Antioxidant potential of hydrolyzed polyphenolic extracts from tara (Caesalpinia spinosa) pods

Industrial Crops and Products 47 (2013) 168–175

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Antioxidant potential of hydrolyzed polyphenolic extracts from tara (Caesalpinia spinosa) pods Flor Chambi a,b , Rosana Chirinos a , Romina Pedreschi c , Indira Betalleluz-Pallardel a , Frédéric Debaste b , David Campos a,∗ a

Instituto de Biotecnología (IBT), Universidad Nacional Agraria La Molina-UNALM, Av. La Molina s/n, Lima, Peru Transfers, Interfaces and Processes (TIPs), Chemical Engineering Unit, Université Libre de Bruxelles-ULB, 50 Avenue F.D. Roosevelt, C.P. 165/67, 1050 Brussels, Belgium c Fresh, Food & Chains, Wageningen UR Food & Biobased Research, Bornse Weilanden 9, 6708WG, The Netherlands b

a r t i c l e

i n f o

Article history: Received 5 October 2012 Received in revised form 7 March 2013 Accepted 9 March 2013 Keywords: Tara Caesalpinia spinosa Gallotannins Hydrolysis degree Chemical hydrolysis Antioxidant potential

a b s t r a c t The antioxidant potential of tara pod extracts rich in gallotannins submitted to chemical hydrolysis was evaluated. The increase in the release of gallic acid from the tara pod extracts during the hydrolysis process reached a maximum ratio of free gallic acid/total phenolics of 94.1% at 20 h, at this point, 100% hydrolysis degree (HD) was obtained. After 4 h of hydrolysis (38.8% of HD) the highest antioxidant capacity was obtained reaching values of 25.9, 23.8 and 8.8 ␮mol trolox equivalent/mg gallic acid equivalent measured by ABTS, FRAP and ORAC methods. Lipophilicity diminished from 0.8 to 0.3 (log P value). In addition, the antioxidant efficacy of 100 ppm total phenolics of hydrolyzates at 9 h (93.7% of HD) and 20 h showed to be significantly more efficient than a similar concentration of the synthetic antioxidant TBHQ to retard soybean oil oxidation. These results indicate that 4 and 9 h of chemical hydrolysis of tara pod extracts under the tested conditions are sufficient to obtain a product with good antioxidant properties to be used as an alternative source of antioxidants. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Tannins are phenolic compounds of relative high molecular weight. They are classified as condensed and hydrolyzable tannins. The hydrolyzable tannins are readily hydrolyzed by acids, alkalis or enzymes (tannases) into a sugar or a related polyhydric alcohol (polyol) and a phenolic carboxylic acid (Hagerman, 1998). Depending on the nature of the phenolic carboxylic acid, hydrolyzable tannins are subdivided into gallotannins and ellagitannins. Hydrolysis of gallotannins yields gallic acid while hydrolysis of ellagitannins yields hexahydroxydiphenic acid which is isolated as ellagic acid (Hagerman, 1998). Hydrolyzable tannins are considered as one of the most potent antioxidants from plant sources. They are ready to form complexes with reactive metals, avoiding free radical generation which results in oxidative damage of cellular membranes and DNA (Khan et al., 2000). Hydrolyzable tannins, in addition, clean free radicals within the body by neutralizing them before cellular damage occurs (Hagerman, 1998). Thus, the in vitro antimutagenic and anticarcinogenic activity of tannic acid has been previously reported (Gülc¸in et al., 2010).

∗ Corresponding author. Tel.: +51 1 3495764; fax: +51 1 3495764. E-mail address: [email protected] (D. Campos). 0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.03.009

Tara (Caesalpinia spinosa (Molina) Kuntze) is a native leguminous tree from South America consisting of red or pale yellow pods of 8–10 cm length. It is spread from the region of Venezuela, Colombia, Ecuador, Peru, Bolivia, until the north of Chile. Tara wildly grows in the Peruvian coast and Andean region at altitudes from 1000 to 2900 m above see level (De la Cruz, 2004). Peru is considered the most important worldwide producer of tara with more than 80% of the world production (Mancero, 2008). Tara infusions have been traditionally and extensively used by the Peruvian folk medicine to treat inflamed tonsils, fever, cold and stomachaches (Bussmann and Sharon, 2006). Tara pods (without seeds) represent approximately 65% (w/w) of the fruit. Ground tara pods concentrate a high tannin content (∼40–60% (w/w)). Tara pods are a good source to produce tannic, gallotannic and gallic acid. Tara tannins are used in the manufacture of leather furniture, plastics and adhesives, as wine clarifier, as malt substitute, as source to obtain the antioxidant gallic acid used in the oil industry (De la Cruz, 2004). Tara tannins are also employed as component of gastroenterological medicaments to cure ulcers and help cicatrization. Astringent, anti-inflammatory, antifungal, antibacterial, antiseptic, antidiarrheal properties have been attributed to tara tannins (Bussmann and Sharon, 2006; De la Cruz et al., 2007). Tara pods contain gallotannins. Gallotannins are mainly composed of polygalloyl esters of quinic acid. Complete hydrolysis which involves rupture of depside and ester bonds yields quinic and

