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Olea europaea leaf (Ph.Eur.) extract as well as several of its isolated phenolics inhibit the gout-related enzyme xanthine oxidase.

ABSTRACT

In Mediterranean folk medicine Olea europaea L leaf (Ph.Eur.) preparations are used as a common remedy for gout. In this in vitro study kinetic measurements were performed on both an 80% ethanolic (v/v) Olea europaea leaf dry extract (OLE) as well as on nine of its typical phenolic constituents in order to investigate its possible inhibitory effects on xanthine oxidase (XO), an enzyme well known to contribute significantly to this pathological process. Dixon and Lineweaver-Burk plot analysis were used to determine [K.sub.i] values and the inhibition mode for the isolated phenolics, which were analysed by RP-HPLC for standardisation of OLE. The standardised OLE as well as some of the tested phenolics significantly inhibited the activity of XO. Among these, the flavone aglycone apigenin exhibited by far the strongest effect on XO with a [K.sub.i] value of 0.52 [micro]M. In comparison, the known synthetic XO inhibitor allopurinol, used as a reference standard, showed a [K.sub.i] of 7.3 [micro]M. Although the phenolic secoiridoid oleuropein, the main ingredient of the extract (24.8%), had a considerable higher [K.sub.i] value of 53.0 [micro]M, it still displayed a significant inhibition of XO. Furthermore, caffeic acid ([K.sub.i] of 11.5 [micro]M; 1.89% of the extract), luteolin-7-O-[beta]-D-glucoside ([K.sub.i] of 15.0 [micro]M; 0.86%) and luteolin ([K.sub.i] of 2.9 [micro]M; 0.086%) also contributed significantly to the XO inhibiting effect of OLE. For oleuropein, a competitive mode of inhibition was found, while all other active substances displayed a mixed modeof inhibition. Tyrosol, hydroxytyrosol, verbascoside,andapigenin-7-0-[beta]-D-glucoside, which makes up for 0.3% of the extract, were inactive in all tested concentrations. Regarding the pharmacological in vitro effect of apigenin-7-O-[beta]-D-glucoside, it has to be considered that it is transformed into the active apigenin aglycone in the mammalian body, thus also contributing substantially to the anti-gout activity of olive leaves. For the first time, this study provides a rational basis for the traditional use of olive leaves against gout in Mediterranean folk medicine.

[C] 2010 Elsevier GmbH. All rights reserved.

ARTICLE INFO

Keywords:

Olive leaf extract

Olea europaea

Phenolic compounds

Xanthine oxidase inhibition

Gout

Introduction

The 290 kDa protein xanthine oxidase (XO, EC 1.1.3.22) catalyses the oxidation of hypoxanthine to xanthine and subsequently to uric acid (Eqs. (1)-(3)) (Candan 2003; Cos et al. 1998; Mittal et al. 2008). During these reactions, superoxide anion radicals ([O.sub.2*.sup.-]) and [H.sub.2][O.sub.2] are formed (Kelley et al. 2010). Superoxide anion radicals dis-mutate to [H.sub.2][O.sub.2] and dioxygen either spontaneously or catalysed by the enzyme superoxide dismutase (SOD) (Eq. (4)).

Hypoxanthine + [O.sub.2] + [H.sub.2]O [right arrow] Xanthine + [H.sub.2][O.sub.2] (1)

Xanthine + 2[O.sub.2] + [H.sub.2]O [right arrow] Uric acid + 2[O.sub.2*.sup.-] + 2[H.sup.+] (2)

Xanthine + [O.sub.2] + [H.sub.2]O [right arrow] Uricacid + [H.sub.2][O.sub.2] (3)

2[O.sub.2*.sup.-] + 2[H.sup.+] [H.sub.2][O.sub.2] + [O.sub.2] (4)

While an inherited XO deficiency is often asymptomatic (Harrison 2002), a pathological high XO activity is strongly correlated to hyperuricemy and gout (Cos et al. 1998). The prevalence of this disease is two to nine percent depending on age and gender and increases continuously in Western European countries (Pacher et al. 2006; Umamaheswari et al. 2009). The pathological symptoms of gout emerge from the extracellular precipitation of monosodium urate crystals in different tissues (e.g. joints) followed by an inflammatory response (Mittal et al. 2008; Pacher et al. 2006). An anti-hyperuremic therapy often includes the application of XO inhibitors like allopurinol. Upon reaction with the enzyme, allopurinol is oxidised to oxypurinol (Pacher et al. 2006). Whereas allopurinol is a weak competitive XO inhibitor, oxypurinol exhibits a strong non-competitive inhibitory effect (Mittal et al. 2008). Unfortunately, the use of the purine analog allopurinol in gout therapy shows adverse effects by inhibiting other enzymes involved in purine metabolism, making the search for alternative XO inhibitors necessary (Pacher et al. 2006; Umamaheswari et al. 2009).

