Contribution of phenolic compounds to the antioxidant potential and type II diabetes related enzyme inhibition properties of Pongamia pinnata L. Pierre seeds

Process Biochemistry 46 (2011) 1973–1980

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Contribution of phenolic compounds to the antioxidant potential and type II diabetes related enzyme inhibition properties of Pongamia pinnata L. Pierre seeds Vellingiri Vadivel ∗ , Hans K. Biesalski Institute for Biological Chemistry and Nutrition, University of Hohenheim, D-70593 Stuttgart, Germany

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Article history: Received 5 February 2011 Received in revised form 17 June 2011 Accepted 5 July 2011 Keywords: Pongamia pinnata Karanja seeds Total free phenolics Antioxidant activity ␣-Amylase inhibition ␣-Glucosidase inhibition

a b s t r a c t The methanolic extract of Pongamia pinnata L. Pierre (locally called as karanja) seed materials, an underutilized food legume collected from India was analyzed for antioxidant and type II diabetes related enzyme inhibition properties. The methanolic extract of raw seeds contained total free phenolic content of 14.85 ± 0.32 g catechin equivalent/100 g extract DM. Encouraging levels of ferric reducing/antioxidant power (FRAP, 1179 mmol Fe[II]/mg extract), inhibition of ␤-carotene degradation (41.13%) and radical scavenging activity against DPPH (54.64%) and superoxide (54.53%) were exhibited by the raw sample. Further, it also recorded 77.92% of ␣-amylase and 86.50% of ␣-glucosidase enzyme inhibition characteristics under in vitro starch digestion bioassay. Sprouting + oil-frying caused a apparent increase on the total free phenolic content and also significant improvement on the antioxidant and free radical scavenging capacity of P. pinnata seeds, while soaking + cooking as well as open-pan roasting treatments showed diminishing effects. Moreover, inhibition of ␣-amylase and ␣-glucosidase enzyme activities was declined to 24.24 and 45.14%, respectively during sprouting + oil-frying treatment, which are more desirable for the dietary management of type II diabetic patients. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Pongamia pinnata (L.) Pierre (Fabacae), popularly known as ‘Karanja’ (in Hindi), Pongam (in Tamil) and ‘Indian beech’ (in English), is native to India and widely distributed along Southeast Asia to the West Pacific and North Australia. It is a medium-sized tree with a short crooked trunk and a broad crown of spreading or drooping branches. It is naturally distributed along the coasts and river banks in India and Myanmar [1]. It is introduced to humid tropical lowlands in the Philippines, Malaysia, Australia, the Seychelles, the United States of America and Indonesia [2]. It is one of the widely grown forest trees with 0.11 million tons of seeds collected every year in Andhra Pradesh, Karnataka and Tamil Nadu states of India [3]. The tree is known for its multipurpose benefits and as a potential source of biodiesel [4], beside its application as animal fodder, green manure, timber and fish poison. The cooked mature seeds are consumed as a food by certain tribal sects, including Lambadi, Uraali and Dravidian in India. The seeds are found to possess 22% of crude protein, 33.4% of crude lipid, 6.8% of crude fiber, 3.3% of ash and 26% of carbohydrates and all the essential amino acids, except cystein, methionine and tyrosine, in addition to very high protein digestibility (92%) [3]. Kesari et al.

∗ Corresponding author. Tel.: +49 71145922948; fax: +49 71145923822. E-mail address: [email protected] (V. Vadivel). 1359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2011.07.007

[4] reported that the seeds contain on an average about 28–34% oil with high percentage of polyunsaturated fatty acids. Historically P. pinnata is used as folk medicinal plant, particularly in Ayurvedha and Siddha systems of Indian medicine [4]. All parts of the plant are used as a crude drug for the treatment of tumors, piles, skin diseases, itches, abscess, painful rheumatic joints, ulcers, diarrhea, bronchitis, whooping cough and quench dipsia in diabetes [4,5]. P. pinnata is being reported to use by traditional healers of Theni District, Tamilnadu India as an antiseptic, blood purifier and also to treat cuts and wounds [6]. Recently, P. pinnata has been exploited as a source of biomedicine, specifically as antimicrobial and therapeutic agents [4,7]. Further, experimental studies also demonstrated its anti-inflammatory, antioxidative, analgesic and antiulcer effects [8–10]. The effects of ethanolic root extract of P. pinnata on the antioxidant status and histopathological changes in acute ischemia-reperfusion injured rat model are investigated [11]. The crude decoction of P. pinnata leaves is known to exhibit selective antidiarrheal action with efficacy against cholera and enteroinvasive bacterial strains causing bloody diarrheal episodes [7]. The leaves are scientifically validated for antinociceptive as well as antipyretic activities for the treatment of pain and pyretic disorders [12]. The flowers of P. pinnata had a protective effect against cisplatin and gentamicin induced renal injury through antioxidant property [13]. The antihyperglycaemic activity of stem bark extract is proved in alloxan-induced diabetic rats [14]. Pongamol and karanjin isolated from the fruits of P. pinnata are reported to

