| Cortex Mori radicis is a traditional Chinese medicine, which is the dry roots ofMorus alba L.. A series of chemical investigations revealed that Diels-Alder typeadduct compounds and flavonoid were the major compositions of Morus alba L.. Inaddition, there are coumarins and terpenoid as well as sterols in it. It clinically usedin the treatment of edema, pleuritis, trachitis, pertussis and diabetes mellitus etc.In this research, six compounds were seperated from the roots of Morus alba L..Various spectral methods, such as NMR, MS, IR and UV, were used to identify theirstructures, and they were elucidated as mulberrofuran G (S1), cyclomorusin (S2),α-amyranol(S3), Ursolic acidl (S4), sitosterol (S5)and (2,6,8,10-butamethyl-6,10-epoxy-[3.2.2]tricycloundecane.In this paper, the quantification analytical method of Diels-Alder type adduct (S1)using HPLC were also investigated in order to improve the quality standards for theroots of Cortex Mori radicis.Extraction, separation and identification20kilograms of dried roots of Morus alba L. were crushed and extracted by heatreflux in85%ethanol (the volume fraction,160L) three times (1.5h,1.0h and0.5hrespectively). The combined extracted soluion was evaporated to dryness, then it wasdiluted with water and flowed through the anion-exchange column andcation-exchange column. After that, the efflux was concentrated and extracted byn-butanol and the solvent was recovered,900g of extraction was obtained. By usingsilica gel, polyamide and ODS column chromatography repeatedly, six compoundspreviously described were separated. The mobile phase was made of methanol,petroleum ether, ethyl acetate, chloroform and so on in certain ratio. Combine theirphysical and chemical properties, IR, UV, MS and NMR were used to identifystructures of the compounds isolated after comparing with the literature reports. Structures of the six isolated compounds were identified, they aremulberrofuran G (S1), cyclomorusin (S2), α-amyranol(S3), Ursolic acidl (S4),sitosterol (S5) and2,6,8,10-butamethyl-6,10-epoxy-[3.2.2] tricycloundecane (S6).Contents determination of mulberrofuran GThe research was performed on Agilent1200HPLC instrument and EclipseXDB-C18coloum (4.6×250mm,5μm), using a mixture of methanol-0.1%Aceticacid in ratio of55:45as the mobile phase. The flow rate was1.0mL.min-1at35℃,the wavelength of VWD detector was set at220nm.ResultsⅠ Theoretical plate number and ResolutionThe Theoretical plate number of mulberrofuran G was16373；The Resolution ofmulberrofuran G was1.61.Ⅱ Precision testSolution of mulberrofuran G was injected into HPLC for six times in one day, theRSD of peak area and retention time for mulberrofuran G were0.15%and0.37%.Ⅲ The standard curveThe linearity was in the range of0.1～10μg for mulberrofuran G, and thecorrelation coefficients was1.0000. The regression equations wasY=3548.9X+44.596.Ⅳ RepeatabilitySix copies of the sample solution were injected into HPLC, the RSD of peak areaand content for mulberrofuran G was1.77%and2.50%respectively.Ⅴ Stability testThe same sample solution was injected into HPLC in2h,4h,6h,8h,10h and12h,the RSD of peak area and retention time for mulberrofuran G were0.22%and0.20%respectively.Ⅵ LOD and LOQFor mulberrofuran G，the experiment gave their detection limi（tLOD）in0.02ng and quantification limit(LOQ) in0.06ng.Ⅶ RecovriesThe average recovery for mulberrofuran G was100.93%and the RSD (n=6) was2.11%.Ⅷ Contents determination of samplesFive batches of samples of the roots of Morus alba L. were determined, and theresults showed that the average content of mulberrofuran G was0.018%、0.022%、0.014%、0.024%、0.024%, respectively.In this paper, the chemical constituents of Cortex Mori radicis was furtherlystudied, and new index of content determination was offered to perfect qualitystandard of the roots of Morus alba L.. The results of this paper provided atheoretical support for further study and a scientific basis for development andutilization of Cortex Mori radicis. |
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