1. Introduction
Herb and spices have been used in food as food additives for their natural antioxidant. Plant phenolics have potential health benefits mainly due to their antioxidant properties. A number of plants have been documented for their health promoting benefits (Scalbert. A., 2000). Fenugreek (Trigonella foenum-gracum ) is an annual leguminous bean belongs to Fabaceae family. Its seeds and green leaves used in many medicinal applications (Thomas et al., 2011; Paridar et al., 2011; Vaidya et al., 2013) that include wound healing, aid in digestion, treatment of sinus and lung congestion, inflammation and infection, mitigation, hair treatment, breast enhancement and aphrodisiac effects (Kumar et al, 2013a). In India, it is extensively used as Ayurvedic medicine and in China as traditional medicine (Prasad et al., 2014). Interestingly, in herbal medicine, it is used in the treatment of diabetes (Leela and Shafeekh,2008).Fenugreek leaves provide a good amount of various minerals and vitamins. They are rich in choline.
Medicinally, seeds are the most important and useful parts of fenugreek plant .Seeds are aromatic, bitter, carminative, galactogogue and antibacterial. These seeds are golden-yellow in colour, small in size, hard, having four-faced stone like structure. The seeds of fenugreek contain lysine and L-tryptophan rich proteins, mucilaginous fiber and other rare chemical constituents such as saponins, coumarin, fenugreekine, nicotinic acid, sapogenins, phytic acid, scopoletin and trigonelline. Fenugreek seeds have high protein (25 %), lysine (5.7 g/16 g N), soluble (20 %) and insoluble (28 %) dietary fiber besides being rich in calcium, iron and betacarotene. The biological and pharmacological actions of fenugreek seeds are mostly attributed to the variety of its bioactive chemical constituents that serve as raw materials for the manufacture of various hormonal and therapeutic drugs (Mehrafarin et al., 2010; Priya et al., 2011).Their application are limited due to bitter taste (Sharma 1986). However these days various processing are applied for debittering of fenugreek seeds.
Fig.1 Different form of fenugreek seeds
Green leaves Dry seeds Germinated seeds
In India, seeds are used as boiled, pressure cooked, roasted or germinated. These processing are done to make seeds soft and to remove their bitterness (Mathur and Chaudhary., 2009).
Plant cell cultures are an alternative source for the production of high value secondary metabolites. In vitro propagated callus cultures can be alternative source to plants grown in their environment under controlled condition. Plant tissue can produce significant amounts of metaboliets (Bolda.V.,2011). However, a considerable progress has been made to stimulate production and accumulation of secondary metabolites.
Secondary metabolites flavonoids and phenolic compounds are widely distributed in plant and it exert multiple biological effects including antioxidant, free radical scavenging, antiinflammatory, and anticarcinogenic (Miller.A.L., 1996). T. foenum-graecum contain high phenolic contents with very high antioxidant activity (Kaur.C. et. al., 2002). Among the plethora of bioactive compounds found in fenugreek seeds the major chemical constituents are polyphenolic compounds, galactomannan (fiber), diosgenin (saponin) , quercetin (flavonoid), trigonelline (alkaloid) and 4-hydroxyisoleucine (unusual amino acid). Germinated fenugreek seeds rich in polyphenolic compounds. Being a rich source of mucilaginous fiber and other dietary essential, fenugreek seeds used as a functional and nutritional foods.
Terefore, the objective of this study was to evaluate raw fenugreek seeds, germinated fenugreek seeds and roasted fenugreek seed extracts as a source of natural antioxidant. In this study, the effect of germination and roasting of fenugreek seeds on their chemical compounds was investigated.
2. Review of Literature
2.1 Fenugreek
Fenugreek (Trigonella foenum graecum) is an annual plant belongs to the family Leguminosae. It is the famous spices in human food. The seeds and green leaves of fenugreek are used in food and also in medicinal application. It has been used to increase the flavoring and color, and also modifies the texture of food materials. The leaves contain seven saponins, known as graecunins. These compounds are glycosides of diosgenin. Leaves contain about 86.1% moisture, 4.4% protein, 0.9% fat, 1.5% minerals,
1.1% fiber, and 6% carbohydrates. The mineral and vitamins present in leaves include calcium, zinc iron, phosphorous, riboflavin, carotene, thiamine, niacin and vitamin C (Rao., 2003).
