- Email: [email protected]
Biosynthesis of large-sized silver nanoparticles using Angelica keiskei extract and its antibacterial activity and mechanisms investigation
Microchemical Journal 147 (2019) 333–338
Contents lists available at ScienceDirect
Microchemical Journal journal homepage: www.elsevier.com/locate/microc
Biosynthesis of large-sized silver nanoparticles using Angelica keiskei extract and its antibacterial activity and mechanisms investigation☆
T
Juan Dua,b,c, Zheyuan Hud, Wei-jie Donge, Yubo Wanga, Shujing Wua, Yanhong Baia,b,c,
⁎
a
College of Food and Biological Engineering, Zhengzhou University of Light Industry, Zhengzhou 450001, China Henan Key Laboratory of Cold Chain Food Quality and Safety Control, Zhengzhou 450001, China c Henan Collaborative Innovation Center of Food Production and Safety, Zhengzhou 450001, China d College of Electrical and Information Engineering, Zhengzhou University of Light Industry, Zhengzhou 450001, China e The Isotope Research Institute Ltd of Henan Academy of Science, Zhengzhou 450001, China b
ARTICLE INFO
ABSTRACT
Keywords: Silver nanoparticles Antibacterial property Listeria monocytogenes Antibacterial mechanism Angelica keiskei
This study demonstrates a novel approach for synthesizing large-sized silver nanoparticles (AgNPs) using Angelica keiskei water extract. The AgNPs exhibited maximum absorbance at 438 nm and were mostly spherical in shape, with an average particle size of 130.1 ± 2.1 nm. The bacteriostatic and bactericidal effects of AgNO3 against the food-borne pathogen Listeria monocytogenes were explored by measuring the minimum inhibitory concentration and minimum bactericidal concentration. The antibacterial activity mechanism of the AgNPs was further investigated. The results show that the AgNPs could damage the membrane integrity of Listeria monocytogenes cells and induce the release of nucleic acids from the cells, thereby disrupting cell reproduction.
1. Introduction Bacterial pathogens are major etiological agents of diseases related to the consumption of dairy products, accounting for 90% of all cases [1]. Listeria monocytogenes (L. monocytogenes) is one of the most dangerous food-borne pathogens and is responsible for many outbreaks related to food consumption. Taking L. monocytogenes-contaminated food may lead to listeriosis, and the fatality rate is as high as 30%, far exceeding that of other foodborne pathogens [2,3]. Antimicrobial agents play an important role in food production and storage, with a long shelf life, providing good assurance by reducing the contamination risk from pathogenic and spoilage microorganisms [4]. Silver nanoparticles (AgNPs) are considered a new class of antimicrobial agents owning to their outstanding antibacterial activity against a wide range of pathogenic bacteria [5]. Although multifarious physical and chemical methods are available for synthesizing AgNPs, green and low cost protocols are needed [6,7]. Recently, biological approaches employing plant extract have been developed and are considered an economical and environmentally friendly process [8]. Various plant parts, including leaves, fruits, roots, peel and their extracts, have been used for the synthesis of AgNPs [9]. Components such as proteins, amino acids, enzymes, polysaccharides, alkaloids, tannins, phenolics, saponins, terpenoids and vitamins are responsible for plant-
mediated synthetic AgNPs [10]. Active components in plant extract are act as both reducing agents and capping agents in the biosynthesis process of AgNPs, make the AgNPs more stable and have the potential in biological applications [11,12]. A large number of plants are reported to syntheses of AgNPs, such as Vitis vinifera (with average size 30–40 nm), Carica papaya (25–50 nm), Citrus sinensis (10–35 nm), Allium sativum (4–22 nm), Acalypha indica (20–30 nm), Thevetia peruviana (10–30 nm), Limonia acidissima (20–40 nm) and Tamarindus indica (6–8 nm) [13–19]. In most of the studies, plant extracts are prepared by deionized distilled water, and antibacterial activity is further studied. The average size of AgNPs synthesized by plant extract is tend to be small, most of them are less than 100 nm [20]. However, particle size plays a critical role in the cytotoxicity of AgNPs, which showed greater cytotoxicity with smaller average size [21]. There is a needed for the synthesis of large-sized AgNPs via biological method for the biological applications. Angelica keiskei (A. keiskei) is a traditional medicine plant which is mainly distributed in Japan, Korean and China. Angelica keiskei has been widely used as a diuretic, laxative and analeptic, the aerial parts are also consumed as a health food and used in tea, wine, flour, and cosmetics [22,23]. Over 100 constituents have been identified in this plant, including various types of flavonoids, coumarins, phenolics, diterpene and triterpenes [24,25]. Extracts of A. keiskei have been
Declarations of interest: none. Corresponding author at: KeXue Road, College of Food and Biological Engineering, Zhengzhou University of Light Industry, Zhengzhou 450001, China. E-mail address: [email protected] (Y. Bai).
☆ ⁎
https://doi.org/10.1016/j.microc.2019.03.046 Received 8 November 2018; Received in revised form 14 March 2019; Accepted 14 March 2019 Available online 15 March 2019 0026-265X/ © 2019 Elsevier B.V. All rights reserved.
J. Du, et al.
Microchemical Journal 147 (2019) 333–338
reported to have anti-bacterial [26], anti-tumor [27], anti-oxidative [28], anti-diabetic [29], anti-inflammatory [30], and anti-coagulant activities [31]. However, there is no report for the utilization of A. Keiskei extract on nanoparticle synthesis. Hence, this study highlights the green synthesis of large-sized AgNPs using Angelica keiskei water extract (AKE) and aims to understand the antibacterial property and membrane damage mechanism of the synthesized AgNPs against pathogens. Strain L. monocytogenes is introduced as a Gram-positive model strain for the evaluation. 2. Materials and methods 2.1. Materials Angelica keiskei powder was purchased from Xi'an Qing Zhi Biotechnology Co., Ltd. (China). Fluorescent dye FDA and PI were purchased from Solarbio Co. Ltd. (China). The pathogenic bacterial culture, L. monocytogenes (ATCC 15313), was obtained from American Type Culture Collection (ATCC). Silver nitrate was purchased from Sigma-Aldrich, USA. Unless otherwise stated, the solvents and inorganic salts were purchased from Sinopharm Chemical Reagent Company, China.
