Review of Literature

3. REVIEW OF LITERATURE

3.1 Introduction

Recently, there has been a noticeable increase of information published in the

literature regarding the food related health problems including aflatoxicosis and their

control measures. Mostly it is driven by the increasing rates of chronic food borne

illnesses, the rising awareness on public health and societal costs of diet-related

problems. A growing coalition of public health professionals is advocating for food

and agriculture policies that promote public health as a means to address these issues

(Muller et al., 2009). The following section abridges the most relevant literature

pertinent to the present work.

3.2 Culinary Oil and Fat

Edible oils and fats are indispensable ingredients for a healthy and balanced

diet and are very important stuff of bulk consumption. Culinary oil refers to edible

vegetable oils, such as those used in food preparation and production. Any of various

oils obtained from plants, used in food products and industrially is called vegetable

oil. The main productions of vegetable oils are from oilseeds (e.g. rapeseed and

sunflower seed) as well as from legumes (e.g. peanut and soybean), nuts (e.g. walnut

and almond) and the flesh of some fruits (e.g. olives). The vegetable oils from these

plants are released by pressing, processed and refined to produce high-quality oils

suitable for use as an ingredient in recipes, for frying, in salad dressings and in the

production of margarines as well as spreads. The main nutrient they provide is fat.

The only other nutrient that is present in appreciable amounts is vitamin E

(tocopherols and tocotrienols). Sometimes they contain health benefits providing plant

components in minor amounts (Foster et al., 2009). Fats contain long fatty acid

chains, and depending on the number of hydrogen atoms in the chain, a fat will be

either „saturated‟ (containing the maximum number of hydrogens) or „unsaturated‟

containing less than the maximum. Unsaturated, saturated, polyunsaturated or trans,

one gram of fat will provide nine calories of energy; considerably more calories than

provided by the same weight of protein or carbohydrate (Donaldson, 2009). Vegetable

oils now constitute a major component of daily diet consumption and its growth in the

market is now considered based on functionality, economy, and acceptability

(Anyasor et al., 2009).

3.3 Nutritional Value of Oil and Its Usage

The annual plant oilseeds, such as soybean, cottonseed, rapeseed (canola),

sunflower seed and peanut, are the largest source of vegetable oils even though most

oil-bearing tree fruits provide the highest oil yields (e.g. olive, coconut and palm

trees). These seeds contain energy for the sprouting embryo mainly as oil, compared

with cereals, which contains the energy in the form of starch (Mckevith, 2005). Olive

oil is the most important dietary fat source of the Mediterranean diet. In Italy, olive oil

consumption is 12-12 kg per person per year for a total of 750,000 tons. Olives are

often stored for a long time in conditions that promote the growth of moulds, such as

prolonged contact with the ground, in bags of jute, in little ventilated plates, all

favouring the toxinogenic moulds colonization and hence olive oil became

contaminated (Ferracane et al., 2007). The Mediterranean diet is rich in vegetables,

cereals, fruit, fish, milk, wine and olive oil and has salutary biological functions. The

relationship between the structure of olive oil polyphenolic compounds and their

antioxidant activity when analyzed revealed that these compounds possible beneficial

effects are due to their antioxidant activity, which is related to the development of

atherosclerosis and cancer, and to anti-inflammatory and antimicrobial activity

(Tripoli et al., 2005). Accordingly, new research and developments on the role of

oilseeds and their by products in human health and disease may provide even more

benefits for health in the future (Mckevith, 2005).

3.4 Groundnut and Other Common Indian Oils: Use and Production

Indian edible oils are obtained principally from two sources, the primary

source and secondary source. The nine principal oilseeds viz. groundnut, rapeseed

mustard, soya bean, sunflower, sesame, niger, safflower, castor and linseeds comprise

the primary source. Whereas edible oils obtained through secondary source, consist of

coconut, cottonseed, rice bran and oilseed cakes. The production of major oilseeds

and net availability of edible oils from domestic sources (primary source and

secondary source) for the years 2007-2008, 2008-09, 2009-10 and estimated

production for 2010-11 are available (DVVO&F, Ministry of Consumer Affairs, Food

and Public Distribution, GOI, 2010) (Table 3.1).

Among oil seeds, groundnut is the world‟s principal oilseed crops. Since it can

be consumed in diverse ways and can be consumed directly as well, it occupies a

unique position among oilseeds. About two thirds of the total groundnuts produced in

the world are utilized in India, China and the US (Misra, 2004). India is one of the

world‟s largest edible oil economies with 15,000 oil mills, 689 solvent extraction

units, 251 Vanaspati plants and over 1,000 refineries employing more than one

million people. The total market size is at Rs. 600,000 million and import export trade

is worth Rs.130, 000 million. India being deficient in oils has to import 40% of its

consumption requirements. With an annual consumption of about 11 million tonnes,

the per capita consumption is at 11.50 kgs, which is very low when world average of

20 kgs is compared. India is also a leading producer of oilseeds, contributing 8-10%

of world oilseed production. India is estimated to account for around 6% of the

world‟s edible oil production. Though it has the largest cultivated area under oilseeds

in the world, crop yields tantamount to only 50-60% of the world‟s average. It is the

fifth largest producer of oilseeds in the world, behind US, China, Brazil, and

Argentina. Three oilseeds-groundnut, soybean and rapeseed/ mustard-together

account for over 80 per cent of aggregate cultivated oilseeds output. Mustard seed

alone contributes Rs.120, 000 million. turnover out of Rs.600, 000 million. oilseed

based sector domestic turnover. Cottonseed, copra and other oil-bearing material too

contribute to domestic vegetable oil pool. Currently, India accounts for 7.0% of world

oilseeds output; 7.0% of world oil meal production; 6.0% of world oil meal export;

6.0% of world vegetable oil production; 14% of world vegetable oil import; and 10%

of the world edible oil consumption. With steady growth in population and personal

income, Indian per capita consumption of edible oil has been growing steadily.

However, oilseeds output and in turn, vegetable oil production have been trailing

consumption growth, necessitating imports to meet supply shortfall (MOFPI, 2010).

Name of oilseed

2007-08 2008-09 2009-10

Total

Oil

Oilseed

Oil

Oilseed

Oil

Oilseed

Kharif Rabi

A. Primary Source

Groundnut 91.83 21.12 71.68 16.48 38.52 15.76 54.29 12.49

Rapeseed/Mustard 58.33 18.08 72.01 22.32 - 66.08 66.08 20.48

Soybean 109.68 17.55 99.05 15.85 99.65 - 99.65 15.94

Sunflower 14.63 4.83 11.58 3.82 2014 6.36 8.51 2.81

Seaame 7.57 2.35 6.40 1.98 5.88 - 5.88 1.82

Niger Seed 1.11 0.33 1.17 0.35 1.00 - 1.00 0.30

Safflower Seed 2.25 0.68 1.89 0.57 - 1.79 1.79 0.54

Castor 10.53 4.21 11.71 4.68 10.09 - 10.09 4.04

Linseed 1.63 0.49 1.69 0.51 - 1.54 1.54 0.46

Sub Total 297.56 69.64 277.19 66.56 157.29 91.53 248.82 58.88

B. Secondary Source

Coconut 4.50 4.50 - - - 4.50

Cottonseed 8.00 7.60 - - - 8.00

Rice Bran 7.20 7.70 - - - 7.20

Solvent Extracted Oils 4.00 4.00 - - - 4.20

Tree & Forest Origin 1.20 1.20 - - - 1.20

Sub Total 24.90 25.00 - - - 25.10

Total (A+B) 94.54 91.56 - - - 83.98

C. Less: Export & Industrial use

8.00

7.00

-

-

-

4.52

D. Net Domestic availability

86.54

84.56

-

-

-

79.46

E. Import of Edible Oils 56.08 81.83 - - - 167.69

F. Total

Availability/ Consumption of

Edible oils from Domestic and

Import Sources

142.62

166.39

-

-

-

167.53

Table 3.1 Estimated domestic production, import and total availability of edible

oils (Oil-wise) during the years 2007-08, 2008-09 and 2009-10 (November to October) Source: MOFPI, Govt Of India. (Qty in million tonnes)

3.5 Regulatory Guidelines, Nutritional Value and Groundnut Oil Processing

Directorate of vanaspati, vegetable oils and fats (DVVO&F) Ministry of

Consumer Affairs, Food and Public Distribution, GOI monitors the quality of edible

oils and fats including groundnut oil in India. The regulatory functions such as

approval limits for production, manufacture of vegetable oil products supply, import

and export of vegetable oil including purchase of raw material, verification of stocks,

labeling requirements, laboratory facilities and drawal of samples of vegetable oil

products for checking, its conformity with prescribed standards, permission to process

oil bearing material for export of deoiled meal, directing any producer or registered

user to maintain records of solvent extracted oils etc. are performed basically through

the following three orders administered by the DVVO&F:- i) Edible Oils Packaging

(Regulation) Order, 1998; ii) Vegetable Oil Products (Regulation) Order, 1998; and

iii) Solvent Extracted Oil, De-Oiled Meal and Edible Flour (Control) Order, 1967

(DVVO & F, Ministry of Consumer Affairs, Food and Public Distribution, GOI,

2010).

