To develop a simple and highly sensitive high performance liquid chromatography with electrospray ionization mass spectrometric(LC-ESI-MS) method for the simultaneous determination of madecassoside and its major metab...To develop a simple and highly sensitive high performance liquid chromatography with electrospray ionization mass spectrometric(LC-ESI-MS) method for the simultaneous determination of madecassoside and its major metabolite madecassic acid in rat plasma, and compare the pharmacokinetics of the two compounds in normal and collagen-induced arthritis(CIA) rats. Glycyrrhetinic acid was used as the internal standard(IS). Chromatographic separation was accomplished on an Inertsil ODS-3 column, using a gradient elution with the mobile phase composed of acetonitrile and water acidified with 0.1%(V/V) formic acid. Detection was achieved by ESI-MS under the negative selected ion monitoring(SIM) mode. In normal and CIA rats, madecassoside(30 mg·kg-1) was orally administered for 21 consecutive days from the day of arthritis onset. For madecassoside, the linear range was 10–1 000 ng·mL-1 with the square regression coefficient(r) of 0.998 9, while for madecassic acid, the linear range was 10–500 ng·mL-1 with the square regression coefficient(r) of 0.996 1. The lower limit of quantification was 10 ng·mL-1 for both analytes. The intra- and inter-day precision ranged from 1.78% to 13.42% for madecassoside and 2.30% to 14.90% for madecassic acid, and the accuracy was between –0.95% and 6.30% for madecassoside and between –1.48% and 5.34% for madecassic acid. The average recoveries of madecassoside, madecassic acid and IS from spiked plasma samples were > 81%. The developed method was successfully applied to the pharmacokinetic study of madecassoside and madecassic acid in rats after an oral administration of madecassoside. During initial 7 days of dosing, the cmax and AUC of madecassoside were greatly decreased and Vd/F was markedly increased in CIA rats, and no significant difference was observed on the first day of dosing. In contrast, the T1/2, cmax and AUC of madecassic acid were significantly increased, and Ke of madecassic acid was greatly decreased in CIA rats compared with normal rats. Along with repeated administration of madecassoside, the differences of pharmacokinetic parameters of both madecassoside and madecassic acid between CIA and normal rats gradually subsided. The pharmacokinetic characteristics of both madecassoside and madecassic acid in rats were significantly altered by arthritis status, and the differences of pharmacokinetic parameters between arthritis and normal rats coincide with the severity of arthritis.展开更多
Background:Overproduction and activation of osteoclasts result in various bone diseases,such as osteoporosis,Paget's disease,and rheumatoid arthritis.Thus,inhibiting osteoclast formation and overactivation may eff...Background:Overproduction and activation of osteoclasts result in various bone diseases,such as osteoporosis,Paget's disease,and rheumatoid arthritis.Thus,inhibiting osteoclast formation and overactivation may effectively prevent osteoclast-related bone diseases,especially osteoporosis.Madecassic acid,one of the most important active ingredients in Centella asiatica,has various biological effects,but its role in osteoclastogenesis remains unknown.Methods:RAW 264.7 cells were stimulated with receptor activator of nuclear factor(NF)-κΒligand(RANKL,25 ng/mL)to differentiate into multinucleated osteoclasts.Subsequently,osteoclasts were treated with or without varying concentrations of madecassic acid(1,2.5,5,and 10μmol/L).Results:Madecassic acid significantly inhibited RANKL-induced osteoclastogenesis in a concentration-dependent manner.In addition,it reduced the percentage of bone resorptive area compared with the control,confirming that madecassic acid can inhibit osteoclast function.Furthermore,luciferase reporter gene studies indicate that madecassic acid could decrease the transcriptional activity of NF of activated T cells(NFAT)and NF-κB in a dose-dependent manner.Quantitative real-time polymerase chain reaction results show that madecassic acid attenuated the expression of osteoclast-associated genes,including V-ATPase-d2,cathepsin K,tartrate-resistant acid phosphatase(TRAP),NFAT cytoplasmic 1(NFATc1).Western blot analysis shows that madecassic acid inhibited RANKL-mediated degradation of IκBαand NFATc1 expression,as well as phosphorylation of c-Jun N-terminal kinase(JNK)in RAW 264.7 cells.Conclusion:Madecassic acid inhibited osteoclast formation and function in vitro by suppressing NF-κB,JNK,and NFAT signaling pathways,indicating its potential as a novel drug for the treatment of osteoclast-related bone diseases,especially osteoporosis.展开更多
