Abstract
The methanol extracts of fruits and leaves of Athamanta turbith subsp. hungarica (Borbás) Tutin (Umbelliferae) and A. turbith subsp. haynaldii (Borbás & Uechtr.) Tutin were analyzed for their antimicrobial and antioxidant properties. Phenolic compounds (flavonoids and phenolic acids) of the extracts were examined using HPLC. All the extracts were characterized by the presence of caffeic acid derivates, luteolin and its glycosides, with luteolin 7-O-glucoside as one of the main compounds. Luteolin 7-O-rutinoside was detected only in A. turbith subsp. haynaldii extracts. Investigation of antimicrobial activity was performed against six bacteria and two fungal strains, using the agar diffusion technique and broth microdilution assay. The extracts of investigated A. turbith subspecies exerted similar antimicrobial activity, whereas the best activity was detected against Candida albicans. In order to investigate antioxidant properties, ferric ion reducing antioxidant power (FRAP), radical scavenging capacity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radical (HO·), and the effect on lipid peroxidation (LP) were examined. All the examined extracts showed moderate antioxidant capacity, whereas the fruit extracts were more active than the extracts of leaves. Also, the extracts of A. turbith subsp. hungarica exerted higher antioxidant capacity than corresponding A. turbith subsp. haynaldii extracts. The relationship between estimated activity and chemical composition of the extracts is discussed.
Introduction
The genus Athamanta L. (Umbelliferae) consists of about nine species, which are mainly distributed in southeastern Europe. Athamanta turbith (L.) Brot. inhabits limestone rock crevices in southeastern Europe (Italy, Balkan and Carpathians). It is currently considered to contain three subspecies: A. turbith subsp. turbith, A. turbith subsp. hungarica (Borbás) Tutin and A. turbith subsp. haynaldii (Borbás & Uechtr.) Tutin (CitationTutin, 1968). In the flora of FR Serbia, A. turbith subsp. hungarica is registered as the species A. hungarica Borbás (CitationObradović, 1986), and A. turbith subsp. haynaldii as A. haynaldii Borbás & Uechtr. (CitationNikolić, 1973).
A. turbith subsp. hungarica is distributed in gorges of the southern Carpathians and northeastern Serbia. A. turbith subsp. haynaldii is an endemic Dinaric alpine plant. These two plants have very similar habitus. Morphologically, the only difference that can be noticed is the stylopodium shape: each half of A. turbith subsp. hungarica stylopodium is 1.5–2 times as long as it is wide, while each half of A. turbith subsp. haynaldii stylopodium is about as long as it is wide (CitationTutin, 1968).
In traditional medicine, Athamanta cretensis and Athamanta macedonica are used in the therapy of sclerosis, Athamanta oreoselinum as an antiseptic and diuretic (CitationDuke, 2004), while Athamanta sicula is used to expel renal calculi (CitationLentini, 2000).
It has been recognized that naturally occurring substances in plants have antimicrobial activity. There is a growing interest in research of antimicrobial properties of plant extracts, since resistance to antibiotics has become an important and pressing global problem. Over the past decade, great interest has also been devoted to the antioxidant activity of phenolic compounds from plant extracts, which is due to their ability to reduce free radical formation and to scavenge free radicals. In this study, in vitro antimicrobial and antioxidant activity of methanol extracts of mature fruits and leaves of A. turbith subsp. hungarica and A. turbith subsp. haynaldii were investigated.