Groundnuts (as the seeds develop beneath the ground), the seeds of the plant

Arachis hypogaea L. contain 40–50% oil, which is expressed from the seeds of the

plant. The groundnuts are prepared for the oil extraction process by being shelled and

cleaned. Simple application of some type of pressure extracts the oil, which is filtered.

Hence, groundnut oil generally requires limited refining, and hydrogenation is not

usually a requisite for the majority of groundnut oil users (Sanders, 1982). Variability

in oil quality was observed in four groundnut mutants (TG-1, TG-3, TG-14, TG-t6)

induced by y-irradiation. The fatty acid composition of the mutants differed from their

parent Spanish improved. All the mutants had lower linoleic and higher oleic acid

than the parent Spanish improved. TG-3 and TG-14 had substantially higher linoleic

acid and lower oleic acid as compared with TG-1 and TG-16. Other fatty acids also

showed minor variation amongst mutants and Spanish improved. The ratio of oleic to

linoleic acid, which is an index of oil stability, was 2.7 and 3.3 fold respectively in

TG-16 and TG-1 as compared with Spanish improved. Protein percent in TG-1, TG-

14 and TG-16 was higher than in Spanish Improved. Amino acid analysis of protein

from mutants indicated decreases in methionine and cystine and an increase in

tryptophan as compared with Spanish improved. Other amino acids did not show any

major variation between the mutants and Spanish improved. In all varieties except

TG-16, lysine was the first limiting amino acid; threonine was second limiting in all

mutants. The essential amino acid content per kernel was higher in mutants than in

Spanish improved (Sharma et al., 1981). Caja et al. (2000) analyzed the volatile

compounds in edible oils using simultaneous distillation-solvent extraction and direct

coupling of liquid chromatography with gas chromatography. They are of the opinion

that the aroma plays an important role not only in the overall quality of the product

but also in its consumer acceptance. Though many methods have been developed for

isolation of volatile compounds from foods, analyzing oil volatile compounds is

difficult because their fat solubility resulting significant reduction of their isolation

(Caja et al., 2000).

Aluyor et al. (2009) studied the effect of refining on the quality and

composition of groundnut oil. They found that the vegetable oil, groundnut oil

contains only a small proportion of non-glyceride constituents. Additional compounds

such as diglyceride, tocopherols, sterols and sterol esters need no removal during

processing. The compounds such as phosphatide, free fatty acids, odiferous volatiles,

colorant, waxes as well as metal compounds negatively affecting taste, smell

appearance and storage stability of the refined oil needs their removal. The specific

gravity of groundnut oil decreases from 0.916 to 0.915 after refining. The decrease

was not significant hence refining does not have significant effect on the specific

gravity. They also noted that refining only slightly affects the physical and chemical

characteristics of groundnut oil. There was decrease from the crude form to the

refined form value. The major and minor constituents, refining did not have much

effect on the fatty acid except for some slight inconsistent decrease in saturated and

unsaturated fatty acids.

3.6 Aflatoxin: Types and Significance

The secondary metabolite mycotoxins produced by fungi in food and feed,

which, upon ingestion, can result in severe diseases of animals, including humans;

these pathologies are commonly referred to as mycotoxicosis, and are classified

according to their symptoms (Cuccioloni et al., 2009). Amongst mycotoxins,

aflatoxins continue to receive major attention as they demonstrated to be potent

genotoxic carcinogens in susceptible laboratory animals and because of their acute

toxicological effects in humans and farm animals that produce economic losses (Rojas

et al., 2005). These aflatoxins have turn out to be recognized as ubiquitous

contaminants of the human food supply throughout the economically developing

world since their discovery 50 years ago. The public health impact of aflatoxin

exposure is omnipresent. Reddy and Raghavender (2007) summarized the various

outbreaks of aflatoxicosis reported throughout India (Table 3.2) in which either the

raw material groundnut or the byproduct groundnut cake or groundnut oil was

implicated as a causative agent.

Type of

Aflatoin

Commodity

Fungus Organism

affected

Symptoms

Location

Year

Aflatoxin

B1, B2

Maize,

Groundnut

A. flavus,

A. parasiticus

Humans,

Dogs,

Cirrhosis in

children

Rapidly

developing ascites,

oedema of the

lower limbs, portal

hypertension,

higher mortality

rate

Banswada and

Panchmahals

districts of

Rajasthan and

Gujarat

respectively

1974,

1975

Children

Hepatomeagaly

South Canara

district of

Karnataka

974,

1976

Ranga Reddy

district,Andhra

Pradesh

994

Chickens/

fowls

High mortality Chittoor district,

Andhra Pradesh

1982

Groundnut

cake

Fatty liver

syndrome

Warangal,

Andhra Pradesh

985

Groundnut

cake

Mortality in

younger chicks

Andhra Pradesh 965

Mysore

Karnataka

966

Aflatoxin

M1

Buffalo milk

Murrah

buffaloes,

humans,

ruminents

Post-mortem

lesions and liver

damage,

subcutaneous

hemorrhage

Kakinada,

Andhra Pradesh,

Mysore,

Karnataka

1973,

1966,

1969

Table 3.2 Summary of various aflatoxicosis outbreaks reported in India (Reddy

and Raghavender, 2007)

In the word aflatoxin, the first syllable “a” was derived from the genus

Aspergillus, the second one, “fla”, from the species flavus, and the term, “toxin”,

came from the adjective “toxic”. In the aflatoxin group, about 16 compounds are

known, but only aflatoxin B1, B2, G1 and G2 (Table 3.3) are chemical derivatives of

difurancoumarin (Papp et al., 2002). Hartley et al. (1963) examined fluorescence and

other properties of these A. flavus toxic metabolites and identified them as aflatoxins

B and G. The identification of aflatoxins B1, B2, G1 and G2 is based on their blue or

green fluorescent colour under ultraviolet light and differences in melting point. Most

naturally occurring aflatoxins consisted mainly of B1 (AFB1) with some B2. The

major aflatoxins comprising aflatoxins B1, B2, G1 and G2 are produced by selected

isolates (not all isolates are toxicogenic) of either A. flavus or A. parasiticus. Rasooli

et al. (2004) described the rapid growth of the secondary metabolite aflatoxins

producing toxigenic strains of A. flavus as well as A. parasiticus on a variety of

natural substrates and consumption of contaminated food can pose serious health

hazards to human and animals. These aflatoxins are primarily hepatotoxic or cause

liver damage in animals; aflatoxin B1 is the most potent. The susceptibility for them

varies with breed, species, age, dose, length of exposure and nutritional status

(Richard, 2007).

3.7 Aflatoxin – A Hazardous Contaminant of Foods and Feeds

Aflatoxins, metabolic products of the molds A. flavus and A. parasiticus, could

occur in food and feeds. They greatly affect the food and feed industry because they

are highly toxic and carcinogenic in a variety of animal species (Woloshuk and Prieto,

1998). Because of the current agronomic and manufacturing processes, these toxins

possibly will not be entirely avoided or eliminated from foods and feeds, so are

considered unavoidable contaminants (Wood, 1989).

STRUCTURE OF AFLATOXINS

AFLATOXIN B1 B2 G1 G2 M1 M2

Molecular Formula C17H12O6 C17H14O6 C17H12O7 C17H14O7 C17H12O7 C17H14O7

Molecular Weight 312.27358 [g/mol] 314.28946 [g/mol] 328.27298 [g/mol] 330.28886

[g/mol]

328.27298 [g/mol] 330.28886 [g/mol]

2D Structure

3D Structure

Table 3.3 Molecular formula, molecular weight and structures of aflatoxins

29

30

3.8 Aflatoxins in Food

Food contaminated with very small quantities of aflatoxins can render it unfit

for animal or human consumption. Buchanan et al. (1975) experiments showed the

A. flavus infection and aflatoxin production in fig fruits. According to them, the

aflatoxin contamination has been prevalent in plant seeds where A. flavus is a

facultative parasite often preferentially colonizing embryos. Klich and Pitt (1988)

noted that aflatoxins are mycotoxins produced by contaminant molds like A. flavus,

A. parasiticus, A. nomius, they most frequently invade corn, peanuts, cottonseed,

almonds, cashews and pistachio nuts, though, not all strains are able to produce

aflatoxin. Wilson and Payne (1994) showed that 77% contamination of grains was

due to aflatoxin B1, while the rest with AF types. Guerzoni et al. (1999) reported the

aflatoxin contaminating molds isolation even from processed food products such as

bread, macaroni, cooked meat, cheese and flour. A. flavus is one of the storage fungi

that develop on a wide variety of stored grains such as wheat, peanuts, soybeans, and

corn (Suberu, 2004). A. flavus and A. parasiticus cause preharvest rots on corn,

peanuts, various tree nuts, cotton. They not only cause disease, but also have ability to

contaminate the food with AF. Toxic secondary metabolites mycotoxins can be

present in a wide range of food and feed commodities including cereal grains, oil

seeds, dried fruits, apple juice, wine and meat products and animals feeds (Adanyi

et al., 2006). Rice has been contaminated with mycotoxin such as aflatoxins B1, B2,

G1, G2, citrinin, deoxynivalenol (DON), fumonisin B1, B2, B3 (FMS), fusarenon-x

(fus.-x), nivalenol (NIV), ochratoxin A (OTA), sterigmatocystin (STE), and

zearalenone (Tanaka et al., 2007). Also aflatoxin (AF), the naturally occurring

aflatoxins B1, B2, G1 and G2 ( AFB1, AFB2, AFG1, AFG2 ) and ochratoxin A (OTA)

have been found in ginseng, ginger, dicorice, turmeric and kava-kava which are used

for the determination of activity (Truckesess et al., 2008). The main toxin aflatoxins

produced by the A. flavus, A. parasiticus, A. nomius group of fungi could be found in

corn, cotton seed, peanuts and other nuts, and spices. Aflatoxins can be controlled by

co-storing whole sweet basil leaves with aflatoxin-infected foods. The economic

value of the study lies in the simplified technique for control of aflatoxins

contamination in agricultural products and the benefits derivable from the use of local

resources (Atanda et al., 2005). The aflatoxin B1 could be found in hazelnuts, walnuts,

31

peanuts, and almonds except roasted chickpeas in high levels, a major public health

concern requires investigations into the reasons for these high levels and means of

minimizing or eliminating them (Gurses, 2006). Rajasinghe et al. (2009) studied

aflatoxigenic A. flavus and aflatoxin formation in selected spices during storage. Since

they isolated toxigenic A. flavus from all spice sample tested, they advised to store the

spices such as chilli powder, turmeric, pepper powder etc, only for a relatively short

period to avoid build up of aflatoxins.