基金financially supported by the National Natural Science Foundation of China(No.81374038,81173631)the Jiangsu Overseas Research&Training Program for University Prominent Young&Middle-aged Teachers and Presidents+1 种基金sponsored by Qing Lan Project,College Students Innovation Project for the R&D of Novel Drugs(No.J1030830)partially funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions
文摘To develop a simple and highly sensitive high performance liquid chromatography with electrospray ionization mass spectrometric(LC-ESI-MS) method for the simultaneous determination of madecassoside and its major metabolite madecassic acid in rat plasma, and compare the pharmacokinetics of the two compounds in normal and collagen-induced arthritis(CIA) rats. Glycyrrhetinic acid was used as the internal standard(IS). Chromatographic separation was accomplished on an Inertsil ODS-3 column, using a gradient elution with the mobile phase composed of acetonitrile and water acidified with 0.1%(V/V) formic acid. Detection was achieved by ESI-MS under the negative selected ion monitoring(SIM) mode. In normal and CIA rats, madecassoside(30 mg·kg-1) was orally administered for 21 consecutive days from the day of arthritis onset. For madecassoside, the linear range was 10–1 000 ng·mL-1 with the square regression coefficient(r) of 0.998 9, while for madecassic acid, the linear range was 10–500 ng·mL-1 with the square regression coefficient(r) of 0.996 1. The lower limit of quantification was 10 ng·mL-1 for both analytes. The intra- and inter-day precision ranged from 1.78% to 13.42% for madecassoside and 2.30% to 14.90% for madecassic acid, and the accuracy was between –0.95% and 6.30% for madecassoside and between –1.48% and 5.34% for madecassic acid. The average recoveries of madecassoside, madecassic acid and IS from spiked plasma samples were > 81%. The developed method was successfully applied to the pharmacokinetic study of madecassoside and madecassic acid in rats after an oral administration of madecassoside. During initial 7 days of dosing, the cmax and AUC of madecassoside were greatly decreased and Vd/F was markedly increased in CIA rats, and no significant difference was observed on the first day of dosing. In contrast, the T1/2, cmax and AUC of madecassic acid were significantly increased, and Ke of madecassic acid was greatly decreased in CIA rats compared with normal rats. Along with repeated administration of madecassoside, the differences of pharmacokinetic parameters of both madecassoside and madecassic acid between CIA and normal rats gradually subsided. The pharmacokinetic characteristics of both madecassoside and madecassic acid in rats were significantly altered by arthritis status, and the differences of pharmacokinetic parameters between arthritis and normal rats coincide with the severity of arthritis.
基金Basic and Applied Basic Research Foundation of Guangdong Province,Grant/Award Number:2019A1515110723Key Laboratory of Guangdong Higher Education Institutes,Grant/Award Number:2021KSYS009。
文摘Background:Overproduction and activation of osteoclasts result in various bone diseases,such as osteoporosis,Paget's disease,and rheumatoid arthritis.Thus,inhibiting osteoclast formation and overactivation may effectively prevent osteoclast-related bone diseases,especially osteoporosis.Madecassic acid,one of the most important active ingredients in Centella asiatica,has various biological effects,but its role in osteoclastogenesis remains unknown.Methods:RAW 264.7 cells were stimulated with receptor activator of nuclear factor(NF)-κΒligand(RANKL,25 ng/mL)to differentiate into multinucleated osteoclasts.Subsequently,osteoclasts were treated with or without varying concentrations of madecassic acid(1,2.5,5,and 10μmol/L).Results:Madecassic acid significantly inhibited RANKL-induced osteoclastogenesis in a concentration-dependent manner.In addition,it reduced the percentage of bone resorptive area compared with the control,confirming that madecassic acid can inhibit osteoclast function.Furthermore,luciferase reporter gene studies indicate that madecassic acid could decrease the transcriptional activity of NF of activated T cells(NFAT)and NF-κB in a dose-dependent manner.Quantitative real-time polymerase chain reaction results show that madecassic acid attenuated the expression of osteoclast-associated genes,including V-ATPase-d2,cathepsin K,tartrate-resistant acid phosphatase(TRAP),NFAT cytoplasmic 1(NFATc1).Western blot analysis shows that madecassic acid inhibited RANKL-mediated degradation of IκBαand NFATc1 expression,as well as phosphorylation of c-Jun N-terminal kinase(JNK)in RAW 264.7 cells.Conclusion:Madecassic acid inhibited osteoclast formation and function in vitro by suppressing NF-κB,JNK,and NFAT signaling pathways,indicating its potential as a novel drug for the treatment of osteoclast-related bone diseases,especially osteoporosis.