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produce antihyperglycaemic activity. Further, the seed extract is reported to completely inhibit the growth of herpes simplex viruses [15]. Even though, the nutritional value of seeds and medicinal properties of different parts of P. pinnata plant are reported earlier, the information regarding the antioxidant and type II diabetes related enzyme inhibition properties of seed materials are very scarce. In current situation, research in this direction is of prime importance to combat with free radical mediated chronic diseases. Hence, in the present study was conducted to analyze the total free phenolics content, antioxidant and type II diabetes related enzyme inhibition properties of methanolic extract of raw and traditionally processed P. pinnata seeds with a view to promote them as a dietary ingredient in the supplementary foods to be formulated with therapeutic value. 2. Materials and methods 2.1. Chemicals (+)-Catechin hydrate, polyvinylpolypyrrolidone, butylated hydroxytoluene (BHT), 2,4,6-tris-(2-pyridyl)-s-triazine, 2,2-diphenyl-1-picryl-hydrazyl (DPPH), ˇcarotene, linoleic acid, tween-40, riboflavin, methionine, nitro-blue tetrazolium, starch, ␣-amylase, ␣-glucosidase and p-nitrophenyl-␣-d-glucopyranoside were procured from Sigma-Aldrich Chemicals, USA and all other chemicals were received from Merck, Darmstadt, Germany. 2.2. Seed samples The seed materials of P. pinnata were collected from different locations of Tamil Nadu, India (Kondayampalayam, Gunderipallam, Vellore, Karaikudi and Coimbatore). Seed sample from each location (about 500 g) was aggregated from 8 to 15 plants and mixed together to obtain a representative sample. After acquisition, the seed samples were frozen immediately and stored at −80 ◦ C until the analysis. Then, each seed sample was randomly divided into four batches with five replicates (each consist of 25 g seeds) for implementing different processing methods. The first batch was stored without any treatment and considered as raw seeds and the remaining three batches were processed as described below.

standard curve prepared with (+)-catechin hydrate (20–100 ␮g/ml), the total phenolic content was calculated and expressed as g catechin equivalent/100 g extract on dry matter (DM) basis. 2.6. Reducing power The ferric reducing/antioxidant power (FRAP) of the methanolic extract of P. pinnata seeds was determined according to the procedure described by Pulido et al. [17]. FRAP reagent (900 ␮l) was prepared freshly and incubated at 37 ◦ C and mixed with 90 ␮l of distilled water and 30 ␮l of methanolic extract (1 mg ml) or methanol (for the reagent blank) or BHT (positive control). The contents were incubated at 37 ◦ C for 30 min and then the absorbance readings were taken immediately at 593 nm. From the calibration curve prepared from different concentrations of Fe [II] (FeSO4 ·7H2 O, 100–2000 ␮M), the reducing power was calculated. 2.7. ˇ-Carotene bleaching assay An aliquot of ␤-carotene solution (20 ␮l, 2 mg ml in chloroform) was added to a flask containing 40 ␮l of linoleic acid, 1.0 ml of chloroform and 0.4 ml of Tween-40 and mixed well [18]. The chloroform was evaporated to dryness under nitrogen gas (Turbo-LV Evaporator, Zymark, USA) and the final volume was made up to 100 ml with oxygenated distilled water. From this mixture, 2.9 ml was added with 100 ␮l of methanolic extract (1 mg ml) and incubated for 2 h at 50 ◦ C. In control, 100 ␮l of methanol was added instead of extract. Then the absorbance of the solution was measured at 450 nm and the inhibition of ␤-carotene oxidation was calculated and expressed on percent basis. 2.8. DPPH radical scavenging activity The DPPH radical scavenging activity was analyzed by following SanchezMoreno et al. [19] method. The methanolic extract (100 ␮l, 1 mg ml) was added to 3.9 ml of DPPH solution (0.025 g/L) and the reactants were incubated at 25 ◦ C for 30 min. Instead of extract, in positive control 100 ␮l of BHT (1 mg ml) was used, while 100 ␮l of DPPH solution was added in negative control. After incubation, the absorbance was measured for test (A1 ), positive control (A1 ) as well as negative control (A2 ) at 517 nm. The radical scavenging activity was calculated based on the following equation and expressed on per cent basis. Scavenging activity (%) = (1 − A1 /A2 ) × 100 2.9. Superoxide radical scavenging activity

The whole seeds of second batch (25 g in each replicate) was soaked in distilled water in a ratio of 1:10 (w/v) for 8 h at 25 ◦ C and then cooked with fresh distilled water at 85–90 ◦ C (about 30 min). Third batch of samples was added to the redsoil suspension (1:5, w/v) and kept for 2 days in dark under moist cloth. Then the sprouts were separated, thoroughly washed and fried in sunflower oil at 185–190 ◦ C for 10 min. The fourth batch of seed materials was roasted in an iron pot for 30 min at 120–130 ◦ C and then the seeds were separated using a sieve and allowed to cool to room temperature.

