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; (Q.H.X.); (Y.J.M.)
* Correspondence: or; 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

Nanomaterials 2016, 6, 160; doi:10.3390/nano6090160

10]. Fu (Taxaceae). callus and  commercial value of paclitaxel and the scarcity of T.    cytotoxic activity against human epidermoid larynx carcinoma cells [12]. yunnanensis focused on enhancing paclitaxel production by means  on cell culture of T.5 cm 0. Results  Results 2. However. 409 and 450 nm. 8.16].9.  Taxus yunnanensis Cheng et L. 411. as acallus.1. and color intensity of the extracts incubated with solution of  silver ions at the beginning (b). Due to the Due  enormous enormous commercial value of paclitaxel and the scarcity of T.  follow-up to ouras  efforts on exploring AgNPs exploring AgNPs for controlling microbes [17] and biotechnological application of T. yunnanensis focused on enhancing paclitaxel production by means of elicitation. an evergreen tree endemic to Yunnan of China. The antibacterial and cytotoxic activity of the biosynthesized AgNPs was also investigated. There was a blue shift of absorbance from absorbance at 439.8. The Synthesis of AgNPs 2. 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). AgNPs synthesized by calli of Citrullus colocynthis were  microorganisms [9.  and  after  120  min  of  reaction  (d).16].  a  follow‐up  to  our  efforts for on  AgNP using Taxus tree or callus.) . ultrasound treatment and medium renewal [15. Fu (Taxaceae). indicating the formation of smaller size nanoparticles [19]. yunnanensis [16]. 2. yunnanensis trees in China.2. Therefore we chose pH 10 as the pH 7 to 10.u .1. little has been reported  ultrasound treatment and medium renewal [15. The AgNPs [10].5 cm 0. yunnanensis (a). 411. of elicitation.) (c) 350 pH = 10.K. Callus of T. AgNPs synthesized by calli of Citrullus colocynthis were reported to have reported to have cytotoxic activity against human epidermoid larynx carcinoma cells [12]. (e) 5 (b) (a) 3 2 1 10 min 0 0.7. There was a blue shift of absorbance from  pH 7 to 10. yunnanensis trees in China.10]. and after 120 min of reaction (d).0 pH = 8. The Synthesis of AgNPs  Color change of callus extracts incubated with 1 mM AgNO 3 was observed. 160    2 of 15 2 of 15  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]. absorbance of AgNPs synthesized with the extracts under different reaction times (e) and different  pH (f). an evergreen tree endemic to Yunnan of China. Taxus yunnanensis Cheng et L.u.  The antibacterial and cytotoxic activity of the biosynthesized AgNPs was also investigated.production  Many studies Many studies on cell culture of T.0 2 pH = 7. after 10 min (c). indicating the formation of smaller size nanoparticles [19]. AgNPs AgNPs with with pH pH of of 7.  to  the  promising sourcesource  of potent anticancer paclitaxel and taxane-type diterpenesditerpenes  [13].0 pH = 9. 6. respectively. Therefore we chose pH 10 as  optimal condition. 409 and 450 nm. Callus of T.5 cm 60 min 90 min 120 min 30 min 20 min 4 300 400 450 500 550 600 500 550 600 Wavelength (nm) (d) (f) 4 0. Therefore. yunnanensis. as shown in Color change of callus extracts incubated with 1 mM AgNO 3 was observed. UV-visible absorbance silver  ions  at  the  beginning  (b).  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.  1a–d. T. and color intensity of the extracts incubated with solution of Figure 1. 9. the optimal condition. 160 Nanomaterials 2016. is a is  a  promising  of  potent  anticancer  paclitaxel  and  taxane‐type  [13]. callus and cell cultures cell  cultures  have  been  as  a  practical  alternative  source production for  paclitaxel  [14].  A bsorb an ce (a. However.  controlling microbes [17] and biotechnological application of T.    . 1e).K. 6. 425.5 cm Ab so rb an ce (a. yunnanensis. yunnanensis (a).0 3 1 0 300 350 400 450 Wavelength (nm)   Figure 1. as shown in Figure  Figure The reaction solution showed a UV-visible absorbance 449nm  nmbecause  because of  of the 1a–d.  have been established as established  a practical alternative source for paclitaxel [14].0 pH = 11. 10 10 and and 11 11 showed showed maximum maximum  absorbance at 439. respectively. yunnanensis [16]. little has been reported on biosynthesized on  biosynthesized  AgNP  using  Taxus  tree  or  Therefore. 425.  UV‐visible  of AgNPs synthesized with the extracts under different reaction times (e) and different pH (f).  after  10  min  (c).Nanomaterials 2016. 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.

8. 6.   Figure 2.8. 1400 cmspectrum indicating most groups attached to AgNPs could not be removed by washing with water or medium. 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). Besides.3 and −6.  different solution. − 1 FTIR spectra of AgNPs showed the main peaks at 1625 and 1518 cm . 1456 and  could be attributed to C-H out of plane bend of aromatic phenol. 1456 and 1400 cm−1 corresponded −1 were assigned to CO adsorbed and the  stretching of C–H bonds.  Figure  3b  spectra aqueous callus extracts.64 mV.06°. indicating most groups attached to AgNPs could not be removed by  alcohol.  X‐ray  diffraction  (XRD) and Fourier  transform infrared  spectroscopy  (FTIR) (Figure 3). methylene scissoring vibration and methyl group of proteins. the FTIR spectrum of synthesized AgNPs was similar with that of water‐washed  and cytotoxicity experiments.  ranged from 6. two-week-old AgNPs and alcohol-washed AgNPs were −28. −28.  which  reached  the  highest  content of 2. respectively (Figure 3c). respectively (Figure 3c).  The  results  showed  that  AgNPs  could  release  Ag+  slowly. no band was observed  two weeks but the stability decreased after washed by alcohol. −28. the FTIR −1 corresponded to amide II. 04-0783). the visible precipitates could be seen and after alcohol washing the dry powder of AgNPs  could not be dispersed in water (Figure S1). Thus. was  stable in  the showed water  even  after could two weeks  but  the  stability  decreased after washed  by  alcohol.12 and 0. Transmission electron microscope (TEM) analysis of AgNPs synthesized with callus extracts Figure 2. 6.  accordance with(111).  . respectively. −1 could be attributed to C‐H out of plane bend of aromatic  respectively. That is. groups around AgNPs were stable and would not release into the medium in both antibacterial phenol.  (Figure 2). This result indicated most groups around AgNPs could be removed by alcohol. Peaks at 2924 cm−1 corresponded to asymmetric stretching of C–H−1.4 to 27.2 nm measured by Image J software. 160 3 of 15 3 of 15  2.79 ppm in NB and RPMI 1640 medium. The particle size of the synthesized AgNPs shown by TEM ranged from 6. 160    Nanomaterials 2016. 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). Transmission electron microscope (TEM) analysis of AgNPs synthesized with callus extracts  from T. indicating the colloidal stability of AgNPs was broken. which was in corresponded  to  the  (200).33°  were  to the (111). Besides. methylene scissoring vibration and methyl group of proteins. Peaks at 2924 cm−1 corresponded to asymmetric  was due to the overtone of C–H out of plane bending. Besides.  The  content  of  Ag+  in  nutrient  broth  (NB)  and  RPMI  1640  medium  was  detected  in  24  h    (Figure  4). FTIR spectra of AgNPs showed the main peaks at 1625 and 1518 cm bonds. 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. which could be assigned to showed the FTIR spectra of aqueous callus extracts.  (220)  and  (311)  faces  of  face‐centered  silver. The particle size of the synthesized AgNPs shown by TEM  The nanoparticles were in spherical shape.2 nm measured by Image J software. Another band at 800–600 cm Thus. the visible precipitates could be when AgNPs was washed with alcohol. groups around AgNPs were stable and would not release into  zeta potentials of water-washed AgNPs. The nanoparticles were in spherical shape.2. (220) and (311) crystal faces of face-centered silver.06◦ . The bands at 2162 and 2033 cm to amide II.Nanomaterials 2016. respectively. That is. two‐week‐old AgNPs and  indicating the colloidal stability of AgNPs was broken. our AgNPs was stable in the water even after the medium in both antibacterial and cytotoxicity experiments. 64. (200). no band was observed when AgNPs was washed with and medium‐washed AgNPs. which  with different solution. as well as AgNPs withNo. + slowly. Peaks at 1541. Besides.  The results that AgNPs release Ag which reached the highest content of 2. The surface zeta potentials of water‐washed AgNPs. X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) (Figure 3). Diffraction peaks at 2θ values of 38.  of synthesized AgNPs was similar with that of water-washed and medium-washed AgNPs.  the values in standard silver card crystal  (JCPDS No. respectively. Another band band round 1979 cm at 800–600 cm−−11  was due to the overtone of C–H out of plane bending.  Diffraction  peaks  at  2θ  values  of  38.43°  and  77. respectively. Furthermore.  respectively. The surface washing with water or medium.64 mV.11°.2. Peaks at 1541.33◦ were corresponded ranging  from  10  to  70.3 and −6.79 ppm in NB and RPMI 1640 medium. 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.11◦ .43◦ and 77. alcohol‐washed AgNPs were −28. our AgNPs  The content of Ag+ in nutrient broth (NB) and RPMI 1640 medium was detected in 24 h (Figure 4).  44. Furthermore. yunnanensis and AgNO from T.12 and 0. Figure 3b showed the FTIR which  was  in of accordance  with  the synthesized values  in AgNPs standard  silver  card washed (JCPDS  04‐0783). synthesized AgNPs as well as AgNPs washed  frame vibration of aromatic ring.  64.  3 .4 to 27. Characterization of AgNPs  2. removed by alcohol. yunnanensis and AgNO 3. 44.

