| Pulsatilla chinensis, the dried roots of Pulsatilla chinensis (Thunb.) Bercht. et Opiz., has been widely used as traditional Chinese medicine (TCM) for thousands of years in China. Modern pharmacological studies demonstrated that Pulsatilla chinensis extracts had a number of effects, such as hypoglycaemic, antitumour, cognition-enhancing, neuroprotective, cvtotoxic and antiendotoxin activities. Pulsatilla chinensis is known for containing a large number of compounds, including triterpenoidal saponins, phytosterone and anthocyanins. Among these, triterpenoidal saponins are generally considered to be the major components, and many of them have been found to have biological activities. In recent years, more and more triterpenoidal saponins have been isolated from Pulsatilla chinensis and they exhibit a variety of bioactivities.Therefore, quality control analysis of the active components is an important issue for the safe and effective use of herbal medicines and their preparations. The constituents in Pulsatilla chinensis are complex, and some of them usually are of low content. Therefore, a more sensitive, selective and rapid method is demanded. The emergence of high performance liquid chromatography connected with tandem mass spectrometry (HPLC-MS/MS) makes the determination possible.In this research, the nine main components were isolated and identified from Pulsatilla chinensis. A rapid, efficient and accurate HPLC-ESI-MS/MS method provided a new basis for overall assessment on quality of Pulsatilla chinensis and should be considered as a suitable quality control method. Then37triterpenoid saponins in Pulsatilla chinensis were identified by HPLC-ESI-MS/MS. Finally, a novel method was developed to determined triterpenoidal saponins in rat plasma and rat bile by HPLC-MS. At the same time, drug-protein binding and absorption kinetics of betulic acid were studies.Part one Studies on chemical components of Pulsatilla chinensisObjective:To study the chemical constituents of Pulsatilla chinensis.Methods:Air-dried herbal of Pulsatilla chinensis (10kg) were extracted by diffusion for three times with70%alcohol.The residue was pretreated by AB-8macroporous resin, and then collected the part of70%alcohol eluant. Finally we got the extractum for500g. The compounds were isolated and purified by macroporous resin, silicagel, dextrane gel and reversed-phase column chromatographies. Their structures were identified by various spectral data.Results:Nine compounds were isolated from the70%ethanol extract of Pulsatilla chinensis, including5triterpenoidal saponins,2triterpenic acid and2phytosterone. Their structures were elucidated as oleanolic acid, anemoside A3, anemoside B4,23-hydroxybetulinic acid, cirenshenoside S, pulsatilloside B, pulsatilloside C, ajugasterone C and (3-ecdysterone, respectively.Conclusion:Nine compounds were isolated and identified from Pulsatilla chinensis. Compounds5,8and9were isolated from Pulsatilla chinensis for the first time.Part two Quantitative determination of9components in Pulsatilla chinensis by HPLC-ESI-MS/MSObjective:The present study describes the development, validation and a practical application of a fully automated analytical method based on high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) for the simultaneous determination of7triterpenoidal saponins, anemoside B4(1), anemoside A3(2),23-Hydroxybetulinic acid (3), cirenshenoside S (4), pulsatilloside C (5), pulsatilloside B (6) and oleanolic acid (7), together with two other phytosterone named β-ecdysterone (8) and ajugasterone C (9) in Pulsatilla chinensis.Methods:A Sapphire C18(250mm×4.6mm,5μm) and gradient elution were used during the analysis. A binary gradient elution was performed:0min (75%A),0-6min (95%A),6-14min (95%A). Then quickly returned to initial75%A and maintained until21min for column balance. The identification and quantification of those analytes were achieved on a hybrid quadrupole linear ion trap mass spectrometer. Multiple-reaction monitoring scanning was employed for quantification with switching electrospray ion source polarity between positive and negative modes in a single run. The ion spray voltage was set to5500V and-4500V, respectively. The turbo spray temperature was maintained at500℃. Nebulizer gas (gas1) and heater gas (gas2) was set at40and50psi, respectively.Results:All calibration curves showed good linearity (r2>0.9948) within the test ranges. The intra-day and inter-day variations were less than2.78%and2.68%, respectively. The developed method was successfully applied to determine the investigated compounds in20samples of Pulsatilla chinensis.Conclusion:Compared with the reported analytical methods of Pulsatilla chinensis, this rapid, efficient and accurate HPLC-ESI-MS/MS method provided a new basis