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gallic acids. Tannins present in other members of the hydrolyzable tannin group contain a galloylated or ellagoylated hexose (Garro et al., 1997). By means of a partial or complete hydrolysis, it is feasible to obtain gallic acid or remaining tannins from tara tannins. Both gallic acid and remaining tannins display higher antioxidant capacity than tannins (Salminen et al., 2002; Wang et al., 2007) due to the exposition of hydroxyl groups of gallic acid released after hydrolysis. The use of strong acid conditions (2 N sulfuric acid at 100 ◦ C) and prolonged hydrolysis time (26 h) has been previously reported (Inoue and Hagerman, 1988) to completely hydrolyze the gallotannin molecule. The present study, however, aims to demonstrate the feasibility to obtain tara hydrolysis products with high antioxidant capacity without incurring in a complete hydrolysis. Thus, the transformation of tara tannins into phenolic compounds of low molecular weight is an alternative process to obtain high added value extracts. The demand of this alternative product is in rise due to its increasing application as feeding supplement, and the possibility to be employed by the pharmaceutical and food industry. Thus, this work aims: (1) to study the chemical hydrolysis of gallotannin extracts from tara, (2) to evaluate the hydrolysis degree (HD) of tara extracts regarding in vitro antioxidant capacity and (3) to evaluate the performance of these extracts by differential scanning calorimetry to retard soybean oil oxidation.

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contents were determined by colorimetric methods (Section 2.3.1) in all extracts. The HD was calculated as follows: HD (%) =

GAHE − GAI × 100 GAHC − GAI

where GAHE , gallic acid concentration in the hydrolyzed tara extract at certain time (mg GA/mL); GAI , gallic acid concentration in the initial tara extract (mg GA/mL); GAHC , gallic acid concentration in the totally hydrolyzed tara extract (mg GA/mL).

2. Materials and methods

2.2.3. Clean up of tara hydrolyzed extracts The tara hydrolyzed extracts were submitted to a liquid–liquid partition using ethyl acetate (González et al., 2004) to eliminate the sulfuric acid present in those extracts. The hydrolyzed extracts were mixed with equal volumes of ethyl acetate. The partition included 10 min agitation in darkness. Then, the mixture was let to stand until two phases were observed and the organic phase was collected. A second partition was performed under the same conditions. The organic phases were assembled; vacuum concentrated at 37 ◦ C until dryness and the remaining pellet was dissolved in absolute ethanol. This extract is referred as tara purified and hydrolyzed extract (TPHEs). The remaining aqueous phase enriched in sulfuric acid was discarded. TPHEs were flushed with nitrogen and stored at −20 ◦ C until analyses. Gallic acid, total phenolics, in vitro antioxidant capacity and HPLC-PDA phenolic profiles were determined in the TPHEs.

2.1. Material and chemicals

2.3. Quantitative analysis

Fresh tara pods were purchased from a local market in Caraz (Ancash, Peru). Tara pods (without seeds) were washed and airdried at 55 ◦ C until a final humidity of ∼3% was reached. Tara pods were subsequently milled and sieved (#80 mesh) and stored at −20 ◦ C for further analysis. Gallic acid, pyrogallol, trolox (6-hydroxy-2,5,7,8tetramethyl chroman-2-carboxylic acid), ABTS (2,2 -azinobis (3-ethylbenzothiazoline-6-sulfonic acid)), TPTZ (2,4,6tripyridyl-s-triazine), AAPH (2,2 -azinobis(2-amidinopropane) dihydrochloride), fluorescein sodium salt, 2 N Folin-Ciocalteu reagent, rhodanine (2-thio-4-ketothiazolidine) and TBHQ (tertbutylhydroquinone) were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). All solvents and other chemicals of analytical grade were purchased from Merck (Darmstadt, Germany) and Fischer Scientific (Fair Lawn, NJ, USA).

