ANTIOXIDANT ACTIVITY AND PHENOLIC CONTENT OF WATER EXTRACTS OF BORAGO OFFICINALIS L.: INFLUENCE OF PLANT PART AND COOKING PROCEDURE

Headnote

ABSTRACT

Water extracts of borage (Borago officinalis L.) were prepared from raw stems and leaves and from cooked (boiled and steamed) stems. Antioxidant activity (AA) was determined by ABTS and DPPH after their respective calibration with Trolox as standard and the results of both assays are expressed as µg Trolox/g fresh plant. Total phenolic compounds (TPC) were also determined for each extract. The DPPH and ABTS values in leaf extracts were similar and approximately 3.5 times higher than the values obtained for stem extracts. The high antioxidant activity of leaf extracts might be attributed to the high amount of phenolic compounds (2.36 mg GAE/g fresh plant for leaves and 0.57 mg GAE/g fresh plant for stems). Boiling significantly decreased the antioxidant activity (51-52% decrease) and total phenolic compounds (67% decrease). Steaming had no significant effect on the antioxidant activity values, whereas the total phenolic compound values showed approximately half of the decrease found for boiling (35%). Borage water extracts, particularly those of their by-products (leaves), showed high antioxidant activity, that could potentially be used for different applications in the food industry.

- Key words: ABTS7 boiling, borage, DPPH, steaming -

INTRODUCTION

Borage (Borago officinalis L.) is an herbaceous plant that belongs to the Boraginaceae family. It is consumed in many Mediterranean countries, and in some countries is considered to be a luxury product because of its characteristic taste. The stems are the edible part of the plant and are cooked prior to consumption, whereas the leaves are discarded. Borage seeds are traditionally known for their oil, which has certain beneficial effects associated with the high γ-linolenic acid content (KHAN and SHAHIDI, 2000), However, the non-lipidic fraction of borage has received less attention and little information is available about it. Defatted borage seeds were subjected to ethanol extractions in order to evaluate the presence of antioxidant phenolic compounds, which were further evaluated in a meat model system (WETTASINGHE and SHAHIDI, 1999), BANDONIENE and MURKOVIC (2002) analyzed the presence of radical scavenging compounds in crude methanolic extracts of borage leaves and observed a high radical-quenching ability due to the presence of rosmarinic acid, along with other phenolic compounds.

Antioxidant compounds protect against harmful free radicals and have been strongly associated with the reduced risk of chronic diseases, such as cardiovascular disease, cancer, diabetes, Alzheimer's disease, cataracts and age-related functional decline, as well as other health benefits (GUNDGAARD et al, 2003), Vegetable waste materials have been successfully explored as sources of natural antioxidants because, like other desirable compounds, higher concentrations are available in the residues (SCHEIBER et al, 2001; OKONOGI et al, 2007), In relation to borage leaves, BANDONIENE et al. (2002) were the first to demonstrate a strong antioxidant activity in crude acetone extracts of borage.

Information on the antioxidant components, antioxidant activity and their changes during cooking is still limited (ZHANG and HAMAUZU, 2004). In general it is known that thermal processing leads to losses in the bioactive compounds of plant products due to their heat-induced instability. However, if water is used as the heat-transfer medium, different and sometimes contradictory effects have been seen in the antioxidant activity of plant material. Nevertheless, it is very important to find the best way to preserve the bioactive compounds and antioxidant activities of processed plant materials (GORINSTEIN et al, 2009).

The total antioxidant capacity of a given food is not the sum of each single compound, but rather may result from the integrated and synergistic action of different compounds (DANESI and BORDONI, 2008). No single assay can be considered a "total antioxidant capacity assay" even though it could be performed in both an aqueous solution and a lipophilic environment (PRIOR et al, 2005). Different methods have been used to perform DPPH or ABTS assays in plant products, so it is often difficult to compare the results from different papers. Furthermore, different ways of expressing concentrations also complicate the comparison of results (dry or fresh weight, molar or mass units, etc.). For DPPH, the results can be shown as the remaining DPPH, (PRIOR et al, 2005), % Inhibition (PESCHEL et al., 2006), IC50 (concentration that causes a 50% decrease in the initial DPPH concentration), (OKONOGI et al, 2007) or as the antiradical efficiency AE = 1/(IC50*TIC) (SANCHEZMORENO et al, 1998). ABTS results are usually expressed as Trolox equivalents (either µM or µg) per amount of sample (RIVERO-PEREZ et al, 2007) using either absorbance values at a certain time or measuring the area under the curve resulting from the absorbance decrease during that time (PEREZ-JIMENEZ and SAURA-CALIXTO 2006; RE et al, 1999). Trolox, a water soluble analogue of vitamin E, has been the choice reference standard in most cases due to its effectiveness in both lipophilic and hydrophilic systems (NENADIS et al, 2007). A more objective comparison of results could be possible by applying the same interpretation procedure with the same common standard and a unified standardization procedure (STRATIL et al, 2006).

