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LWT - Food Science and Technology

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Chemical composition and antioxidant activity of the husk and endosperm of fenugreek seeds
M. Madhava Naidu*, B.N. Shyamala, J. Pura Naik, G. Sulochanamma, P. Srinivas
Plantation Products, Spices and Flavour Technology, Central Food Technological Research Institute, Council of Scientic and Industrial Research, Mysore 570020, India

a r t i c l e i n f o
Article history: Received 10 February 2010 Received in revised form 17 August 2010 Accepted 17 August 2010 Keywords: Fenugreek Husk Endosperm Antioxidant activity Polyphenols Dietary ber

a b s t r a c t
Fenugreek (Trigonella foenum-graecum) is used as a spice, vegetable and a medicinal plant. In the present study, fenugreek seeds were separated into husk and endosperm. The proximate composition of fenugreek seeds, husk and cotyledons showed that endosperm had the highest saponin (4.63 g/100 g) and protein (43.8 g/100 g) content. In contrast, husk had higher total polyphenols (103.8 mg of gallic acid equivalent/g, and TDF (77.1 g/100 g), comprising IDF (31.9 g/100 g) and SDF (45.2 g/100 g). At 200 mg concentration, extracts of husk, fenugreek seed, and endosperm exhibited 72%, 64%, and 56% antioxidant activity respectively by free-radical scavenging method. The study indicated that separation of fenugreek seeds into husk and endosperm could have advantage of process viability with respect to prior selective fractionation of bioactive components for their effective isolation. 2010 Elsevier Ltd. All rights reserved.

1. Introduction Fenugreek (Trigonella foenum-graecum) is a member of the legume family, also known as methi, Greek hayseed, and birds foot. Fenugreek is originally from southeastern Europe and western Asia, but today it is grown in many parts of the world, including India, northern Africa, and the United States (Altuntas, Engin Ozgoz, & Taser, 2005). Fenugreek is known for its pleasantly bitter, slightly sweet seeds. The seeds, available in whole and ground form, are used to avor many foods including curry powders, spice blends and teas. The fenugreek seed contains a central hard, yellow embryo surrounded by a corneous and comparatively large layer of white, semi-transparent endosperm (Betty, 2008), which contains the galactomannan gum. Emulsion and rheological properties of galactomannans of fenugreek are reported (Wu, Cui, Eskin, & Goff, 2009). A tenacious and darkbrown husk surrounds the endosperm. The color of the gum fraction depends upon the amount of outer husk (brown color) and cotyledon (yellow color) present. Fenugreek is known to have several pharmacological effects such as hypoglycemia (Sharma, Raghuram, & Rao, 1990; Zia, Nazrul Hasnain, & Hasan, 2001), hypocholesterolemia (Stark & Madar, 1993; Srinivasan, 2006), gastroprotective (Suja

* Corresponding author. Tel.: 91 821 2512352 (O), 91 821 2413069 (R); fax: 91 821 2517233. E-mail address: madhavanaidu45@yahoo.com (M. Madhava Naidu). 0023-6438/$ e see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2010.08.013

Pandian, Anuradha, & Viswanathan, 2002), chemopreventive (Amin, Aysha Alkaabi, Shamaa Al-Falasi, & Sayel Daoud, 2005), antioxidant (Hettiarachchy, Glenn, Gnanasambandam, & Johnson, 1996; Kavirasan, Naik, Gangabhagirathi, Anuradha, & Priyadarsini, 2007), anti-inammatory, antipyretic (Ahmadiani, Javan, Semnanian, Barat, & Kamalinejad, 2001), laxative (Riad & El-Baradie, 1959) and appetite stimulation (Petit et al., 1993) attributes. This plant is known to contain alkaloids (Jain & Madhu, 1988), avonoids (Kamal & Yadav, 1991), salicylate (Swain, Dutton, & Truswell, 1985), and nicotinic acid (Rajalakshmi, Nanavaty, & Gumashta, 1964). Dietary ber consisting of non-digestible carbohydrates and lignin that are intrinsic and intact in traditional plants, has received much attention due to its health benets (Slavin, 2005; Kochar, Nagi, & Sachdeva, 2006; Anderson et al., 2009). Soluble ber lowers serum cholesterol and helps to reduce the risk of heart attack and colon cancer. It dissolves in the gut to form a viscous gel that lowers the absorption of released glucose. Consumption of dietary ber is shown to reduce the risk of diseases such as cardiovascular disease, colon cancer and obesity (Chau & Huang, 2004). Soluble and insoluble dietary bers are the storage and cell wall polysaccharides of plants that cannot be hydrolyzed by human digestive enzymes. A ber rich meal is metabolized more slowly and nutrient absorption occurs over a longer period (Jenkins et al., 1993). They have been reported to decrease the absorption of carbohydrates and reduce post-prandial serum glucose levels (Ou, Kwok, Li, & Fu, 2001). Optimal intake of total dietary ber