Table 1: Proximate Composition (%) of Fenugreek Seeds (Daniel Zohary,2000)
Component Whole Seeds
Moisture 9.0
Ash 3.0
Lipids 8.0
Protein 26.0
Starch 6.0
Total Fiber 48.0
Gum 20.0
Yadav and Sehgal (1997) observed that fresh leaves of fenugreek contain ascorbic acid of about 220.97 mg/100g and b-carotene was present about 19 mg/100 g. It was reported that ascorbic acid were reduced in dried fenugreek leaves
2.2 Physico-chemical properties
Physico-chemical properties of fenugreek seeds were studied by R.S.Agrawal (2015). Physical properties were evaluated for storage and equipment design and chemical properties were investigated for nutritional analysis and product development. The average length, width, thickness, seed mass and geometric mean of the seed ranged as 3.461 mm, 2.061 mm, 1.067 mm, 0.0177 g and 1.990 mm respectively. Chemical properties such as moisture, fat, protein and ash content (% dry basis) were determined to be 11.21%, 07%, 23.30% and 03% respectively in raw fenugreek seeds and 13.50%, 6.24%, 24.12% and 3.14% respectively in germinated fenugreek seeds. Higher moisture, protein and ash content was observed in germinated fenugreek seeds. Fat content was decreased as compared to raw fenugreek seeds. Chemical properties of germinated fenugreek seeds was investigated by S. Shakuntala et.al, (2011).Germinated fenugreek seeds were divided into endosperm, seed coat and sprouts for nutritive evaluation. Germinated endosperm, sprouts had less protein content than raw endosperm which contained 48.20% protein. Germinated seed coat had higher amount of total dietary fibre than raw seed coat. Sprouts were rich in polyphenols (97.55 mg/100 g). At 200 ppm concentration, the extracts of seed coat, sprouts and endosperm from germinated fenugreek seeds had higher antioxidant activity than seed coat and endosperm of raw fenugreek seeds. The GC analysis of the ester derivatives of the fixed oils of above fractions showed discernable changes in fatty acid profile. Atomic absorption spectroscopy analysis of the above fractions showed overall differences in their mineral content particularly calcium, zinc, copper, iron, potassium, manganese and magnesium.
Pal.K.S et. al., (2013) studied the biochemical contents in fenugreek seeds of different varieties. Results revealed minimum polyphenol content in genotype NDM-278. Genotype HM-267 had the maximum phytic acid while RM-185 resulted in maximum saponin content. The maximum carbohydrate was determined in the genotypes NDM-12 and ACC-012 and the maximum protein content in genotype NDM-33 were estimated. Genotype HM-258-1 determined the maximum moisture content. Ash content was maximum in genotype UM-132, dietary fiber in genotype UM-113, zinc content in RM-189, iron content in HM-271, manganese content in UM-116 and magnesium content in genotype RM-190.
2.3 Effect of processing
Pandey & Awasthi ,(2013) reported that nutritional and therapeutic quality of fenugreek seeds can be improved through processing methods viz. soaking, germination and roasting. Antioxidant activity increased significantly after processing which was found to be responsible for medicinal properties of processed fenugreek seed flour. They concluded that raw fenugreek seed flour contained crude protein (32.7 %), crude fat (4.8 %), ash (3.7 %) and crude fiber (6.0 %). Raw fenugreek seed flour had higher amount of dietary fiber (45.4 %), phytic acid (552.3 mg/100 g) as compared to processed fenugreek seed flours. Significantly lower phenolic content (45.4 mg gallic acid equivalents/g of sample) and antioxidant activity (18.1 %) has been observed in raw fenugreek seed flour as compared to processed. Therefore, the use of processed fenugreek seed flour can be exploited in functional foods as well as a therapeutic agent on a regular basis.
From a study by Ahmed F.A. ,(2015) it was concluded that sprouting process results increases in moisture, protein, ash, crude fiber, protein solubility, free amino acids, total, reducing and nonreducing sugars. But oil content and antinutritional factors decreased. Phenolic content increased from 1341.13 mg/100 g DW in raw lentil seeds to 1510.10 in germinated sample. Sprouted seeds had higher DPPH radical scavenging and reducing power activities. As germination period increased TPC also increased as well as flavonoid content. Based on these resuts germination process recommended to increase nutritive value and antioxidant activity of lentil seeds.