The objective of this paper was to evaluate the antioxidant activity and total phenolic content of different parts of borage (stems and leaves) and to study the changes induced by different cooking treatments of the edible part of this plant. Furthermore, a standardization procedure for expressing these results was attempted.

MATERIAL AND METHODS

Reagents

ABTS(2,2-Azino-bis(3-ethylbenzothiazoline-6sulfonicacid) diammonium salt), DPPH (2,2-Diphenyl-1-picryl-hydrazyl) and Folin-Ciocalteu reagent were purchased from SIGMA-ALDRICH Chemie GmbH (Steinheim, Germany), Trolox (6-hydroxy-2,5,7,8 tetramethylchromon-2-carboxylic acid) was purchased from SIGMA-ALDRICH Chemie GmbH (Steinheim, Germany). Gallic acid (Gallic acid 1 -hydrate) and methanol were purchased from PANREAC Quimica SAU (Barcelona, Spain) and ethanol from OPPAC, S.A. (Noain, Spain).

Plant material and sample preparation

Fresh borage Borago officinalis L.) (5 kg) was purchased from a local supermarket. The plants were separated into leaves and stems; they were cleaned, washed, and comminuted separately. The moisture content of the raw samples was measured according to the Official AOAC Method. The water extraction procedure was carried out as follows: 100 g of raw leaves were weighed and put into 100 mL of distilled water, preheated at 96°C. The mixture was subjected to sonication for 30 min at room temperature, and filtered with a metallic filter. The extraction process was repeated with another 100 mL of distilled water, and the two extracts were united and adjusted with distilled water to a final volume of 250 mL. The same procedure was used for the raw stem extracts, using Whatman n. 3 filter paper instead of a metallic filter.

Two different cooking procedures were applied to borage stems: a) 100 g of fresh cut stems were boiled with 1000 mL of distilled water for 30 minutes at 96°C to simulate domestic processing; b) 100 g of fresh stems were steam cooked with a conventional steam pot using 1,000 mL of distilled water for 30 min at 96°C. The cooked samples were subsequently subjected to an extraction procedure using the same method as that used for the raw stem samples.

Each extract was diluted with distilled water to obtain different solutions with concentrations ranging from 0.0009 to 0.4 g of fresh plant/mL, which were subsequently used to evaluate the antioxidant activity and the total phenolic content. All extractions were performed in duplicate.

Determination of total phenolic content (TPC)

TPC was determined spectrophotometrically following the Folin-Ciocalteu colorimetrie method (SINGLETON and ROSSI, 1965). Dilutions of borage extracts ranging from 0.004 to 0.4 g/mL were chosen in order to obtain readings within the standard calibration curve made from dilutions between 0,005 and 2 mg of gallic acid. The reaction mixture consisted of 0. 1 mL of suitably diluted sample, 7.9 mL of distilled water, 0.5 mL of Folin-Ciocalteu reagent, and 1.5 mL of a 20% sodium carbonate anhydrous solution (added 2 min after the Folin-Ciocalteu reagent). After the initial mixing, the tubes were allowed to stand at room temperature for 2 h in the dark. The optical density of the blue-colored resulting solution was measured at 765 nm using a Lambda 5-UV-VIS spectrophotometer (Perkin Elmer, Paris, France). The total phenolic content is expressed as mg of gallic acid equivalents (mg GAE) /g fresh plant, using the corresponding calibration curve and taking into account the concentration of the diluted extracts. Absorbance measurements were made in duplicate for each diluted solution.