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reduces the risk of obesity, blood pressure, appendicitis and many other diseases (Rehiman, Rashid, & Shah, 2004). Further, a diet that provides adequate ber is usually less energy dense and larger in volume and thus may bring a feeling of satiety sooner (Saris, 2003). Indeed the, National Advisory Committee in Great Britain has recommended a ber intake of 25e30 g/day per person (Dashti, AlAwadi, Khalafawi, Sawaya, & Al Amiri, 2003). The total dietary ber content of infant foods plays a central role in meeting the recommendations [19 g/day] as well as stabilizing the intestinal population by stimulating the proliferation of bacteria capable of digesting dietary ber and lowering the colonic pH (Brooks, Mongeau, Deeks, Lampi, & Brassard, 2006). Cereal brans are used as a source of dietary ber, but alternative sources of dietary ber and data on their nutritional input is also required. Spices are reported to contain 15e55% crude ber and, except for a few, scanty information is available on dietary ber of spices. Though there are several studies on the medicinal properties of fenugreek seeds, their antioxidant abilities have not been examined separately with respect to fenugreek husk and cotyledon fractions. Objective of this study was to isolate and investigate the signicance of fenugreek husk as a source of antioxidants as well as dietary ber. It was envisaged that information on the total phenolics and antioxidant activities of different fractions of fenugreek and their individual phenolic compounds could be helpful in their application to retard or prevent lipid oxidation in a variety of food products. The aim of the present investigation was (i) to isolate and characterize the functional components from fenugreek fractions and (ii) to study their antioxidant properties. 2. Materials and methods 2.1. Materials Fenugreek (Trigonella foenum-graecum) seeds used in the present study were procured from Rajasthan, India through a wellestablished indigenous supplier. Linoleic acid, 1-1-diphenyl-2-picrylhydrazyl (DPPH), b-carotene, potassium ferricyanide, trichloroacetic acid, BHA (Butylated hydroxyl anisole), and ferric chloride were purchased from SigmaeAldrich. All the solvents/chemicals used were of analytical grade and obtained from SD Fine Chemicals, Mumbai, India. 2.2. Methods 2.2.1. Separation of husk and endosperm The fenugreek seeds were milled in a laboratory plate mill (1440 rpm) to obtain mixture containing husk and endosperm. Husk was separated from endosperm by sieving (British standard Sieve, size 10 equivalents to aperture size of 1.70 mm.) and used for the studies. AOAC methods (1995) were followed for determining the proximate composition viz., moisture, fat and ash content of the fenugreek seeds and fractions viz., husk and endosperm. 2.2.2. Moisture Milled fenugreek samples were accurately weighed in an aluminum dish and dried overnight (about 16 h) in an oven at 72  C. It was covered and cooled in a desiccator and then weighed. Sample was further dried for 2 h and reweighed until a constant weight was obtained. 2.2.3. Crude fat The crude fat was determined by extracting the sample in a Soxhlet apparatus for 16 h using petroleum ether. The solvent was evaporated and the residue was weighed to determine the fat content.

2.2.4. Total ash About 5 g of the sample was weighed accurately into a platinum (or porcelain) crucible and heated rst over a low ame till all the materials were completely charred and then heated in a mufe furnace to a constant weight to ensure complete conversion to ash.

