Study On Chemical Constituents Of The Leaves Of Nelumbo Nucifera Gaetrn

Folium nelumbinis is the dry leaves of Nelumbo nucifera Gaertn, which is highly valuedin traditional medicine field and can be widely used in many aspects. It is stated that Foliumnelumbinis can be introduced into the effective treatment of stroke, acute diarrhea,hematochezia and phalacrosis etc.The objective of this research is to systematically separate and determine the chemicalcomposition of Folium nelumbinis. Seven compounds were extracted from Folium nelumbinis,the structures of which were identified by employing several spectral methods such as NMR,MS, UV and IR, combining with their physical and chemical properties. The seven isolatedcompounds are identified as quercetin-3-O-β-D-xylopyranosyl(1â†'2)-β-D-glucopyranoside,isoquercitrin,daucosterol,quercetin-3-O-β-D-glucuronide,sitosterol,2-hydroxy-1-methoxyaporphine and nuciferine.In this paper, high pressure liquid chromatography(HPLC) was used to establish thequantification analysis method of the two flavonoids, quercetin-3-O-β-D-xylopyranosyl(1â†'2)-β-D-glucopyranoside and Quercetin-3-O-β-D-glucuronide, the contents of which werealso determined on three batches of samples of Folium nelumbinis.1. Extraction, separation and structural identification1.1Extraction6Kg dried leaves of Nelumbo nucifera Gaertn were crushed and then extracted by refluxin80%(V/V) ethanol solution three times (1h,0.5h and0.5h, respectively). The mixed filtratewas evaporated under vacuum distillation to recycle ethanol and finally condensed tonon-ethanol.1.2Separation and purificationDilute ten folds of the above condensed solution and then rinsed on the macroporousadsorption resin with0.1%NaOH solution until to colorless. The macroporous adsorption resin was first rinsed by distilled water to colorless（pH=7.0） and then eluted with80%ethanol. The left elution was condensed to extract the540g part A. The alkali leftovers wasprocessed to pH=7.0by0.1%hydrochloric acid. The left elution was condensed to extract the205g part B.1.2.1A1and A2were isolated from A by silica gel column chromatography with eluent I.White powder precipitated in A1and then we rinsed it with ethyl acetate, finally,compound LL-3(58mg) was isolated.A2′and A2″were isolated from A2by alkaline aluminum oxide columnchromatography with eluent II. The two extracts were evaporated to dryness and thenheated to dissolve in elutant II. When it was cooled to room temperature, there are someprecipitations and then it was filtered. Finally, LL-S1(62mg) and LL-S2(480mg) wereisolated after recrystallization in ethyl acetate.1.2.2B1, B2, B3and B4were isolated from B by silica gel column chromatography whenusing III as mobile phase.B1′was isolated from B1by polyamide column chromatography when usingdeveloping solvent VIII as mobile phase. B1〞was isolated from B1′by ODS columnchromatography when using developing solvent IX as mobile phase. The compoundLL-2(140mg) was separated from B1〞by gel filtration chromatography.B2′was isolated from B2by silica gel column chromatography when usingdeveloping solvent VI as mobile phase. The compound LL-1(130mg) was finallyseparated from B2′by dry column chromatography repeatedly when using developingsolvent VII as mobile phase.B3′was isolated from B3by silica gel column chromatography when usingdeveloping solvent IV as mobile phase. The compound LL-4(120mg) was finallyseparated from B3′by dry column chromatography repeatedly when using developingsolvent V as mobile phase.White powder precipitated in B4and then it was filtered under vacuum. Thecompound LL-5(87mg) was separated after recrystallization in chloroform.1.3Identification On the basis of the physical and chemical properties with various spectral methods suchas UV, IR, MS, NMR, all the isolated compounds were identified