| Radix Angelicae dahuricae and Radix glehniae, belonging to umbelliferae family, are commonly used traditional Chinese medicine and widely cultivated in Hebei. As reported previously, coumarins are generally considered as the most abundant components in both of herbs. In the past decades, there have been some preliminary researches about quality control, pharmacologic action and mechanism of action. In this paper, the chemical constituents, quality control and pharmacokinetics of Radix Angelicae dahuricae and Radix glehniae were systematically investigated in order to provide scientific evidence for the clarification of its active constituents and for the establishment of suitable quality control standard.The chemical constituents in 75% ethanol extracts of Radix Angelicae dahuricae were systematically investigated and six coumarins were isolated. The HPLC-MS-ESI method was developed for simultaneous qualitative and quantitative determination of 11 major coumarins in Radix Angelicae dahuricae. Simultaneous determination and pharmacokinetics study of five coumarins after oral administration of Radix Angelicae dahuricae extract in rats by HPLC. The analytical method of HPLC fingerprint of Radix glehniae was developed and validated. The HPLC method was developed to simultaneously determine the six major coumarins of Radix glehniae at 310 nm wavelength.This paper utilized the knowledge and technologies of phytochemistry, pharmaceutical analysis and pharmacokinetics and the methods of chemical constituents, quality control and pharmacokinetics for Radix Angelicae dahuricae and Radix glehniae were developed. The current research work provided a beneficial exploration for the modernization of traditional Chinese medicine.Part one Study on chemical constituents, quality control and pharmacokinetics for coumarins in Radix Angelicae dahuricae from Hebei ProvinceObjective: To establish a separation method of from Radix Angelicae dahuricae. 2. To establish a reliable and rapid HPLC-MS method for qualitatively identify and quantitatively determine the 11 major active coumarins in Angelica dahurica. 3. To develop and validate a simple, fast and reliable HPLC method detection for the determination of psoralen, bergapten, imperatorin, cnidilin and isoimperatorin in rat plasma.Methods: 1. Chemical constituents: After extracted with 75% alcohol, the extract was isolated and purified by silica gel column chromatography and preparative HPLC. Psoralen, bergapten, oxypeucedanin, imperatorin, cnidilin and isoimperatorin were identified by TLC, UV, HPLC, 1H NMR, 13C NMR and MS. 2. Assay: (1) Optimization of the extraction method: In order to obtain satisfactory extraction efficiency, extraction method, extraction solvent and extraction time were investigated. (2) Optimization of the chromatographic conditions: Chromatographic conditions such as mobile phase systems, MS conditions and analytical columns were tested for optimization of the chromatographic conditions. (3) Qualitative analysis of 11 coumarins: The 11 coumarins were identified by compared their retention time, quasi-molecular ions and fragment ions with standards. Fragmentation behaviors of coumarins in MS were investigated and applied to the structure characterization of similar compounds. (4) Methodology: calibration curves, precision, recovery and stability were determined. (5) Sample analysis: under above mentioned conditions, determine the contents of 11 coumarins in 12 batches of Radix Angelicae dahuricae. 3. Pharmacokinetics: The chemical constituents in 75% ethanol extracts of Radix Angelicae dahuricae was coarsely eluted by column chromatography on silica gel with petroleum ether-ethyl acetate (5:1,4:1,3:1,2:1,1:1). Fr. 5:1 and Fr. 3:1 was collected and suspended in 0.5% carboxymethylcellulose sodium. Radix Angelicae dahuricae extract was orally administered to the rats, and then blood samples were collected into heparinized centrifuge tubes from the via fossa orbitalis vein at 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 24 h. Sample was pretreated by a single-step protein precipitation with methanol. The analytical column was a C18 column, the column temperature was 30℃. The mobile phase for HPLC analysis consisted of acetonitrile-water using gradient elution with the flow rate of 1.0 ml/min. The detection wavelength was set at 310 nm and the sample injection volume was 60μl. Calibration curves for the five compounds in plasma were generated by plotting the peak area versus those nominal concentrations in standard plasma. The calculation of pharmacokinetics parameters was according to the pharmacopoeia of China (2005 edition).Results: 1. Chemical constituents: 6 compounds were separated from Radix Angelicae dahuricae and identified as: psoralen, bergapten, oxypeucedanin, imperatorin, cnidilin and