Antioxidant and Antimicrobial Activity of Ferulago trojana E. Akalin & Pimenov.
Many medicinal plants and raw extracts, due to their traditional use in vitro antioxidants and antibacterial activities have been screened by many researchers. These activites have been observed on plants containing specially phenolic compounds (Wang et al. 1999; Kahkonen et al. 1999; Pietta 2000; Cushine & Lamb 2005; Rios & Recio 2005).
The genus Ferulago (Apiaceae/Umbelliferae) includes 49 species occuring throughout the Northern hemisphere. It is naturally grown mainly in Europe, Northwest and Central Asia, Caucasus, North and Northwest Africa and Turkey. 34 Ferulago species (18 endemic) are naturally grow in Turkey (Pesmen 1972; Davis et al. 1988; Pimenov and Leonov 1993; Ozhatay and Akalin 2000; Solanas et al. 2000; Akalin and Pimenov 2004; Kandemir and Hedge 2007).
In different regions in Turkey, Ferulago species are known by different names, as the most common "caksirotu", "kisnis", "asaotu", "kuzubasi" ve "kuzukemirdi". Since Dioscorides, Ferulago species are used for the purpose tonic, digestive, carminative, aphrodisiac as well as in the treatment of intestinal worms and hemorrhoids (Baytop 1999; Akalin 1999).
Ferulago trojana is endemic to Mount Ida which was first identified in 2004. On ISTE's samples, collected in Canakkale and Balikesir, previously determined as F. sylvatica (also this name was specified from same areas in the Flora of Turkey and East Aegean Islands). Later studies showed that they were different from the European sample. The results of the detailed examination of the Turkish 'F. sylvatica' samples, they have been identified as a new species, F. trojana. The species of F. sylvatica is not considered to be in Turkey (Akalin and Pimenov 2004).
To date, there have been some studies on chemical composition of various Ferulago species. Essential oils, coumarins, flavonoids, sesquiterpenes, fatty acids and phytosterols were reported as the chemical constituents of the Ferulago plants. In these studies with respect to the essential oils of Ferulago genus, coumarins, monoterpenes and sesquiterpenes were characterized as the main components (Miski et al. 1990; Doganca et al. 1991; Yoti&Assenov 1995; Rustaiyan et al. 1999; Jimenez et al. 2000; Baser et al.; 2001; 2002; Erdurak et al. 2006; Kilic et al. 2006; Erdemoglu et al. 2008; Alkhatib et al. 2009).
In previous study, five compounds have been isolated from F.trojana. From these compounds bergapten, isoimperatorin, 3'-epidecursin and isomaltol are known compounds, isomaltol-3[beta]-O-glucoside and 3,6-dimethoxy-7-isopropilcoumarin-4-tetradeca-13"-one have been isolated for the first time. Also antioxidant activities and anticholinesterase activities of dichloromethane and methanol extracts of F. trojana were determined (Cakar 2010). GS/MS analysis has resulted in the characterization of 19 compounds representing 99.3% of the oil with p-cymene (45.8%) as the main costituents. Monoterpenes and sesquiterpenes were reported from aerial parts of the essential oil of F. sylvatica (Chalchat et al. 1992).
The aim of this study was to evaluate and compare the antioxidant and antimicrobial activities of methanol extracts from the aerial parts (HFT) and rhizomes (RFT) of F. trojana. There are no reports on antimicrobial activity of F.trojana. The most active extract and parts of the plant will be detected and the compounds responsible for the antimicrobial activity in another study will be isolated.
MATERIALS AND METHODS
Ferulago trojana E. Akalin & Pimenov a species growing in Turkey was collected from Kaz Daglari (Balikesir) in June 2007, and identified by Dr. Emine Akalin. A voucher specimen (ISTE No: 74316) is deposited in the Herbarium of Faculty of Pharmacy, Istanbul University (ISTE).
Preparation of extracts
The dried and powdered aerial parts (30 g) (HFT) and rhizomes (30 g) (RFT) of the F.trojana were percolated with 600 mL methanol. The methanol extracts were evaporated to dryness under reduced pressure and controlled temperature (45 to 50[degrees]C) in a rotary evaporator. The extracts were lyophilized. The obtained methanol extracts (HFT, RFT) were used for antioxidant and antimicrobial activity determinations.
