Antioxidant activity , total phenolic content and flavonoid concentrations of different plant parts of Teucrium polium L . subsp . polium

Recently, there is growing interest in finding naturally occurring antioxidants for use in foods or medicinal materials to replace synthetic antioxidants, which are being restricted due to their carcinogenicity [1]. Antioxidants are secondary constituents or metabolites found naturally in plants. They are mainly phenolics serving in plant defense mechanisms to counteract reactive oxygen species (ROS) in order to avoid oxidative damage. Their antioxidant activity is related to their redox properties, ability to scavenge a variety of reactive species such as superoxide, hydroxyl and peroxyl radicals and hypochlorous acid, singlet oxygen quenching, metal ion chelation [2,3]. It was reported that the antioxidant activity of plant materials was well correlated with the content of their phenolic compounds [4]. Teucrium polium L. subsp. polium (Felty germander) – belongs to the family Lamiaceae Lindely, subfamily Ajugoideae Kostel and section Polium (Miller) Schreber. T. polium is a wild-growing perennial herbaceous flowering plant with little branched stems up to 40 cm high. Leaves are without or with very short petiole, oval-elongated and slightly incised. Flowers are white to light pink, densely clustered at the top of the branch. Inhabit rocky limestones and dry mountain meadows in the Mediterranean region and Middle East [5]. T. polium is best popular species of this genus in the folk medicine, very often used for tea and tonic preparing during treatment of appetite loss, gastrointestinal ailments and as a spice plant. Numerous in vitro and in vivo tests of biological activity prove antibacterial, antifungal, antiinflammatory, antiproliferative, apoptotic, antinociceptive, antihypertensive, hepatoprotective and hypoglycemic properties [6]. Extracts from T. polium potentiates the cytotoxic and apoptotic effects of anticancer drugs: vincristine, vinblastine and doxorubicin, against a panel of cancerous cell lines [7]. The therapeutic benefits of T. polium extracts are usually attributed to their ability to suppress oxidative processes [8]. Numerous investigations showed the antioxidant activity of T. polium extracts, it was reported that the alcoholic extract of T. polium had a suppressing effect on hydrogen peroxide-induced lipid peroxidation in red blood cells [9], the ability of water extract to suppress Fe2+-induced lipid peroxidation in rat liver homogenates [10]. The objective of this study was to examine the distribution of phenolic compounds, as the carriers of antioxidant activity, in different vegetative parts of the plant, and what are the most suitable solvents for the production of extracts that are rich in phenolic compounds from T. polium. Separate examination of plant parts allows a significant contribution to medicinal plant study and their pharmaceutical applications [11]. Twenty Abstract


Introduction
Recently, there is growing interest in finding naturally occurring antioxidants for use in foods or medicinal materials to replace synthetic antioxidants, which are being restricted due to their carcinogenicity [1].Antioxidants are secondary constituents or metabolites found naturally in plants.They are mainly phenolics serving in plant defense mechanisms to counteract reactive oxygen species (ROS) in order to avoid oxidative damage.Their antioxidant activity is related to their redox properties, ability to scavenge a variety of reactive species such as superoxide, hydroxyl and peroxyl radicals and hypochlorous acid, singlet oxygen quenching, metal ion chelation [2,3].It was reported that the antioxidant activity of plant materials was well correlated with the content of their phenolic compounds [4].
Teucrium polium L. subsp.polium (Felty germander) -belongs to the family Lamiaceae Lindely, subfamily Ajugoideae Kostel and section Polium (Miller) Schreber.T. polium is a wild-growing perennial herbaceous flowering plant with little branched stems up to 40 cm high.Leaves are without or with very short petiole, oval-elongated and slightly incised.Flowers are white to light pink, densely clustered at the top of the branch.Inhabit rocky limestones and dry mountain meadows in the Mediterranean region and Middle East [5].T. polium is best popular species of this genus in the folk medicine, very often used for tea and tonic preparing during treatment of appetite loss, gastrointestinal ailments and as a spice plant.Numerous in vitro and in vivo tests of biological activity prove antibacterial, antifungal, antiinflammatory, antiproliferative, apoptotic, antinociceptive, antihypertensive, hepatoprotective and hypoglycemic properties [6].Extracts from T. polium potentiates the cytotoxic and apoptotic effects of anticancer drugs: vincristine, vinblastine and doxorubicin, against a panel of cancerous cell lines [7].
The therapeutic benefits of T. polium extracts are usually attributed to their ability to suppress oxidative processes [8].Numerous investigations showed the antioxidant activity of T. polium extracts, it was reported that the alcoholic extract of T. polium had a suppressing effect on hydrogen peroxide-induced lipid peroxidation in red blood cells [9], the ability of water extract to suppress Fe 2+ -induced lipid peroxidation in rat liver homogenates [10].
The objective of this study was to examine the distribution of phenolic compounds, as the carriers of antioxidant activity, in different vegetative parts of the plant, and what are the most suitable solvents for the production of extracts that are rich in phenolic compounds from T. polium.Separate examination of plant parts allows a significant contribution to medicinal plant study and their pharmaceutical applications [11].Twenty extracts were obtained from T. polium using different solvents for extraction of whole plant and its vegetative parts, separately.For each extract total phenolic content and flavonoid concentrations were determined using spectrophotometric methods.2,2-dyphenyl-1-picrylhydrazyl (DPPH) scavenging activity of each extract was also determined and compared to reference natural and synthetic antioxidants [chlorogenic acid, rutin and 3-tert-butyl-4-hydroxyanisole (BHA)].The obtained results were also compared with the values of standard synthetic antioxidants.A parallel analysis of Ginkgo (Ginkgo biloba L.) and Green tea [Camellia sinensis (L.) Kuntze] as most popular plants rich in natural antioxidants was carried out and compared with the values related to T. polium.