Materials and methods
Chemicals
1,1-Diphenyl 2-picryl hydrazyl (DPPH), Folin-Ciocalteu reagent, 2,4,6-tripyridyl-s-triazine (TPTZ), apigenin and caffeic acid were obtained from Sigma Chemical Co. (St. Louis, MO); thiobarbituric acid (TBA) from Merck (Darmstadt, Germany); trichloroacetic acid (TCA), ethylenediaminetetraacetic acid (EDTA) and l-ascorbic acid from Lachema (Neratovice, Czech Republic); 2- deoxyribose from Acros Organics (New Jersey, USA); rutin, quercetin, umbelliferone and kaempferol from Fluka AG (Buchs, Switzerland); cynarine from Roth (Karlsruhe, Germany); all other reagents used were of analytical grade. Luteolin, luteolin 7-O-glucoside and luteolin 7-O-rutinoside were isolated from Hieracium gymnocephalum Griseb. ex Pant. (CitationPetrović et al., 1999). Scopoletin and 3,5-di-O- caffeoylquinic acid were isolated from Anthemis triumfetti (L.) DC. (CitationPavlović et al., 2006). Ampicillin, amikacin and nystatin were obtained from Galenika (Belgrade, Serbia); Müeller-Hinton agar and Sabouraud agar from Institute of Immunology and Virology, Torlak (Belgrade, Serbia).
Plant material
The plants were collected in July 2004 from two localities in Serbia: đerdap gorge (Iron Gate) (A. turbith subsp. hungarica) and Ovčar-Kablar gorge (A. turbith subsp. haynaldii), and were identified by Marjan Niketić. Voucher specimens (ko2004071 and ko2004072, respectively) were deposited at the Herbarium of the Natural History Museum in Belgrade (BEO).
Extraction
Air-dried, powdered plant material (mature fruits and leaves, separately) was macerated with chloroform for three days, and after filtration, for two more days. The extracts were filtered and the residues extracted with methanol, under the same conditions. The methanol extracts were evaporated to dryness under vacuum and were used for further analyses. Yields of methanol extracts of fruits and leaves of A. turbith subsp. hungarica were 6% and 7.2%, and of A. turbith subsp. haynaldii 6.5% and 10.9%, respectively.
High Performance Liquid Chromatography (HPLC)analysis
Phenolic compounds of the extracts were assayed by HPLC on an Agilent 1100 series system consisting of a G1312A binary pump, a G1328B injector (20 μL sample loop) and G1315B DAD detector, equipped with ZORBAX Eclipse XDB-C18 column (4.6 × 250 mm, 5 μm). A gradient elution was performed with solvent A (H3PO4 in H2O, pH = 2.75) and solvent B (solvent A: acetonitrile = 10:90; solvent B: H3PO4 in H2O, pH = 2.75: acetonitrile = 10:90) as follows: in 0 min 10% B; in 5 min 25% B; in 15 min 25% B; in 20 min 30% B; in 25 min 50% B, in 30 min 70% B and in 35 min 10% B. The column temperature was 25°C, flow rate 0.8 mL/min. Luteolin, luteolin 7-O-glucoside, luteolin 7-O-rutinoside, apigenin, quercetin, kaempferol, caffeic acid, 3,5-dicaffeoylquinic acid, cynarine, scopoletin and umbelliferone were used as standards ( 1 mg/mL in acetonitrile:H2O = 1:1). Dry methanol extracts were dissolved in methanol in concentration of 1.5%. Chromatograms of extracts and standards were recorded under the same conditions. Detection was performed at 350 nm. Identification of compounds was carried out by comparing their spectra and their retention times with those of standards when available. UV spectra were also compared with literature data (CitationMabry et al., 1970).
Antimicrobial activity
Microbial strains
Methanol extracts were tested against six bacteria and two fungal strains, listed in . Microorganisms were provided by the Institute for Immunology and Virology, Torlak, Belgrade.
Table 1. HPLC analysis of Athamanta turbith subsp. hungarica and A. turbith subsp. haynaldii methanol extracts.
Agar diffusion assay
Antimicrobial activity was assayed using the agar diffusion method (CitationAcar & Goldstein, 1996), in comparison to ampicillin, amikacin and nystatin.
