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Green synthesis, antibacterial, antioxidant and cytotoxic effect of gold nanoparticles using Pistacia Atlantica extract
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Green synthesis, antibacterial, antioxidant and cytotoxic effect of gold nanoparticles using Pistacia Atlantica extract Mona Hamelian a, Saba Hemmati b, Kambiz Varmira a,∗, Hojat Veisi b,∗ a b
Research Center of Oils and Fats, Kermanshah University of Medical Sciences, Kermanshah, Iran Department of Chemistry, Payame Noor University, Tehran, Iran
a r t i c l e
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Article history: Received 14 March 2018 Revised 12 July 2018 Accepted 13 July 2018 Available online xxx Keywords: Gold nanoparticles Green Pistacia Atlantica Antibacterial Antioxidant Cytotoxic effect
a b s t r a c t A simple, affordable and ecofriendly method for synthesis gold nanoparticles (Au NPs) using Pistacia Atlantica (leave and fruit) extract at the room temperature, has been carry out in the present study. Higher reaction rate about a minute is so noticeable and Au NPs synthesis with this method provided nontoxic carrier for application. Various analytical technique including UV–vis absorption spectroscopy approved presence of Au NPs in the solution, the functional groups of Pistacia Atlantica extract in the reduction and capping process of Au NPs is determined by FT-IR, crystallinity with the fcc plane approved from the X-ray diffraction (XRD) pattern, energy dispersive spectroscopy (EDS) determined existence of elements in the sample, surface morphology, diverse shapes and size of present Au NPs were showed by using scanning electron microscopy (SEM), atomic force microscopy (AFM) and Transmission electron microscopy (TEM). In addition, antibacterial, antioxidant and cytotoxicity properties of Au NPs were studied. Agar disk and agar well diffusion are the methods to determined antibacterial properties of synthesized Au NPs. Also MIC (Minimum Inhibitory Concentration) and MBC (Minimum Bactericidal Concentration) were recognized by macro broth dilution assay. DPPH free radical scavenging assay was used for antioxidant property and compare to butylated hydroxytoluene (BHT) as standard antioxidant. Synthesized Au NPs have great cell viability in a dose depended manner and demonstrate that this method for synthesis Gold nanoparticles provided nontoxic. The average diameter of synthesized Au NPs was about 50–60 nm. © 2018 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction Nano science, science of materials with smallest dimensions less than 100 nm, is noteworthy according to applications in medical, drug delivery, engineering, molecular imaging, chemistry, physics and etc. [1–3]. Despite of various methods using for nanoparticles synthesis and due to harmful effects of chemical and physical methods for environment [5], nowadays development of efficient green chemistry for synthesis metal nanoparticles provided safe and affordable methods by using tea [4], coffee [6], microorganism [7] and different parts of plants extract [8–11]. Nanoparticles synthesized by plant extract are more stable and the rate of synthesis is faster and easier than another [12]. Labeling, delivering, heating and sensing are important characteristics of gold nanoparticle that make it noteworthy in compare to other metal nanoparticles [13]. Important application of gold nanoparticles in human contact area, and because of its cy-
totoxicity [14,15] and anti-bacterial activity [1617] recommended ecofriendly method to synthesis nontoxic nanoparticles. Recently, plants such as Cassia auriculata leaf [18], Rosa rugosa leaf [19], Aloe Wera [20], and Thyme [21] were used for reducing AuCl3 in aqueous solutions. Pistacia Atlantica is useful plant that antioxidant and antimicrobial activity of different parts of this plant including fruit, leave and resin extract were determined [22]. In addition, wide application such as antiseptic, anti-inflammatory, antitumor, antinociceptive, antidiabetic, wound healing and etc. of Pistacia Atlantica make it valuable plant for green synthesis Au NPs [23,24]. Due to impressive properties of different parts of Pistacia Atlantica and various applications of nontoxic gold nanoparticles, present study synthesis Au NPs by using this plant as reducing agent, cytotoxicity and antioxidant activity of synthesized Au NPs were studied. 2. Experimental 2.1. Materials
∗
Corresponding authors. E-mail addresses: [email protected] (K. Varmira), [email protected] (H. Veisi).
All materials were obtained from Sigma Aldrich chemicals. Pistacia Atlantica plant (leave& fruit) were purchased from vegetable market at Kermanshah province (west of Iran).
https://doi.org/10.1016/j.jtice.2018.07.018 1876-1070/© 2018 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Please cite this article as: M. Hamelian et al., Green synthesis, antibacterial, antioxidant and cytotoxic effect of gold nanoparticles using P istacia At lant ica extract, Journal of the Taiwan Institute of Chemical Engineers (2018), https://doi.org/10.1016/j.jtice.2018.07.018
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Fig. 1. Effect of the time on solution containing HAuCl4 × H2 O and Pistacia Atlantica extract. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
Fig. 2. UV–vis absorption spectra of different solution containing Au NPs. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
2.2. Preparation of plant extract
2.3. Synthesis of gold nanoparticles
Pistacia Atlantica plant (leave& fruit) were washed several time with deionized water and dried at 25 °C in the oven, then separately powdered with mortar to smallest size. Prepared powder once alone and once again together in equal amount of each one (10 g) was added to 300 mL deionized water and heated to boiling for 30 min, until solution color changed to light yellow. Each extract was cooled in the room temperature and filtered through Whatman filter no.1 and maintained at 4 °C for further studied.
