High Plant Growth Temperatures Increase Antioxidant Capacities in Strawberry Fruit

The influence of four day/night growing temperature combinations (18/12, 25/12, 25/22, 30/22 C) on phenolic acid, flavonol, and anthocyanin content and their antioxidant activities against peroxyl radicals (ROO ), superoxide radicals (O2 ), hydrogen peroxide (H2O2), hydroxyl radicals (OH ), and singlet oxygen (O2,) in fruit juice of ‘Earliglow’ and ‘Kent’ strawberry (Fragaria x ananassa Duch.) were studied. The content of cyanidinbased anthocyanins, were much lower than pelargonidinbased anthocyanins. High day/night temperature conditions significantly enhanced pcoumaroylglucose, dihydroflavonol, quercetin 3-glucoside, quercetin 3-glucuronide, kaempferol 3-glucoside, kaempferol 3-glucuronide, cyanidin 3-glucoside, pelargonidin 3-glucoside, pelargonidin 3-rutinoside, cyanidin 3-glucoside-succinate and pelargonidin 3-glucoside-succinate content in strawberry juice. Plants grown at low day and night temperatures (18/12 C) generally had the lowest anthocyanin contents. Plants grown at the highest day/night temperatures (30/22 C) produced fruit with the most phenolic content as well as antioxidant capacity. Fruit of ‘Kent’ strawberry had higher contents of phenolic acid, flavonols, anthocyanins and antioxidant capacities compared to fruit of ‘Earliglow’ strawberry under all temperature regimes. INTRODUCTION Fruits and vegetables contain high levels of antioxidant compounds, which provide protection against harmful free radicals and have been associated with lower incidence and mortality rates of cancer and heart disease in addition to a number of other health benefits (Ames et al., 1993; Velioglu et al., 1998). Our previous studies have shown that thornless blackberries (Rubus sp.), blueberries (Vaccinium spp.), cranberries (Vaccinium macrocarpon Aiton), raspberries (Rubus idaeus L. and Rubus occidentalis L.) and strawberries (Fragaria x ananassa Duch.) have high antioxidant capacities (Wang and Jiao, 2000; Wang and Lin, 2000). However, no information is available on the effect of environmental factors such as growing temperatures on scavenging capacity of strawberry against active oxygen species. The present study evaluated the effects of four day/night growing temperatures (18/12, 25/12, 25/22 and 30/22 C) on antioxidant activities against ROO , O2 C, H2O2 , OH , and O2 radicals associated with changes in anthocyanins and other phenolic compounds of strawberry. MATERIALS AND METHODS Plant Materials and Experimental Plans : Uniform sized, approximately one-year-old plants of Earliglow and Kent cultivars were used. The plants were propagated by runnertip cuttings in June and plants were grown in 2liter plastic pots containing Pro Mix BX (Premier Brands Inc., Stamford, Conn.) in a greenhouse. Radiation sources in the greenhouse consisted of natural daylight and Watt-Miser incandescent lamps (Nela Park, Cleveland, OH) that provided a PAR around 700 Fmol ms for 14h/d (0600-2000h). Temperatures were set at around 25EC during the day and 20EC at night. During the growing season, all plants were watered daily and fertilized biweekly with 150 ml/ plant of Peters fertilizer (20-20-20, N/P/K). Prior to initiation of temperature treatments, plants were exposed to ambient winter temperatures in Beltsville, Maryland, USA, in an Proc. XXVI IHC – Berry Crop Breeding Eds. P. Hicklenton and J. Maas Acta Hort. 626, ISHS 2003 Publication supported by Can. Int. Dev. Agency (CIDA) 58 unheated greenhouse from October to February. Plants were then moved to a heated greenhouse (25EC during the day and 20EC at night) for approximately 1.5 months to force flowering. Blossoms were self-pollinated by hand using a small brush. Plants with the most fruit (at least ten fruits per plant) at their green fruit stage were selected for the growth chamber experiments. Forty plants each of ‘Earliglow’ and ‘Kent’ were removed from the greenhouse in March and divided into lots of 10 plants. One lot of each cultivar was randomly placed in four growth chambers set at day/night temperatures of 18/12, 25/12, 25/22 and 30/22EC. The plants were in growth chambers for 1.5 months. The photoperiod for each growth chamber was 14 hr (6:00-20:00 hr) with a PAR around 700 Fmol ms at plant height. Firm red-ripe fruits free from defects or decay were harvested from each cultivar in each growth chamber during fruit ripening and the berries were cut into small slices, mixed, and then used for analyses. Fruit Sample Preparation : To prepare the juice samples, three-100g samples of berries from three replicates of each cultivar of each treatment were pulverized and then centrifuged at 14,000 g for 20 minutes at 4EC. The supernatants were transferred to vials, stored at -80EC and then used for analyses. Peroxyl Radicals (ROO . ) ORAC Assay : The procedures for the ORAC assay on strawberries were modified from a previously described method by Cao et al. (1993). The ORAC value refers to the net protection area under the quenching curve of R-PE in the presence of an antioxidant. The final results (ORAC value) were calculated and expressed using Trolox equivalents per gram on a fresh weight basis. Superoxide Radical (O2 ) Assay : The assay for superoxide radical (O2 ) was determined using the methods of Elstner and Heupel (1976). The final results were expressed as percent inhibition of O2 Cproduction in the presence of fruit juice. The scavenging capacity of "-tocopherol at various concentrations (1 to10 Fg) on superoxide radical (O2 ) was measured and used for determining the O2 Cscavenging capacity of fruit juice. The antioxidant capacity of fruit juice against the O2 Cvalue was expressed as Fmole of "-tocopherol equivalent per gram fresh weight. Hydroxyl Radical (OH @) Assay : The assay for hydroxyl radical (OH@) was determined using the methods of Richmond et al. (1981). Relative scavenging efficiency (% inhibition of hydroxylation) of fruit juice was estimated from the difference in absorbance (OD) with and without addition of the fruit juice. The scavenging capacity of chlorogenic acid at various concentrations (1 to10 Fg) on hydroxyl radical (OH@) was measured and used for determining the OH@ scavenging capacity of fruit juice. The antioxidant capacity of fruit juice against OH@ value was expressed as Fmole of chlorogenic acid equivalent per gram fresh weight. Hydrogen Peroxide (H2O2) Assay : The assay for hydrogen peroxide in fruit juices of blackberry, raspberry, cranberry, blueberry and strawberry was carried out following procedures previously described by Patterson et al. (1984). The scavenging capacity of ascorbate at various concentrations (1 to10 Fg) on hydrogen peroxide (H2O2) was measured and used for determining the H2O2 scavenging capacity of fruit juice. The antioxidant capacity of fruit juice against H2O2 value was expressed as Fmole of ascorbate equivalent per gram fresh weight. Singlet Oxygen (O2) Assay : The production of singlet oxygen (O2) by sodium hypochloride and H2O2 was determined by using a spectrophotometric method according to Chakraborty and Tripathy (1992). Relative scavenging efficiency (% inhibition production of O2) of fruit juice was estimated from the difference in absorbance of N, N, dimethyl-pnitrosoaniline with and without the addition of fruit juice. The scavenging capacity of $-carotene at various concentrations (1 to10 Fg) on singlet oxygen (O2) was