Anti-inflammatory, Gastroprotective, and Cytotoxic Effects of Sideritis scardica Extracts

• Abstract
• Introduction
• Materials and Methods
• General
• Plant material and the procedure for plant material extraction
• Animals
• Determination of total phenols content
• Tannins content
• Total flavonoids content
• HPLC procedure
• Determination of the free radical scavenging activity
• Carrageenan-induced rat paw edema
• Absolute ethanol-induced stress ulcer in rats
• Cell lines
• Determination of cell viability
• Analysis of apoptosis and cell cycle
• ROS measurement
• Lactate dehydrogenase release assay
• Statistical analysis
• Results
• Discussion
• Acknowledgements
• References

Abstract

Sideritis scardica Griseb. (ironwort, mountain tea), an endemic plant of the Balkan Peninsula, has been used in traditional medicine in the treatment of gastrointestinal complaints, inflammation, and rheumatic disorders. This study aimed to evaluate its gastroprotective and anti-inflammatory activities. Besides, continuously increasing interest in assessing the role of the plant active constituents preventing the risk of cancer was a reason to make a detailed examination of the investigated ethanol, diethyl ether, ethyl acetate, and n-butanol extracts regarding cytotoxicity. Oral administration of the investigated extracts caused a dose-dependent anti-inflammatory effect in a model of carrageenan-induced rat paw edema. Gastroprotective activity of the extracts was investigated using an ethanol-induced acute stress ulcer in rats. The cytotoxic activity of plant extracts was assessed on PBMC, B16, and HL-60 cells and compared to the cytotoxicity of phenolic compounds identified in extracts. Apoptotic and necrotic cell death were analyzed by double staining with fluoresceinisothiocyanate (FITC)-conjugated annexin V and PI. The developed HPLC method enabled qualitative fingerprint analysis of phenolic compounds in the investigated extracts. Compared to the effect of the positive control, the anti-inflammatory drug indomethacine (4 mg/kg), which produced a 50 % decrease in inflammation, diethyl ether and n-butanol extracts exhibited about the same effect in doses of 200 and 100 mg/kg (53.6 and 48.7 %; 48.4 and 49.9 %, respectively). All investigated extracts produced dose-dependent gastroprotective activity with the efficacy comparable to that of the reference drug ranitidine. The diethyl ether extract showed significant dose-dependent cytotoxicity on B16 cells and HL-60 cells, decreasing cell growth to 51.3 % and 77.5 % of control, respectively, when used at 100 µg/mL. It seems that phenolic compounds (apigenin, luteolin, and their corresponding glycosides) are responsible for the diethyl ether extract cytotoxic effect. It also appears that induction of oxidative stress might be involved in its cytotoxicity, since B16 and HL-60 cells increased their ROS production in response to treatment with diethyl ether extract. Neither of the tested extracts nor any phenolic compounds showed significant cytotoxic effect to human PBMC. These results demonstrated the potent anti-inflammatory and gastroprotective activities, as well as the promising cytotoxicity.

Introduction

The promising new source of therapeutic agents refers to plant secondary metabolites, irregularly occurring compounds that characterize certain plants or plant groups. There is continuously increasing interest in assessing the role of the phenolic compounds which show antioxidative properties and may act with beneficial health effects, reducing the risk of chronic diseases (inflammation, cancer, osteporosis, and cardiovascular diseases). Among them, flavonoids, as a large group of plant secondary metabolites, have been produced in the plant for the purpose of protection from photosynthetic stress, reactive oxygen species (ROS), wounds, and herbivores. Studies of flavonoids have revealed the most compelling data for cytotoxic activities in various types of cancers, and several flavonoids have been shown to inhibit cancer development while exhibiting antioxidant activities in different animal models. Furthermore, some studies suggest that the most promising use of these compounds may be as an adjuvant to currently used therapies in antitumor treatment [1].

The results of numerous preliminary investigations of plants belonging to the genus Sideritis L. revealed a plant-derived source of particular pharmacological and nutritional interest. The genus Sideritis L. (Lamiaceae) includes approximately 150 species of annual and perennial plants distributed mainly in the Mediterranean region. This genus is divided into two subgenera, Sideritis and Marrubiastrum, formed by the European and Macaronesian species, respectively. So far, different biological activities of Sideritis species have been reported: anti-inflammatory, antiulcer, analgesic, antimicrobial and antifungal [2], [3], [4], [5], [6], immunomodulating [7], macrophage NOS-2-expression inhibiting [8], and hypoglycemic [5]. Recently, aldose reductase inhibiting activity [9], antiproliferative, anticholinesterase, and selective estrogen receptor modulator-like effects have been reported [10], [11], [12]. The previous studies of Sideritis species reported the presence of flavonoid aglycones and glycosides, phenolic acids, di- and triterpenoids, fatty acids, coumarines and iridoid glycosides [3], [9], [11], [13], [14], [15], [16], and essential oil as well [2]. Most of the studies on Sideritis species attributed the previously cited biological activities mainly to phenolic compounds [9], [13]. Rios et al. [17] reported that flavonoids were reducing agents able to interact with free radical species (of relevance to autoxidation mechanism) and could prevent generation of inflammatory mediators.

The genus Sideritis is represented in Serbia by one species only, S. montana L. [18], but because of its pro-oxidant properties this plant has not been used in traditional medicine [19]. S. scardica Griseb. (ironwort, mountain tea) is an endemic plant of the Balkan Peninsula belonging to the Empedoclea section. Aerial parts of mountain tea are traditionally known for their anti-inflammatory, antimicrobial, antibacterial, antirheumatic, and gastroprotective properties. S. scardica is used as a loosening agent in bronchitis and bronchial asthma, against common cold and lung emphysema. It has been imported in Serbia from the former Yugoslav Republic of Macedonia and Albania and widely used in the treatment of inflammation, gastrointestinal disorders, and coughs, as well as an active constituent of dietary supplements for the prevention of anemia. In the literature, all previously cited biological activities are mainly attributed to the phenolic content of this plant [14].

The present study aimed to investigate anti-inflammatory and gastroprotective activities of S. scardica extracts in order to examine the above-stated folkloric utilizations and to establish the correlation between observed activities and phenolic constituents of the extracts based on previous studies which have recognized flavonoids in S. scardica as potent biologically active substances. Besides, in the present study we investigated the in vitro cytotoxic action of S. scardica extracts in order to establish the connection of significant antitumor potential and the polyphenol components present in the examined extracts. Qualitative and quantitative fingerprint analyses of polyphenolic compounds in the investigated extracts were also conducted applying the HPLC method.

