Volume 4 Special Issue 1 2010
Antioxidant Properties of Crops II

Guest Editor

Robert D. Hancock

The Scottish Crop Research Institute, UK


www.scri.ac.uk/

CONTENTS AND ABSTRACTS

Natalia Hounsome, Barry Grail, Deri Tomos, Barry Hounsome, Gareth Edwards-Jones (UK) High-Throughput Antioxidant Profiling in Vegetables by Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry (pp 1-10)

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Original Research Paper: Vegetables are an important source of dietary antioxidants. Electrospray ionisation Fourier-transform ion cyclotron resonance mass spectrometry was applied to study the antioxidant profiles of beans, broccoli, cabbage, endive, peas and 3 varieties of lettuces. Metabolite extracts from vegetables were analysed in positive and negative ion mode in the m/z range <1000. Mass peaks were assigned by comparing measured and theoretical molecular masses with an accuracy of 0.5 mDa using a metabolite reference database. More than 300 mass peaks were identified, and 100 of them were assigned as potential antioxidant compounds. Antioxidants were grouped into 7 metabolic categories. Phenolic compounds were the most numerous group of antioxidants (45-62%), followed by organic acids (12-24%), amino acids and amines (11-17%), pyridines and purines (5-10%), organosulphur compounds, terpenoids, and alkaloids (<5%). Phenolic compounds included flavonoids, phenolic acids, lignans, coumarins, stilbenes, phenylpropanoid glycosides and quinones. Flavonoids and phenolic acids accounted for more than half of all phenolic compounds. About 20% of identified antioxidants were found in all analysed vegetables, while 16% of compounds were variety-specific. Hierarchical cluster analysis revealed similarities in antioxidant profiles between cabbage and broccoli, beans and peas and lettuces and endive. With respect to occurrence in vegetable species, antioxidants can be classified as ubiquitous (appearance frequency >90%), common (appearance frequency between 10% and 90%), and rare (appearance frequency <10%). The results of this study demonstrate that ubiquitous antioxidants include primary metabolites and key compounds in secondary metabolic pathways, while rare antioxidants represent the end products of secondary metabolism.

Patricia García-Salas, David Arráez-Roman, Antonio Segura-Carretero, Belen García-Villanova, Eduardo Guerra-Hernández, Alberto Fernández-Gutiérrez (Spain) Garcinia mangostana Fruit: Determination of the Total Antioxidant Activity in Extracts and Phenolic Compound Characterization using CE-MS(TOF) (pp 11-16)

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Original Research Paper: Xanthones are an important family of polyphenols present in Garcinia mangostana fruit. Products of G. mangostana have begun to be commercialized as a dietary supplement because of their potent antioxidant properties. Interest in their beneficial health effects such as antioxidant characteristics, lipid profile and anticarcinogenic activity, etc., has encouraged scientific research to determine compounds responsible for these properties. The antioxidant capacity determination and the phenolic compound characterization in extracts of G. mangostana were investigated. High antioxidant capacity was determined in whole fruit dried extract by ORAC, FRAP and ABTS^{• +} assays. The values obtained by FRAP, ORAC and ABTS assays were 1222 and4088 and 3590µM TE/g, respectively. Phenolic compound identification in these extracts was carried out by capillary electrophoresis coupled to ionization by electrospray-mass spectrometry (CE-ESI-MS) with a time of flight analyzer (Micro TOF), and a sensible, fast and efficient method was developed. The CE-ESI parameters were optimized to obtain good separation and suitable sensitivity. The CE parameters were: 80 mM ammonium acetate, pH 10.5, 30 kV, 15 s for injection and fused-silica capillary of 50 μm i.d. and 100 cm in length. The ESI-MS parameters were also optimized: drying gas flow of 4L/min, 300°C, nebulization gas pressure at 4 psi, sheath liquid composition and flow (60:40) isopropanol/water + 0.1% triethylamine to 0.18 mL/h. Spectrometric determination was achieved in negative polarity and the mass range was between 100-800 m/z. Considering the scarcity of commercial standards and the scanty bibliography references, some of the most important xanthones from G. mangostana fruit have been identified using CE-ESI-MS (micro TOF), such as Garcinine C, Garcinone E, Mangostenone C and β-mangostin.