and (c) zeta potential of AgNPs.  AgNPs. 160 4 of 15 Nanomaterials 2016. water‐washed AgNPs. 2.Nanomaterials 2016. synthesized AgNPs. NB medium‐washed AgNPs. 6. NB medium-washed AgNPs. 6. 2. callus extract. water-washed AgNPs. (b) Fourier transform infrared spectroscopy (FTIR) spectra (1. (a) X‐ray diffraction (XRD) pattern. RPMI 1640 medium‐washed AgNPs. alcohol-washed AgNPs). 4. 3. and (c) zeta potential of AgNPs. 6. synthesized  (1. 160    4 of 15    Figure 3. 4. (a) X-ray diffraction (XRD) pattern. 3. RPMI 1640 medium-washed AgNPs. alcohol‐washed AgNPs). 6. . callus extract. 5. 5. (b) Fourier transform infrared spectroscopy (FTIR) spectra  Figure 3.

  was inhibited3 control at 1699 μg/mL (10 mM) showed no antibacterial activity against E. 160 Nanomaterials 2016. aureus. and 1 mM gentamycin sulfate (6). The release of Ag Figure 4. 8 µg/mL (3).  The  control  of  callus  extracts (Figure  at  different  (0. The growth of Staphylococcus aureus. The control gentamycin sulfate at 1 mM had greater inhibitory effect effect  on  gram‐negative  bacteria  E. Salmonella paratyphi B and Bacillus subtilis was  2. (c) S.      Figure 5.0 0 1. 1. ++ The  AgNPs  had  concentration‐dependent  inhibitory  effect  on  all  testing  human  pathogenic  2. paratyphi B but weaker effect on gram-positive positive  bacteria  S.1%–10%)  had  no  inhibition  effects  against  all  tested  bacteria  6). We further tested antibacterial activity of antibacterial activity of AgNPs by the minimum inhibitory concentration (MIC). subtilis) was stronger than that of Gram-negative bacteria (Escherichia coli and and lower activity against other three bacteria.coli  by AgNPs even at 2 µg/mL.  E. coli inhibitory  effect  on  gram‐negative  bacteria  E. subtilis. Activity of AgNPs against human pathogen: (a) E. coli.5 0. B. aureus. (d) B. B.0 0. callus  paratyphi  B  but  weaker  effect  on  gram‐ positive  bacteria  S.3.1%–10%) (0. 160  1. AgNO aureus  and  B. 32 μg/mL (2).3.  The had inhibitory effect on human bacteria (Figure 5). S.  (S. and (d) B.  1699 μg/mL AgNO3 control (5). subtilis. (a) (a) 1 (b) (b) 1 2 4 4 2 3 3 5 5 (c) 6 4 4 5 4 2 3 3 6 6 5 (d) 2 1 1 2 1 6 (c) 1 4 2 5 5 3 3 6 6 (d) 1 2 41 52 4 3 5 6 3 6   Figure 5. AgNO 3 control at 1699 µg/mL (10 mM) showed no antibacterial activity against E. subtilis and S. MIC of AgNPs was 8. coli. (b) S. B. 6. respectively. paratyphi B.coli  S. paratyphi B. depicting zones of inhibition of: 128 μg/mL (1). The growth of Staphylococcus aureus. aureus.5 18 24 Time (h)   0. 1 and 4 μg/mL against E. aureus and B. 32 μg/mL (2). coli. 1 and 4 μg/mL against E.  paratyphi  B  but  weaker  effect  on  gram‐ and lower activity against other three bacteria. MIC of AgNPs was 8. and  (d) B. subtilis.  bacteria (Figure 5). paratyphi B. The release of Ag+ from AgNPs in NB and RPMI 1640 medium. Antibacterial Activity of AgNPs  bacteria (Figure 5). MIC of AgNPs was 8.5   5 of 15 5 of 15  2.  1.  aureus  and  B.5 Ag+ concentration (ppm) 1. antibacterial activity 7. paratyphi B.0 Nanomaterials 2016. Activity of AgNPs against human pathogen: (a) E. The control gentamycin sulfate at 1 mM had greater  paratyphi B). (c) S.0 NB RPMI 1640 2. 6. and 1 mM gentamycin sulfate (6). The control of callus extracts at different concentrations (0. 8 μg/mL (3).1%–10%)  had  no  inhibition  effects  against  all  tested  bacteria  (Figure  6). (b) S. depicting zones of inhibition of: 128 μg/mL (1). 32 µg/mL (2). Antibacterial Activity of AgNPs    Figure 4.0 6 12 0. 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.  coli  and  S. and  Figure 5. Gram‐negative  bacteria  (Escherichia  coli  and  S. and 1 mM gentamycin sulfate (6).  coli  S. coli.  2 µg/mL (4).  0 6 12 18 24 Time (h) 2. 1 and 4 µg/mL against 1. coli. The control gentamycin sulfate at 1 mM had greater  and lower activity against other three bacteria. Activity of AgNPs against human pathogen: (a) E. antibacterial activity increased with the increasing concentration of AgNPs. S. The  inhibition  of AgNPs against  Gram‐positive  bacteria  (S.5 NB RPMI 1640 2. respectively.3 control (5).  subtilis)  was  that  of aureus.  subtilis. aureus and B.  7.  subtilis. Thestronger  growth of than  Staphylococcus Salmonella paratyphi B and Bacillus subtilis inhibited  by AgNPs  even at  2  μg/mL. respectively. 8 μg/mL (3). AgNO 3 control at 1699 μg/mL (10 mM) showed no antibacterial activity against E. As shown in Figure 7.5 1. S. Salmonella paratyphi B and Bacillus subtilis was  aureus  and  B. As shown in Figure  antibacterial activity of AgNPs by the minimum inhibitory concentration (MIC). antibacterial activity increased with the increasing concentration of AgNPs. aureus. depicting zones of inhibition of: 128 µg/mL (1).  subtilis)  was  stronger  than  that  of  Gram‐negative  bacteria  (Escherichia  coli  and  S.3. The  inhibition  of AgNPs against  Gram‐positive  bacteria  (S. subtilis. paratyphi B). aureus. 2 μg/mL (4). The inhibition of AgNPs against Gram-positive bacteria paratyphi B).  1699 µg/mL AgNO3 control (5). concentrations  We  further  tested  bacteria S.  We  further  tested  had no inhibition effects against all tested bacteria (Figure 6).  The  control  of  extracts  at  different  concentrations  inhibitory on gram-negative bacteria E. 2 μg/mL (4). coli.  increased with the increasing concentration of AgNPs. subtilis and S.  aureus  and  B. paratyphi B. coli and and  S.  from AgNPs in NB and RPMI 1640 medium. subtilis and S. paratyphi B.Ag+ concentration (ppm) 2. (b) S. 1699 μg/mL AgNO   . (c) S. As shown in Figure  AgNPs by the minimum inhibitory concentration (MIC). aureus.0 Figure 4. The release of Ag  from AgNPs in NB and RPMI 1640 medium.