Aspergillus spp. are the most common among toxigenic fungi affecting the

food chain. These fungi infect commodities such as corn, peanuts, cotton, tree nuts,

sorghum and other oil seeds. When one of tree nuts, peanuts, and other oilseeds such

as corn and cottonseed crops are infected with toxigenic strains of the A. flavus,

contamination by aflatoxins occurs under certain environmental conditions (Liang

et al., 1997). Specifically A. flavus is of enormous importance due to its bearing on

agriculture and human health. Aflatoxin contamination of crops can also result in

significant economic losses to both producers and processors in the USA and

worldwide (Duran et al., 2007). When the dry-roasted groundnut samples purchased

from street hawkers, markets and retail shops in southwestern Nigeria were analyzed

for moisture content, fungal populations and aflatoxin contamination, 2.9 × 102

to 6.3

× 102

colony-forming units of mould counts were obtained for the moisture content

2.1 to 3.6 percent. Aflatoxin B1 was found in 64.2 percent of samples with a mean of

25.5 ppb. Aflatoxins B2, G1 and G2 were detected in 26.4, 11.3 and 2.8 percent of the

samples with mean levels of 10.7, 7.2 and 8 ppb respectively in contaminated samples

(Bankole and Eseigbe, 2004). Due to large-scale observation, some African

governments have insisted food safety regulations to control mycotoxin, especially

aflatoxin, contamination of the national food supply and, research into natural

occurrence of aflatoxins in a range of local foods is widely conducted. It emphasizes

that much of the research effort has been performed in South Africa, Egypt and in

various countries in West Africa including Ghana, Nigeria and Gambia. Levels of

aflatoxin contamination in staple foods such as maize and groundnuts, other

particularly local foods such as cured and smoke-dried fish have been implicated as

sources of dietary aflatoxin in various areas of Africa. Aflatoxin exposure remains an

important aspect of food safety, which needs to be addressed by African communities

32

(Shephard, 2003). Aflatoxins have adverse effects on humans, animals and crops that

result in illnesses and economic losses. The largest risk of aflatoxins to humans is

usually as the result of chronic dietary exposure. Such dietary aflatoxin exposures

have been associated with human hepatocellular carcinoma. Therefore, it is important

to prevent the pre- and post-harvest mould contamination and growth (Gurses, 2006).

Since aflatoxin B1 is the most potent naturally occurring carcinogen in animals and

several healths, economic, industrial, and environmental problems are associated with

their crop contamination. For example, epidemiological studies implicated aflatoxin

as one of the most important risk factors associated with the incidence of

hepatocarcinoma and lung cancer in humans (Roze et al., 2011).

3.9 Animal Feed Contamination

The presence of aflatoxins is a problematic one in animal husbandry also.

When cows are fed aflatoxin contaminated feed, a hydroxylated derivative of

aflatoxin B1 is found in the milk named aflatoxin M1 (AFM1) (Hartley et al., 1963).

Veldman et al. (1992) during their examination found the carryover of aflatoxin from

feed (AFB1) into cow‟s milk (AFM1) and concluded that maximum levels of AFB1 in

feed should be set to limit AfM1 in milk. Feeds and forages for ruminant animals with

protein supplements such as cottonseed cake and groundnut meal likely to contain

mycotoxins, the aflatoxins need careful attention, since at least two of these (AFB1

and AFM1) are endowed with carcinogenic properties with dangers for the human

consumer (D‟Mello and Macdonald, 1997). In 1997, Smith noted the synthesis of

cyclopiazonic acid, a potent mycotoxin by A. flavus that is generally accepted only as

the producers of AFB1 and AFB2. According to Gowda et al. (2004) “among the

different types of aflatoxins produced, aflatoxin B1 as both the most prevalent and

potent and is often found in high concentrations in peanut meal and cereal grains”.

Ingestion of aflatoxin contaminated feeds not only affects the animal health and

production but is potentially dangerous to human as the toxin metabolites are excreted

in animal milk, meat and eggs. The aflatoxins can pass through the food chain from

animal feeds into milk as aflatoxin M1, after fewer metabolisms in the cow. Allowed

concentrations in raw milk are usually <100 ng/l in Europe but may be > 1000 ng/l in

other parts of the world such as India and Equador (Moss, 2002).

33

Diaz and Espitia (2006) found that the level of AFM1 contamination in the

milk sample ranged from 10.7 to 213.0 ngl-1

in 2004 and from 10.6 to 288.9 ngl-1

in

2005. They noticed contamination of most of the analyzed milk samples, although at

very low levels, suggests that only a few contaminated samples entering bulk milk

supply. Therefore, they recommended a continuous monitoring of AFM1 levels in

milk is necessary in order to ensure that milk lots do not exceed the maximum

regulatory levels of 400 ngl-1

. Due to carcinogenicity, AFM1 compound contaminated

milk can be a major public health concern. AFM1 residues in milk are regulated in

many parts of the world and can cost dairy farmers significantly due to lost milk sales

(Masoero et al., 2007). The hydroxylated metabolite of aflatoxin B1, AFM1 might be

found in milk and dairy products such as cheese. A study conducted to determine the

AFM1 levels in Turkish white, kashar and tulum cheese found that 51.3 % of them

were found to be contaminated (Hampikyan et al., 2010).

3.10 Aflatoxin Contamination of Edible Oils

Woloshuk and Prieto (1998) observed the common occurrence of aflatoxins in

oil seed crops such as corn, cotton, peanuts and tree nuts prior to harvest. Olive oil

can be significantly contaminated by mycotoxins and confirm that a scrupulus

application of European regulation 1019/2002 (European commission, 2002), which

prohibits the sale of non-labeled olive oil, is strongly recommended. Conventional

qualitative parameters such peroxide number, spectrophotometric evaluation and acid

values were not correlated with mycotoxin occurrence. OTA was detected with high

frequency (80%) in samples from both geographical areas (up to 17.0 µgg-1

). While

AFB1 was found from three of four samples from North Africa (up to 2.4 µgg-1

). In

addition „not labeled‟ oil samples proved to be more contaminated by OTA then

„labeled‟ samples (mean values of 2.47 and 0.66 µgg-1

, respectively) (Ferracane et al.,

2007). Idris et al. (2010) documented the first data on the determination of the

aflatoxin levels in Sudanese edible oils. They found that majority of Sudanese edible

oil samples used in their study are safe.

3.11 Groundnut Oil Contamination

One of the problems with groundnuts and groundnut oil production is the

potential for contamination with aflatoxin. The aflatoxigenic A. flavus is a strict

34

saprophyte and hence the faulty or inadequate methods of handling, drying or storage,

permitting fungus colonization of the dried commodity (Buchanan et al., 1975) like

groundnut leading to their toxin contamination. The concentrations measured toxins

are usually quite low in peanuts produced from developed countries, where control

and testing programmes are in place. However, this is not always the case in many

groundnut oil producing and consuming developing countries. Aflatoxin is usually

found in the protein component of groundnuts and therefore not usually found in

refined oil (Foster et al., 2009).

These are very potent acute poisons to animals, and humans, aflatoxins

pose the most serious problem in groundnut, but they can occur in all of them

(Mirghani et al., 2001). Tiger nuts meant for human use oil production are also one of

the commodities susceptible to aflatoxin contamination (Arranz et al., 2006). A. flavus

and A. parastiticus have a particular affinity for nuts and oil seeds. Pre-harvest

infection of fruits by Aspergillus spp. is a serious problem. Aspergillus species are

regarded as weak plant pathogens. Systemic infection of developing peanut plant by

A. flavus has been reported. The plant infection by Aspergillus could probably be

initiated frequently by pre-harvest penetration by the fungus. These fungi generally

infect plant hosts wounded by insects or other agents. Penetration of healthy tissue of

plants causing systemic infection has been demonstrated under conditions of stress

e.g. drought (Gugnani, 2003). The contamination with Aspergillus spp of dried fruits

such as peanut, hazelnut, walnut and almond occurs generally during harvest,

processing and storage. Although these types of hard crust fruits are less susceptible

to mould contamination, aflatoxin formation still perhaps seen (Gurses, 2006).