Kuppusamy Asokkumar, Muthuswamy Umamaheswari, Abdulkadar Bahrudeen, Andichettiyar Thirumalaisamy Sivashanmugam, Varadharajan Subhadradevi, Thengungal Kochupapy Ravi (India) Antioxidant and Hepatoprotective Activities of the Fractions of Ficus microcarpa using in Vitro and ex Vivo Models (pp 17-27)

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Original Research Paper: The aim of the present study was to investigate the antioxidant and hepatoprotective activities of the various fractions of the hydromethanolic extract of the leaves of Ficus microcarpa L.f (Moraceae). The antioxidant activities of the fractions were evaluated using H-donor activity, reducing power ability, nitric oxide, superoxide anion, hydroxyl radical, hydrogen peroxide scavenging activities, metal chelating ability, b-carotene bleaching and total peroxy radical assays, and total antioxidant activity by thiocyanate method. The pro-oxidant activity was measured using bleomycin-dependent DNA damage. Ex vivo models like lipid peroxidation, erythrocyte haemolysis and total antioxidant capacity in human plasma were also studied. Total phenolic and flavonoid content of the fractions were determined as gallic acid and quercetin equivalents, respectively. Increasing concentration of all the fractions showed increased antioxidant potential in a dose-dependent manner and the antioxidant activities were compared with standard drugs such as ascorbic acid, α-tocopherol, curcumin, quercetin and butylated hydroxy toluene (BHT) in different assays and the results were found to be statistically significant (P<0.001). None of the fractions exerted an obvious pro-oxidant activity when compared to ascorbic acid. The hepatotoxicity of carbon tetrachloride (CCl₄) on rat liver slice culture in vitro was also decreased by the leaf fraction which was evidenced by an increase of rat liver catalase, superoxide dismutase, glutathione peroxidase, reduced glutathione, and significant decrease of LDH and lipid peroxidation end products, and malondialdehyde levels. All the four fractions offered strong antioxidant and hepatoprotective activity justifying the claim of traditional use and serve as a potential source of natural antioxidants.

Rajangam Udayakumar (Korea/India), Sampath Kasthurirengan, Thankaraj Salammal Mariashibu, Jesudass Joseph Sahaya Rayan (India), Sei Chang Kim, Chang Won Choi (Korea), Andy Ganapathi (India) Antioxidant Activity of Phenolic Compounds Extracted from the Roots and Leaves of Withania somnifera (L.) from Different Geographical Locations in India (pp 28-33)

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Original Research Paper: Phenolic compounds and antioxidant activity were determined in the extracts of Withania somnifera root (RE) and leaf (LE) from five different geographical locations like Coimbatore, Gujarat, Kambam, Lucknow and Nagappattinam of India. The highest amount of total phenolic compounds at 29.25 mg/g DW (dry weight) of plant tissue and total flavonoids at 6.3 mg/g DW were found in RE-Lucknow. The maximum amount of kaempferol at 4.472 mg/100 g DW and quercetin at 1.340 mg/100 g DW were found in RE-Lucknow and LE-Lucknow, respectively. Kaempferol was found in all root extracts and LE-Lucknow, while quercetin was found in all leaf extracts and two roots extracts like RE-Coimbatore and RE-Gujarat. RE-Lucknow showed the best radical scavenging activity with the IC₅₀ value of 105.06 ± 1.65 μg/mL in the DPPH assay. In addition, RE-Lucknow also showed maximum percentage of ABTS absorption inhibition activity with the IC₅₀ value of 204.03 ± 14.88 μg/mL. Therefore, the present study reveals that the W. somnifera leaf and rootextracts from five geographical locations possess different antioxidant capacities due to various amounts of phenolic compounds.

Jo Whitson, Gordon J. McDougall, Heather A. Ross, Victoria A. Lund, Carlene A. Hamilton, Anna F. Dominiczak, Derek Stewart (UK) Bioactive Berry Components: Potential Modulators of Health Benefits (pp 34-39)

