Food Chemistry 146 (2014) 299–307
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Analytical Methods
Koelreuteria formosana extract impedes in vitro human LDL and prevents oxidised LDL-induced apoptosis in human umbilical vein endothelial cells Chin-Yin Lin a, Pei-Ni Chen a,b, Yih-Shou Hsieh a,b, Shu-Chen Chu c,⇑ a b c
Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan Clinical Laboratory, Chung Shan Medical University Hospital, Taichung, Taiwan Institute and Department of Food Science, Central Taiwan University of Science and Technology, Taichung, Taiwan
a r t i c l e
i n f o
Article history: Received 7 September 2012 Received in revised form 2 September 2013 Accepted 4 September 2013 Available online 12 September 2013 Keywords: Atherogenesis Koelreuteria formosana oxLDL HUVECs ROS
a b s t r a c t Koelreuteria formosana ethanol extract (KFEE) is obtained from natural plants that are endemic in Taiwan. A study showed that KFEE has antioxidant activity in DPPH assay. In the current study, the antioxidative activity of KFEE, which contains polyphenols including gallic acid and caffeic acid, was evaluated. The manner by which KFEE protects human umbilical vein endothelial cells (HUVECs) from oxidised LDL (oxLDL)-mediated dysfunction in vitro was investigated as well. The results indicate that the antioxidative activity of KFEE is defined by the relative electrophoretic mobility of oxLDL, the fragmentation of ApoB, conjugated diene production, and malondialdehyde production through Cu2+-mediated oxidation of LDL. KFEE also inhibited ROS generation as well as the subsequent mitochondrial membrane potential collapse, chromosome condensation, cytochrome C release, and caspase-3 activation induced by oxLDL in HUVECs. Our results also indicate that KFEE may protect LDL oxidation and prevent oxLDL-induced cellular dysfunction in HUVECs. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Atherosclerosis is widely regarded as a chronic inflammatory disease, and the oxidisation of low-density lipoprotein (LDL) is a crucial risk factor for accelerating atherogenesis in the arterial intima (Vaidya et al., 2011). Several studies demonstrated that oxidised LDL (oxLDL) or its products can affect several components of the atherogenic process, thrombosis, and lesion initiation as well as progression. oxLDL shows an enhanced uptake in macrophages by scavenger receptors, and it can lead to the accumulation of cholesterol esters and the formation of foam cells (Min, Cho, & Kwon, 2012; Witztum and Steinberg, 1991). Accumulation of oxLDL and induction of gene expression in endothelial cells cause an endothelial barrier by a variation in the function and structural integrity (Ross, 1993). Apoptosis is caused by an increase in reactive oxygen species (ROS) production, a decrease in mitochondrial membrane potential with the concomitant release of the mitochondrial protein cytochrome C, and the activation of caspase-3 and poly ADP-ribose polymerase (PARP) (Nazzal et al., 2006).
⇑ Corresponding author. Address: Institute and Department of Food Science, Central Taiwan University of Science and Technology, No. 11 Pu-tzu Lane, Pu-tzu Road, Taichung 406, Taiwan. Tel.: +886 4 2239 1647x3504; fax: +886 4 2324 8195. E-mail address: [email protected] (S.-C. Chu). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.09.018
Koelreuteria formosana, commonly known as the Chinese rain tree, is a deciduous tree native to Taiwan. Earlier reports indicated that K. formosana contain kaempferol, quercetin, and two kaempferol glycosides (Abou-Shoer et al., 1993). Another study indicated that K. formosana have protein-tryrosine kinase inhibitors, including anthraquinone, stilbene, and flavonoids, such as galangin, morin, myricetin, and apigenin. K. formosana may be an oncogene-based anticancer drug (Chang, Ashendel, Geahlen, McLaughlin, & Waters, 1996). A prior study showed that K. formosana extracts exhibit superior 2,2-diphenyl-1-picrylhydrazyl (DPPH)-scavenging activities compared with other plants (Lee, Jiang, Juan, Lin, & Hou, 2006). A recent report showed that the leaves of K. formosana have a new flavonol, galloylrhamnoside, and a new lignin, glycoside (Lee, Chiang, Chen, Chen, & Lee, 2009). In addition, the ethanol extract of K. formosana has kaempferol and quercetin, with potential protein-tyrosine kinase inhibitory activity (Abou-Shoer et al., 1993). Other studies indicated three classes of protein-tyrosine kinase inhibitors from K. formosana: anthraquinone, stilbene, and flavonoid (Chang et al., 1996). The extracted component of K. formosana is a more powerful antioxidant than xanthine oxidase, lipoxygenase, and tyrosinase (Chen et al., 2009). However, the protective effect of K. formosana on LDL oxidation and endothelial cells is unclear, especially with oxLDL-induced endothelial dysfunction. The effects of K. formosana extract on LDL oxidation and oxLDL-induced apoptosis as well as
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the mitochondrial changes in endothelial cells were investigated in this study. 2. Materials and methods 2.1. Materials and chemicals Ethylenediaminetetraacetic acid (EDTA), cupric sulphate, sodium dodecyl sulphate (SDS), 3-(4,5-dimethylthiazol-2-y1)-2,5diphenyltetrazolium bromide (MTT), 40 ,6-diamidino-2-phenylindole (DAPI), and polyacrylamide were purchased from Sigma Chemical Co. (St. Louis, MO). M199, fetal bovine serum, and trypsin–EDTA were obtained from GibcoBRL (Grand Island, NY). Low serum growth supplement (LSGS) and monoclonal antibody against b-actin were purchased from Cascade Biologicals (Portland, OR) and Sigma (St. Louis, MO), respectively. Mouse monoclonal antibodies against Bcl-2, Bax, and cytochrome C and goat polyclonal antibody against caspase-3 were both purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Rabbit polyclonal antibody against PARP was purchased from BD Transduction Laboratories (San Diego, CA). ECL Plus detection kit was obtained from Amersham Life Sciences, Inc. (Millipore). 2.2. Total phenolic contents Total phenolic content was measured by photometric assay using Folin–Ciocalteu reagent (Chen et al., 2009). Folin–Ciocalteu reagent (Fluka, Buchs, Switzerland) and sodium carbonate were successively added to each sample. The specific absorbance at 760 nm was immediately measured with a spectrophotometer (Thermo Biomate 5, Thermo Electron Corporation, San Jose, CA). Gallic acid was used as a standard phenolic compound for the calibration curve (y = 0.7758x + 0.0582, R2 = 0.9982). Total phenolic content was expressed as milligram of gallic acid equivalents per gram of dry weight of plant (mg GA/g DW). 2.3. Preparation of KFEE The K. formosana branches with leaves fallen off that were used in this study were purchased from local herb stores in Taichung. K. formosana peduncle extracts were initially prepared by condensation followed by lyophilization, as described previously. Briefly, 100 g of air-dried branch were boiled at 70 °C for 24 h in 50% ethanol. Then, the solvent was removed from the combined extract with a vacuum rotary evaporator. The filtrate was then lyophilized and stored at 20 °C. 2.4. Endothelial cell cultures HUVECs were isolated from human umbilical cords with collagenase (Jaffe, Nachman, Becker, & Minick, 1973). After dissociation, the cells were collected and cultured on gelatin-coated culture dishes in medium 199 with LSGS, 100 IU/mL penicillin, and 0.1 mg/mL streptomycin. 2.5. Lipoprotein separation and oxidation LDL was isolated from normal human plasma of six people. The human plasma was obtained from Taichung Blood Bank. LDL was isolated using the sequential ultracentrifugation method (Havel, Eder, & Bragdon, 1955). LDL was diluted with PBS to the final concentration of 1 mg protein/mL and incubated at 37 °C in the presence of CuSO4 (10 lM) for 16 h followed by the removal of Cu2+ by gel filtration (PD-10 gel) with 10 mM PBS.
2.6. Conjugated diene determination Conjugated diene formation was monitored by second derivative spectroscopy (220–300 nm) based on the method described by Bourne and Rice-Evans (Wallin, Rosengren, Shertzer, & Camejo, 1993). The second derivative spectrum was subtracted from the second derivative spectrum of the matching control sample without Cu2+. The increase in conjugated dienes expressed in relative unit was obtained from the amplitude of the peak at 254 nm.
2.7. Thiobarbituric acid reactive substances (TBARS) MDA production was assessed as an indicator of lipid peroxidation according to the procedures of Wallin et al. (1993). To each tube containing 0.55 mL of the incubated LDL was added 0.5 mL of 25% (w/v) trichloroacetic acid for protein denaturation. Then, the samples were centrifuged (10,000 rpm) at 10 °C for 30 min to remove the pellets. TBA (1%, 0.5 mL) in 0.3% NaOH was added to the supernatant, and the mixed reagents were allowed to react at 95 °C for 40 min in the dark. After the reaction was completed, samples were analysed using a Hitachi F2000 spectrophotofluorimeter (excitation wavelength at 532 nm and emission wavelength at 600 nm). The concentration of MDA or TBA-reacting substance was expressed as equivalents of 1,1,3,3-tetraethoxypropane that served as a standard.
