nanomaterials Article
Biosynthesis of Silver Nanoparticles Using Taxus yunnanensis Callus and Their Antibacterial Activity and Cytotoxicity in Human Cancer Cells Qian Hua Xia, Yan Jun Ma and Jian Wen Wang * College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China; [email protected] (Q.H.X.); [email protected] (Y.J.M.) * Correspondence: [email protected] or [email protected]; Tel.: +86-512-6956-1430 Academic Editor: Eleonore Fröhlich Received: 12 May 2016; Accepted: 26 August 2016; Published: 1 September 2016
Abstract: Plant constituents could act as chelating/reducing or capping agents for synthesis of silver nanoparticles (AgNPs). The green synthesis of AgNPs has been considered as an environmental friendly and cost-effective alternative to other fabrication methods. The present work described the biosynthesis of AgNPs using callus extracts from Taxus yunnanensis and evaluated their antibacterial activities in vitro and potential cytotoxicity in cancer cells. Callus extracts were able to reduce silver nitrate at 1 mM in 10 min. Transmission electron microscope (TEM) indicated the synthesized AgNPs were spherical with the size range from 6.4 to 27.2 nm. X-ray diffraction (XRD) confirmed the AgNPs were in the form of nanocrystals. Fourier transform infrared spectroscopy (FTIR) suggested phytochemicals in callus extracts were possible reducing and capping agents. The AgNPs exhibited effective inhibitory activity against all tested human pathogen bacteria and the inhibition against Gram-positive bacteria was stronger than that of Gram-negative bacteria. Furthermore, they exhibited stronger cytotoxic activity against human hepatoma SMMC-7721 cells and induced noticeable apoptosis in SMMC-7721 cells, but showed lower cytotoxic against normal human liver cells (HL-7702). Our results suggested that biosynthesized AgNPs could be an alternative measure in the field of antibacterial and anticancer therapeutics. Keywords: Taxus yunnanensis; callus extract; silver nanoparticles; antibacterial; cytotoxicity
1. Introduction As an important antibacterial agent, silver has been used for many years [1]. Recently, silver nanoparticles (AgNPs) promoted the usage of silver in biomedical applications with its unique physic-chemical, optical and biological properties [2,3]. Several methods have been developed for the synthesis of AgNPs, including physical, chemical and biological methods [4,5]. The biological synthetic methods are preferred because they are safe, cheap and environment-friendly, whereas other physical and chemical methods require energy, high pressure, temperature and chemicals toxic to biological systems. Plants and microbes are currently used for AgNP synthesis among the biological synthetic methods [6]. Recently, a novel use of plant callus to synthesize AgNPs has been widely investigated. Many kinds of plant callus induced from papaya, alfalfa, Catharanthus roseus and Sesuvium portulacastrum have been applied in the green synthesis of AgNPs [7–10]. Applying plant callus in synthesizing AgNPs avoids various drawbacks including low yield, high expense, energy consumption in the physical approaches, and contamination in the wet-chemical procedure [11]. As an incessant source in laboratory, callus culture overcomes the difficulties of scarcity in wild plant source [8]. Interestingly, callus extracts were more efficient in the fabrication of AgNPs and the synthesized AgNPs were more
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AgNPs were more distinct and scattered in distribution than those synthesized by intact leaf extract distinct and scattered in distribution than those synthesized by intact leaf extract [10]. The AgNPs [10]. The AgNPs biologically synthesized by callus showed excellent antibacterial against biologically synthesized by callus showed excellent antibacterial effect against humaneffect pathogenic human pathogenic microorganisms [9,10]. AgNPs synthesized by calli of Citrullus colocynthis were microorganisms [9,10]. AgNPs synthesized by calli of Citrullus colocynthis were reported to have reported to have cytotoxic activity against human epidermoid larynx carcinoma cells [12]. cytotoxic activity against human epidermoid larynx carcinoma cells [12]. Taxus yunnanensis Cheng et L.K. Fu (Taxaceae), an evergreen tree endemic to Yunnan of China, Taxus yunnanensis Cheng et L.K. Fu (Taxaceae), an evergreen tree endemic to Yunnan of China, is a is a promising of potent anticancer paclitaxel and taxane‐type [13]. to the promising sourcesource of potent anticancer paclitaxel and taxane-type diterpenesditerpenes [13]. Due to the Due enormous enormous commercial value of paclitaxel and the scarcity of T. yunnanensis trees in China, callus and commercial value of paclitaxel and the scarcity of T. yunnanensis trees in China, callus and cell cultures cell cultures have been as a practical alternative source production for paclitaxel [14]. have been established as established a practical alternative source for paclitaxel [14].production Many studies Many studies on cell culture of T. yunnanensis focused on enhancing paclitaxel production by means on cell culture of T. yunnanensis focused on enhancing paclitaxel production by means of elicitation, of elicitation, ultrasound treatment and medium renewal [15,16]. However, little has been reported ultrasound treatment and medium renewal [15,16]. However, little has been reported on biosynthesized on biosynthesized AgNP using Taxus tree or Therefore, a follow‐up to our efforts for on AgNP using Taxus tree or callus. Therefore, as acallus. follow-up to ouras efforts on exploring AgNPs exploring AgNPs for controlling microbes [17] and biotechnological application of T. yunnanensis [16], controlling microbes [17] and biotechnological application of T. yunnanensis [16], we therefore wish we therefore wish to report a biological method to synthesize AgNPs using the callus extracts from to report a biological method to synthesize AgNPs using the callus extracts from T. yunnanensis. T. yunnanensis. The antibacterial and cytotoxic activity of the biosynthesized AgNPs was also investigated. The antibacterial and cytotoxic activity of the biosynthesized AgNPs was also investigated. 2.2. Results Results 2.1. The Synthesis of AgNPs 2.1. The Synthesis of AgNPs Color change of callus extracts incubated with 1 mM AgNO 3 was observed, as shown in Color change of callus extracts incubated with 1 mM AgNO 3 was observed, as shown in Figure Figure The reaction solution showed a UV-visible absorbance 449nm nmbecause because of of the 1a–d. 1a–d. The reaction solution showed a UV‐visible absorbance at at 449 the surface surface plasmon resonance of AgNPs [18] and the absorbance increased with the reaction time but decreased plasmon resonance of AgNPs [18] and the absorbance increased with the reaction time but decreased after after 60 60 min min ofof the the reaction reaction (Figure (Figure 1e). 1e). AgNPs AgNPs with with pH pH of of 7,7, 8,8, 9,9, 10 10 and and 11 11 showed showed maximum maximum absorbance at 439, 425, 411, 409 and 450 nm, respectively. There was a blue shift of absorbance from absorbance at 439, 425, 411, 409 and 450 nm, respectively. There was a blue shift of absorbance from pH 7 to 10, indicating the formation of smaller size nanoparticles [19]. Therefore we chose pH 10 as the pH 7 to 10, indicating the formation of smaller size nanoparticles [19]. Therefore we chose pH 10 as optimal condition. the optimal condition.
