Keywords: Teucrium polium, antioxidant, superoxide radical, hydroxyl radical, glutathione
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Copyright © 2006 The Author(s) Aqueous Extracts of Teucrium polium Possess Remarkable Antioxidant Activity In Vitro For reprints and all correspondence: Dr Arieh Bomzon, Department of Pharmacology, Bruce and Ruth Rappaport Faculty of Medicine, Technion—Israel Institute of Technology, PO Box 9649, Haifa, Israel 31096. Tel: +972-4-8295259; Fax:+972-4-8524978; E-mail: bomzon/at/tx.technion.ac.il Received September 12, 2005; Accepted May 25, 2006. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commerical use, distribution, and reproduction in any medium, provided the original work is properly cited. | |||||||||||
Abstract Teucrium polium L. (Lamiaceae) (RDC 1117) is a medicinal plant whose species have been used for over 2000 years in traditional medicine due to its diuretic, diaphoretic, tonic, antipyretic, antispasmodic and cholagogic properties. The therapeutic benefit of medicinal plants is often attributed to their antioxidant properties. We previously reported that an aqueous extract of the leaves and stems of this plant could inhibit iron-induced lipid peroxidation in rat liver homogenate at concentrations that were not toxic to cultured hepatic cells. Others have reported that organic extracts of the aerial components of this plant could inhibit oxidative processes. Against this background, we felt further investigation on the antioxidant action of the extract of T. polium prepared according to traditional Arab medicine was warranted. Accordingly, we assessed (i) its ability to inhibit (a) oxidation of β-carotene, (b) 2,2′-azobis(2-amidinopropan) dihydrochloride (AAPH)-induced plasma oxidation and (c) iron-induced lipid peroxidation in rat liver homogenates; (ii) to scavenge the superoxide ( Keywords: Teucrium polium, antioxidant, superoxide radical, hydroxyl radical, glutathione | |||||||||||
Introduction The medicinal use of plants dates back to ancient times. Teucrium polium L. (Lamiaceae) (RDC 1117) is a plant that has been used for over 2000 years in traditional medicine due to its diuretic, diaphoretic, tonic, antipyretic, antispasmodic and cholagogic properties (1–3). In addition, the plant possesses hypoglycemic, insulinotropic and anti-inflammatory activities (4–6), reduces body weight and lowers high blood pressure (7,8) and has hypolipidemic, antinociceptive and antioxidant properties (9–11). The therapeutic benefit of medicinal plants is often attributed to their antioxidant properties (12–15). In this regard, a methanolic extract of T. polium protected red blood cells (RBCs) against lipid peroxidation induced by 10 mM hydrogen peroxide (16). In another study, Kadifkova-Panovska and co-workers (17) demonstrated that extracts of T. polium prepared using different organic solvents (diethyl ether, ethyl acetate and n-butanol) were effective inhibitors of β-carotene oxidation. In a previous study, we showed that the aqueous extracts prepared from the foliage of T. polium suppressed iron (Fe2+)-induced lipid peroxidation in rat liver homogenates to the same extent as Trolox, the water soluble analog of vitamin E (18). Additionally, we showed that the extract was not cytotoxic because it did not adversely affect cell membrane integrity or suppress mitochondrial respiration of cultured Hep G2 and PC12 cells following 24 h exposure (18). In another study, we reported that adding fertilizer caused a significant concentration-dependent increase in antioxidant activity of the cultivated T. polium compared with the wild-type (19). Based on these findings, we felt further investigation on the antioxidant action of the aqueous extract of T. polium prepared according to traditional Arab medicine was warranted. In this report, we present our findings on the effects of increasing concentrations up to 1 mg ml−1 of aqueous extracts prepared from the leaves and stems of T. polium on (i) inhibition of oxidative processes, namely, inhibition of oxidation of β-carotene and plasma oxidation and Fe2+-induced lipid peroxidation; (ii) its ability to scavenge reactive oxygen species (ROS), namely the superoxide anion ( | |||||||||||
