Biosynthesis of silver nanoparticles

We obtained the vegetal extract by boiling (95 °C) for 5 min 2 g of Lagg^® green tea (Camellia sinensis) in 100 mL of deionized water, cooled to 22 °C and filtered with Whatman No. 4 paper. From a 10 mM solution of AgNO₃ (Sigma Aldrich from Mexico, ACS ≥ 99.0%) prepared with deionized water, we took 5 mL, added 3 mL of aqueous extract of Camellia sinensis, and then adjusted the solution to 15 mL with deionized water (modification by ^{Carrillo, 2014} and ^{Nakhjavani et al., 2017}) and exposed to sunlight for 5 minutes.

Characterization of Ag NPs

We evaluated the formation of the NPs with spectroscopy (UV-vis) in an HP 845x UV-visible System Spectrophotometer in a range of 350 to 700 nm, to verify the presence of the surface plasmon resonance (SPR). The stability over time of NPs was verified at 1 h, 2 h, 3 h, 4 h, 5 h, 24 h, 48 h, 72 h, 5 d, 12 d, 30 d, 60 d, 90 d, 130 d, 160 d, 200 d, and 9, 10, 11, 12, 13 and 14 months. We studied the morphology and dimensions of the Ag NPs with an Electronic Transmission Microscope (MET, Tecnai 2 Spirit, Thermo Fisher Scientific-Fei Copany), in bright field mode, at 150000 and x300,000 magnifications and 120 kV.) The largest (DMa) and smallest (DMe) diameters were measured in 251 particles with the TIA 4.7 SP3 image processor (Tecnai: Imagining & Analysis, USA) to obtain the average diameter and the roundness index (IR = DMa/DMe).

Biological material and establishment of the experiment

We obtained stems of Rosa hybrida cv. Freedom from the commercial greenhouse of the company “Flores Selectas of Tequexquinahuac”, S. de P. R. de R. L. (19° 28' 51.26" N, 98° 50' 23.03" W), in Tequexquinahuac, Texcoco, State of Mexico. The floral stems were cut the same day, with a harvest index of 2, according to ^{De la Cruz et al., (2015)} and they were used without receiving any postharvest treatment. We adjusted the stem lengths to 35 cm ± 2 and 6 leaves.

In a preliminary study, the concentration of 1 ppm NPs appeared to be the most efficient in reducing vascular tissue obstruction and the bacterial population in the preservative solution, so we used this concentration for the present study. We prepared the solutions with distilled water from a solution with 1 ppm of Ag NPs, with and without sucrosesucrose (2%) and two pH (5.8 and 3.4) by the addition of citric acid. The control treatment was distilled water pH 5.6.

Experimental design and statistical analysis

The experimental design was completely randomized with the following treatments: 1) 1 ppm Ag NPs, 2) 1 ppm Ag NPs + 2% sucrosesucrose (pH 6), 3) 1 ppm Ag NPs + citric acid (pH 3), 4) 1 ppm Ag NPs + citric acid + 2% sucrosesucrose and 5) control (distilled water, pH 6). The experimental unit was a floral stem in a vase (250 mL) with 200 mL of preservative solution, with 15 replicates per treatment. The stems were placed in a room at 25 ± 2 °C and 60% relative humidity, and 2 cm were cut at the base on days 4 and 9. The vases were covered with parafilm to avoid evaporation. Results were analysed with ANOVA and the treatment means were compared with the Tukey test (p ≤ 0.05) using the SAS System 9.0 software.

