Plant Foods Hum Nutr (2010) 65:105–111 DOI 10.1007/s11130-010-0156-6
ORIGINAL PAPER
Antioxidant Activity and Phenolic Content of Betalain Extracts from Intact Plants and Hairy Root Cultures of the Red Beetroot Beta vulgaris cv. Detroit Dark Red Vasil Georgiev Georgiev & Jost Weber & Eva-Maria Kneschke & Petko Nedyalkov Denev & Thomas Bley & Atanas Ivanov Pavlov
Published online: 27 February 2010 # Springer Science+Business Media, LLC 2010
Abstract Betalains are water-soluble plant pigments that are widely used as food colorants, and have a wide range of desirable biological activities, including antioxidant, antiinflammatory, hepatoprotective, anti-cancer properties. They can be produced from various plants, notably beetroot, but betalain products obtained in this way also have some undesirable properties and are difficult to standardize. A potentially attractive alternative is to use hairy root cultures. In the study reported here, we found that betalain extracts obtained from hairy root cultures of the red beetroot B. vulgaris cv. Detroit Dark Red also had higher antioxidant activity than extracts obtained from mature beetroots: six-fold higher 2,2-dyphenyl-1-picrylhydrazyl radical scavenging ability (90.7% inhibition, EC50 = 0.11 mg, vs 14.2% inhibition, EC50 =0.70 mg) and 3.28fold higher oxygen radical absorbance capacity (4,100 µM V. G. Georgiev : A. I. Pavlov (*) Department of Microbial Biosynthesis and Biotechnologies, Laboratory in Plovdiv, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 139 Ruski Blvd., 4000 Plovdiv, Bulgaria e-mail:
[email protected] e-mail:
[email protected] J. Weber : E.-M. Kneschke : T. Bley Institute of Food Technology and Bioprocess Engineering, Technische Universität Dresden, 01062 Dresden, Germany P. N. Denev Laboratory of Biologically Active Substances, Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, 139 Ruski Blvd., 4000 Plovdiv, Bulgaria
TE/g dry extract, vs 1,250 µM TE/g dry extract). The high antioxidant activity of the hairy root extracts was associated with increased concentrations (more than 20-fold) of total phenolic concomitant compounds, which may have synergistic effects with betalains. The presence of 4-hydroxybenzoic acid, caffeic acid, catechin hydrate, and epicatechin were detected in both types of extract, but at different concentrations. Rutin was only present at high concentration (1.096 mg.g−1 dry extract) in betalain extracts from the hairy root cultures, whereas chlorogenic acid was only detected at measurable concentrations in extracts from intact plants. Keywords Antioxidant activities . Betalains . DPPH . Food colorant . Hairy roots . HPLC . Phenolics . ORAC . Red beetroot Abbreviations FL Fluorescein TE Trolox equivalents FAE Ferulic acid equivalents DE Dry extract
Introduction Red beetroot betalain extract, consisting mostly of betanin (E162), is widely used as a natural colorant in many dairy products (e.g. milk, ice creams, yogurt, and kefir), beverages (e.g. juices and Burakovyi kvas), candies (e.g. cookies and desserts) and cattle products (cooked, smoked, semi-dry or fermented sausages) [1–3]. In addition to their red color, betalains possess several desirable biological activities, including antioxidant, antiinflammatory, hepatoprotective, and antitumor properties [4–6]. The bioavail-
106
ability of betalains was reported to be high in humans, and they remain stable in the gastrointestinal tract without any significant loss in antioxidative properties, which increases their value as health food additives [7, 8]. The mechanism of the antioxidant action of the pure pigments was poorly studied. The high antioxidant activity of betanin was associated with an increasing of its electron donation ability [9]. Recently, it was reported that the high antioxidant and free radical scavenging activities of betaxanthins were not linked to the presence of hydroxy groups or aromaticity, but are enhanced with the presence of phenolic hydroxy groups in their structure [10]. Tough amaranthaceous plants and cactus fruits were the initial foci of researchers seeking to produce betanin, but its most important commercial sources as a colouring agent today are juice concentrates or powders from field-cultivated red beetroot (Beta vulgaris ssp.) [11, 12]. However, betalain products obtained in this way have some undesirable properties, including an unpleasant flavor, due to the presence of geosmin and pyrazine derivatives, and high concentrations of soil-borne microbes that may contaminate food products. Furthermore, there are difficulties with standardizing the product in terms of concomitant metabolite contents, due to variations in climatic and environmental conditions [1, 13]. The application of plant in vitro systems for betalain production may enable these problems to overcome in the future [14]. A promising option is to use hairy root cultures, which can be readily obtained following genetic transformation of plant cells by Agrobacterium rhizogenes, and are considered to be fast-growing, genetically stable cultures with secondary metabolite biosynthetic potential equal to that of their parent plants [14]. These in vitro cultures and their products are not classified as genetically modified organisms (GMOs), as defined by the European Parliament Directive 2001/18/EC, and hence are not subject to EU regulations regarding GMOs (OJ, L 106, 17.4.2001, p.1–38). In previous work, we showed that hairy root cultures of the red