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Figure 1. Callus of T. yunnanensis (a); and color intensity of the extracts incubated with solution of Figure 1. Callus of T. yunnanensis (a); and color intensity of the extracts incubated with solution of silver ions at the beginning (b); after 10 min (c); and after 120 min of reaction (d). UV-visible absorbance silver ions at the beginning (b); after 10 min (c); and after 120 min of reaction (d). UV‐visible of AgNPs synthesized with the extracts under different reaction times (e) and different pH (f). absorbance of AgNPs synthesized with the extracts under different reaction times (e) and different pH (f).
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2.2. Characterization of AgNPs 2.2. Characterization of AgNPs
The biosynthesized AgNPs were characterized using transmission electron microscope (TEM) The biosynthesized AgNPs were characterized using transmission electron microscope (TEM) (Figure 2), X‐ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) (Figure 3). (Figure 2), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) (Figure 3). The nanoparticles were in spherical shape. The particle size of the synthesized AgNPs shown by TEM The nanoparticles were in spherical shape. The particle size of the synthesized AgNPs shown by TEM ranged from 6.4 to 27.2 nm measured by Image J software. ranged from 6.4 to 27.2 nm measured by Image J software.
Figure 2. Transmission electron microscope (TEM) analysis of AgNPs synthesized with callus extracts Figure 2. Transmission electron microscope (TEM) analysis of AgNPs synthesized with callus extracts from T. yunnanensis and AgNO from T. yunnanensis and AgNO 3. 3 .
Theof XRD of AgNPs (Figure 3a) 3a) exhibited intense peaks peaks in the whole spectrum of spectrum 2θ value ranging The XRD AgNPs (Figure exhibited intense in the whole of 2θ value from 10 to 70. Diffraction peaks at 2θ values of 38.06◦ , 44.11◦ , 64.43◦ and 77.33◦ were corresponded ranging from 10 to 70. Diffraction peaks at 2θ values of 38.06°, 44.11°, 64.43° and 77.33° were to the (111), (200), (220) and (311) crystal faces of face-centered silver, respectively, which was in corresponded to the (200), (220) and (311) faces of face‐centered silver, respectively, accordance with(111), the values in standard silver card crystal (JCPDS No. 04-0783). Figure 3b showed the FTIR which was in of accordance with the synthesized values in AgNPs standard silver card washed (JCPDS 04‐0783). Figure 3b spectra aqueous callus extracts, as well as AgNPs withNo. different solution. − 1 FTIR spectra of AgNPs showed the main peaks at 1625 and 1518 cm , which could be assigned to showed the FTIR spectra of aqueous callus extracts, synthesized AgNPs as well as AgNPs washed frame vibration of aromatic ring. Peaks at 2924 cm−1 corresponded to asymmetric stretching of C–H−1, which with different solution. FTIR spectra of AgNPs showed the main peaks at 1625 and 1518 cm bonds. The bands at 2162 and 2033 cm−1 were assigned to CO adsorbed and the band round 1979 cm−1 could be assigned to frame vibration of aromatic ring. Peaks at 2924 cm−1 corresponded to asymmetric was due to the overtone of C–H out of plane bending. Peaks at 1541, 1456 and 1400 cm−1 corresponded −1 were assigned to CO adsorbed and the stretching of C–H bonds. The bands at 2162 and 2033 cm to amide II, methylene scissoring vibration and methyl group of proteins, respectively. Another band band round 1979 cm at 800–600 cm−−11 was due to the overtone of C–H out of plane bending. Peaks at 1541, 1456 and could be attributed to C-H out of plane bend of aromatic phenol. Besides, the FTIR −1 corresponded to amide II, methylene scissoring vibration and methyl group of proteins, of synthesized AgNPs was similar with that of water-washed and medium-washed AgNPs, 1400 cmspectrum indicating most groups attached to AgNPs could not be removed by washing with water or medium. −1 could be attributed to C‐H out of plane bend of aromatic respectively. Another band at 800–600 cm Thus, groups around AgNPs were stable and would not release into the medium in both antibacterial phenol. Besides, the FTIR spectrum of synthesized AgNPs was similar with that of water‐washed and cytotoxicity experiments. Furthermore, no band was observed when AgNPs was washed with and medium‐washed AgNPs, indicating most groups attached to AgNPs could not be removed by alcohol. This result indicated most groups around AgNPs could be removed by alcohol. The surface washing with water or medium. Thus, groups around AgNPs were stable and would not release into zeta potentials of water-washed AgNPs, two-week-old AgNPs and alcohol-washed AgNPs were −28.8, −28.3 and −6.64 mV, respectively (Figure 3c). That is, our AgNPs was stable in the water even after the medium in both antibacterial and cytotoxicity experiments. Furthermore, no band was observed two weeks but the stability decreased after washed by alcohol. Besides, the visible precipitates could be when AgNPs was washed with alcohol. This result indicated most groups around AgNPs could be seen and after alcohol washing the dry powder of AgNPs could not be dispersed in water (Figure S1), removed by alcohol. The surface zeta potentials of water‐washed AgNPs, two‐week‐old AgNPs and indicating the colloidal stability of AgNPs was broken. alcohol‐washed AgNPs were −28.8, −28.3 and −6.64 mV, respectively (Figure 3c). That is, our AgNPs The content of Ag+ in nutrient broth (NB) and RPMI 1640 medium was detected in 24 h (Figure 4). + slowly, was stable in the showed water even after could two weeks but the stability decreased after washed by alcohol. The results that AgNPs release Ag which reached the highest content of 2.12 and 0.79 ppm in NB and RPMI 1640 medium, respectively. Besides, the visible precipitates could be seen and after alcohol washing the dry powder of AgNPs could not be dispersed in water (Figure S1), indicating the colloidal stability of AgNPs was broken. The content of Ag+ in nutrient broth (NB) and RPMI 1640 medium was detected in 24 h (Figure 4). The results showed that AgNPs could release Ag+ slowly, which reached the highest content of 2.12 and 0.79 ppm in NB and RPMI 1640 medium, respectively.
