Antioxidant and antimicrobial activities of essential oil and extracts of fennel

Research Article Received: 19 September 2008, Revised: 11 March 2009, Accepted: 16 March 2009, Published online 14 April 2009 in Wiley Interscience (www.interscience.wiley.com) DOI 10.1002/ffj.1929 Antioxidant and antimicrobial activities John Wiley & Sons, Ltd. of essential oil and extracts of fennel (Foeniculum vulgare Mill.) seeds from Pakistan Farooq Anwar a *, Muhammad Ali a,b, Abdullah Ijaz Hussain a and Muhammad Shahid a ABSTRACT: The present study was conducted to examine the antioxidant and antimicrobial activities of essential oil, methanol and ethanol extracts of fennel (Foeniculum vulgare Mill.) seeds native to Pakistan. The seed essential oil and extract yields from fennel seeds were found to be 2.81 and 6.21 15.63% w/w, respectively. GC and GC MS analysis of the fennel essential oil revealed the presence of 23 compounds, with trans-anethol (69.87%), fenchone (10.23%), estragole (5.45%) and limonene (5.10%) as the major components. The fennel seed extracts contained appreciable levels of total phenolic contents (627.21 967.50 GAE, mg/100 g) and total flavonoid contents (374.88 681.96 CE, mg/100 g). Fennel essential oil and extracts also exhibited good DPPH radical scavenging activity, showing IC 50 32.32 and 23.61 26.75 mg/ml, and inhibition of peroxidation 45.05 and 48.80 70.35%, respectively. Of the fennel essential oil and solvent extracts tested, 80% ethanol extract exhibited the maximum antioxidant activity, whereas the essential oil showed appreciable antimicrobial activity against selected strains of bacteria and pathogenic fungi. The results of the present investigation demonstrated significant (p < 0.05) variations in the antioxidant and antimicrobial activities of fennel essential oil and extracts. Copyright 2009 John Wiley & Sons, Ltd. Keywords: Foeniculum vulgare; trans-anethol; GC MS; total phenolics; IC 50 ; total flavonoids; MIC 170 Introduction Antioxidants act as radical scavengers, inhibit lipid peroxidation and other free radical-mediated processes and are able to protect the human body as well as processed foods from oxidative damage attributed to the reaction of free radicals. The use of synthetic antioxidants, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and tertiary butylhydroquinone (TBHQ), in foods is discouraged due to their perceived carcinogenic potential and safety concerns. [1] Currently, the use of plant-based natural antioxidants, such as those of phenolic substances like flavonoids and phenolic acids and tocopherols in foods, as well as preventive and therapeutic medicine, is gaining much recognition. Such natural substances are believed to exhibit anticarcinogenic potential and offer diverse health-promoting effects because of their antioxidant attributes. [1,2] Food-borne diseases are a severe health problem in the world, even in well-developed nations. [3] The consumption of food contaminated with food-borne microorganisms can pose a serious threat to human health. The existence of microorganisms causes spoilage and results in reduction of the quality and quantity of processed foods. [4] Some biologically active compounds isolated from spices and herbs have been in use for the inhibition of growth of pathogenic microorganisms because of the resistance that microorganisms have built against antibiotics. [5] Herbal spices, being a promising source of phenolics, flavonoids, anthocyanins and carotenoids, are usually used to impart flavour and enhance the shelf-life of dishes and processed food products. [6] Essential oils, extracts and bioactive constituents of several spices and herbs are well known to exert antioxidant and antimicrobial activities. [7] Fennel (Foeniculum vulgare Miller), a plant belonging to the family Apiaceae, has a long history of herbal uses. Traditionally, fennel seeds are used as anti-inflammatory, analgesic, carminative, diuretic and antispasmodic agents. [8] Recently there has been considerable interest in the antioxidant potential and antimicrobial activities of fennel seed extracts and essential oil. [8 11] The purpose of the present study was to evaluate the antioxidant and antimicrobial effectiveness of the