Müller-Hinton agar and Sabouraud agar were used to test sensitivity of bacteria and yeasts, respectively. The diffusion technique was carried by pouring agar into Petri dishes to form 4 mm thick layers and adding dense inocula of the tested microorganisms (106 microorganisms/mL) in order to obtain semiconfluent growth. Dry methanol extracts were dissolved in methanol in the concentration of 500 and 250 μg/mL. One drop of each extract solution was poured on the agar prepared as required. Incubation lasted 18 h at 37°C for bacteria and 48 h at 26°C for Candida albicans. Reading of results was carried out by measuring diameters of zones of inhibitions in mm (in quadruplicate). Each assay in this experiment was repeated twice.
Broth microdilution assay
For the determination of minimal inhibitory concentration (MIC), a broth microdilution assay was used, as recommended by the National Committee for Clinical Laboratory Standards (CitationNCCLS, 2001). Tests for antibacterial activity were performed in Müller-Hinton broth, and in Sabouraud dextrose broth for antifungal activity. Test strains were suspended in broth to give a final density of 5 × 105 cfu/mL. Dilutions ranging from 500 to 62.5 μg/mL of the methanol extracts were prepared in 96-well plates. As a positive control of growth, the wells containing only microorganisms in the broth were used. The contents of each well were mixed and incubated for 18 h at 37°C for bacteria and 48 h at 26°C for yeast. Bacterial growth was indicated by the presence of a pellet on the well bottom. The extracts were screened twice against each microorganism. The MIC of ampicillin, amikacin and nystatin were determined in parallel experiments.
Antioxidant activity
In order to investigate the antioxidant properties of the examined extracts, ferric ion reducing antioxidant power (FRAP), radical scavenging capacity (RSC) against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radical (HO˙), and the effect on lipid peroxidation (LP) were examined. All the tests were carried out in triplicate.
FRAP assay
FRAP reagent was freshly prepared by mixing 25 mL acetate buffer (300 mM, pH 3.6), 2.5 mL 2,4,6-tris(2-pyridyl)-S-triazine (TPTZ) solution (10 mM TPTZ in 40 mmol/L HCl) and 2.5 mL FeCl3 (20 mM) water solution. Each sample (150 μL) (0.5 mg/mL) dissolved in methanol was added in 4.5 mL of freshly prepared FRAP reagent and stirred, and after 5 min, absorbance was measured at 593 nm, using FRAP working solution as blank (CitationSzőllősi & Szőllősi Varga, 2002). A calibration curve of ferrous sulfate (100-1000 μmol/L) was used, and results were expressed in μmol Fe2+/mg dry weight extract. The relative activity of the samples was compared to l-ascorbic acid, quercetin, and rutin.
DPPH radical assay
For measuring RSC against DPPH radical, 1 mL of 0.5 mM methanol solution of DPPH was added to 4 mL of methanol solution of the investigated extract, in four different concentrations. Absorbance at 517 nm was determined after 30 min, using methanol as blank (CitationCuendet et al., 1997). Scavenging of DPPH radical was calculated using the equation: S(%) = 100 × (A0–As)/A0, where A0 is the absorbance of the control (containing all reagents except the test compound), and As is the absorbance of the tested sample. Results were compared with the activity of l-ascorbic acid, quercetin, and rutin.
2-Deoxyribose assay
RSC against HO˙ radical was determined in non-site-specific deoxyribose assay. Methanol solution (20 μL) of the examined extract (five different concentrations) was mixed with 100 μL of 0.004 M FeCl3, 100 μL 0.004 M ethylenediaminetetraacetic acid (EDTA), 200 μL of 2-deoxyribose (0.05 M), 20 μL of 1.5% H2O2 and 100 μL of l-ascorbic acid (0.004 M) in phosphate buffer, pH = 7.4 (4 mL final solution). The mixtures were then incubated at 37°C for 60 min. When adding 1 mL of 1% (w/v) solution of thiobarbituric acid (TBA) in 0.05 M NaOH and 1 mL of 2.8% (w/v) solution of trichloroacetic acid (TCA), the mixtures were heated at 100°C for 15 min, and then cooled on ice. Absorbance of samples was measured at 532 nm (CitationLee et al., 2003). Inhibition of 2-deoxyribose degradation was calculated the same way as described in DPPH radical assay. Quercetin and rutin were used as standards.
