Selection and validation of reference genes for quantitative real-time PCR analysis in Paeonia veitchii / 中国中药杂志

Paeonia veitchii and P. lactiflora are both original plants of the famous Chinese medicinal drug Paeoniae Radix Rubra in the Chinese Pharmacopoeia. They have important medicinal value and great potential in the flower market. The selection of stable and reliable reference genes is a necessary prerequisite for molecular research on P. veitchii. In this study, two reference genes, Actin and GAPDH, were selected as candidate genes from the transcriptome data of P. veitchii. The expression levels of the two candidate genes in different tissues(phloem, xylem, stem, leaf, petiole, and ovary) and different growth stages(bud stage, flowering stage, and dormant stage) of P. veitchii were detected using real-time fluorescence quantitative technology(qRT-PCR). Then, the stability of the expression of the two reference genes was comprehensively analyzed using geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that the expression patterns of Actin and GAPDH were stable in different tissues and growth stages of P. veitchii. Furthermore, the expression levels of eight genes(Pv-TPS01, Pv-TPS02, Pv-CYP01, Pv-CYP02, Pv-CYP03, Pv-BAHD01, Pv-UGT01, and Pv-UGT02) in different tissues were further detected based on the transcriptome data of P. veitchii. The results showed that when Actin and GAPDH were used as reference genes, the expression trends of the eight genes in different tissues of P. veitchii were consistent, validating the reliability of Actin and GAPDH as reference genes for P. veitchii. In conclusion, this study finds that Actin and GAPDH can be used as reference genes for studying gene expression levels in different tissues and growth stages of P. veitchii.

Quality evaluation of Huocao based on UPLC fingerprint and multi-component content determination / 中国中药杂志

Huocao(a traditional Chinese herbal medicine) moxibustion is a characteristic technology in Yi medicine suitable for cold-dampness diseases. Huocao, as the moxibustion material, is confusedly used in clinical practice and little is known about its quality control. In this study, UPLC method was used to establish the chemical fingerprint of non-volatile components in Huocao, and the contents of eight phenolic acids such as chlorogenic acid were determined. Multivariate statistical analysis was performed to obtain the indicator components of Huocao for quality evaluation, and thus a comprehensive evaluation system for the quality of Huocao was built. The UPLC fingerprints of 49 batches of Huocao were established, and there were 20 common peaks, of which eight phenolic acids including neochlorogenic acid and chlorogenic acid were identified. Except for three batches of Huocao, the similarity of the other 46 batches was higher than 0.89, suggesting that the established fingerprint method could be used for quality control of the medicinal herb. The correlation coefficient between entropy weight score of the eight phenolic acids and comprehensive fingerprint score in Huocao was 0.875(P<0.01), which indicated that the eight phenolic acids could be used as indicator components for the quality evaluation of Huocao. Furthermore, in multivariate statistical analysis on the common peaks of fingerprint and the contents of the eight phenolic acids, chlorogenic acid, isochlorogenic acid A and isochlorogenic acid C were screened to be the indicator components. The results revealed that the proposed method achieved a simple and accurate quality control of Huocao based on UPLC fingerprint and multi-component content determination, which provided useful data for establishing the quality standard of Huocao.

UPLC fingerprint and indicator components of Boenninghausenia albiflora var. albiflora / 中国中药杂志

The fingerprint of Boenninghausenia albiflora var. albiflora was established by ultra performance liquid chromatography(UPLC), and the content of 12 active components including chlorogenic acid was determined. Multivariate statistical analysis was used to explore the indicator components of B. albiflora var. albiflora and a comprehensive evaluation system was created for the quality of B. albiflora var. albiflora. In this study, 33 batches of B. albiflora var. albiflora with different sources were collected and studied, and the UPLC fingerprint of B. albiflora var. albiflora was developed. There were 37 common peaks, of which 12 components were identified, and the content of these 12 components was measured. In combination of the common peaks and the content of chemical components, multivariate statistical analysis was performed, and the results showed that 6 components [daphnoretin, isoimperatorin, astragalin, imperatorin, neochlorogenic acid, and isoquercitrin(weight coefficient>0.1)] were selected as chemical markers for the quality of B. albiflora var. albiflora. Technique for order of preference by similarity to ideal solution(TOPSIS) analysis and chemometrics revealed that the quality of S32, S28 and S29 were superior, while that of S12, S7 and S16 were inferior. The quality evaluation method of B. albiflora var. albiflora constructed in this study was accurate and reliable, with simpleness and easiness to operate. It is suggested that the 6 above-mentioned active components could be used as indicator components for quality control of B. albiflora var. al-biflora. The samples were harvested during the flowering and fruiting period, which is from the beginning of July to the end of August.