For each synthesis, Pistacia Atlantica (leave & fruit) extract once separately and another time together (10 mL) was added to aqueous solution of (1 mM) HAuCl4 × H2 O (100 mL) at room temperature and stirred, color of the solution with fruit quickly about 10 s, fruit and leave about 20 s and leave during a minute turned to dark red (Fig. 1), that indicated formation of gold nanoparticles. The solution stirred for 1 h to complete reduction process, then centrifuge at 12,0 0 0 rpm for 15 min and upper phase was
Please cite this article as: M. Hamelian et al., Green synthesis, antibacterial, antioxidant and cytotoxic effect of gold nanoparticles using P istacia At lant ica extract, Journal of the Taiwan Institute of Chemical Engineers (2018), https://doi.org/10.1016/j.jtice.2018.07.018
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Fig. 3. FT-IR spectra of (a): Pistacia Atlantica extract and (b): Au NPs synthesized using Pistacia Atlantica extract.
Fig. 4. XRD spectrum of Au NPs synthesized using Pistacia Atlantica extract.
Please cite this article as: M. Hamelian et al., Green synthesis, antibacterial, antioxidant and cytotoxic effect of gold nanoparticles using P istacia At lant ica extract, Journal of the Taiwan Institute of Chemical Engineers (2018), https://doi.org/10.1016/j.jtice.2018.07.018
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Fig. 5. EDS image of Au NPs synthesized by Pistacia Atlantica extract.
Fig. 6. SEM image of (a) Pistacia Atlantica extract; and (b–d) different magnification of biosynthesized Au NPs.
removed, obtained Au NPs was washed several time with deionized water to remove all uncoordinated biological materials, then put it in the oven at 50 °C to dried.
3. Antioxidant assay 3.1. DPPH free radical scavenging assay 1, 1-diphenyl-2-picryl-hydrazil (DPPH), free radical scavenging activity of Au NPs prepared using Pistacia Athlantica extract was determined by method reported by Hosseinimehr, et al. [25]. Different concentration of present Au NPs (20, 40, 60, 80 and 100 μg/mL) were added at 1 mL–3 mL methanol solution of DPPH (10 mg/250 mL). The mixture was shaken, then incubated in dark place at room temperature for 30 min. Finally, the absorbance of each mixture was recorded at 517 nm. The absorbance of DPPH (containing no sample) was recorded as control. The experiment was done in triplicated. Butylated hydroxytoluene (BHT) was performed as the standard antioxidant. The percentage of free radical scavenging was calculated using this formula:
DPPH free radical scavenging (% ) =
Fig. 7. AFM images of Au NPs biosynthesized.
control − test × 100 control
Please cite this article as: M. Hamelian et al., Green synthesis, antibacterial, antioxidant and cytotoxic effect of gold nanoparticles using P istacia At lant ica extract, Journal of the Taiwan Institute of Chemical Engineers (2018), https://doi.org/10.1016/j.jtice.2018.07.018
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Fig. 8. TEM images of Au NPs biosynthesized at different magnification.
Fig. 9. Antioxidant activity of Au NPs synthesized by Pistacia Atlantica extract.
Fig. 10. Percent viability measured on Hela cells after treatment with present Au NPs for 48 h by MTT assay.
4. Evaluation of cytotoxicity assay by MTT
4.2. MTT assay
4.1. Cell lines and culture conditions
Cytotoxicity effect on hela cell lines by Au NPs synthesized using Pistacia Athlantica extract were evaluated by MTT 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide assay and the method of [26]. The cell lines were plated in 96 (1 × 105 cell/well) in complete medium and incubated at 37 °C under humidified atmosphere with 5% CO2 for 48 h, then cells were washed with PBS buffer and treated with present gold nanoparticles in 1% DMSO and different concentrations (0, 25, 50, 10 0 and 20 0 μg/mL),
Hela NCBI-C115 (Human cervix carcinoma), were purchased from Pasture Institute of Tehran-Iran. Cell lines were culture in Dulbecco, s Modified Eagle, s Medium (DMEM), 10% Fetal Bovine Serum (FBS) and antibiotics including Penicillin 100 IU/mL and Streptomycin 100 μL/mL, then incubated at 37 °C with 5% CO2 Cells were under culture for the experiment.
Please cite this article as: M. Hamelian et al., Green synthesis, antibacterial, antioxidant and cytotoxic effect of gold nanoparticles using P istacia At lant ica extract, Journal of the Taiwan Institute of Chemical Engineers (2018), https://doi.org/10.1016/j.jtice.2018.07.018
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Fig. 11. The diameters of growth inhibition zones of Gram negative bacteria (EC and PA) in agar disk diffusion test in different dilutions of synthesized Au NPs.
Fig. 12. The diameters of growth inhibition zones of Gram positive bacteria (SA and BS) in agar disk diffusion test in different dilutions of synthesized Au NPs.
