Biosci. Biotechnol. Biochem., 72 (10), 2750–2755, 2008 Lignans from the Fruits of Forsythia suspensa (Thunb.) Vahl Protect High-Density Lipoprotein during Oxidative Stress Min-Jung C HANG,1 Tran Manh H UNG,2 Byung-Sun M IN,1; y Jin-Cheol K IM,3 Mi Hee WOO,1 Jae Sue C HOI,4 Hyeong Kyu L EE,2 and KiHwan B AE5 1 College of Pharmacy, Catholic University of Daegu, Gyeongsan 712-702, Korea Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Korea 3 Korea Research Institute of Chemical Technology, Daejeon 305-600, Korea 4 Faculty of Food Science and Biotechnology, Pukyoung National University, Busan 608-737, Korea 5 College of Pharmacy, Chungnam National University, Daejeon 305-764, Korea 2 Received June 11, 2008; Accepted July 7, 2008; Online Publication, October 7, 2008 [doi:10.1271/bbb.80392] The objective of the present study was to investigate the beneficial properties lignan compounds obtained from the fruits of Forsythia suspensa (Thunb.) Vahl (Oleaceae) for protecting human high-density lipoprotein (HDL) against lipid peroxidation. The isolated compounds (1–8) inhibited the generation of thiobarbituric acid-reactive substances (TBARS) in a dosedependent manner with IC50 values from 8.5 to 18.7 M, since HDL oxidation mediated by catalytic Cu2þ . They also exerted an inhibitory effect against thermo-labile radical initiator (AAPH)-induced lipid peroxidation of HDL with IC50 values from 12.1 to 51.1 M. Compounds 1 and 5 exerted inhibitory effects against the Cu2þ -induced lipid peroxidation of HDL, as shown by an extended lag time prolongation at the concentration of 3.0 M. These results suggest that the antioxidative effects of F. suspensa are due to its lignans and that these constituents may be useful for preventing the oxidation of HDL. Key words: Forsythia suspense; Oleaceae; lignan; human high-density lipoprotein; lipid peroxidation The oxidative modification of low-density lipoprotein (LDL) and the accelerated uptake of oxidized LDL by artery wall macrophages have been implicated in the formation of atherosclerotic plaque. LDL oxidation is a crucial step in atherosclerosis. However, this process can be inhibited by high-density lipoprotein (HDL) through its oxidizable components or by associated enzymes like paraoxonase and platelet-activating factor acetylhydrolase.1) The ability of HDL to protect LDL from y oxidation may consequently influence some of the pathological processes mediating the development of atherosclerosis. Numerous prospective studies have demonstrated the protective nature of an elevated level of HDL and the high risk associated with a low level of this class of lipoproteins.2,3) However, similar to LDL, the lipids contained in HDL are susceptible to oxidation by a variety of pro-oxidants such as redox-active copper ions, metal ion-independent oxidants including reactive oxygen, and nitrogen species.4) In general, the oxidation of HDL has been found to result in a loss of cardioprotective properties and loss of paraoxonase activity.4) It is known that most of the measurable lipid peroxides in plasma can be found in the HDL fraction.5) Surprisingly, in spite of having several important pathophysiological implications, studies on the HDL oxidation process have received less attention than those on LDL oxidation.6) As a part of our screening program to find antioxidants from natural sources, we have attempted to determine the HDL oxidation inhibitory constituents from fruits of Forsythia suspensa (Thunb.) Vahl (Oleaceae). This species is a perennial herb that is cultivated for its beautiful yellow flowers, and is distributed throughout Korea, Japan, and China. The fruits have been