| Isodon rubescens (Hemsl.) Hara (labiatae) is a perennial herb that is native to the Yellow River valley and the Yangtze River in China. Isodon rubescens (formally named Rabdosia rubescens) is the entire dried plant of R. rubescens (Hemsl) Hara. It is a well-known traditional Chinese medicine (TCM), called "Dong Ling Cao" in Chinese. This herb has long been used as a folk remedy for respiratory and gastrointestinal bacterial infections, inflammation, and cancer. Phytochemical studies of Isodon rubescens have revealed that it contains diterpenoids, flavonoids, phenolic acids, triterpenoids and volatile oils, as well as other chemicals. Some preparations that are extracted from Isodon rubescens have existed for a long time, including anti-cancer tablets and buccal tablets for sore throat treatment. It has been reported that diterpenoids are the most important active constituents that contribute to the pharmacological efficacy of Isodon rubescens. However, for a large number of studies only in Isodon rubescens extraction and separation stages, and its quality control methods and pharmacokinetics of research are few.In this paper, the main components was isolated and purified in Isodon rubescens. HPLC-MS was performed to analyze chemical components in Isodon rubescens extract and biological samples. By HPLC-MS method, diterpenoids were characterized in crude extract of Isodon rubescens with the summarized fragmentation rules. Meanwhile, a novel sensitive and selective HPLC-ESI-MS/MS method was developed and validated to simultaneously determinate and identify constituents in Isodon rubescens samples. Then, a sensitive and selective LC-ESI-MS method for the simultaneous determination of diterpenoids in rat plasma was firstly developed and validated to analyze plasma samples of the four analytes after oral administration of Isodon rubescens. Finally, a HPLC-MS method was established for the quantification of diterpenoids in rat bile after oral administration of Isodon rubescens extract. At the same time, drug-protein binding and absorption kinetics of oridonin were studies. These results of this work will contribution to its development and utilization.Part one Studies on the chemical constituents of Isodon rubescensObjective: To isolate and purify the compounds in Isodon rubescens. by the technologies of Silica gel column chromatography, Sephadex LH-20 column chromatography, preparative TLC, HPLC and recrystallization and to characterize the structures of the isolated pure compounds by using the spectroscopic methods including MS, 1H-NMR, (13)C-NMR.Methods: The pulverized dried aerial parts of Isodon rubescens (10 Kg) were macerated for 7 days at room temperature with 95% alcohol for three times. The leachate was heated and activated carbon was added to absorb and then filtered and alcohol was evaporated in vacuum to yield the total crud extract of 340 g. Then, the crude extract was suspended in saturated brine and extracted successively with petroleum ether, trichloromethane, ethyl acetate and n-butyl alcohol in order to obtain four fractions: the petroleum ether fraction 8 g, the trichloromethane fraction 32 g, the ethyl acetate fraction 48 g and the n-butyl alcohol fraction 36 g. The ethyl acetate fraction and n-butyl alcohol fraction were applied to silica gel column chromatography for preliminary fractionation in turn; each fraction was monitored with TLC and the similar fractions were combined. The subfractions was combined and subjected to Silica gel column chromatography, preparative TLC and reversed phase preparative HPLC for further separation and purification to get pure compounds. The spectroscopic methods including NMR methods were used for the structural identification of these compounds.Results: 7 compounds were yielded by systemical separation of the aerial parts of Isodon rubescens. The structures of 7 compounds were identified on the basis of chemical and spectral analysis, including 1 diterpenoid, 5 phenolic acids and a phytostero. They were oridonin (1), caffeic acid (2), ferulic acid (3), protocatechuic aldehyde (4), salicylic acid (5), chlorogenic acid (6), β-sitosterol (7).Conclusion: The result indicated that the solvents and methods of extraction and isolation used in this experiment were practicable. Silica gel column chromatography, preparative TLC and HPLC were employed to isolate and purify the components of the aerial parts of Isodon rubescens, and spectroscopic methods were used to establish the structures of the compounds.7 compounds were obtained and identified with the aid of spectroscopic methods and chlorogenic acid was fistly obtained in this herb.Part two Screening and characterization of