Inorganic Chemistry Communications 132 (2021) 108808

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Inorganic Chemistry Communications

journal homepage: www.elsevier.com/locate/inoche

Green synthesis of nano silver by tea extract with high

antimicrobial activity
Yun Fang, Ci-Qing Hong, Fang-Rong Chen, Fang-Ze Gui, Yun-Xin You, Xiong Guan *,
Xiao-hong Pan *
State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Key Lab of Biopesticide and Chemical Biology, Ministry of Education, College of Plant
Protection, Fujian Agriculture and Forestry University, Fuzhou 350002, China

A R T I C L E I N F O A B S T R A C T

Keywords: In this study, a facile synthetic pathway was proposed to fabricate silver nanoparticles by using tea extract as
Nano silver reducing agent and stabilizer to reduce silver nitrate, which was further evaluated for the antimicrobial activity
Tea extract against bacterium (Escherichia coli) and pathogenic fungus of tea (Pseudopestalotiopsis theae). The average particle
Antimicrobial
size of silver nanoparticles is 15.41 nm by X-ray diffraction (XRD) and Scherrer equation, the morphology is
relatively regular and well dispersed by scanning electron microscope (SEM) and transmission electron micro­
scope (TEM), the zeta potential is − 13.91 ± 0.89 mV and the aggregate particle size distribution in water is
around 773.49 nm. In addition, the synthesized nano silver had good antimicrobial effect on Pseudopestalotiopsis
theae (P. theae) and Escherichia coli (E. coli), and the inhibition ratio was 96.05% and 61.87%, respectively. And
the reaction of silver nanoparticles with bacteria and fungi could not be driven by the electrostatic interactions,
but might by intermolecular forces. This study provides a facile and effective method for the preparation of nano
silver, and broadens the application of nano silver in antimicrobial agent.

Silver not only has the best conductivity and ductility in precious
metals, but also has high safety and good sterilization effect. Compared
with bulk silver, nano silver particles show better properties because of
their small size and large specific surface area[1]. Moreover, nano silver
has better antibacterial activity than traditional silver fungicides. Nano
silver has been widely used in many fields such as electronics, bio-
sensing, biomedical materials, catalyzer, clothing, household water fil­
ters, and cosmetics due to its special magnetic and antibacterial prop­
erties in the past few years[2–7]. The preparation of silver nanoparticles
has attracted extensive attention, which can be prepared by photo­
chemical reduction, ultraviolet irradiation, electrochemical reduction
and reverse micelles[8–11]. A facile method to prepare silver nano­
particles with highly uniform morphologies and narrow size distribu­
tions was established, and the antibacterial activity increased with the
decreasing particle size[12]. Ag triangle nanoplates and nanospheres
could be synthesized by liquid chemical reduction method, and the as-
synthesized silver nanoparticles with different morphologies exhibited
excellent antibacterial properties[13].
As a new technology for the synthesis of nano materials, the plant
reduction method has become a research hotspot in recent years because
of its simple conditions, low cost and environmental friendly[14]. It was
reported that AgNPs could be synthesized by fresh melon extract, and
the obtained AgNPs exhibited high insecticidal activity on Musca
domestica [15]. Azadirachta indica plant fruit extract were used as raw
material to prepare Ag nanoparticles, AgCl-NPs and Ag/AgCl nano­
composites, the obtained Ag/AgCl nanocomposites exhibited an
enhanced photocatalytic and antibacterial activities in comparison with
pure Ag and AgCl nanomaterials[16]. However, it should be noted that
the prepared silver nanoparticles by plant reduction method have some
disadvantages, such as wide particle size distribution and uneven par­
ticle size. Thus a simple one-step formation method of AgNPs with better
control over the size is the major focus of the researchers.
Previous study indicated that tea contains the phenolics, terpenoids,
polysaccharides, flavonoids and other phytochemicals, these substances
can reduce silver nitrate to obtain nano silver[17]. Our results indicated
that the tea extract contained total phenolics, soluble sugar and flavo­
noids, and the contents were 11.25 mg/ml, 5.29 mg/g and 15.61 mg/g,
respectively by UV–Vis spectroscopy using Kit assay (Table 1). In addi­
tion, these phytochemicals can also interact with the surface of nano­
particles to stabilize them[18]. In this study, nano silver was prepared

* Corresponding authors.
E-mail addresses: guanxfafu@126.com (X. Guan), xhpan@fafu.edu.cn (X.-h. Pan).