The light-induced reaction was performed using 20 W fluorescent lamp [20]. The total volume of the reaction mixture was 5.0 ml and the concentrations of the riboflavin, methionine and nitro-blue tetrazolium (NBT) were 3 × 10−6 , 1 × 10−2 and 1 × 10−4 mol/l, respectively. The reactants were illuminated at 25 ◦ C for 25 min, so that the photochemically reduced riboflavin generates superoxide radicals, which reduce NBT to form a blue formazan. The methanolic extract (100 ␮l, 1 mg ml) was added to the reaction mixture, in which superoxide radicals are scavenged, thereby inhibit the NBT reduction. The un-illuminated reaction mixture was used as a blank and the absorbance was measured at 560 nm. Based on the absorbency values, the superoxide radical scavenging activity was calculated and expressed on per cent basis.

2.4. Preparation of methanolic extract

2.10. ˛-Amylase inhibition assay

All the processed as well as raw samples were frozen at −80 ◦ C and freezedried for 10 h and then powdered in a grinder to 1 mm particle size. One gram of defatted seed flour was extracted sequentially with 10 ml of 100%, 80%, 70% and 50% methanol acidified with 1% conc. HCl in an ultra-sonic bath for 10 min followed by extraction in magnetic stirrer for 30 min. After centrifugation, the supernatants were pooled and made up to a known volume. The extract was treated with 5 g of polyvinylpolypyrrolidone at 0 ◦ C for 30 min and the supernatant was collected and then purified using a Solid Phase Catridge (Strata-x-33 um polymeric sorbent, L1001105, 200 mg/6 ml sample, 8B-S100-FCH-S from Phenomenex, USA). The phenolics were eluted with 10 ml of 50% and 100% methanol and the solvent was evaporated using rotary vacuum evaporator (Büchi Rotavapor–R, CH-9230, Switzerland) at 40 ◦ C and dried in lyophilizer (Virtis Freeze mobile 25 EL, New York) for 1 h. Finally the residue was weighed and the total dry yield of extract was calculated and then the extract was re-dissolved in water:methanol:formic acid (47.5:47.5:5%, v/v/v) solution in the ratio of 1 mg ml of solvent and used for further analysis.

Methanolic extract (100 ␮l, 1 mg ml) was mixed with 100 ␮l of 0.02 M sodium phosphate buffer (pH 6.9) and 100 ␮l of ␣-amylase solution (4.5 Units/ml/min) and pre-incubated at 25 ◦ C for 10 min [21]. Then, 100 ␮l of 1% starch solution was added and incubated at 25 ◦ C for 30 min and the reaction was stopped by the addition of 1.0 ml of dinitrosalicylic acid reagent. The test tubes were then incubated in a boiling water bath for 5 min and then cooled to room temperature. The reaction mixture was then diluted (10-fold) with distilled water and the absorbance was measured at 540 nm. The readings were related with the control, which contained buffer instead of extract and the per cent of ␣-amylase enzyme inhibition was calculated.

2.3. Processing methods

2.5. Total phenolic content The total phenolic content of methanolic extracts from raw and processed samples was estimated according to the method of Singleton et al. [16]. The extract (500 ␮l) was mixed with 500 ␮l of freshly prepared Folin-Ciocalteu reagent and 6 ml of distilled water. Then, 2 ml of 15% sodium carbonate was added and shaken for 0.5 min. Finally the solution was brought up to a volume of 10 ml with distilled water. After 2 h of incubation at 37 ◦ C in dark, the absorbance was measured at 750 nm in a UV–visible Spectrophotometer (Perkin-Elmer, Lambda 35). Based on the

2.11. ˛-Glucosidase inhibition assay Methanolic extract (100 ␮l, 1 mg ml) was mixed with 100 ␮l of 0.1 M phosphate buffer (pH 6.9) and 100 ␮l of ␣-glucosidase solution (1 Unit/ml/min) and pre-incubated at 25 ◦ C for 5 min [21]. Then, 100 ␮l of p-nitrophenyl-␣-dglucopyranoside (5 mM) was added and the reaction mixture was incubated at 25 ◦ C for 10 min. After the incubation period, the absorbance readings were recorded at 405 nm and allegorized to a control that had 100 ␮l of buffer in place of the extract. The results were calculated and expressed on per cent basis. 2.12. Statistical analysis All the data were analyzed and expressed as means ± standard deviation of five separate determinations (n = 5). One-way ANOVA with Dunnett’s post test to determine the significant differences between the experimental batches as well

V. Vadivel, H.K. Biesalski / Process Biochemistry 46 (2011) 1973–1980

Fig. 1. Total free phenolic content in methanolic extract of raw and traditionally processed seed material of Pongamia pinnata.

as correlation analysis were performed using GraphPad PRISM® version 5.00 for Windows, San Diego, California, USA.

