Aim To develop and determine pinoresinol diglucopyranoside in Qing'e Pill, atraditional Chinese compound preparation containing Eucommia ulmoides Oliv. as the principal drug,by a reverse-phase high-performance liq...Aim To develop and determine pinoresinol diglucopyranoside in Qing'e Pill, atraditional Chinese compound preparation containing Eucommia ulmoides Oliv. as the principal drug,by a reverse-phase high-performance liquid chromatographic method (RP-HPLC) . Methods The extract ofQing'e Pill was refluxed with 75% ethanol, purified on an AB-8 macroporous adsorption resin columnand then injected into HPLC system. The HPLC assay was performed on an ODS analytical column with amixture of methanol-acetonitrile-water (24:3:78, V/V/V) as the mobile phase at a flow-rate of 1.0mL·min^(-1), and a UV detector set at 227 nm. Results Good linearity between peak area andconcentration was found in the range of 5.5 - 170 μg·mL^(-1) for pinoresinol diglucopyranoside ( r> 0.9998) . The average recovery was 99.3%. The intra-day assay RSD and the inter-day assay RSDwere 1.3% and 2.8%, respectively (n = 5). The content of pinoresinol diglucopyranoside in Qing'ePill was determined to be 0.446 +- 0.012 mg·g^(-1) (n = 10). Conclusion The RP-HPLC method wasproved to be sensitive, specific, accurate and precise for the determination of pinoresinoldiglucopyranoside in Qing' e Pill.展开更多
UDP-glycosyltransferases (UGTs) constitute the largest glycosyltransferase family in the plant kingdom,regulating many metabolic processes by transferring sugar moieties onto various small molecules. How-ever, their p...UDP-glycosyltransferases (UGTs) constitute the largest glycosyltransferase family in the plant kingdom,regulating many metabolic processes by transferring sugar moieties onto various small molecules. How-ever, their physiological significance in plants remains largely unknown. Here, we reveal the functionsand mechanisms of two Arabidopsis UGT genes, UGT73C3 and UGT73C4, which are strongly induced byPseudomonas syringae pv. tomato (Pst) DC3000. Overexpression of these genes significantly enhancedplant immune response, whereas their loss of function in double mutants led to increased sensitivity topathogen infections. However, single mutants showed no obvious alteration in pathogen resistance. Tofurther investigate the regulatory mechanisms of UGT73C3/C4 in plant immunity, we conducted compre-hensive secondary metabolome analyses and glycoside quantification. Overexpression lines accumulatedhigher levels of pinoresinol diglucosides than wild-type plants, both before and after Pst DC3000 treatment,whereas double mutants accumulated lower levels. Furthermore, in vitro and in vivo experiments demon-strated that UGT73C3 and UGT73C4 can glycosylate pinoresinol to form pinoresinol monoglucoside anddiglucoside. Moreover, pinoresinol glycosylation promotes the plant immune response by increasing reac-tive oxygen species production and callose deposition. Additionally, the transcription factor HB34 wasfound to activate UGT73C3 and UGT73C4 transcription and play a key role in plant immunity. Overall,this study reveals a novel pathway in which UGT73C3/C4-mediated pinoresinol glycosylation, regulatedby HB34, enhances the plant immune response.展开更多
Objective Pinoresinol di-glucopyranoside（PDG） is one of the main active lignans of Eucommiae Cortex considered to be a high-quality antihypertensive drug. In this study the pharmacokinetic process of PDG and its pri...Objective Pinoresinol di-glucopyranoside（PDG） is one of the main active lignans of Eucommiae Cortex considered to be a high-quality antihypertensive drug. In this study the pharmacokinetic process of PDG and its primary in vivo metabolite pinoresinol glucoside（PG） in the portal and jugular vein were surveyed and evaluated simultaneously. Methods A sensitive high-performance liquid chromatography coupled with tandem quadruple mass spectrometry（HPLC-MS/MS） method and sample preparation protocol were developed and validated in method of selectivity, sensitivity, precision, stability, and extraction recovery for the simultaneous determination of PDG and its primary metabolite PG in rat plasma. The double intubation technique was used to simultaneously collect blood from common jugular vein and hepatic portal vein after single ig administration of PDG. Results Using this method, the quantification linearity ranges of PDG and PG in rat plasma were both 0.05-100 ng/mL. This method was successfully applied to the evaluation of the absolute oral bioavailability of PDG and determination of the pharmacokinetic properties of PDG and PG after ig administration of single dose in rats. The bioavailability of PDG at common jugular vein was 51.3% compared to that of 91.6% at hepatic portal vein. Conclusion We conclude that liver is the major conversion site of PDG to PG.展开更多