https://doi.org/10.1016/j.inoche.2021.108808
Received 10 April 2021; Received in revised form 19 July 2021; Accepted 20 July 2021
Available online 26 July 2021
1387-7003/© 2021 Elsevier B.V. All rights reserved.
Y. Fang et al.
Table 1
The content of total phenolics, soluble sugar and flavo­
noids in tea extract.
compositions content
total phenolics 11.25 mg/ml
soluble sugar 5.29 mg/g
flavonoids 15.61 mg/g
by reducing silver nitrate with tea extract containing reducing sub­
stance. Moreover, it was used to control fungal diseases in the tea garden
and explored its antibacterial effect on E. coli.
Subsequently, the preparation method of nano silver was optimized.
Different concentrations of AgNO3 (0.05 mol/L, 0.08 mol/L) were pre­
pared. The tea extract was reacted with different concentrations of
AgNO3 by volume ratio of 0.5:1, 1:1, 1.5:1, and the yield of nano silver
was calculated. When the volume ratio of tea extract to 0.08 mol/L
AgNO3 was 1.5:1, the highest yield was 17.36%.
6 g oolong tea was soaked in 480 mL distilled water for 30 min, and
then bathed it at 90 ◦ C for 30 min. Subsequently, the tea dregs were
separated from the liquid by 0.22 μm filters, and the obtained liquid was
mixed with 0.08 mol/L AgNO3 in a volume ratio of 1.5:1. Afterwards,
the mixture was stirred in a magnetic stirrer at 40 ◦ C for 2 h. The ob­
tained nano silver material (Fig. 1a) was centrifuged for precipitation,
washed with distilled water for three times, dried at 50 ◦ C (Fig. 1b) and
ground into fine powder (Fig. 1c). Finally, the powdered silver nano­
particles were stored in a refrigerator at 4 ℃. Moreover, the obtained
nano silver was characterized using UV–Visible Spectroscopy (UV–Vis)
recorded by UV2600 (Shimadzu, Japan) under the 300 nm to 600 nm
region, and the absorbance peak was observed at 479 nm (Fig. 1d),
which is similar to previous studies[19]. According to previous studies
[20], we speculate that the obtuse absorption peak of nano silver is
possibly assigned to little tea extract coated on its surface thus leading to
the wide particles size distribution.
Fig. 1. The image of silver nanoparticles (a, b and c) and UV–Vis analysis (d).
2
Inorganic Chemistry Communications 132 (2021) 108808
In addition, the silver nanoparticles were characterized by XRD
(Fig. 2a), the diffraction pattern of XRD could be well indexed to crys­
talline nature (no more impurity peak), and the average crystalline size
values was 15.41 nm by Scherrer equation. SEM technology was used to
study the surface morphology of silver nanoparticles (Fig. 2b), the
synthesized silver nanoparticles have small particle morphology and
agglomeration behavior. The TEM results also indicated that a few
agglomerated silver nanoparticles were also perceive, and this data is in
agreement with the average size of the silver evaluated from the XRD
diffraction peaks (Fig. 2c). Meanwhile, it was found that the size dis­
tribution of the as-prepared silver nanoparticles is quite wide and the
particle size is not uniform, which indicated plant reduction of nano­
particles is flexible in producing the nanoparticle with different particle
size and size distribution[21]. Previous studies reported that the stirring
and sonication process might crack the agglomeration of particles and
makes them uniform[21], or used sodium borohydride (NaBH4) and
sodium citrate as the reducing agent and modifying agent to prepare
silver nanoparticles with highly uniform morphology and narrow size
distribution[12].
Moreover, the functional group of tea extract and silver nano­
particles was identified by FT-IR. The band of silver nanoparticles
assigned at 3434 cm− 1 might be corresponded to O-H stretching vibra­
tion[22], the peak around at 1630 cm− 1 corresponds to C = O stretching
vibration, while 1021 cm− 1 corresponds to C-O phenolic compounds
[23,24]. Comparing with the band of silver nanoparticles, there are
many infrared peaks of natural products in tea extracts, which might
corresponding to different groups, such as C = C stretching vibration
(1695 cm− 1), C-H deformation vibration (1452 cm− 1), and C-O
stretching vibration (1044 cm− 1). In addition, Zeta potential and DLS
showed that the potential was − 13.91 ± 0.89 mV, and the average
particle size in water was 773.49 nm (Table 2).
E. coli were used for antimicrobial test, and the bacterial plate count
was the standard methods to determine antibacterial activity. All the
experiments were repeated three times. E. coli was cultured in LB me­
dium at 37 ◦ C with aeration, and the shaking speed was 200 r/min. After
24 h, bacteria were centrifugation at 6000 r/min for 5 min at 4 ◦ C, the
supernatant was removed. Then the precipitate was washed twice with
PBS buffer to remove the residual medium, the obtained wet bacteria
were mixed with sterile water, doxycycline hyclate soluble powder (set
as positive control) and 500 mg/L nano silver solution at a mass to
volume ratio of 1:100 respectively for 12 h. The bacterial suspensions
were cultured on LB solid medium by proper diluted multiple, and
calculated the bacterial colony after one day culture. The results indi­
cated that doxycycline hyclate soluble powder and nano silver had
obvious inhibitory effect on E. coli, and the inhibition rate of doxycycline
hyclate soluble powder and nano silver were 44.75% and 61.87%,
respectively (Fig. 3). The antibacterial rate of Ag NPs can be lower than
previous studies[25], and we speculated that the particle size and ag­
gregation behavior of silver nanoparticles might affect its antibacterial
effect. Moreover, the zeta potentials of bacteria were further measured
in order to explore the antibacterial mechanism of silver nanoparticles,
it was found that the E. coli bacteria carried negative charges (-45.71 ±
2.52 mV). As the mean potential of silver nanoparticles was − 13.91 mV,
the negative bacteria is hardly to contact with negatively charged silver
nanoparticles through electrostatic adsorption, and the reaction be­
tween bacteria and silver nanoparticles might be driven by intermo­
lecular forces[26].
In addition, Pseudopestalotiopsis theae isolated from diseased leaves of
tea was used in antifungal experiment, and P. theae is the main pathogen
of tea gray blight. The mycelial growth rate method was the standard
methods to determine antifungal activity. The nano silver was prepared
into 0.1 g/10 mL concentration with sterile water and ultrasonically for
20 min, and diluted into 500, 1000 and 1500 mg/L adding into PDA
medium, and the cake of tea pathogenic fungi with diameter of 8 mm
was inoculated on PDA medium. The PDA plate without nano silver was
used as blank control, and the plate with 75% chlorothalonil wettable
Y. Fang et al. Inorganic Chemistry Communications 132 (2021) 108808