Fig. 2. FE-SEM image of the AgNPs.
2.4. Antimicrobial testing The antimicrobial activity of the AgNPs was studied by measuring the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the AgNPs against L. monocytogenes. The MIC was determined using a broth microdilution assay, which was modified based on a previously described method [32]. Briefly, twofold serial dilutions of the AgNPs were prepared in sterile trypticase soy broth (TSB) from a top concentration of 200 μg/mL to 3.125 μg/mL. 100 μL of the AgNPs dilutions was placed in sextuplicate in 100-well plate, and inoculate with 100 μL of the L. monocytogenes culture to a final concentration of 5 × 105 CFU/mL. 200 μL/well of TSB was used as a blank control to monitor sterility and 200 μL/well of the L. monocytogenes culture was used as positive control. The MIC was taken as the lowest concentration of the AgNO3 at which growth of the bacterium was complete inhibited after 24 h of incubation at 37 °C. The optical density at 600 nm was measured and recorded every 30 min using a Microbiology Reader Bioscreen C (BioScreen, Finland). The wells of 1× MIC and 2× MIC from the plate were spread (100 μL) on trypticase soy agar (TSA), and the lowest concentration of the AgNPs not showing any growth on TSA was referred to as the MBC. MIC and MBC of AgNO3 (at final concentration range from 1.56 to 100 μg/mL) and AKE (25 to 1600 μg/mL) against L. monocytogenes were performed in the same manner.
2.2. Preparation of Angelica keiskei water extract (AKE) and synthesis of AgNPs Angelica keiskei powder was added to Milli-Q water (1:50, w/v) and boiled at 95 °C for 20 min. The extract solution was filtered and freeze dried to a powder form. For AgNP synthesis, the AKE was treated with an aqueous solution of AgNO3 (at concentration of 2 mM) at 65 °C. The reaction parameters were optimized by varying the concentrations of AKE and AgNO3. 2.3. Characterization of AgNPs The bio-reduction of the AgNPs was observed visually by color change and further confirmed using a Multiskan Go microplate spectrophotometer (Thermo Scientific, USA) at a range of 300–800 nm. The morphology of the AgNPs was analyzed using FE-SEM (JSM-7001F, JEOL, Japan). The crystal structure of the synthesized AgNPs was detected using XRD (D8 Advance, Bruker, Germany). The particle size distribution and charge on the AgNPs were investigated by DLS analysis and zeta potential, respectively, using Malvern Zetasizer Nano ZS90 (Malvern Instruments, UK). To check salt stability, AgNPs was diluted with Milli-Q water to a concentration of 0.4 μg/mL. NaCl solution with same volume of AgNPs solution was added to obtain a final concentration range from 1 mM to 2 M, AgNPs added with water was used as control. The LSPR spectra of mixture were measured after 120 min, respectively.
2.5. Antibacterial mechanisms of cell membrane The effect of AgNPs on the cell membrane integrity was determined using FDA and PI fluorescent dye to distinguish living cells from dead
Fig. 1. UV–Visible spectroscopic measurements of biosynthesized AgNPs as a function of amount of plant extract (A) and AgNO3 (B). 334
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2.6. Determination of extracellular nucleic acids The effect of the synthesized AgNO3 on the release of intracellular nucleic acid was determined by measuring the release of nucleic acids from L. monocytogenes into the medium [33]. Cells in the logarithmic growth phase were collected and washed three times. The bacterial suspensions were incubated at 37 °C in the presence of 1× MIC and 1× MBC of AgNO3. Subsequently, the extracellular nucleic acids were measured using NanoReady Micro UV–Vis spectrophotometer at OD260 (Life Real Co., Ltd., China), after 0, 30, 60, 90, and 240 min from incubation. 3. Results and discussion 3.1. Characterization of the AgNPs The present work first synthesized AgNPs using AKE, and the color in the reaction mixture changed from pale yellow to dark brown. In the UV–visible spectra, the AKE at 400 μg/mL shows a peak at 400 nm, indicating the synthesis of AgNPs (Fig. 1A). With the increase in the amount of AgNO3, the absorbance and breadth of the peaks increase (Fig. 1B). The peak intensities help explain the logarithmic trend in the formation of AgNPs from lower to higher concentration. Therefore, the AKE at a concentration of 400 μg/mL treated with 8 mM AgNO3 was considered as the optimized condition. The FE-SEM images reveal that most of the AgNPs formed are mostly spherical in shape (Fig. 2). In the XRD pattern (Fig. 3), the diffraction peaks at 2θ values of 38.07, 46.21, 64.37, 77.36, and 81.54° are identified and matched with the JCPDS databases of standard silver (file No. 04-0783), indexed to (111), (200),
Fig. 3. XRD spectrum of the AgNPs.
cells. L. monocytogenes in the logarithmic growth phase was collected and washed three times. The bacterial suspensions were incubated at 37 °C in the presence of 1× MIC and 1× MBC AgNO3 for 1 h. The cells treated with 0.85% NaCl were used as the control. The cells were then collected and suspended in a PBS buffer, fixed with FDA (final concentration: 50 μg/mL) and PI (final concentration: 20 μg/mL) for 20 min in the dark. The cells were observed under a Nikon Ni-E upright research-grade microscope (NIKON, Japan), in the phase contrast field and fluorescence field.
Fig. 4. Particle size distribution of the AgNPs.
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(220), (311), and (222) lattice planes of face-centered cubic silver, respectively. The DLS analysis (Fig. 4) shows that the zeta potential and average size of the AgNPs are 130.1 ± 2.1 nm (PDI: 0.229) and −34.3 ± 0.2 mV, respectively. This signifies the good quality and stability of the synthesized AgNPs. LSPR peak (425 nm) of AgNPs shows no change at NaCl concentration ranging between 1 mM to 2 M, as compared with control (Fig. 5). However, absorbance value is significant decreasing in 2 M NaCl, indicating the AgNPs is stable at NaCl concentration as high as 1 M. 3.2. Bacteriostatic and bactericidal property of the AgNPs Bacteriostatic and bactericidal property of the AgNPs, AgNO3 and AKE against L. monocytogenes are investigated by Mic and MBC. Fig.6 shows that with an increasing of AgNPs and AgNO3 concentration, the growth of L. monocytogenes is delayed and decreased. When the AgNPs and AgNO3 concentration reached 12.5 μg/mL, no cell growth is detected, indicating the MICs of the AgNPs and AgNO3 are both 12.5 μg/
Fig. 5. Stability of AgNPs in the presence of increasing NaCl concentrations.