Ash contentg=100 g sample

Weight of the ash Weight of the sample taken 100

2.2.5. Total proteins The estimation of nitrogen was done by Kjeldahl method wherein the protein content was obtained by multiplying the nitrogen value with 6.25. 2.2.5.1. Reagents. Digestion mixture, 40% NaOH, 0.1 mol equi/L NaOH, 0.1 mol equi/L H2SO4, methyl red indicator. 2.2.5.2. Procedure. The sample (1 g) was weighed into a dry Kjeldahl ask. About 2 g of digestion mixture and 20 mL of pure conc. H2SO4 were added to the same sample and the mixture digested by heating for 4e5 h. Glass beads were added to prevent bumping. After the contents of the ask became clear, the digestion was continued for at least 1 h. The contents of the Kjeldahl ask were cooled, diluted with distilled water and the mixture made alkaline by adding excess of 40% NaOH (about 75 mL). A small quantity of pumice powder was added to prevent bumping during distillation. The ammonia liberated was distilled into a receiver containing 25 mL of 0.1 mol equi/L H2SO4. The excess of acid in the receiver was back-titrated against 0.1 mol equi/L NaOH using 3 drops of methyl red indicator. A reagent blank was similarly digested and distilled. This titer value was subtracted from the value obtained for the sample to get the true titer value b.

Protein content in g=100 g sample c b 14 d 6:25 100=a 1000

where a g of the sample taken and if b and c mL of alkali of normality d required for back-titration and to neutralize 25 mL of 0.1 mol equi/L H2SO4 respectively. 2.2.6. Dietary ber The total dietary ber (TDF), a measure of the sum of insoluble and soluble dietary ber, based on digestion of food samples (1 g) with enzymes, was determined as described by Asp, Johnos, Hollmer, and Slijestrom (1983). 2.2.6.1. Reagents. 0.1 mol/L Sodium phosphate buffer pH 6.0; 4 mol/L HCl; 4 mol/L NaOH; 95% ethanol, technical grade; 78% ethanol and acetone AR grade. 2.2.6.2. Enzymes. Termamyl 60 L (a higher grade, 120 L is now available and preferred); pepsin NF; Pancreatin 4 NF. 2.2.6.3. Procedure. 1 g of defatted sample was weighed with 0.1 mg accuracy (W) and transferred to an Earlenmeyer ask. Sodium phosphate buffer (0.1 mol/L, pH 6.0, 25 mL) was added and the contents were mixed thoroughly. Termamyl (100 mL) was added. Flask was covered with Al lm and incubated in a boiling water bath for 15 min (with occasional shaking). Contents were allowed to cool, distilled water (20 mL) was added and the pH was adjusted to 1.5 with HCl. 100 mg of pepsin was added and the ask was covered, incubated in water bath maintained at 40  C with agitation for 60 min. Distilled water (20 mL) was then added and pH adjusted to 6.8 with NaOH. 100 mg of pancreatin was added and the ask

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incubated in water bath again at 40  C with agitation for further 60 min. The pH adjusted to 4.5 with HCl. Precipitate was ltered through a dry and weighed crucible (porosity 2) containing 0.5 g of dry celite (exact weight known) as the lter aid and washed with 2 10 mL of distilled water. 2.2.6.4. Residue (insoluble ber). Residue was washed with 2 10 mL of 95% ethanol and 2 10 mL of acetone. Residue was dried at 105  C to constant weight (or overnight). It was weighed after cooling in a desiccator (D1), then incinerated at 550  C for 5 h and weighed, after cooling in a desiccator (I1). 2.2.6.5. Filtrate (soluble ber). Volume of the combined ltrate and washings was made up to 100 mL with water. Warm (60  C) 95% ethanol (400 mL) was added and allowed to precipitate for 1 h, ltered through a dried and weighed crucible (porosity 2) containing 0.5 g celite. The residue washed with 78% ethanol (2 10 mL), followed by 96% ethanol (2 10 mL), and acetone (2 10 mL). The precipitate was dried at 105  C overnight cooled in a desiccator and weighed (D2). It was then incinerated at 550  C for 5 h, cooled in a desiccator and then weighed (I2). 2.2.6.6. Blank. Insoluble and soluble blank values were obtained by running the procedure without sample (B1 and B2). 2.2.6.7. Calculation. W Sample weight (g). D weight after drying (g). I Weight after incineration (g). B weight of ash free blank (g).