Ramakrishna.V., (2006 ) concluded that raw dry Indian bean having a very high trypsin inhibitory activity which progressively decrease by soaking, boiling, roasting, pressure cooking and germination processes However, decreased amount of polyphenols , tannins ,phytic acids, phytate phosphorus and in cabohydrates were noticed. Maximum reduction in roasting process was observed in TIA and phytic acids whereas the boiling and pressure cooking decreases the levels of polyphenols and tannins. Germination was more effective method in reducing trypsin inhibitor activity, tannins, polyphenols and phytic acid than the other cooking treatments. (Mansour and El-Adway., (1994) reported that germination and soaking of fenugreek seeds caused significant decrease in fat content as compared to raw seeds. Reduction in fat during germination may be due to its consumption as an energy source in germination process of germination. Hooda and Jood., (2003) examined that germination process caused decrease in fat content along with decrease in free fatty acids, monoglycerides and lipids. The protein content increased after germination. The changes in sugar content may be due to mobilisation and hydrolysis of polysaccharides during soaking and germination processes. The increased content of protein on germination might be due to reduction of seed nitrates into ammonium compound
Kumar.E.K.et.al., (2015) investigated that different processing methods like wet roasting, boiling and soaking and boiling effect mineral content e.g calcium, iron, copper, zinc, magnesium. Among all the States of India, Lathyrus sativus cultivated in Andhra Pradesh showed higher amounts of minerals compared to other States of India. In Andhra Pradesh, the soaked and boiled processed seeds had a better amount of mineral values as compared to raw. The seeds have a higher concentration of magnesium followed by calcium. The trace minerals were also found in high amount and the levels of phytic acid was significantly altered. Mubarak. A.E., (2014) observed the effects of traditional processes like dehulling, soaking, germination, boiling, autoclaving and microwave cooking, on the nutritional composition and antinutritional factors of mung bean seeds were studied. Germination and cooking processes results decreases in fat, carbohydrate fractions, antinutritional factors and total ash contents. It can be concluded that these processes decreased the concentrations of lysine, tryptophan, threonine and sulfur-containing amino acids. Dehulling, soaking and germination processes were less effective than cooking processes in reducing trypsin inhibitor, tannins contents. Germination was more effective in reducing phytic acid, stachyose and raffinose. Germination resulted retention of all minerals compared to other processes. In vitro protein digestibility and protein efficiency ratio were improved by all processes
El-Shimi et. al., (1984) reported that change in starch content of endosperm of fenugreek seeds on germination was negligible. In seed coat of germinated fenugreek seeds, the starch content was lower by 29% when compared to seed coat of ungerminated fenugreek seeds. These findings are in agreement with the literature report that decreases in starch content after soaking and germination. Comparatively sprouts contain very small amount of starch (3.12%).
(Mathur and Chaudhary., (2009) studied that reduction in total dietary fiber, insoluble dietary fiber and soluble dietary fiber has determine in roasting process. Reduction in IDF content after roasting might be due to retrogradation of starch molecules.
2.4 Total polyphenolic content
2.4.1 Total phenolic content and antioxidant properties
In a study by Mashkor. A.L., (2014) three types of solvent extracts of fenugreek seeds were used to determine the effects of extraction solvent on total phenolics content (TPC), 1,1-diphenyl-2-picryl hydrazyl radical scavenging (DPPH) and ferric reducing antioxidant power (FRAP) were determined From their study it can be concluded that extraction solvent had significant effects on TPC and antioxidant activity. The highest content of TPC and antioxidant activity (FRAP and DPPH) were found in 50% acetone extracts. Acetone 50% and methanol 50% solvent showed the greatest capability in extracting antioxidants and had greatest inhibiting the free radicals produced. It can be concluded that extraction solvent play important roles on the phenolic compounds and their antioxidant activity of fenugreek seeds extract.
Saxena et.al., (2011) observed high antioxidant activity of ethanol extract in fenugreek seed but phenolic content was less than methanol and distilled water extract. Methanol extract had good antioxidant activity with high phenolic content in fenugreek seeds. Ethanol extract showed high flavonoid content with high antioxidant activity. From the above it can be concluded that solvent used for extraction is very important for effective extraction of plant phytochemicals.