Fig. 6. Growth curves of L. monocytogenes cultured with different concentrations of the AgNPs (A) and AgNO3 (B), and the growth of L. monocytogenes cultured with 1× MIC and 2× MIC AgNPs and AgNO3 on trypticase soy agar plates.
Fig. 7. A, B, and C are fluorescence images of L. monocytogenes cells treated with 0.85% NaCl, 1× MIC AgNPs and 1× MBC AgNPs, respectively. Cells treated with 0.85% NaCl were performed as control. Cells staining by FDA dye show green fluorescence, and cells staining by PI dye show red fluorescence. 336
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Acknowledgments This work was supported by the National Key Research and Development Program of China [2018YFD0401204]; the Major Science & Technology Project of Henan Province [grant number 161100110900]; the Scientific and Technological Project of Henan Province [grant number 182102110392]; and Doctoral Scientific Research Foundation of Zhengzhou University of Light Industry. Conflict of interest The authors declare no competing financial interests. References [1] M.M.J. Franco, A.C. Paes, M.G. Ribeiro, A.C.B. Santos, M. Miyata, C.Q.F. Leite, et al., Occurrence of mycobacteria in bovine milk samples from both individual and collective bulk tanks at farms and informal markets in the southeast region of Sao Paulo, Brazil, BMC Vet. Res. 9 (2013) 85. [2] V. Coroneo, V. Carraro, N. Aissani, A. Sanna, A. Ruggeri, S. Succa, et al., Detection of virulence genes and growth potential in Listeria monocytogenes strains isolated from ricotta salata cheese, J. Food Sci. 81 (2015) M114–M120. [3] B. Jagadeesan, V. Bastic Schmid, B. Kupski, W. McMahon, A. Klijn, Detection of Listeria spp. and L. monocytogenes in pooled test portion samples of processed dairy products, Int. J. Food Microbiol. 289 (2018) 30–39. [4] C. Pablos, A. Romero, A. de Diego, C. Vargas, I. Bascon, F. Perez-Rodriguez, et al., Novel antimicrobial agents as alternative to chlorine with potential applications in the fruit and vegetable processing industry, Int. J. Food Microbiol. 285 (2018) 92–97. [5] R. Salomoni, P. Leo, A.F. Montemor, B.G. Rinaldi, M. Rodrigues, Antibacterial effect of silver nanoparticles in Pseudomonas aeruginosa. Nanotechnology, Science and Applications, 10 (2017), pp. 115–121. [6] D. Nayak, S. Ashe, P.R. Rauta, M. Kumari, B. Nayak, Bark extract mediated green synthesis of silver nanoparticles: evaluation of antimicrobial activity and antiproliferative response against osteosarcoma, Mater. Sci. Eng. C 58 (2016) 44–52. [7] H.E. Emam, H.B. Ahmed, Polysaccharides templates for assembly of nanosilver, Carbohydr. Polym. 135 (2016) 300–307. [8] S.V. Otari, R.M. Patil, S.J. Ghosh, S.H. Pawar, Green phytosynthesis of silver nanoparticles using aqueous extract of Manilkara zapota (L.) seeds and its inhibitory action against Candida species, Mater. Lett. 116 (2014) 367–369. [9] P. Singh, Y.J. Kim, D.B. Zhang, D.C. Yang, Biological synthesis of nanoparticles from plants and microorganisms, Trends Biotechnol. 34 (2016) 588–599. [10] S. Ahmed, M. Ahmad, B.L. Swami, S. Ikram, A review on plants extract mediated synthesis of silver nanoparticles for antimicrobial applications: a green expertise, J. Adv. Res. 7 (2016) 17–28. [11] J. Du, H. Singh, T.H. Yi, Antibacterial, anti-biofilm and anticancer potentials of green synthesized silver nanoparticles using benzoin gum (Styrax benzoin) extract, Bioprocess Biosyst. Eng. 39 (2016) 1923–1931. [12] H. Singh, J. Du, P. Singh, T.H. Yi, Ecofriendly synthesis of silver and gold nanoparticles by Euphrasia officinalis, leaf extract and its biomedical applications, Artif. Cell Nanomed. B. 46 (2018) 1163–1170. [13] G. Gnanajobitha, K. Paulkumar, M. Vanaja, S. Rajeshkumar, C. Malarkodi, G. Annadurai, et al., Fruit-mediated Synthesis of Silver Nanoparticles Using Vitis vinifera and Evaluation of Their Antimicrobial Efficacy, 3 (2013), p. 67. [14] S. Kaviya, J. Santhanalakshmi, B. Viswanathan, J. Muthumary, K. Srinivasan, Biosynthesis of silver nanoparticles using Citrus sinensis peel extract and its antibacterial activity, Spectrochim. Acta A Mol. Biomol. Spectrosc. 79 (2011) 594–598. [15] M. Ahamed, M.A.M. Khan, M.K.J. Siddiqui, M.S. Alsalhi, Alrokayan SAJPEL-dS, Nanostructures. Green Synthesis, Characterization and Evaluation of Biocompatibility of Silver Nanoparticles, 43 (2011), pp. 1266–1271. [16] C. Krishnaraj, E.G. Jagan, S. Rajasekar, P. Selvakumar, P.T. Kalaichelvan, N. Mohan, Synthesis of Silver Nanoparticles Using Acalypha indica Leaf Extracts and Its Antibacterial Activity Against Water Borne Pathogens, 76 (2009), pp. 50–56. [17] N.R. Ni, A. Aher, S. Gosavi, P.B. Vidyasagar, Green Synthesis of Silver Nanoparticles Using Latex Extract of Thevetia peruviana: A Novel Approach Towards Poisonous Plant Utilization, 423 (2013), p. 012032. [18] V. Annavaram, V.R. Posa, V.G. Uppara, S. Jorepalli, A.R. Somala, Facile Green Synthesis of Silver Nanoparticles Using Limonia acidissima Leaf Extract and Its Antibacterial Activity, 5 (2015), pp. 97–103. [19] N. Jayaprakash, J.J. Vijaya, K. Kaviyarasu, K. Kombaiah, L.J. Kennedy, R.J. Ramalingam, et al., Green Synthesis of Ag Nanoparticles Using Tamarind Fruit Extract for the Antibacterial Studies, 169 (2017), pp. 178–185. [20] K.S. Siddiqi, A. Husen, R.A.K. Rao, A review on biosynthesis of silver nanoparticles and their biocidal properties, J. Nanobiotechnol. 16 (2018) 14. [21] Y. Hung-Jen, H. Shan-Hui, T.J.S. Ching-Lin, Cytotoxicity and Immunological Response of Gold and Silver Nanoparticles of Different Sizes, 5 (2010), pp. 1553–1561. [22] K. Baba, M. Taniguchi, K. Nakata, Studies on Angelica keiskei “Ashitaba”, Foods
Fig. 8. Measurement of nucleic acid released from L. monocytogenes into extracellular during the incubation with the synthesized AgNPs. Values are mean ± SD of three determinations.