essential oil and methanol and ethanol extracts of fennel seeds grown in the central Punjab region of Pakistan. Materials and Methods Collection and Pretreatment of Plant Material Fully ripened fennel (Foeniculum vulgare Mill.) seeds (var. Dulce) were collected from cultivated plants during May June 2006 from the Botanical Garden, University of Agriculture, Faisalabad, * Correspondence to: F. Anwar, Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad-38040, Pakistan. E-mail: fqanwar@yahoo.com a b Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad-38040, Pakistan National Institute for Biotechnology and Genetic Engineering (NIBGE), PO Box 577, Faisalabad, Pakistan Copyright 2009 John Wiley & Sons, Ltd. Flavour Fragr. J. 2009, 24, 170 176

Pakistan. The collections were made from the fully matured plants and only brown fruits were picked. The specimens were further identified and authenticated by a taxonomist, Dr Mansoor Hameed, Assistant Professor, Department of Botany, University of Agriculture, Faisalabad, Pakistan [voucher specimen code, Eclipta alba (L). Hassk (AS21)]. Collected specimens were dried at 35 C in a hot air oven (IM-30, Irmec, Germany) and stored in polyethylene bags at 4 C. Chemicals and Reagents Linoleic acid, 2,2-diphenyl-1-picrylhydrazyl, gallic acid, Folin Ciocalteu reagent, ascorbic acid, trichloro-acetic acid, sodium nitrite, aluminium chloride, ammonium thiocyanate, ferrous chloride, ferric chloride, potassium ferricyanate, butylated hydroxytoluene (99.0%), homologous series of C 9 C 24 n-alkanes and various reference chemicals (α-pinene, camphene, β- pinene, β-myrcene, α-phellandrene, p-cymene, limonene, β- ocimene, γ-terpinene, 1,8-cineol, fenchone, fenchyl alcohol, estragole, fenchyl acetate, anethole, p-anisaldehyde and β- caryophyllene) used to identify the constituents were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals (analytical grade), i.e. anhydrous sodium carbonate ferrous chloride, ammonium thiocyanate, chloroform and methanol, used in this study were purchased from Merck (Darmstadt, Germany), unless stated otherwise. All culture media and standard antibiotic discs were purchased from Oxoid Ltd. (Hampshire, UK). essential oil composition was reported as a relative percentage of the total peak area. Furthermore, the four major components of the fennel essential oils, trans-anethole, fenchone, estragole and limonene, were quantified by mean of the internal standard addition method. [12] Gas chromatography/mass spectrometry analysis. GC MS analyses of the essential oils were performed using an Agilent Technologies (Little Falls, CA, USA) 6890 N Network gas chromatographic (GC) system, equipped with an Agilent Technologies 5975 inert XL mass selective detector and Agilent Technologies 7683B series auto-injector. Compounds were separated on an HP-5 MS capillary column (30 m 0.25 mm i.d., film thickness 0.25μm; Agilent Technologies). A sample of 1.0 μl was injected in the split mode with split ratio 1:100. For GC MS detection, an electron ionization system, with ionization energy of 70 ev, was used. Column oven temperature programme was the same as in GC analysis. Helium was used as the carrier gas at a flow rate of 1.5 ml/min. Mass range was 50 550 m/z, while injector and MS transfer line temperatures were set at 220 C and 290 C, respectively. Compounds identification. The identification of components was based on comparison of their mass spectra with those of NIST mass spectral library, [13,14] and those described by Adam, [15] as well as on comparison of their retention indices either with those of authentic compounds or with literature values. [9,15 17] Preparation of Fennel Seed Extracts Ground (80 mesh) seed sample (20 g) was extracted separately with 200 ml 100% methanol, 80% methanol (80:20, methanol:water, v/v), 100% ethanol, 80% ethanol (80:20, ethanol:water, v/v) using an orbital shaker (Gallenkamp, UK) for 8 h at room temperature. The extracts were separated from solids by filtering through Whatman No. 1 filter paper. The remaining residue was re-extracted twice and the extracts were pooled. The solvent was removed under vacuum at 45 C, using a rotary vacuum evaporator (N-N Series, Eyela, Rikakikai Co. Ltd, Tokyo, Japan) and