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The monoterpene terpinolene from the oil of Pinus mugo L. in concert with [alpha]-tocopherol and [beta]-carotene effectively prevents oxidation of LDL.

Abstract

Antioxidants from several nutrients, e.g. vitamin E, [beta]-carotene, or flavonoids, inhibit the oxidative modification of low-density lipoproteins. This protective effect could possibly retard atherogenesis and in consequence avoid coronary heart diseases. Some studies have shown a positive effect of those antioxidants on cardiovascular disease. Another class of naturally occurring antioxidants are terpenoids, which are found in essential oils.

The essential oil of Pinus mugo and the contained monoterpene terpinolene effectively prevent low-density lipoprotein (LDL)-oxidation. In order to test the mechanism by which terpinolene protects LDL from oxidation, LDL from human blood plasma enriched in terpinolene was isolated. In this preparation not only the lipid part of LDL is protected against copper-induced oxidation--as proven by following the formation of conjugated dienes, but also the oxidation of the protein part is inhibited, since loss of tryptophan fluorescence is strongly delayed. This inhibition is due to a retarded oxidation of intrinsic carotenoids of LDL, and not, as in the case of some flavonoids, attributable to a protection of intrinsic [alpha]-tocopherol. These results are in agreement with our previous results, which showed the same effects for a monoterpene from lemon oil, i.e. [gamma]-terpinene.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Antioxidants; Low-density lipoproteins (LDL); Essential oil; Monoterpenes; Terpinolene; Carotenoids

Introduction

Oxidation of LDL is believed to play a key role in atherogenesis (Halliwell and Gutteridge, 1999; Chisolm and Steinberg, 2000; Steinberg, 1997). For this reason, a sufficient protection of LDL by antioxidants may provide protection from atherosclerosis (Niki and Noguchi, 2002; Mashima et al., 2001). In this context, [alpha]-tocopherol and ubichinol are well known for their ability to protect LDL from oxidation and particularly for their synergistic effects (Schneider and Elstner, 2000; Thomas et al., 1995; Upstone et al., 1999; Esterbauer et al., 1991; Kaikkonen et al., 2000; Raitakari et al., 2000). Since some years additionally flavonoids are under investigation and have been shown to possess good antioxidatve capacity (Pietta, 2000, Harborne and Williams, 2000), depending on individual structures and lipophilicity (Heijnen et al., 2002; Brown and Rice-Evans, 1998). We, however, reported on the high antioxidative properties of essential oils (EOs) (Grassmann et al., 2000, 2001, 2003), another class of secondary plant metabolites. These non-fat-oils are obtained by steam distillation or extraction of a wide range of parts of plants such as needles, bark and are used as drugs for curing bronchial, skin--and muscle-disorders of infectious, rheumatic or neuralgic origin or as ointments and bathing oils (Kohlert et al., 2000; Bhattaram et al., 2002). Monoterpenes (10 C-atoms) such as [alpha]- and [beta]-pinene, [[DELTA].sup.3]-carene, limonene, terpinolene, [gamma]-terpinene and myrcene are major components of EOs such as the EO from Pinus mugo L. (Bruneton, 1999; Leung and Foster, 1996). These substances are highly lipophilic and possess good antioxidative capacity in lipophilic test systems (Grassmann et al., 2003) and can be very effectively enriched in LDL and protect it from oxidation (Grassmann et al., 2001, 2003). Additionally, we showed synergistic effects between the flavonoid rutin and the monoterpene [gamma]-terpinene (Milde et al., 2004). Very recently, we proved the antioxidative potential of PMEO regarding LDL-oxidation (Grassmann et al., 2003). In this communication, we present detailed data on the kinetics of the LDL-protective effect of terpinolene (TPO). We compare protein oxidation and diene conjugation of LDL, as well as the "protective" effects on the intrinsic antioxidants [alpha]-tocopherol and [beta]-carotene. Their oxidation is analyzed and compared in the presence of added terpinolene.

Materials and methods

Materials

Chemicals

All chemicals were obtained from Merck, Darmstadt if not otherwise stated.

