Stevia Metabolism And Transport:Mechanistic Investigations On Steviol And Its Glucuronide

Stevia glycosides, extracted from stevia rebaudiana, are a class of diterpenoid compounds that have been used as natural sweeteners due to their intense sweetness and low caloric intakes. As exemplified byrebaudioside A, components of stevia extracts also exhibit pharmacological activities such as asanti-hyperglycemic, anti-hypertensive, anti-diabetic, anti-inflammatory, and anti-tumor activities. In the present study, we investigated the metabolism of steviol, the aglycone of rebaudioside A, using various in vitro systems. Mechanisms of the transmembrane transport of steviol glucuronide (SVG) were examined using stably transfected cell lines and rat liver primary hepatocytes. In conjunction with in vitro and in vivo interaction studies, current findings would lead to safe and effective use of stevia glycosides.In order to achieve all intended studies, steviol glucuronide (the authentic standard) was synthesized and its purity and structural integrity were verified by HPLC and NMR. A simple and sensitive LC-MS/MS method was developed for the quantitation of SVG in biological matrices. Using a short C18 column (2.1 mm×50 mm,5μm) with linear gradient, the method afforded a baseline resolution of SVG from other components with a total run time of 6 min. The method was linear from 2.0 to 1000nM with good sensitivityand reproducibility.Upon oral ingestion, rebaudioside A could be hydrolyzed by intestinal microflora to steviol, its aglycone. Steviol was then absorbed into the circulation and subject to glucuronidation. Using UDPGA fortified liver and intestinal microsomal preparations from human and rat, the intrinsic clearance of steviol was first investigated based on the formation of steviol glucuronide.Data showed that there was no apparent specie difference, whereas liver appeared to play a major role in steviol glucuronidation due to its high intrinsic clearance. Further studies using recombinant human UGT enzymes revealed that UGT2B7 was more predominant in steviol glucuronidation at low steviol concentrations (<2μM), while both UGT2B7 and UGT1A3 produced high levels of the steviol glucuronide at a high steviol concentration (20μM). Other UGT enzymes such as UGT1A3 and UGT2B4 also contributed to steviol glucuronidation albeit with much lower activities. Enzyme kinetic studies of UGT2B7 and UGT1A3 revealed similar substrate inhibition characteristics as observed in human and rat liver microsomes.Steviol glucuronide is the major metabolite identified in human urine after rebaudioside A ingestion, suggesting transporters might play a role in its renal clearance. To examine the transmembrane transport of SVG, major efflux and uptake transporters were studied using a series of stably transfected cell lines. For the efflux transport, assays were performed using MDCKII cells transfected with human MDR1 (P-gp), hBCRP, hMRP2 or hMATE1, and the results indicated that SVG was not a substrate of these human efflux transporters. For the uptake transport, a number of human uptake transporters were used and data suggested that the intracellular uptake of SVG was mainly mediated by hOAT3 at low SVG concentrations, with contributions fromhOATP1B1, hOATP1B3, and hOATP2B1. Based on uptake transport kinetics, the role of transporters in SVG uptake followed the order of OAT3> OATP1B3> OATP1B1> OATP2B1. Given the fact that the circulating SVG concentrations in humans was below 5μM, the above data indicated that hOAT3 might play a predominant role in SVG’s renal clearance.Botanical-drug interactions have received considerable attention among researchers in academia, industries and regulatory agencies. Based on the current findings that UGT2B7 was the major UGT enzyme involved in steviol glucuronidation and OAT3 was important in SVG’s uptake clearance, potential mechanism-based interactions were further evaluated. Among drugs that could inhibit UGT2B7 activity, diclofenac displayed a relatively strong inhibition of steviol glucuronidation in human liver microsomes, whereas haloperidol, lamotrigine and zidovudine showed weak inhibition. Kinetic analysis revealed that diclofenac had a Ki of 4.2μM against UGT2B7-mediated steviol glucuronidation in human liver microsomes. On the other hand, steviol (20μM) exhibited a moderate inhibitory effect against diclofenac glucuronidation. The interactions mediated by OAT3 were examined with multiple drugs and natural products and data indicated that quercetin,telmisartan, diclofenac and mulberrin exhibited the highest inhibition potency with IC50 values of 1.82μM,2.92μM,8.01μM and 9.97μM, respectively. Additional studies showed that rutin and atorvastatin had obvious inhibition (>50%) against OATP1B1-mediated uptake of SVG, whereas minimal or no inhibition was observed for OATP2B1-mediated uptake.Studies using primary rat hepatocytes displayed that the uptake of SVG was transporter mediated and interactions with telmisartan and mulberrin demonstrated IC50 values of 2.0μM and 7.1μM against SVG uptake, respectively. Further interaction studies in vivo in rats showed that telmisartan could increase the systemic exposure of SVG with minimal effect on the pharmacokinetics of RA, suggesting that rat might be an appropriate species to study transporter-mediated botanical-drug interactions.Based on the current findings and literature reports, the disposition of stevia glycosides (such as rebaudioside A) could be proposed as the following after oral ingestion: intestinal hydrolysis to the aglycone, steviol, which was then absorbed and glucuronidated in the liver, leading to the formation of steviol glucuronide; steviol glucuronide was then subjected to transporter mediated uptake and eventually cleared via renal excretion. The present study demonstrated that UGT2B7 might play a predominant role in the hepatic steviol glucuronidation and OAT3 might be the major uptake transporter that mediated SVG’s renal clearance. In addition, mechanism-based interactions were also observed as multiple drugs or compounds were capable of inhibiting UGT2B7 activity and OAT3-mediated transmembrane transport. The current findings not only filled an important knowledge gap in the disposition of stevia glycosides, but more importantly provided scientific foundation for the safe and effective use of stevia glycosides in combination with appropriate drugs for the blood sugar control particularly in diabetic patients.However, combinatory use should be avoided in patients with kidney diseases.