mL. To determine the MBCs of AgNPs and AgNO3, L. monocytogenes cultured with 1× MIC and 2× MIC were spread on TSA and incubated at 37 °C for two days. As shown in Fig. 6, a lot of colonies are observed on the TSA with the presence of 1× MIC AgNPs, while no visible colony was observed for 2× MIC AgNPs, indicating the MBC of the AgNO3 is 25 μg/mL; several colonies are observed on the TSA with the presence of 1× MIC AgNO3, no visible colony was observed for 2× MIC AgNO3. AKE have no antibacterial activity against L. monocytogenes (data not shown). The AgNPs exhibited same MIC and MBC with AgNO3, this low MIC and MBC value demonstrate that the AgNPs have a strong suppression of proliferation for L. monocytogenes. 3.3. Effect of the AgNPs on membrane integrity of L. monocytogenes The effect of AgNPs on the cell membrane integrity was examined by fluorescence observation of the treated cells after staining them with FDA and PI fluorescent dye. With the mixture staining of FDA and PI, the cells with intact membranes exhibit fluorescence green, and the cells with damaged membranes exhibit fluorescence red. Fig. 7 shows that the AgNPs induce the damage of L. monocytogenes cell membrane and increase the membrane permeability in a dose-dependent manner. In particular, the cells treated with 1× MBC show a large part of red fluorescence, and the dark points at the center of the cell aggregate are regarded as AgNPs (Fig. 7C). The effect of the AgNO3 on the release of intracellular nucleic acid was further examined. At 240 min, the nucleic acids released from 0.85% NaCl, 1× MIC, and 1× MBC AgNPs cultures are found to be 53.2 ± 0.83, 73.5 ± 0.3, and 159.6 ± 3.08 ng/μL, respectively, showing a positive correlation with the treatment of AgNPs (Fig. 8). DNA is one of the most important genetic materials, and the data show that L. monocytogenes cells released a significant amount of nucleic acids after treating with the AgNPs, thereby disrupting the reproduction of the cells, even leading to death. 4. Conclusion A biosynthesis approach for large-sized AgNPs was developed using AKE; the synthesis was done in an ecofriendly and relatively new manner. This study showed that AgNPs have a strong antibacterial activity against food-borne pathogens and that they could damage the membrane integrity and induce the release of nucleic acids, thereby disrupting cell reproduction.
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J. Du, et al. Food Ingredients J. Jpn. (1988) 52–60. [23] Y.S. Kil, S.T. Pham, E.K. Seo, M. Jafari, Angelica keiskei, an emerging medicinal herb with various bioactive constituents and biological activities, Arch. Pharm. Res. 40 (6) (2017) 655–675. [24] Y. Kimura, K. Baba, Antitumor and antimetastatic activities of Angelica keiskei roots, part 1: isolation of an active substance, xanthoangelol, Int. J. Cancer 106 (3) (2003) 429–437. [25] T. Akihisa, H. Tokuda, D. Hasegawa, M. Ukiya, Y. Kimura, F. Enjo, ... H. Nishino, Chalcones and other compounds from the exudates of Angelica keiskei and their cancer chemopreventive effects, J. Nat. Prod. 69 (2006) 38–42. [26] K. Sugamoto, Y.-I. Matsusita, K. Matsui, C. Kurogi, T. Matsui, Synthesis and antibacterial activity of chalcones bearing prenyl or geranyl groups from Angelica keiskei, Tetrahedron 67 (2011) 5346–5359. [27] K. Motani, K. Tabata, Y. Kimura, S. Okano, Y. Shibata, Y. Abiko, H. Nagai, T. Akihisa, T. Suzuki, Proteomic analysis of apoptosis induced by xanthoangelol, a major constituent of Angelica keiskei, in neuroblastoma, Biol. Pharm. Bull. 31 (2008) 618–626. [28] L. Luo, R. Wang, X. Wang, Z. Ma, N. Li, Compounds from Angelica keiskei with NQO1
[29] [30] [31] [32] [33]
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induction, DPPH·scavenging and α-glucosidase inhibitory activities, Food Chem. 131 (2012) 992–998. T. Enoki, H. Ohnogi, K. Nagamine, Y. Kudo, K. Sugiyama, M. Tanabe, E. Kobayashi, H. Sagawa, I. Kato, Antidiabetic activities of chalcones isolated from a Japanese herb, Angelica keiskei, J. Agric. Food Chem. 55 (2007) 6013–6017. H.R. Chang, H.J. Lee, J.H. Ryu, Chalcones from Angelica keiskei attenuate the inflammatory responses by suppressing nuclear translocation of NF-κB, J. Med. Food 17 (2014) 1306–1313. D.J. Son, Y.O. Park, C. Yu, S.E. Lee, Y.H. Park, Bioassay-guided isolation and identification of anti-platelet-active compounds from the root of Ashitaba (Angelica keiskei Koidz.), Nat. Prod. Res. 28 (2014) 2312–2316. E. Mohamed, A. Syed, F. Scott, D. Paul, M.G. Mark, M. Roger, et al., Resazurinbased 96-well plate microdilution method for the determination of minimum inhibitory concentration of biosurfactants, Biotechnol. Lett. 38 (2016) 1015–1019. W.R. Diao, Q.P. Hu, H. Zhang, J.G. Xu, Chemical composition, antibacterial activity and mechanism of action of essential oil from seeds of fennel (Foeniculum vulgare Mill.), Food Control 35 (2014) 109–116.
