Simultaneous determination of 7 components in Dipsacus asper by HPLC / 中国药房

OBJECTIVE To establis h the method for the simultaneous determination of six iridoids （loganic acid ，loganin， sweroside，dipsanoside B ，dipsanoside A ，sylvestroside Ⅰ）and one triterpene saponin （asperosaponin Ⅵ）in Dipsacus asper . METHODS High performance liquid chromatography （HPLC） method was adopted. The determination was performed on Symmetry® C18 column with mobile phase consisted of acetonitrile- 0.1％ phosphoric acid solution （gradient elution ）at the flow rate of 1.0 mL/min. The detection wavelengths were set at 212 nm（asperosaponin Ⅵ）and 237 nm（dipsanoside B ，dipsanoside A ， sweroside，loganic acid ，sylvestroside Ⅰ，loganin）. The column temperature was set at 30 ℃，and sample size was 20 μL. RESULTS The linear range of loganic acid ， loganin， sweroside， sylvestroside Ⅰ ， dipsanoside B ， dipsanoside A and asperosaponin Ⅵ were 399.24-931.56，50.30-150.90，48.24-168.84，27.00-70.20，12.93-38.80，40.64-121.92，42.08-147.28 µg/mL （all r＞0.999 0）. RSDs of precision ，reproducibility and stability tests （24 h）were all less than 2％. Average recoveries were 104.43％（RSD＝0.63％，n＝6），101.74％（RSD＝1.11％，n＝6），100.76％（RSD＝1.06％，n＝6），98.00％（RSD＝1.58％，n＝6）， 99.03％（RSD＝2.31％，n＝6），102.93％（RSD＝2.26％，n＝6），102.31％（RSD＝1.00％，n＝6），respectively，The contents were 142.5-280.6，5.5-49.0，28.0-112.9，7.2-35.8，4.4-16.9，17.2-79.3，0.8-54.5 mg/g，respectively. CONCLUSIONS Established method is accurate and reliable ，and can be used for the content determination of 7 components in D. asper .

Identification and expression analysis of the DaNAC transcription factor family in Dipsacus asper / 药学学报

italic>NAC transcription factor genes play an important role in regulating plant adversity stress tolerance and secondary metabolism. To explore DaNAC transcription factor participation in the synthesis of asperosaponin Ⅵ in Dipsacus asper, we analyzed the expression of DaNAC genes based on full-length transcriptome data from different tissues (root, stem, leaf, flower, seed) to provide a theoretical foundation for regulating the metabolism of D. asper. RNA-seq data was used to identify open reading frames. Bioinformatic methods were used to identify the conserved domain motifs and construct an evolutionary tree. qRT-PCR was carried out to analyze tissue-specific and adversity-stressed expression. Twenty-nine DaNAC sequences were identified, all of which contain the conserved NAM domain and conserved motif 1 and motif 2 at the N terminal. Five DaNAC genes are closely related to the NAC genes in Arabidopsis thaliana and rice that are involved in adversity stress and are clustered in the Group Ⅰ subfamily. qRT-PCR revealed that DaNAC genes are differentially expressed between tissues. The expression levels were highest in leaves, followed by roots, stems and petioles, and the lowest in flowers and seeds. Compared with normal growth conditions, the expression of four NAC genes was up-regulated by treatment with low temperature (15 ℃). The expression of three genes (34564NAC2, 33883NAC48, 6727NAC14) was up-regulated and one gene (34480NAC22) was down-regulated by 150 μmol·L-1 MeJA. The results illustrate that the expression of NAC genes is induced by adversity stress, which provides a foundation for further study on the role of NAC family members in adversity stress in D. asper.

Content changes of triterpene saponins in crude and sweated Dipsacus asper:an iTRAQ-based analysis / 中国中药杂志

The present study aimed to explore the mechanism of the sweating of Dipsacus asper on content changes of triterpene sa-ponins by detecting the total triterpene saponins and the index component asperosaponin Ⅵ in the crude and sweated D. asper, and analyzing the differentially expressed proteins by isobaric tags for relative and absolute quantification(iTRAQ) combined with LC-MS/MS. After sweating, the content of total triterpene saponins decreased manifestly, while that of asperosaponin Ⅵ increased significantly. As revealed by the iTRAQ-LC-MS/MS analysis, 140 proteins with significant differential expression were figured out, with 50 up-regulated and 90 down-regulated. GO analysis indicated a variety of hydrolases, oxido-reductases, and transferases in the differential proteins. The results of activity test on two differentially expressed oxido-reductases were consistent with those of the iTRAQ-LC-MS/MS analysis. As demonstrated by the analysis of enzymes related to the triterpene saponin biosynthesis pathway, two enzymes(from CYP450 and UGT families, respectively, which are involved in the structural modification of triterpene saponins) were significantly down-regulated after sweating. The findings suggested that sweating of D. asper presumedly regulated triterpene saponins by affecting the expression of downstream CYP450 s and UGTs in the biosynthesis pathway of triterpene saponins of D. asper.

Methyl jasmonate stimulates the synthesis and accumulation of asperosaponin VI in the roots of Dipsacus asper / 药学学报

We investigated the effect of methyl jasmonate (MeJA) on the content of asperosaponin VI and the expression of genes involved in its synthesis. Dipsacus aspero seedlings were treated with MeJA at different concentrations of 50, 100, 150, 200 and 300 μmol·L-1, and leaves and roots were sampled following treatment for 1, 3 and 5 days. The content of asperosaponin VI and superoxide anion in the roots, malondialdehyde (MDA) content in leaves and superoxide dismutase were determined. The results show that 150 μmol·L-1 MeJA significantly increased the accumulation of asperosaponin VI in roots. The content of asperosaponin VI was greatest after treatment for 3 days, and was 2.16 times higher than the control. After MeJA treatment, SOD activity decreased and MDA content increased in leaves. Moreover, superoxide anion content in roots increased. The expression of squalene epoxidase (DaSE1) and geranyl diphosphate synthase (DaGPS), key enzymes in the synthesis of asperosaponin VI, were up-regulated compared with the control group. These results indicate that an optimal concentration of 150 μmol·L-1 MeJA increases the accumulation of asperosaponin VI by up-regulating the expression of key enzymes involved in the synthesis of asperosaponin VI, which facilitates resistance to adversity stress stimulated by MeJA.

