromatography was successfully applied to the preparative separation of five quaternary ammonium alkaloids from the crude extract of Coptis chinensis Franch. The separation was performed with a two-phase solvent system composed of chloroform-methanol-water (4:3:3, v/v), where the upper aqueous stationary phase was added with 60mM of hydrochloric acid and the lower organic mobile phase with 5mM of triethylamine. From 1.0 g of crude extract, 5.4 mg of columbamine at 96.6% purity, 6.1mg of jateorhizine at 98.8% purity, 58.3mg of coptisine at 99.5% purity, 25.6 mg of palmatine at 98.4% purity and 503.9 mg of berberine at 99.5% purity were obtained. The purities of the isolated alkaloids were analyzed by HPLC and the chemical structures were identified by electrospray ionization-mass spectrometry and (1)H NMR.

Neuroprotection of total steroid saponins from Dioscorea zingiberensis against transient focal cerebral ischemia-reperfusion injury in rats via anti-inflammatory and antiapoptotic effects.

Total steroid saponins isolated from Dioscorea zingiberensis have shown a variety of beneficial bioactivities. However, there are no reports about their neuroprotective effects, until now. Therefore, in the present study, we explored the neuroprotective effects of the total steroid saponins from D. zingiberensis on rats against transient focal cerebral ischemia-reperfusion and their underlying mechanisms. Healthy adult Sprague-Dawley rats were randomly assigned to six groups. After pretreatment with D. zingiberensis total steroid saponins intragastrically for six days, the rats were subjected to an ischemia injury by surgery on the middle cerebral artery occlusion for 90 min. Compared to the ischemia-reperfusion group, the D. zingiberensis total steroid saponin group of rats, especially those given a 30-mg/kg dose, showed not only a marked reduction in neurological deficit scores, cerebral infarct volume, and brain edema, but also an increase in neuron survival (Nissl bodies) in the hippocampal cornuammons 1 and cortex hemisphere of the ipsilateral ischemia. At the same time, the inflammatory cytokines in serum induced by the middle cerebral artery occlusion were significantly decreased by the preadministration of D. zingiberensis total steroid saponins. Furthermore, the increase of caspase-3 was evidently reduced in the hippocampal cornuammons 1 and cortex of the hemisphere injured brain. Finally, the downregulating antiapoptotic Bcl-2 and upregulating proapoptotic Bax proteins were obviously suppressed. Taken together, the current findings suggest that D. zingiberensis total steroid saponins played a potential neuroprotective role against a severe injury induced by transient focal cerebral ischemic reperfusion in a rat experimental model, and this role may be mediated by its anti-inflammatory and antiapoptotic actions.

Enrichment and purification of pedunculoside and syringin from the barks of Ilex rotunda with macroporous resins.

Jiubiying, the dried barks of Ilex rotunda Thunb. (Aquifoliaceae), has been used as herbal tea and traditional Chinese medicine for heat-clearing, detoxifying, dehumidification, and odynolysis. Pedunculoside and syringin are two main bioactive components. For the new drug development, we tried to isolate and purify several chemical constituents from Jiubiying by high-speed counter-current chromatography (HSCCC). The two-phase solvent system used was composed of ethyl acetate-n-butanol-water (1:6:7, v/v/v). From 1.0 g of Jiubiying extracts syringaresinol 4',4″-bis-O-ß-D- glucopyranoside (I, 20.2 mg), syringin (II, 56.8 mg), sinapaldehyde glucoside (III, 26.2 mg), syringaresinol 4'-O-ß-D-glucopyranoside (IV, 20.4 mg), and pedunculoside (V, 45.1 mg) were obtained by one run of TBE-1000A HSCCC machine with 1000 mL of column volume. Their structures were identified by IR, MS, and extensive NMR studies. Syringaresinol 4',4″-bis-O-ß-D-glucopyranoside (I) was isolated from this plant for the first time.

Therapeutic effects of total steroid saponin extracts from the rhizome of Dioscorea zingiberensis C.H.Wright in Freund's complete adjuvant induced arthritis in rats.