Plant material
In August 2009 aerial flowering parts of T. polium were collected from natural populations in the region of Suva Planina Mt. in Southeast Serbia: (position: 43°19'14.82"N, 22°10'21.42"E, altitude: 302.00 m, exposition: W, substratum: limestone).The voucher specimen of T. polium L. 1753.subsp.polium No. 2-2212, UTM 34 TEN 98, August 9th, 2009, det: Milan Stanković; rev: Goran Anačkov, were confirmed and deposited at the Herbarium of the Department of Biology and Ecology (BUNS Herbarium), Faculty of Natural Science, University of Novi Sad.The collected plant material was air-dried in darkness at ambient temperature (20°C).The dried material was cut up and stored in tightly sealed dark containers until needed.

Chemicals
Organic solvents and sodium hydrogen carbonate were purchased from "Zorka pharma" Šabac, Serbia.Gallic acid, rutin hydrate, chlorogenic acid and DPPH were obtained from Sigma Chemicals Co., St Louis, MO, USA.Folin-Ciocalteu phenol reagent, butylated hydroxyanisole (BHA) and aluminium chloride hexahydrate (AlCl 3 ) were purchased from Fluka Chemie AG, Buchs, Switzerland.All other solvents and chemicals were of analytical grade.The samples of Green tea (Camellia sinensis) were purchased from a local pharmacy.A standardized extract of Ginkgo biloba was obtained from Pharmaceutical Company "Ivančić i Sinovi", Belgrade, Serbia (base for dietary products Ginkgo biloba extract, produced by Sichuan Xieli Pharmaceutical.Co. Ltd., Sichuan, China).

Preparation of plant extracts
Prepared plant material (10 g) was transferred to dark-coloured flasks and with 200 ml of solvent (water, methanol, ethyl acetate, acetone, petroleum ether) respectively and stored at room temperature.After 24 h, infusions were filtered through Whatman No. 1 filter paper and residue was re-extracted with equal volume of solvents.After 48 h, the process was repeated.Combined supernatants were evaporated to dryness under vacuum at 40°C using Rotary evaporator.The obtained extracts were kept in sterile sample tubes and stored in a refrigerator at 4°C.

Determination of total phenolic contents in the plant extracts
The total phenolic content was determined using spectrophotometric method [12].The reaction mixture was prepared by mixing 0.5 ml of methanolic solution (1 mg/ml) of extract, 2.5 ml of 10% Folin-Ciocalteu's reagent dissolved in water and 2.5 ml 7.5% NaHCO 3 .The samples were incubated at 45°C for 15 min.The absorbance was determined at λ max = 765 nm.The samples were prepared in triplicate and the mean value of absorbance was obtained.Blank was concomitantly prepared, with methanol instead of extract solution.The same procedure was repeated for the gallic acid and the calibration line was construed.The total phenolic content was expressed in terms of gallic acid equivalent (mg of GaA/g of extract).