Contents lists available at ScienceDirect
Microchemical Journal journal homepage: www.elsevier.com/locate/microc
Biosynthesis of large-sized silver nanoparticles using Angelica keiskei extract and its antibacterial activity and mechanisms investigation☆
T
Juan Dua,b,c, Zheyuan Hud, Wei-jie Donge, Yubo Wanga, Shujing Wua, Yanhong Baia,b,c,
⁎
a
College of Food and Biological Engineering, Zhengzhou University of Light Industry, Zhengzhou 450001, China Henan Key Laboratory of Cold Chain Food Quality and Safety Control, Zhengzhou 450001, China c Henan Collaborative Innovation Center of Food Production and Safety, Zhengzhou 450001, China d College of Electrical and Information Engineering, Zhengzhou University of Light Industry, Zhengzhou 450001, China e The Isotope Research Institute Ltd of Henan Academy of Science, Zhengzhou 450001, China b
ARTICLE INFO
ABSTRACT
Keywords: Silver nanoparticles Antibacterial property Listeria monocytogenes Antibacterial mechanism Angelica keiskei
This study demonstrates a novel approach for synthesizing large-sized silver nanoparticles (AgNPs) using Angelica keiskei water extract. The AgNPs exhibited maximum absorbance at 438 nm and were mostly spherical in shape, with an average particle size of 130.1 ± 2.1 nm. The bacteriostatic and bactericidal effects of AgNO3 against the food-borne pathogen Listeria monocytogenes were explored by measuring the minimum inhibitory concentration and minimum bactericidal concentration. The antibacterial activity mechanism of the AgNPs was further investigated. The results show that the AgNPs could damage the membrane integrity of Listeria monocytogenes cells and induce the release of nucleic acids from the cells, thereby disrupting cell reproduction.
1. Introduction Bacterial pathogens are major etiological agents of diseases related to the consumption of dairy products, accounting for 90% of all cases [1]. Listeria monocytogenes (L. monocytogenes) is one of the most dangerous food-borne pathogens and is responsible for many outbreaks related to food consumption. Taking L. monocytogenes-contaminated food may lead to listeriosis, and the fatality rate is as high as 30%, far exceeding that of other foodborne pathogens [2,3]. Antimicrobial agents play an important role in food production and storage, with a long shelf life, providing good assurance by reducing the contamination risk from pathogenic and spoilage microorganisms [4]. Silver nanoparticles (AgNPs) are considered a new class of antimicrobial agents owning to their outstanding antibacterial activity against a wide range of pathogenic bacteria [5]. Although multifarious physical and chemical methods are available for synthesizing AgNPs, green and low cost protocols are needed [6,7]. Recently, biological approaches employing plant extract have been developed and are considered an economical and environmentally friendly process [8]. Various plant parts, including leaves, fruits, roots, peel and their extracts, have been used for the synthesis of AgNPs [9]. Components such as proteins, amino acids, enzymes, polysaccharides, alkaloids, tannins, phenolics, saponins, terpenoids and vitamins are responsible for plant-
mediated synthetic AgNPs [10]. Active components in plant extract are act as both reducing agents and capping agents in the biosynthesis process of AgNPs, make the AgNPs more stable and have the potential in biological applications [11,12]. A large number of plants are reported to syntheses of AgNPs, such as Vitis vinifera (with average size 30–40 nm), Carica papaya (25–50 nm), Citrus sinensis (10–35 nm), Allium sativum (4–22 nm), Acalypha indica (20–30 nm), Thevetia peruviana (10–30 nm), Limonia acidissima (20–40 nm) and Tamarindus indica (6–8 nm) [13–19]. In most of the studies, plant extracts are prepared by deionized distilled water, and antibacterial activity is further studied. The average size of AgNPs synthesized by plant extract is tend to be small, most of them are less than 100 nm [20]. However, particle size plays a critical role in the cytotoxicity of AgNPs, which showed greater cytotoxicity with smaller average size [21]. There is a needed for the synthesis of large-sized AgNPs via biological method for the biological applications. Angelica keiskei (A. keiskei) is a traditional medicine plant which is mainly distributed in Japan, Korean and China. Angelica keiskei has been widely used as a diuretic, laxative and analeptic, the aerial parts are also consumed as a health food and used in tea, wine, flour, and cosmetics [22,23]. Over 100 constituents have been identified in this plant, including various types of flavonoids, coumarins, phenolics, diterpene and triterpenes [24,25]. Extracts of A. keiskei have been
Declarations of interest: none. Corresponding author at: KeXue Road, College of Food and Biological Engineering, Zhengzhou University of Light Industry, Zhengzhou 450001, China. E-mail address: [email protected] (Y. Bai).
☆ ⁎
https://doi.org/10.1016/j.microc.2019.03.046 Received 8 November 2018; Received in revised form 14 March 2019; Accepted 14 March 2019 Available online 15 March 2019 0026-265X/ © 2019 Elsevier B.V. All rights reserved.
J. Du, et al.
Microchemical Journal 147 (2019) 333–338
reported to have anti-bacterial [26], anti-tumor [27], anti-oxidative [28], anti-diabetic [29], anti-inflammatory [30], and anti-coagulant activities [31]. However, there is no report for the utilization of A. Keiskei extract on nanoparticle synthesis. Hence, this study highlights the green synthesis of large-sized AgNPs using Angelica keiskei water extract (AKE) and aims to understand the antibacterial property and membrane damage mechanism of the synthesized AgNPs against pathogens. Strain L. monocytogenes is introduced as a Gram-positive model strain for the evaluation. 2. Materials and methods 2.1. Materials Angelica keiskei powder was purchased from Xi'an Qing Zhi Biotechnology Co., Ltd. (China). Fluorescent dye FDA and PI were purchased from Solarbio Co. Ltd. (China). The pathogenic bacterial culture, L. monocytogenes (ATCC 15313), was obtained from American Type Culture Collection (ATCC). Silver nitrate was purchased from Sigma-Aldrich, USA. Unless otherwise stated, the solvents and inorganic salts were purchased from Sinopharm Chemical Reagent Company, China.
Fig. 2. FE-SEM image of the AgNPs.