Variables evaluated

We measured water consumption every day with a digital balance (Setra, S1-2000, USA), to calculate the volume (mL) of water consumed by the stem. We estimated the change in fresh weight from the difference between the initial fresh weight (PF0) and the fresh weight at 24 h (PF1) x 100 until the end of the vase life. The vase life (d) or longevity was measured at the beginning of the experiment until the senescence of the stems, which was determined considering the presence of any of the following characteristics: wilting of the petals, darkening of ≥ 30% of the edge of external petals, neck bending and relative weight of 85% (^{Hernández et al., 2009}). We evaluated the floral opening and the maximum floral opening by daily measuring the diameter of the button (mm) with a digital vernier scale (Trupper, Mexico). The bacterial count (Log 10 CFU mL^-1) in the preservative solution was measured on the third and seventh days in 5 vases per treatment per date, for which we used 3M^TM Petrifilm^TM plates (3M MEXICO S.A. DE C.V.); on this account, we placed 1 mL of vase solution per plate and incubated it at 22 °C for 24 h, under aseptic conditions. In cases of excessive bacterial colonies, we diluted 1:10 and 1:20 the solution with distilled water, depending on the case.

Characterization of nanoparticles

^{Brahmachari et al. (2014)} suggest that in Oncimum sanctum eugenol is the predominant chemical compound, so under solar radiation the phenolic OH bond undergoes homolytic cleavage to form a radical that eventually transfers its electron to the silver ion (Ag⁺) to form Ag NPs. The UV-Vis absorption spectra provide information on the optical properties dependent on the size, distribution and surface properties of metallic NPs (^{Ider et al., 2016}).

With the biosynthesis method with 5 mL of 10 mM AgNO₃ + 3 mL of Camellia sinensis aqueous extract, the absorption peak was 436.8 ± 2.154 nm from the first hour of reaction, which increased in time, and the highest absorbance was obtained at month 14 (Figure 1). This indicates an increase of Ag NPs synthesized over time (^{Carrillo et al., 2014}). The amplitude of the plasmon peak depends on the size distribution of NPs (^{Pastoriza et al., 2002}). ^{Vilchis-Nestor et al. (2008)} observed that in the solutions with AgNO₃, 10 mM and aqueous extract of C. sinensis from 1 to 10 mL the reduction of silver ions and the formation of stable NPs occurred within the first 4 h of reaction. The wavelengths of the maximum absorption of Ag NPs at 4 h (440, 445 and 430 nm) were similar to those obtained by ^{Hussain and Khan (2014)}, ^{Kamal et al. (2010)} and ^{Vilchis-Nestor et al. (2008)}. The maximum absorbance (20.89 u.a.) was higher in our study compared with those already mentioned. The experiment with black tea (C. sinensis) conducted by ^{Begum et al. (2009)} shows a similar maximum absorbance (460 nm) to that reported. The maximum absorbances increased over time. At 60 min (1 h) the maximum absorbance was 11.99 u.a. (λ of 433 nm), but at 14 months the absorbance increased to 31.06 u.a. (λ 446 nm). The SPR were symmetric, indicating the stability of the system over time (^{Ider et al., 2016}; ^{Carrillo et al., 2014}). The symmetric shape of the PSR peak indicates the existence of spherical-shaped Ag NPs and a uniform distribution (^{Ider et al., 2016}).

Figure 1 Spectra of absorption (SPR) of silver nanoparticles synthesized with 10 mM AgNO₃ (5 mL) and aqueous extract of C. sinensis (3 mL) at 24 °C taken at different times. 

A greater absorption registered in the peak of the SPR reflects a greater reduction of silver ions and, in turn, a higher concentration of Ag NPs (^{Ider et al., 2016}; ^{Carrillo et al., 2014}).

Eighty percent of the Ag NPs synthesized and stored for 14 months at 4 °C showed diameters between 10-40 nm and a quasi-spherical shape (roundness index 0.893 ± 0.006) (Figure 2).

Figure 2 Transmission Electron Microscopy of Ag NPs synthesized with + 5 mL 10 mM AgNO₃ + 3 mL of C. sinensis extract. A and B, morphology and distribution. D, measurement of the largest and smallest diameter for roundness index. Tecnai 2G Spirit, at 120 kV. 