of water, and washed with 100 mL water and 100 mL 40% methanol successively. Saponin was eluted with 100 mL 95% methanol solution. The solvent in the saponin fraction was removed under reduced pressure. The residue was dissolved with methanol and made up to 20 mL for the hydrolysis step. 2.4.2. Hydrolysis Methanolic solution of saponin (5e20 mL) was placed in a short neck Kjeldhal ask. Methanol was removed under reduced pressure. HCl (2 mL/L, 10 mL) and ethanol (10 mL) were added to the residue and hydrolyzed for 3 h at 90  C, cooled, and extracted twice with diethyl ether (80 mL). The saponin fraction in the ether layer was washed with water (20 mL) and organic layer dried over anhydrous sodium sulfate. The ether was removed under reduced pressure and the residue, which contained sapogenin was dissolved with ethyl acetate and made upto 10 mL for spectrophotometric assay. 2.4.3. Spectrophotometric determination Color developing reagent solutions (A) 0.5 mL p-anisaldehyde and 99.5 mL ethyl acetate and (B) 50 mL concentrated sulfuric acid and 50 mL ethyl acetate were prepared. The ethyl acetate solution containing sapogenin was diluted with ethyl acetate to contain 2.5e10 mg/mL sapogenins. Two mL diluted sapogenin solution was placed in a 10 mL test tube. One mL each of reagent solutions (A) and (B) were added and the test tube sealed with a glass stopper. After stirring, the test tube was placed in a water bath maintained at 60  C for 10 min in a water point maintained at room temperature. The absorbance of the solution was measured with ethyl acetate as blank. As a reagent blank, 2 mL ethyl acetate was placed in a test tube and assayed in a similar way as the sapogenin solution. Solutions containing standard sapogenin (2e40 mg) in ethyl acetate (2 mL) were used to obtain a calibration curve. 2.5. Determination of polyphenols

Insoluble dietary fiber IDF Soluble dietary fiber SDF

D1 I1 B1 100 W

D 2 I 2 B2 100 W

2.3. Preparation of antioxidant conserves Fenugreek seeds, husk and endosperm were grinded using lab grinder and powder obtained was passed through passed through 30 mesh (500 mm) sieve. This powder was used as the raw material for preparation of extracts. These samples, 100 g each, were loaded into separate glass columns and extracted with selected solvents, at a material to solvent ratio of 1:10 solvent mixtures of ethanol and water. Fenugreek extract at different proportions of ethanol and water (40:60, 50:50, 60:40 and 70:30) were employed and the extracts were analyzed for their constituents. The data indicated that the highest level of extracts yield and polyphenols obtained with ethanol plus water mixture in 70:30 ratio, which was used for further studies. The solvent was added in 10 installments of 100 mL each allowing 30 min contact time over a period of 5 h. The extracts were drained and pooled. The pooled extracts were concentrated using rotavapor at 50  C under reduced pressure (40 mbar) and the product stored at 4  C. Recovery of extracts with reference to yield of conserves, and polyphenol content were determined. 2.4. Determination of saponins 2.4.1. Sample preparation Fenugreek samples were analyzed for saponin content according to the method of Yoko, Keiko, and Kazuo (2000). Twenty mL of HP-20 resin was soaked in methanol overnight and packed in a glass column of 15 mm diameter with methanol. The resin was washed with 100 mL methanol and 200 mL water successively. Fenugreek sample was charged to the column with a small amount

Samples were analyzed for total polyphenol content according to the Folin-Ciocalteu method (Singleton & Rossi, 1965). A known concentration of the extract was dissolved in water. A 0.5 mL aliquot of the resulting solution was added to 0.2 mL of Folin-Ciocalteau reagent, and a saturated solution of Na2CO3 (0.5 mL) was added. This was diluted to 10 mL with distilled water and incubated at 27  C for 1 h. Optical density was measured at 765 nm using a spectrophotometer. The concentration was calculated using gallic acid as a standard, and the results were expressed as gallic acid equivalents per gram of extract. 2.6. Antioxidant activity 2.6.1. b-caroteneelinoleate model system The antioxidant activity of fenugreek seed, husk and endosperm extracts was evaluated by the b-carotene method (Adegako et al., 1998). b-Carotene (0.2 mg) in 0.2 mL of chloroform, linoleic acid (20 mg), and Tween-40 (polyoxyethylenesorbitan monopalmitate) (200 mg) were mixed. Chloroform was removed at 40  C under reduced pressure (40 mmHg), and the resulting mixture was diluted with 10 mL of water and mixed well. To this emulsion was added 40 mL of oxygenated water. Aliquots (4 mL) of the emulsion were transferred into different test tubes containing 0.2 mL solutions of fenugreek extracts and BHA of different concentrations (50, 100, and 200 mg). A control containing 0.2 mL of ethanol and 4 mL of the above emulsion was prepared. The tubes were placed in a water bath maintained at 50  C, and the absorbance was measured at 470 nm at zero time (t 0). Measurement of absorbance was continued until the color of the b-carotene