Authentic P. mugo EO was obtained from ALLGA-Pharma (Fischen/Allgau). A gas chromatographic analysis is reported by Grassmann et al. (2003). Main constituents are [[DELTA].sup.3]-carene, [alpha]-pinene, (+)-limonene and terpinolene. The content of terpinolene is between 3% and 8%. Pure terpinolene was obtained from Fluka, Germany.

Instruments

Spectrophotometer: Kontron Instruments Uvikon 922.

Fluorospectrometer: Hitachi F-4500.

Ultra centrifuge: Beckmann Optima LE-70, Swinging Bucket Rotor SW 40 Ti

Test systems and methods

Plasma preparation

The plasma used for the shown experiments was gained from blood of 10 healthy volunteers ([female] = 5, [male] = 5) aged 17-38. The preparation of the plasma was as follows: to 100 ml blood 4 ml EDTA stock solution (25 mg/ml) is added immediately after receiving the blood. The blood is centrifuged at 10[degrees]C for 20 min at 3000 rpm. The plasma is withdrawn and after addition of 1 ml saccharose solution (60%) per 100 ml plasma, it is stored at -70[degrees]C in [N.sub.2]-atmosphere for a maximum of 6 months.

LDL-preparation

The preparation of LDL (d = 1.019-1.063 g/ml) by density gradient ultra-centrifugation was described by Giessauf et al. (1995) and modified by Schlussel and Elstner (1995). Plasma (3 ml) was adjusted to a density of 1.41 g/ml with KBr in Beckmann Polyallomer Centrifuge tubes (No. 331372) and stratified with approximately 2.5 ml density solution A (1080 g/ml; 1 g EDTA/l), B (1050 g/ml; 1 g EDTA/l) and C (1000 g/ml; 1 g EDTA/l) per layer. After 22 h centrifugation at 10[degrees]C with 40,000 rpm (285,000 g) three different layers appear above the plasma: lowest HDL, then LDL, on the surface VLDL and chylomicrones. After gaining the LDL layer, it was filtrated with a sterile filter (Nalgene 0.22 [micro]m) and stored at 4[degrees]C. Before use the LDL was desalted with an EconoPac DG-10 column and its concentration defined by standard protein determination of BioRad.

The copper-induced LDL-oxidation

LDL is the main lipoprotein fraction involved in atherogenesis, which in turn is the major cause of heart disease and stroke. One process of modification of LDL is its oxidation, which can be measured as an increase in absorption at 234 nm, reflecting lipid peroxidation associated diene conjugation. Thereby, a prolongation of the lag-phase (time until rapid extinction increase occurs) means higher antioxidative capacity of the substance investigated (Esterbauer et al., 1989, 1991). To enrich LDL with the test substances we incubated human blood plasma with terpinolene for 1.5 h at 37[degrees]C and subsequently isolated the LDL.

For monitoring the diene conjugation as an indicator for the oxidation of LDL an assay contains in a final volume of 1 ml: PBS pH 7.4, CuS[O.sub.4] 1.67 [micro]M, LDL 25 [micro]g protein, sample(s) (amounts indicated). The change of the extinction was measured photometrically at 234 nm every 10 min for 1000 min at 37 [degrees]C.

Tryptophane fluorescence as indicator of protein oxidation

Loss of tryptophan fluorescence is a marker for oxidations at the protein core of LDL (Reyftmann et al., 1990; Giessauf et al., 1995). The measurements were conducted at 282 nm excitation and 331 nm emission wavelength. The assays contained 50 [micro]g protein of the different LDL-samples per ml and 3.36 [micro]M [Cu.sup.2+] ions to induce oxidation. The cuvettes had to be removed from the excitation light between the single measurements to avoid photooxidation of the tryptophane residues; fluorescence was measured every 10 min.

