Phenolic extracts of clove (Syzygium aromaticum) with novel antioxidant and antibacterial activities

Accepted Manuscript Title: Phenolic extracts of clove (Syzygium aromaticum) with novel antioxidant and antibacterial activities Author: Mohamed F. Abo El-Maati Samir A. Mahgoub Salah M. Labib Ali M.A. Al-Gaby Mohamed Fawzy Ramadan PII: DOI: Reference:

S1876-3820(16)30012-9 http://dx.doi.org/doi:10.1016/j.eujim.2016.02.006 EUJIM 510

To appear in: Received date: Revised date: Accepted date:

9-6-2015 18-2-2016 18-2-2016

Please cite this article as: El-Maati Mohamed F Abo, Mahgoub Samir A, Labib Salah M, Al-Gaby Ali MA, Ramadan Mohamed Fawzy.Phenolic extracts of clove (Syzygium aromaticum) with novel antioxidant and antibacterial activities.European Journal of Integrative Medicine http://dx.doi.org/10.1016/j.eujim.2016.02.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Phenolic extracts of clove (Syzygium aromaticum) with novel antioxidant and antibacterial activities

Mohamed F. Abo El-Maatia, Samir A. Mahgoubb, Salah M. Labiba, Ali M. A. Al-Gabya, Mohamed Fawzy Ramadana* aAgricultural

Biochemistry Department, Faculty of Agriculture, Zagazig University, Zagazig 44519, Egypt.

bAgricultural

Microbiology Department, Faculty of Agriculture, Zagazig University, 44519 Zagazig, Egypt.

Correspondence to: Prof. Dr. Mohamed Fawzy Ramadan Agricultural Biochemistry Department, Faculty of Agriculture, Zagazig University, Zagazig 44519, Egypt. Fax: +2 055 2287567 or +2 055 2345452 Tel: +2 055 2320282 or +2 055 2313940 E-mail: [email protected]

Abstract Introduction: clove (Syzygium aromaticum) is a rich source of bioactive compounds. The goal of this study was to test different extracts of clove in the term of their content of phenolics, their antioxidant potential and their antibacterial action against pathogenic bacteria. Methods: Ethyl acetate, ethanol (80%) and water were used to extract bioactive phytochemicals from clove. Recovered extracts were studied in terms of total phenolic compounds, total flavonoids, antioxidant properties and antibacterial activity. Scanning 1

and transmission electron microscopy was applied to study the effect of ethanol extract on the morphology and membranes of tested bacterial cells. Results: Ethanol and water were the best solvents for extracting phenolics (ca. 230 mg GAE g−1 extract) wherein water was also the best solvent for extracting flavonoids (17.5 mg QE g−1 extract). Antioxidant potential of clove extracts was estimated using DPPH• (1,1-diphenyl-2 picrylhydrazyl), ABTS•+ (2, 2`azino bis-(3-ethylbenzthiazoline-6sulfonic acid), β-carotene-linoleic bleaching assay and ferric reducing antioxidant power (FRAP). Ethanol and water extracts showed comparable antioxidant activity to the synthetic antioxidant tert-butylhydroquinone (TBHQ). The DPPH• radical quenching activity varied from 25.3 to 91.4%, while clove extracts showed ABTS•+ scavenging activities from 49.4 to 99.4%. Clove extracts inhibited the bleaching of β-carotene wherein the order of decreasing activity was water> ethanol> ethyl acetate extracts as compared with TBHQ. Extracts showed strong antibacterial activities against Staphylococcus aureus ATCC 6538, Listeria monocytogenes Scott A, Salmonella enteritidis PT4, Serratia marcescens and Escherichia coli ATCC 8739. Clove extracts exhibited antibacterial activities against the growth of Staph. aureus and E. coli in concentration range from 50 to 100 μg/mL. The results indicated that the extracts with stronger antibacterial capacity also had higher phenolic content. Scanning and transmission electron microscopy showed that ethanol extract damaged the morphology and membranes of tested bacterial cells. Conclusions: Applying clove and its extracts in food or pharmaceutical products may be an effective antioxidant and antimicrobial control strategy. Data from this study might be used for developing natural preservative and bioactive agents with health promoting activities. Keywords: Natural antioxidants, antiradical activity, phenolic compounds, pathogenic bacteria, electron microscope.

