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Synergistic inhibition of low-density lipoprotein oxidation by rutin, [gamma]-terpinene, and ascorbic acid.

Summary

Low-density lipoprotein (LDL) oxidation may play a significant role in atherogenesis. Flavonoids are well-known for their excellent antioxidative capacity in various model systems, therefore we examined the behaviour of rutin, a quercetin-3-rutinosid, in the copper-mediated LDL oxidation. Rutin alone has been shown to protect LDL against oxidation.

Furthermore we investigated the combination of rutin with a hydrophilic (ascorbate) and a lipophilic antioxidant ([gamma]-terpinene) in copper-mediated LDL oxidation. In both cases we found a synergistic effect on lag phase prolongation.

To elucidate whether this effect mainly depends on the copper chelating ability of rutin we examined its reaction in more detail. Although inhibiting the oxidation of [alpha]-linolenic acid in the "rose bengal system" no direct influence of a copper-rutin-complex was determined. We conclude that a redox active copper-rutin-complex is still able to initiate the LDL oxidation but may prevent copper from a reaction at the binding sites of apoB-100. The synergistic effect in preventing LDL oxidation is due to this trapping of copper in a complex in the case of ascorbate. The synergistic action of rutin and [gamma]-terpinene can be explained by different distribution of rutin and [gamma]-terpinene in, and around the LDL-particle, respectively.

Key words: LDL oxidation, copper, rutin, [gamma]-terpinene, ascorbate, synergistic, cooperative, antioxidants, terpenoids, flavonoids

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Introduction

Polyphenols represent a large group of secondary plant metabolites and are discussed to possess health promoting properties. The beneficial health effects of these substances are probably associated, at least in part, with their ability to prevent oxidative damage. An important pathological process involving oxidative stress is the oxidation of low-density lipoproteins. These lipoproteins are the main carrier of cholesterol and cholesterolester in plasma and their oxidation is believed to play a key role in atherogenesis (Berliner and Heinecke, 1996; Chisolm and Steinberg, 2000; Iuliano et al. 2001; Navab et al. 2002; Niki and Noguchi, 2002; Steinberg, 1997; Witztum and Steinberg, 2001). Once vascular lesions are formed, the LDL can migrate into the intima where it may be oxidized by different mechanisms, e.g. macrophages, lipoxygenases, myeloperoxidase or transition metals (Carr et al. 2000; Cathcart and Folcik, 2000; Chisolm et al. 1999; Patel et al. 1999). Oxidized LDL contributes to the formation of fatty streaks which is assumed to represent the first step in atherogenesis. LDL-particles usually are protected from oxidation by several endogenous antioxidants, such as [alpha]-tocopherol and carotenoids, and by high-density lipoprotein associated paraoxonase (Mackness and Durrington, 1995; Stein and Stein, 1999). In case of increased oxidative stress and/or decreased antioxidative defense LDL is oxidized nevertheless. LDL oxidation is just one of several pathological processes in which oxidative stress is involved. Therefore research on antioxidants has achieved much importance in recent years and several secondary plant metabolites have been found to comprise good antioxidative properties. The best investigated metabolites in this context are polyphenols and for a wide range of this substances antioxidative properties are proven (Duthi and Crozier, 2000; Fukumoto and Mazza, 2000; Heijnen et al. 2002; Nakatani, 2000; Nijveldt et al. 2001; Pietta, 2000; Rice-Evans et al. 1996).

Rutin, a quercetin-3-rutinosid, is derived for example from buckwheat and possesses good antioxidative capacity (Oomah and Mazza, 1996; Quettier-Deleu et al. 2000; Watanabe et al. 1997), particularly in combination with other antioxidants (Negre-Salvayre et al. 1991; Negre-Salvayre et al. 1995). Tea from buckwheat has been known for long times for its vascular protective and antiinflammatory properties and is utilized for vascular stabilization (Ihme et al. 1996). So rutin should be a promising flavonoid for reducing the risk of atherosclerosis. It has recently been shown that ascorbic acid in combination with phytoestrogens shows a synergistic inhibition of LDL oxidation (Hwang et al. 2000). For this reason we investigated whether this is also true for ascorbate in combination with rutin.

