Preparation of Piper betle leaf extracts (PbLE)
Piper betle (ver. Kali Bangla) leaves have obtained from landrace of Paschim Medinipur, West Bengal, India. The material was washed, dried in hot air oven (40 °C ± 1 °C) for 48 h. The dried leaves were grounded into fine powder with mortar and pestle and stored at − 20 °C. The ground powder (1 g) was Soxhlet extracted using 80% ethanol for overnight. Final crude extract was concentrated and dried by rotary vacuum evaporator at a temperature less than 50 °C following concentrated extract which was maintained at 4 °C. Working PbLE stock solution of concentration 10 mg/ml was prepared by dissolving 100-mg dried crude extract in 500 μL of dimethyl sulfoxide (DMSO) and further diluted with distilled water (Maity et al. 2014).
Synthesis of silver nanoparticles from Piper betle leaf extracts (PbLE)
Silver nitrate (AgNO3) was obtained from Sigma Aldrich chemicals. The reduction reaction of pure silver ions (Ag+) into silver (Ag0) was carried out with PbLE using the method by Kumar et al. (2014). The protocol used involved drop-wise addition of 250 μL of PbLE (1 mg/ml) to 50 ml freshly prepared 1 mM of silver nitrate aqueous solution in a Teflon container. This was stirred continuously at different temperature (30 °C, 40 °C, 50 °C, 60 °C, and 70 °C) for 4 h using a magnetic stirrer. This reduction reaction was confirmed by the color change of the solution from colorless to yellowish-brown. When nanoparticles have been formed, the pH of the aqueous solution was changed using 0.1 (N) KOH in different (pH -6, pH -7, pH -8, pH -9 and pH -10) and incubated for overnight at room temperature.
Characterization of silver nanoparticles
The UV-vis spectrum of the reduced product was measured in the range of visible region (300–800 nm) with the help of UV-VIS spectrophotometer (Genesys 10S UV-VIS, Thermo Scientific). The size and morphology of the Piper betle mediated silver nanoparticles were studied by the transmission electron microscopy (TEM) image by using (Philips CM12) at Central Research Facility, Indian Institute of Technology, Kharagpur, India.
Plant material and treatments
Fresh cut spikes of Gladiolus (Gladiolus grandiflorus) were obtained from a local commercial market (Haldia, West Bengal, India) and brought to the laboratory early in the morning. The spikes were cut uniformly having a length of 65 cm with six pairs of florets per spike.
The experiments were conducted in completely designed setups by placing the spikes in a 250-ml bottle containing 100 ml of different vase solutions. To prevent the surface evaporation of the vase solution, non-absorbent cotton was used to plug the bottles. The bottles were placed at a room temperature of 25 ± 2 °C, relative humidity 70 ± 3% under continuous illumination (range 400–700 nm) system. At every 48-h intervals, the vase solutions in the bottles were changed and the remaining volume of vase solution was measured. The six vase solutions used were as follows: (1) control, distilled water; (2) T1, 4% sucrose; (3) T2, 4% sucrose + 100 ppm 5-SSA; (4) T3, 4% sucrose + 2 ppm PbSNPs; (5) T4, 4% sucrose + 4 ppm PbSNPs; (6) T5, 4% sucrose + 6 ppm PbSNPs.
Observations of vase life, fresh weight (FW), height of spike, total solution uptake, flower diameter, relative water content (RWC), number of opened flower, activities of SOD and CAT, lipid peroxidation, total soluble protein, total soluble sugar, chlorophylls and carotenoids content, microbial growth, and vascular blockage were recorded.
All the morphological and biochemical estimations were done in triplicate using flower petal and leaf sample from the post-harvest gladiolus cut spikes at different stages (initial day, 2nd, 5th, 7th, 9th, 12th days of vase life) of flower development.
Vase life
Vase life of the cut spikes depends on various physical appearances such as slumping down of spike head, flower discoloration, and shedding of petals. Vase life was calculated as the time period during which the fresh weight of the spikes was retained the same to that at the initial day of harvest (Ezhilmathi et al. 2007).
The percentage increase or decrease in fresh weight and height of spikes were recorded at initial day, 2nd, 5th, 7th, 9th, and 12th days of vase life.
