Assessment of Antimycotoxigenic and Antioxidant Activity of Star Anise (Illicium Verum) in Vitro

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FULL LENGTH ARTICLE Assessment of antimycotoxigenic and antioxidant activity of star anise (Illicium verum) in vitro

Soher E. Aly a, Bassem A. Sabry a,*, Mohamed S. Shaheen b, Amal S. Hathout a

a Food Toxicology and Contaminants Dept., National Research Centre, Dokki, Cairo, Egypt b Flavour and Aroma Dept., National Research Centre, Dokki, Cairo, Egypt

Received 10 February 2014; revised 19 May 2014; accepted 21 May 2014 Available online 29 May 2014

KEYWORDS Abstract In recent years scientists have focused on the identification and the application of natural Star anise; products for inactivation of mycotoxins. Essential oils with antimicrobial properties are probably Phenolic compounds; the most promising method for the prevention of potentially toxigenic fungi. Thus the aim of this Flavonoid content; work is to characterise star anise (Illicium verum) and to assess its antioxidant and antifungal and Antioxidants; antimycotoxigenic properties using different methods. Results revealed that the major components Antifungal; of star anise essential oil identified by GC/MS were trans-anethole (82.7%), carryophyllene (4.8%) Aflatoxin; and limonene (2.3%). Total phenolics of ethanol and methanol extracts recorded 112.4 and Fumonisin 96.3 mg GAE/g DW respectively, whereas higher total flavonoid content was recorded for the ethanol extract than the methanol extract. Star anise essential oil showed lower antioxidant activity (55.6 mg/mL) than the extracts using DPPH-scavenging and b-carotene/linoleic acid assays. Results revealed growth reduction of Aspergillus flavus, Aspergillus parasiticus and Fusarium verticillioides by 83.2%, 72.8% and 65.11%, respectively when using 100 ppm of the star anise essential oil, where a complete inhibition was achieved at 200 ppm for A. flavus and A. parasiticus respectively. Afla- toxin B1 and Fumonisin B1 production were inhibited completely at 100 and 200 ppm respectively. It could be concluded that star anise extracts could be considered an important substance that should be explored for the discovery and development of newer and safer food supplements as well as drug products. ª 2014 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.

