ANTIOXIDANT AND ANTIMICROBIAL ACTIVITY OF

FENUGREEK SEEDS
A Project Report

Submitted to the
Centre of Food Technology
University of Allahabad, Allahabad
In partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE
(FOOD TECHNOLOGY)
By
Niharika Dubey
(12 AU/787)
Under the supervision of
Dr. Pinki saini
CENTRE OF FOOD TECHNOLOGY
UNIVERSITY OF ALLAHABAD





Centre of Food Technology
University of Allahabad
Allahabad 211002

Certificate of Evaluation Committee
This project entitled Antioxidant and antimicrobial activity of fenugreek seeds has been
prepared and submitted by Niharika Dubey in partial fulfillment of the requirement for the
award of degree of Master of Science, Centre of Food Technology, University of Allahabad,
Allahabad and is hereby accepted for the award of degree.
Members of Evaluation Committee Signature
1. Prof. G. K. Rai
Coordinator, Centre of Food Technology
2. Dr. Neelam Yadav
Reader, Centre of Food Technology
3. Mrs.Vinita Puranik
Course Coordinator, Centre of Food Technology
4. Dr. Pinki Saini
Lecture, Centre of Food Technology
5. Dr. Devinder Kaur
Lecture, Centre of Food Technology

ACKNOWLEDGEMENT
Sometimes words are not enough to express our worship and revere to those who have made
us capable and brought us to this commendable position in our life. This acknowledgement is
just an effort by me to convey my heartfelt gratitude to those who believed in me and made
me the person that I am today.
First and foremost, I thank the Almighty, by whose grace I accomplished my task
successfully.
It is unforgettable experience in my life to be fortunate enough to work under the inspiring
guidance of Dr. pinki saini ,cft, allahabad for his valuable guidance, continuous
encouragement, and co-operation, critical suggestions, believing in me and making me
capable in my dissertation work. Without his guidance and assistance it would not have been
possible to complete this work.
I acknowledge my sincere and indebted thanks to Dr. G.K.Rai Director, CFT, Allahabad,
for rendering me an opportunity to carry out my project work in this esteemed institute,
CFT,Allahabad
I would like to express my warm gratitude to Dr. Neelam Yadav, Head of the department,
Centre of Food technology, University of Allahabad for providing me a platform entering in
the field of Food Technology and excelling in this field.
I am greatly indebted to my respected teacher Mrs. Vinita Puranik, Dr. Devinder Kaur,
Dr. Pinki Saini and Mr. Shubham Kaushik Centre of Food Technology, University of
Allahabad for giving me an opportunity to do work in one of the most prestigious laboratory
in India and for their constant support and encouragement.

I am also deeply grateful to Miss Ankita, Lovy Ayushi, Asma,etc project students for
making this work successful, and making gloomy days happy for me, ideas and most
importantly being my friends throughout the study and dissertation work.
Finally, I am highly indebted to my ever-loving parents, for their constant motivation,
inspiration, encouragement, love, patience, sacrifice and it was impossible for me to achieve
this position without them.

CONTENTS
Sl. No. Chapter Page No.
1. INTRODUCTION
2. REVIEW OF LITERATURE
3. MATERIALS AND METHODS
4. RESULTS AND DISCUSSION
5. SUMMARY AND CONCLUSION
6. REFERENCES












INTRODUCTION
Fenugreek is a legume, originally from south eastern Europe and western Asia, but
grown now mainly in India and also in certain parts of Asia, northern Africa, Europe and the
United States (Altuntas et al., 2005). It is known as methi in India and also as Fenugrec
(France), Bockshorklee (Germany), Koroha (Japan), Hulba (Arab), Pazhitnik (Russia) and
Ku-Tou (China). The seeds find extensive use in Indian cuisine to flavour many foods
including curry powders, spice blends and tea. Its leaves are also used as a green leafy
vegetable in the diet. Fenugreek seeds are aromatic but bitter with carminative, galactogouge,
antibacterial and antiviral properties. The seeds contain a central hard yellow embryo
surrounded by a corneous and comparatively large layer of white, semi-transparent
endosperm. In Ayurveda, both fenugreek seeds and leaves are used to prepare extract or
powder for medicinal use (Daniel, 2006).
The scientific name of fenugreek seeds is Trigonella foenum graecum. Fenugreek
seeds are traditionally used for antioxidant properties. Many medicinal properties are
attributed to fenugreek seeds and leaves. It is known to have several pharmacological
attributes such as hypoglycaemic, hypercholesterolaemia, gastro protective, chemo-
preventive, antioxidant, laxative and appetite stimulation (Sathyanarayana, 2011).
Rheological properties and emulsions of galactomannans of fenugreek seed endosperm are
also reported. The plant is known to contain alkaloids, flavonoids, salicylate, and nicotinic
acid and used in treatment of many diseases. Studies show that the soluble fibre derived from
fenugreek seeds has been identified as galactomannan similar to other soluble fibre of guar
seeds, psyllium husk and other species.
The fenugreek galactomannan has the maximum content of mannans and galactose.
The galactose to mannan ratio being 1:1; as they are uniformly linked and hence they provide
maximum hydration and solubility, the reasons for the low viscosity of fenugreek gum
solution and desegregation of molecular aggregates in solutions at any given temperature.
This property makes it an excellent ingredient for various food applications compared to
other natural hydrocolloids (Ref).
The overproduction of reactive oxygen species like hydroxyl radicals, superoxide
anion radical, hydrogen peroxide radical can contribute to oxidative stress. Oxidative damage
of proteins, DNA and lipids is associated with chronic degenerative diseases including
diabetes, hypertension, coronary heart diseases, cancer, etc. Most of the reactive oxygen
species are scavenged by endogenous defences systems but these systems may not be
completely efficient requiring them to depend on exogenous antioxidant from natural sources
(Braca et al., 2002). Antioxidant compounds in food play an important role as a health
protecting factor. Scientific evidence suggests that antioxidants reduce the risk for chronic
diseases including cancer and heart disease. Primary sources of naturally occurring
antioxidants are whole grains, fruits and vegetables. Plant sourced food antioxidants like
vitamin C, vitamin E, carotenes, phenolic acids, phytate and phytoestrogens have been
recognized as having the potential to reduce disease risk. Most of the antioxidant compounds
in a typical diet are derived from plant sources and belong to various classes of compounds
with a wide variety of physical and chemical properties. Some compounds, such as gallates,
have strong antioxidant activity, while others, such as the mono-phenols are weak
antioxidants. The natural food antioxidants are readily accepted by consumers and do not
require safety tests if they are components of food and are generally recognized as safe.
New antimicrobial agents are needed to treat diseases in humans and animals caused
by drug resistant microorganisms. Interest in plant-derived drugs has been increasing, mainly
due to the current widespread belief that green medicine is safer and more dependable than
costly synthetic drugs, many of which have adverse side effects. The general belief that the
advent of antibiotics will bring end to the occurrence of infectious diseases was cut short with
the occurrence of resistance to antimicrobial drug. The incidence and increasing frequency of
microorganisms that are resistant to common and generally accepted effective first choice
drugs is on the increase. A significant opportunity exists to identify new, natural plant derived
antimicrobial agents for treatment of diseases or as food or cosmetic preservatives (Ref).
The aim of the present study is to investigate the effects of processing methods (roasting,
soaking, germinating and pressure cooking) on the phenolic contents and antioxidant
activities of fenugreek seeds. The study has been planned with the following objectives:
1. To analyse the proximate composition and mineral content of fresh seed samples.
2. To analyze the antioxidant components such as total phenolics, tannin, anthocyanins,
flavonoids and beta carotene content of the fenugreek seeds.
3. To analyse the antioxidant capacity of seeds on the basis of total ascorbic acid content,
DPPH radical scavenging activity and Ferric reducing antioxidant power (FRAP) assay.
4. To study the effect of processing on proximate composition and mineral content of the
fenugreek seeds.
5. To study the effect of processing on the antioxidant components and activity of fenugreek
seeds.
6. To study the anti microbial activity of fenugreek seeds in different solvents.
REVIEW OF LITERATURE

Fenugreek is one of the well known spices in human food. Its seeds and green leaves are
used in food as well as in medicinal application which is an old practice of human history.
It provides natural food fibre and other nutrients required in human body [1]. Fenugreek
has strong spicy and seasoning type sweet flavor [2]. Aromatic and flavourful fenugreek
is a popular spice and is widely used for well recognized culinary and medicinal
properties [3]. Kasuri Methi is very famous for its appetizing fragrance and it is used
for culinary preparations [4]. In recent trend, fenugreek is also used as spice adjunct [5].
India is a major producer of fenugreek and also a major consumer of it for its culinary
uses and medicinal application. It is used in functional food, traditional food,
nutraceuticals as well as in physiological utilization such as antibacterial, anticancer,
antiulcer, anthelmintic, hypocholesterolemic, hypoglycemic, antioxidant, and antidiabetic
agent. It has beneficial influence on digestion and also has the ability to modify food
texture.
Fenugreek is cultivated all over the world as a semi-arid crop. It has got different names
as per the locality. Table 1 shows various names of fenugreek in different languages with
their country of origin.

