1.Effect of processing on the alkaloids in Aconitum tubers by HPLC-TOF/MS
Min Liu ; Yan Cao ; Diya Lv ; Wen Zhang ; Zhenyu Zhu ; Hai Zhang ; Yifeng Chai
Journal of Pharmaceutical Analysis 2017;7(3):170-175
According to the Chinese Pharmacopoeia 2015, only processed Aconitum tubers can be clinically applied, and the effect of processing is unclear. This research aimed to explore the effect of processing on cardiac efficacy of alkaloids in Aconitum tubers. First, the chemical ingredients in unprocessed and processed Aconitum tubers were identified and compared by using high performance liquid chromatography time-of-flight mass spectrometry (HPLC-TOF/MS) and multivariate pattern recognition methods. Then the representative alkaloids in Aconitum tubers, aconitine, benzoylaconine, and aconine, which belong to diester-diterpenoid alkaloids, monoester-diterpenoid alkaloids, and amine-diterpenoid alkaloids, respectively, were selected for further validation of attenuated mechanism. Subsequent pharmacological experiments with aconitine, benzoylaconine, and aconine in SD rats were used to validate the effect of processing on cardiac functions. After processing the Aconitum tubers, it was found that the contents of diester-diterpenoid alkaloids were reduced, and those of monoester-diterpenoid alkaloids and amine-diterpenoid alkaloids were increased, suggesting that diesterditerpenoid alkaloids were transformed into monoester-diterpenoid alkaloids and amine-diterpenoid alkaloids. Through further decocting the aconitine in boiling water, it was confirmed that the three alkaloids could be progressively transformed. Pharmacological experiments with aconitine, benzoylaconine, and aconine in SD rats showed that aconitine at a dose of 0.01 mg/kg and aconine at a dose of 10 mg/kg enhanced the cardiac function, while benzoylaconine at a dose of 2 mg/kg weakened the cardiac function. The effect of processing is attributed to the transformation of the most toxic diester-diterpenoid alkaloids into less toxic monoesterditerpenoid alkaloids and amine-diterpenoid alkaloids.
2.Rapid identification of chemical components in Artemisiae argyi folium by RRLC-TOFMS
Ling LI ; Lei L ; Xin DONG ; Diya LV ; Ziyang LOU
Journal of Pharmaceutical Practice 2014;(6):448-452
Objective To identify the chemical components in Artemisiae argyi folium by rapid resolution liquid chromatog-raphy-time of flight mass spectrometry (RRLC-TOFMS).Methods The separation was performed on an Agilent Eclipse C18 column (2.1 mm ×100 mm,1.8 μm ).The mobile phase consisted of acetonitrile (A) and 0.1% formic acid (B) were in gradient elu-tion.The flowing rate was 0.35 ml/min, the injection volume was 1μl and the temperature of column was 40℃.Time of flight mass spectrometer ( TOFMS) with electro spray ion source ( ESI) was applied to qualitative analysis under the positive ion mode, and mass scan range was m/z 100-1 500 .Results 31 chemical compounds in Artemisiae argyi folium were identified unequivocally .Conclu-sion A rapid and efficient RRLC-TOFMS approach for identifying the chemical constituents of Artemisiae argyi folium had been suc-cessfully established,which paved a way for quality control and further in vivo studies of Artemisiae argyi folium.
3.A strategy of screening and binding analysis of bioactive components from traditional Chinese medicine based on surface plasmon resonance biosensor
Lv DIYA ; Xu JIN ; Qi MINYU ; Wang DONGYAO ; Xu WEIHENG ; Qiu LEI ; Li YINGHUA ; Cao YAN
Journal of Pharmaceutical Analysis 2022;12(3):500-508
Elucidating the active components of traditional Chinese medicine(TCM)is essential for understanding the mechanisms of TCM and promote its rational use as well as TCM-derived drug development.Recent studies have shown that surface plasmon resonance(SPR)technology is promising in this field.In the present study,we propose an SPR-based integrated strategy to screen and analyze the major active components of TCM.We used Radix Paeoniae Alba(RPA)as an example to identify the compounds that can account for its anti-inflammatory mechanism via tumor necrosis factor receptor type 1(TNF-R1).First,RPA extraction was analyzed using an SPR-based screening system,and the potential active in-gredients were collected,enriched,and identified as paeoniflorin and paeonol.Next,the affinity con-stants of paeoniflorin and paeonol were determined as 4.9 and 11.8 μM,respectively.Then,SPR-based competition assays and molecular docking were performed to show that the two compounds could compete with tumor necrosis factor-α(TNF-α)while binding to the subdomain 1 site of TNF-R1.Finally,in biological assays,the two compounds suppressed cytotoxicity and apoptosis induced by TNF-α in the L929 cell line.These findings prove that SPR technology is a useful tool for determining the active in-gredients of TCM at the molecular level and can be used in various aspects of drug development.The SPR-based integrated strategy is reliable and feasible in TCM studies and will shed light on the eluci-dation of the pharmacological mechanism of TCM and facilitate its modernization.