Bukhari S.B. et. al., (2008) prepared extracts of fenugreek in methanol, ethanol, dichloromethane, acetone, hexane and ethyl acetate and these extract were subjected for the measurement of total phenolic content as well as flavonoid content, chelating activity, reducing power and antioxidant/radical scavenging activity. From this it was determined that ethanol extract had highest total phenolic content as well as flavonoid content, chelating activity and antioxidant/radical scavenging activity. It can be concluded that the antioxidant activity could be correlated with the polyphenolic components present in the extract
The phytochemical analysis observed by Seasotiya.L. et.al., (2014) and they reported
the presence of alkaloids, flavonoids, saponins, phenols and tannins in fenugreek seeds. Antioxidant activity, phenolic contents and phytochemical of various extracts (methanol, chloroform, ethyl acetate and hexane) of fenugreek seeds were also investigated. Ethyl acetate extract (100 μg/ml) resulted highest inhibitory potential with higher antioxidant assay. The differences in antioxidant activity of extracts were attributed to the presence of various phytochemicals in the extract. The IC50 values of different extracts were also calculated which had positive correlation between the total phenolic content and the antioxidant activity of extracts .
In a research by Palash M. et. al., (2014) the effect of some nitric oxide e.g sodium nitroprusside (SNP), sodium nitrite (NN) and potassium ferricyanide (FCN) on the antioxidant activity of fenugreek was studied during germination. The treated seeds were germinated in dark for 72 h and the antioxidant activity were determined at interval of 24 h up to 3 days. The in vitro antioxidant activity and polyphenolic compounds was determined. It can be concluded that the seeds treated with nitric oxide donors showed high antioxidant potential from 24 h to 48 h and then declined at 72 h stages. Overall, the seeds treated with sodium nitroprusside and potassium ferricyanide had higher antioxidant potential particularly at concentration 80 mM and 40 mM respectively
2.4.2 Total flavonoids content
Priya et al., (2011) reported more than 5000 different flavonoids which providing flavour
and colour to fruits and vegetable. These flavonoids have been found to have biological
activities e.g inhibitory effects on enzymes, modulation of cell , antiallergic, antibacterial,
antifungal, antiviral, anti-malarial, antioxidant, anti-inflammatory and anticarcinogenic
properties.
Nanjundan et al., (2009) concluded that fenugreek seeds contains five different types of
flavonoids which are as vitexin, tricin, naringenin, quercetin, and tricin-7-O-β-D-
glucopyranoside. Amoung them quercetin and kaempferol are flavonols; luteolin is a
flavone; naringenin is a flavanone whereas vitexin is a glycosylated flavone. Isoflavanoid
phytoalexins are also found in fenugreek seeds.
Arivalagan et al., (2013) revealed that saponins include a diverse group of compounds characterized by their structure containing a steroidal or triterpenoid aglycone. Their structural diversity is related to their physicochemical and biological properties. Fenugreek seeds contain 4.8% saponins in the form of diosgen yamogenin,tigogenin, neotigogenin,yuccagenin, lilagenin, gitogenin, neogitogenin, sarsapogenin and smilagenin. Among them diosgenin is major the steroidal saponin.
2.5 Medicinal properties
In a study conducted by Laila and Murtaza (2014 ) reported that fenugreek have an enormous potential to cure diabetes as compared to other plant species which could be due to the presence of unique chemical constituents including quercetin, diosgenin, trignolline, galactomannan and unusual amino acid 4 hydroxy isolucine. However, due to lack of enough scientific or clinical studies the use of fenugreek as hypoglycaemic official drug remains to be explored. It is proposed that a close attention be paid for preventive and curative properties of this potent herb against diabetes and its complications. Fenugreek seeds possess antioxidant activity and can be applied for preservation of lipid peroxidation in biological systems.
Ahmoud Y.M, (2012) studied the effect of wheat flour supplemented with germinated fenugreek powder at 5 to 10% levels on iron deficiency anemia in rats. It can be concluded that fenugreek flour is a good source of protein, fat, fiber and minerals (Fe, Ca and Zn). Biscuits supplemented with germinated fenugreek (GF) results highest content of polyphenols. Fenugreek flour also increased the vitamin B2 and -carotene contents of biscuits. Wheat flour supplemented by 5 and 10% GF produced acceptable and high nutritive values of biscuits. The biological examination concluded that the fortified diets with GF, fenugreek leaves (FL), germinated fenugreek biscuits(GFB) and fenugreek seeds(FS) produce the greatest improved effect on body weight gain. The hematological and biochemical analysis showed that the changes in blood picture, total iron binding capacity, proteins and minerals (iron and zinc) levels were in favor of fortification with fenugreek products when compared to the positive control. It can be concluded that fenugreek products have good nutritive value and positive response on blood and serum biochemical parameters in anemic rats. Therefore, this study recommended that intake of fenugreek products may be beneficial for patients who suffer from iron deficiency anemia due to their nutritional properties.