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1. Introduction Medical plant, herbs, spices and oilseeds are rich sources of natural antioxidants such as flavonoids, curcumanoids, tannins, terpenoids and lignans. There is growing interest in characterizing and extracting of natural bioactive compounds from these sources for using them in different food and pharmaceutical applications (1-3). Solvents with different polarities were used to extract phenolics and bioactive compounds from plant materials (4, 5). Extraction yield depend on the solvent polarity and the technique of extraction (6, 7). Water, aqueous ethanol (70%), aqueous methanol (50%) and acetone are used commonly in the extraction of bioactive compounds (8). For extracting flavonoids from tea, aqueous ethanol was better than methanol and acetone (9). For extracting tea catechins, water was better solvent than aqueous methanol and aqueous ethanol (10). Clove (Syzigium aromaticum L., family Myrtaceae) is considered an enormous potential food preservative against spoilage and pathogenic bacteria. Commercial cloves are the dried and unexpanded flower buds which are used as a condiment or spice in many food and pharmaceutical applications. The essential oil of S. aromaticum buds is widely used in medicinal applications especially in dental care. The essential oil is active against oral bacteria associated with dental caries and periodontal disease (11, 12) and effective against other bacteria including Listeria monocytogenes, Escherichia coli, Salmonella enterica (13), Salmonella enteritidis, Campylobacter jejuni, and Staphylococcus aureus (14-16). Studies have reported antifungal (17), antiallergic, anticarcinogenic and antimutagenic activities (18) of clove essential oil. Clove contains a variety of bioactive compounds such as sesquiterpenes and triterpenoids (11). Eugenol (4-allyl-2methoxyphenol), the main bioactive compound of clove oil, displayed strong insecticidal (19) and antioxidant (20) properties. As a food additive, eugenol was classified by Food and Drug Administration (FDA) to be a substance that is generally regarded as safe (21). The extract from dried clove buds (50 μg/mL) inhibited lipid oxidation for 30 days. In addition, clove bud extract (160 μg/mL) inhibited malonaldehyde formation from cod liver oil by 93%. Major compounds identified in the extracts of clove buds by GC-MS were eugenol (ca. 24.3 mg/g) and eugenyl acetate (ca. 2.35 mg/g). The antioxidant potential of clove extract and its main bioactive components (eugenol and eugenyl 3

acetate) were comparable to

α-tocopherol (22). Clove essential oil, at 15 μg/mL

concentration, inhibited 97% lipid oxidation of linoleic acid. Moreover, clove oil had an effective ABTS•+ scavenging, DPPH˙ scavenging, hydrogen peroxide scavenging, ferrous ions (Fe2+) chelating activities and ferric ions (Fe3+) reducing power (23). Many medicinal plants were studied in the term of phenolic bioactive compounds and antioxidant activity. However, most of these studies focused on using only one solvent to extract bioactive compounds. In this study, we tested different extracts of clove in the term of their content of phenolic compounds, total flavonoids and their antioxidant potential. The second aim of the study was to specify the potential antibacterial action of clove extracts against pathogenic bacteria including Staphylococcus aureus ATCC 6538, Salmonella enterica enteritidis PT4, Listeria monocytogenes Scott A, Serratia marcescens and Escherichia coli ATCC 8739.

2. Materials and methods 2.1. Reagents and strains Clove (Syzygium aromaticum) buds were obtained from a local market (Zagazig, Egypt). Tert-butylhydroquinone (TBHQ), 1,1-diphenyl-2-picrylhydrazyl (DPPH˙), 2, 2azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS•+), β-carotene, gallic acid and quercetin were obtained from Sigma (St. Louis, MO, USA). Other chemicals and reagents were of analytical grade and of the highest purity available. Bacterial strains of Staphylococcus aureus ATCC 6538, Listeria monocytogenes Scott A, Salmonella enterica serovar enteritidis PT4, Serratia marcescens and Escherichia coli ATCC 8739 were obtained from Egyptian Culture Collection MERCIN (Ain Shams University, Cairo, Egypt). 2.2. Preparation of bacterial strains inoculum The culture strain was activated for 24 h by three transfers in tryptic soy broth (Difco laboratories) at 37°C. Cells were recovered by centrifugation (10000 x g for 10 min at 4°C), washed three times and re-suspended to a final volume of 10 mL in Ringer’s solution (Lab M, Bury, UK). A final inoculum was prepared by dilution in Ringer’s solution to reach a final concentration of 5 log CFU mL-1 as measured by optical density (OD) at 600 nm using UV spectrophotometer (Jenway-6405-UV/VIS) as well as the confirmation by plate counting on the selective media. Aliquot of 0.1 mL of each pathogen was inoculated into three sterile tubes containing Mueller Hinton broth with 100 µg/mL of clove extract so that the final OD600 becomes ca. 0.002. 4