Please cite this article as: M. Hamelian et al., Green synthesis, antibacterial, antioxidant and cytotoxic effect of gold nanoparticles using P istacia At lant ica extract, Journal of the Taiwan Institute of Chemical Engineers (2018), https://doi.org/10.1016/j.jtice.2018.07.018
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Fig. 13. The diameters of growth inhibition zones of Gram negative bacteria (EC and PA) in agar well diffusion test in different dilutions of synthesized Au NPs.
then incubated at 37 °C for 48 h The experiment was done in triplicated. Further the samples were washed with PBS (pH 7.4), again incubated for 4 h with 20 μL/well MTT (5 mg/mL) to made formazan crystals. After that dissolved formazan crystals by adding 0.04 M HCl/Isopropanol to each well. The absorbance at 570 nm was measured with UV-Spectroscopy. Well containing cells and no nanoparticles was measured as the control. Cell viability was calculated by following formula:
Percentage of cell viability (% ) =
sample absorbance × 100 control absorbance
4.3. Evaluation of antimicrobial activities Four bacterial species namely Escherichia coli (ATCC No. 25,922) (EC), Pseudomonas aeruginosa (PTCC No. 1707) (PA), Staphylococcus aureus (ATCC No. 25,923) (SA) and Bacillus subtilis (ATCC No. 21,332) (BS) were procured from Iranian Research Organization for Science and Technology as lyophilized. Each bacterial strain was activated on Tryptic Soy broth, constant at 37 °C for 18 h Then 60 μL of the broth was transferred to Nutrient agar and incubated at 37 °C for another 24 h; cell concentration was 108 cfu/mL. Agar disk and well diffusion tests were used as screen tests to assess antibacterial effects of Au NPs based on standard protocol. The solution of the Au NPs was produced in 1 g/mL from which six fold serial dilutions (v/v) were prepared. 70 μL of each dilution was
poured on each disk and well in order. After a period of 24 h incubation, the diameters of growth inhibition zones around the disks wells were measured. Distilled water was used as negative control whereas Kanamycin and Cephalexin were used as positive control in negative and positive bacteria, respectively. Minimum Inhibitory Concentration (MIC) and Minimum Bacterial Concentration (MBC) were specified by macro broth dilution assay based on Clinical Laboratory Standard Institute (CLSI) guidelines1 . 4.4. Statistical analysis Antibacterial activities were determined by one-way variance analysis (ANOVA), using the SPSS 22 software package. Data were analyzed statistically significant at p ≤ 0.01. 5. Result and discussion Formation of gold nanoparticle synthesis with Pistacia Atlantica (leave & fruit) extract, first separately and then mixture of both, determined by UV–vis spectroscopy (Perkin-Elmer, Lambda 25) which directly related to nanoparticle size [27]. Synthesized Au NPs that used leave extract have red color solution and peaks at
1
Clinical and laboratory standards institute (CLSI), M7-A7, 2016; 26 (2).
Please cite this article as: M. Hamelian et al., Green synthesis, antibacterial, antioxidant and cytotoxic effect of gold nanoparticles using P istacia At lant ica extract, Journal of the Taiwan Institute of Chemical Engineers (2018), https://doi.org/10.1016/j.jtice.2018.07.018
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Fig. 14. The diameters of growth inhibition zones of Gram positive bacteria (SA and BS) in agar well diffusion test in different dilutions of synthesized Au NPs.
520 nm that indicated size of 30 nm, using mixture of leave and fruit extract, cause size of nanoparticle increased to 40 nm, solution color turns to light purple and synthesized Au NPs peaks shift to longer wave length at 530 and when used fruit extract separately, solution color change to dark purple and its peak shift to 540 nm that shows size of 50 nm (Fig. 2) [28,29]. The FT-IR spectrum of samples were analyzed using ABB Bomem MB 100 instrument that this measurement was used to identify and study functional groups of extract and synthesized Au NPs. Fig. 3a; shows the spectrum of Pistacia Atlantica extract, the peak at 3452 cm−1 can be attributed to the stretching vibration of hydroxyl/amine functional group in phenolic compound, 2981 cm−1 (C–H stretch), 1704 cm−1 (C = O stretch), 1623 and 1454 cm−1 related to amine III and amine I bands of proteins, 1261 cm−1 (C–C stretch) and 1083 cm−1 shows C–N stretching vibration of aliphatic amines. Fig 3b, spectrum of synthesized Au NPs shows strong band at 3382 cm−1 shows O–H stretch, 3186 cm−1 (C–H stretch), 1716 cm−1 (C = O stretch), 1623 and 1454 cm−1 (amine III and amine I), 1211 cm−1 (C–C stretch) and 1060 cm−1 (C–N stretch). Comparing both spectra shows peak at 3452 cm−1 related to hydroxyl/amine group moves to lower wavelength in 3382 cm−1 which demonstrate the binding of Au NPs to the surface. Structure of synthesized Au NPs has been performed using XRD analysis which was done on Bruker AXS-D8 Advance instrument, operating at voltage of 40 KV and current of 30 MA with CuK radiation. In X-Ray diffraction by crystal (Fig. 4), observed that XRay intensity which reflected from crystals are maximum at certain angles. The charactrastic peak corresponding to (111), (200), (220) and (311) of Au NPs are located at 38.067, 44.215, 64.291
and 77.592 that demonstrate fcc structure of gold nanoparticles (JCPDS.NO.004-0784) and indicated that synthesized Au NPs using Pistacia Atlantica extract are crystalline [30]. Energy Dispersive X-ray Spectroscopy (TESCAN Vega, USA) is an analytical technique used for elemental analysis to confirmed existence of element in the samples. Metallic gold nano crystal show typical optical observation peak approximately at 3 keV due to surface plasmon resonance. Fig. 5 shows EDX spectrum of Au NPs synthesized using Pistacia Atlantica extract. Strong gold signal confirming the formation Au NPs. Weak signals relating to carbon, oxygen and nitrogen which may have originated from biomolecules are observed. Scanning Electron Microscope (TESCAN Vega Model) was carry out to recognized morphology and size of nanoparticles by SEM image of Pistacia Atlantica extract (Fig. 6a) and different magnification of biosynthesized gold nanoparticles (Fig. 6b–d) that indicated formation of homogenous and relative capping of Au NPs with diameter range 40–50 nm. The larger gold nanoparticles may be due to aggregation of the smallest one due to SEM instrument. Atomic force of microscopy (DME-95-50 E) was used to determine morphology and agglomeration of nanoparticles. AFM images (Fig. 7a–c) showed surface morphology of Au NPs synthesized using Pistacia Athlantica extract and Fig. 7d–f images express agglomeration of Au NPs biosynthesized in the surface. High resolution transmission electron microscopy (HRTEM) has been used to identify the size and shape of nanoparticles. Typical TEM images obtained for colloids are shown in Fig. 8. From the images, it is clear that the morphology of gold nanoparticles is almost spherical. This result is in agreement with the shape of SPR bands centered at 530 nm of colloid.