2.3.1. Total phenolics, gallic acid, gallotannin content and ratio gallic acid/total phenolics Total phenolics were determined following the method of Singleton and Rossi (1965) using gallic acid as a standard. Absorbance was measured at 755 nm and the results were expressed as mg of gallic acid equivalents (GAE)/g or mL. Gallic acid and gallotannin contents were determined using the rhodanine assay reported by Inoue and Hagerman (1988) and Salminen (2003). Briefly, 1.5 mL of 0.667% (w/v) methanolic rhodanine solution was mixed with 1 mL of 0.2 N H2 SO4 tara extract. After 5 min incubation, 1 mL of 0.5 N KOH was added. After 2.5 min the mixture was diluted to 25 mL with distilled water and the absorbance at 520 nm was measured after 5–10 min. Gallic acid was calculated as mg of GAE/g or GAE/mL from a standard curve developed with gallic acid. Gallotannins were estimated as the difference between the absorbance of the totally hydrolyzed tara extract (20 h hydrolysis) and the initial absorbance of free gallic acid in the initial tara extract. Gallotannin content was calculated as mg of GAE/g or GAE/mL. The ratio gallic acid/total phenolics was calculated by dividing them and the results were expressed in percentage (%).

2.2. Extract preparation 2.2.1. Extraction Phenolic compounds were extracted employing 80% (v/v) acetone/water as solvent (Tian et al., 2009) and a material/solvent ratio of 1/100 (w/v). Extraction was carried out at 4 ◦ C for 20 h. Then, the mixture was centrifuged at 10,000 × g for 15 min and the supernatant was vacuum concentrated at 38 ◦ C until dryness. The resulting product was dissolved with Milli-Q water. This aqueous solution was cooled at ∼4 ◦ C for 16 h and then again centrifuged at 10,000 × g for 10 min to obtain a clarified solution. This remaining product is referred as the whole extract. 2.2.2. Hydrolysis of tara extracts The whole extract was mixed with sulfuric acid until reaching a final concentration of 2 N H2 SO4 and a phenolic compound concentration of 20 mg gallic acid equivalent/mL. The mixture was let to stand at 100 ◦ C for 0, 0.5, 1, 1.5, 2, 4, 5, 6, 8, 9, 12, 16, 20, 24 and 28 h. The resulting mixture referred as the hydrolyzed extracts were centrifuged at 10,000 × g for 10 min. Then, total phenolic and gallic acid

2.3.2. In vitro antioxidant capacity and specific antioxidant capacity The antioxidant capacity was determined with the ABTS, FRAP and ORAC assays. For ABTS assay, the procedure followed was the same procedure described by Campos et al. (2006). Samples (150 ␮l) were allowed to react with 2850 ␮l of ABTS+ solution in ethanol until a steady absorbance was reached at room temperature and dark conditions. The decrease in absorbance due to antioxidants was recorded at 734 nm. The FRAP assay was conducted according to Benzie and Strain (1996) with minor modifications. The FRAP reagent consists of acetate buffer (300 mM, pH 3.6), TPTZ (10 mM in HCl 40 mM) and FeCl3 ·6H2 O (20 mM) (10:1:1, v/v/v). A total of 2850 ␮l of FRAP reagent was mixed with 150 ␮l of sample at 37 ◦ C. Then, the

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absorbance at 593 nm decreased to a stable value, implicating that Fe3+ -2,4,6-tri-pyridyl-S-triazine was reduced by the samples. The ORAC assay was performed in a 96-well microplate fluorometer (Bio Tek Instruments), essentially as described by Ou et al. (2001). Peroxyl radical was generated using AAPH and fluorescein was used as the substrate. To all experimental wells, 250 ␮l of sodium fluorescein solution 48 nM was added. In addition, blank, standard and samples received 25 ␮l of 75 mM phosphate buffer (pH 7.4), trolox and extract dilutions respectively, and were incubated for 10 min at 37 ◦ C. Reactions were initiated by the addition of 25 ␮l of AAPH. Fluorescence was collected at 520 nm on excitation at 480 nm, taking measurements from each sample at 60 s intervals. The specific antioxidant capacity (SAC) was determined by dividing the antioxidant capacity by the total phenolic content and the results were expressed as ␮mol of TE/mg GAE. 2.3.3. Differential scanning calorimetry (DSC) assay The antioxidant efficacy of TPHEs in crude soybean oil was determined by using a Perkin Elmer differential scanning calorimeter Pyris 6. The equipment was calibrated with pure indium and an aluminum pan was used as reference in order to obtain a baseline. Oil samples of 5.0 ± 0.1 mg were individually weighed in an open aluminum pan and placed into the sample chamber. The isothermal temperature was programmed at 140 ◦ C and then 99.8% pure oxygen was flushed through the sample at 35 mL/min. The onset time of oxidation reaction referred as induction period (IP) closely corresponded to the intersection of the extrapolated baseline and the tangent line (leading edge) of the exothermal function (Tan et al., 2002). The effectiveness of the TPHEs against soybean oil oxidation was tested by measuring the induction periods (IP) of the TPHEs samples containing a concentration of 100 ppm total phenolics for the different HD and 100 ppm TBHQ (positive control). A negative control (soybean oil without added antioxidant) was also evaluated. From the induction periods, the stabilization factors were calculated (Farhoosh et al., 2007). The stabilization factor was estimated as follow: Stabilization factor =