2.8. Relative electrophoretic mobility (REM) shift assay LDL (200 lg/mL) was pretreated with the indicated concentrations of KFEE for 2 h followed by incubation with 10 lM CuSO4 at 37 °C for 16 h. LDL modifications were assessed by agarose electrophoresis (0.6% agarose) to detect the increase in electrophoretic mobility of the modified LDL relative to native LDL accordingly (Miyakawa, Honma, Qi, & Kuramitsu, 2004). The gel was fixed in 75% ethanol and 5% acetic acid for 15 min, stained with 1% oil red O (in 60% isopropanol) for 30 min, and rinsed with 30% isopropanol to visualise LDL bands. The distance migrated by each LDL band was measured and expressed as an arbitrary REM value compared with the native LDL.
2.9. Electrophoresis of ApoB fragmentation After oxidation with or without KFEE, samples were denatured with 3% SDS, 10% glycerol, and 5% 2-mercaptoethanol at 95 °C for 5 min. SDS polyacrylamide gel electrophoresis (7.5% SDS–PAGE) was performed to detect ApoB fragmentation, with subsequent staining with Coomassie brilliant blue R250 (Lee et al., 2002).
2.10. DPPH radical scavenging assay DPPH assay was performed to measure the antiradical activity of KFEE. KFEE and Trolox storage solutions were added to DPPH solution to obtain the indicated concentrations. The absorbance was measured at 517 nm after 2 min of reaction at room temperature.
2.11. Cell growth assay After treatment with oxLDL (200 lg/mL) for 16 h in the presence or absence of KFEE, cells were harvested, and the viable cell number was counted in a hemocytometer by trypan blue exclusion.
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2.12. Cell cycle analysis HUVECs were pretreated with the indicated concentrations of KFEE for 2 h followed by oxLDL (200 lg/mL) for 16 h. Briefly, 1 105 cells were fixed in 70% ethanol and then suspended in propidium iodide (PI) solution (25 lg/mL PI, 0.1 mM EDTA, and 10 lg/ mL RNase A in PBS) for 30 min. The cell cycle distribution was then analysed by flow cytometry analysis using Cell-Quest software (Becton–Dickinson, CA). 2.13. Measurement of mitochondrial membrane potential After treatment with oxLDL (200 lg/mL) for 16 h in the presence or absence of KFEE, cells (3 105 cells/6-well plate) were rinsed with medium, followed by the addition of JC-1 (5 lM). After incubation at 37 °C for 20 min, cells were examined under a fluorescent microscope (Bedner, Li, Gorczyca, Melamed, & Darzynkiewicz, 1999). 2.14. Measurement of ROS production To investigate the effect of KFEE on oxLDL-induced ROS production in HUVECs (3 105 cells/6-cm cell culture dish), a fluorometric assay using DCFH-DA was conducted to detect the presence of hydroxyl radical. HUVECs were pretreated with or without the indicated concentrations of KFEE for 2 h followed by incubation with oxLDL for 16 h. After the removal of media from the wells, cells were incubated with 10 lM DCFH-DA for 1 h and then centrifuged and resuspended for immediate determination of ROS generation by flow cytometry using 488 nm for excitation and 525 nm for emission (Yen, Hsieh, Chou, & Lau, 2001). 2.15. Determination of cell viability (MTT assay) After the indicated treatments, the treated HUVECs (3 104 cells/24-well plate) were incubated with 0.5 mg/mL MTT in culture medium for an additional 4 h, and the blue formazan crystals of viable cells were dissolved in isopropanol and then measured spectrophotometrically at 563 nm (Chen et al., 2005). 2.16. DAPI staining Single-cell suspensions of treated HUVECs (3 105 cells/6-well plate) were washed with PBS and fixed by 70% ethanol. Afterward, cells were treated with DAPI stain (0.6 lg/mL in PBS). Chromatin fluorescence was observed under a UV-light microscope. Apoptotic cells were morphologically defined by cytoplasmic and nuclear shrinkage as well as chromatin condensation (Yen et al., 2001). 2.17. Western blot analysis Samples of cell lysates or cytosolic fractions were separated in 12.5% polyacrylamide gel and transferred onto a nitrocellulose membrane, as previously described (Chen et al., 2005). The protein was loaded to the gel for Western blot analysis (30 lg/well). The blot was subsequently probed with antibodies. Immobilon Western Chemiluminescent HRP substrate kit was obtained from Millipore (Burlington, MA), and the relative intensities of the signals were quantified by densitometry using a gel documentation and analysis system (LAS-3000 Image Reader, Fujifilm, Stamford, CT). 2.18. High-performance liquid chromatography (HPLC) analysis
Fig. 1. The chemical profile of KFEE was analysed by HPLC-mass spectrometry. (A) Chromatographic patterns from HPLC analysis of KFEE extracts showed peaks corresponding to the retention times (min). (B) HPLC chromatogram of nine kinds of standard compounds. Peaks: 1, gallic acid; 2, gallocatechin; 3, (+)catechin; 4, caffeic acid; 5, rutin; 6, naringin; 7, quercetin; 8, kaempferol; 9, anthraquinone. (C) Peaks: 10, myricetin; 11, morin; 12, apigenin; 13, galangin. (D) Combination of 100 lg KFEE with two kinds of standard compound (gallic acid and caffeic acid). Absorbance was monitored at 254 nm.
HPLC analysis (Waters 600 with a 2998 Photodiode Array detector) was performed using a LiChroCART RP-18 reversed phase col-
umn (200 4 mm, 5 lm) with mobile phase consisting of water/acetic acid (0.05%, v/v) (solvent A) and acetic acid/water/
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acetonitrile (0.05%, v/v) (solvent B). Elution was carried out in a programmed gradient elution as follows: 0 min to 30 min, with 0% to 100% B.
2.19. Statistical analysis Statistical significance was analysed by one-way analysis of variance (ANOVA) with post-hock Dunnett’s test. A P-value 6 0.05 was considered statistically significant (Sigma-Stat 2.0, Jandel Scientific, San Rafael, CA, USA).
3. Results 3.1. Polyphenol contents in KFEE Quantitative phytochemical analysis revealed the presence of 38.8 ± 0.4 mg/g of KFEE polyphenols. To evaluate the bioactive compound of KFEE, consecutive extractions of KFEE with 50% ethanol were performed. Chromatographic patterns from the HPLC analysis of KFEE (Fig. 1A) and 13 kinds of standard compounds, including gallic acid, gallocatechin, catechin, caffeic acid, rutin, naringin, quercetin, kaempferol, anthaquinone, myricetin, morin, apigenin, and galangin (Fig. 1B and C), showed peaks corresponding to the retention times. Absorbance was monitored at 254 nm. Fig. 1D showed that gallic acid and caffeic acid are included in the composition of KFEE.
3.2. KFEE inhibited copper-induced oxidation of LDL and ApoB fragmentation As shown in Fig. 2A and B, the REM of the control was assigned a mobility of 1.0, whereas chemical oxidation shifted the REM to 2.8. Pretreatment with KFEE (5, 10, 15, and 20 lg/mL) and Trolox (a water-soluble analogue of vitamin E; as a positive control) substantially reduced electrophoretic mobility in a dose-dependent manner. The inhibitory effects of KFEE on ApoB fragmentation were examined using SDS–PAGE in a similar manner (Fig. 2C). The ApoB band (arrow) in native LDL disappeared because of the fragmentation caused by CuSO4 incubation for 4 h. The presence of KFEE (5, 10, 15, and 20 lg/mL) and Trolox (10 and 50 lM) reversed copper-induced ApoB fragmentation substantially (Fig. 2C and D).
3.3. KFEE attenuated copper-induced lipid peroxidation of LDL The effects of KFEE on Cu2+-mediated LDL oxidation are shown in Fig. 2E. The lag time was 100 min for the control group with LDL and Cu2+. However, LDL oxidation was inhibited, and the lag time increased when KFEE was added. Results showed statistical differences (P < 0.001), and 20 lg/mL of KFEE increased the lag time in a similar manner with the control group at 600 min. MDA concentration, which is a lipid peroxidation indicator, was measured to assay LDL oxidation. Fig. 2B shows that LDL incubated with 10 lM CuSO4 increased MDA production (lane 2, Fig. 2F). LDL treatment with KFEE (25, 50, 75, and 100 lg/mL) or Trolox (as a positive control; 10 and 50 lM) reduced MDA formation in a dose-dependent manner. This result shows that KFEE exerted an inhibitory effect on the lipid peroxidation of LDL. Furthermore, to investigate the radical scavenging activity of KFEE, the reactivity toward the stable free radical DPPH was measured at 517 nm. As shown in Fig. 2G, 5 lg/mL of KFEE showed statistical differences (P < 0.001), and 20 lg/mL of KFEE demonstrated the strong activity of a free radical scavenger.