Methods Plant Material and Powder Preparation Leaves and stems of T. polium L. (Lamiaceae) (RDC 1117) were collected during the spring (May–June) from the hills of the Galilee region of Israel. After collection, plant parts were dried for 7–10 days in the shade at room temperature. They were then ground and the powder was stored in cloth bags at 5°C until transfer to the laboratory for preparing dry water-soluble plant powders, as described previously (18). Briefly, dried plant material (25 g) was stirred in 250 ml of distilled water for 15 min at 95°C, followed by rapid filtration through a crude cellulose filter and then Whatman #1 filter paper. The average w/w yield was 11.5%.The resulting filtrate was freeze-dried and the powder was stored at −18°C in a desiccant until required.Chemicals and Reagents All the materials for the experiments involving cultured cells were purchased from Biological Industries Ltd, Beit Haemek, Israel. All chemicals and reagents of the highest purity were purchased from the Sigma Chemical Co., St Louis, MO, USA.Analytical Procedures and Experimental Protocols Does T. polium Inhibit ß-Carotene Oxidation? The ability of T. polium extract to suppress β-carotene oxidation was assayed using the coupled oxidation of β-carotene and linoleic acid developed by Marco (20) and subsequently modified by Miller (21) and Pratt and Miller (22). The assay measures the rate of consumptive oxidation of β-carotene at 50°C. Two minor assay modifications were made as follows: the plant powder was dissolved in water instead of alcohol to a final concentration of 100 μg ml−1 and spectrophotometric readings were taken at 454 nm. The OD454 readings of the rate of β-carotene bleaching were recorded at 20 min intervals for 2 h against water blank. The antioxidant butylated hydroxyanisole (BHA) (100 μg ml−1) served as a positive control. The assay was repeated three times.Suppression of 2,2′-Azobis(2-amidinopropane) dihydrochloride-Induced Plasma Oxidation The assay relies on ability of the hydrophilic radical generator 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) to generate peroxyl radicals, thereby oxidizing plasma (23,24) and measuring the extent of plasma oxidation by the thiobarbituric acid reactive substances (TBARS) assay (25). Human plasma from healthy donors, obtained from the Haifa branch of Israel Blood Bank, was diluted (1:4) with phosphate-buffered saline (PBS). Aliquots (2 ml) of diluted plasma were incubated with freshly prepared 50 mM AAPH in the absence (control) and presence of different concentrations of T. polium extract (133, 333 and 667 μg ml−1, final concentrations) at 37°C for 2 h. BHA (100 μg ml−1) was used as a positive control. At the end of incubation period, the samples were rapidly cooled to stop the plasma oxidation. The generated malondialdehyde (MDA) was measured by the TBARS assay (see later for details). Results were expressed in terms of spectrophotometric readings of the samples at OD532 against water blank. The assay was done three times.Testing for Iron-Induced Lipid Peroxidation in Liver Homogenates The antioxidant activity of the extract was evaluated by quantifying the ability of different concentrations of plant extract to suppress iron (Fe2+)-induced lipid peroxidation in rat liver homogenates. Liver homogenates were prepared from male Sprague–Dawley rats weighing between 250 and 300 g scheduled to be killed by decapitation as ‘healthy untreated controls’ in institute-approved animal-based investigations. Briefly, this involved excision of the liver, its washing in ice-cold 0.15 M saline and cutting into small pieces using scissors before homogenization in ice-cold 0.1 M phosphate buffer, pH 7.4, at 4°C with a Potter–Elvehjem glass homogenizer. The resultant homogenate was filtered through four layers of wet gauze and the filtrate collected. The protein concentration of the homogenate was determined by the method described by Lowry and co-workers (26) using bovine serum albumin as a standard. The final protein concentration was adjusted to 10 mg protein per ml.Aliquots of liver homogenates were incubated with 1 μg ml−1 to 1 mg ml−1 concentrations of extracts of T. polium in a shaking water bath at 37°C for 1 h in the absence and presence of 100 μM FeSO4, as described previously (18). The incubation was stopped by adding ice-cold 20% trichloroacetic acid and the incubates were centrifuged at 1000 g for 10 min. The assessment of the extent of lipid peroxidation relied on individual determinations of MDA contents in sample supernatants by performing the TBARS assay, as described by Draper and Hadley (25). MDA is an end product of peroxidative decomposition of polyenoic fatty acids in the lipid peroxidation process and its accumulation in tissues is indicative of the extent of lipid peroxidation (25). TBARS reagent (1 ml) was added to a 0.5 ml of sample and the sample was heated for 20 min at 100°C. The antioxidant, butylated hydroxytoluene, was added before heating the samples. After cooling on ice, the absorbance of the samples was read at OD532 nm. Blanks for each sample were