TBA assay
For measuring the inhibitory effect on Fe2+/ascorbate induced lipid peroxidation (LP) in liposomes, the TBA test was used. Liposomes were prepared from the commercial preparation “Lipotech 10”, containing 10% lecithin, diluted with distilled water in ultrasonic bath for 30 min. Liposomes (0.03 mg lecithin/mL) were incubated at 37°C for 60 min with 20 μL of FeSO4 (0.075 M), 10 μL of l-ascorbic acid (0.1 M) and 10 μL methanol solution of the examined extract (five different concentrations) in phosphate buffer, pH = 7.4 (4 mL final solution). LP was terminated by adding 0.2 mL 0.1 M EDTA and 1.5 mL of TBA reagent (3 g TBA, 120 g TCA and 10.4 mL HClO4 in 800 mL of distilled water). Mixtures were heated for 15 min at 100°C. After cooling on ice, samples were centrifuged for 10 min (3000 rpm) and absorbance of supernatants was measured at 532 nm (CitationAfanas’ev et al., 1989). Inhibition of LP was calculated the same way as described in DPPH radical assay. Results were compared with the activity of quercetin.
Statistical analysis
Results were expressed as means ± standard deviations (SD). Statistical analysis was performed by ANOVA. Differences were accepted as statistically significant when p < 0.05.
Results and discussion
HPLC analysis
The extracts of both A. turbith subspecies are characterized by the presence of luteolin, its glycosides and caffeic acid derivates (). Luteolin 7-O-glucoside and 3,5-dicaffeoylquinic acid were detected in all four investigated extracts. Between the extracts of fruits and leaves of each subspecies there are only quantitative differences. In the extracts of fruits, free luteolin is the dominant compound, while in the extracts of leaves, luteolin glycosides are present in larger quantity. However, between investigated subspecies there are qualitative differences: in the A. turbith subsp. haynaldii extracts more luteolin glycosides have been detected, with luteolin 7-O-rutinoside being one of the major compounds, while in A. turbith subsp. hungarica extracts this component has not been detected. Similar flavonoid patterns of the investigated plants support their taxonomic classification in the same species. Also, concerning estimated differences, flavonoids could be regarded as good chemotaxonomic markers for the investigated A. turbith subspecies.
Antimicrobial activity
The extracts of investigated A. turbith subspecies exerted similar antimicrobial activity. All the extracts showed strong activity against Candida albicans. As for antibacterial activity, at the applied concentrations, the ext racts exerted moderate activity against Staphylococcus aureus, slightly weaker against Enterococcus faecalis and Escherichia coli, and weak against Pseudomonas aeruginosa and Klebsiella pneumoniae ( and ). In previous investigations on antibacterial properties of luteolin, the best activity was also detected against S. aureus, while its activity against E. faecalis, E. coli, P. aeruginosa and K. pneumoniae was weaker (CitationTshikalange et al., 2005; CitationSato et al., 2000). CitationZhu et al. (2004) reported that 3,5-dicaffeoylquinic acid and luteolin 7-O-rutinoside were active against S. aureus, E. coli, and C. albicans, but had no effect against P. aeruginosa.
Table 2. Antimicrobial activity of Athamanta turbith subsp. hungarica and A. turbith subsp. haynaldii methanol extracts tested by agar diffusion assay.
Table 3. Antimicrobial activity of Athamanta turbith subsp. hungarica and A. turbith subsp. haynaldii methanol extracts tested by broth microdilution assay.