Fig. 2. The XRD patterns (a), SEM image (b), TEM image (c) and FT-IR of nano silver samples and tea extract (d).

calculated by cross method. The results showed that 75% chlorothalonil

Table 2
wettable powders had excellent inhibitory effect on the growth of
Potential and diameter of nano silver.
P. theae, and the inhibition rate was 93.99% (Fig. 4 & Table 3). The
Min Max Mean antifungal effect of nano silver was increased with the concentration of
Potential (mV) − 14.91 − 13.23 − 13.91 nano silver, and the highest inhibition rate was 96.05% when the con­
Diameter (nm) 726.40 807.94 773.49 centration was 1000 mg/L. This result indicated that the antifungal
activity of nano silver is lower than chlorothalonil at 500 mg/L, but the
antifungal activity is significantly higher than that of commercially
powders (500 mg/L) as positive control. The inverted samples was
available nano silver (Aladdin, S110974 and S110971, CAS: 7440–22-4,
cultured at 26 ◦ C for 2–5 days. The growth of fungi was recorded and
the highest inhibition rate was only 25.08 ± 0.48% and 20.90 ± 1.60%,
observed every day, and the inhibition rate of mycelium growth was
respectively, data not shown). Meanwhile, the zeta potentials of fungi

Fig. 3. Antibacterial plate experiments for E. coli. The positive control was doxycycline hyclate soluble powder.

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Y. Fang et al. Inorganic Chemistry Communications 132 (2021) 108808

Fig. 4. Antifungal plate experiments for P. theae. The positive control was 75% chlorothalonil wettable powders (500 mg/L).

Declaration of Competing Interest

Table 3
The inhibiting rate of P. theae by nano silver and chlorothalonil.
The authors declare that they have no known competing financial
Days/ Inhibiting rate/% interests or personal relationships that could have appeared to influence
d 500 mg/L 100 mg/ 250 mg/ 500 mg/ 1000
the work reported in this paper.
chlorothalonil L L L mg/L

