In vitro antioxidant effects and cytotoxicity of polysaccharides extracted from Laminaria japonica

In vitro antioxidant effects and cytotoxicity of polysaccharides extracted from Laminaria japonica

International Journal of Biological Macromolecules 50 (2012) 1254–1259 Contents lists available at SciVerse ScienceDirect International Journal of B...

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International Journal of Biological Macromolecules 50 (2012) 1254–1259

Contents lists available at SciVerse ScienceDirect

International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

In vitro antioxidant effects and cytotoxicity of polysaccharides extracted from Laminaria japonica Zhenfei Peng a,b , Min Liu a,b , Zhexiang Fang a , Qiqing Zhang a,c,∗ a b c

Institute of Biomedical and Pharmaceutical Technology, Fuzhou University, Fuzhou, China College of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou, China Institute of Biomedical Engineering, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, China

a r t i c l e

i n f o

Article history: Received 14 February 2012 Received in revised form 2 April 2012 Accepted 5 April 2012 Available online 12 April 2012 Keywords: Laminaria japonica polysaccharides Antioxidant activity Cytotoxicity

a b s t r a c t A water-soluble crude polysaccharide (WPS) was obtained from Laminaria japonica by hot water extraction. Three major polysaccharide fractions (WPS-1, WPS-2 and WPS-3) were purified from WPS by anion-exchange chromatography. Monosaccharide components analysis indicated that galactose was the predominant monosaccharide in WPS and WPS-3, accounting for 56.25% and 54.11%, respectively. And fucose was the predominant monosaccharide in WPS-1 and WPS-2, accounting for 46.91% and 45.1%, respectively. Antioxidant activity tests revealed that WPS-2 showed significant function of scavenging hydroxyl free radical and WPS-1 exhibited the highest inhibitory effects on superoxide radical. Cytotoxicity of all polysaccharide fractions was evaluated by MTT assay and Hoechst 33258 staining. Results showed that WPS-1 and WPS-2 significantly inhibited the growth of A375 cells and low anti-proliferative effects of WPS-2 on vascular smooth muscle cells (VSMCs) were observed. These results suggested that the polysaccharide fraction of WPS-2 might be explored as a potential safe antioxidant and antitumor agent. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Oxidation is an essential biological process to many organisms for the production of energy. However, the uncontrolled production of oxygen derived free radicals triggers many health problems such as Alzheimer’s disease [1], diabetes mellitus [2], atherosclerosis [3] and cancer [4]. Accumulated evidences have indicated that reactive oxygen species (ROS) could also promote tumor heterogeneity, invasion and metastasis, through inhibiting antiproteases and injuring local tissues [5,6]. Therefore, it is essential to develop and utilize effective antioxidants to scavenge free radicals in the human bodies. In recent decades, large amounts of investigations revealed that algal polysaccharides acted as free radical scavengers in vitro, antioxidants for the prevention of oxidative damage in living organisms as well as growth inhibitors on carcinoma cells [7–10]. Laminaria japonica is a popular edible seaweed in oriental countries and applies in food preparation as a health protection. Polysaccharides isolated from Laminaria japonica have attracted a great deal of attention because of their excellent bioactivities with fewer

∗ Corresponding author at: Institute of Biomedical and Pharmaceutical Technology, Fuzhou University, Fuzhou, China. Tel.: +86 591 83725260. E-mail address: [email protected] (Q. Zhang). 0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2012.04.006

side-effects. Recent studies have shown that Laminaria japonica polysaccharides could protect endogenous antioxidant enzymes and prevent CCl4 -induced liver damage [11], as well as scavenge free radicals [12]. Some sulfated polysaccharides isolated from different Laminariales species have been demonstrated to possess antitumor activities [13,14]. However, so far there is little information published about antitumor activity of Laminaria japonica polysaccharides. Therefore, in this paper, the purification, characteristics and bioactivities of purified polysaccharides of Laminaria japonica were investigated.