Panhwar (2005) studied anti-nutritional factors in oil seeds such as aflatoxin in

groundnut. Anti-nutritional factors are those substances found in most foods, and they

are poisonous or in some way limit the nutrients available to the body. Anti-

nutritional substances destroy some vitamins in food. According to him, the aflatoxin

found in them is a poisonous toxin. Jolly et al. (2006) also found these most potent

carcinogenic aflatoxins in staple foods such as groundnuts, maize and other oil seeds.

Aflatoxin contamination of peanut is one of the most important constraints to peanut

production worldwide (Sandosskumar et al., 2007).

35

Diener et al. (1987) determined that aflatoxin contamination of peanuts

(A. hypogaea L.) results from growth in peanut kernels by toxigenic strain of the

fungi, A. flavus Link and A. parasiticus Speare. Since contaminated lots of peanuts

could not be used for human consumption, they represent great economic losses for

the peanut industry. Owing to aflatoxins potent hepatotoxic and carcinogenic nature,

their presence in peanuts is heavily monitored and regulated to ensure a safe food

supply. Cole et al. (1995) noted that aflatoxin contamination of peanuts could occur in

the field (preharvest) when severe late season drought stress occurs and during storage

(post-harvest) when improper conditions of moisture and temperature exist. In 2002,

Dorner took the commission of simultaneous quantitation of A. flavus/A. parasiticus

and aflatoxins in peanuts. Infection of peanuts by A. flavus and A. parasiticus can

occur in the field as peanuts are forming and maturing, but he concluded that infection

does not necessarily result in aflatoxin contamination. According to Gurses (2006)

adverse pre-harvest conditions of temperature and humidity in the field, improper

post-harvest handling and storage are the main causes of contamination. The

prevention of contamination with toxigenic fungi of foods during harvest, processing

and storage is the best way to control aflatoxin formation. The mold growth and toxin

formation may be significantly limited by packaging, removal of the damaged and

moldy fruits and mechanical drying prior to storage. Idris et al. (2010) survey on fifty-

six samples of groundnut, sesame and cottonseed oils form factories, and traditional

mills collected from several localities in Kordofan, Gezira and Khartoum states,

Sudan and assessed for AFB1, AFB2, AFG1 and AFG2 using high performance liquid

chromatography (HPLC) detected only aflatoxin B1. It was found in eight samples,

corresponding to 14.3% of the total samples analyzed and found in the range of 0.2-08

mg/kg. These results indicate that unrefined oils were more susceptible to aflatoxins

contamination. These were confirmed using corn oil obtained from corn germ

deliberately contaminated in the laboratory with A. flavus. The detected aflatoxin B1

level in this study was found to be lower than the legal limit that regulated in Sudan

(Idris et al., 2010). Despite improved handling, processing and storage, the toxin

contamination remains as a problem in the peanut industry causing huge economic

losses like in many other agricultural crops. Therefore, new ways of detoxification

produce limiting economic/health impacts and adding values to the peanut industry

are needed (Proctor et al., 2004).

36

3.12 Factors Influencing Fungal and Aflatoxin Contamination

Proctor et al. (2004) found that under certain favourable conditions of

temperature and humidity A. flavus and A. parasiticus grow on particular foods and

produce aflatoxins. The major aflatoxins of health concern are aflatoxins B1, B2, G1

and G2 with B1 being the most toxic. The nitrogen and simple sugar organic sources

favors aflatoxin production, while inorganic nitrogen source and complex

carbohydrates retard their biosynthesis. Also the optimal growth temperature of 370C

inhibits aflatoxin biosynthesis completely, while 28o C allows production. The growth

optimal pH 4.5 is conducive for aflatoxin biosynthesis whereas pH 8.0 inhibits its

production.

Parra and Magan (2004) made a detailed modeling study on the effect of

temperature and water activity on growth of A. niger strains and applications for food

spoilage moulds. The most important environmental parameters that determine the

ability of moulds to grow on foods are water activity (aw) and temperature (T). The

minimal water activity was obtained with species of A. flavus, A. niger, A. ochraceus

and A. parasiticus at 250c. Moreover, good agreement was found with A. candidus,

A. nidulans, A. versicolor at 300c and Eurotium ripens at 35

0c.

The soil moisture and soil temperature were also have an effect on pre-harvest

infection with A. flavus and production of aflatoxin. Average air and soil temperatures

of 28-34o

C were favorable for aflatoxin contamination. Infection and aflatoxin

concentration in peanut can be related to the occurrence of soil moisture stress during

pod-filling when soil temperatures are near optimal for A. flavus (Craufurd et al.,

2006). Yu et al. (2007) noted that many internal and external factors, such as nutrition

and environment affect aflatoxin biosynthesis. When the grains such as corn grows

during warm ambient temperature, especially noted during drought conditions, the

grain becomes more susceptible to aflatoxin formation (Richard, 2007). Fluctuations

in climate also influence predisposition of hosts to contamination by altering crop

development and by affecting insects that create wounds on which aflatoxin-

producers proliferate. Aflatoxin contamination is prevalent both in warm humid

climates and in irrigated hot deserts. In temperate regions, contamination may be

severe during drought. The contamination process is frequently broken down into two

37

phases with the first phase occurring on the developing crop and the second phase

affecting the crop after maturation. Rain and temperature influence the phases

differently with dry, hot conditions favouring the first and warm, wet conditions

favouring the second. Contamination varies with climate both temporally and

spatially (Cotty and Jaime-Garcia, 2007).

3.13 Aflatoxin – A Health Hazard

Sargeant et al. (1961) described the first demonstrated case of mycotoxicosis

is in Great Britain in 1960 where more than 100,000 turkeys died of severe liver

necrosis and biliary hyperplasia caused by feed Aspergillus aflatoxins contaminated

peanut meal imported from Brazil. The research that followed this event eventually

led to the identification and isolation of the aflatoxins (D‟Mello and Macdonald,

1997).

Maron and Ames (1983) explained that these carcinogens have specific

properties related with DNA. They are mutagenic in the classic S. typhimurium,

involving in DNA repair in freshly explanted hepatocytes.

The AF is biotransformed in the liver by monooxygenases, where the

cytochrome P450 turns them into an electrophilic highly active compound known as

aflatoxin 8, 9 epoxide to be then conjugated with glutathione and excreted through

bile and urine (Wilson and Payne, 1994).

AFB1 has been shown to produce G-T transversions at codon 249 of P53

tumor

suppressor gene, whose altered sequence has been associated with a number of human

cancers (Woloshuk and Prieto, 1998).

Guerzoni et al. (1999) referred that aflatoxins are a particular concern for

populations with a high incidence of hepatitis B because the relative rate of liver

cancer in people with hepatitis B is up to 60 times greater than normal when those

peoples are exposed to aflatoxin.

Quezada et al. (2000) studied the effects of AFB1 on the liver and kidney of

broiler chickens during development. They found the negative influence of AFB1 on

chicken growth. The aflatoxin was found to be firstly as a hepatotoxin causing an

38

excessive build up of hepatic lipids with enlargement of liver, proliferation of biliary

ducts, hepatocellular carcinoma. Secondly, aflatoxin causes enlargement of renal

tissue and alternation of its function.

These best known hepatocarcinogen AFB1 also targets respiratory system.

AFB1 is bioactivated by cytochromes P450, primarily in nonciliated bronchiolar

epithelial (Clara) cells. The DNA binding AFB1 epoxide metabolite can leave the

cells of origin, and potentially interact with other cell types. AFB1-induced AC3F1

mouse lung tumors contain point mutations at guanine residues in K-ras, with the

anticipated bias for the Alf allele. Following AFB1 treatment but prior to tumor

development, K-ras mutations occur preferentially in mouse Clara cells. Over

expression of p53

protein as well as p53

point mutations, suggesting a carcinogen-

specific response. Human peripheral lung bioactivates AFB1 primarily by

prostaglandin H synthase and/or lipoxygenase catalyzed cooxidation, with activity

concentrated in macrophages. In addition, although glutathione S-transferase M1-1

has high specific activity for AFB1 epoxide conjugation, lung tissues from GSTM1-

null individuals do not demonstrate diminished rates of conjugation, compared to

tissues from GSTM1-positive individuals. AFB1 tumorigenesis in mice demonstrates

unique properties, and processes of bioactivation show significant species differences

(Massey et al., 2000).

These known mutagenic, carcinogenic and teratogenic aflatoxins compounds

intake over a long period in very low concentrations may be highly dangerous. After

ingestion, aflatoxin B1 is metabolized by enzymes to generate a reactive 8, 9-epoxide

metabolite that can be bound to DNA as well as to serum albumin forming aflatoxin-

Nt- guanine and lysine adducts, respectively. Covalent binding to DNA is considered

a critical step in aflatoxin hepatocarcinogenesis (Papp et al., 2002).

Sun and Chen (2003) carried out an experiment on aflatoxin-induced

worldwide common cancer hepatocarcinogenesis for which Hepatitis B virus (HBV)

and aflatoxin exposure represent the main risk factor. Metabolic activation of AFB1

has been studied using human cell lines that expressed individual cytochrome P450S.