3.4. KFEE reduced oxLDL-induced cytotoxicity on HUVECs The protective effect of KFEE was examined by monitoring the oxLDL-induced morphological features of cultured HUVECs in the absence and presence of KFEE under phase-contrast microscopy. Treatment of cultured endothelial cells with oxLDL (200 lg/mL) for 16 h led to cell shrinkage or membrane blebbing, which were reduced substantially by adding KFEE (Fig. 3A). This observation was further supported by the KFEE-induced increment of cell viability; KFEE did not exhibit cytotoxicity to HUVECs (Fig. 3B), indicating that KFEE had proliferative activities (Fig. 3C) in oxLDL-treated HUVECs. To investigate whether KFEE prevented oxLDL-induced apoptosis in HUVECs, DAPI staining and flow cytometry analysis were performed. Cells incubated with oxLDL for 16 h exhibited typical features of apoptosis, such as formation of condensed chromatin (Fig. 3D) and accumulation of a sub-G1 population (Fig. 3E). However, this effect was decreased substantially by KFEE pretreatment. This result indicates that KFEE protected endothelial cells from oxLDL-induced cytotoxicity and apoptosis. oxLDL-induced HUVEC cell death occurred mainly through the apoptotic pathway by Annexin V and PI stain. Cell necrosis rate also decreased with KFEE treatment (data not shown).
3.5. KFEE perturbed oxLDL-induced ROS generation in HUVECs The effects of KFEE on oxLDL-mediated ROS generation were determined by pretreating endothelial cells with KFEE before oxLDL exposure. As shown in Fig. 4A–F, oxLDL produced an increase in ROS generation (Fig. 4B, dark trace), compared with the control (Fig. 4A, gray trace). Pretreatment with KFEE (25, 50, and 75 lg/mL) inhibited (left shift) oxLDL-induced ROS formation considerably (Fig. 4C–E), whereas 100 lg/mL of KFEE almost completely inhibited ROS production (Fig. 4F).
3.6. KFEE reverted oxLDL-induced alterations in mitochondrial transmembrane permeability The effects of oxLDL on mitochondrial permeability were examined to determine whether mitochondrial disruption is involved in the anti-apoptotic effect of KFEE. When HUVECs were exposed to oxLDL, DWm of the mitochondria was depolarised, as shown by the decrease in red fluorescence and the increase in green fluorescence. Pretreatment with KFEE reduced the change in DWm, as indicated by the repression of green fluorescence and the restoration of red fluorescence (Fig. 4G). The disruption of mitochondrial membrane function results in the release of mitochondrial enzyme cytochrome C into the cytosol, which can be detected by Western blot. The incubation of HUVECs with oxLDL for 16 h induced a threefold increase in the release of cytochrome C into the cytosolic fraction, compared with that of the control cells (Fig. 5A). KFEE considerably reduced the oxLDL-induced release of cytochrome C.
3.7. KFEE prevented oxLDL-induced changes in apoptotic protein expression in HUVECs KFEE reduced the oxLDL-induced expression of Bax considerably, and it increased the levels of antiapoptotic protein Bcl-2 at the highest concentration of 100 lg/mL (Fig. 5B). The expression levels of active caspase-3 and cleaved-PARP were increased substantially in HUVECs treated with oxLDL. In contrast, caspase-3 activation and PARP cleavage by oxLDL were suppressed in a dose-dependent manner in cells pretreated with KFEE.
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Fig. 2. Effects of KFEE on Cu2+-mediated shift of electrophoretic mobility, he ApoB fragmentation in LDL, and copper-mediated LDL oxidation, DPPH scavenged capability. (A) LDL was incubated with CuSO4 in the presence or absence of KFEE or Trolox, as positive control, and it was applied to agarose gels as described in Section 2. (B) The results from the agarose gel electrophoresis were quantified and expressed in the form of REM. The distance travelled in the agarose gel by the native LDL was assigned the arbitrary unit 1. (C) LDL (200 lg/mL) was incubated with a 10-lM CuSO4 at 37 °C in the absence or presence of KFEE or Trolox for 4 h and applied to SDS–PAGE as described in Section 2. (D) Quantification of the ApoB fragmentation assay using densitometry. Results were statistically evaluated by using one-way ANOVA with post hoc Dunnett test (⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001). The signal intensity of the native LDL was assigned as 100% arbitrarily. #P < 0.001 compared with control. ⁄P < 0.05; ⁄⁄P < 0.01; and ⁄⁄⁄ P < 0.001 compared with the oxLDL treated group. (E) LDL pre-incubated with increasing concentration of KFEE. Oxidation was induced with the addition of CuSO4 (10 lM), and conjugated dienes were formed. #P < 0.001 compared with control. ⁄⁄⁄P < 0.001 compared with the oxLDL-treated group. (F) LDL (200 lg/mL) was incubated with 10-lM CuSO4 at 37 °C for 16 h in the presence or absence of KFEE or Trolox, and then MDA formation was measured as described in Section 2. (G) The radical-scavenging activities of KFEE and Trolox were evaluated by using the DPPH radical scavenging assay. The absorbance of the sample without adding KFEE or Trolox was assigned as 100%, and its radical scavenging rate was assigned as 0% consequently. The quantitative data were presented as means.
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Fig. 3. Effect of KFEE on oxLDL-induced endothelial cell apoptosis. (A) Photomicrographs of the treated HUVECs were observed using phase-contrast microscopy. (B) The viability of treated HUVECs was detected using MTT assay as described in Section 2. (C) Viable cells and dead cells were counted using the trypan blue exclusion assay. The quantitative data were presented as means. (D) Nuclear morphology of the treated cells was observed by fluorescence microscopy using DAPI stain at a magnification of 200. Arrows showed areas of intense fluorescence staining with condensed nuclei. (E) The hypodiploid cell population (sub G1 phase) of the treated HUVECs was analysed by flow cytometry using PI stain, and 10,000 events of total cells were analysed for each experimental treatment. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. Effects of KFEE on oxLDL-induced ROS production and on mitochondrial membrane potential of HUVECs. ROS levels of (A) the HUVECs without treatment, control (without oxLDL; gray trace); (B) HUVECs with treatment of oxLDL (200 lg/mL), control (with oxLDL; dark trace); (C) HUVECs with treatment of oxLDL and KFEE (25 lg/mL), KFEE 25 lg/mL (dark trace); (D) HUVECs with treatment of oxLDL and KFEE (50 lg/mL), KFEE 50 lg/mL (dark trace); (E) HUVECs with treatment of oxLDL and KFEE (75 lg/ mL), KFEE 75 lg/mL (dark trace); and (F) HUVECs with treatment of oxLDL and KFEE (100 lg/mL), KFEE 100 lg/mL (dark trace) was measured by flow cytometry using DCFH staining. (G) JC-1 is selectively accumulated within intact mitochondria to form multimer J-aggregates emitting fluorescence light at 590 nm (red) at a higher membrane potential (left), and monomeric JC-1 emits light at 530 nm (green) at a low membrane potential (right). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
4. Discussion oxLDL is a crucial clinical risk factor for atherosclerosis. The reduction of oxLDL formation may prevent heart diseases. The data show that KFEE can reduce LDL ApoB fragmentation (Fig. 2C) and increase REM (Fig. 2A); conversely, it also inhibits the formation of conjugated dienes in LDL (Fig. 2E). These data suggest that KFEEs have a strong antioxidative ability to reduce LDL oxidation. The increase in serum LDL cholesterol levels is an essential factor in atherogenesis formation (Holvoet and Collen, 1997). Endothelial cell apoptosis is vital in the progress of several cardiovascular diseases
(Simionescu, 2007). A study showed that accumulated LDL in the arterial wall becomes oxLDL, which leads to endothelial function damage in the early stages of atherogenesis in hypercholesterolemic patients (Das, Snehlata Das, & Srivastava, 2006). Other studies also showed that LDL becomes oxLDL when LDL is incubated with endothelial cells, smooth muscle cells, or macrophages. A previous study showed that extract from K. formosana had inhibitory activities for xanthine oxidase, tyrosinase, and lipoxygenase (Chen et al., 2009; Lee et al., 2006). In the current study, KFEE was found to prevent oxidative damage to endothelial cells in vitro (Figs. 3 and 4), and this protective effect is concomitant with a
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Conversely, the extract from K. formosana had biological effects on heme oxygenase-1 induction (Lee et al., 2006), and three classes of protein-tyrosine kinase inhibitors were isolated from K. Formosana, that is, anthraquinone, stilbene, and flavonoid (Chang et al., 1996). Thus, KFEE protection against oxLDL-induced endothelial dysfunction may be associated with decrease in ROS overproduction. Compared with direct scavenging ROS activity attributed to its chemical structure, this effect may control the expression or activity of the ROS-generating enzyme and the ROS-scavenging enzyme. However, the exact mechanism by which KFEE protects against oxidative stress remains unclear and requires further examination. Other studies showed that Caesalpinia sappan extract could decrease inflammatory responses in HUVECs (Lee et al., 2010), and extracts of Ginkgo biloba and Solanum lyratum have the ability to protect HUVECs against oxLDL-induced injury (Kuo et al., 2009; Ou et al., 2009). The results of these studies and our results provide a possible molecular mechanism underlying plant crude extract suppression of oxLDL-mediated vascular endothelial dysfunction. 5. Conclusion Many reports have demonstrated that the branch and stem of K. formosana contain flavonoids, polyphenols and quinone, such as quercetin, kaempferol (Abou-Shoer et al., 1993), catechin (Lin, Deng, Lei, Fu, & Li, 2002), and anthraquinone (Chang et al., 1996). The composition of KFEE was assessed by HPLC. Results showed that KFEE contains gallic acid and caffeic acid, but it does not have kaempferol, catechin, and anthraquinone. These results may be attributed to the use of different solvent conditions for the purification of K. formosana. Moreover, the KFEE in the present study was extracted from branches after the leaves have fallen off, which differed from the extraction location and method of the other studies (Abou-Shoer et al., 1993; Chen et al., 2009; Lin et al., 2002). In the future, the actions of other components of KFEE should be further examined. Furthermore, understanding the signalling pathways of the intracellular are crucial, as is the manner by which the oxidative damage induced by oxLDL is modulated in endothelial cultures. This knowledge may enable us to devise therapeutic tools for preventing atherosclerotic progression. Our results indicate a mechanism that links ROS production and membrane damage, which leads to apoptosis caused by oxLDL in HUVECs. These findings may provide additional information on the anti-inflammatory properties of KFEE, which may contribute to its anti-atherosclerotic effect and anti-inflammatory action. However, further animal model testing is required to validate the relevance of these results. Fig. 5. Effects of KFEE on oxLDL-induced caspase-3 and PARP activation. Cell lysates were subjected to SDS–PAGE followed by Western blot with (A) anti- cytochrome C, (B) anti-Bcl-2, anti- BAX, anti-caspase-3, and anti-PARP antibodies with b-actin as internal control. Signals of proteins were visualised with an ECL detection system. The results were obtained from three independent experiments. #P < 0.001 compared with control. ⁄P < 0.05; ⁄⁄P < 0.01; and ⁄⁄⁄P < 0.001 compared with the oxLDLtreated group.