2.4. Antimicrobial testing The antimicrobial activity of the AgNPs was studied by measuring the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the AgNPs against L. monocytogenes. The MIC was determined using a broth microdilution assay, which was modified based on a previously described method [32]. Briefly, twofold serial dilutions of the AgNPs were prepared in sterile trypticase soy broth (TSB) from a top concentration of 200 μg/mL to 3.125 μg/mL. 100 μL of the AgNPs dilutions was placed in sextuplicate in 100-well plate, and inoculate with 100 μL of the L. monocytogenes culture to a final concentration of 5 × 105 CFU/mL. 200 μL/well of TSB was used as a blank control to monitor sterility and 200 μL/well of the L. monocytogenes culture was used as positive control. The MIC was taken as the lowest concentration of the AgNO3 at which growth of the bacterium was complete inhibited after 24 h of incubation at 37 °C. The optical density at 600 nm was measured and recorded every 30 min using a Microbiology Reader Bioscreen C (BioScreen, Finland). The wells of 1× MIC and 2× MIC from the plate were spread (100 μL) on trypticase soy agar (TSA), and the lowest concentration of the AgNPs not showing any growth on TSA was referred to as the MBC. MIC and MBC of AgNO3 (at final concentration range from 1.56 to 100 μg/mL) and AKE (25 to 1600 μg/mL) against L. monocytogenes were performed in the same manner.
2.2. Preparation of Angelica keiskei water extract (AKE) and synthesis of AgNPs Angelica keiskei powder was added to Milli-Q water (1:50, w/v) and boiled at 95 °C for 20 min. The extract solution was filtered and freeze dried to a powder form. For AgNP synthesis, the AKE was treated with an aqueous solution of AgNO3 (at concentration of 2 mM) at 65 °C. The reaction parameters were optimized by varying the concentrations of AKE and AgNO3. 2.3. Characterization of AgNPs The bio-reduction of the AgNPs was observed visually by color change and further confirmed using a Multiskan Go microplate spectrophotometer (Thermo Scientific, USA) at a range of 300–800 nm. The morphology of the AgNPs was analyzed using FE-SEM (JSM-7001F, JEOL, Japan). The crystal structure of the synthesized AgNPs was detected using XRD (D8 Advance, Bruker, Germany). The particle size distribution and charge on the AgNPs were investigated by DLS analysis and zeta potential, respectively, using Malvern Zetasizer Nano ZS90 (Malvern Instruments, UK). To check salt stability, AgNPs was diluted with Milli-Q water to a concentration of 0.4 μg/mL. NaCl solution with same volume of AgNPs solution was added to obtain a final concentration range from 1 mM to 2 M, AgNPs added with water was used as control. The LSPR spectra of mixture were measured after 120 min, respectively.
2.5. Antibacterial mechanisms of cell membrane The effect of AgNPs on the cell membrane integrity was determined using FDA and PI fluorescent dye to distinguish living cells from dead
Fig. 1. UV–Visible spectroscopic measurements of biosynthesized AgNPs as a function of amount of plant extract (A) and AgNO3 (B). 334
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2.6. Determination of extracellular nucleic acids The effect of the synthesized AgNO3 on the release of intracellular nucleic acid was determined by measuring the release of nucleic acids from L. monocytogenes into the medium [33]. Cells in the logarithmic growth phase were collected and washed three times. The bacterial suspensions were incubated at 37 °C in the presence of 1× MIC and 1× MBC of AgNO3. Subsequently, the extracellular nucleic acids were measured using NanoReady Micro UV–Vis spectrophotometer at OD260 (Life Real Co., Ltd., China), after 0, 30, 60, 90, and 240 min from incubation. 3. Results and discussion 3.1. Characterization of the AgNPs The present work first synthesized AgNPs using AKE, and the color in the reaction mixture changed from pale yellow to dark brown. In the UV–visible spectra, the AKE at 400 μg/mL shows a peak at 400 nm, indicating the synthesis of AgNPs (Fig. 1A). With the increase in the amount of AgNO3, the absorbance and breadth of the peaks increase (Fig. 1B). The peak intensities help explain the logarithmic trend in the formation of AgNPs from lower to higher concentration. Therefore, the AKE at a concentration of 400 μg/mL treated with 8 mM AgNO3 was considered as the optimized condition. The FE-SEM images reveal that most of the AgNPs formed are mostly spherical in shape (Fig. 2). In the XRD pattern (Fig. 3), the diffraction peaks at 2θ values of 38.07, 46.21, 64.37, 77.36, and 81.54° are identified and matched with the JCPDS databases of standard silver (file No. 04-0783), indexed to (111), (200),
Fig. 3. XRD spectrum of the AgNPs.
cells. L. monocytogenes in the logarithmic growth phase was collected and washed three times. The bacterial suspensions were incubated at 37 °C in the presence of 1× MIC and 1× MBC AgNO3 for 1 h. The cells treated with 0.85% NaCl were used as the control. The cells were then collected and suspended in a PBS buffer, fixed with FDA (final concentration: 50 μg/mL) and PI (final concentration: 20 μg/mL) for 20 min in the dark. The cells were observed under a Nikon Ni-E upright research-grade microscope (NIKON, Japan), in the phase contrast field and fluorescence field.
Fig. 4. Particle size distribution of the AgNPs.
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(220), (311), and (222) lattice planes of face-centered cubic silver, respectively. The DLS analysis (Fig. 4) shows that the zeta potential and average size of the AgNPs are 130.1 ± 2.1 nm (PDI: 0.229) and −34.3 ± 0.2 mV, respectively. This signifies the good quality and stability of the synthesized AgNPs. LSPR peak (425 nm) of AgNPs shows no change at NaCl concentration ranging between 1 mM to 2 M, as compared with control (Fig. 5). However, absorbance value is significant decreasing in 2 M NaCl, indicating the AgNPs is stable at NaCl concentration as high as 1 M. 3.2. Bacteriostatic and bactericidal property of the AgNPs Bacteriostatic and bactericidal property of the AgNPs, AgNO3 and AKE against L. monocytogenes are investigated by Mic and MBC. Fig.6 shows that with an increasing of AgNPs and AgNO3 concentration, the growth of L. monocytogenes is delayed and decreased. When the AgNPs and AgNO3 concentration reached 12.5 μg/mL, no cell growth is detected, indicating the MICs of the AgNPs and AgNO3 are both 12.5 μg/
Fig. 5. Stability of AgNPs in the presence of increasing NaCl concentrations.