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Table 1 Proximate chemical composition of fenugreek seeds, husk and endosperm. Samples Fenugreek Husk Endosperm Moisture content (g/100 g) 11.44 0.01 11.53 0.01 10.78 0.02 Total Ash (g/100 g) 3.9 0.14 2.17 0.03 4.58 0.14 Protein (g/100 g) 27.57 0.09 7.9 0.10 43.78 0.16 Fat (g/100 g) 6.71 0.01 1.30 0.07 6.59 0.01 Soluble dietary ber (g/100 g) 30.6 0.14 45.2 0.14 20.75 0.07 Insoluble dietary ber (g/100 g) 20.6 0.14 31.9 0.14 13.57 0.14

Mean values of ve readings with three replications. Values are expressed as mean standard deviation.

disappeared in the control tubes (t 180 min) at intervals of 15 min. A mixture prepared as above without b-carotene served as the blank. All determinations were carried out in triplicate. The antioxidant activity (AA) of the extracts was evaluated in terms of bleaching of the b-carotene using the following formula: AA 100 1 A0 At =A0 A0 , where A0 and A0 are the absorbance t 0 0 values measured at zero time of the incubation for test sample and t and A0 are the absorbance values control, respectively, and A t measured in the test sample and control, respectively, after incubation for 180 min. 2.6.2. Free-radical scavenging activity Free-radical scavenging activity was measured by DPPH (1,1diphenyl-2-picrylhydrazil) method (Oktay, Culcin, & Kufrevioglu, 2003). Different concentrations (50, 100, and 200 mg) of fenugreek extracts were taken in different test tubes. The volume was adjusted to 1000 mL by adding MeOH. Methanolic solution of DPPH (0.1 mM, 4 mL) was added to these test tubes and shaken vigorously. The tubes were then incubated in the dark at room temperature for 20 min. A control sample was prepared as above without extract and methanol, which was used for the baseline correction. Changes in the absorbance of the samples were measured at 517 nm. All analyses were run in triplicate and the values averaged. Radical scavenging activity was expressed as the inhibition percentage calculated using the formula:

Radical scavenging activity % Control OD Sample OD=Control OD 100:

2.7. Statistical analysis The data were analyzed by two-way analysis of variance (ANOVA) to study the differences in the antioxidant activity of extracts prepared from husk and endosperm and between the concentrations. Probability level was xed to P < 0.05. The analyses were carried out using Microsoft Excel XP. 3. Results and discussion 3.1. Proximate composition The yields of husk and endosperm separated from fenugreek seeds were 45 and 55 g/100 g respectively. Proximate analysis of

