Plant materials and treatments
Tea [Camellia sinensis (L.) Kuntze] cultivar Chin-Shin Oolong was used in the experiments in this study. All seedlings of the tea plant were grown in the same area in Nantou, Central Taiwan, over a 2-year period. The tea plant seedlings were approximately 50 cm high. For ACC treatment, 2-year-old tea seedlings were irrigated with 20 mL of 100 µM ACC per day for 5 days; the control (CK) samples were irrigated with 20 mL of water instead. Fresh samples each with one tip and two leaves were collected from tea seedlings, frozen immediately in liquid nitrogen, and stored at − 80 °C until analysis.
Preparation of extracts from young tea seedling leaves
The samples were weighed, and lyophilized samples were homogenized to powder by using a disposable pestle. The extraction of tea was prepared with 1.0 mL of 75% ethanol and shaking the mixture vigorously with an Intelli-Mixer (MyLabTM RM-2 M) for 2 h at room temperature. The mixture extraction of tea was filtered by 0.45 µm polyvinylidene difluoride membrane filter (Pall Corporation, Glen Cove, NY, USA) before HPLC analysis.
HPLC analysis
Tea extracts were analyzed on a liquid chromatography system coupled with a Model 600E photodiode array detector (Waters Corporation, Milford, MA, USA). The chromatographic separation was performed on a Mightysil RP-18 GP column (250 × 4.6 mm i.d., 5 µm; Kanto Chemical Co., Tokyo, Japan). The mobile phase consisted of water containing 0.5% acetic acid (solvent A) and acetonitrile (solvent B).
The samples were eluted using the following gradient program, 0 min, 95% A and 5% B; 100 min, 75% A and 25% B. The column was kept at room temperature, the volume of each injection was 10 µL, and the flow rate was 1 mL/min. The PDA detector was set at 280 nm. The four major catechins (EC, EGC, ECG, and EGCG) shown in the HPLC profiles were identified as described in a previous study (Chen et al. 2014).
Determination of anthocyanins, flavonoids, and total phenol
The tea seedling samples (0.1 g) were excised and immediately used for anthocyanin, flavonoid, and total phenol content assays. For the anthocyanin and flavonoid content assays, the sample extracts in 2 mL of potassium phosphate buffer (100 mM, pH 7.8) were ground into powder with liquid nitrogen. The mixture of tea extraction was centrifuged at 16,000g and 4 °C for 30 min, and the resultant supernatant was measured at 600 and 320 nm by using a spectrophotometer (Metertec SP8001). One absorbance unit was defined as the quantity of anthocyanins and flavonoids that exhibits an absorbance of 1 at 600 and 320 nm. The total phenolic content assay was performed using a modified form of the procedure described by Singleton and Rossi (1965). The sample extracts in 1.0 mL of 75% ethanol were ground into powder with liquid nitrogen. The homogenate was centrifuged at 10,000g and 4 °C for 10 min, and 50 μL of the resultant supernatant was diluted with distilled water and added to 250 μL of the Folin–Ciocalteu reagent (0.2 mol/L). The reaction mixture reacted for 5 min before 50 μL of 20% Na2CO3 was added. The samples were then incubated at room temperature in darkness for 60 min, and the absorbance was measured at 760 nm. A calibration curve was obtained using 0–4000 mg of gallic acid per milliliter and used to calculate the total phenolic content of the tea seedling leaves.
Determination of antioxidant enzyme activity
The fresh leaves (0.2 g) of tea seedling were used immediately for enzyme extraction after excised, and subsequently homogenized with liquid nitrogen. Sodium phosphate buffer (50 mM; pH 6.8) was used as extraction buffer. The mixture of tea leaves extraction was then centrifuged at 15,000g for 30 min, and the resultant supernatant was used in the following enzyme activity assays. CAT activity was analyzed as described in previous studies (Chao et al. 2012; Kato and Shimizu 1985). Decreasing in absorbance at 240 nm was observed using a spectrophotometer (Metertec SP8001) which implied the decrease of H2O2. Activity of CAT was calculated on the basis of the extinction coefficient (40 mM−1 cm−1 at 240 nm) of H2O2. One unit of CAT was defined as the enzyme amount that degraded 1 μmol of H2O2 per minute. SOD activity assay was conducted based on Chao et al. (2012). The reaction buffer was Triethanolamine–diethanolamine buffer (100 mM; pH 7.4) containing ethylenediaminetetraacetic acid/MnCl2 (100 mM/50 mM, pH 7.4), 7.5 mM β-nicotinamide adenine dinucleotide (β-NADH), and 10 mM 2-mercaptoethanol and mixed with enzyme extract. The enzyme reaction was initiated by the addition of β-NADH, and detected the absorbance at 340 nm for 10 min. One unit of SOD was defined as the enzyme amount that inhibited the rate of β-NADH oxidation by 50%. For APX activity assay, the decrease in the ascorbic acid (AsA) concentration was determined according to the decline in absorbance at 290 nm, and the activity was calculated based on the extinction coefficient (2.8 mM−1 cm−1 at 290 nm) for AsA (Chao et al. 2012; Nakano and Asada 1981). Both soluble and ionically bound peroxidase (Total POX) activity assay was modified from previous studies (Lin and Kao 1999; MacAdam et al. 1992; Wu and Yang 2016). Samples were extracted by homogenized in liquid nitrogen with 50 mM potassium phosphate buffer (pH 5.8) containing 0.8 M KCl buffer. The 50 mM potassium phosphate buffer (pH 5.8), 21.6 mM guaiacol, and 39 mM H2O2 were added into enzyme extract for reaction. The enzyme reaction was initiated by adding H2O2, the absorbance was measured at 470 nm for 3 min. Total POX activity was calculated on the basis of the extinction coefficient of 26.6 mM−1 cm−1 at 470 nm for tetraguaiacol. One unit of POX was defined as the enzyme amount that caused the formation of 1 μmol of tetraguaiacol per minute.