2. Materials and methods 2.1. Materials and reagents Dried Laminaria japonica was obtained from Fuzhou, Fujian province of China. The human melanoma cell line A375 and vascular smooth muscle cells (VSMCs) were obtained from College of Biological Science and Technology of Fuzhou University. Dulbecco’s Modified Eagle’s Medium (DMEM, low glucose) was purchased from GIBCO-BRL (Grand Island, New York, USA). Fetal bovine serum (FBS) was purchased from Sangong (Shanghai, China). Dimethylsulphoxide (DMSO), glucose and 3-methyl-1-phenyl-2-pyrazole-5-one (PMP) were purchased from Sigma Co. (St. Louis, USA). Thiazolyl blue

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(MTT) was purchased from Fluka Chemie (Buchs, Switzerland). All chemicals used in this study have a purity of 95% or greater. 2.2. Preparation of polysaccharides The powdered Laminaria japonica (100.0 g) were defatted with anhydrous ethanol at 60 ◦ C for 3 h under stirring, then dipped in 5 L distilled water at 100 ◦ C for 3 h. The aqueous extractions were concentrated and precipitated with 20% ethanol containing 0.2% CaCl2 . After separating the precipitate by filtrating, the ethanol was added to the supernatant to yield a 75% ethanolic solution, and the suspension was kept at 4 ◦ C overnight. The precipitate was collected and lyophilized to yield the crude polysaccharide (WPS). Further fractionation was performed in anion-exchange chromatography. WPS (0.3 g) was dissolved in Tris–HCl (20 mmol/L, pH 7.4) buffer and loaded onto a DEAE-A25 chromatography column (26 mm × 300 mm) equilibrated with Tris–HCl buffer. The column was eluted with successive elution of 0–2.0 mol/L NaCl aqueous solution at a flow rate of 3 mL/5 min. The fractions were detected by the phenol–sulfuric acid method [15], using glucose as standard. Three fractions of polysaccharide were obtained and lyophilized for further study. 2.3. Analysis of monosaccharide composition Polysaccharides of Laminaria japonica were hydrolyzed with 150 ␮L of 2 mol/L trifluoroacetic acid (TFA) for 4 h at 110 ◦ C in a sealed eppendorf tube. The residual acid was removed through decompression and distillation with methanol for three times. The standard monosaccharides and polysaccharide hydrolysates were pre-column derivated with PMP at 70 ◦ C for 100 min. The resulting solutions were extracted with 1.0 mL chloroform and the process was repeated three times. Then the aqueous layer was subjected to high performance liquid chromatography (HPLC) fitted with RP-C18 column. 2.4. Infrared analysis The IR spectra of polysaccharide fractions were determined using a Fourier transform infrared spectroscopy (FT-IR) spectrophotometer. The polysaccharide was ground with KBr powder and then pressed into pellets for FT-IR measurement in the frequency range of 4000–500 cm−1 [16].

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2.5.2. Hydroxyl radical-scavenging activity The hydroxyl radicals scavenging activity of polysaccharide fractions were measured according to the method of Smironoff and Cumbes [18], with slight modifications. The sample solutions were incubated with EDTA-Fe2+ (0.945 mmol/L), H2 O2 (1%) and crocus (40 ␮g/mL) in Tris–HCl buffer (0.15 mol/L, pH 7.4) for 30 min at 37 ◦ C. The presence of the hydroxyl radical was detected by monitoring absorbance at 520 nm. In the control, sample was substituted with distilled water. The capability of scavenging hydroxyl radical was calculated using following equation:



Scavenging effect (%) =

1−

Asample Acontrol



× 100

2.6. Cytotoxicity assay 2.6.1. MTT assay The growth inhibition assays of polysaccharides on A375 cells and VSMCs were evaluated in vitro by MTT method. Briefly, cells (1 × 105 cells/mL) were incubated in 96-well plates. Each well was incubated with 100 ␮L culture medium containing 10% FBS at 37 ◦ C in a humidified atmosphere with 5% CO2 . After 24 h, cells were treated with different concentrations of polysaccharides (0.03125, 0.125, 0.5 and 2.0 mg/mL). The wells were further incubated for 20 h at 37 ◦ C. Then 20 ␮L of MTT stock solution (5 mg/mL) were added and incubated for 4 h. After removing the medium, 100 ␮L DMSO were added to terminate the reaction. The amount of purple formazan was determined by measuring the absorbance at 578 nm. For treated cells, cytotoxicity was expressed as follow:



Growth inhibition rate (%) =

1−

Atreated Anegative control



× 100

where Atreated is the absorbance of cells with sample treated, Anegative control is the absorbance of cells without sample treated. 2.6.2. Nuclear staining with Hoechst 33258 The nuclear morphology of the cells was evaluated using the cell-permeable DNA dye, Hoechst 33258. A375 cells from exponential phase cultures (1 × 106 mL–1 ) were treated with polysaccharides at 1 mg/mL and incubated simultaneously in DMEM (low glucose) medium containing 10% FBS under an atmosphere of 5% CO2 at 37 ◦ C. After 24 h, cells were collected and stained with DNA dye Hoechst 33258 (5 ␮g/mL) for 15 min. The stained cells were observed under an inverted phase fluorescence microscope.

