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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.
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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.
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AIM To establish an HPLC method for the content determination of six constituents in Shiyifang Vinum (Notoginseng Radix et Rhizoma,Dipsaci Radix,Carthami Flos,etc.).METHODS The content determination of notoginsenoside R1,ginsenoside Rg1,ginsenoside Rb1 and asperosaponin Ⅵ was performed on a 30℃ thermostatic Inertsil(R) ODS-3 C18column (4.6 mm ×250 mm,5 μm),with the mobile phase comprising of acetonitrile-0.1％ phosphoric acid flowing at 1.0 mL/min in a gradient manner,and the detection wavelength was set at 203 nm.The content determination of brucine and strychnine was conducted on a 30 ℃ thermostatic Geminni(R) C18 110(A) column (4.6 mm × 250 mm,5 μm),with the mobile phase comprising of acetonitrile-mixed solution of 0.01 mol/L sodium heptanesulfonate and 0.02 mol/L potassium dihydrogen phosphate flowing at 1.0 mL/min in an isocratic elution manner,and the detection wavelength was set at 260 nm.RESULTS Six constituents showed good linear relationships within their own ranges (r ＞ 0.999 0),whose average recoveries were 98.52％-99.96％ with the RSDs of 2.0％-2.3％.CONCLUSION This simple,accurate and reproducible method can be used for the quality control of Shiyifang Vinum.
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To establish the HPLC fingerprint and determine five index components (loganic acid， chlorogenic acid， loganin， sweroside and asperosaponin Ⅵ) of Zishen Yutai pills by high performance liquid chromatography, and provide a scientific basis for its quality control. The fingerprint chromatogram was analysed by the chromatographic fingerprint similarity evaluation system for tradition Chinese medicine (2012), fifteen common peaks were obtained at the wavelength of 254 nm. Different batches of Zishen Yutai pills showed a similarity of above 0.90 in HPLC fingerprint profiles. For the quantitive analysis method, The separation of five components showed good regression (>0.999 2) with linear ranges, and the mean recoveries were in the range of 97.62%-101.9%, with the RSD (=9) less than 3%. The established fingerprint and quantitative analysis methods are highly specific, simple and accurate, which can reflect the quality of Zishen Yutai pills more comprehensively, and can be used for its quality control.
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Objective To establish and compare HPLC fingerprint chromatograms of Dipsaci Radix decoction pieces, aqueous decoction and formula granules.Methods The HPLC analysis was carried out in Wondasil C18 column (4.6 mm × 250 mm, 5 μm) with a mobile phase of acetonitrile-0.1% phosphoric acid by gradient elution. The flow rate was 1.0 mL/min; the detection wavelength was set at 212 nm; the column temperature was kept at 30℃. Results The fingerprint chromatograms from 12 batches of Dipsaci Radix decoction pieces, aqueous decoction and formula granules were established respectively. 14 common peaks in the fingerprint chromatogram in the formula granules could be tracked in the aqueous decoction, and 13 common peaks in the fingerprint chromatogram could be tracked in the decoction pieces. 2 chemical compounds were identified, such as asperosaponinⅥ and chlorogenic acid.ConclusionThe method of HPLC fingerprint chromatograms is stable and with good repeatability. Dipsaci Radix decoction pieces, aqueous decoction and formula granules are basically the same chemical composition.