文摘Aim To develop and determine pinoresinol diglucopyranoside in Qing'e Pill, atraditional Chinese compound preparation containing Eucommia ulmoides Oliv. as the principal drug,by a reverse-phase high-performance liquid chromatographic method (RP-HPLC) . Methods The extract ofQing'e Pill was refluxed with 75% ethanol, purified on an AB-8 macroporous adsorption resin columnand then injected into HPLC system. The HPLC assay was performed on an ODS analytical column with amixture of methanol-acetonitrile-water (24:3:78, V/V/V) as the mobile phase at a flow-rate of 1.0mL·min^(-1), and a UV detector set at 227 nm. Results Good linearity between peak area andconcentration was found in the range of 5.5 - 170 μg·mL^(-1) for pinoresinol diglucopyranoside ( r> 0.9998) . The average recovery was 99.3%. The intra-day assay RSD and the inter-day assay RSDwere 1.3% and 2.8%, respectively (n = 5). The content of pinoresinol diglucopyranoside in Qing'ePill was determined to be 0.446 +- 0.012 mg·g^(-1) (n = 10). Conclusion The RP-HPLC method wasproved to be sensitive, specific, accurate and precise for the determination of pinoresinoldiglucopyranoside in Qing' e Pill.
文摘UDP-glycosyltransferases (UGTs) constitute the largest glycosyltransferase family in the plant kingdom,regulating many metabolic processes by transferring sugar moieties onto various small molecules. How-ever, their physiological significance in plants remains largely unknown. Here, we reveal the functionsand mechanisms of two Arabidopsis UGT genes, UGT73C3 and UGT73C4, which are strongly induced byPseudomonas syringae pv. tomato (Pst) DC3000. Overexpression of these genes significantly enhancedplant immune response, whereas their loss of function in double mutants led to increased sensitivity topathogen infections. However, single mutants showed no obvious alteration in pathogen resistance. Tofurther investigate the regulatory mechanisms of UGT73C3/C4 in plant immunity, we conducted compre-hensive secondary metabolome analyses and glycoside quantification. Overexpression lines accumulatedhigher levels of pinoresinol diglucosides than wild-type plants, both before and after Pst DC3000 treatment,whereas double mutants accumulated lower levels. Furthermore, in vitro and in vivo experiments demon-strated that UGT73C3 and UGT73C4 can glycosylate pinoresinol to form pinoresinol monoglucoside anddiglucoside. Moreover, pinoresinol glycosylation promotes the plant immune response by increasing reac-tive oxygen species production and callose deposition. Additionally, the transcription factor HB34 wasfound to activate UGT73C3 and UGT73C4 transcription and play a key role in plant immunity. Overall,this study reveals a novel pathway in which UGT73C3/C4-mediated pinoresinol glycosylation, regulatedby HB34, enhances the plant immune response.
基金Doctoral Fund of Ministry of Education of China(20131210120010)Important Drug Develop of MOST,China(2013ZX09401-004)+2 种基金Important Drug Develop of MOST,China(2012ZX09103201-046)Program for Innovative Research Team in Universities of Tianjin(TD12-5033,TD12-5036)Tianjin Science and Technology Innovation System and Conditions Platform Construction Plan(14TXZYJC00440)
文摘Objective Pinoresinol di-glucopyranoside（PDG） is one of the main active lignans of Eucommiae Cortex considered to be a high-quality antihypertensive drug. In this study the pharmacokinetic process of PDG and its primary in vivo metabolite pinoresinol glucoside（PG） in the portal and jugular vein were surveyed and evaluated simultaneously. Methods A sensitive high-performance liquid chromatography coupled with tandem quadruple mass spectrometry（HPLC-MS/MS） method and sample preparation protocol were developed and validated in method of selectivity, sensitivity, precision, stability, and extraction recovery for the simultaneous determination of PDG and its primary metabolite PG in rat plasma. The double intubation technique was used to simultaneously collect blood from common jugular vein and hepatic portal vein after single ig administration of PDG. Results Using this method, the quantification linearity ranges of PDG and PG in rat plasma were both 0.05-100 ng/mL. This method was successfully applied to the evaluation of the absolute oral bioavailability of PDG and determination of the pharmacokinetic properties of PDG and PG after ig administration of single dose in rats. The bioavailability of PDG at common jugular vein was 51.3% compared to that of 91.6% at hepatic portal vein. Conclusion We conclude that liver is the major conversion site of PDG to PG.