used as a folk medicine for the treatment of inflammation, ulcers, pharyngitis, pyrexia, and tonsillitis.7) Previous studies on this plant have reported the isolation of caffeoyl glycosides, cyclohexylethanes, flavonoids, iridoid glycosides, lignans and triterpenes, together with their anti-inflammatory, antibacterial, antioxidative, weight loss, blood pressurereducing and cyclic adenosine monophosphate phos- To whom correspondence should be addressed. Fax: +82-53-850-3602; E-mail: bsmin@cu.ac.kr Abbreviations: LDL, low-density lipoprotein; HDL, high-density lipoprotein; PBS, phosphate-buffered saline; MDA, malondialdehyde; TBARS, thiobarbituric acid-reactive substances; EDTA, ethylenediaminetetraacetic acid; AAPH, 2,20 -azobis-(2-amidinopropane)hydrochloride; IC50 , concentration for 50% inhibition Lignans from Forsythia suspensa Protect against High-Density Lipoprotein Oxidation 8–11) phodiesterase inhibitory effects. An ethanol extract of F. suspensa has recently protected against enzymatic and non-enzymatic lipid peroxidation in membranes and showed scavenging activity toward the superoxide radical.12) Even though the antioxidative activities of some lignans from this plant have been assessed by evaluating their protective effects against peroxynitrileinduced oxidative stress,13–15) no studies have specifically investigated the capacity of those components to protect HDL from oxidation. We therefore examined the susceptibility of HDL to in vitro copper (Cu2þ ) and thermo-labile radical initiator, 2,20 -azobis(2-amidinopropane) dihydrochloride (AAPH)-induced lipid peroxidation in the presence of an extract, fractions and isolated compounds from F. suspensa. Materials and Methods Plant materials. The fruits of F. suspensa were purchased from a local market in Daegu, Korea in March 2006, and were identified by Prof. Byung-Sun Min. A voucher specimen (CUD-2477-2) has been deposited in the herbarium of the College of Pharmacy, Catholic University of Daegu, Korea. Extraction and isolation. Dried plant material (10.0 kg) was extracted with 70% EtOH at room temperature (4
5 liters) to obtain 1.85 kg of a solid extract. The 70% EtOH extract was suspended in H2 O and successively extracted with hexane (3
3 liters), CHCl3 (3
3 liters), EtOAc (3
3 liters), and BuOH (3
3 liters) to give the hexane-(520 g), CHCl3 -(320 g), EtOAc-(250 g), and BuOH-soluble fractions (504 g), respectively. These fractions were screened for antioxidative activity, and the CHCl3 and EtOAc active fractions were subjected to fractionation by column chromatography. The CHCl3 soluble fraction was chromatographed in a silica gel column, eluting with hexane–EtOAc (5:1 to 1:2) and then with CHCl3 –MeOH (5:1 to 4:1) to afford fourteen fractions (C1–14). Fraction C13 was chromatographed in a silica gel column, eluting with CHCl3 –MeOH (50:1 to 10:1), to give eight subfractions (C13.1–8). Subfraction C13.1 was rechromatographed on silica gel with hexane–acetone (2:1) and then with CHCl3 –MeOH (20:1) to yield compounds 1 (3470.9 mg) and 2 (3491.7 mg). Subfraction C13.6 was purified in an RPC18 column, using MeOH–H2 O (1:1), and further applied to preparative HPLC-RP-C18 , using acetonitrile–H2 O (1:4), to yield compounds 3 (10 mg) and 4 (7.9 mg). Subfraction C13.7 was rechromatographed in an RP-C18 column, using MeOH–H2 O (1:1), to afford compounds 5 (140.2 mg) and 6 (16.5 mg). The EtOAcsoluble fraction was chromatographed in a silica gel column eluting with a gradient of hexane–EtOAc (7:1 to 3:1) and CHCl3 –MeOH (50:1 to 4:1) to give eleven fractions (E1–11). Fraction E9 was re-chromatographed in an RP-C18 column eluting with a gradient of MeOH– H2 