3. Materials and methods
Fenugreek seeds were purchased from local market of Hissar, Haryana. Seeds were cleaned to remove any extraneous material. Raw seeds were dried at 40±5 °C in a hot air oven to increase its keeping quality and stored in air tight containers at ambient temp.
Raw fenugreek seeds
Germination
Fenugreek seeds (20 g) were soaked overnight in water at the ratio of 1:5 (w/v). The excess water was drained and seeds were germinated (tied in a muslin cloth) at room temperature for 24 h, 36 h and 48 h. The germinated seeds were dried in an oven at 50 °C until the constant weight. The average length of sprouts was 1.1cm,1.7cm and 2.6cm which was germinated for 24 hr,36 hr and 48 hr respectively.
Fig 3.1 Germinated fenugreek seeds
Germination (24 hr)
Germination (36 hr)
Germination (48 hr)
Roasting
Fenugreek seeds (20 g) were roasted in an open pan at 130±5 °C,150±5 °C and 200±5°C for 7 min,5 min and 2 min respectively. It was continuously stirred with laddle for proper and uniform roasting. The roasted seeds was then cooled at room temperature and filled in air tight pouches.
Fig 3.2 Roasted fenugreek seed powder
Roasted at 130°C Roasted at150°C Roasted at 200°C
Preparation of fenugreek seeds extract
Raw and processed (germination and roasting) fenugreek seeds were ground in Wall Mill. Ground sample were collected in air tight container separately for further analysis at room temperature. The prepared sample were extracted in absolute methanol and 80% methanol in orbital shaker for 4 hrs at 45°C. In extraction process 3 g of prepared sample were weigh in universal bottle and 75 ml solvent was added. After extraction process supernatant were collected for further analysis.
Determination of total phenolic content
The total phenolic content in extracts was measured by UV spectrophotometry based on a Colorimetric oxidation/reduction reaction. The oxidizing agent used was Folin-Ciocalteu reagent (AOCS,1990). To 0.2 ml of extract,7.5 ml water was added in a test tube and after that 0.5 ml of Folin-Ciocalteu reagent (diluted 2 times with water) was added and, then, 1 ml of Na2CO3 (40 %) were added. The sample was incubated for 30 min at room temperature . For a control sample, 0.2 ml of distilled water was used. The absorbance of the resulting blue-colored solutions was measured at 760 nm. Quantitative measurements were performed, based on a standard calibration curve of gallic acid in water. The results were expressed as gallic acid equivalents (GAE) in mg/g of sample.
Total phenolic content (mg/100g) = (C*V*100)/(W*v*10000)
where, C = concentration
V = volume make up
W = weight of sample (mg)
v = volume of aliquot
Determination of total flavonoid content
Total flavonoid content was determined using a method described by (Liu et al., 2008).Briefly, 2 ml of the extracts was taken in a test tube and 0.2 ml of 5% sodium nitrite was mixed.after 5 min 0.2 ml of 10% aluminium chloride was added and after 6 min 1ml of 1M sodium hydroxide was added. The absorbance was measured at 510 nm. The results were expressed as Quarcetin equivalents (QE) in mg/g of sample.
Total flavanoid content (mg/100g) = (C*V*100)/(W*v*10000)
where, C = concentration
V = volume make up
W = weight of sample (mg)
v = volume of aliquot
Total Antioxident Activity
Antioxidant activity was estimated by DPPH free radical scavenging method (Maizura et al., 2011). To 0.2 ml of extract was taken, 2 ml DPPH(3.94mg/100ml) in methanol was added and made volume upto 3 ml. After that sample was incubated for 30 min in dark. The absorbance was measured at 517nm.
% DPPH Radical Scavenging activity =(Ac-A)/Ac * 100
where, Ac = Absorbance of the control
A = Absorbance of the sample
Total Dietary Fiber
The dietary fiber content in processed fenugreek seeds was estimated by rapid enzymatic assay method. Briefly, 1 g processed fenugreek seeds sample was subjected to sequential enzymatic digestion by heat-stable α-amylase, protease and amyloglucosidase. Insoluble dietary fiber (IDF) filtered, and then residue were washed with warm distilled water. Combined solution of filtrate and water washings was precipitated with 4 volumes of 95% ethanol (EtOH) for soluble dietary fiber (SDF) determination. Precipitates was filtered and dried. Both SDF and IDF residues was estimated for protein, ash and blank, for the final calculation of SDF and IDF values. SDF was precipitated with EtOH, and residue was filtered, dried and weighed. Total dietary fiber (TDF) value was calculated for protein and ash content ( AOAC, 1991 ).