1. Introduction

In areas, such as Egypt, harvested grains are infected by various species of fungi, such as Aspergillus flavus, Aspergillus par- * Corresponding author. Tel.: +2 02 01123417755. asiticus and Fusarium verticillioides (syn. Fusarium E-mail address: [email protected] (B.A. Sabry). moniliforme) leading to deterioration and mycotoxin produc- Peer review under responsibility of King Saud University. tion (Reddy and Raghavender, 2007). These mycotoxins attract worldwide attention because of the significant eco- nomic losses associated with their impact on human health, animal productivity and trade (Wagacha and Muthomi, Production and hosting by Elsevier 2008). Aflatoxins (AFs) and Fumonisins (FUM) are the most 1658-077X ª 2014 King Saud University. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jssas.2014.05.003 Antimycotoxigenic and antioxidant activity of star anise 21 important mycotoxins (Liu et al., 2006; Mangala et al., 2006). obtained from TCI AMERICA (Portland, OR 97203, USA). AFs induce mutagenic, teratogenic and carcinogenic effects The authentic compounds were obtained from Takata (Rustom, 1997), whereas FUM have been found to be associ- Koryo Co. Ltd. (HYOGO 6610001, Osaka, Japan). Clean, ated with several animal diseases such as leukoencephalomala- mature star anise was purchased from local market (Haraz, cia in horses (Kellerman et al., 1990), pulmonary edema in pigs Egypt). (Harrison et al., 1990), while in humans, their occurrence has been associated with high incidences of oesophageal cancer 2.2. Fungal cultures (Rheeder et al., 1992; Chu and Li, 1994) and liver cancer (Ueno et al., 1997). Fusarium species (F. verticillioides, F. solani F. oxysporum and Current protective measures rely heavily on the chemical F. graminearum) and Aspergillus species (A. Parasiticus, A. fla- control of pathogens, with severe and undesirable environmen- vus) used in this study were isolated in a previous study from tal consequences (Chen and Zhou, 2009). Thus there is an maize samples collected in Egypt. All cultures were maintained increasing demand for safe and organic foods, without chem- on Potato Dextrose Agar (PDA, Difico). ical preservatives. Most plants produce antimicrobial secondary metabolites, either as part of their normal programme of 2.3. Preparing essential oil and extracts growth and development or in response to pathogen attack or stress (Bajpai et al., 2008). A novel way to reduce the pro- Essential oil was prepared by hydro-distillation using a Cle- liferation of microorganism and/or their toxins production is venger type apparatus, and was dried over anhydrous sodium the use of essential oils. They are composed mainly of terpe- sulphate. Essential oil was kept in a closed dark glass bottle noids and phenylpropanoids, including polyketides and very and stored at 4 C until use. few alkaloids. The terpenoids are classified depending on the Ethanol and methanol extracts were prepared according to number of isoprene units as hemiterpenes, monoterpenes, ses- the method of Shyamala et al. (2005) with some modifications. quiterpenes, diterpenes, sesterterpenes, triterpenes and tetra- Briefly, 15 g of dried star anise fruits were extracted with terpenes (de Sousa, 2011). 100 mL of ethanol and methanol for 24 h with occasional Phenolic compounds are ubiquitously distributed through- shaking. Both extracts were filtered and evaporated to dryness out the plant kingdom (Naczk and Shahidi, 2004). Phenolic in vacuum. phytochemicals are known to exhibit several health beneficial activities such as antioxidant, anti-inflammatory, antihepato- 2.4. Gas chromatography–mass spectrometry analysis (GC/MS) toxic, antitumor, and antimicrobial (Middleton et al., 2000). The wide spectrum of bioactivities displayed by phenolic compounds isolated from different foods or food products has dic- The essential oil was analysed by gas chromatography (Perkin tated a demand for accurate determination of phenolic Elmer Auto XL GC, Waltham, MA, USA) equipped with a compounds in different food matrices (Luthria et al., 2008). flame ionisation detector, and the GC conditions were Inhibition of fungal growth and AF production by phenol EQUITY-5 column (60 m · 0.32 mm · 0.25 lm); H2 was the compounds has been a subject of many studies (Kim et al., carrier gas; column head pressure 10 psi; oven temperature 2005, 2006). programme isotherm 2 min at 70 C, 3 C/min gradient Star anise (Illicium verum) is an aromatic evergreen tree 250 C, isotherm 10 min; injection temperature, 250 C; detec- bearing purple-red flowers and anise-scented star-shaped fruit. tor temperature 280 C. The identification of individual com- It grows almost exclusively in southern China and Vietnam. Its pounds was based on their retention times relative to those fruit is an important traditional Chinese medicine as well as a of authentic samples and matching spectral peaks available commonly used spice (Loi and Thu, 1970; Jiangsu New with the published data (Adams, 2007). Medical College, 1977). Few reports were published for char- acterisation of star anise and its efficiency as antifungal activity 2.5. Determination of total flavonoid content especially against mycotoxigenic fungi. Therefore, the aim of the current study is to identify the chemical composition and Total flavonoid content of star anise extracts was determined determine total phenolic and flavonoid content, as well as using the aluminium chloride colorimetric method as described investigate the antioxidant and antimicotoxigenic activity of by Willet (2002), with some modifications. Methanol and star anise against toxigenic strains of Aspergillus and Fusarium ethanol extracts (0.5 mL), 10% aluminium chloride (0.1 mL), species and discuss the mechanism of action with references to 1 M potassium acetate (0.1 mL) and distilled water (4.3 mL) its main components. were mixed, and the absorbance was measured at 415 nm using a Spectrophotometer (Shimadzu, Japan). Cata- 2. Materials and methods chine was used to make the calibration curve. The calculation of total flavonoids in the extracts was carried out in triplicate. 2.1. Chemicals and reagents 2.6. Determination of total phenolic compounds