O (1:1.7 to 3:1) to afford fifteen subfractions (E9.1– 2751 15). Subfraction E9.3 was applied to preparative HPLCC18 , using a gradient of acetonitrile–H2 O (1:3 to 1:1) to yield compound 7 (5.1 mg). Further chromatography of E10 in a silica gel column, eluting with CHCl3 – MeOH (8:1) gave three sub-fractions (E10.1–3). Compound 10 (48.8 mg) was obtained from E10.3 after chromatographed on a silica gel column with CHCl3 – MeOH (7:1). Pinoresinol (1): yellow amorphous powder; ½ D 22 +88.5 (c 0.18, MeOH); UV (MeOH) max nm (log "): 232 (4.51), 281 (4.14); EI-MS (rel. int.) m=z: 358 [M]þ (39), 327 (65), 221 (6), 205 (14), 151 (100), 137 (68), 131 (23); mol. formula: C20 H22 O6 . Phillygenin (2): yellow crystals; mp: 136–138  C; ½ D 22 : +90.0 (c 0.2, MeOH); UV (MeOH) max nm (log "): 232 (4.12), 280 (3.66); EI-MS (rel. int.) m=z: 372 [M]þ (80), 341 (9), 151 (100), 137 (41), 131 (16); mol. formula: C21 H24 O6 . 8-Hydroxypinoresinol (3): white amorphous powder; ½ D 22 : +23.9 (c 0.16, MeOH); UV (MeOH) max nm (log "): 233 (4.45), 281 (4.04); EI-MS (rel. int.) m=z: 374 [M]þ (45), 222 (26), 207 (34), 193 (20), 165 (41), 151 (100), 137 (67), 131 (46); mol. formula: C20 H22 O7 . 70 -epi-8-Hydroxypinoresinol (4): white amorphous powder; ½ D 22 : +70.7 (c 0.16, MeOH); UV (MeOH) max nm (log "): 232 (4.45), 281 (4.05); EI-MS (rel. int.) m=z: 374 [M]þ (57), 222 (28), 207 (61), 193 (20), 165 (48), 151 (99), 137 (100), 131 (61); mol. formula: C20 H22 O7 . Lariciresinol (5): yellow amorphous powder; ½ D 22 : +25.3 (c 0.21, MeOH); UV (MeOH) max nm (log "): 231 (4.41), 282 (4.05); EI-MS (rel. int.) m=z: 360 [M]þ (69), 311 (3), 194 (30), 175 (18), 137 (100), 122 (14); mol. formula: C20 H24 O6 . Isolariciresinol (6): yellow amorphous powder; ½ D 22 : +24.0 (c 0.18, MeOH); UV (MeOH) max nm (log "): 232 (sh), 284 (4.15); EI-MS (rel. int.) m=z: 360 [M]þ (25), 311 (1), 267 (72), 137 (44), 98 (100); mol. formula: C20 H24 O6 . Olivil (7): white amorphous powder; ½ D 22 : 46:5 (c 0.17, MeOH); UV (MeOH) max nm (log "): 232 (4.67), 281 (4.32); EI-MS (rel. int.) m=z: 376 [M]þ (10), 326 (6), 311 (2), 137 (100); mol. formula: C20 H24 O7 . Cedrusin (8): brown amorphous powder; ½ D 22 : +10.0 (c 0.16, MeOH); UV (MeOH): max nm (log ") 232 (sh), 283 (4.21); EI-MS (rel. int.) m=z: 346 [M]þ (31), 328 (79), 316 (100), 296 (17), 137 (35); mol. formula: C19 H22 O6 . HDL preparation. Blood from healthy normolipemic donors was obtained by venipuncture and collected in EDTA-containing vacutainer tubes. To isolate HDL, plasma was prepared by centrifugation at 3,000 rpm for 10 min and thereafter used for the preparation of plasma lipoproteins. HDL was isolated from the plasma by ultracentrifugation for 1.5 h with a vertical rotor as described previously.16,17) After dialyzing at 4  C for 24 h against 10 mM phosphate-buffered saline (PBS) at 2752 M.-J. C HANG et al. pH 7.4, the HDL protein concentration (mg protein/ml) was determined as described previously.18) HDL oxidation. The oxidation of HDL was assessed by the formation of conjugated dienes which was determined as the change in UV absorbance at 232 nm at 10-min intervals over 5 h at 37  C, using a UV-1240 spectrophotometer (Shimadzu, Tokyo, Japan). The lag time was measured as the intercept between the baseline and the tangent to the absorbance curve during the propagation phase.19) The oxidation of HDL to malondialdehyde (MDA) was measured by using a thiobarbituric acid reactive substances (TBARS) assay. HDL in PBS (pH 7.4) was pre-incubated with each compound, and then Cu2þ or one of the thermo-labile radical initiators (AAPH) was added to initiate the oxidation process. The reaction mixture