Besides the newly developed and licensed synthetic XO-inhibitor febuxostat (Baker and Schumacher 2010), only a few ethnopharmacological approaches have been described (Cos et al. 1998; Lespade and Bercion 2010), finding gallic and ellagic acids as well as several flavonoids as inhibitors of XO. Recently, testing of the pharmacological potential of Mediterranean plants by the consortium "Local Food-Nutraceuticals" also included XO inhibitory studies (Heinrich et al. 2006). In Mediterranean folk medicine, e.g. in Spain (Cecchini 1992) and Tuscany (Leporatti et al. 1985), olive leaf preparations such as aqueous decocts are used against gout and hypertension. Olive leaves are newly incorporated in the Ph.Eur.; their corresponding 80% ethanolic extract was recently evaluated as a L-type [Ca.sup.2+] channel blocker by our group (Scheffler et al. 2008). This included the standardisation by a newly developed gradient elution HPLC method for six characteristic phenolic leaf constituents, which we also examined in the present paper. At all, the following nine phenolic constituents of OLE were tested: the flavones apigenin and luteolin and their corresponding 7-O-[beta]-d-glucosides, tyrosol, hydroxytyrosol, caffeic acid, verbascoside and the phenolic secoiridoid oleuropein, the dominant leaf compound (Scheffler et al. 2008) also used as analytical marker in Ph.Eur.

It is noteworthy that the few medical plants traditionally used for gout treatment (e.g. Erythrina stricta Roxb., Cunonia macrophylla Brongn. & Gris., Olea europaea) also exhibit antiinflammatory effects (Cecchini 1992; Fogliani et al. 2005; Leporatti et al. 1985; Scheffler et al. 2008; Umamaheswari et al. 2009). This fact as well as structural complexity, specialised tissue distribution, and manifold regulatory mechanisms of XO strongly suggest a (pathophysiological XO function beyond purine metabolism (Harrison 2002; Pacher et al. 2006).

Some of the above mentioned single phenolics such as apigenin, hydroxytyrosol and luteolin were already tested for their XO inhibitory and/or radical scavenging properties by means of non-kinetic approaches (Cos et al. 1998; Lavelli 2002). Yet, to date no focused study on the XO inhibitory activity of the olive leaves and their broad spectrum of phenolic constituents has been carried out. Applying enzyme kinetic approaches, this in vitro investigation was performed to clarify potential pharmaceutical reasons for the traditional use of olive leaves against gout and other inflammatory diseases.

Materials and methods

Olea leaf extract, reference substances, and HPLC standardisation

An 80% ethanolic Olea europaea leaf (Ph.Eur.) dry extract (OLE) containing 13.4 [micro]M/100 mg oleuropein was obtained from Burger Ysatfabrik, Bad Harzburg (certified charge no. 968701). Standardisation of OLE was achieved in our laboratory by means of gradient elution RP-HPLC-UV/DAD for quantitative assessment of single phenolics; for HPLC separation and determination see Fig. 1. Corresponding tested reference substances: oleuropein, hydroxytyrosol (Rauwald et al. 1991) and caffeic acid (Ritter et al. 2010) were isolated in our laboratory; tyrosol ([greater than or equal to]98%; Merck, Darmstadt), apigenin, apigenin-7-O-[beta]-D-glucoside,luteolin-7-0-P-D-glucoside ([greater than or equal to]96.4%, 96.7%, 98.8%; Roth, Karlsruhe), luteolin, verbascoside ([greater than or equal to]95%, 92%; Phytolab, Vestenbergsgreuth).