IPinh IP0

where IPinh is the induction period duration in the presence of an inhibitor, and IP0 is the induction period duration of the noninhibited system. 2.3.4. Measurement of lipophilicity of tara purified hydrolyzed extracts To estimate the lipophilicity of the extracts, the partition coefficient was measured (P, ratio of concentrations of a unionized compound in organic and in aqueous phases; log Pow ) between n-octane and water by a widespread technique, the shake-flask method (Takàcs-Novak and Avdeel, 1996; Poaty et al., 2010). To ensure the minimal ionization of phenolic compounds, the Britton–Robinson buffer at pH 1.5 was used as the aqueous phase. Buffer and n-octane were saturated with each other at 25 ◦ C for 3 h. The phases were allowed to separate and were then filtered (aqueous phase on analytical filter-paper and n-octanol on a G4 glass filter under vacuum). The TPHEs were dissolved in Britton–Robinson buffer at a final concentration of 0.02 mg GAE/mL and shaken with n-octanol for 10 min at ambient temperature (∼25 ◦ C). Then, the two phases were separated by centrifugation and the concentration of the phenolic compounds was determined in each phase by UV analysis at 280 nm. The log Pow values were calculated by using the formula: Log Pow = log

 [S]

octanol

[S]water



where [S] is the phenolic compound concentration. 2.3.5. HPLC-PAD analysis of phenolic compounds Phenolic profiles were determined according to the procedure proposed by Chirinos et al. (2008) with slight modifications. TPHEs were separated on a reversed-phase HPLC column on a Waters 2695 Separation Module (Waters, Milford, MA) equipped with an autoinjector, a 996 photodiode array detector (PDA) and the Empower software. Spectral data were recorded from 200 to 700 nm during the whole run. An X-terra RP18 (5 ␮m, 250 mm × 4.6 mm) column (Waters, Milford, MA) and a 4.6 mm × 2.0 mm guard column were used for phenolic separation at 30 ◦ C. The mobile phase was composed of solvent (A) water:formic acid (95:5, v/v, pH 2) and solvent (B) acetonitrile. The solvent gradient was as follows: 0–15% B in 40 min, 15–45% B in 45 min, and 45–100% B in 10 min. A flow rate of 0.5 mL/min was used and 20 ␮l of sample was injected. Samples and mobile phases were filtered through a 0.22 ␮m Millipore filter, type GV (Millipore, Bedford, MA) prior to HPLC injection. Phenolic compounds were identified by comparing their retention time and UV–visible spectral data to known previously injected standards. 2.4. Statistical analyses All quantitative analysis was performed at least in triplicate. Values of different results were expressed as the mean ± standard deviation (SD). Results were tested for statistical significance by one-way ANOVA. A Duncan test was used to assess statistical significant differences among treatments (p < 0.05). The SPSS software for Windows 14.0 (SPSS, Chicago, IL) was used for statistical calculations. 3. Results and discussion 3.1. Gallotannins, free gallic acid and total phenolics of tara pods Hydrolyzable tannins are synthesized by a wide variety of plants and can occur in wood, bark, leaves, fruits and galls (MuellerHarvey, 2001). Free gallic acid and gallotannins contents in tara pods were 1.7 and 50.4 g GAE/100 g, respectively. These values are in accordance with gallic acid and gallotannin contents in tara pods of 2.6 and 53.1 g GAE/100 g, previously reported by Garro et al. (1997). Barthomeuf et al. (1994) reported a tara pod tannin content of 56 g/100 g. Gallotannin content in tara pods was compared with other important sources (Table 1). Total phenolics in tara pods corresponded to 55.1 g GAE/100 g. The sum of gallotannins and gallic acid corresponded to 52.1 g GAE/100 g. These two compounds represented ∼95% of the total phenolics present in tara pods. The remaining 5% of tara phenolics might be composed of ellagitannins and others phenolic compounds. Ellagitannins have been previously reported in minor amounts in tara pods (Garro et al., 1997). 3.2. Hydrolysis of tara gallotannins Total phenolics, free gallic acid contents, and the HD of tara pod extracts during the course of the chemical hydrolysis are displayed in Fig. 1a and b, respectively. Total phenolics remained almost constant during the whole hydrolysis process (Fig. 1a). The maximum amount of released gallic acid occurred after 20 h of hydrolysis (18.8 mg GAE/mL) and then a slight reduction was observed after 24 h and 28 h (Fig. 1a). The HD increased considerably during the hydrolysis process. A HD of 93.7% was achieved after 9 h (Fig. 1b). After 8 h hydrolysis, the slope of the curve decreased. Maximum HD (100%) was achieved after 20 h, however nonsignificant differences (p > 0.05) in HD were obtained between 16 and 20 h of hydrolysis.