Please cite this article as: M. Hamelian et al., Green synthesis, antibacterial, antioxidant and cytotoxic effect of gold nanoparticles using P istacia At lant ica extract, Journal of the Taiwan Institute of Chemical Engineers (2018), https://doi.org/10.1016/j.jtice.2018.07.018
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M. Hamelian et al. / Journal of the Taiwan Institute of Chemical Engineers 000 (2018) 1–10 Table 1 MIC and MBC of synthesized Au NPs.
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7. Conclusion
Microorganism
EC
PA
SA
BS
MIC (μg/mL) MBC (μg/mL)
1/128 (7.81) 1/64 (15.62)
1/256 (3.9) 1/16 (62.5)
1/128 (7.81) 1/64 (15.62)
1/32 (31.25) 1/16 (62.5)
5.1. Antioxidant activity Free radical which cause damage and mutation cells are harmful to human health. Antioxidant play significant role against them, recently consumption of natural materials including cereals, pulses, nuts, fruits, vegetables that having antioxidant activity, was increased [31]. DPPH is well-known method to determine antioxidant activity due to its potential on free radical’ s reduction [32]. DPPH free radical scavenging activity of present Au NPs demonstrate effective inhibition in compare to BHT as the standard antioxidant (Fig. 10). As it was showed free radical scavenging activity of Au NPs biosynthesized by Pistacia Atlantica extract was increased due to increase in dose depended method (Fig. 9). In addition, color of the mixture was changed. Both results may refer to antioxidant potential of the Pistacia Atlantica extract that was used to Au NPs synthesized. 5.2. Cytotoxicity survey The cells which treated with different concentrations (0, 25, 50, 10 0 and 20 0 μg/mL) of present Au NPs were examined in term of cytotoxicity effect on hela cells by MTT assay for 48 h The absorbance was measured at 570 nm and showed excellent viability even up to 200 μg/mL (Fig. 10). This result determined that present Au NPs, providing nontoxic coating on cells. Fig. 11; showed different cell viability percentage in varying of concentrations. Lack of any significant toxicity of Au NPs, provided many safe applications in medical and pharmaceutical fields. 6. Antibacterial survey 6.1. Agar disk diffusion test EC, PA (Gram negative bacteria), SA and BS (Gram positive) were sensitive to synthesized Au NPs. The most sensitivity about ES, PA, SA and BS is around 21, 23, 20 and 20 mm in diameter related to dilution 31 μg/mL. in addition, there was inhibition zone even in dilution 3 μg/mL around 8 mm due to all bacteria. Growth inhibition zone due to different dilution are listed in Figs. 11 and 12. There was not inhibition zone in dilution 2 μg/mL. 6.2. Agar well diffusion test Figs. 13 and 14 indicated that widest zone was seen in 15 μg/mL due to ES, PA, SA and BS bacterium (10, 10, 9 and 10 mm) and there wasn’t any inhibition zone in dilution 2 μg/mL. 6.3. MIC and MBC The value of MIC for ES, PA, SA and BS are 7.81, 3.9, 7.81 and 3.25 μg/mL but the value of MBC are 15.62, 62.5, 15.62 and 62.5 μg/mL (Table 1). As the result of Table 1 showed synthesized Au NPs by Pistacia Atlantica extract have prevented the growth of mention bacteria. Increasing concentration of Au NPs increased the inhibition zone (p ≤ 0.01), this conclusion showed that bacteria in used have a significant difference (p ≤ 0.01) in terms of sensitivity to concentration of synthesized Au NPs.