reduction in ROS level (Fig. 4A–F) and steady mitochondrial integrity (Fig. 4G). Mitochondrial cytochrome C (Fig. 5A), cleaved caspase-3, and PARP (Fig. 5B) levels were down-regulated, verifying that KFEE can reduce the extent of apoptosis. In summary, KFEE can decrease endothelial cell apoptosis induced by oxLDL, and it exhibits anti-atherosclerotic ability through cytochrome C-mediated pathways. We also showed that 25 lg/mL of KFEE protected HUVECs from oxLDL-induced cytotoxicity because of its pharmacokinetics and the efficiency level tested in vitro. Similar to Trolox, KFEE can prevent LDL oxidation (Fig. 2). KFEE can substantially inhibit oxLDL-induced ROS overproduction (Fig. 3B).
Acknowledgements This study was supported by grants from the National Science Council, Republic of China (NSC 98-2313-B-166-004-MY3 and NSC 98-2313-B-040-004-MY3). HPLC was performed in the Instrument Center of Chung Shan Medical University, which is supported by the National Science Council, Ministry of Education, and Chung Shan Medical University. References Abou-Shoer, M., Ma, G. E., Li, X. H., Koonchanok, N. M., Geahlen, R. L., & Chang, C. J. (1993). Flavonoids from Koelreuteria henryi and other sources as proteintyrosine kinase inhibitors. Journal of Natural Products, 56, 967–969. Bedner, E., Li, X., Gorczyca, W., Melamed, M. R., & Darzynkiewicz, Z. (1999). Analysis of apoptosis by laser scanning cytometry. Cytometry, 35, 181–195. Chang, C. J., Ashendel, C. L., Geahlen, R. L., McLaughlin, J. L., & Waters, D. J. (1996). Oncogene signal transduction inhibitors from medicinal plants. In Vivo, 10, 185–190.
C.-Y. Lin et al. / Food Chemistry 146 (2014) 299–307 Chen, C. H., Chan, H. C., Chu, Y. T., Ho, H. Y., Chen, P. Y., Lee, T. H., et al. (2009). Antioxidant activity of some plant extracts towards xanthine oxidase, lipoxygenase and tyrosinase. Molecules, 14, 2947–2958. Chen, P. N., Chu, S. C., Chiou, H. L., Chiang, C. L., Yang, S. F., & Hsieh, Y. S. (2005). Cyanidin 3-glucoside and peonidin 3-glucoside inhibit tumor cell growth and induce apoptosis in vitro and suppress tumor growth in vivo. Nutrition and Cancer, 53, 232–243. Das, S., Snehlata Das, N., & Srivastava, L. M. (2006). Role of ascorbic acid on in vitro oxidation of low-density lipoprotein derived from hypercholesterolemic patients. Clinica Chimica Acta, 372, 202–205. Havel, R. J., Eder, H. A., & Bragdon, J. H. (1955). The distribution andchemical composition of ultracentrifugally separated lipoproteins in human serum. The Journal of Clinical Investigation, 34, 1345–1353. Holvoet, P., & Collen, D. (1997). Beta-VLDL hypercholesterolemia relative to LDL hypercholesterolemia is associated with higher levels of oxidized lipoproteins and a more rapid progression of coronary atherosclerosis in rabbits. Arteriosclerosis, Thrombosis, and Vascular Biology, 17, 2376–2382. Jaffe, E. A., Nachman, R. L., Becker, C. G., & Minick, C. R. (1973). Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. The Journal of Clinical Investigation, 52, 2745–2756. Kuo, W. W., Huang, C. Y., Chung, J. G., Yang, S. F., Tsai, K. L., Chiu, T. H., et al. (2009). Crude extracts of Solanum lyratum protect endothelial cells against oxidized low-density lipoprotein-induced injury by direct antioxidant action. Journal of Vascular Surgery, 50, 849–860. Lee, T. H., Chiang, Y. H., Chen, C. H., Chen, P. Y., & Lee, C. K. (2009). A new flavonol galloylrhamnoside and a new lignan glucoside from the leaves of Koelreuteria henryi Dummer. Journal of Natural Medicines, 63, 209–214. Lee, M. J., Chou, F. P., Tseng, T. H., Hsieh, M. H., Lin, M. C., & Wang, C. J. (2002). Hibiscus protocatechuic acid or esculetin can inhibit oxidative LDL induced by either copper ion or nitric oxide donor. Journal of Agricultural and Food Chemistry, 50, 2130–2136. Lee, M. H., Jiang, C. B., Juan, S. H., Lin, R. D., & Hou, W. C. (2006). Antioxidant and heme oxygenase-1 (HO-1)-induced effects of selected Taiwanese plants. Fitoterapia, 77, 109–115. Lee, M. J., Lee, H. S., Jung, H. J., Lee, C. S., Kim, J. E., Moon, H. I., et al. (2010). Caesalpinia sappan L. ameliorates hypercholesterolemia in C57BL/6 mice and suppresses inflammatory responses in human umbilical vein endothelial cells
307
(HUVECs) by antioxidant mechanism. Immunopharmacol Immunotoxicol, 32, 671–679. Lin, W. H., Deng, Z. W., Lei, H. M., Fu, H. Z., & Li, J. (2002). Polyphenolic compounds from the leaves of Koelreuteria paniculata Laxm. Journal of Asian Natural Products Research, 4, 287–295. Min, K. J., Cho, K. H., & Kwon, T. K. (2012). The effect of oxidized low density lipoprotein (oxLDL)-induced heme oxygenase-1 on LPS-induced inflammation in RAW 264.7 macrophage cells. Cell Signal, 24, 1215–1221. Miyakawa, H., Honma, K., Qi, M., & Kuramitsu, H. K. (2004). Interaction of Porphyromonas gingivalis with low-density lipoproteins: implications for a role for periodontitis in atherosclerosis. Journal of Periodontal Research, 39, 1–9. Nazzal, D., Cantero, A. V., Therville, N., Segui, B., Negre-Salvayre, A., Thomsen, M., et al. (2006). Chlamydia pneumoniae alters mildly oxidized low-density lipoprotein-induced cell death in human endothelial cells, leading to necrosis rather than apoptosis. The Journal of Infectious Diseases, 193, 136–145. Ou, H. C., Lee, W. J., Lee, I. T., Chiu, T. H., Tsai, K. L., Lin, C. Y., et al. (2009). Ginkgo biloba extract attenuates oxLDL-induced oxidative functional damages in endothelial cells. Journal of Applied Physiology, 106, 1674–1685. Ross, R. (1993). The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature, 362, 801–809. Simionescu, M. (2007). Implications of early structural-functional changes in the endothelium for vascular disease. Arteriosclerosis, Thrombosis, and Vascular Biology, 27, 266–274. Vaidya, D., Szklo, M., Cushman, M., Holvoet, P., Polak, J., Bahrami, H., et al. (2011). Association of endothelial and oxidative stress with metabolic syndrome and subclinical atherosclerosis: multi-ethnic study of atherosclerosis. European Journal of Clinical Nutrition, 65, 818–825. Wallin, B., Rosengren, B., Shertzer, H. G., & Camejo, G. (1993). Lipoprotein oxidation and measurement of thiobarbituric acid reacting substances formation in a single microtiter plate: its use for evaluation of antioxidants. Analytical Biochemistry, 208, 10–15. Witztum, J. L., & Steinberg, D. (1991). Role of oxidized low density lipoprotein in atherogenesis. The Journal of Clinical Investigation, 88, 1785–1792. Yen, C. H., Hsieh, C. C., Chou, S. Y., & Lau, Y. T. (2001). 17Beta-estradiol inhibits oxidized low density lipoprotein-induced generation of reactive oxygen species in endothelial cells. Life Sciences, 70, 403–413.