2 79.54 ± 0.11 10.97 ± 23.22 84.21 96.05

± ± ±
Acknowledgements
0.06 0.05 7.44 5.58
3 90.04 ± 0.05 11.74 ± 25.80 ± 73.36 ± 74.25 ±
0.07 0.05 4.02 6.28 This work was financially supported by the National Key R&D Pro­
4 93.99 ± 0.04 12.47 ± 20.99 ± 74.01 ± 77.85 ± gram of China (2017YFE0121700), the National Natural Science Foun­
0.09 0.08 0.00 0.64 dation of China, the Natural Science Foundation of Fujian Province,
5 93.36 ± 0.04 9.76 ± 12.10 80.36 77.95
China (2020 J01522), Science Fund for Distinguished Young Scholars of
± ± ±
0.06 0.04 7.58 0.8
Fujian Agriculture and Forestry University (Grant xjq201719), and the
Special Fund for Scientific and Technological Innovation of Fujian
were also measured, and the potential of P. theae is − 35.11 ± 1.70 mV, Agriculture and Forestry University (Grants CXZX2019005S and
which means that the silver nanoparticles might also be reacted with CXZX2020024A).
P. theae by intermolecular forces not by electrostatic interactions.
In conclusion, we propose to use tea extract as reducing agent and References
stabilizer, reduce silver nitrate to synthesize nano silver, and the ob­
tained nano silver exhibited good antimicrobial activity. This work [1] B. Ajitha, Y.A.K. Reddy, H.J. Jeon, C.W. Ahn, Synthesis of silver nanoparticles in an
eco-friendly way using Phyllanthus amarus leaf extract: Antimicrobial and catalytic
provides a green and environment-friendly synthesis method of nano activity, Adv. Powder Technol. 29 (2018) 86–93.
silver by plant reduction method, and we expected that this study can [2] F. Wang, P. Mao, H. He, Dispensing of high concentration Ag nano-particles ink for
offer significant reference for the real antimicrobial agent application of ultra-low resistivity paper-based writing electronics, Sci. Rep. 6 (2016) 21398.
[3] N. Srikhao, P. Kasemsiri, N. Lorwanishpaisarn, M. Okhawilai, Green synthesis of
silver nanoparticles. silver nanoparticles using sugarcane leaves extract for colorimetric detection of
ammonia and hydrogen peroxide, Res. Chem. Intermed. 47 (2021) 1269–1283.
CRediT authorship contribution statement [4] C. Li, R. Fu, C. Yu, Z. Li, H. Guan, D. Hu, D. Zhao, L. Lu, Silver nanoparticle/
chitosan oligosaccharide/poly(vinyl alcohol) nanofibers as wound dressings: a
preclinical study, Int. J. Nanomedicine 8 (2013) 4131–4145.
Yun Fang: carried out most of characterization and antimicrobial [5] Y. Tao, X.B. Cheng, Z.J. Wang, R.W. Tan, X.Q. Yu, Z.W. Zhai, J.G. Han, The
experiments in this work. Ci-Qing Hong: conducted the experiments on application possibility of acellular dermal matrix decorated with nano-silver in the
reconstruction of contaminated abdominal wall, Mater. Sci. Eng. C 119 (2021),
microbiology. Fang-Rong Chen: carried out the synthesis experiments.
111645.
Fang-Ze Gui: conducted the zeta potentials testing. Yun-Xin You: iso­ [6] H. Yun, J. Kim, W. Choi, M.H. Han, J.H. Park, H. Oh, D.H. Won, K. Kwak, Y.
lated the fungi from diseased leaves of tea. Xiong Guan: Writing - re­ J. Hwang, Understanding morphological degradation of Ag nanoparticle during
view & editing. Xiao-hong Pan: Writing - review & editing. electrochemical CO2 reduction reaction by identical location observation,
Electrochim. Acta. 371 (2021), 137795.
[7] M. Ahamed, M.S. Alsalhi, M.K.J. Siddiqui, Silver nanoparticle applications and
human health, Clin. Chim. Acta. 411 (2010) 1841–1848.