5 32 64 128 32 64 128 32 32 64 64 128 128 (d)  (d)  120 0 16 AgNPs (g/mL) (c)  (c)  30 8 60 60 30 30 0 0. aureus. subtilis (c). The cellular morphology of  SMMC‐7721 cells were observed and photographed under microscopy (Figure 8c). 500 µg/mL (2). (c) S.5‐diphenyltetrazolium  bromide  (MTT)  assay  (Figure  7a). 1% (5).  (b). (b)  Figure 6. and callus extracts at: 10% (4). SMMC‐7721 cells were the most sensitive tumors cells with  viability in a dose dependent manner.1% Callus extracts 1% 0. coli. subtilis (c).1% 10% Callus extracts AgNO3 (g/mL)   Figure 6. Cytotoxicity Experiments  shown  Figure  8a. The cellular morphology of  the IC 50 of 27. aureus. 1% (5).  Apoptosis  rate  was  apoptosis  of  SMMC‐7721  cells  was  measured  by  flow  cytometry  (Figure  9).5 0.5‐diphenyltetrazolium  bromide  (MTT)  assay  (Figure  7a). paratyphi B.  viability). Activity (a–d) and growth inhibition (e) of: AgNO 3 at 1699 μg/mL (1). coli (a).Nanomaterials 2016. aureus (b).79  ppm  had  cytotoxic  effect on  on human  human colon  colon adenocarcinoma  adenocarcinoma  cells  control  at  0. and (d) B. 6. subtilis.  dimethylthiazol‐2‐yl)‐2. 500 μg/mL (2). and S.5‐ presented at AgNPs concentration of 40 and 50 μg/mL. 500 μg/mL (2). coli (a). paratyphi B (d). The shape of cells  SMMC‐7721 cells were observed and photographed under microscopy (Figure 8c). 6. aureus  Figure 7. against human pathogen: (a) E.  μg/mL (3). coli. (c) S.4.75 μg/mL thus we chose SMMC‐7721 for further research.5 0. aureus  B.5 1 1 2 2 4 4 8 16 8 16 AgNPs ( g/mL) AgNPs (g/mL)    Figure 7. subtilis. 160    6 of 15  Nanomaterials 2016. (c) S. (b) S.5 1 2 4 1 2 4 AgNPs8(g/mL)16 AgNPs (g/mL) 120 120 120 90 90 90 Growth inhibition (%) Growth inhibition (%) Growth inhibition (%) Growth inhibition (%) 90 60 60 30 0 0. and 0. SMMC‐7721 cells were the most sensitive tumors cells with  the IC 50 of 27.  (a)  (a)  (b)  (b)  120 120 Growth inhibition (%) Growth inhibition (%) Growth inhibition (%) Growth inhibition (%) 120 90 90 60 60 30 30 0 120 90 90 60 60 30 30 0 0.  10%  (v/v) callus  callus extracts  extracts had  had no  no  obvious  cytotoxicity  3  3  As As  shown  in in  Figure  8a.   and 2 µg/mL (3). and callus extracts at: 10% (4).  10%  (v/v)  cytotoxicity while  while AgNO AgNO control  at  0.  Furthermore.4. subtilis (c).  apoptosis  of  SMMC‐7721  cells  was  measured  by  flow  cytometry  (Figure  9). coli. 6.8%  reduce  all  all tested  tested cancer  cancer cells  cells  viability in a dose dependent manner. and callus extracts at: 10% (4).79  ppm  had  no no  cytotoxic  effect  cells (LS174T). S. B.1% (6).  Apoptosis  rate  was  .5‐ dimethylthiazol‐2‐yl)‐2. paratyphi B. which was in accord with the results of 3‐(4. and 0. 1% (5). paratyphi B. (b)  S. coli (a). subtilis. Activity (a–d) and growth inhibition (e) of: AgNO Figure 6. and (d) B. and 2  3 at 1699 µg/mL (1). B. against human pathogen: (a) E. paratyphi B (d).1% (6). AgNPs  AgNPs  could  could  reduce  hepatoma  cells  (SMMC‐7721)  (87. and 0. Determination of the minimum inhibitory concentration (MIC) against: E. and 2  (a) E. 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. against human pathogen: μg/mL (3). Cytotoxicity Experiments  2. S. S. (b).8% cell  cell viability). and S. aureus. and S. paratyphi B (d).1% (6).  (LS174T). 160  (b) (a) 2 4 (a) 6 5 1 2 4 2 3 4(b) 5 6 1 2 3 5 6 1 3 3 6 5 4 1 2 (c) 4 5 3 6 1 2 4 5 1 2 3 (d) 4 5 6 3 6 Staphylococcus aureus 12 (e) (d) (c) 1 2 3 4 5 6 6 of6 of 15  15 16 Growth inhibition (%) Growth inhibition (%) 1 (e) 8 Bacillus subtilis 16 Salmonella paratyphi B Staphylococcus aureus Escherichia coli Bacillus subtilis 12 Salmonella paratyphi B 4 8 Escherichia coli 0 4 1699 500 -4 0 2 AgNO3 (g/mL) 500 1699 -4 10% 2 1% 0. Figure 7. Activity (a–d) and growth inhibition (e) of: AgNO3 at 1699 μg/mL (1). which was in accord with the results of 3‐(4. Determination of the minimum inhibitory concentration (MIC) against: E. 160  Nanomaterials 2016. and (d) B. 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.5 1 1 2 2 4 8 16 4 8 16 AgNPs (g/mL) AgNPs (g/mL) 32 32 64 64 128 128 0 0.  2. S.5 2 1 4 8 16 AgNPs (g/mL) 4 8 2 32 16 64 32 128 64 0 128 0.  Furthermore. Determination of the minimum inhibitory concentration (MIC) against: E.75 μg/mL thus we chose SMMC‐7721 for further research.5 0 1 0.

79 ppm had no cytotoxic effect on human colon adenocarcinoma cells (LS174T).39 μg/mL. 50.5-diphenyltetrazolium bromide (MTT) assay (Figure 7a). AgNPs could reduce all tested cancer cells viability in a dose dependent manner. IC50 value of AgNPs on HL-7702 cellsindicated  was 81. which was in accord with the results of 3-(4.  (c) Cells were treated without (1). MCF-7 and SMMC-7721 cells were incubated with 0. 10% (v/v) callus extracts as well as 0. 75. * p < 0. 40 (5).Nanomaterials 2016. * p < 0. 30. HL‐7702. Data presented  presentedare  are the  the means  means± ±SD  SDof  ofresults  resultsfrom  fromthree  threeindependent  independentexperiments. Nanomaterials 2016. 160 7 of 15 2. A549. 40 (5). we investigated the cytotoxicity increased  as  the normal concentration  of  AgNPs  increased. and 50 (6) µg/mL AgNPs. 10.8% cell viability). and with 10 (2). 25. 20 (3).  On  the  other  hand.  (c) Cells were treated without (1).5-dimethylthiazol-2-yl)-2.  we  investigated  the  of AgNPs against human liver cells HL-7702. 40 and 50 μg/mL AgNPs.01  HL-7702 and SMMC-7721 cells were incubated with 0.01 vs.  Cell viability (%) (a) (b) 120 LS174T * 90 * * 60 A549 * ** * MCF-7 7721 * 30 ** * ** 0 CK Extracts AgNO3 10 20 30 40 1 4 2 5 3 6 50 AgNPs (μg/mL) (c) 100 Cell viability (%) 7702 7721 75 * 50 25 * * * * 0 25 50 75 100 125 AgNPs (μg/mL) Figure 8. and with 10 (2). 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). 10. 75.  SMMC-7721 cells (b). Furthermore.4. 8. 25.  . 30 (4).  Dose-dependent Dose‐dependent  cytotoxic  Figure cytotoxic activity  activityof  ofAgNPs  AgNPs(a). (a) LS174T. Apoptosis rate was increased as the concentration of AgNPs increased. On the other hand.  Data  vs.39AgNPs  µg/mL. 6. inhibited the growth of HL‐7702 at higher concentration (Figure 8b). 20 (3).  experiments. 100 and 125 µg/mL AgNPs. MCF‐7 and SMMC‐7721 cells were incubated with 0.  HL-7702. 10% (v/v) callus extracts had no obvious cytotoxicity while AgNO3 control at 0.79 ppm AgNO 40 and 50 µg/mL AgNPs.01). 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. 100 and 125 μg/mL AgNPs. 20. The difference of induced cell viability between cancer cells (SMMC‐7721) and  was significant (p < 0. control.and  andthe  theinduced  inducedmorphology  morphologyinjury  injuryof of SMMC‐7721 cells (b). A549.01 vs. 10% (v/v) callus extracts as well as 0.01). control. SMMC-7721 cells were the most sensitive tumors cells with the IC50 of 27. * p < 0. * p < 0. (a) LS174T.75 µg/mL thus we chose SMMC-7721 for further research. 50. 160    7 of 15  apoptosis of SMMC-7721 cells was measured by flow cytometry (Figure 9). 20. 6. 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. HL‐7702 and SMMC‐7721 cells were incubated with 0. and 50 (6) μg/mL AgNPs.01 vs. 30.79 ppm AgNO33. 30 (4). normal HL‐7702 cells was significant (p < 0. The cellular morphology of SMMC-7721 cells were observed and photographed under microscopy (Figure 8c).  (a). Cytotoxicity Experiments As shown in Figure 8a. lung adenocarcinoma cells (A549) and breast cancer cells (MCF-7) but weak cytotoxic effect on human hepatoma cells (SMMC-7721) (87.    .