prepared and assessed in the same way to exclude any A532 contribution due to the background. The TBARS results were expressed as MDA equivalents using 1,1,3,3-tetraethoxypropane as the standard. The extent of suppression of Fe2+-induced lipid peroxidation was compared with equivalent data obtained using 1 mg ml−1 Trolox, the hydrophilic analog of vitamin E. The assay was done 11 times. Superoxide Anion Scavenger Potential The xanthine–xanthine oxidase (X–XO) enzymatic reaction is a suitable system to generate superoxide anion (Influence on the Activity of XO Compounds that interact with XO can affect the kinetics of reaction of oxidation of xanthine to uric acid (28). Accordingly, we assessed the effect of 1 μg ml−1 to 1 mg ml−1 extracts of T. polium on XO activity by spectrophotometrically monitoring the rate of uric acid formation at OD295 nm for 3 min at 25°C in the absence and presence of extracts using the method described by Marcocci et al. (29). The rate of uric acid formation was compared in the absence and presence of extracts. The specificity of the reaction was confirmed using 100 μg ml−1 allopurinol. In this experiment, duplicate measurements were done five times at each concentration.Hydroxyl Radical Scavenger Potential The ability of T. polium to scavenge OH• radical was assessed using the classic deoxyribose degradation assay described by Halliwell et al. (30). When EDTA chelated iron-(III) ions are incubated with reducing agent and H2O2 in the assay, OH• radicals are generated in free solution that attack the deoxyribose substrate and fragmenting it into TBARS. The generated TBARS reflect the extent of generation of OH•. OH• is generated in a 1 ml reaction mixture containing 0.1 mM phosphate buffer, pH 7.4, 2.8 mM deoxyribose, 20 μM FeCl3–100 μM EDTA (premixed immediately before its addition to the reaction mixture) and 500 μM H2O2 in the absence or presence of 5 μg to 1 mg T. polium extract. The reaction was commenced by adding 100 μM ascorbic acid (AA). The samples were incubated for 60 min at 37°C in shaking water bath followed by the TBARS assay. The extent of inhibition was compared with equivalent data obtained using 1 mM mannitol, the well-known OH• radical scavenger.The assay was repeated eight times. Prior to execution of each assay, the stability of the H2O2 solution was checked spectrophotometrically using the equation that 10 mM H2O2 solution gives an OD reading of 0.436 absorbance units at 240 nm.Can T. polium Bind with Iron? The deoxyribose degradation assay can also be used to assess the ability of a compound to bind iron when the assay is performed in the absence of EDTA (31,32). When FeCl3 is added to the deoxyribose assay in the absence of EDTA, Fe3+ reacts with deoxyribose generating OH• radicals that immediately attack the deoxyribose. Any molecule that can prevent the process of deoxyribose degradation under such conditions are those that bind iron in weakly reactive forms to generate OH• radicals. Accordingly, incubations were done in the absence and presence of 5 μg to 1 mg T. polium in the deoxyribose assay system with the omission of EDTA followed by the TBARS assay. The extent of inhibition was compared with equivalent data obtained using 1 mM mannitol. The experiment was repeated seven times.Changes in Cellular GSH Content and its Redox State The effects of T. polium extracts on GSH content and redox state were assessed in cultured Hep G2 cells. Hep G2 cells are derived from a human hepatoblastoma and retain many of the differentiated features of mature hepatocytes (33). The cells were grown in Dulbecco's modified Eagle's medium (DMEM) with a high glucose content (4.5 g l−1) supplemented with 10% vol/vol inactivated fetal calf serum, 1% non-essential amino acids, 1% glutamine, 100 U ml−1 penicillin and 10 μg ml−1 streptomycin, pH 7.4, and maintained in a humidified atmosphere of 95% O2:5% CO2 at 37°C. In order to assess the effects of the extract on cellular GSH content and redox state, the cells were seeded and grown in cell flasks under the already described conditions. When 70–80% confluence was reached, the cells were trypsinized, centrifuged (250 g for 5 min at 40C), resuspended in fresh medium and plated in 6-well dishes (106 cells ml−1). After attachment, the medium was replaced and cells were incubated in fresh serum-free medium containing 10 μg ml−1 to 1 mg ml−1 extract for 24 h at 37°C. At the end of incubation period, the cells were washed three times with ice-cold physiological buffered saline. After washing, cells were scraped into 1 ml of ice-cold PBS. To extract cellular GSH, the cells were sonicated at medium speed with three 20 s bursts and