EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 37, No. 5, 436-446, October 2005 Natural compounds, fraxin and chemicals structurally related to fraxin protect cells from oxidative stress Wan Kyunn Whang1, Hyung Soon Park2, genes expressed differentially by fraxin and to InHye Ham1, Mihyun Oh1, compare antioxidative effect of fraxin with its struc- Hong Namkoong3, Hyun Kee Kim3, turally related chemicals. Of the coumarins, protective 2 4 effects of fraxin against cytotoxicity induced by H2O2 Dong Whi Hwang , Soo Young Hur , were examined in human umbilical vein endothelial 4 5 Tae Eung Kim , Yong Gyu Park , cells (HUVECs). Fraxin showed free radical scavenging Jae-Ryong Kim6 and Jin Woo Kim3,4,7 effect at high concentration (0.5 mM) and cell protective effect against H2O2-mediated oxidative stress. Fraxin 1 College of Pharmacy, Chung-Ang University recovered viability of HUVECs damaged by H2O2- 221, Heukseok-dong, Dongjak-gu treatment and reduced the lipid peroxidation and the Seoul 156-861, Korea internal reactive oxygen species level elevated by H2O2 2KeyGene Life Science Institute treatment. Differential display reverse transcrip- KeyGene Science, Corp. tion-PCR revealed that fraxin upregulated antiapo- Ansan, Gyeonggido 425-791, Korea ptotic genes (clusterin and apoptosis inhibitor 5) and 3Molecular Genetic Laboratory tumor suppressor gene (ST13). Based on structural Research Institute of Medical Science similarity comparing with fraxin, seven chemicals, 4Department of Obstetrics and Gynecology fraxidin methyl ether (29.4% enhancement of viability), 5Department of Biostatistics prenyletin (26.4%), methoxsalen (20.8 %), diffratic acid College of Medicine (19.9%), rutoside (19.1%), xanthyletin (18.4%), and The Catholic University of Korea kuhlmannin (18.2%), enhanced more potent cell via- Seoul 137-040, Korea bility in the order in comparison with fraxin, which 6Department of Biochemistry and Molecular Biology showed only 9.3% enhancement of cell viability. These Aging-assoiciated Vascular Disease Research Center results suggest that fraxin and fraxin-related che- College of Medicine, Yeungnam University micals protect HUVECs from oxidative stress. Daegu 705-717, Korea 7 G Corresponding author: Tel, 82-2-590-2389; Keywords: antioxidants; apoptosis; coumarins; fraxin; oxidative stress; plants, medicinal Fax, 82-2-593-2389; E-mail [email protected] Accepted 29 August 2005 Introduction Abbreviations: CAT, catalase; DCFH-DA, 2',7'-dichlorodihydrofluo- Coumarins comprise a very large class of phenolic rescein-diacetate; DDRT-PCR, differential display reverse tran- substances found in plants and are made of fused scription-PCR; GPX, glutathione peroxidase; HUVECs, human benzene and -pyrone rings. To date, at least 1,300 umbilical vein endothelial cells; LPO, lipid peroxidation; MDA, coumarins have been identified, principally as se- malondialdehyde; RNS, reactive nitrogen species; ROS, reactive condary metabolites in green plants but also in fungi oxygen species; SOD, superoxide dismutase and bacteria (Murray, 1989; Hoult and Paya, 1996). The prototypical compound is coumarin itself (also known as 1,2-benzopyrone or less commonly, as Abstract o-hydroxycinnamic acid-8-lactone), and it has been well studied. The pharmacological and biochemical Coumarins comprise a group of natural phenolic properties and therapeutic applications of simple compounds found in a variety of plant sources. In view coumarins depend upon the pattern of substitution of the established low toxicity, relative cheapness, (Hoult and Paya, 1996). presence in the diet and occurrence in various herbal Several natural products with a coumarinic moiety remedies of coumarins, it appears prudent to evaluate have been reported to have multiple biological acti- their properties and applications further. The purpose vities (Ivanovska et al., 1994; Paya et al., 1994; of this study is to investigate cellular protective activity Chang and Chiang, 1995; Chang et al., 1996; of coumarin compound, fraxin extracted from Weigela Fylaktakidou et al., 2004). It is to be expected that florida var. glabbra, under oxidative stress, to identify coumarins might affect the formation and sca- Antioxidative activityof fraxin 437 venging of ROS and influence processes involving fanova et al ., 1995). In this study, as the constituent free radical-mediated injury. Coumarin can reduce of the leaves of Weigela florida var. glabra (Capri- tissue edema and inflammation. Moreover coumarin foliaceae), fraxin was studied phytochemically be- and its 7-hydroxy-derivative inhibit prostaglandin cause the leaves of Weigela florida var. glabra have biosynthesis, which involves fatty acid hydroperoxy not been studied as medicinal plant. intermediates. Natural products like esculetin, fra- ROS