2.3. Preparation of clove extracts Clove bud was dried in a vacuum oven (40°C) and ground to fine powder in a mill. Milled material (10 g) was extracted individually with solvents with different polarities (100 mL) including ethyl acetate, ethanol 80% and distilled water using magnetic stirrer at room temperature (ca. 22°C) and by filtration via filter paper (Whatman No.1). The extraction period was 60 min for each solvent. The residues were re-extracted under the same extraction conditions. The ethyl acetate filtrate was evaporated using rotary evaporator (BÜCHI-water bath-480) below 40°C. Water and ethanol (80%) extracts were freeze-dried (Thermo-Electron Corporation-Heto power dry LL300 freeze dryer). The dried extracts were weighed to determine the dried yield then stored at -20°C for further analyses. 2.4. Total phenolic compounds (TPC) The concentration of TPC in all extracts were measured using UV spectrophotometer (Jenway-6405-UV/VIS), based on oxidation/reduction reaction, as reported by Škerget et al. (24) using Folin-Ciocalteu reagent (25). To 500 μL of diluted extract (10 mg in 10 mL solvent) 2.5 mL of Folin-Ciocalteu reagent (diluted 10 times with distilled water) and 2 mL of Na2CO3 (75 g/L) were added. The sample was incubated for 5 min at 50˚C then cooled. For a control sample, 500 μL of distilled water was used. The absorbance was recorded at 760 nm. TPC content expressed as gallic acid equivalent (GAE) was calculated based on the calibration curve using the following linear equation: y = 0.015x + 0.0533 R² = 0.9966 Where y is the absorbance, x is the concentration (mg GAE g−1 extract) and R2 is the correlation coefficient. 2.5. Total flavonoids Total flavonoids content was measured according to the method of Ordon et al. (26) with some modification. A 1.5 mL aliquot of 20 g/L AlCl3 ethanol solution was added to 0.5 mL of the extract (10 mg in 10 mL solvent). After 60 min, the absorbance at 420 nm was recorded. Extracts were evaluated at a final concentration of 1 mg/mL. Total flavonoids content expressed as quercetin equivalent (QE) was calculated based on the calibration curve using the following equation: y = 0.02248x R² = 0.992 5

Where x is the absorbance, y is the concentration (µg QE) and R2 is the correlation coefficient.

2.6. Antioxidant potential of different extracts 2.6.1. DPPH• radical scavenging activity The electron donation ability of each extract was recorded by bleaching of the DPPH• purple colored solution according to Hanato et al. (27). One hundred µL of each extracts (10 mg extract/10 mL solvent) was added to 3 mL of 0.1 mM DPPH• dissolved in ethyl acetate or ethanol or methanol according to the solvent used for extraction. After incubation time of 30, 60 and 120 min at room temperature, the absorbance was recorded against the control at 517 nm (28). Percentage of antioxidant potential of DPPH• radicals was calculated as follow: Antioxidant activity (Inhibition) % = [(Acontrol – Asample)/Acontrol] × 100 Where Acontrol is the absorbance of the control and Asample is the absorbance in the presence of clove extract. TBHQ was used as a positive control for comparison wherein the measurements were performed in triplicate. 2.6.2. ABTS•+ radical scavenging activity The method of Re et al. (29) was adopted for the ABTS•+ assay. The stock solutions were 7 mM/L ABTS•+ solution and 2.4 mM/L potassium persulphate solution. The solution was prepared by mixing in equal quantities the stock solutions and allowing them to react for 12–16 h in the dark. One mL of the ABTS•+ solution was diluted with 60 mL of methanol. ABTS•+ solution was prepared freshly for each assay. Ten µL of each clove extract (10 mg extract in 10 mL solvent) and (TBHQ solution) were reacted with 5 mL of ABTS•+ solution for 7 min then the absorbance was recorded at 734 nm. A control without extract was also analyzed. Antiradical activity was calculated as follows: ABTS radical-scavenging activity (%) = [(Abscontrol − Abssample)/ Abscontrol] × 100 Wherein Abscontrol is the absorbance of ABTS•+ radical in methanol and Abssample is the absorbance of ABTS radical with added clove extract or TBHQ. 2.6.3. β-Carotene/linoleic acid bleaching method The ability of clove extracts and TBHQ to prevent the bleaching of β-carotene was tested as reported by Keyvan et al. (30). In summary, 0.2 mg β-carotene dissolved in 1 mL chloroform, 20 mg linoleic acid and 200 mg Tween 20 were mixed in a round-bottom flask. After removal of chloroform, 50 mL of distilled water were added and the mixture 6