Materials and Methods

General

Sodium bicarbonate (analytical grade), DPPH (1,1′-diphenyl-2-picrylhydrazyl; analytical grade), indomethacin (purity ≥ 99.0 %), carrageenan (EP grade), and trolox (purity ≥ 99.0 %) were purchased from Sigma-Aldrich. Analytical grade reagents 2,6-di-tert-butyl-4-methylphenol (BHT, purity ≥ 99.8 %), ether, petrol, dimethyl sulfoxide (DMSO), ethyl acetate, n-butanol (BuOH), acetone, and absolute ethanol (96 %, v/v) were purchased from Merck. Acetonitrile (MeCN), water, and methanol were of HPLC grade and also from Merck. Reference HPLC standards p-coumaric (purity ≥ 99.0 %), protocatechuic (purity ≥ 99.0 %), chlorogenic (purity ≥ 99.0 %), vanillic (purity ≥ 95.0 %), caffeic (purity ≥ 90.0 %), ferulic (purity ≥ 99.0 %), and syringic (purity ≥ 95.0 %) acids, luteolin-7-O-β-glucoside (purity ≥ 98.0 %), apigenin-7-O-β-glucoside (purity ≥ 99.0 %), luteolin (purity ≥ 99.0 %), chrysoeriol (purity ≥ 99.0 %), apigenin (purity ≥ 99.0 %), hyperoside (purity ≥ 99.0 %), gallic acid (purity ≥ 99.0 %), pyrogallol (purity ≥ 99.0 %), and cisplatin [cis-diamineplatinum(II) dichloride, purity ≥ 99.9 %] were purchased from Sigma or from Extrasynthese. Their purity was declared as stated previously, based on the manufacturer's internal high-precision HPLC method. Ranitidine, purity ≥ 95.0 % (Ranisan ampoules), was purchased from Zdravlje-Actavis Company.

Plant material and the procedure for plant material extraction

The wild growing species Sideritis scardica Griseb., Lamiaceae, was collected on Shara Mountain (at the hill foot of the Ljuboten, at ca. 1300 m) during the time of flowering. Plant material was air dried, packed in paper bags and kept in a dark and cool place until analysis. Plant material was verified, and the voucher specimen of the plant (SS/08) was deposited at the Herbarium of the Botanical Garden, Jevremovac, Belgrade, Serbia. The identification was provided by Prof. Dmitar Lakušić (Institute of Botany and Botanical Garden, Faculty of Biology, University of Belgrade). The shade-dried and powdered aerial parts of S. scardica (600 g) were coarsely extracted using 70 % (V/V) ethanol. The yield of the final extract (crude extract, 1) in terms of starting crude material was determined to be 16.7 %. The crude ethanol extract (1) was redissolved in distilled water, shaken vigorously and extracted with 600 mL of diethyl ether, 600 mL ethyl acetate, and 600 mL saturated n-butanol in a separating funnel, successively. The obtained extracts were: diethylether extract, 2 (2.8 g); ethyl acetate extract, 3 (1.3 g); and n-butanol extract, 4 (4.4 g). The yield of extraction for extracts 2, 3, and 4 was 16.7, 7.5, and 26.3 % in crude extracts, or 0.46, 0.21, and 0.73 % of dry plant, respectively.

Animals

Adult, male Wistar rats weighing 200–300 g were used for estimating mountain tea ethanol extract anti-inflammatory (carrageenan-induced paw edema test) and gastroprotective activities (absolute ethanol-induced stress ulcer test). Experimental groups consisted of 6–10 animals each. The animals were deprived of food for 18–20 h before the beginning of experiments with free access to tap water.

This study was performed after approval from the local Institutional Animal Care and Use Committee and run in accordance to the statements of the European Union regarding handling of experimental animals (approval number 86/609/EEC, 31.01.2008).

Determination of total phenols content

The total phenolic content was determined by the Folin-Ciocalteu method [20]. One hundred microliters of the MeOH solution of the dry investigated extracts 1, 2, 3, and 4 (15.75, 31.5, and 63; 27.75, 55.5, and 138.75; 7.28, 14.56, and 19.13; and 7.06, 14.13, and 28.25 µg/mL final quantity, respectively) were mixed with 0.75 mL of Folin-Ciocalteu reagent (previously diluted 10-fold with distilled water) and allowed to stand at 22 °C for 5 min; 0.75 mL of sodium bicarbonate (60 g/L) solution was added to the mixture. After 90 min at 22 °C, absorbance was measured at 725 nm. Gallic acid (0–100 mg/L) was used for calibration of a standard curve. The calibration curve showed the linear regression at r > 0.99, and the results were expressed as milligrams of gallic acid equivalents per gram of plant extracts dry weight (mg GAE/g DW). Triplicate measurements were taken, and data were presented as mean ± standard deviation (SD).

Tannins content

The percentage content of tannins was calculated using the method described in the European Pharmacopoeia, Ph. Eur. 6.0 [21]. Shortly, decoctions prepared from the investigated extracts were treated with phosphomolybdotungstic reagent in alkaline medium after and without treatment with hide powder. The absorbance was measured by UV-VIS spectrophotometer HP 8453 (Agilent Technologies) at λ _max 760 nm. From the difference in absorbance of total polyphenols and polyphenols not adsorbed by hide powder, the percentage content of tannins expressed as pyrogallol (%, w/w) was calculated from the expression:

where m₁ represents mass of the sample to be examined, in grams; and m₂ is mass of pyrogallol, in grams. The results represent the mean ± SD of three determinations.

Total flavonoids content

The percentage content of flavonoids was calculated using the method described in the European Pharmacopoeia, Ph. Eur. 6.0 [21]. Briefly, the sample was extracted with acetone/HCl under reflux condenser; the AlCl₃ complex of the flavonoid fraction extracted by ethyl acetate was measured by UV-VIS spectrophotometer HP 8453 at 425 nm. The content of flavonoids, expressed as hyperoside percentage, was presented as the mean ± standard deviation of three determinations.

HPLC procedure

Chromatographic fingerprint of the extract and quantification of identified compounds were achieved by HPLC (Agilent Technologies 1200). Detection was performed using diode array detector (DAD), and the chromatograms were recorded at λ = 260 nm (for protocatechin and syringic acid), 280 nm (for chlorogenic, vanilic, p-coumaric, and caffeic acids), 325 nm (for ferulic acid), and 360 nm (for flavonoids). HPLC separation of components was achieved using a LiChrospher 100 RP 18e (5 µm), 250 × 4 mm i. d. column, with a flow rate of 1 mL/min and mobile phase, A [500 mL of H₂O plus 9.8 mL of 85 % H3PO4 (w/w)], B (MeCN), elution, combination of gradient mode: 90–75 % A, 0–25 min; isocratic 75 % A, 25–30 min; 75–55 % A, 30–46 min. The sample was prepared dissolving 118.6, 49.4, 9.4, and 53.0 mg of the extracts 1, 2, 3, and 4, respectively (obtained by the procedure previously described) in 10 mL of MeOH, filtered through 0.2 µm PTFE filters prior to HPLC analysis. The injected volume was 4 µL. Standard solutions for the determination of flavonoids and polyphenolic acid were prepared at a final concentration of 0.01 mg/mL (protocatechin, p-coumaric, vanilic, ferulic, and syringic acids, as well as luteolin and chrysoeriol), 0.05 mg/mL (chlorogenic and caffeic acids, and apigenin), or 0.12 mg/mL (apigenin-7-O-glycoside and luteolin-7-O-glycoside) in methanol. For the purpose of the phenolic compounds identification and determination in the investigated extracts, three mixtures of the standards were prepared with the already mentioned concentrations: mix 1 with caffeic acid, apigenin-7-O-glycoside, and apigenin; mix 2 with chlorogenic acid, luteolin-7-O-glycoside, luteolin, and chrysoeriol; mix 3 contained the rest of the investigated phenolic compounds. The volume injected was 4 µL, the same as the investigated extract. The identification was carried out based on retention time and spectral matching. Once spectral matching succeeded, results were confirmed by spiking with respective standards to achieve a complete identification by means of the so-called peak purity test. Those peaks not fulfilling these requirements were not quantified. Quantification was performed by external calibration with standards.

