Quantitative Analysis of Multi-components in Gnaphalium affine Based on UPLC-Q-Exactive Orbitrap MS / 中国实验方剂学杂志

ObjectiveTo establish a rapid and stable liquid chromatography-mass spectrometry（LC-MS） for simultaneous analysis of 17 chemical components in Gnaphalium affine aboveground parts with flowers， so as to provide experimental basis for improving the quality standard of this herb. MethodUltra performance liquid chromatography-quadrupole/electrostatic field orbitrap mass spectrometry（UPLC-Q-Exactive Orbitrap MS） was used for the quantitative analysis of 17 constituents in 15 batches of G. affine from different origins， the separation was performed on an ACQUITY UPLC® BEH C18 column（2.1 mm×100 mm， 1.7 μm） with the mobile phase of methanol（A）-0.1% formic acid aqueous solution（B） for gradient elution（0-1.0 min， 8%A； 1.0-4.0 min， 8%-26%A； 4.0-9.0 min， 26%A； 9.0-14.0 min， 26%-34%A； 14.0-14.5 min， 34%-45%A； 14.5-15.0 min， 45%-60%A； 15.0-18.0 min， 60%-90%A； 18.0-19.0 min， 90%A； 19.0-19.01 min， 90%-8%A； 19.01-20.0 min， 8%A）， the flow rate was 0.3 mL·min-1， the column temperature was 40 ℃ and the injection volume was 2 μL. And the electrospray ionization was used with full scanning in both positive and negative ion modes， and the scanning range was m/z 100-1 000. ResultThe established method has been verified by the methodology and could be used for the simultaneous quantification of 17 components in G. affine. The content ranges of the 17 components（quinic acid， gallic acid， protocatechuic acid， neochlorogenic acid， chlorogenic acid， cryptochlorogenic acid， caffeic acid， 1，3-O-dicaffeoylquinic acid， isochlorogenic acid A， isoquercitrin， 1，5-O-dicaffeoylquinic acid， apigenin-7-O-glucoside， astragalin， isochlorogenic acid C， luteolin， apigenin and hispidulin） in 15 batches of G. affine samples was 39.60-179.12， 0.17-0.84， 2.41-8.38， 4.33-31.50， 13.63-180.38， 2.43-14.75， 1.16-19.68， 0.49-5.63， 55.77-445.16， 0.23-10.26， 62.04-530.10， 1.11-18.01， 11.36-90.61， 12.22-65.98， 7.22-69.84， 3.37-45.65， 0.30-2.59 μg·g-1， respectively. The content of organic acids was higher than that of flavonoids in G. affine， and the contents of 1，5-O-dicaffeoylquinic acid， isochlorogenic acid A， quinic acid and chlorogenic acid were higher. Meanwhile， the content of flavonoids in the samples from Guizhou was higher than that from Jiangsu， while the content of organic acids in the samples from Jiangsu was higher than that from Guizhou. ConclusionThe established method can be used for the rapid and accurate determination of 17 components in G. affine， which clarifies the content range of the main components in this herb， and can provide a reference for the selection of quality control markers of G. affine.

Screening of anti-COPD quality biomarkers of Gnaphalium affine / 中国药房

OBJECTIVE To screen the quality biomarkers of Gnaphalium affine with anti-chronic obstructive pulmonary disease （COPD） effect and determine their contents. METHODS The effective components and targets of “G. affine” with anti- COPD effect were predicted by using network pharmacology as a search criterion. HPLC fingerprints for 10 batches of G. affine were established by using Similarity Evaluation System of TCM Chromatographic Fingerprint （2012 edition）； common peak identification and similarity evaluation were conducted； cluster analysis （CA）， principal component analysis （PCA）， and orthogonal partial least squares-discriminant analysis （OPLS-DA） were performed to screen differential components as quality maker that affected the quality of G. affine using variable importance projection （VIP）＞1 as the standard. The same HPLC method was adopted to determine the contents of the differential components in 10 batches of samples. RESULTS A total of 10 flavonoids （such as quercetin， luteolin， and chlorogenic acid） and organic acid components， were identified through network pharmacology search， with 91 targets closely related to anti-COPD. A total of 9 common peaks were identified in 10 batches of samples， with similarity greater than 0.90. Among them， the differential components included chlorogenic acid， caffeic acid， 1，3-O- dicaffeoylquinic acid and apigenin 7-O-β-D-glucopyranoside； S3， S4， S6， S7 and S10 were clustered into one category， S2， S5， S8 and S9 clustered into one category， and S1 clustered into one category. The contents of chlorogenic acid， caffeic acid， 1，3-O- dicaffeoylquinic acid， and apigenin 7-O-β-D-glucopyranoside in 10 batches of G. affine ranged 0.070-7.653， 0.010-0.097， 0.001- 0.036， 0.508-6.627 mg/g， respectively. CONCLUSIONS Chlorogenic acid， caffeic acid， 1，3-O-dicaffeoylquinic acid， apigenin 7- O-β-D-glucopyranoside can serve as the potential quality marker for the anti-COPD effect of G. affine， with the highest content of chlorogenic acid in G. affine produced in Ji’an， Jiangxi province， and the highest content of caffeic acid in G. affine produced in Ji’an， Jiangxi province and Sanming， Fujian province. The contents of the last two components are highest in G. affine produced in Chaoshan， Guangdong province.