Agnes and Akbarsha (2003) predicted the effect of AFB1 on sperm with the

background that the food borne mycotoxin AFB1 could be toxic to the male

39

reproductive mechanism in man as well as wild and domestic animals. They found

that the fertility of the aflatoxin treated mice was reduced drastically. Sperm

concentration in the epididymis and motility decreased whereas sperm abnormalities

increased. In particular, sperm abnormalities like two axonemes in a common,

sticking together of heads/tails, etc., were noted. A higher percentage of cauda

epididymidal spermatozoa than in the control mice retained the cytoplasmic droplet

(CD) and such retention was dependent on the duration of the treatment. Spermatozoa

retaining the CD were inhibited in motility. Sperm CD of AFB1 treated mice

contained electron-dense spherical inclusions, which are hypothesized as lipid

inclusions produced from the lamellae through the spherical vesicles of the CD.

Hence, they observed disruption of AFB1 mediated spermatogenic as well as

androgenic compartments of the testis.

Bingham et al. (2004) described that aflatoxins are structurally similar group

of naturally occurring harmful fungal byproducts that are strongly implicated in

diseases and death of man and animals. These aflatoxins left substantial agricultural

and economic losses due to their potent liver carcinogenicity. Existing considerable

evidence indicates that low-level aflatoxin exposure could suppress the immune

system and increase the disease susceptibility. Exposure during pregnancy has

resulted in transplacental transfer of aflatoxin to and immune dysfunction in the

offspring.

These potent hepatocarcinogenic aflatoxins, especially AFB1 induce liver

tumors in many species of animals, including rodents, nonhuman primates, and fish.

Human primary liver cancer, mainly hepatocellular carcinoma, is one the most

common diseases in Asia, Africa, and in populations of Asian and Hispanic-

Americans (Wang and Tang, 2004).

Aflatoxin B1 (AFB1) is a highly toxin and carcinogenic metabolite produced

by Aspergillus species on agricultural commodities (Rasooli et al., 2004).

The worldwide harmful effects of aflatoxins on animals and humans have

been well documented (Juvvadi and Chivukula, 2006).

40

Most of mycotoxicosis occur after consumption of mycotoxin contaminated

grain or products made from such grains but other routes of exposure exist. The

diagnosis of mycotoxicosis may prove to be difficult because of the similarity of signs

of disease to those caused by other agents. Therefore, diagnosis of a mycotoxicosis is

dependent upon adequate testing for mycotoxins involving sampling, sample

preparation and analysis (Richard, 2007).

Excretion of aflatoxins in the milk of exposed lactating mothers in the form of

AFM1 was observed in breast milk from a group of Egyptain mothers attending the

New E1-Qalyub Hospital, Qalyubiyah governorate, Egypt (Polychronaki et al., 2007).

These toxins are hepatocarcinogens and mutagenic agents and in 1993, they were

declared as Group 1 carcinogens by the International Agency for Research on Cancer

(IARC) (Amadasi et al., 2007).

AFB1, a metabolite produced by A. flavus and A. parasiticus, is a known

causal agent of human HCC (Yen et al., 2009). A number of epidemiological

evidences are currently available for human HCC. AFB1 is the most carcinogenic

aflatoxin, and the understanding of its metabolism was essential in the molecular

epidemiology studies that established this relationship. AFB1 has been reported to

form adducts with DNA, thus being crucial in the development of extra-hepatic

cancers. In this regard, early works implicated AFBl in the development of lung

cancer (Cuccioloni et al., 2009).

3.14 Aflatoxin Toxicity Evaluation

Tung et al. (1975) explained the inhibition of protein synthesis in the liver and

plasma proteins as well as lipoproteins decrease during aflatoxicosis. Inhibition of

plasma enzyme activities has also been reported. Serum proteins, SDH and Glu DH

are sensitive early indicators of toxicity that was more severe in developing chickens.

They suggested that early and suitable indicator of the deleterious effect of this

mycotoxin in developing chickens would be decrease in serum albumin.

The excretion of aflatoxin residues in quail eggs might occur at relatively low

concentrations under conditions of long-term exposure of quail to low levels of AFB1.

Evaluation of the excretion of residues of AFB1, AFM1, AFB2a and aflatoxicol (AFL)

41

in eggs of laying Japanese quail fed rations with low levels of aflatoxin B1 for 90 days

revealed that average egg production and feed consumption were not affected

(P<0.05) by AFB1. Egg weight was significantly lower (P<0.05) only for groups

exposed to 100 µg AFB1 kg-1

. Residues of aflatoxins were detected in eggs at levels

that ranged from 0.01 to 0.08 µgkg-1

(AFB1), 0.03-0.37 µgkg-1

(AFM1), 0.01-1.03

µgkg-1

(AFB2a) and 0.01-0.03 µgkg-1

(AFL) (Oliveira et al., 2000).

Liu et al. (2006) utilized the Ames test, employing S. typhimurium tester

strains TA98 and TA100 to evaluate the residual toxicity of the AFB1 sub products in

peanut oil. The results indicated that the mutagenic activity of UV-treated samples

(800 µw/cm2

x 30 min) was completely lost compared with that of untreated samples,

providing clues to the assessment of safety issues of UV method applied in AFB1

decontamination.

Celik et al. (2000) employed modified chick embryotoxicity screening test

(CHEST) determine the embroyotoxicity of mixed AF and AFB1. They also

investigated their adverse effects on the early embryonic development of thymus and

bursa of Fabricius by light microscopy. The higher doses of both AF and AFB1

caused higher embryonic mortality and an increase in early deaths. In the groups

received 100 ng/egg AF and AFB1, an abnormal development was seen, with a

protruded central region, corresponding to the area pellucid of the blastoderm. No

other developmental abnormality attributable to AF or AFB1 was found.

Richard (2007) reported that, “During the late 1800s and early 1900s there

was considerable recognition of the ability of fungi to carry out fermentations and a

number of investigators recognized the myriad of “Secondary metabolites” produced

by fungi in both solid state and liquid fermentations. Because a few of the products of

such fermentations were consumed by humans, some interest, in the toxicity of these

products was developed.

3.15 Research on Aflatoxin Adverse Effect Amelioration

The effects of dietary administration of 3,5-di-tert-butyl-4-hydroxytoluene

(BHT), 2(3)-tert-butyl-4-hydroxyanisole (BHA), ethoxyquin (EQ) and

5-(2-pyrizinyl)-4-methyl-1,2-dithiol-3-thione(oltipraz) on aflatoxin B1 (AFB1)-DNA

42

adduct formation in vivo in livers and kidneys of rats were investigated. An excellent

correlation (r = 0.95) between the degree of inhibition of DNA binding by AFB1 and

the induction of hepatic glutathione S-transferase activities by four antioxidants was

observed. Male F344 rats were treated with 1mg/kg AFB1 by i.p. administration and

nucleic acids isolated 2 h post dosing. Animals were fed a semipurified diet

supplemented with either 0.5% EQ, 0.45% BHT, 0.45% BHA or 0.1% oltipraz for 2

weeks prior to AFB1 treatment. Analysis of nucleic acid bases by HPLC showed that

several AFB1 metabolite-DNA adducts were formed in both tissues. The principle and

related adducts of 8, 9-dihydro-8-(N7-guanyl)-9- hydroxyaflatoxin B1 represented

~80-90% of all adducts in both tissues and in all treatment groups. However,

inclusion of the antioxidants in the diet resulted in substantial reductions in overall

AFB1 modified DNA levels. EQ, BHT, BHA and oltipraz reduced the covalent

binding of AFB1 to liver DNA by 91, 85, 65 and 76% and to kidney DNA by 80, 35,

62, and 64% respectively. The specific activities of hepatic enzymes of

presumed importance to AFB1 detoxification, epoxide hydrase, and glycuronyl and

glutathione transferases were significantly elevated by all antioxidants. Reduced

glutathione levels were unchanged except by olitipraz, although activities of enzymes

contributing to the maintenance of reduced glutathione pools, glutathione reductase

and glucose-6-phosphate dehyrogenase were elevated in most treatment groups

(Kensler et al., 1985).

A dramatic effect of grapefruit juice in blocking the oxidation of

dihydropyridine calcium channel blockers was studied. The flavonoid naringin is the

most abundant natural product specific for grapefruit and related citrus- the aglycone

naringenin, known to be readily formed from naringin in humans, was found to inhibit

the oxidation of the dihydropyridines nifedipine and felodipine in human liver

microsomal preparations. The human liver cytochrome p450 (IIIA4) appears to be a

major catalyst in both nifedipine oxidation and aflatoxin B1 activation. Several

flavones inhibited the in vitro activation of alfatoxin B1 in a system employing umuC

gene activation due to DNA damage in S. typhimurium TA1535/PSK1002, with

naringenin being as effective as any. The high concentration of derivatives of

naringenin in certain citrus fruits may be of relevance to cancer chemoprevention

43

involving those carcinogens that are activated by cytochrome P450IIIA4 (Guengerich

and Kim,1990).