was vigorously stirred. Aliquots (3 mL) of the emulsion were transferred to tubes containing clove extract or TBHQ. After mixing 0.5 mL of extract (10 mg extract in 10 mL solvent), an aliquot from each tube was transferred to cuvette and the absorbance at 470 nm was measured (Abs0). The remaining samples were placed at 50˚C in a water bath for 120 min, then the absorbance was recorded at 470 nm (Abs120). A control without added extract or TBHQ was analyzed. Antioxidant potential was calculated as follows: Antioxidant activity (%) = [1 − (Abs0sample − Abs120sample)/ (Abs0control − Abs120control)] × 100 Wherein Abs0sample is the absorbance of sample at zero time, Abs120sample is the absorbance after 120 min, Abs0control is the absorbance of control at zero time and Abs120control is the absorbance of control after 120 min. 2.6.4. Ferric reducing antioxidant power (FRAP) method Reducing power of clove extracts was measured according to Gülçin et al. (21). The reduction of Fe+3 to Fe+2 was assayed by measuring the absorbance of Perl's Prussian blue complex. 100 μL of each extracts (10 mg extract in10 mL solvent) was mixed with 1 mL of 0.2 M sodium phosphate buffer (pH 6.6) and 1 mL of 1% K3Fe(CN)6. The mixture was incubated at 50˚C for 20 min. After 20 min of incubation, the mixture was acidified with 1 mL 10% of trichloroacetic acid then 250 µL of FeCl3 (0.1%) was added to the solution. Water was used as blank and for control. Absorbance at 700 nm of this mixture was measured. Low absorbance indicates ferric reducing power activity of the sample.

2.7. Antibacterial activity 2.7.1. Disc assay S. enteritidis PT4, L. monocytogenes Scott A, S. marcescens, Staph. aureus ATCC 6538 and E. coli ATCC 8739 were grown at 37˚C in Brain Heart Infusion liquid medium. After 6 h, each microorganism, at 105 cells/mL was inoculated on the surface of MuellerHinton agar plates. Filter paper discs (6 mm in diameter) saturated either with clove extract (1 mg/mL) or the solvent or TBHQ (30 μL/disc) were placed on the surface of each inoculated plate. The plates were incubated for 24 h at 37˚C. After 24 h, it was possible to record inhibition zone. When necessary, DMSO and Tween 40 were used to dissolve clove extracts in the culture media. The controls were the solvents used for dissolving each extract and the TBHQ wherein they showed no inhibitions in preliminary studies.

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2.7.2. Minimum inhibition concentration (MIC) Ethanol extract of clove was assayed for the antibacterial activity by common broth dilution assay against Staph. aureus and E. coli (31). Staph. aureus and E. coli were grown in broth for 6 h. An aliquot (100 μL) of 105 cells/mL was inoculated in tubes with broth supplemented with different levels (25-100 μL) of clove ethanol extract, TBHQ and ethanol. After 48 h at 37˚C, the MIC was recorded by measuring the OD (600 nm), comparing the sample readout with non inoculated nutrient broth. 2.7.3. Scanning electron microscopy (SEM) Scanning electron microscopy screening was performed according to Benli et al. (32) to explore the mode of action of clove ethanol extract on S. enteritidis, Staph. aureus and E. coli cell morphology. An aliquot (0.1 mL) of bacteria cultures were inoculated in 10 mL MHB and incubated at 37˚C with gentle agitation for 12 h. The cells were centrifuged at 4500 × g for 15 min at 4°C. Cells were washed three times and resuspended in peptone buffer solution (PBS, pH 7.4) at the same volume. The ethanol extract (100 μg/mL) was added to the cell suspension and incubated at 37˚C with agitation for 2 h. Bacterial cells were obtained by centrifugation at 4500 × g for 15 min at 4˚C, washed with PBS (pH 7.4) and fixed in PBS in 2.5% glutaraldehyde. Fixed bacterial pellet was dehydrated in graded alcohol series, dried, mounted onto stubs using double sided carbon tape and coated with thin layer of gold. Cell samples were assayed using SEM (JEOL-SEM, Japan). 2.7.4. Transmission electron microscopy (TEM) S. enteritidis PT4, Staph. E. coli ATCC 8739 and aureus ATCC 6538 were grown on MHB and incubated to reach maximum level of 1 × 108 CFU/mL at 37˚C. The ethanol extract (100 μg/mL) was added to cell suspension and incubated for 4 h at 37˚C. Control samples were prepared in the same manner without treatments. Bacterial cells were collected after centrifugation at 4500 ×g for 10 min, fixed in 2.5% glutaraldehyde (in 0.1 M phosphate buffer, pH 7.4) for 60 min, rinsed three times for 10 min with 0.1 M phosphate buffer (pH 7.4) and fixed with 1% osmium tetraoxide at 4˚C for 2 h. After fixation, cells were rinsed 3 times for 10 min with 0.1 M phosphate buffer (pH 7.4) then dehydrated using 30%, 50%, 70% and 95% acetone for 15 min. Cells were dehydrated 3 times for 30 min with 100% acetone. Subsequently, cells were treated with propylene oxide 2 times for 10 min at 4˚C then infiltrated with a mixture of propylene oxide and Durcupan's ACM epoxy resin (3:1, 1:1 and 1:3) for 45 min. Polymerization of the resin to form specimen blocks was carried out at 60˚C for 72 h. Specimen blocks 8