3. Results and discussion 3.1. Total free phenolics The phenolic compounds constitute one of the most numerous and ubiquitously distributed groups of plant secondary metabolites, which are ranges from simple molecules (e.g. simple phenols, phenolic acids, phenyl-propanoids and flavonoids) to highly polymerized compounds (e.g. lignins, lignans, tannins, suberins and cutins). The phenolic compounds have been demonstrated to prevent the development of many chronic diseases, which might be associated with their powerful antioxidant and free radical scavenging properties [22]. Although the dietary intake of phenolics varies considerably between 20 mg and 1 g among the geographical regions, the mean daily intake of total free phenolics was higher than that of vitamin E. The total free phenolics content of methanolic extract of defatted raw seeds of P. pinnata was found to be 14.85 g catechin equivalent/100 g extract DM (Fig. 1). This value is higher when compared to the previous report on total phenolic content of P. pinnata flower extracts (0.14–0.47 mg/g extract) [23] and other legume grains [24] such as white bean (1.08 g catechin equivalent/100 g extract), pea (3.48 g catechin equivalent/100 g extract), faba bean (8.09 g catechin equivalent/100 g extract), lentil (6.01 g catechin equivalent/100 g extract) and broad bean (6.01 g catechin equivalent/100 g extract). Such high yield of total free phenolics might be due to the repeated extraction of phenolic compounds using different concentrations of acidified methanol as a solvent. Because, recovery of phenolic compounds from food samples are mainly depend upon the type of solvent used and method of extraction. It is interesting to notice that the seed coat colour of P. pinnata samples is brown. Relationship between seed coat colour and phenolics level are still controversial. While Barampama and Simard [25] found a positive correlation between the seed coat colour and phenolic content, Guzman-Maldonado et al. [26] did not find any connection. However, there are some reports available with high correlation between cultivar lines and phenolic content [27]. In addition to seed coat colour, the quantity of phenolic compounds in

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Fig. 2. Ferric reducing/antioxidant potential (FRAP) of methanolic extract (1 mg ml) of raw and differentially processed Pongamia pinnata seeds.

seed samples is influenced by soil, environmental conditions, genotype (cultivar/variety), agronomic practices (irrigation, fertilization and pest management), maturity level at harvest and post-harvest storage. For instance, low temperature during the onset and duration of seed fill were shown to increase the isoflavone content by several folds in soybean [28]. Since, P. pinnata grows wildly in adverse environmental conditions such as drought, poor soil etc., a high phenolic content in the seed materials contribute to the resistant function. Now-a-days food technologists are keen to harness the nutritional benefits of phenolics, namely their antioxidant or free radical scavenging, food preservative, antimicrobial, anti-mutagenic, therapeutic and pharmaceutical properties. The seed coat of legume grains is reported to contain numerous types of phenolics, which are suggested to play an important protective role against oxidative damage in consumer’s body [29]. Hence, in recent years, research efforts are under-way to incorporate these wild type legume grains in the formulation of supplementary therapeutic foods for the dietary management of various chronic diseases, including diabetes, obesity and cardiovascular diseases. 3.2. Reducing power Antioxidants can be explained as reductants and inactivation of oxidants by reductants can be described as redox reactions, in which one reactive species (oxidant) was reduced at the expense of oxidation of other. The FRAP assay measures the antioxidant effect of any substance in the reaction medium in term of its reducing ability. FRAP reflects total antioxidant power involving the single electron transfer reaction. Antioxidant potential of methanolic extracts of P. pinnata seeds was estimated from their ability to reduce TPTZ-Fe(III) complex to TPTZ-Fe(II) complex. The reducing power of methanolic extract of raw seed materials of P. pinnata (1179 mmol Fe [II]/mg extract DM, Fig. 2) was found to be higher when reviewed earlier report on seed samples of Tamarindus indica (517 mmol Fe [II]/mg extract DM) [30]; moth bean (618 mmol Fe [II]/mg extract DM) [31]; light brown (545 mmol Fe [II]/mg extract DM) and dark brown (487 mmol Fe [II]/mg extract DM) varieties of cowpea [32]. However, this value was lower in comparison to the positive control, BHT (2182 mmol Fe[II]/mg extract DM, Fig. 2) and other underutilized legume grains

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Fig. 3. Inhibition of ␤-carotene degradation by the methanolic extract (1 mg ml) of raw and differentially processed Pongamia pinnata seeds.

such as Vigna vexillata (1967 mmol Fe[II]/mg extract DM) [33] and brown variety seeds of horse gram (1724 mmol Fe [II]/mg extract DM) [34]. Earlier in vitro research investigation proved that the dark chocolate containing phenolics, in particular (−) epicatechin possess potential antioxidant activity through FRAP assay, which was also evidenced later to increase the total antioxidant capacity of blood plasma in human beings [35]. Hence, the reducing power of P. pinnata methanolic extract perceived from the FRAP results is also expected to demonstrate potential antioxidant activity in consumer’s body. 3.3. Inhibition of ˇ-carotene bleaching The methanolic extract of raw P. pinnata seeds demonstrated 41.13% of inhibition of ␤-carotene degradation, which is comparable to that of positive control BHT (Fig. 3). This value is higher than that of former reports on Lathyrus filiformis (28%) [36] and large black soybean (25%) [37], but lower when compared to pseudo-cereals including Amaranthus cruentus var. R-104 (73.5%) and Chenopodium quinoa var. JQ (70.4%) [38]. The ␤-carotene bleaching method is associated with rancidity reaction in fats and oils at room temperature. This method is based on the loss of yellow colour of ␤-carotene due to its reaction with radicals, which are formed by linoleic acid oxidation in an emulsion. The capacity of antioxidant compounds to prevent the discoloration/degradation of ␤-carotene during the auto-oxidation of linoleic acid was measured. In this assay, the oxidation of linoleic acid generated peroxyl free radicals due to the abstraction of hydrogen atom from di-allylic methylene groups of linoleic acid. The free radicals oxidized the highly unsaturated ␤-carotene and consequently the orange coloured chromophore of ␤-carotene was degraded and the results were monitored spectrophotometrically. The results indicate that the presence of phenolic compounds in methanolic extract of P. pinnata seeds can moderately prevent the degradation of ␤-carotene caused by radical reactions. Thus, consumption of such underutilized legume sample can protect the oxidation/degradation of cellular macromolecules due to free radical attacks. 3.4. DPPH radical scavenging activity According to the mode of action, the antioxidants can be classified as free radical terminators, chelators of metal ions capable of catalyzing lipid oxidations or as oxygen scavengers that react with oxygen in closed systems. Among the various methods to evaluate the radical scavenging activity of natural compounds, the latest DPPH method received more attention due to its fast and reliable results. DPPH (2,2-diphenyl-1-picrylhydrazyl), a stable organic free radical has a maximum absorption at 517 nm but upon reduc-

tion by an antioxidant, the absorption disappears. This method is based on the reduction of alcoholic DPPH solution in the presence of hydrogen donating antioxidant compound due to the formation of a non-radical form (DPPH-H). The DPPH radical scavenging activity of methanolic extract (1 mg ml) obtained from raw P. pinnata seeds was found to be 54.64% (Fig. 4), which is lower when compare to the radical scavenging ability (28.11%) of P. pinnata leaf extract (100 ␮g/ml) [39] as well as ethanolic extract (100 ␮g/ml) of P. pinnata flower (10.4%) [23]. Further, this value is comparable with the previous report on an under-utilized legume, Mucuna pruriens (50%) [40], but slightly higher than certain common legumes like Phaseolus vulgaris var. Bayo Victoria (40%) [41]; Vigna radiata (25%) [42]; soybean (44%) [43]; Navy bean (14%) and Pinto bean (43%) [44]. However, the free radical quenching property of Vicia faba (60%) [45]; kidney bean (62%) [43] and Caesalpinia bonducella (69%) [46] were higher than P. pinnata seeds. The interaction of phenolic compounds with DPPH was depending upon their structural conformation. Certain compounds can react very rapidly with the DPPH radicals and reduce a large number of DPPH molecules corresponding to the number of available hydroxyl groups. The free radical scavenging activity of the phenolic compounds was depends on different structural features such as O–H bound dissociation energy, resonance delocalization of the antioxidant and steric-hindrance derived from bulky groups substituting hydrogen in the antioxidant compound. Potential radical scavenging activity revealed by methanolic extract of P. pinnata seeds of the present study against a synthetic DPPH radical might confirm its hydrogen donating capacity and also its proposed or claimed ability to protect the consumers health from various freeradical related diseases including aging, cancer, atherosclerosis, Alzheimer disease, diabetes, etc.

3.5. Superoxide radical scavenging activity Superoxide radicals, a biologically quite toxic oxygen molecule with one unpaired electron and deployed by the immune system to kill the invading microorganisms. In phagocytes, superoxide is produced in large quantities by the enzyme NADPH-oxidase for the implementation in oxygen-dependent killing mechanisms of invading pathogens, but also deleterious to cellular macromolecules on the other hand. Although 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. Presently investigated methanolic extract of P. pinnata raw seeds exhibited 54.53% of superoxide radical scavenging activity (Fig. 4). This is prominent than that of an earlier report in ethanolic extract of P. pinnata flower (46%) [23]; brown (37.23%) and black (23.43%) varieties of horse gram [34]; moth bean (19.73%) [31]; light brown (32%) and dark brown (32.8%) varieties of cowpea [32]. However, it is beneath when matched to the seeds of Bauhinia vahlii (82.6%) [47] and Caesalpinia bonducella (74.71%) [46]. Superoxide was deleteriously produced as a byproduct of mitochondrial respiration, most notably by complexes I and III, as well as several other enzymes like xanthine oxidase. Superoxide contributes to the pathogenesis of many chronic diseases and perhaps aging via the oxidative damage. Superoxide radicals were observed to kill cells, inactivate enzymes and degrade DNA, cell membranes and polysaccharides. The superoxide was also play an important role during peroxidation of unsaturated fatty acids and possibly other susceptible substances. Therefore, the study on superoxide radical scavenging activity of natural compounds is one of the most important ways to prove their antioxidant mechanism.

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Fig. 4. DPPH and superoxide radical scavenging activities of methanolic extract (1 mg ml) of raw and differentially processed Pongamia pinnata seeds.

In this connection, the methanolic extract of P. pinnata seeds deciphered a moderate scavenging activity against superoxide radicals. Thus, incorporation of such non-traditional legume grains with notable phenolic content in the regular diets of human population could play a preventive role against the dangerous free radicals like superoxide, and thus confers the alleviation of oxidative stress and ultimately disease protection in human body. 3.6. ˛-Amylase inhibition activity Research priorities on type II diabetes are now-a-days becoming more prevalent with increased emphasis on its management through dietary practice. Considering the diet-linked challenge of type II diabetes consumption of foods rich in ␣-amylase and ␣-glucosidase inhibitors, the so-called hypoglycemic foods are receiving more attention and being investigated extensively to manage type II diabetic patients. ␣-Amylase and ␣-glucosidase are well-known key enzymes, playing a vital role in the management of hyperglycemia linked type II diabetes. ␣-Amylase catalyzes the hydrolysis of glycosidic linkages in the starch and release the hydrolyzed products, which constitute the first step in enzymatic degradation of this polymer. ␣-Amylase inhibitors are starch blockers, which can binds with the reactive sites of amylase enzyme and alter its catalytic activity and thus reducing the blood sugar level. Acarbose, miglitol and metformin are certain examples of commercially available enzyme inhibitors for the clinical treatment of type II diabetes. However, these drugs are reported to cause various side effects such as abdominal distention, flatulence and possibly diarrhea due to the excessive inhibition of pancreatic ␣amylase, which resulted in the abnormal bacterial fermentation of undigested carbohydrates in the colon [48]. Hence, at present there is an increasing interest among the food scientists to find out an alternative natural source of ␣-amylase inhibitor with potential antioxidant activity – without any side effects - for the dietary management of type II diabetic patients [48–50]. The methanolic extract (1 mg ml) of raw seed materials of P. pinnata showed 77.92% of ␣-amylase inhibition (Fig. 5). This value is lower than Mucuna pruriens seeds (87%) [40] and higher than mung bean (65%) [42] and certain cereal grains such as wheat, buckwheat, corn and oats (38–55%) [51]; Foxtail millet (32%), Proso millet (55%) and finger millet (55%) [52,53]. The low level of ␣-amylase inhibitors from natural fruits, vegetables and legume grains are reported to offer a good strategy to control postprandial hyperglycemia [49,54], since high amylase inhibition results in many harmful side effects in human beings. In this connection, the ␣-amylase inhibition activity observed in raw

P. pinnata seed seems to be not suitable to implement in the dietary practice of type II diabetes. 3.7. ˛-Glucosidase inhibition activity The cells lining the small intestine release the ␣-glucosidase that results in the cleavage of di- and oligosaccharides into glucose and its absorption in the intestine. The delay of glucose absorption could have a beneficial effect in controlling the postprandial blood sugar level. ␣-Glucosidase inhibitor can retard rate of glucose absorption in the intestine, by inhibiting the cleavage of di- and oligosaccharides through competitive and reversible inhibition of intestinal ␣-glucosidase enzyme. A high level of ␣-glucosidase inhibition (86.50%) was noticed in methanolic extract of P. pinnata seed, which is comparable to that of acarbose (Fig. 5). This value is higher than that of antecedent results give for wheat, buckwheat, corn and oats (18–31%) [51], but comparable when look at Foxtail millet (82.5%), Proso millet (77%), sorghum (95%) and finger millet (78%) [52,53]; Mucuna pruriens seeds (79%) [40] and Psoralea corylifolia seeds (77.5%) [55]. However, it should be noted that these results are based on in vitro biochemical tests and are indicative of anti-glycemic effects in the prevention/management of type II diabetes and have limited implications to what happens under in vivo. 3.8. Effect of soaking + cooking Significant level of reduction of total free phenolics was noticed during soaking + cooking treatment (17%) (Fig. 1). In contrast 60 and 28% of losses of phenolics were observed during soaking + autoclaving in light brown and dark brown coloured seeds of cowpea, respectively [32]; 82% loss in Vigna vexillata [33] and 48% in Bauhinia vahlii seeds [47]. Such significant loss of total free phenolics during this treatment might be due to the leaching out of this compound into the soaking medium by increased permeability of the seed coat and also due to degradation of phenolics with the high temperature during subsequent cooking for a longer period (30 min). Soaking + cooking treatment was found to affect the antioxidant activities (Figs. 2–4). Similarly, Xu and Chang [56] reported the loss of antioxidant activity during soaking (6–34%) as well as cooking (33–82%) in food legumes such as green pea, yellow pea, chickpea and lentil. Further, even cooking alone was recognized to reduce the antioxidant activity in Lathyrus sativus [57]; Phaseolus vulgaris [58] and Chenopodium quinoa [59]. Such a decreased antioxidant property in wild legume grain of the present analysis might be due

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Fig. 5. ␣-Amylase anda-glucosidase inhibition activities of methanolic extract (1 mg ml) of raw and differentially processed Pongamia pimuiici seeds.

to the degradation of phenolic compounds under cooking at elevated temperature. Hence, such soaking + cooking treatment is not recommendable to utilize the P. pinnata seeds as a natural source of antioxidants and type II diabetes related enzyme inhibitors. 3.9. Effect of sprouting + oil-frying An appreciable level of increase of total free phenolics (13%) was observed during sprouting + oil-frying in P. pinnata legume grains (Fig. 1). Similarly, sprouting for 2 days + autoclaving was reported to increase the total free phenolics by 9%, 20%, 27% and 50% in wheat, buckwheat, corn and oats, respectively [51]. In agreement, a very high increase on the level of total free phenolics was noticed in Vigna radiata (217%) after 7 days of germination [60]. Further, Zielinski [61] also reported that germination of Glycine max caused an increase of total free phenolics (from 2.6 to 3.1 mg/g extract DM). A major portion of phenolic compounds was stored in seeds as soluble conjugate or insoluble forms. Hence, significant level of increase exhibited by the P. pinnata seeds under sprouting + oilfrying treatment might be due to mobilization of stored phenolics by the activation of enzymes like polyphenol oxidase during sprouting and also due to the release of free phenolics from bounded form by the breakdown of cellular constituents and cell walls during subsequent thermal process (oil-frying). Further, dissociation of conjugated phenolic constituents due to oil-frying followed by some polymerization/oxidation reactions may also be responsible for this improvement. Sprouting + oil frying was found to increase the antioxidant activity at significant level (Figs. 2–4). Similarly, germination was reported to increase the antioxidant activity of Mucuna pruriens seeds, lupin seeds, mung bean seeds, faba bean, peas and common beans [40,42,62–64]. Sprouting + autoclaving was also reported to significantly increase the antioxidant activity in wheat, buckwheat, corn and oats [51]. On the other hand, reduction of antioxidant activity after germination was noticed in Lens culinaris [63]. Such significant increase of antioxidant properties of P. pinnata seeds was obviously attributed to elevation of their phenolic content during sprouting + oil-frying treatment. A significant level of positive correlation was noticed between the phenolic content and antioxidant properties of P. pinnata seeds (Table 1). The results of the present analysis emphasize that the chemical and biological properties of phenolic compounds were not affected by this treatment. Even though, the seed samples were exposed to elevated heat under oil-frying, the duration is very short (10 min). Also, improvement of antioxidant activities was explained by the fact that release of free phenolics from the bound form, which could possess high antioxidant potential that may counteract the degradation of phenolics, if any during oil-frying. Further, the Mail-

lard reaction products that formed during oil-frying might also be responsible for such a rise in the antioxidant characteristics of P. pinnata grains. Nonetheless, significant level of loss of ␣-amylase and ␣glucosidase inhibition activities were observed in P. pinnata grains after sprouting + oil-frying treatment (Fig. 5). This is in agreement with that of earlier observation on Mucuna pruriens seeds during germination [40]. In contrast, increase of ␣-amylase inhibition activity in wheat grains and ␣-glucosidase inhibition property in certain cereal grains during sprouting + autoclaving were reported [51]. However, the ␣-amylase and ␣-glucosidase inhibition levels recorded in sprouting + oil-fried seeds were similar to that of synthetic antidiabetic agent, acarbose and hence, more desirable for the dietary maintenance of type II diabetes. Such low level of ␣amylase and moderate inhibition of ␣-glucosidase can regulate the blood sugar level of the diabetic patients without any adverse side effects. Thus, sprouting + oil frying could be considered as a mild and favourable treatment to exploit P. pinnata seeds as a therapeutic food source. 3.10. Effect of open-pan roasting Open-pan roasting caused a drastic loss of total free phenolics (27%) in P. pinnata seeds (Fig. 1). Similarly, removal of phenolic content was recorded in moth bean (11%) [31]; dark brown seed coated cowpea (16%) [32] and Bauhinia vahlii (20%) [47] and drastic levels of loss of 48 and 64% were also reported in light brown coloured cowpea and Vigna vexillata seeds, respectively [32,33]. In contrast dry-heat treatment was reported to increase the phenolics content at significant level in brown and black varieties of horse gram [34]. Degradation of phenolic compounds as a result of direct heat exposure might be a reason for drop in phenolic content observed in P. pinnata seeds under open-pan roasting. Severe losses of antioxidant as well as enzyme inhibition properties were caused by open-pan roasting (Figs. 2–5). Similarly, decrease of antioxidant activity during roasting was reported in