Aﬂatoxin B1 (AFB1) and Fumonisin B1 (FB1) were purchased from Sigma–Aldrich Co. (St. Louis, MO 63103, USA). Sol- Total phenolic content was determined by the Folin–Ciocal- vents were HPLC grade (Merck, Germany). Folin–Ciocalteau teau method (Ordonez et al., 2006). Star anise extracts reagents, 2,2-diphenylpicrylhydrazyl (DPPH), b-carotene, (100 ll) were mixed with 4 mL of diluted Folin–Ciocalteau Tween 40, linoleic acid, sodium carbonate, sodium sulphate reagent (1:9, v/v) for 5 min and 2.0 mL of 75 g/L Na2CO3 anhydrous, aluminium chloride and sodium nitrite were was then added. The absorbance was measured at 760 nm after 22 S.E. Aly et al.

2 h of incubation at room temperature. Results were expressed evaporation of chloroform, 100 mL oxygen-saturated distilled as lg/mL of gallic acid equivalents (GAE). water was added with vigorous shaking. Then 3 mL aliquots were dispensed into the test tubes, 100 ll of the essential oil, 2.7. Identification of phenolic compounds using HPLC ethanol and methanol extracts of different concentrations were added and the emulsion system was incubated for 48 h at room Quantification of phenolic compounds in fractions was carried temperature. The same procedure was done on both BHT (as out as described by Konczak et al. (2010). One g of dried star positive control) and blank. In turn, absorbance of the mix- anise was extracted by 100 mL ethanol. The extract was evap- tures was measured at 490 nm. Antioxidant activity of the orated under vacuum at room temperature to dryness. The dry essential oil and extracts was compared with those of BHT extracts were dissolved in ethanol and chromatographed under and blank. All tests were done in triplicate, and values of the gradient conditions, with a flow rate of 0.8 mL/min. The gra- extracts and essential oil were compared with those values of dient started with mobile phase A, methanol; and mobile phase BHT. B, 2% acetic acid; at a flow rate of 1 mL/ min; and at a fixed wavelength of 280 nm, with gradient elution program: A%/ 2.9. Antifungal activity B% (5/95) in 0 min; A%/B% (25/75) in 5 min; A%/B% (30/ 70) in 10 min; A%/B% (35/65) in 20 min; A%/B% (100/0) Antifungal activity of star anise essential oil was tested by two in 30 min and back to the initial ratio in 10 min. The injection methods:- volume was 50 ll and chromatogram was acquired at 280 nm. Solutions of available pure known compounds were chromato- A- The agar diffusion method is described in European graphed as external standards. All standards were dissolved in Pharmacopoeia (2001). The tested essential oil was used ethanol before injection in the analytical HPLC system (HP at different concentrations: 0%, 0.05%, 0.1%, 0.2% and 1100 chromatograph Agilent Technologies, Palo Alto, CA, 0.4%. 100 ll of suspension containing 106 spore/mL USA) equipped with an auto-sampler, quaternary pump and fungi (Fusarium species) was spread using a glass rod a diode array detector were used. The quantitation was inte- spreader on PDA. At the end of the incubation period, grated by Chemstation chromatographic software interfaced antifungal activity was evaluated by measuring the zone to a personal computer. The analytical column was ZORBAX of inhibition (mm) against the test fungus. Eclipse XDB C18 column (15 cm · 4.6 mm I.D., 5 lm, USA). B- Dry mycelium weight method were performed in flasks Their ranges of concentration used were 0.7–15.0 mg/l. Pheno- containing yeast extract sucrose broth (Aspergillus med- lic compounds of plant extracts were identified by comparing ium) and corn (Fusarium medium) inoculated with spore their retention times with those of pure standards. The results suspension containing 106 spore/mL fungi and incu- were expressed as lg/100 g DW. bated for 8 d at 28 ± 2 C. Flasks containing mycelia were filtered through a pre weighed Whatman filter 2.8. Antioxidant activity No. 1 and washed with distilled water. The mycelia were placed on pre weighed Petri plates and allowed to dry at 2.8.1. DPPH scavenging assay 50 C for 6 h and then at 40 C for overnight. The net 100 ll of the essential oil, ethanol and methanol extracts of dif- dry weight of mycelia was then determined (Prakash ferent concentrations (1, 2.5, 5 and 10 mg/mL) were added to et al., 2011). 5 mL of the DPPH solution (0.005% methanol solution) as described by Burits and Bucar (2000). After 30 min incubation at room temperature, the absorbance was read against pure 2.9.1. Extraction AFB1 methanol at 520 nm. The radical-scavenging activities of the AFB1 was extracted from the cultures that showed mycelia samples were calculated as percentages of inhibition according growth by taking 25 mL of liquid media (broth) and using to the following equation: 25 mL chloroform twice. After shaking for 30 min, the lower layer was filtered through filter paper No. 4 containing anhy- A blank A sample DPPH% ¼ 100 drous sodium sulphate then the filtrate was evaporated to dry- A blank ness under a stream of nitrogen. The filtrate was passed where A blank is the absorbance of the control (containing all through an immunoaffinity column (Afla B, VICAM, USA) reagents except the test compound), and A sample is the absor- for clean-up by 5 mL of purified water at a rate of about bance of the test compound. All tests were done in triplicate. 2 drops/s. AFB1 was eluted with 1.0 mL methanol at a rate Values of the extracts and essential oil were compared with of 1–2 drops/s and then evaporated to dryness under a stream those values of BHT. of nitrogen. Samples were quantitatively analysed using HPLC. 2.8.2. b-Carotene–linoleic acid assay