Calculation of blank (B):
B (blank) = wt. of residue – PB – PA
where, wt. of residue = average residue weight (mg) for duplicate blank determination.
PB & PA = weight (mg) of protein and ash, respectively determined in first and second blank residue.
Calculation of total dietary fiber (%):
Total dietary fiber (%) = [(wt. of residue) – P – A – B) wt. of sample] * 100
where, wt. of residue = average of weight (mg) for duplicate blank determination.
P & A = weight (mg) of protein and ash, respectively in first and second sample residue.
B = blank
wt. of sample = average of two sample weight (mg) taken.
Estimation of phytic acid
Phytic acid was estimated by the method by Davies and Reid (1979). One g of material was ground and extracted with 0.2 N HCl by continuous shaking. To 0.2 ml of the filtrate, distilled water to make volume 1.4 ml was added. After that1ml of ferric ammonium sulphate solution (21.6 mg in 100 ml water) was added, mixed and placed in a boiling water bath for 20 min. The contents were cooled and 5 ml of isoamyl alcohol was added and mixed. To this, 0.1 ml ammonia solution was added, shaken thoroughly and centrifuged at 3000 rpm for 10 min. The alcoholic layer was separated and the colour intensity was read at 465 nm against amyl alcohol blank after 15 min. Sodium phytate standards were run along with the sample. The results were expressed as mg/100 g of sample.
Phytic acid (mg/100g) = (C*V*100)/(W*v*10000)
where, C = concentration
V = volume make up
W = weight of sample (mg)
v = volume of aliquot
Estimation of tannins
Tannins were estimated by Vanillin-HCl method ( Price et al.,1978). One ml of suitably diluted extract was taken in a test tube and 5 ml of freshly prepared vanillin-HCl reagent was added slowly with mixing and colour developed was read at 525 nm. Catechin standards were run simultaneously along with sample. The results were expressed as mg/100g of sample.
Tannins (mg/100g) = (C*V*100)/(W*v*10000)
where, C = concentration
V = volume make up
W = weight of sample (mg)
v = volume of aliquot
Statistical analysis
Each experiment was conducted trice. The results were expressed as mean±SD. Analysis of variance was applied to analyze data and significance was accepted at p≤0.05 level.
4. Result and Discussion
Total phenol content
Data regarding total phenolic contents presented in Table 1 of Appendix. Total phenolic content of raw and processed fenugreek seeds significantly (P ≤ 0.05) different in absolute methanolic extract and 80% methanolic extract. This difference might be due difference in polarity of extracting solvent (Rostagno.M.A. et.al., 2003). Total phenolic content of raw fenugreek seeds was 67.32 mg/g and 59.72 mg/g in 80% methanolic extract and absoulte methanolic extract respectively. Total phenolic contents was significantly (P ≤ 0.05) increased by germination process. In 80% methanolic extract phenolics content increased from 67.32 mg/g in raw fenugreek seeds to 75.17 mg/g, 80.25 mg/g and 93.27 mg/g in those samples germinated for 24 hr, 36hr and 48 hr respectively. In absolute methanolic extract phenolic content increased from 59.72 mg/g to 89.54 mg/g in germinated fenugreek seeds. The highest level of phenolic content (93.27 mg/g) was observed for fenugreek seeds germinated for 48 hr in 80% methanolic extract. These increases could be due to the biosynthesis of phenolic compounds during germination process (Randhir et al., 2004).
Processed seeds
Figure 4.1: Total phenolic content in seeds extracts of fenugreek.
Absoulte methanol ; 80% methanolic extract
Total phenolic content in roasted fenugreek seeds increased significantly (P ≤ 0.05) from 73.12mg/g to 86.43mg/g in 80% methanolic extract as presented in table 1. In roasting process TPC increased with roasting temp. and maximum level (86.43 mg/g) at 200°C temp (Jeong et al.,2004).
Antioxident Activity
Antioxidant activities of raw and germinated fenugreek seeds, as determined by the DPPH radical scavenging method, shown in Table 2 of Appendix. DPPH radical-scavenging activity expressed in % inhibition of raw and germinated fenugreek seeds ranged from 39.11% to 73.65% in 80% methanolic extract. Antioxident activity is significantly (P ≤ 0.05) different in both extracting solvent of raw and processed fenugreek seeds. In absolute methanolic extract antioxidant activity ranged from 16.47% to 63.72%. Germinated fenugreek seeds had significantly (P ≤ 0.05) higher DPPH radical-scavenging activity compared to raw seeds. Polyphenols have high free radical scavenging activity. This increase might be due to the synthesis of compounds like vitamin C and tocopherols which are responsible for antioxidant activity (Sharma and Gujral, 2010). Antioxident activity increased from 39.11% in raw fenugreek seeds to 49.03%, 54.19% and 61.29% roasted at 130°C,150°C and 200°C respectively in 80% methanolic extract. In absoulte methanolic extract antioxidant activity ranged from 29.11% to 44.31%..