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Table 1 Gallotannin content in tara (Caesalpinia spinosa) and other important plant sources. Specie

Part of the plant

Gallotannin content

Reference

Galla chinensis Caesalpinia spinosa Acer saccharum Liquidambar stiraciflua Betuna pubescens Terminalia chebula Quercus persica Quercus infectoria Quercus libani

Gall Pods Leaves Leaves Leaves Fruit Leaves Leaves Leaves

61.6 g GAE/100 g 55.1 g GAE/100 g 3.48 g/100 g 1.5 g/100 g 6.0 g/100 g 40 g/100 g 4.6 g/100 g 8.7 g/100 g 6.2 g/100 g

Tian et al. (2009) Experimental Inoue and Hagerman (1988) Inoue and Hagerman (1988) Salminen (2003) Lokeswari and Jaya Raju (2007) Yousef Elahi and Rouzbehan (2008) Yousef Elahi and Rouzbehan (2008) Yousef Elahi and Rouzbehan (2008)

Based on these results, 20 h was considered as total hydrolysis time of tara tannins. For leaves of Betula pubescens and maple, employing the same hydrolysis conditions of this study, Ossipov et al. (1997) and Inoue and Hagerman (1988), found total hydrolysis times for gallotannins of 4 and 26 h, respectively. Thus, hydrolysis time of gallotannins is structure-dependent on each plant species (Ossipov et al., 1997). Hydrolysis of gallotannins in general involves the rupture of depside and ester bonds releasing a mixture of penta-, tetra, and tri-galloil glucose, gallic and digallic acids, and a poliol group (hexose or quinic acid). After 20 h hydrolysis, a small decrease of phenolic compounds, free gallic acid and HD was observed (Fig. 1a and b), probably due to the prolonged exposure time to high temperatures (100 ◦ C). Thus, a maximum hydrolysis time of 20 h was considered. In tara purified and hydrolyzed extracts (TPHEs), the ratio of free gallic acid/total phenolics during the hydrolysis process is displayed in Table 2. The corresponding TPHEs at 0 h hydrolysis

Fig. 1. Total phenolics and gallic acid (a) and hydrolysis degree (b) evolution during chemical hydrolysis of tara extracts (with 2 N H2 SO4 , 20 mg of GAE/mL and 100 ◦ C). Different capital and short letters on each curve indicate significant differences (p < 0.05) as revealed by a Duncan test.