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Original Research Paper: This article focuses on biological activities that have been ascribed to berry antioxidant components other than vitamin C. We illustrate activities of berry polyphenol components, such as anthocyanins, flavonols and tannins, in model systems with potential relevance to cardiovascular health, cancers and glycaemic control. We present evidence that raspberry extracts but not blackcurrant extracts protected nitric oxide bioavailability in ex vivo rat carotid artery models. Nitric oxide is a key regulator of blood vessel dilation and can influence cardiovascular fitness. The raspberry extracts were more effective at lower concentrations than ascorbic acid and were as effective as quercetin metabolites known to be effective in vivo. Raspberry extracts also showed positive effects on platelet aggregation which could have beneficial effects on cardiovascular health. We present evidence that raspberry and cloudberry extracts can inhibit the proliferation of cancer cells grown in vitro and that raspberry extracts are more effective than cloudberry. As the cloudberry extracts are essentially similar in polyphenol composition to raspberry but much lower in anthocyanins and proportionally higher in ellagitannins, this raises questions about previous findings that suggested that ellagitannins were the major antiproliferative agents in raspberry. We screened berry extracts for their ability to inhibit pancreatic α-glucosidase activity, a key enzyme in starch digestion and blood glucose control. Raspberry, rowan, lingonberry and pomegranate juice extracts caused significant inhibition whilst strawberry, cloudberry, blackcurrant, blackberry and blueberry caused no inhibition at the concentrations used. A red wine extract significantly increased activity. A comparison of the polyphenol composition of the effective extracts suggests that the active agents may be more complex than previously thought.

Paul G. Walker (United Kingdom), Roberto Viola (Italy), Mary Woodhead, Linzi Jorgensen, Sandra L. Gordon, Rex M. Brennan, Robert D. Hancock (United Kingdom) Ascorbic Acid Content of Blackcurrant Fruit is Influenced by Both Genetic and Environmental Factors (pp 40-52)

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Original Research Paper: Blackcurrant (Ribes nigrum L.) is a widely grown commercial crop valued for its high ascorbic acid content. In the present study we report large year-to-year variation in the ascorbic acid content of four different blackcurrant cultivars grown at the same site demonstrating strong environmental influence on fruit ascorbic acid concentration. All cultivars examined showed the same trend in ascorbic acid concentration on a year-to-year basis and cultivar hierarchy in fruit ascorbic acid was generally maintained. These data suggest strong underlying genetic determinants for fruit vitamin C concentration and demonstrate that different blackcurrant cultivars show similar responses to prevailing environmental conditions. Linear regression analysis of fruit ascorbic acid content versus several environmental parameters (total solar radiation, total precipitation and average air temperature) suggested a complex environmental interaction with multiple environmental parameters affecting fruit ascorbate. However, pre-harvest solar radiation showed the strongest correlation and data from fruit grown on north or south facing slopes further highlighted the importance of irradiation. Expression analysis of four genes encoding ascorbate biosynthetic enzymes failed to identify any correlations between gene expression and fruit ascorbate content although specific alleles of the gene encoding GDP-d-mannose 3,5-epimerase (E.C. 5.1.3.18) were associated with high fruit ascorbate.

Michael J. Davies, June M. Taylor, Y-Yannick Ford, Phillippa A. A. Dodds, Helen Longbottom, Neil A. Hipps, Christopher J. Atkinson (UK) Opportunities for Agronomic Manipulation of Ascorbic Acid Yield in Fruit Crops: A Case Study with Ribes nigrum L. (pp 53-65)

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Original Research Paper: This work focuses on developing an understanding of how agronomic manipulation might enhance fruit L-ascorbic acid (AsA) yields of blackcurrant and how this information might be best used, in field-grown crops. A series of pot and field experiments were carried out to determine the effects of manipulating plant nitrogen, soil moisture and plant leaf canopy light interception, in interaction with the use of reflective mulches. Results show that using ‘excessive’ nitrogen (ammonium nitrate) enhanced leaf canopy area, but at the expense of fruit AsA concentration which declined, in ‘Hedda’ and ‘Baldwin’ grown in pots. In the field, nitrogen application to ‘Baldwin’ and ‘Ben Lomond’ showed no stimulation of bush growth or fruit yield with increasing concentration, while fruit AsA concentration and total AsA yield declined. To evaluate the impacts of soil moisture, different rates of trickle irrigation were applied to field-grown ‘Baldwin’ and ‘Ben Lomond’. Plants not receiving irrigation had reduced shoot growth compared with those receiving irrigation, but drought stress did not influence fruit yield per bush. Optimal irrigation did have a cumulative positive effect on fruit yield. There was no impact of drought stress on berry AsA concentration, but a large positive enhancement on total AsA yield per bush. Enhancement of bush solar radiation interception was achieved using light reflective plastic soil mulches. Mulches increased bush canopy vegetative growth and were accompanied by small increases in fruit yield. There was no influence on fruit AsA concentration, or total bush yield of AsA initially, but in the second growing season total bush AsA yield increased. A combination of irrigation and light reflective mulch, in field-grown ‘Baldwin’ and ‘Ben Lomond’, enhanced fruit yields and the total AsA yield per bush after two consecutive years of treatment. For ‘Baldwin’, irrigation was the factor mainly responsible for this enhancement, while for ‘Ben Lomond’ it was the light reflective mulch that was most important. The implications of how field agronomy might influence the processes which determine AsA biosynthesis are discussed.