for overall assessment on quality of Pulsatilla chinensis and should be considered as a suitable quality control method.Part three Characterization of triterpenoid saponins in Pulsatilla chinensis using liquid chromatography coupled with hybrid triple quadruple linear ion trap mass spectrometryObjective:Eight triterpenoid saponins in crude extracts from Pulsatilla chinensis have been investigated by electrospray ionization multi-stage tandem mass spectrometry. To summarize fragmentation rules and develop a high sensitive and efficient liquid chromatography-mass spectrometry (LC-MS) method for detection and characterization of the trace triterpenoid saponins in Pulsatilla chinensis.Methods:The triterpenoidal saponins in a crude extract of Pulsatilla chinensis were characterized by the combined use of the MRM-IDA-EPI and PREC-IDA-EPI modes on a hybrid triple quadrupole-linear ion trap mass spectrometer. The chromatographic separation was performed on a Sapphire C18column (250mm×4.6mm,5μm) with linear gradient elution with methanol containing0.1%formic acid-0.1%aqueous formic acid at a flow rate of0.8mL·min-1. The ion spray voltage was set to5500V and-4500V, respectively. The turbo spray temperature was maintained at650℃. Nebulizer gas (gas1) and heater gas (gas2) was set at40and50psi, respectively. The curtain gas was kept at25psi and interface heater was on. Nitrogen was used in all cases.Results:According to the retention times, molecular weight and characteristic fragment ions, compounds6,10,14,18,20,21,35and36were identified anemoside B4, pulchinenoside C,23-hydroxy-lup-20(29)-en-28-oic acid3-0-[rhamnopyranosyl-arabinopyranosyl]28-0-[glucopyranoside], pulchinenoside B,23-hydroxy-lup-20(29)-en-28-oic acid3-O-[arabinopyranosyl]28-O-[glucopyranoside], hederagenin28-O-[glucopyranosyl-glucopyranoside], anemoside A3and hederagenin, respectively. Six fragmentation rules were summarized. Finally,29triterpenoid saponins in a crude extract of Pulsatilla chinensis were characterized.Conclusion:In this study, a high sensitive, accurate and effective HPLC-MS method for on-line qualitative analysis of the trace triterpenoid saponins in Pulsatilla chinensis has been developed. This study has demonstrated the unprecedented advantage of the combination use of the MRM-IDA-EPI and PREC-IDA-EPI mode. It will play an important role in controlling the quality of medicinal herb in the future.Part four Development of a novel method for triterpenoidal saponins in rat plasma by HPLC-MSObjective:A novel method using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) has been developed for the quantification of four triterpenoidal saponins (anemoside B4, pulsatilloside B, anemoside A3and23-hydroxybetulinic acid) in rat plasma following solid phase extraction (SPE).Methods:The optimized procedure utilized off-line extraction of the analytes from plasma using polymeric (Strata-X) SPE cartridges. Detection and quantitation was performed by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) in a novel multi-switching monitoring mode. The analytes and internal standard (scutellarin) were analyzed using a Sapphire C18column (250mm×4.6mm,5μm) with a linear gradient elution. The mass transition ion pairs of the triterpenoidal saponins were executed as the following:m/z1219.7/749.4for anemoside B4, m/z819.4/347.2for pulsatilloside B, m/z749.6/471.2for anemoside A3, m/z471.4/471.4for23-hydroxybetulinic acid and m/z461.1/285.0for the internal standard. the negative ionization mode was switched to the negative mode from0.0to6.998min to detect the IS and anemoside B4. Then, the negative ionization mode was switched to the positive mode from6.998to7.028min. Within the time range of7.028to10.027min, pulsatilloside B was detected in the positive ionization mode. Subsequently, the ESI converted back to the negative ionization mode once again from10.027to10.057min. In the last section, anemoside A3and23-hydroxybetulinic acid were detected in the negative mode from10.057to15.059min.Results:The specificity, linearity, accuracy, precision, recovery, matrix effect, and stabilities were validated for all analytes in the plasma samples.the four analytes showed good linearity over the low and high concentration range with r2>0.99. The LLOQ of the assay was defined as the lowest concentration of the standard curve that could be quantified accurately within a20%bias of the nominal concentration, with the precision not exceeding20%. Both the precision and accuracy of the four analytes at LLOQ were less than9%. The analytes in rat plasma were all stable for24h at room temperature, three cycles of freeze-thaw,30days at-20℃with accuracy in the range from-6.82to6.18%. At three concentration levels of these analytes, the extraction recoveries were all between82.16and104.5%. The