1. Singh K. G; Colaco M; Misquith S
“AN IN-VITRO STUDY OF THE ANTIOXIDANT PROPERTIES OF GERMINATED FENUGREEK SEEDS”
Singh K. G; Colaco M; Misquith S
Department of Chemistry, St. Joseph’s College, Bangalore
ABSTRACT
Trigonella foenum-graecum (Fenugreek) is one of the oldest medicinal plant known and has been used as a medicinal herb and culinary additive in both Asia and the Mediterranean. It is believed that fenugreek seeds contain many active compounds with pharmaceutical applications such as saponins, flavanoids, phenols, lecithin, tannins, iron, vitamin A, vitamin B, vitamin C, phosphates, trigonelline, other alkaloids and also some antioxidants. They are present in varying amounts in the sprouted and dried seeds of fenugreek. The medicinal properties were studied by isolating and estimating the various components present in sprouted fenugreek and dry fenugreek seeds which include minerals like calcium, sodium, potassium, protein, carbohydrates, ascorbic acid, phenols, vitamin A, riboflavin, thiamin and carotenoids. There was an increase in the phytochemicals in germinated seeds compared to dry seeds. A detailed study of the antioxidant properties was conducted by sprouting the seeds for 5 days followed by isolating and estimating the amount of antioxidants like proline, flavonoids, catalase and superoxide dismutase. Flavonoids exhibit biological activities, including antiallergenic, antiviral, anti-inflammatory, and vasodilating actions. However, attention has mainly been focused on the antioxidant activity of flavonoids, which is due to their ability to reduce free radical formation and to scavenge free radicals. A comparative study of flavonoids on sprouted seeds was done using GC-MS. The number of types of flavonoids was observed to have increased in sprouted seeds in comparison to dry seeds. Sprouted seeds can also be used for the isolation and purification of the medicinally important naturally occurring phytochemicals and phytonutrients which can be further used in drug formulation instead of synthetic chemicals which may have side effect.
INTRODUCTION
SAMPLE USED
Sprouted Seeds of Trigonella foenum-graecum
Trigonella foenum-graecum is one of the oldest medicinal plants dating back to ancient Egyptians, Greeks and Romans who used it as a culinary and medicinal herb (1). It also has a long history of medicinal uses in Ayurvedic, Chinese, Siddha, Unani and Tibetian medicine.
Seeds are aromatic carminative,emmenagogue,diuretic,galactogogue,demulcent,bechic,astringent,aphrodisiac, emollient, tonic; useful in colic, flatulence, dysentery, diarrhoea, dyspepsia with loss of appetite, chronic cough, dropsy, enlargement of liver and spleen, bronchitis, colinitis, fever, vomiting and as soother of mucous membrane, relieves irritation of skin and alleviates pain and swelling; promotes lactation in nursing mothers; used as poultice for boils, abscesses and ulcer; given internally as an emollient in intestinal inflammation, an infusion given to small pox patients as a cooling drink; powdered seeds used as application in veterinary medicine; aqueous extract of seeds possess anti-bacterial property; used in Israel as an oral insulin substitute (2,3,4). The oxidation of low-density lipoproteins (LDL) can result in the formation of atherosclerotic plaques, leading to cardiovascular disease and other stress related disorders (5, 6). Fenugreek seeds are found to help in reduction of lipid level in the body, which in turn is related to atherosclerosis. Our intention is to study the antioxidant property of fenugreek concerned to flavonoids, proline, catalase and superoxide dismutase.
Day Day Day Day Day 5
METHODOLOGY AND RESULTS
AIMS AND OBJECTIVES
CATALASE
Catalase in an antioxidant enzyme, that is capable of cleaving Hydrogen Peroxide present in the system. Its activity is of high importance in assessing the variations caused by stress. During stressed conditions, its activity is altered depending upon the variation seen in free radicals (15). The enzyme activity is measured by estimating the residual H2O2 in the reaction mixture. Determination of its activity is done by using a U V Spectrophotometer (16).
In the present scenario stress is the main reason for various ailments like aging, atherosclerosis, cancer, neuro degenerative diseases like Alzheimer’s disease and Parkinson’s disease because stress releases lots of free radicals, these free radicals are detoxified by antioxidants, and there are many natural antioxidants present in various medicinal plants and spices.
There is also a growing interest in understanding the biological effect of time-tested folk medicinal plants.
Sprouted fenugreek seeds were selected because of their medicinal value. We would like to see how effective they are in the removal of lipid peroxides and compare it with dried fenugreek which is already widely used.
To study the nature of the antioxidants present in sprouted fenugreek.
To study the contribution of antioxidants in vitro.
PROLINE
Proline is a α-imino-acid; it is unique among the twenty protein forming amino acids because the α-amino group is secondary. Proline may also function as a protein compatible hydrotope and as a hydroxyl radical scavenger. Exogenously supplied protein also can be osmoprotective (17) and has an important role as an osmolyte, recent studies have shown that it has a protective role against free radical-induced damage (18). Proline accumulation increased drastically after lipid peroxidation level reached its highest value while remaining unchanged until the end of the stress treatment.. Proline was estimated using sulphosalicylic acid (18, 19).
RESULTS
ESTIMATION OF PROTEIN USING LOWRY’S METHOD
The Folin-Lowry method (7) is one of the commonly used methods to determine the concentration of proteins in the given sample. The assay is about 10 times more sensitive than the Biuret method. The developed blue color absorbs maximally at 660 nm. Bovine serum albumin is used to prepare the standard stock protein solution.