was incubated at 37  C for 2 h. At the end of this incubation, the reaction was terminated by adding 20% trichloroacetic acid (TCA) and 1% thiobarbituric acid (TBA). After boiling at 95  C for 15 min, the mixture was centrifuged at 10,000 rpm for 10 min. The absorbance of the supernatant was measured at 532 nm.16,20) In this experiment, vitamin C and vitamin E were used as positive controls.21) Statistical analysis. Student’s t-test and a two-way analysis of variance were used to determine the statistical significance of differences between values for the experimental and control groups. Each result is expressed as the mean value  S.D. of three experiments conducted in triplicate. A p < 0:05 value was considered statistically significant. Results and Discussion The fruits of F. suspensa were extracted with 70% EtOH at room temperatures and the alcoholic extract obtained was partitioned into hexane-, CHCl3 -, EtOAc-, BuOH- and aqueous fractions. Repeated column chromatography led to the isolation of eight compounds (1– 8). A spectroscopic analysis and comparison of physical constants with those in the literature allowed us to identify these compounds as pinoresinol (1), phillygenin (2), 8-hydroxypinoresinol (3), 70 -epi-8-hydroxypinoresinol (4), lariciresinol (5), isolaraciresinol (6), olivil (7) and cedrusin (8) (Fig. 1).8–11) In the primary study, we tested the inhibitory activity of the 70% EtOH extract and fractions (hexane-, CHCl3 -, EtOAc-, and BuOH-soluble) at the concentration of 100 mg/ml against the oxidation of HDL, which was initiated by Cu2þ to form the malondialdehyde (MDA), by using a thiobarbituric acid reactive substances (TBARS) assay. The results showed that the CHCl3 and EtOAc-soluble fractions were approximately 2.5and 2.3-fold more potent than the alcoholic extract, while the hexane- and BuOH-solube fractions showed very weak activity at the same tested concentration (Table 1). Considering that the CHCl3 and EtOAc fractions were the most potent, these were selected for isolation of the active constituents. In the next stage of this study, we tested the inhibitory activity of all the isolated compounds against the oxidation of HDL initiated by both Cu2þ and AAPH. As shown in Table 1, the tested compounds markedly reduced the formation of TBARS. Under Cu2þ -mediated oxidation, compounds 1–8 showed HDL-antioxidative activities in a dose-dependent manner, the concentration required for 50% inhibition (IC50 ) ranging from 8.5 to 18.7 mM. Under AAPH-mediated oxidation, isolated compounds 1, 2, 4–6 and 8 also exhibited HDL oxidation activities, with IC50 values ranging from 12.1 to 51.1 mM. Vitamin C and vitamin E were used as the positive controls,21) vitamin C showing inhibitory activity with IC50 values of 11.7 and 18.9 mM, while vitamin E showed inhibitory activity with IC50 values of 2.0 and 5.8 mM, under Cu2þ - and AAPH-mediated oxidation. Since the formation of conjugated dienes represents the propagation phase of HDL oxidation, the extent of the lag time indicates the oxidation resistance. As shown in Fig. 2, a spectrophotometric analysis of Cu2þ -induced HDL oxidation based on the formation of conjugated diene indicated the presence of unsaturated lipids. When HDL was incubated with Cu2þ alone, the lag time was 48 min, whereas, in the presence of pinoresinol (1, 3.0 mM) and lariciresinol (5, 3.0 mM), the lag phase was delayed to 87 and 90 min, respectively. The lag phases of vitamin C (3.0 mM) and vitamin E (3.0 mM) were delayed to 85 and 128 min, respectively. Certain forms of oxidized HDL may actually enhance protection by stimulating the delivery of intracellular cholesterol to