stored at 4 o C until used for further analyses. Isolation of Essential Oil The oven-dried and ground fennel seeds (80 mesh) were subjected to hydrodistillation for 3 h using a Clevenger-type apparatus. The obtained essential oil was dried over anhydrous sodium sulphate, filtered and stored at 4 C until tested and analysed. Analysis of the Essential Oil Gas chromatography. The essential oils were analysed using a Perkin-Elmer gas chromatograph, Model 8700, equipped with a flame ionization detector (FID) and an HP-5MS capillary column (30 m 0.25 mm i.d., film thickness 0.25 μm). Injector and detector temperatures were set at 220 C and 290 C, respectively. Column oven temperature was programmed from 80 C to 220 C at a rate of 4 C/min; initial and final temperatures were held for 3 and 10 min, respectively. Helium was used as carrier gas at a flow rate of 1.5 ml/min. A sample of 1.0 μl was injected, using split mode (split ratio, 1:100). All quantification was done by a built-in data-handling program provided by the manufacturer of the gas chromatograph (Perkin-Elmer, Norwalk, CT, USA). The Antioxidant Activity Determination of total phenolic contents (TPC). The total phenolic content (TPC) in the fennel seed extracts was calculated using the Folin Ciocalteu reagent method as described by Sultana et al. [18] Determination of total flavonoid contents (TFC). The total flavonoid content (TFC) in the fennel seed extracts was determined following the procedure as described by Sultana et al. [18] Determination of reducing power. The reducing power of the fennel seed extracts was determined according to the procedure described by Yen et al. [19] DPPH radical scavenging assay. The 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assay was carried out spectrophotometrically as described by Tepe et al. [20] Aliquots (50 μl) of various concentrations (10 100 μg/ml) of the essential oil and extract samples was added to 5 ml of a 0.004% methanol solution of DPPH. After a 30 min incubation period at room temperature, the absorbance was read against a blank at 517 nm: I(%) = 100 (A blank A sample /A blank ) where A blank is the absorbance of the control reaction (containing all reagents except the test compound) and A sample is the absorbance of the test compound. Extract concentration providing 50% inhibition (IC 50 ) was calculated from a graph plotting percentage inhibition against extract concentration. Antioxidant activity determination in linoleic acid system. The antioxidant activities of fennel essential oil, methanol and ethanol extracts were also determined in terms of measurement 171 Flavour Fragr. J. 2009, 24, 170 176 Copyright 2009 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/ffj

F. Anwar et al. 172 of percentage inhibition of peroxidation in the linoleic acid system, following the method described by Iqbal et al. [2] with some modifications. Essential oil and extracts (5 mg) were added to a solution mixture of linoleic acid (0.13 ml), 99.8% ethanol (10 ml) and 10 ml 0.2 M sodium phosphate buffer, ph 7. The total mixture was diluted to 25 ml with distilled water. The solution was incubated at 40 C for 175 h. The extent of oxidation was measured by the peroxide value, using the colorimetric method as described by Yen et al. [19] Antimicrobial Activity Microbial strains. The fennel essential oil and extracts were individually tested against a panel of microorganisms, including two bacteria, Escherichia coli B10 and Bacillus subtilis SPS2, and three pathogenic fungi, Aspergillus niger ATCC 10 575, Fusarium solani ATCC 36 031 and Rhizopus solani. The pure bacterial and fungal strains were obtained from the Biological Division of the Nuclear Institute for Agriculture and Biology (NIAB), Faisalabad, Pakistan. The purity and identity of the strains were verified by the Department of Veterinary Microbiology, University of Agriculture, Faisalabad, Pakistan. Bacterial strains were cultured overnight at 37 C in nutrient agar (Oxoid, Hampshire, UK) while fungal strains were cultured overnight at 30 C using potato dextrose agar (Oxoid). Disc diffusion method. The antimicrobial activity of the fennel essential oil and extracts was determined by the disc diffusion