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Figure 3. (a) X‐ray diffraction (XRD) pattern; (b) Fourier transform infrared spectroscopy (FTIR) spectra Figure 3. (a) X-ray diffraction (XRD) pattern; (b) Fourier transform infrared spectroscopy (FTIR) spectra (1, callus extract; 2, NB medium‐washed AgNPs; 3, RPMI 1640 medium‐washed AgNPs; 4, synthesized (1, callus extract; 2, NB medium-washed AgNPs; 3, RPMI 1640 medium-washed AgNPs; 4, synthesized AgNPs; 5, water‐washed AgNPs; 6, alcohol‐washed AgNPs); and (c) zeta potential of AgNPs. AgNPs; 5, water-washed AgNPs; 6, alcohol-washed AgNPs); and (c) zeta potential of AgNPs.
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The AgNPs had concentration‐dependent inhibitory effect on all testing human pathogenic 2.3. Antibacterial Activity of AgNPs bacteria (Figure 5). The growth of Staphylococcus aureus, Salmonella paratyphi B and Bacillus subtilis was 2.3. Antibacterial Activity of AgNPs The AgNPs AgNPs had concentration-dependent concentration‐dependent inhibitory effect on all all testing testing human pathogenic pathogenic inhibited by AgNPs even at 2 μg/mL. The inhibition of AgNPs against Gram‐positive bacteria (S. The had inhibitory effect on human bacteria (Figure 5). The growth of Staphylococcus aureus, Salmonella paratyphi B and Bacillus subtilis was aureus and B. subtilis) was that of aureus, Gram‐negative bacteria (Escherichia coli and S. bacteria (Figure 5). Thestronger growth of than Staphylococcus Salmonella paratyphi B and Bacillus subtilis inhibited by AgNPs even at 2 μg/mL. The inhibition of AgNPs against Gram‐positive bacteria (S. was inhibited3 control at 1699 μg/mL (10 mM) showed no antibacterial activity against E.coli by AgNPs even at 2 µg/mL. The inhibition of AgNPs against Gram-positive bacteria paratyphi B). AgNO aureus and B. subtilis) was stronger than that of Gram‐negative bacteria (Escherichia coli and S. (S. aureus and B. subtilis) was stronger than that of Gram-negative bacteria (Escherichia coli and and lower activity against other three bacteria. The control gentamycin sulfate at 1 mM had greater paratyphi B). AgNO 3 control at 1699 μg/mL (10 mM) showed no antibacterial activity against E.coli S. paratyphi B). AgNO 3 control at 1699 µg/mL (10 mM) showed no antibacterial activity against E. coli inhibitory effect on gram‐negative bacteria E. coli and S. paratyphi B but weaker effect on gram‐ and lower activity against other three bacteria. The control gentamycin sulfate at 1 mM had greater and lower activity against other three bacteria. The control gentamycin sulfate at 1 mM had greater inhibitory effect effect on gram‐negative bacteria E. coli S. callus paratyphi B but weaker effect on gram‐ positive bacteria S. aureus and B. subtilis. The control of extracts at different concentrations inhibitory on gram-negative bacteria E. coli and and S. paratyphi B but weaker effect on gram-positive positive bacteria S. aureus and B. subtilis. The control of callus extracts (Figure at different (0.1%–10%) had no inhibition effects against all tested bacteria 6). concentrations We further tested bacteria S. aureus and B. subtilis. The control of callus extracts at different concentrations (0.1%–10%) (0.1%–10%) had no inhibition effects against all tested bacteria (Figure 6). We further tested had no inhibition effects against all tested bacteria (Figure 6). We further tested antibacterial activity of antibacterial activity of AgNPs by the minimum inhibitory concentration (MIC). As shown in Figure antibacterial activity of AgNPs by the minimum inhibitory concentration (MIC). As shown in Figure AgNPs by the minimum inhibitory concentration (MIC). As shown in Figure 7, antibacterial activity 7, antibacterial activity increased with the increasing concentration of AgNPs. MIC of AgNPs was 8, 7, antibacterial activity increased with the increasing concentration of AgNPs. MIC of AgNPs was 8, increased with the increasing concentration of AgNPs. MIC of AgNPs was 8, 1, 1 and 4 µg/mL against 1, 1 and 4 μg/mL against E. coli, S. aureus, B. subtilis and S. paratyphi B, respectively. 1, 1 and 4 μg/mL against E. coli, S. aureus, B. subtilis and S. paratyphi B, respectively. E. coli, S. aureus, B. subtilis and S. paratyphi B, respectively.