The fenugreek (Trigonella foenum-gracum) seeds sown in well prepared soil sprouts in
three days. Seedling grows erect, semi-erect or branched based on its variety and attains a
height of 30 to 60 cm. It has compound pinnate, trifoliate leaves, axillary white to yellow
flowers, and 3-15 cm long thin pointed hoop-like beaked pods. Every pod contains 10-20
oblong greenish-brown seeds with unique hooplike groves [6]. Pods, number of seeds in a
pod, seed shape-size and plant height varies from one fenugreek variety to another.
Fenugreek is a self pollinating annual leguminous bean which belongs to Fabaceae family
[7]. It is one of the most ancient medicinal herbs [8]. The leaves, flower,

Green fenugreek leaves are one of the most ancient medicinal herbs [1]. Yadav and
Sehgal (1997) [11] found that fresh fenugreek leaves contain ascorbic acid of 220.97 mg
per 100 g of leaves and -carotene of 19 mg per 100 g of leaves. On the other hand, they
reported that 84.94 and 83.79 % ascorbic acid was reduced in sun and oven-dried
fenugreek leaves respectively. The green fenugreek leaves (fresh or dried) are used as
herb. The fresh leaves are used in the vegetables as green leafy vegetable in the diets.
They suggested that for better retention of nutrients in fenugreek leaves, it should be
stored in refrigeration, dried in oven, blanched for a short period of time (5 min) and
should be cooked in pressure cooker. Their studies in 1999 (Yadav and Sehgal) further
showed that there is no change in the calcium and zinc content of the processed fenugreek
leaves whereas. Food Process Technol
ISSN:2157-7110 JFPT, an open access journal
Volume 3 Issue 9 1000181
during blanching and cooking HCL extractability for these minerals has increased.
Medicinally, fenugreek leaves has been found to have little effect on glycemia [12].
These leaves provide -carotene, fibre, calcium and zinc [13]. Jani et al. [14] tested about
the mineral content of various food items like pulses (dal), bread (chapatti) and fenugreek
leaf vegetables by feeding them to children of 13-24 months of age group and found that
fenugreek leaves had high calcium, iron and zinc content compared to those available in
other food items chosen for this study.
The seeds of fenugreek -
Fenugreek seeds are the most important and useful part of fenugreek plant. These seeds
are golden-yellow in colour, small in size, hard and have four-faced stone like structure
[15]. Fenugreek seed is 3-6 mm long, 2-5 mm wide and 2 mm thick in geometry. Raw
fenugreek seeds have maple flavour and bitter taste but by the process of roasting, their
bitterness can be reduced and flavour can be enhanced. Fenugreek seeds are used as
spices. The whole seed or its ground powder is used in pickles, vegetable dishes and spice
powder. Dried seeds are used as condiments. Fenugreek seeds are gummy, fibrous, sticky
and gummy in nature. Biologically, its seeds are endospermic in nature [14]. The
fenugreek seeds, as a thumb rule, are harvested 150 to 170 days after sowing or 30 to 35
days after flowering [16]. In fenugreek, saponin and alkaloids are anti-nutritional factors
[14]. Defatted fenugreek seeds are not bitter in taste and can be easily consumed by those
who have problems to consume fenugreek without removing fat, especially by patients.
The bitterness of fenugreek can be masked by making
.
Volatile content -
Fenugreek seed contains volatile oil and fixed oil in small quantities [18]. Blank et al.
(1997) [19] has reported the following odour active compounds based on the fenugreek
aroma detection with the help of Gas Chromatograph : Olfactometry diacetyl; 1-Octene-
3-one; (Z)- 1,5-Octadiene-3-one; 3-isopropyl-2-methoxypyrazine; acetic acid; 3-Isobutyl-
2-methoxypyrazine; linalool; butanoic acid; isovaleric acid; caproic acid; eugenol; 3-
Amino-4,5-dimethyl-3; 4-dihydro-2(5H)- Furanone; sotolon with characteristic aroma of
buttery, mushroom like, metallic, roasty / earthy, pungent, paprika like, flowery, sweaty/
rancid, musty, spicy, seasoning like, respectively but out of all these compounds, sotolon
was reported to be found predominantly in (5s)- enantiomeric form (95%) in fenugreek.
Mabazaa et al. (2011) studied sweat of humanMabazaa et al. (2011) studied sweat of
human after fenugreek ingestion and concluded that compounds responsible for the strong
maple-syrup odour present in sweat after fenugreek ingestion are due to the following
compounds: 2,5-dimethylpyrazine; -pinene; 3-octen- 2-one; camphor; terpinen-4-ol; 4-
isopropyl-benzaldehyde; neryl acetate and -caryophyllene but confirmed 2,5-
dimethylpyrazine to be a major sweat odour contributing compound.

CLASSIFICATION OF FENUGREEK SEEDS

Kingdom- Plantae
Division- Magnoliophyta(flowering plant)
Class-Magnoliopsida
Order-Fabales
Family-Fabaceae
Genus-Trigonella
Species- Trigonella foenum graecum














