Study On Chemical Constituents From Ramulus Mori

Ramulus Mori，as a Chinese herbal medicine， is the dried tender branches of Morusalba L. The main chemical constituents of Ramulus Mori are flavonoids and alkaloids. Inaddition, it also contains steroids, terpenoids, coumarin, stilbene and sterols, and so on. It canbe used primarily to treat the arches and numbness of the shoulder and arm joins. It clinicallyused in the treatment of many diseases, such as swelling and pain of the joints, numbness ofthe hands and feet, rheumatoid-paining and paralyzed etc.In this experiment, the chemical constituents of Ramulus Mori system has been separatedsystematically by using the methods of macroporous adsorption resins, silica, ODS andgelatin column chromatography etc. Seven compounds were isolated from Ramulus Mori.Various spectral methods, such as NMR and MS etc. were used to identify their chemicalstructures before they were elucidated as3,4,5-trimethoxyphenyl-1-O-β-D–apiofuranyl(1â†'6)-β-D-glucopyranoside(T-1),mulberrosideA(T-2),cis-mulberrosideA(T-3),coumarin-7-O-α-L-rhamnopyranosyl-(1â†'6)-O-β-D-glucopyranoside (T-4), oxyresveratrol-2-O-β-D-glupyranoside (T-5),β-sitosterol(T-6) and β-daucosterol(T-7). Compound T-1，T-3and T-4wereisolated from Ramulus Mori for the first time.In this experiment, the quantification analytical mathod of3,4,5-trimethoxyphenyl-1-O-β-D-apiofuranosyl(1â†'6)-β-D-glucopyranoside and cis-mulberroside A by HPLC wereestablished and the contents of the two compounds in four batches of the Ramulus Morisamples were dedermined.Extraction, separation and identificationCrushed20kilograms of dried Ramulus Mori were extracted by heat reflux in85%ethanol (liquid-solid ratio was8:1,8:1,6:1, V/W) for three times,2hours every times. Theextraction solution was combined and filtered, then concentrated and diluted with water,flowed through AB-8macroporous adsorption resin column, eluted with85%ethanol. Theeluent was concentrated,300g extractions was obtained. By using silica gel, ODS and gelcolumn chromatography repeatedly, seven compounds previously described were isolated. The NMR, MS, IR and UV spectra combined the physical and chemical properties were usedto identify the chemical structures of these compounds after compared with the literaturereports.The structures of these seven isolated compounds has been identified as3,4,5–trimethoxyphenyl-1-O-β-D-apiofuranosyl(1â†'6)-β-D-glucopyranoside(T-1),mulberrosideA(T-2), cis-mulberrosideA(T-3), coumarin-7-O-α-L-rhamnopygranosyl-(1â†'6)-O-β-D-glucopyside(T-4),oxyresveratro-2-O-β-D-glucopyranoside(T-5),β-sitosterol(T-6), β-daucosterol(T-7).Contents determination of compound T-1and T-3The research was performed on Agilent1200HPLC instrument and CosmosilC18-MS-II (4.6×250mm ODS,5μm), using a mixture of methanol-water (22:78) as themobile phase. The flow rate was1.0mL.min-1at25℃. The wavelength of VWD detectorwas set at210nm, the injection volume is20μL.ResultsI Theoretical plate number and ResolutionThe theoretical plate number of T-1and T-3was10254and10144respectively; Theresolution of T-1and T-3were2.60and8.44respectively.II LOD and LOQFor compound T-1and T-3, the experiment gave their detection limit (LOD) in0.1ng and1.0ng, quantification limit (LOQ) in1.5ng and2.0ng, respectively.III Precision testReference solution of T-1and T-3were injected into HPLC for six times in one day, theresults showed that RSD of peak area and retention time were1.01%and0.72%respectivelyfor T-1, and were1.05%and0.74%respectively for T-3.Reference solution of T-1and T-3were injected into HPLC for six times during six days,the RSD of peak area and retention time were1.90%and0.80%respectively for T-1, andwere1.77%and1.49%respectively for T-3.IV The standard curveThe linearity was in the range of0.02~5.0μg for both compound T-1and T-3. Theregression equations were y=2299.5x+17.924(R=0.99999) for compound T-1and y=2604.5x-10.581（R=099998）for compound T-3respectively.V RepeatabilityInjected the six copies of the sample solution into HPLC, the RSD were0.88%and1.37%for compound T-1and T-3respectively.VI Stability testThe same sample solution was injected into HPLC six times in different duration (2h,4h,6h,8h,10h,12h) after parepared, the RSD of peak area and retention time for T-1were1.27%and1.18%respectively, the RSD of peak area and retention time for T-3were0.27%and0.35%respectively.VII RecoveriesThe average recovery for T-1was99.63%and the RSD (n=6) was1.18%. The averagerecovery for T-3was100.12%and the RSD (n=6) was0.81%.VIII Contents determination of samplesFour batches of samples of Ramulus Mori were determined, the results showed that theaverage content of T-1and T-3were0.030%and0.011%respectively.In the paper, the chemical constituents of Ramulus Mori were studied deeply and newindex of content determination was offered to perfect quality standard of Ramulus Mori. Theresults of this experiment provided index of contents determination for the perfect qualitystandard of Ramulus Mori and establish a scientific basis for development and utilization ofRamulus Mori.