DPPH method

The DPPH assay was performed according to the method of BLOIS (1958) with some modifications. Briefly, a DPPH solution of approximately 20 mg/mL was prepared in methanol and subsequently diluted to obtain an absorbance of 0.8 at 516 nm (working solution). Two mL of diluted water extracts of borage of different concentrations (0.0009 g/mL - 0.2 g/mL) were allowed to react with 2 mL of DPPH working solution for 30 min in the dark, at room temperature. A control sample was prepared with 2 mL of methanol. The final absorbance of the reaction mixture was measured at 5 16 nm with a Lambda 5 UV-VIS Spectrophotometer (Perkin Elmer, Paris, France). The radical scavenging capacity of each dilution was calculated as the percent of inhibition (% I), calculated according to the formula:

% I = (Abs^sub control^ - Abs^sub sample^ )/Abs^sub control^ *100

Where Abs^sub control^ was the absorbance of the control control after 30 min of reaction and Abs^sub sample^ was the absorbance of the sample after 30 mm of reaction. The percent of inhibition was plotted versus the concentration of the extracts. A calibration curve with Trolox (0.1-200 µg/mL) was used to calculate the antioxidant capacity in µg Trolox/mL. Results were finally expressed as µg Trolox/g fresh plant. Absorbance measurements were made in duplicate for each diluted solution.

ABTS method

For the ABTS assay, the procedure described by RE et al (1999) was used with some modifications. Briefly, the ABTS + chromogenic radical was generated by a chemical reaction mixing an aqueous solution of ABTS with K2S2O4 (140 mM) to reach a 7 mM final concentration of ABTS. The mixture was kept in the dark for 12-16 h at room temperature (stock solution). Before use, 1 mL OfABTS+ stock solution was diluted with ethanol (50%) to an absorbance of 0.70 (+/-0.02) at 741 nm (working solution). Three mL of ABTS + working solution were allowed to react with 300 µL of suitably diluted water borage extracts (0.0009 g/mL - 0.2 g/mL) or control (ethanol-50%) for 6 min, and then the absorbance was measured at 741 nm (Lambda 5 UV-VIS Spectrophotometer, Perkin Elmer, Paris, France). The decrease in absorbance was recorded as percent of inhibition (% I) and was calculated according to the formula:

% I = (Abs^sub control^ - Abs^sub sample^ )/Abs^sub control^ *100

Where Abs^sub control^ was the absorbance of the control after 6 min of reaction and Abs^sub sample^ was the absorbance of the sample after 6 min of reaction. The percent of inhibition was plotted versus the concentration of the extracts. A calibration curve with Trolox (0.1-60 µg/mL) was used to calculate the antioxidant capacity. Results were finally expressed as µg Trolox/g fresh plant. Absorbance measurements were made in duplicate for each diluted solution.

Statistical analysis

Differences between raw, boiled and steamed stems were evaluated by a one-way ANOVA and the Tukey b Post hoc test was applied when appropriate. A Student-t test was used (p>0.05) to evaluate differences between raw samples (leaves and stems). A Pearson correlation test was performed to study the correlation between phenolic compounds and both ABTS and DPPH. The correlation between ABTS and DPPH was also evaluated (SPSS 15.0, Inc., Chicago, IL, U.S.A.).

RESULTS AND DISCUSSION

Methodology (ABTS versus DPPH)

Calibration curves were plotted for both ABTS and DPPH assays using Trolox as the reference compound (Fig. 1 a, b) in order to express the results of the antioxidant activity of the samples using the same units for both methods. Within the range of Trolox concentrations that nearly reached 100% of% 1, ABTS fit to a linear model (R^sup 2^=0.999), whereas DPPH fit an exponential model (R^sup 2^=0.994).

Increasing concentrations of different diluted borage extracts (g fresh plant/mL) were subjected to both assays, and plotting the results of % I. vs. concentration (Figs. 2 and 3) were plotted. As occurred with Trolox, ABTS fit a linear model and DPPH fit an exponential model. These% I were transformed to their corresponding µg Trolox/mL values after applying the required calibration equations (shown in Figs. 1 a, b). Taking into account the concentration (g fresh plant/ mL) the antioxidant activity was then given for each diluted extract (µgTrolox/g fresh plant). After a careful examination of these data, only percentages of inhibition values between 20% and 80% were considered representative of each extract and were used to calculate the final mean value expressed as µg Trolox/g fresh plant. RE et al (1999) also selected this range for the ABTS evaluation of different antioxidant solutions. At the concentrations tested in this study, in general the ABTS results did not reach 80%. For the DPPH results, the concentration of samples that had a % I greater than 80% could not be reproduced and consequently were not used to calculate the mean antioxidant activity of the borage extracts. To our knowledge, this is the first time that this approach has been used to obtain the antioxidant activity value of a plant extract. Most papers in the literature report only evaluation of one or two extract concentrations in order to make comparisons with fixed amounts of standard compounds.