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Please cite this article as: M. Hamelian et al., Green synthesis, antibacterial, antioxidant and cytotoxic effect of gold nanoparticles using P istacia At lant ica extract, Journal of the Taiwan Institute of Chemical Engineers (2018), https://doi.org/10.1016/j.jtice.2018.07.018
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Please cite this article as: M. Hamelian et al., Green synthesis, antibacterial, antioxidant and cytotoxic effect of gold nanoparticles using P istacia At lant ica extract, Journal of the Taiwan Institute of Chemical Engineers (2018), https://doi.org/10.1016/j.jtice.2018.07.018
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Journal of the Taiwan Institute of Chemical Engineers journal homepage: www.elsevier.com/locate/jtice
Green synthesis, antibacterial, antioxidant and cytotoxic effect of gold nanoparticles using Pistacia Atlantica extract Mona Hamelian a, Saba Hemmati b, Kambiz Varmira a,∗, Hojat Veisi b,∗ a b
Research Center of Oils and Fats, Kermanshah University of Medical Sciences, Kermanshah, Iran Department of Chemistry, Payame Noor University, Tehran, Iran
a r t i c l e
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Article history: Received 14 March 2018 Revised 12 July 2018 Accepted 13 July 2018 Available online xxx Keywords: Gold nanoparticles Green Pistacia Atlantica Antibacterial Antioxidant Cytotoxic effect
a b s t r a c t A simple, affordable and ecofriendly method for synthesis gold nanoparticles (Au NPs) using Pistacia Atlantica (leave and fruit) extract at the room temperature, has been carry out in the present study. Higher reaction rate about a minute is so noticeable and Au NPs synthesis with this method provided nontoxic carrier for application. Various analytical technique including UV–vis absorption spectroscopy approved presence of Au NPs in the solution, the functional groups of Pistacia Atlantica extract in the reduction and capping process of Au NPs is determined by FT-IR, crystallinity with the fcc plane approved from the X-ray diffraction (XRD) pattern, energy dispersive spectroscopy (EDS) determined existence of elements in the sample, surface morphology, diverse shapes and size of present Au NPs were showed by using scanning electron microscopy (SEM), atomic force microscopy (AFM) and Transmission electron microscopy (TEM). In addition, antibacterial, antioxidant and cytotoxicity properties of Au NPs were studied. Agar disk and agar well diffusion are the methods to determined antibacterial properties of synthesized Au NPs. Also MIC (Minimum Inhibitory Concentration) and MBC (Minimum Bactericidal Concentration) were recognized by macro broth dilution assay. DPPH free radical scavenging assay was used for antioxidant property and compare to butylated hydroxytoluene (BHT) as standard antioxidant. Synthesized Au NPs have great cell viability in a dose depended manner and demonstrate that this method for synthesis Gold nanoparticles provided nontoxic. The average diameter of synthesized Au NPs was about 50–60 nm. © 2018 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction Nano science, science of materials with smallest dimensions less than 100 nm, is noteworthy according to applications in medical, drug delivery, engineering, molecular imaging, chemistry, physics and etc. [1–3]. Despite of various methods using for nanoparticles synthesis and due to harmful effects of chemical and physical methods for environment [5], nowadays development of efficient green chemistry for synthesis metal nanoparticles provided safe and affordable methods by using tea [4], coffee [6], microorganism [7] and different parts of plants extract [8–11]. Nanoparticles synthesized by plant extract are more stable and the rate of synthesis is faster and easier than another [12]. Labeling, delivering, heating and sensing are important characteristics of gold nanoparticle that make it noteworthy in compare to other metal nanoparticles [13]. Important application of gold nanoparticles in human contact area, and because of its cy-
totoxicity [14,15] and anti-bacterial activity [1617] recommended ecofriendly method to synthesis nontoxic nanoparticles. Recently, plants such as Cassia auriculata leaf [18], Rosa rugosa leaf [19], Aloe Wera [20], and Thyme [21] were used for reducing AuCl3 in aqueous solutions. Pistacia Atlantica is useful plant that antioxidant and antimicrobial activity of different parts of this plant including fruit, leave and resin extract were determined [22]. In addition, wide application such as antiseptic, anti-inflammatory, antitumor, antinociceptive, antidiabetic, wound healing and etc. of Pistacia Atlantica make it valuable plant for green synthesis Au NPs [23,24]. Due to impressive properties of different parts of Pistacia Atlantica and various applications of nontoxic gold nanoparticles, present study synthesis Au NPs by using this plant as reducing agent, cytotoxicity and antioxidant activity of synthesized Au NPs were studied. 2. Experimental 2.1. Materials
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Corresponding authors. E-mail addresses: [email protected] (K. Varmira), [email protected] (H. Veisi).
All materials were obtained from Sigma Aldrich chemicals. Pistacia Atlantica plant (leave& fruit) were purchased from vegetable market at Kermanshah province (west of Iran).
https://doi.org/10.1016/j.jtice.2018.07.018 1876-1070/© 2018 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Please cite this article as: M. Hamelian et al., Green synthesis, antibacterial, antioxidant and cytotoxic effect of gold nanoparticles using P istacia At lant ica extract, Journal of the Taiwan Institute of Chemical Engineers (2018), https://doi.org/10.1016/j.jtice.2018.07.018
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Fig. 1. Effect of the time on solution containing HAuCl4 × H2 O and Pistacia Atlantica extract. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
Fig. 2. UV–vis absorption spectra of different solution containing Au NPs. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
2.2. Preparation of plant extract
2.3. Synthesis of gold nanoparticles
Pistacia Atlantica plant (leave& fruit) were washed several time with deionized water and dried at 25 °C in the oven, then separately powdered with mortar to smallest size. Prepared powder once alone and once again together in equal amount of each one (10 g) was added to 300 mL deionized water and heated to boiling for 30 min, until solution color changed to light yellow. Each extract was cooled in the room temperature and filtered through Whatman filter no.1 and maintained at 4 °C for further studied.