2.5. Antioxidant activity assay 2.7. Statistical analysis 2.5.1. Superoxide anion-scavenging activity The assay was based on the method of Elstner and Heupel [17], with slight modifications. The reaction mixture contained 1.0 mL of PBS buffer (75 mmol/L, pH 7.8), 200 ␮L samples with different concentrations, 100 ␮L hydroxylamine hydrochloride (0.1 mol/L), 100 ␮L xanthine (0.75 mmol/L) and 100 ␮L xanthine oxidase (0.1 U/mL). The mixture solution was incubated at 37 ◦ C for 30 min. The chromogenic reaction was triggered by color agent (3 mmol/L ␣-naphthylamine and 23 mmol/L sulfanilic acid) and processed at room temperature for 10 min. The optical density of the mixture was determined at 530 nm against blanks that had been prepared similarly but without sample. The scavenging rate of superoxide radical production was calculated using following equation:

 Scavenging effect (%) =

1−

Asample Ablank

 × 100

The data were presented as means ± SD. Statistical analyses were performed using Student’s t-test and one-way analysis of variance. Multiple comparisons of means were done by the least significance difference test. All computations were done by employing the statistical software (SAS, version 8.0). 3. Results and discussion 3.1. Isolation and purification of polysaccharide fractions The water-soluble crude polysaccharide, named WPS, was isolated from Laminaria japonica by hot water extraction, ethanol precipitation and lyophilization. The final yield of WPS was about 2.5% and WPS was fractionated on a DEAE-A25 column. After elution in a successive manner, three main fractions of WPS were obtained from NaCl eluent, named WPS-1, WPS-2 and WPS-3 (Fig. 1).

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3.4. Antioxidant activities of polysaccharides

Fig. 1. Elution profiles of Laminaria japonica polysaccharide by DEAE-A25 anionexchange chromatograph.

Table 1 Monosaccharide compositions of the polysaccharide fractions from Laminaria japonica. Contents of the sugar residues (%)

WPS WPS-1 WPS-2 WPS-3

Mana

Rhab

Gluc

Gald

Xyle

Fucf

Unknown

2.56 11.01 10.2 1.08

2.97 3.00 20.0 nd

5.10 2.93 nd 9.45

56.25 13.49 20.9 54.11

6.04 20.55 nd 2.84

25.74 46.91 45.1 32.51

1.32 2.12 3.8 nd

nd, not detectable below the limit at 0.01. a Mannose. b Rhamnose. c Glucose. d Galactose. e Xylose. f Fucose.

3.2. Monosaccharide compositions Monosaccharide compositions of WPS and its fractions were summarized in Table 1. WPS and WPS-3 were mainly composed of galactose and fucose with molar ratios of 2.2:1 and 1.7:1, respectively. WPS-2 consisted of rhamnose, galactose and fucose with a molar ratio of 1:1.05:2.3. WPS-1 was mainly composed of xylose and fucose with a molar ratio of 1:2.3. In particular, galactose was the predominant monosaccharide in WPS and WPS-3, accounting for 56.25% and 54.11%, respectively. However, fucose was the predominant monosaccharide in WPS-1 and WPS-2, accounting for 46.91% and 45.1%, respectively. 3.3. Infrared spectra of polysaccharide fractions The FT-IR spectra of the three polysaccharide fractions were presented in Fig. 2a–c. All samples exhibited a broad stretching intense characteristic peak at around 3436 cm−1 and 3345 cm−1 for the hydroxyl group, and a weak C H band at around 2944 cm−1 . The absorption bands at 1631–1647 cm−1 were caused by C O asymmetric stretching vibration. Each particular polysaccharide has a specific band in the 1200–1000 cm−1 region, this region was dominated by ring vibrations overlapped with stretching vibrations of (C O H) side groups and the (C O C) glycosidic band vibration. A characteristic peak at around 851 cm−1 was found in WPS-2, indicating the ␣-configuration of sugar units. The relatively strong absorption peak at 964 cm−1 of WPS-3 reflected the absorption of the furan ring.