NUTRIENT CONTENT IN FENUGREEK SEEDS

Fenugreek seeds are the most important and useful part of fenugreek plant. These seeds are
golden-yellow in colour, small in size, hard and have four-faced stone like structure [15].
Fenugreek seed is 3-6 mm long, 2-5 mm wide and 2 mm thick in geometry. Raw fenugreek
seeds have maple flavour and bitter taste but by the process of roasting, their bitterness can be
reduced and flavour can be enhanced. Fenugreek seeds are used as spices. The whole seed or
its ground powder is used in pickles, vegetable dishes and spice powder. Dried seeds are used
as condiments. Fenugreek seeds are gummy, fibrous, sticky and gummy in nature.
Biologically, its seeds are endospermic in nature [14]. The fenugreek seeds, as a thumb rule,
are harvested 150 to 170 days after. sowing or 30 to 35 days after flowering [16]. In
fenugreek, saponin and alkaloids are anti-nutritional factors [14]. Defatted fenugreek seeds
are not bitter in taste and can be easily consumed by those who have problems to consume
fenugreek without removing fat, especially by patients. The bitterness of fenugreek can be
masked by making formulations with other food ingredients [17].
Fenugreek seeds are rich source of soluble dietary fibre content [26]. Raju et al. (2001)
[27] reported that the fibre content of fenugreek extract plays a role in its ability to moderate
metabolism of glucose inthe digestive tract. Water absorption on the outer surface makes
seeds coat soft and mucilaginous. The 100 g of seeds provide more than 65% of dietary fibre.
Non-starch polysaccharides constitute fibre content of the fenugreek. Fenugreek contains
saponins, hemicelluloses, mucilage, tannins and pectin and these compounds help to decrease
the level of low density lipoprotein-cholesterol (LDL) in blood by inhibiting bile salts re-
absorption in the colon. Following are the advantages of fibre present in fenugreek seed:
a. It binds to toxins in the food and helps to protect the colon mucus membrane from
cancer causing toxins.
b. It has been established that the amino-acid, 4-hydroxyisoleucine present in the fenugreek
seed has facilitator action on insulin secretion.
c. Fibre helps to lower rate of glucose absorption in the intestines controlling blood sugar
levels.
d. The higher content of soluble fibre in fenugreek enhances its strength for glucose level
tolerance.
Fenugreek seeds are rich in carbohydrates and especially mucilaginous fibre which is
comprised of galactomannans. The ability of fenugreek to improve glucose tolerance is
further enhanced by its rich content of soluble fibre (Table 4).
Galactomannan is a major soluble fibre of the fenugreek seeds; it decreases the bile salts
uptake in intestine and also reduces the digestion and absorption of starch in body [28].
Mathern et al. [29] has made studies on trend of food consumption in obese human beings
and found that fenugreek fibre significantly increased satiety and reduced energy intake at
lunch, suggesting thatit may have short-term beneficial effects in obese subjects. Naidu et al.
(2011) [30] reported that fenugreek husk is a valuable source of dietary fiber and phenolic
acids; therefore, it could be an effective source of natural antioxidants and natural ingredients
in functional
Alkaloid, flavonoids and saponin in fenugreek -
Fenugreek contains different alkaloids, flavonoids and saponins [31-33] but out of all these,
saponins are found to be in maximum concentration in the fenugreek [34]. Alkaloid is natural
bases containing at least one nitrogen atom in its heterocyclic ring and is found in plants [31].
Alkaloid and volatiles of fenugreek seed are two major constituents which causes bitter taste
and bad odour due to which people try to avoid consumption of fenugreek seed and its
products [35]. Fenugreek contains 35% alkaloids, primarily trigonelline [36], whereas
saponin was found to be 4.8% [14,37]. One hundred gram of Fenugreek endosperm is
reported to be containing 4.63 g saponin. The alkaloids, flavonoids and saponins of fenugreek
have pharmacological effect. They act as antilipidemic, hypoglycaemic and cholagogic agent
and their use should be promoted to manage diabetes mellitus, hypercholesterolemia because
clinical evidence shows promising results in reducing serum cholesterol level. At the same
time, care should be taken to avoid minor gastrointestinal symptoms and allergic reactions on
its consumption [38]. Murlidhara et al. [14] however, considered alkaloid and saponins as
anti-nutritional factors in human food though the extract of fenugreek containing saponins is
found to be enhancing hunger, reducing plasma cholesterol level and hypocholesterolemia in
rats [39]. Fenugreek also contains flavonoid more than 100 mg per g of seed [40].
Protein in fenugreek -
Fenugreek endosperm is rich in protein such as globulin, histidine, foods. albumin and
lecithin [41] and 100 g endosperm is found to contain protein of 43.8 g [40]. However, 100 g
of fenugreek seed contains 25.4 g protein [14]. Isikli et al. (2005) [22] reported that it has a
high proportion of protein ranging from 20 to 30% as well as amino acid 4-hydroxyisoleucine
in particular, which has high potential for insulin-stimulating activity. The fenugreek protein
fraction is found to be lysine-rich and comparable in quality to that of soybean protein. Garti
et al. (1997) [25] concluded that proteinaceous matter does not have any significant effect on
the surface activity of the fenugreek gum. Youssef et al. [20] reported that residual proteins
played an important role in decreasing the tension at the oil-water interface. The molecular
weight of fenugreek gum is increased by removing the attached proteins. Viscosity of
fenugreek gum increases with increasing gum concentration or with reduction of the residual
protein attached.
Interestingly, studies concluded so far [36] reveal that considering the relatively low levels
of spices that are added to foods, the thresholds for sensitization and provocation of allergic
incidents are probably low. Therefore, the food manufacturers should consider adding
fenugreek to lists of ingredients. However, it should be warned that fenugreek seed powder is
allergic and potentially cross-reactive with peanut allergens. This may be why the
consumption of fenugreek containing foods represents a risk for persons having peanut
allergy. It is also to be noted that digestibility in rat has decreased with increasing protein and
dry matter of fenugreek seed in their diet. This indicates that a judicious use of fenugreek
seed in diet may be helpful for those who have no symptoms of allergy as such.
Nasri and Tinay [42] studied functional properties of fenugreek protein concentrate and
measured emulsion and foaming properties and showed that they are greatly affected by pH
levels and salt (NaCl) concentration. Minimum values of both emulsion and foam properties
are attained at pH 4.5 which is the isoelectricpoint of the protein. Further to this, their results
showed that fenugreek protein concentrate has high oil absorption capacity (1.56 ml oil per g
protein), water absorption capacity (1.68 ml H2O per g protein) and bulk density (0.66 g per
ml). The protein of fenugreek seeds is found to be more soluble(18.5%, 91.3%) at acidic (4.5)
and alkaline (11) pH respectively than at nearly neutral pH. The gums containing high protein
show the ability to decrease the surface tension of water. However, it is also indicated that
removal of proteins by an enzyme protease affected the surface activity of fenugreek gum.
Hence, enzymatic removal of protein reduces the ability of fenugreek gum to reduce the
interfacial tension [22]. In another study on fenugreek protein, Srinivasan (2006) [43]
reported that cooking does not affect the quality of fenugreek seed proteins. It is evidenced in
the animal study that the replacement of casein diet up to 10% by fenugreek seeds did not
produce any harmful effect in protein quality of casein as studies on animal subjects has been
evidenced for protein efficiency ratio, protein digestibility and net protein utilization [25].
However, debitterized fenugreek seeds are rich in protein and lysine [12].
Vitamins in fenugreek -
Fenugreek is especially rich in choline [12]. Vitamins - A (1040 IUer 100 g), B1 (0.41 mg per
100 g), B2 (0.36 mg per 100 g), C (12.0 mg per 100 g), niacin (6.0 mg per 100 g) and
nicotinic acid (1.1 mg per 100 g) are reported in fenugreek seed where as germinating seeds
contain pyridoxine, cyanocobalamine, calcium pantothenate, biotin and vitamin C [44].
Fenugreek leaves contain Vitamin C (43.10 mg per 100 g), calcium, -carotene but by boiling
in water, or steaming and frying, the vegetable loses 10.8 and 7.4% of the vitamin,
respectively and exposure of the germinating seeds to - and -radiation reduces the vitamin
C content. Srinivasan (2006) [6] reported vitamin C, -carotene, thiamine, riboflavin,
nicotinic acid, folic acid contents as 52 mg, 2.3 mg, 40 g, 310 g, 800 g, 0 m in leaves
and 43 mg, 96 g, 340 g, 290 g, 1.1 mg, 84 g in seeds respectively. Poole et al. [45]
showed that fenugreek consumption has potentially improved body composition in particular
body fat percentage and vitamin C of fenugreek seed has an important role.
Minerals in fenugreek -
Fenugreek does not contain so many minerals but it has some of them such as it has good
amount of phosphorus and sulphur [42]. Jani et al. (2009) [46] has reported higher occurrence
of calcium, iron and zinc in curry made from fenugreek compared to the curry made from
potato.
Grinding of fenugreek -
To preserve the volatile and aroma content and also to take care of protein content of
fenugreek in processes such as grinding, one can opt for cryogenic grinding because of its
beneficial effects as mentioned in the flow chart (Figure 1).
Medical Uses of Fenugreek -
Anticarcinogenic activities and complementary cancer therapy -
Fenugreek is a promising protective medicinal herb for complementary therapy in cancer
patients under chemotherapeutic interventions because fenugreek extract shows a protective
effect by modifying the cyclophosphamide induced apoptosis and free radical-mediated lipid
peroxidation in the urinary bladder of mice [47]. Diosgenin (C27H42O3) is a crystalline
steroid sapogenin found in fenugreek and used as a starting material for the synthesis of
steroid hormones such as cortisone and progesterone. It has been found to be potentially
important in treatment cancer [48]. It has the ability to prevent invasion, suppress
proliferation and osteoclastogenesis


Particulars Contents(g
per 100 g)

