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Objective:To evaluate and compare the identification of several DNA barcoding candidate sequences on Illicium difengpi and its fake I. jiadifengpi. Method:Samples from different origins of I. difengpi and I. jiadifengpi, were collect extraction of total DNA,nuclear gene ITS2 sequence,chloroplast rbcL,matK gene sequence were selected for PCR amplification,product purification and sequencing,and CondonCode Aligner V3.7.1 was used to proofread stitching. Result:PCR amplification and sequencing of rbcL sequences of I. difengpi and I. jiadifengpi were not satisfactory. It is assumed that their rbcL sequences were too long with slow evolution,which was unsuitable for interbreeding. The success rate of matK sequencing of I. difengpi and I. jiadifengpi was 0 and 76.8%,which may be because primer standards were different for matK sequences of different groups. The results of PCR amplification and sequencing of ITS2 on I. difengpi and I. jiadifengpi were successful,with the success rate of sequencing was 89.3% and 91.2%. Analysis sequencing results, the total length of ITS2 sequences was 268 bases,and there were 2 variation sites of I. difengpi. The total length of ITS2 sequences was 430 bases,and there were 4 or 3 variation sites of I. jiadifengpi. It shows that ITS2 sequences of I. difengpi and I. jiadifengpi were short and has obvious variability and can be amplify,that ITS2 sequence was better than rbcL and matK sequence in molecular identification of I. difengpi and I. jiadifengpi. Conclusion:DNA barcoding based on ITS2 sequence was a powerful and efficient tool for identification of I. difengpi and its fake I. jiadifengpi.
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Objective: To isolate and identify bioactive constituents from the stem barks of Illicium difengpi. Methods: The chemical constituents were isolated and purified by repeated silica gel, Sephadex LH-20, recrystallization, and preparative HPLC techniques. The structures of the compounds were identified on the basis of spectral data including NMR, MS, and IR. Results: Two sesquiterpene lactones, majucin (1) and anisatin (2), two steroids, β-sitosterol (3) and daucosterol (4), three carboxylic acids, 2-ethyldecanoic acid (5), shikimic acid (6), and 3,4-dihydrobenzoic acid (7), and a flavonoid, quercetin (8), were successively isolated from the stem barks of I. difengpi. Conclusion: Apart from compound 3, other seven compounds are reported in this plant for the first time. The results suggested that the current studies on I. difengpi is still far from being well known and therefore more studies need to be done for better understanding of this plant. © 2014 Tianjin Press of Chinese Herbal Medicines.
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Objective: To establish a stable, reproducible, and suitable reaction system for ISSR analysis of genetic differences in Illicium difengpi. Methods: The ISSR-PCR amplification system on I. difengpi in five factors (Mg2+, dNTPs, primers, Taq DNA polymerase, and DNA template) was optimized by orthogonal design, and the PCR result was analyzed by SPSS. Then based on the optimal ISSR-PCR amplification system, the annealing temperature and cycle times in PCR were proposed by gradient determenation. Results: Most of the factors in different levels had the significant effects on the result of PCR, and the most remarkable factor was the quantity of Taq DNA polymerase. The optimized ISSR-PCR reaction system (20 μL) for I. difengpi was constructed of Mg2+ (1.60 mmol/L), dNTP (0.22 mmol/L), primer (0.90 μmol/L), Taq polymerase (0.50 U), and DNA template (70.00 ng). The optimized annealing temperature and cycle times were 51.8 °C and 40 cycles, respectively. Thirteen ISSR primers with stable amplification and abundant polymorphism were selected from 62 ISSR primers. Conclusion: The established and optimized ISSR reaction system is stable and credible according to the testing results of 16 samples of I. difengpi, and provides the basis for the genetic analysis of I. difengpi.