Study On Chemical Constituents Of The Zingiberis Rhizoma

Zingiberis Rhizoma is the dried roots of Zinger officinale Rosc,which is herbalmonocotyledon grow all the year round.Zingiberis Rhizoma, as a kind of widely used traditional Chinese medicine,hasbeen included in the “Pharmacopoeia of the People’s Republic of China”. It is statedthat Zingiberis Rhizoma can be introduced into the effective treatment of stomachdisease, inflammation,central nervous,vomiting,antioxidant function and stroke etc.The objective of this research is to systematically isolate and determine thechemical composition of Zingiberis Rhizoma. Five compounds were isolated fromthe ethanol extract of Zingiberis Rhizoma, the chemical structures of which wereidentified by spectral methods such as NMR,MS,UV and IR, combining with theirphysical and chemical properties. The five isolated compounds are identified asmethyl-6-gingerol,6-shogaol,β-eudesmol,2,5–dihydroxybisabola-3,10-diene and4-gingerol. Among them, the monomer compounds of methyl-6-gingerol, β-eudesmoland2,5-dihydroxybisabola-3,10-diene are first isolated from the Zingiberis Rhizoma.In this paper, HPLC was used to establish the quantification analysis method ofthe methy-6-gingerol, the content of which were also determined on three batches ofsamples of Zingiberis Rhizoma.1. Extraction, isolation and structural identification1.1Extraction and isolationThirty kilograms of Zingiberis Rhizoma were extracted by heat reflux in95%alcohol(150L,100L,100L, respectively)for three times(1.5h,1h,1h,respectively).Theextraction was subjected to silica gel and ODS column chromatography and elutedrepeatedly to afford compoundsⅠ(1.2g),Ⅱ(500mg),Ⅲ(460mg),Ⅳ(90mg),Ⅴ（1.5g)。1.2IdentificationOn the basis of the physical and chemical properties with various spectralmethods such as UV,IR,MS and NMR, all the isolated compounds were identified as methyl-6-gingerol,6-shogaol, β-eudesmol,2,5-dihydroxybisabola-3,10-dieneand4-gingerol.2. The Determination of methyl-6-gingerol2.1Equipments,reagents,control samples and reference substancesChromatograph:Agilent1200Series HPLC(UV detector);Column: Agilent Zorbax SB-C-18(4.6mm×250mm,5μm)Mobile phase:methanol-H2O(56:44)Flow rate:1.0mL/minChromatography column temperature:30℃Wave length:208nmReference substance::methyl-6-gingerol(99.5%)2.2Determine of methyl-6-gingerol in Zingiberis Rhizoma2.2.1Intra-day precision testThe solution of methyl-6-gingerol with the same volume was injected into theHPLC continuously for six times in one day. It was shown that the RSD(n=6) was0.41%for the peak area and1.25%for the retention time.2.1.2Inter-day precision testThe same solution was injected into the HPLC once a day for six days and theresults showed that the RSDs(n=6) of peak area and retention time were0.21%and0.33%respectivelyt2.2.3The Linear testIn the range of0.01-5.0μg, the linearity of compound methyl-6-gingerol were good.The regression equations of methyl-6-gingerol were y=3721.1x-49.487with itscoefficient of correlation at0.9999.2.2.4Repeatability testSix copies of the sample solution were injected into HPLC and the RSD of thecontent of methyl-6-gingerol was0.59%。2.2.5Stability testThe same sample solution was injected into the HPLC once every two hours during12h.The RSDs of peak area and retention time were0.30%and0.50%respectively. 2.2.6LOD and LOQThe LOD and LOQ of methyl-6-gingerol were5ng and10ng respectively.2.2.7Recovery testThe average recovery for methyl-6-gingerol was99.4%and the RSD(n=6) was2.02%.3. Contents determination of methyl-6-gingerolThree batches of samples of the Zingiberis Rhizoma were determined and itshowed that the average content of compound methyl-6-gingerol were66.27,63.05and63.72(μg/g) respectively.