Protein (gm)
SAMPLE
MEAN ± SD
Dry seed
25.1  0.5
Sprouted sample
32.3  0.5
Ascorbic acid (mg)
ESTIMATION OF ASCORBIC ACID
Ascorbic acid reduces the 2, 6 dichlorophenol dye to a colourless leuco-base. The ascorbic acid gets oxidized to dehydro ascorbic acid. Though the dye is a blue coloured compound, the end point is the appearance of pink colour. The dye is pink coloured in acid medium. Oxalic acid is used as the titrating medium (8)
SAMPLE
MEAN  SD
Dry Seed
2.65  0.05
Sprouted Sample
4.18 
FLAVONOIDS
Flavonoids are polyphenolic compounds that are ubiquitous in nature and are categorized according to their chemical structure into flavonols, flavones, flavonones, isoflavones, catechins, anthocyanidins and chalcones. They have been reported to have antiviral, anti-allergic, anti-platelet, anti- inflammatory, anti-tumor and antioxidant activity. Flavonoids may help provide protection against these diseases by contributing along with antioxidant vitamins and enzymes to the total antioxidant defense system of the human body (20, 21).
Vitamin A (IU)
ESTIMATION OF VITAMIN A
Vitamin A reacts with trichloroacetic acid to form a blue coloured complex which is measured at 620 nm. (9) .
SAMPLE
MEAN  SD
Dry Seed
61.3  0.6
Sprouted Sample
178.1  2.0
SUPEROXIDE DISMUTASE
Superoxide dismutase (SOD) is a class of closely related enzymes that catalyze the breakdown of the superoxide anion into oxygen and hydrogen peroxide. The reduction of NBT by superoxide radicals results in blue colored formazan, which was followed at 560nm.
One unit of SOD activity is designed as that amount of enzyme required to inhibit the reduction of Nitro blueTetrazolium by 50% under specific conditions.
Proline (mg)
ESTIMATION OF PROLINE
Proline present in the tissue is estimated by selectively extracting proline in sulphosalicylic acid. Its concentration is quantitatively measured by using Ninhydrin reagent. The higher the concentration of proline, more is the color intensity of the solution. This is measured using a spectrophotometer (10).
SAMPLE
MEAN  SD
Dry Seed
2.45  0.2
Sprouted Sample
7.34  0.25
Thiamine (mg)
ESTIMATION OF THIAMINE
Alkaline potassium ferricyanide oxidizes thiamine to thio chrome which is a fluorescent compound (9). The thiochrome is extracted in isobutyl alcohol and measured in fluorimeter..
SAMPLE
MEAN  SD
Dry Seed
0.332  0.010
Sprouted Sample
0.750  0.002
DISCUSSION
Sprouting is the practice of germinating seeds to be eaten either raw or cooked. They are a convenient way to have fresh vegetables for salads, or otherwise, in any season and can be germinated at home or produced industrially. Sprouts are believed to be highly nutritious and rich in enzymes which promote good health. There is an amazing increase in nutrients in sprouted foods when compared to their dried embryo. In the process of sprouting, the vitamins, minerals and protein increase substantially with corresponding decrease in calories and carbohydrate content. These comparisons are based on equivalent water content in the foods measured. Analysis of dried seeds, grains and legumes shows a very low water content. Very complex qualitative changes are reported to occur during soaking and sprouting of seeds. The conversion of storage proteins of cereal grains into albumins and globulins during sprouting may improve the quality of cereal proteins. An increase in proteolytic activity during sprouting is desirable for nutritional improvement of cereals because it leads to hydrolysis of prolamins and the liberates amino acids such as glutamic acid and proline. Most reports agree that sprouting treatment of cereal grains generally improves their vitamin value, especially the B-group vitamins. Certain vitamins such as α-tocopherol (Vitamin-E) and β-carotene (Vitamin-A precursor) are produced during the growth process
“Sprouts provide a good supply of Vitamins A, E & C plus B complex. Like enzymes, vitamins serve as bioactive catalysts to assist in the digestion and metabolism of feeds and the release of energy. They are also essential for the healing and repair of cells. However, vitamins are very perishable, and in general, the fresher the feeds eaten, the higher the vitamin content. The vitamin content of some seeds can increase by up to 20 times their original value within several days of sprouting.”A tremendous increase in the phytonutrients is found in the third day sample and again there is a gradual decrease in the nutritional content from third day.
Β-Carotene (μg)
ESTIMATION OF CAROTENOIDS
Carotenoids are extracted in methanol and are separated from other interfering compounds. They are coloured compounds and hence show a yellow to orange colouration depending on their concentration. The higher the concentration of carotenoids present, higher is the colour intensity. This is recorded using a spectrophotometer and the concentration is determined (11).
SAMPLE
MEAN  SD
Dry Seed
97.2  0.52
Sprouted Sample
178 
Riboflavin (mg)
SAMPLE
MEAN  SD
Dry Seed
0.30  0.015
Sprouted Sample
1.09  0.068
ESTIMATION OF RIBOFLAVIN
Riboflavin fluoresces at wave length 440 – 500 nm. The intensity of fluorescence is proportional to the concentration of riboflavin in dilute solutions (132). The riboflavin is measured in terms of the difference in florescence before and after chemical reduction.
Total phenols (gm)
SAMPLE
MEAN  SD
Dry Seed
246  2
Sprouted Sample
343.3  1.52
ESTIMATION OF TOTAL PHENOLS
It is estimated using folin-ciocalteau reagent. The blue colour developed was measured at 765nm. Gallic acid was used as standard (13).
Phospholipids (μg)
ESTIMATION OF PHOSPHOLIPIDS
Phospholipids are extracted with ethanolic ether and are digested with per chloric acid to liberate inorganic phosphorus which is estimated by the Fiske- Subbarow method (14).
SAMPLE
MEAN  SD
Dry Seed
 7.83
Sprouted Sample
 25.16