Antioxidant effect of astragalin isolated from the leaves of Morus alba L. against free radical-induced oxidative hemolysis of human red blood cells
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- Choi, J., Kang, H.J., Kim, S.Z. et al. Arch. Pharm. Res. (2013) 36: 912. doi:10.1007/s12272-013-0090-x
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We evaluated the antioxidant properties of mulberry leaves extract (MLE) and flavonoids isolated from MLE. MLE was prepared by extraction with methanol. Flavonoids were analyzed by high-performance liquid chromatography. Oxidative hemolysis of normal human red blood cells (RBCs) was induced by the aqueous peroxyl radical [2,2′-Azobis (2-amidinopropane) dihydrochloride, AAPH]. MLE contained three flavonoids in the order quercetin (QC) > kaempferol (KF) > astragalin (AG). Oxidative hemolysis of RBCs induced by AAPH was suppressed by MLE, AG, KF, and QC in a time- and dose-dependent manner. MLE and these three flavonoids prevented the depletion of cystosolic antioxidant glutathione (GSH) in RBCs. AG had the greatest protective effect against AAPH-induced oxidative hemolysis and GSH depletion in RBCs.
KeywordsAntioxidant activityAstragalinFlavonoidMulberry leavesOxidative damageRed blood cells
Oxidative stress is disruption of the balance between the generation of reactive oxygen species (ROS) and the activity of antioxidant defense systems (Ray et al. 2012). ROS consist of free radicals such as the superoxide anion (·O2−), hydroxyl radical (HO·) and hydrogen peroxide (H2O2). ROS react readily with many biological molecules, including proteins, amines, lipids and deoxyribonucleic acid (DNA). Excessive production of ROS can lead to arthritis, diabetes mellitus (DM), inflammation and vascular disease (Csiszar et al. 2009; Loeser 2011; Sharma et al. 2012; Tang et al. 2012). Red blood cells (RBCs) are more frequently exposed to oxygen than other tissue and are more susceptible to oxidative damage; invasion of RBC membranes by peroxidants may lead to cell hemolysis (Dai et al. 2006). Oxidative damage to the erythrocyte membrane may be implicated in hemolysis associated with oxidative drugs, radiation, and deficiencies in some erythrocyte antioxidant systems (Paiva-Martins et al. 2009; Rizzo et al. 2012). Antioxidants regulate various oxidative reactions occurring naturally in cells and tissues, and can terminate or retard oxidation by scavenging free radicals, chelating free catalytic metals, and by acting as electron donors (Ray et al. 2012). Antioxidants have been used widely as food additives to provide protection from the oxidative degradation of foods and medicinal plants (Kaefer and Milner 2008). Hence, antioxidants are used to protect food quality mainly by the prevention of oxidative damage. The antioxidant activity of flavonoids affects the activities of the oxidative free radical-scavenging enzymes superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-px), glutathione reductase (GR), and reduced GSH, and reduces the oxidative damage to cells and biomolecules caused by ROS (Luangaram et al. 2007; Stevenson and Hurst 2007).
Mulberry (Morus alba L.) leaves are cultivated in China, Korea and Japan. Their leaves contain many nutritional components. Mulberry leaves have been used in traditional Chinese medicine (TCM) to treat fever, prevent DM, strengthen bone joints, facilitate discharge of urine, and lower blood pressure (Asano et al. 2001; Enkhmaa et al. 2005). Mulberry leaves has been reported to be rich in flavonoids that have different biological activities, including antioxidant activity (Zhishen et al. 1999; Enkhmaa et al. 2005; Katsube et al. 2010; Khan et al. 2013). The chemical composition of leaves includes kaempferol-3-O-β-D-glucopranoside (AG), 3,5,7-Trihydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one (KF), 3,3′,4′,5,7-pentahydroxyflavone (QC) and other flavonoids (Chan et al. 2010). However, reports on the antioxidant activity of mulberry leaves extract (MLE) and the effects of flavonoids from MLE on aqueous peroxyl radicals [2,2′-Azobis (2-amidinopropane) dihydrochloride, AAPH]-induced hemolysis of human RBCs are lacking.
Therefore, the purpose of this study was to determine of the content of flavonoids in MLE and to investigate the antioxidant capacity of its components AG, KF and QC in vitro.
Materials and methods
Heparin, 2,2′-Azobis (2-amidinopropane) dihydrochloride (AAPH), hemoglobin, Drabkin’s reagent, Brij® L23 solution, meta-phosphoric acid, 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) and other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Mulberry (M. alba L.) leaves were collected on 20 May 2011 from Manduk Mountain, Wanju-gun, Jeollabuk-do, Republic of Korea. The plant was identified and authenticated by Professor Hong-Jun Kim at the College of Oriental Medicine, Woosuk University (Jeollabuk-do, Korea). A voucher specimen (ML-11-02) has been deposited in the author’s (Professor Seon-Il Jang) laboratory.
