Study On Chemical Constituents From The Ipecac

Ipecac is defined as the dried roots of either Cephaelis ipecacuanha A.Richard orCephaelis acuminata Karsten belonging to the family Rubiaceae.Eight compounds were isolated from the ipecac (Cephaelis ipecacuanha (Brot.)A.Rich.), according to the analysis of physicochemical properties of compounds andspectral data, they were identified as emetine (TG-1), O-methylpsychotrine (TG-2),ursolic acid (TG-3),1,6-anhydro-β-D-Glucose (TG-4), β-sitosterol(TG-6),β-daucosterol (TG-7), cephaeline (TG-8). The compound ursolic acid and1,6-anhydro-β-D-Glucose were obtained from the medicinal plant for the first time.The experiment also established the HPLC method to determine the content ofo-methylpsychotrine in ipecac, this method has the features of high precision, goodsensitivity and high selectivity.1. Extraction, isolation and structural identification of chemicalcomponents1.1ExtractionSeventeen kilograms of dry ipecac were crushed into powders (20mesh) andloaded into chromatographic column,then percolated by ethyl acetate solution，whichdiethylamine was added and adjusted the pH at9-10. Thin layer chromatography（TLC）was used to detect extraction progress. The percolate was combined beforethe solvent was evaporated by rotary vaporization under reduced pressure, then850gcrude extract of ipecac was obtained.1.2The separation and purificationThe extracts were subjected to silica column chromatography. The eluent waspetroleum ether-acetic ether-diethylamine-water (2.0:1.0:0.4:0.1). Combining withthin layer chromatography as the inspection method, the same components weremerged and concentrated. Finally five parts (part1-5) were obtained. The second part was subjected to silica column chromatography. The eluent waspetroleum ether-acetic ether-diethylamine-water (7:1:0.1:0.05）. Part2-1,2-2and2-3were obtained. The three parts subjected to silica column chromatography again andrecrystallized. Finally compound TG-1(150mg), TG-2(600mg), TG-8(450mg) wereobtained respectively.The part third was subjected the silica column chromatography. The eluent waspetroleum ether-acetone-water (6:1:1). Part3-1,3-2,3-3and3-4were obtained. Thefour parts were subjected into silica column chromatography, ODS columnchromatography and recrystallized. Finally compound TG-3(20mg), TG-4(280mg),TG-5(800mg) and TG-6(1.2g) were obtained respectively.The part5th was subjected into the silica column chromatography andrecrystallized before the TG-7(1.5g) was obtained.1.3Structural identificationBased on the physical and chemical properties of the compounds, nuclearmagnetic resonance spectroscopy (NMR) and mass spectrometry (MS) data, thechemical structures of eight compounds were identified after compared with theliteratures. They were emetine (TG-1), O-methylpsychotrine (TG-2), ursolic acid(TG-3),1,6-anhydro-β-D-Glucose (TG-4), psychotrine (TG-5), β-sitosterol (TG-6),β-daucosterol (TG-7), cephaeline(TG-8).2. The determination of the content of O-methylpsychotrine(TG-2) in Ipecac by HPLC2.1Instruments, chromatographic conditions and referencesubstanceChromatograph-Detector: Agilent1200HPLC DAD Detector;Column: Zorbax Extend-C18(4.6mm*250mm,5μm);Mobile phase: acetonitrile-triethylamine aqueous solution (0.05%)=40:60; Flow rate:1.0mL/minColumn temperature:25℃Detection wavelength:310nmReference substance: O-methylpsychotrine (TG-2) was isolated from ipecac andits purity was99.5%determined and normalized by HPLC method.2.2Methodological InvestigationI Intra-day precision testReference solution of TG-2were injected into HPLC for six times during one day,the results showed that RSD of the peak area of o-methylpsychotrine were0.84%.Theexperimental results show that the intra day precision was good.II Inter-day precision testReference solution of TG-2was injected into HPLC once a day for six days. TheRSD of the contents of compound TG-2was1.72%.The experimental results showthat the intra day precision was good.III Limit of detection and quantificationUnder the experimental condition, the detection limit of TG-2was0.805ng andthe limit of quantification was16ng.IV Linear relationship testIn the concentration range of0.00805~1.6mg/mL for compound TG-2(X) andpeak area(Y) showed a good linear relationship. Linear equation wasY=12673X+113.04, R2=0.9999.V Repeatablity testThe same sample was determined by established HPLC method for six times.The RSD of the contents of compound TG-2was1.3%, showing that the method wasreproducible.VI Stability testWithin10hours, The same sample solution was injected into the HPLC onceevery two hours. The RSD of the contents of compound TG-2was1.9%, visible forthe test solution was stable within10hours. VII Recovery rate testThe average recovery rate of TG-2is97.16%and its RSD was1.3%, so therecovery of this method was good.2.3Determination of the content of TG-2in the sample of ipecacThe average content of TG-2in ipecac was0.014%.3ConclusionThis experiment studied the chemical constituents of ipecac and established amethod for determination of the content of O-methylpsychotrine in ipecac by HPLC.Our results provided new scientific basis for the development, utilization andquality standard establishment of ipecac.