Soybean lecithin (L-a-phosphatidylcholine Type IV-S), 2,2-diphenyl-1-picrylhydrazyl (DPPH), quercetin and ascorbic acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt ([ABTS.sup.+]), 6-hydroxy-2,5,7,8,-tetramethylchroman-2-carboxylic acid (Trolox) were purchased from Fluka Chemical Co. (Bushs, Switzerland). Thiobarbituric acid (TBA), trichloroacetic acid (TCA) and ferric chloride were purchased from Merck (Darmstadt, Germany). All other chemicals used were of analytical grade.
Determination of total phenolic compounds
Total soluble phenolics in the methanol extract of F.trojana were determined with Folin-Ciocalteu reagent according to the method of (Slinkard and Singleton 1977) with some modifications. Aliquots (0.1 mL) of extracts were transferred into the test tubes and their volumes made up to 4.6 mL with distilled water. After addition of 0.1 mL Folin-Ciocalteu reagent (previously diluted 3-fold with distilled water) and 0.3 mL 2% [Na.sub.2]C[O.sub.3] solution, tubes were vortexed and absorbance of mixture recorded after 2 h at 760 nm against a blank containing 0.1 mL of extraction solvent. Gallic acid (0.05 mg/mL-0.4 mg/mL) was used for calibration of a standard curve. The results were expressed as gallic acid equivalents (GAE)/g of extract. The data were presented as the average of triplicate analyses.
Determination of total flavonoid content
Total flavonoid content was determined by using a colorimetric method described by (Sakanaka et al. 2005). Briefly, 0.25 mL of the extract (0.625 mg/mL extract or (+)-catechin standard solution was mixed with 1.25 mL of distilled water or solvent in a test tube, followed by addition of 75 [micro]L of a 5% (w/v) sodium nitrite solution. After 6 min, 150 [micro]L of a 10% (w/v) Al[Cl.sub.3] solution was added and the mixture was allowed to stand for a further 5 min before 0.5 mL of 1 M NaOH was added. The mixture was brought to 2.5 mL with distilled water and mixed well. The absorbance was measured immediately at 510 nm, using a spectrophotometer (Shimadzu UV-1208). (+)-Catechin standard solution (15-250 [micro]L/mL) was used for the calibration of a standard curve. The results were expressed as means ([+ or -] SD) mg of (+)-catechin equivalents per gram of extract.
Antioxidant activity on liposome peroxidation
Lipid peroxidation assay was based on the method described by (Duh et al. 1999). Lecithin (300 mg) was suspended in 30 mL phosphate buffer (10 mmol/L, pH 7.4). This suspension was then sonicated with a rod using an ultrasonic homogenizer (Bandelin, Berlin, Germany) at 30 s intervals for 10 min until an opalescent suspension was obtained.
The sonicated solution (10 mg/mL), Fe[Cl.sub.3], ascorbic acid and the extracts (from 0.625 to 10 mg/mL) or quercetin (from 0.005 to 0.08 mg/mL) used as a reference antioxidant were mixed to produce a final concentration of 3.08 mg liposome/mL, 123.2 [micro]mol Fe[Cl.sub.3] and 123.2 [micro]mol ascorbic acid. After 1 h incubation at 37[degrees]C, the formation of lipid peroxidation products was assayed by the measurement of malondialdehyde (MDA) levels on the basis that MDA reacted with TBA at 532 nm according to (Buege and Aust 1978). Briefly, 500 [micro]L of this reaction mixture was mixed with 1000 [micro]L TCA-TBA reagent (consisting of 15% w/v TCA and 0.375% TBA in 0.25 N HCl) and 14 [micro]L BHT (2% in absolute ethanol). The mixture was vortexed and heated for 10 min in a boiling water bath. After cooling, an equal volume of n-butanol was added and the mixture was shaken vigorously. The n-butanol layer was separated by centrifugation at 3000 rpm for 5 min. The absorbance of the sample was read at 532 nm against a blank which contained all reagents except lecithin. The percentage inhibition of lipid peroxidation was calculated by comparing the results of the samples with those of controls not treated with the extract using the following equation: Inhibition effect (%) = [1-(Absorbance of sample at 532 nm/Absorbance of control at 532 nm)] x 100.
DPPH radical scavenging activity
The DPPH radical scavenging activity of the extract was measured according to the procedure described by (Brand-Williams et al. 1995). A 0.1 mL aliquot of extracts (from 0.16 to 15 mg/mL) or quercetin (from 0.01 to 0.16 mg/mL) in methanol was added to 3.9 mL of 6 x [10.sup.-5] M methanolic solution of DPPH. The mixture was shaken vigorously and allowed to stand in the dark at room temperature for 30 min. The decrease in absorbance of the resulting solution was then measured spectrophotometrically at 517 nm against methanol. All measurements were made in triplicate and averaged. Two controls were used for this test, a negative control (containing all reagents except the test sample) and positive controls (using the reference antioxidants). The ability to scavenge DPPH radical was calculated by the following equation:
DPPH radical scavenging activity (%) = [1 - (Absorbance of sample at 517 nm/Absorbance of control at 517 nm)] x 100.
