ABSTRACT
Objective To characterize the metabolic kinetics of aloe emodin in human liver microsomes(HLM)and rat liver microsomes(RLM)and identify the CYP phenotyping of phase-metabolism. Methods Aloe emodin was incubated at 37° with HLM and RLM in the presence or absence of NADPH, UDGPA or NADPH+UDGPA. The remaining aloe emodin was determined with a validated LC-MS/MS method to assess the metabolic stability and enzymatic kinetics. A panel of rCYP isoforms(CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4)and HLM with specific inhibitors of CYP isoforms were used to identify the CYP phenotyping of aloe emodin. Results In HLM and RLM, aloe emodin was metabolically eliminated in the presence of NADPH, with 85.8% and 81.7% of the parent compounds eliminated in 30 min, respectively. The t1/2 were(10.3±0.3)and(11.5±3.3)min, and the CLint were(420.1±10.9) and(573.4±188.2)ml/(min·kg), respectively. The apparent Km and Vmax for HLM and RLM were obtained and found to be(2.4±0.9) and(3.9±1.4)µmol/L, (1492±170.5)and(2783±595.8)nmol/(min·g protein), respectively. In RLM with UDPGA, 38.5% of aloe emodin was metabolized in 30 min with t1/2 of 31.6 min and CLint of(197.1±15.5)ml/(min·kg). The results of CYP phenotyping indicated that CYP1A2, 2B6, 2C19 and 3A4 were the major enzymes involved in the metabolism of aloe emodin. By using the method of total normalized rate, the contributions of the major enzymes were assessed to be 35.4%, 6.6%, 2.2% and 21.9%, respectively. Conclusion Aloe emodin is mainly eliminated by CYP mediated metabolism in HLM and RLM. CYP1A2 and 3A4 are the major responsible enzymes of aloe emodin, and the contributions are above 20%. Species differences in liver metabolism of aloe emodin are observed. It undergoes notable glucuronidation in RLM only.
ABSTRACT
<p><b>OBJECTIVE</b>To determine the effects of KH2PO4 on the odonto- and osteogenic differentiation potential of human stem cells from apical papillae (SCAP) in vitro.</p><p><b>METHODS</b>SCAP were isolated and cultured respectively in alpha minimum essential medium (α-MEM) or α-MEM containing 1.8 mmol/L KH2PO4. Alkaline phosphatase (ALP) activity, alizarin red staining, real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to examine the odonto and osteogenic potential of SCAP in the two media.</p><p><b>RESULTS</b>SCAP cultured in α-MEM containing 1.8 mmol/L KH2PO4 exhibited a higher ALP activity [(0.370 ± 0.013) Sigma unit×min(-1)×mg(-1)] at day 3 than control group [(0.285 ± 0.008) Sigma unit×min(-1)×mg(-1)] and KH2PO4-treated SCAP formed more calcified nodules at day 5 [(0.539 ± 0.007) µg/g] and day 7 [(1.617 ± 0.042) µg/g] than those in normal medium [(0.138 ± 0.037) µg/g, P < 0.01]. The expression of odonto- and osteogenic markers were significantly up-regulated after the stimulation of KH2PO4 at day 3 and 7 respectively, as compared with control group.</p><p><b>CONCLUSIONS</b>1.8 mmol/L KH2PO4 can promote the odonto and osteogenic differentiation potential of human SCAP.</p>