[FIGURE 1 OMITTED]

For HPLC, we used a C-18 nucleosil 100-5, EC250/4 column with a C-18 nucleosil 100-5, CC8/4 precolumn for analysis together with a Waters 600 controller and pump, and a Waters UV-DAD and Waters tunable 486 absorbance detector. Measurements were performed at 280 nm using two mobile phases A and B (A: 15% acetonitrile, 85% of a 2.5% acetic acid watery solution; B: 80% acetonitrile, 20% of a 2.5% acetic acid aqueous soluation). Extract was analysed using a gradient elution (time [minutes]/mobile phase A [% V/V]/mobile phase B [%, v/v]: 0-5/85-80/15-20; 5-13/80-78/20-22; 13-15/78-60/22-40; 15-18/60-60/40-40; 18-20/60-50/40-50; 20-25/50-85/50-15; flow rate: 1 ml/min, atemperature: 38 [degrees]C; injection volume: 20 [micro]l).

Biochemicals and stock solutions

Stock solutions of xanthine (5 mM; Sigma-Aldrich, X4002), uric acid (5 mM; Sigma-Aldrich, U2625), cytochrome c (5 mM; Sigma-Aldrich, 30398) and allopurinol (4 mM; Sigma-Aldrich, A8003) were prepared in 0.1 N NaOH. A stock solution of xanthine oxidase from bovine milk (Sigma-Aldrich, X4500) was prepared by dilution in Millipore water. OLE was diluted in Millipore water to give a stock solution of 100 mg/ml. Oleuropein (100 mM stock solution) has been diluted in Millipore water as well and was freshly prepared on a daily basis. Stock solutions of caffeic acid (10.3 mM in 10% DMSO), luteolin (34.25 [micro]M in 50% ethanol and 25% DMSO), luteolin-7-O-[beta]-D-glucoside (5.26 mM in 50% DMSO), apigenin (12.58 mM in DMSO), apigenin-7-O-[beta]-D-glucoside (0.975 mM in 50% DMSO), verbascoside (1.6 mM in 50% methanol), tyrosol, and hydroxyryrosol (each 11 mM in Millipore water) were also prepared immediately before usage. The final concentration of these solvents did not exceed 1% (v/v). Control experiments excluded an influence of such low solvent amounts on the xanthine oxidase activity.

Determination of xanthine oxidase activity

The activity of the xanthine oxidase was determined spec-trophotometrically by measuring the formation of uric acid at 292 nm using an absorption coefficient of 9600 [M.sup.-1] [cm.sup.-1] (Chen et al. 2008). 50 mU xanthine oxidase (bovine milk) were added to xanthine (50 to 150 [micro]M) in a final volume of 500 [micro]l in air-saturated 50 mM phosphate buffer, 0.1 mM ethylene diamine tetraacetic acid (EDTA), pH 7.4. All concentrations are final ones. Kinetic measurements were performed for 3 to 5 min under temperature control at 22 [degrees]C. Michaelis-Menten conditions were tested by using various amounts of xanthine (5-200 [micro]M) and XO (25-100 mU/ml). Lineweaver-Burk plots were used for [K.sub.m] and [V.sub.max] value determination.

Cytochrome c test

Superoxide anion radicals formed during the reaction of xanthine oxidase can be quantified using cytochrome c. The reaction mixture contained 20 [micro]M xanthine, 50 [micro]M cytochrome c, in an air-saturated sodium-phosphate buffer, pH 7.4 with 0.1 mM EDTA. The reaction was started with 20 mU/ml xanthine oxidase. All concentrations indicated are final ones. The reduction of cytochrome c was followed spectrophotometrically at 550 nm using an absorption coefficient of 21, 100[M.sup.-1] [cm.sup.-1] (Saxena et al. 2009). The sensitivity of the reaction was determined by using bovine erythrocyte superoxide dismutase (330 U/ml, final concentration).

Inhibition studies

Allopurinol as well as OLE and its individual constituents were pre-incubated with xanthine for about two minutes. Afterwards the reaction was started by adding xanthine oxidase. Kinetic measurements were performed using at least three different inhibitor concentrations and two or three different xanthine concentrations (50-150 [micro]M). In the case of OLE 50-100 [micro]g/ml were used, allopurinol and the isolated compounds were used in concentrations up to about 100 [micro]M. Only oleuropein was tested up to 1 mM. The data collected during these measurements were analysed using Dixon plots to determine [K.sub.i] values for the tested compounds. Lineweaver-Burk plots revealed the inhibition mode.