Cloning and expression stability analysis of Actin, Tubulin and GAPDH genes in Dipsacus asper / 中草药

Objective: To clone and screen the stable internal reference genes from Dipsacus asper for qRT-PCR analysis correction, so as to provide a preliminary basis for future research on expression analysis and regulation mechanism of D. asper functional genes. Methods: The internal reference genes of Actin, Tubulin and GAPDH gene families were screened and cloned from D. asper transcriptome database. The D. asper plants from different origins, different tissues and different developmental stages were used to obtain expression information of each gene by qRT-PCR. The expression stability of each gene was analyzed by geNorm, NormFinder, BestKeeper, Delta CT and RefFinder, and the best genes were synthetically evaluated and screened. Results: Ten core fragments for candidate internal reference genes were cloned, belonging to three gene families: Actin, Tubulin and GAPDH, with high homology among them. The results of stability analysis showed that the expression of DaACT103 was stable and relatively high in different regions and tissues, while the expression of DaTUB5 was stable and relatively low in different developmental stages. Conclusion:s DaACT103 and DaTUB5 are suitable as the internal reference genes for D. asper. DaACT103 is used as the internal reference gene with high abundances and DaACT105 is used as the internal reference gene with low abundances.

Study of Transfer Rate of Standard Decoction of Dipsacus asper Decoction Pieces / 中国药房

OBJECTIVE:To study transfer rate of asperosaponinⅥ in standard decoction of Dipsacus asper decoction pieces. METHODS:The content of asperosaponin Ⅵ in Dipsacus asper decoction pieces and its standard decoction was determined by HPLC. The determination was performed on SinoChrom ODS-AP with mobile phase consisted of acetonitrile-water(30∶70,V/V)at the flow rate of 1.0 mL/min. The detection wavelength was set at 212 nm,and column temperature was 30℃. The sample size was 10 μL. By the ingredients content obtained,the transfer rate of asperosaponin Ⅵ was calculated during decoction piece to standard decoction. RESULTS:The linear range of asperosaponin Ⅵ was 0.484-4.84 μ g(r＝0.999 9). RSDs of precision,stability and reproducibility tests were all lower than 2%. The limits of quantification and detection were 0.3 and 0.1 μ g,respectively. The average recoveries in D. asper decoction pieces and standard decoction were 95.13% -100.22%(RSD＝1.78%,n＝6), 97.07%-100.08%(RSD＝0.98%,n＝6). RSD of durability test was lower than 1%.The transfer rate of asperosaponin Ⅵ in standard decoction of D. asper decoction pieces ranged 26.3%-49.5%. CONCLUSIONS:The method is simple,accurate,precise, stable,reproducible and durable,and can be used for transfer rate of asperosaponin Ⅵ in standard decoction of D. asper decoction pieces.

Comparisons on chemical constituents of crude and wine-processed Dipsacus asper by using UPLC-Q-TOF/MS / 中国中药杂志

To compare the quality control indexes and chemical constituents of crude and wine-processed Dipsacus asper. According to Chinese Pharmacopoeia 2015 edition, water content, total ash, acid-insoluble ash and water soluble extract of different processed products were detected. UPLC-Q-TOF/MS approach was established to compare the contents of major constituents in crude and wine-processed D. asper. Moreover, the linearity, precision, stability, repeatability and recoveries of the approach were well studied. The results of water content, total ash, acid-insoluble ash and water-soluble extract of crude and wine-processed D. asper were all in line with the requirements of the Chinese Pharmacopoeia 2015 edition. Meanwhile, 20 main chemical constituents were identified by using UPLC-Q-TOF/MS. After wine-processing, the contents of asperosaponin Ⅵ, acetylate analogues and caffeic acid were significantly increased, while the contents of other phenolic components such as dicaffeoylquinic acid were decreased significantly, which may be which may be the main reason for different clinical efficacy of crude and wine-processed D. asper.

SRAP analysis on genetic diversity for germplasm resources of Dipsacus asper / 中草药

Objective: To study the genetic diversity of Dipsacus asper from different populations and provide a reference for the rational utilization of its germplasm. Methods: The genetic diversity of the 14 populations of D. asper was analyzed by SRAP molecular markers. Results: Ten pairs of primers produced 124 sites, among which 102 were polymorphic sites. The percentage of polymorphic loci (PPL) was 82.26%. The Nei's genetic diversity index (H) and the Shannon's information index (I) were 0.2800 and 0.4353, respectively. At the population level, PPL was 53.92%, H was 0.1212-0.2440, and I was 0.1796-0.3611. The genetic diversity values of the five populations were relatively high, and the populations had the characteristics of high altitude and microhabitat. Genetic differentiation coefficient (Gst) was 0.2930, gene flow (Nm) was 1.2064. Cluster analysis based on genetic similarity indicated that the 14 populations could be divided into three groups. Conclusion: The genetic diversity among the populations of D. asper was at relatively high level. The genetic variance of D. asper mainly existed within the populations. The high genetic diversity could be attributed to the geographical position (altitude) and climate, while geographic isolation (microhabitat) was another important factor for the genetic variance within the populations.