 extracts of  compounds such as taxane diterpenoids and polysaccharides [20–22]. Discussion  Discussion Extracts  yunnanensis  have  been  used  extensively  produce  various  active  anticancer  Extracts ofof  T. metastatic ovary carcinoma.that  yunnanensis extracts contain metabolites metabolites  including including  taxane  diterpenoids. we  agent [24]. * p < 0.  application of extracts of T.27. Other researchers found the extracts could be used as an anti-osteoporotic [24].  Cymbopogan  synthesized by as  plant extracts such as Alternanthera dentate (50–100 nm).28]. 6. The biosynthesized AgNPs had a size  research. we used the callus extract of T. In previous studies. 3. and  carcinoma Hela cells. In our experiment (Figure 3b. human chronic myeloblastic leukemia cells (K562) [22]. (b) A dose‐dependent increase in the percentage of apoptosis rate. 10. which was much smaller than that of many AgNPs extracts  such  Alternanthera  dentate  (50–100  nm).  yunnanensis  extracts secondary contain  secondary  Previous studies havehave  shown that T. The excellent anticancer effect caused itsits  widely application in clinic.  Acorus  calamus  (31. which was much smaller than that of many AgNPs synthesized by plant  AgNPs had a size range from 6. head and non-small cell lung cancer [23]. Acorus calamus (31. an antiallergic reagent [25] as well as a hypoglycemic agent for diabetes [26]. Herein. 20.4 to 27.83  nm). 3. the novel of T. polysaccharides and phenolic compounds [25. Apoptosis analysis of SMMC-7721 cells treated by AgNPs.01 vs.  agents for green synthesis of AgNPs [5]. 6. yunnanensis to synthesize AgNPs. human fibroma carcinoma HT1080 cells [21]. The synthesized AgNPs had significant activity of growth inhibition against human and cancer cells with lower cytotoxicity against normal cells. human chronic myeloblastic leukemia cells (K562) [22]. 160 Nanomaterials 2016.  The  excellent  anticancer  effect  caused  widely  colon.0. the novel application of extracts  pathogenic bacteria and cancer cells with lower cytotoxicity against normal cells.01 vs.c). control.4 to 27.   (32 nm).T. yunnanensis in green synthesis of AgNPs was reported for the first time. Psoralea corylifolia (100–110 nm) and Melia dubia synthesized AgNPs had significant activity of growth inhibition against human pathogenic bacteria  (35 nm) [11]. Herein. 10. The biosynthesized range from 6. It was presumed that plant polysaccharides and phenolic compounds may act  It was presumed that plant polysaccharides and phenolic compounds may act as reducing and capping as reducing and capping agents for green synthesis of AgNPs [5]. yunnanensis and the purified compounds showed excellent cytotoxic effect against human breast  yunnanensis and the purified compounds showed excellent cytotoxic effect against human breast adenocarcinoma cells (MCF‐7).c). but  could be removed by alcohol. human cervix  adenocarcinoma cells (MCF-7). Apoptosis analysis of SMMC‐7721 cells treated by AgNPs. 40 and 50 μg/mL AgNPs. 20. and colon. metastatic ovary carcinoma.  lignans. (a) Flow cytometry analysis of  Figure 9. The  Cymbopogan citrates (32 nm). rearranged abietanes. Other researchers found the extracts could be used as an anti‐osteoporotic agent  application in clinic. Previous  studies  shown  T.2 nm. 30.Nanomaterials 2016. T. Cells were incubated with 0.  head  and  non‐small  cell  lung  cancer  [23]. human fibroma carcinoma HT1080 cells [21]. In our used the callus extract of T. extracts of T.27. 160    8 of 15 8 of 15  (a) 0 μg/mL 10 μg/mL 20 μg/mL 30 μg/mL 40 μg/mL 50 μg/mL Annexin V PI (b) Apoptosis rate (%) 100 * * 75 * 50 25 * * 0 CK 10 20 30 AgNPs (μg/mL) 40 50   Figure 9. (b) A dose-dependent increase in the percentage of apoptosis rate.  rearranged  abietanes. Centella asiatica (30–50 nm). yunnanensis to synthesize AgNPs. an antiallergic reagent [25] as well as a hypoglycemic agent for diabetes [26]. yunnanensis in green synthesis of AgNPs was reported for the first time. Centella asiatica (30–50 nm). compounds [25. Cells were incubated with 0. lignans. AgNPs was stable in the water even after two weeks but the stability  . * p <  and 50 µg/mL AgNPs.  control. In our experiment (Figure 3b. human cervix carcinoma Hela cells.83 citrates  nm). (a) Flow cytometry analysis of apoptosis in SMMC‐7721 cells stained with Annexin V‐FITC/PI.  yunnanensis have been used extensively toto  produce various active anticancer compounds such as taxane diterpenoids and polysaccharides [20–22].28]. Psoralea corylifolia (100–110 nm) and Melia dubia (35 nm) [11].  apoptosis in SMMC-7721 cells stained with Annexin V-FITC/PI.  polysaccharides  and  phenolic  taxane diterpenoids. 4030. In previous studies.2 nm. groups such as aromatic ring groups such as aromatic ring attached to AgNPs could not be removed by washing with water. In our research.