were hand trimmed with a razor blade then sectioned with a diamond knife in a Reichert Ultracut R ultramicrotome (Leica, Wetzler, Germany). Sections (70–80 nm) were placed on 300 mesh copper grids, stained for 15–20 min in uranyl: ethanol (1:1, v/v), then washed 3 times with saline solution for 2 min and incubated in a drop of Reynold's lead citrate for examination using a TEM (JEOL-JEM-1400, Japan). 2.8. Statistical analysis Experiments were repeated three times and the results were expressed as mean ± standard error. Results were statistically analyzed with ANOVA variance analysis through general linear models (GLM) method of statistical analysis system software (SAS version 9.1, SAS Institute, 2003). Significant differences were used to separate means at p< 0.05

3. Results and discussion Lipid oxidation is one of the main causes of food deterioration. Oxidation results in induction of reactive oxygen species (ROS) and free radicals which are associated with carcinogenesis, inflammation, DNA damage and cardiovascular diseases. Addition of antioxidants in lipid-containing foods is one way to minimize rancidity, retard the formation of toxic oxides, maintain nutritional quality and increase the shelf life of food products (33). Although their safety has been questioned, synthetic antioxidants including butylated hydroxyanisole, butylated hydroxytoluene and TBHQ are used in the food processing because they are more effective than natural antioxidants (34). Therefore, searching for safer and effective natural antioxidants is under way wherein numerous natural sources were investigated. 3.1. Yield and phenolics content of clove extracts The yield of clove dry extracts recovered from different solvents under study ranged from 3.9 to 11.7 g extract/100 g dried clove bud. Ethanol extracted the highest amount (11.7 g extract/100 g) followed by water (7.5 g extract/100 g) and finally ethyl acetate (3.9 g extract/100 g). Variation in the extraction yields from different solvents is attributed to the differences in the polarity of constituents found in plant materials (35). The amount of TPC varied in different extracts, ranging from 58.5 to 293 mg GAE g−1 extract. The results showed that ethanol and water were better solvents for extracting TPC from clove (ca. 230 mg GAE g−1 extract) than ethyal acetate (58.8 mg GAE g−1 extract). Flavonoids have a broad spectrum of biochemical and biological activities, including antiradical activity. Hence, clove extracts were assayed for total flavonoid 9