4
Y. Fang et al.
[8] S. Xiong, X. Cao, H. Fang, H. Guo, B. Xing, Formation of silver nanoparticles in
aquatic environments facilitated by algal extracellular polymeric substances:
Importance of chloride ions and light, Sci. Total Environ. 775 (2021), 145867.
[9] J. Zhu, X. Liao, H. Chen, Electrochemical preparation of silver dendrites in the
presence of DNA, Mater. Res. Bull 36 (2001) 1687–1692.
[10] R.A. Salkar, P. Jeevanandam, S.T. Aruna, Y. Koltypin, A. Gedanken, The
sonochemical preparation of amorphous silver nanoparticles, J. Mater. Chem. 9
(1999) 1333–1335.
[11] J.N. Solanki, Z.V.P. Murthy, Highly monodisperse and sub-nano silver particles
synthesis via microemulsion technique, Colloids Surf. A 359 (2010) 31–38.
[12] Y. Wu, Y. Yang, Z. Zhang, Z. Wang, Y. Zhao, L. Sun, A facile method to prepare size-
tunable silver nanoparticles and its antibacterial mechanism, Adv. Powder
Technol. 29 (2018) 407–415.
[13] M. Gao, L. Sun, Z. Wang, Y. Zhao, Controlled synthesis of Ag nanoparticles with
different morphologies and their antibacterial properties, Mater. Sci. Eng. C 33
(2013) 397–404.
[14] S. Prabhu, E.K. Poulose, Silver nanoparticles: mechanism of antimicrobial action,
synthesis, medical applications, and toxicity effects, Int. Nano Lett. 2 (2012) 1–10.
[15] S. Gul, M. Ismail, M.I. Khan, S.B. Khan, A.M. Asiri, I.U. Rahman, M.A. Khan, M.
A. Kamboh, Novel synthesis of silver nanoparticles using melon aqueous extract
and evaluation of their feeding deterrent activity against housefly Musca domestica,
Asian Pac. J. Tropa. Disease 6 (2016) 311–316.
[16] P. Panchal, P. Meena, S.P. Nehra, A rapid green synthesis of Ag/AgCl-NC
photocatalyst for environmental applications, Environ. Sci. Pollut. R. 28 (2021)
3972–3982.
[17] Q. Sun, X. Cai, J. Li, M. Zheng, Z. Chen, C. Yu, Green synthesis of silver
nanoparticles using tea leaf extract and evaluation of their stability and
antibacterial activity, Colloids Surf. A 444 (2014) 226–231.
Inorganic Chemistry Communications 132 (2021) 108808
[18] P. Kouvaris, A. Delimitis, V. Zaspalis, D. Papadopoulos, S.A. Tsipas, N. Michailidis,
Green synthesis and characterization of silver nanoparticles produced using
Arbutus unedo leaf extract, Mater. Lett. 76 (2012) 18–20.
[19] M. Aravind, A. Ahmad, I. Ahmad, M. Amalanathan, K. Naseem, S.M.M. Mary,
C. Parvathiraja, S. Hussain, T.S. Algarni, M. Pervaiz, M. Zuber, Critical green
routing synthesis of silver NPs using jasmine flower extract for biological activities
and photocatalytical degradation of methylene blue, J. Environ. Chem. Eng. 9
(2021), 104877.
[20] Z. Ni, Z. Wang, L. Sun, B. Li, Y. Zhao, Synthesis of poly acrylic acid modified silver
nanoparticles and their antimicrobial activities, Mat. Sci. Eng. C 41 (2014)
249–254.
[21] M. Nakhjavani, V. Nikkhah, M.M. Sarafraz, S. Shoja, M. Sarafraz, Green synthesis
of silver nanoparticles using green tea leaves: Experimental study on the
morphological, rheological and antibacterial behaviour, Heat Mass Transfer 53
(2017) 3201–3209.
[22] X. Pan, Z. Chen, Y. Cheng, D. Pan, S. Yin, F. Huang, X. Guan, Z. Lin, The analysis of
the immobilization mechanism of Ni(II) on Bacillus cereus, J. Nanosci. Nanotechnol.
11 (2011) 3597–3603.
[23] I. Fatimah, Green synthesis of silver nanoparticles using extract of Parkia speciosa
Hassk pods assisted by microwave irradiation, J. Adv. Res. 7 (2016) 961–969.
[24] G. Bagherzade, M.M. Tavakoli, M.H. Namaei, Green synthesis of silver
nanoparticles using aqueous extract of saffron (Crocus sativus L.) wastages and its
antibacterial activity against six bacteria, Asian Pac. J. Trop. Bio. 7 (2017)
227–233.
[25] Y.P. Wu, Y. Yang, Z.J. Zhang, Z.H. Wang, Y.B. Zhao, L. Sun, Fabrication of cotton
fabrics with durable antibacterial activities finishing by Ag nanoparticles, Text.
Res. J. 89 (2019) 867–880.
[26] X.H. Pan, S.L. Chen, D.Z. Li, W.H. Rao, Y.L. Zheng, Z.Y. Yang, L. Li, X. Guan,
Z. Chen, The Synergistic Antibacterial Mechanism of Gentamicin-Loaded CaCO3
Nanoparticles, Front. Chem. 5 (2018) 130.