fenugreek seed, husk and endosperm is presented in Table 1. Moisture content of samples ranged from 10.78 to 11.53 g/100 g, which is optimum for storage of spices. Total ash content was slightly higher in endosperm (4.58 g/100 g) followed by whole fenugreek (3.9 g/100 g) and husk (2.17 g/100 g). Protein content was high in endosperm (43.78 g/100 g) compared to husk (7.9 g/100 g). The husk showed lower fat content (1.3 g/100 g) compared to endosperm (6.5 g/100 g). Fenugreek husk was rich in total dietary ber (TDF, 77.1 g/100 g) of which soluble (SDF) and insoluble dietary bers (IDF) were 45.2 and 31.9 g/100 g respectively. The endosperm was relatively low in ber with respective TDF of 34.32, SDF and IDF values being 20.75 and 13.57 respectively. Comparison of these values with that of total fenugreek seeds clearly indicated fenugreek husk is the main source for ber. The yield of the aqueous ethanolic extract of husk, endosperm and whole fenugreek seeds were found to be 14.84, 36.56 and 22.63 g/100 g respectively. The husk showed lowest content of saponins (1.12 g/100 g) followed by endosperm (4.63 g/100 g) and fenugreek seeds (5.12 g/100 g). The total polyphenol content of fenugreek seeds, husk and endosperm was 85.88 0.01, 103.88 0.01, 65.81 0.007, respectively, as gallic acid equivalents (Table 2). Earlier studies by Gupta and Nair (1999) showed that the fenugreek seeds were rich in avonoids (>100 mg/g). It is known that the antioxidant activity of plants is due to phenolic and avonoid compounds present in them (Cook & Samman, 1996; Tung, Wu, Kuo, & Chang, 2007). Therefore, phenolic contents of different fractions clearly showed that fenugreek husk abounded in phenolic constituents and this was also substantiated by higher antioxidant activity of its extracts in different assays. 3.2. b-caroteneelinoleate model system The mechanism of bleaching of b-carotene is a free-radical mediated phenomenon resulting from the hydroperoxides formed from linoleic acid. b-carotene in this model system undergoes rapid discoloration in the absence of an antioxidant. The linoleic acid free radical formed upon the abstraction of a hydrogen atom from one of its diallylic methylene groups attacks the highly unsaturated b-carotene molecules. As b-carotene molecules lose their double bonds by oxidation, the compound loses its chromophore and characteristic orange color, which can be monitored spectrophotometrically. The presence of different extracts can hinder the extent of b-carotene bleaching by neutralizing the linoleate free radical and other free radicals formed in the system. The antioxidant activity of

Table 2 Yield, Saponin and Polyphenols content of fenugreek seeds, husk and endosperm. Samples Fenugreek Husk Endosperm Fractions Yield (g/100 g) e 45 55 Extract Yield (g/100 g) 22.63 0.01 14.84 0.01 36.56 0.01 Saponin (g/100 g) 5.12 0.01 1.12 0.01 4.63 0.01 Total Polyphenols (mg of gallic acid equivalents/g of sample) 85.88 0.01 103.88 0.01 65.81 0.01

Values are expressed as mean standard deviation. Mean values of ve readings with three replications.