Antioxidant activity
Results obtained from antioxidant capacity assays are given in . In the FRAP assay, the extracts of A. turbith subsp. hungarica exerted higher activity than A. turbith subsp. haynaldii extracts. Investigated extracts exhibited dose-dependent scavenging of DPPH radical. SC50 values for A. turbith subsp. hungarica extracts suggest high antiradical activity (<50 μg/mL), while activity of A. turbith subsp. haynaldii extracts could be characterized as moderate (SC50 values were slightly above 50 μg/mL) (CitationCho et al., 2003). The extracts of A. turbith subsp. hungarica also exhibited higher HO˙ radical scavenging capacity in 2-deoxyribose assay. In the TBA assay, none of the extracts reached 50% of inhibition.
Table 4. Antioxidant properties of Athamanta turbith subsp. hungarica and A. turbith subsp. haynaldii methanol extracts.
Antioxidant properties of luteolin and its glycosides were previously confirmed (Heim et al., 2002; Wu et al., 2006). It has been found that presence of the 3′,4′-dihydroxyl phenyl substitution pattern in B-ring, as well as presence of 2,3-double bond in conjugation with a 4-oxo group in C-ring is very important for antioxidant activities of luteolin. Luteolin was also shown to be a significantly stronger antioxidant than its glycosides (CitationIgile et al., 1994; CitationCao et al., 1997; CitationArora et al., 1998; CitationHeim et al., 2002; CitationWu et al., 2006). Higher content of luteolin in the extracts of fruits could explain their higher antioxidant activity in comparison to the extracts of leaves.
Although flavonoid content in A. turbith subsp. hungarica extracts was about two times lower, their higher antioxidant capacity in comparison to A. turbith subsp. haynaldii extracts could be explained by the presence of other phenolic compounds such as caffeic acid derivates. In general, caffeic acid derivates are considered as effective antioxidants because the catechol structure donates the phenolic hydrogens or electron to the acceptors such as lipid peroxyl groups or reactive oxygen species (CitationChakraborty & Mitra, 2007). Caffeic acid was shown to have the same antioxidant capacity as l-ascorbic acid (CitationKim & Lee, 2004).
Acknowledgement
This research was supported by the Ministry of Science and Environmental Protection, Republic of Serbia (Grants No. 143012 and 143015).
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
References
- Acar JF, Goldstein FW (1996): Disc susceptibility test. In: Lorian V, ed., Antibiotics in Laboratory Medicine, 4th edn. Baltimore, Williams & Wilkins, pp. 1–51.
- Afanas’ev IB, Dorozhko AI, Brodskii AV, Kostyuk VA, Potapovitch AI (1989): Chelating and free radical scavenging mechanisms of inhibitory action of rutin and quercetin in lipid peroxidation. Biochem Pharmacol 38: 1763–1769.
- Arora A, Nair MG, Strasburg GM (1998): Structure-activity relationships for antioxidant activity of a series of flavonoids in liposomal system. Free Rad Biol Med 24: 1355–1363.
- Cao G, Sofic E, Prior RL (1997): Antioxidant and prooxidant behavior of flavonoids: Structure-activity relationship. Free Rad Biol Med 22: 749–760.
- Chakraborty M, Mitra, A (2008): The antioxidant and antimicrobial properties of the methanolic extracts from Cocos nucifera mesocarp. Food Chem 107:994–999.
- Cho EJ, Yokozawa T, Rhyu DY, Kim SC, Shibahara N, Park JC (2003): Study on the inhibitory effects of Korean medicinal plants and their main compounds on the 1,1-diphenyl-2-picrylhydrazyl radical. Phytomedicine 10: 544–551.
- Cuendet M, Hostettmann K, Potterat O (1997): Iridoid glucosides with free radical scavenging properties from Fagraea blumei. Helv Chim Acta 80:1144–1152.
- Duke JA (2004): Phytochemical Database, US Department of Agriculture, Beltsville Agricultural Research Center, MA, USA. Available at: http://www.ars-grin.gov/duke/ethnobot.html
- German Pharmacopoeia (1996): DAB 10 (Deutsches Arzneibuch) Amtliche Ausgabe, Frankfurt a.m./Eschborn, Deutscher Apotheker Verlag Stuttgart, Govi-Verlag.