method. [21] The discs (6 mm in diameter) were impregnated with 15 μl essential oils or 30 mg/ml extracts (300 μg/disc) placed on the inoculated agar. Amoxycillin (30 μg/disc) (Oxoid) and Flumequine (30 μg/disc) (Oxoid) were used as positive reference for bacteria and fungi, respectively. Disc without samples was used as a negative control. Antimicrobial activity was evaluated by measuring the inhibition zone. Microdilution broth method. For calculation of minimum inhibitory concentration (MIC), which represents the concentration that completely inhibits the growth of microorganisms, a microdilution broth susceptibility assay was used, as reported by NCCLS. [22] A series of dilutions were prepared in the range 0.01 72.0 mg/ml of the fennel essential oil in a 96-well microtitre plate, including one growth control (NB/SDB + Tween 80) and one sterility control (NB/SDB + Tween 80 + test oil). 160 μl NB and SDB for bacteria and fungi, respectively, were added onto the microplates with 20 μl of the tested solution. Then, 20 μl 5 10 5 CFU/ml (confirmed by viable count) of standard microorganism suspension was inoculated onto the microplates. The plates were incubated at 37 C for 24 h for bacteria and at 30 C for 48 h for fungi. Amoxycillin was used as a reference compound for antibacterial and flumequine for antifungal activities. The growth was indicated by the presence of a white pellet on the well bottom. The MIC was calculated as the highest dilution showing complete inhibition of the tested strains. Statistical Analysis All the experiments were conducted in triplicate unless stated otherwise and statistical analysis of the data was performed by analysis of variance (ANOVA), using STATISTICA 5.5 (Stat Soft Inc, Tulsa, Oklahoma, USA) software. A probability value of p 0.05 was considered to denote a statistically significance difference. All data are presented as mean values ± standard deviation (SD). Results and Discussion Essential Oil and Extracts Yields Yields (g/100 g) of fennel seeds essential oil and various extracts are given in Table 1. The yield of hydrodistilled fennel seed essential oil was found to be 2.81%. Maximum extract yield was obtained with 80% ethanol (15.63%) and the minimum with 100% methanol (6.21%). Based on these results, the extracting ability of different solvents followed the order: 80% ethanol > 80% methanol > absolute ethanol > absolute methanol. The present results demonstrated a significant (p <0.05) difference in the extract yields among the solvent systems used. Our results regarding higher extract yield with ethanol are in good agreement with the findings of Oktay et al., [8] who reported 10.95% yield of fennel seed extracts with absolute ethanol. Conforti et al. [23] reported 10.95% and 15.78% extract yields, respectively, from cultivated and wild fennel seeds using methanol. Mata et al. [11] found the yield of essential oil and ethanol extract of fennel seed from Portugal to be 0.1% and 6.9%, respectively. Mimica-Dukic et al. [9] determined the yield of the essential oils obtained from F. vulgare seeds by steam distillation, range 2.82 3.38%. Chemical Composition of the Essential Oil The results of the chemical composition of fennel essential oil are presented in Table 2. Overall, 23 compounds representing 95.53% of the oil were identified with the aid of GC MS. The major constituents of the essential oil tested were transanethole (69.87%), fenchone (10.23%), estragole (5.45%) and limonene (5.10%). In addition, the tested fennel essential oil also contained considerable amounts of various minor constituents whose contribution was <10%. Regarding the groups of chemical constituents represented, the fennel essential oil mainly consisted of oxygenated monoterpenes (87.30%), followed by monoterpene hydrocarbons (7.88%) and sesquiterpene hydrocarbons (0.35%). Trans-anethole, fenchone and estragole were the main oxygenated monoterpenes, while limonene was the major monoterpene. A literature search revealed trans-anethole (62.0%), fenchone (20.3%), estragole (4.90%) and limonene (3.15%) to be the main Table 1. Yield of fennel seeds essential oil, methanol and ethanol extracts Samples Yield (g/100 g) a Essential oil 2.81 ± 0.14 100% methanol extract 6.21 ± 0.37 d 80% methanol extract 12.11 ± 0.73 b 100% ethanol extract 8.84 ± 0.44 c 80% ethanol extract 15.63 ± 0.62 a a Values are mean ± SD of three separate experiments. Different letters in superscript indicate significant differences within solvents. www.interscience.wiley.com/journal/ffj Copyright 2009 John Wiley & Sons, Ltd. Flavour Fragr. J. 2009, 24, 170 176