cell surface sites where it becomes available for removal by other (non-oxidized) HDL particles.4,16) Lipid oxidation in HDL is promoted by a variety of factors. Although there have been a number of reports on the design and development of synthetic peroxidation inhibitors,22–24) only a few studies have been reported on HDL oxidation inhibitors derived from plants. Our results clearly demonstrate that the HDL oxidation inhibitors of isolated lignans significantly inhibited the lipid oxidation of HDL which had been exposed to such sources of oxidant stress as metal ionindependent (Cu2þ ) and peroxyl radicals (AAPH). Interestingly, such lignans as pinoresinol (1), 8-hydroxypinoresinol (3), 70 -epi-8-hydroxypinoresinol (4), lariciresinol (5), isolariresinol (6) and olivil (7) were more effective than vitamin C, but less effective than vitamin E from evidence obtained by the TBARS method (Table 1). Pinoresinol (1) and lariciresinol (5) were selected to examine the inhibitory ability in the propagation phase through a delay in the lag time oxidation. Despite the important role of HDL, the absence of a lag phase before the initiation of oxidation in HDL is associated with several antioxidative enzymes, this suggests that HDL may protect LDL from Lignans from Forsythia suspensa Protect against High-Density Lipoprotein Oxidation OCH 3 OCH 3 OH OH OH O H 2753 OCH 3 O H O H H3CO H H H3CO O O HO OH H3CO O H3CO HO Pinoresinol (1) 8-Hydroxypinoresinol (3) Phillygenin (2) OCH 3 OH H3CO OH HO OH O O H HO H3CO OH HO H3CO O OCH 3 OH HO OH OCH 3 Lariciresinol (5) 7'-epi-8-Hydroxypinoresinol (4) Isolariciresinol (6) HO OH O H3CO HO HO HO H3CO O OH OH OCH 3 HO Cedrusin (8) Olivil (7) Fig. 1. Chemical Structure of the Isolated Compounds. Table 1. Effects of the Extracts and Isolated Compounds on the Oxidation of HDL Extract/Compound c 70% EtOH extract Hexane fraction CHCl3 fractionc EtOAc fractionc n-BuOH fraction Pinoresinol (1) Phillygenin (2) 8-Hydroxypinoresinol (3) 70 epi-8 Hydroxypinoresinol (4) Lariresinol (5) Isolariciresinol (6) Olivil (7) Cedrusin (8) Vitamin Cd Vitamin Ed a a 2þ (min) Cu -mediated AAPH-mediated ND ND ND ND ND 87 ND ND ND 90 ND ND ND 85 128 42:6  4:2 — 88:3  4:1 75:5  5:6 — 9:7  0:6;y 14:5  1:1;y 10:5  0:5;y 10:2  0:3;y 8:5  0:4;y 8:8  0:2;y 8:8  1:0;y 18:7  1:3;y 11:7  0:5 2:0  0:1 ND ND ND ND ND 18:2  1:5;y 25:4  2:2;y ND 24:8  0:9;y 12:7  1:0;y 12:1  0:6;y ND 51:1  4:3;y 18:9  1:6 5:8  0:5 The lag time of the blank was estimated to be 48 min. Each value represents the mean  S.D. of five experiments performed on different days. c % inhibition at a concentration of 100 mg/ml. d Compounds used as positive controls. () weak activity. ND, not determined.  P < 0:05 vs. vitamin C y P < 0:05 vs. vitamin E b TBARS, IC50 (mM)b Lag time 2754 M.-J. C HANG et al. 0.6 OD at 232 nm 0.5 0.4 HDL HDL + Vitamin C 0.3 HDL + Vitamin E HDL + 1 HDL + 5 0.2 0 50 100 150 200 250 300 Time (min) Fig. 2. Effects of 1 and 5 on the Generation of Conjugated Dienes during Cu2þ -Mediated Oxidation of HDL. The formation of conjugated dienes was determined as the change in UV absorbance at 232 nm. HDL (200 mg/ml) in PBS (pH 7.4) was preincubated in either the absence (control) or presence of 1 (3.0 mM), 5 (3.0 mM), vitamin C (3.0 mM), or vitamin E (3.0 mM), and then Cu2þ (5.0 mM) was added to initiate the oxidation at 37  C. The absorbance at 232 nm was continuously monitored at 10-min intervals for 5 h at 37  C. The lag time was measured as the intercept between the base-line and the tangent to the absorbance curve during the propagation phase. oxidation in part by acting as a sacrificial target for oxidation until the antioxidants