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Koelreuteria formosana extract impedes in vitro human LDL and prevents oxidised LDL-induced apoptosis in human umbilical vein endothelial cells Chin-Yin Lin a, Pei-Ni Chen a,b, Yih-Shou Hsieh a,b, Shu-Chen Chu c,⇑ a b c
Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan Clinical Laboratory, Chung Shan Medical University Hospital, Taichung, Taiwan Institute and Department of Food Science, Central Taiwan University of Science and Technology, Taichung, Taiwan
a r t i c l e
i n f o
Article history: Received 7 September 2012 Received in revised form 2 September 2013 Accepted 4 September 2013 Available online 12 September 2013 Keywords: Atherogenesis Koelreuteria formosana oxLDL HUVECs ROS
a b s t r a c t Koelreuteria formosana ethanol extract (KFEE) is obtained from natural plants that are endemic in Taiwan. A study showed that KFEE has antioxidant activity in DPPH assay. In the current study, the antioxidative activity of KFEE, which contains polyphenols including gallic acid and caffeic acid, was evaluated. The manner by which KFEE protects human umbilical vein endothelial cells (HUVECs) from oxidised LDL (oxLDL)-mediated dysfunction in vitro was investigated as well. The results indicate that the antioxidative activity of KFEE is defined by the relative electrophoretic mobility of oxLDL, the fragmentation of ApoB, conjugated diene production, and malondialdehyde production through Cu2+-mediated oxidation of LDL. KFEE also inhibited ROS generation as well as the subsequent mitochondrial membrane potential collapse, chromosome condensation, cytochrome C release, and caspase-3 activation induced by oxLDL in HUVECs. Our results also indicate that KFEE may protect LDL oxidation and prevent oxLDL-induced cellular dysfunction in HUVECs. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Atherosclerosis is widely regarded as a chronic inflammatory disease, and the oxidisation of low-density lipoprotein (LDL) is a crucial risk factor for accelerating atherogenesis in the arterial intima (Vaidya et al., 2011). Several studies demonstrated that oxidised LDL (oxLDL) or its products can affect several components of the atherogenic process, thrombosis, and lesion initiation as well as progression. oxLDL shows an enhanced uptake in macrophages by scavenger receptors, and it can lead to the accumulation of cholesterol esters and the formation of foam cells (Min, Cho, & Kwon, 2012; Witztum and Steinberg, 1991). Accumulation of oxLDL and induction of gene expression in endothelial cells cause an endothelial barrier by a variation in the function and structural integrity (Ross, 1993). Apoptosis is caused by an increase in reactive oxygen species (ROS) production, a decrease in mitochondrial membrane potential with the concomitant release of the mitochondrial protein cytochrome C, and the activation of caspase-3 and poly ADP-ribose polymerase (PARP) (Nazzal et al., 2006).
⇑ Corresponding author. Address: Institute and Department of Food Science, Central Taiwan University of Science and Technology, No. 11 Pu-tzu Lane, Pu-tzu Road, Taichung 406, Taiwan. Tel.: +886 4 2239 1647x3504; fax: +886 4 2324 8195. E-mail address: [email protected] (S.-C. Chu). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.09.018
Koelreuteria formosana, commonly known as the Chinese rain tree, is a deciduous tree native to Taiwan. Earlier reports indicated that K. formosana contain kaempferol, quercetin, and two kaempferol glycosides (Abou-Shoer et al., 1993). Another study indicated that K. formosana have protein-tryrosine kinase inhibitors, including anthraquinone, stilbene, and flavonoids, such as galangin, morin, myricetin, and apigenin. K. formosana may be an oncogene-based anticancer drug (Chang, Ashendel, Geahlen, McLaughlin, & Waters, 1996). A prior study showed that K. formosana extracts exhibit superior 2,2-diphenyl-1-picrylhydrazyl (DPPH)-scavenging activities compared with other plants (Lee, Jiang, Juan, Lin, & Hou, 2006). A recent report showed that the leaves of K. formosana have a new flavonol, galloylrhamnoside, and a new lignin, glycoside (Lee, Chiang, Chen, Chen, & Lee, 2009). In addition, the ethanol extract of K. formosana has kaempferol and quercetin, with potential protein-tyrosine kinase inhibitory activity (Abou-Shoer et al., 1993). Other studies indicated three classes of protein-tyrosine kinase inhibitors from K. formosana: anthraquinone, stilbene, and flavonoid (Chang et al., 1996). The extracted component of K. formosana is a more powerful antioxidant than xanthine oxidase, lipoxygenase, and tyrosinase (Chen et al., 2009). However, the protective effect of K. formosana on LDL oxidation and endothelial cells is unclear, especially with oxLDL-induced endothelial dysfunction. The effects of K. formosana extract on LDL oxidation and oxLDL-induced apoptosis as well as
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the mitochondrial changes in endothelial cells were investigated in this study. 2. Materials and methods 2.1. Materials and chemicals Ethylenediaminetetraacetic acid (EDTA), cupric sulphate, sodium dodecyl sulphate (SDS), 3-(4,5-dimethylthiazol-2-y1)-2,5diphenyltetrazolium bromide (MTT), 40 ,6-diamidino-2-phenylindole (DAPI), and polyacrylamide were purchased from Sigma Chemical Co. (St. Louis, MO). M199, fetal bovine serum, and trypsin–EDTA were obtained from GibcoBRL (Grand Island, NY). Low serum growth supplement (LSGS) and monoclonal antibody against b-actin were purchased from Cascade Biologicals (Portland, OR) and Sigma (St. Louis, MO), respectively. Mouse monoclonal antibodies against Bcl-2, Bax, and cytochrome C and goat polyclonal antibody against caspase-3 were both purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Rabbit polyclonal antibody against PARP was purchased from BD Transduction Laboratories (San Diego, CA). ECL Plus detection kit was obtained from Amersham Life Sciences, Inc. (Millipore). 2.2. Total phenolic contents Total phenolic content was measured by photometric assay using Folin–Ciocalteu reagent (Chen et al., 2009). Folin–Ciocalteu reagent (Fluka, Buchs, Switzerland) and sodium carbonate were successively added to each sample. The specific absorbance at 760 nm was immediately measured with a spectrophotometer (Thermo Biomate 5, Thermo Electron Corporation, San Jose, CA). Gallic acid was used as a standard phenolic compound for the calibration curve (y = 0.7758x + 0.0582, R2 = 0.9982). Total phenolic content was expressed as milligram of gallic acid equivalents per gram of dry weight of plant (mg GA/g DW). 2.3. Preparation of KFEE The K. formosana branches with leaves fallen off that were used in this study were purchased from local herb stores in Taichung. K. formosana peduncle extracts were initially prepared by condensation followed by lyophilization, as described previously. Briefly, 100 g of air-dried branch were boiled at 70 °C for 24 h in 50% ethanol. Then, the solvent was removed from the combined extract with a vacuum rotary evaporator. The filtrate was then lyophilized and stored at 20 °C. 2.4. Endothelial cell cultures HUVECs were isolated from human umbilical cords with collagenase (Jaffe, Nachman, Becker, & Minick, 1973). After dissociation, the cells were collected and cultured on gelatin-coated culture dishes in medium 199 with LSGS, 100 IU/mL penicillin, and 0.1 mg/mL streptomycin. 2.5. Lipoprotein separation and oxidation LDL was isolated from normal human plasma of six people. The human plasma was obtained from Taichung Blood Bank. LDL was isolated using the sequential ultracentrifugation method (Havel, Eder, & Bragdon, 1955). LDL was diluted with PBS to the final concentration of 1 mg protein/mL and incubated at 37 °C in the presence of CuSO4 (10 lM) for 16 h followed by the removal of Cu2+ by gel filtration (PD-10 gel) with 10 mM PBS.
2.6. Conjugated diene determination Conjugated diene formation was monitored by second derivative spectroscopy (220–300 nm) based on the method described by Bourne and Rice-Evans (Wallin, Rosengren, Shertzer, & Camejo, 1993). The second derivative spectrum was subtracted from the second derivative spectrum of the matching control sample without Cu2+. The increase in conjugated dienes expressed in relative unit was obtained from the amplitude of the peak at 254 nm.
2.7. Thiobarbituric acid reactive substances (TBARS) MDA production was assessed as an indicator of lipid peroxidation according to the procedures of Wallin et al. (1993). To each tube containing 0.55 mL of the incubated LDL was added 0.5 mL of 25% (w/v) trichloroacetic acid for protein denaturation. Then, the samples were centrifuged (10,000 rpm) at 10 °C for 30 min to remove the pellets. TBA (1%, 0.5 mL) in 0.3% NaOH was added to the supernatant, and the mixed reagents were allowed to react at 95 °C for 40 min in the dark. After the reaction was completed, samples were analysed using a Hitachi F2000 spectrophotofluorimeter (excitation wavelength at 532 nm and emission wavelength at 600 nm). The concentration of MDA or TBA-reacting substance was expressed as equivalents of 1,1,3,3-tetraethoxypropane that served as a standard.