showed 6% of free gallic acid per total phenolics. This ratio constantly increased during the hydrolysis process reaching a value of ∼94% at 20 h (100% of HD). Thus, the phenolics present in TPHE after 20 h hydrolysis were mainly gallic acid. 3.3. In vitro antioxidant capacity and specific antioxidant capacity The in vitro antioxidant capacity increased during the hydrolysis process, reaching a maximum value of ∼500 ␮mol TE/mL with the ABTS and FRAP assays at 8 h of hydrolysis and ∼160 ␮mol TE/mL at 4 h with the ORAC assay (Fig. 2). A slight decrease was observed after these times, this decrease is due to the lower yield of extraction of phenolic compounds (%) during the liquid–liquid separation with ethyl acetate (Fig. 2). For the specific antioxidant capacity (␮mol TE/mg GAE) during the hydrolysis process, increases were evidenced (Table 2). Maximum values of ∼25 ␮mol TE/mg GAE (with ABTS and FRAP assays) and ∼9 ␮mol TE/mg GAE (with ORAC assay) were obtained at 4 h hydrolysis (38.8% of HD) and these values remained constant until 20 h hydrolysis. Hagerman (1998) mentioned that when gallotannins are hydrolyzed, bonds between gallic acid and the polyol center (ester bonds) are broken, as well as its depside bonds formed between gallic acids in meta or para-position. The depside bond is more easily hydrolyzed than the ester bond (Mueller-Harvey, 2001). Depside bonds involved a hydroxyl functional group of gallic acid that when hydrolyzed, is exposed to react with free radicals. However, when an ester bond is hydrolyzed, an exposition of the hydroxyl group of gallic acid is not possible, because in this bond this group is not involved. Several studies have demonstrated that the antioxidant capacity of a phenolic compound increases as the number of hydroxyl groups increases in the molecule. Thus, the antioxidant capacity depends on the number of hydroxyl groups present in the phenolic compound molecule, especially when they are in the ortho or para position (Balasundram et al., 2006; Tian et al., 2009). The increase in antioxidant capacity is due to the capacity of hydroxyl groups to donate electrons or hydrogen ions stabilizing free radicals (Murthy et al., 2002). Gallic acid possesses three hydroxyl groups, this feature increases its ability to donate electrons or hydrogens. Therefore, the antioxidant capacity of tara hydrolyzed extracts increases when gallic acid is released. The newly formed hydroxyl groups on the galloyl group as a result of hydrolysis also contributed to the increase in antioxidant capacity. Verma et al. (2009) reported that gallic acid is one of the phenolic acids with highest antiradical capacity against ABTS. As expected values measured with the ABTS and FRAP assays were similar, but the values measured with the ORAC assay were significantly different, this is due to differences in the methods and the pH of the medium. The pH influences the deprotonation of acids to give anions, which has big influence on the radicalscavenging behaviors. As the pKa values of gallic acids are around 4.0, the anions derived from proton dissociation will dominate in the neutral system (occurrence >99.5%). In fact, carboxyl itself

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Table 2 Ratio gallic acid/total phenolic, specific antioxidant capacity and lipophilicity of hydrolyzed tara extracts at different hydrolysis degrees.a Hydrolysis time (h)

Gallic acid/total phenolic (%)

Specific antioxidant capacity (␮mol TE/mg GAE)

ABTS 0.0 0.5 1.5 4.0 5.0 6.0 8.0 9.0 20.0 Gallic acid a

6.4 15.4 26.6 45.4 65.6 73.2 78.2 90.0 94.1

± ± ± ± ± ± ± ± ±

0.2a 0.3b 0.5c 0.5d 2.1e 0.6f 0.1g 0.6h 0.2i

17.6 18.7 20.5 25.9 25.6 25.3 25.6 25.8 25.7 25.5

FRAP ± ± ± ± ± ± ± ± ± ±

0.2a 0.2b 0.3c 0.6d 0.4d 0.3d 0.4d 0.6d 0.2d 1.9d

17.2 18.6 22.0 23.8 24.0 24.7 25.6 24.8 25.2 24.0

Lipophilicity log P (octanol/water) ORAC

± ± ± ± ± ± ± ± ± ±

0.9a 0.7a 0.6b 0.8c 0.5c 1.6c 0.9c 1.7c 1.4c 0.3c

7.4 7.5 7.7 8.8 7.5 7.1 7.3 8.1 7.5 7.6

± ± ± ± ± ± ± ± ± ±

0.4ab 0.4a 0.4ab 0.8b 0.5a 0.3a 0.2a 0.2ab 0.2ab 0.5ab

0.85 0.65 0.42 0.38 0.34 0.33 0.36 0.34 0.36 0.36

± ± ± ± ± ± ± ± ± ±

0.02a 0.01b 0.01c 0.05cd 0.04d 0.01d 0.01d 0.03d 0.03d 0.02d

Results are means ± SD (n = 3). Means within a column with the same letter are not significantly different (p > 0.05) as revealed by a Duncan test.