have been depleted from LDL. Even at a low concentration (3.0 mM), 1 and 5 could exert a protective effect against Cu2þ -induced lipid peroxidation of HDL, as shown by the delayed lag time of the conjugated diene process in comparison with the control (87 and 90 min versus 48 min, Fig. 2). The results from the TBARS method implicate that those lignans were more effective antioxidants in the metaldependent pro-oxidant system than in the peroxyl radical system; metal ion-chelating properties may underlie their apparent antioxidative effect towards HDL oxidation in vitro. Incubating HDL in the presence of Cu2þ -oxidized erythrocyte membranes increased the content of lipid hydroperoxides in HDL, this suggests transport of the phospholipids containing hydroperoxides from oxidized erythrocyte membranes to lipoprotein. It has previously been reported that the 4-hydroxy3-methoxy substitution pattern of such guaiacyl lignans as pinoresinol (1), 8-hydroxypinoresinol (3), 70 -epi-8hydroxypinoresinol (4), lariciresinol (5), isolaraciresinol (6), and olivil (7) showed slightly stronger anti-lipid peroxidation capacity.25,26) Their antioxidative effects via the initial stage might involve the reversible donation of a phenolic hydrogen radical.26) The results demonstrate that, by reducing oxidative stress, the lignan constituents of the fruits of F. suspensa may prevent the development and progression of HDL oxidation. However, the cardio-protective ability of the HDL lipoprotein fraction in combination with active compounds to prevent atherogenic modification should be investigated. Acknowledgment This research was supported by a grant (PF06219-00) from the Plant Diversity Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology of the Korean government. References 1) 2) 3) 4) 5) Stampfer, M. J., Sacks, M. D., Salvini, S., Willett, S. C., and Hennekens, C. H., A prospective study of cholesterol, apolipoproteins, and the risk of myocardial infarction. N. Engl. J. Med., 325, 373–381 (1991). Rubins, H. B., Robins, S. J., Iwane, M. K., Boden, W. E., Elam, M. B., Fye, C. L., Gordon, D. J., Schaefer, E. J., Schectman, G., and Wittes, J. T., Rationale and design of the department of veterans affairs high density lipoprotein cholesterol intervention trial (HIT) for secondary prevention of coronary artery disease in men with low high density lipoprotein cholesterol and desirable low density lipoprotein cholesterol. Am. J. Cardiol., 71, 45– 52 (1993). Rubin, E. M., Krauss, R. M., Spangler, E. A., Verstuyft, J. G., and Clift, S. M., Inhibition of early atherogenesis in transgenic mice by human apolipoprotein AI. Nature, 353, 265–267 (1991). Francis, G. A., High density lipoprotein oxidation: in vitro susceptibility and potential in vivo consequences. Biochim. Biophys. Acta, 1483, 217–235 (2000). Nofer, J. R., Kehrel, B., Fobker, M., Levkau, B., Assmann, G., and von Eckardstein, A., HDL and arteriosclerosis: beyond reverse cholesterol transport. Atherosclerosis, 161, 1–16 (2002). Lignans from Forsythia suspensa Protect against High-Density Lipoprotein Oxidation 6) 7) 8) 9) 10) 11) 12) 13) 14) 15) 16) 17) Alsheikh-Ali, A. A., Kuvin, J. T., and Karas, R. H., High-density lipoprotein cholesterol in the cardiovascular equation: does the ‘‘good’’ still count? Atherosclerosis, 180, 217–223 (2005). Xinyixueyuan, ‘‘Dictionary of Traditional Chinese Medicine,’’ Shanghai Scientific & Technologic Press, Shanghai, pp. 1111–1113 (1977). Nikaido, T., Ohmoto, T., Kinoshita, T., Sankawa, U., Nishibe, S., and Hisada, S., Inhibition of cyclic AMP phosphodiesterase by lignans. Chem. Pharm. Bull., 29, 3586–3592 (1981). Kitagawa, S., Hisada, S., and