Antioxidant capacity of essential oil and extracts was deter- 2.9.2. Extraction of FB1 mined by measuring the inhibition of volatile organic com- The extraction of FB1 from corn cultures was performed pounds and the conjugated dienehydroperoxides arising from according to Rottinghaus et al. (1992). Fifty g corn sample linoleic acid oxidation according to the method of was extracted with 100 mL acetonitrile/water (1:1 v/v) for Dapkevicius et al. (1998). In this respect a stock solution of 30 min. Portion of the extract was filtered through a filter b-carotene–linoleic acid mixture was prepared as follows: paper. Two mL of the filtered extract was combined with 0.5 mg b-carotene was dissolved in 1 mL of chloroform and 5 mL aqueous 1% potassium chloride (KCl) and applied to 40 ll linoleic acid and 200 mg Tween 40 were added. After a preconditioned C18 Sep-Pak column (Waters). The column Antimycotoxigenic and antioxidant activity of star anise 23 was washed with 5 mL 1% aqueous KC1 followed by 5 mL acetonitrile. Samples were quantitatively analysed using HPLC. The HPLC system (Waters, Milford, MA, USA) consisted of a model 1525 binary pump, model 1500 Rheodyne-manual injector, model 2475 multi-wavelength fluorescence detector and data workstation with Breeze software, version 3.3 (Waters, Breeze Software, Milford, MA, USA). AFB1 was eluted with acetonitrile/water/methanol (1:6:3 v/v), and Fumonisins were eluted from the column with 1 mL of HPLC grade methanol and the separation was performed at ambient Figure 1 Total phenolic and total flavonoid content of star anise temperature at a flow rate of 1.0 mL/min. ethanol and methanol extracts. Bars indicate means ± SD obtained from three independent measurements. 2.10. Statistical analysis