For each synthesis, Pistacia Atlantica (leave & fruit) extract once separately and another time together (10 mL) was added to aqueous solution of (1 mM) HAuCl4 × H2 O (100 mL) at room temperature and stirred, color of the solution with fruit quickly about 10 s, fruit and leave about 20 s and leave during a minute turned to dark red (Fig. 1), that indicated formation of gold nanoparticles. The solution stirred for 1 h to complete reduction process, then centrifuge at 12,0 0 0 rpm for 15 min and upper phase was
Please cite this article as: M. Hamelian et al., Green synthesis, antibacterial, antioxidant and cytotoxic effect of gold nanoparticles using P istacia At lant ica extract, Journal of the Taiwan Institute of Chemical Engineers (2018), https://doi.org/10.1016/j.jtice.2018.07.018
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Fig. 3. FT-IR spectra of (a): Pistacia Atlantica extract and (b): Au NPs synthesized using Pistacia Atlantica extract.
Fig. 4. XRD spectrum of Au NPs synthesized using Pistacia Atlantica extract.
Please cite this article as: M. Hamelian et al., Green synthesis, antibacterial, antioxidant and cytotoxic effect of gold nanoparticles using P istacia At lant ica extract, Journal of the Taiwan Institute of Chemical Engineers (2018), https://doi.org/10.1016/j.jtice.2018.07.018
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Fig. 5. EDS image of Au NPs synthesized by Pistacia Atlantica extract.
Fig. 6. SEM image of (a) Pistacia Atlantica extract; and (b–d) different magnification of biosynthesized Au NPs.
removed, obtained Au NPs was washed several time with deionized water to remove all uncoordinated biological materials, then put it in the oven at 50 °C to dried.
3. Antioxidant assay 3.1. DPPH free radical scavenging assay 1, 1-diphenyl-2-picryl-hydrazil (DPPH), free radical scavenging activity of Au NPs prepared using Pistacia Athlantica extract was determined by method reported by Hosseinimehr, et al. [25]. Different concentration of present Au NPs (20, 40, 60, 80 and 100 μg/mL) were added at 1 mL–3 mL methanol solution of DPPH (10 mg/250 mL). The mixture was shaken, then incubated in dark place at room temperature for 30 min. Finally, the absorbance of each mixture was recorded at 517 nm. The absorbance of DPPH (containing no sample) was recorded as control. The experiment was done in triplicated. Butylated hydroxytoluene (BHT) was performed as the standard antioxidant. The percentage of free radical scavenging was calculated using this formula:
DPPH free radical scavenging (% ) =
Fig. 7. AFM images of Au NPs biosynthesized.
control − test × 100 control
Please cite this article as: M. Hamelian et al., Green synthesis, antibacterial, antioxidant and cytotoxic effect of gold nanoparticles using P istacia At lant ica extract, Journal of the Taiwan Institute of Chemical Engineers (2018), https://doi.org/10.1016/j.jtice.2018.07.018
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Fig. 8. TEM images of Au NPs biosynthesized at different magnification.
Fig. 9. Antioxidant activity of Au NPs synthesized by Pistacia Atlantica extract.
Fig. 10. Percent viability measured on Hela cells after treatment with present Au NPs for 48 h by MTT assay.
4. Evaluation of cytotoxicity assay by MTT
4.2. MTT assay
4.1. Cell lines and culture conditions
Cytotoxicity effect on hela cell lines by Au NPs synthesized using Pistacia Athlantica extract were evaluated by MTT 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide assay and the method of [26]. The cell lines were plated in 96 (1 × 105 cell/well) in complete medium and incubated at 37 °C under humidified atmosphere with 5% CO2 for 48 h, then cells were washed with PBS buffer and treated with present gold nanoparticles in 1% DMSO and different concentrations (0, 25, 50, 10 0 and 20 0 μg/mL),
Hela NCBI-C115 (Human cervix carcinoma), were purchased from Pasture Institute of Tehran-Iran. Cell lines were culture in Dulbecco, s Modified Eagle, s Medium (DMEM), 10% Fetal Bovine Serum (FBS) and antibiotics including Penicillin 100 IU/mL and Streptomycin 100 μL/mL, then incubated at 37 °C with 5% CO2 Cells were under culture for the experiment.
Please cite this article as: M. Hamelian et al., Green synthesis, antibacterial, antioxidant and cytotoxic effect of gold nanoparticles using P istacia At lant ica extract, Journal of the Taiwan Institute of Chemical Engineers (2018), https://doi.org/10.1016/j.jtice.2018.07.018
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Fig. 11. The diameters of growth inhibition zones of Gram negative bacteria (EC and PA) in agar disk diffusion test in different dilutions of synthesized Au NPs.
Fig. 12. The diameters of growth inhibition zones of Gram positive bacteria (SA and BS) in agar disk diffusion test in different dilutions of synthesized Au NPs.