Establish and optimization of inter-simple sequence repeat PCR reaction system of Gnaphalium affine / 药学实践杂志

Objective To provide the experimental basis for the subsequent genetic diversity research through establishing and optimizing the inter-simple sequence repeat PCR (ISSR-PCR) reaction system of Gnaphalium affine. Methods The single-factor experimental method and full experimental method were used to optimize the ISSR-PCR reaction system of Gnaphalium affine. Under the optimal system, after screening primers and corresponding annealing temperatures, the systematic feasibility was verified. Results The optimal ISSR-PCR reaction system was consisted of 10 μl Premix Taq DNA polymerase, 0.3 μmol/L primer, 10 ng DNA template, and sterilized water added to 20 μl. Finally, 10 primers were screened from 100 universal primers, and verification results indicated the system had high stability, good reproducibility, and the selected primers had good polymorphism. Conclusion The ISSR-PCR amplification system of Gnaphalium affine was established for the first time and the primers with appropriate annealing temperatures were filtered out, which provided a reference for the subsequent genetic diversity research of Gnaphalium affine.

Simultaneous determination of seven constituents in Gnaphalium affine / 中成药

AIM To establish an HPLC method for the simultaneous content determination of seven constituents in Gnaphalium affine D.Don.METHODS The analysis of 80％ methanol of G.affine was performed on a 30 ℃ Atlantis (C) T3 column (4.6 mm× 250 mm,5 μm),with the mobile phase comprising of acetonitrile-formic acid flowing at 1.0 mL/min in a gradient elution manner,and the detection wavelength was set at 288 nm.RESULTS Seven constituents showed good linear relationships within their own ranges (R2 ≥0.999 8),whose average recoveries were 98.58％-103.8％ with the RSDs of 0.88％-1.74％.CONCLUSION This accurate,stable and reproducible method can be used for the quality control of G.affine.

Chemical constituents from Gnaphalium affine and their xanthine oxidase inhibitory activity / 中国天然药物

Gnaphalium affine D. Don, a medicinal and edible plant, has been used to treat gout in traditional Chinese medicine and popularly consumed in China for a long time. A detailed phytochemical investigation on the aerial part of G. affine led to the isolation of two new esters of caffeoylquinic acid named (-) ethyl 1, 4-di-O-caffeoylquinate (1) and (-) methyl 1, 4-di-O-caffeoylquinate (2), together with 35 known compounds (3-37). Their structures were elucidated by spectroscopic data and first-order multiplet analysis. All the isolated compounds were tested for their xanthine oxidase inhibitory activity with an in vitro enzyme inhibitory screening assay. Among the tested compounds, 1 (IC 11.94 μmol·L) and 2 (IC 15.04 μmol·L) showed a good inhibitory activity. The current results supported the medical use of the plant.

Chemical constituents from Gnaphalium affine and their xanthine oxidase inhibitory activity / 中国天然药物

Gnaphalium affine D. Don, a medicinal and edible plant, has been used to treat gout in traditional Chinese medicine and popularly consumed in China for a long time. A detailed phytochemical investigation on the aerial part of G. affine led to the isolation of two new esters of caffeoylquinic acid named (-) ethyl 1, 4-di-O-caffeoylquinate (1) and (-) methyl 1, 4-di-O-caffeoylquinate (2), together with 35 known compounds (3-37). Their structures were elucidated by spectroscopic data and first-order multiplet analysis. All the isolated compounds were tested for their xanthine oxidase inhibitory activity with an in vitro enzyme inhibitory screening assay. Among the tested compounds, 1 (IC 11.94 μmol·L) and 2 (IC 15.04 μmol·L) showed a good inhibitory activity. The current results supported the medical use of the plant.

Simultaneous Determination of Quercetin and Mignonette in Gnaphalium affine by HPLC / 世界科学技术-中医药现代化

Objective To establish a HPLC method for the simultaneous determination of quercetin and mignonette in Gnaphalium affine. Methods The determination was performed on Diamonsil C18 (2) column (4.6 mm×250 mm, 5 μm), mobile phase was methanol-0.4％ phosphoric acid solution (42:58), detection wavelength was set at 360 nm, column temperature was 30℃, flow rate was 1 mL · min-1 and the injection volume was 20 μL. Results The linear rages of quercetin and mignonette were 3.588-35.880 μg · mL-1 (R2=0.999 6) and 1.294-12.940 μg · mL-1 (R2=0.999 4) respectively. Their average recoveries (n=6) were 99.51％ (RSD=0.31％) and 99.74％ (RSD=2.28％) respectively.Quercetin Content was 0.39-0.58 mg·g-1, mignonette content was 0.17-0.27 mg·g-1.Conclusion The method is simple, accurate, reliable and repeatable, thus it can be used for the quality control of Gnaphalium affine.

Study on chemical constituents from Gnaphalium affine and their anti-oxidative activities / 中草药

Objective: To study the chemical constituents from Gnaphalium affine and their anti-oxidative activities. Methods: Chemical constituents were isolated by column chromatography and semi-prepared HPLC, the structures were elucidated by spectral data and physicochemical properties. DPPH free radical scavenging activities of compounds 1,3-11, and 16 were determined. Results: Seventeen compounds were isolated and respectively identified as apigenin (1), dihydroapigenin (2), luteolin (3), chrysin (4), wogonin (5), stimasterol (6), β-sitosterol (7), ursolic acid (8), oleanolic acid (9), 19α-hydroxyl-oleanolic acid (10), 2α, 3α, 19α-trihydroxy-28-norurs-12ene (11), α-amyrin acetate (12), β-amyrin acetate (13), patriscabratine (14), aurantiamide acetate (15), 4'-hydroxydehydrokawain (16), and isovanillin (17). Compounds 1 and 3-5 had significant anti-oxidative activities. Conclusion: Compounds 2,4,5,11-15, and 17 are isolated from G. affine for the frist time.