subtilis) was stronger than that of Gram-negative bacteria (E. In our study. We also tested the cytotoxic effect of AgNPs against human colon adenocarcinoma cells (LS174T) and human hepatoma cells (SMMC-7721). paratyphi B). paratyphi [33]. aureus [34]. Gossypium hirsutum and Cleistanthus collinus [33.41] and IC50 values of 17. AgNO3 control showed little antibacterial activity against the tested bacteria and the concentration of AgNO3 control (1699 µg/mL. found the antibacterial activity of AgNPs synthesized by Origanum vulgare against E. phosphorus moieties in DNA. Kim et al. The AgNPs of less than 10 nm diameters attached to cell wall caused perforation of the cell membrane of green fluorescent protein (GFP)-expressing recombinant E. Ruparelia et al. Various factors including concentration of plant extracts and Ag+ . the inhibition of AgNPs against Gram-positive bacteria (S.2 µg/mL for MCF-7. indicating other factors might play roles besides membrane structure [37].75 µg/mL) while Ag+ control had no cytotoxicity in LS174T. A549 and MCF-7 but weak cytotoxic effect on SMMC-7721. Sankar et al. Previous studies revealed both nanoparticles and dissolved Ag+ can induce cytotoxicity synergistically [45]. It has been reported that the IC50 value against A549 cells was 100. 160 9 of 15 attached to AgNPs could not be removed by washing with water. AgNPs could reduce . The AgNPs synthesized by Acacia leucophloea extract had antibacterial activity against S. paratyphi B) as shown in Figures 5 and 6. In our research. AgNPs promoted the usage of silver as an important antibacterial agent because of its smaller sizes. E. reported that the average size of AgNPs decreased as pH of the reaction system increased from 6. suggesting a moderate cytotoxic effect on both cell lines. AgNPs was stable in the water even after two weeks but the stability decreased after washed by alcohol. coli. Furthermore. 50 and 7. but could be removed by alcohol. equal to 10 mM) was much higher than that of Ag+ released in NB medium (Figure 4). callus extract had no growth inhibition effects against the tested bacteria (Figure 6).Nanomaterials 2016. respectively. 40 and 30 µg/mL. coli and S. NB medium with high concentration of sodium chloride (5. Interestingly. B. The IC50 of AgNPs was 40. coli and S. coli and S. AgNPs themselves can produce reactive oxygen species (ROS) and oxidative stress as well as the process to release Ag+ [46]. Our study demonstrated that the biosynthesized AgNPs had significant dose-dependent growth inhibition in four human cancer cells. higher surface areas and greater activities [1]. On the other hand. for different AgNPs synthesized by extracts of Origanum vulgare.41.30]. the absorption peak shift towards the shorter wavelength side from pH 7 to 10. while small sized AgNPs could create pores on bacterial wall. aureus and B.3 µg/mL for A549 and 42.5 [31]. In our research. indicating the decrease of AgNPs average size [19]. the maximum absorption wavelength of mixture at pH 11 showed a red shift relative to that at pH 10. found Gram-negative strains were more resistant to AgNPs compared to Gram-positive strains. However. Annona squamosa leaf and Cassia fistula flowers.40. It has been assumed that the released Ag+ could reacted with sulfur-containing amino acids inside or outside the cell membrane.0 g/L) could interfere with the action of released Ag+ by precipitating of Cl− . aureus. The AgNPs exhibited excellent cytotoxic effect on SMMC-7721 cells (IC50 = 27. Moreover. which may cause by the accumulation of AgNPs [32]. coli and B. Those results suggested that the antibacterial activity of AgNPs in our experiments was not related to the release of Ag+ ions or callus extracts. Qin et al. subtilis. the control of callus extracts also showed no cytotoxic effect on all tested cells. Our study showed that the biosynthesized AgNPs had significant antibacterial activity against the clinical bacteria strains (S. temperature and exposure time could affect the biosynthesis of AgNPs [29. which lead to the cell death [39].0 to 10.19 µg/mL for MCF-7 cells were presented by the fabricated AgNPs with bacteria extracts. reported Gram-negative strains were more sensitive to AgNPs than Gram-positive strains because of their difference on membrane structure [36]. However. which was in accordance with Ruparelia’s results [37]. respectively [42–44]. These results indicated lignans or other secondary metabolites in the extracts might play roles in the stability of AgNPs. AgNPs synthesized by pine mushroom (Tricholoma matsutake) extracts could be a potential antimicrobial agent against E. It remains to be further explored whether our fabricated AgNPs can penetrate and disrupt bacterial membranes. subtilis [35]. 6. thereby affecting bacterial cell viability [38].

yunnanensis were induced from young stems and subcultured on solid MS medium supplemented with 1. Although AgNPs as an antitumor agent can provide new opportunity for medical science.000 g for 30 min [57] and the produced pellets were washed three times with distilled water and dried in a freeze drier to remove excess silver ions. our fabricated AgNPs have exhibited significant cytotoxic effects. . further studies are needed in order to analyze the probable mechanism of induced apoptosis and the effects of AgNPs on mammalian immune system.39 µg/mL) of AgNPs to human liver cells (HL-7702) suggested our AgNPs were more cytotoxic to cancer cells than to normal cells in vitro. Braun et al developed etching technique to enable AgNPs to target tumor cells. Japan) to find the absorbance peak [10]. Notably. boiled for 5 min and filtered through Whatman No. In our present study. 1 filter paper [7]. The extracts were stored at 4 ◦ C and used in 1 week. The reaction mixture was stirred properly and incubated for different time (10. It has been reported that AgNP surface could be functionalized with hydrophilic molecules such as polyethylene glycol (PEG) to avoid the removal by macrophage [51]. The AgNPs may be conjugated to both a chemotherapeutic agent such as docetaxel or methotrexate. 90 and 120 min) and at different pH (7. Therefore. Materials and Methods 4. showed that AgNPs had a 44-time stronger inhibitory effect on HepG2 cells compared with normal cells [50].5 mg/L kinetin. Although cytotoxicity itself can be useful for cancer therapies. through a poly(ethylene glycol) linker [56]. In the present study. 4. developed successfully a multifunctional magnetic Fe3 O4 /AgNPs with a monoclonal antibody C225 targeting to the epidermal growth factor receptor (EGFR). Combining monoclonal antibodies such as EGFR-specific antibody C225 with the fabricated AgNPs is also a good strategy to improved efficacy and reduce toxicity on neighboring healthy tissues. Zhao et al.1. yunnanensis Callus cultures of T. Considering the biocompatible nature of the green synthesized AgNPs and potent cytotoxicity towards cancer cells. 6. Callus extracts were prepared by grinding 50 g fresh callus in 250 mL sterile distilled water. Reduction of silver ions was monitored by measuring the absorbance of the reaction mixture from 300 to 600 nm using UV-vis spectrophotometer (UV-2600. 20. 60. The mixture solution was centrifuged at 22.5 mg/L 2. more studies are still needed to advance to clinical translation. 160 10 of 15 adenosine triphosphate content of the cell. and a targeting ligand such as folic acid to certain breast cancer and ovarian cancer cells of folate overexpression [55] or chlorotaxin with a high affinity for tumors of neuroectodermal origin. We believe further cellular and molecular investigations on the different responses to AgNP exposure between cancer cells and normal cells might be helpful for a better understanding of unique mechanisms of cytotoxic actions of AgNPs. Kyoto. Faedmaleki et al. caused mitochondria damage and increased ROS production in a dose-dependent manner [47]. which minimized the off-target toxicity [52]. 8. Preparation of Callus Extracts of T. an attractive target of many cancers [54]. 10 and 11) to optimize the reaction condition.2. 30. we expect that our fabricated AgNPs could be conjugated peripherally with targeting moieties.4-dichlorophenoxy acetic acid and 0. 4. The apoptosis of SMMC-7721 cells induced by cerium oxide nanoparticles was proved to be associated with oxidative stress and the activation of mitogen-activated protein kinase (MAPK) signaling pathways [48]. 9. the higher IC50 value (81. Synthesis of AgNPs Callus extracts (1 mL) were added to 9 mL of 1 mM aqueous AgNO3 solution. Callus were cultured for 4 weeks and then harvested. and rapidly disassemble and remove AgNPs outside living cells. The apoptotic effects of AgNPs synthesized by mycelia extracts were confirmed by activation of caspase 3 and DNA nuclear fragmentation [49]. Shimadzu Corporation. nonspecific oxidative damage is one of the greatest concerns for the application of AgNPs. The treatment of targeted human ovary cancer cells by AgNPs conjugated with folic acid was conducted through site specificity by irradiating cells with a continuous wave near-infrared laser [53].Nanomaterials 2016.