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Objective To assay Icariine, Epimedin C, asperosaponin Ⅵ, psoralen and angelicin in Xianlinggubao capsules via multi-wavelength HPLC method.Methods Separation was carried out on Welch Ultimate○R XB-C18 column.The mobile phase was acetonitrile-water system and a linear gradient elution was used.The column temperature was 30 ℃.The detection wavelength for Icariine, Epimedin C,asperosaponin Ⅵ was set at 212 nm, psoralen and angelicin at 246 nm.Results Five components reached baseline separation, the linearity was good when sample size was in the range of 0.008 2-0.328 μg for Icariine(r=0.999 5), 0.055 6-2.224 μg for Epimedin C (r=0.999 6), 0.144 1-5.764 μg for asperosaponin Ⅵ(r=0.999 6), 0.005 4-0.215 2 μg for psoralen(r=0.998 0), 0.006 6-0.265 6 μg for angelicin(r=0.998 5).The average recoveries were 97.59％, 98.58％, 98.11％, 97.86％, 98.22％ respectively.The RSDs of recovery were all less than 2.0％.Conclusion This method is simple, accurate, with good separation, high sensitivity for the assay of multiple components in Xianlinggubao capsule.
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Objective To establish the method for simultaneous determination of epimedium glycoside and other 4 kinds of active ingredients in Xianling Gubao Capsules by three wavelength switching method. Methods An Waters Atlantis T3 C18 column (4.6 mm × 250 mm, 5 μm) was used with the mixture of acetonitrile-0.05％ formic acid solution as the mobile phase in gradient elution (0–5 min, 12％–20％ A; 5–15 min, 20％–55％ A; 15–35 min, 55％ A;35–55 min, 55％–76％ A; 55.1 min, 12％ A; 55.1–60 min, 12％ A). Detection wavelength was as follow: 0–30 min, 212 nm; 30–42 min, 246 nm; 42–60 min, 270 nm. The flow rate was 1.0 mL/min. The column temperature was 30 ℃. Results The calibration curves of asperosaponin Ⅵ, psoralen, angelicin, epimedin C, and icariin were in good linearity among the ranges of 101.6–3048 ng, 8.7–261 ng, 7.9–237 ng, 117.2–3516 ng, 78–2340 ng, respectively. In the instrument precision test, stability test, repeatability test, the RSD was less than 3％. The average recoveries were 99.83％, 100.35％, 100.59％, 100.60％, 99.72％, respectively, and all the RSD were less than 3％. Conclusion The method is sensitive, accurate, and separation effect is good, which can provide a basis for quality evaluation standard of Xianling Gubao Capsules.
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Objective The effect of Asperosaponin Ⅵ(ASAⅥ)on adipocyte differentiation and the involvement of Wnt signal pathway was investigated. Methods The murine bone marrow stromal cell line ST-2 were divided into 6 groups:control group, adipocyte differentiation group, and 4 different doses of ASAⅥgroups. Control group was exposed to the vehicle, adipocyte differentiation group was exposed to adipogenic reagent, and those 4 ASAⅥgroups were treated with different concentration(10-7, 10-6, 10-5, 10-4 mol/L)of ASAⅥafter adipocyte differentiation induction. 5 days later, oil red O staining was performed to calculate adipocyte rate. Then mRNA transcription levels of PPARγ, FABP4 genes andβ-catenin that were Wnt/β-catenin signaling pathway proteins were examined by FQ-PCR. Then Wnt pathway inhibitor DKK1 was supplemented into ST-2 cells treated with 10-4 mol/L ASAⅥfor 5 days. After that FQ-PCR was used to detect whether tran?scription levels of PPARγ, FABP4 andβ-catenin in ST-2 cells were changed. Results Compared with adipocyte differenti?ation group 10-5 mol/L and 10-4 mol/L ASAⅥtreatments greatly down-regulated the number of lipid droplets and markedly inhibited transcription levels of adipocyte characterization transcription factors included PPARγ, FABP4 while up-regulat?ed transcription level ofβ-catenin in ST-2 cells. DKK1 can reverse the inhibitory effect of ASAⅥon adipocyte differentia?tion in ST-2 adipocyte. The transcription levels of PPARγand FABP4 were up-regulated significantly while transcription level ofβ-catenin was inhibited. Conclusion ASAⅥblocks adipocyte differentiation in ST-2 cells which might be medi?ated through activating Wnt/β-catenin signaling pathway.