Furthermore in our laboratory it could be demonstrated that some essential oils and their components, i.e. monoterpenes, above all [gamma]-terpinene, reveal an antioxidative capacity including the ability to delay LDL oxidation (Grassmann et al. 2000; Grassmann et al. 2001; Grassmann et al. 2002). These monoterpenes are lipohilic in contrast to the more hydrophilic rutin. Thus our aim was to investigate whether rutin alone or in combination with these monoterpenes is able to protect LDL from oxidation in a synergistic manner.

Materials and Methods

Materials

Lemon oil was obtained from Pohl Boskamp (Hohenlockstedt, Germany). Main component is (+)-limonene besides other terpenes and hydrocarbons. All other chemicals were either purchased from Sigma (Deisendorf, Germany), Merck (Darmstadt, Germany) or Fluka (Buchs, Switzerland).

Methods

* Preparation of LDL: Ethylenediaminetetraacetic acid (EDTA)-containing plasma samples (1 mg/ml) were prepared from blood of 10 healthy donors ([male] = 5, [female] = 5, 23-40 years old), pooled and supplemented with sucrose (0.6% final concentration). In vitro loading of LDL with lemon oil or [gamma]-terpinene was performed by addition of 7.5 [micro]l and 1.5 [micro]l respectively of the different substances to 3 ml of human blood plasma and ensuing incubation at 37 [degrees]C for 1.5 h.

LDL (d = 1.019-1.063 g/ml) was isolated by a single step ultracentrifugation procedure as described previously (Kogl et al. 1994; Schlussel and Elstner, 1995). After centrifugation LDL samples were filtered through a 0.22 [micro]m filter (Millex[R]-GS, Millipore Corporation, Bedford, Ireland) and desalted by gel filtration (Econo-Pac 10 DG desalting column, BioRad). LDL content was determined by protein quantification using the BioRad protein assay and assuming a formular weight of 540 kDa for apoB-100.

* Oxidation of LDL: Formation of conjugated dienes (CD) was followed at 37 [degrees]C using a Kontron Instruments Uvikon 922 spectrophotometer by monitoring the increase in absorbance at 234 nm every 10 min for 1000 min. The assays contained 25 [micro]g/ml protein of the different LDL samples and 1.68 [micro]M Cu(II) to induce oxidation ad 1 ml phosphate buffered saline (PBS) (Esterbauer et al. 1989 modified by Schlussel and Elstner 1995).

Loss of tryptophane fluorescence is a marker for oxidation of apoB-100 of LDL. Measurements were conducted at 282 nm excitation and 331 nm emission wavelength using a Hitachi F-4500 fluorescence spectrometer. The assays contained 50 [micro]g protein of the different LDL samples per ml and 3.36 [micro]M Cu(II) to induce oxidation ad 1 ml PBS (Giessauf et al. 1995; Reyftmann et al. 1990). All cuvettes had to be removed from excitation light into darkness between the single measurements to avoid photooxidation of tryptophane residues. Fluorescence was measured every 20 min.

* Extraction and quantification of terpenoids: The terpenoid content of the different LDL samples was determined as follows: 250 [micro]l of the LDL sample were mixed thoroughly with 250 [micro]l of ethanol in order to precipitate the proteins. The ethanol contained (+)-limonene as internal standard except in case of lemon oil enriched LDL samples. Subsequently 500 [micro]l of hexane were added and the sample was vortexed for 1 min. After centrifugation (4000 X g, 3 min) 1 [micro]l of the hexane phase was used for chromatographic analysis of terpenoids. These gaschromatographic analyses were conducted on a Fisons DB-225 capillary column in a GC 86.10 (DANI, Mainz, Germany) with programmed temperature vaporizer injection and flame ionisation detector (FID). The temperature programme was as follows: 5 min isothermal at 65 [degrees]C, 5 [degrees]C/min [right arrow] 70 [degrees]C, 10 [degrees]C/min [right arrow] 200 [degrees]C, 3 min isothermal at 200 [degrees]C.

