| Rauvolfia verticillata is the roots of plants, belongs to Rauvolfia genus of Apocynaceae family, mainly produced in China, India, Vietnam, with antihypertensive and heat-clearing and detoxicating effect. The chemical constituents, pharmacological activity, analytical methods of the roots of Rauvolfia, and the pharmacokinetics of the active compound, ajmalicine were investigated in this study.The chemical constituents of 75% ethanol of Rauvolfia were isolated by using multiple column chromatographic techniques, and 30 compounds were obtained. The structures of 19 compounds were fully elucidated by chemical and spectroscopic methods (MS, UV, NMR). Among them, two are new compounds, rauvox A (4), which is the acridone alkaloid isolated from Rauvolfia genus for the first time, and rauvox B (10); two compounds were isolated from Rauvolfia genus for the first time:seselin (1) and xanthyletin (2), which are coumarins compounds isolated from Rauvolfia genus; eleven indole alkaloids compounds:sandwicine (5), reserpine (6), raunescine (7), yohimbine (14), a-yohimbine (9),3-epi-a-yohimbine (11), ajmalicine (8), ajmaline (16), serpentine (17), sarpagine (18), harmane (15); two steroidal compounds:daucosterol (19) andβ-sitoterol (12); one lignans compound:syringaresinol (3); one phenolic acid esters compound:methyl 3,4,5-trimethoxycinnamate (13).The following activities of compound 4 were investigated:1. The antibiotic activities of compound 4 of various concentrations against (Staphyloccus aureus) CMCC(B)26003, (Bacillus subtilis)CMCC(B)63501,(Escherichia coli)CMCC(B)44102, (Candida albicans) CMCC(F) 98001, (Aspergillius niner)CMCC(F)98003 were investigated by liquid dilution method, the results indicated that compound 4 had weak antibacterial activities to Bacillus subtilis and Staphyloccus aureus, but the value of MIC was too high. The others were no antimicrobial active.2. We investigated the relaxant effect of compound 4 on isolated small intestinal smooth muscle of rabbits using DMSO as the blank control and loperamide as positive control. The result showed that compound 4 can reduce the tension and amplitude cause of spontaneous contractions of isolated small intestinal smooth muscle of rabbits dose-dependently; 3. We investigated the vitro antitumor activity of compound 4 to human breast tumor lines MCF-7 and human promyelocytic leukemia HL-60 using MTT method, the result showed that compound 4 decreased MCF-7 cell proliferation in a statistically IC50 significant manner at 201.1μmol/L, however, compound 4 was shown to be inactive against human promyelocytic leukemia HL-60, even the dose has been set at 300μmol/L, so the IC50 value wasn’t calculated. The chemical compositions of Rauvolfia were studied systematically through the following method:1. High performance liquid chromatography with diode array detector and time of flight tandem mass spectrometry (HPLC-Q-TOF/MS) was applied to study the alkaloids components of Rauvolfia. The analysis was performed on a Diamonsil C18 column using gradient elution with the mobile phase consisting of 0.1% formic acid in acetonitrile and water (pH 2.3), the flow rate was 1.0 mL/min. The detection was performed under ESI positive mode. Standards, references and other methods were used to identify the 22 alkaloids of Rauvolfia; 2. A simple and sensitive gas chromatography tandem mass spectrometry (GC-MS) method was developed to study the volatile constituents of Rauvolfia. The seperation of the 39 volatile compounds of Rauvolfia was conducted on DB-1 quartz capillary column with programmed column temperature, and the identification was based on the retention time and MS library in the computer. The relative content of common peaks was more than 94.6% of the total content of the volatile constituents in Rauvolfia by area normalization. Research on the chemical constituents of Rauvolfia by liquid chromatography and gas chromatography complemented previous studies of the indole alkaloids only and revealed the structures of the compounds compositively, which was made for the recognization of the pharmacological effect and the development of new efficacy of Rauvolfia.In order to improve the quality control standard of Rauvolfia, the following research were performed:1. The 18 elements related to hypertension in different plant parts of Rauvolfia were determined by inductive coupled plasma emission spectrometer (ICP-OES). The samples needed to be digested in nitric acid-perchloric acid at 230℃on the heating pad before measured. The relationship between the content of elements and hypertension may reveal the antihypertension principle of Rauvolfia. The method was simple and accurate, which could be used in the determination of elements in traditional Chinese medicine. The results showed that, although the quite different elemental contents in different parts, the contents of Mg, K, Ca are the largest, and the three elements have a strong antihypertensive effect, which further proved that Rauvolifa has the efficacy of the treatment of hypertension; 2. A simple, accurate and sensitive GC method to determine 11 residue pesticides with ECD detection was developed and validated for the research on pesticide residues in Rauvolfia. The pesticides were extracted from plant samples by ultrasonic extraction and column extraction. Chromatographic separation was conducted on OV-1701 quartz capillary column (30 m×0.32 mm,0.45μm). The research provided the theoretical basis for the safety monitoring and quality control of Chinese herbal medicines.Indole alkaloids are main active compounds in Rauvolfia. A specific, simple and sensitive HPLC method with UV detection was developed and validated for simultaneous quantitative determination of five indole alkaloids in Rauvolfia. The analysis was performed on Diamonsil C18(250 mm×4.6 mm i.d.,5μm, Dalian Zhonghuida, China) using gradient elution with the mobile phase methanol and water. The flow rate was 1 mL/min. The calibration curve of the five indole alkaloids showed good linearity. The method has been successfully applied to quantitative determination of Rauvolfia from four different areas. The results showed that the alkaloids content is different because of the resources. The content of reserpine and ajmalicine in Yuanyang, Yunnan and yohimbine, sarpagine and ajmaline in Jinghong, Yunnan are higher than others.Ajmalicine is an active compound of Rauvolfia with antihypertensive effect. A HPLC method was developed and validated for pharmacokinetic study of ajmalicine in rat plasma. Ajmalicine, with osthole as the internal standard, was extracted from plasma samples by protein precipitation liquid-liquid extraction. The analysis was carried out on a Hypersil-C18 column (250 mm×4.6 mm id,5μm) with mobile phase of methanol and water (70:30, v/v). The validated method was successfully applied to a pharmacokinetic study in rats after vena caudalis administrations of dose of the extract of Rauvolfia. Ajmalicine distributed and eliminated in rats rapidly, and its biological half-life is 45 min.In conclusion, by utilizing various professional knowledge and analytical techniques, the constituents in Rauvolfia were investigated, quality control methods based on HPLC and GC were established, the antibiotic activity of compound 4 was determined and the pharmacokinetic study on ajmalicine in Rauvolfia was conducted. This study provided useful information for the exploration of active constituents and utilization of Rauvolfia. |
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