diterpenoids in Isodon rubescens using liquid chromatography coupled with hybrid triple quadruple linear ion trap mass spectrometryObjective: 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 diterpenoids in Isodon rubescens.Methods: First, we studied the mass fragmentation patterns of 16 diterpenoids standards in the negative ion mode by full scan mass spectra, product ion scan (PI) and precursor ion scan (PREC). On the basis of the summarized new rules, diterpenoids in a crude extract of Isodon rubescens were characterized by the combined use of the MIM-IDA-EPI and NL-IDA-EPI modes on a hybrid triple quadrupole-linear ion trap mass spectrometer, for the first time. The chromatographic conditions: Diamonsil C18 column (250mm×4.6mm, 5μm), and the column temperature was kept at room temperature. The mobile phase was composed of 0.1% aqueous formic acid (A) -method (B) with gradient elution.Results: Seven fragmentation rules were summarized. According to the summarized fragmentation rules, polarity rules of isomer, retention times, accurate molecular weights and characteristic fragment ions, 40 diterpenoids in crude extract of Isodon rubescens were characterized.Conclusion: In this study, a high sensitive, accurate and effective LC- MS method for on-line qualitative analysis of the trace diterpenoids in Isodon rubescens has been developed. This study has demonstrated the unprecedented advantage of the combination use of the MIM-IDA-EPI and NL-IDA-EPI mode. The MIM-IDA-EPI mode is sensitive and no pre-acquisition of MS/MS spectra of the parent ion due to the same precursor ion and product ion. Therefore, the characterization of the trace diterpenoids has become very easy and accurate by combination use of the two modes. It has played an important role in controlling the quality of medicinal herb and supplied a method for other trace diterpenoids compounds from natural and synthetic source in the future.Part three Simultaneous qualitative and quantitative analysis of 28 components in Isodon rubescens by high performance liquid chromatography-electrospray ionization tandem mass spectrometryObjective: To develop a novel qualitative and quantitative method using high performance liquid chromatography coupled with tandem mass spectrometry for simultaneous analysis of 28 components including 19 diterpenoids, 6 phenolic acids and 3 flavonoids in Isodon rubescens, an important traditional Chinese medicine.Methods: First, an information-dependent acquisition (IDA) method was employed to trigger product ion scans above the MRM signal threshold so that the 28 components could be identified through enhanced product ion (EPI) scans. Second, multiple-reaction monitoring (MRM) was employed in positive and negative mode at the same time in single analysis process and validated to simultaneously determinate and identify 28 constituents in 21 batches of Isodon rubescens. The instrument operated using electrospray ionization source in positive and negative mode simultaneously. The ion spray voltage was set to 5500V and -4500V, respectively. The turbo spray temperature was maintained at 600°C. Nebulizer gas (gas 1) and heater gas (gas 2) was set at 40 and 50 psi, respectively. The curtain gas was kept at 25 psi and interface heater was on. Nitrogen was used in all cases. The chromatographic separation was performed on a Diamonsil C18 column with linear gradient elution with 0.1% aqueous formic acid/methanol containing 0.1% formic acid at a flow rate of 0.7mL·min-1 for 15.1 min and the column temperature set at 25°C.Results: The linear relationships, linearity, precision, accuracy, limit of detection and limit of quantification of the method were good for the 28 components. And we successfully applied it to analyze 21 Isodon rubescens samples from different sources. The results demonstrated that a number of reasons such as plant origin, growth circumstance and harvest time might contribute to the differences in the level of active constituents among various Isodon rubescens samples. Plant origin and growth circumstance were principal reasons. These all suggested the quality of Isodon rubescens could be assured if locality and collecting time should be standardized.Conclusion: A novel sensitive and selective HPLC-ESI-MS/MS method operating both negative and positive scanning modes in single analysis process was developed and validated to simultaneously determinate and identify 28 constituents in Isodon rubescens samples. The satisfactory results demonstrated that the HPLC-MS/MS method offered a good alternative for routine analysis due to its rapidness, sensitivity and specificity and could be applied as a reliable quality evaluation method for