levels. Water was the best solvent for extracting flavonoids from clove (17.5 mg QE g−1 extract), followed by ethanol (12 mg QE g−1 extract) and ethyl acetate (4.70 mg QE g−1 extract). It is hard to find information in the scientific literature to compare the obtained results with published data. No previous data has been found so this would provide an ample data for future references. 3.2. Antioxidant activity of clove extracts As reported by Huang et al. (36), no single assay is enough for testing the antioxidant activity of plant extracts, since different tests can lead to diverging results. Thus, several tests based on different mechanisms should be assayed. For studying the antioxidant potential of clove extracts we applied different methods including ABTS•+ antiradical activity, DPPH antiradical activity and β-carotene/linoleic acid bleaching test. 3.2.1. DPPH• radicals scavenging activity Free radicals involved in the process of lipid oxidation are playing the main role in chronic pathologies such as cardiovascular diseases and cancer (37). The impact of antioxidants on DPPH• and ABTS•+ radicals scavenging is considered to be due to their ability of hydrogen-donating. DPPH• and ABTS•+ are free radicals that accepts hydrogen radical or an electron to become a stable molecule (28). DPPH• is a model of a stable lipophilic radicals wherein a chain reaction of lipophilic radicals is initiated by lipid oxidation. Antioxidants can react with DPPH• resulting in reducing the number of DPPH• radicals to the number of their available hydroxyl groups. Hence, the absorption at 517 nm is proportional to the amount of residual DPPH• wherein the scavenging potential of plant extracts against DPPH• was concentration-dependent (38). The results of DPPH• antiradical activities of clove extracts are shown in Figure 1. The results indicated that all clove extracts exhibited antiradical activity. The extracts that contained high amount of TPC showed high antiradical activity when compared with TBHQ (Figure 1: A, B, and C). It has been reported that the antioxidant potential of plant extracts is ascribable to the concentration of phenolics in the extract (39). The antiradical activity of clove extracts varied from 25.3% to 91.4% after 120 min incubation. High antiradical activity was recorded with water and ethanol extracts.

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3.2.2. ABTS•+ radical scavenging activity Different clove extracts showed a wide range of ABTS•+ scavenging activities from 49.45% to 99.49% (Figure 2). The highest radical scavenging activity was recorded for water extract followed by ethanol extract with respective values of 99.4% and 98.5%, respectively. Scavenging of ABTS•+ radicals by clove extracts was found to be higher than that of DPPH• radicals. Factors such as the stereoselectivity of radicals and extracts solubility in different systems were reported to affect the activity of the extracts to quench radicals. The ABTS•+ radical is used when issues of solubility or interference arise wherein the application of DPPH• based test becomes inappropriate (37, 40). ABTS•+, a protonated radical, has an absorbance maximum at 734 nm which decreased with the quenching of proton radicals (41). 3.2.3. β-Carotene/linoleic acid bleaching As shown in Figure 3, all clove extracts inhibited the bleaching of β-carotene by scavenging linoleate-derived radicals. The order of decreasing activity at 200 μg/mL was water> ethanol> ethyl acetate extracts as compared with TBHQ. According to the data of bleaching test, clove extracts were capable of quenching free radicals in a complex heterogeneous medium. This suggests that the extracts might have potential application as natural antioxidant preservatives in emulsions. Synthetic radical quenching models (i.e., ABTS•+ and DPPH•) are good tools to test the antioxidant potential of plant extracts; however, those systems do not use a food or biologically-relevant oxidizable substrate. Therefore, no direct information on an extracts’ protective action can be obtained (37). Thus, it is important to test the extracts in a lipid–water emulsion system such as βcarotene/linoleic acid bleaching test (42). In this test, oxidation of linoleic acid induces hydroperoxide-derived radicals which attack β-carotene chromophore, leading to bleaching in the reacted emulsion. An extract able to retarding or inhibiting β-carotene oxidation might be described as a free radical quencher or primary antioxidant (43). 3.2.4. Ferric reducing antioxidant power (FRAP) As shown in Figure 4, ethanol extracts of clove showed higher ferric reducing power when compared with TBHQ. Both ferric reducing power and TPC of clove ethanol extract were higher than those of the ethyl acetate and water extracts. It could be concluded that TPC and ferric reducing power are related. Fe+3 reductions are usually 11