Fig. 6. Growth curves of L. monocytogenes cultured with different concentrations of the AgNPs (A) and AgNO3 (B), and the growth of L. monocytogenes cultured with 1× MIC and 2× MIC AgNPs and AgNO3 on trypticase soy agar plates.
Fig. 7. A, B, and C are fluorescence images of L. monocytogenes cells treated with 0.85% NaCl, 1× MIC AgNPs and 1× MBC AgNPs, respectively. Cells treated with 0.85% NaCl were performed as control. Cells staining by FDA dye show green fluorescence, and cells staining by PI dye show red fluorescence. 336
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Acknowledgments This work was supported by the National Key Research and Development Program of China [2018YFD0401204]; the Major Science & Technology Project of Henan Province [grant number 161100110900]; the Scientific and Technological Project of Henan Province [grant number 182102110392]; and Doctoral Scientific Research Foundation of Zhengzhou University of Light Industry. Conflict of interest The authors declare no competing financial interests. References [1] M.M.J. Franco, A.C. Paes, M.G. Ribeiro, A.C.B. Santos, M. Miyata, C.Q.F. Leite, et al., Occurrence of mycobacteria in bovine milk samples from both individual and collective bulk tanks at farms and informal markets in the southeast region of Sao Paulo, Brazil, BMC Vet. Res. 9 (2013) 85. [2] V. Coroneo, V. Carraro, N. Aissani, A. Sanna, A. Ruggeri, S. Succa, et al., Detection of virulence genes and growth potential in Listeria monocytogenes strains isolated from ricotta salata cheese, J. Food Sci. 81 (2015) M114–M120. [3] B. Jagadeesan, V. Bastic Schmid, B. Kupski, W. McMahon, A. Klijn, Detection of Listeria spp. and L. monocytogenes in pooled test portion samples of processed dairy products, Int. J. Food Microbiol. 289 (2018) 30–39. [4] C. Pablos, A. Romero, A. de Diego, C. Vargas, I. Bascon, F. Perez-Rodriguez, et al., Novel antimicrobial agents as alternative to chlorine with potential applications in the fruit and vegetable processing industry, Int. J. Food Microbiol. 285 (2018) 92–97. [5] R. Salomoni, P. Leo, A.F. Montemor, B.G. Rinaldi, M. Rodrigues, Antibacterial effect of silver nanoparticles in Pseudomonas aeruginosa. Nanotechnology, Science and Applications, 10 (2017), pp. 115–121. [6] D. Nayak, S. Ashe, P.R. Rauta, M. Kumari, B. Nayak, Bark extract mediated green synthesis of silver nanoparticles: evaluation of antimicrobial activity and antiproliferative response against osteosarcoma, Mater. Sci. Eng. C 58 (2016) 44–52. [7] H.E. Emam, H.B. Ahmed, Polysaccharides templates for assembly of nanosilver, Carbohydr. Polym. 135 (2016) 300–307. [8] S.V. Otari, R.M. Patil, S.J. Ghosh, S.H. Pawar, Green phytosynthesis of silver nanoparticles using aqueous extract of Manilkara zapota (L.) seeds and its inhibitory action against Candida species, Mater. Lett. 116 (2014) 367–369. [9] P. Singh, Y.J. Kim, D.B. Zhang, D.C. Yang, Biological synthesis of nanoparticles from plants and microorganisms, Trends Biotechnol. 34 (2016) 588–599. [10] S. Ahmed, M. Ahmad, B.L. Swami, S. Ikram, A review on plants extract mediated synthesis of silver nanoparticles for antimicrobial applications: a green expertise, J. Adv. Res. 7 (2016) 17–28. [11] J. Du, H. Singh, T.H. Yi, Antibacterial, anti-biofilm and anticancer potentials of green synthesized silver nanoparticles using benzoin gum (Styrax benzoin) extract, Bioprocess Biosyst. Eng. 39 (2016) 1923–1931. [12] H. Singh, J. Du, P. Singh, T.H. Yi, Ecofriendly synthesis of silver and gold nanoparticles by Euphrasia officinalis, leaf extract and its biomedical applications, Artif. Cell Nanomed. B. 46 (2018) 1163–1170. [13] G. Gnanajobitha, K. Paulkumar, M. Vanaja, S. Rajeshkumar, C. Malarkodi, G. Annadurai, et al., Fruit-mediated Synthesis of Silver Nanoparticles Using Vitis vinifera and Evaluation of Their Antimicrobial Efficacy, 3 (2013), p. 67. [14] S. Kaviya, J. Santhanalakshmi, B. Viswanathan, J. Muthumary, K. Srinivasan, Biosynthesis of silver nanoparticles using Citrus sinensis peel extract and its antibacterial activity, Spectrochim. Acta A Mol. Biomol. Spectrosc. 79 (2011) 594–598. [15] M. Ahamed, M.A.M. Khan, M.K.J. Siddiqui, M.S. Alsalhi, Alrokayan SAJPEL-dS, Nanostructures. Green Synthesis, Characterization and Evaluation of Biocompatibility of Silver Nanoparticles, 43 (2011), pp. 1266–1271. [16] C. Krishnaraj, E.G. Jagan, S. Rajasekar, P. Selvakumar, P.T. Kalaichelvan, N. Mohan, Synthesis of Silver Nanoparticles Using Acalypha indica Leaf Extracts and Its Antibacterial Activity Against Water Borne Pathogens, 76 (2009), pp. 50–56. [17] N.R. Ni, A. Aher, S. Gosavi, P.B. Vidyasagar, Green Synthesis of Silver Nanoparticles Using Latex Extract of Thevetia peruviana: A Novel Approach Towards Poisonous Plant Utilization, 423 (2013), p. 012032. [18] V. Annavaram, V.R. Posa, V.G. Uppara, S. Jorepalli, A.R. Somala, Facile Green Synthesis of Silver Nanoparticles Using Limonia acidissima Leaf Extract and Its Antibacterial Activity, 5 (2015), pp. 97–103. [19] N. Jayaprakash, J.J. Vijaya, K. Kaviyarasu, K. Kombaiah, L.J. Kennedy, R.J. Ramalingam, et al., Green Synthesis of Ag Nanoparticles Using Tamarind Fruit Extract for the Antibacterial Studies, 169 (2017), pp. 178–185. [20] K.S. Siddiqi, A. Husen, R.A.K. Rao, A review on biosynthesis of silver nanoparticles and their biocidal properties, J. Nanobiotechnol. 16 (2018) 14. [21] Y. Hung-Jen, H. Shan-Hui, T.J.S. Ching-Lin, Cytotoxicity and Immunological Response of Gold and Silver Nanoparticles of Different Sizes, 5 (2010), pp. 1553–1561. [22] K. Baba, M. Taniguchi, K. Nakata, Studies on Angelica keiskei “Ashitaba”, Foods
Fig. 8. Measurement of nucleic acid released from L. monocytogenes into extracellular during the incubation with the synthesized AgNPs. Values are mean ± SD of three determinations.
