Microbial metabolism. Part 13. 1 Metabolites of hesperetin Wimal Herath a , Ikhlas Ahmad Khan a,b,⇑ a National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, University, MS 38677, USA b Department of Pharmacognosy, Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, University, MS 38677, USA article info Article history: Received 2 June 2011 Revised 29 July 2011 Accepted 1 August 2011 Available online 9 August 2011 Keywords: Hesperitin Microbial metabolism Mucor ramannianus abstract The fungal culture, Mucor ramannianus (ATCC 2628) transformed hesperitin (1) to four metabolites: 4 0 - methoxy-5,7,8,3 0 -tetrahydroxyﬂavanone (8-hydroxyhesperetin) (2), 5,7,3 0 ,4 0 -tetrahydroxyﬂavanone (eriodictyol) (3), 4 0 -methoxy-5,3 0 -dihydroxyﬂavanone 7-sulfate (hesperetin 7-sulfate) (4) and 5,7,3 0 -tri- hydroxyﬂavanone 4 0 -O-a-quinovopyranoside (eriodictyol 4 0 -O-a-quinovopyranoside) (5). The structures were established by spectroscopic methods. Ó 2011 Elsevier Ltd. All rights reserved. The ﬂavonoid, hesperetin is the aglycone of hesperidin found in sweet oranges, other citrus fruits and some herbs. 2–4 It is the most consumed ﬂavonoid amounting to about 30% of the total intake. 4 Both compounds are associated with beneﬁcial health effects. 2 Bio- logical activities of hesperetin include antioxidant, bone-sparing and lipid-lowering effects. 5 The antioxident property together with the ability to penetrate the blood–brain barrier is suggested to help in neuroprotection against oxidative damage. 6 Hesperetin also plays a signiﬁcant role in inﬂammation and cancer inhibition. 2 It is known that the Nuclear Factor-kappa B (NF-jB) found in the cytoplasm promotes inﬂammation-associated cancer. NF-jB, in addition, activates the genes responsible for inﬂammation, among others and prevents cancer cell destruction by inactivating tumor suppressor proteins. 7,8 It also plays a major role in the aging pro- cess. 9 Animal experiments show the importance of antioxidants as inhibitors of NF-jB. 10 Thus, phytochemicals like ﬂavanoids with such properties may hold promise for cancer prevention, 7 provided that they block speciﬁc signal partways which depend on NF-jb activation to prevent adverse side effects. 10 One such compound is hesperitin. In vitro biological activities of hesperitin are well doc- umented but little is known about its in vivo efﬁcacy. Despite many investigations carried out to date, its bioavailability is poorly understood. 3,4,11–16 Using an experiment where hesperetin itself is administered orally to humans, 11 some conclusions would have been drawn as to the fate of hisperetin in vivo, if the chemical structures of the blood plasma metabolites were elucidated. 12 It is vital to learn the bioavailability and selectivity of hesperetin and its metabolites on choosing NF-jB as the target to prevent can- cer. 10 Since, microorganisms can be used as predictive models for mammalian drug metabolism we investigated retrospectively the microbial transformation of hesperetin (1) 17 to isolate and charac- terize metabolites which may help to predict its fate in mamma- lian systems. 18,19 Forty microorganisms were screened to evaluate the ability to transform hesperitin (1) to its metabolites. 20 Fermentations were carried out according to a two-stage procedure 21 in medium a. 22 Suitable controls were used to ensure that the metabolites were a result of enzyme activity and not a consequence of non-meta- bolic transformation. 22 Mucor ramannianus (ATCC 2628) was se- lected for the preparative stage due to its higher transformation efﬁciency compared to several other organisms which showed conversion capability. M. ramannianus transformed hesperitin (1)(Fig. 1) to 4 0 -meth- oxy-5,7,8,3 0 -tetrahydroxyﬂavanone (8-hydroxyhesperetin) (2), 5,7,3 0 ,4 0 -tetrahydroxyﬂavanone (eriodictyol) (3), 4 0 -methoxy-5,3 0 - dihydroxyﬂavanone 7-sulfate (hesperetin 7-sulfate) (4) and 5,7,3 0 -trihydroxyﬂavanone 4 0 -O-a-quinovopyranoside (eriodictyol 4 0 -O-a-quinovopyranoside) (5). The molecular formulae of all the metabolites were determined by HR-ESI-MS. Structures were elucidated by spectroscopic methods. 23 Metabolite 2 24 (0.9 mg, 0.03%) was isolated as a white solid with a molecular formula, C 16 H 14 O 7 . It was more polar than hes- peretin (1). The NMR data were similar to those of 1 except for the presence of an additional hydroxyl group in ring A. NMR corre- lations along with the published data were used to characterize the compound as 4 0 -methoxy-5,7,8,3 0 -tetrahydroxyﬂavanone (8-hydroxyhesperitin). 25 The white solid 3 26 (10 mg, 0.27%) with a molecular ion peak at m/z 287.0450 [MH] + in its HR-ESI-MS corresponded to the 0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2011.08.004 ⇑ Corresponding author. Tel.: +1 662 915 7821; fax: +1 662 915 1006. E-mail address: ikhan@olemiss.edu (I.A. Khan). Bioorganic & Medicinal Chemistry Letters 21 (2011) 5784–5786 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl