Effect of different processing methods on antioxidant activity of underutilized legumes, Entada scandens seed kernel and Canavalia gladiata seeds

Food and Chemical Toxicology 50 (2012) 2864–2872

Contents lists available at SciVerse ScienceDirect

Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox

Effect of different processing methods on antioxidant activity of underutilized legumes, Entada scandens seed kernel and Canavalia gladiata seeds Gopalakrishnan Sasipriya, Perumal Siddhuraju ⇑ Bioresource Technology Lab, Department of Environmental Sciences, Bharathiar University, Coimbatore 641 046, Tamilnadu, India

a r t i c l e

i n f o

Article history: Received 24 March 2012 Accepted 28 May 2012 Available online 7 June 2012 Keywords: Gila bean Sword bean Polyphenols Processing DPPH

a b s t r a c t The present study is proposed to determine the antioxidant activity of raw and processed samples of underutilized legumes, Entada scandens seed kernel and Canavalia gladiata seeds. The indigenous processing methods like dry heating, autoclaving and soaking followed by autoclaving in different solutions (plain water, ash, sugar and sodium bicarbonate) were adopted to seed samples. All other processing methods than dry heat showed significant reduction in phenolics (2.9–63%), tannins (26–100%) and flavonoids (14–67%). However, in processed samples of E. scandens, the hydroxyl radical scavenging activity and b-carotene bleaching inhibition activity were increased, whereas, 2,2-azinobis (3-ethyl benzothiazoline-6-sulfonic acid) diammonium salt (ABTS+), ferric reducing antioxidant power (FRAP), metal chelating and superoxide anion scavenging activity were similar to unprocessed ones. In contrary, except dry heating in C. gladiata, all other processing methods significantly (P < 0.05) reduced the 2,20 -diphenyl-1picryl-hydrazyl (DPPH) (20–35%), ABTS+ (22–75%), FRAP (34–74%), metal chelating (30–41%), superoxide anion radical scavenging (8–80%), hydroxyl radical scavenging (20–40%) and b-carotene bleaching inhibition activity (15–69%). In addition, the sample extracts of raw and dry heated samples protected DNA damage at 10 lg. All processing methods in E. scandens and dry heating in C. gladiata would be a suitable method for adopting in domestic or industrial processing. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Oxidative stress is defined as an imbalance between production of free radicals and reactive metabolites, so-called oxidants or reactive oxygen species (ROS), and their elimination by protective mechanisms, referred to as antioxidants (Durackova, 2010). The continued oxidative stress can lead to chronic inflammation, which in turn could mediate most chronic diseases including cancer, diabetes, and cardiovascular, neurological, aging and pulmonary diseases. Oxidative stress can activate a variety of transcription factors can lead to the expression of over 500 different genes, including those for growth factors, inflammatory cytokines, chemokines, cell cycle regulatory molecules, and anti-inflammatory molecules (Reuter et al., 2010). ROS can control the expression of various tumor suppressor genes and also implicated in the chemopreventive and anti-tumor action of nutraceuticals derived from fruits, vegetables, spices, and other natural products used in traditional medicine (Gupta et al., 2012). During endogenous metabolic reactions, aerobic cells produce ROS such as superoxide anion (O2), hydrogen peroxide (H2O2), hydroxyl radical (OH), and

⇑ Corresponding author. Tel.: +91 4222428394; fax: +91 4222422387. E-mail address: [email protected] (P. Siddhuraju). 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.05.048

organic peroxides as normal products of the biological reduction of molecular oxygen (Fridovich, 1978). In such conditions, external supply of antioxidants is essential to countervail the deleterious consequences of oxidative stress (Reuter et al., 2010). The bioactive compounds derived from legumes, fruit and vegetables can modulate inflammatory pathways and thus affect the survival, proliferation, invasion, angiogenesis and metastasis of the tumor (Gupta et al., 2010). The consumption of plant foods like fruits, vegetables, legumes and cereals has been linked with reduction in the risk of developing chronic diseases, such as cancer, diabetes, obesity and cardiovascular diseases (Adams and Standridge, 2006). It is becoming clear that the regular intake of these plant foods in our daily life can be able to prevent free radical mediated degenerative diseases. Among them, legumes (Family: Fabaceae), the third largest family is recognized as a second most valuable plant source for human and animal nutrition (Doyle, 1994). They are excellent sources of protein, dietary fiber, starch, micronutrients and bioactive compounds with low level of fat. In addition to their nutritive value, legumes contain significant quantities of polyphenolic compounds such as flavonoids, isoflavones, phenolic acids and lignans (Lin and Lai, 2006). In fact, 650 genera and 20,000 species of legumes are available, but only few species like common beans (Phaseolus vulgaris), soya bean (Glycine max) and cowpea (Vigna unguiculata)

G. Sasipriya, P. Siddhuraju / Food and Chemical Toxicology 50 (2012) 2864–2872

are commonly available in the market. Due to their low production rate compared with consumption, an ever increasing demand has been witnessed (Ali and Kumar, 2000). The dependence on these certain plant species can lead to high price, scarcity and cause malnutrition in developing countries. Thus, many researchers are interested in the exploration of cost effective underutilized legumes. Entada phaseoloides Merrill (Gila bean), is an important tribal pulse with a variety of medicinal uses. It occurs throughout the sub-Himalayan tract, from Nepal eastwards ascending to 4000 ft. in Sikkim, Assam, Bihar and Orissa, and in the monsoon forest of Western and Eastern Ghats and it is abundant in Andaman Islands. The soaked and boiled seed kernels are consumed by tribal peoples in India (Janardhanan and Nalini, 1991). Due to its wide array of chemical compounds in the seeds, it is used as an alexiteric, narcotic, tonic, emetic, anthelmintic, antipyretic, stomachache, edema and diabetes mellitus (Das, 1994) (Zheng et al., 2012). A potent Kunitz type trypsin inhibitor from seed acts as a potential candidate molecule for the development of insect resistant transgenic plants (Lingaraju and Gowda, 2008). Canavalia gladiata (sword bean), is a leguminous plant originated in the Asian continent and spread throughout the tropics. They are cultivated on a limited scale throughout Asia, the West Indies, Africa and South America and have been introduced into tropical parts of Australia (Herklots, 1972). Their immature pod is consumed as a vegetable and the mature dry beans may be consumed after cooking because of their presence of antinutritional factors (Purseglove, 1968). The nutritional and antinutritional properties of Entada scandens and C. gladiata were reported by Siddhuraju et al. (2002), Sridhar and Seena (2006) and Vadivel et al. (2008). In spite of these health benefits, the utilization of these raw legumes is limited by the presence of antinutrients like phenolics, tannins, phytic acid, protease inhibitors, lectins, a-amylase inhibitors, canavanine and saponins and their consumption can lead to nausea, vomiting and diarrhea. The removal of undesirable components is therefore essential to improve the nutritional quality of legumes and effectively utilize their full potential as human food. In order to inactivate or reduce the above mentioned antinutrients, various conventional, simple processing methods have been used such as dry heating, roasting, boiling, soaking in water, alkali and acid, solvent extraction, germination and fermentation (Khokhar and Apenten, 2003; Siddhuraju and Becker, 2003). Heat treatment has been used to deactivate the thermo-labile antinutritional factors and soaking could be one of the processes for removal of soluble antinutritional compounds, which can be eliminated with the discarded soaking solution (Vidal-Valverde et al., 1992). The nutritive quality of most tropical legume grains, particularly cowpea, soybean, pigeon pea, lima bean and winged beans was notably improved by heat treatment (Akande and Fabiyi, 2010) and the amino acid, canavanine content in C. gladiata was reduced in overnight soaking and boiling in excess water (Ekanayake et al., 2007). Moreover, the processing methods like dry heating, autoclaving, soaking following by autoclaving in plain water, ash and sodium bicarbonate solution were found to improve the nutritional value of Mucuna pruriens by reducing maximum level of antinutrients present in it (Siddhuraju and Becker, 2003; Vadivel et al., 2010; Emenalom et al., 2005). In other parts of Asia, sword beans are often soaked in water over-night, boiled in water to which a small quantity of sodium bicarbonate has been added, rinsed, boiled, pounded and used in curries, or as a substitute for mashed potato (Eknayake et al., 1999). Moreover, the processing (dry heating, soaking and autoclaving in various solutions like plain water, ash and sugar solution) of C. ensiformis seeds showed higher antioxidant activity than raw seeds (Sowndhararajan et al., 2011). The increase or decrease in antioxidant activity of samples will depend upon our method of processing. This study is therefore

2865

centered to evaluate the antioxidant and free radical scavenging activity of raw and traditionally processed E. scandens and C. gladiata seeds. 2. Materials and methods 2.1. Sample collection The mature and dry raw seeds of E. scandens were collected from Kolli hills, Tamil Nadu, India and C. gladiata (red color) was purchased from Bangalore, India. Botanical identity of both the samples was established based on the morphology of the seeds, vegetative and floral parts. The identity was confirmed by comparing voucher specimens available in the botanical Survey of India, Coimbatore. After collection and purchase, the immature and damaged seeds were removed. The kernel was separated from whole seed of E. scandens and the following processing methods were adopted. 2.2. Processing methods E. scandens kernel and whole seed of C. gladiata were cracked into similar size. Both of the samples were randomly divided into 7 batches. The first batch was kept raw without any treatment. The second batch of seeds was dry heated in a Petri plate containing sand in a hot air oven at 120 °C for 30 min. The third batch of seeds was autoclaved using sample: water in the ratio of 1:5 (W/V) at a 15 lbs pressure for 15 min. The other four batches were subsequently soaked separately in various solutions like water, ash (Dried male inflorescences of sugar palm tree were ignited in a muffle furnace at 650 °C for 4 h, cooled and 0.1% of this ash solution was prepared using water and filtered), sodium bicarbonate (0.1%) and palm sugar solution (Bud of a coconut tree was made into slit and sap was collected, boiled and poured into bamboo tubes and left to solidify. Then, 1% of this sugar solution was prepared using water) at a ratio of 1:7 (w/v) for 12 h at room temperature. After 12 h of soaking, the samples were autoclaved with freshly prepared respective solutions (1:5) (w/v) at 15 lbs pressure for 15 min. After discarding the autoclaved liquid, the samples were dried, powdered and stored separately at 4 °C until further analysis. 2.3. Sample extraction The raw and all processed samples were subjected to extraction. Before extraction, the samples were defatted with petroleum ether. Then the samples were extracted with 80% methanol (1:5 w/v) for 48 h at room temperature. The extract were filtered, air dried and stored at 4 °C for further analysis. 2.4. Chemicals 2,20 -Azobis (2-amidinopropane) dihydrochloride (AAPH), Butylated hydroxyanisole (BHA), 2,20 -diphenyl-1-picryl-hydrazyl (DPPH), b-carotene, a-tocopherol, catechin, linoleic acid, 2,4,6-tripyridyl-s-triazine (TPTZ), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) and 2,2-azinobis (3-ethyl benzothiazoline6-sulfonic acid) diammonium salt (ABTS) were procured from Sigma (Bangalore, Karnataka, India). All other chemicals like nitro blue tetrazolium (NBT), ethylenediamine tetra acetic acid (EDTA), pBR 322, Ferrozine and linoleic acid were purchased from HiMedia (Mumbai, Maharastra, India). 2.5. Determination of total phenolics and tannin contents Total phenolics and tannins were measured as tannic acid equivalents (Makkar et al., 2007) from tannic acid standard curve (3–15 lg range). One milliliter of the sample extract was transferred to a test tube and 0.5 mL of Folin–Ciocalteu reagent and 2.5 mL of sodium carbonate solution (20% w/v) were added. After an incubation period of 40 min in dark, the absorbance was recorded at 725 nm with UV–visible spectrophotometer (Cyberlab-UV100, USA) against the reagent blank. Using the same extracts and method, the tannins were estimated after treatment with polyvinylpolypyrrolidone (PVPP). 2.6. Estimation of total flavonoids Total flavonoid content was measured according to the method of Zhishen et al. (1999). Sample extract was added with 0.3 mL of 5% sodium nitrite and well mixed. After 5 min of incubation, 0.3 mL of 10% aluminum chloride solution was added. Then, after 6 min, 2 mL of 1 M sodium hydroxide was added to the mixture and made up the volume to 10 mL with water. The absorbance was measured at 510 nm with UV–visible spectrophotometer. Total flavonoids were measured from rutin (20–100 lg) standard curve and expressed as mg rutin equivalents/g extract. 2.7. Free radical scavenging activity on 2,2-diphenyl-1-picrylhydrazyl (DPPH) The antioxidant activity of extracts and standards (BHA, rutin and tannic acid) was measured in terms of hydrogen donating ability using a stable, commercially available organic and nitrogen centered DPPH radical by the method of Brand-Wil-

2866

G. Sasipriya, P. Siddhuraju / Food and Chemical Toxicology 50 (2012) 2864–2872

liams et al. (1995) with slight modifications. Sample extracts prepared in methanol were mixed with 3.9 mL of methanol containing DPPH (0.025 g/L) and incubated in dark for 30 min. The absorbance was measured at 515 nm with UV–visible spectrophotometer. The trolox standards were prepared in the range of 0–2.5 mM. The concentration of DPPH was calculated from trolox standard curve and expressed as mmol trolox equivalents/g extract. 2.8. Antioxidant activity by the ABTS+ assay The ABTS+ radical cation decolorization assay was performed to evaluate the radical scavenging ability of crude extracts by the method of Re et al. (1999) with slight modification made by Siddhuraju and Becker (2003). ABTS radical cation (ABTS+) was generated by adding 2.45 mM potassium persulfate to 7 mM ABTS and incubated in dark at room temperature for 12–16 h. This stock solution of ABTS+ was diluted with ethanol to give an absorbance of 0.70 (± 0.02) at 734 nm, which act as a positive control. Ten microliters of crude extract (prepared in ethanol) was mixed with 1.0 mL of diluted ABTS+ solution and incubated at 30 °C for 30 min. The absorbance value was measured at 734 nm with UV–visible spectrophotometer. Trolox standard were also prepared (in ethanol: 0–1.5 mM) to get the concentration response curve. The unit of trolox equivalent antioxidant activity (TEA) was defined as the concentration of Trolox having the equivalent antioxidant activity expressed as mmol/g of extracts. The TEA of BHA, rutin and tannic acid was also measured by ABTS+ method for comparison. 2.9. Ferric reducing antioxidant power assay (FRAP) FRAP assay can be used to evaluate the electron donating ability of antioxidants according to the method of Benzie and Strain (1996) with slight modifications made by Pulido et al. (2000). An aliquot of 30 lL sample was mixed with 90 lL of water and 900 lL of FRAP reagent (2.5 mL of 20 mmol/L of TPTZ in 40 mM of HCl, 2.5 mL of 20 mmol/L of ferric chloride, 25 mL of 0.3 mol/L of acetate buffer (pH 3.6)) and incubated at 37 °C for 30 min. After incubation the absorbance values were recorded at 593 nm with UV–visible spectrophotometer. Known ferrous sulfate concentrations ranging from 400 to 2000 lmol were used to generate the calibration curve. From the curve, the ferrous ions reduced by the sample were calculated using regression equation. The antioxidant activity was expressed as amount of extract required to reduce 1 mmol of ferrous ions. The antioxidant activity of samples was compared with the following standards: BHA, rutin and tannic acid. 2.10. Metal chelating activity The chelating activities of samples, standards like BHA and a-tocopherol were estimated by the method of Dinis et al. (1994). An aliquot of 0.1 mL sample, 0.6 mL of distilled water and 0.1 mL of 0.2 mM ferrous chloride were well mixed and incubated for 30 s. Then, 0.2 mL of 1 mM ferrozine was added to the above mixture and incubated for 10 min at room temperature and the absorbance was recorded at 562 nm with UV–visible spectrophotometer. EDTA (0–2 lg) was used as standard for the preparation of calibration curve. Metal chelating ability of antioxidant was expressed as mg EDTA/g extract. 2.11. Superoxide anion radical scavenging assay This assay is used to evaluate the superoxide anion radical scavenging ability of plant extracts according to the method of Beauchamp and Fridovich (1971) as described by Zhishen et al. (1999). All the solutions used for this assay were prepared in phosphate buffer, 0.05 M, pH 7.8. All standards and samples (150 lg/mL) prepared in phosphate buffer 0.05 M, pH 7.8 were mixed with 1 mL of NBT (104 mol/L), 1 mL of methionine (102 mol/L) and 3 mL of riboflavin (106 mol/L) solution. The mixtures were kept in an aluminum foil lined box with two 20 W fluorescent lamps. The reactants were kept in such a way that the light should reach the contents with approximately 4000 lux intensity. Control (assay mixture without sample) was also treated as above. The absorbance was recorded at 560 nm with UV–visible spectrophotometer. All the samples and standards (BHA, catechin, trolox and rutin) were run in triplicates and in both illuminated and non-illuminated conditions. The differences in control (A) and sample absorbance (A1) between the illuminated and non-illuminated condition were recorded in order to avoid interferences. The degree of superoxide anion radical scavenging activity was calculated as,

%SRSA ¼ ½ðA  A1 Þ=A  100

2.12. Hydroxyl radical scavenging activity Hydroxyl radical scavenging ability of extract and standard (catechin) was measured according to the method of Klein et al. (1981). Standards (200 lg/mL), C. gladiata (200 lg/mL) and E. scandens (2000 lg/mL) were mixed with 1 mL of iron–EDTA solution (0.13% ferrous ammonium sulfate in 0.26% EDTA), 0.5 mL of 0.018% EDTA and 1 mL of DMSO solution (0.85% in phosphate buffered saline 0.1 M, pH 7.4). The reaction was terminated by the addition of 1 mL of ice cold TCA (17.5 w/v).

Three milliliters of Nash reagent (7.5 g ammonium acetate, 0.3 mL of glacial acetic acid, 0.2 mL of acetyl acetone and distilled water – 100 mL) was added to the above mixture and incubated at room temperature for 15 min and the absorbance values were recorded at 412 nm with UV–visible spectrophotometer. Sample control was also run with the substitution of phosphate buffer instead of ascorbic acid. The% hydroxyl radical scavenging activity (HRSA) was calculated using the following formula,

%HRSA ¼ 1  ðDifferences in absorbance of sample= Difference in absorbance in blankÞ  100

2.13. b-Carotene linoleic acid system The antioxidant activity of sample extracts and standards (BHA, rutin and trolox) were analyzed according to the method of Taga et al. (1984) with slight modifications. Two milligram of b-carotene was dissolved in 1 mL of chloroform containing 40 mg of linoleic acid and 400 mg of Tween 40. The chloroform was removed by rotary vacuum evaporator at 45 °C for 4 min and 100 mL of distilled water was added slowly to the semisolid residue with vigorous agitation to form an emulsion. A 5 mL aliquot of the emulsion was added to a tube containing standards (50 lg/mL) and sample extracts (100 lg/mL) and the absorbance was measured at 470 nm with UV–visible spectrophotometer, immediately, against a blank, consisting of the emulsion without b-carotene. The tubes were placed in a water bath at 50 °C and the absorbance measurements were conducted again at 30 min intervals up to 120 min. All determinations were carried out in triplicates. The antioxidant activity (AA) of the extracts was evaluated in terms of bleaching of b-carotene using the following formula: AA = [1  (A0–At)/(A0 0–A0 t)]  100, where A0 and A0 0 are absorbance of values measured at zero time of the incubation for test sample and control, respectively and At and A0 t are absorbances measured in the test sample and control, respectively, after incubation for 120 min. 2.14. DNA protection assay The DNA protection assay was performed using pBR 322 plasmid DNA as described by Benedetti et al. (2011) with some modifications. The reaction mixture contained 2 lL plasmid DNA (0.1 mg/mL) in phosphate buffered saline (pH 7.4) and sample extracts. Then it was incubated at room temperature for 10 min. Peroxyl radical was generated by adding a final concentration of 5 mM AAPH in PBS. The final volume was brought up to 20 lL using distilled water. The mixture was incubated for 10 min at 37 °C. Upon completion of incubation 3 lL loading dye (consisting of 0.25% bromophenol blue, 0.25% xylene cyanol and 40% sucrose in distilled water) was added and the DNA was analyzed on 1% agarose gel (0.5 g of agarose in 50 mL of 1 TBE Buffer). The DNA bands were visualized under UV light using gel documentation system (Gel doc lab image ID L 320 (Medicare scientific, India)). 2.15. Statistical analysis Results were expressed as the mean ± standard deviation (SD) of at least three independent experiments. Differences were estimated by the analysis of variance (ANOVA) followed by Duncan’s multiple-range test. Differences were considered to be significant at P < 0.05. Correlation analysis was performed between phenolics, tannins and flavonoids with antioxidant activity using Pearson correlation-Two tailed. All statistical analyzes were performed using the statistical software SPSS 13.0 version (SPSS Inc., Chicago, Illinois, USA).

3. Results and discussion 3.1. Total phenolics and tannins Phenolic compounds can act as excellent radical scavengers and metal chelators that reduce oxidation because phenolic groups are nucleophiles and are able to inhibit lipid peroxidation, avoiding oxidation reaction by binding to free radicals generated through lipid peroxidation (Bravo, 1998). Thus, the contents of total phenolics and tannins of E. scandens (35.87–63 mg/g extract) (3.59– 5.38 mg/g extract) and C. gladiata (39–104.19 mg TAE/g extract) (13.97–47.28 mg TAE/g extract) were estimated and the results are shown in Table 1. The phenolics and tannins of both of the samples were higher in raw and dry heated samples. After processing, the phenolic content was reduced in autoclaved samples (2.9% and 14%) and soaked followed by autoclaved samples (12–43% and 48– 63%). Although, the phenolics and tannins of E. scandens and C. gladiata were greatly reduced in soaked and autoclaved samples.

G. Sasipriya, P. Siddhuraju / Food and Chemical Toxicology 50 (2012) 2864–2872 Table 1 Total phenolics, tannins and flavonoids of raw and processed samples of E. scandens and C. gladiata. Samples

Phenolicsa

Tanninsa

Flavonoidsb

E. scandens R D W A AS SB S

63.01 ± 3.19a 62.94 ± 3.30a 55.36 ± 1.16b 61.21 ± 5.09a 35.87 ± 1.17d 42.65 ± 1.84c 47.34 ± 1.67c

3.59 ± 1.55 5.38 ± 3.92 ND ND ND ND ND

21.07 ± 1.09a 21.67 ± 2.06a 18.16 ± 1.48b 17.52 ± 0.47b 13.14 ± 1.16c 14.19 ± 1.12c 14.47 ± 0.28c

C. gladiata R D W A AS SB S

104.19 ± 3.39e 98.98 ± 8.84e 54.46 ± 2.41h 89.67 ± 8.16f 49.17 ± 2.47h 47.01 ± 2.69gh 39.00 ± 1.90h

47.28 ± 3.80e 44.28 ± 3.52e 23.26 ± 2.00g 35.03 ± 3.27f 20.45 ± 2.02g 21.78 ± 2.12g 13.97 ± 2.09g

32.69 ± 3.03f 42.00 ± 2.52e 20.06 ± 0.71g 34.69 ± 2.32f 15.33 ± 0.53gh 18.48 ± 1.67g 10.78 ± 0.47h

Values in columns (mean ± SD) with different letters (a–d: E. scandens, e–h: C. gladiata) are significantly different (P < 0.05). ND, not detected; R, raw; D, dry heating; W, soaking in water + autoclaving; A, autoclaving alone; AS, soaking in ash solution + autoclaving; SB, soaking in sodium bicarbonate solution + autoclaving; S, soaking in sugar solution + autoclaving. a mg tannic acid equivalents/g extract. b mg rutin equivalents/g extract.

It might be due to the leaching out of water soluble phenolics into the soaking and cooking medium, breakdown of phenolics during processing, chemical transformation, decomposition of phenolics and formation of phenolic-protein complex under thermal and pressure conditions (Xu and Chang, 2008; Barroga et al., 1985). Similar reductions in processed legume samples were reported by Bressani and Elias (1980), Siddhuraju (2006), Xu and Chang (2008) and Boateng et al. (2008). The total phenolic contents of unprocessed E. scandens and C. gladiata were higher than C. ensiformis and Phaseolus spp. but lower than their respective immature samples of E. scandens and C. gladiata (Sowndhararajan et al., 2011; Boateng et al., 2008; Vadivel et al., 2011). The level of tannins in C. gladiata was higher than C. ensiformis (Sowndhararajan et al., 2011). Plant extracts rich in phenolic compounds are of great importance in the maintenance of health in food industry because of having antiproliferative, antibacterial, antiinflammatory and antiallergic effects (Liu, 2003). These compounds retard oxidative degradation of lipids and thereby improve the quality and nutritional value of food. 3.2. Flavonoids Plant flavonoids are a large group of polyphenolic compounds that are characterized by benzo-y-pyrone structure that are important for human health because of their high pharmacological activities as radical scavengers (Cook and Samman, 1996). The flavonoid contents of E. scandens (13.14–21.67 mg rutin/g extract) and C. gladiata (10.78–42 mg rutin/g extract) are presented in Table 1. When compared to raw sample, the flavonoid content of dry heated sample was similar in E. scandens and increased up to 22% in C. gladiata. It was noted that soaking followed by autoclaving caused a decrease of total flavonoids by 14–37% in E. scandens and 39–67% in C. gladiata than their raw sample. Similar decreases in flavonoid content in legumes and increases in oak acorns after processing were observed by Franke et al. (1994) and Rakic et al. (2006). The reported flavonoid contents were higher than green pea, yellow pea, chick pea, lentil, yellow soybean, black soybean, red kidney and black bean (Xu and Chang, 2007). The decrease in flavonoid content might be due to the leaching out of soluble compounds into the cooking medium. The stability of phenolics and

2867

flavonoids during heating may be due to the formation of antioxidant compounds like Maillard reaction products such as hydroxymethylfurfuraldehyde (HMF) (Siddhuraju, 2006). Flavonoids have been shown to possess antibacterial, anti-inflammatory, antiallergic, antimutagenic, antiviral, antineoplastic, anti-thrombotic, cholesterol biosynthesis modulating, vasodilatory and anticancer activity. 3.3. DPPH and ABTS+ scavenging activity DPPH and ABTS+, are free radicals used to determine the antioxidant activity of plant extracts. When a solution of DPPH (violet color) and ABTS (blue–green chromophore) is mixed with the plant extracts that can donate a hydrogen atom, then this gives rise to the reduced form with the loss of their color (Molyneux, 2004). Table 2 show the results of DPPH and ABTS+ scavenging activity. In E. scandens, autoclaved, dry heated and water soaked followed by autoclaved samples showed higher DPPH activity than raw sample. The ABTS radical scavenging activities of all processed samples were significantly (P < 0.05) similar in E. scandens. In C. gladiata, both activities were reduced in all processed samples up to 20–35% in DPPH and 22–75% in ABTS+ scavenging activity. Raw, dry heated and water soaked followed by autoclaved samples of C. gladiata showed similar ABTS+ activity to rutin. However, processed methods like sugar, sodium bicarbonate and ash soaked following by autoclaved samples resulted in a significant loss of DPPH activity than other methods. Phenolics (r2 = 0.808, 0.774, P < 0.05) and tannins (r2 = 0.815, 0.825, P < 0.05) in C. gladiata can donate more hydroxyl groups for reducing a number of DPPH and ABTS+ radical efficiently (Table 3) whereas, in E. scandens, the significant correlation was not observed. Hence, compounds other than phenolics, tannins and flavonoids may be responsible for scavenging activity in E. scandens. The correlation in C. gladiata showed that the loss of DPPH activity in processed samples was only due to the leaching out of phenolics and tannins in cooking medium. The DPPH activity of E. scandens (300 lg: 55%) was lower than immature whole E. scandens (100 lg: 78.46%), whereas, C. gladiata (15 lg: 82.4%) was higher than their immature samples (100 lg: 69.73%) (Vadivel et al., 2011). All samples showed higher activity than Cajanuscajan, V. unguiculata and Sphenostylis stenocarpa (Oboh, 2006). The ABTS+ scavenging activity was found to be higher than Vigna vexillata (Sowndhararajan et al., 2011). Phenolics can act as efficient free radical scavengers by donating their alcoholic hydrogen or one of their delocalized electrons to radicals for their stabilization (Brown, 1995). The similar antioxidant activity after cooking was noted in peas (Turkmen et al., 2005) and reduction in cool season food legumes was observed by Xu and Chang (2008). The DPPH and ABTS radical scavenging activities of sample extracts indicating that some of the compounds present in the extracts were electron donors and could react with free radicals to terminate radical chain reactions and, therefore, were able to boost the natural antioxidant defense mechanism. 3.4. FRAP assay The FRAP assay is a simple and inexpensive procedure that measures the total antioxidant level in a sample. It utilizes the reducing potential of the antioxidants to react with a ferric tripyridyltriazine (FeIII-TPTZ) complex and produce a colored ferrous tripyridyltriazine (FeII-TPTZ) form (Benzie and Strain, 1996). The FRAP activities of raw and processed E. scandens (4606.6–6229.6 mmol Fe(II)/g extract) and C. gladiata (18231.9–80305.4 mmol Fe(II)/g extract) are presented in Table 2. Similar to DPPH and ABTS radical scavenging activity, the reducing power of E. scandens was significantly similar between autoclaved, dry heated and water soaked followed by autoclaved samples. In C. gladiata, the reducing

2868

G. Sasipriya, P. Siddhuraju / Food and Chemical Toxicology 50 (2012) 2864–2872

Table 2 DPPH, ABTS+, FRAP and metal chelating activity of raw and processed samples of E. scandens and C. gladiata. Samples

DPPH (mmol TE/g extract)a

ABTS (mmol TE/g extract)

a

BHA RUT TAN a-TOC

814172.7 ± 187 748175.2 ± 598 848540.1 ± 547 –

654356.1 ± 617.1 432942.7 ± 233.1 751041.7 ± 632.3 –

350278.7 ± 735.7 172898.6 ± 272.7 565217.4 ± 427.7 –

10.49 ± 0.06 – – 12.68 ± 0.26

E. scandens R D W A AS SB S

6559.2 ± 31c 6843.1 ± 64b 6788.3 ± 18b 7023.5 ± 21a 6437.6 ± 9d 6362.5 ± 18e 6405.1 ± 61de

125921.8 ± 418.8a 154924.2 ± 275.69a 149774.8 ± 329.4a 143650.8 ± 336.4a 121279.8 ± 270.8a 108796.3 ± 116.5a 123366.0 ± 168.1a

5540.6 ± 77.6abc 5762.0 ± 26.5ab 5892.8 ± 32.8ab 6229.6 ± 18.8a 4606.6 ± 44.8d 4928.5 ± 28.9cd 5276.1 ± 24.1bcd

4.11 ± 0.8a 4.01 ± 0.42a 3.05 ± 0.6a 3.81 ± 0.2a 3.04 ± 0.43a 3.19 ± 0.04a 3.49 ± 0.81a

C. gladiata R D W A AS SB S

170802.9 ± 729f 131995.1 ± 170h 131711.3 ± 141h 136536.9 ± 670g 123276.6 ± 225i 110867.8 ± 237J 112692.6 ± 548J

496428.6 ± 498.9f 495535.7 ± 193.9f 389682.5 ± 346.9gh 217105.3 ± 424.8h 146825.4 ± 323.6fgh 140833.3 ± 106.3fg 125809.1 ± 183.8h

69758.5 ± 46.8g 80305.4 ± 51.7f 28612.8 ± 14.8i 45919.1 ± 18.4h 26749.5 ± 29.8i 18231.9 ± 20.5J 21640.6 ± 13.5J

3.87 ± 0.13fg 2.64 ± 0.16gh 2.3 ± 0.09h 2.71 ± 0.07gh 2.33 ± 0.3h 4.26 ± 0.56f 2.39 ± 0.24h

FRAP (mmol Fe(II)/g extract)

b

Metal chelating(mg EDTA/g extract)

c

Values in columns (mean ± SD) with different letters among samples (a–e: E. scandens, f–j: C. gladiata) are significantly different (P < 0.05). BHA, Butylated hydroxyanisole; RUT, rutin; TAN, tannic acid; a-TOC, a-tocopherol. R, raw; D, dry heating; W, soaking in water + autoclaving; A, autoclaving alone; AS, soaking in ash solution + autoclaving; SB, soaking in sodium bicarbonate solution + autoclaving; S, soaking in sugar solution + autoclaving. a mmol of trolox equivalents/g extract. b mmol of ferrous equivalents/g extract. c mg of EDTA equivalent/g extract.

Table 3 Correlation between phenolics, tannins, flavonoids and different antioxidant parameters of E. scandens and C. gladiata. Parameters

Phenolics ES

DPPH ABTS FRAP MCA SRSA HRSA CBS

0.746 0.704 0.879b 0.811a 0.849 a 0.615 0.824

Tannins CG a

a

Flavonoids

ES

0.808 0.774 a 0.942 b 0.211 0.983 b 0.884 b 0.304

0.227 0.378 0.241 0.786 a 0.871 a 0.422 0.851

CG a

a

0.815 0.825 a 0.951 b 0.304 0.965 b 0.839 a 0.279

ES

CG

0.609 0.683 0.685 0.774 a 0.890 a 0.491 0.945b

0.602 0.733 0.911 a 0.146 0.942 b 0.852 a 0.523

ES – Entada scandens, CG – Canavalia gladiata. FRAP, Ferric reducing antioxidant power assay; MCA, Metal chelating activity; SRSA, Superoxide anion radical scavenging activity; HRSA, Hydroxyl radical scavenging activity; CBS-b, carotene/linoleic acid bleaching activity. a Correlation is significant at the 0.05 level (2-tailed). b Correlation is significant at the 0.01 level (2-tailed).

activity was increased in dry heated sample (15%), whereas, the decrease of activity was noted in all other processed samples (34–74%). From the correlation analysis, it is conceived that phenolics (r2 = 0.879, P < 0.01) of E. scandens and phenolics (r2 = 0.942, P < 0.01), tannins (r2 = 0.951, P < 0.01) and flavonoids (r2 = 0.911, P < 0.05) of C. gladiata were the main contributors for their reducing activity (Table 3). The increase of activity in dry heated samples may be due to the formation of reductones from the thermolysis of Amadori products in the primary stage of Maillard reaction (Yamaguchi and Yashito, 1967). The reducing activity was higher than immature E. scandens, C. gladiata and Vigna aconitifolia (Siddhuraju, 2006; Vadivel et al., 2011). 3.5. Metal chelating activity Transition metals can generate hydroxyl and superoxide radicals and lead to lipid peroxidation, protein modification and DNA damage. Chelating agents may inactivate these metal ions and potentially inhibit the metal-dependent processes (Finefrock

et al., 2003). Both extracts interfered with the formation of ferrous and ferrozine complex, suggesting that they have chelating activity and captures ferrous ion before ferrozine. The metal chelating activities of E. scandens and C. gladiata were 3–4.11 mg EDTA/g extract and 2.3–4.26 mg EDTA/g extract (Table 2). When compared to raw, the metal chelating activities of processed samples were significantly similar in E. scandens and the activities were maintained in sodium bicarbonate soaked followed by autoclaved samples and it is reduced in all other samples up to 30–41% in C. gladiata. However, the metal chelating activities of samples were lower than the standards. In contrary to DPPH and ABTS, phenolics (r2 = 0.811, P < 0.05), tannins (r2 = 0.786, P < 0.05) and flavonoids in E. scandens (r2 = 0.774, P < 0.05) were responsible for metal chelating activity. While in C. gladiata, the significant correlation was not observed (Table 3). Sample extracts with metal chelating activity have ability to convert metal ions into insoluble metal complexes or generate steric hindrance, which can prevent the interactions between metals and lipid intermediates, thus inhibiting lipid oxidation. 3.6. Superoxide anion radical scavenging activity Superoxide radicals, the first reduction product of molecular oxygen, have been observed to kill cells, inactivate enzymes and degrade DNA, cell membranes and polysaccharides (Fridovich, 1978). Both extracts, E. scandens (22–38%) and C. gladiata (12– 58%) exhibited scavenging activity on superoxide radicals (Fig. 1). When compared to raw samples, the scavenging activities of E. scandens and C. gladiata were increased in dry heated samples, whereas, it reduced in autoclaved (8–19%) and soaked followed by autoclaved samples (19–80%). The scavenging activities of raw, dry heated and autoclaved samples in C. gladiata were comparable to standard, trolox. Phenolics (r2 = 0.849, P < 0.05; r2 = 0.983, P < 0.01), tannins (r2 = 0.871, P < 0.05; r2 = 0.965, P < 0.01) and flavonoids (r2 = 0.890, P < 0.05; r2 = 0.942, P < 0.01) in E. scandens and C. gladiata may be responsible for scavenging superoxide radicals (Table 3). Within accordance to correlation, the lower activity of hydrothermally processed samples may be attributed

G. Sasipriya, P. Siddhuraju / Food and Chemical Toxicology 50 (2012) 2864–2872

2869

Fig. 1. Superoxide anion radical scavenging activity of raw and processed samples of E. scandens and C. gladiata values of triplicate determinations (mean ± SD) with different letters (a–d: E. scandens) (e–i: C. gladiata) are significantly different (P < 0.05). BHA, butylated hydroxyanisole; CAT, catechin; TRO, trolox; RUT, rutin; R, raw; D, dry heating; W, soaking in water + autoclaving; A, autoclaving alone; AS, soaking in ash solution + autoclaving; SB, soaking in sodium bicarbonate solution + autoclaving; S, soaking in sugar solution + autoclaving (standards, ; E. scandens, ; C. gladiata, ).

to the partial loss of phenolics, tannins and flavonoids during the soaking as well as decanting of autoclaved liquid. The values were comparable with that of certain legume grains, raw and dry heated samples of V. aconitifolia (16% and 20%) (Siddhuraju, 2006), immature samples of C. gladiata (54%), M. pruriens (45%) and E. scandens (60%) (Vadivel et al., 2011). Superoxide anion is a weak oxidant, it gives rise to the generation of powerful and dangerous hydroxyl radicals as well as singlet oxygen, both of which contribute to the oxidative stress and lead to the genesis of several chronic diseases in human beings. Thus, superoxide anion scavenging activity of plant extracts is important to protect oxidative stress and their free radical mediated degenerative diseases. 3.7. Hydroxyl radical scavenging activity Hydroxyl radicals are short-lived, most damaging radicals within the body formed from superoxide radicals and hydrogen peroxide and oxidize biological macromolecules including lipids, proteins and nucleic acids (Imlay and Linn, 1988). The hydroxyl radical scavenging activities of E. scandens (9–46%) and C. gladiata (40–71%) are shown in Fig. 2. When compared to raw samples, the activity was maintained in autoclaved samples and all other

processed samples showed higher activity (up to 2–3 times) in E. scandens. In C. gladiata, the activity was increased in dry heated sample and decreased in soaked followed by autoclaved samples (20–40%). Phenolics (r2 = 0.884, P < 0.01), tannins (r2 = 0.839, P < 0.05) and flavonoids (r2 = 0.852, P < 0.05) in C. gladiata contribute to hydroxyl radical scavenging activity but it was negatively correlated in E. scandens (Table 3). The potential scavenging ability of phenolic substances might be due to the active hydrogen-donating ability of hydroxyl substitutions. C. gladiata exhibited higher activity than V. aconitifolia (33%) (Siddhuraju, 2006). The increase of hydroxyl radical scavenging activity in dry heated sample than raw was reported in tamarind seed coat by Siddhuraju (2007). The results suggest that the extracts display a scavenging effect on hydroxyl radical generation that could help to protect proteins, DNA, polyunsaturated fatty acids in membranes, and almost any biological molecule from free radical attack. 3.8. b-carotene linoleic acid emulsion assay In this assay, linoleic acid in the reaction mixture forms hydroperoxide during incubation at 50 °C, thereby it attacks the b-carotene chromophore resulting in a bleaching effect (Koleva et al.,

Fig. 2. Hydroxyl radical scavenging activity of raw and processed samples of E. scandens and C. gladiata values of triplicate determinations (mean ± SD) with different letters (a–c: E. scandens) (d–g: C. gladiata) are significantly different (P < 0.05). CAT, catechin; R, raw; D, dry heating; W, soaking in water + autoclaving; A, autoclaving alone; AS, soaking in ash solution + autoclaving; SB, soaking in sodium bicarbonate solution + autoclaving; S, soaking in sugar solution + autoclaving (standards, ; E. scandens, ; C. gladiata, ).

2870

G. Sasipriya, P. Siddhuraju / Food and Chemical Toxicology 50 (2012) 2864–2872

Fig. 3. Antioxidant activity of raw and processed samples of E. scandens and C. gladiata in b-carotene/linoleic acid system Values of triplicate determinations (mean ± SD) with different letters (a–e: E. scandens) (e–j: C. gladiata) are significantly different (P < 0.05). BHA, butylated hydroxyanisole; RUT, rutin; TRO, trolox; R, raw; D, dry heat; W, soaking in water + autoclaving; A, autoclaving alone; AS, soaking in ash solution + autoclaving; SB, soaking in sodium bicarbonate solution + autoclaving; S, soaking in sugar solution + autoclaving (standards, ; E. scandens, ; C. gladiata, ).

2002). The antioxidants present in the sample extracts were able to prevent the bleaching of b-carotene (E. scandens – 7–71% and C. gladiata – 16–53%) by scavenging hydroperoxides formed in it (Fig. 3). When compared to raw, the peroxidation inhibition activity was increased in all processed samples in E. scandens. In C. gladiata, all processed samples other than sugar decreased the peroxidation inhibiting activity up to 15–69%. The negative correlation existed between phenolics, tannins, flavonoids and b-carotene bleaching activity in both of the plant extracts (Table 3). Apart from these bioactive compounds, Maillard reaction products, L-Dopa and phytic acid may be responsible for inhibition of lipid peroxidation (Seiquer et al., 2008; Gulcin, 2007; Singh et al., 2003). The peroxidation inhibition activities of raw, sodium bicarbonate and sugar soaked followed by autoclaved samples in C. gladiata, and all samples, except raw and dry heated in E. scandens were comparable to standards. The peroxidation inhibition activities were comparable to V. aconitifolia (39%) and P. vulgaris (16–32%) (Siddhuraju, 2006; Cardador-Martinez et al., 2002). Thus, consumption of such underutilized legume samples can prevent lipid peroxidation mediated diseases like coronary heart disease, atherosclerosis, cancer and the aging process. 3.9. DNA protection Reactive oxygen species (ROS) such as superoxide anion (O2), hydrogen peroxide (H2O2) and hydroxyl radical (OH) can cause damage to biological macromolecules leading to lipid peroxidation, protein oxidation, and DNA base modification and strand breaks (Halliwell and Gutteridge, 1984). Thus, this assay was performed to verify the ability of sample extracts to protect supercoiled plasmid DNA. Overall, the best processed method in E. scandens and C. gladiata was observed to be dry heated sample. The raw and dry heated samples of both extracts were further tested for DNA protection ability. Different concentrations of sample extracts were used for testing DNA protection, at last, the protection ability on plasmid DNA was found at 10 lg (Fig. 4). The predominate form of pBR 322 plasmid DNA in the absence of AAPH was supercoiled form (Lane 1). Peroxyl radicals generated from AAPH cleave supercoiled plasmid DNA to a nicked circular form. This was clearly seen in Lane: 2 where the reaction mixture did not contain any antioxidants. The presence of antioxidants in raw and dry heated sample of E. scandens (Lanes: 3 and 4) and C. gladiata (Lanes: 5 and 6) resulted in a partial inhibition of the conversion of supercoiled DNA to open circular and linear forms, indicating that the extracts

Fig. 4. Effect of raw and processed samples of E. scandens and C. gladiata on protection of supercoiled pBR 322 plasmid DNA against AAPH induced radical Lane 1, pBR 322 DNA; Lane 2, pBR 322 DNA + AAPH; Lane 3, pBR 322 DNA + AAPH + E. scandens raw (10 lg); Lane 4, pBR 322 DNA + AAPH + E. scandens dry heating (10 lg); Lane 5, pBR 322 DNA + AAPH + C. gladiata raw (10 lg); Lane 6, pBR 322 DNA + AAPH + C. gladiata dry heating (10 lg). Supercoiled form, (b) Open circular form.

were able to protect plasmid DNA against AAPH-induced oxidative damage. A possible mechanism behind the DNA protection may be polyphenols have ability to scavenge lipid free radicals, hydroxyl radicals, superoxide radicals and singlet oxygen (Guo et al., 1999). Sample extracts possess peroxyl radical scavenging activity and thus the supplementation of these plant extracts can protect from peroxyl radical induced diseases like carcinogenesis, mutagenesis and cytotoxicity, respectively. From the above observations, it may be clear that, in E. scandens, the phenolics, tannins and flavonoid contents were decreased in soaked followed by autoclaved samples. Overall, dry heated method enhanced the activity and all other processed samples showed similar activity in ABTS, hydroxyl radical scavenging activity, FRAP, metal chelating activity and b-carotene bleaching inhibition activity. In addition, the significant losses of DPPH and superoxide radical scavenging activity were noted in processed samples in E. scandens, but it is observed to be minimum. Apart from phenolics, tannins and flavonoids, the similar antioxidant activities of raw and processed samples of E. scandens could be due to the additive and synergistic effects between other phytochemicals and thermally altered phenolics and formation of Maillard reaction products (Bondet et al., 1997). Maillard reaction is known as a major source of compounds related to enhanced antioxidant activity by heat treatment in various foods (Chen and Kitts, 2008). It has been reported that thermal processing deactivates the oxidative and hydrolytic enzymes which are responsible for destroying the antioxidants (Chism and Haard, 1996). Another possible reason might due to the presence of heat stable antinutrients such as phytic acid and L-Dopa in E. scandens (Vadivel and Biesalski, 2010; Gautam et al., 2012). L-Dopa and phytic acid were found to be

G. Sasipriya, P. Siddhuraju / Food and Chemical Toxicology 50 (2012) 2864–2872

effective antioxidants in different in vitro assays (Gulcin, 2007; Graf and Eaton, 1990). Similar observations were made in V. aconitifolia, where the antioxidant activities were similar in raw and dry heated samples (Siddhuraju, 2006). In other sample C. gladiata, dry heated sample showed higher antioxidant activity than other processed methods. Over all, the antioxidant activity in C. gladiata arises in the order of raw > dry heated > autoclaved > water soaked > ash, sodium bicarbonate and sugar soaked followed by autoclaved samples. The observed order of activity was also seen in phenolics, tannins and flavonoids. Hence, the decrease in antioxidant activities in soaked followed by autoclaved samples might have been due to the softening of cell wall tissues which is usually accompanied by solubilisation of bound polyphenols into the soaking water and discarded it (Boateng et al., 2008). The antioxidant activities were greatly influenced in soaking in ash, sodium bicarbonate and sugar solutions than water alone. It might be due to the increase in solubility nature of bound polyphenolics into the soaking solutions than water alone. Therefore, dry heating, autoclaving and soaking followed by autoclaving in various solutions (plain water, ash, sodium bicarbonate and sugar) of E. scandens and dry heating of C. gladiata would be considered as most suitable method to preserve bioactive compounds and antioxidant activity. These wild legume grains have excellent potential as antioxidant additives in foods because they can inhibit lipid oxidation through multiple pathways including inactivation of reactive oxygen species, scavenging free radicals and chelation of prooxidative transition metals. It can be used as natural preservatives in foodstuffs and in living tissues. These wild legumes have capability to scavenge major free radicals responsible for neurodegenerative diseases, liver damage, diabetes, cancer, aging, inflammation and atherosclerosis. It is estimated that 75–85% of all chronic illnesses and diseases are linked to lifestyle and cannot be explained by differences in genetic makeup (Wong et al., 2005). Hence, the consumption of these wild legumes would help to reduce the losses from oxidation disturbances and their related diseases. The identification of phytochemicals responsible for antioxidant activity of E. scandens kernel and C. gladiata might be useful for drug development for chronic diseases. 4. Conclusions The results depicted that the increase or decrease in antioxidant activity will depend upon the processing methods adopted to the samples. The incorporation of the present reported processing methods of E. scandens and dry heating of C. gladiata would helpful in the formulation of supplementary therapeutic foods for the dietary management of various chronic diseases, including diabetes, obesity and cardiovascular diseases. The exploitation of these wild legumes would help us to overcome present scarcity of conventional legumes in the market and also it protect against free radical mediated degenerative diseases. Future studies will be conducted on identification of bioactive constituents, molecular mechanisms involved in antioxidant activity, determination of their efficacy by in vivo studies and demonstration of their safety and effectiveness in clinical trials. Conflict of Interest The authors declare that there are no conflict of interest. References Adams, S.M., Standridge, J.B., 2006. What should we eat? Evidence from observational studies. Southern Med. J. 99, 744–748.

2871

Akande, K.E., Fabiyi, E.F., 2010. Effect of processing methods on some antinutritional factors in legume seeds for poultry feeding. Int. J. Poult. Sci. 9, 996–1001. Ali, M., Kumar, S., 2000. Problems and prospects of pulses research in India. Indian Farm. 50, 4–13. Barroga, F.C., Laurena, A.C., Mendoza, E.M.T., 1985. Polyphenols in Mung bean (Vigna radiata (L.)) Wilczek): determination and removal. J. Agric. Food Chem. 33, 1006–1009. Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44, 276–287. Benedetti, S., Catalani, S., Palma, F., Canestrari, F., 2011. The antioxidant protection of CELLFOOD Ò against oxidative damage in vitro. Food Chem. Toxicol. 49, 2292– 2298. Benzie, I.F.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of ‘‘antioxidant power’’: the FRAP assay. Anal. Biochem. 239, 70–76. Boateng, J., Verghese, M., Walker, L.T., Ogutu, S., 2008. Effect of processing on antioxidant contents in selected dry beans (Phaseolus spp. L.). LWT – Food Sci. Technol. 41, 1541–1547. Bondet, V., Brand-Williams, W., Berset, C., 1997. Kinetics and mechanisms of antioxidant activity using the DPPH free radical method. LWT – Food Sci. Technol. 30, 609–615. Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of a free radical method to evaluate antioxidant activity. LWT – Food Sci. Technol. 28, 25–30. Bravo, L., 1998. Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance. Nutr. Rev. 56, 317–333. Bressani, A., Elias, L.G., 1980. The nutritional role of polyphenols in beans. In: Hulse, J.H. (Ed.), Polyphenols in Cereal and Legumes. International Development Research Center, Ottawa, Canada, pp. 61–68. Brown, H.W., 1995. Organic Chemistry. Saunders College Publishing, New York, NY, USA, 521. Cardador-Martinez, A., Loarca-Pina, G., Dave Oomah, B., 2002. Antioxidant activity in common beans (Phaseolus vulgaris L.). J. Agric. Food Chem. 50, 6975–6980. Chen, X.M., Kitts, D.D., 2008. Antioxidant activity and chemical properties of crude and fractionated Maillard reaction products derived from four sugaramino acid Maillard reaction model systems. Ann. N. Y. Acad. Sci. 1126, 220–224. Chism, G.W., Haard, N.F., 1996. Characteristics of edible plant tissues. In: Fennema, O.R. (Ed.), Food Chemistry, third ed. Dekker, New York, pp. 943–1011. Cook, N.C., Samman, S., 1996. Review: flavonoids – chemistry, metabolism, cardioprotective effects, and dietary sources. J. Nutr. Biochem. 7, 66–76. Das, C.R., 1994. Rare and beautiful crawling climbers of special interest in India. J. Living World 1, 85–88. Dinis, T.C.P., Madeira, V.M.C., Almeida, L.M., 1994. Action of phenolic derivatives (acetoaminophen, salycilate and 5-aminosalycilate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Arch. Biochem. Biophys. 315, 161–169. Doyle, J.J., 1994. Phylogeny of the legume family: an approach to understanding the origins of nodulation. Annu. Rev. Ecol. Syst. 25, 325–349. Durackova, Z., 2010. Some current insights into oxidative stress. Physiol. Res. 59, 459–469. Eknayake, S., Jansz, E.R., Nair, B.M., 1999. Proximate composition, mineral and amino acid content of mature Canavalia gladiata seeds. Food Chem. 66, 115– 119. Ekanayake, S., Skog, K., Asp, N.G., 2007. Canavanine content in sword beans (Canavalia gladiata): analysis and effect of processing. Food Chem. Toxicol. 45, 797–803. Emenalom, O.O., Orji, V.C., Ogbonna, N.C., 2005. Effect of velvet bean (Mucuna pruriens) seeds cooked in maize-cob ash solution on the performance of broiler starter chickens. Livest. Res. Rural Dev. 17. Finefrock, A.E., Bush, A.I., Doraiswamy, P.M., 2003. Current status of metals as therapeutic targets in Alzheimer’s disease. J. Am. Geriatr. Soc. 51, 1143–1148. Franke, A.A., Custer, L.J., Cerna, C.M., Narala, K.K., 1994. Quantitation of phytoestrogens in legumes by HPLC. J. Agric. Food Chem. 42, 1905–1913. Fridovich, I., 1978. The biology of oxygen radicals. Science 201, 875–880. Gautam, B., Vadivel, V., Stuetz, W., Biesalski, H.K., 2012. Bioactive compounds extracted from Indian wild legume seeds: antioxidant and type II diabetesrelated enzyme inhibition properties. Int. J. Food Sci. Nutr. 63, 242–245. Graf, E., Eaton, J.W., 1990. Antioxidant functions of phytic acid. Free Radical Biol. Med. 8, 61–69. Gulcin, I., 2007. Comparison of in vitro antioxidant and antiradical activities of Ltyrosine and L-Dopa. Amino Acids 32, 431–438. Guo, Q., Zhao, B., Shen, S., Hou, J., Hu, J., Xin, W., 1999. ESR study on the structure– antioxidant activity relationship of tea catechins and their epimers. Biochim. Biophys. Acta 1427, 13–23. Gupta, S.C., Kim, J.H., Prasad, S., Aggarwal, B.B., 2010. Regulation of survival, proliferation, invasion, angiogenesis and metastasis of tumor cells through modulation of inflammatory pathways by nutraceuticals. Cancer Metastasis Rev. 29, 405–434. Gupta, S.C., Hevia, D., Patchva, S., Park, B., Koh, W., Aggarwal, B.B., 2012. Upsides and downsides of reactive oxygen species for cancer: the roles of reactive oxygen species in tumorigenesis, prevention, and therapy. Antioxid. Redox. Signal. 16, 1295–1322. Halliwell, B., Gutteridge, J.M., 1984. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem. J. 219, 1–14. Herklots, G.A.C., 1972. Beans and Peas, Vegetables in South-East Asia. George Allen and Unwind Ltd., London, pp. 225–236. Imlay, J.A., Linn, S., 1988. DNA damage and oxygen radical toxicity. Science 240, 1302–1309.

2872

G. Sasipriya, P. Siddhuraju / Food and Chemical Toxicology 50 (2012) 2864–2872

Janardhanan, K., Nalini, K., 1991. Studies on the tribal pulse, Entada scandens Benth: chemical composition and antinutritional factors. J. Food Sci. Technol. 28, 249– 251. Khokhar, S., Apenten, R. K.O., 2003. Antinutritional factors in food legumes and effects of processing. In Squires, R.V. (Ed.), The Role of food, Agriculture, Forestry and Fisheries in Human Nutrition. Encyclopedia of Life Support Systems (EOLSS). Publishers Co Ltd., Oxford, UK, pp. 82–116. Klein, S.M., Cohen, G., Cederbaum, A.I., 1981. Production of formaldehyde during metabolism of dimethyl sulfoxide by hydroxyl radical-generating systems. Biochemistry 20, 6006–6012. Koleva, I.I., Van Beek, T.A., Linssen, J.P.H., De Groot, A., Evstatieva, L.N., 2002. Screening of plant extracts for antioxidant activity: a comparative study on three testing methods. Phytochem. Anal. 13, 8–17. Lin, P.Y., Lai, H.M., 2006. Bioactive compounds in legumes and their germinated products. J. Agric. Food Chem. 54, 3807–3814. Lingaraju, M.H., Gowda, L.R., 2008. A Kunitz trypsin inhibitor of Entada scandens seeds: another member with single disulfide bridge. Biochim. Biophys. Acta 1784, 850–855. Liu, R., 2003. Health benefits of fruit and vegetables are from additive and synergistic combinations of phytochemicals. Am. J. Clin. Nutr. 78, 517S–520S. Makkar, H.P.S., Siddhuraju, P., Becker, K., 2007. Plant Secondary Metabolites, first ed. Humana Press, New Jersy, pp. 70. Molyneux, P., 2004. The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity. Songklanakarin J. Sci. Technol. 26, 211–219. Oboh, G., 2006. Antioxidant properties of some commonly consumed and underutilized tropical legumes. Eur. Food. Res. Technol. 224, 61–65. Pulido, R., Bravo, L., Saura-Calixto, F., 2000. Antioxidant activity of dietary polyphenols as determined by a modified ferric reducing/antioxidant power assay. J. Agric. Food Chem. 48, 3396–3402. Purseglove, J.W., 1968. Canavalia gladiata (Jacq.) DC. In Tropical Crops: Dicotyledons. Longmans, Green and Co., London, pp. 245. Rakic, S., Povrenovic, D., Tesevic, V., Simic, M., Maletic, R., 2006. Oak acorn, polyphenols and antioxidant activity in functional food. J. Food Eng. 74, 416– 423. Re, R., Pellegrini, N., Pannala, A., Yang, M., Rice-Evans, C.A., 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical. Biol. Med. 26, 1231–1237. Reuter, S., Gupta, S.C., Chaturvedi, M.M., Aggarwal, B.B., 2010. Oxidative stress, inflammation, and cancer: how are they linked? Free Radic. Biol. Med. 49, 1603–1616. Seiquer, I., Ruiz-Roca, B., Mesías, M., Muñoz-Hoyos, A., Galdó, G., Ochoa, J.J., Navarro, M.P., 2008. The antioxidant effect of a diet rich in Maillard reaction products is attenuated after consumption by healthy male adolescents. In vitro and in vivo comparative study. J. Sci. Food Agric. 88, 1245–1252. Siddhuraju, P., Becker, K., Makkar, H.P.S., 2002. Chemical composition, protein fractionation, essential amino acid potential and antimetabolic constituents of an unconventional legume, gila bean (Entada phaseoloides Merrill) seed kernel. J. Sci. Food Agric. 82, 192–202. Siddhuraju, P., Becker, K., 2003. Studies on antioxidant activities of Mucuna seed (Mucuna pruriens var. utilis) extracts and certain non-protein amino/imino acids through in vitro models. J. Sci. Food Agric. 83, 1517–1524.

Siddhuraju, P., 2006. The antioxidant activity and free radical-scavenging capacity of phenolics of raw and dry heated moth bean (Vigna aconitifolia) (Jacq.) Marechal seed extracts. Food Chem. 99, 149–157. Siddhuraju, P., 2007. Antioxidant activity of polyphenolic compounds extracted from defatted raw and dry heated Tamarindus indica seed coat. LWT – Food Sci. Technol. 40, 982–990. Singh, B., Bhat, T.K., Singh, B., 2003. Potential therapeutic applications of some antinutritional plant secondary metabolites. J. Agric. Food Chem. 51, 5579– 5597. Sowndhararajan, K., Siddhuraju, P., Manian, S., 2011. Antioxidant activity of the differentially processed seeds of jack bean (Canavalia ensiformis L. DC). Food Sci. Biotechnol. 20, 585–591. Sridhar, K.R., Seena, S., 2006. Nutritional and antinutritional significance of four unconventional legumes of the genus Canavalia – A comparative study. Food Chem. 99, 267–288. Taga, M.S., Miller, E.E., Pratt, D.E., 1984. Chia seeds as a source of natural lipid antioxidants. J. Am. Oil Chem. Soc. 61, 928–931. Turkmen, N., Sari, F., Velioglu, S., 2005. The effect of cooking methods on total phenolics and antioxidant activity of selected green vegetables. Food Chem. 93, 713–718. Vadivel, V., Pugalenthi, M., Megha, S., 2008. Biological evaluation of protein quality of raw and processed seeds of gila bean (Entada scandens BENTH). Trop. Subtrop. Agroecosyst. 8, 125–133. Vadivel, V., Biesalski, H.K., 2010. HPLC analysis of bioactive compounds in ten different wild type under-utilized legume grains. IIOAB J. 1, 17–24. Vadivel, V., Doss, A., Pugalenthi, M., 2010. Evaluation of nutritional value and protein quality of raw and differentially processed sword bean [Canavalia gladiata (Jacq.) DC.] seeds. Afr. J. Food Agric. Nutr. Dev. 10, 2850–2865. Vadivel, V., Stuetz, W., Scherbaum, V., Biesalski, H.K., 2011. Total free phenolic content and health relevant functionality of Indian wild legume grains: effect of indigenous processing methods. J. Food Comp. Anal. 24, 935–943. Vidal-Valverde, C., Frias, J., Valverde, S., 1992. Effect of processing on the soluble carbohydrate content of lentils. J. Food Prot. 55, 301–304. Wong, A.H., Gottesman, I.I., Petronis, A., 2005. Phenotypic differences in genetically identical organisms: the epigenetic perspective. Hum. Mol. Genet. 14, R11–R18. Xu, B.J., Chang, S.K.C., 2007. A comparative study on phenolic profiles and antioxidant activities of legumes as affected by extraction solvents. J. Food Sci. 72, S159–S166. Xu, B., Chang, S.K.C., 2008. Total phenolics, phenolic acids, isoflavones, and anthocyanins and antioxidant properties of yellow and black soybeans as affected by thermal processing. J. Agric. Food Chem. 56, 7165–7175. Yamaguchi, N., Yashito, K., 1967. Browning reaction products produced by the reaction between sugars and amino acids. V. Effect of browning reaction products and legal antioxidants on stability of fats. Nippon Shokuhin Kogyo Gakkaishi 14, 281–285. Zheng, T., Shu, G., Yang, Z., Mo, S., Zhao, Y., Mei, Z., 2012. Antidiabetic effect of total saponins from Entada phaseoloides (L.) Merr. in type 2 diabetic rats. J. Ethnopharmacol. 139, 814–821. Zhishen, J., Mengcheng, T., Jianming, W., 1999. The determination of flavanoid contents on mulberry and their scavenging effects on superoxide radical. Food Chem. 50, 6929–6934.