is an electron-withdrawing group, which does not benefit radical scavenging. However, the deprotonated carboxyl becomes an electron-donating group, which favors H-atom-transfer- and electron-donation-based radical scavenging. According to the current understanding of radical-scavenging processes of phenolic antioxidants, H-atom donation is the dominant mechanism (Ji et al., 2006). 3.4. Lipophilicity Lipophilicity of the TPHEs is displayed as log P values (P is the partition coefficient of the molecule in the water/octanol system) (Table 2). Data analysis allows concluding that the TPHEs hydrolysis

leads to a significant decrease in their lipophilicity until 4 h hydrolysis (at 38.8% DH). As expected, lipophilicity decreased with bond hydrolysis, release of gallic acid and exposition of the hydroxyl groups. 3.5. Antioxidant efficacy of TPHEs against soybean oil oxidation The IP for the different TPHEs tested at 100 ppm phenolics corresponding to different HD are displayed in Table 3. A negative control (no antioxidant added) and a positive control (100 ppm TBHQ) were tested. A progressive increase in the IP values for the different TPHEs was observed as the HD increased. In general, IP values for all TPHEs were higher than the IP value of the control.

Fig. 2. Antioxidant capacity ABTS (a), FRAP (b) and ORAC (c) of TPHEs at differents hydrolysis degree. Different capital and short letters on each curve indicate significant differences (p < 0.05) as revealed by a Duncan test.

F. Chambi et al. / Industrial Crops and Products 47 (2013) 168–175 Table 3 Induction periods and stabilization factors for hydrolyzed tara extracts at different hydrolysis times using differential scanning calorimetry.a Hydrolysis time (h)

Induction period (min)

0 0.5 1.5 4.0 5.0 6.0 8.0 9.0 20.0 TBHQ Control

40.17 42.03 43.35 47.27 47.67 48.38 49.11 50.42 50.24 49.45 37.35

± ± ± ± ± ± ± ± ± ± ±

0.28b 0.37c 0.36d 0.29e 0.29e 0.13f 0.0g 0.1h 0.4h 0.2g 0.19a

Stabilization factor 1.07 1.12 1.16 1.26 1.27 1.29 1.31 1.35 1.34 1.32 1.00

± ± ± ± ± ± ± ± ± ± ±

0.01b 0.01c 0.01d 0.01e 0.00e 0.00f 0.0g 0.01h 0.0h 0.0g 0.00a

a Results are means ± SD (n = 3). Means within a column with the same letter are not significantly different (p > 0.05) as revealed by a Duncan test.

After 8 h hydrolysis corresponding to 88.4% HD, the IP of this TPHE showed to be similar to the IP value of the positive control (100 ppm TBHQ) but lower than the IP values of the TPHEs after 9 and 20 h hydrolysis (93.7 and 100% HD, respectively) (Table 3). Thus, using similar concentrations, significant better protective effects were observed for the TPHEs at 9 and 20 h hydrolysis than TBHQ. Similar results were observed with the stabilization factor (SF) values. TPHEs at 9 and 20 h hydrolysis displayed higher stabilization factors than TBHQ (Table 3). Nonsignificant differences in SF were observed between TPHEs at 9 and 20 h (93.3 and 100% HD, respectively). The stabilization factors for the different TPHEs exhibited a good correlation with respect to HD during soybean oil oxidation. A polynomial equation was found to describe this relationship (SF = −2 × 10−5 HD2 (%) + 0.0049 HD (%) + 1.077); (r2 = 0.979). Our results agree with those reported by Kim et al. (2010) who showed that thermal processing of tannic acid (as a medium to hydrolyze tannic acid) favored a higher antioxidant capacity than fresh tannic acid. In this study of Kim et al. (2010), it was also demonstrated

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that increasing thermal processing time (which involved increases in HD) of thermally processed tannic acid favored the stability of soybean oil to oxidation. Thermal processing enhanced hydrolysis of tannic acid producing free gallic acid and galloyl groups on the remaining gallotannins, which were responsible for the displayed biological activity. The high antioxidant capacity of gallic acid and gallotannins has been reported in several studies. Thus, Lu et al. (2006) and Kosar et al. (2007) reported the great antioxidant potential against lipid oxidation of gallic acid in liposome and linoleic acid systems. Moore et al. (2005) showed that gallotannin acid 3,4,5 tri-O-galloylquinic, present in high amounts in Myrothamnus flabellifolius leaves, acted as a good antioxidant preventing the oxidation of linoleic acid. Similar results were reported for gallotannins of Galla chinensis (Tian et al., 2009). Gallotannins from Pistacia weinmannifolia leaves containing two units of gallic acid referred as pistafolin A and B, showed high inhibition level of liposome oxidation. The protective effects of these two gallotannins against oxidative damage were due to their strong free radical scavenging ability (Zhao et al., 2005). Gallic acid is a very polar phenolic compound due to the presence of three hydroxylations in its structure. Gallic acid was present in all TPHEs at different concentrations. It performed as a good antioxidant in an apolar system such as soybean oil. There is a polar paradox mentioned by Pokorny et al. (2005) who state that the more polar antioxidants exert better protective effects in less polar systems (e.g., oils) and less polar antioxidants exert better protective effects in polar systems (e.g., emulsions). Gallotannins display different polarities due to the differences in structure, substitution and size. High molecular weight gallotannins present low polarity (Tian et al., 2009). Gallic acid is more polar than gallotannins because when gallotannins are hydrolyzed into gallic acid, this one increases the polarity of the system and this is in accordance with the lipophilicity assay. This is one of the reasons why better protective effects are observed in soybean oil containing TPHEs than with the initial tara tannin extract.