2.8. Relative electrophoretic mobility (REM) shift assay LDL (200 lg/mL) was pretreated with the indicated concentrations of KFEE for 2 h followed by incubation with 10 lM CuSO4 at 37 °C for 16 h. LDL modifications were assessed by agarose electrophoresis (0.6% agarose) to detect the increase in electrophoretic mobility of the modified LDL relative to native LDL accordingly (Miyakawa, Honma, Qi, & Kuramitsu, 2004). The gel was fixed in 75% ethanol and 5% acetic acid for 15 min, stained with 1% oil red O (in 60% isopropanol) for 30 min, and rinsed with 30% isopropanol to visualise LDL bands. The distance migrated by each LDL band was measured and expressed as an arbitrary REM value compared with the native LDL.
2.9. Electrophoresis of ApoB fragmentation After oxidation with or without KFEE, samples were denatured with 3% SDS, 10% glycerol, and 5% 2-mercaptoethanol at 95 °C for 5 min. SDS polyacrylamide gel electrophoresis (7.5% SDS–PAGE) was performed to detect ApoB fragmentation, with subsequent staining with Coomassie brilliant blue R250 (Lee et al., 2002).
2.10. DPPH radical scavenging assay DPPH assay was performed to measure the antiradical activity of KFEE. KFEE and Trolox storage solutions were added to DPPH solution to obtain the indicated concentrations. The absorbance was measured at 517 nm after 2 min of reaction at room temperature.
2.11. Cell growth assay After treatment with oxLDL (200 lg/mL) for 16 h in the presence or absence of KFEE, cells were harvested, and the viable cell number was counted in a hemocytometer by trypan blue exclusion.
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2.12. Cell cycle analysis HUVECs were pretreated with the indicated concentrations of KFEE for 2 h followed by oxLDL (200 lg/mL) for 16 h. Briefly, 1 105 cells were fixed in 70% ethanol and then suspended in propidium iodide (PI) solution (25 lg/mL PI, 0.1 mM EDTA, and 10 lg/ mL RNase A in PBS) for 30 min. The cell cycle distribution was then analysed by flow cytometry analysis using Cell-Quest software (Becton–Dickinson, CA). 2.13. Measurement of mitochondrial membrane potential After treatment with oxLDL (200 lg/mL) for 16 h in the presence or absence of KFEE, cells (3 105 cells/6-well plate) were rinsed with medium, followed by the addition of JC-1 (5 lM). After incubation at 37 °C for 20 min, cells were examined under a fluorescent microscope (Bedner, Li, Gorczyca, Melamed, & Darzynkiewicz, 1999). 2.14. Measurement of ROS production To investigate the effect of KFEE on oxLDL-induced ROS production in HUVECs (3 105 cells/6-cm cell culture dish), a fluorometric assay using DCFH-DA was conducted to detect the presence of hydroxyl radical. HUVECs were pretreated with or without the indicated concentrations of KFEE for 2 h followed by incubation with oxLDL for 16 h. After the removal of media from the wells, cells were incubated with 10 lM DCFH-DA for 1 h and then centrifuged and resuspended for immediate determination of ROS generation by flow cytometry using 488 nm for excitation and 525 nm for emission (Yen, Hsieh, Chou, & Lau, 2001). 2.15. Determination of cell viability (MTT assay) After the indicated treatments, the treated HUVECs (3 104 cells/24-well plate) were incubated with 0.5 mg/mL MTT in culture medium for an additional 4 h, and the blue formazan crystals of viable cells were dissolved in isopropanol and then measured spectrophotometrically at 563 nm (Chen et al., 2005). 2.16. DAPI staining Single-cell suspensions of treated HUVECs (3 105 cells/6-well plate) were washed with PBS and fixed by 70% ethanol. Afterward, cells were treated with DAPI stain (0.6 lg/mL in PBS). Chromatin fluorescence was observed under a UV-light microscope. Apoptotic cells were morphologically defined by cytoplasmic and nuclear shrinkage as well as chromatin condensation (Yen et al., 2001). 2.17. Western blot analysis Samples of cell lysates or cytosolic fractions were separated in 12.5% polyacrylamide gel and transferred onto a nitrocellulose membrane, as previously described (Chen et al., 2005). The protein was loaded to the gel for Western blot analysis (30 lg/well). The blot was subsequently probed with antibodies. Immobilon Western Chemiluminescent HRP substrate kit was obtained from Millipore (Burlington, MA), and the relative intensities of the signals were quantified by densitometry using a gel documentation and analysis system (LAS-3000 Image Reader, Fujifilm, Stamford, CT). 2.18. High-performance liquid chromatography (HPLC) analysis
Fig. 1. The chemical profile of KFEE was analysed by HPLC-mass spectrometry. (A) Chromatographic patterns from HPLC analysis of KFEE extracts showed peaks corresponding to the retention times (min). (B) HPLC chromatogram of nine kinds of standard compounds. Peaks: 1, gallic acid; 2, gallocatechin; 3, (+)catechin; 4, caffeic acid; 5, rutin; 6, naringin; 7, quercetin; 8, kaempferol; 9, anthraquinone. (C) Peaks: 10, myricetin; 11, morin; 12, apigenin; 13, galangin. (D) Combination of 100 lg KFEE with two kinds of standard compound (gallic acid and caffeic acid). Absorbance was monitored at 254 nm.
HPLC analysis (Waters 600 with a 2998 Photodiode Array detector) was performed using a LiChroCART RP-18 reversed phase col-
umn (200 4 mm, 5 lm) with mobile phase consisting of water/acetic acid (0.05%, v/v) (solvent A) and acetic acid/water/
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acetonitrile (0.05%, v/v) (solvent B). Elution was carried out in a programmed gradient elution as follows: 0 min to 30 min, with 0% to 100% B.
2.19. Statistical analysis Statistical significance was analysed by one-way analysis of variance (ANOVA) with post-hock Dunnett’s test. A P-value 6 0.05 was considered statistically significant (Sigma-Stat 2.0, Jandel Scientific, San Rafael, CA, USA).
3. Results 3.1. Polyphenol contents in KFEE Quantitative phytochemical analysis revealed the presence of 38.8 ± 0.4 mg/g of KFEE polyphenols. To evaluate the bioactive compound of KFEE, consecutive extractions of KFEE with 50% ethanol were performed. Chromatographic patterns from the HPLC analysis of KFEE (Fig. 1A) and 13 kinds of standard compounds, including gallic acid, gallocatechin, catechin, caffeic acid, rutin, naringin, quercetin, kaempferol, anthaquinone, myricetin, morin, apigenin, and galangin (Fig. 1B and C), showed peaks corresponding to the retention times. Absorbance was monitored at 254 nm. Fig. 1D showed that gallic acid and caffeic acid are included in the composition of KFEE.
3.2. KFEE inhibited copper-induced oxidation of LDL and ApoB fragmentation As shown in Fig. 2A and B, the REM of the control was assigned a mobility of 1.0, whereas chemical oxidation shifted the REM to 2.8. Pretreatment with KFEE (5, 10, 15, and 20 lg/mL) and Trolox (a water-soluble analogue of vitamin E; as a positive control) substantially reduced electrophoretic mobility in a dose-dependent manner. The inhibitory effects of KFEE on ApoB fragmentation were examined using SDS–PAGE in a similar manner (Fig. 2C). The ApoB band (arrow) in native LDL disappeared because of the fragmentation caused by CuSO4 incubation for 4 h. The presence of KFEE (5, 10, 15, and 20 lg/mL) and Trolox (10 and 50 lM) reversed copper-induced ApoB fragmentation substantially (Fig. 2C and D).
3.3. KFEE attenuated copper-induced lipid peroxidation of LDL The effects of KFEE on Cu2+-mediated LDL oxidation are shown in Fig. 2E. The lag time was 100 min for the control group with LDL and Cu2+. However, LDL oxidation was inhibited, and the lag time increased when KFEE was added. Results showed statistical differences (P < 0.001), and 20 lg/mL of KFEE increased the lag time in a similar manner with the control group at 600 min. MDA concentration, which is a lipid peroxidation indicator, was measured to assay LDL oxidation. Fig. 2B shows that LDL incubated with 10 lM CuSO4 increased MDA production (lane 2, Fig. 2F). LDL treatment with KFEE (25, 50, 75, and 100 lg/mL) or Trolox (as a positive control; 10 and 50 lM) reduced MDA formation in a dose-dependent manner. This result shows that KFEE exerted an inhibitory effect on the lipid peroxidation of LDL. Furthermore, to investigate the radical scavenging activity of KFEE, the reactivity toward the stable free radical DPPH was measured at 517 nm. As shown in Fig. 2G, 5 lg/mL of KFEE showed statistical differences (P < 0.001), and 20 lg/mL of KFEE demonstrated the strong activity of a free radical scavenger.