A range of potential chemoprotective agents, most of them natural dietary

constituents, has been examined for ability to modulate both phase I (cytochrome

P450 1A1, 1A2, 2B1/2, 2C11, 2E1, 3A 4A) and phase II drug metabolizing enzymes

(glutathione S-transferases, in particular subunits Yc2 and p, aflatoxin B1-aldehyde

reductase and quinone reductase) in rat liver. In addition to assays of total enzyme

activity and western blots for individual isozyme, the ability of microsomes to

metabolize aflatoxin B1 and of cytosols to conjugate aflatoxin B1 (AFB1)-epoxide to

GSH and to produce AFB1-dialcohol, were measured. Induction of y-glutamyl

transpeptidase activity was examined by histochemistry. Differing patterns of

induction were observed, reflecting differences in the control of expression of the

individual enzymes studied. Of the compounds examined, butylated hydroxytoluene,

ethoxyquin, indole-3- carbinol and phenethyl isothiocyanate were the most potent

bifunctional agents (inducing both phase I and II activities). Oltipraz, while only

weakly inducing CYP1A2 and 2B1/2 was a potent inducer of phase II enzymes.

Caffeic acid, garlic oil, sinigrin and propyl gallate all showed some ability to induce

phase II enzymes. 4- Methyl catechol, a-tocopherol and red wine decreased certain

phase I enzyme activities, while inducing total GST activity. Butylated

hydroxytoluene, ethoxyquin, garlic oil and indole-3-carbinol induced gamma

glutamyltranspeptidase in periportal hepatocytes. Particularly because of their ability

to induce the detoxifying activities of glutathione S-transferase Yc2 and aldehyde

reductase, butylated hydroxytoluene, ethoxyquin, indole-3-carbinol, oltipraz,

phenethyl isothiocyanate and sinigrin will be effective blocking agents in rodents, if

administered prior to AFB1 (Manson et al., 1997).

The effect of the active principles in garlic–dially sulfide (DAS) and diallyl

disulfide (DADS)–on AFB1 induced DNA damage in primary rat hepatocytes was

investigaed. The results of LDH leakage, 0.5 and 2 mM of DAS or 0.5 and 1 mM of

DADS significantly increased the viability of hepatocytes compared with the AFB1

controls after 4, 8 and 24 h treatment (P<0.05). According to the results of

unscheduled DNA synthesis (UDS) test, 0.5 and 2 mM of DAS or 0.5 and 1 mM of

DADS could significantly decrease the DNA damage induced by AFB1 (P<0.05).

44

Furthermore, 0.5 and 2 mM DAS or 0.5 and 1 mM DADS could increase the

glutathione S-transferase (GST) and glutathione peroxidase (GPx) activities as

compared with the AFB1 controls after 24 hours treatment (P<0.05). Results of

immunoblot analysis of cytosolic GST isoenzyme indicate that the levels of GST

isoform Ya, Yb2 and Yc were markedly increased after treatment with 0.5 and 2 mM

DAS or 0.5 and 1 mM DADS compared with the AFB1 control. That 0.5 and 2 mM

DAS or 0.5 and 1 mM DADS might protect hepatocytes from AFB1-induced DNA

damage via increasing the activities of GST and Gpx (Sheen et al., 2001).

Hydrated sodium calcium aluminosilicate (HSCAS), a sorbent compound

obtained from natural zeolite, has an ability to sorb AFs with a high affinity. Addition

of this compound to feedstuffs contaminated with AFs has shown a protective effect

against the development of aflatoxicosis in farm animals (Abdel-Wahhab et al.,

2002).

The polyphenols present in fruits, vegetables and legumes are found to have

antimutagenic and anticarcinogenic properties. Phenolic compounds extracted from

the common bean (Phaseolus vulgaris) showed their antimutagenic effect on the

S. typhimurium tester strains TA98 and TA100 against the mutagen AFB1. The

inhibitory effect of PE against AFB1 mutagenicity was dose-dependent at the lower

concentrations tested and interestingly greatest inhibitory effect of the PE on AFB1

mutagenicity occurred when PE and AFB1 were incubated together. When the

bacteria were first incubated with PE followed by a second incubation with AFB1,

lower inhibition was observed. Lower inhibition was also observed when the bacteria

were first incubated with AFB1 followed by a second incubation with PE. The

mechanism of inhibition could involve the formation of a chemical complex between

of PE and AFB1 (Cardador-Martinez et al., 2002).

Garlic (Allium sativum) well known for its beneficial effects on health and

particularly for its chemopreventive potential against cancer, partly exerts its

anticarcinogenic effects through increasing enzymes involved in AFB1 detoxification.

The chemopreventive efficacies of several garlic powders with various levels of alliin,

a precursor of active sulfur compounds were compared. The medium term

hepatocarcinogenesis protocol (resistant hepatocyte model), which allows the

45

detection of preneoplasic foci expressing the placental form of glutathione

S-transferase (GST-P) as an end-point was used. Rats were fed diets containing three

garlic powders (5% of the diet) with various alliin contents for 3 weeks. Garlic

powders were obtained from bulbs grown on soils with different levels of sulfur

fertilization. During the period of garlic feeding hepatocarcinogenesis was initiated by

administration of 10 I.P. injections of 0.025 mg/kg body weight AFB1. The rats were

later submitted to 2-acetlaminofluorene treatment and partial hepatectomy, and GST-

P foci were detected and quantified. Consumption of diets containing garlic powders

decreased the appearance and size of hepatic GST-P foci. A strong reduction was

observed in rats fed garlic containing the highest level of alliin. In addition, increased

alliin content of the garlic powder was associated with a proportional decrease in the

number and area of preneoplastic foci. Elsewhere, garlic powder ingestion increased

hepatic ethoxyresorufin deethylase, glutathione S- transferase and UDP glucuronosyl

transferase activities while no modification of nifedipine oxidase activity was found.

An increase in the levels of GST A5 and AFB1 aldehyde reductase was observed

(Berges et al., 2004).

Effect of amount and source of supplemental dietary vegetable oil on broiler

chickens exposed to aflatoxicosis was studied. The dietary inclusion of SFO or SBO

at 30 g/kg alleviates the adverse effects of 0.3 µg/g of AFB1 in commercial broiler

chickens. Groundnut oil, although showing beneficial effects on some biochemical

variables, failed to improve growth performance. Addition of sunflower oil (SFO) at

30 or 60 g/kg or three vegetable oils, namely SFO, soybean (SBO) or groundnut

(GNO), at 30 g/kg to isocaloric and isonitrogenous broiler chicken diets were

evaluated for possible counteractive effects against aflatoxin (AF) (0.3 µg B1/g diet)

from 0 to 42 d of age. Body weight, food intake and serum concentration of protein

were lower in the AF group than in the control, whereas in the SFO and SBO

supplemented groups they were comparable with those of the control. Sunflower oil at

both concentrations exerted similar effects on growth. Groundnut oil did not improve

growth or food intake in AF-fed birds. The serum concentration of cholesterol and

triglycerides decreased with AF feeding and was increased by supplementation of any

of the three oils both in the control and in AF-fed groups. Liver and giblet weight and

46

liver fat content were increased by AF; these effects were countered by dietary oil

inclusion, except for liver weight at 60 g/kg SFO. Weights of pancreas and gall

bladder were increased by AF. Oil supplementation reduced the weight of pancreas in

chicken given AF. Humoral immune response was depressed by AF and dietary oil

supplementation (particularly SFO or SBO) countered this effect. Other variables,

namely, serum gamma glutamyl transferase activity, bone mineralization, weights of

lymphoid organs, kidney and adrenals, ready–to-cook yields and fat content in muscle

and skin showed little or no effect of dietary oil supplementation (Raju et al., 2005).

3.16 Dietary Aflatoxin Exposure Level and Their Limits

Kraybill and Shimkin (1964) have shown that the known epidemiology of

human liver cancer strengthened the case made by several authors at this time in

which aflatoxins were implicated. However, when the FAO and WHO established an

upper limit of 30 µgkg-1

aflatoxin for famine relief programme foods, it was estimated

that the aflatoxin produced liver cancer danger was not greater than the malnutrition.

Carter et al. (1997) found that detection of AFB, from food sources is necessary to

protect the public from chronic low dosage exposures, which may play a role in

production of certain cancers. Higher dosages of AFB1 over a period of years have

been proven to increase the formation of hepatic cancers in humans.

The fungal contamination of food and feeds is a public health problem

throughout the world. Due to its potential human health concerns, most countries have

expensive monitoring programs and legislation restricting marketing of aflatoxin-

contaminated grain (Woloshuk and Prieto, 1998). Whitaker (2003) described

outcomes of 1995 global survey conducted on aflatoxins. According to him, at least

77 nations had mycotoxins regulation for controlling aflatoxin levels in both food and

feed (FAO, 1997) which also varies significantly among these 77 countries.

Mycotoxin regulations are generally implemented using a defined maximum limit and

a sampling plan to detect and divert mycotoxin-contaminated products from food and

feed markets. Variation among maximum limits and sampling plans make it difficult

for exporters and importers to market commodities in the world market. To show that

47

standardization can work, FAO/WHO working through the CODEX committee

structure has developed an aflatoxin-sampling plan for raw shelled peanuts traded in

the export market and destined for further processing. The sampling plan calls for a

20 kg sample and a maximum limit of 15 mg/g total aflatoxins. Currently, the

CODEX committee on methods of analysis and sampling and committee on food

additives and contaminants are developing a document for member nations that

demonstrates how to implement the FAO/WHO aflatoxin sampling for raw shelled

peanuts. WHO aflatoxin sampling contains directions for how to select a

representative sample from a bulk lot, the sample preparation and sets minimum

performance standards for analytical methods (Whitaker, 2003). Considerably more

data are available for AFB1 than for other carcinogens evaluated by the expert group.