Determination of flavonoid concentrations in the plant extracts
The concentrations of flavonoids was determined using spectrophotometric method [13].The sample contained 1 ml of methanolic solution of the extract in the concentration of 1 mg/ml and 1 ml of 2% AlCl 3 solution dissolved in methanol.The samples were incubated for an hour at room temperature.The absorbance was determined at λ max = 415 nm.The samples were prepared in triplicate and the mean value of absorbance was obtained.The same procedure was repeated for the rutin and the calibration line was construed.Concentration of flavonoids in extracts was expressed in terms of rutin equivalent (mg of Ru/g of extract).

Evaluation of DPPH scavenging activity
The ability of the plant extract and reference substance to scavenge DPPH free radicals was assessed using the method described by Tekao et al. [14], adopted with suitable modifications from Kumarasamy et al. [15], The stock solution of the plant extract was prepared in methanol to achieve the concentration of 1 mg/ml.Dilutions were made to obtain concentrations of 500, 250, 125, 62.5, 31.25,15.62, 7.81, 3.90, 1.99, 0.97 µg/ml.Diluted solutions (1 ml each) were mixed with 1 ml of DPPH methanolic solution (80 µg/ml).After 30 min in darkness at room temperature (23°C), the absorbance was recorded at 517 nm.The control samples contained all the reagents except the extract.The percentage inhibition was calculated using equation: % inhibition = 100 × (A of control − A of sample)/A of control, whilst concentrations of extracts required to inhibit radical by 50% (IC 50 ) values were estimated from the % inhibition versus concentration sigmoidal curve, using a non-linear regression analysis.The data were presented as mean values ± standard deviation (N = 3).

Statistical analysis
All experimental measurements were carried out in triplicate and are expressed as average of three analyses ± standard deviation.The magnitude of correlation between variables was done using a SPSS (Chicago, IL) statistical software package (SPSS for Windows, ver.XII, 2004).