The mixture was cultured at 37 ◦ C for 24 h then the optical density at 600 nm was measured by a spectrophotometer (PC22. B. The overnight grown bacterial suspensions were swabbed on NB plates. Cell Viability Assay The cell viability was measured by the modified (MTT) method [60]. 5% CO2 environment and then 10% sodium dodecyl . UK) was used to measure the zetapotential of the AgNPs. Malvern. CA. Cells were collected. 4. The cells were then incubated for another 4 h in a 37 ◦ C.5. 5. The growth inhibition was further determined by turbidimetric study. human breast cancer cells (MCF-7). pH 7. 32 and 128 µg/mL solution of AgNPs in sterile water. St. 5. The discs were evaporated and then impregnated on the plates. sodium chloride.1. 16. China). Louis.3. 6. Eindhoven. Zhejiang. USA) as well as RPMI1640 medium (Gibco. 1. aureus CMCC(B)26003.79 ppm AgNO3 were used as control. Cells in 100 µL medium were seeded per well in 96-well plates and exposed with AgNPs of different concentrations for 24 h after adhering to the plates.. Palo Alto.0. 4. USA) were also analyzed by FTIR. Ten-percent (v/v) callus extracts and 0. paratyphi BCMCC(B)50094 were purchased from China General Microbiological Culture Collection Center (CGMCC) and cultured in NB media at 37 ◦ C. Japan). Cytotoxicity Experiments 4. USA). The samples were taken at different time. centrifuged and adjusted the density to 1. The AgNPs washed by NB medium (peptone. Philips. Bacterial was suspended in NB media and the bacterial suspension (1 mL. XRD measurement was carried out using Cu-Kα radiation source in power diffractometer (X’Pert Pro MPD. Osaka. 1) discs of 6 mm diameter were impregnated with 20 µL of 2. Characterization of AgNPs The size and morphology of the synthesized nanoparticles were recorded by TEM (H-600. water-washed AgNPs and alcohol-washed AgNPs were ground with KBr pellets and subjected to FTIR analysis (Model FTS 7000. Gaithersburg. human hepatoma cells (SMMC-7721) and human liver cells (HL-7702) were grown in RPMI 1640 medium in a 37 ◦ C. 160 11 of 15 4. The plates containing bacteria and discs were held at 37 ◦ C for 24 h then inhibition zones were observed to evaluate antibacterial activity of AgNPs preliminary.Nanomaterials 2016. 1. 4.0 g/L. coli CMCC(B)44102. Antibacterial Activity The antibacterial activity of AgNPs against pathogenic microorganisms was examined using the standard disc diffusion method [58].5 × 105 /mL with the medium. 8. Varian Inc. S. Lengguang Tech. USA).0 g/L. 100 U/mL penicillin and 100 µg/mL streptomycin (Beyotime Biotechnology.5. Zetasizer Nano (ZEN3600. Worchestershire. The medium was added 10% (v/v) fetal bovine serum (Tianhang Biotechnology Company. 4.5. human lung adenocarcinoma cells (A549). Varian Inc. 128 and 256 µg/mL. The Netherlands). meat extract.0 g/L. CA. Whatman filter paper (No. 8. Yeast extract. Sigma-Aldrich. 2.4. Hitachi. subtilis CMCC(B)63501 and S. Palo Alto. Haimen. 106 colony-forming units/mL) were mixed with 1 mL solution of AgNPs with the concentration of 0. 10 µL MTT (5 mg/mL in phosphate buffer saline PBS) was added to each well. AgNO3 at 1699 µg/mL and gentamycin sulfate at 1 mM were used as control. 2. 64. The release of Ag+ from AgNPs in the medium was determined by atomic absorption spectroscopy (SpetrAA 240. 32.. the freeze-dried powder of callus extracts. 5% CO2 environment. To obtain information about chemical groups present around the AgNPs for their stabilization. Shanghai. Cell Culture Human colon adenocarcinoma cells (LS174T). AgNPs were dissolved with NB and RPMI 1640 medium at 100 µg/mL.2. MD. Bacterial strains E. The antibacterial activity of AgNPs was tested by determining its MIC [59]. China). China). centrifuged and digested with 5% nitrate acid for 24 h. Then.0 g/L. MO..

yunnanensis callus and suggested that the AgNPs synthesized by environment-friendly method could be used as antibacterial and anticancer agents. XRD pattern confirmed the presence of AgNPs and FTIR spectra indicated phytochemicals in the extracts may be involved in the synthesis and stability of AgNPs. 40 and 50 µg/mL AgNPs. Acknowledgments: The authors are grateful to the Graduate Program of Higher Education in Jiangsu Province (No. Q. [CrossRef] [PubMed] . paratyphi B and B. FL. 160 12 of 15 sulfate was added to each well. CXLX13_84).com/2079-4991/6/9/160/s1. Author Contributions: J.5-Dimethylthiazol-2-yl)-2. The 570 nm absorbance was detected with theKLx808microplate reader (Bio-Tek.5-diphenyltetrazolium bromide Phosphate buffered saline Reactive oxygen species Transmission electron microscopy X-ray diffraction References 1. MCF-7 and SMMC-7721 cancer cells but lower activity against normal human liver cells.5. and metal.01. 81473183) for financial support of this work. 14317363) and the projects sponsored by the NSFC (No. 20. Silver as antibacterial agent: Ion. 1636–1653. Chernousova. 4. S. Epple. Beckman-Coulter. Hialeah. S. 2013.W wrote the paper. aureus. 10. nanoparticle. SMMC-7721 cells (2 × 105 per well) were seeded into 6-well plates and treated with 0. The experiment was repeated three independent times and the cells survival rate was calculated. 52.X. coli. Our research explored a new application of T. yunnanensis callus was described. A549. and Q. USA)..H. 6. performed the experiments on AgNP fabrication and characterization.3. Suzhou Scholar Program (No. Int. VT. subtilis while cytotoxicity assays exhibited excellent activity of AgNPs against LS174T. Differences in means were considered to be significant for p values < 0. Antibacterial assays using AgNPs revealed creditable activity against E. Angew. the cells were analyzed by flow cytometry (FC500. Chem. USA) [48].W.X. Abbreviations The following abbreviations are used in this manuscript: AgNPs FTIR MIC MTT PBS ROS TEM XRD Silver nanoparticles Fourier transform infrared spectroscopy Minimum inhibitory concentration 3-(4. M. Supplementary Materials: The following are available online at www. Conclusions In conclusion.2 nm.4 to 27. TheAnnexin V-PI assay evaluates phosphatidylserine translocation from the inner to the outer layer of the plasma membrane which is an event typically associated with apoptosis.J.mdpi. Conflicts of Interest: The authors declare no conflict of interest. Flow Cytometry Analysis of Apoptosis The number of apoptotic cell induced by AgNPs with different concentrations was measured by flow cytometry using Annexin V-FITC-PI kit. Y. The biosynthesized AgNPs had spherical shapes with particle size ranging from 6.M analyzed the data on bioactivities. cells were harvested. Winooski. Ed.W. the biosynthesis of AgNPs by extracts from T. Data are the mean ± standard deviation (SD) and a student’s t-test was used for statistical comparisons of two means. 5.Nanomaterials 2016.H. and J. Finally. After treatment with AgNPs for 24 h. Two microliters Annexin V-FITC and 2 µL PI were added and then the mixture was incubated at room temperature for 10 min in the dark. S. 30. rinsed twice with ice-PBS and resuspended in 200 µL binding buffer. conceived and designed the experiments.W.