Total radical antioxidant potential (TRAP) assay
The total radical antioxidant potential of the extract was measured using the Trolox equivalent antioxidant coefficient (TEAC) assay as described by (Re et al. 1999) with minor modifications. ABTS was dissolved in water to a 7 mM concentration. ABTS radical cation was produced by reacting [ABTS.sup.+] stock solution with 2.45 mM potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 12-16 hours before use. At the begining of the analysis day, an [ABTS.sup.*+] working solution was obtained by the dilution in 96% ethanol of the stock solution to an absorbance of 0.70 ([+ or -] 0.02) at 734 nm. After addition of 990 [micro]L of [ABTS.sup.*+] solution to 10 [micro]L of the extracts (from 0.625 to 15 mg/mL) or quercetin (from 0.01 to 0.16 mg/mL) or trolox standards (final concentration 0 - 20 [micro]M/l) in absolute ethanol the decrease in absorbance at 734 nm was monitored exactly 6 min after the initial mixing. Appropriate methanol blanks were run in each assay. All determinations were carried out in triplicate.
The ability to scavenge [ABTS.sup.*+] radical was calculated by the following equation: [ABTS.sup.*+] radical scavenging activity (%) = [1 - (Absorbance of sample at 734 nm/Absorbance of control at 734 nm)] x 100.
The total antioxidant capacity value in a sample was assessed as TEAC. The TEAC value was calculated by using a regression equation between the Trolox concentration and the percentage of inhibition of absorbance at 734 nm at 6 minutes of incubation and was expressed as mmol TEAC.
Ferric reducing antioxidant power (FRAP) assay
The FRAP assay was carried out according to the procedure of (Benzie and Strain 1996). The FRAP reagent contained 2.5 mL of10 mM TPTZ solution in 40 mM HCl plus 2.5 mL of 20 mM Fe[Cl.sub.3].6[H.sub.2]O and 25 mL of 0.3 M acetate buffer, pH 3.6. FRAP reagent (900 [micro]L), prepared freshly and incubated at 37[degrees]C, was mixed with 90 [micro]L of distilled water and 30 [micro]L of the extracts (from 1.25 to 10 mg/mL) or quercetin (from 0.02 to 0.31 mg/mL) or water for the reagent blank. The increase in absorbance at 593 nm was measured at 4 min. The standard curve was constructed using iron sulfate heptahydrate solution (125-2000 [micro]M), and the results were expressed as mM [Fe.sup.2+] equivalents. All the measurements were taken in triplicate and the mean values were calculated.
All measurements were made in triplicate. The results were statistically analyzed using GraphPad Prism version 7.00. Results were considered significant at p<0.05.
Antimicrobial activity against Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis ATCC 12228, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 4352, Pseudomonas aeruginosa ATCC 27853, Proteus mirabilis ATCC 14153, Methicillin-resistant (MRSA) ATCC 43300 and Candida albicans ATCC 10231 were determined by the microbroth dilutions technique the Clinical Laboratory Standards Institute (CLSI) recommendations (CLSI 2008; 2015). Mueller-Hinton broth for bacteria, RPMI-1640 medium buffered to PH 7.0 with MOPS for yeast strain were used as the test medium. Serial two-fold dilutions ranging from 5000 [micro]g/mL to 4.9 [micro]g/mL were prepared in medium. The inoculum was prepared using a 4-6h broth culture of each bacteria and 24h culture of yeast strains adjusted to a turbidity equivalent to a 0.5 Mc Farland standard, diluted in broth media to give a final concentration of 5x[10.sup.5] cfu/mL for bacteria and 0.5x[10.sup.3] to 2.5x[10.sup.3] cfu/mL for yeast in the test tray. The trays were covered and placed in plastic bags to prevent evaporation. The trays containing Mueller-Hinton broth were incubated at 35 [degrees]C for18-20h and the trays containing RPMI-1640 medium were incubated at 35 [degrees]C for 46-50h. The Minimum Inhibitory Concentration (MIC) was defined as the lowest concentration of compound giving complete inhibition of visible growth. As control, antimicrobial effects of the solvents were investigated against test microorganisms. According to values of the controls, the results were evaluated.