* Photodynamic lipidperoxidation of [alpha]-linolenic acid: A 71 mM stock solution of [alpha]-linolenic acid was prepared dissolving 0.1 ml of the pure fatty acid in 5 ml borate buffer (20 mM, pH 9.0) which contained 0.1 ml Tween 20. 0.26 ml of a 1 M sodium hydroxide solution were added until a clear solution was obtained.

The photodynamic peroxidation of [alpha]-linolenic acid was performed in reaction tubes containing 1 ml phosphate buffer (0.1 M, pH 7.2), 3.55 mM [alpha]-linolenic acid, 5 [micro]M Cu(II) and 20 [micro]M rose bengal. The final concentrations of rutin in the reaction mixture are given under results. Water was added to give a final volume of 2 ml. The tubes were sealed with gas tight rubber stoppers and incubated for 30 min at 37 [degrees]C in the light (500 [micro]E/[m.sup.2] X sec).

After incubation 1 ml of the gaseous headspace was withdrawn with gas tight syringes and analysed using a Varian GC (Varian Star 3400 CX) which was equipped with a 1/8 inch X 60 cm aluminum oxide column and a FID. The gaschromatograph was set up as follows: Column temperature 80 [degrees]C, injector temperature 80 [degrees]C and FID temperature 225 [degrees]C (Heiser et al. 1998).

This system is used to measure copper chelation and its effect on lipidperoxidation (LPO). Rose bengal produces mainly singlet oxygen leading to ethane as oxidation product of [alpha]-linolenic acid, whereas in combination with Cu(II) also ethene as the main oxidation product is released. Using exactly the mentioned concentrations of Cu(II) and rose bengal the emerged amounts of ethane and ethene are nearly equal so the ratio is about 1. Is this ratio shifted in the direction of ethane a lowered redox activity and subsequently a copper chelation by the tested substance can be concluded.

[FIGURE 1 OMITTED]

Results

LDL oxidation

After incubating the isolated LDL with different concentrations of rutin (0.5 [micro]M-10 [micro]M) in the presence of Cu(II) a concentration-dependent increase in the lag phase was observed. A 0.5 [micro]M rutin solution led to a lag phase prolongation of about 70 min, a 10 [micro]M solution inhibited the oxidation completely within reaction time of 1000 min. Lag phase extension and rutin concentration could be implicated in a linear regression (Fig. 1). The measured lag phase extension as well as the beginning of the late protein oxidation caused by a certain concentration of an antioxidant, here for example rutin, will differ in each experiment. The reasons are different plasma pools for the LDL isolation, LDL is per se an inhomogeneous solution, and the different oxidative states of each LDL sample, e.g. unequal lipid hydroperoxides levels.

When monitoring loss of tryptophane fluorescence a similar antioxidative effect of rutin was observed. Rutin in a 1 [micro]M concentration already delayed the late protein oxidation (time at which fast loss of fluorescence sets on) up to 58 min (Table 1). This method is an indicator for oxidation at the protein part of the LDL particle. These results implicate that rutin is capable of reducing lipid peroxidation as well as protecting apoB-100 from oxidative destruction.

Incubating plasma with lemon oil or [gamma]-terpinene extends the resulting lag phase in the copper-mediated formation of CD in LDL by the factor 1.5 or 2.7 respectively. Data for terpenoid contents in LDL are represented in Table 2. Loading LDL with terpenoids is a process which depends mainly on the properties of the plasma used. In consequence the content of terpenoids and as a result also the lag phase prolongations will differ. The concentration dependent prolongation of the lag time or of the late protein oxidation of these terpenoids was already shown by Grassmann and coworkers (2001).

By adding rutin (1 [micro]M) to these samples the lag phase is prolonged in both cases more than additive (Fig. 2). In the first case (preincubated with lemon oil) the lag phase prolongation was pre-estimated for 170 min. In fact it occurred a lag phase prolongation of 265 min, an extension of 56%. In case of LDL loaded with [gamma]-terpinene an extension of 22% is observed.

Monitoring the behaviour of terpenoid loaded LDL during copper-induced loss of tryptophane fluorescence we focused on [gamma]-terpinene. Its content was 18 mol/mol LDL. The result is at least an additive protection of the LDL protein part. 237 min was the estimated time for extension of late protein oxidation and 270 min (14% more) were measured (Fig. 3).