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the samples were not signicant as per analysis of variance (F 0.2517 ns). The antioxidant activity appears to be directly correlated to the polyphenol contents of fenugreek fractions; thus, the husk showed higher activity as compared to the fenugreek seeds and endosperm. The inuence of fenugreek seed powder supplementation in the diet on lipid peroxidation and antioxidant status has been studied in alloxan-diabetic rats (Anuradha & Ravikumar, 2001). The activities of antioxidant enzymes catalase, superoxidase dismutase and glutathione peroxidase as well as the oxidative damage were examined in the tissues of diabetic rats treated with fenugreek (Genet, Kale, & Baquer, 2002). Several health benecial attributes of the fenugreek have been experimentally evidenced in recent decades, demonstrating the potential of possible therapeutic applications of fenugreek. 4. Conclusions The distribution of phenolic acids and dietary ber content varies clearly among the fenugreek seeds, husk and endosperm fractions, with the husk containing the highest total level of dietary ber and phenolic acids. On the other hand, the endosperm is enriched in saponins and protein. Hence, separation of fenugreek seeds into husk and endosperm affords advantage of process viability for facile isolation of respective bioactive components. This study also indicated that fenugreek husk, being a valuable source of dietary ber and phenolic acids could be an effective source of natural antioxidants and natural ingredients in functional foods. Our ndings now provide a valuable basis for developing fenugreek fractions as valuable food additives to enhance human nutrition via their phenolic acids, dietary ber, saponins and protein. Acknowledgements We thank Dr. V. Prakash Director, CFTRI, Mysore, India for his keen interest in this work and the facilities provided. The nancial support from CSIR, New Delhi is gratefully acknowledged. References
Adegako, G. O., Vijay Kumar, M., Gopal Krishna, A. G., Vardaraj, M. C., Sambaiah, K., & Lokesh, B. R. (1998). Antioxidants and lipid oxidation in foods. Journal of Food Science and Technology, 35, 283e298. Ahmadiani, A., Javan, M., Semnanian, S., Barat, E., & Kamalinejad, M. (2001). Antiinammatory and antipyretic effects of Trigonella foenum-graecum leaves extract in the rat. Journal of Ethnopharmacology, 75, 283e286. Altuntas, E., Engin Ozgoz, O., & Taser, F. (2005). Some physical properties of fenugreek (Trigonella foenum-graceum) seeds. Journal of Food Engineering, 71, 37e43. Amin, A., Alkaabi, A., Al-Falasi, S., & Daoud, S. A. (2005). Chemopreventive activities of Trigonella foenum-graecum (Fenugreek) against breast cancer. Cell Biology International, 29, 687e694. Anderson, J. W., Baird, P., Davis, R. H., Jr., Ferreri, S., Knudtson, M., Koraym, A., et al. (2009). Health benets of dietary ber. Nutrition Reviews, 67, 188e205. Anuradha, C. V., & Ravikumar, P. (2001). Restoration of tissue antioxidants by fenugreek seeds in alloxan-diabetic rats. Indian Journal of Physiology and Pharmacology, 45, 408e420. AOAC. (1995). Ofcial methods of analysis of AOAC international (15th ed.).. Washington, DC. Asp, N. G., Johnos, C. G., Hollmer, H., & Slijestrom, M. (1983). Rapid enzymatic assay of dietary ber. Journal of Agricultural and Food Chemistry, 31(3), 476e482. Betty, R. I. (2008). The many healing virtues of fenugreek. Spice India, 1, 17e19. Brooks, S. P. J., Mongeau, R., Deeks, R., Lampi, B. J., & Brassard, R. (2006). Dietary ber in baby foods of major brands sold in Canada. Journal of Food Composition and Analysis, 19, 59e66. Chau, C. F., & Huang, Y. L. (2004). Characterization of passion fruit seed bres e a potential bre source. Food Chemistry, 85, 189e194. Cook, N. C., & Samman, S. (1996). Flavonoids d chemistry, metabolism, cardioprotective effects, and dietary sources. Nutrition Biochemistry, 7, 66e76. Dashti, B., Al-Awadi, F., Khalafawi, M. S., Sawaya, W., & Al Amiri, H. (2003). Soluble and insoluble dietary bre in thirty-two Kuwaiti dishes. Food Chemistry, 83, 557e561. Genet, S., Kale, R. K., & Baquer, N. Z. (2002). Alteration in antioxidant enzymes and oxidative damage in experimental diabetic rat tissues: effect of vanadate and fenugreek. Molecular Cell Biochemistry, 236, 7e12.

Fig. 1. Antioxidant activity of fenugreek fraction extracts by b-carotene method.

fenugreek fractions as measured by the bleaching of b-carotene is presented in Fig. 1. At 200 mg, fenugreek fractions viz., husk, fenugreek seeds, and endosperm exhibited 73%, 22%, and 6% activity in comparison to corresponding values of 95% for BHA. The bleaching capacity varied among the different fenugreek fractions. IC50 values of fenugreek fractions were found to be 454 mg (whole fenugreek extract) 136 mg (husk extract), and 1666 mg (endosperm extract). The differences among the samples were found to be extremely significant as per analysis of variance (F 12.86647*** (P < 0.001)). 3.3. Free-radical scavenging activity Fenugreek extracts exhibited good free-radical scavenging activities from 50 to 70% (Fig. 2), the activity increasing with higher concentrations. Of the fractions, fenugreek husk exhibited the highest activity followed by whole fenugreek seed and endosperm extracts. This result indicated that the fenugreek fractions such as husk had a noticeable effect on scavenging free radicals. From the above results, it is clear by both assays that antioxidant activities of different compounds vary to a large extent and the activity of fenugreek fraction extract depends largely on the proportion of various phenolic compounds present. The ability to scavenge DPPH radical by fenugreek fractions was in the order of husk > whole fenugreek seeds > endosperm. IC50 values of fenugreek fractions were found to be 156 mg (whole fenugreek extract) 138 mg (husk extract), and 178 mg (endosperm extract). The differences among

Fig. 2. Radical scavenging activity of fenugreek fraction extracts by DPPH method.

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