| Objective: The principal aim of this article is to isolate and purify alkaloids. All thisifacilitate its better research, development and utilization. To develop a HPLC method forquality evaluation of Euodiae Fructus and related species by simultaneous determinationlimonin, indole alkaloids （14-fomyldihydroxyrutaecarpine, evodiamine, rutaecarpine）, andquinolone alkaloids （1-methyl-2-undecyl-4(1H)-quinolone, evocarpine，dihydroevocarpine） in the fruits of five Evodia species. In this work, the volatilecomponents of Evodiae Fructus from three official sources and twelve habitats at differentstages of maturity were detected and identified for the first time using gaschromatography-mass spectrometry (GC-MS) and related statistical analysis.Method: By repeated chromatographic methods. Totally,20compounds were isolatedand purified, and by physicochemical properties and spectroscopic methods, the structureswere elucidated (UV, EI-MS,1H-NMR,13C-NMR,1H-1HCOSY, HMBC, HSQC, NOESY).Samples were analyzed on a YMC C18column (4.6mm×250mm,5μm) eluted with mobilephases of acetonitrile (A), tetrahydrofuran (B), and a buffer solution of5mmol·L-1ammonium acetate (PH3.8)(C) in a linear gradient mode. The column temperature was30℃and the flow rate was1.0mL·min-1. The PDA detector wavelengths were set at220,250nm.The essential oils of evodia rutaecarpa were extracted by steam distillation and gaschromatography-mass spectrometry (GC-MS) techniques were used to analyze itschemical composition and relative content.Results: Totally20compounds were isolated and purified, Including five indolealkaloids,11quinolone alkaloids,1degraded limonoid, two amide,a sterol. The sevencompounds were well separated and showed good linearity (r=0.9999) within theconcentration ranges tested. The mean recoveries were between96.7%-102.4%(RSD 1.4%-3.1%). All the seven constituents were detected in the fruits of five species, but thecontents of them varied widely in different samples. The total contents of seven constituentsin16batches of Euodiae Fructus were9.46-69.9mg·g-1, and the mean content was28.2mg·g-1. The total content of seven constituents in E. compacta and E. fargesii was25.8and7.69mg·g-1, respectively. Thirty-nine volatile components were identified, of whichtwenty-one were present in all the specimens analyzed. On the basis of maturity, the sampleis divided into three clusters (dendrogram) or distributed in the three regions (scatter plot).The results obtained from the two analytical methods showed good consistence with eachother in overall trend and ripeness was the key factor that influence the volatile compositionof the fruits. In nearly mature fruits, β-myrcene(19.83%-29.54%),D-limonene(19.99%-26.18%) and β-cis-ocimene(10.31-31.77%) were the main components.Mature fruits had very high contents of β-cis-ocimene(26.86-51.80%), but lower contents ofβ-myrcene(3.00%-5.01%) and D-limonene(0.24%-3.57%). Immature fruits contained thehighest contents of caryophyllene(10.22%-14.85%), but lower contents ofβ-myrcene(0.02%-6.50%), D-limonene(0.02%-7.40%) and β-cis-ocimene(0.02%-8.68%).Therefore, β-myrcene, D-limonene, β-cis-ocimene and caryophyllene could be used aschemical markers for differentiation and evaluation the quality of essential oils of thesamples in terms of maturity.Conclusion: The separation and identification of alkaloid compositions which canprovide theoretical and experimental basis for quality evaluation and rational utilization ofevodia rutaecarpa.Through the validation, the method was proved to be accurate andrepeatable. We can elucidate rational use of two related species from the perspective ofchemical composition. GC-MS combined with chemometrics methods can classify andidentify12batches of samples effectively and account for the cause of the changes inchemical composition. It is a significant finding that ripeness is the key factor that influencesthe main volatile ingredients of fruits. From above studies, it can be suggested that a periodof two to three weeks after blossom fall were the preferable harvest time. |
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