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Figure 5. Activity of AgNPs against human pathogen: (a) E. coli; (b) S. aureus; (c) S. paratyphi B; and (d) B. subtilis, depicting zones of inhibition of: 128 μg/mL (1); 32 μg/mL (2); 8 μg/mL (3); 2 μg/mL (4); 1699 μg/mL AgNO3 control (5); and 1 mM gentamycin sulfate (6).
Figure 5. Activity of AgNPs against human pathogen: (a) E. coli; (b) S. aureus; (c) S. paratyphi B; and Figure 5. Activity of AgNPs against human pathogen: (a) E. coli; (b) S. aureus; (c) S. paratyphi B; and (d) B. subtilis, depicting zones of inhibition of: 128 µg/mL (1); 32 µg/mL (2); 8 µg/mL (3); (d) B. subtilis, depicting zones of inhibition of: 128 μg/mL (1); 32 μg/mL (2); 8 μg/mL (3); 2 μg/mL (4); 2 µg/mL (4);3 control (5); and 1 mM gentamycin sulfate (6). 1699 µg/mL AgNO3 control (5); and 1 mM gentamycin sulfate (6). 1699 μg/mL AgNO
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Figure 7. Determination of the minimum inhibitory concentration (MIC) against: E. coli (a); S. aureus (b); Figure 7. Determination of the minimum inhibitory concentration (MIC) against: E. coli (a); S. aureus Figure 7. Determination of the minimum inhibitory concentration (MIC) against: E. coli (a); S. aureus B. subtilis (c); and S. paratyphi B (d).
(b); B. subtilis (c); and S. paratyphi B (d). (b); B. subtilis (c); and S. paratyphi B (d).
2.4. Cytotoxicity Experiments 2.4. Cytotoxicity Experiments shown Figure 8a, 10% (v/v) callus callus extracts extracts had had no no obvious cytotoxicity 3 3 As As shown in in Figure 8a, 10% (v/v) cytotoxicity while while AgNO AgNO control at 0.79 ppm had cytotoxic effect on on human human colon colon adenocarcinoma adenocarcinoma cells control at 0.79 ppm had no no cytotoxic effect cells (LS174T), (LS174T), lung lung adenocarcinoma cells (A549) and breast cancer cells (MCF‐7) but weak cytotoxic effect on human adenocarcinoma cells (A549) and breast cancer cells (MCF‐7) but weak cytotoxic effect on human hepatoma cells (SMMC‐7721) (87.8% cell cell viability). viability). AgNPs AgNPs could could reduce hepatoma cells (SMMC‐7721) (87.8% reduce all all tested tested cancer cancer cells cells viability in a dose dependent manner. SMMC‐7721 cells were the most sensitive tumors cells with viability in a dose dependent manner. SMMC‐7721 cells were the most sensitive tumors cells with the IC 50 of 27.75 μg/mL thus we chose SMMC‐7721 for further research. The cellular morphology of the IC 50 of 27.75 μg/mL thus we chose SMMC‐7721 for further research. The cellular morphology of SMMC‐7721 cells were observed and photographed under microscopy (Figure 8c). The shape of cells SMMC‐7721 cells were observed and photographed under microscopy (Figure 8c). The shape of cells was altered obviously when 30 μg/mL AgNPs was applied and a large number of dead cells were was altered obviously when 30 μg/mL AgNPs was applied and a large number of dead cells were presented at AgNPs concentration of 40 and 50 μg/mL, which was in accord with the results of 3‐(4,5‐ presented at AgNPs concentration of 40 and 50 μg/mL, which was in accord with the results of 3‐(4,5‐ dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay (Figure 7a). Furthermore, dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay (Figure 7a). Furthermore, apoptosis of SMMC‐7721 cells was measured by flow cytometry (Figure 9). Apoptosis rate was apoptosis of SMMC‐7721 cells was measured by flow cytometry (Figure 9). Apoptosis rate was
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2.4. Cytotoxicity Experiments As shown in Figure 8a, 10% (v/v) callus extracts had no obvious cytotoxicity while AgNO3 control at 0.79 ppm had no cytotoxic effect on human colon adenocarcinoma cells (LS174T), lung adenocarcinoma cells (A549) and breast cancer cells (MCF-7) but weak cytotoxic effect on human hepatoma cells (SMMC-7721) (87.8% cell viability). AgNPs could reduce all tested cancer cells viability in a dose dependent manner. SMMC-7721 cells were the most sensitive tumors cells with the IC50 of 27.75 µg/mL thus we chose SMMC-7721 for further research. The cellular morphology of SMMC-7721 cells were observed and photographed under microscopy (Figure 8c). The shape of cells was altered obviously when 30 µg/mL AgNPs was applied and a large number of dead cells were presented at AgNPs concentration of 40 and 50 µg/mL, which was in accord with the results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Figure 7a). Furthermore, Nanomaterials 2016, 6, 160 7 of 15 apoptosis of SMMC-7721 cells was measured by flow cytometry (Figure 9). Apoptosis rate was increased as the concentration of AgNPs increased. On the other hand, we investigated the cytotoxicity increased as the normal concentration of AgNPs increased. On the other hand, we investigated the of AgNPs against human liver cells HL-7702. The results indicated AgNPs inhibited the growth cytotoxicity AgNPs against normal liver cells HL‐7702. The results of HL-7702 atof higher concentration (Figurehuman 8b). IC50 value of AgNPs on HL-7702 cellsindicated was 81.39AgNPs µg/mL. inhibited the growth of HL‐7702 at higher concentration (Figure 8b). IC 50 value of AgNPs on HL‐7702 The difference of induced cell viability between cancer cells (SMMC-7721) and normal HL-7702 cells cells was 81.39 μg/mL. The difference of induced cell viability between cancer cells (SMMC‐7721) and was significant (p < 0.01). normal HL‐7702 cells was significant (p
Biosynthesis of Silver Nanoparticles Using Taxus yunnanensis Callus and Their Antibacterial Activity and Cytotoxicity in Human Cancer Cells Qian Hua Xia, Yan Jun Ma and Jian Wen Wang * College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China; [email protected] (Q.H.X.); [email protected] (Y.J.M.) * Correspondence: [email protected] or [email protected]; Tel.: +86-512-6956-1430 Academic Editor: Eleonore Fröhlich Received: 12 May 2016; Accepted: 26 August 2016; Published: 1 September 2016