Figure 4.2: Antioxident activity in seeds extracts of fenugreek.
Absoulte methanol ; 80% methanolic extract
Total Flavonoid content
Data pertaining total flavonoids content is presented in Table 2. Total flavonoids content of raw fenugreek seeds was 3.48 mg/ g in 80% methanolic extract.
.
Figure 4.2: Total flavonoids content (TFC) in seeds extract of fenugreek.
Absoulte methanol ; 80% methanolic extract
Total flavonoids content in 80% methanolic extract was increased to 5.29 mg/g and in absoulte methanolic extract it increased to 5.10 mg/g in processed fenugreek seeds. Total flavonoids content of raw and processed fenugreek seeds significantly (P ≤ 0.05) different in two different extracting solvent. Flavonoids content in raw fenugreek seeds was significantly (P ≤ 0.05) lower than germinated seeds. Significantly (P ≤ 0.05) increase in the total flavonoids content of fenugreek seeds was observed as a result of germination process. A gradual increase was observed in flavanoid content with increase in germination time from3.48 mg/g to 5.29 mg/g . The highest level (5.29 mg/g ) was recorded for fenugreek seeds germinated for 48 hr. Roasting process also increased the flavonoids content from 3.48 mg/g to 3.92 mg/g in 80%methanolicextract.In absoulte methanolic extract, flavonoids content also increased from 3.15 mg/g to 3.54 mg/g.
Antinutritional factors:
Tannins content is different significantly (P ≤ 0.05) in both extracting solvent as shown in Table 4 of Appendix. Tannins content was higher in absoulte methanolic extract which is 41.76 mg/100g and reduced to 27.23 mg/100g during germination process. In germination process, tannins are decrease to a large extent from 12.52 mg/100g to 9.40 mg/100g in fenugreek seed in 80% methanolic extract. As the germination period increased tannin content gradually decreases. The reduction of tannins during germination might be due to leaching of tannins into water (Shimelis and Rakshit, 2007) and bonding of polyphenols with carbohydrate or protein (Saharan et al., 2002).
In roasting process, tannins content decreased from 12.52 mg/100g to 8.42 mg/100g in 80% methanolic extract and from 41.76 mg/100g to 20.37 mg/100g in absolute methanolic extract. During roasting breakdown of the bond between phytate and P takes place which results destruction of phytates, tannins and oxalates (Reddy et al.,1978).
Germination of fenugreek seeds reduced the phytic acid content from 190.30-117.74 mg/100 g. It was found that phytase activity originates after germination and the phosphatase activity was increased in the germinated seeds which results in the reduction of phytic acid content in fenugreek seeds after germination and roasting ( El Mahdy and El-Sebaiy .,1982).During germination enzymatic hydrolysis of phytate phosphorus takes place which decreases phytic acid content (Gupta et al., 2001). During roasting phytic acid reduced from 298.77-177.18 mg/100g. Decrease in phytic acid during roasting may be due to thermoability of phytic acid. Germination effects phytic acid more than roasting as shown in Table.5 of Appendix.
Figure 4.2: Tannin content in seeds extract of fenugreek.
Absoulte methanol ; 80% methanolic extract
Total Dietary Fibre:
Data regarding TDF, IDF and SDF presented in Table 5 of Appendix. Significant (P ≤ 0.05) reduction was noted in total dietary fibre (TDF),soluble dietary fibre (SDF) and insoluble dietary fibre (IDF) upon germination as shown in Table 4. Total dietary fibre reduced from 47.35% to 40.20% during germination process. There was no significant (P ≤ 0.05) change in insoluble dietary fibre germinated for 36 hr and 48 hr. Soluble dietary fibre also significantly (P ≤ 0.05) reduced from 17.60% to 11.50% upon germination process. Reduction in dietary fiber content during germination may be due to enzymatic breakdown of the galactomannan units. Shakuntala et al.(2011) concluded that decrease in soluble dietary fiber content on germination of fenugreek seeds. An enzyme α-galactosidase during germination of fenugreek seeds partially attacks galactomannan to produce galactose.
