Study On Chemical Constituents Of The Plantaginis Semen

Plantaginis Semen is dried and mature seeds of Plantago asiatica L. or P.depressaWilld. It is widely used in traditional Chinese herbal medicine which is clearing heat,diuresis, treating stranguria, improving eyesight and eliminating phlegm. Thismedicine has effect on relieving pain, and it treats heat stranguria. At the same time itcan cure edema, diarrhoea, hot eyes, distending pain and sputum and heat cough.Thechemical constituents of Plantaginis Semen are mainly phenylethanoid glycosides,iridoids,flavones, polysaccharide etc.In this experiment, Plantaginis Semen is extracted by80%ethanol and the ethanolextraction was separated and purified by silica gel, ODS column chromatography andrecrystallization. Finally,8compounds were isolated and identified by MS and NMRas adenosine, cannabiside B, rhoifolin, ethyl--glycoside,1-Octen-3-ol3-O-β-D-xylopyranosyl(1â†'6)-β-D-glucopyranoside,geniposidic acid, apigenin-7-O--glucoside and acteoside，among which， adenosine, cannabiside B, ethyl—glycosidewere gain from Plantaginis Semen for the first time.This experiment also established a HPLC method to determin the contents ofadenosine.1. Extraction, isolation and structural identification of the ChemicalConstituents1.1ExtractionTen kilograms of Plantaginis Semen were extracted by heat reflux for threetimes in80%ethanol. The ratio of80%ethanol to raw material is10,8,8times(V/W)in turn. The extracting solution was combined after filerted. When the solvent wasevaporated by rotary vaporization under reduced pressure, we obtain crude extract ofPlantaginis Semen.1.2Isolation The crude extract of Plantaginis Semen are separeted by silica gel column witheluents I. After this separation,we get seven fractions marked A,B,C,D,E,F,G.1) Fraction C was subjected on silica gel column chromatography and eluted by ethylacetate-ethanol–water, and then were purified by ODS column chromatography fortwo times before CQZ-8was purified.2) Fraction D was separated into D1, D2,D3by silica gel column chromatogra-phy and eluted by eluentsâ…¢. Compound CQZ-1and CQZ-2were obtained from D2and D3by recrystallization. Compound CQZ-3and CQZ-5were purified from D1byODS column chromatography.3) Fraction E were performanced on silica gel column chromatography with eluentâ…¥ eluting, two parts named E1and E2were separated. Compound CQZ-4wasobtained from part E1by silica gel column chromatography and eluted by eluent â…¦,and ompound CQZ-6isolated from part E2by silica gel column chromatographyeluted by eluent â…§.4）Compound CQZ-7was isolated and purified by silica gel column and ODSchromatography for many times.1.3Structural identificationThe seven monomers are identified by MS and NMR spectra as adenosine,cannabiside B,rhoifolin, ethyl-glycoside,1-Octen-3-ol3-O-β-D-xylopyranosyl-β-D-glucopyranoside,geniposidic acid, apigenin-7-O—glucoside and acteosiderepectively.2. Content determination of adenosine in Plantaginis Semen byHPLC2.1Equipment and Chromatographic conditionsChromatography instrument: Agilent1200HPLCColumn: Prevail C18（250mm×4.6mm，5mm）Conditions:Mobile phase: Acetonitrile-water(5:95);Flow rate:1.0mL/min; Column temperature:25℃;Detection wavelength:255nm.Injection volume:20μL;2.2Methodology Investigation2.2.1Linear relationThe peak areas (S) and the sample concentration (X) was in a good linear rela--tionship within the range of0.012-1.21μg of adenosine. Linear regressionequation and correlation coefficient is showed as follows:y=3211x-6.5467, R2=0.99902.2.2Limit of detection and quantificationDilute the solution of adenosine, until the corresponding S/N ratio are3.0and10.0respectively. Detection limit of CQZ-1was13ng and quantification limit was40ng.2.2.3PrecisionThe solution of the adenosine is measured by HPLC. The result showed thatintraday and interday RSD of peak area were0.13%and0.20%respectively. Thisshowed the equipment is in good precision.2.2.4RepeatabilitySix solutions were measured and the RSD was1.34%, which showed theexperiment in good precision.2.2.5RecoveriesThe average recovery of adenosine is99.85%and the RSD was0.90%. The RSDis less than2%, so the recoveries are good.3. Contents determinationThe average contents of adenosine are50.3,44.8,46.9μg/g in Plantaginis Semenproduced in differents regions.4. SummaryWe study deeply on the chemical constituents of Plantaginis Semen in this thesis.Eight compounds are isolated and identified，among which，adenosine, cannabiside B, ethyl—glycoside were first discovered. Adenosine is analyzed quantitatively byHPLC. This study further improved the establishment of quality standards, andprovided favorable scientific basis for research, development of Plantaginis Semen.