asquercetin-3-O-β-D-xylopyranosyl(1â†'2)-β-D-glucopyranoside(LL-1),isoquercitrin(LL-2),daucosterol(LL-3), quercetin-3-O-β-D-glucuronide(LL-4), sitosterol(LL-5),2-hydroxy-1-methoxyaporphine(LL-S1) and nuciferine(LL-S2).2. The Contents Determination of two kinds of Flavonoids Compounds inNelumbo nucifera Gaertn. by HPLC2.1Equipments, Chromatographic conditions and control samplesChromatograph: Agilent1200Series HPLC (UV detector);Balance: Mettlertoledo AG135electronicbalance;Chromatography column: ZORBAX Extend C18column（250mm×4.6mm，5um）;Mobile phase: methanol-acetonitrile-acetic acid (4:1:15)Flow rate:1.0mL/min;Chromatography column temperature:35℃;Controlsamples:quercetin-3-O-β-D-xylopyranosyl(1â†'2)-β-D-glucopyranoside(LL-1) andquercetin-3-O-β-D-glucuronide(LL-4). According to the HPLC normalized method, weconcluded that the purities of the two compounds are98.5%and99.6%, respectively.2.2Contents determination and methodologyI. Theoretical plate number and ResolutionThe results show that the theoretical plate of number of LL-1and LL-4are4803and6612, respectively. The resolution of LL-1and LL-4are2.03and1.51, respectively.II. Intra-day precision testThe solution of compounds LL-1and LL-4with the same volume was injected into theHPLC for six times in one day. It was shown that the RSD(n=6) of peak area for thecompounds LL-1and LL-4were1.22%and1.34%, respectively, and the retention time were0.58%and0.14%, respectively.III. Inter-day precision testThe same solution was injected into the HPLC once a day for six days and the resultsshowed that the RSD(n=6) of peak area for LL-1and LL-4were0.84%and0.50%, respectively, while the retention time were0.20%and0.14%, respectively.IV. The standard curveThe linearity was in the concentration range of0.1μg-1μg for compounds LL-1andLL-4, and the correlation coefficients were0.9995and0.9999, respectively. The regressionequations of the two compounds were y=3417.6x-45.795(LL-1) andy=3761.2x-4.7947(LL-4).V. Repeatability testSix copies of the same compound were injected into HPLC and the RSD of peak area forLL-1and LL-4were0.14%and0.11%, respectively.VI. Stability testThe same solution of samples was injected into the HPLC once every two hours in24h.The RSD of peak area for compounds LL-1and LL-4were0.58%and0.25%, respectively,while the retention time were0.43%and0.46%, respectively. The above results clearlydemonstrated the good stability of the two compounds in24h.VII. LOD and LOQFor both compounds LL-1and LL-4，based on the experiments conditions, theirdetection limit（LOD）was5ng, while quantification limit（LOQ）was also5ng.VIII. Recoveries testThe average recovery for compound LL-1was100.4%and the RSD (n=6) was0.50%,while the average recovery for compound LL-4was100.1%and the RSD (n=6) was0.33%.â…¨. Contents determination of three batches of samplesThree batches of samples of the leaves of Folium nelumbinis were determined, and itwas showed that the average content of compound LL-1was0.357%,0.328%and0.371%,and the average content of compound LL-4was0.994%,0.965%and1.029%, respectively.All in all, this research provided valid and scientific theoretical evidence to furtherexplore and promote the technological upgrading in determination, extraction and purificationof chemical compositions in Folium nelumbinis.3. The lotus two flavonoids study on the free radical scavenging effectsThe experimental results show that, quercetin-3-O-β-D-xylopyranosyl-(1â†'2)- β-D-glucopyranoside(LL-1) and quercetin-3-O-β-D-glucuronide have the ability ofscavenging capacity. quercetin-3-O-β-D-glucuronide is better than quercetin-3-O-β-D-xylopyranosyl(1â†'2)-β-D-glucopyranoside.In this paper, new index of content determination was offered to improve qualitystandard of the leaves of Nelumbo nucifera Gaertn. The results of this paper provided atheoretical support for further study and a scientific basis for development and utilization ofOroxylum indicum.