used as an indicator of electron-donating potential, which is an important mechanism of antioxidant activity (37). The extracts that contained high levels of TPC showed relatively high antioxidant potential. It has been reported that ferric reducing power of plant extracts is ascribable to the levels of TPC (39). Fe+3 reducing power of clove solvents ranged from 0.377 to 1.646. The highest antioxidant potential was observed with ethanol and water extracts of clove. From the results of previous assays, clove extracts had high antioxidant activity. Strong antioxidant activity was observed with ethanol and water extracts. TPC can explain the high antioxidant activity (44, 45), although some researched have mentioned that there is no correlation between TPC and antioxidant capacity. The antioxidant activity of phenolics is mainly due to their redox characteristics, which could play an important role in neutralizing radicals, quenching singlet and triplet oxygen or decomposing oxides (46). 3.3. Antibacterial activity of clove extracts Using agar disc assay (Figure 5), the tested clove extracts induced inhibition zones against five tested bacteria. Generally, there were no significant differences between the Gram-positive (Staph. aureus) and Gram-negative bacteria (E. coli) in their susceptibility to the tested extracts referring to broad specificity antimicrobial action of different extracts. Ethanol extract of clove exhibited inhibition zone diameter ca. 10-17 mm with Staph. aureus, E. coli, S. marcescens, L. monocytogenes and S. enteritidis. The MIC of clove was 100 μg/mL against Gram-negative and Gram-positive bacteria. These results indicating that the antimicrobial action of clove extracts begins at low concentrations. The extracts that contained high amount of TPC showed relatively high antibacterial activity. Ethanol followed by ethyl acetate extract showed good inhibition zone diameter with Gram-positive and Gram-negative bacteria (Figure 5). It has been reported that the antibacterial potential of extracts is ascribable to the concentration of phenolics in the plant extract. 3.3.1. Minimum inhibition concentration (MIC) The effect of extract concentration (25–100 μg/mL) on the growth of Staph. aureus and E. coli as compared to TBHQ was evaluated during 48 h of incubation using broth dilution assay (Figure 6). The time-growth curves (Figure 6) of both bacteria were monitored during 48 h as influenced by extracts concentration (25–100 μg/mL) effect. The extracts exhibited activities in concentration range from 50 to 100 μg/mL (Table 1). 12

Similar reducing effects of the ethanol extract were screened against both bacteria starting after 6-48 h of incubation at all concentrations. TBHQ was more effective at low level (25 μg/mL) against Gram-positive bacteria. All extracts were less effective against the growth curve of Gram negative (G-, E. coli) than Gram positive (G+, Staph. aureus) bacteria. This results indicating that the Gram negative bacteria are more resistance than Gram positive bacteria when treated with clove extracts. The Gram negative bacteria are containing outer membrane that protects these bacteria from chemical and drugs. Saxena et al. (47) reported that MIC varying from 12.5 to 1 μg/mL when assaying different levels of Rhus glaba extracts on Gram-negative and Gram-positive bacteria. 3.3.2. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) The morphology changes of Staph. aureus, E. coli and S. enteritidis were evaluated using SEM. Figure 7 shows images of Staph. aureus, E. coli and S. enteritidis (~105 CFU/mL) treated with or without clove ethanol extract at 37°C for 2 h. The cells of Staph. aureus treated with extract exhibited irregularly wrinkled outer surface, adhesion and aggregation of damaged cells or cellular debris. Moreover, these cells were not uniform in size and distribution. However, bacterial cells in control without treatment had regular and spherical shape morphology. Similarly, as seen in Figure 7 the bacterial cells of E. coli and S. enteritidis without clove extract displayed typical bacilliform morphology with uniform in size and distribution. In contrast, E. coli and S. enteritidis cells treated with clove extract had irregular, withered, coarse surfaces, forming aggregations and adhesions. Furthermore, these cells were not uniform in size and distribution. This result showed that the ethanol extract led to the damage of the tested pathogenic bacteria. Similarly, Kaya et al. (48) observed using SEM that the cells of Pseudomonas aeruginosa, Shigella sp., Listeria monocytogenes and Staph. aureus treated with plant methanol extracts were damaged. The same impact on pathogenic bacteria was reported by Sitohy et al. (49), who examined B. subtilis cells with subunit of soybean protein and Li et al. (50) who tested the Staph. aureus and E. coli with ε-poly-lysine. Both studies showed irregular wrinkled outer surface, with fragmentation and adhesion. TEM images of Staph. aureus, E. coli and S. enteritidis were also taken after treating with clove ethanol extract (100 μg/mL) at 37°C for 2 h (Figure 8). Different signs of cell membrane and cell wall deformation might be seen. The most deformation signs were manifested by loss of regular cellular shapes, cell wall and cell membrane disintegration, 13

bigger cell sizes as well as separation of cell wall from cell membrane. Sitohy et al. (49) found that L. monocytogenes and S. enteritidis treated with protein fractions resulted in loss of regular cellular shapes, disintegration of cell membrane, bigger cell sizes and separation of cell wall from cell membrane.