Extraction and quantification of antioxidants and terpinolene

In the isolated LDL the terpenoids can be quantified by hexane extraction as follows: To 250 [micro]l LDL-sample 250 [micro]l ethanol and 500 ml hexane are added. After vortexing for 1 min and centrifugation (4000g, 3 min) 1 [micro]l of the hexane phase was used for the gaschromato-graphic analysis of the terpenoids which was conducted on a Fisons DB-225 capillary column in a GC 86.10 (DANI, Mainz, Germany) with PTV injection and FID detection. The temperature program was as follows: 5 min isothermal at 65 [degrees]C//5 [degrees]C/min[right arrow]70 [degrees]C//1 min isothermal at 70 [degrees]C//10 [degrees]C/min[right arrow]200 [degrees]C//3 min isothermal at 200 [degrees]C.

Statistics

In case of LDL-oxidation plasma preincubation was conducted on two different days, from each plasma incubation LDL was isolated and copper-induced oxidation was conducted twice. The presented results are from one representative experiment.

[FIGURE 1 OMITTED]

Results

Uptake of terpenoides into LDL and corresponding activities on copper-induced oxidation

If human blood plasma is incubated with different amounts of terpinolene (0.01% up to 0.25%), retardation of dieneconjugation is observed in LDL moieties stemming from incubations with 0.025% terpinolene and higher (Fig. 1, see also Grassmann et al., 2003). The effect is clearly concentration dependent and can be explained by the enrichment of terpinolene in LDL (Table 1): Incubation of human blood plasma with 0.01% terpinolene leads to an enrichment of 14 molecules of terpinolene per LDL-particle, after incubation of plasma with 0.25% terpinolene the LDL-particle is loaded with nearly 350 molecules terpinolene.

Terpinolene is not only able to protect the lipophilic part of LDL from oxidation, it also inihibits oxidation of the proteinaceous part of the lipoprotein. As shown in Fig. 2, the loss of tryptophan fluorescence is retarded in a concentration-dependent manner. Already, the lowest terpinolene-concentration in plasma, i.e. 0.01%, brings about a delay in protein oxidation. In the samples, which stem from incubations with 0.1% or 0.25% the oxidation is almost completely inhibited.

[FIGURE 2 OMITTED]

Consumption of endogenous antioxidants during copper-induced oxidation

To get information about the mechanism, by which terpinolene exhibits its protection of LDL we studied the consumption of the main endogenous antioxidants in LDL, namely [alpha]-tocopherol and [beta]-carotene. For this purpose, LDL from plasma, which was incubated with 0.05% terpinolen was subjected to a copper-induced oxidation and aliqouts were taken according to the time scale indicated. In these aliqouts conjugated dienes, tryptophane fluorescence and antioxidants ([alpha]-tocopherol, [beta]-carotene, terpinolene) were quantified as shown in Fig. 3.

The presence of TPO in LDL clearly separates the kinetics into two "time windows":

(i) a first section (without TPO, hollow symbols) from start of the reaction until 100 min and

(ii) a second section (with TPO, solid symbols) from ca. 180 min until the end of measurement at approximately 300 min.

First section (without TPO):

(A) In this samples, initial protein oxidation measured as decrease of tryptophan fluorescence penetrates (after a short fast phase of approximately 15 min) the so-called "slow phase" which is followed by the "rapid phase" after approximately 50 min which in turn is finished after approximately 90 min. Exactly after 50 min diene conjugation is initiated.

(B) TOC content of LDL decreases rapidly from the very beginning in a reaction, which seems to be even faster than in presence of TPO. This is due to the lower content of TOC at the beginning (5 mol/mol LDL in LDL with TPO compared to 6.5 mol/mol in LDL without TPO). TOC oxidation is finished after 50 min.

(C) [beta]-Carotene consumption starts at 50 min and is finished after 90 min, in analogy to tryptophan fluorescence.

Second section (with TPO):

(A) In this section, the "slow phase" of protein oxidation is penetrating the "rapid phase" after approximately 180 min, again exactly when diene conjugation starts.

(B) TPO content is slowly decreasing finally ending at "not detectable" at 300 min. TOC consumption is similar to that in LDL without TPO.

(C) [beta]-Carotene content of LDL is gradually decreasing from the very beginning ending at approximately 180 min at no detectable levels. Compared to LDL without TPO, [beta]-carotene consumption is considerably slowed down.