a small part of was taken for protein determination (26). The rest of sonicate was immediately acidified with 5% sulfosalycilic acid (2:1 v/v) to prevent spontaneous oxidation of GSH. After standing for 10 min on ice, the sonicate was centrifuged in a refrigerated tabletop centrifuge at 10 000 g for 10 min to remove denaturated proteins. The resultant protein-free supernatants were transferred into 1.7 ml plastic tubes and the acidified samples were frozen at −70°C for a maximum of 1 week. The non-treated cells served as control.Determination of GSH levels was performed using the DTNB–GSSG reductase recycling assay described by Tietze (34) with minor modifications. Before measurement of GSH, the sample was thawed and back titrated to pH 7 with 0.2 N NaOH. The GSH content (the sum of the reduced and oxidized forms of GSH) and the oxidized form (GSSG) were measured separately. The GSH assay was performed in a cuvette containing 0.01 M sodium phosphate buffer (pH 7.5), 5 mM EDTA, 10 mM 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB), 2 mM NADPH, 10 U ml−1 GSSG reductase and sample corresponding to ~100 μg of cell protein in a final volume of 1.0 ml. The reaction kinetics was followed spectrophotometrically at OD412 nm for 5 min by monitoring increase in absorbance. For determination of GSSG content, the same DTNB–GSSG reductase recycling assay was performed after the alkylation of the SH groups of reduced GSH by 10 mM N-ethylmaleimide (NEM) to remove reduced GSH from the reaction. To avoid the adverse effects of excess NEM on the assay used to determine GSSG levels, excess NEM was removed by separation on a Sep-Pak (C18) column (Sigma Chemical Corp., MO, USA). The increments in absorbance at OD412 nm were converted to GSH and GSSG concentrations using a standard curve (0–2.5 nmol GSSG). Duplicate GSH and GSSG determinations were repeated three times. The results were expressed in nmol GSH per mg protein. Statistical Analysis In order to calculate the concentration at which the plant inhibited Fe2+-induced lipid peroxidation or scavenge | |||||||||||
RESULTS Decreased Rate in Oxidation of β-carotene Initially, we evaluated the antioxidant potential of the extract by determining the ability of 100 μg ml−1 extract to inhibit β-carotene oxidation. The rate of β-carotene oxidation was reduced by 60.0 ± 2.4% at this concentration, whereas BHA almost completely inhibited the rate of β-carotene oxidation (Fig. 1, upper).
Inhibition of AAPH-Induced Plasma Oxidation Increasing concentrations of the extract cause a significant concentration-dependent inhibition of AAPH-induced plasma oxidation reaching 84% at the highest concentration (Fig. 1, lower).Iron-Induced Lipid Peroxidation Prevented in Liver Homogenate The addition of 100 (M FeSO4 to liver homogenates for 1 h caused almost a 30-fold increase (P < 0.001) in the extent of lipid peroxidation compared to the control samples (0.20 ± 0.15 nmol MDA per mg protein versus 5.41 ± 0.87 nmol MDA per mg protein) (Fig. 2). When different concentrations of the extract was added to the Fe2+-containing homogenates, we observed a significant concentration-dependent inhibition of the extent of lipid peroxidation. At concentrations of the extract >50 μg ml−1, inhibition of the extent of lipid peroxidation was 90% and equal to the inhibition obtained using 1 mg ml−1 Trolox. The calculated IC50 value of the response curve was 7 ± 2 μg ml−1 (Fig. 2, inset).
The Excellent Figure 3 (upper) summarizes the experiments whose aim was to establish whether the extract contained an
d-ribose is Protected Against OH•-induced Degradation Increasing concentrations of extracts scavenge OH• in a concentration-dependent manner (Fig. 4, upper). The calculated IC50 value of the response curve was 36 ± 20 μg ml−1 (Fig. 4, upper, insert).
Is d-ribose Degradation Prevented by Iron Binding? When we used the deoxyribose degradation assay in the absence of EDTA to assess the ability of the extracts to bind iron, we detected a concentration-dependent effect (Fig. 4, lower). The calculated IC50 value of the response curve was 79 ± 17 μg ml−1 (Fig. 4, lower, insert).Reduced GSH Concentrations Increase in Cultured Hepatocytes Figure 5 (upper) shows the effects of the plant extract on the GSH levels in Hep G2 cells. The extract did not have any significant effect on GSH levels except at its highest concentration (1 mg ml−1) where GSH content was five times higher than the GSH content in untreated cells. effect on GGSG content (Fig. 5, middle). When the GSSG/GSH ratio was calculated, we observed a concentration-dependent decrease in this ratio that was statistically non-significant (Fig. 5,lower).