Please cite this article as: M. Hamelian et al., Green synthesis, antibacterial, antioxidant and cytotoxic effect of gold nanoparticles using P istacia At lant ica extract, Journal of the Taiwan Institute of Chemical Engineers (2018), https://doi.org/10.1016/j.jtice.2018.07.018
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Fig. 13. The diameters of growth inhibition zones of Gram negative bacteria (EC and PA) in agar well diffusion test in different dilutions of synthesized Au NPs.
then incubated at 37 °C for 48 h The experiment was done in triplicated. Further the samples were washed with PBS (pH 7.4), again incubated for 4 h with 20 μL/well MTT (5 mg/mL) to made formazan crystals. After that dissolved formazan crystals by adding 0.04 M HCl/Isopropanol to each well. The absorbance at 570 nm was measured with UV-Spectroscopy. Well containing cells and no nanoparticles was measured as the control. Cell viability was calculated by following formula:
Percentage of cell viability (% ) =
sample absorbance × 100 control absorbance
4.3. Evaluation of antimicrobial activities Four bacterial species namely Escherichia coli (ATCC No. 25,922) (EC), Pseudomonas aeruginosa (PTCC No. 1707) (PA), Staphylococcus aureus (ATCC No. 25,923) (SA) and Bacillus subtilis (ATCC No. 21,332) (BS) were procured from Iranian Research Organization for Science and Technology as lyophilized. Each bacterial strain was activated on Tryptic Soy broth, constant at 37 °C for 18 h Then 60 μL of the broth was transferred to Nutrient agar and incubated at 37 °C for another 24 h; cell concentration was 108 cfu/mL. Agar disk and well diffusion tests were used as screen tests to assess antibacterial effects of Au NPs based on standard protocol. The solution of the Au NPs was produced in 1 g/mL from which six fold serial dilutions (v/v) were prepared. 70 μL of each dilution was
poured on each disk and well in order. After a period of 24 h incubation, the diameters of growth inhibition zones around the disks wells were measured. Distilled water was used as negative control whereas Kanamycin and Cephalexin were used as positive control in negative and positive bacteria, respectively. Minimum Inhibitory Concentration (MIC) and Minimum Bacterial Concentration (MBC) were specified by macro broth dilution assay based on Clinical Laboratory Standard Institute (CLSI) guidelines1 . 4.4. Statistical analysis Antibacterial activities were determined by one-way variance analysis (ANOVA), using the SPSS 22 software package. Data were analyzed statistically significant at p ≤ 0.01. 5. Result and discussion Formation of gold nanoparticle synthesis with Pistacia Atlantica (leave & fruit) extract, first separately and then mixture of both, determined by UV–vis spectroscopy (Perkin-Elmer, Lambda 25) which directly related to nanoparticle size [27]. Synthesized Au NPs that used leave extract have red color solution and peaks at
1
Clinical and laboratory standards institute (CLSI), M7-A7, 2016; 26 (2).
Please cite this article as: M. Hamelian et al., Green synthesis, antibacterial, antioxidant and cytotoxic effect of gold nanoparticles using P istacia At lant ica extract, Journal of the Taiwan Institute of Chemical Engineers (2018), https://doi.org/10.1016/j.jtice.2018.07.018
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Fig. 14. The diameters of growth inhibition zones of Gram positive bacteria (SA and BS) in agar well diffusion test in different dilutions of synthesized Au NPs.
520 nm that indicated size of 30 nm, using mixture of leave and fruit extract, cause size of nanoparticle increased to 40 nm, solution color turns to light purple and synthesized Au NPs peaks shift to longer wave length at 530 and when used fruit extract separately, solution color change to dark purple and its peak shift to 540 nm that shows size of 50 nm (Fig. 2) [28,29]. The FT-IR spectrum of samples were analyzed using ABB Bomem MB 100 instrument that this measurement was used to identify and study functional groups of extract and synthesized Au NPs. Fig. 3a; shows the spectrum of Pistacia Atlantica extract, the peak at 3452 cm−1 can be attributed to the stretching vibration of hydroxyl/amine functional group in phenolic compound, 2981 cm−1 (C–H stretch), 1704 cm−1 (C = O stretch), 1623 and 1454 cm−1 related to amine III and amine I bands of proteins, 1261 cm−1 (C–C stretch) and 1083 cm−1 shows C–N stretching vibration of aliphatic amines. Fig 3b, spectrum of synthesized Au NPs shows strong band at 3382 cm−1 shows O–H stretch, 3186 cm−1 (C–H stretch), 1716 cm−1 (C = O stretch), 1623 and 1454 cm−1 (amine III and amine I), 1211 cm−1 (C–C stretch) and 1060 cm−1 (C–N stretch). Comparing both spectra shows peak at 3452 cm−1 related to hydroxyl/amine group moves to lower wavelength in 3382 cm−1 which demonstrate the binding of Au NPs to the surface. Structure of synthesized Au NPs has been performed using XRD analysis which was done on Bruker AXS-D8 Advance instrument, operating at voltage of 40 KV and current of 30 MA with CuK radiation. In X-Ray diffraction by crystal (Fig. 4), observed that XRay intensity which reflected from crystals are maximum at certain angles. The charactrastic peak corresponding to (111), (200), (220) and (311) of Au NPs are located at 38.067, 44.215, 64.291
and 77.592 that demonstrate fcc structure of gold nanoparticles (JCPDS.NO.004-0784) and indicated that synthesized Au NPs using Pistacia Atlantica extract are crystalline [30]. Energy Dispersive X-ray Spectroscopy (TESCAN Vega, USA) is an analytical technique used for elemental analysis to confirmed existence of element in the samples. Metallic gold nano crystal show typical optical observation peak approximately at 3 keV due to surface plasmon resonance. Fig. 5 shows EDX spectrum of Au NPs synthesized using Pistacia Atlantica extract. Strong gold signal confirming the formation Au NPs. Weak signals relating to carbon, oxygen and nitrogen which may have originated from biomolecules are observed. Scanning Electron Microscope (TESCAN Vega Model) was carry out to recognized morphology and size of nanoparticles by SEM image of Pistacia Atlantica extract (Fig. 6a) and different magnification of biosynthesized gold nanoparticles (Fig. 6b–d) that indicated formation of homogenous and relative capping of Au NPs with diameter range 40–50 nm. The larger gold nanoparticles may be due to aggregation of the smallest one due to SEM instrument. Atomic force of microscopy (DME-95-50 E) was used to determine morphology and agglomeration of nanoparticles. AFM images (Fig. 7a–c) showed surface morphology of Au NPs synthesized using Pistacia Athlantica extract and Fig. 7d–f images express agglomeration of Au NPs biosynthesized in the surface. High resolution transmission electron microscopy (HRTEM) has been used to identify the size and shape of nanoparticles. Typical TEM images obtained for colloids are shown in Fig. 8. From the images, it is clear that the morphology of gold nanoparticles is almost spherical. This result is in agreement with the shape of SPR bands centered at 530 nm of colloid.