Table 2. Fennel essential oil composition (%), obtained by GC MS a Compounds b RI c RI from literature Composition Identification d Monoterpene hydrocarbons α-pinene 939 939 [16] 0.55 ± 0.02 RT, RI, MS Camphene 954 954 [16] 0.13 ± 0.03 RT, RI, MS Sabinene 975 975 [16] 0.19 ± 0.04 RT, RI, MS β-pinene 979 979 [16] 0.09 ± 0.02 RT, RI, MS β-myrcene 991 991 [16] 0.87 ± 0.10 RT, RI, MS α-phellandrene 1003 1003 [9] 0.19 ± 0.02 RT, RI, MS p-cymene 1025 1025 [16] t RT, RI, MS Limonene 1029 1029 [16] 5.10 ± 0.10 RT, RI, MS, CI (Z)-β-Ocimene 1037 1037 [9,16] 0.60 ± 0.02 RT, RI, MS (E)-β-ocimene 1049 1048 [9] t RT, RI, MS γ-terpinene 1060 1059 [9] 0.16 ± 0.02 RT, RI, MS Oxygenated monoterpenes 1,8-Cineol 1031 1031 [16] 0.23 ± 0.02 RT, RI, MS Fenchone 1088 1088 [16] 10.23 ± 0.20 RT, RI, MS, CI Fenchyl alcohol 1114 1113 [9] 0.40 ± 0.04 RT, RI, MS Estragole 1197 1199 [9] 5.45 ± 0.20 RT, RI, MS, CI Fenchyl acetate (endo) 1220 1222 [17] 0.12 ± 0.03 RT, RI, MS Fenchyl acetate (exo) 1232 1232 [9] 0.54 ± 0.10 RT, RI, MS cis-anethole 1252 1252 [9] 0.27 ± 0.03 RT, RI, MS p-anisaldehyde 1256 1256 [17] 0.19 ± 0.01 RT, MS trans-anethole 1288 1287 [17] 69.87 ± 0.65 RT, RI, MS, CI Sesquiterpene hydrocarbons β-caryophyllene 1421 1420 [17] 0.26 ± 0.00 RT, RI, MS Germacrene D 1485 1485 [16] 0.09 ± 0.00 RT, RI, MS Total 95.53 a Values are mean ± SD of two independent experiments. b Compound listed in the order of elution from a HP-5MS column. c Retention indices relative to C 9 C 24 n-alkanes on the HP-5MS column. d RI, identification based on retention index; MS, identification based on comparison of mass spectra; RT, identification based on retention time; CI, co-injection with authentic standards; t, trace (<0.05%). components of essential oils from wild-growing fennel seed native to the Podgorica region, central south Montenegro. [24] Mimica-Dukic et al. [9] also reported trans-anethole (74.18%), fenchone (11.32%), estragole (5.29%), limonene (2.53%) and α- pinene (2.77%) as the major compounds identified in the essential oil from Foeniculum vulgare Mill. Ozcan et al. [10] and Ozcan and Chalchat [25] reported estragole (61.08% and 40.49%), fenchone (23.46% and 16.90%) and limonene (8.68% and 17.66%), respectively, as the major constituents in the essential oil of bitter fennel (F. vulgare spp. piperitum) grown in Turkey. Such variations in the chemical composition of essential oil across countries might be attributed to the varied agroclimatic (climatical, seasonal, geographical) conditions of the regions, stage of maturity and adaptive metabolism of plants. On a quantitative basis, the amounts of the four main components, calculated using calibrated curves with pure standard compounds, were: trans-anethole, fenchone, estragole and limonene in the essential oil tested, 68.1, 9.50, 4.92 and 4.50 g/ 100 g oil, respectively (Table 3). There are no previous data available in the literature on the quantitative analysis of fennel essential oil s components with which to compare our present results. Table 3. Amounts of the major constituents in fennel essential oil Compounds Content (g/100 g) a trans-anethole 68.1 ± 0.8 Fenchone 9.50 ± 0.40 Estragole 4.92 ± 0.18 Limonene 4.50 ± 0.22 a Values are mean ± SD of three independent experiments. Antioxidant Activity DPPH radical scavenging assay. We investigated the free radical scavenging activity and lipid oxidation inhibition of fennel seeds essential oil and extracts. Free radical scavenging activities of the fennel essential oil and extracts were measured by DPPH assay. Free radical scavenging capacity increased with increasing extract and essential oil concentration (Table 4). Fennel seed 173 Flavour Fragr. J. 2009, 24, 170 176 Copyright 2009 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/ffj