Results

Formation of uric acid by xanthine oxidase

The formation of uric acid from xanthine upon addition of xanthine oxidase was followed at 292 nm over five minutes. Its rate was calculated from the initial slope of the time trace. Variations of the xanthine concentration revealed a Michaelis-Menten like dependence of the initial enzymatic rate on substrate concentration with a [K.sub.m] value of 6.8 [micro]M and a [V.sub.max] value of 21 [micro]M/min as calculated from the Lineweaver-Burk plot. Using 100 [micro]M or more xanthine, the maximum initial reaction rates were directly proportional to the applied amount of XO (data not shown).

Inhibition of xanthine oxidase by the standardised olive leaf extract

An aqueous solution of the standardised OLE inhibited the production of uric acid from xanthine by xanthine oxidase in a concentration dependent manner. A selected example for this inhibition is shown in (Fig. 2A). Analysis of inhibitory data using a Lineweaver-Burk plot (Fig. 2B) revealed an intersection region between the straight lines at about -0.017 [micro][M.sup.1] (1/c) and 0.03 min/[micro]M (1/v). This resembles a mixed inhibition mode. However, OLE did not display a clear [K.sub.i] value as there was no clear intersection point in the Dixon plot (Fig. 2C).

[FIGURE 2 OMITTED]

OLE also inhibited the superoxide anion radical-induced cytochrome c reduction. Yet, the inhibition of superoxide radical generation was identical to those determined for the production of uric acid. Applying 50-100 [micro]g/ml extract, comparable decreases in the initial reaction rates were found in the cytochrome c and the uric acid system (Fig. 2D). Thus, the effects of OLE may be explained solely by an inhibition of XO. This shows clearly that any potential scavenging of superoxide anion radicals by OLE components does obviously not play any significant role.

Inhibition of xanthine oxidase by isolated Olea phenolics

Subsequently, nine isolated individual Olea components were analysed for their ability to inhibit the production of uric acid from xanthine by xanthine oxidase. An overview about the effects of these substances on XO activity is given in (Table 1). Oleuropein, apigenin, luteolin, luteolin-7-O-[beta]-D-glucoside, and caffeic acid inhibited the enzyme significantly, while tyrosol, hydroxytyrosol, and apigenin-7-O-[beta]-D-glucoside, were ineffective at concentrations up to 200 [micro]M. Verbascoside, up to concentrations of about 20 [micro]M, did not inhibit the XO activity either.
Table 1
Inhibitory effect of olive leaf components on xanthine oxidase in the
present in vitro study. The data received from the experiments with
allopurinol were included in the table for the purpose of comparison.

            Compound                 Amount in OLE      Inhibition mode
                                 ([micro]M/100 mg) (a)

Oleuropein                               24.8             Competitive
Luteolin-7-O-[beta]-D-glucoside          0.86             Mixed
Caffeic acid                             1.89             Mixed
Luteolin                                 0.086 (b)        Mixed
Apigenin                                 0.033 (b)        Mixed
Tyrosol                                  0.42
Hydroxytyrosol                           0.58
Verbascoside                             1.68
Apigenin-7-O-[beta]-D-glucoside          0.33
Allopurinol                              -                Competitive

             Compound            [K.sub.i] ([micro]M)  Net effect (c)

Oleuropein                           53                Strong
Luteolin-7-O-[beta]-D-glucoside      15                Moderate
Caffeic acid                         11.5              Strong
Luteolin                             2.9               Weak
Apigenin                             0.52              Moderate
Tyrosol                              No inhibition
Hydroxytyrosol                       No inhibition
Verbascoside                         No inhibition
Apigenin-7-O-[beta]-D-glucoside      No inhibition
Allopurinol                          7.3               -

(a) The amounts were calculated from a recent quantitative HPLC
analysis (Scheffler et al. 2008).
(b) The amounts of apigenin and luteolin were estimated to be about 10%
of the corresponding glycosides (Benavente-Garcia et al. 2000).
(c) The net effect was calculated from the [K.sub.i] value by
considering the amount of the component in the extract.