Hélène Gautier, Félicie Lopez-Lauri, Capucine Massot, Ramzi Murshed, Isabelle Marty, Dominique Grasselly, Cyril Keller, Huguette Sallanon, Michel Génard (France) Impact of Ripening and Salinity on Tomato Fruit Ascorbate Content and Enzymatic Activities Related to Ascorbate Recycling (pp 66-75)

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Original Research Paper: Our objectives were i) to determine the impact of ripening and salinity on fruit ascorbate content in a cherry tomato ‘Cervil’, a medium sized tomato ‘Levovil’ and a larger sized tomato ‘Marmara’, ii) to compare the impact of increasing salinity by adding NaCl alone (Na) or NaCl plus CaCl₂ (Ca+Na) as calcium may limit the salinity stress and iii) to determine how increased salinity and ripening modified the activity of antioxidant enzymes related to ascorbate recycling. Tomato fruits were harvested at three ripening stages: mature green (MG), pink (P) and red ripe (RR), on plants irrigated with a control solution (3 dS m^-1) or with increased salinity solutions (Na or Ca+Na at 7.6 dS m^-1). During ripening, ascorbate content and oxidative parameters increased whereas antioxidant activities of enzymes did not show any obvious trend. ‘Cervil’ had higher ascorbate and oxidative parameters but lower ascorbate peroxydase (APX) activity compared to ‘Marmara’ and ‘Levovil’. Salinity increased the reduced ascorbate content (except for RR fruits of ‘Cervil’), and the dehydroascorbate content in ‘Cervil’ and ‘Marmara’. Despite qualitatively similar impacts on the ascorbate pools, adding Na had a stronger impact on the oxidative parameters (hydrogen peroxide and lipid peroxidation) compared to adding Ca+Na. Increasing salinity did not increase the activity of antioxidant enzymes in Marmara, but it significantly enhanced them in ‘Levovil’ or ‘Cervil’. The stronger antioxidant enzyme activities observed in ‘Cervil’ when adding Ca+Na could be linked to a lower accumulation of Na⁺ compared to other genotypes. We discuss a possible link between the oxidative parameters, the ascorbate content and the activity of ascorbate recycling enzymes in genotypes harvested at different ripening stages and receiving different salinity solutions.

Noé Gest, David Page, Simona Birtić, Barbara Gouble, Louise Gilbert, Cécile Garchery, Mathilde Causse, Rebecca Stevens (France) Response of the Fruit Antioxidant System to the Post-Chilling Period in Two Different Tomato Lines (pp 76-83)

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Original Research Paper: Antioxidant systems play an important role in prevention of chilling injury, and chilling-tolerant fruit may initially contain more antioxidants or be able to maintain their antioxidant levels during stress. Tomato fruit are chilling sensitive and chilling symptoms generally appear after fruit are transferred to non chilling temperatures. The aim of this study was to characterise the events occurring at 20°C, immediately after a storage period at 4°C, and to establish the importance of tomato fruit ascorbic acid levels in the response to chilling. Two different tomato lines were used: a processing cultivar (M82) with low to average ascorbic acid levels and an introgression line (IL9.2.5) containing elevated ascorbic acid levels due to the introgression of wild Solanum pennellii alleles on chromosome 9. We show a rapid response (0.5 hours) of the antioxidant system in tomato to re-exposure to normal temperatures and light following chilling. The ascorbate redox state undergoes rapid changes and is followed by changes in the redox state of the glutathione pool. The response is different in the two tomato lines studied: fruits from IL9.2.5 have a higher ascorbic acid and glutathione redox state compared to the parent M82 where the pool of both antioxidants becomes oxidised. The redox state of the ascorbate pool is correlated with the activity of enzymes involved in ascorbate regeneration (monodehydroascorbate reductase and dehydroascorbate reductase) in the fruit. Furthermore the ability of IL9.2.5 to maintain reduced antioxidant levels is correlated with a decreased loss of fruit firmness when compared to M82.