F. Anwar et al. Table 4. Antioxidant activities of fennel seeds essential oil, ethanol and methanol extracts a Antioxidant assays Essential oil Extracts BHT 100% Methanol 80% Methanol 100% Ethanol 80% Ethanol DPPH, IC 50 (μg/ml) 32.32 ± 0.77 a 26.75 ± 1.06 b 24.25 ± 0.72 bc 26.10 ± 0.90 b 23.61 ± 0.89 c 19.00 ± 0.95 d Inhibition in linoleic 45.05 ± 1.01 d 48.80 ± 0.90 c 55.31 ± 1.04 c 50.76 ± 0.99 c 70.35 ± 1.74 b 92.07 ± 2.24 a acid system (%) Total phenolic contents b 627.21 ± 18.36 c 635.84 ± 12.01 b 870.93 ± 20.82 b 967.50 ± 35.51 a (mg/100 g of seed extracts) Total flavonoid contents c (mg/100 g of seed extracts) 374.88 ± 12.89 d 454.99 ± 16.33 b 529.41 ± 15.81 c 681.96 ± 24.11 a a Values are mean ± SD of three separate experiments. b Total phenolic contents expressed as gallic acid equivalent. c Total flavonoid contents expressed as catechin equivalent Different letters in superscript indicate significant differences within solvents. 174 essential oils and extracts showed excellent radical scavenging activity, with IC 50 (the extract concentration providing 50% of inhibition) values of 32.32 and 23.61 26.75 μg/ml, respectively. The free radical scavenging activity of ethanol extract was superior to that of essential oil. Furthermore, 80% ethanol and methanol extracts exhibited more scavenging activity than absolute ethanol and methanol extracts. When compared with the synthetic antioxidant BHT, both essential oil and extracts offered slightly lower antioxidant activity. No earlier reports are available regarding the DPPH radical scavenging activity of fennel seed essential oils with which to compare the results of our present analysis. However, according to Conforti et al. [23] the IC 50 value for methanol extract of wild and cultivated fennel seeds was determined to be 31 and 83 μg/ml, respectively. Mata et al. [11] reported that ethanol extract of fennel seeds exhibited stronger radical scavenging activity (IC 50 =12.0μg/ml) than the synthetic antioxidant BHT (IC 50 = 15.7 μg/ml). Percentage inhibition of linoleic acid oxidation. Table 4 shows the percentage inhibition of linoleic acid oxidation as exhibited by the fennel essential oil and extracts. 80% ethanol extract offered significantly (p < 0.05) higher inhibition of peroxidation (70.35%) than fennel essential oil (45.05%) and other extracts (48.8 55.31%). When the inhibitions of linoleic acid oxidation of fennel essential oil and extracts were compared with BHT, all the extracts and essential oil exhibited significantly (p < 0.05) lower antioxidant activity than that shown by BHT (92.1%). The order of inhibition of linoleic acid oxidation offered by essential oil and various extracts of fennel seeds were as follows: BHT > 80% ethanol > 80% methanol > absolute ethanol > absolute methanol > essential oil. Due to lack of data on the percentage inhibition of linoleic acid oxidation of fennel seed essential oil, we could not compare the results of our present study with the literature. However, the percentage inhibitions of fennel seed extracts determined in our study are in close agreement with the findings of Parejo et al., [26] who reported the linoleic acid peroxidation for crude fennel extract as being in the range 45.35 45.79% and that of BHT as 89.24%. Contrary to our results, Ozcan et al. [25] reported that the inhibition of an ethanol extract of fennel seed was found to be superior (98.6%) to that of BHT (97.8%). Total phenolic and total flavonoid contents. The total phenolic contents (TPC) and total flavonoid contents (TFC) of fennel seed extracts are presented in Table 4. The amounts of TPC and TFC extracted from fennel seeds in different solvent systems were in the ranges 627.21 967.50 GAE (mg/100 g) and 374.88 681.96 CE (mg /100 g), respectively. Ethanol extract (80%) of the fennel seeds showed the highest TPC and TFC, 967.50 and 681.96 mg/100 g, respectively. These differences in the amount of TPC and TFC may be due to varied efficiency of the extracting solvents to dissolve endogenous compounds. The ability of different solvents to extract TPC and TFC was of the order: 80% ethanol > 80% methanol > absolute ethanol > absolute methanol. Ethanol is preferred for the extraction of antioxidant compounds mainly because of its lower toxicity. [27,28] Conforti et al. [24] reported TPC (chlorogenic