Measuring formation of CD after incubation of LDL samples with ascorbic acid there was a minimal prooxidative influence of a low concentration (0.5 [micro]M) and a little antioxidative effect of 2.5 [micro]M ascorbate observed. A concentration dependent prolongation of lag time is asserted for higher concentrations (>2.5 [micro]M). Addition of 1 [micro]M rutin inhibited the prooxidative influence completely and beyond converted it into an antioxidative effect which led to an obvious synergistic lag time prolongation. Rutin (1 [micro]M) also caused a drastic lag phase prolongation in the case of the 2.5 [micro]M ascorbic acid (Fig. 4). At higher ascorbate concentrations an additive effect with rutin is recognised (data not shown).

[FIGURE 2 OMITTED]

Monitoring the loss of tryptophane fluorescence of such LDL samples similar results are observed. Ascorbate (<2 [micro]M) leads to an acceleration of the loss of tryptophane fluorescence, whereas at higher concentrations (>5 [micro]M) a slowdown can be registered. However, the concentration where the prooxidative effect switches into an antioxidative one has moved (Fig. 5). This phenomenon can be explained by different LDL and Cu(II) concentrations in the samples (see methods). All in all we estimate a synergistic antioxidative effect in the lipid part as well as in the protein part of LDL by using ascorbic acid and rutin.

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

Photodynamic [alpha]-linolenic acid-peroxidation

Rutin also prevents the oxidation of [alpha]-linolenic acid in a concentration dependent manner. This effect can be followed in the produced amounts of ethane and ethene (Fig. 6). Addition of 5 [micro]M rutin reduces release of these gases by 22% and adding 75 [micro]M rutin the inhibition reaches 62%. The ratio of generated ethane and ethene varies only between 1.45 and 1.22 (Table 3).

Discussion

Rutin is a flavonoid found in buckwheat in relative high concentrations. Extracts from buckwheat, e.g. buckwheat tea, are known in traditional medicine. As a flavonoid rutin should also possess a comparative antioxidative capacity (Pietta, 2000; Nijveldt et al. 2001; Cotelle, 2001; Viana, 1996). Since in course of atherogenesis impairment of vessels occurs as well as oxidation of lipoproteins we tested whether rutin not only provides protection from vascular damage but also from LDL oxidation.

[FIGURE 6 OMITTED]

Our results show that rutin is able to inhibit copper induced formation of conjugated dienes in isolated LDL in a concentration-dependent manner. A concentration of 1 [micro]M rutin already prolongs the lag phase for 145 minutes. A similar effect is observed when monitoring the loss of tryptophane fluorescence, a marker for protein oxidation. A concentration of 1 [micro]M rutin inhibits late protein oxidation, which proofs the ability of rutin not only to protect the lipophilic core of the LDL-particles from oxidation but also the surrounding protein. This protection of LDL is mainly due to the ability of rutin to chelate copper, as proofed for different flavonoids (Mira et al. 2002) and by the different absorption-spectra of rutin alone, rutin in presence of Cu(II) and in presence of Cu(II) plus EDTA, respectively (Brown et al. 1998). To investigate the mechanism of antioxidative and copper chelating action of rutin in more detail we chose another model of lipidperoxidation, namely the "rose bengal system". In this model of LPO [alpha]-linolenic acid is oxidized photodynamically by the photosensitizer rose bengal which yields singlet oxygen in the light. Singlet oxygen leads to formation of lipid-hydroperoxides which decay in a further reaction thereby forming ethane and ethene beside other products (Heiser et al. 1998). The ratio of ethane and ethene formed depends on the presence of redox active copper and can be used to determine copper chelating agents. The presence of rutin during this lipid peroxidation results in an inhibition of peroxidation (i.e. total amount of ethane + ethene) but does not change the ethane/ethene ratio. This proves that a possible copper chelation has no effect on lipidperoxidation. Thus inhibition of ethane and ethene release must be attributed to radical scavenging properties of rutin.