Total solution uptake
The vase solution remaining in the bottles was recorded. Hence, the water uptake was calculated using the following formula.
$$ {N}^{\mathrm{th}}\mathrm{day}\ \mathrm{water}\ \mathrm{uptake}\ \left(\mathrm{ml}\right)={N}^{\mathrm{th}}\mathrm{day}\ \mathrm{total}\ \mathrm{volume}\ \left(\mathrm{ml}\right)-{\left(N\hbox{-} 1\right)}^{\mathrm{th}}\mathrm{day}\ \mathrm{total}\ \mathrm{volume}\ \left(\mathrm{ml}\right) $$
$$ \mathrm{Water}\ \mathrm{uptake}\ \mathrm{rate}\ \left(\mathrm{ml}\right)=\mathrm{amount}\ \mathrm{of}\ \mathrm{water}\ \mathrm{consumed}\ \mathrm{per}\ \mathrm{day}\ \left(\mathrm{ml}/\mathrm{day}\right) $$
$$ \mathrm{Total}\ \mathrm{water}\ \mathrm{uptake}=\mathrm{water}\ \mathrm{uptake}\ \mathrm{on}\ N+\left(N+1\right)+\left(N+2\right)\kern0.5em {\left(N+n\right)}^{\mathrm{th}}\mathrm{day}..\left(\mathrm{where}\ n\ge 1\right) $$
Flower diameter
The mean diameters of the bloomed flowers were recorded on 2nd, 5th, 7th, 9th, and 12th days of vase life. Flower diameter was measured by taking three measurements, which crossed at the center of the opened flowers, and finally, the mean was calculated.
Relative water content
RWC of the fresh flower petals was recorded by using a 0.5-g fresh petal sample which was dipped in water for 4 h to find the turgid weight. Consequently, they were dried in a hot air oven at 80 °C to achieve a constant weight (Weatherley 1950).
$$ \mathrm{RWC}=\left[\left(\mathrm{Fresh}\ \mathrm{weight}-\mathrm{dry}\ \mathrm{weight}\right)/\left(\mathrm{turgid}\ \mathrm{weight}-\mathrm{dry}\ \mathrm{weight}\right)\right]\times 100 $$
Antioxidant enzymes activity
The enzyme extract for SOD and CAT was prepared by grinding of a 0.5-g petal tissue in ice cold extraction buffer (0.1 M potassium phosphate buffer of pH 7.6 containing 0.5 M EDTA) with a pre-chilled mortar and pestle. The homogenate was centrifuged at 4 °C in refrigerated centrifuge (Remi C-24 plus) for 15 min at 15,000 rpm, and the supernatant was used to determine enzyme activity.
SOD activity was estimated by recording the decrease in absorbance of nitro-blue tetrazolium dye (Dhindsa et al. 1981; Datta Gupta and Datta 2003). Three milliliters of the reaction mixture was prepared which contained 100 mM potassium phosphate buffer (pH − 7.6), 100 mM methionine, 1 mM nitro-blue tetrazolium (NBT), 0.5 M EDTA, 0.1 ml enzyme, and distilled water for making up mixture volume. The reaction was started by adding l00 μM riboflavin and placing the tubes under three 5 W fluorescent lamps for 15 min. The reaction was stopped on the withdrawal of the light source. The reaction mixture without enzyme was used as a standard. A non-irradiated reaction mixture containing enzyme served as a blank. The tubes which were enzyme less showed the maximum color, and the blank showed no color development. Finally, absorbance was recorded at 560 nm, and one unit of enzyme activity was taken as the quantity of enzyme which reduced the absorbance reading of samples to 50% in comparison with the reaction mixture lacking enzyme.
CAT activity was obtained by monitoring the decrease in absorbance due to hydrogen peroxide (H2O2) at 240 nm (Aebi 1984; Datta Gupta and Datta 2003). The reaction mixture for measuring the CAT activity consisted of 3-mL 50 mM potassium phosphate buffer (pH -7), 5 μl H2O2, and 50 μl enzyme extract, whereas only 3-ml 50 mM potassium phosphate buffer (pH -7) was served as blank. The reaction began by the addition of H2O2, and the decrease in the absorbance value was recorded for 30-s interval of 10 min at 240 nm. Hence, by taking the standard curve of known concentrations of H2O2 as a reference, the amount of H2O2 decomposed was calculated and thus the amount of enzyme activity.
The level of lipid peroxidation depends on the terms of thiobarbituric acid reactive substances (TBARS). Thus, the amount of TBARS was used to determine the lipid peroxidation level (Heath and Packer 1968). The protocol for estimation of lipid peroxidase activity involved homogenization of the petal sample (0.5 g) in 10 ml 0.1% trichloroacetic acid (TCA). The homogenate was centrifuged for 15 min at 10,000 rpm. The supernatant aliquot (2 ml) was added in 4 ml of 0.5% thiobarbituric acid (TBA) in 20% TCA. The mixture was then heated at 95 °C for 30 min following which it was quickly cooled in an ice bath. The mixture was further centrifuged at 10,000 rpm for 10 min. The absorbance spectrum of supernatant was recorded at 532 nm and 600 nm. The TBARS content was calculated according to its extinction coefficient [155 mM−1 cm−1]. The final result was obtained by subtracting the non-specific absorbance at 600 nm.