Moisture content
Leaves 86
Seeds 7.49
Carbohydrates 42.3
Gum (Seed) 20.9
Fats (leaves) 1
Fats (Seed) 7.9
Protein (fresh
leaves)
4.4
Protein (seed) 25.4
Fibre (Seeds) 50
a) Soluble
1) Raw 21.7
2) Germinated 10.3
b) Insoluble
1) Raw 26.8
2) Germinated 23.9
Fibre (Leaves)
a) Soluble 0.7
b)Insoluble 4.2
Dietary fibre 48
Ash (Seeds) 3.38
through inhibition of necrosis factor NF-kappa B-regulated gene expression and enhances
apoptosis induced by cytokines and chemotherapeutic agents [49]. The seed powder in the
diet due to the presence of fibre, flavonoids and saponins decreased the activity of -
glucuronidase significantly and prevented the free carcinogens from acting on colonocytes
whereas mucinase helped in hydrolysing the protective mucin.
Sur et al. [50] found that intra-peritoneal administration of the alcohol seed extract before
and after inoculation of Ehrlich ascites carcinoma cell in mice prevented tumor cell growth
and this treatment enhanced peritoneal exudates and macrophage cell counts. Protodioscin of
fenugreek exhibited a strong inhibitory effect against leukemic cell line HL-60 and a weak
growth inhibitory effect on gastric cell line KATO-III [51]. Diosgenin in fenugreek prevented
cell growth and induced apoptosis in the H-29 human colon cancer cell line [52] and
fenugreek seed was found to have hepatoprotective properties [53]. Kaviarasan and Anuradha
[54] concluded that polyphenolic extract of fenugreek seed acts as a protective agent against
ethanol induced abnormalities in the liver having similar affects as that of silymarin, a known
hepatoprotective agent.
Hypocholesterolemic activities -
The abnormal deficiency of cholesterol level in the blood is known as
hypocholesterolemic problem and oral administration of methanolic and aqueous extracts of
seeds at a dose of one gram per kilogram body weight resulted in hypoglycaemic effect in
mice [55]. Singhal et al. [56] showed hypocholesterolemic effects of fenugreek seeds and
[57] reported that fenugreek seeds have lowered serum cholesterol, triglyceride and low-
density lipoprotein in hypercholesterolemia suffering patients and experimental models.
Fenugreek consumption in diet reduced triglyceride accumulation in the liver but do not
interfered with the plasma insulin or glucose levels obesity suffering rats [57].
Hypoglycemic activities -
Hypoglycemia is a condition of human body in which there is an abnormal decrease in the
sugar level of the blood. Singh and Garg (2006) [58] reexperiment on animals. It improves
peripheral glucose utilization, contributing to improvement in glucose tolerance and exerts its
hypoglycemic effect by acting at the insulin receptor level as well as at the gastrointestinal
level. Raghuram et al. (1994) [59] reported increased erythrocyte insulin reception due to
fenugreek consumption and they concluded with the help of intravenous glucose tolerance
test that fenugreek in the diet reduced the area under the plasma glucose curve significantly
and shortened the half-life of plasma glucose by the increased metabolic clearance.
Sharma (1986) [13] reported that injection of fenugreek seed extracts improved plasma
glucose and insulin responses and reduced urinary concentrations. Daily administration of 25
g fenugreek seed powder in diabetic insulin suffering patient reduced fasting plasma glucose
profile, glycosuria and daily insulin requirement (56 to 20 units) and resulted in significant
reductions in serum cholesterol concentrations [13]. The post-prandial blood glucose levels in
targeted subjects were reduced significantly by giving raw and germinated fenugreek
compared to those without fenugreek or boiled fenugreek [60].
Antioxidant -
Fenugreek contains phenolic and flavonoid compounds which help to enhance its antioxidant
capacity [61]. Table 5 shows the major medicinal and general uses and application of the
fenugreek.
Balch [7] suggested that fenugreek has powerful antioxidant property that has beneficial
effect on liver and pancreas; since antioxidant properties have been linked to health benefits
of natural products; such properties are studied with germinated fenugreek seeds which are
observed to be more beneficial than dried seeds because of the fact that germinated seed
increases the bioavailability of different constituents of fenugreek. An aqueous fraction of
fenugreek exhibits the highest antioxidant activity compared to other fractions and the
quantity of phenolic and flavonoid compounds are related to antioxidant activity. These
studies reveal significant antioxidant activity in germinated fenugreek seeds which may be
due to the presence of flavonoids and polyphenols . Furthermore, Grover et al. (2002) [62]
reported that mustard and fenugreek seeds showed hypoglycemic and antihyperglycemic
activity in diabetic mice and they have attributed that the health benefits may be due to the
presence of antioxidant carotenoids in those spices.
Antibacterial