The aim of our present study is to explore the anti-arthritic potential effect of total steroid saponins (TSSNs) extracted from the rhizome of Dioscorea zingiberensis C.H.Wright (DZW) and to investigate the underlying mechanisms. This work was performed using adjuvant-induced arthritis (AIA) rats in vivo and lipopolysaccharide (LPS) simulated 264.7 macrophage cells in vitro. In AIA-induced arthritic rats, TSSN significantly alleviated the arthritic progression through evaluating arthritic score, immune organ indexes, paw swelling, and body weight. This phenomenon was well correlated with significant suppression of the overproduction of inflammation cytokines (IL-1, IL-1ß, IL-6, and TNF-α), oxidant stress makers (MDA and NO), eicosanoids (LTB4 and PGE2), and inflammatory enzymes (5-LOX and COX-2) versus the AIA rats without treatment. On the contrary, the release of SOD and IL-10 was profoundly increased. What's more, TSSN could obviously ameliorate the translocation of NF-κB to the nucleus through phosphorylation of the p65 and IκBα in vivo and in vitro. The current findings demonstrated that TSSN could protect the injured ankle joint from further deterioration and exert its satisfactory anti-arthritis properties through anti-inflammatory and anti-oxidant effects via inactivating the NF-κB signal pathway. This research implies that DZW may be a useful therapeutic agent for the treatment of human arthritis.

Neuroprotective effects of total steroid saponins on cerebral ischemia injuries in an animal model of focal ischemia/reperfusion.

Total steroid saponins isolated from Dioscorea zingiberensis are a unique traditional Chinese medicine known for its potential usage in various types of diseases. However, there is little evidence about its neuroprotective effect in transient focal ischemia-reperfusion cerebral injury. Therefore, the current study was carried out to investigate the effect of total steroid saponins on neuroprotection and its potential mechanisms in the rat ischemia-reperfusion model by middle cerebral artery occlusion for 90 min. The rats were each treated with total steroid saponins (30 mg/kg, 10 mg/kg, and 3 mg/kg) or nimodipine (20 mg/kg) daily for 6 days before middle cerebral artery occlusion. Then, the neurological deficit score, cerebral infarct volume, and brain water content were measured at 24 h after reperfusion. Meanwhile, the histopathological changes and AQP-4 protein activities were examined in hippocampal CA1 and the cortex of ipsilateral ischemic cerebral hemisphere by hematoxylin-eosin staining and immunohistochemistry, respectively. The indices of oxidative stress in the serum were also obtained, and NF-κB and ERK 1/2 protein expressions in the injured brain were evaluated by Western blotting. The results indicated that the pre-treatment with these drugs not only significantly reduced cerebral infarct volume, brain water content and improved neurological deficit score, but also restored neuronal morphology and decreased the AQP-4 positive cells in CA1 and the cortex. Moreover, it markedly restored the level of oxidant stress markers (CAT, SOD, MDA, NO and iNOS) to their normal range in serum. In addition, the increased NF-κB and ERK 1/2 protein expressions were alleviated as compared with the ischemia-reperfusion group. These findings demonstrate that total steroid saponins exhibit promising neuroprotection effects against the transient focal ischemia-reperfusion cerebral injury in the rat experimental model and the underlying mechanisms might be mediated through inhibition of anti-edema as well as anti-oxidative effects by inactivation of NF-κB and ERK 1/2 signalling pathway.

Development of a method to extract and purify target compounds from medicinal plants in a single step: online hyphenation of expanded bed adsorption chromatography and countercurrent chromatography.

Pure compounds extracted and purified from natural sources are crucial to lead discovery and drug screening. This study presents a novel two-dimensional hyphenation of expanded bed adsorption chromatography (EBAC) and high-speed countercurrent chromatography (HSCCC) for extraction and purification of target compounds from medicinal plants in a single step. The EBAC and HSCCC were hyphenated via a six-port injection valve as an interface. Fractionation of ingredients of Salvia miltiorrhiza and Rhizoma coptidis was performed on the hyphenated system to verify its efficacy. Two compounds were harvested from Salvia miltiorrhiza, one was 52.9 mg of salvianolic acid B with an over 95% purity and the other was 2.1 mg of rosmarinic acid with a 74% purity. Another two components were purified from Rhizoma coptidis, one was 4.6 mg of coptisine with a 98% purity and one was 4.1 mg of berberine with a 82% purity. The processing time was nearly 50% that of the multistep method. The results indicate that the present method is a rapid and green way to harvest targets from medicinal plants in a single step.

Quality control and identification of steroid saponins from Dioscorea zingiberensis C. H. Wright by fingerprint with HPLC-ELSD and HPLC-ESI-Quadrupole/Time-of-fight tandem mass spectrometry.