Determination of the free radical scavenging activity

The DPPH scavenging assay was carried out according to the procedure described by Blois, with some modifications [22]. Various concentrations of the samples (100 µL) were mixed with 900 µL of 0.04 mg/mL methanolic solution of DPPH. UV spectra were recorded on a UV-VIS spectrophotometer HP 8453. Absorbance at 517 nm was measured after 20 min. The inhibition percentage was calculated using the following equation:

I = [(A_c − A_s)/A_c] × 100

where I was the inhibition percentage, A_c was the absorbance of the negative control (contained 100 µL of MeOH instead of the samples), and A_s was the absorbance of the samples. Synthetic antioxidants, trolox, and tert-butyl hydroxytoluene (BHT) were used as positive controls. The inhibition percentage was plotted against concentration of the samples, and IC₅₀ values, determined by linear regression analysis, were presented as the mean ± standard deviation of three determinations.

Carrageenan-induced rat paw edema

The carrageenan-induced rat paw edema test was used as an experimental model for screening the anti-inflammatory activity according to the modified method of Oyanagui and Sato [23]. The extracts were administered p. o. in doses of 50, 100, and 200 mg/kg. Indomethacin, dissolved in DMSO, was used as a reference in a dose of 4 mg/kg p. o., which was a dose producing 50 % reduction of rat paw edema. The control animals were given DMSO in a dose of 1 mL/kg p. o. Carrageenan-saline solution (0.5 % in a volume of 0.1 mL) was injected into the plantar surface of the right hind paw 1 h after the oral administration of the extracts or indomethacin. A pure saline solution (0.9 % NaCl, 0.1 mL) was injected into the left hindpaw, which served as a control (non-inflamed paw). The animals were killed 3 h after the carrageenan injection, and the paws were cut off for weighing. The difference in weight between the right and left paw, treated versus untreated (control) rats, served as an indicator of the inflammatory response intensity (i.e., anti-inflammatory activity). The percent of anti-inflammatory effect was calculated from the expression

where Δk represents the difference in the paw weight in the control group; Δe is the difference in the paw weight in the treatment group.

Absolute ethanol-induced stress ulcer in rats

To study the gastroprotective activity of the investigated extracts, an experimental model of acute gastric mucosa damage induced by absolute ethanol (1 mL/rat p. o.) was used. The investigated extracts, dissolved in DMSO, were administered p. o. in doses of 50–200 mg/kg 60 min prior to ethanol. Ranitidine given in doses of 5–20 mg/kg p. o. was used as a reference drug. The control animals were given the vehicle in a dose of 1 mL/kg p. o., also 60 min before ethanol. The animals were sacrificed 1 h after giving ethanol, and their stomachs were removed and opened along the greater curvature. Lesions were examined under an illuminated magnifier (3×). The intensity of gastric lesions was assessed according to a modified scoring system of Adami et al. [24].

Cell lines

Mouse melanoma cell line (B16) and the human promyelocytic leukemia cell line (HL-60) were obtained from the European Collection of Cell Cultures (ECACC) while peripheral blood mononuclear cells (PBMC) were obtained from healthy blood donors after written informal consent. This study was approved by the Ethical Committee of the School of Medicine, University of Belgrade (approval number 89/101/EC, 24.02.2011). All cell lines were maintained at 37 °C in a humidified atmosphere with 5 % CO₂. B16 mouse melanoma cell lines were cultured in DMEM supplemented with 5 % fetal calf serum-FCS while the HL-60 human leukemia cell line and PBMC were cultivated in RPMI supplemented with 10 % FCS.

The adherent cells were prepared for experiments using the conventional trypsinization procedure with trypsin/EDTA and incubated in 96-well flat-bottom plates (2 × 10⁴ cells/well) for viability and LDH analyses and in 6-well flat-bottom plates for flow cytometry analyses (3 × 10⁵ cells/well). Cells were rested for 24 h and then treated with plant extracts and different flavonoids: apigenin, luteolin, chlorogenic acid, apigenin 7-O-glucoside, luteolin 7-O-glucoside, chrysoeriol, and ferrulic acid. Suspension cells were cultivated in 96-well flat-bottom plates (3.5 × 10⁴ cells/well) for assessing cell viability, and in 24-well flat-bottom plates for flow cytometry analyses (1.5 × 10⁵ cells/well). Cells were rested for 2 h and then treated with plant extracts or flavonoids. The extracts and flavonoids were dissolved in dimethyl sulfoxide-DMSO and diluted in appropriate medium. Final concentration of DMSO in the incubation mixture did not exceed 0.1 % and did not have any influence on cell viability. Cisplatin (25 µM) was used as the positive control in all methods except for lactate dehydrogenase (LDH) release assay where Triton X-100 (3 %) was used.

Determination of cell viability

Cell viability was assessed using acid-phosphatase method. Briefly, after treatment (24 h), adherent cells were washed twice with phosphate buffered saline, and 100 µL of reaction mixture (0.1 M acetate buffer pH 5.5, containing para-nitrophenyl phosphate PNPP and 0.1 % Triton-X) was added to each well. After 90 minutes, the reaction was stopped by adding 50 µL of 0.1 M NaOH. The absorbance of the developed yellow color, which was directly proportional to the cells viability [25], was measured by an automated microplate reader at 405 nm. The results were presented as percent of the control value (untreated cells), which was arbitrarily set to 100 %. For determination of suspension cell viability, we used a modified acid-phosphatase method. After treatment, 50 µL of reaction mixture (0.3 M acetate buffer pH 5.5, containing PNPP and 0.2 % Triton-X) was added to each well. After 60 minutes, the reaction was stopped by the addition of 50 µL of 0.3 M NaOH into each well, and absorbance was read as described. At the same time blanks, containing cell culture medium (without cells), were prepared to achieve the correction for the absorbance caused by medium color (at 405 nm).