Discussion

In the early events of atherogenesis LDL oxidation is assumed to represent one characteristic mechanism (Chisolm and Steinberg, 2000; Parthasarathy et al., 1999).

[FIGURE 3 OMITTED]

So a sufficient maintenance of LDL with antioxidants should protect LDL from oxidation and in turn inhibits development of atheroslcerosis. Copper--catalyzed diene conjugation and decrease of tryptophane fluorescence have been used as sensitive and relevant model reactions for testing potential biochemical mechanisms for this initiative events (Giessauf et al., 1995; Patel and Darley-Usmar, 1999). These test systems allow to investigate several natural compounds to be tested on their antioxidative capacity regarding LDL oxidation. We recently reported on the effects of terpenoids from lemon oil or P. mugo EO on this reaction (Grassmann et al., 2001, 2003; Grassmann and Elstner, 2003). The main results were that terpenoids can be enriched effectively in LDL after incubation of human blood plasma with EOs and that some terpenoids possess outstanding capacity in protecting LDL from oxidation. These results may help to explain the results from Bordia et al. who reported that EOs from Allium species (onion and garlic) protect rabbits from atherosclerosis (Bordia et al., 1975, 1977). Also, some other researchers found EOs to be protective against LDL-oxidation but mostly investigated only the wellknown phenolic compounds and did not enrich the compounds in LDL (Teissedre and Waterhouse, 2000; Takahashi et al., 2003).

In this communication, we show experiments proving that incubation of blood plasma with terpinolene, a terpenoid from P. mugo EO, leads to enrichment of LDL with this terpene and that LDL, loaded by this means, is protected from oxidation. Also, other monoterpenes from P. mugo EOs can be enriched in LDL, but they are only weak or no antioxidants in the copper-induced LDL-oxidation (Grassmann et al., 2003); the monoterpene terpinolene is superior to the other mentioned compounds from P. mugo essential oil (PMEO). As described already for [gamma]-terpinene, up to 350 molecules terpinolene can be enriched in LDL. This leads to a very strong delay of both diene conjugation and tryptophane fluorescence (Figs. 1 and 2). Preincubation fo plasma with 0.025% terpinolene leads to an enrichment of 32 mol terpinolene/mol LDL and to a prolongation of the lag phase by 50 min. For comparison, enrichment of LDL with [alpha]-tocopherol, the most common antioxidant in this context, up to a content of 30 mol [alpha]-tocopherol/mol LDL, increases lag phase between 53 and 110 min, dependent on the donor (Esterbauer et al., 1991). So the effect of terpinolene is a little bit weaker than that of [alpha]-tocopherol.

If we compare the kinetics of protein oxidation and dieneconjugation with [alpha]-tocopherol, [beta]-carotene- and terpinolene-oxidation in LDL, we observe that immediately after incubation of LDL with copper ions protein oxidation (measured as decrease of tryptophane fluorescence) is initiated (slow phase). This reaction proceeds parallel to rapid oxidation of TOC. After 50 min, when TOC is quantitatively consumed, protein oxidation penetrates the rapid phase, diene conjugation is initiated and [beta]-carotene oxidation starts. [beta]-Carotene is quantitatively consumed after approximately 90 min.

In the presence of TPO in LDL the same sequence of events can be observed, however with a delay of approximately 100 min. The fast phase of protein oxidation together with the initiation of diene conjugation starts after 180 min, just when the internal [beta]-carotene supply is getting exhausted. TOC oxidation in TPO-supplemented LDL proceeds even faster than in the absence of TPO. This must be explained by the lower content of TOC at the beginning (5 mol/mol LDL in LDL with TPO compared to 6.5 mol/mol in LDL without TPO). TPO in this experiment is consumed by 100% after 300 min just when protein oxidation and diene conjugation exhibit their fastest rates.