The analysis of different borage extracts using these methodologies revealed similar values of µg Trolox/g fresh plant for ABTS and DPPH (p>0.05); the coefficients of variation were low in both cases (Table 1). In wines, the results for both tests, expressed in mM Trolox, gave higher values for ABTS than for DPPH. This was attributed to the capacity of ABTS to measure lipophilic and hydrophilic activity, whereas DPPH seems to be more specific for lipophilic antioxidants (RIVERO-PEREZ et al, 2007). However, THAIPONG et al. (2006) found that the data for ABTS and DPPH were comparable in guava methanol extracts. This finding shows that the solvent used for the extractions plays a critical role in allowing comparisons between tests, as reported by VENSKUTONIS et al (2007). STRATIL et al. (2006) obtained nearly the same results for TEAC and DPPH, while the values obtained with the FRAP method were about 50% higher when testing the antioxidant activity of fruits and cereals. In that paper, significant correlations were found between total phenolic compounds and the total antioxidant activity of plants measured by different methods. In the present study, although some discrepancies were found in the decrease of TPC and AA as a consequence of steaming, high correlations were found between each of the AA parameters and TPC (R^sup 2^=0.971 for ABTS and TPC; R^sup 2^=0.979 for DPPH and TPC). Good correlation values were also obtained between the DPPH and ABTS results (R^sup 2^=0.985).

Antioxidant activity: leaves and stems

As shown in Table 1, raw borage leaf extracts showed significantly higher antioxidant activity than raw stem extracts (p<0.05). Values for both DPPH and ABTS in leaf extracts were 1236.71 µg Trolox/g fresh plant and 1128.38 µg Trolox/g fresh plant respectively; approximately 3.5 times higher than the values obtained for stem extracts (342.34 µg Trolox/g fresh plant and 324.53 µg Trolox/g fresh plant). These results suggest that, since borage leaves are not used for human consumption and are usually discarded before cooking, they could be a byproduct used as a good source of bioactive compounds. BANDONIENE et al (2002) showed that an acetone extract of borage leaves efficiently reduced the oxidation rate of rapeseed oil at 80°C in terms of peroxides. PYO et al (2004) also found significantly higher antioxidant activity in the leaves of Swiss chard compared to the stems, although in that case both plant parts are consumed. Significant differences in the antioxidant activity and the levels of phenolics between leaves and stems or other plant parts of other vegetables have also been reported (PESCHEL et al, 2006).

The high antioxidant activity of borage leaf extracts could be due to the high levels of phenolic compounds (2.36 mg GAE/g fresh plant for leaves and 0.57 mg GAE/g fresh plant for stems) (Fig. 4). Although there is an overestimation of total phenolic compounds when the plants are analyzed by the Folin-Ciocalteu method due to interference with some reducing components such as ascorbic acid and sugars (PADDA and PICHA, 2007), a linear correlation between radical scavenging activity and polyphenolic concentration has been found in different vegetables and fruits (KAHKONEN et al, 1999; MEZADRI et al, 2008). This correlation was also found in this study, especially in raw extracts. It can therefore be concluded that the antioxidant activity of each extract is mostly related to the concentration of phenolic hydroxyl groups, as also reported by PYO et al (2004). TURKMEN et al (2005) reported that total phenolic content of different fresh vegetables (pepper, squash, green beans, peas, leek broccoli and spinach) ranged from 183.2 to 1344.7 mg GAE/ 100 g on dry weight basis. These values are three times lower than those found for borage extracts in this work (602 mg GAE/ 100 g dry weight for stems and 4,262 mg GAE/ 100 g dry weight for leaves).

Many studies report the phenolic content of different vegetables in mg GAE/g or 100 g (WU et al, 2004; ZHANG and HAMAUZU, 2004; PATTAHAMAKANOKPORN et al, 2008). In these studies different mixtures of solvents were used; acetone/water/acetic acid gave the highest values for fresh broccoli (337 mg GAE/ 100 g fresh plant) (PYO et al, 2004), The other results in those papers did not exceed 236 mg GAE/ 100 g fresh plant, the value obtained with the borage leaf water extracts in the current work.