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Discussion In a previous study, we found that an aqueous extract prepared from the aerial components of T. polium, according to the time-honored method of preparing a decoction in traditional Arabic medicine, was very effective in suppressing iron-induced lipid peroxidation (18). In the present study, we extended this observation to further investigate its antioxidant properties using a variety of cell and cell-free in vitro assays. In this regard, we found that the extract (i) inhibited (a) oxidation of β-carotene, (b) AAPH-induced plasma oxidation and (c) Fe2+-induced lipid peroxidation in rat liver homogenates; (ii) scavenged Our results are unequivocal in establishing that an aqueous extract of T. polium can effectively inhibit oxidative processes. This finding was based on a series of experiments involving three different antioxidant assays, namely oxidation of β-carotene, AAPH-induced plasma oxidation and Fe2+-induced lipid peroxidation in rat liver homogenates in which we were able to progressively refine the relationship between the ability of the extract to inhibit an oxidative process and its concentration. Previously, we reported that the IC50 of the extract to inhibit Fe2+-induced lipid peroxidation in rat liver homogenates was 16 ± 2 μg ml−1 (18). In the present experiment using the identical assay, we established that the IC50 of the extract was 7 ± 2 μg ml−1 with maximal inhibition (90%) already evident at the 0.1 mg ml−1 extract concentration. One may ascribe the different IC50 values to the current study using a different batch of collected plant material for the powder preparation, albeit using the same method of preparation. We also found that the extract is an efficient scavenger of Our results are in agreement with those reported by other groups of investigators using a variety of other assays to assess the antioxidant activity of extracts of T. polium (11,16,17). In their study, Couladis and her co-workers (11) screened Greek aromatic plants from the Lamiaceae family for the antioxidant activity. Of the 21 plants tested, they found ethanol extracts prepared from T. polium, as well as 9 other plants, exhibited the same antioxidant activity as α-tocopherol in their ability to inhibit bleomycin-Fe(II) complex-induced arachidonic acid superoxidation to MDA. Regrettably, these authors did not include any data about the plant extract concentration(s) in their report. In another study, Suboh and co-workers (16) investigated the antioxidant activity effects of various concentrations of methanol extracts of T. polium and the six other medicinal plants as high as 1 mg ml−1, the identical concentration to the highest one used in our study. They reported that the extract significantly reduced 10 mM hydrogen peroxide-induced lipid peroxidation in human erythrocytes. In contrast, the extract failed to protect erythrocytes against protein oxidation and loss of cell deformability of the oxidatively stressed erythrocytes. Kadifkova-Panovska and co-workers (17) also investigated the antioxidant activity of extracts of T. chamaedrys, T. montanum and T. polium prepared using different organic solvents, namely diethyl ether, ethyl acetate and n-butanol. Using such extracts, they assessed their antioxidant abilities in a series of assays that included inhibition of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH assay), inhibition of hydroxyl radical (d-ribose assay) and inhibition of β-carotene oxidation. They found that a 0.4 mg ml−1 extract of T. polium was very effective in inhibiting β-carotene oxidation. Based on our results and these reports, there can be little doubt that extracts of T. polium, irrespective of the mode of preparation, can inhibit oxidative processes leading us to conclude that the extract has substantial antioxidant activity in vitro. We did not attempt to identify the chemical constituents that could account for the antioxidant action of the extract. However, other investigators have reported that the aerial parts of T. polium are rich in flavonoids (35,36). Therefore, it would be reasonable to assume that the antioxidant properties of an aqueous extract of T. polium can be attributed to presence of these bioactive components. In our previous study, we reported that an aqueous extract of T. polium was not toxic to cultured hepatic cells since mitochondrial respiration was fully preserved and the cell membrane integrity remained intact when cells were exposed for 24 h to the plant powder up to 1 mg ml−1 concentration (17). Based on the results of this study and our previous report, we conclude that an aqueous extract of T. polium possesses remarkable antioxidant activities at the concentrations that are not toxic to cultured hepatic cells. Accordingly, we propose that the therapeutic benefit of the extract as used in traditional Arab medicine might be due to its antioxidant activity. Further investigations are needed to verify whether this antioxidant effect occurs in vivo. | |||||||||||
Acknowledgments This research was supported by grants from the Ministry of Science and Technology, Jerusalem, Israel, The Center for Absorption in Science of the Ministry of Immigrant Absorption and the Committee for Planning and Budgeting of the Council for Higher Education under the framework of the KAMEA and GILADI Programs and the Technion Vice-President Research Fund for Promotion for Research at the Technion. | |||||||||||
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