Fenugreek (Trigonella foenum-graecum L. Leguminosae) is one of the oldest medicinal
plants, originating in India and Northern Africa, and is grown native in Kurdistan (Iraq) .The
leaves and seeds, which mature in long pods, are used to prepare extracts or powders for
medicinal use, having properties of reducing blood sugar level. Leaves, bark, flowers and
fruits of plants derived phytoalexin(1) isothiocynates(2) allicins, anthocya- nins (3) and
essentials oils (4)tannins and polyphenols and terpenoids (5,6,7) have demonstrated
antibacterial and/or
antifungal activities. These compounds are bactericidal and/or bacteriostatic influencing lag
time, growth rate and maximum growth of microorganisms (8), anthelmentic, antibacterial
(9) anti-inflammatory, antipyretic (10) and antimicrobial (11).
In Kurdistan region, fenugreek is still used as a supplement in wheat and maize
flour for bread-making.
New antimicrobial agents are needed to treat diseases in humans and animals caused by drug
resistant microorganisms. Interest in plant-derived drugs has been increasing, mainly due to
the current widespread belief that green medicine is safer and more dependable than costly
synthetic drugs, many of which have adverse side effects(12).Antimicrobial compounds of
plant origin may occur in stems, roots,
The general belief that the advent of antibiotics will bring end to the occurrence of infectious
diseases was cut short with the occurrence of resistance to antimicrobial drug. The incidence
and increasing frequency of microorganisms that are resistant to common and generally
accepted effective first choice drugs is on the increase.
A significant opportunity exists to identify new, natural plant derived antimicrobial agents for
treatment of diseases or as food or cosmetic preservatives. Our objective was to evaluate the
antibacterial activity of aerial parts of native Fenugreek plant cultivated in Sulaimany city
against strains of bacteria isolated from sputum samples of hospitalized patients.
ulaimany city- Iraq. The samples were collected and transported to the laboratory for analysis
using .
Influence on enzymatic activities -
Several researchers as mentioned in [63,64] and [28] have shown in human subject and
animal models that fenugreek has the ability to some extent to restore the actions of key
enzymes in particular lipids and carbohydrates. Baquer et al. (2011) [65] reported that
trigonella administration in rats restored the changed enzyme activities and partially
normalized hyperglycemia. Concluded from their experiments that the altered levels of
superoxide dismutase, antioxidant enzymes catalase and glutathione peroxidase in liver and
kidney of diabetic rats were corrected by treating with insulin, vanadate, fenugreek and the
combined dose of vanadate and fenugreek. It showed that the activities of glucose-6-
phosphatase and fructose-1, 6-biphosphatase in the liver and kidneys of diabetic rats are
reduced by administration of fenugreek.
The -galactosidase enzyme of germinated fenugreek seeds act on galactomannan to convert
it into galactose [66]. Giving of fenugreek seed polyphenol per day that reduced the levels of
lipid peroxidation products and protein carbonyl content. On the other hand, it promoted
mode of action of antioxidant enzymes, and restored content of thiol groups.
Yadav et al. [67] has exhibited that by the combined treatment of fenugreek and sodium-
orthovanadate, activities of nicotinamide adenine dinucleotide phosphate-linked enzymes
such as glucose-6- phosphate dehydrogenase, malic enzyme, isocitrate dehydrogenase , and
the activities of lipogenic enzymes such as adenosine triphosphate-citrate lyase and fatty acid
synthase were decreased significantly in liver and increased in kidney during diabetes as
compared to those of control.
Immunomodulatory effect -
An agent that intensifies or diminishes the immune responses is known as immunomodulator
and such effect is called as immunomodulatory effect. Research work in this effect of
fenugreek is scanty but showed stimulatory immunomodulatory effect (as evidenced from
body weight, relative thymus weight, cellularity of lymphoid organs, delayed type of
hypersensitivity response, plaque forming cell assay, haemagglutination titre, quantitative
haemolysis assay, phagocytosis, lymph proliferation and a significant increase in phagocytic
index and phagocytic capacity of macrophages) of aqueous extract of fenugreek at three
doses 50, 100 and 200 mg per kg of body weight for 10 days on the immune system of Swiss
albino mice.
Antifertility effect -
Evaluated the potential antifertility activity of feeding diets containing 30% fenugreek
seeds to male and female white rabbits of New Zealand and reported the following results: a)
an anitifertility effect of fenugreek seed in female rabbit; b) toxicity effect in male rabbit; c)
testis weight in male reduced with damage to the seminiferous tubules and interstitial tissues;
d) in the treated animals, the plasma concentration of the androgen hormone and sperm
concentrations were halved; f) in the females rabbits, significant reduction of developing
foetuses and g) in the treated animals, the circulation of plasma progesterone concentrations
at 10 and 20 days of gestation significantly increased with no significant effect on the pre-
breeding estrogen concentrations [68].
Diabetes management -
Das et al. [69] has reported that 25-50 g fenugreek seeds were given to diabetic patients
daily in diet to prevent and manage long term complications of diabetes whereas studies have
been made about the glycemic index of fenugreek recipes and found that the soluble
fenugreek fibre has significantly reduced the glycemic index [18] and therefore, they
recommended the inclusion of fenugreek recipes in daily diet to provide at least 25 g
fenugreek seeds that helps in diabetes management. On the other hand, water extract of
fenugreek seeds has higher hypoglycemic and antihyperglycemic potential and for this reason
it may be used as a supplementary medicine to treat the diabetic population by significantly
reducing the dose of standard drugs. Since fenugreek seeds are a source of protein, they can
replace pulses in the diets of diabetics. 25-50 g fenugreek in the diet of diabetic patients
(taken daily)can be an effective supportive therapy in the management of diabetes [70].
Fenugreek has been well known to be used as antidiabetic remedyfor both type I and II
diabetes. Sharma et al. [27] studied in non insulin dependent diabetic patients by
incorporating 100 g of defatted fenugreek seed powder in their diet for 10 days and found
fasting food glucose levels to decrease and reported an improvement in theglucose tolerance
test, urinary glucose excretion reduction by 64% in 2 h, decrease in serum total cholesterol,
low density lipoprotein and very low density lipoprotein cholesterol and triglyceride levels
without alteration in the high density lipoprotein cholesterol fraction.
In type-I diabetic rats, administration of fenugreek and sodium-orthovanadate orally [71]
concluded that sodium-orthovanadate and fenugreek administration to diabetic animals
prevent development of hyperglycemia and alteration in lipid profile in plasma and tissues
and maintain it near normal but maximum prevention can be observed in the combined
treatment with lower dose of sodium-orthovanadate, whereas in another studies, in mild type-
2 diabetic patients adjunct use of fenugreek seeds found to improve glycemic control and
decrease insulin resistance [72]. Kocchar and Nagi [73] concluded that to lower blood
glucose level in diabetics, 2 g of a powdered assortment of bittergourd, jamun seed and
fenugreek seed, either raw or cooked, can be used successfully. Studies of [74] on mechanism
of anti-diabetic action, efficacy and safety profile of GII (anti-hyperglycemic compound)
purified from fenugreek seeds in sub-diabetic and moderately diabetic rabbits shows that GII
seems to decrease lipid content of liver and stimulate the enzymes of glycolysis and inhibit
enzymes of gluconeogenesis in the liver of diabetics, especiallymoderately diabetic rabbits.
4-hydroxyisoleucine is a type of isomer, an atypical branched-chain amino acid, found in
fenugreek has effect on glucose and lipid metabolism and can be used for control of type- II
diabetes, obesity and dyslipidemia, because it has been clinically evidenced that 4-
hydroxyisoleucine stimulates glucose-dependent insulin secretion, decreases insulin
resistance in muscles and liver by activating insulin receptor substrate-associated
phosphoinositide 3-kinase activity, reduces body weight (plasma insulin and glucose levels)
in diet-induced obese mice and decreases elevated plasma triglyceride and total cholesterol
levels in hamster model of diabetes [75].
Kariarasan et al. (2009) [76] concluded that ethanol-induced liver cell damage can be
protected by cytoprotective action of fenugreek seed polyphenolic extract possibly due to its
enhancing cellular redox status. Fenugreek reduced significantly the blood sugar in fasting
and post prandial subjects but it did not affect platelet aggregation, fibrinolytic activity and
fibrinogen [77].
Antiulcer -
The aqueous extract and a gel fraction, isolated from the seeds showed significant ulcer
protective effects. It has soothing effect on gastric and gastritis ulcer [6].
Beneficial Influence of Fenugreek on Digestion -
Spices consumed in diet positively influenced the pancreatic digestive enzymes. Platel and
Srinivasan [78] experimentally showed that dietary curcumin, capsaicin, piperine, ginger,
fenugreek and asafoetida prominently enhanced pancreatic lipase activity withhelp of feeding
rats with spicy diets for eight weeks.
Non-starchy polysaccharides increase the bulk of the food and augments bowel movements.
Also, non-starchy polysaccharides assist in smooth digestion whereas high fibre of fenugreek
helps in relieving constipation ailments.
Fenugreek as food stabilizer, food adhesive, food emulsifier and gum -
The interaction of fenugreek protein with the food constituentsdetermines its ability to
stabilize and emulsify the food constituents. Hefnawy and Ramadan [79] evaluated the effect
of fenugreek gum on solubility and emulsifying properties of soy protein isolate and they
reported that the emulsifying activity of soy protein isolate with fenugreek gum was four
times higher than that of soy protein isolate with fenugreek gum or fenugreek gum alone and
the results were comparable to those of bovine serum albumin. The emulsifying stability of
soy protein isolate with fenugreek gum dispersions was respectively three times higher than
that of soy protein isolate with fenugreek gum and bovine serum albumin. The solubility and
emulsifying properties of soy protein isolate with fenugreek gum dispersions were also stable
over wide range of pH, ion strength and high temperature. The higher dietary fibre content of
fenugreek acts as probiotic in functional food [9]. Sowmya and Rajyalakshmi (1999) [19]
reported that the soluble fibre of fenugreek acts as an excellent substrate for fermentation
done by the microorganisms in the large intestine. Garti et al (1997) [26] demonstrated that
fenugreek gum shows an emulsifying capability for stabilizing oil-in-water emulsions and
they further concluded from their study that critical coverage of gum/oil ratio for stable non-
coalesced emulsion was smaller than the ratio obtained for guar or other gum, indicating its
emulsification properties to be superior to those of other galactomannans. Fenugreek gum has
very good application in making soups because it modifies the rheological properties and
interaction of starch and other soup ingredients.
Losso et al. [80] incorporated fenugreek in bread making and demonstrated that fenugreek
in food helps to reduce blood sugar but use of fenugreek is a barrier due to its bitter taste and
strong odd flavor. They did not find significant variation in color, texture, proximate
composition, firmness, and flavor intensity between the fenugreek and wheat bread, whereas
glucose and insulin was found to be lower in the bread with fenugreek. Bread maintained
fenugreeks functional property of reducing insulin resistance. Therefore, it is evident from
this study that fenugreek can be incorporated in baked products in acceptable limit which will
reduce insulin resistance and treat diabetes patients as well.
Fenugreek in traditional food -
Fenugreek paste, locally termed as Cemen is a popular food in Turkey [23], which is
prepared from ground fenugreek seeds. Crushed fenugreek seed or coarse fenugreek powder
is used to make ball forOther benefits, beneficial uses and application of fenugreek
Fenugreek has the ability to lower the hepatic lipids in body because of its potential to modify
the activities of several enzymes such as enzymes related to glucose and lipid metabolism
[28]. Fenugreek is antihelmintic (ability to cause the evacuation of parasitic intestinal worms)
in nature. As fenugreek has a high fibre content, which helps in controlling cholesterol in
human body, it takes care of some of the problems related to heart. It is diuretic
emmenagogue, emollient in nature and controls heart diseases.






