In this study, a fingerprint of steroid saponins, the major bioactive constituents in the crude extracts from Dioscorea zingiberensis C. H. Wright (DZW), has been established for the first time by combined use of the following two methods: high-performance liquid chromatography coupled with evaporative light scattering detector (HPLC-ELSD) and the simultaneous characterization of the steroid saponins by high-performance liquid chromatography coupled with electrospray ionization-mass spectrometry and quadrupole tandem time-of-fight mass analyzers detection (HPLC-ESI-Q/TOF). All HPLC analyses were carried out on a Welchrom C18 column (250mm×4.6mm I.D., 5µm) with a mobile phase composed of water and acetonitrile under gradient elution. There were 68 common characteristic peaks in the fingerprints, in which 12 of them were confirmed by comparing their mass spectra and retention times with those of the reference compounds. In order to identify other unknown peaks, their fragmentation behaviors characteristic of the major groups of steroid saponins from DZW with six types of aglycone skeletons were discussed in detail, and possible MS/MS fragmentation pathways were proposed for aiding the structural identification of these components. According to the summarized fragmentation patterns, these peaks were tentatively assigned by matching their empirical molecular formula with those of the published compounds, or by elucidating their quasi-molecular ions and fragment ions referring to available literature information when the reference standards were unavailable. As a result, 22 new steroid saponins were found in DZW for the first time. In addition, the quantitative analysis of the nine (except for the reference compounds A, B, and C) known peaks was accomplished at the same time which indicated that there was a great variability in the amount of these active compounds in different batches in the crude extracts. This approach could demonstrate that the fingerprint could be considered to be a suitable tool to comprehensively improve the quality control of DZW. The identification and structural elucidation of the peaks in the fingerprint may provide important experimental data for further pharmacological and clinical researches.

Determination of major components and fingerprint analysis of Flaveria bidentis (L.) Kuntze.

A sensitive high-performance liquid chromatography method coupled with photodiode array detection was developed for the simultaneous determination of six major constituents in Flaveria bidentis (L.) Kuntze: hyperoside, patuletin-3-O-glucoside, isorhamnetin 3-sulfate, astragalin, 6-methoxykaempferol-3-O-galactoside and α-terthienyl. The chemical fingerprint of Flaveria bidentis (L.) Kuntze leaves was established using raw materials of 12 batches in China. The chromatographic separations were obtained by using an Eclipse XDB-C18 reserved-phase column (150 × 4.6 mm i.d., 5 µm) using gradient elution with water (0.0125% trifluoroacetic acid, v/v) and acetonitrile at a flow rate of 1.0 mL/min, an operating temperature of 30°C and a detection wavelength of 360 nm. The new method was validated by linearity, limits of detection and quantification, precision, reproducibility, stability and recovery, and was also successfully applied to the simultaneous determination of components in Flaveria bidentis (L.) Kuntze. The results indicate that this multi-component determination method in combination with chromatographic fingerprint analysis is suitable for the quantitative analysis and identification of Flaveria bidentis (L.) Kuntze.

Rapid preparative separation of six bioactive compounds from Gentiana crassicaulis Duthie ex Burk. using microwave-assisted extraction coupled with high-speed counter-current chromatography.

A rapid method combining microwave-assisted extraction (MAE) and high-speed counter-current chromatography (HSCCC) was applied for preparative separation of six bioactive compounds including loganic acid (I), isoorientin-4'-O-glucoside (II), 6'-O-ß-d-glucopyranosyl gentiopicroside (III), swertiamarin (IV), gentiopicroside (V), sweroside (VI) from traditional Tibetan medicine Gentiana crassicaulis Duthie ex Burk. MAE parameters were predicted by central composite design response surface methodology. That is, 5.0 g dried roots of G. crassicaulis were extracted with 50 mL 57.5% aqueous ethanol under 630 W for 3.39 min. The extract (gentian total glycosides) was separated by HSCCC with n-butanol/ethyl acetate/methanol/1% acetic acid water (7.5:0.5:0.5:3.5, v/v/v/v) using upper phase mobile in tail-to-head elution mode. 16.3, 8.8, 12., 25.1, 40.7, and 21.8 mg of compounds I-VI were obtained with high purities in one run from 500 mg of original sample. The purities and identities of separated components were confirmed using HPLC with photo diode array detection and quadrupole TOF-MS and NMR spectroscopy. The study reveals that response surface methodology is convenient and highly predictive for optimizing extraction process, MAE coupled with HSCCC could be an expeditious method for extraction and separation of phytochemicals from ethnomedicine.