4.Detection of malachite green in natural indigo by surface enhanced Raman spec-troscopy based on monolithic column
Binxing ZHENG ; Diya LV ; Fang FANG ; Yanhua LIU ; Dan LI ; Feng LU ; Jiyang XU
Journal of Pharmaceutical Practice 2015;(5):426-428,459
Objective To develop a rapid SERS detection method based on monolithic column for detection of dye adul-terated natural indigo .Methods The dyes in natural indigo were extracted and mixed with silver colloid .The spectra were re-corded after applying the mixture solution to the monolithic column since the intertwined pores in monolithic column could con-tribute for the distribution of silver nanoparticles .Results SERS signals of malachite green dyed natural indigo at quantity as low as 500 μg/kg could be obtained .Conclusion This simple ,fast and specific SERS detection method based on monolithic col-umn could be used for rapid detection of stained natural indigo .
5.Development of a surface plasmon resonance biosensor for accurate and sensitive quantitation of small molecules in blood samples
Minyu QI ; Diya LV ; Ying ZHANG ; Dongyao WANG ; Xiaofei CHEN ; Zhenyu ZHU ; Zhanying HONG ; Yifeng CHAI ; Hai ZHANG ; Yan CAO
Journal of Pharmaceutical Analysis 2022;12(6):929-936
Therapeutic drug monitoring(TDM)has played an important role in clinical medicine for precise dosing.Currently,chromatographic technology and immunoassay detection are widely used in TDM and have met most of the needs of clinical drug therapy.However,some problems still exist in practical appli-cations,such as complicated operation and the influence of endogenous substances.Surface plasmon resonance(SPR)has been applied to detect the concentrations of small molecules,including pesticide residues in crops and antibiotics in milk,which indicates its potential for in vivo drug detection.In this study,a new SPR-based biosensor for detecting chloramphenicol(CAP)in blood samples was developed and validated using methodological verification,including precision,accuracy,matrix effect,and extraction recovery rate,and compared with the classic ultra-performance liquid chromatography-ultraviolet(UPLC-UV)method.The detection range of SPR was 0.1-50 ng/mL and the limit of detec-tion was 0.099±0.023 ng/mL,which was lower than that of UPLC-UV.The intra-day and inter-day ac-curacies of SPR were 98%-114%and 110%-122%,which met the analysis requirement.The results show that the SPR biosensor is identical to UPLC-UV in the detection of CAP in rat blood samples;moreover,the SPR biosensor has better sensitivity.Therefore,the present study shows that SPR technology can be used for the detection of small molecules in the blood samples and has the potential to become a method for therapeutic drug monitoring.
6.I n situ synthesis and unidirectional insertion of membrane proteins in liposome-immobilized silica stationary phase for rapid preparation of microaffinity chromatography.
Yanqiu GU ; Rong WANG ; Panpan CHEN ; Shengnan LI ; Xinyi CHAI ; Chun CHEN ; Yue LIU ; Yan CAO ; Diya LV ; Zhanying HONG ; Zhenyu ZHU ; Yifeng CHAI ; Yongfang YUAN ; Xiaofei CHEN
Acta Pharmaceutica Sinica B 2022;12(9):3682-3693
Cell membrane affinity chromatography has been widely applied in membrane protein (MP)-targeted drug screening and interaction analysis. However, in current methods, the MP sources are derived from cell lines or recombinant protein expression, which are time-consuming for cell culture or purification, and also difficult to ensure the purity and consistent orientation of MPs in the chromatographic stationary phase. In this study, a novel in situ synthesis membrane protein affinity chromatography (iSMAC) method was developed utilizing cell-free protein expression (CFE) and covalent immobilized affinity chromatography, which achieved efficient in situ synthesis and unidirectional insertion of MPs into liposomes in the stationary phase. The advantages of iSMAC are: 1) There is no need to culture cells or prepare recombinant proteins; 2) Specific and purified MPs with stable and controllable content can be obtained within 2 h; 3) MPs maintain the transmembrane structure and a consistent orientation in the chromatographic stationary phase; 4) The flexible and personalized construction of cDNAs makes it possible to analyze drug binding sites. iSMAC was successfully applied to screen PDGFRβ inhibitors from Salvia miltiorrhiza and Schisandra chinensis. Micro columns prepared by in-situ synthesis maintain satisfactory analysis activity within 72 h. Two new PDGFRβ inhibitors, salvianolic acid B and gomisin D, were screened out with K D values of 13.44 and 7.39 μmol/L, respectively. In vitro experiments confirmed that the two compounds decreased α-SMA and collagen Ӏ mRNA levels raised by TGF-β in HSC-T6 cells through regulating the phosphorylation of p38, AKT and ERK. In vivo, Sal B could also attenuate CCl4-induced liver fibrosis by downregulating PDGFRβ downstream related protein levels. The iSMAC method can be applied to other general MPs, and provides a practical approach for the rapid preparation of MP-immobilized or other biological solid-phase materials.