ANTIOXIDANT AND THEIR MECHANISM


The oxidative metabolism of all the living organisms plants, microorganisms and humans
continuously produces oxygen centered free radicals and other reactive oxygen species (ROS) in
vivo, causing cell death and tissue damage which ultimately leads to many health problems like
carcinogenesis, coronary heart disease, ageing, atherosclerosis, stroke, diabetes, cancer and
neurodegenerative diseases such as Alzheimer and Parkinsonism. Every living organism has a
defense system of endogenous antioxidants to combat the reactive species. These are the
substances which terminate direct ROS attacks and radical-mediated oxidative reactions, and appear
to be of primary importance in the prevention of diseases. But due to limited amount of endogenous
antioxidants or excess production of reactive species, exogenous antioxidants are required.
Minimizing the oxidative damage may well be one of the most important approaches for primary
prevention of these age-associated diseases and health problems.
Most of the commercially available antioxidants are synthetic ones and reported for their
toxicity and carcinogenicity. So products from natural origin are came in to the light and are now
considered as an alternative to the arsenal of synthetic antioxidants for the treatment of severe
chronic diseases. Lichens are the symbiotic organisms composed of a fungal partner (mycobiont) in
association with one or more photosynthetic partner (photobiont). They are supposed to be the
most unexploited organisms on our planet. The adaptability of these organisms to extreme
environmental conditions, particularly to temperature-induced ones, is of much interest for the new
and effective therapeutic compounds. newline The majority of lichen secondary metabolites
originate from the fungal partners, but produced primarily when the organisms are in their symbiotic
association. Other compounds are produced when the mycobiont is isolated from its symbiont.
Oxygen is a toxic mutagenic gas that appeared in significant amount in the earths
atmosphere over 2.5 billion years ago (Harman, 1986). The rise in atmospheric oxygen led to the
formation of the ozone layer that filtered out enough of the solar ultraviolet radiation to allow living
organisms to leave the sea and inhabit the land. At the same time, the more primitive anaerobic
organisms died out with the exception of those that evolved a defense mechanism to protect
themselves from the toxicity of oxygen. Other surviving organisms continued to evolve by oxygen for
metabolic transformations and energy production in mitochondria (Willcox & all, 2004). Mammals
have developed mechanism to ensure that oxygen is transported to all the cells that need it. About
85% to 90% of the oxygen taken up from the plasma by animal cells is utilized by the mitochondria
to produce adenosine triphosphate(ATP) (Halliwell, 1994). The essence of metabolic energy
production involves the oxidation of food materials, i.e., the loss of electrons that are accepted by
electroncarriers such as nicotinamide adenine dinucleotide (NAD+), flavin mononucleotide (FMN),
and flavin adenine dinucleotide (FAD). These reduced compounds in turn arere-oxidized by oxygen
in the mitochondria, producing ATP. The terminal enzyme in this electron transport chain,
cytochrome oxidase, adds four electrons to oxygen. It is through a stepwise process that partially
reduced oxygen species are generated (Willcox & all, 2004).
The presence of unpaired electrons makes free radicals highly reactive. When a single electron is
added to the ground state oxygen (O2) molecule, the superoxide radical is produced. Several
organic molecules oxidize in the presence of O2 produce the superoxide radical, including
glyceraldehydes, the reduced flavins, Ldopa, dopamine, cysteine etc. (Willcox & all, 2004)
The most important source of superoxide radicals in vivo are the electron transport chains
present within the mitochondria, endoplasmic reticulum, and nuclear membranes of all the living
organisms. Most of these radicals are produced by the incomplete transfer of electrons on oxygen
(O2) molecule prior to the terminal cytochrome oxidase step. This production of radicals is increased
as the O2 concentration increases. It is estimated that 1% to 3% of the O2 reduced in mitochondria
may form the superoxide radical (O2Turrens, 1997). Superoxide can decrease the activity of
certain enzymes including some antioxidant defense enzymes such as catalase (CAT), glutathion
peroxidase (GPx), and several in the energy metabolism scheme such as nicotinamide adenine
dinucliotide (NADH) dehydrogenase. Another enzymatic target of superoxide damage is the
ribonucleotide reductase that makes the precursors required for DNA synthesis (Willcox & all, 2004).
Apart from direct damage, superoxide can be more cytotoxic by generating other reactive
species, such as hydrogen peroxide (H2O2), by the addition of one more electron. H2O2 is not a
radical since the additional electron fills the orbital, but it can attack certain enzymes such as
glyceraldehydes-3-phosphate dehydrogenase, an enzyme in the glycolytic pathway. It can also
oxidize certain keto-acids such as pyruvate. H2O2 leads to depletion of adenosine triphosphate
(ATP), reduced glutathione, and reduced nicotinamide adenine dinucliotide (NADPH). It induces a
rise in free cytosolic calcium ion (Ca2+) and activates a polymerase that leads to cell death. Finally
H2O2 can cross cell membranes to react with iron and copper ions toform much more damaging
species such as hydroxyl radical (OH) and nitric oxide(NO) (Roberford & Calderon, 1995).
Other environmental factors also lead to the production of free radicals. Exposure to
ultraviolet radiation can generate free radicals, i.e. air pollution and cigarette smoke. Nitric oxide
(NO), one of the major oxidants found in cigarette smoke, which is capable to reduce oxygen to
superoxide radical (Chow, 1993)
The living organisms have evolved a highly complicated defense system with antioxidant
compounds composed of enzymes and vitamins against oxidative stress in the course of their
evolution. These defense systems are mainly classified as (i) suppression of generation of ROS, (ii)
scavenging of ROS, (iii) clearance, repairing and reconstitution of damage and (iv) induction of
antioxidant proteins and enzymes (Noguchi & all, 2000).
An antioxidant has been defined by Halliwell & Gutteridge (1999) as any substance that, when
present at low concentrations compared with those of an oxidizable substrate, significantly delays or
prevents oxidation of that substrate.
When free radicals are generated in vivo as a first line of defense, the preventive
antioxidants such as peroxidases and metal chelating proteins suppress the generation of free
radicals in order to defend the organism from oxidative damage. Next, the radical-scavenging
antioxidants such as vitamin C and vitamin E scavenge radicals to inhibit the oxidation chain
inhibition and prevent chain propagation as a second line of defense. This may also include the
termination of a chain by the reaction of two radicals. The repair and de novo enzyme act as the
third line of defense by repairing damage and reconstitution membranes. These include lipases,
proteases, DNA repair enzymes, and
transferases (Niki, 1997).
Endogenous antioxidants: There is a vast network of intracellular and extracellular
antioxidants with diverse roles within each area of defense. Catalase (CAT) converts the hydrogen
peroxide (H2O2) to oxygen (O2) and water (H2O) while superoxide dismutase (SOD) converts the
superoxide radical (O2) to H2O2 and O2. Some of the antioxidant enzymes exist in several forms.
For example, membrane, cytosolic, and plasma forms of glutathione peroxidase (GPx) and
superoxide dismutase (SOD) have been isolated. The levels and locations of these antioxidants must
be tightly regulated for cell survival. The antioxidant enzymes, SOD, GPx and CAT, work within the
cells to remove most superoxides and peroxides before they react metal ions to form more reactive
free radicals. Peroxidative chain reactions initiated by free radicals that escaped the antioxidant
defenses are terminated by chain-breaking water or lipid soluble antioxidants (Mates & all,1999).
Exogenous antioxidants: Different protective devices present under the normal
physiological conditions are sufficient only to cope with the normal threshold of physiological rate of
free radical generation. Therefore, any additional burden of free radicals, either from an indigenous
or exogenous source on the animal (human) physiological system can tip free radical (prooxidant)
and antioxidant balance leading to oxidative stress (Tiwari, 2001).
Natural antioxidant: Diet plays a vital role in the production of the antioxidant defense system by
providing essential nutrient antioxidants such as vitamin E, C, and -carotene, other antioxidant such
as plant phenols including flavanoids, and essential minerals that form important antioxidant
enzymes. For example, superoxide dismutase (SOD) contains zinc and glutathione peroxidase (GPx)
contains selenium (Punchard & all, 1997). Diet also plays an important role in the oxidation process
by affecting the substrates that are subject to oxidation e.g. oxidation of lipids (Willcox & all, 2004).

Synthetic antioxidant: Antioxidants have been widely used as food additives to provide protection,
to human body from oxidative stress, and oxidative degradation of foods and oils. The most
extensively used synthetic antioxidants are butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ) and propyl gallate (PG) (Sherwin, 1990). They
are used widely in the food industry because of their effectiveness and generally being less
expensive than natural antioxidants. However, BHT and BHA have been suspected of being
responsible for liver damage and carcinogenesis (Hettiarachchy & all, 1996). Concerns regarding
toxicological effects and carcinogenic potential of synthetic antioxidants have prompted the need
for natural alternatives in the last few decades (Lu & Foo, 2001; Aligiannis & all, 2003; Vgi & all,
2005; Es-Safi et al, 2007). Due to the increasing concerns regarding safety issues of using synthetic
antioxidants, continuously research has focused on the development and utilization of antioxidants
from natural sources. Natural antioxidants have appeared as a healthier and safer alternative to
synthetic antioxidants. Various kinds of fruits, vegetables and medicinal plants were examined and
their abundance in antioxidant came to light. More and more antioxidant substances have become
known to provide the biomolecules an effective antioxidant protection (Khknen & all, 1999;
Yanishlieva, 2001; Dimitrios, 2006).
Natural antioxidants are found in almost all plants, microorganisms, fungi, and even in
animal tissues and they possess multifacetedness and the magnitude of the activities, which
provides enormous scope in preventing the ROS induced diseases. Therefore, the importance of
search for the exploitation of effective natural antioxidants, especially of plant origin, has greatly
increased in recent years (Jayaprakash & Jaganmohan, 2000; Pokorny, 2001).