3.4. KFEE reduced oxLDL-induced cytotoxicity on HUVECs The protective effect of KFEE was examined by monitoring the oxLDL-induced morphological features of cultured HUVECs in the absence and presence of KFEE under phase-contrast microscopy. Treatment of cultured endothelial cells with oxLDL (200 lg/mL) for 16 h led to cell shrinkage or membrane blebbing, which were reduced substantially by adding KFEE (Fig. 3A). This observation was further supported by the KFEE-induced increment of cell viability; KFEE did not exhibit cytotoxicity to HUVECs (Fig. 3B), indicating that KFEE had proliferative activities (Fig. 3C) in oxLDL-treated HUVECs. To investigate whether KFEE prevented oxLDL-induced apoptosis in HUVECs, DAPI staining and flow cytometry analysis were performed. Cells incubated with oxLDL for 16 h exhibited typical features of apoptosis, such as formation of condensed chromatin (Fig. 3D) and accumulation of a sub-G1 population (Fig. 3E). However, this effect was decreased substantially by KFEE pretreatment. This result indicates that KFEE protected endothelial cells from oxLDL-induced cytotoxicity and apoptosis. oxLDL-induced HUVEC cell death occurred mainly through the apoptotic pathway by Annexin V and PI stain. Cell necrosis rate also decreased with KFEE treatment (data not shown).
3.5. KFEE perturbed oxLDL-induced ROS generation in HUVECs The effects of KFEE on oxLDL-mediated ROS generation were determined by pretreating endothelial cells with KFEE before oxLDL exposure. As shown in Fig. 4A–F, oxLDL produced an increase in ROS generation (Fig. 4B, dark trace), compared with the control (Fig. 4A, gray trace). Pretreatment with KFEE (25, 50, and 75 lg/mL) inhibited (left shift) oxLDL-induced ROS formation considerably (Fig. 4C–E), whereas 100 lg/mL of KFEE almost completely inhibited ROS production (Fig. 4F).
3.6. KFEE reverted oxLDL-induced alterations in mitochondrial transmembrane permeability The effects of oxLDL on mitochondrial permeability were examined to determine whether mitochondrial disruption is involved in the anti-apoptotic effect of KFEE. When HUVECs were exposed to oxLDL, DWm of the mitochondria was depolarised, as shown by the decrease in red fluorescence and the increase in green fluorescence. Pretreatment with KFEE reduced the change in DWm, as indicated by the repression of green fluorescence and the restoration of red fluorescence (Fig. 4G). The disruption of mitochondrial membrane function results in the release of mitochondrial enzyme cytochrome C into the cytosol, which can be detected by Western blot. The incubation of HUVECs with oxLDL for 16 h induced a threefold increase in the release of cytochrome C into the cytosolic fraction, compared with that of the control cells (Fig. 5A). KFEE considerably reduced the oxLDL-induced release of cytochrome C.
3.7. KFEE prevented oxLDL-induced changes in apoptotic protein expression in HUVECs KFEE reduced the oxLDL-induced expression of Bax considerably, and it increased the levels of antiapoptotic protein Bcl-2 at the highest concentration of 100 lg/mL (Fig. 5B). The expression levels of active caspase-3 and cleaved-PARP were increased substantially in HUVECs treated with oxLDL. In contrast, caspase-3 activation and PARP cleavage by oxLDL were suppressed in a dose-dependent manner in cells pretreated with KFEE.
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Fig. 2. Effects of KFEE on Cu2+-mediated shift of electrophoretic mobility, he ApoB fragmentation in LDL, and copper-mediated LDL oxidation, DPPH scavenged capability. (A) LDL was incubated with CuSO4 in the presence or absence of KFEE or Trolox, as positive control, and it was applied to agarose gels as described in Section 2. (B) The results from the agarose gel electrophoresis were quantified and expressed in the form of REM. The distance travelled in the agarose gel by the native LDL was assigned the arbitrary unit 1. (C) LDL (200 lg/mL) was incubated with a 10-lM CuSO4 at 37 °C in the absence or presence of KFEE or Trolox for 4 h and applied to SDS–PAGE as described in Section 2. (D) Quantification of the ApoB fragmentation assay using densitometry. Results were statistically evaluated by using one-way ANOVA with post hoc Dunnett test (⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001). The signal intensity of the native LDL was assigned as 100% arbitrarily. #P < 0.001 compared with control. ⁄P < 0.05; ⁄⁄P < 0.01; and ⁄⁄⁄ P < 0.001 compared with the oxLDL treated group. (E) LDL pre-incubated with increasing concentration of KFEE. Oxidation was induced with the addition of CuSO4 (10 lM), and conjugated dienes were formed. #P < 0.001 compared with control. ⁄⁄⁄P < 0.001 compared with the oxLDL-treated group. (F) LDL (200 lg/mL) was incubated with 10-lM CuSO4 at 37 °C for 16 h in the presence or absence of KFEE or Trolox, and then MDA formation was measured as described in Section 2. (G) The radical-scavenging activities of KFEE and Trolox were evaluated by using the DPPH radical scavenging assay. The absorbance of the sample without adding KFEE or Trolox was assigned as 100%, and its radical scavenging rate was assigned as 0% consequently. The quantitative data were presented as means.
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Fig. 3. Effect of KFEE on oxLDL-induced endothelial cell apoptosis. (A) Photomicrographs of the treated HUVECs were observed using phase-contrast microscopy. (B) The viability of treated HUVECs was detected using MTT assay as described in Section 2. (C) Viable cells and dead cells were counted using the trypan blue exclusion assay. The quantitative data were presented as means. (D) Nuclear morphology of the treated cells was observed by fluorescence microscopy using DAPI stain at a magnification of 200. Arrows showed areas of intense fluorescence staining with condensed nuclei. (E) The hypodiploid cell population (sub G1 phase) of the treated HUVECs was analysed by flow cytometry using PI stain, and 10,000 events of total cells were analysed for each experimental treatment. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. Effects of KFEE on oxLDL-induced ROS production and on mitochondrial membrane potential of HUVECs. ROS levels of (A) the HUVECs without treatment, control (without oxLDL; gray trace); (B) HUVECs with treatment of oxLDL (200 lg/mL), control (with oxLDL; dark trace); (C) HUVECs with treatment of oxLDL and KFEE (25 lg/mL), KFEE 25 lg/mL (dark trace); (D) HUVECs with treatment of oxLDL and KFEE (50 lg/mL), KFEE 50 lg/mL (dark trace); (E) HUVECs with treatment of oxLDL and KFEE (75 lg/ mL), KFEE 75 lg/mL (dark trace); and (F) HUVECs with treatment of oxLDL and KFEE (100 lg/mL), KFEE 100 lg/mL (dark trace) was measured by flow cytometry using DCFH staining. (G) JC-1 is selectively accumulated within intact mitochondria to form multimer J-aggregates emitting fluorescence light at 590 nm (red) at a higher membrane potential (left), and monomeric JC-1 emits light at 530 nm (green) at a low membrane potential (right). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
4. Discussion oxLDL is a crucial clinical risk factor for atherosclerosis. The reduction of oxLDL formation may prevent heart diseases. The data show that KFEE can reduce LDL ApoB fragmentation (Fig. 2C) and increase REM (Fig. 2A); conversely, it also inhibits the formation of conjugated dienes in LDL (Fig. 2E). These data suggest that KFEEs have a strong antioxidative ability to reduce LDL oxidation. The increase in serum LDL cholesterol levels is an essential factor in atherogenesis formation (Holvoet and Collen, 1997). Endothelial cell apoptosis is vital in the progress of several cardiovascular diseases
(Simionescu, 2007). A study showed that accumulated LDL in the arterial wall becomes oxLDL, which leads to endothelial function damage in the early stages of atherogenesis in hypercholesterolemic patients (Das, Snehlata Das, & Srivastava, 2006). Other studies also showed that LDL becomes oxLDL when LDL is incubated with endothelial cells, smooth muscle cells, or macrophages. A previous study showed that extract from K. formosana had inhibitory activities for xanthine oxidase, tyrosinase, and lipoxygenase (Chen et al., 2009; Lee et al., 2006). In the current study, KFEE was found to prevent oxidative damage to endothelial cells in vitro (Figs. 3 and 4), and this protective effect is concomitant with a
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Conversely, the extract from K. formosana had biological effects on heme oxygenase-1 induction (Lee et al., 2006), and three classes of protein-tyrosine kinase inhibitors were isolated from K. Formosana, that is, anthraquinone, stilbene, and flavonoid (Chang et al., 1996). Thus, KFEE protection against oxLDL-induced endothelial dysfunction may be associated with decrease in ROS overproduction. Compared with direct scavenging ROS activity attributed to its chemical structure, this effect may control the expression or activity of the ROS-generating enzyme and the ROS-scavenging enzyme. However, the exact mechanism by which KFEE protects against oxidative stress remains unclear and requires further examination. Other studies showed that Caesalpinia sappan extract could decrease inflammatory responses in HUVECs (Lee et al., 2010), and extracts of Ginkgo biloba and Solanum lyratum have the ability to protect HUVECs against oxLDL-induced injury (Kuo et al., 2009; Ou et al., 2009). The results of these studies and our results provide a possible molecular mechanism underlying plant crude extract suppression of oxLDL-mediated vascular endothelial dysfunction. 5. Conclusion Many reports have demonstrated that the branch and stem of K. formosana contain flavonoids, polyphenols and quinone, such as quercetin, kaempferol (Abou-Shoer et al., 1993), catechin (Lin, Deng, Lei, Fu, & Li, 2002), and anthraquinone (Chang et al., 1996). The composition of KFEE was assessed by HPLC. Results showed that KFEE contains gallic acid and caffeic acid, but it does not have kaempferol, catechin, and anthraquinone. These results may be attributed to the use of different solvent conditions for the purification of K. formosana. Moreover, the KFEE in the present study was extracted from branches after the leaves have fallen off, which differed from the extraction location and method of the other studies (Abou-Shoer et al., 1993; Chen et al., 2009; Lin et al., 2002). In the future, the actions of other components of KFEE should be further examined. Furthermore, understanding the signalling pathways of the intracellular are crucial, as is the manner by which the oxidative damage induced by oxLDL is modulated in endothelial cultures. This knowledge may enable us to devise therapeutic tools for preventing atherosclerotic progression. Our results indicate a mechanism that links ROS production and membrane damage, which leads to apoptosis caused by oxLDL in HUVECs. These findings may provide additional information on the anti-inflammatory properties of KFEE, which may contribute to its anti-atherosclerotic effect and anti-inflammatory action. However, further animal model testing is required to validate the relevance of these results. Fig. 5. Effects of KFEE on oxLDL-induced caspase-3 and PARP activation. Cell lysates were subjected to SDS–PAGE followed by Western blot with (A) anti- cytochrome C, (B) anti-Bcl-2, anti- BAX, anti-caspase-3, and anti-PARP antibodies with b-actin as internal control. Signals of proteins were visualised with an ECL detection system. The results were obtained from three independent experiments. #P < 0.001 compared with control. ⁄P < 0.05; ⁄⁄P < 0.01; and ⁄⁄⁄P < 0.001 compared with the oxLDLtreated group.