The inhibition mode of the substances given in Table 1 was determined by analysing the data by Lineweaver-Burk plots. For oleuropein, the most characteristic and major component in Olea extract and leaves, a competitive inhibition mode (no changes in the [V.sub.max] value, increasing [K.sub.m] values) was observed. All other active single OLE constituents exhibited a mixed inhibition mode (increasing [K.sub.m] values, decreasing [V.sub.max] values). Under our experimental conditions allopurinol also exhibited a competitive inhibition mode. The inhibition constants of the isolated OLE phenolics as well as of allopurinol were determined using Dixon plot analysis. As can be seen in Fig. 3, the [K.sub.i] values can be directly obtained from the intersection of the straight lines representing constant substrate concentrations.

[FIGURE 3 OMITTED]

For oleuropein (Fig. 3A), a [K.sub.i] value of 53 [micro]M has been obtained. Having a [K.sub.i] of about 0.52 [micro]M, apigenin (Fig. 3B) showed the strongest XO inhibitory effect of all tested substances thus displaying a stronger inhibition than allopurinol (Fig. 3C) for which a [K.sub.i] value of 7.3 [micro]M was calculated.

In order to evaluate the relative impact of the single components on the XO inhibitory effect of the olive leaf extract, the [K.sub.i] value of each substance was related to its relative amount in the extract. Thereby for oleuropein (53 [micro]M, 24.8%) a value of 2.1 [micro]M can be calculated which indicates a considerable impact of this compound on the global XO inhibitory activity of the extract. In contrast, apigenin, despite its low [K.sub.i] value (0.52 [micro]M), gave a value of 15.8 [micro]M and thus revealed only a moderate "net effect" due to its small abundance in the extract (0.033%). In Table 1 compounds with values lower than 10 [micro]M (oleuropein, caffeic acid) were indicated to have a strong net effect, inhibitors with moderate net effects (apigenin, luteolin-7-glucoside) showed values up to 20 [micro]M. Values higher than 30 indicated a weak net effect of the inhibitor (luteolin) in respect to the XO inhibitory activity of the whole extract.

Discussion

The 80% ethanolic OLE showed an inhibitory activity against xanthine oxidase. The inhibition of uric acid formation in the presence of 50 [micro]g/ml extract by 60%, which corresponds to an [IC.sub.50] of 42 [micro]g/ml, is well comparable to a corresponding value for the XO inhibition by the extract of Erythrina stricta leaves (21.2 [micro]g/ml) (Umamaheswari et al. 2009). While Olea europaea is used in Mediterranean folk medicine for gout treatment (Cecchini 1992; Leporatti et al. 1985), Erythrina is a common anti-gout plant in Indian and Chinese traditional medicine (Leporatti et al. 1985; Umamaheswari et al. 2009). From the few plants investigated with an ethnopharmacological approach, XO inhibitory activities have been found in Centaurium erythraea ([IC.sub.50]:73.2 [micro]g/ml), Chrysanthemum sinense ([IC.sub.50]:5.1 [micro]g/ml), and Pinus pinaster (Moinietal. 2000; Nguyen et al. 2004; Valentao et al. 2001), while Rhus coriaria exhibited both radical scavenging ([IC.sub.50]: 232 [micro]g/ml) and XO inhibiting ([IC.sub.50]:172.5 [micro]g/ml) effects (Candan 2003). For OLE, a strong radical scavenging activity can be excluded as the inhibitory effect on the [O.sub.2*.sup.-] formation did not exceed its XO inhibitory effect.