beetroot B. vulgaris cv. Detroit Dark Red have considerable potential for producing betalain pigments with high antioxidant activities [15, 16]. The successful cultivation of hairy roots in shake flasks and several types of bioreactors demonstrated the potential for developing suitable large-scale biotechnological systems for betalain production [17–20]. However, the in vitro cultivation of plant cells could significantly change their metabolic profiles as a result of stresses that they are exposed to under in vitro conditions. Further, it has been found recently that hairy root cultures undergo more endoreduplication cycles than the tissue of the intact plants they are obtained from [21–23], and the observed differences in ploidy profiles of hairy root cultures may cause changes in their phytochemical composition. This could alter their biological activities, via synergistic interactions between some compounds or the synthesis of new,
Plant Foods Hum Nutr (2010) 65:105–111
biologically-active compounds that are not produced by intact plants. However, the chemical composition and antioxidant activities of extracts from hairy root cultures and intact plants have not been previously compared in any published studies. In the present study, we compared the antioxidant activities of betalain extracts from hairy root cultures and intact plants of the beetroot Beta vulgaris cv. Detroit Dark Red. Antioxidant activities of extracts were evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and oxygen radical absorbance capacity (ORAC) assays. The former is a widely used method for the initial screening of antioxidants, based on the two main mechanisms of antioxidant action; single electron transfer (SET) and hydrogen atom transfer (HAT). In contrast, the ORAC method is used to assess the antioxidant properties of complex mixtures of antioxidants, based solely on the HAT mechanism [24]. The phenolic compounds and flavonoid contents of both types of extracts were determined and are discussed according to their antioxidant properties. We also discussed the advantages of hairy root culture for the production of biologicallyactive betalain colorant.
Materials and Methods Plant Material Seeds of beetroot Beta vulgaris cv. Detroit Dark Red (Heirloom, Seeds of Change™ Certified Organic, USA) were sown in the field and the resulting plants were cultivated for 3 months. The mature beetroots were collected, the leaves were removed and the soil was cleaned from the roots, which were washed with water, sliced and then used for betalain extraction. Hairy Root Culture Hairy root culture was obtained from young leaves of Beta vulgaris cv. Detroit Dark Red after transformation with Agrobacterium rhizogenes ATCC 15834, as reported by Pavlov et al. [16]. To collect root biomass, fresh hairy roots were cultivated by incubation in 500 mL conical flasks, filled with 100 mL liquid MS medium supplemented with 30 g/L sucrose, on a shaker (11 rad/s) in darkness at 26 °C. After 18 days, the hairy roots were collected, washed with water and used for betalain extraction. Preparation of Betalain Extracts To prepare dry betalain extracts, 0.5 kg of fresh biomass from either beetroots or hairy roots was homogenized with sand and extracted three times with 70% ethanol (100 mg/L). The extracts were centrifuged at 10,000 g for 30 min and the
Plant Foods Hum Nutr (2010) 65:105–111
supernatants were evaporated at 40 °C under vacuum until dry. The residues were dissolved in 500 mL of 70% methanol, then the methanol-sample mixtures were refrigerated at −20 °C for 24 h, thawed, and the supernatants were carefully collected from the precipitate. The methanol was removed from the supernatants by evaporation at 40 °C under vacuum, then the aqueous fractions containing betalains were lyophilized (Christ, Alpha 1-2, Germany) and used as dry betalain extracts. The total amounts of obtained final dry extracts from hairy roots and intact plant were 6.5 and 5.9 g dry extracts, respectively. Betalain Content Fifty milligram portions of dry lyophilized intact plant and hairy root extracts were each dissolved in 10 mL of 70% ethanol. The betacyanins and betaxanthins content of the extracts were then determined spectrophotometrically (Shimadzu UV mini 1240, Japan) following the method described by Pavlov et al. [20]. DPPH Radical Scavenging Activity Assay Lyophilized dry extracts from plants or hairy roots were dissolved in 40% ethanol to a final concentration of 1.0 mg/ mL, and their DPPH radical scavenging activity was determined as described by Pavlov et al. [16] and Pavlov et al. [8] with modifications, briefly as follows. Each assay mixture contained 0.2 mL of extract, 2.0 mL of 0.1 mM DPPH (Sigma, Germany) solution and 0.8 mL of 40% ethanol (total volume, 3.0 mL). These mixtures were incubated for 10 min in darkness at room temperature, and the reduction in absorbance at 517 nm was measured against that of a control sample, which contained the same amount of extract as the assay sample and 2.8 mL of 40% ethanol. A blank sample (with no added antioxidant) containing 2 mL 0.1 mM DPPH and 1.0 mL of 40% ethanol was prepared and measured before every measurement of corresponding extracts. The radical scavenging activity, which was expressed as percentage inhibition of DPPH formation, was calculated as described by Pavlov et al. [16]. The halfmaximal effective concentrations (EC50) of the extracts were calculated as described by Molyneux [25].