Abstract: Plant constituents could act as chelating/reducing or capping agents for synthesis of silver nanoparticles (AgNPs). The green synthesis of AgNPs has been considered as an environmental friendly and cost-effective alternative to other fabrication methods. The present work described the biosynthesis of AgNPs using callus extracts from Taxus yunnanensis and evaluated their antibacterial activities in vitro and potential cytotoxicity in cancer cells. Callus extracts were able to reduce silver nitrate at 1 mM in 10 min. Transmission electron microscope (TEM) indicated the synthesized AgNPs were spherical with the size range from 6.4 to 27.2 nm. X-ray diffraction (XRD) confirmed the AgNPs were in the form of nanocrystals. Fourier transform infrared spectroscopy (FTIR) suggested phytochemicals in callus extracts were possible reducing and capping agents. The AgNPs exhibited effective inhibitory activity against all tested human pathogen bacteria and the inhibition against Gram-positive bacteria was stronger than that of Gram-negative bacteria. Furthermore, they exhibited stronger cytotoxic activity against human hepatoma SMMC-7721 cells and induced noticeable apoptosis in SMMC-7721 cells, but showed lower cytotoxic against normal human liver cells (HL-7702). Our results suggested that biosynthesized AgNPs could be an alternative measure in the field of antibacterial and anticancer therapeutics. Keywords: Taxus yunnanensis; callus extract; silver nanoparticles; antibacterial; cytotoxicity
1. Introduction As an important antibacterial agent, silver has been used for many years [1]. Recently, silver nanoparticles (AgNPs) promoted the usage of silver in biomedical applications with its unique physic-chemical, optical and biological properties [2,3]. Several methods have been developed for the synthesis of AgNPs, including physical, chemical and biological methods [4,5]. The biological synthetic methods are preferred because they are safe, cheap and environment-friendly, whereas other physical and chemical methods require energy, high pressure, temperature and chemicals toxic to biological systems. Plants and microbes are currently used for AgNP synthesis among the biological synthetic methods [6]. Recently, a novel use of plant callus to synthesize AgNPs has been widely investigated. Many kinds of plant callus induced from papaya, alfalfa, Catharanthus roseus and Sesuvium portulacastrum have been applied in the green synthesis of AgNPs [7–10]. Applying plant callus in synthesizing AgNPs avoids various drawbacks including low yield, high expense, energy consumption in the physical approaches, and contamination in the wet-chemical procedure [11]. As an incessant source in laboratory, callus culture overcomes the difficulties of scarcity in wild plant source [8]. Interestingly, callus extracts were more efficient in the fabrication of AgNPs and the synthesized AgNPs were more
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AgNPs were more distinct and scattered in distribution than those synthesized by intact leaf extract distinct and scattered in distribution than those synthesized by intact leaf extract [10]. The AgNPs [10]. The AgNPs biologically synthesized by callus showed excellent antibacterial against biologically synthesized by callus showed excellent antibacterial effect against humaneffect pathogenic human pathogenic microorganisms [9,10]. AgNPs synthesized by calli of Citrullus colocynthis were microorganisms [9,10]. AgNPs synthesized by calli of Citrullus colocynthis were reported to have reported to have cytotoxic activity against human epidermoid larynx carcinoma cells [12]. cytotoxic activity against human epidermoid larynx carcinoma cells [12]. Taxus yunnanensis Cheng et L.K. Fu (Taxaceae), an evergreen tree endemic to Yunnan of China, Taxus yunnanensis Cheng et L.K. Fu (Taxaceae), an evergreen tree endemic to Yunnan of China, is a is a promising of potent anticancer paclitaxel and taxane‐type [13]. to the promising sourcesource of potent anticancer paclitaxel and taxane-type diterpenesditerpenes [13]. Due to the Due enormous enormous commercial value of paclitaxel and the scarcity of T. yunnanensis trees in China, callus and commercial value of paclitaxel and the scarcity of T. yunnanensis trees in China, callus and cell cultures cell cultures have been as a practical alternative source production for paclitaxel [14]. have been established as established a practical alternative source for paclitaxel [14].production Many studies Many studies on cell culture of T. yunnanensis focused on enhancing paclitaxel production by means on cell culture of T. yunnanensis focused on enhancing paclitaxel production by means of elicitation, of elicitation, ultrasound treatment and medium renewal [15,16]. However, little has been reported ultrasound treatment and medium renewal [15,16]. However, little has been reported on biosynthesized on biosynthesized AgNP using Taxus tree or Therefore, a follow‐up to our efforts for on AgNP using Taxus tree or callus. Therefore, as acallus. follow-up to ouras efforts on exploring AgNPs exploring AgNPs for controlling microbes [17] and biotechnological application of T. yunnanensis [16], controlling microbes [17] and biotechnological application of T. yunnanensis [16], we therefore wish we therefore wish to report a biological method to synthesize AgNPs using the callus extracts from to report a biological method to synthesize AgNPs using the callus extracts from T. yunnanensis. T. yunnanensis. The antibacterial and cytotoxic activity of the biosynthesized AgNPs was also investigated. The antibacterial and cytotoxic activity of the biosynthesized AgNPs was also investigated. 2.2. Results Results 2.1. The Synthesis of AgNPs 2.1. The Synthesis of AgNPs Color change of callus extracts incubated with 1 mM AgNO 3 was observed, as shown in Color change of callus extracts incubated with 1 mM AgNO 3 was observed, as shown in Figure Figure The reaction solution showed a UV-visible absorbance 449nm nmbecause because of of the 1a–d. 1a–d. The reaction solution showed a UV‐visible absorbance at at 449 the surface surface plasmon resonance of AgNPs [18] and the absorbance increased with the reaction time but decreased plasmon resonance of AgNPs [18] and the absorbance increased with the reaction time but decreased after after 60 60 min min ofof the the reaction reaction (Figure (Figure 1e). 1e). AgNPs AgNPs with with pH pH of of 7,7, 8,8, 9,9, 10 10 and and 11 11 showed showed maximum maximum absorbance at 439, 425, 411, 409 and 450 nm, respectively. There was a blue shift of absorbance from absorbance at 439, 425, 411, 409 and 450 nm, respectively. There was a blue shift of absorbance from pH 7 to 10, indicating the formation of smaller size nanoparticles [19]. Therefore we chose pH 10 as the pH 7 to 10, indicating the formation of smaller size nanoparticles [19]. Therefore we chose pH 10 as optimal condition. the optimal condition.