matrix effects of this study were evaluated by analyzing samples at three concentration levels (low, medium and high), which were in the ranges of77.69-108.2%.Conclusion:A novel analytical approach, multiswitching monitoring mode, was presented that applies different ESI modes in a single HPLC ESI-MS/MS run. In addition, the use of polymeric (Strata-X) SPE cartridges has been applied to the off-line extraction of triterpenoidal saponins from rat plasma samples for the first time. Combining the rapid and efficient off-line drug SPE with the specific and sensitive multiswitching monitoring mode, a new, robust analytical method for the analysis of triterpenoidal saponins in rat plasma has been developed and validated. This validated method is a novel technique for sample preparation and quantitation and was successfully applied to estimate the pharmacokinetics of triterpenoidal saponins.Part five Quantitative analysis of six triterpenoidal saponins in rat bile after oral administration of Pulsatilla chinensis extract by high performance liquid chromatography-electrospray ionization tandem mass spectrometryObjective:The aim of this study was to develop a liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous quantitative determination of the following triterpenoidal saponins:anemoside A3, anemoside B4,23-hydroxybetulinic acid, pulsatilloside B, pulsatilloside C and cirenshenoside S in rat bile.Methods:The chromatographic separation was performed using a Sapphire C18column (250mm×4.6mm,5μm) and gradient elution was used during the analysis. Anemoside A3, anemoside B4and23-hydroxybetulinic acid was detected with the mass spectrometer in negative ion mode monitoring at m/z749.6/471.2,1219.7/749.4,471.4/471.4and pulsatilloside B, pulsatilloside C and cirenshenoside S were in positive ion mode monitoring at m/z819.4/347.2,965.5/493.2and1097.9/493.1, respectively. As an internal standard, scutellarin was used.Results:The calibration curves were indicative of good linearity (r2≥0.9973) in the range of interest for each analyte. Intra-day and inter-day precision (C.V.,%) was less than9.1%and9.0%, respectively. Accuracy was between-9.5%and9.2%. Recovery was80.03%～115.6%. The method is very rapid, simple, reliable and suitable for pharmacokinetic analysis. It can be routinely used for simultaneous determination of six triterpenoidal saponins in rat bile.Conclusion:These results indicated that the newly developed method can also be applied to studies after administration of extraction of saponins from Pulsatilla chinensis to rats. Part six Determination of plasma protein binding rate of betulic acidObjective:To develop high performance liquid chromatography (HPLC) method to determine the protein binding rates of betulic acid in human plasma, rat plasma and bovine serum albumin (BSA).Methods:Plasma protein binding rate studies were conducted by equilibrium dialysis method. The influence of drug concentration and plasma in different species on plasma protein binding rate were studied.Results:There was no significant difference in the plasma protein binding rates at low, middle and high betulic acid concentrations in dilution medium. The protein binding rate of betulic acid in human plasma was higher than in the rat plasma.Conclusion:Betulic acid has higher protein binding extent with both rat plasma and human plasma. Part seven Studies on Absorption Kinetics of Betulic acid in Rat’s IntestinesObjective:To investigate the in vivo absorption kinetics of betulic acid at different intestine segments in rats and develop a high performance liquid chromatography coupled with diode array detection (HPLC-DAD) method to determine the contents of betulic acid and phenolsulfonphthalein.Methods:The rat single-pass intestinal perfusion technique was applied. The effects of absorption site, drug concentration and pH value on betulic acid absorption were studied. The samples were separated on a Diamonsil ODS C18column (250mm×4.6mm,5μm), by a gradient elution using acetonitrile and0.1%v/v aqueous acetic acid as mobile phase at a flow rate of1.0mL·min-1. The column temperature was30℃Results:When the concentration was increased from75to125μg·mL the uptake of betulic acid in was increased linearly. The concentration had no effect on the permeability coefficient. The permeability coefficients of betulic acid at duodenum, jejunum, ileum and colon were (0.151±0.0049),(0.156±0.0056),(0.149±0.0083),(0.159±0.0041) h-1, respectively.Conclusion:Betulic acid has no specific absorption site in the intestine. The absorption mechanism of the drug conforms to passive transport mechanism and first-order kinetics. The bioavailability of betulic acid preparation can be increased by increasing the dissolution rate and the solubility. |
PDF Full Text Request