Table 1 Relationship between phenolic content and antioxidant and type II diabetes related enzyme inhibition properties of Pongamia pinnata seeds. Compound

Total phenolics

Pearson coefficient (r) FRAP

BCB

DPPH

SO

AI

GI

0.9111*

0.9310*

0.9236*

0.8774*

0.0369

0.2958

FRAP – ferric reducing/antioxidant potential; BCB – ␤-carotene bleaching assay; DPPH – DPPH free radical scavenging activity; SO – super-oxide radical scavenging activity; AI – ␣-amylase inhibition activity; GI – ␣-glucosidase inhibition activity. * Correlation is significant at 0.01 (p two-tailed).

V. Vadivel, H.K. Biesalski / Process Biochemistry 46 (2011) 1973–1980

black-eyed peas, kidney beans and pinto beans [43]; amaranth, quinoa, wheat and buckwheat [65] and almond nut [66]. On the other hand, increase of antioxidant activity was reported in dry-heated seed materials of Tamarindus indica [30] and Vigna aconitifolia [31]. As a result of the significant adverse effects of the open-pan roasting on the total free phenolic content, diminishing effect were observed on antioxidant and enzyme inhibition properties of P. pinnata samples. This might be due to the disintegration of phenolic compounds by the direct action of high temperature during roasting. There is no significant correlation between the enzyme inhibition properties and phenolic content, even though a positive correlation was established between phenolic content and antioxidant properties of P. pinnata seeds (Table 1). Therefore, open-pan roasting could be considered as a most aggressive practice and not a suitable method to preserve the phenolic compounds and their antioxidant and health relevant functionalities in P. pinnata seeds. 4. Conclusion Methanolic extract of P. pinnata seeds was found to contain appreciable levels total free phenolics with promising antioxidant and type II diabetes related enzyme inhibition properties. A significant correlation was recognized between the phenolic content and antioxidant properties, while it was lacking in the case of enzyme inhibition characteristics. Considering the effect of different indigenous processing methods, soaking + cooking has exhibited substantial loss of total free phenolics, antioxidant and type II diabetes related enzyme inhibition properties. Open-pan roasting showed significant level of reduction of total free phenolics and thereby drastically affected the antioxidant and starch digestive enzyme inhibition characteristics, and thus considered to be the most aggressive practice. Finally, sprouting + oil-frying extensively increased the total free phenolics content as well as antioxidant properties of P. pinnata grain. Such viable processing technique could offers a good strategy to improve the phenolic content in P. pinnata seeds for enhanced antioxidant activity and type II diabetes related enzyme inhibition properties. Therefore, such suitably processed underutilized legume grain could be envisaged as a dietary ingredient in the formulation of supplementary foods with therapeutic value. Further, identification of phenolic constituents using LC–MS and evaluation of in vivo antioxidant and antidiabetic characteristics of methanolic extract from P. pinnata seeds are under progress. Acknowledgements One of the authors (VV) is thankful to Alexander von Humboldt (AvH) Foundation, Bonn, Germany for the award of Post Doctoral Research Fellowship. References [1] Janardhanan K, Vadivel V, Pugalenthi M. Biodiversity in Indian underexploited/tribal pulses. In: Jaiwal PK, Singh RP, editors. Improvement strategies for leguminosae biotechnology. The Netherlands: Kluwer Academic Publishers; 2003. p. 353–405. [2] Mukta N, Sreevalli Y. Propagation techniques, evaluation and improvement of the biodiesel plant, Pongamia pinnata (L.) Pierre – a review. Ind Crop Prod 2010;31:1–12. [3] Vinay BJ, Kanya TCS. Effect of detoxification on the functional and nutritional quality of proteins of karanja seed meal. Food Chem 2008;106:77–84. [4] Kesari V, Das A, Rangan L. Physico-chemical characterization and antimicrobial activity from seed oil of Pongamia pinnata, a potential biofuel crop. Biomass Bioenerg 2010;34:108–15. [5] Punitha R, Manoharan S. Antihyperglycemic and antilipidperoxidative effects of Pongamia pinnata (Linn.) Pierre flowers in alloxan induced diabetic rats. J Ethnopharmacol 2006;105:39–46. [6] Pandikumar P, Chellappandian M, Mutheeswaran S, Ignacimuthu S. Consensus of local knowledge on medicinal plants among traditional healers in Mayiladumparai block of Theni District, Tamil Nadu, India. J Ethnopharmacol. In press.

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