Determination of DPPH radical scavenging activity
Potential antioxidant activity was determined using DPPH (2,2-diphenyl-1-picrylhydrazyl) according to Tadolini et al. (2000) with some modifications. The sample (0.1 g) extract was added to 1.0 mL of 75% ethanol. The mixture was shaken for 120 min through vortexing and left to centrifuge at 10,000g and room temperature in darkness for 10 min. The absorbance for the sample was measured using a SpectraMax M2 spectrophotometer at 521 nm against an ethanol blank. A control sample (ΔADPPH) was extracted after adding 0.19 mM DPPH solution to 0.2 mL of the respective extraction solvent. Every sample was extracted in triplicate, and the results were calculated based on four biologically independent experiments. The percentage of DPPH free radicals scavenged in the sample was calculated using the following equation:
$${\text{DPPH free radical scavenging ratio }}\left( \% \right) \, = \, \left[ {{{\left( {\Delta {\text{A}}_{\text{DPPH}} - \Delta {\text{A}}_{{{\text{sample}} + {\text{DPPH}}}} } \right)} \mathord{\left/ {\vphantom {{\left( {\Delta {\text{A}}_{\text{DPPH}} - \Delta {\text{A}}_{{{\text{sample}} + {\text{DPPH}}}} } \right)} {\left( {\Delta {\text{A}}_{\text{DPPH}} } \right)}}} \right. \kern-0pt} {\left( {\Delta {\text{A}}_{\text{DPPH}} } \right)}}} \right] \, \times 100$$
Oxygen radical absorbance capacity (ORAC) measurement
The ORAC assay was by using a modified form of the method described by Ou et al. (2001). The ORAC assay was measured the capacity of antioxidative compounds in test materials to inhibit the decrease in fluorescence induced by the peroxyl radical 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH). The samples (0.1 g) were homogenized with liquid nitrogen and added to 1 mL of double distilled H2O for incubation 10 min at 100 °C. The 20 μL extract was added to 120 μL 120 nM disodium fluorescein solution (72 nM, final concentration) and preincubated for 15 min at 37 °C. The mixture was rapidly added to 60 μL of 40 mM AAPH in the well of the microplate and the microplate was immediately placed in the reader, and the fluorescence was recorded at an 485 nm (excitation wavelength) and 535 nm (emission wavelength) every 1 min for 60 min. The blank (FL + AAPH) with phosphate buffer instead of the antioxidant solution and Trolox was used as the standard. All the reaction mixtures were detected in duplicate, and at least three independent assays were performed for each sample. Fluorescence was measured and recorded every 1 min (f0, f1, f2, f3,…, f60) until the fluorescence began to decline. The curves of antioxidant were initially normalized to the blank curve corresponding to the same assay by the factor fluorescenceblank,t= 0/fluorescencesample,t=0. Based on the normalized curves, the area under the fluorescence decay curve (AUC) was calculated as follows:
$${\text{AUC = 1 + }}\sum\limits_{i = 1}^{i = 60} {{{fi} \mathord{\left/ {\vphantom {{fi} {f0}}} \right. \kern-0pt} {f0}}}$$
$${\text{ORAC}}_{\text{FL}} = \frac{{{\text{AUC}}_{\text{sample}} - {\text{AUC}}_{\text{blank}} }}{{{\text{AUC}}_{\text{trolox}} - {\text{AUC}}_{\text{blank}} }} \times {\text{Trolox}}\;{\text{molarity}} \times {\text{sample}}\;{\text{dilution}} .$$
Each sample was calculated by subtracting the AUC corresponding to the blank. Equations for regression between the net AUC and antioxidant concentration were calculated for all samples. The concentrations are expressed as micrograms of Trolox equivalents per 100 mg of fresh weight.