RESULTS AND DISCUSSION
It was reported that the oil, rhizomes, stems, leaves, and flowers of different Ferulago species contain phenolic compounds and show antioxidant activity (Azarbani et al. 2012; Mileski et al. 2015; Dikpinar 2017; Kiziltas et al. 2017).
The results given in Table 1 showed that the amount of extractable phenolic compounds and flavonoids in HFT extract is higher than that detected in RFT extract (p<0.05), so the aerial parts of Ferulago trojana is a rich source of phenolics and flavonoids. A similar content of flavonoids was reported by (Kiziltas et al. 2017) for the flowers of Ferulago angulata (Schlecht.) Boiss. (Apiaceae).
The antioxidant activity was tested using four in vitro assays including, lipid peroxidation inhibition, scavenging effect on DPPH and ABTS radicals, and FRAP assays. For comparison, Table 2 presents the results of the antioxidant activities, expressed as [EC.sub.50], TEAC and FRAP values. As can be seen from the [EC.sub.50] values, the methanol extract of aerial parts showed a higher scavenging effect on DPPH and ABTS radicals, and reducing power when compared to its capability to inhibit lipid peroxidation. TEAC value was similar to the FRAP value, which indicates that the extract is effective in donating of electrons. The aerial parts showed the better antioxidant activity than rhizomes in DPPH and FRAP assays. However, the results showed a weak antioxidant activity of both the extract compared to the referance antioxidant quercetin. Although the extract was less active than the quercetin (p<0.05), it was seen that it has hydrogen and a single electron donor activities, thus could serve as antioxidant.
These results are in accordance with previous studies, which reported the efficiacy of Ferulago species to scavenge free radicals (Azarbani et al. 2012; Mileski et al. 2015; Dikpinar 2017; Kiziltas et al. 2017). Antioxidant activity against lipid peroxidation (LPO) has been reported for the dichloromethane extract of F. trojana, which is attributed to the richness of the coumarin in the extract (Cakar, 2010). Cakar (2010) also reported that the pure compounds isolated from the Ferulago trojana (bergapten, isoimperatorin, 3'-epidecursin, isomaltol, isomaltol-3[beta]-O-glucoside and 3,6-dimethoxy-7-isopropilcoumarin-4-tetradeca-13"-one) showed anti-oxidant activity against lipid peroxidation investigated in a [beta]-carotene-linoleic acid model system, but do not have free radical scavenging ability.
The antimicrobial activity of F. trojana has been studied for the first time. The antimicrobial activity results of methanol extracts prepared from aerial parts (HFT) and rhizomes (RFT) of F. trojana are shown in Table 3. In this study, both methanol extracts from aerial parts and rhizomes of F. trojana showed antibacterial activity against Gram positive bacteria such as S. aureus, MRSA, S. epidermidis while no activity was observed against E. coli, K. pneumoniae and P. aeruginosa for any of the extracts. Methanol extract from rhizomes of F. trojana showed antibacterial activity against P. mirabilis and antifungal activity the yeast C. albicans. When the results of the antimicrobial activity were evaluated, it was found that the RFT extract showed moderate antimicrobial activity against 4 bacteria and 1 fungal strain while the HFT extract showed activity against three bacterial strains. When compared with positive control results, the best activity was the HFT extract against the S. aureus strain with an MIC value of 4.8 mg/L; S. epidermidis with 78 mg/L. It is planned to identify the active compounds and to exhibit antimicrobial activity of the HFT extract.
It was concluded that methanol extract from the aerial parts of the plant, containing the highest amount of total phenolics and flavonoids, has the antioxidative potential for chain-breaking inhibition of lipid peroxidation and shows the strongest hydrogen and a single electron donor activities, thus could serves as free radical scavenger, acts as reductant and provide protection against oxidative stress. Although the methanol extract from rhizomes did not show any inhibitory effect on lipid peroxidation, it may also be expected to protect against oxidative damage by scavenging free radicals and acting as reductant. The results demonstrated the health promoting potential of aerial parts from F. trojana. On the other hand, because of the high antimicrobial activity of the plant, the aerial parts of the plant and its rhizomes could be source of antimicrobial effective new molecules.