Figure 4.2: Dietary fibre in seeds extract of fenugreek.
Absoulte methanol ; 80% methanolic extract
Figure 4.2: Phytic Acid in seeds extract of fenugreek.
Absoulte methanol ; 80% methanolic extract
Decrement in total dietary fiber, insoluble dietary fiber and soluble dietary fiber also noticed during roasting process of fenugreek seed as shown in Table 4.Total dietary fibre (TDF) reduced from 45.45% to 32.60% during roasting process. Significant (P ≤ 0.05) reduction in soluble dietary fibre (SDF) and insoluble dietary fibre (IDF) also determined as listed in table 5 of Appendix. Reduction in IDF content during roasting might be due to retrogradation of starch (Mathur and Chaudhary., 2009).
Appendix
Table 1. Effect of processing on Total Phenolic Content in processed fenugreek seeds.
Total Phenolic Content (mg/g)
Solvent
Raw Germination
24 hr 36 hr 48 hr Roasting
130°C 150°C 200°C
Methanol
(80%)
Methanol 67.32±0.22b
59.72±0.01a 75.17±0.06b 80.25±0.13b 93.27±0.33b
66.64±0.14a 72.60±0.10a 89.54±0.05a 73.12±0.06b 81.30±0.44b 86.43±0.16b
61.34±0.36a 64.12±0.09a 76.64±0.22a
Means in the same column with different superscripts differ significantly at (P < 0.05).
Table 2. Effect of processing on Antioxident Activity in processed fenugreek seeds.
Antioxident Activity(%)
Solvent
Raw Germination
24 hr 36 hr 48 hr Roasting
130°C 150°C 200°C
Methanol
(80%)
Methanol 39.11±0.08a
16.47±0.19b 50.74±0.14a 53.87±0.12a 73.65±0.29a
35.37±0.13b 53.41±0.21b 63.72±0.31b 49.03 ±0.14a 54.19 ±0.09a 61.29 ±0.04a
29.11 ±0.11b 41.06±0.07b 44.31±0.23b
Means in the same column with different superscripts differ significantly at (P < 0.05).
Table 3. Effect of processing on total flavonoids content of processed fenugreek seeds.
Total Flavonoids Content (mg/g)
Solvent
Raw Germination
24 hr 36 hr 48 hr Roasting
130°C 150°C 200°C
Methanol
(80%)
Methanol 3.48±0.08b
3.15±0.09a 4.98±0.04b 5.17 ±0.06b 5.29±0.10b
4.20 ±0.14a 4.40 ±0.09a 5.10±0.04a 3.61±0.08b 3.72 ±0.23b 3.92 ±0.01b
3.19±0.17a 3.38 ±0.02a 3.54±0.19a
Means in the same column with different superscripts differ significantly at (P < 0.05).
Table 4. Effect of processing on tannins content in processed fenugreek seeds.
Tannins(mg/100g)
Solvent
Raw Germination
24 hr 36 hr 48 hr Roasting
130°C 150°C 200°C
Methanol
(80%)
Methanol 12.52±0.21a
41.76±0.30b 11.22±0.22a 10.31±0.38a 9.40±0.57a
33.37±0.28b 31.75±0.34b 27.23±0.15b 9.22±0.23a 8.45±0.49a 8.42 ±0.15a
38.61±0.07b 32.17 ±0.14b 20.37±0.24b
Means in the same column with different superscripts differ significantly at (P < 0.05).
Table 5. Effect of processing on Dietary Fibre and phytic acid in processed fenugreek seeds.
Sample TDF (%) IDF (%) SDF (%) PA (mg/100g)
Raw 50.20±0.14ͣa 31.70±0.14a 18.40±0.14b 317.18±0.14a
Germination
24 hr 47.35±0.21b 30.30±0.14b 17.60±0.14c 190.30±0.25c
36 hr 43.40±0.28d 28.20±0.14c 15.65±0.21d 157.44±0.14f
48 hr 40.20±0.14f 28.45±0.35c 11.50±0.28e 117.74±0.24g
Roasting
130°C 45.45±0.2c 24.45±0.35c 20.75±0.21a 298.77±0.31b
150°C 41.40±0.28e 23.45±0.35e 17.45±0.35c 182.25±0.07d
200°C 32.60±0.28g 20.65±0.21f 11.55±0.35e 177.18±0.21e
Means in the same column with different superscripts differ significantly at (P < 0.05).
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Seeds