Antioxidant activity of quercetin and myricetin in liposomes

Chemistry and Physics of Lipids 97 (1998) 79 – 85

Antioxidant activity of quercetin and myricetin in liposomes Michael H. Gordon *, Andrea Roedig-Penman Department of Food Science and Technology, Hugh Sinclair Unit of Human Nutrition, The Uni6ersity of Reading, Whiteknights PO Box 226, Reading GG6 6AP, UK Received 29 June 1998; received in revised form 21 September 1998; accepted 7 October 1998

Abstract The antioxidant activity during storage at 30°C of quercetin, myricetin and a-tocopherol in small unilamellar liposomes has been investigated. Myricetin was more effective than a-tocopherol as an antioxidant in liposomes under all conditions studied. At pH 5.4 with a concentration of 10 − 2 mol/mol phospholipid, myricetin has been shown to be the strongest antioxidant followed by quercetin and a-tocopherol. Cupric chloride and ferric chloride strongly reduced the antioxidant activity of myricetin and quercetin with cupric chloride causing a stronger reduction in activity than ferric chloride. At a pH of 7.4, quercetin was less effective than a-tocopherol at a concentration of 10 − 2 mol/mol phospholipid, but it’s activity increased more strongly with concentration and it was very effective at a concentration of 5 ×10 − 2 mol/mol phospholipid. © 1998 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Antioxidant; Flavonols; Liposomes; Myricetin; Quercetin

1. Introduction Quercetin and myricetin are important dietary constituents belonging to the flavonol class. Quercetin is the flavonol that is consumed at highest levels due to it’s presence in tea, onions and apple skins (Hertog et al., 1993) whereas myricetin is consumed at lower levels in products

* Corresponding author. Tel.: + 44-1189-316723; fax: +441189-310080; e-mail: [email protected].

including blackcurrants, black grapes, cranberries, bilberries, broad beans, red wine and grape juice (Hertog et al., 1992). The consumption of flavonols and flavones varies widely around the world from 64 mg/day in Japan to 6 mg/day in Finland (Hertog and Hollman, 1996). In the Netherlands, 23 mg/day was found to be the average intake, with quercetin contributing 16 mg/day to this figure (Hertog and Hollman, 1996). Interest in flavonols has increased in recent years because of their presence as antioxidants in

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food and the suggestion that they may help to reduce the incidence of coronary heart disease (CHD). This suggestion was partly based on experimental evidence of their antioxidant activity in vitro (Takahama, 1985; Limasset et al., 1993), especially their ability to inhibit LDL oxidation by macrophages (De Whalley et al., 1990). Flavonols were also shown to have an anti-thrombotic action in vivo (Gryglewski et al., 1987). Epidemiological evidence for the importance of flavonols in reducing mortality from CHD was provided by the Zutphen Elderly Study (Hertog et al., 1993), but the significance of flavonols is still not proven (Katan, 1997). Most studies of the antioxidant activity of quercetin and myricetin have shown that myricetin is more effective than quercetin as an antioxidant in oils (Mehta and Seshadri, 1959; Shahidi and Wanasundara, 1995), in emulsions (Pratt, 1976; Taya et al., 1984; Das and Pereira, 1990) and in LDL oxidation (Frankel et al., 1995; Teissedre et al., 1996), although the reverse order has also been found for oxidation in emulsions (Chen et al., 1996) and LDL (Vinson et al., 1995). However, comparative studies of the activity of these flavonols in liposomes are limited, with quercetin being reported as a more active antioxidant than myricetin in fish phospholipid liposomes (Ramanathan et al., 1994). Since, liposomes have a phospholipid bilayer structure which is similar to that of biological membranes, it was considered important to investigate the activity of these flavonols in liposomes in more detail. A pH of 5.4 was used for most of the studies since this pH is typical of foods such as low-fat spreads that may contain liposomes. In addition chronic inflammatory sites, where freeradical challenges occur, have an acidic pH. This is shown by the pH of 7.2 found for bulk synovial fluid in rheumatoid arthritis compared to the normal physiological pH of 7.4. The pH is even lower close to the synovial membrane (Farr et al., 1985). Anaerobic glycolysis, which produces lactic acid, may cause significant reductions in pH in ischemic regions of tissues including atherosclerotic lesions (Morgan and Leake, 1995). A pH as low as 5.2 may occur in tumours of hyperglycemic animals (Jahde and Rajewski, 1982).