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Sevda Suzgec Selcuk (1,*), Nurten Ozsoy (2), Berna Ozbek Celik (3), Emine Akalin Urusak (4)
(1) Deparment of Pharmacognosy, Faculty of Pharmacy Marmara University, 34668, Istanbul, Turkey
(2) Department of Biochemistry, Faculty of Pharmacy Istanbul University, 34416, Istanbul, Turkey
(3) Department of Pharmaceutical Microbiology, Faculty of Pharmacy Istanbul University, 34416, Istanbul, Turkey
(4) Department of Pharmaceutical Botany, Faculty of Pharmacy Istanbul University, 34416, Istanbul, Turkey
Address for Correspondence:
Sevda Suzgec Selcuk, e-mail: firstname.lastname@example.org
Cite this article as: Suzgec Selcuk S, Ozsoy N, Ozbek Celik B, Akalin Urusak E (2017). Antioxidant and Antimicrobial Activity of Ferulago trojana E. Akalin & Pimenov. Istanbul J Pharm 47 (3): 101-106.
Table 1. Total phenolic compounds (PC) (as gallic acid equivalents) and total flavonoids (as catechin equivalents) in methanol extracts from F. trojana PC Flavonoids Extract (mg/g extract) (mg/g extract) HFT 64.49[+ or -]4.47 (a) 58.89[+ or -]4.11 (a) RFT 6.51[+ or -]0.82 (b) 4.56[+ or -]0.70 (b) HFT: Aerial parts of F. trojana RFT: Rhizomes of F. trojana Values were the means of three replicates [+ or -] standard deviation. Values with different letters in the same column were significantly (p<0.05) different. Table 2. [EC.sub.50], TEAC and FRAP values of methanol extracts from F. trojana Lipid peroxidation (a) DPPH (a) [EC.sub.50] [EC.sub.50] Extracts (mg/mL) (mg/mL) HFT 3.01[+ or -]0.045 (a) 1.35[+ or -]0.069 (a) RFT N.d 16.69[+ or -]0.34 (b) quercetin 0.034[+ or -]0.006 (b) 0.069[+ or -]0.001 (f) Reducing ABTS (a) power (a) [EC.sub.50] [EC.sub.50] Extracts (mg/mL) (mg/mL) HFT 1.79[+ or -]0.22 (a) 1.18[+ or -]0.34 (a) RFT N.d. 10.73[+ or -]0.54 (b) quercetin 0.113[+ or -]0.002 (d) 0.019[+ or -]0.003 (e) Total Antioxidant FRAP potential (b*) value (c*) Extracts (mM/L TEAC) (mM/L [Fe.sup.2+]) HFT 2.15[+ or -]0.005 (a) 1.87[+ or -]0.08 (a) RFT 0.26[+ or -]0.03 (b) 0.27[+ or -]0.03 (b) quercetin 2.15[+ or -]2.42 (a) 2.15[+ or -]0.011 (a) (at 0.16 mg/mL) (at 0.16 mg/mL) HFT: Aerial parts of F. trojana; RFT: Rhizomes of F.trojana (a) [EC.sub.50] value: The effective concentration at which the antioxidant activity was 50%; DPPH and ABTS radicals were scavenged by 50% and the absorbance was 0.5 for reducing power. [EC.sub.50] value was obtained by interpolation from linear regression analysis. (b) Expressed as mmol Trolox equivalents per gram of dry weight (c) Expressed as mmol ferrous ions eqivalents per gram of dry weight (*) - Determined at 5 mg/mL N.d. Not determined Values were the means of three replicates [+ or -] standard deviation Table 3. Antimicrobial activities (MIC in mg/L) of methanol extracts from F. trojana Methicillin Extracts/ Staphylococcus -resistant Staphylococcus Pozitive aureus Staphylococcus epiddermiddis control ATCC aureus ATCC (mg/L) 6538 ATCC 33591 12228 HFT 4.8 48 78 RFT 78 19 625 Cefuroxime-Na 1.2 nt 9.8 Vancomycin nt 2 nt Ceftazidime nt nt nt Klotrimazole nt nt nt Extracts/ Escherichia Klebsiella Pseudomonas Proteus Candida Pozitive coli pneumoniae aeruginosa mirabilis albicans control ATCC ATCC ATCC ATCC ATCC (mg/L) 8739 4352 1539 14153 10231 HFT - - - - - RFT - - - 156 312 Cefuroxime-Na 4.9 4.9 nt 2.4 nt Vancomycin nt nt nt nt nt Ceftazidime nt nt 2.4 nt nt Klotrimazole nt nt nt nt 4.9 MIC: miniumum inhibitory concentration; HFT: Aerial parts of F.trojana; RFT: Rhizomes of F.trojana (-): Not active (nt): Not tested
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|Title Annotation:||Original Article|
|Author:||Selcuk, Sevda Suzgec; Ozsoy, Nurten; Celik, Berna Ozbek; Urusak, Emine Akalin|
|Publication:||Journal of the Faculty of Pharmacy of Istanbul University|
|Date:||Dec 1, 2017|
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