Nishibe, S., Phenolic compounds from Forsythia leaves. Phytochemistry, 23, 1635–1636 (1984). Shamsur-Rouf, A. S., Ozaki, Y., Rashid, M. A., and Rui, J., Dammarane derivatives from the dried fruits of Forsythia suspensa. Phytochemistry, 56, 815–818 (2001). Ozaki, Y., Rui, J., and Tang, Y. T., Anti-inflammatory effect of Forsythia suspensa V(AHL) and its active principle. Biol. Pharm. Bull., 23, 365–367 (2000). Schinella, G. R., Tournier, H. A., Prieto, J. M., Mordujovich de Buschiazzo, P., and Rı́os, J. L., Antioxidant activity of anti-inflammatory plant extracts. Life Sci., 18, 1023–1033 (2002). Steffan, B., Wätjen, W., Michels, G., Niering, P., Wray, V., Ebel, R., Edrada, R., Kahl, R., and Proksch, P., Polyphenols from plants used in traditional Indonesian medicine (jamu): uptake and antioxidative effects in rat H4IIE hepatoma cells. J. Pharm. Pharmacol., 57, 233– 240 (2005). Piao, X. L., Jang, M. H., Cui, J., and Piao, X., Lignans from the fruits of Forsythia suspensa. Bioorg. Med. Chem. Lett., 18, 1980–1984 (2008). Qu, H., Zhang, Y., Wang, Y., Li, B., and Sun, W., Antioxidant and antibacterial activity of two compounds (forsythiaside and forsythin) isolated from Forsythia suspensa. J. Pharm. Pharmacol., 60, 261–266 (2008). Ferretti, G., Bacchetti, T., Menanno, F., and Curatola, G., Effect of genistein against copper-induced lipid peroxidation of human high density lipoproteins (HDL). Atherosclerosis, 172, 55–61 (2004). Hung, T. M., Lee, J. P., Min, B. S., Choi, J. S., Na, M., Zhang, X., Ngoc, T. M., Lee, I., and Bae, K., Magno- View publication stats 18) 19) 20) 21) 22) 23) 24) 25) 26) 2755 florine from Coptidis Rhizoma protects high density lipoprotein during oxidant stress. Biol. Pharm. Bull., 30, 1157–1160 (2007). Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, P. J., Protein measurement with the folin phenol reagent. J. Biol. Chem., 193, 265–275 (1951). Puhl, H., Waeg, G., and Esterbauer, H., Methods to determine oxidation of low-density lipoproteins. Methods Enzymol., 233, 425–441 (1994). Sobal, G., and Sinzinger, H., Effect of simvastatin on the oxidation of native and modified lipoproteins. Biochem. Pharmacol., 70, 1185–1191 (2005). Rifici, V. A., and Khachadurian, A. K., Effects of dietary vitamin C and E supplementation on the copper mediated oxidation of HDL and on HDL mediated cholesterol efflux. Atherosclerosis, 127, 19–26 (1996). Garner, B., Witting, P. K., Waldeck, A. R., Christison, J. K., Raftery, M., and Stocker, R., Oxidation of high density lipoproteins. I. Formation of methionine sulfoxide in apolipoproteins AI and AII is an early event that accompanies lipid peroxidation and can be enhanced by alpha-tocopherol. J. Biol. Chem., 273, 6080–6087 (1998). Albertini, R., De Luca, G., Passi, A., Moratti, R., and Abuja, P. M., Chondroitin-4-sulfate protects high-density lipoprotein against copper-dependent oxidation. Arch. Biochem. Biophys., 365, 143–149 (1999). Nguyen, S. D., and Sok, D. E., Effect of 3,4-dihydroxyphenylalanine on Cu(2+)-induced inactivation of HDL-associated paraoxonasel and oxidation of HDL; inactivation of paraoxonasel activity independent of HDL lipid oxidation. Free Radic. Res., 38, 969–976 (2004). Chen, C. C., Chen, H. Y., Shiao, M. S., Lin, Y. L., Kuo, Y. H., and Ou, J. C., Inhibition of low density lipoprotein oxidation by tetrahydrofurofuran lignans from Forsythia suspensa and Magnolia coco. Planta Med., 65, 709–711 (1999). Eklund, P. C., Långvik, O. K., Wärnå, J. P., Salmi, T. O., Willför, S. M., and Sjöholm, R. E., Chemical studies on antioxidant mechanisms and free radical scavenging properties of lignans. Org. Biomol. Chem., 3, 3336–3347 (2005).