Identification and quantification of phenolic compounds
The air-dried leaves of the mulberry plant (1.23 kg) were ground mechanically, macerated with n-hexane for 7 days, and extracted with MeOH (10 days). The MeOH extract (190 g) was suspended in H2O and partitioned sequentially with n-hexane, CH2Cl2, EtOAc and n-BuOH with saturated H2O. The EtOAc fraction (42.2 g) was fractionated on a Sephadex LH-20 column and eluted with H2O-MeOH (1:0–0:1) to give six fractions (M1–M6). Fraction M4 (2.3 g) was subjected to silica-gel column chromatography and eluted with toluene-EtOAc-formic acid (5:4:1) to yield eight fractions (M41–M48). Fraction M42 (187 mg) was subjected to preparative high-performance liquid chromatography (prep-HPLC; GS 310 column, 3 mL/min, and detection at 254 nm) and eluted with MeOH to give pure compound 1 (KF) (24.7 mg), the spectral data of which were in agreement with literature data (Xiao et al. 2006). Subfraction M423 (68 mg) underwent prep-HPLC in the same manner as previous fractions to afford compound 2 (11 mg) whose mass spectrometry (MS) and nuclear magnetic resonance (NMR) data were identical to those of 3,3′,4′,5,7-pentahydroxyflavone (QC). Fraction M44 (223 mg) was subjected to prep-HPLC (GS 310 column, 3 mL/min, and detection at 254 nm) and eluted with MeOH to yield 3 (38.4 mg). Spectral analyses confirmed compound 3 to be identical to kaempferol-3-O-β-D-glucopyranoside (AG) (Wang et al. 2007).
Preparation of erythrocyte suspensions
Heparinized blood samples were obtained from healthy volunteers via venipuncture. RBCs were isolated by centrifugation at 1,500×g for 10 min at 4 °C, washed thrice with phosphate-buffered saline (PBS; pH 7.4) and resuspended using the same buffer to a hematocrit level of 5 %. The RBC suspension was preincubated with MLE (0–100 μg/mL) and flavonoids (AG, KF and QC; 0–10 μg/mL) for 15 min at 37 °C. Samples were incubated with AAPH (dissolved in PBS; final concentration, 50 mM) for ≤6 h at 37 °C to induce the oxidation of free radical chains in RBCs.
At the indicated time, an aliquot of the reaction mixture (1 mL) was removed and centrifuged at 3,000×g for 2 min at 4 °C. The absorbance of the supernatant solution at 540 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader (Molecular Devices, Sunnyvale, CA, USA). Reference values (100 % hemolysis) were obtained using the same amount of RBCs in distilled water. Percentage hemolysis was calculated using the ratio of the readings (absorbance of sample supernatant/reference value) × 100.
Determination of GSH content in human RBCs
After centrifugation of the reaction mixture (2 mL), 0.6 mL of distilled water was added to the RBC pellet to lyse the cells. Then, 0.5 mL of the lysate was precipitated by the addition of 0.5 mL meta-phosphoric acid solution. After 5 min, the protein precipitate was separated from the remaining solution by centrifugation at 18,000×g for 10 min at 4 °C. We then combined 0.45 mL of the solution with 0.45 mL of 300 mM Na2HPO4. Then, GSH content in the lysate was determined at 412 nm by titration with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) (Hseu et al. 2007).
Data were analyzed using SPSS ver10 (SPSS, Chicago, IL, USA) and the Student’s t test. The hypothesis-testing method was one-way analysis of variance (ANOVA). Values are the mean ± SD for five sets of experiments. p < 0.05 was considered significant.
Ethical approval of the study protocol
The study protocol was approved by the Ethics Committee of Jeonju University (Jeonju, Korea). Written informed consent was obtained from all participants.
Results and discussion
HPLC fingerprint analyses of flavonoids from MLE
Inhibitory effects of MLE, AG, KF and QC on AAPH-induced hemolysis in human RBCs
Moreover, the suppressive effect of AG in hemolysis was better than for other flavonoids. KF and QC have antioxidant and anti-inflammatory activities, and can prevent the oxidative damage induced by several oxidizing agents in RBCs (Kim and Jang 2011; Asgary et al. 2005). AG has various health benefits and biological activities: antioxidant, anti-inflammatory and anti-allergic (Enkhmaa et al. 2005). It has strong effects on the inhibition of histamine release in human blood cells (Kotani et al. 2000), protects against dysfunction in the vascular endothelium (Peng et al. 2011) and is typically contained in small amounts in plants (Matsumoto et al. 2002). However, AG content in MLE is relatively high compared with other plants (Katsube et al. 2006). Studies focusing on the ability of AG from MLE to prevent free radical-induced oxidative damage in RBCs are lacking. We found that not only KF and QC but also AG from MLE has good antioxidant activity against free radical-induced hemolysis by AAPH in RBCs.
Effects of MLE, AG, KF and QC on GSH levels in AAPH-damaged human RBCs
In conclusion, this is the first report demonstrating that mulberry leaves efficiently protect human RBCs against free radical-induced oxidative damage, and that QC and KF were the predominant antioxidants in mulberry leaves. Moreover, AG had the greatest protective effect against AAPH-induced oxidative hemolysis and GSH depletion in RBCs. Taken together, the results of the present study suggest that MLE as well as flavonoids from MLE have strong antioxidant activity against free radical-induced oxidative damage due to the prevention of GSH depletion, and may be useful in diminishing the damage to RBCs. Further studies are needed to explore the potential of AS from MLE as a chemopreventive and therapeutic agent.
This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No. 00966303)” Rural Development Administration, Republic of Korea.