Analysis of apoptosis and cell cycle

Apoptotic and necrotic cell death were analyzed by double staining with fluoresceinisothiocyanate (FITC)-conjugated annexin V and PI, in which annexin V bound to the apoptotic cells with exposed phosphatidylserine, while PI labeled the necrotic cells with membrane damage. This staining was performed according to the manufacturer's instructions (BD Pharmingen).

The cell cycle was analyzed by measuring the amount of propidiumiodide (PI)-labeled DNA in ethanol-fixed cells, exactly as previously described [26]. DNA fragmentation, as another marker of apoptosis, was determined during cell cycle analysis by counting the hypodiploid cells in the sub-G0/G1 cell cycle phase.

The green (FL1) and red (FL2) fluorescences of annexin/PI-stained live cells and PI stained fixed cells were analyzed with FACSCalibur flow cytometer (BD).

The number of viable (annexin−/PI−), apoptotic (annexin+/PI−), and necrotic (annexin+/PI+) cells as well as the proportion of cells in different cell cycle phases were determined with Cell Quest Pro software (BD). Ten thousand cells (gated to exclude cell debris) were analyzed in each sample.

ROS measurement

Intracellular production of ROS was determined by measuring the intensity of green fluorescence emitted by redox-sensitive dye dihydrorhodamine 123 (DHR; Invitrogen), which was added to cell cultures (2.5 µM) at the beginning of the treatment. At the end of incubation, cells were detached by trypsinization, washed in PBS, and the green fluorescence (FL1) of DHR-stained cells was analyzed using a FACSCalibur flow cytometer. The results are expressed as mean intensity of DHR fluorescence.

Lactate dehydrogenase release assay

The release of the cytosolic enzyme lactate dehydrogenase (LDH) reflects a loss of membrane integrity in dying cells, and it was assessed by a colorimetric assay as previously described [27]. Briefly, 100 µL of cell culture supernatant after treatment (cells grown in colorless medium) was mixed with 100 µL of solution containing 54 mM lactic acid, 0.28 mM phenazine methosulfate, 0.66 mM p-iodonitrotetrazolium violet, and 1.3 mM NAD⁺. The pyruvate-mediated conversion of 2,4-dinitrophenylhydrazine into visible hydrazone precipitate was measured using an automated microplate reader at 492 nm. The total loss of membrane integrity resulting in complete loss of cell viability was determined by lysing the cells with 3 % Triton X-100 and using this sample as a positive control. The cytotoxicity in LDH release test was calculated using the formula: (E-C)/(T-C) × 100, where E is the experimental absorbance of cell cultures, C is the control absorbance of cell culture medium, and T is the absorbance corresponding to the maximal (100 %) LDH release of Triton X-100-lysed cells (positive control).

Statistical analysis

The statistical significance of the observed differences was analyzed by the Mann-Whitney U- test and the Kruskal–Wallis test (in tests for anti-inflammatory and gastroprotective activities, respectively) or by t-test or ANOVA followed by the Student-Newman-Keuls test. A value of p < 0.05 was considered significant.

Results

The current study evaluated the anti-inflammatory and gastroprotective activities of various extracts (1–4) of mountain tea and explored further perspectives in the investigation of mountain tea cytotoxic activity.

Each extract was analyzed phytochemically. Total phenolic amount ranged from 84.2 to 345.6 mg of gallic acid equivalents per gram of plant extracts dry weight; the content of flavonoids, expressed as hyperoside percentage, ranged from 0.4 to 1.1, while the percentage contents of tannins were found to be from 0.5 to 5.7 (data summarized in [Table 1]). Quantitative analysis of total phenolics, flavonoids, and tannins content, presented in [Table 1], pointed out a high amount of phenolic compounds in the investigated extracts. The total phenolics content in crude ethanolic extract 1 was smaller in comparison to the one of the methanolic extract of S. condensata Boiss. & Heldr. and S. erythrantha var. erythrantha Boiss. & Heldr. [28], but the extracts 3 and 4 were much richer in total phenols ([Table 1]). The total phenolics content in S. scardica methanolic extract reported by Tunalier et al. [29] was comparable to the results presented here regarding the total phenolics content in the investigated extracts 3 and 4 of S. scardica extract 1. An HPLC method has been developed for analysis of phenolic compounds, and as analyzed by HPLC, ferulic acid was the dominant component in all investigated samples and composed 0.36, 2.34, and 2.92 % of the extracts 2, 3, and 4, respectively ([Table 2]). The identified compounds are presented in [Table 2] regarding their retention times. Extract 2 appeared to be more abundant in flavonoid aglycones in comparison to the more polar extracts 3 and 4. Other identified components were protocatechuic (1), chlorogenic acid (2), vanillic (3), caffeic (4), syringic (5), p-coumaric and ferulic (6) acid, luteolin-7-O-β-glucoside (8), apigenin-7-O-β-glucoside (9), luteolin (10), chrysoeriol (11), and apigenin (12) ([Figs. 1] and [2], [Table 2]). The percentage contents of hydroxycinnamic (2, 4, 6, and 7), 4-hydroxybenzoic acid derivatives (1, 3, and 5), and flavonoids (8-12) in extracts were different (data summarized in [Fig. 2 C]).

Antioxidant activity of S. scardica extracts of different polarities was investigated by the DPPH⁺ free radical scavenging method. The results demonstrated that the extracts tested possessed DPPH free radical scavenging activity. When the extracts were applied in the concentration range of 466.0–27.5 µg/mL, their DPPH free radical scavenging activity varied approximately from 20 to 90 %, respectively, with IC₅₀ values from 147.0 to 5.7 µg/mL ([Table 1]). Trolox and BHT, known as potent antioxidants, served as positive controls. n-Butanol extract (4) showed antioxidant activity comparable to positive controls ([Table 1]).

Investigated extracts applied in the doses of 50, 100, and 200 mg/kg significantly reduced the carrageenan rat paw edema. Diethyl ether extract, 2, possessed the strongest anti-inflammatory activity, reducing the paw edema in a dose-dependent manner. The reduction of the edema, achieved by the doses of 100 and 200 mg/kg used, was statistically significant, and at a level comparable to the one of the positive control, indomethacine, applied in a dose of 4 mg/kg producing 50 % reduction ([Fig. 3]).

All tested extracts exhibited significant gastroprotective activity, with the most effective proven to be n-butanol extract, 4, whose effect at a dose of 100 mg/kg was even significantly better than ranitidine, which served as a positive control ([Fig. 4]).