2. Materials and methods Myricetin was purchased from Aldrich Chemical Company, Gillingham, UK, Quercetin, a-tocopherol, phosphatidyl glycerol (PG), cholesterol, egg phosphatidyl choline (PC) (type XVIF), tris (hydroxymethyl)aminomethane, sodium acetate and acetic acid were purchased from Sigma Chemical Company, Poole, UK. Small unilamellar liposomes were prepared by a method based on that described in the literature (New, 1990). PC (50 mg) in chloroform, cholesterol (20 mg) and PG (5 mg) were added to a round-bottom flask. A mixture of chloroform– methanol (2.5 ml, 2:1) was added. Any antioxidant being studied was added in methanol. The solvent was evaporated on a rotary evaporator at 30°C, with rotation at about 60 rpm. Atmospheric pressure was re-established with argon. The flask was transferred to an oil-pump and high vacuum (B 0.5 mmHg) was maintained for 1 h. The vacuum was released with argon and degassed buffer solution (Tris, pH 7.4, or acetate, pH 5.4, 0.05 M) containing any water-soluble additives (2.5 ml) was added with glass beads (0.25 g, 2–3 mm diameter). The flask under argon atmosphere was hand shaken in a water bath at 30°C for 5 min, until all lipid had been removed from the walls and a milky suspension free of visible particles was present. The suspension was allowed to swell for 2 h in a dark room at room temperature. The suspension was transferred to a test tube under argon and sonicated with a VC-50 ultrasonic processor (Jencons Scientific, Leighton Buzzard, UK) for 26 min in an ice-bath, using successive cycles of sonication at full power (1 min) and resting for 30 s. Additional buffer solution (2.5 ml) was added and sonication was continued in a similar manner for a further 4 min. The liposomal suspension was then almost transparent. It was filtered through a disk filter (0.2 mm) and stored in the dark at 30°C in a 10 ml vial, closed with a septum, which held a needle to allow air to enter. Samples (0.3 ml) were removed periodically for analysis. All liposome samples were prepared and stored in duplicate, except for samples containing myricetin or quercetin with cupric chloride, which

M.H. Gordon, A. Roedig-Penman / Chemistry and Physics of Lipids 97 (1998) 79–85

were prepared four times. Statistical analysis of the times to an oxidative index of 0.7 involved ANOVA one-way analysis to determine the pooled standard deviation. The individual means were compared by a two-sample t-test using the pooled standard deviation to determine differences significant at the 5% level. Transmission electron microscopy of a platinum image of the liposomes indicated the particle size was about 22 nm, although 10% of the liposomes were in the range 40 – 130 nm. UV analysis of liposome suspensions was performed by dissolving the liposomal suspension (0.3 ml) in absolute ethanol (3 ml). The UV absorbance at 233 and 215 nm was determined and the oxidative index was calculated as the ratio of the absorbance values. Oxidation was monitored until the ratio became about 1, after which it no longer increased with oxidation of the fatty acids (Konings, 1983). Fatty acid methyl esters were prepared from the phospholipids using boron trifluoride containing butylated hydroxytoluene in methanol and the fatty acid composition was determined by gas chromatography using a CP-Sil 88 column (50 m ×0.25 mm, 0.2 mm film thickness) at 180 – 195°C.

3. Results and discussion The fatty acid composition of the egg PC was 33.7% 16:0, 12.5% 18:0, 30.2% 18:1, 15.8% 18:2, 3.5% 20:4. Many of the studies concerned with investigation of the antioxidant activity of flavonols have involved metal-catalysed oxidation. However, it is known that metals may convert antioxidants into strong pro-oxidants as shown for flavonols in oil-in-water emulsions (Roedig-Penman and Gordon, 1998). To assess the antioxidant activity of quercetin and myricetin in the absence of free metal ions, citric acid (100 mg/kg), which is a strong metal chelator, was added to the aqueous phase of the liposomes at pH 5.4. It was found that the order of activity was myricetin \ quercetin\ a-tocopherol under these conditions (Fig. 1). The times to an oxidative index of 0.7

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Fig. 1. Oxidative stability of liposomes containing antioxidants (10 − 2 mol/mol phospholipid) at pH 5.4 in the presence of citric acid (100 mg/kg).

(Table 1) provided clear evidence for the strong radical-scavenging activity of myricetin in liposomes, with the time to an oxidative index of 0.7 being \ 50% higher for myricetin than for quercetin.. Since, the liposomes were prepared by sonication with a titanium probe, it is likely that samples without added citric acid would contain a considerable proportion of free titanium ions. Without added citric acid at pH 5.4, the sample containing myricetin was still extremely stable (Fig. 2). However, the oxidation times for the control and samples containing a-tocopherol and quercetin were all shortened, compared to the samples containing citric acid (Table 1). For the samples lacking flavonols, the time to a conjugated diene value of 0.7 was reduced from 93 h in the presence of citric acid to 27 h in the control sample at pH 5.4. This indicates the increased generation of radicals in the aqueous phase. Myricetin-containing liposomes were considerably more stable than quercetin-containing liposomes, which indicates the strong radical-scavenging properties of myricetin towards radicals generated in the aqueous phase. The stronger radical-scav-

Fig. 2. Oxidative stability of liposomes containing antioxidants (10 − 2 mol/mol phospholipid) at pH 5.4.

Citric acid – FeCl3 (5× 10−3M) CuCl2 (5× 10−3M) – –

5.4 5.4 5.4 135c 85b \3100c

97b \3100d \3100c

30a 21a 21a

164c 421b

61a,b

10−2 5×10−2

10−2

10−2 10−2 10−2

491b 117b 67a

\3144d \3144d 571c

93a 27a 25a

1951c 340c 221b

Concentration of lipid additive (mol/ mol PL)

Time (h) in presence of Time (h) in presence of Time (h) in presence of amyricetin quercetin tocopherol

Time (h) for control

* Different superscripts within a row indicate samples that were significantly different (pB0.05).