4. Conclusion It was observed that clove extracts with higher antioxidant capacity were in parallel to their higher TPC. It could be concluded that extracting bioactive chemicals from clove with solvents with high-polarity is more effective than the extraction with low-polarity solvents. Ethanol showed better characteristics than water as a solvent for extracting phenolic compounds and flavonoids from clove bud. Thus, for applications in the food or pharmaceutical industry, ethanol might be a more appropriate solvent. Further research is required to identify and elucidate the structure of phenolic compounds found in different extracts. Authors contribution All research has been done by the authors.

Funding Zagazig University funded this research.

Conflict of Interest None.

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18

120

Ethyl acetate extract

Inhibition of DPPH %

100

TBHQ

80 60 40 20 0 Zero time

30 min

60 min

120 min

A

Inhibition of DPPH %

120

Ethanol extract

TBHQ

100 80 60 40 20 0 Zero time

30 min

60 min

120 min

B Inhibition of DPPH %

120

Water extract

100

TBHQ

80 60 40 20 0 Zero time

30 min

60 min

120 min

C Figure 1. Scavenging activity of (A) ethyl acetate, (B) ethanol, and (C) water extracts against DPPH• radical compared with TBHQ

19

120

Clove extract

TBHQ

Scavenging of ABTS•+ (%)

100 80 60 40 20 0 Ethyl acetate extract

Ethanol 80% extract

Water extract

Figure 2. Scavenging activity of clove extracts against ABTS•+ radical as compared with TBHQ

20

Antioxidant activity (%)

120

Clove extract

TBHQ

100 80 60 40 20 0 Ethyl acetate extract

Ethanol 80% extract

Water extract

Figure 3. Antioxidant activity of clove extracts in β-carotene-linoleic acid emulsion as compared with TBHQ

21

1.8

Clove extract

TBHQ

Absorbane at 700 nm

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 Ethyl acetate extract

Ethanol 80% extract

Water extract

Figure 4. Antioxidant activity of clove extracts in ferric reducing test as compared with TBHQ

22

20

Ethyl acetate extract

Ethanol 80 % extract

Water extract

inhibation zone (mm)

18 16 14 12 10 8 6 4 2 0 L. monocytogenes

E. coli

S. aureus

S. Enteritidis

S. marcescens

Figure 5. Inhibition zones diameter (mm) as recorded by different clove extracts against Gram positive and Gram negative bacteria

23

2.5

clove

TBHQ

ethanol

S. aureus 2.5

(25 µg/mL)

2

2

1.5

1.5

1

1

0.5

0.5

0 zero

6h

12 h

24 h

TBHQ

ethanol

E. coli

(25 µg/mL)

0

48 h

zero

2

6h

12 h

24 h

48 h

12 h

24 h

48 h

12 h

24 h

48 h

2.5

(50 µg/mL)

(50 µg/mL) 2

1.5

O.D 600

clove

1.5 1 1 0.5

0.5

0

0 zero

6h

12 h

24 h

48 h

zero

2.5

2.5

(100 µg/mL) 2

2

1.5

1.5

1

1

0.5

0.5

0

0 zero

6h

12 h

24 h

48 h

S. aureus (G+)

6h

(100 µg/mL)

zero

6h

E. coli (G-)

Figure 6. Growth curve of Staph. aureus and E. coli during 48 h at 37˚C in the presence of 25-100 µg/mL of clove extract

24

Control

Clove ethanol extract (100 µg/mL) 5000x

7500x

7500x

5000x

7500x

7500x

5000x

7500x

S. Enteritidis

E. coli

Staph. aureus

7500 x

Fig. 7 Scanning electron microscope (SEM) images of Staph. aureus (G+), E. coli (G-) and S. enteritidis (G-) treated with ethanol extract (100 µg/mL) of clove

25

Control

Clove ethanol extract (100 µg/mL) 25000x

25000x

20000x

15000x

25000x

30000x

25000x

25000x

S. Enteritidis

E. coli

Staph. aureus

25000x

Fig. 8 Transmission electron microscope (TEM) images of Staph. aureus (G+), E. coli (G-) and S. enteritidis (G-) treated with ethanol extract (100 µg/mL) of clove

26

Table 1. Minimal Inhibition Concentration (MIC) and Minimal Bactericidal Concentration (MBC) of clove ethanol extract and TBHQ against tested pathogenic bacteria MIC (µg/mL)

MBC

G+

G-

G+

G-

Clove ethanol extract

50

100

100

150

TBHQ

25

50

100

100

Ethanol

50

100

ND

ND

MIC; Minimal Inhibition Concentration MBC; Minimal Bactericidal Concentration ND; not determined

27