LLORACH et al (2003) evaluated water extracts of cauliflower by-products as a possible source of antioxidant phenolics; 1.8 g of total phenolic compounds per kg of cauliflower byproduct were obtained. Water extracts showed total phenolic values that were more similar than other conventional solvent extracts used with by-products (PESCHEL et al, 1999). The advantages obtained were an easier manipulation with a lower cost and a safer extraction process. PESCHEL et al (1999) found that the total phenolic content of extracts from different plants obtained with organic solvents were: 48.6, 251.4 and 514.2 mg GAE/g dry extract, for apple, golden rod and artichoke, respectively. However, these authors pointed out that more nutritional studies are needed to determine the amount of extracts needed to obtain beneficial effects.

Cooking effect

Clear differences in the antioxidant activity and phenolic compounds were observed depending on the type of thermal process applied to the borage stems. When stems were boiled, both ABTS and DPPH decreased significantly (51-52%) (Table 1), and the total phenolic compounds decreased about 67% (Fig. 4). It can be concluded that boiling reduces the potential health benefits associated with the antioxidant compounds present in fresh borage. The water used in this type of cooking was probably enriched with, at least, some of these compounds due to a dilution effect. This aspect was confirmed by GLISZCZYNSKA-SWIGLO et al (2006) in a similar work carried out with broccoli. These authors also found that cooking in water significantly decreased most of the health-promoting compounds of broccoli. They also confirmed that the loss of both vitamin C and polyphenols was mainly due to being leached into the cooking water.

When steam cooking of borage stems was applied, no effect was observed in the antioxidant activity values. The ABTS and DPPH showed results that were similar to those obtained for raw stems (Table 1). Total phenolic compounds decreased with this treatment (around 35%, approximately half of the decrease found for boiling (Fig. 4). In comparison to boiling, this milder heat treatment, with no leaching-effect, resulted in a higher antioxidant value and phenolic content in the edible portions of borage. In both cooking methods, the relative loss of phenolic compounds in the treated samples compared to the fresh ones was more evident than that observed for the antioxidant activity. TURKMEN et al (2005) also reported different results for the phenolic compounds and antioxidant activity depending on the vegetables being analyzed and the cooking treatments applied. Some authors suggest that there is the possibility of formation of new antioxidant compounds (e.g. Maillard reaction products) during heat treatment (NICOLI et al, 1999).

The literature shows that the effect of thermal treatment on the potential antioxidant activity of vegetables is different depending on several factors such as species, intensity and modality of the treatment. Analyzing the influence of heat treatment on the antioxidant activities and polyphenols compounds of Shiitake mushroom extracts CHOI et al (2006) found that they increased as heating temperature and time increased. These results are in agreement with those found by DEWANTO et al (2002), who showed that thermal processing enhanced the nutritional value of tomatoes by increasing the bio-accessible lycopene content and total antioxidant activity.

On the contrary, some studies on broccoli and cauliflower showed that heat treatments (blanching and cooking) had a detrimental effect on the polyphenols and antioxidant activity in comparison with the raw products (GEBCZYNSKI and KMIECIK, 2007). ZHANG and HAMAUZU (2004) also found that the antioxidant components (including phenolic compounds) and antioxidant activity in broccoli decreased markedly during conventional and microwave cooking. RACCHI et al (2002) found that boiling had different effects on the antiradical activity of water-soluble components depending on the type of vegetable. Testing the effect of thermal treatment on the water-soluble fraction of different vegetables, ROY et al (2007) concluded that normal cooking temperatures (75°- 100°C, 10-30 min) were detrimental to the phenolic content and to the antiradical and antiproliferative activities of many vegetable juices; however mild heating (50°C, 10-30 min) preserved 80-100% of the phenolic content.

All of these findings confirm that more research is needed to determine the effects of cooking on total phenolics, as stated by RICKMAN et al (2007) in a recent review of the nutritional value of different fresh and processed fruits and vegetables.

In summary, borage water extracts, particularly, those of their by-products (leaves) showed great antioxidant activity, that could potentially be used for different applications in the food industry , Steam cooking of borage was a better technology than traditional boiling for preserving the antioxidant activity and total phenolic content of borage.

ACKNOWLEDGEMENTS

The authors are grateful to CONSOLIDER-INGENIO 2010 CARNISENUSA CSD2007-00016, to project AGL 200801099/ALI (Ministerio de Ciencia y Tecnología, Spain), to the Government of Navarra (Plan Tecnológico), to PIUNA (Plan de Investigación de la Universidad de Navarra) and to OLUS Tecnología for their financial support of this research project.

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Paper received June 10, 2009 Accepted October 2, 2009