Isodon rubescens. In the future, HPLC-MS/MS method would be more and more popular in analyzing herb medicine.Part four Simultaneous determination of lasiodonin, oridonin, ponicidin and rabdoternin A in rat plasma by liquid chromatography-electrospray ionization mass spectrometric method and its application to pharmacokinetic study of Isodon rubescens extractObjective: To develop a sensitive, specific and rapid liquid chromatography-mass spectrometry (LC-MS) method and validate for the simultaneous determination of lasiodonin, oridonin, ponicidin and rabdoternin A in rat plasma, using sulfamethoxazole as an internal standard (IS).Methods: Blood samples were collected into heparinized centrifuge tubes from the fossa orbitalis vein at 10, 30, 60, 90, 105, 120, 150, 180, 210, 240, 300 and 540min after single oral administration of Isodon rubescens decoction (10mL·kg-1). Within 30min after blood withdrawal, the samples were centrifuged at 4 000 rpm for 10min and the separated plasma samples were frozen in polypropylene tubes at -20°C prior to analysis. The plasma samples were pretreated by liquid–liquid extraction (LLE) with acetic ether and chromatographic separation was performed on a C18 column with linear gradient elution using water and methanol, which were both acidified with 0.1% aqueous formic acid, at a flow rate of 0.8mL·min-1.The detection was accomplished by multiple-reaction monitoring (MRM) scanning via electrospray ionization (ESI) source operating. Higher sensitivity was achieved by setting three scanning periods in a novel detection mode. The optimized mass transition ion-pairs (m/z) for quantitation were 365.3/347.3 for lasiodonin and oridonin, 361.2/343.2 for ponicidin, 363.2/283.1 for rabdoternin A, and 254.1/156.0 for IS. The total run time was 13.50 min between injections.Results: The calibration curves were linear over the investigated concentration range: 2.24～2240 ng·mL-1(lasiodonin), 4.92～4920 ng·mL-1 (oridonin), 5.32～5320 ng·mL-1(ponicidin) and 1.36～1360 ng·mL-1 (rabdoternin A), with all correlation coefficients higher than 0.998. The lower limits of quantitation (LLOQ) of these analytes were less than 1.36 ng·mL-1. The intra- and inter-day RSD were no more than 9.1% and the relative errors were within the range of -8.1% to 4.1%. The average extraction recoveries for all compounds were between 70.2 %-108.0%. The four analytes have parallel pharmacokinetic parameters in vivo, such as t1/2α, t1/2β and MRT. All of the four analytes were absorbed rapidly and eliminated quickly with the similar rate. But the T(max0 of lasiodonin, oridonin, rabdoternin A was longer than that of ponicidin.Conclusion: A sensitive and selective LC-ESI-MS method for the simultaneous determination of lasiodonin, oridonin, ponicidin and rabdoternin A in rat plasma was firstly developed and validated. The proposed method showed appropriate accuracy and repeatability and was successfully applied to a plasma samples analysis of the four analytes after oral administration of Isodon rubescens extract, which maybe provide some references to the apprehension of the action mechanism and clinical application of Isodon rubescens.Part five Quantitative analysis of ten diterpenoids in rat bile after oral administration of Isodon rubescens extract by high performance liquid chromatography-electrospray ionization tandem mass spectrometryObjective: A sensitive and selective HPLC–ESI–MS/MS method for simultaneous determination of ten major diterpenoids (effusanin A, lasiodonin, oridonin, epinodosin, nodosin, ponicidin, rabdoternin A, enmenol, lasiokaurin and lasiokaurinol) in rat bile. Using this method, the biliary excretion profiles of these diterpenoids were further investigated after a single oral administration of Isodon rubescens extract.Methods: Eight healthy rats were divided into experimental group and blank group. For experimental group, six rats were administered with Isodon rubescens extract at a single oral dosage of 10mL·kg-1, while the other two rats were administered with deionized water at an equal dose for blank. After these rats had been anesthetized, an abdominal incision was made and the common bile duct was cannulated with PE-10 tubing for the collection of bile samples. A heating lamp was used for maintaining the body temperature during the experimental procedure to prevent hypothermic alterations of the bile flow. Bile samples were collected during 0-1h, 1-3h, 3-5h, 5-8h, 8-12h, 12-20h and 20-24h periods. Blank bile samples were collected to check whether they were free of interfering components. All samples were stored at -20°C until additional extraction and analysis. A simple liquid-liquid extraction method was applied