In context of LDL-oxidation this means that the rutin-copper-complex remains redox active, i.e. the chelated copper-ion is still able to initiate oxidations. The antioxidative effect of rutin on LDL oxidation therefore must be explained by the hydrophilic surrounding which is formed around the copper-ion and prevents it from oxidizing the lipophilic LDL-particle. Since the copper-ions are complexed by rutin they are not able to bind at the protein part and therefore cannot initiate oxidation. Additionally rutin--as a flavonoid--possesses good radical scavenging abilities, which has been shown for a wide group of different flavonoids (Pietta, 2000; Nijveldt et al. 2001; Kerry and Abbey, 1998; Rice-Evans et al. 1996).

To determine the effects of rutin in combination with other antioxidants we first chose ascorbate. Low ascorbate concentrations in combination with Cu(II) show a prooxidative effect (Buettner and Jurkiewicz, 1996), which is also true for copper induced LDL oxidation: 1 [micro]M ascorbate leads to a reduction of lag time and accelerate late protein oxidation. This is due to the formation of Cu(I) from ascorbate and Cu(II), Cu(I) can now advance the lipidperoxidation in LDL. This effect was also shown for other watersoluble antioxidants like uric acid or ferulic acid (Bagnati et al. 1999; Bourne and Rice-Evans, 1997). In higher concentrations ascorbate shows antioxidant properties towards LDL oxidation, which is explained by the reduction of hydroperoxides by ascorbate (Hwang et al. 2000; Hwang et al. 2001). Another mode of action may be the oxidation of histidine residues to 2-oxohistidine by ascorbate which prevents binding of copper to these amino acids and therefore inhibits oxidation of the lipoprotein (Retsky et al. 1999). As we showed, rutin in combination with ascorbate not only prevents the prooxidative effect of ascorbate, but results in a synergistic protection of LDL. This is, from our point of view, caused by the copper-chelation by rutin, which in consequence does not allow the reaction of ascorbate with Cu(II), so the formation of the prooxidative Cu(I) can not take place and only the antioxidative properties of ascorbate emerge. At some degree also structural variations, caused by interaction of rutin with apoB-100 will contribute to the synergistic effect, as described by Hwang and coworkers. This group investigated the inhibition of LDL oxidation by ascorbic acid and phytoestrogens (Hwang et al. 2000; Hwang et al. 2001) and discusses a structural basis for inhibition of LDL oxidation by phytoestrogens and therefore a higher efficacy of the additionally tested ascorbic acid. The potentiation of antioxidative capacities of [alpha]-tocopherol and ascorbic acid by rutin was also described by Negre-Salvayre and co-workers (Negre-Salvayre et al. 1991; Negre-Salvayre et al. 1995).

In further investigations we studied the influence of more lipophilic antioxidants like lemon oil and [gamma]-terpinene on copper induced LDL oxidation. By incubating human blood plasma with lemon oil LDL-particles can be loaded with monoterpenes (limonene, [alpha]- and [beta]-pinene, [gamma]-terpinene). Incubation with [gamma]-terpinene alone leads to a very effective enrichment of this monoterpene in LDL (up to 264 mol [gamma]-terpinene/mol LDL). These enriched LDL-particles show a high resistance towards copper-mediated oxidation (Grassmann et al. 2001). Addition of rutin to monoterpene-enriched LDL again results in a synergistic inhibition of CD-for-mation, which must be explained by different places of action of rutin and monoterpenes, respectively. The lipophilic monoterpenes penetrate into the inner core of the lipoprotein where they protect carotenoids from oxidation (Grassmann et al. 2001) whereas the hydrophilic rutin remains near the surface where it may protect [alpha]-tocopherol as it was shown for other flavonoids (Zhu et al. 2000; Hirano et al. 2001; Brown and Rice-Evans, 1998). A second effect caused by the accumulation of monterpenes in the core may be the displacement of [alpha]-tocopherol to the outer surface. Here the antioxidative action of [alpha]-tocopherol will be much more effective, partly due to its better regeneration by rutin, which is dissolved in the hydrophilic surrounding. However regarding the protective actions of this combination of antioxidants on the apoB-100 no obvious synergistic behaviour was detectable. The measured delay of late protein oxidation was only 10% over the pre-estimated value what is within the range of standard deviation of this test system. One reason for this weak effect may be the low degree of loading with [gamma]-terpinene of LDL (18 mol/mol LDL) in comparison to our investigations on CD formation (164 mol/mol LDL). This might be due to the use of another plasma pool in the first case. In consequence a different distribution of [gamma]-terpinene within the lipoprotein fractions might occure.