Occurrence data for aflatoxins were described at the 68th meeting of the Joint

FAO/WHO Expert Committee on Food Additives (JECFA). The international

estimates of AFB1 dietary exposure analysed by JECFA based on the 13 GEMS/Food

(Global Environment Monitoring System-Food Contamination Monitoring and

Assessment Programme) consumption cluster diets, the patterns of contamination of

food commodities with AFB1 described elsewhere (Table 3.4). The given table (3.4)

showed that estimated dietary intakes ranged from 0.3–0.5 ng/kg-bw/day (Cluster K,

South America) to 2.3–2.8 ng/kg-bw/ day (Cluster J, Africa), assuming a body weight

of 60 kg using the lower–upper bound approach. It is also found that the mean total

dietary exposure to AFB1 from maize, groundnuts, oilseeds, cocoa products and

pistachios made the greatest contribution to total exposure in all cluster diets (Benford

et al., 2010).

Commodity No. of

Samples Groundnuts 9132

n < LOR

(%) Mean ub

83 2.4

CV (%)

834

P5

0.03

P25

0.10

P50

0.10

P60

0.20

P75

0.20

P90

1.00

P95

4.64

P97.5

13.03

Maximum

935 Maize 961 86 0.3 194 0.04 0.10 0.14 0.20 0.20 0.50 0.70 1.02 8

Rice 541 86 0.8 391 0.04 0.10 0.20 0.50 0.80 1.00 2.00 3.00 57

Cocoa Products 266 68 1.9 587 0.04 0.10 0.20 0.35 0.50 0.76 1.86 7.31 120

Oilseeds 339 89 0.4 441 0.02 0.10 0.10 0.10 0.20 0.23 0.91 1.29 22

Dried figs 40,822 8 0.6 821 0.06 0.20 0.20 0.20 0.20 0.30 1.02 2.21 424

Brazil nuts 329 85 8.5 406 0.50 0.5 0.5 0.5 0.6 14.9 44.7 71.6 425

Almonds 2390 82 1.3 1189 0.1 0.1 0.2 0.2 0.5 0.5 1.5 4.1 575

Hazelnuts 3383 76 1.0 783 0.04 0.1 0.2 0.2 0.5 1.3 2.8 4.5 334

Pistachios 1849 24 49.2 216 0.2 1.2 8.6 17.8 46.8 133.6 243.7 331.0 1411.0

Dried fruits other than figs 1477 93 0.3 388 0.03 0.10 0.10 0.20 0.20 0.80 1.00 1.00 33

Other nuts 1177 93 1.0 1356 0.04 0.10 0.10 0.10 0.20 0.23 1.00 1.20 385

Butter of Karite nut 29 28 3.7 157 0.50 0.70 1.62 1.87 3.33 8.32 16.0 21.40 25

Oil of groundnut 496 62 0.6 284 0.05 0.10 0.20 0.21 0.50 1.07 2.03 3.22 22

Spices 4704 60 1.5 312 0.04 0.10 0.20 0.40 1.00 3.10 6.60 9.30 96

Groundnuts 9132 83 2.4 834 0.03 0.10 0.10 0.20 0.20 1.00 4.64 13.03 935

Maize 961 86 0.3 194 0.04 0.10 0.14 0.20 0.20 0.50 0.70 1.02 8

Rice 541 86 0.8 391 0.04 0.10 0.20 0.50 0.80 1.00 2.00 3.00 57

Cocoa Products 266 68 1.9 587 0.04 0.10 0.20 0.35 0.50 0.76 1.86 7.31 120

Oilseeds 339 89 0.4 441 0.02 0.10 0.10 0.10 0.20 0.23 0.91 1.29 22

Dried figs 40,822 8 0.6 821 0.06 0.20 0.20 0.20 0.20 0.30 1.02 2.21 424 Brazil nuts 329 85 8.5 406 0.50 0.5 0.5 0.5 0.6 14.9 44.7 71.6 425

Almonds 2390 82 1.3 1189 0.1 0.1 0.2 0.2 0.5 0.5 1.5 4.1 575

Hazelnuts 3383 76 1.0 783 0.04 0.1 0.2 0.2 0.5 1.3 2.8 4.5 334

Pistachios 1849 24 49.2 216 0.2 1.2 8.6 17.8 46.8 133.6 243.7 331.0 1411.0

Dried fruits other than figs 1477 93 0.3 388 0.03 0.10 0.10 0.20 0.20 0.80 1.00 1.00 33

Other nuts 1177 93 1.0 1356 0.04 0.10 0.10 0.10 0.20 0.23 1.00 1.20 385

Butter of Karite nut 29 28 3.7 157 0.50 0.70 1.62 1.87 3.33 8.32 16.0 21.40 25

Oil of groundnut 496 62 0.6 284 0.05 0.10 0.20 0.21 0.50 1.07 2.03 3.22 22

Spices 4704 60 1.5 312 0.04 0.10 0.20 0.40 1.00 3.10 6.60 9.30 96

Table 3.4 Summary of the statistical distribution of aflatoxin B1 content (in µg/kg) in groundnut and other foods from 2000 to 2006

48

49

3.17 Aflatoxin Assay Methods

In 1965, Nabney and Nesbitt developed a method for determining the

aflatoxin, particularly aflatoxin B1 based on the intensity of the ultraviolet absorption

at 363 nm (the long-wavelength absorption maximum) after purification by thin-layer

chromatography.

Vorster (1969) proposed a suitable treatment of single sample extract for the

three-mycotoxigenic analysis of aflatoxin, ochratoxin and sterigmatocystins. The

results obtained demonstrated to have ±20 percent accuracy based on the subjective

evaluation of thin-layer chromatograms of the extract. The method is considered to be

satisfactory for the purposes of a field survey when the determination of the

approximate level of mycotoxin contamination of cereals and groundnuts in the

shortest possible time. Appropriate modifications of the method could deal problems

encountered with samples having high oil contents or darkly pigmented.

Broadbent et al. (1963) described a more rapid method for detection aflatoxin

B in which the defatted meal is extracted with methanol, any toxin present in the

methanol phase is extracted into chloroform. Aliquots of the chloroform extract are

chromatographed on thin layers of aluminium oxide, and the chromatogram is

examined in ultraviolet light. Over pressured layer chromatographic (OPLC) and high

performance liquid chromatographic methods most often used for the analysis of

aflatoxins (Papp et al., 2002).

Carter et al. (1997) used competitive ELISA to quantitate AFB1, zearalenone

and deoxynivalenol in cereal grain foods, which were detected colorimetrically. Fiber-

optic biosensors, based on the membrane bound ELISA formats can acts as an

alternative method for aflatoxin detection in field usage without expensive

instrumentation.

According to Mirghani et al. (2001) FTIR-ATR spectroscopy can be used to

determine aflatoxin contents in groundnut and groundnut cake. They found that the

FTIR spectroscopic method was the more accurate as the TLC method presents

difficult for visually estimating small differences in intensity on the TLC plates.

50

Papp et al. (2002) compared the HPLC method for aflatoxin analysis of

various food stuffs (maize, wheat, peanut, fish meat, rice and sunflower seeds)

developed in their laboratory and found its competitiveness. It is more rapid and low

cost and allows the simultaneous determination of up to seven samples.

Blesa et al. (2003) developed new method for aflatoxin extraction based on

matrix solid phase dispersion (MSPO) extraction.

Gaag et al. (2003) reported that the aflatoxin B1, zearalenone, ochratoxin

A, deoxynivalenol and fumonisin B1 could be analyzed by an immunosensor.

Ammida et al. (2004) proposed new methods for the detection of aflatoxins

such as the application of surface Plasmon resonance biosensors, flow infection

monitoring, fibre optic sensors, capillary electro kinetics and electrochemical

transduction. According to them, ELISA methods have many potential advantages

over the other procedures owing to their simplicity, sensitivity, low cost and the safe

reagents use.

The in vitro digestion model, combined with Caco-2 cells, is a powerful

experimental tool, which can aid to a more accurate risk assessment of ingested

contaminants. Bio accessibility of aflatoxin B1 from peanut slurry and ochratoxin A

from buckwheat was high, 94% and 100% respectively, and could be determined

reproducibly. With the in vitro digestion model, the bio accessibilities of aflatoxin B1

and ochratoxin A in the presence of four different absorption modulators were in five

out of six situations in accordance with the in vivo effects in humans and animals. By

determining the effect of chlorophyllin on the transport of aflatoxin B1 across the

intestinal Caco-2 cells, also the sixth combination was in agreement with data in

humans (Versantvoort et al., 2005).

Fazekas et al. (2005) analyzed 91 commercial outlets origin spice samples for

AFB1, AFB2, AFG1, AFG2 and ochratoxin A (OTA) content by high-performance

liquid chromatography (HPLC) after immuoaffinity column clean-up. They found the

presence of aflatoxin in almost all samples and called attention to the importance of

consistently screening imported batches of ground red pepper for aflatoxin and

51

ochratoxin A content and strictly prohibiting the use of batches containing mycotoxin

concentrations exceeding the maximum permitted level.

Liu et al. (2006) developed a new bio-electro catalytic reaction on micro comb

electrode fabricated by means of self-assembling horseradish peroxidase (HRP) and

AFB1 antibody molecules based immunoassay concept. They found that developed

immune biosensor showed an acceptable accuracy compared with those obtained from

ELISA.