Total soluble protein
The protein sample was prepared by grinding of a 0.5-g petal tissue in ice cold extraction buffer (0.1 M potassium phosphate buffer of pH 7.6 containing 0.5 M EDTA) with a pre-chilled mortar and pestle. The homogenate was centrifuged at 4 °C in refrigerated centrifuge (Remi C-24 plus) for 15 min at 15,000 rpm, and the supernatant was used to determine total soluble protein using the Bradford Assay method (Bradford 1976). The absorbance of blue color was read at 595 nm using UV-VIS spectrophotometer. The amount of protein was quantified by using a standard curve of known concentration.
Total soluble sugar
The total soluble sugar contents were carried out by hydrolyzing into simple sugars using dilute hydrochloric acid (Sadasivam and Manickam 1996). In the presence of hot acidic medium, glucose is dehydrated to hydroxymethyl furfural. This compound forms with anthrone, a green color product. A 0.5-g petal sample was hydrolyzed by keeping in a boiling water bath for 3 h with 5 ml of 2.5 N HCl. After cooling to room temperature, aqueous sample was neutralized with solid sodium carbonate until the effervescence ceases. The homogenize was centrifuged at 15,000 rpm for 15 min after making the volume up to 10 ml with distilled water. Five hundred-microliter supernatant was mixed gently with 4 ml of anthrone reagent (2 mg/ml) and heated for 8 min in a boiling water bath. The mixture was cooled rapidly and read the green to dark green color at 630 nm. The amount of total soluble sugars present in the sample was calculated using a standard curve of known concentration.
Chlorophyll and carotenoid
Chlorophyll was extracted with 80% acetone, and the absorption at 663 nm, 645 nm, and 470 nm was read in a spectrophotometer after being centrifuged twice at 5000 rpm for 5 min at room temperature. The amount of chlorophyll present in the supernatant was calculated using the following equations (Arnon 1949),
$$ \mathrm{Total}\ \mathrm{chlorophyll}\ \mathrm{a}\ \left(\mathrm{mg}\;\mathrm{g}\hbox{-} {\;}^1\mathrm{FW}\right)=\left[12.7\left({A}_{663}\right)\hbox{-} 2.69\left({A}_{645}\right)\right]\times \left(V/{W}^{\ast }1000\right) $$
$$ \mathrm{Total}\ \mathrm{chlorophyll}\ \mathrm{b}\ \left(\mathrm{mg}\;\mathrm{g}\hbox{-} {\;}^1\mathrm{FW}\right)=\left[22.9\left({A}_{645}\right)\hbox{-} 4.68\left({A}_{663}\right)\right]\times \left(V/{W}^{\ast }1000\right) $$
$$ \mathrm{Total}\ \mathrm{chlorophyll}\ \left(\mathrm{mg}\;\mathrm{g}\hbox{-} {\;}^1\mathrm{FW}\right)=\left[20.2\left({A}_{645}\right)+8.02\left({A}_{663}\right)\right]\times \left(V/{W}^{\ast }1000\right) $$
$$ \mathrm{Total}\ \mathrm{carotenoid}\ \left(\mathrm{mg}\;\mathrm{g}\hbox{-} {\;}^1\mathrm{FW}\right)=\left[1000\times {A}_{470}-3.27\ \left(\mathrm{chl}\ \mathrm{a}\right)-104\ \left(\mathrm{chl}\ \mathrm{b}\right)\right]\div 229\ \left(V/{W}^{\ast }1000\right) $$
(where A = absorbance at specific wavelength; V = Final volume of chlorophyll extract in 80% acetone; W = Fresh weight of tissue extracted; chl = Chlorophyll).
Microbial growth
The evaluation of microbes was studied to determine the effectiveness of PbSNPs in controlling the microbial growth in the vase solution of the cut flowers gladiolus. Ten-microliter vase solutions from each of the bottles were analyzed after 10 times dilution to check the presence of microbes after the 6th day of vase life by placing it on nutrient agar (Himedia) media. The bacterial colony was observed and counted after overnight incubation at 37 °C.
Vascular blockage
Vascular blockage was determined by microscopic observation of the transverse section of xylem vessels. The spike ends were cut on the 6th day of vase life and immediately stained with safranin dye and observed under microscope (Olympus).
Statistical analysis
Standard errors of the means were calculated, and LSD (P ≤ 0.05) was performed to check the level of significance of the difference with the help of ANOVA test using Microsoft Excel data analysis tools.