. N. 2013.R.. [CrossRef] [PubMed] Satyavani. Microbiol. Chem.. Nanomed. Yin. Morones. Y...H. Q. P. Appl..A. Adv. G..L. 17–28. R.N. Nanotechnol. W.T. C. 814–818. Samim. F.F. Tan. Y. Sunlight-induced biosynthesis of silver nanoparticles by animal and fungus biomass and their characterization..) Schrad.. Liu. [CrossRef] . C.M. Sorkhabadi. Protoplast culture and paclitaxel production by Taxus yunnanensis. Gade. Adv. M. 12.P. Alikunhi..L. Kim. 6.S. K. C. Res.. [CrossRef] Zhang.. 2014. Nguyen.. 1991.. Chem.Y. Gurudeeban. A.. J. Zhang..P... J. Eng. Tagahara. 257–262. Safekordi. Xu.. 19. H. 83–86. 2785–2788. 25–29. 1141–1147. 2010.A. Surface plasmon spectroscopy of nanosized metal particles. M.Nanomaterials 2016. L.S. T. Int. Carbohyd. 2014..H. 10. J. Chem. 5. 6. Lett. Y. R.A. 6.. 25. A. 2–8..J..R. Gay. S.. Yacaman.R.. J. Fukamiya. 488–493. 2011. Biomedical potential of silver nanoparticles synthesized from calli cells of Citrullus colocynthis (L. K. P.. [PubMed] Wang. Yan.. Mhatre. Nanomedicine 2010. J. 2011. 396–402. Pharm. 9. Polym. 69–78.. H.. J.M. A. [CrossRef] Wei. 1591–1603... 59. and evaluation of antimicrobial and cytotoxic effect of silver and titanium nanoparticles. Luo. Langmuir 1996. 15. Polysaccharides and phytochemicals: A natural reservoir for the green synthesis of gold and silver nanoparticles. [CrossRef] Hegazy. Okano. M. Olive. Camacho. 2012. Br. L. Ingle. Mujeeb.. M.. Attar. Balasubramanian.. Verma. B.H. Polym.. Uchiyama.. J.K. W.. Zhang. K.. Nitric Oxide 2006.. [CrossRef] Shinozaki.T. K. Kandasamy. H. Bastow.. Ikram. [CrossRef] [PubMed] Singla. Orrantia. Biological and non-biological methods for silver nanoparticles synthesis. Plant Cell Tissue Org. characterization. Garg. 64. R. Involvement of nitric oxide in oxidative burst.. [CrossRef] [PubMed] Ahmed. Ruiz. L. Ramírez.. Carbohyd. 543–548.. [CrossRef] Yan.. Yu. Holt. 2010. M.B. Nanotechnology 2005. Technol.H. 11. 2346–2353. H.. S. J. A. A. Life Sci. 2011. alba and assessment of its antimicrobial activity. Biochem. Raj. Biological synthesis of metallic nanoparticles. A. Y.X. Plant Biochem. R. F. 179–192. Yang.. 4.W. 20. Kadota. 160 2. Mu. 11. Zhang. Colloid Surface B 2010. Park. E. 2002. 82. Rai. 96. 9. Linhardt. A review on plants extract mediated synthesis of silver nanoparticles for antimicrobial applications: A green expertise. 2014. A. [CrossRef] [PubMed] Thakkar. M.. S. Song. Yuan. 23.. J. R. J. Res. Y. Tezuka. Ahmad. T. G.. H. [CrossRef] [PubMed] Mude. He.. Multidrug resistant cancer cells susceptibility to cytotoxic taxane diterpenes from Taxus yunnanensis and Taxus chinensis. 2002. Sesuvium portulacastrum L. 788–800.M. J. [CrossRef] [PubMed] Ghorbani. A. 16. Y. K. L. Aggarwal. N. Banuelos. M. J.. Mater. Biotechnol. Ramanathan. Y. 3. Q. Raie. J. 4. F. 7. 235. J. Banskota. Biosynthesis of silver nanoparticles using callus extract of Catharanthus roseus var.. Synthesis of silver nanoparticles using callus extract of Carica papaya—A first report... 21. phenylalanine ammonia-lyase activation and taxol production induced by low-energy ultrasound in Taxus yunnanensis cell suspension cultures. A..H. S. 22... Wang. 6. 79. N.... Biotechnol. N... Diterpenes and sesquiterpenes from the bark of Taxus yunnanensis. 12. Antifungal properties of Ag-SiO2 core-shell nanoparticles against phytopathogenic fungi. Gu. 14. T. The bactericidal effect of silver nanoparticles. [CrossRef] [PubMed] Zheng.Y.S. Structural characterization and anti-tumor activity of a novel heteropolysaccharide isolated from Taxus yunnanensis. Med.Y. 89. 2002. 18. 317–326. Y. C. 5... 18. 7. D. S. Wu. Hong. Bach. Phytochemistry 2003. A.. Zhou. 2016.L. 681–688. 13 of 15 Gutierrez. Weyers. Biotechnol. 389–395. B. X. 476–478.. S. 1211–1214. H. A. Paclitaxel and its formulations. Nanobiotechnol. Rabie. N. 12.N. E. Lee. Effects of inoculum size and age on biomass growth and paclitaxel production of elicitor-treated Taxus yunnanensis cell cultures.Y.. Luo. IET Nanobiotechnol. K. Zheng. J.P. Yang. Y. Synthesis.. Pharm. D. 305–311. Yu.S.W. 16. [CrossRef] Nabikhan.. Nino. Wu. Res. M. D. 15. [CrossRef] Mulvaney.. Swami. 17... 13. [CrossRef] Yin. K. S. Z. Barkat..D. Bioorg. 8. Sanchez. Kouri. Nobukawa.. J. Structural characterization and in vitro antitumor activity of a novel polysaccharide from Taxus yunnanensis.. 2009. Elechiguerra. 60. L.J. Synthesis of antimicrobial silver nanoparticles by callus and leaf extracts from saltmarsh plant. Shaaban. Extracellular synthesis of silver nanoparticles by callus of Medicago sativa. 351–358. Parikh..M.

. Nguyen.P. 25.. K. S. Nanomed.. Antiallergic activity of aqueous extracts and constituents of Taxus yunnanensis. B... 2405–2410.H. Ind. Acta Biomater. Y..I.J.A. P.. A feasible approach to phyto-mediated synthesis of silver nanoparticles using industrial crop Gossypium hirsutum (cotton) extract as stabilizing agent and assessment of its in vitro biomedical potential...K. K. Senbagam. A. Karthik.Nanomaterials 2016.. Park. [CrossRef] [PubMed] Qin. Crop. Nanoparticles and their potential application as antimicrobials. [CrossRef] [PubMed] Kanipandian. R. Int.J. Kannan. Nanomed. Nanotechnol.. R. Kim. Strain specificity in antimicrobial activity of silver and copper nanoparticles. J. 1–10. C. S. Mater. Han. 425–427.. H.. Productions of taxol and related taxanes by cell suspension cultures of Taxus yunnanensis. H. 4.L. 80–84. Ji. Prod. E.K. V. P.. 2015. Ravikumar. M. Anwar. [CrossRef] [PubMed] Ruparelia. Biol. 29... S. Process Biochem. 22. Characterization. Dayem.. R. Y. 45. 1488–1491.. Lett.H. 2014. S. P.M.. J. Chem.. [CrossRef] Kanipandian.Y. [CrossRef] [PubMed] Murugan. 2003.. R. K. Mandal. M. 13. Shi... B. Chattopadhyay.. Nobukawa. A.H. [CrossRef] [PubMed] Koyama. Synthesis of silver nanoparticles using pine mushroom extract: A potential antimicrobial agent against E.J.. 2325–2331. Rathinam. S. 14 of 15 Yin. antioxidant and cytotoxicity evaluation of green synthesized silver nanoparticles using Cleistanthus collinus extract as surface modifier... 2009. W.. J. A. 37–42. R. M. J. Hirai. 172–176. Chatterjee. 41. Phytomedicine 2006. Prod... Yang. 32. Langmuir 2006. F. 2014. M. S. 3. J. Bull. 9923–9941.. D. Cho. J. H. S. Gunasekaran. Kobayashi. R. K. Role of pH in the green synthesis of silver nanoparticles. 2012. J.. Simamura.. 2310–2312. Nobukawa. Formatex: Badajoz. J.A. Ghosh. Pharm.. 47.. Int. Med.. Li. Tezuka. H. Shivashangari. J. Thirumurugan. Karthik.Y. 33.. X. 2014. Sci. S.H. 13. Hypoglycemic effects of the wood of Taxus yunnanensis on streptozotocin-induced diabetic rats and its active components.S. N. 29. H. antimicrobial and urease inhibitory activities against Helicobacter pylori. Duttagupta.J. Nobukawa. M... [CrossRef] Singh. 2006..G. 27. Colloids Surf. Kim. 2011. Green fluorescent protein-expressing Escherichia coli as a model system for investigating the antimicrobial activities of silver nanoparticles. [CrossRef] Sankar. A. Harperc. Eng. In vivo anti-osteoporotic activity of isotaxiresinol. 2013. Jamuna. Kannan. Ramesh. R. K.. Antimicrobial effects of silver nanoparticles.. Paul. Biosynthesis of silver nanoparticles using Acacia leucophloea extract and their antibacterial activity.H. Morita. S. Ind.. K. Ind. Y. S. [CrossRef] Kim. 55.D. Green synthesis of silver nanoparticles through reduction with Solanum xanthocarpum L. Thangam. Park. S. Mukherji. Mater. N. [CrossRef] Gurunathan.K. Wu. Prod. Nobukawa. Mol. 707–716.. 9322–9328. 2007.. 30. 197–209. [CrossRef] [PubMed] Ravishankar.. A. R. pp. Kadota. 40. I. Ind.. Sangiliyandi. S.. J. [CrossRef] [PubMed] Banskota.. T.. Kim. S. Kalaichelvana. Phytomedicine 2006. C. 20. 28.K. Park. Jeyaraj. [CrossRef] . F.. P. R. Ed.S. H.S. Eng.. Kuk. 19. A. A.. V... Bull. N.. J. Lee. Shen. J.A. Méndez-Vilas.S. Communicating Current Research and Technological Advances.. E. Kadota.. Zhang. 9.K. 6. S.S.. Yunb. 13. T. Ding... Sin. subtilis. 2431–2438. 1600–1605. A. Jing. P. Green biosynthesis of silver nanoparticles from Annonasquamosa leaf extract and its in vitro cytotoxic effect on MCF-7 cells. Murugan.. 38.N.. Chandranb. Liu. N. Anthony. Senthilkumar...A. 31. S. 26. Fong. B 2013. L. T. J. 63. [CrossRef] Amin.H. Ramesh. Rearranged taxanes from the bark of Taxus yunnanensis.. Acta Bot. 108. A.. Colloids Surf.. 109–114.H. 372.J. Kaveri. 63.P.. S. I. In Science against Microbial Pathogens.. G. A.. 35. Yao..P. a lignan from wood of Taxus yunnanensis. Gan. H. Chem.M.W. Y.S. J...V.. Lee. K. Y. Nat. Sun. K. Eppakayala. 70.H... [CrossRef] [PubMed] Hu.. Iqbal. Spain. 49..S. Biol. 2012. Res..T. Sinha. 494–502. 39. [CrossRef] [PubMed] Rajana.. coli and B... Green synthesis of anisotropic silver nanoparticles and its potential cytotoxicity in human breast cancer cells (MCF-7). Yu. Kadota. 43. Prabu.. N. [CrossRef] Vivek.. 2000. Origanum vulgare mediated biosynthesis of silver nanoparticles for its antibacterial and anticancer activity. S. A 2010. K. Rashid. Tezuka. Gopinath. 37. Lu. berry extract: Characterization. U. Crop. 160 24. Janjua.T. 356–373. Y. Park. Saleh.. Thirumurugan.H. et al. 42.. 373–378. Y. M. 36. 34. 2008. M. Gogoi.R. 95–101.. Hwang. Muthuchelian. Plant extract synthesized silver nanoparticles: An ongoing source of novel biocompatible materials. Size control over spherical silver nanoparticles by ascorbic acid reduction. 2014. Subramanian.