7.4 7.4

5.4

Additive in aqueous phase

pH

Table 1 Times to oxidative index of 0.7 for liposome preparations*

82 M.H. Gordon, A. Roedig-Penman / Chemistry and Physics of Lipids 97 (1998) 79–85

M.H. Gordon, A. Roedig-Penman / Chemistry and Physics of Lipids 97 (1998) 79–85

enging activity of myricetin compared to quercetin is consistent with the order of scavenging activity towards peroxyl radicals (Cao et al., 1997). However, in the trolox-equivalent antioxidant capacity method, which involves radicalscavenging of 2,2%-azinobis(3-ethylbenzothiazoline)-6-sulfonate radical cation (ABTS + ), quercetin was found to be more effective as a radical scavenger than myricetin (Rice-Evans et al., 1996). Although an earlier report using ferrous ion catalysed oxidation of liposomes describes quercetin as a more effective antioxidant than myricetin (Ramanathan et al., 1994), it is clear that myricetin is more effective than quercetin as an antioxidant in the absence of metal ions. Oxidation times for liposomes containing no citric acid were reduced by factors of 3.4, 4.2 and 5.7 for the control and the samples containing a-tocopherol and quercetin respectively, compared with the analogous samples containing citric acid at pH 5.4. If quercetin increased the stability of liposomes by metal chelation, it would be expected that the fall in oxidation time between liposomes containing citric acid and those lacking this component would be less for quercetin than for a-tocopherol. However, since the fall in oxidation time is greater for liposomes containing quercetin, there is no evidence that quercetin increases the stability of the liposomes by metalchelation. In order to assess the effects of metal ions on the oxidative stability of liposomes containing flavonols, the oxidation of samples containing ferric chloride or cupric chloride in the aqueous phase at a concentration of 5× 10 − 3 M was monitored (Figs. 3 and 4). It is clear that cupric and ferric ions were strongly prooxidant and the loss of stability due to the metal ions was greater with liposomes containing flavonols than with liposomes containing a-tocopherol (Table 1). This suggests that the decomposition of the flavonols is strongly catalysed by the metal ions. The loss of stability was stronger for liposomes containing myricetin than those containing quercetin, especially in the presence of copper. The strong interaction between flavonols and metal ions is consistent with the flavonols being located near

83

Fig. 3. Oxidative stability of liposomes containing antioxidants (10 − 2 mol/mol phospholipid) at pH 5.4 in the presence of ferric chloride (5 ×10 − 3 M).

the surface of the liposome, since the metal ions are unlikely to penetrate into the hydrocarbon region of the liposome (Terao et al., 1994). The oxidative stability of liposomes lacking added metal ions was also investigated at pH 7.4 (Fig. 5). Under these conditions, at a concentration of 10 − 2 mol/mol phospholipid, quercetin was less effective than a-tocopherol as an antioxidant but myricetin was still very effective. Increasing the quercetin concentration from 10 − 2 mol/mol phospholipid to 5× 10 − 2 mol/mol phospholipid caused a strong increase in oxidative stability with the liposomes still being stable at 3100 h, whereas the same increase in concentration of a-tocopherol only increased the time to an oxidative index of 0.7 to 421 h. The stronger radical-scavenging activity of quercetin compared to a-tocopherol in liposomes is in contrast to the behaviour in solution, where a-tocopherol was a more effective radical-scavenger than quercetin by a factor of 5–6 (Terao et al., 1994). This finding is consistent with the suggestion that quercetin is located

Fig. 4. Oxidative stability of liposomes containing antioxidants (10 − 2 mol/mol phospholipid) at pH 5.4 in the presence of cupric chloride (5 × 10 − 3 M).

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84

Fig. 5. Oxidative stability of liposomes containing antioxidants (10 − 2 and 5×10 − 2 mol/mol phospholipid) at pH 7.4.

near the surface of the phospholipid bilayer, whereas a-tocopherol is located further into the bilayer (Terao et al., 1994). Hence, quercetin would be more effective at scavenging radicals generated in the aqueous phase of the liposomes. The antioxidant activity of a-tocopherol was reported to increase only weakly with concentration above about 3×10 − 3 mol/mol phospholipid in multilamellar liposomes (Yamaoka et al., 1991), which is consistent with our results for unilamellar liposomes. It is reported that myricetin was consumed rapidly when liposomes were stored for a few hours in the dark without a radical initiator at pH 7.4 (Terao et al., 1994). However, a very strong antioxidant effect was observed for myricetin in our study at pH 7.4, since it prevented significant oxidation until the experiment was stopped at 3100 h. The flavonol: PC ratio was only 0.2 × 10 − 2 mol/mol in Terao’s experiment, compared with 10 − 2 mol/mol in our study, but the difference in stability of myricetin between the two experiments is probably due to contamination of Terao’s samples with components that catalysed myricetin decomposition.

Acknowledgements The authors acknowledge a grant from the European Union Copernicus Program.

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