DW, whereas the ethanol extract showed lower TFC Results were expressed as means (±standard deviation n = 3). 46.8 ± 0.64 mg CAT. Eq./g DW. Flavonoids are potent anti- Statistical analysis was performed using SPSS statistical pro- oxidants, free radical scavengers and metal chelators, they gramme for windows (Version 16) (SPSS Inc., Chicago, IL, inhibit lipid peroxidation and exhibit various physiological USA). All data were statistically analysed using analysis of activities, including antihypertensive and anti-arthritic activi- variance and the results were considered significant at ties (Wang, 2000; Harborne and Williams, 2000). Phenolics P <0.05. and flavonoids possess many useful biological properties, such as anti-inflammatory, antimicrobial, enzyme inhibition, anti- 3. Results and discussion allergic, and antioxidant activities (Raina et al., 2003), whereas their antioxidant activities are the result of the hydroxyl group 3.1. Chemical composition (–OH) in the aromatic ring, which mediates redox reaction, and is capable of scavenging free radicals. Chemical analysis of the components of the star anise essential oil by GC/MS led to identification of 13 components, repre- 3.3. Total phenolic content (TPC) senting 95.74% of the hydro-distilled essential oils (Table 1). The major components were trans-anethole (82.7%), carryo- The assay used for the determination of TPC employs Folin phyllene (4.8%) and limonene (2.3%). Similar results were and Ciocalteu’s phenol reagent depending on the chemical reported by Zhai et al. (2009). Our results are in agreement structure of phenolics (i.e. the higher the number of functional with Wichtl (2004) who reported that the major compound –OH group the higher the total phenolics content), and were of star anise oil is trans-anethole, which ranged from 86.0 to reported as gallic acid equivalents by reference to a standard 93.0%. In the same trend, Padmashree et al. (2007) and curve (y = 0.007 · +0.087; r2 = 0.987). Results illustrated Ming et al. (2013) reported that trans-anethole is the major in Fig. 1 show that the TPC of ethanol and methanol extracts component in star anise oil, and ranged between 86.66% and recorded 112.4 ± 0.95 and 96.3 ± 0.51 mg GAE/g DW, 94.21%. respectively. In agreement, Padmashree et al. (2007) reported that ethanol/water extract of star-anise contained higher 3.2. Total flavonoid content (TFC) amounts of TPC with (10,025 ppm). Recent studies have also shown that many polyphenols contribute significantly to the Data in Fig. 1 revealed that the methanol extract of star-anise total antioxidant activity in fruits and vegetables (Luo et al., fruits exhibited high TFC with 65.9 ± 0.9 mg CAT. Eq./g 2002). Bluma et al. (2008) reported that higher content of phe-

Table 1 Chemical composition of star anise essential oil. Peak No. Compound LRI Area % Identiﬁcation methods 1 a-Pinene 939 0.18 KI and MS 2 b-Pinene 985 0.2 KI and MS 3 Myrcene 993 0.4 KI and MS 4 a-Phellandrene 1006 0.24 KI and MS 5 Limonene 1036 2.3 KI and MS 6 c-Terpineol 1072 0.45 KI and MS 7 Linalool 1107 0.67 KI and MS 8 a-Terpineol 1190 0.3 KI and MS 9 Estragole 1195 1.8 KI and MS 10 trans-Anethole 1286 82.7 KI and MS 11 a-Cubebene 1392 0.3 KI and MS 12 Carryophyllene oxide 1573 4.8 KI and MS 13 a-Humulene 1597 1.4 KI and MS LRI: linear retention index, MS: mass spectra. 24 S.E. Aly et al.

