Archives of Pharmacal Research

, Volume 36, Issue 7, pp 912–917

Antioxidant effect of astragalin isolated from the leaves of Morus alba L. against free radical-induced oxidative hemolysis of human red blood cells

Authors

  • Jiwon Choi
    • Department of Radiological SciencesJeonju University
    • Department of Physiology, School of MedicineChonbuk National University
  • Hyun Ju Kang
    • Department of Healthcare & ScienceJeonju University
  • Sung Zoo Kim
    • Department of Physiology, School of MedicineChonbuk National University
  • Tae Oh Kwon
    • College of Life Science and Natural ResourcesWonkwang University
  • Seung-Il Jeong
    • Jeonju Biomaterials Institute
    • Department of Healthcare & ScienceJeonju University
Research Article

DOI: 10.1007/s12272-013-0090-x

Cite this article as:
Choi, J., Kang, H.J., Kim, S.Z. et al. Arch. Pharm. Res. (2013) 36: 912. doi:10.1007/s12272-013-0090-x

Abstract

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.

Keywords

Antioxidant activityAstragalinFlavonoidMulberry leavesOxidative damageRed blood cells

Introduction

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

Chemicals

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).

Plant material

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.

Hemolysis assay

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).

Statistical analyses

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

To confirm the quality of mulberry leaves, HPLC analyses were undertaken with a reverse-phase Agilent 1200 series. Separation was carried out using a Phenomenex Luna C18 (4.6 × 250 mm; particle diameter, 5 μm) column at 30 °C. The mobile phase consisted of 0.1 % formic acid in water in pump A, and 100 % acetonitrile solution in pump B at a flow rate of 0.7 mL/min. The gradient elution program was: 20 % B (0–5 min), 20–30 % B (5–7 min), 30 % B (7–12 min), 30–45 % B (12–17 min), 45 % B (17–25 min), 45–60 % B (25–30 min), maintained at 60 % B to 35 min, and returned to 20 % B in 5 min. The injection volume was 15 μL. The constituents of MLE, i.e., AG (0.113 ± 0.072 mg/g), KF (0.503 ± 0.026 mg/g) and QC (0.603 ± 0.032 mg/g), were identified by known standards at 330 nm in the HPLC analysis system (Fig. 1a). The total flavonoid content of MLE was in the order: QC > KF > AG (Fig. 1b). The flavonoids in MLE comprise glycosides and aglycones such as AG, KF, QC, and isoquercitrin has been identified as the main flavonoid in mulberry leaves (Kim and Jang 2011). Mulberry leaves contain various phenolic antioxidants, and several authors have supported supplementation with phenolic antioxidants for reducing the level of oxidative stress and inflammation as well as preventing DM (Enkhmaa et al. 2005; Katsube et al. 2010; Kim and Jang 2011).
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Fig. 1

HPLC chromatograms of active components from mulberry leaves. a Standard chemicals. b Extract of mulberry leaves (MLE). Peaks: (1) astragalin (AG), (2) quercetin (QC), and (3) kaempferol (KF)

Inhibitory effects of MLE, AG, KF and QC on AAPH-induced hemolysis in human RBCs

The hemolytic effects of MLE (12.5–100 μg/mL) on human RBCs exposed to a water-soluble initiator of free radicals, AAPH, were investigated. RBCs incubated at 37 °C as a 5 % suspension were stable, with little hemolysis observed within 6 h (6.2 ± 0.4 %). When AAPH (50 mM) was added to RBC suspensions, it induced hemolysis in a time-dependent manner (>80 % at 4 h). However, the onset of AAPH-induced hemolysis was delayed appreciably in the presence of MLE, AG, KF and QC. When MLE (100 μg/mL) was incubated with AAPH, ≈26 % hemolysis was observed. Treatment with KF, QC and AG (all 10 μg/mL) from MLE markedly suppressed AAPH-induced hemolysis in the order AG > QC > KF (Fig. 2). Jani et al. (2012) showed that GSH depletion by AAPH-induced hemolysis may limit the ability to detect an antioxidant effect of such compounds, and GSH is known to be involved in recycling antioxidants back to their active form. We showed that AAPH with 1 mM GSH did not affect the initial increase in hemolysis but did delay the time for complete hemolysis. Therefore, the antioxidant activity of MLE, AG, QC, and KF could serve as AAPH-induced hemolysis involving the complete and rapid depletion of intracellular reduced GSH. Treatment of MLE, AG, QC and KF significantly suppressed AAPH-induced hemolysis in a dose-dependent manner (Fig. 3). The inhibitory effect of MLE was not significantly different at <25 μg/mL but a big difference was noted at >50 μg/mL.
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Fig. 2

Time-dependent protection of MLE, AG, KF, QC and GSH against AAPH-induced hemolysis. Human RBC suspensions (10 %) were incubated for 0–6 h with 25 mM AAPH alone or in the presence of MLE (100 μg/mL), flavonoids (10 μg/mL AG, KF and QC) and GSH (1 mM) under air pressure at 37 °C

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Fig. 3

Dose-dependent protection of MLE, AG, KF and QC against AAPH-induced hemolysis. Human RBC suspensions (10 %) were incubated for 6 h with 25 mM AAPH alone or in the presence of MLE and flavonoids (AG, KF and QC) under air pressure at 37 °C

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

GSH plays an important part as a RBC antioxidant on different levels. Thus, one method of protection against oxidative hemolysis is preventing GSH depletion (Zou et al. 2001) and reduced GSH levels lead to a decrease in thiol groups (Maritim and Sanders 2003). AAPH-induced reduction in GSH levels was inhibited in the presence of MLE, AG, QC, and KF in a dose-dependent manner and AAPH treatment dramatically decreased the level of GSH (≈50 %) compared with the normal control at 6 h (Fig. 4). The percentage recovery of 25–100 μg/mL MLE was 50–60 %. The effects of AG and QC were better than those seen with KF. GSH directly protects membrane proteins and preserves their stability (Asano et al. 2001). Decreased levels of GSH result in oxidization of membrane GSH groups and loss of membrane stability (Stevenson and Hurst 2007). Anti-free radical properties are linked to the formation of stable radicals that can react with GSH, and GSH oxidation by stable free radicals may also reflect regeneration of oxidized scavengers in their active reduced form (Jones 2008). Therefore, we provided evidence that MLE and flavonoids from MLE have excellent antioxidant activity against AAPH-induced GSH depletion in RBCs. However, their mechanism of action in cell signaling-related molecules warrants future investigation.
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Fig. 4

Dose-dependent protection of MLE, AG, KF and QC against AAPH-induced cytosolic GSH depletion. Human RBC suspensions (10 %) were incubated for 6 h with 25 mM AAPH alone or in the presence of MLE and flavonoids (AG, KF and QC) under air pressure at 37 °C

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.

Acknowledgments

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.

Supplementary material

12272_2013_90_MOESM1_ESM.pdf (40 kb)
Supplementary material 1 (PDF 40 kb)

Copyright information

© The Pharmaceutical Society of Korea 2013