Chemical studies on antioxidant mechanism of garcinol: analysis of radical reaction products of garcinol and their antitumor activities Shengmin Sang, a Min-Hsiung Pan, b Xiaofang Cheng, c Naisheng Bai, a Ruth E. Stark, c Robert T. Rosen, a Shoei-Yn Lin-Shiau, b Jen-Kun Lin b and Chi-Tang Ho a,p a Department of Food Science, Center for Advanced Food Technology, Rutgers University, 65 Dudley Road, New Brunswick, NJ 08901-8520, USA b InstituteofBiochemistryandToxicology,CollegeofMedicine,NationalTaiwanUniversity,1Section1,Jen-aiRoad,Taipei,Taiwan,ROC c Department of Chemistry, College of Staten Island, City University of New York, New York, NY 10314, USA Received 7 August 2001; accepted 18 September 2001 Abstract ÐGarcinol 1), a polyisoprenylated benzophenone, puri®ed from Garcinia indica fruit rind, displays antioxidant properties and is thought to act as an antioxidant in biological systems. However, the mechanisms of its antioxidant reactions remain unknown. The objective of this study was to characterize the reaction products of garcinol with a stable radical, 2,2-diphenyl-1-picrylhydrazyl DPPH). Structural elucidationoftheseproductscanprovideinsightsintospeci®cmechanismsofantioxidantreactions.Twomajorreactionproducts,GDPPH-1 2) and GDPPH-2 3), were isolated and identi®ed for the ®rst time. Their structures were determined on the basis of detailed high ®eld 1D and 2D spectral analysis. The identi®cation of these products provides the ®rst unambiguous proof that the principal sites of antioxidant reactionsareonthe1,3-diketoneandthephenolicringpartof 1.TheinductionofapoptosisinhumanleukemiaHL-60cells,theinhibitionof NO generation, the effects on the activity of MMP, and the inhibitory effects on H 2 O 2 production of TPA-stimulate HL-60 cells by 1 and its twooxidantproducts2 and 3)wereinvestigatedandwouldalsobediscussedinthispaper. q 2001ElsevierScienceLtd.Allrightsreserved. 1. Introduction Free radicals and reactive oxygen species play an important physiological role, they are essential for production of energy, synthesis of biologically essential compounds, and phagocytosis, a critical process of immune system. 1 They also play a vital role in signal transduction. On the other hand, there is now increasing evidence that these reactive oxygen species may play a causative role in a variety of diseases including heart disease, aging and cancer. 2,3 Free radicals and reactive oxygen species attack lipids, sugars, proteins, and DNA and induce their oxidation, which may result in oxidative damage such as membrane dysfunction, protein modi®cation, enzyme inactivation, and breakage of DNA strands and modi®cation of its bases. 1 Consequently, the role of antioxidants has received increased attention during the past decade. It is important to elucidate the mechanisms and dynamics of the oxidative damage in order to understand its biological signi®cance and develop strategies for its prevention. Both active oxygen species and antioxidants are double-edged swords and the balance of their bene®cial and toxic effects is determined by the relativeimportanceofmanycompetingbiologicalreactions. Humans have evolved with antioxidant systems to protect against free radicals. These systems include some anti- oxidants produced in the body endogenous) and others obtained from the diet exogenous). Over the past decade, evidence has been accumulated that plant polyphenols are an important class of defense antioxidants. These compounds are widespread virtually in all plant foods, often at high levels, and include phenols, phenolic acids, ¯avonoids, tannins, and lignans. Garcinol 1) is a polyisoprenylated benzophenone deriva- tive from Garcinia indica and other species. 4±8 The dried rindof G.indica cv.Kokum)whichisusedasagarnishfor curry and in some of the folklore medicine in India contains 2±3% garcinol. 4,5 Garcinol is structurally similar to a well- known antioxidant-curcumin, which contains both phenolic hydroxyl groups and a b-diketone moiety. Garcinol has been reported to possess antibiotic activities, 8 antiulcer activity, 10 suppressed colonic aberrant crypt foci ACF) formation, 11 and induction of apoptosis through cytochrome c release and activation of caspases in human leukemia HL-60 cells. 12 It also showed strong antioxidant activity. In the H 2 O 2 ±NaOH±DMSO system, garcinol suppressed superoxide anion, hydroxyl radical, and methyl radical; in the Fenton reaction system, garcinol suppressed hydroxyl Tetrahedron 57 2001) 9931±9938 Pergamon TETRAHEDRON 0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII:S0040-402001)01015-8 Keywords: garcinol; antioxidant mechanism; antitumor activities. p Corresponding author. Tel.: 11-732-932-9611x235; fax: 11-732-932- 6776; e-mail: ho@aesop.rutgers.edu