3.4.1. Scavenging effects on superoxide radical The superoxide radical scavenging activities of the polysaccharides and Vitamin C were evaluated and shown in Fig. 3a. At a concentration of 0.1–1.0 mg/mL, the polysaccharides exhibited varying degrees of antioxidant activities. WPS-2 had a low-level of radical scavenging effect. The highest scavenging ability of 30% was obtained at the concentration of 1 mg/mL. Results indicated that the superoxide radicals could be scavenged by WPS-1 at a concentration as low as 0.1 mg/mL. At the concentration of 0.5 mg/mL, the inhibition ratio of WPS-1 reached 70%, significantly higher than WPS and WPS-3 (39% and 27%, respectively), but lower than Vitamin C (92.1%). Although, superoxide radical was a weak oxidant in most organisms, it could produce precursors of hydroxyl radical in vivo [19]. Moreover, superoxide radical and its derivatives could cause damage to DNA and membrane of cell [20]. Therefore, superoxide radical scavenging was extremely important to antioxidant work. The above results clearly indicated that WPS-1 had a relatively higher effect on superoxide radical scavenging. 3.4.2. Scavenging effects on hydroxyl radical As shown in Fig. 3b, the scavenging activities of polysaccharides and Vitamin C on hydroxyl radical improved with the increase of concentration. Polysaccharides showed higher scavenging effects on hydroxyl radical than Vitamin C at the concentration of 1.0 mg/mL. The inhibition ratios of polysaccharides (WPS, WPS-1, WPS-2 and WPS-3) were 43.1%, 42.9, 83.1% and 44.6%, respectively. However, at the same concentration, the scavenging ratio of Vitamin C was 35.3%. Notably, WPS-2 presented significantly high scavenging ratio of more than 99% at the concentration of 3 mg/mL, indicating hydroxyl radical produced by Fenton reaction was completely scavenged. Hydroxyl radical is mainly responsible for the oxidative injury of biomolecules [21]. It could easily cross cell membrane and readily react with most biomolecules, causing tissue damage or cell death [22]. Thus, removing hydroxyl radical is important for the protection of living systems. Data on the hydroxyl radical scavenging activities of polysaccharides suggested that polysaccharides, especially the fraction of WPS-2, had appreciable scavenging power on hydroxyl radicals. 3.5. Antitumor activities of polysaccharides The antitumor activities of polysaccharides were shown in Fig. 4. All polysaccharides presented growth inhibition than did blank control groups, and the inhibition abilities were dose-dependent. The growth of A375 cells could be inhibited by WPS-1, WPS-2 and WPS-3 at a concentration as low as 0.125 mg/mL, and the inhibition ratios reached 20.5%, 13.6% and 13.9%, respectively. WPS-1 and WPS-2 exhibited higher antitumor activities than WPS at the range of 0.03125–2.0 mg/mL. In particular, at the concentration of 2.0 mg/mL, the inhibition ratios of WPS-1 and WS-2 on A375 cells were more than 40%, significantly higher than WPS and WPS-3 (33.5% and 28.3%, respectively). Since WPS-1 and WPS-2 showed significantly high antitumor activities as described above, fluorescence staining with Hoechst was used for analysis morphological changes of cells treated with WPS-1 and WPS-2 (Fig. 5). Hoechst is able to stain the nuclei of cells. Generally, living cells appear with normal nuclei, with blue pale chromatin having an organized structure. Apoptotic cells can be identified by the presence of chromatin condensation and intact nuclear boundaries, and bright blue chromatin that is highly condensed [23]. As shown in Fig. 5b and c, apoptotic cells with fragmented nuclei were observed after exposure to WPS-1 and WPS-2, respectively, while the control group was mainly living cells (Fig. 5a).

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Fig. 2. FT-IR spectra of polysaccharide fractions of WPS-1 (a), WPS-2 (b) and WPS-3 (c).