Please cite this article as: M. Hamelian et al., Green synthesis, antibacterial, antioxidant and cytotoxic effect of gold nanoparticles using P istacia At lant ica extract, Journal of the Taiwan Institute of Chemical Engineers (2018), https://doi.org/10.1016/j.jtice.2018.07.018
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M. Hamelian et al. / Journal of the Taiwan Institute of Chemical Engineers 000 (2018) 1–10 Table 1 MIC and MBC of synthesized Au NPs.
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7. Conclusion
Microorganism
EC
PA
SA
BS
MIC (μg/mL) MBC (μg/mL)
1/128 (7.81) 1/64 (15.62)
1/256 (3.9) 1/16 (62.5)
1/128 (7.81) 1/64 (15.62)
1/32 (31.25) 1/16 (62.5)
5.1. Antioxidant activity Free radical which cause damage and mutation cells are harmful to human health. Antioxidant play significant role against them, recently consumption of natural materials including cereals, pulses, nuts, fruits, vegetables that having antioxidant activity, was increased [31]. DPPH is well-known method to determine antioxidant activity due to its potential on free radical’ s reduction [32]. DPPH free radical scavenging activity of present Au NPs demonstrate effective inhibition in compare to BHT as the standard antioxidant (Fig. 10). As it was showed free radical scavenging activity of Au NPs biosynthesized by Pistacia Atlantica extract was increased due to increase in dose depended method (Fig. 9). In addition, color of the mixture was changed. Both results may refer to antioxidant potential of the Pistacia Atlantica extract that was used to Au NPs synthesized. 5.2. Cytotoxicity survey The cells which treated with different concentrations (0, 25, 50, 10 0 and 20 0 μg/mL) of present Au NPs were examined in term of cytotoxicity effect on hela cells by MTT assay for 48 h The absorbance was measured at 570 nm and showed excellent viability even up to 200 μg/mL (Fig. 10). This result determined that present Au NPs, providing nontoxic coating on cells. Fig. 11; showed different cell viability percentage in varying of concentrations. Lack of any significant toxicity of Au NPs, provided many safe applications in medical and pharmaceutical fields. 6. Antibacterial survey 6.1. Agar disk diffusion test EC, PA (Gram negative bacteria), SA and BS (Gram positive) were sensitive to synthesized Au NPs. The most sensitivity about ES, PA, SA and BS is around 21, 23, 20 and 20 mm in diameter related to dilution 31 μg/mL. in addition, there was inhibition zone even in dilution 3 μg/mL around 8 mm due to all bacteria. Growth inhibition zone due to different dilution are listed in Figs. 11 and 12. There was not inhibition zone in dilution 2 μg/mL. 6.2. Agar well diffusion test Figs. 13 and 14 indicated that widest zone was seen in 15 μg/mL due to ES, PA, SA and BS bacterium (10, 10, 9 and 10 mm) and there wasn’t any inhibition zone in dilution 2 μg/mL. 6.3. MIC and MBC The value of MIC for ES, PA, SA and BS are 7.81, 3.9, 7.81 and 3.25 μg/mL but the value of MBC are 15.62, 62.5, 15.62 and 62.5 μg/mL (Table 1). As the result of Table 1 showed synthesized Au NPs by Pistacia Atlantica extract have prevented the growth of mention bacteria. Increasing concentration of Au NPs increased the inhibition zone (p ≤ 0.01), this conclusion showed that bacteria in used have a significant difference (p ≤ 0.01) in terms of sensitivity to concentration of synthesized Au NPs.
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Please cite this article as: M. Hamelian et al., Green synthesis, antibacterial, antioxidant and cytotoxic effect of gold nanoparticles using P istacia At lant ica extract, Journal of the Taiwan Institute of Chemical Engineers (2018), https://doi.org/10.1016/j.jtice.2018.07.018