The cytotoxic activity of plant extracts was assessed in PBMC, B16 melanoma, and HL-60 leukemic cells and compared to the cytotoxic activity of the main phenolic compounds of extracts. After 24 h of incubation, only diethyl ether extract – extract 2, showed significant dose-dependent cytotoxicity in B16 cells, decreasing cell viability to 51.3 % of the control at a concentration of 100 µg/mL ([Fig. 5]). Among the main phenolic compounds of the extracts, the most cytotoxic were luteolin, apigenin-7-O-β-glycoside, apigenin, and luteolin-7-O-β-glycoside, decreasing B16 cell viability to 48.8 %, 67.3 %, 77.2 %, and 82.0 % of the control, respectively, when used at a concentration of 100 µM ([Fig. 5]). The other phenolic compounds, chlorogenic acid, ferrulic acid, and chrysoeriol, did not affect cell viability. Extract 2 and extract 4 expressed significant cytotoxic potential against HL-60 cells, decreasing their viability to 77.5 % and 81.9 % of the control, respectively, at a concentration of 100 µg/mL ([Fig. 5]). However, the most toxic compounds for HL60 cells were apigenin, which decreased their viability to 34.4 %, luteolin (47.1 %), and their glycosides, luteolin-7-O-β-glucoside and apigenin-7-O-β-glucoside, decreased viability to 66.6 % and 78.4 % compared to the control, respectively ([Fig. 5]). Neither of the tested extracts nor any phenolic compounds showed a significant cytotoxic effect to human PBMC. After 24 h treatment with cisplatin (25 µM), viability of PBMC, B16, and HL-60 cell lines decreased to 73.6 %, 56.7 %, and 59.8 % of the control (untreated cells), respectively.

As the most potent cytotoxic agents for further assessment of the cell death mechanisms, we selected extract 2 (100 µg/mL) and aglycone phenolic compounds apigenin and luteolin (both at 100 µM), because the glycosylated phenolic compounds in vivo enter extensive deglycosylation pathways producing aglycone components that further exhibit biological activity [30]. The LDH assay that reflects loss of membrane integrity in dying cells revealed a dose-dependent increase in LDH release after 24 h treatment with all tested compounds in both cell lines ([Fig. 6]). Accordingly, double staining of HL60 leukemic cells with anexin V-FITC (Ann) and propidium iodide (PI) revealed that extract 2 and its main constituents apigenin and luteolin significantly increased numbers of both early (Ann⁺/PI) and late apoptotic/necrotic cells with membrane damage (Ann⁺/PI⁺) ([Fig. 7]). Similar although somewhat less prominent results were obtained in B16 melanoma cells ([Fig. 7]). DNA content analysis using PI staining showed that all three compounds caused an increase of percentage of HL60 cells in the subG₀ phase, indicating DNA fragmentation ([Fig. 8]). Cisplatin tretated cells (positive control) also showed a significant increase in the number of early and late apoptotic cells (14.6 % Ann⁺/PI ⁻ and 22.6 % Ann⁺/PI ⁺ for B16 and 22.3 % Ann⁺/PI ⁻ and 28.3 % Ann⁺/PI ⁺ for HL-60 cells) as well as increase in the percentage of cells in the subG₀ phase (27.4 % for B16 and 36.5 % for HL-60 cells).

On the other hand, no increase in percentage of cells in subG₀ phase (hypodiploid-apoptotic cells) was observed in less sensitive B16 cells treated with apigenin, luteolin, or extract 2. However, B16 cells treated with apigenin or luteolin displayed a cell cycle block in S/G₂ M phase ([Fig. 8]).

To further investigate the mechanisms underlying cytotoxic action of extract 2, apigenin, and luteolin, we investigated their ability to induce oxidative stress in HL60 and B16 cells. DHR staining demonstrated that both cell types significantly increased their ROS production in response to treatment with extract 2, apigenin, or luteolin ([Fig. 9]). Also, cisplatin-treated cells (positive control) demonstrated the increase in ROS production of 1.8 and a 2.1-fold increase for B16 and HL-60 cells, respectively, compared to untreated cells (negative control). However, the effects of apigenin and extract 2 were more pronounced in B16 cells ([Fig. 9]). It therefore appears that induction of oxidative stress might be involved in the cytotoxic activity of extract 2 and phenolic compounds.

Discussion

The ethanol extract (1) of S. scardica, mountain tea, and diethyl ether (2), ethyl acetate (3), and n-butanol (4) extracts of the crude ethanol extract were evaluated for their traditionally known but here for the first time confirmed anti-inflammatory and gastroprotective activities applying in vivo tests. Further, we investigated the cytotoxicity of the extracts as well, as a worldwide ongoing investigation and survey for novel natural constituents and drugs with cytotoxic properties. In order to establish the correlation of the anti-inflammatory and gastroprotective activities and cytotoxic potential verified in this work to the polyphenol components present in the investigated extracts, the total phenolic content and radical scavenging capacity were determined. Besides, HPLC analysis enabled the identification of 12 phenolic compounds present in different percentages in the investigated extracts.

An antioxidant, which can quench reactive free radicals, can prevent the oxidation of other molecules and may therefore have health-promoting effects in the prevention of degenerative diseases. Namely, free radicals are considered to play an important role in numerous chronic pathologies, such as inflammation, cardiovascular and cancer diseases among others, and are implicated in the aging process. Therefore, the extracts were assessed against DPPH radicals serving as the oxidizing substrate to determine their free radical properties. The antioxidant activity of the tested extracts by the DPPH method was expressed by the parameter IC₅₀, the amount of antioxidant necessary to decrease the initial DPPH concentration by 50 %, where the lower its value, the more efficient the antioxidant ([Table 1]). The antioxidant capacity of the tested extracts of S. scardica aerial parts could be attributed to their phenolic content. A good correlation between phenolic content and antioxidant activity was established ([Table 1]). Though phenolic content served as a reasonable indicator of an extract's overall antioxidant potential, activity in individual assays depends on the quantities and properties of specific phenolics in the tested extracts. The phenolic substances are capable of scavenging reactive oxygen species (O⁻² and OH⁻) and act like hydrogen donors. Apigenin and apigenin glycosides are antioxidant agents due to the acidic 4′-hydroxyl group. Furthermore, the presence of hydroxycinnamic acids enriched the antioxidant capacity. The –CH=CH–CO– group ensures great hydrogen-donating ability and thus enforces the antioxidant capacity [31]. The investigated extracts were shown to be rich in hidroxycinnamic derivatives, which, in respect of identified constituents, were the most abundant group of phenolics in all investigated extracts ([Table 2] and [Fig. 3 C]). The presented results were in good accordance with the data available in the literature [29].