reduction in ROS level (Fig. 4A–F) and steady mitochondrial integrity (Fig. 4G). Mitochondrial cytochrome C (Fig. 5A), cleaved caspase-3, and PARP (Fig. 5B) levels were down-regulated, verifying that KFEE can reduce the extent of apoptosis. In summary, KFEE can decrease endothelial cell apoptosis induced by oxLDL, and it exhibits anti-atherosclerotic ability through cytochrome C-mediated pathways. We also showed that 25 lg/mL of KFEE protected HUVECs from oxLDL-induced cytotoxicity because of its pharmacokinetics and the efficiency level tested in vitro. Similar to Trolox, KFEE can prevent LDL oxidation (Fig. 2). KFEE can substantially inhibit oxLDL-induced ROS overproduction (Fig. 3B).
Acknowledgements This study was supported by grants from the National Science Council, Republic of China (NSC 98-2313-B-166-004-MY3 and NSC 98-2313-B-040-004-MY3). HPLC was performed in the Instrument Center of Chung Shan Medical University, which is supported by the National Science Council, Ministry of Education, and Chung Shan Medical University. References Abou-Shoer, M., Ma, G. E., Li, X. H., Koonchanok, N. M., Geahlen, R. L., & Chang, C. J. (1993). Flavonoids from Koelreuteria henryi and other sources as proteintyrosine kinase inhibitors. Journal of Natural Products, 56, 967–969. Bedner, E., Li, X., Gorczyca, W., Melamed, M. R., & Darzynkiewicz, Z. (1999). Analysis of apoptosis by laser scanning cytometry. Cytometry, 35, 181–195. Chang, C. J., Ashendel, C. L., Geahlen, R. L., McLaughlin, J. L., & Waters, D. J. (1996). Oncogene signal transduction inhibitors from medicinal plants. In Vivo, 10, 185–190.
C.-Y. Lin et al. / Food Chemistry 146 (2014) 299–307 Chen, C. H., Chan, H. C., Chu, Y. T., Ho, H. Y., Chen, P. Y., Lee, T. H., et al. (2009). Antioxidant activity of some plant extracts towards xanthine oxidase, lipoxygenase and tyrosinase. Molecules, 14, 2947–2958. Chen, P. N., Chu, S. C., Chiou, H. L., Chiang, C. L., Yang, S. F., & Hsieh, Y. S. (2005). Cyanidin 3-glucoside and peonidin 3-glucoside inhibit tumor cell growth and induce apoptosis in vitro and suppress tumor growth in vivo. Nutrition and Cancer, 53, 232–243. Das, S., Snehlata Das, N., & Srivastava, L. M. (2006). Role of ascorbic acid on in vitro oxidation of low-density lipoprotein derived from hypercholesterolemic patients. Clinica Chimica Acta, 372, 202–205. Havel, R. J., Eder, H. A., & Bragdon, J. H. (1955). The distribution andchemical composition of ultracentrifugally separated lipoproteins in human serum. The Journal of Clinical Investigation, 34, 1345–1353. Holvoet, P., & Collen, D. (1997). Beta-VLDL hypercholesterolemia relative to LDL hypercholesterolemia is associated with higher levels of oxidized lipoproteins and a more rapid progression of coronary atherosclerosis in rabbits. Arteriosclerosis, Thrombosis, and Vascular Biology, 17, 2376–2382. Jaffe, E. A., Nachman, R. L., Becker, C. G., & Minick, C. R. (1973). Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. The Journal of Clinical Investigation, 52, 2745–2756. Kuo, W. W., Huang, C. Y., Chung, J. G., Yang, S. F., Tsai, K. L., Chiu, T. H., et al. (2009). Crude extracts of Solanum lyratum protect endothelial cells against oxidized low-density lipoprotein-induced injury by direct antioxidant action. Journal of Vascular Surgery, 50, 849–860. Lee, T. H., Chiang, Y. H., Chen, C. H., Chen, P. Y., & Lee, C. K. (2009). A new flavonol galloylrhamnoside and a new lignan glucoside from the leaves of Koelreuteria henryi Dummer. Journal of Natural Medicines, 63, 209–214. Lee, M. J., Chou, F. P., Tseng, T. H., Hsieh, M. H., Lin, M. C., & Wang, C. J. (2002). Hibiscus protocatechuic acid or esculetin can inhibit oxidative LDL induced by either copper ion or nitric oxide donor. Journal of Agricultural and Food Chemistry, 50, 2130–2136. Lee, M. H., Jiang, C. B., Juan, S. H., Lin, R. D., & Hou, W. C. (2006). Antioxidant and heme oxygenase-1 (HO-1)-induced effects of selected Taiwanese plants. Fitoterapia, 77, 109–115. Lee, M. J., Lee, H. S., Jung, H. J., Lee, C. S., Kim, J. E., Moon, H. I., et al. (2010). Caesalpinia sappan L. ameliorates hypercholesterolemia in C57BL/6 mice and suppresses inflammatory responses in human umbilical vein endothelial cells
307
(HUVECs) by antioxidant mechanism. Immunopharmacol Immunotoxicol, 32, 671–679. Lin, W. H., Deng, Z. W., Lei, H. M., Fu, H. Z., & Li, J. (2002). Polyphenolic compounds from the leaves of Koelreuteria paniculata Laxm. Journal of Asian Natural Products Research, 4, 287–295. Min, K. J., Cho, K. H., & Kwon, T. K. (2012). The effect of oxidized low density lipoprotein (oxLDL)-induced heme oxygenase-1 on LPS-induced inflammation in RAW 264.7 macrophage cells. Cell Signal, 24, 1215–1221. Miyakawa, H., Honma, K., Qi, M., & Kuramitsu, H. K. (2004). Interaction of Porphyromonas gingivalis with low-density lipoproteins: implications for a role for periodontitis in atherosclerosis. Journal of Periodontal Research, 39, 1–9. Nazzal, D., Cantero, A. V., Therville, N., Segui, B., Negre-Salvayre, A., Thomsen, M., et al. (2006). Chlamydia pneumoniae alters mildly oxidized low-density lipoprotein-induced cell death in human endothelial cells, leading to necrosis rather than apoptosis. The Journal of Infectious Diseases, 193, 136–145. Ou, H. C., Lee, W. J., Lee, I. T., Chiu, T. H., Tsai, K. L., Lin, C. Y., et al. (2009). Ginkgo biloba extract attenuates oxLDL-induced oxidative functional damages in endothelial cells. Journal of Applied Physiology, 106, 1674–1685. Ross, R. (1993). The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature, 362, 801–809. Simionescu, M. (2007). Implications of early structural-functional changes in the endothelium for vascular disease. Arteriosclerosis, Thrombosis, and Vascular Biology, 27, 266–274. Vaidya, D., Szklo, M., Cushman, M., Holvoet, P., Polak, J., Bahrami, H., et al. (2011). Association of endothelial and oxidative stress with metabolic syndrome and subclinical atherosclerosis: multi-ethnic study of atherosclerosis. European Journal of Clinical Nutrition, 65, 818–825. Wallin, B., Rosengren, B., Shertzer, H. G., & Camejo, G. (1993). Lipoprotein oxidation and measurement of thiobarbituric acid reacting substances formation in a single microtiter plate: its use for evaluation of antioxidants. Analytical Biochemistry, 208, 10–15. Witztum, J. L., & Steinberg, D. (1991). Role of oxidized low density lipoprotein in atherogenesis. The Journal of Clinical Investigation, 88, 1785–1792. Yen, C. H., Hsieh, C. C., Chou, S. Y., & Lau, Y. T. (2001). 17Beta-estradiol inhibits oxidized low density lipoprotein-induced generation of reactive oxygen species in endothelial cells. Life Sciences, 70, 403–413.