acid equivalents) from extracts of cultivated and wild fennel to be 100 and 151 mg/g extract, respectively. According to Mata et al., [11] the ethanol extract of fennel seed revealed 63.1 mg/g TPC (pyrogallol equivalents). No earlier reports are available on the TFC of fennel seeds extracts with which to compare the results of our present analysis. Reducing power. The trends of reducing potential of different fennel seed extracts are presented in Figure 1; the greater the intensity of the colour, the greater will be the absorption; consequently, the greater will be the antioxidant activity. [18] The reducing potential of the tested fennel extracts was observed at concentrations of 2.5 10 mg/ml. The absorbance recorded for the tested extract solutions in this assay were noted to be in the range 0.20 1.85, indicating a high correlation index (r 2 = 0.9416 0.9954). The maximum absorbance value (1.85) was recorded for 80% ethanol, while the minimum was for absolute methanol (0.20). The reducing power of different solvent extracts lowered in the order: 80% ethanol > 80% methanol > absolute methanol > absolute ethanol. The variations in the reducing powers of different fennel seed extracts were statistically significant (p < 0.05). When these results were compared with standard ascorbic acid, all the extracts showed significantly (p <0.05) less reducing power. Our results are comparable with the investigations of Oktay et al., [8] who found the reducing power of ethanol and water extracts of fennel seeds at a concentration of 250 μg/ml to be 1.09 and 0.509, respectively. www.interscience.wiley.com/journal/ffj Copyright 2009 John Wiley & Sons, Ltd. Flavour Fragr. J. 2009, 24, 170 176

Figure 1. Reducing potential of methanol and ethanol extracts of fennel seeds Table 5. Antimicrobial activity in terms of inhibition zones and minimum inhibitory concentration of fennel essential oil, ethanol and methanol extracts against the selected strains of bacteria and fungi a Tested microorganisms Essential oil Extracts Amoxicillin Flumequine 100% methanol 80% methanol 100% ethanol 80% ethanol Diameter of inhibition zone b Escherichia coli 14 ± 1 28± 2 Bacillus subtilis 29 ± 1 32± 2 Aspergillus niger 28 ± 2 31± 2 Fusarium solani 26 ± 1 29± 1 Rhizopus solani 19 ± 1 28± 2 Minimum inhibitory concentration (MIC) c Escherichia coli 259.3 ± 4.9 80.3 ± 3.2 Bacillus subtilis 62.6 ± 1.9 20 ± 0.9 Aspergillus niger 80.6 ± 1.4 30.3 ± 1.3 Fusarium solani 91.1 ± 5.0 40.4 ± 1.2 Rhizopus solani 110.3 ± 3.3 43.9 ± 1.7 a Values are mean ± SD of three separate experiments. b Diameter of inhibition zone (mm) including disc diameter of 6 mm. c Minimum inhibitory concentration, MIC (mg/ml). Antimicrobial Activity The antibacterial activity of the essential oil and extracts from fennel seeds against a panel of food-borne and pathogenic microorganisms were assessed. The results are presented in Table 5. Fennel essential oils exhibited considerable antimicrobial activity against all the strains tested, particularly against Gram-positive bacteria. The results from the disc diffusion method, followed by measurement of minimum inhibitory concentration (MIC), indicated that B. subtilis and A. niger were the most sensitive microorganisms tested, showing the largest inhibition zones (29 and 28 mm) and the lowest MIC values (62.6 and 80.6 μg/ml), respectively. Least activity was exhibited against E. coli, with the smallest inhibition zones (14 mm) and the highest MIC value (259.3 μg/ml). In general, the antimicrobial activity of the tested fennel essential oil is comparable with the standard drugs, amoxicillin and flumequine. In agreement with our results, Cantore et al. [29] reported that the Gram-negative strains of bacteria, especially E. coli, have less sensitivity to fennel essential oils. Ozcan et al. [30] found that fennel essential oils exhibit an inhibitory effect against a wide range of Bacillius species. Mimica-Dukic et al. [9] also reported that the essential oils of fennel are active against Aspergillus species. Ozcan et al. [10] reported the antifungal activity of essential oils from bitter fennel. As expected, fennel seed extracts offered no antimicrobial activity. The results of the present study demonstrated that essential oil and various extracts from fennel (F. vulgare) show good antioxidant and free radical scavenging activities. Furthermore, fennel essential oil also exhibited appreciable antimicrobial activity. The production of such essential oil and bioactive components from indigenous resources and their utilization as potential natural food preservatives could be of economic value. However, further investigations involving more detailed in vitro 175 Flavour Fragr. J. 2009, 24, 170 176 Copyright 2009 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/ffj