isoimperatorin. The purity of six compounds was more than 98% by normalization method of HPLC. 2. Assay: (1) Optimization of the extraction method: The dried powders of Radix Angelicae dahuricae samples were accurately weighed and extracted by ultrasonic with 75% methanol solution for 30 min. The extraction method of the major chemical components of Radix Angelicae dahuricae was efficacy and stable. (2) Optimization of the chromatographic conditions: Chromatographic separation was carried out using Waters SunFireTM C18 column (150 mm×4.6 mm, 5μm) at 25℃. The flow-rate of mobile phase was maintained at 0.8 ml/min and the injection volume was 5μl. The mobile phase was methanol/0.1% formic acid water (75:25, v/v). The HPLC system was connected to 3200 Q TRAP LC/MS/MS System, a hybrid triple quadrupole/LIT (linear ion trap) mass spectrometer equipped with an ESI ion source (Applied Biosystems/MDS Sciex, USA). The temperature was set at 400℃in the positive ion mode with a ionSpray voltage 5500 V. Nitrogen gas was used as nebulizer gas (40 psi), auxiliary gas (40 psi), collision gas (Medium), curtain gas (20 psi). Detection was operated in the multiple reaction monitoring (MRM). The instrument control and data acquisition were carried out by the analyst 1.4.2 software. (3) Qualitative analysis of 11 coumarins: The positive-ion mode was found to be more sensitive. The 11 coumarins exhibited their quasi-molecular ions [M+H]+, [M+Na]+, [M+NH4]+, [M+K]+ and fragment ions [M+H-CO]+, [M+H-C5H9O]+, [M+H-C5H8]+, [M+H-C5H8-CO]+, [M+H-C5H8-CO2]+, [M+H-CH3]+. (4) Methodology: The calibration curves of 11 coumarins have good linearity (r > 0.99). Concentration range : 340-10880,62.4-1996,216-6912,45.4-1452,19.8~635,222~7104,1680~53760,1330~42560,744~23808,2.7~87.6,735~23520 ng/ml. The intra-day and inter-day precisions calculated as the relative standard deviation (R.S.D.) were within the range of 1.2% to 4.5% and 0.4% to 4.0%. The coumarins showed the overall recoveries ranging from 92.1–105.6%. The analytes were found to be very stable in 75% methanol solution within 24 h. (5) Sample analysis: The results showed that content of the total coumarins fell in the range 132.8~589.3μg/g. 3. Pharmacokinetics: Calibration curves of psoralen, bergapten, imperatorin, cnidilin and isoimperatorin were generated over the range 26.0~833.3,28.1~897.8,74.9~2396.2,48.0~1536.1 and 45.5~1360.1 ng/ml, respectively. The intra-day and inter-day precisions calculated as the relative standard deviation (RSD) were 2.7%~14.8% and 0.1%~14.5%. The accuracies of five coumarins were within the range of 97.1–114.6%. The mean extraction recoveries were within the range of 87.1–109.2%. The main pharmacokinetics parameters of five coumarins were as follows: Cmax: 811.4 ng/ml, 588.9 ng/ml, 2070.2 ng/ml, 753.8 ng/ml, 870.9 ng/ml. Tmax: 4.0 h, 4.0 h, 5.0 h, 5.0 h, 5.0 h. T1/2: 8.57 h, 6.13 h, 6.47 h, 6.86 h, 6.91. k: 0.0809 1/h, 0.1130 1/h, 0.1070 1/h, 0.1010 1/h, 0.1003 1/h. AUC0?t: 4157.4 ng h/ml, 2777.6 ng h/ml, 11322.1 ng h/ml, 3948.5 ng h/ml, 5110.8 ng h/ml. AUC0?∞: 4791.8 ng h/ml, 2918.3 ng h/ml, 12037.4 ng h/ml, 4210.0 ng h/ml, 5323.6 ng h/ml.Conclusion: 1. Chemical constituents: The developed method is simple and at low production cost. The products can be used as reference substance for quality control and pharmacokinetics study. 2. Assay: The developed method was simple, sensitive and reproducible. It demonstrated that qualitative and quantitative analysis in plant material and commercial products was of great importance and it could be used for the comprehensive evaluation of Radix Angelicae dahuricae. 3. Pharmacokinetics: The present study described a simple and reliable HPLC method for the simultaneous determination of psoralen, bergapten, imperatorin, cnidilin and isoimperatorin in blood samples. It was successfully applied to the pharmacokinetics studies of active components of Radix Angelicae dahuricae. The method with high sensitivity, specificity, good precision and accuracy was fit to the needs of bio-sample analysis. Part two Studies on fingerprints and simultaneous determination of six coumarins in Radix Glehniae from Hebei ProvinceObjective: To establish HPLC fingerprints of Radix Glehniae from Hebei province and get reference fingerprint, compare the fingerprints of Radix Glehniae collected from different producing areas. To establish a method for simultaneous determination of 6 major coumarins in Radix Glehniae, namely psoralen, xanthotoxin, bergapten, imperatorin, cnidilin and isoimperatorin.Methods: 1. Fingerprints: (1) Optimization of the extraction method: In order to obtain satisfactory extraction efficiency, extraction method, extraction solvent and extraction time were investigated. (2) Optimization of the chromatographic conditions: Chromatographic conditions such as mobile phase systems, wavelength and column temperature were tested for optimization of the chromatographic conditions. (3) System suitability test: Under the above conditions, resolution and theoretical plate number the peak of psoralen was calculated. (4) Methodology: Precision, reproducibility and stability were determined. (5) Establishment of fingerprint: Prepare each sample solution of Radix Glehniae, and get their relevant fingerprints. 