| The usage of Rhizoma Curcumae was first recorded in Yao Xing Lun in 627-649 B.C., the dry rhizomes derived from Curcuma phaeocaulis, Curcuma kwangsiensis and Curcuma wenyujin. The described functions were to remove blood stasis and to alleviate pain. Rhizoma Curcumae (rhizome of Curcuma; Ezhu in Chinese) is a common traditional Chinese medicine (TCM) that has been used to treat hepatitis, menstrual disorders, and epilepsy. The essential oils of Ezhu are considered as the active constituents, which have reported to possess antitumor, antiviral activities, antioxidation. According for most of the known pharmacological properties of essential oils in Rhizoma Curcumae, the major components within the essential oils are curcumol, ?-elemene, curdione and so on.About 20 species occur in China, it is recorded that it is easy to acquire Ezhu from Sichuan, Fujian, Zhejiang and Guangxi Province. Up to data, it has been only reported its pharmacological effects. Therefore, our point is to establish a method to identification and quantitation of different species of Curcuma Rhizomes. For further study in curdione, we will establish a method of pharmacokinetic studies in SD rats. Details as follows:1. Headspace-GC/MS combined with multivariate statistical analysis technique for identification of components in CurcumaCurcuma from different geographical locations were first analyzed and classified via HS-GC/MS combing with multivariate statistical analysis technique for the first time. A capillary column (30 m×0.32 mm×0.25μm) was used for separation. The column temperature was set at 50°C and held for 3 min for injection, then programmed at 20°C/min to 150°C and then held for 2°C/min to 200°C/min, hold 10 min. Split injection 1.5 ml was conducted with a split ration for 10:1 flow-rate and high-purity helium was used as carrier gas. The spectrometers were operated in electron-impact (EI) mode, the scan range was 50-550 ammu. The inlet, ionization source temperature were 250°C and 230°C, respectively. Finally, we have identified 68 components in 24 min. Combining with multivariate statistical analysis technique, our point is to distinguish the different species and find out the active ingredients of Curcuma.2. Supplementary identification of Curcuma by UPLC-TOF/MS and HPLC-QIT/MSAlthough we can use GC-MS to separate volatile components in curcuma, there are many similar molecular weights or same chemical composition in curcuma to hamper the analysis. UPLC-QTOF/MS technology was applied for the initial identification of chemical composition of curcuma. An Agilent Extend-C18 column (1.8μm, 100 mm×2.1 mm I.D.) was used for separation, solvents that constituted the mobile phase were A (acetonitrile) and B (0.1% formic acid). The column temperature was 30°C, flow rate 0.4 ml/min. he spectrometers were operated in ESI mode in positive ion mode, the scan range was 100-1100 m/z. The conditions of ESI source were as follows: drying gas (N2) flow rate 11 l/min; drying gas temperature 350°C; Nebulizer 50 psig; capillary voltage 4500 V; fragmentor voltage 150V. We have identified 12 components, and combing with HPLC-QIT/MS to get multiple stage crushing from standard substance of curcumol, germacrone and curdione. An Agilent ZORBAX SB-C18 column (3.5μm, 100 mm×2.1 mm I.D.) was used for separation, solvents that constituted the mobile phase were A (acetonitrile) and B (0.5% formic acid). The column temperature was 30°C, flow rate 0.3 ml/min. The work studied the mass spectrum characterization of the 3 compounds and speculated on the fragmentation pathways.3. Simultaneous determination of 5 biological active ingredients by GC-MSWe established a method to quantitation of five sesquiterpenes in different species of Ezhu and Yujin, such asа-pinene,β-elemene, curcumol, germacrone, curdione. The method is sensitive, stable, practical, and can be used for routine quality control of Curcuma. The calibration curves, which obtained from the selected ions peak area, forа-pinene, ?-elemene, curcumol, germacrone, curdione were linear over the range 0.0113-0.5631μg/ml, 0.2501-12.5061μg/ml, 0.5155-25.7339μg/ml, 3.3326-166.6294μg/ml, 1.9348-96.7398μg/ml, respectively. The coefficients of correlation(r) were between 0.9993-0.9997. Standard curve of 5 compounds were y = 0.3387x + 0.0036, y = 0.0636x - 0.0104, y = 0.0285x - 0.0148, y = 0.1138x - 0.0932, y = 0.1161x - 0.073. Thus, the quantitation of 5 compounds in Ezhu cound be performed fast, simple and effective.4. The Pharmacokinetics studies of curdioneIt has been reported that the other main component curdione have the pharmacological effects of anti-inflammatory analgesic. For further study curdione in vivo absorption, metabolism, we established a method of protein precipitation with methanol and UPLC-QTOF/MS to the pharmacokinetic studies in SD rats. An Agilent Extend-C18 column (1.8μm, 100 mm×2.1 mm I.D.) was used for separation, solvents that constituted the mobile phase were A (acetonitrile) and B (0.1% formic acid). The gradient elution conditions applied were 0-5 min, 40-55% B; 5-15min, 55-85% B. The column temperature was 30°C, flow rate 0.3 ml/min. The spectrometers were operated in ESI mode in positive ion mode, the scan range was 100-1100 m/z. The conditions of ESI source were as follows: drying gas (N2) flow rate, 11 l/min; drying gas temperature 350°C; nebulizer 50 psig; capillary voltage 4500 V; fragmentor voltage 150V. The linear range of components 0.12-12.26μg/ml, the precision and stability results are in line with requirements. Plasma samples of endogenous substances do not interfere with the determination of the peak, the material limit of quantification was 6.5 ng/ml. The method is simple, fast, reliable and suitable for the determination of biological samples, and successfully used in SD rats after oral administration and intraperitoneal injection The method can perform a follow-up guidance to the clinical use of drugs . |
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