to extract the ten diterpenoids and IS from rat bile. The chromatographic separation was performed on a Diamonsil C18 column (250mm×4.6mm, 5μm), and the column temperature set at 25°C. A linear gradient elution of methanol and water was used for the separation. The analyses were performed using an electrospray ionization source in positive and negative mode respectively. Multiple–reaction monitoring (MRM) mode was carried out for obtaining the maximum sensitivity for the detection of the target compounds.Results: A rapid HPLC-ESI-MS/MS method was established for the simultaneous quantification concentrations of ten diterpenoids in bile. The cumulative biliary excretion of diterpenoids (effusanin A, lasiodonin, oridonin, epinodosin, ponicidin,) over the dose administered was 10.67, 11.23, 9.77, 20.81, 8.55%, respectively, while compound (nodosin, rabdoternin A, enmenol, lasiokaurin, lasiokaurinol)was 20.61, 17.22, 15.63, 8.87, 19.42%, respectively.Conclusion: The specificity, linearity, accuracy, precision, recovery, matrix effect and several of stabilities have been validated for diterpenoids in rat bile samples. The results showed that this method is robust, specific and sensitive and it can successfully fulfill the requirement of excretion study of the ten diterpenoids in Isodon rubescens. These amounts gradually decreased with time. At different times, bile excretion of these diterpenoids was the highest in 0-1h. These amounts gradually decreased with time. The concentration of these diterpenoids increased in 3-5h, which may be caused by these diterpenoids absorbed into the blood in different parts of the intestine, and then decreased gradually.Part six Drug-protein binding determination of oriodoninObjective: To develop a high performance liquid chromatography (HPLC) to determine the protein binding rates of oridonin in human plasma, rat plasma, bovine serum albumin (BSA), and to calculate the correlate parameters of oridonin to different genera plasma proteins.Methods: The binding rates of oridonin with different genera plasma proteins were determined by equilibrium dialysis method. The concentrations of oridonin were assayed by HPLC.Results: The binding rates of oridonin with rat, human plasma and BSA 69.66±12.8%, 59.62±12.6%, 57.94±4.1%, 78.15±3.6%, 77.92±8.8%, 76.72±7.3%, 35.58±7.2%, 34.59±10.8%, 32.03±6.0%, respectively.Conclusion: The equilibrium dialysis method was applied to study the the protein binding rates of oridonin. The binding rates of oridonin to human plasma protein, rat plasma protein and BSA were in middle level. They were a kind of middle plasma protein binding rate drug and most of the drug molecules act on body with free type. It would not made the obvious change of free fraction with pharmacological action that the protein binding of oridonin is not dependent on the doses.It is suggested that the above results may ensure to have safety of oridonin in clinic.Part seven Studies on absorption kinetics of oridonin in intestines in ratsObjective: To develop a high-performance liquid chromatography coupled with diode array detection (HPLC/DAD) method for simultaneous determination of oridonin and phenolsulfonphthalein in the circulation solution and to investigate the absorption kinetics of oridonin at different intestine segments of rats and the influence of the drug solution concentration on the absorption kinetics.Methods: The intestine in rats was cannulated for in situ recirculation. Oridonin concentration in the flux was measured by the reversed phase HPLC. The chromatographic procedure was carried out with Diamonsil C18 (250 mm×4.6mm, 5μm) as an analytic column and a mixture consisting of methanol- acetonitrile-0. 5% phosphoric acid(55:15:30)as mobile phase at the temperature of column of 30?C. The detection wavelength was set at 238 nm for oridonin and phenolsulfonphthalein and the flow rate was 1.0mL·min-1.Results: When the concentration was raised from 5.0 to 15.0μg·mL-1, the uptake of oridonin was increased linearly. Concentration had no effect on the permeability coefficient. The permeability coefficients of oridonin at duodenum, jejunum, ileum and colon were ( 0.0475±0.0062 ),(0.0468±0.0051),(0.0346±0.0037),(0.0435±0.0023)h-1, respectively.Conclusion: It is the first time to use sensitive, accurate, and simple HPLC/DAD method to determine oridonin and phenolsulfonphthalein in the circulation solution simultaneously. The absorption of oridonin in rat's intestine is a first-order process with the passive diffusion mechanism. Oridonin can be absorbed in whole intestinal segments. So oridonin sustained-released formulations can be prepared.
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