F. Anwar et al. and in vivo studies to establish which components of the essential oil or extracts offer the best antioxidant activity are recommended. Overall, this study presents valuable information on the composition and antioxidant attributes of fennel essential oil from Pakistan. It advocates its consumption in food and pharmaceutical preparations local industries. In addition, fennel showing antioxidant activity might be explored for functional food and nutraceutical applications, besides its traditional uses. Acknowledgements Authors are highly thankful to Higher Education Commission (HEC), Islamabad, Pakistan for funding this research under the PYI programme. We would also like to extend our sincere gratitude to Professor Dr. Muhammad lqbal Bhanger, Director, National Centre of Excellence in Analytical Chemistry, University of Sindh, Jamshoro, Pakistan for providing us GC-MS facility. References [1] Q. Liu, H. Yao, Food Chem. 2007, 102, 732. [2] S. Iqbal, M. I. Bhanger, F. Anwer, LWT-Food Sci. Tech. 2007, 40, 361. [3] A. Sokmen, G. Vardar-Unlu, M. Polissiou, D. Daferera, M. Sokmen, E. Donmez, Phytother. Res. 2003, 17(9), 1005. [4] K. M. Soliman, R. I. Badeaa, Food Chem. Toxicol. 2002, 40, 1669. [5] T. Essawi, M. Srour, J. Ethnopharmacol. 2000, 70, 343. [6] E. Cieslik, A. Greda, W. Adamus, Food Chem. 2006, 94, 135. [7] L. Majhenic, M. Skerget, Z. Knez, Food Chem. 2007, 104(3), 1258. [8] M. Oktay, I. Gulcin, O. I. Ufrevioglu, Lebensm. Wiss. Technol. 2003, 36, 263. [9] N. Mimica-Dukic, S. Kujundzic, M. Sokovic, M. Couladis, Phytother. Res. 2003, 17, 368. [10] M. M. Ozcan, J. C. Chalchat, D. Arslan, A. Ates, A. Unver, J. Med Food. 2006, 9, 552. [11] A. T. Mata, C. Proenca, A. R. Ferreira, M. L. M. Serralheiro, J. M. F. Nogueira, M. E. M. Araujo, Food Chem. 2007, 103, 778. [12] R. Kowalski, Flavour Fragr. J. 2008, 23, 164. [13] Y. Massada, Analysis of Essential Oils by Gas Chromatography and Mass Spectrometry. Wiley: New York, 1976. [14] Mass Spectral Library. NIST/EPA/NIH: USA, 2002, http:// www.nist.gov/srd/nistla.htm. [15] R. P. Adams, Identification of Essential Oil Components by Gas Chromatography/Quadrupole Mass Spectroscopy. Allured: Carol Stream, IL, 2001. [16] K. Vagionas, K. Graikou, O. Ngassapa, D. Runyoro, I. Chinou, Food Chem. 2007, 103, 319. [17] G. Singh, S. Maurya, M. P. De-Lampasona, C. Catalan, Food Cont. 2006, 17, 745. [18] B. Sultana, F. Anwar, R. Przybylski, Food Chem. 2007, 104, 1106. [19] G. C. Yen, P. D. Duh, D. Y. Chuang, Food Chem. 2007, 70, 307. [20] B. Tepe, D. Daferera, A. Sokmen, M. Sokmen, M. Polissiou, Food Chem. 2005, 90, 333. [21] National Committee for Clinical Laboratory Standards (NCCLS). Approved Standard M2-A6, 5th edn. NCCLS: Wayne, PA, 1997. [22] National Committee for Clinical Laboratory Standards (NCCLS). M100-S9. NCCLS: Wayne, PA, 1999. [23] F. Conforti, G. Statti, D. Uzunov, F. Menichini, Biol. Pharm. Bull. 2006, 29, 2056. [24] B. Damjanavic, Z. Lepojevic, V. Zivkovic, A. Tolic, Food Chem. 2005, 92, 143. [25] M. M. Ozcan, J. C. Chalchat, Eur. Food Res. Tech. 2006, 224, 279. [26] I. Parejo, F. Viladomat, J. Bastida, G. Schmeda-Hirschmann, G. S. Burillo, C. Codina, J. Agric. Food Chem. 2004, 52, 1890. [27] F. Karadeniz, H. S. Burdurlu, N. Koca, Y. Soyer, J. Agric. Food Chem. 2005, 29, 297. [28] Y. T. Tung, J. H. Wu, Y. H. Kuo, S. T. Chang, Biores. Tech. 2007, 98(5), 1120. [29] P. L. Cantore, N. S. Iacobellis, A. D. Marco, F. Capasso, F. Senatore, J. Agric. Food Chem. 2004, 52, 7862. [30] M. M. Ozcan, O. Sagdic, G. Ozkan, J. Med. Food. 2006, 9(3), 418. 176 www.interscience.wiley.com/journal/ffj Copyright 2009 John Wiley & Sons, Ltd. Flavour Fragr. J. 2009, 24, 170 176