mL. To determine the MBCs of AgNPs and AgNO3, L. monocytogenes cultured with 1× MIC and 2× MIC were spread on TSA and incubated at 37 °C for two days. As shown in Fig. 6, a lot of colonies are observed on the TSA with the presence of 1× MIC AgNPs, while no visible colony was observed for 2× MIC AgNPs, indicating the MBC of the AgNO3 is 25 μg/mL; several colonies are observed on the TSA with the presence of 1× MIC AgNO3, no visible colony was observed for 2× MIC AgNO3. AKE have no antibacterial activity against L. monocytogenes (data not shown). The AgNPs exhibited same MIC and MBC with AgNO3, this low MIC and MBC value demonstrate that the AgNPs have a strong suppression of proliferation for L. monocytogenes. 3.3. Effect of the AgNPs on membrane integrity of L. monocytogenes The effect of AgNPs on the cell membrane integrity was examined by fluorescence observation of the treated cells after staining them with FDA and PI fluorescent dye. With the mixture staining of FDA and PI, the cells with intact membranes exhibit fluorescence green, and the cells with damaged membranes exhibit fluorescence red. Fig. 7 shows that the AgNPs induce the damage of L. monocytogenes cell membrane and increase the membrane permeability in a dose-dependent manner. In particular, the cells treated with 1× MBC show a large part of red fluorescence, and the dark points at the center of the cell aggregate are regarded as AgNPs (Fig. 7C). The effect of the AgNO3 on the release of intracellular nucleic acid was further examined. At 240 min, the nucleic acids released from 0.85% NaCl, 1× MIC, and 1× MBC AgNPs cultures are found to be 53.2 ± 0.83, 73.5 ± 0.3, and 159.6 ± 3.08 ng/μL, respectively, showing a positive correlation with the treatment of AgNPs (Fig. 8). DNA is one of the most important genetic materials, and the data show that L. monocytogenes cells released a significant amount of nucleic acids after treating with the AgNPs, thereby disrupting the reproduction of the cells, even leading to death. 4. Conclusion A biosynthesis approach for large-sized AgNPs was developed using AKE; the synthesis was done in an ecofriendly and relatively new manner. This study showed that AgNPs have a strong antibacterial activity against food-borne pathogens and that they could damage the membrane integrity and induce the release of nucleic acids, thereby disrupting cell reproduction.
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J. Du, et al. Food Ingredients J. Jpn. (1988) 52–60. [23] Y.S. Kil, S.T. Pham, E.K. Seo, M. Jafari, Angelica keiskei, an emerging medicinal herb with various bioactive constituents and biological activities, Arch. Pharm. Res. 40 (6) (2017) 655–675. [24] Y. Kimura, K. Baba, Antitumor and antimetastatic activities of Angelica keiskei roots, part 1: isolation of an active substance, xanthoangelol, Int. J. Cancer 106 (3) (2003) 429–437. [25] T. Akihisa, H. Tokuda, D. Hasegawa, M. Ukiya, Y. Kimura, F. Enjo, ... H. Nishino, Chalcones and other compounds from the exudates of Angelica keiskei and their cancer chemopreventive effects, J. Nat. Prod. 69 (2006) 38–42. [26] K. Sugamoto, Y.-I. Matsusita, K. Matsui, C. Kurogi, T. Matsui, Synthesis and antibacterial activity of chalcones bearing prenyl or geranyl groups from Angelica keiskei, Tetrahedron 67 (2011) 5346–5359. [27] K. Motani, K. Tabata, Y. Kimura, S. Okano, Y. Shibata, Y. Abiko, H. Nagai, T. Akihisa, T. Suzuki, Proteomic analysis of apoptosis induced by xanthoangelol, a major constituent of Angelica keiskei, in neuroblastoma, Biol. Pharm. Bull. 31 (2008) 618–626. [28] L. Luo, R. Wang, X. Wang, Z. Ma, N. Li, Compounds from Angelica keiskei with NQO1
[29] [30] [31] [32] [33]
338
induction, DPPH·scavenging and α-glucosidase inhibitory activities, Food Chem. 131 (2012) 992–998. T. Enoki, H. Ohnogi, K. Nagamine, Y. Kudo, K. Sugiyama, M. Tanabe, E. Kobayashi, H. Sagawa, I. Kato, Antidiabetic activities of chalcones isolated from a Japanese herb, Angelica keiskei, J. Agric. Food Chem. 55 (2007) 6013–6017. H.R. Chang, H.J. Lee, J.H. Ryu, Chalcones from Angelica keiskei attenuate the inflammatory responses by suppressing nuclear translocation of NF-κB, J. Med. Food 17 (2014) 1306–1313. D.J. Son, Y.O. Park, C. Yu, S.E. Lee, Y.H. Park, Bioassay-guided isolation and identification of anti-platelet-active compounds from the root of Ashitaba (Angelica keiskei Koidz.), Nat. Prod. Res. 28 (2014) 2312–2316. E. Mohamed, A. Syed, F. Scott, D. Paul, M.G. Mark, M. Roger, et al., Resazurinbased 96-well plate microdilution method for the determination of minimum inhibitory concentration of biosurfactants, Biotechnol. Lett. 38 (2016) 1015–1019. W.R. Diao, Q.P. Hu, H. Zhang, J.G. Xu, Chemical composition, antibacterial activity and mechanism of action of essential oil from seeds of fennel (Foeniculum vulgare Mill.), Food Control 35 (2014) 109–116.