2. Assay: (1) Optimization of the extraction method: In order to obtain satisfactory extraction efficiency, extraction method, extraction solvent and extraction time were investigated. (2) Optimization of the chromatographic conditions: Chromatographic conditions such as mobile phase systems, wavelength and column temperature were tested for optimization of the chromatographic conditions. (3) System suitability test: Under the above conditions, resolution and theoretical plate number the peak of xanthotoxin was calculated. (4) Methodology: calibration curves, precision, recovery and stability were determined. (5) Sample analysis: Under above-mentioned conditions, determine the contents of 6 coumarins in 19 batches of Radix Glehniae.Results: 1. Fingerprints: (1) Optimization of the extraction method: The dried powders of radix glehniae samples were accurately weighed and extracted by ultrasonic with 75% methanol solution for 30 min. (2) Optimization of the chromatographic conditions: The analytical column was DiamonsilTM C18 (250 mm×4.6 mm I.D., 5μm). The flow rate was 1.0 ml/min and the column temperature was 30℃. The detection wavelength was set at 295 nm and the sample injection volume was 20μl. The mobile phase for HPLC analysis consisted of methanol-acetonitrile-H3PO4 (0.05%) using gradient elution. (3) System suitability test: Under above conditions, the peak of psoralen was separated well with the resolution of more than 1.5 and about 6.0×103 of theoretical plate. (4) Precision: Designating the major peaks for reference peak, the RSD values of relative retention time and peak areas were less than 0.9% and 1.5%. Reproducibility: Designating the major peaks for reference peak, the RSD values of relative retention time and peak areas were less than 0.3% and 2.4%. Stability: Designating the major peaks for reference peak, the RSD values of relative retention time and peak areas were less than 0.3% and 2.2%. (5) Establishment of fingerprint and data analysis: We have got 17 common peaks in reference fingerprint from 13 batches of Radix Glehniae. Used software for similarity analysis, the results showed that the similarities of 13 batches of samples from Hebei province were above 0.9. There was significant difference among samples which collected from Hebei province and different sources. The similarities were above 0.1~0.4. 2. Assay: (1) Optimization of the extraction method: The dried powder of radix glehniae samples was accurately weighed and extracted by ultrasonic with 75% methanol solution for 30 min. (2) Optimization of the chromatographic conditions: The analytical column was UltimateTM C18 (250 mm×4.0 mm I.D., 5μm). The flow rate was 1.0 ml/min and the column temperature was 30℃. The detection wavelength was set at 310 nm and the sample injection volume was 20μl. The mobile phase for HPLC analysis consisted of acetonitrile-water using gradient elution. (3) System suitability test: Under above conditions, the peak of xanthotoxin was separated well with the resolution of more than 1.5 and about 8.0×103 of theoretical plate. (4) Methodology: The calibration curves of 6 coumarins have good linearity (r>0.99). Concentration range: 0.52~10.42, 0.89~17.84, 0.32~6.40, 0.26~5.27, 0.05~0.96, 0.17~3.49μg/ml. The intra-day and inter-day precisions calculated as the relative standard deviation were within the range of 0.2%~2.7% and 0.3%~1.7%. The coumarins showed the overall recoveries ranging from 91.7%–107.6%. The analytes were found to be very stable in 75% methanol solution within 24 h. (5) Sample analysis: The results showed that content of the total coumarins fell in the range 338.3~1353.2μg/g.Conclusion: 1. Fingerprints: The analytical method of HPLC fingerprint of Radix Glehniae was developed and validated. Relative retention system and the software for similarity analysis were applied to evaluate the variability of those fingerprints from different sources. The method could be able to effectively control the quality of Radix Glehniae. 2. Assay: In this paper, a rapid, reliable and sensitive method was developed for the determination of bioactive constituents in by HPLC-DAD. It was the first time to report the simultaneous quantification of 6 major coumarins in Radix Glehniae. The newly established method could be applied as a reliable and sensitive quality control procedure for radix glehniae.
|
PDF Full Text Request