Anti-inflammatory activity has been reported in different kinds of extracts of several Sideritis species so far [4]. In the present study, the results ([Table 2], [Figs. 3] and [4]) indicated that the ethyl ether extract, 2, of S. scardica exhibited the highest anti-inflammatory, while n-butanol extract, 4, possessed the most prominent gastroprotective activity. These results are in good accordance with the uses of this genus and another survey study [9]. Recently, a number of flavonoids, such as apigenin, luteolin, and quercetin, have been reported to exhibit anti-inflammatory activity [31]. The high total phenolic content and capability of the extracts tested for scavenging free radicals might partly be responsible for both their anti-inflammatory and gastroprotective activities as demonstrated in carrageenan-induced paw edema test and ethanol-induced acute gastric damage, respectively. It is thought that in the early phase of the anti-inflammatory response (within the first hour after injecting carrageenan), many vasoactive substances (e.g. histamin, 5-hydroxytryptamin, bradykinins, and prostaglandins) are released. On the contrary, the second phase is related to neutrophil infiltration as well as to the maintaining of the production of arachidonic acid metabolites. In the second phase of acute inflammation, activated polymorphonuclear cells produce a great amount of free radical species that additionally may damage the tissue caught by inflammation. Numerous investigations have shown that flavonoids and phenolcarbonic acids, preventing neutrophil infiltration in the inflammed area and neutralizing free radical species, act as anti-inflammatory agents [32]. Regarding the DPPH-scavenging capacity of the S. scardica extracts tested and high total phenol content, it could be hypothesized that its anti-inflammatory effect in the model of carrageenan-induced acute inflammation is a consequence, at least partly, of their flavonols and phenolcarbonic acid content. Based on the mentioned investigations, our assumption addressed phenolic compounds as the potential carriers of anti-inflammatory activity of investigated extracts.

The results of the present study demonstrated that the investigated S. scardica extracts offered significant protection against the ulcerogenic effect of absolute ethanol in rats, and that this effect was very close to that achieved by the current antiulcer drug ranitidine. As known, the absolute ethanol is noxious for the stomach and that affects the gastric mucosa topically by disrupting its barrier and thus causing hydrogen back diffusion that leads to necrosis. As a result of the disturbed barrier function of the gastric mucosa, a rapid and strong vasoconstriction accompanied by rapid and vigorous arterial dilation occurs. As a consequence, oxyradical-mediated injury of the gastric mucosa results from ischemia followed by reperfusion. The oxygen-derived radicals are directly implicated in that mechanism, and their remotion stimulates the healing of ethanol-induced gastric lesions [33]. Many studies have demonstrated that flavonoids and phenolic acids, known substances with antioxidant properties, may protect against gastric damaging effects of absolute ethanol and possess antiulcerogenic activity. Since the extracts of S. scardica tested in this study contained the phenolic components ([Tables 1] and [2], [Fig. 2]) and showed antioxidant activity, it could be suggested that the significant gastroprotective effect of the extracts, similarly to their anti-inflammatory effect, might at least in part be consequences of the presence of phenolic compounds in this extract.

Today, cancer is becoming one of the most important causes of mortality worldwide. Cancer screening and cancer prevention are a main challenge for the health care system in the developed world. Many studies showed that flavonoids – widespread phytochemicals – have strong cytotoxic properties against different forms of cancer [34], [35]. According to this, we further assessed the in vitro cytotoxic activity of the extracts and their phenolic compounds. We chose the adherent melanoma B16 cell line based on previous results showing that some flavonoids (apigenin, quercetin) inhibit melanoma cell growth and metastatic potential in vivo [35]. In parallel, experiments on leukemia suspension cell line HL60 and on human normal PBMC were performed. The main constituents of plant extracts were also assessed for their cytotoxic activity in order to explain anticancer activity of the extracts. None of the tested extracts showed toxicity to human PBMC, but diethyl ether extract (extract 2), as well as its main constituents, flavonoids apigenin and luteolin, showed significant cytotoxicity to both tumor cell lines. The mechanisms of their anticancer activity apparently included induction of apoptotic cell death characterized by phosphatidylserine exposure and DNA fragmentation, which correlated with the induction of ROS generation. Overproduction of ROS and ensuing oxidative stress are well-known factors able to trigger cell death [36] and might be crucial for the cytotoxic activity of extract 2 and its flavonoid ingredients.

Apigenin and luteolin have been shown to inhibit proteasome activity and induce apoptosis in human leukemia cells [37]. Proteasome inhibitors induce accumulation of proteasome target proteins and subsequent activation of caspases as well as cleavage of poly-ADP ribose polymerase finally leading to apoptosis in transformed but not in normal cells. Decreasing viability of HL60 cells could also be attributed to cell differentiation, as apigenin and luteolin induce morphological differentiation of HL60 cells into granulocytes. It could be expected that extract 2 has even greater potential to induce differentiation because of high apigenin-7-O-β-glucosyde content which previously has been shown to have very high ability to induce differentiation in HL60 cells [38]. Moreover, it has been shown that flavonoids can directly bind to some protein kinases including Akt, Fyn, Janus kinase 1, mitogen-activated protein kinase (MAPK) kinase 1, MAPK kinase 4, phosphoinositide 3-kinase, and Raf1, and then alter their phosphorylation state to regulate multiple cell signaling pathways in carcinogenic processes [39].

The type of tumor cell death induced by some agents could influence therapy response. Even though the majority of anticancer drugs promote apoptotic cell death, the resistance to chemotherapy-induced apoptosis seems to be a hallmark of most common cancers. Necrotic tumor cells potentiate macrophage-mediated antitumor response in vitro, while apoptosis has the opposite effect. Necrotic cell death acts as an important stimulus for the induction and maintenance of an efficient immune response mediated by dendritic cells [40]. Numerous data describing immunostimulatory properties of necrotic cell death have fostered a hypothesis that necrosis might be more efficient than apoptosis in inducing tumor regression. Thus, the ability of a plant extract to induce necrotic cell death could be an advantage to classical antitumor agents and potentially a very useful adjunctive therapy in cancer treatment. While we have observed an increase in cell membrane permeability of cancer cells treated with extract 2, apigenin and luteolin, the possibility that necrotic cell death was secondary to apoptosis induction could not be completely excluded. We are currently investigating the contribution of necrosis to the anticancer activity of extract 2 and its flavonoid constituents.

A multitude of cytotoxic effects of flavonoids deserve attention and further investigation as adjuvant anticancer drugs, as well as very potent chemicals for cancer chemoprevention. Plant-based diet, rich in phytochemicals, has been long considered to have an important role in cancer prevention. Many studies showed that flavonoids as widespread phytochemicals have strong cytotoxic properties against different forms of cancer [35]. Plants rich in flavonoids, used for centuries in traditional medicine, could be a very good source for providing appropriate daily intake of flavonoids components as a simple and cheap tool in cancer prevention. The data presented here support further exploration of flavonoids as chemotherapeutic and chemopreventive agents.

Acknowledgements

The authors wish to thank the Serbian Ministry of Science and Technological Development for financial support, projects N° III 45017 and N° III 41025.

Conflict of Interest

There are no conflicts of interest among all authors.

