Abstract
To discriminate the feasible differences and find potential similarities and relationships of Citri Reticulatae Pericarpium (CRP), this work was accomplished by a comprehensive and reliable method using gas chromatography–mass spectrometer (GC–MS) to analyze the volatile oils and high‐performance liquid chromatography (HPLC) simultaneously to determine the contents of five bioactive flavonoids, namely hesperidin, nobiletin, 3,5,6,7,8,3′,4′‐heptamethoxyflavone, tangeretin, and 5‐hydroxy‐6,7,8,3′,4′‐pentamethoxyflavone in 25 batches of CRP samples of 10 cultivars collected from different regions in China. The GC–MS analyses indicated that 98 compounds were successfully identified from the volatile oils obtained and the major constituents of volatile oil are d‐limonene, γ‐terpinene, α‐pinene, linalool, and myrcene. Even 2‐(methylamino) benzoate was found in all cultivar samples harvested at maturation stage. Under the optimal condition, the quantitative analyses of five bioactive flavonoids were successfully performed by HPLC and hierarchical cluster analysis (HCA). Results showed significant differences among cultivars in the contents of five bioactive flavonoids mentioned earlier. The HCA and GC–MS results provided a convenient approach which might be applied for rapid similarity evaluation and also holds the potential for analysis of compounds present in other plants. Therefore, this work obtained offers scientific basis to control quality and develop medicinal value of the medicinal materials in CRP.
Highlights
| MATERIALS AND METHODSTwenty-five samples including 10 different cultivars (collected between October 2013 and December 2015) were collected from different major citrus-producing areas in China
For all the examined compounds, all five calibration curves exhibited good linearity (R2 > .9990), the intraday precisions, repeatability, stability, recovery calculated as relative standard deviation (RSD) were all
The results revealed the developed method was applied to the determination of the five flavonoids in samples collected from different regions in China
Summary
Twenty-five samples including 10 different cultivars (collected between October 2013 and December 2015) were collected from different major citrus-producing areas in China. Must (Chengdu, China) were isolated and purified from “Chachi” CRP by conventional column chromatography, and their structures were identified by EI-MS, 1HNMR, and 13C NMR in comparison with the data from the literature Their purities were determined to be 98% based on HPLC analysis using a peak area normalization method. To build the calibration curves, the mixed standard stock solution was prepared by dissolving the reference compounds (C1–C5) in methanol with the final concentrations of each compound at 483.6, 203.2, 160.8, 163.6, and 160.8 μg/ml, respectively. Resulting solutions were filtered through a 0.22-μm nylon syringe filters, and aliquots of 20 μl were injected in the chromatographic system for analyses These solutions were stored at 4°C for further HPLC analysis. The calibration curves were constructed by plotting the peak areas versus the concentrations of standards
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