107
comparing the area under the fluorescence decay curves obtained from preparations containing the samples and controls in which no antioxidant is present. For these assays, stock solutions of sample (20 mg DE/mL), AAPH, FL and Trolox (Sigma, Germany) were prepared in phosphate buffer (75 mM, pH=7.4), then mixed as follows. First, 170 μL of FL solution (final concentration = 5.36×10−8 mM) and 10 μL of the sample solution were incubated at 37 °C for 30 min in darkness directly in the FLUOstar plate reader, then 20 μL of AAPH (final concentration = 51.51 mM) was added and the mixture was incubated for another 30 s before the initial fluorescence was read. Further fluorescence readings were then taken at 1-min intervals (FLUOstar OPTIMA Multifunction Microplate Reader, BMG LABTECH, Offenburg, Germany). For blank samples, 10 μL of phosphate buffer was used instead of the sample. Antioxidant activity was expressed in Trolox equivalents, by comparison to a fluorescence calibration curve obtained using 6.25, 12.5, 25, 50 and 100 mM Trolox solutions. One ORAC unit was defined as the net protection provided by 1 mM of Trolox, and final ORAC values were calculated from the regression between the Trolox concentrations and the net areas under the curves, as described by Prior et al. [27]. Results are expressed as μM Trolox equivalents (TE) per gram dry extract. Determination of Total Phenolic Content Lyophilized dry extracts from intact plants or hairy roots were dissolved in 70% methanol at concentrations of 1.0 mg/mL, and their total phenolic contents were measured using FolinCiocalteu reagent (Fluka, Germany) according to the method described by Stintzing et al. [28], except that 0.2 mL of sample extract solution was mixed with 2.0 mL of FolinCiocalteu reagent and 0.8 mL of sodium carbonate (7.5%). The reaction time was 30 min at room temperature in darkness and the absorption of the solution was measured at 765 nm (Shimadzu UV mini 1240, Japan). The total phenolic content was expressed as ferulic acid equivalents (FAE) in milligrams per gram dry extract. For calibration, solutions with 5, 10, 20, 40 and 80 mg/L of ferulic acid (Fluka, Germany) were used instead of the extract. HPLC Analysis of Phenolic Compounds
Oxygen Radical Absorbance Capacity (ORAC) Assay ORAC assays measure the antioxidant scavenging activity of samples by determining their ability to scavenge peroxyl radicals generated from 2,2′-asobiz (2-amidinopropane) dihydrochloride (AAPH), at 37 °C, using fluorescein (FL) as a fluorescent probe [26]. The reaction between peroxyl radicals and FL causes loss of fluorescence, thus the protective effect of antioxidants present in the samples can be estimated by
Fifty milligram portions of lyophilized intact plant or hairy root extract were each dissolved in 25 mL ddH2O and filtered through 0.22 µm filters before HPLC analysis, using an HPLC system consisting of: a Smartline 1000 pump, controlled by Smartline Manager software (Knauer, Berlin, Germany); a GAT LCD 500 detector; a GAT CH 150 column thermostat (GAT GmbH, Bremerhaven, Germany); and an ET250/4 Nucleosil 100-5 C18 AB column (Machery
108
and Nagl, Germany) at an operating temperature of 25 °C. The phenolic compounds were separated and quantified using the method described by Schieber et al. [29], with modifications, briefly as follows. The mobile phase consisted of a gradient of 0.5% acetic acid (A) and 0.5% acetic acid: acetonitrile (1:1, v/v; B), from 5% B (held for 1 min), rising to 55% B after 50 min and 100% B after 60 min, then falling to 5% B after 65 min, which was held for 15 min for reequilibration. Eluting compounds were detected by monitoring the eluate at 280 nm, and calibration curves were constructed using aqueous solutions of the following standards gallic acid, 4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, ferulic acid, epicatechin, catechin hydrate, rutin, hesperidin, quercetin hydrate and kaempferol at concentrations of 0.5, 1.0, 2.0, 5.0, 10.0, and 20 µg/mL.
Plant Foods Hum Nutr (2010) 65:105–111
vulgaris cv. Egyptian have different patterns of mixoploidy compared with intact plants [21, 23]. This may be an important factor causing changes in the secondary metabolite profiles in these in vitro systems.
Statistical Analysis All data were collected from three independent experiments repeated twice, and results are presented as mean values with a standard deviation. Data were analyzed by unpaired t-test with a level of significance of P≤0.05 using SigmaPlot v.7.0 software.
Results and Discussion Cultivation of plant cells and tissues under in vitro conditions can result in significant changes in metabolism, especially secondary metabolism, and hence contents of metabolites. For instance, changes in betacyanin/betaxanthin ratios and concentrations of betacyanins have been reported in various betalain-producing plants cultured in in vitro systems [30, 31]. Further, flow cytometric analysis has recently shown that hairy root cultures from the beetroot B.
Fig. 1 Betalain contents of extracts from intact plants (1) and hairy root cultures (2) of B. vulgaris cv. Detroit Dark Red. Bars indicate standard deviations (level of significance P