- Heim KE, Tagliaferro AR, Bobilya DJ (2002): Flavonoid antioxidants: Chemistry, metabolism and structure-activity relationships. J Nutr Biochem 13: 572–584.
- Igile GO, Oleszek W, Jurzyst M, Burda S, Fafunso M, Fasanmade AA (1994): Flavonoids from Veronia amygdalina and the antioxidant activities. J Agric Food Chem 42: 2445–2448.
- Kim DO, Lee CY (2004): Comprehensive study on vitamin C equivalent antioxidant capacity (VCEAC) of various polyphenolics in scavenging a free radical and its structural relationship. Crit Rev Food Sci Nutr 44: 253–273.
- Lee JC, Kim J, Park JK, Chung GH, Jang YS (2003): The antioxidant, rather than prooxidant, activities of quercetin on normal cells: Quercetin protects mouse thymocyte from glucoseoxidase-mediated apoptosis. Exp Cell Res 291: 386–397.
- Lentini F (2000): The role of ethnobotanics in scientific research. State of ethnobotanical knowledge in Sicily. Fitoterapia 71: 83–88.
- Mabry TJ, Markham KR, Thomas MB (1970): The Systematic Identification of Flavonoids. Berlin, Heidelberg, New York, Springer-Verlag, pp. 262–275.
- NCCLS (National Committee for Clinical Laboratory Standards) (2001): Document M100–S11. National Committee for Clinical Laboratory Standard, Wayne, PA, USA. Performance Standards for Antimicrobial Susceptibility Testing, 11th informational supplement.
- Nikolić V (1973): Athamanta L. In: Josifović M, ed., The Flora of FR Serbia, Vol. 5. Belgrade, SANU, pp. 258–260.
- Obradović M (1986): Athamanta hungarica Borb. In: Sarić MR, Diklić N, eds., The Flora of FR Serbia, Vol. 10. Belgrade, SANU, pp. 150–152.
- Pavlović M, Kovačević N, Couladis M, Tzakou O (2006): Phenolic constituents of Anthemis triumfetti (L.) DC. Biochem Sys Ecol 34: 449–452.
- Petrović SD, Gorunović MS, Wray V, Merfort I (1999): A taraxasterol derivative and phenolic compounds from Hieracium gymnocephalum. Phytochemistry 50: 293–296.
- Sato Y, Syzaki S, Nishikawa T, Kihara M, Shibata H, Higuti T (2000): Phytochemical flavones isolated from Scutellaria barbata and antibacterial activity against methicillin-resistant Staphylococcus aureus. J Ethnopharmacol 72: 483–488.
- Szőllősi R, Szőllősi Varga I (2002): Total antioxidant power in some species of Labiatae (Adaptation of FRAP method). Acta Biol Szeged 46: 125–127.
- Tshikalange TE, Meyer JJM, Hussein AA (2005): Antimicrobial activity, toxicity and the isolation of a bioactive compound from plants used to treat sexually transmitted diseases. J Ethnopharmacol 96: 515–519.
- Tutin TG (1968): Athamanta L. In: Tutin TG, Heywood VH, Burges NA, Moore DM, Valentine DH, Walters SM, Webb DA, eds., Flora Europaea 2. Cambridge, Cambridge University Press, pp. 340–341.
- Wu C, Chen F, Wang X, Kim HJ, He G, Haley-Zitlin V, Huang G (2006): Antioxidant constituents feverfew (Tanacetum parthenium) extract and their chromatographic quantification. Food Chem 96: 220–227.
- Zhu X, Zhang H, Lo R (2004): Phenolic compounds from the leaf extracts of artichoke (Cynara scolymus L.) and their antimicrobial activities. J Agric Food Chem 52: 7272–7278.