4399–4413.A. 103... 10.. 330–338.T..R. In Vitro 2009.. 543–552.. T. T. 58. 2012. Ryu.. 56. Vikineswary. H. 60. Potara. Philos.. Astilean. R. S. L. R.L. Free radical scavenging and apoptotic effects of Cordyceps sinensis fractionated by supercritical carbon dioxide. Fang. N. J..N. O. functionalization and bioconjugation of colloidal inorganic nanoparticles.. Kotamraju. Q. G. Cytotoxic potential of silver nanoparticles. Pallaoro. John..G. Majidi. A 2011.-S. H.R. Liu. 13. Mater. A 2010. Baldeck. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons. 8. R. Folic acid-conjugated.H. 51. J. 2014. Osawa.. Salarian. S.R. 51. 48. An investigation on cytotoxic effect of bioactive AgNPs synthesized using Cassia fistula flower extract on breast cancer cell MCF-7. Parak. R. Environ. Y. S. [CrossRef] [PubMed] Liu. Arfan.. Acta Biochim. A. 160 44. [CrossRef] [PubMed] Zhao. 13. Faedmaleki. Sarada. 6. 54. Chen. M. 15 of 15 Remya. 57. Y.. C...J. Li.F.. [CrossRef] [PubMed] © 2016 by the authors. Ai.. Int. H. Zhou.B. [CrossRef] Zhang. 391–399. 55. N.H.0/). Food Chem. M. 23. Suman. J. Chen. R. Ali.. 53. Simon.. Kong.R. X.. Rehman. Stephen. S. L. J. [CrossRef] [PubMed] Gurunathan. T. [PubMed] Sperling. H.. H. Park. A.J.. A.. Ellenbogen. 44.. Yu... Wang.Y. Tong.. [CrossRef] [PubMed] Wang. Willmore. S. S. V.... [CrossRef] [PubMed] Zarchi. Feng. Sin. Song. 6046–6051. T..Nanomaterials 2016. C. R... 11.. 2013.. Z. Juhem. Folic acid conjugated nanoparticles of mixed lipid monolayer shell and biodegradable polymer core for targeted delivery of Docetaxel... S. Sugahara. Ashtiani. Soc. Toxicol. 2013. In Vitro 2013. A. Wang.. She. 3.H. Choi.A. Li. K. A. 2012. Switzerland... R.. L. Won.A. P.. 59.. 2014. Rajasree. Biophys.. Appl. et al. 50.... Chen. Characterization and antimicrobial activity of gold and silver nanoparticles synthesized using saponin isolated from Trianthema decandra L. Green synthesis of silver nanoparticles using Ganoderma neo-japonicum Imazeki: A potential cytotoxic agent against breast cancer cells. D.E. 2014. Phys. [CrossRef] Geethalakshmi. B. Veiseh. Lee. W. Nanomed.R. C. 8. 52.. D.V.. Aranganathan. 43.. Res.J.. Toxicity effect of silver nanoparticles on mice liver primary cell culture and HepG2 cell line. Pan. [CrossRef] [PubMed] Cheng.. Chen.B. 55.. Biotechnol. W. Pang.. 49.. Oxidative stress dependent toxicity of silver nanoparticles in human hepatoma cells.N. 107–115.. J. 2005. . 283–291.. Shahverdi. Sun. 1082–1088. In vitro toxicity of silver nanoparticles at noncytotoxic doses to HepG2 human hepatoma cells.. Zhang. P. Cirillo. Z.. K. Nanobiotechnol. Han.Y. Antibacterial activities of gold and silver nanoparticles against Escherichia coli and bacillus Calmette-Guérin. Okabe. R. Rep. J. G.. Mann. Rastegar. A.K. T. [CrossRef] [PubMed] Kim.. Toxicol. licensee MDPI. J. Z. [CrossRef] [PubMed] Kawata. Friman. H. X. Fan.. Yonsei Med.A. Olson. S.. S. B.. Sci. 349–353. de Mendoza. [CrossRef] [PubMed] Boca-Farcau. 2009. 45.. K. M.. Hansen. [CrossRef] [PubMed] Sun. Crop. Su. L. SERS-labelled silver nanotriangles for multimodal detection and targeted photothermal treatment on human ovarian cancer cells. J. Biomaterials 2010. Xu. Kong. C. Ma. Pharm. Mokhtari... Amini. Surface modification.A. A. Etchable plasmonic nanoparticle probes to image and quantify cellular internalization.L. Tumor-targeted drug delivery and MRI contrast enhancement by chlorotoxin-conjugated iron oxide nanoparticles... Nat. M.R. 495–505. Pharm. Nanomedicine 2008. 1333–1383. 46. J. F. S. Kundu. Technol. Raman. F. S. Toxicol.Y. Cerium oxide nanoparticles induce cytotoxicity in human hepatoma SMMC-7721 cells via oxidative stress and the activation of MAPK signaling pathways. Guo. Ind.. Mol. W. E. 43. 904–911. L.A. J. Transl.D. Y. 1076–1084.G. D. [CrossRef] [PubMed] Braun. Shirazi.. Yi. 47. Malek.. Basel.. A novel multifunctional nanocomposite C225-conjugated Fe3 O4 /Ag enhances the sensitivity of nasopharyngeal carcinoma cells to radiotherapy. 2015. M. K. M. Prod. A sunlight-induced method for rapid biosynthesis of silver nanoparticles using an Andrachnea chordifolia ethanol extract. Iran. K. 110–115. 678–684. 368. 2014. 27. 31. T. S. D.. J. Chung.A.L.. Bu. 235–242...P. Liang.