Conclusion

We showed that the flavonoid rutin can inhibit copper-mediated LDL oxidation. Combination of rutin with other--hydrophilic or lipophilic--antioxidants leads to a synergistic protection of LDL against oxidation of the lipid part as well as of the protein part. In case of rutin and [gamma]-terpinene the action is at least additive. These results again support the aspect, that one should not only investigate on single antioxidants alone, but further research should concentrate on the combination of different antioxidants.

Rutin in combination with other antioxidants may be helpful to reduce atherosclerosis-risk, particularly since the vascular protective and antiinflammatory properties of rutin are known for a long time.
Table 1. Extension of late protein oxidation measured by the loss of
tryptophane fluorescence of LDL.

rutin [[micro]M] delayed start of late protein oxidation [min]*

0.5 0
1 58
2.5 320

*results shown are means of two independent experiments (n = 2)

Table 2. Content of terpenoids of LDL samples isolated from plasma
preincubated with lemon oil or [gamma]-terpinene.

plasma
preincubated mol (+)-limonene/ mol [gamma]-terpinene/
with mol LDL mol LDL

-* 0 ([dagger]) 0 ([dagger])
lemon oil 56 [+ or -] 13 ([dagger]) 10 [+ or -] 2 ([dagger])
[gamma]-terpinene 0 ([dagger]) 162 [+ or -] 91 ([dagger])

*control LDL, ([dagger]) results shown are means of three independent
experiments (n = 3), sd is given as [[sigma].sub.n-1]

Table 3. Ethane and ethene production during photodynamic
lipidperoxidation of [alpha]-linolenic acid and ratio of ethane and
ethene.

rutin ethane* ethene* [SIGMA]* ratio
[micro]M [nmol] [nmol] [nmol] ethane/
 ethene

 0 6.3 [+ or -] 0.8 4.4 [+ or -] 0.4 10.7 [+ or -] 1.0 1.45
 5 4.7 [+ or -] 0.8 3.6 [+ or -] 0.4 8.3 [+ or -] 1.1 1.28
 25 3.8 [+ or -] 0.1 3.1 [+ or -] 0.1 7.0 [+ or -] 0.1 1.22
 75 2.3 [+ or -] 0.1 1.8 [+ or -] 0.0 4.1 [+ or -] 0.1 1.29
125 1.5 [+ or -] 0.0 1.1 [+ or -] 0.0 2.6 [+ or -] 0.1 1.29
175 1.0 [+ or -] 0.1 0.7 [+ or -] 0.1 1.7 [+ or -] 0.2 1.37

* results shown are means of four samples in one experiment (n = 4), sd
is given as [[sigma].sub.n-1]


Acknowledgements

Thanks are offered to Dr D. Weiser (Steigerwald Arzneimittel GmbH, Darmstadt, Germany) for stimulating discussions and financial support.

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J. Milde (1), E. F. Elstner, (1) and J. Grassmann (2)

(1) Department of Plant Sciences, Institute of Phytopathology, Laboratory for Applied Biochemistry, Munich Technical University, Freising-Weihenstephan, Germany

(2) Institute of Vegetable Sciences, Munich Technical University, Freising-Weihenstephan, Germany

Address

J. Milde, Technische Universitat Munchen, Fakultat fur Ernahrung, Landnutzung und Umwelt, Department fur Pflanzenwissenschaften, Lehrstuhl fur Phytopathologie, Am Hochanger 2, D-85350 Freising-Weihenstephan, Germany

Tel.: ++49-8161-713737; Fax: ++49-8161-714538; e-mail: j.milde@lrz.tu-muenchen.de
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Author:Milde, J.; Elstner, E.F.; Grassmann, J.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:4EUGE
Date:Feb 1, 2004
Words:5111
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