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Figure 1. Callus of T. yunnanensis (a); and color intensity of the extracts incubated with solution of Figure 1. Callus of T. yunnanensis (a); and color intensity of the extracts incubated with solution of silver ions at the beginning (b); after 10 min (c); and after 120 min of reaction (d). UV-visible absorbance silver ions at the beginning (b); after 10 min (c); and after 120 min of reaction (d). UV‐visible of AgNPs synthesized with the extracts under different reaction times (e) and different pH (f). absorbance of AgNPs synthesized with the extracts under different reaction times (e) and different pH (f).
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2.2. Characterization of AgNPs 2.2. Characterization of AgNPs
The biosynthesized AgNPs were characterized using transmission electron microscope (TEM) The biosynthesized AgNPs were characterized using transmission electron microscope (TEM) (Figure 2), X‐ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) (Figure 3). (Figure 2), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) (Figure 3). The nanoparticles were in spherical shape. The particle size of the synthesized AgNPs shown by TEM The nanoparticles were in spherical shape. The particle size of the synthesized AgNPs shown by TEM ranged from 6.4 to 27.2 nm measured by Image J software. ranged from 6.4 to 27.2 nm measured by Image J software.
Figure 2. Transmission electron microscope (TEM) analysis of AgNPs synthesized with callus extracts Figure 2. Transmission electron microscope (TEM) analysis of AgNPs synthesized with callus extracts from T. yunnanensis and AgNO from T. yunnanensis and AgNO 3. 3 .
Theof XRD of AgNPs (Figure 3a) 3a) exhibited intense peaks peaks in the whole spectrum of spectrum 2θ value ranging The XRD AgNPs (Figure exhibited intense in the whole of 2θ value from 10 to 70. Diffraction peaks at 2θ values of 38.06◦ , 44.11◦ , 64.43◦ and 77.33◦ were corresponded ranging from 10 to 70. Diffraction peaks at 2θ values of 38.06°, 44.11°, 64.43° and 77.33° were to the (111), (200), (220) and (311) crystal faces of face-centered silver, respectively, which was in corresponded to the (200), (220) and (311) faces of face‐centered silver, respectively, accordance with(111), the values in standard silver card crystal (JCPDS No. 04-0783). Figure 3b showed the FTIR which was in of accordance with the synthesized values in AgNPs standard silver card washed (JCPDS 04‐0783). Figure 3b spectra aqueous callus extracts, as well as AgNPs withNo. different solution. − 1 FTIR spectra of AgNPs showed the main peaks at 1625 and 1518 cm , which could be assigned to showed the FTIR spectra of aqueous callus extracts, synthesized AgNPs as well as AgNPs washed frame vibration of aromatic ring. Peaks at 2924 cm−1 corresponded to asymmetric stretching of C–H−1, which with different solution. FTIR spectra of AgNPs showed the main peaks at 1625 and 1518 cm bonds. The bands at 2162 and 2033 cm−1 were assigned to CO adsorbed and the band round 1979 cm−1 could be assigned to frame vibration of aromatic ring. Peaks at 2924 cm−1 corresponded to asymmetric was due to the overtone of C–H out of plane bending. Peaks at 1541, 1456 and 1400 cm−1 corresponded −1 were assigned to CO adsorbed and the stretching of C–H bonds. The bands at 2162 and 2033 cm to amide II, methylene scissoring vibration and methyl group of proteins, respectively. Another band band round 1979 cm at 800–600 cm−−11 was due to the overtone of C–H out of plane bending. Peaks at 1541, 1456 and could be attributed to C-H out of plane bend of aromatic phenol. Besides, the FTIR −1 corresponded to amide II, methylene scissoring vibration and methyl group of proteins, of synthesized AgNPs was similar with that of water-washed and medium-washed AgNPs, 1400 cmspectrum indicating most groups attached to AgNPs could not be removed by washing with water or medium. −1 could be attributed to C‐H out of plane bend of aromatic respectively. Another band at 800–600 cm Thus, groups around AgNPs were stable and would not release into the medium in both antibacterial phenol. Besides, the FTIR spectrum of synthesized AgNPs was similar with that of water‐washed and cytotoxicity experiments. Furthermore, no band was observed when AgNPs was washed with and medium‐washed AgNPs, indicating most groups attached to AgNPs could not be removed by alcohol. This result indicated most groups around AgNPs could be removed by alcohol. The surface washing with water or medium. Thus, groups around AgNPs were stable and would not release into zeta potentials of water-washed AgNPs, two-week-old AgNPs and alcohol-washed AgNPs were −28.8, −28.3 and −6.64 mV, respectively (Figure 3c). That is, our AgNPs was stable in the water even after the medium in both antibacterial and cytotoxicity experiments. Furthermore, no band was observed two weeks but the stability decreased after washed by alcohol. Besides, the visible precipitates could be when AgNPs was washed with alcohol. This result indicated most groups around AgNPs could be seen and after alcohol washing the dry powder of AgNPs could not be dispersed in water (Figure S1), removed by alcohol. The surface zeta potentials of water‐washed