3.6. Effects of polysaccharides on VSMCs proliferation In vitro inhibition ratios of VSMCs growth by polysaccharides at different concentrations were shown in Fig. 6. WPS-2 showed poor

inhibition against the proliferation of VSMCs. The inhibition ratios of WPS-2 on VSMCs at the concentrations from 0.031 to 2.0 mg/mL were no more than 13%. On the contrary, WPS exhibited relatively higher growth inhibition against VSMCs. At the concentration of

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Fig. 3. Scavenging effects on superoxide radical and hydroxyl radical of different polysaccharide fractions from Laminaria japonica. Values are mean ± SD of three separated experiments. (a) Superoxide radical and (b) hydroxyl radical.

Fig. 4. Inhibition of proliferation of A375 cells by polysaccharide fractions at different concentrations. Values are mean ± SD of six separated experiments. Significant differences compared to control group are designated as *p < 0.05 and **p < 0.01.

0.125 mg/mL, the inhibition ratio of WPS on VSMCs was 15.95%. And the highest inhibition ratio of WPS on VSMCs was 24.54% at the concentration of 2.0 mg/mL. WPS-1 and WPS-3 exhibited growth inhibition at high concentration of 2.0 mg/mL, the inhibition ration were 17.6% and 20.1%, respectively. The results showed that WPS2 had selective cytotoxicity. The proliferation of A375 could be effectively inhibited by WPS-2, at the same time little obviously inhibitory proliferation activity on VSMCs was observed. Amounts of investigations have suggested that bioactivities of algal polysaccharides depended on chemical composition and type of sugar [24,25]. In this study, WPS-1 and WPS-2 exhibited significant antioxidant and antitumor activities. Results of

Fig. 6. Inhibition of proliferation of VSMCs by polysaccharide fractions at different concentrations. Values are mean ± SD of six separated experiments. Significant differences compared to control group are designated as *p < 0.05 and **p < 0.01.

monosaccharide compositions assay showed that fucose was main monosaccharide composition of WPS-1 and WPS-2, accounting for 46.91% and 45.1%, respectively. However, the ratio of fucose in WPS and WPS-3 were 25.74% and 32.51%, respectively. It has been revealed that fucose or fucan were received as critical factor in algal polysaccharides bioactivities [26,27]. Therefore, fucose contents in Laminaria japonica polysaccharides might provide some contributions to their bioactivities. Cytotoxicity assay showed that WPS-2 had higher antitumor activity and lower cytotoxicity on normal cells. Possibly, more order conformation of WPS-2 in aqueous solution had certain effect on its selective cytotoxicity.

Fig. 5. Nuclear morphology of the various type of A375 cells death was visualized by fluorescence microscopy with Hoechst staining following exposure to the control group (a), the fraction of WPS-1 (b) and the fraction of WPS-2 (c) (200×).

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4. Conclusion In the paper, three main fractions (WPS-1, WPS-2 and WPS-3) of Laminaria japonica were purified by anion-exchange chromatography. Chemical analysis indicated that polysaccharide fractions showed varying monosaccharide compositions. Among the polysaccharide fractions, WPS-1 exhibited the highest scavenging effect on superoxide radical and WPS-2 presented a powerful scavenging effect on hydroxyl radicals. Furthermore, higher antiproliferative effects of both WPS-1 and WPS-2 on carcinoma cells (A375 cells) were observed. However, WPS-2 possessed lower cytotoxicity on non-cancerous cells (VSMCs). The results suggested that the Laminaria japonica polysaccharide fraction of WPS-2 could be considered as a potential candidate for developing safe antioxidant and antitumor agent. Acknowledgment This research was supported by Technology Development Fund of Fuzhou University (Grant 2010-XY-16). References [1] X. Zhu, A.K. Raina, H.G. Lee, G. Casadesus, M.A. Smith, G. Perry, Brain Research 1000 (2004) 32–39. [2] P. Dandona, K. Thusu, S. Cook, Lancet 347 (1996) 444–445. [3] M. Yokoyama, Current Opinion in Pharmacology 4 (2004) 110–115. [4] J.A. Knight, Annals of Clinical and Laboratory 25 (1995) 111–121. [5] H. Wiseman, B. Halliwell, Biochemical Journal 313 (1996) 17–29. [6] T.P. Szatrowski, C.F. Nathan, Cancer Research 51 (1991) 794–798. [7] H. Song, Q. Zhang, Z. Zhang, J. Wang, Carbohydrate Polymers 80 (2010) 1057–1061.

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