Results and discussion
The results of the total phenolic content determination of the twenty different extracts from whole plant, leaves, flowers and stems of T. polium, obtained using five different solvents (water, methanol, ethyl acetate, acetone and petroleum ether), are presented in Fig. 1.The content of total phenolic compounds in extracts, expressed as gallic acid equivalents per gram of dry extract, ranged between 14.57 to 157.84 mg of GaA/g.The result in the Fig. 1 indicates a significant difference between concentrations of phenolics in different plant parts.Total phenolic content was the highest in methanolic extracts from different parts of T. polium, among which methanol extract of leaves (157.84 mg of GaA/g) contained the highest concentracion of phenolic compound.Water and acetone extracts also contained high levels of phenolics, while contents of phenolics in ethyl acetate and petroleum ether extracts was lower.The results revealed that total phenolic contents in flowers extracts were significantly lower than those obtained from other plant parts, with the exception of petroleum ether extracts.The petroleum ether extract of flowers had higher total phenolic content (63.45 mg of GaA/g) than whole plant, leave and stem extracts obtained from the same solvent.Phenolic content for Green tea samples analyzed in the study ranged from 16.02 to 233.68 mg of GaA/g dry extract (Tab.1).Among the analyzed extracts, methanolic extract of Green tea had the highest phenolic content (233.68 mg/g) followed by acetone (147.77mg/g) and water (140.11mg/g) extracts.The petrol ether extracts of whole plant, leaves and flovers of T. polium had a higher concentrations of phenolic compounds in relation to the petrol ether extract of Green tea.The extract from other solvents of T. polium had significantly lower concentration of phenolic compounds than those obtained from Green tea.Phenolic content of G. biloba standardized extract was 140.18 mg of GaA/g dry sample (Tab.2).Methanol extract of T. polium leaves (157.84 mg of GaA/g) had a higher concentration of phenolic compounds than G. biloba standardized extract, while the levels of phenolic compounds in other extracts of T. polium were less.
The variability in the total phenolic contents in different extracts could be the result of the varying solubility of the phenolic compounds; this variation in solubility may be driven by the solvent polarity [16].Some studies showed that methanol and ethanol were better extraction solvents for phenolics from plant materials than less polar solvents including acetone and hexane [17,18].The solubility of phenolics is governed by the chemical nature of the plant sample, as well as the polarity of the solvents used.Plant materials may contain phenolics varying from simple (e.g., phenolic acids, anthocyanins) to highly polymerized substances (e.g., tannins) in different quantities.Moreover, phenolics may also be associated with other plant components such as carbohydrates and proteins.Therefore, there is no universal extraction procedure suitable for extraction of all plant phenolics.Solvents, such as methanol, ethanol, acetone, ethyl acetate, and their combinations have been used for the extraction of phenolics from plant materials, often with different proportions of water [19].According to another study, a less polar solvent such as acetone could extract more phenolic compounds from the flowers than more polar solvents, including methanol and water.These differences may be due to the types of phenolic compounds in the plant materials.In general, a good balance in polarity is needed in extracting phenolics from plant sources [20,21].In particular, methanol has been generally found to be more efficient in extraction of lower molecular weight polyphenols while the higher molecular weight flavanols are better extracted with aqueous acetone.Ethanol is another good solvent for polyphenol extraction and is safe for human consumption [22,23].
The concentration of flavonoids in various extracts of T. polium was determined using spectrophotometric method with aluminum chloride.The content of flavonoids was expressed in terms of rutin equivalents (mg of Ru/g of extract).The summary of quantities of flavonoids identified in the tested extracts is shown in Fig. 2. The concentrations of flavonoids in plant extracts ranged from 6.48 to 139.87 mg of Ru/g.The acetone extract of leaves showed higher amounts of flavonoid content (139.87 mg of Ru/g) than the other examined extracts.In contrast, flavonoid concentrations of water and petroleum ether extracts were the lowest.The obtained values of the flavonoid concentrations in Green tea varied from 34.18 to 335.40 mg of Ru/g (Tab.1).In the comparison of flavonoid concentrations between Green tea and T. polium only the methanolic extracts of T. polium showed higher or approximately equal values than methanolic extract of Green tea.Water, acetone, ethyl acetate and petroleum ether extracts of Green tea had greater concentration of flavonoids than T. polium extracts obtained from the same solvents.Based on the flavonoid contents of the extracts, acetone was the best extraction solvent to extract flavonoids from T. polium and Green tea.The flavonoid concentration in G. biloba standardized extract was 192.69 mg of Ru/g, (Tab.2) -higher than the values of all T. polium extracts.Based on the flavonoid contents of the extract, acetone was the best extraction solvent to extract flavonoids from T. polium and Green tea.
The concentration of each extract required to inhibit radical by 50% (IC 50 ) is shown in Tab. 3. Parallel to the examination  Tab. 1 Antioxidant (DPPH scavenging) activity of investigated plant extracts from T. polium presented as IC 50 values (µg/ml).
Each value is the average of three analyses ± standard deviation.