AgNPs, two‐week‐old AgNPs and indicating the colloidal stability of AgNPs was broken. alcohol‐washed AgNPs were −28.8, −28.3 and −6.64 mV, respectively (Figure 3c). That is, our AgNPs The content of Ag+ in nutrient broth (NB) and RPMI 1640 medium was detected in 24 h (Figure 4). + slowly, was stable in the showed water even after could two weeks but the stability decreased after washed by alcohol. The results that AgNPs release Ag which reached the highest content of 2.12 and 0.79 ppm in NB and RPMI 1640 medium, respectively. Besides, the visible precipitates could be seen and after alcohol washing the dry powder of AgNPs could not be dispersed in water (Figure S1), indicating the colloidal stability of AgNPs was broken. The content of Ag+ in nutrient broth (NB) and RPMI 1640 medium was detected in 24 h (Figure 4). The results showed that AgNPs could release Ag+ slowly, which reached the highest content of 2.12 and 0.79 ppm in NB and RPMI 1640 medium, respectively.
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Figure 3. (a) X‐ray diffraction (XRD) pattern; (b) Fourier transform infrared spectroscopy (FTIR) spectra Figure 3. (a) X-ray diffraction (XRD) pattern; (b) Fourier transform infrared spectroscopy (FTIR) spectra (1, callus extract; 2, NB medium‐washed AgNPs; 3, RPMI 1640 medium‐washed AgNPs; 4, synthesized (1, callus extract; 2, NB medium-washed AgNPs; 3, RPMI 1640 medium-washed AgNPs; 4, synthesized AgNPs; 5, water‐washed AgNPs; 6, alcohol‐washed AgNPs); and (c) zeta potential of AgNPs. AgNPs; 5, water-washed AgNPs; 6, alcohol-washed AgNPs); and (c) zeta potential of AgNPs.
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The AgNPs had concentration‐dependent inhibitory effect on all testing human pathogenic 2.3. Antibacterial Activity of AgNPs bacteria (Figure 5). The growth of Staphylococcus aureus, Salmonella paratyphi B and Bacillus subtilis was 2.3. Antibacterial Activity of AgNPs The AgNPs AgNPs had concentration-dependent concentration‐dependent inhibitory effect on all all testing testing human pathogenic pathogenic inhibited by AgNPs even at 2 μg/mL. The inhibition of AgNPs against Gram‐positive bacteria (S. The had inhibitory effect on human bacteria (Figure 5). The growth of Staphylococcus aureus, Salmonella paratyphi B and Bacillus subtilis was aureus and B. subtilis) was that of aureus, Gram‐negative bacteria (Escherichia coli and S. bacteria (Figure 5). Thestronger growth of than Staphylococcus Salmonella paratyphi B and Bacillus subtilis inhibited by AgNPs even at 2 μg/mL. The inhibition of AgNPs against Gram‐positive bacteria (S. was inhibited3 control at 1699 μg/mL (10 mM) showed no antibacterial activity against E.coli by AgNPs even at 2 µg/mL. The inhibition of AgNPs against Gram-positive bacteria paratyphi B). AgNO aureus and B. subtilis) was stronger than that of Gram‐negative bacteria (Escherichia coli and S. (S. aureus and B. subtilis) was stronger than that of Gram-negative bacteria (Escherichia coli and and lower activity against other three bacteria. The control gentamycin sulfate at 1 mM had greater paratyphi B). AgNO 3 control at 1699 μg/mL (10 mM) showed no antibacterial activity against E.coli S. paratyphi B). AgNO 3 control at 1699 µg/mL (10 mM) showed no antibacterial activity against E. coli inhibitory effect on gram‐negative bacteria E. coli and S. paratyphi B but weaker effect on gram‐ and lower activity against other three bacteria. The control gentamycin sulfate at 1 mM had greater and lower activity against other three bacteria. The control gentamycin sulfate at 1 mM had greater inhibitory effect effect on gram‐negative bacteria E. coli S. callus paratyphi B but weaker effect on gram‐ positive bacteria S. aureus and B. subtilis. The control of extracts at different concentrations inhibitory on gram-negative bacteria E. coli and and S. paratyphi B but weaker effect on gram-positive positive bacteria S. aureus and B. subtilis. The control of callus extracts (Figure at different (0.1%–10%) had no inhibition effects against all tested bacteria 6). concentrations We further tested bacteria S. aureus and B. subtilis. The control of callus extracts at different concentrations (0.1%–10%) (0.1%–10%) had no inhibition effects against all tested bacteria (Figure 6). We further tested had no inhibition effects against all tested bacteria (Figure 6). We further tested antibacterial activity of antibacterial activity of AgNPs by the minimum inhibitory concentration (MIC). As shown in Figure antibacterial activity of AgNPs by the minimum inhibitory concentration (MIC). As shown in Figure AgNPs by the minimum inhibitory concentration (MIC). As shown in Figure 7, antibacterial activity 7, antibacterial activity increased with the increasing concentration of AgNPs. MIC of AgNPs was 8, 7, antibacterial activity increased with the increasing concentration of AgNPs. MIC of AgNPs was 8, increased with the increasing concentration of AgNPs. MIC of AgNPs was 8, 1, 1 and 4 µg/mL against 1, 1 and 4 μg/mL against E. coli, S. aureus, B. subtilis and S. paratyphi B, respectively. 1, 1 and 4 μg/mL against E. coli, S. aureus, B. subtilis and S. paratyphi B, respectively. E. coli, S. aureus, B. subtilis and S. paratyphi B, respectively.