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Objective:To establish a method for the determination of asperosaponinⅥin Dieda Cuyu tablets by HPLC. Methods:A Hypersil C18(250 mm ×4.6 mm,5 μm)column was used. The mobile phase was acetonitrile-water(30∶70) with a flow rate of 1.0 ml·min-1 . The detection wavelength was 212 nm, the column temperature was room temperature,and the injection volume was 10μl. Results:AsperosaponinⅥ showed a good linear relationship within the range of 0. 04-0. 32 μg(r=0. 999 6). The average recovery was 97. 84%(RSD=1. 70%, n=6). Conclusion:The method is simple,accurate and reproducible, which can be used in the deter-mination of asperosaponinⅥ in Dieda Cuyu tablets.
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AIM: To compare the dissolution of asperosaponin Ⅵ between the ultra-micro powder and the fine powder of Radix Dipsaci.METHODS : The real contents,in vitro release and releasing rate of asperosaponin Ⅵ were determined by HPLC for the ultra-micro powder and the fine powder.RESULTS: In the ultra- micro powder and the ordinary powder,the real content of asperosaponin Ⅵ were 4.87%,4.74%,respectively; in vitro release in 1 h were 48.2 mg/g,47.5 mg/g,respectively; releasing rate parameter T_(0.9) were 0.23 min,10.41 min,respectively.CONCLUSION: The ultra- micro porphyrization could not influent the real content and in vitro release of asperosaponin Ⅵ in Radix Dipsaci.But it could improve the releasing rate of asperosaponin Ⅵ.
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Objective:In order to discuss the influence of processed methods to the contents of total saponins and asperosaponin Ⅵ in Dipsacus asperoids,the content alternation of total saponins and asperosaponin Ⅵ in crude and processed Dipsacus asperoids was investigated.Methods:The content of total saponins was determined by spectrophotometry using vanillin-acetic acid-perchloric acid as the chromogenic reagent.The content of asperosaponin Ⅵ in crude and processed Dipsacus asperoids was determined by HPLC.Results: Contracted to the crude Dipsacus asperoids,the content of total saponins in the processed is higher.The content of asperosaponin Ⅵ in the processed Dipsacus asperoids is higher than that in the crude significantly.Conclusion:Different processed mothods affected the content alternation of asperosaponin Ⅵ in Dipsacus asperoids.The processing of Dipsacus asperoids is reasonable.
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OBJECTIVE:To establish HPLC method for the content determination of asperosaponin Ⅵ in Yulin mixture.ME-THODS:The separation was performed on Zorbax EcLipse XDB-C18(150 mm?4.6 mm,5 ?m) column with mobile phase consisted of methanol-0.01 mol?L-1 hydrochloric acid (69:31) and flow rate of 1 mL?min-1.The detection wavelength was set at 212 nm and column temperature was 20 ℃.RESULTS:The linear range of asperosaponin Ⅵ was 0.022～0.23 mg?mL-1(r=0.998 9) with an average recovery of 101.33% (RSD=2.35%,n=9).CONCLUSION:The method is convenient,simple,accurate and reproducibility for the quality control of Yulin mixture.
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AIM:To compare the dissolution of asperosaponin Ⅵ between the ultra-micro powder and the fine powder of Radix Dipsaci.METHODS:The real contents,in vitro release and releasing rate of asperosaponin Ⅵwere determined by HPLC for the ultra-micro powder and the fine powder.RESULTS:In the ultra-micro powder and the ordinary powder,the real content of asperosaponin Ⅵ were 4.87%,4.74%,respectively;in vitro release in 1 h were 48.2 mg/g,47.5 mg/g,respectively;releasing rate parameter T_ 0.9 were 0.23 min,10.41 min,respectively.CONCLUSION:The ultra-micro porphyrization could not influent the real content and in vitro release of asperosaponin Ⅵ in Radix Dipsaci.But it could improve the releasing rate of asperosaponin Ⅵ.