Sara Tulipani, Stefania Romandini, Franco Capocasa, Bruno Mezzetti, Maurizio Battino (Italy) The Nutritional Quality of Strawberries (Fragaria x ananassa) after Short-refrigeration: Genetic Influences (pp 84-89)

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Original Research Paper: The strawberry (Fragaria x ananassa,Duch.)has recently received great commercial development and represents the most commonly consumed berry. The strawberry is also a relevant source of micronutrients and phenolic substances, most of which are natural antioxidants and contribute to the high nutritional quality (NQ) of the fruit. In addition to genetic heritable differences, several pre- and post-harvest environmental conditions seem to strongly influence the NQ of strawberry. The short-refrigeration of the fruits is the most common method to control the decay of strawberries. However, little is known on the influence of cold storage on the antioxidant, phenolic and micronutrient contents in strawberries, and on the genetic influence on the storability of these fruits. In this work, five strawberry cultivars were analyzed for total antioxidant capacity, total phenolics, flavonoids and anthocyanins, and for micronutrient contents of both fresh and stored fruits. Three consecutive years of harvest were studied to assess the combined effect of genotype and pre-harvest environmental conditions on the NQ and storability of the fruits. Significant cultivar-to-cultivar differences were observed in the NQ parameters studied, confirming how the genetic background may significantly affect the nutritional value of strawberries. Short-refrigeration did not seem to affect either negatively or positively the main NQ attributes of the strawberries, with the exception of the folate content, which significantly increased after storage in all three years. These findings are particularly concerned with some varieties in this study, suggesting a genetic influence on fruit response to storage.

Ryszard Amarowicz, Stanisław Weidner, Izabela Wójtowicz, Magdalena Karamać, Agnieszka Kosińska, Anna Rybarczyk (Poland) Influence of Low-Temperature Stress on the Changes in the Composition of Grapevine Leaf Phenolic Compounds and their Antioxidant Properties (pp 90-96)

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Original Research Paper: The impact of low-temperature stress on the content of phenolic compounds of grapevine leaves and their antioxidant activity was examined. The experiment with seedlings was performed under optimal conditions and under low-temperature stress. Phenolic compounds were extracted from leaves of Vitis vinifera using 80% (v/v) acetone. After lyophilization, the extracts were characterized by their content of total phenolics, condensed tannins, and individual phenolic acids. The reducing power and antiradical activity against DPPH^· as well as ABTS^·+ were also investigated. Low-temperature stress decreased the content of total phenolics and condensed tannins in the leaf extract, but the quantity of some phenolic acids (i.e., gallic, caffeic, and ferilic acids) was higher than in the control extract. Caffeic acid was determined to be the dominant phenolic acid. Although phenolic acids were present in free, esterified, and glycosylated forms, the majority were esterified. The extract of the leaves treated with low-temperature stress was characterized by a lower total antioxidant activity, antiradical activity against DPPH^·, and reducing power (e.g., the total antioxidant activity of the extract after low-temperature stress was 0.545 mmol Trolox/g extract whereas the extract from unstressed leaves was 0.808 mmol Trolox/g extract).

Anna Podsędek, Dorota Sosnowska, Barbara Anders (Poland) The Effect of Cooking Conditions on Hydrophilic Antioxidants in Brussels Sprouts (pp 97-101)

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Original Research Paper: Knowledge on the stability and antioxidant activity of natural vegetable antioxidants affected by cooking conditions can help in planning diets for enhanced antioxidant intake. This study evaluates the effect of boiling, microwaving and steaming on antioxidant activity and content of both vitamin C and phenolic compounds present in Brussels sprouts (cvs. ‘Ajax’ and ‘Filemon’). Steaming cooking is recommended to minimise the loss of hydrophilic antioxidants, while conventional cooking significantly reduces the content of vitamin C (up to 53%) and phenolics (up to 64%) in cooked Brussels sprouts. The losses of dietary antioxidants tested caused a decrease of the free radical scavenging activity towards ABTS^·+ and DPPH^· radicals for all the samples cooked.