Fig. 3. Chromatograms of TPHEs at different hydrolysis degrees: 0 h (nonhydrolyzed extract) (a), 4 h (38.8% HD) (b), 9 h (93.7% HD) (c) and 20 h (100% HD) (d).

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3.6. HPLC-PDA analysis of the hydrolyzed tara extracts To elucidate the phenolic profile of tara extracts at 0, 4, 9 and 20 h hydrolysis time, HPLC-PDA analysis was performed. The HPLC chromatograms recorded at 280 nm for the corresponding TPHEs are displayed in Fig. 3. Several peaks within the 50–70 min range were observed in the nonhydrolyzed tara extract (0 h) (Fig. 3a). These peaks could not be identified but presented similar spectral characteristics to gallic acid indicative of the presence of gallic acid derivatives such as gallotannins. Salminen et al. (1999) characterized the gallotannins of B. pubescens by HPLC as highly galloacylated presenting higher retention times that gallotannins of lower molecular weight with maximum absorbances between 273 and 285 nm. Our results are similar to the results found by Salminen et al. (1999). After 4 h of hydrolysis (HD of 38.8%), almost all peaks appearing after 50 min (0% of DH) disappeared and they were mainly transformed into one representative peak that eluted at 13.8 min identified as gallic acid. Peaks eluting between 32 and 50 min also presented similar spectral characteristics of gallic acid (Fig. 3b). This feature provides evidence of the rupture of bonds of tara tannins with the release mainly of gallic acid and other remaining tara gallotannins. After 9 and 20 h hydrolysis time (HD of 93.7 and 100%, respectively), gallic acid was the main peak (Fig. 3c and d). Gallic acid at high temperatures can be degraded to pyrogallol, a toxic crystalline phenol. However pyrogallol was not detected in these samples. 4. Conclusions This study provides strong evidence about the antioxidant potential of TPHEs with different HD. Total hydrolysis (100%) was achieved at 20 h with a concentration of gallic acid/total phenolics of 94.1%. An increase of in vitro antioxidant capacity for the different TPHEs was observed as the HD increased, especially from 4 h hydrolysis. At a concentration of 100 ppm TPHEs at 9 and 20 h hydrolysis (94.1 and 100% HD, respectively) significantly better antioxidant protection against soybean oil oxidation was observed than with 100 ppm TBHQ using the DSC assay. Thus, complete hydrolysis (100%) is not necessary to obtain a tara phenolic extract of excellent antioxidant characteristics. Four hours of hydrolysis using the conditions tested in this study are necessary to obtain a TPHE extract with good radical-scavenging capacity and 9 h to obtain a good performance as antioxidant using soybean oil system. Future studies regarding the stability of tara phenolics at different pHs, temperatures and oxygen contents should be carried out in order to optimize the hydrolysis process and scale it up to industrial applications. Acknowledgments This research was supported by the Peru Biocomercio project (Peru) and by the PIC project of the Belgian Coopération Universitaire au Développement (CUD, Belgium). References Balasundram, N., Sundram, K., Samman, S., 2006. Phenolic compounds in plants and agri-industrial by-products: antioxidant activity, occurrence, and potential uses. Food Chem. 99, 191–203. Barthomeuf, C., Regerat, F., Pourrat, H., 1994. Production, purification and characterization of a tannase from Aspergillus niger LCF 8. J. Ferment. Bioeng. 77, 320–323. Benzie, I., Strain, J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal. Biochem. 239, 70–76. Bussmann, R., Sharon, D., 2006. Traditional medicinal plant use in Northern Peru: tracking two thousand years of healing culture. J. Ethnobiol. Ethnomed. 2, 47.

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