References

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• 7 Navarro A, de Las Heras B, Villar A. Anti-inflammatory and immunomodulating properties of a sterol fraction from Sideitis foetens Clem.  Biol Pharm Bull. 2001;  24 470-473
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• 17 Rios J L, Manez S, Paya M, Alcaraz M J. Antioxidant activity of flavonoids from Sideritis javalabrensis.  Phytochemistry. 1992;  31 1047-1050
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• 19 Koleva I, Linssen J P H, van Beek T A, Evstatieva L N, Kortenska V, Handjieva N. Antioxidant activity screening of extracts from Sideritis species (Labiatae) grown in Bulgaria.  J Sci Food Agric. 2003;  83 809-819
  Search in Google Scholar
• 20 Velioglu Y S, Mazza Gao L, Oomah B D. Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products.  J Agric Food Chem. 1998;  46 4113-4117
  Search in Google Scholar
• 21 Council of Europe .Determinations of tannins in herbal drugs. European Pharmacopoeia 6.0, edition no. 6, Volume 1 Strasbourg: Council of Europe; 2004: 255
  Search in Google Scholar
• 22 Blois M S. Antioxidant determinations by the use of a stable free radical.  Nature. 1958;  181 1199-1200
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• 23 Oyanagui Y, Sato S. Inhibition by nilvadipine of ischemic and carrageenan paw oedema as well as of superoxide radical production from neutrophils and xanthine oxidase.  Drug Res. 1991;  41 469-474
  Search in Google Scholar
• 24 Adami E, Marazzi-Uberti E, Turba C. Pharmacological research on gefarnate, a new synthetic isoprenoid with an anti-ulcer action.  Arch Int Pharmacodyn Ther. 1964;  58 1780-1783
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• 25 Touhey S, Heenan M. Cell culture. In: Davey J, Lord J M, editors Essential cell biology. Oxford: Oxford University Press; 2003
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• 26 Mijatovic S, Maksimovic-Ivanic D, Radovic J, Miljkovic D, Harhaji L, Vuckovic O, Stosic-Grujicic S, Mostarica Stojkovic M, Trajkovic V. Anti-glioma action of aloe emodin: the role of ERK inhibition.  Cell Mol Life Sci. 2005;  62 589-598
  Search in Google Scholar
• 27 Badovinac V, Trajkovic V, Mostarica-Stojkovic M. Nitric oxide promotes growth and major histocompatibility complex-unrestricted cytotoxicity of interleukin-2-activated rat lymphocytes.  Scand J Immunol. 2000;  52 62-70
  Search in Google Scholar
• 28 Özkan G, Sagdiç O, Özcan M, Özçelik H, Ünver A. Antioxidant and antibacterial activities of Turkish endemic Sideritis extracts.  Grasas Aceites. 2005;  56 16-20
  Search in Google Scholar
• 29 Tunalier Z, Kosar M, Ozturk N, Baser K H C, Duman H, Kirimer N. Antioxidant properties and phenolic composition of Sideritis species.  Chem Nat Comp. 2004;  40 206-210
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• 30 Spencer J, Mohsen M, Rice-Evans C. Cellular uptake and metabolism of flavonoids and their metabolites: implications for their bioactivity.  Arch Biochem Biophys. 2004;  423 148-161
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• 34 Shukla S, Gupta S. Apigenin: a promising molecule for cancer prevention.  Pharm Res. 2010;  27 962-978
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Dr Vanja Tadić, Science Advisor

Department of Pharmacy
Institute for Medicinal Plant Research “Dr Josif Pančić”