Among the tested isolated Olea phenolics, the strongest XO inhibitory effect was detected for apigenin, thus confirming recent results in which apigenin and luteolin were found to inhibit xanthine oxidase strongly, this ascribed to the meta-di-hydroxy\ation at C5/C7, while their corresponding glucosides had weaker or no effects (Cos et al. 1998). However, the small amounts of free apigenin and luteolin in OLE indicate only a small net participation in their XO inhibitory activity. For the first time, oleuropein, the main OLE constituent, was tested for its XO inhibitory activity exhibiting only weak effects. However, in respect of its high content in OLE, oleuropein together with caffeic acid, which also inhibited XO moderately, are likely candidates to contribute significantly to the XO inhibitory effect. No inhibitory effect was found for tyrosol, hydroxytyrosol, verbascoside, and apigenin-7-O-[beta]-D-glucoside. Nevertheless, pharmacokinetic investigations have to be considered demonstrating that apigenin-7-O-[beta]-D-glucoside is almost quantitatively transformed into apigenin in the mammalian body (Hanske et al. 2009). These data strongly indicate that the actual in vivo effect of OLE is considerably stronger than indicated by the present in vitro experiments as the prodrug apigenin-7-O-[beta]-D-glucoside, which was inactive in the applied enzyme assay, is transformed into the powerful xanthine oxidase inhibitor apigenin under physiological conditions (Hanske et al. 2009). All tested single phenolics besides oleuropein showed a mixed inhibition mode (Table 1). This is in line with the observation that the apigenin-derived XO inhibitor apigenin-4'-0-(2"-0-p-coumaroyl)-[beta]-D-glucopyranoside from the club moss Palhinhaea cernua exhibits a mixed inhibition mode as well (Jiao et al. 2006).

In most comparable experiments published, XO inhibitors were preincubated with the enzyme before addition of xanthine (Lespade and Bercion 2010). In contrast we applied a more physiological approach, namely preincubation of the inhibitor with the substrate. Therefore, the presented kinetic data cannot be directly compared to the results obtained from different experimental conditions or using a non-kinetic approach (Cos et al. 1998). Under these conditions, some Olea phenolics even exceeded the inhibitory effect of allopurinol (Table 1).

New investigations strongly indicate an important pathophysiological role of XO in various forms of post-ischemic injuries, inflammatory diseases, and chronic heart failure (Eger et al. 2000). The involvement of XO in the described pathologies can be explained by the production of reactive oxygen species by this enzyme which were recently addressed to different proinflammatory effects (Cos et al. 1998; Mittai et al. 2008). Beyond this, xanthine oxidase seems to be actively involved in immune responses as it is activated after bacterial or viral infections by cytokines like TNF-[alpha] and shows bactericidal properties in the presence of xanthine (Harrison 2002; Lespade and Bercion 2010).

Thus, the XO inhibitory properties of olive leaves may also partially explain their general anti-inflammatory properties, corresponding to their application against a wide variety of inflammatory diseases in Mediterranean folk medicine such as liver colic, gastroenteritis, nephritis, cystitis and fever, as well as their use for the treatment of wounds, burns, ulcer, and haemorrhoids (Cecchini 1992; Leporattietal. 1985). Furthermore, OLE decreases blood pressure and heart contractility and thus exhibits vasodilatative and hypotensive effects (Leporatti et al. 1985; Scheffler et al. 2008) due to a strong L-type calcium antagonistic activity of OLE and its constituents like hydroxytyrosol (Rauwald et al. 1991; Scheffler et al. 2008), but in addition, the complex involvement of XO in NO metabolism (Harrison 2002; Pacher et al. 2006) may represent an equal, alternative explanation for this effect. This is an interesting afterthought, as the synthetic XO inhibitor allopurinol has also just recently been clinically examined for the therapy of angina pectoris (Noman et al. 2010).

Acknowledgements

This work was supported by the German Research Foundation (Transregio 67, project A-06). We want to thank the students of pharmacy J. Byun, C. Eckelmann, D. GroBe, N. Hamann, I. Mischenson, and M.A. Nguyen for their help in performing experiments. K. Kuchta wishes to express his gratitude towards the "Studienstiftung des deutschen Volkes" for providing a doctoral scholarship.

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J. Flemmig (a), K. Kuchta (b), J. Arnhold (a), H.W. Rauwald (b), *

(a) Institute for Medical Physics and Biophysics, Medical Faculty, University of Leipzig, Hdrtelstrafie 16-18, 04107 Leipzig, Germany

(b) Department of Pharmaceutical Biology, University of Leipzig, Johannisallee 21-23, 04103 Leipzig, Germany

* Corresponding author. Tel: +49 341 97 36951: fan: +49 341 97 36959. E-mail address: rauwald@rz.uni-leipzig.de (H.W. Rauwald).

doi: 10.1016/j.phymed.2010.10.021
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Author:Flemmig, J.; Kuchta, K.; Arnhold, J.; Rauwald, H.W.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EUGE
Date:May 15, 2011
Words:4607
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