of the antioxidant activity of the plant extracts, the values for two well-known medicinal plants -Green tea (Tab. 1) and G. biloba (Tab.2) -were obtained and compared to the values of the antioxidant activity.The reference antioxidants used were rutin, chlorogenic acid and BHA.The scavenging activity of the reference compounds are summarized in Tab. 4. In comparison to antioxidant activity of pure reference antioxidants (Tab.4), investigated under same conditions, had IC 50 value between 11.65 and 5.39 µg/ml, hence, the activities of the extracts were moderate.The investigated extracts of T. polium demonstrated very different radical-scavenging activities and IC 50 values varied between 2190.75 and 26.30 µg/ml.The methanolic leaves extract showed the greatest potency (IC 50 = 26.30µg/ ml) among the twenty extracts, and exhibited the greatest radical-scavenging activity.When compared to the activity of natural and synthetic standards, such as rutin (IC 50 = 9.28 µg/ ml), chlorogenic acid (IC 50 = 11.65 µg/ml) and BHA (IC 50 = 5.39 µg/ml), DPPH scavenging activity of methanolic leaves extract is very high, considering that the extract is a mixture of a great number of components opposite pure compounds used as standards.Methanolic extract of flowers also showed considerable DPPH scavenging activity with IC 50 value of 41.23 µg/ml.Water (IC 50 = 56.40µg/ml) and methanol (IC 50 = 59.37 µg/ml) extracts of whole plant, as well as water (IC 50 = 59.12 µg/ml) and methanol (IC 50 = 59.28 µg/ml) extracts of stems showed similar antioxidant capacity, while acetone extracts had less DPPH scavenging activity.In average, the petroleum ether and ethyl acetate extracts were the least active in comparison of the corresponding extracts obtained from other solvents.The values of antioxidant activity of Green tea obtained for comparison with T. polium ranged from 14.50 to 238.25 µg/ mL.Results (Tab.4) clearly indicate that all extracts exhibited antioxidant activity as follows: methanol > water > acetone > ethyl acetate > petroleum ether.Comparing the antioxidant activity of Green tea and T. polium, only the methanol extract from leaves of T. polium showed values approximate to Green tea, while the rest of the extracts were with less activity than the corresponding extracts of Green tea.The value of antioxidant activity of G. biloba standardized extract was 33.91 µg/ml, (Tab.2).In comparison, methanol extract from leaves of T. polium showed higher activity than G. biloba, while methanolic extract of flowers T. polium, showed particularly high free radical scavenging activity (IC 50 = 41.23 µg/ml), close to the activity of G. biloba standardized extract.All the other extracts of T. polium showed smaller activity than G. biloba extracts.
In the previous phytochemical screening of T. polium phenylethanoid glycosides (verbascoside and poliumoside), apigenin, 4' ,7 dimethoxy apigenin, 3' ,6 dimethoxy apigenin and rutin were identified as very active compounds.For flavonoids found in a T. polium in a number of studies demonstrated the significant antioxidant activity [24].Phenolic compounds possess ideal structure chemistry for free radical scavenging activities because they have phenolic hydroxyl groups that are prone to donate a hydrogen atom or an electron to a free radical and extended conjugated aromatic system to delocalize an unpaired electron.As an alternative antioxidant property, some phenolic compounds with dihydroxy groups can conjugate transition metals, preventing metal-induced free radical formation [19].Comparing the concentration of phenolic compounds and values for DPPH scavenging activity we found that extracts with the highest concentrations of phenolic compounds also have strong scavenging effect.Based on these results, each extracts of T. polium exhibited phenolic concentration-dependent scavenging effects.Numerous investigations of the antioxidant activity of plant extracts have confirmed a high Tab. 2 Values of antioxidant (DPPH scavenging) activity of standard substances obtained for comparison with the values of T. polium.
Each value is the average of three analyses ± standard deviation.Tab. 3 Values of total phenolic content (mg of GaA/g of extract), flavonoid concentrations (mg of Ru/g of extract) and antioxidant activity (IC 50 -µg/ml) of Green tea extracts obtained for comparison with the values of T. polium.
Each value is the average of three analyses ± standard deviation.
linear correlation between the values of phenolic content and antioxidant activity [25].The difference in morpho-anatomical structure and physiological activity of different plant parts contribute to varying phenolic content and antioxidative activity values.The authors who have comparatively analyzed the antioxidant activity of different parts of other plants have obtained similar results.Our data confirms the results of Özen et al. [26], in their comparative study of Urtica pilulifera: leaf extracts have higher concentrations of phenolic compounds and greater antioxidant activity than other plant parts.
In addition, the phenolic contents of extracts depend on the plant part used in the experiment and solvents used for extraction, and not only the concentration of phenolic contents but also properties of these compounds contribute to the activites of different extracts.The obtained results indicate that the methanolic extract from the leaves of T. polium shows a stronger antioxidant activity than those from the other plant parts also equal in activity with Ginkgo or Green tea.It seems the reason for this difference just is that the phenolic content in the methanolic extract from the leaves is higher than in those from other parts.Comparative analysis of different plant parts can be helpful when estimating the beneficial properties of T. polium extracts as valuable medicinal raw plant materials to be used for natural antioxidants in phytopharmacy.Further studies of this plant species should be directed to a detailed qualitative analysis of all its parts and carry out in vivo evaluation antioxidant of antioxidant properties.
Tab. 4 Values of total phenolic content (mg of GaA/g of extract) and flavonoid concentrations (mg of Ru/g of extract) and antioxidant activity (IC 50 -µg/ml) of G. biloba standardized extract obtained for comparison with the values of T. polium.
Each value is the average of three analyses ± standard deviation.

Fig. 1
Fig. 1 Total phenolic contents in the plant extracts of T. polium expressed in terms of gallic acid equivalent (mg of GaA/g of extract).

Fig. 2
Fig. 2 Flavonoid concentrations in the plant extracts of T. polium expressed in terms of rutin equivalent (mg of Ru/g of extract).