Tadeusa Koscuska 1

11000 Belgrade

Serbia

Phone: +38 11 13 03 16 58

Fax: +38 11 13 03 16 55

Email: vtadic@mocbilja.rs

References

• 1 Lui Z, Tao X, Zhang C, Lu Y, Wei D. Protective effects of hyperoside (Quercetin-3-O-galactoside) to PC12 cells against cytotoxicity induced by hydrogen peroxide and tert-butyl hydro-peroxide.  Biomed Pharmacother. 2005;  59 481-490
  Search in Google Scholar
• 2 Basile A, Senatore F, Gargano R, Sorbo S, Del Pezzo M, Lavitola A, Ritieni A, Bruno M, Spatuzzi D, Rigano D, Vuotto M L. Antibacterial and antioxidant activities in Sideritis italica (Miller) Greuter et Burdet essential oils.  J Ethnopharmacol. 2006;  107 240-248
  Search in Google Scholar
• 3 Charami M, Lazari D, Karioti A, Skaltsa H, Hadjipavlou-Litina D, Souleles C. Antioxidant and antiinflammatory activities of Sideritis perfoliata subsp. perfoliata (Lamiaceae).  Phytother Res. 2008;  22 450-454
  Search in Google Scholar
• 4 Küpeli E, Şahin F P, Çalış I, Yeşilada E, Ezer N. Phenolic compounds of Sideritis ozturkii and their in vivo anti-inflammatory and antinociceptive activities.  J Ethnopharmacol. 2007;  118 356-360
  Search in Google Scholar
• 5 Aboutabl E A, Nassar M I, Elsakhawy F M, Maklad Y A, Osman A F, El-Khrisy E A M. Phytochemical and pharmacological studies on Sideritis taurica Stephan ex Wild.  J Ethnopharmacol. 2002;  82 177-184
  Search in Google Scholar
• 6 Hernàndez-Pérez M, Rabanal R M. Evaluation of the antinflammatory and analgesic activity of Sideritis canariensis var. pannosa in mice.  J Ethnopharmacol. 2002;  81 43-47
  Search in Google Scholar
• 7 Navarro A, de Las Heras B, Villar A. Anti-inflammatory and immunomodulating properties of a sterol fraction from Sideitis foetens Clem.  Biol Pharm Bull. 2001;  24 470-473
  Search in Google Scholar
• 8 de las Heras B, Navarro A, Díaz-Guerra M J, Bermejo P, Castrillo A, Boscá L, Villar A. Inhibition of NOS-2 expression in macrophages through the inactivation of NF-kB by andalusol.  Br J Pharmacol. 1999;  28 605-612
  Search in Google Scholar
• 9 Güvenç A, Okada Y, Küpeli Akkol E, Duman H, Okuyama T, Çalıs I. Investigations of anti-inflammatory, antinociceptive, antioxidant and aldose reductase inhibitory activities of phenolic compounds from Sideritis brevibracteata.  Food Chem. 2010;  118 686-692
  Search in Google Scholar
• 10 Demirtas I, Sahin A, Ayhan B, Tekin S, Telci I. Antiproliferative effects of the methanolic extracts of Sideritis libanotica Labill. subsp. linearis.  Rec Nat Prod. 2009;  3 104-109
  Search in Google Scholar
• 11 Ertaş A, Öztürk M, Boga B, Topçu G. Antioxidant and anticholinesterase activity evaluation of ent-kaurane diterpenoids from Sideritis arguta.  J Nat Prod. 2009;  72 500-502
  Search in Google Scholar
• 12 Kassi E, Papoutsi Z, Fokialakis N, Messari J, Mitakou S, Moutsatsou P. Greek plant extracts exhibit selective estrogen receptor modulator (SERM)-like properties.  J Agric Food Chem. 2004;  52 6956-6961
  Search in Google Scholar
• 13 Plioukas M, Termentzi A, Gabrieli C, Zervou M, Kefalas P, Kokkalou E. Novel acylflavones from Sideritis syriaca ssp. syriaca.  Food Chem. 2010;  123 1136-1141
  Search in Google Scholar
• 14 Petreska J, Stefova M, Ferreres F, Moreno D A, Thomas-Barberan F A, Stefkov G, Kulevanova S, Gil-Izquiredo A. Potential bioactive phenolics of Macedonian Sideritis species used for medicinal “Mountain tea”.  Food Chem. 2011;  125 13-20
  Search in Google Scholar
• 15 Alipieva K I, Kostadinova E P, Evstatieva L N, Stefova M, Bankova V S. An iridoid and a flavonoid from Sideritis lanata L.  Fitoterapia. 2009;  80 51-53
  Search in Google Scholar
• 16 Kilic T. Isolation and biological activity of new and known diterpenoids from Sideritis stricta Boiss. & Heldr.  Molecules. 2006;  11 257-262
  Search in Google Scholar
• 17 Rios J L, Manez S, Paya M, Alcaraz M J. Antioxidant activity of flavonoids from Sideritis javalabrensis.  Phytochemistry. 1992;  31 1047-1050
  Search in Google Scholar
• 18 Diklić N. Genus Sideritis L. In: Josifović M, editor Flora of Serbia, Volume IV. Serbia: Serbian Academy of Science and Art; 1974: 371-372
  Search in Google Scholar
• 19 Koleva I, Linssen J P H, van Beek T A, Evstatieva L N, Kortenska V, Handjieva N. Antioxidant activity screening of extracts from Sideritis species (Labiatae) grown in Bulgaria.  J Sci Food Agric. 2003;  83 809-819
  Search in Google Scholar
• 20 Velioglu Y S, Mazza Gao L, Oomah B D. Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products.  J Agric Food Chem. 1998;  46 4113-4117
  Search in Google Scholar
• 21 Council of Europe .Determinations of tannins in herbal drugs. European Pharmacopoeia 6.0, edition no. 6, Volume 1 Strasbourg: Council of Europe; 2004: 255
  Search in Google Scholar
• 22 Blois M S. Antioxidant determinations by the use of a stable free radical.  Nature. 1958;  181 1199-1200
  Search in Google Scholar
• 23 Oyanagui Y, Sato S. Inhibition by nilvadipine of ischemic and carrageenan paw oedema as well as of superoxide radical production from neutrophils and xanthine oxidase.  Drug Res. 1991;  41 469-474
  Search in Google Scholar
• 24 Adami E, Marazzi-Uberti E, Turba C. Pharmacological research on gefarnate, a new synthetic isoprenoid with an anti-ulcer action.  Arch Int Pharmacodyn Ther. 1964;  58 1780-1783
  Search in Google Scholar
• 25 Touhey S, Heenan M. Cell culture. In: Davey J, Lord J M, editors Essential cell biology. Oxford: Oxford University Press; 2003
  Search in Google Scholar
• 26 Mijatovic S, Maksimovic-Ivanic D, Radovic J, Miljkovic D, Harhaji L, Vuckovic O, Stosic-Grujicic S, Mostarica Stojkovic M, Trajkovic V. Anti-glioma action of aloe emodin: the role of ERK inhibition.  Cell Mol Life Sci. 2005;  62 589-598
  Search in Google Scholar
• 27 Badovinac V, Trajkovic V, Mostarica-Stojkovic M. Nitric oxide promotes growth and major histocompatibility complex-unrestricted cytotoxicity of interleukin-2-activated rat lymphocytes.  Scand J Immunol. 2000;  52 62-70
  Search in Google Scholar
• 28 Özkan G, Sagdiç O, Özcan M, Özçelik H, Ünver A. Antioxidant and antibacterial activities of Turkish endemic Sideritis extracts.  Grasas Aceites. 2005;  56 16-20
  Search in Google Scholar
• 29 Tunalier Z, Kosar M, Ozturk N, Baser K H C, Duman H, Kirimer N. Antioxidant properties and phenolic composition of Sideritis species.  Chem Nat Comp. 2004;  40 206-210
  Search in Google Scholar
• 30 Spencer J, Mohsen M, Rice-Evans C. Cellular uptake and metabolism of flavonoids and their metabolites: implications for their bioactivity.  Arch Biochem Biophys. 2004;  423 148-161
  Search in Google Scholar
• 31 Rice-Evans C A, Miller N J, Paganga G. Structure-antioxidant activity relationships of flavonoids and phenolic acids.  Free Radic Biol Med. 1996;  20 933-956
  Search in Google Scholar
• 32 Maleki N, Garjani A, Nazemiyeh H, Nilfouroushan N, Eftekhar Sadat A T, Allameh Z, Hasannia N. Potent anti-inflammatory activities of hydroalcoholic extract from aerial parts of Stachys inflata in rats.  J Ethnopharmacol. 2001;  75 213-218
  Search in Google Scholar
• 33 Salim A S. Removing oxygen-derived free radicals stimulates healing of ethanol-induced erosive gastritis in the rat.  Digestion. 1990;  47 24-28
  Search in Google Scholar
• 34 Shukla S, Gupta S. Apigenin: a promising molecule for cancer prevention.  Pharm Res. 2010;  27 962-978
  Search in Google Scholar
• 35 Caltagirone S, Rossi C, Poggi A, Ranelletti F O, Natali P G, Brunetti M, Aiello F B, Piantelli M. Flavonoids apigenin and quercetin inhibit melanoma growth and metastatic potential.  Int J Cancer. 2000;  87 595-600
  Search in Google Scholar
• 36 Fiers W, Beyaert R, Declercq W, Vandenabeele P. More than one way to die: apoptosis, necrosis and reactive oxygen damage.  Oncogene. 1999;  18 7719-7730
  Search in Google Scholar
• 37 Chen D, Chen M S, Cui Q C, Yang H, Dou Q P. Structure-proteasome-inhibitory activity relationships of dietary flavonoids in human cancer cells.  Front Biosci. 2007;  12 1935-1945
  Search in Google Scholar
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Dr Vanja Tadić, Science Advisor

Department of Pharmacy
Institute for Medicinal Plant Research “Dr Josif Pančić”

Tadeusa Koscuska 1

11000 Belgrade

Serbia

Phone: +38 11 13 03 16 58

Fax: +38 11 13 03 16 55

Email: vtadic@mocbilja.rs