The Ag NPs produced had an average size of 21.509 ± 0.634 nm and 81.6% were less than 30 nm. The size intervals with the highest frequency were 10 to 20 and 20 to 30 (Figure 3). ^{Hussain and Khan (2014)} obtained nanodiscs 47 nm long and 10 nm wide, synthesized with 1 mM AgNO₃ and 4% extract with catechins; and ^{Sun et al. (2014)} synthesized NPs of 20-90 nm with 10 mM AgNO₃ and with different concentrations of tea extract. In addition, ^{Rastogi and Arunachalam (2011)} obtained spherical Ag NPs of 7.3 ± 4.4 nm by exposing a solution of [Ag (NH₃) ₂] + 0.1 M with extract of Allium sativum (garlic) to sunlight for 15 min.

Figure 3 Size distribution of Ag NPs synthesized with 5 mL of 10 mM AgNO₃ + 3 mL of aqueous extract of C. sinensis and 5 min of exposure to sunlight (n = 251). 

The roundness index (IR) was high, 0.893 ± 0.006, and indicated a quasi-spherical shape.

Changes in fresh weight and water consumption

In the treatment with citric acid (pH 3) we obtained the highest relative fresh weight (PFR) (104.77%) on the third day and during the whole experiment it was significantly higher than the control (pH 6) (PFR, 86.24%), which was reflected in a reduction of the vase life. ^{Safa et al. (2015)}, ^{Liu et al. (2012)} and ^{Lü et al. (2010)} observed that the use of Ag NPs in vase solutions significantly decreases the loss of PFR in stems of Gerbera jamesonii cv. 'Balance', Acacia holasericea and R. hybrida cv. 'Movie Star'. In all these tests, the PFR was significantly lower in the control treatment. The reduction of pH by the addition of citric acid to a solution with NPs improved the preservative effect of the Ag NPs and allowed a greater absorption of water by the stem. The treatment of Ag NPs with sucrosesucrose (pH 6) had an accelerated loss of PF from day 4. The treatments of Ag NPs without sucrosesucrose caused gain of PF until day 3, and later they lost weight progressively (Figure 4).

Figure 4 Stems of Rosa hybrida cv. Freedom after ten days of evaluation. T-1: Water; T-2: 1 ppm of Ag NPs; T-3: 1 ppm Ag NPs + sacchrose; T-4: 1 ppm of Ag NPs + citric acid, and T-5: 1 ppm of Ag NPs + citric acid + sacchrose. 

The water balance results from the difference between water consumption and water loss in the stem (^{Ried and Jiang, 2012}). The absorption of water stops during dry management due to the phenomenon of embolism, in which the water column in the xylem does no longer flow due to a bacterial occlusion or formation of tampons by tyloses, and gels in the vessels (^{Van Doorn and Reid, 1995}). The control and treatments with Ag NPs without sucrosesucrose registered higher water consumption during the first 2 d (54.16 mL) (Table 2). The treatment with Ag NPs + citric acid had the highest cumulative consumption (147.08 mL) on day 8. According to ^{Nazemi and Ramezanian (2013)}, treatments with Ag NPs increase the rate of water absorption in Rosa cv. Avalanche, but in the present study we corroborated the positive effect of acidification of the medium on water absorption due to the reduction of vascular blockage (^{Reid and Jiang, 2012}, ^{Reid and Kofranek, 1980}).

The control and sucrosesucrose treatments had less accumulated water consumption. This variable related to vase life, and stems with a lower water consumption showed early wilting, loss of color and neck bending (Figure 5). The Ag NPs + citric acid treatment favored longer vase life, increased water consumption, and obtained greater fresh weight and better vase life quality (Figure 5). Ethylene is a phytohormone that has negative effects on the floral longevity of species sensitive to this molecule, but this effect can be inhibited by the Ag + ion (^{Reid and Jiang, 2012}).

Figure 5 Effect of Ag NPs with sacchrose and citric acid in the vase life of stems of Rosa hybrida cv. Freedom. Different letters indicate significant differences (Tukey, p ≤ 0.05, n=15). 

