Acclimatization of Experimental Plants
Three species of indoor ornamental plants (Chlorophytum, Dracaena and Ficus) were selected for studying the combined effect of benzene and formaldehyde. Plants were purchased from the nursery and were transferred into new pots containing mixture of coco peat, peat moss and perlite (2:2:1). Pots were then placed in a growth chamber at 24oC temperature and 12 hrs light period, for 2 weeks for acclimatization before application of treatments.
Application of Benzene and Formaldehyde
Experiments were conducted in an airtight glass chamber of volume 0.128 m3 having a fan and an inlet for injection of formaldehyde and benzene as shown in figure 1. The chamber was cleaned properly with a cotton towel and the pots containing plants were covered with aluminum foil to avoid the direct contact of formaldehyde and benzene with media. Individual plant species was then placed in the chamber and the chamber was sealed with a lid covered by teflon tape. Plants were then exposed to different combined concentrations (2 and 4 ppm) of formaldehyde and benzene. After 24 hrs exposure to benzene and formaldehyde, the plants were taken out of the chamber and their biochemical analysis were carried out.
Enzymatic Antioxidants
Catalase, Peroxidase and Superoxide Dismutase
Freshly harvested leaf (0.20 g) was grounded and extracted in 2ml of pH. 7 potassium phosphate buffer (100 mM). The sample was then placed in centrifuge for 5 minutes at 15000 rpm. The supernatant was transferred to a separate eppendorf tube for analysis of catalase, peroxidase and superoxide dismutase. The catalase activity was measured by UV-vis spectrophotometer (T80+ Spectrometer) according to Aebi (1984). The absorbance of reaction mixture containing 50 mM of potassium phosphate buffer sample and 40 mM H2O2 was taken at 240 nm for 3 minutes. For peroxidase activity, absorbance of reaction mixture containing pH. 7 potassium phosphate buffer (50 mM), sample extract, 40 mM H2O2 and 1% guaiacol was taken at 420 nm for 3 minutes. Superoxide dismutase activity of plant extract was determined according to Beyer et al. (1987). The reaction mixture of plant extract, 50 mM phosphate buffer, 2 mM EDTA, 14 mM L-methionine, 55 mM NBT, and 0.025% Triton-X was incubated for 20 minutes under fluorescent light and absorbance was taken at 560 nm.
Ascorbate Peroxidase
In order to assess ascorbate peroxidase activity, freshly harvested leaf sample (0.20 g) was extracted in potassium phosphate buffer pH 7 with 0.5 mM ascorbate. Mixture was then centrifuged at 15000 rpm for almost 5 minutes. Supernatant was take in another eppendorf tube for further use. Firstly, 2 ml of 50 mM pH 7 potassium phosphate buffer was added to glass cuvette, followed by 100 μl of 1 mM ascorbate, 0.1 mM EDTA, 1 mM H2O2, and 100 μl of sample, while absorbance was recorded at 290 nm.
Total Antioxidants
Total antioxidants activity was determined by using DPPH salt method (Brand et al. 1995). Freshly harvested leaf sample (80 mg) was grounded and extracted in 80% methanol. Freshly prepared DPPH solution (0.004%) was added in reaction tubes containing sample extract and incubated in the dark for 50 minutes. A spectrophotometer (T80+ UV/VIS Spectrometer) was used to measure the absorbance at 517 nm. Activity was calculated by measuring the change in sample extract and blank by using following equation.
The radical scavenging activity was expressed as the radical scavenging % using the equation,
DPPH Scavenged (%) = {(A0 –A1)/A0} × 100
Where A0 is the absorbance of control and A1 is the absorbance of the sample extract.
Non-Enzymatic Antioxidants
Results of two-way ANOVA presented in supplementary table 1 describe the significance of main factors (treatments of benzene and formaldehyde, and plants) and their interaction. All the non-enzymatic antioxidants were found statically significant at p≤0.05.
Total Phenolic Content
Plant leaf sample (80 mg) was weighed, ground, and extracted in 97% methanol. Folin-Ciocalteu reagent (10%) was prepared. The reaction tube containing 200 μl of the FC reagent, 200 μl of the sample extract 1.6 mL of 700 mM Na2CO3 incubated for 2 hrs. Absorbance was measured at 765 nm using spectrophotometer. Gallic acid was used as standard to quantify total phenolic contents in samples (Singleton et al. 1999).
Total Flavonoids
Fresh leaf sample (80 mg) was grounded and extracted in 85% methanol. Total flavonoid contents were assessed following the protocol of Olajire et al. (2011).
Proline
The proline content was quantified by ninhydrin method following the protocol of Bates et al. (1973). Freshly harvested plant leaf (250 mg) was weighed, grounded, and extracted in 2 ml sulfosalicylic acid. After centrifugation at 15000 rpm for 5 minutes, the supernatant was taken and used further. A reaction tube containing sulfosalicylic acid (3%), Glacial acetic acid (60%), Ninhydrin and sample was placed in water bath at 97°C for 1 hr. Then the colored phase was separated by adding 2 ml toluene in the reaction tubes. Absorbance of extracted pigmented layer was measured at 520 nm. Standard curve was developed by using proline as a standard.
Total Carotenoids
Total carotenoids content of plant leaves was determined following the protocol of Lichtenthaler (1983). Plant leaf sample (100 mg) was grounded and extracted in 10 ml acetone (80%). Top of the test tubes were covered with aluminum foil and kept in dark for overnight. The sample mixture was centrifuged, and supernatant was taken in separate tube. Absorbance was measured through UV-vis spectrophotometer at 470 nm.
Statistical Analysis
Experiment was conducted in completely randomized design with three biological replicates. The collected data was subjected to two-way analysis of various (ANOVA) followed by Tukey test at ≤ 0.05 by using IBM-SPSS software. Pearson correlation method was used to find out the correlations among studied parameters.