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Figure 5. Activity of AgNPs against human pathogen: (a) E. coli; (b) S. aureus; (c) S. paratyphi B; and Figure 5. Activity of AgNPs against human pathogen: (a) E. coli; (b) S. aureus; (c) S. paratyphi B; and (d) B. subtilis, depicting zones of inhibition of: 128 µg/mL (1); 32 µg/mL (2); 8 µg/mL (3); (d) B. subtilis, depicting zones of inhibition of: 128 μg/mL (1); 32 μg/mL (2); 8 μg/mL (3); 2 μg/mL (4); 2 µg/mL (4);3 control (5); and 1 mM gentamycin sulfate (6). 1699 µg/mL AgNO3 control (5); and 1 mM gentamycin sulfate (6). 1699 μg/mL AgNO
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(b); B. subtilis (c); and S. paratyphi B (d). (b); B. subtilis (c); and S. paratyphi B (d).
2.4. Cytotoxicity Experiments 2.4. Cytotoxicity Experiments shown Figure 8a, 10% (v/v) callus callus extracts extracts had had no no obvious cytotoxicity 3 3 As As shown in in Figure 8a, 10% (v/v) cytotoxicity while while AgNO AgNO control at 0.79 ppm had cytotoxic effect on on human human colon colon adenocarcinoma adenocarcinoma cells control at 0.79 ppm had no no cytotoxic effect cells (LS174T), (LS174T), lung lung adenocarcinoma cells (A549) and breast cancer cells (MCF‐7) but weak cytotoxic effect on human adenocarcinoma cells (A549) and breast cancer cells (MCF‐7) but weak cytotoxic effect on human hepatoma cells (SMMC‐7721) (87.8% cell cell viability). viability). AgNPs AgNPs could could reduce hepatoma cells (SMMC‐7721) (87.8% reduce all all tested tested cancer cancer cells cells viability in a dose dependent manner. SMMC‐7721 cells were the most sensitive tumors cells with viability in a dose dependent manner. SMMC‐7721 cells were the most sensitive tumors cells with the IC 50 of 27.75 μg/mL thus we chose SMMC‐7721 for further research. The cellular morphology of the IC 50 of 27.75 μg/mL thus we chose SMMC‐7721 for further research. The cellular morphology of SMMC‐7721 cells were observed and photographed under microscopy (Figure 8c). The shape of cells SMMC‐7721 cells were observed and photographed under microscopy (Figure 8c). The shape of cells was altered obviously when 30 μg/mL AgNPs was applied and a large number of dead cells were was altered obviously when 30 μg/mL AgNPs was applied and a large number of dead cells were presented at AgNPs concentration of 40 and 50 μg/mL, which was in accord with the results of 3‐(4,5‐ presented at AgNPs concentration of 40 and 50 μg/mL, which was in accord with the results of 3‐(4,5‐ dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay (Figure 7a). Furthermore, dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay (Figure 7a). Furthermore, apoptosis of SMMC‐7721 cells was measured by flow cytometry (Figure 9). Apoptosis rate was apoptosis of SMMC‐7721 cells was measured by flow cytometry (Figure 9). Apoptosis rate was
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2.4. Cytotoxicity Experiments As shown in Figure 8a, 10% (v/v) callus extracts had no obvious cytotoxicity while AgNO3 control at 0.79 ppm had no cytotoxic effect on human colon adenocarcinoma cells (LS174T), lung adenocarcinoma cells (A549) and breast cancer cells (MCF-7) but weak cytotoxic effect on human hepatoma cells (SMMC-7721) (87.8% cell viability). AgNPs could reduce all tested cancer cells viability in a dose dependent manner. SMMC-7721 cells were the most sensitive tumors cells with the IC50 of 27.75 µg/mL thus we chose SMMC-7721 for further research. The cellular morphology of SMMC-7721 cells were observed and photographed under microscopy (Figure 8c). The shape of cells was altered obviously when 30 µg/mL AgNPs was applied and a large number of dead cells were presented at AgNPs concentration of 40 and 50 µg/mL, which was in accord with the results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Figure 7a). Furthermore, Nanomaterials 2016, 6, 160 7 of 15 apoptosis of SMMC-7721 cells was measured by flow cytometry (Figure 9). Apoptosis rate was increased as the concentration of AgNPs increased. On the other hand, we investigated the cytotoxicity increased as the normal concentration of AgNPs increased. On the other hand, we investigated the of AgNPs against human liver cells HL-7702. The results indicated AgNPs inhibited the growth cytotoxicity AgNPs against normal liver cells HL‐7702. The results of HL-7702 atof higher concentration (Figurehuman 8b). IC50 value of AgNPs on HL-7702 cellsindicated was 81.39AgNPs µg/mL. inhibited the growth of HL‐7702 at higher concentration (Figure 8b). IC 50 value of AgNPs on HL‐7702 The difference of induced cell viability between cancer cells (SMMC-7721) and normal HL-7702 cells cells was 81.39 μg/mL. The difference of induced cell viability between cancer cells (SMMC‐7721) and was significant (p < 0.01). normal HL‐7702 cells was significant (p