This experiment shows that the slow rate of protein oxidation always precedes lipid peroxidation in LDL and a that clear "pecking order" of antioxidants (Buettner, 1993) seems to exist in LDL where TOC oxidation precedes carotenes which in turn are survived by monoterpenes. This results are in agreement with our results about the protection of endogenous carotenoids in copper-induced LDL-oxidation by [gamma]-terpinene (Grassmann et al., 2001).

Additionally, these data complete results about the protection of [alpha]-tocopherol in copper-induced LDL-oxidation by flavonoids and/or ubichinol (Brown and Rice-Evans, 1998; Thomas et al., 1995; Zhu et al., 2000) and thereby show that there are at least two ways to inhibit LDL-oxidation: by protecting [alpha]-tocopherol or by retarding oxidation of carotenoids. This is probably due to different polarities of the tested compounds; so the more polar flavonoids remain at the outer surface of LDL, where they protect [alpha]-tocopherol whereas the very lipophilic terpenoids penetrate into the LDL-particle, where also the very lipophilic carotenoids are located. The same effect was reported for glabridin, an isoflavan from licorice (Belinky et al., 1998).

As mentioned above another monoterpene, [gamma]-terpinene, shows an analogous mechanism in protecting LDL from oxidation, however being clearly more protecting by a factor close to two (Grassmann et al., 2001). Since the amounts of terpene--molecules enriched in LDL are similar for both compounds, the different extent of antioxidative capacity is not a question of the amount but of the structure. Bisallylic carbons are known as potential electron donors for free radicals such as alkoxyl and hydroperoxyl thus terminating chain reactions. Terpinolene has only one of those bisallylic carbons and [gamma]-terpinene has two. This reaction mechanism has also been proposed for [gamma]-terpinene by Foti and Ingold (Foti and Ingold, 2003). So a new attribute of structure, namely the bisallylic carbons, must be introduced when discussing antioxidative capacity.

We hypothesized that protection of lipids in skin and other organs may proceed via an analogous mechanism thus providing an appropriate shield against various exogenous attacks (Grassmann et al., 2003). From our data we so far cannot conclude that these events and mechanisms are also operating in vivo since it is not clear whether and to which extent monoterpenes are incorporated into LDL. Animal experiments (Bordia et al. 1975, 1977) however indicate that this might be indeed the case. Additionally, there is epidemiological evidence for a protective function of carotenoids and [alpha]-tocopherol regarding several diseases like cardiovascular disease or some types of cancer (Johnson, 2002; Kohlmeier and Hastings, 1995; Stampfer and Rimm, 1995; Patterson et al., 1997). So protection of carotenoids from degradation by terpenoids may also contribute to prevent these diseases.

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J. Grassmann (a), S. Hippeli (b), R. Spitzenberger (c), E.F. Elstner (c,*)

(a) Institute of Vegetable Science, Quality of Vegetal Foodstuff, Life Science Center Weihenstephan, TUM, Freising, Germany

(b) Isarlab Systems, Zusmarshausen, Germany

(c) Institute of Phytopathologie, Laboratory of Applied Biochemistry, Life Science Center Weihenstephan, TUM, Freising, Germany

Received 8 July 2003; accepted 28 October 2003

Abbreviations: LDL, low-density lipoprotein; ROS, reactive oxygen species; EO, essential oil; PMEO, Pinus mugo essential oil; TPO, terpinolene; TOC, [alpha]-tocopherol

*Corresponding author. Technische Univ. Munchen-Weihensteph, Lehrstuhl fur Phytopathologie, Labor fur Angewandte Biochemie, Am Hochanger 2, D-85350 Freising, Germany. Tel.: +49 0 8161 715626; fax: +49 0 8161 714538.

E-mail address: elstner@lrz.tu-muenchen.de (E.F. Elstner).
Table 1. Molecules terpinolene per LDL particle after incubation of
human blood plasma with different amounts of terpinolene

% Terpinolene in human blood plasma Mol terpinolene/mol LDL

0.01 13.8
0.025 31.6
0.05 87
0.1 223.7
0.25 341.6
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Author:Grassmann, J.; Hippeli, S.; Spitzenberger, R.; Elstner, E.F.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
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Date:Jun 1, 2005
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