and reactive nitrogen species (RNS) constan- xetin, daphnetin and other related coumarin deriva- tly generated in normal condition by aerobic metabo- tives are recognised as inhibitors not only of the lism include free radicals such as superoxide anion, lipoxygenase and cycloxygenase enzymic systems, hydroxyl radicals, nonradical hydrogen peroxide, but also of the neutrophil-dependent superoxide peroxinitrite, nitroxyl anion and nitric oxide (Beck- anion generation (Ivanovska et al ., 1994; Paya et al ., man and Ames, 1998; Curtin et al., 2002; Droge, 1994; Chang and Chiang, 1995; Chang et al ., 1996; 2002). In normal biological systems, redox home- Fylaktakidou et al ., 2004). Coumarin derivatives ostasis is maintained by contro lling the balance bet- have recently attracted much attention because of ween ROS production and various types of sca- their broad pharmacological activities. vengers called antioxidants. Transient changes in Fraxin (7-hydroxy-6-methoxycoumarin 8-glucoside), oxidants-antioxidant balance are normally regulated structurally a derivative of a coumarin glucoside, is a by changing the production of counter species and colorless crystalline substance (C 16H18O10) found in reached to the steady-state over time (Shull et al., the bark of the ash (Fraxinus), and along with es- 1991; Nakamura et al., 1994; Adler et al., 1999; culin in the bark of the horse-chestnut. It shows a Zhang and Storz, 2000; Droge, 2002). The persi- delicate blue-green fluorescence in alkaline solu- stent production of abnormally large amount of ROS tions; - called also paviin and fraxoside. Fraxin shows or RNS, however, may lead to persistent changes in its antioxidative effect through inhibition of cyclo signal transduction and gene expression, which in AMP phosphodiesterase enzyme (Schempp et al., turn may give rise to certain diseases. 2000). It also shows analgesic effect like a non- Various enzymatic and non-enzymatic self defense steroidal anti-inflammatory drugs (von Kruedener et systems against oxidative stress including transi- al., 1995; Klein-Galczinsky, 1999). Previous studies ently over-produced ROS inside cells or exposure to also suggested that fraxin isolated from some plants external ROS inducing species have been identified showed various activities. Fraxin from Fraxinus ex- and studied extensively. Catalase (CAT), SOD, and celsior had anti-inflammatory and antimetastatic glutathione peroxidase (GPX) are the examples of properties, the former probably because of its direct enzymatic defense systems and non-enzymatic sys- action on cells, predominantly on macrophages in- tems include tocopherol, ascorbate, urate and glu- hibitory effect on 5-HETE production (Ivanovska et tathione (Sies, 1993). al., 1994). And the flowering or manna, ash, Fraxi- The endothelium is known to be sensitive to injury nus ornus L. [Fam. Oleaceae], the bark contains caused by ROS (Valen et al ., 1999), and in contrast, hydroxycoumarins, secoiridoid glucosides, pheny- free radicals released by endothelial cells mediate lethanoids and flavonoids. Biological studies of hy- the oxidation of low density lipid (LDL) (Zapolska- droxycoumarins reveal significant antimicrobial, anti- Downar et al., 1999). HUVECs have been used to inflammatory, immunomodulatory, antiviral and diu- study oxidative stress related researches and oxi- retic activities that support the use of this bark in folk dative stress mediated by H 2O2 damages cellular medicine (Guarrera, 1999; Kostova, 2001). These functions of HUVECs via various mechanisms (Val- results show that the traditional use of Fraxinus en et al., 1999; Estrada-Garcia et al., 2002; Waxman ornus stem bark in the treatment of inflammatory et al., 2003). disorders is at least partially due to its coumarins In the present study, we investigated cellular pro- constituents. Fraxin is also known to act as a tective activity of coumarin compound, fraxin (7- choleretic agent for stimulating bile flow and aiding hydroxy-6-methoxycoumarin 8-glucoside), under oxi- digestion, and has noted activity for preventing the dative stress, identified genes expressed differen- development of abnormal growths (Iossifova et al ., tially by fraxine treatment and compared antioxidant 1994). In the study of effect of some hydroxy- effect of fraxin with its structurally related chemicals. coumarins on complement-mediated hemolysis in human serum, the interactions between esculin, esculetin, fraxin, fraxetin, as well as their acetylated Materials and Methods and methylated derivatives and non-cell system participating in inflammatory processes, comprised Chemicals of serum complement proteins, were investigated in Fraxin was extracted from Weigela florida var. glab- vitro. Fraxin was able to enhance hemolysis (Ste- bra