Figure 2 Phenolic acid profile of star anise ethanol extract by HPLC. nolic compounds in star anise is important for building blocks 3.5. Antioxidant activity assays for cell wall structures, serving as defence against pathogens. Phenolics are the most abundant secondary metabolites of A large number of methods have been developed to evaluate plants with more than 8000 known structures ranging from total antioxidant capacity of food and dietary supplements, simple compounds such as phenolic acids to complex struc- herbal extracts or pure compounds. Nevertheless, few of them tures such as tannins (Antolovich et al., 2002; Dai and have been used widely due to the difficulty of measuring total Mumper, 2010). In plants, accumulation of phenolics varied antioxidant capacity owing to limitations associated with from one part to another part and also depended on the age methodological issues and free radical sources (Schauss and developmental stage of the concerned plant parts. It is well et al., 2006). To assess the antioxidant activity of the essential known that the phenolic extracts are always a mixture of dif- oil, ethanol and methanol extracts, two methods were used in ferent classes of phenols, which are selectively soluble in the this study because it is generally accepted that at least two solvents. A phenolic-OH group is very reactive and can easily methods should be used for assessing antioxidant activities form hydrogen bonds with the active sites of enzymes (Rasooli in vitro (Viuda-Martos et al., 2010). et al., 2009). The stable violet DPPH radical was changed into yellow- coloured DPPH by 92.3 and 88.9 mg/mL for ethanol and 3.4. Identification and quantification of phenolic compounds by methanol extracts respectively, whereas star anise essential HPLC oil showed a lower antioxidant activity by 55.6 mg/mL (Fig. 3). Four different concentrations (1, 2.5, 5, and 10 mg/ Selected phenolic compounds in the star anise were separated mL) of the essential oil and extracts of star anise fruits were and tentatively identified by using HPLC (Fig. 2). The major measured in terms of hydrogen donating or radical scavenging groups of phenolic compounds detected were: catechin, gallic ability, using stable b-carotene radical. This method is consid- acid, caffeic acid, cinnamic acid, ferulic acid and rutin with ered to be a valid and easy assay to evaluate scavenging activ- 75.64, 62.89, 46.42, 16.90, 103.23 and 1112.6 mg/100 g DW ity of antioxidants, since the radical compound is stable and respectively. These results were in agreement with those does not have to be generated as in other radical scavenging reported by Sakulnarmrat et al. (2013). assays. The radical scavenging effects of the tested samples

Figure 3 Antioxidant activity of star anise essential oil, ethanol and methanol extracts measured by DPPH assay. Antimycotoxigenic and antioxidant activity of star anise 25

Figure 4 Antioxidant activity of star anise essential oil, ethanol and methanol extracts measured by b-carotene-linoleic assay.

Figure 5 Linear growth inhibition of Fusarium using star anise essential oil. as well as BHT on the b-carotene radical were concentration growth of F. graminearum, F. solani and F. oxysporum at lower dependent (Fig. 4). All samples with the highest concentration concentration of 100 ppm. On the other hand, F. verticillioides of extracts (10 mg/mL) had scavenging effect comparable to was completely inhibited at a concentration of 200 ppm. the BHT, showing b-carotene radical inhibition ability by Data in Table 2 illustrate the antifungal activity at 100 ppm 56.9, 90.3 and 84.9 mg/mL for essential oil, ethanol and meth- which recorded 83.2%, 72.8% and 65.11% inhibition for A. anol extracts respectively. flavus A. parasiticus and F. verticillioides, respectively. Com- Antioxidant properties may be recommended in enhancing plete inhibition (100%) was observed at 200 ppm for both A. shelf life of products such as spices (Prakash et al., 2011). Nat- flavus and A. parasiticus and at 400 ppm for F. verticillioides. ural antioxidants are known to protect cells from damage These results were confirmed by Singh et al. (2006) who induced by oxidative stress, which is generally considered to reported that the volatile oil inhibited the growth of F. verti- be a cause of ageing, degenerative disease, and cancer cillioides completely. Kim et al. (2006) revealed that growth (Ringman et al., 2005; Viuda-Martos et al., 2010). Luo et al. inhibition of A. flavus by the phenolic salicylic acid, thymol, (2002) reported that many polyphenols contribute significantly vanillyl acetone, vanillin and cinnamic acid is via targeting to the total antioxidant activity in fruits and vegetables. the mitochondrial oxidative stress defence system. The antifungal activity of star anise essential oils can be assigned to 3.6. Antifungal and antimycotoxigenic activity their active components, such as anethole. Star anise is one of many spices that contain bioactive compounds as well as Data in Fig. 5 reveal that star anise essential oil showed high a number of phenolic and flavonoid compounds, having anti- antifungal activity and completely inhibited (100%) fungal oxidant, preservative and antimicrobial properties (Shobana and Naidu, 2000). Numerous investigations have confirmed

Table 2 Antifungal activity (%) of star anise essential oil using Zone inhibition. Fungus species Star anise essential oil concentration (ppm) 50 100 200 400 800 A. ﬂavus 48.70 83.20 100 100 100 A. parasiticus 36.21 72.80 100 100 100 F. moniliforme 26.40 65.11 89.37 100 100 26 S.E. Aly et al.

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