In the present study there were no significant differences between treatments regarding the diameter of the maximum floral opening, but we did detect them in the floral opening speed which was delayed in the solution with citric acid + 1 ppm of Ag NPs (Figure 5), variable related to the longevity of the flower.

Vase life

The longest vase life treatment was NPs + citric acid (7.3 d). In Gerbera jamesonii we obtained the maximum vase life only with the addition of Ag NPs to the vase solution (^{Geshnizjany et al., 2014}). In the experiment with the cv. of Rosa Freedom, the addition of sucrosesucrose to the vase solutions with 1 ppm of Ag NPs decreased the vase life, in contrast to the sole presence of Ag NPs (Figure 5). The use of sucrosesucrose in the vase solutions has benefits: longer vase life and relative fresh weight in stems, improvement in floral opening, pigmentation, reduction of sensitivity to ethylene and improved water relations (^{Asgari et al., 2013}; and Jiang, 2012; ^{Hayat et al., 2012}; ^{Lü et al., 2010}). Yet in the present study, sucrosesucrose had an adverse effect on solutions with Ag NPs, which increased the bacterial count in the solution and reduced the flower vase life. This adverse effect was counteracted by citric acid, which decreased the adverse effects of sucrosesucrose by acidifying the solution (Figure 7). The decrease of pH limits the proliferation of microorganisms (^{Hayat et al., 2012}; ^{Jowkar et al., 2012}), related to vessel obstruction and lower water absorption (^{Macnish et al., 2008}; ^{Van Doorn, 1997}).

Bacterial count of the solution

In this study, the addition of sucrosesucrose in the preservative solution with AG NPs promoted bacterial growth in the solution on the third day, but on the seventh day the control (water) recorded the highest bacterial count (Table 3). The treatment with the lowest bacterial count (CFU mL^-1 0.85) was that of Ag NPs + citric acid (pH3). The treatments with citric acid, with pH 3, had lower CFU mL^-1 than the other treatments, with pH 6, so the acidification of the solutions contributed to the inhibition of bacterial growth.

The number of CFUs increased over time, the same as in the experiment by ^{Liu et al. (2009}, ²⁰¹²⁾, who did not find significant differences between the control treatment (distilled water) and the treatment with Ag NPs (5 mg L^-1), in pulse solutions. According to ^{Okafor et al. (2013)}, concentrations of 4 ppm of Ag NPs synthesized with plant extracts (aloe, geranium, magnolia and black cohosh) and 3-9 nm in size, inhibited the bacterial growth of E. coli.

In a previous study all the treatments presented bacterial growth in the solution, but the treatments with 1, 5 and 10 ppm of Ag NPs (21.5 nm) synthesized with aqueous extract of C. sinensis had an antimicrobial activity that prevented the occlusion of a large number of xylem vessels. ^{Okafor et al. (2013)} observed that Ag NPs synthesized with aloe extract had the highest antibacterial activity compared to treatments with 2 and 4 ppm of Ag NPs (3-9 nm) synthesized with extracts of geranium, magnolia and black cohosh. According to ^{Jowkar et al. (2013)}, there was no microbial growth in vase solutions treated with 1, 2.5 and 5% of Ag NPs, but these concentrations are higher than those of our study. However, ^{Carrillo et al. (2016)} reported that vase solutions with 0.01, 0.05, 0.1, 0.5, 1 and 5 mM of Ag NPs (10.3 nm) had no CFU in the solution. ^{Reid and Jiang (2012)} mentioned that reducing the pH of the solution, either with citric acid or Al2 (SO₄) ₃, helps to reduce bacterial growth, but it is insufficient in itself because acidophilic yeasts and bacteria can quickly colonize the vase solution.

It follows that not all species of microorganisms affect vase life in the same way. ^{Zagory and Reid (1986)} observed that an unidentified yeast caused more damage in the vase life of carnation (Dianthus caryophyllus) with an inoculum of 103 CFU mL^-1 than Pseudomonas sp. with an inoculum of 106 CFU mL^-1.