MULTIFACETED NATURE OF ANTIOXIDANTS:
The word antioxidant is increasingly popular in modern society as it gains publicity
through mass media coverage of its health benefits. The dictionary definition of antioxidant is
explains it as: a substance that opposes oxidation or inhibits reactions promoted by oxygen
or peroxides, many of these substances (as the tocopherols) being used as preservatives in
various products (as in fats, oils, food products, and soaps for retarding the development of
rancidity, in gasoline and other petroleum products for retarding gum formation and other
undesirable changes, and in rubber for retarding aging).
A more biologically relevant definition of antioxidants is synthetic or natural
substances added to products to prevent or delay their deterioration by action of oxygen in
air. In biochemistry and medicine, antioxidants are enzymes or other organic substances, such
as vitamin E or -carotene, that are capable of counteracting the damaging effects of
oxidation in animal tissues. The biologically relevant definition fits better to the concept of
antioxidants known to the general public as people are more aware of their health than
prevention of rubber autoxidation. Depending on the scientific discipline, the scope and
protection targets are significantly different. In the chemical industry, antioxidants often refer
to compounds that retard autoxidation of a chemical product such as rubber and plastics. The
auto-xidation is caused primarily by radical chain reactions between oxygen and the
substrates. Effective antioxidants are radical scavengers that break down radical chain
reactions. Sterically hindered phenols and amines are often used as antioxidants in the rubber
and plastic industries. (Dejian Huang, Boxin Ou, and Ronald 2003.). In food science,
antioxidants have a broader scope, in that they include components that prevent fats in food
from becoming rancid as well as dietary antioxidants a substance in foods that significantly
decreases the adverse effects of reactive species, such as reactive oxygen and nitrogen
species, on normal physiological function in humans as defined by the Institute of Medicine.
Like the other definitions, this definition does not provide limitation on the mechanism(s) of
antioxidant action. Therefore, a dietary antioxidant can (sacrificially) scavenge reactive
oxygen/nitrogen species (ROS/RNS) to stop radical chain reactions, or it can inhibit the
reactive oxidants from being formed in the first place (preventive). In living systems, free
radicals attack key biological molecules, leading to many degenerative disease conditions
such as cancer, inflammation, atherosclerosis, and aging. Dietary antioxidants often broadly
include radical chain reaction inhibitors, metal chelators, oxidative enzyme inhibitors, and
antioxidant enzyme cofactors.
Nonetheless, nonenzymatic lipid autoxidation by radical chain reaction may still
occur and lead to oxidative stress. Consequently, biological antioxidants include enzymatic
antioxidants (e.g., superoxide dismutase (SOD), catalase, and glutathione peroxidase) and
nonenzymatic antioxidants such as oxidative enzyme (e.g., cyclooxygenase) inhibitors,
antioxidant enzyme cofactors, ROS/RNS scavengers, and transition metal chelators. Halliwell
defined biological antioxidants as molecules which, when present in small concentrations
compared to the biomolecules they are supposed to protect, can prevent or reduce the extent
of oxidative destruction of biomolecules. (Dejian Huang, Boxin Ou, and Ronald l. Prior)
Selenium is a cofactor of selenoproteins (e.g., glutathione peroxidase), which reduce
peroxides to alcohols and water. Whereas autoxidation of a lifeless matter occurs by radical
chain reactions, oxidation in a biological system is primarily mediated by a host of redox
enzymes. Also, antioxidants have been widely used as food additives to provide protection
against oxidative degradation of foods (Gulc in et al., 2005b, 2004e). Lipid peroxidation
leads to the development of off-flavors and undesirable chemical compounds.
Recently, there has been growing interest in natural antioxidants of plant origin
because they have greater application in the food industry for increasing the stability and
shelf life of food products. Moreover, they also find use as nutraceuticals and phytoceuticals
as they have significant impact on the status of human health and disease prevention.












A3. MATERIALS AND METHODS
3.1. Sources of materials:-
Fenugreek seeds were procured from local market of Allahabad city; they were
washed and dried under fans, grind to powder form and stored at room temperature. All the
chemicals were used for chemical analysis in extracts.

3.2. Processing of samples:-
The seeds were processed in the terms of dry heating at 40-50C under tray drier. The
sample was prepared for analysis of changes in proximate and antioxidant composition from
fresh material.
Soaked:-The clean seeds (50g) were soaked in distilled water at the ratio of 1:5 (w/v)at room
temperature for 12 hrs. The water was intermittently changed every 6 hrs. After 12 hrs ,the
excess water was discarded and seeds were dried in hot air oven at 40
0
C for 6 hrs.
Germinated:- Fenugreek seeds (50g) were soaked overnight in water at the ratio of 1:5 (w/v).
The excess water was drained and seeds were kept in the dark for germination (tied in muslin
cloth) at 27
0
2
0
C temperature for 24 hrs. The germinated seeds were dried in an oven at
40
0
C for 6 hrs.
Roasting:- Fenugreek seeds (50g) were roasted in an open pan at 1305
0
C for 7 min. It was
continuously stirred with ladle for proper and uniform roasting until it became slight brown
and left a peculiar aroma.
Pressure cooked:- Fenugreek seeds (50g) were cooked in pressure cooker for 20 min. Water
taken in a ratio of 1:5 (w/v). Water was discarded and the cooked sample were dried at 50
0
C.
3.3. Proximate Composition:-
The seeds were analysed for moisture, protein, fat, carbohydrates, ash and
crude fibre.
3.3.1. Moisture content:-
Moisture content of the sample was determined by AOAC (1984) procedure. The
sample (5g) was weighed accurately and transferred to a clean, dried and weighed glass dish.
The contents were dried in a hot air oven at 70
o
C until a constant weight is achieved. After
Initial weight of sample - Weight of dried sample
Moisture (%) = 100
Initial weight of sample

cooling the loss in weight was taken as moisture content and expressed in terms of
percentage.

3.3.2. Protein
Protein estimation was done by Micro Kjeldahl method (AOAC, 1984). Two grams
of the sample was transferred to a digestion flask followed by addition of 3g of digestion
mixture (K
2
SO
4
: CuSO
4
: SeO
2
: 100:20:1 ratio) and 40 ml of concentrated sulphuric acid. The
contents were then digested till a bluish green transparent liquid was obtained. The contents
were then quantitatively transferred to a 100 ml volumetric flask and volume was made up to
100 ml using glass distilled water. A 10 ml aliquot of digested mixture was distilled with
excess of 30 per cent NaOH and the liberated ammonia was collected in 30 ml of 2 per cent
boric acid solution containing 2-3 drops of mixed indicator (10 ml of 0.1 per cent
bromocresol green + 2ml of 0.1 per cent methyl red indicator in 95 per cent ethyl alcohol).
The entrapped ammonia was titrated against 0.01N HCl. A blank was also simultaneously
digested and distilled. Nitrogen content in the sample was calculated as follows:


N= Normality of hydrochloric acid (0.01 N)
% Protein = % Nitrogen x 6.25
3.3.3. Fat:-
Fat content of the samples was determined by Soxhlet Extraction Method (AOAC,
1984). Two grams of the dried sample was transferred to a thimble. The thimble was placed
in the Soxhlet apparatus and fat was extracted for 2-2 hrs with petroleum ether (B.P. 60-
14 x (Sample titre-Blank titre) x N x volume made up of digest
% Nitrogen = 100
1000 x Weight of the sample (g) x Aliquot of digest taken for distillation (ml)

80
o
C). Thereafter, the ether was evaporated and the extracted fat was weighed. Fat percentage
was calculated by the following expression:


Weight of ether extract= weight of extract with beaker-weight of empty beaker



3.3.4. Carbohydrate
The carbohydrate content in the samples was calculated by difference. The sum of
moisture, protein, fat and ash content was subtracted from hundred and the result was
designated as per cent carbohydrate content.
3.3.5. Ash
Ash content in the samples was determined according to AOAC (1984) method. Two
grams of sample was weighed in to a silica dish, charred and ignited in muffle furnace at
550
o
C until carbon free ash was obtained. The dish was cooled in dessicator and weighed.
The ash content in the sample was calculated by following formula:-

Weight of residue= weight of residue with silica dish- weight of empty silica dish
3.3.6. Minerals Composition:-
The seeds were analysed for calcium, phosphorous and iron.
3.3.7. Crude fibre
Crude fibre content of the samples was determined according to AOAC (1984)
procedure. Defatted sample was boiled in 1.25 per cent sulphuric acid for exactly 30 minutes,
Weight of the ether extract
Fat (%) = 100
Weight of the sample

Weight of the residue
Ash (%) = 100
Weight of the sample

filtered through Whatman No.54 filter paper and washed with hot distilled water. The residue
was then boiled in 1.25 per cent sodium hydroxide solution for exactly 30 minutes, filtered
through Whatman No. 54 filter paper and washed with hot distilled water. The filter paper
with the residue was dried in oven at 105
o
C for 3-4 hour till constant weight was obtained.
The difference in weight of filter paper with residue minus filter paper was reported as crude
fibre content of the sample and it was expressed as per cent crude fibre using the following
formula:

3.4. Preparation of aqueous decoction:-
The seeds were washed thoroughly with sterile double distilled water to make these
leaves completely free from any possible contamination. Aqueous decoction of seeds was
prepared by boiling 20 grams of seeds in 100 ml sterile distilled water over moderate flame
for 20 minutes. The aqueous extract was cooled, filtered through Whatman No. 1 filter paper
and then kept in sterile screw capped glass vials at 4C. The aqueous extracts were used to
determine antimicrobial activity of leaves. These crude extracts were reconfirmed as free of
any contamination by plating method (APHA, 1992). These crude extracts were diluted with
sterile double distilled water (as negative control) to obtain required concentrations before
experiments.
3.5. Preparation of solvent extract:-
20 g sample of raw and processed fenugreek seeds were soaked in ethanol(80%) for
48 hours at 24C with stirring (Liu and Nakano, 1996). The extracts were centrifuged and
filtered through Whatman No. 1 filter paper and evaporated using vacuum rotary evaporator
or water bath to near dryness and stored in glass vials in dark at 4C and extracts were used to
determine the antioxidants. These crude extracts were diluted with 10% dimethyl sulphoxide
(weight of residue + filter paper) - (weight of the filter paper)
% Crude fibre = 100
Weight of the sample

(DMSO- which is to be used as negative control) to obtain required concentrations before

experiments (for antimicrobial activity).
3.6. Antioxidant Analysis:-
3.6.1. Estimation of DPPH free radical scavenging method
The free radical scavenging activity of the fenugreek seeds extracts was measured by
measuring the decrease in absorbance of ethanolic DPPH solution at 517 nm in the presence
of the extract (Krings and Berger, 2001). The initial concentration of DPPH was 0.1 mM and
the readind was taken after allowing the solution to stand for 30 min. In cases where the
absorbance decreased too much before the 30 minutes period. The sample was appropriately
diluted. The antioxidant activity was expressed as:-




3.6.2. Ferric reducing power of plasma:-
The ferric reducing power of the seeds extracts was determined by using potassium
ferricyanide- ferric chloride method (Oyaizu, 1986). Different dilutions of extracts amounting
to 1 ml were added to 2.5 ml 0.2 M phosphate buffer (pH=6.6) and 2.5 ml potassium
ferricyanide (1%). The mixtures were incubated at 50C for 20 minutes, after which 2.5 ml
trichloroacetic acid (10%) was added. 2.5 ml of the mixture was taken and mixed with 2.5 ml
water and 0.5 ml 1% ferric chloride. The absorbance at 700 nm was measured after allowing
the solution to stand for 30 minutes.
3.6.3. Total Phenolic Content:-
Total phenolic content was determinbed by adopting Folin-Ciocalteu method
(Velioglu et al., 1998). Basically, 0.2 ml of extracts was added with 1.5 ml of Folin-Ciocalteu
reagent and mixture was allowed to stand at room temperature for 5 minutes. Then 1.5 ml of
sodium carbonate solution (6%) was added into the mixture. Absorbance was measured using
spectrophotometer at 725 nm after 1 hours standing at room temperature. Results were
expressed as gallic acid equivalent in mg/100 g dry weight (DW).



3.6.4. Total Tannin Content:-
The tannin content was determined by the method of Ranganna, 2005. Powdered
sample (0.5 g) was boiled with water (75ml) for 30 minutes and centrifuged at 2000 rpm for
20 minutes and the supernatant was collected. Folin Denis reagent and sodium carbonate is
added to the sample extract, solution is diluted tom100ml with water proper shaking and
absorbance is taken at 700 nm after 30 minutes.
3.7. Stastical Analysis:-
All the data were analysed stats by using technique of analysis of various method
Snede or Cochar linear correlation coefficient was calculated using SISS120 software.
3.8. Antibacterial Activity:-
3.8.1. Test Organisms:-
Four pathogenic bacterial strains were selected for the study to assess antimicrobial
activities of herb seeds against those strains. The strains were Escherichia coli, Styphlococcus
aureus, Salmonella enterica, Shigella flexeneri were obtained from Centre of Food
Technology, Institute of Professional Studies, University of Allahabad, Allahabad, India. All
bacterial cultures were maintained on tryptic soy agar (HiMedia) and subcultured regularly.
Standard inoculums was prepared by sub-culturing 4-5 freshly grown isolated colonies of
each strain in Tryptic soy broth (TSB) and incubated at 37C for 24 hours. Inocula were
standardized with sterile TSB to give final cell load

CFU/ml.


3.8.2. Well Agar Diffusion Method:-
The selected strains of bacteria were inoculated into 10 ml of sterile TSB, and
incubated at 37C for 16-18 hours. Using a sterile cotton swab, the TSB cultures were
swabbed on the surface of sterile TSA plates. Agar wells were prepared with the help of
sterilized cork borer or sterilized micropipette tip with 10 mm diameter. Using a
micropipette, 100 l of different concentrations of seeds extracts were added along with 10%
DMSO as negative and streptomycin as positive controls to different wells in the plate. The
plates were incubated in an upright position at 37C for 24 hours. The diameter of inhibition
zones was measured in mm and the results were recorded (Indu et al.,2006).
3.8.3. Determination of MICs:-
Minimum Inhibitory Concentrations (MICs) were determined by broth dilution
method in culture tubes (Jorgensen et al., 1999). Various concentrations (40, 30, 20, 10, 5,
2.5 mg dry weight/ ml) of extracts were added to broth immediately after inoculating with
fresh 0.2 ml cultures of the stain, keeping final volume at 5 ml. The cultures were incubated
on a rotary shaking incubator or normal incubator at 37C for 48 hours. The lowest
concentrations of the leaves extracts showing no visible growth was considered as the MIC.

Supervisor Student
Dr. Pinki Saini


























Niharika Dubey
M.sc in Food TECHNOLOGY
Semester IV
Tables
4.1. Proximate Composition(%) and mineral content(mg) of fresh Fenugreek
Seeds
Sample
Moisture% Fat% Crude
fibre
Ash% Iron Calcium Phosphorus
Raw 8.92

0.15

9.230.75 20.610.53

3.650.15

11.19
0.27

84.17 5.2

0.06 0.017


4.2 Antioxidant Components in fresh Fenugreek seeds
Sample Total phenolic
content(mg Gallic
Acid/gm)
Tannin
content(mg
Tannic Acid/gm)
DPPH % FRAP(mg
Ascorbic
Acid/gm)
Raw 173.38 12.29

0.94 0.13

O.720.011

4.890.2

4.4 Effect of processing on proximate and mineral content of processed
fenugreek seeds
Sample
Moisture% Fat% Crude
fibre
Ash% Iron Calcium Phosphorus
Raw
8.92

0.15
b
9.230.75
c
20.610.53
d
3.650.15
b
11.19
0.27
d

84.17 5.2
a
0.06 0.017
d

Roasted
4.25 0.23
a
7.05 0.56
b
15.86 0.92
c
4.060.081
c
5.170.1
5
a
107 2.54
bc
0.05 0.01
c
Soaked
8.81 1.2
b
0.66 0.28
a
7.22 0.52
a
4.17 0.16
c
9.750.6
2
c
101.101.01
b
0.03 0.02
b
Germinated
11.39 .55
c
1.48 0.015
a
10.26 0.59
b
1.04 0.06
a
6.530.5
8
b
95.2 5.01
b
0.14 0.01
a

4.5 Effect of processing on antioxidant components of Fenugreek seeds
Sample Total phenolic content Tannin content
Raw
173.38 12.29
c
0.94 0.13
a
Roasted 172.25 13.94
c
0.6 0.11
d
soaked 157.92 5.70
b
4.33 0.34
b
Germinated 148.31 7.02
a
5.5 0.13
c
Pressure cooked 178.16 3.7
c
5.34 0.38
c

4.6 Antioxidant activity of fenugreek seeds of processed sample
Sample DPPH FRAP
Raw 0.720.001
a
4.76 0.08
d

Roasted 0.810.04
b
3.44 0.25
a
Soaked 0.850.02
b
6.24 0.17
c
Germinated 0.840.01
b
59 1.33
d
Pressure cooked 0.740.02
a
7.16 0.76
c

Antibacterial activity , indicated by diameter of inhibition zone (DIZ, mm,
for10 mg dry weight/ disc, MeanSD
Sample Extracts S.aureus Salmonella Shigella E.coli
Aqueous
18 3 16 1 14 1 18 1
Acetone
38 1 40 2 43 1 42 1
Hexane
0 0 0 0
Ethanol
34 1 37 1 37 2 39 1
Petroleum
ether
18 1 0 18 2 18 1
Streptomycin
35 41 28 33

Antibacterial Activities, Minimum Inhibitory Concentration (MIC, for mg
dry weight/ml):
Extract S. aureus Salmonella Shigella E.coli
Aqueous
50 30 40 40
Acetone
50 20 30 30
Hexane
50 40 40 40
Ethanol
50 40 40 20
Petroleum
eher
50 40 40 40
Streptomycin 20 10 40 30