Sapsford et al. (2006) reported the competitive immuno assay for detection of

aflatoxin B1 for various corn and nut products using monoclonal mouse CY 5-anti-

AFB1. The fluorescence of aflatoxins itself was employed, so no special labeling was

needed. This assay provided an LOD of 2 ng/mg of AFB1.

Among the various analytical methods currently used for AF screening,

immunoassay methods are well suited for a rapid, routine screening. However, the

detection of concentrations of AFM1 in milk those are very close to the European

Union legal limit (50 ng/l) is affected by significant error, due to the low

reproducibility and sensitivity of the ELISA technique. Chromatographic methods, in

particular reversed-phase HPLC with fluorescence detection, are currently the most

commonly used and are particularly suited for the analysis of complex matrices

(Amadasi et al., 2007).

Pohanka et al. (2007) studied mycotoxin assay using biosensor technology.

They used the aflatoxin B1 metabolizing aflatoxin-detoxify enzyme for bio

recognition in multiwalled carbon nanotubes in three electrodes system where the

optical biosensors are based on antibodies labeled by fluorochrome or the active

fluorescence of some mycotoxins. Ammida et al (2006) studied aflatoxin B1 in barley,

using immunosensor and HPLC.

Bacaloni et al. (2008) demonstrated the sensitive and reliable liquid

chromatography–tandem mass spectrometric with eletrospray ionisation methods for

determining aflatoxins in hazelnuts.

52

Truckesess et al. (2008) developed method for the determination of aflatoxins

B1, B2, G1, G2, and Ochratoxin A in ginseng as well as ginger by multitoxin

immunoaffinity column cleanup and liquid chromatographic quantitation.

Shephard (2008) reviewed the various analytical methods available for the

determination of mycotoxins in food commodities. A number of analytical methods

have been applied to mycotoxin analysis. HPLC methods with UV or fluorimetric

detection are extensively used both in research and for legal enforcement of food

safety legislation and for regulations in international agricultural trade. Other

chromatographic methods, such as TLC and GC, are also employed for the

determination of mycotoxins. Conventional chromatographic methods are generally

time consuming and capital intensive. Recent advances in analytical instrumentation

have highlighted the potential of LC-MS methods, especially for multi-toxin

determination and for confirmation. A range of methods, mostly based on

immunological principles, have been developed and commercialized for rapid

analysis. These methods include enzyme-linked immunosorbent analysis (ELISA),

direct fluorimetry, fluorescence polarization, and various biosensors and strip

methods.

3.18 Current Detoxification Methods of Aflatoxin and Its Draw Backs

The FAO requirements for acceptable decontamination process stipulate that

the procedure must destroy, inactivate or remove aflatoxins without producing or

leaving toxic and/or carcinogenic/mutagenic residues in the final products or in food

products. Hence, the decontamination process must be technically and economically

feasible for their practical application (Piva et al., 1995).

Ghosh et al. (1996) found that out of the three chemicals propionic acid,

sodium bisulfite or sodium hydroxide tested as mold inhibitors, the propionic acid

was found to be most effective followed by sodium bisulfite and sodium hydroxide.

Solvents extraction has been used to remove aflatoxins from the oil seeds and

cotton seeds. The solvents used include 95% ethanol, 90% aqueous acetone, 80%

isopropanol, hexane, methanol, methanol water, acetonitrile water, hexane-ethanol-

water and acetone-hexane-water. Solvent extraction can remove all traces of aflatoxin

53

from oil seed meals with no formation of toxic by-products or reduction in protein

content and quality. Since aflatoxins are sensitive to UV radiation, treatment of peanut

oil with UV light for 2Hrs destroyed 40-45% of aflatoxins initially present in the oil.

Some absorbents can bind and thus remove aflatoxins from aqueous solutions.

Bentonite clay adsorbed AFB1 from a Sorensen buffer solution. Separation of the clay

resulted in the removal of 94-100% of toxin from the solution. Hydrated sodium

calcium aluminosilicate was reported to have a high affinity for AFB1 (Rustom,

1997).

Phillips (1999) suggested that simple and effective approach for the

chemoprevention of aflatoxicosis is to diminish or block aflatoxins exposure via the

inclusion of HSCAS clay in the diet. HSCAS clay acts as an aflatoxin enterosorbent

that tightly and selectively binds these poisons in the gastrointestinal tract of animals,

decreasing their bioavailability and associated toxicities. The molecular mechanism of

its action is that the dicarbonyl system of aflatoxin is essential for tight binding by

HSCAS. Aflatoxins may react at multiple sites on HSCAS particles, especially the

interlayer region, but also at edges and basal surfaces. Since clay and zeolitic minerals

comprise a broad family of functionally diverse chemicals, there may be significant

hidden risks associated with their indiscriminate inclusion in the diet. According to

him, all aflatoxin-binding agents should be rigorously tested, paying particular

attention to their effectiveness and safety in aflatoxin-sensitive animals and their

potential for interactions with critical nutrients.

Neal (2001) explained the aflatoxin removal from peanut meal having an

initial, unacceptable level of contamination to comparable, acceptable levels by two

ammonia based processes, but also he cautioned since they may still have different

effects in vivo when incorporated into animal diets.

Arranz et al. (2006) proposed the commodity washing for contamination

minimization. After collection of the tiger nuts, they are washed and sorted. This is an

important step to minimize possible aflatoxin contamination present in the raw

material and it is nowadays performed with the help of industrial machines.

The inactivation of AFB1, AFM1 and AFB1-dihydrodiol in the extrusion

process using lime together with hydrogen peroxide showed higher elimination of

54

AFB1 than treatments with lime or hydrogen peroxide alone. The extrusion process

with 0.3% lime and 1.5% hydrogen peroxide was the most effective process to

detoxify aflatoxins in corn tortillas, but a high level of those reagents negatively

affected the taste and aroma of the corn tortilla (Elias-Orozco et al., 2002).

Gowda et al. (2004) found that certain organic acids and some herbal

compounds suppresses the growth of Aspergillus fungus and reduces aflatoxin

production.

Aly et al. (2004) found that the commercial HSCAS and the Egyptian

montmorillonite (EM) had an excellent capability of adsorbing AFB1 and FB1 in an

aqueous solution at different levels.

Proctor et al. (2004) suggested that higher levels of peanut aflatoxin

degradation by combining ozonation and temperature. The temperature effect

lessened as the exposure time increased, suggesting that ozonation at room

temperature for 10-15 min could yield degradation levels similar to those achieved at

higher temperature while being more economical.

The aflatoxin are heat-stable but can be degraded by strong acid or alkaline

solution, oxidizing agent, bisulphate. They could be removed from peanut meal by

solvent extraction and inactivated by oxidizing agents and other treatments (Panhwar,

2005).

Razzaghi et al. (2006) investigated an ultra structural evidence of growth

inhibitory effects of a noval biocide, akacid plus, a novel member of guanidine based

polymeric compounds on an aflatoxigenic A. parasiticus. Transmission Electron

Microscopy (TEM) determined their detailed research on the effects as a potent

inhibitor of fungal growth and aflatoxin biosynthesis.

Shi et al. (2006) determined the effects of adding 3 g modified

montmorillonite nanocomposite (MMN)/kg diets containing 0.1 mg aflatoxin/kg

using broiler chicks from 0 to 42 days of age. They found that the addition of 3 g

MMN/kg AF-contaminated diet diminished the adverse effects of AF on most relative

organ weights, hematological values, serum and liver biochemical values and

enzymatic activities associated with aflatoxicosis. MMN can effectively reduce the

55

toxicity of AF in broiler chicks and MMN can be a potential ameliorator of

aflatoxicosis in broiler chicks

Jaynes et al. (2007) determined and compared the adsorption and removal of

aflatoxin B1 (AFB1) from water and from aqueous corn meal by reference clays,

activated carbon and a commercial montmorillonitic clay product, Novasil. Novasil

reduces significantly aflatoxicosis when added to animal feed. They also suggested

that a simple method might be developed to determine extractable aflatoxins in feed

before and after clay addition.

Inan et al. (2007) have recommended many physical and chemical methods

such as microwave heating, treatments with ozone (ozonation) and ammonia for

detoxification of aflatoxin contaminated food.

Inan et al. (2007) have used the high oxidizing power of ozone for

detoxification of aflatoxin. They found that the treatment with ozone could be an

effective method for the degradation of aflatoxin in red peppers. However, they have

not investigated the effect of ozone treatment on quality changes other than color.

Current control measures aiming at controlling fungal growth and mycotoxin

formation in stored grains includes physical methods (aeration, cooling and modified

atmospheres), chemical treatments with ammonia, acids and bases or with food

preservatives and by biological methods. These methods require sophisticated

equipment and expensive chemicals or reagents. Relatively drastic conditions are

necessary when using acids and bases to convert large amount of aflatoxin B1 (AFB1)

and AFG1 to AFB2 and AFG2 respectively (Atanda et al., 2007).

Phillips et al. (2008) have developed an innovative sorption strategy for the

detoxification of aflatoxins. NovaSil clay (NS) shown to prevent aflatoxicosis in a

variety of animals when included in their diet. NS clay binds aflatoxins with high

affinity and high capacity in the gastrointestinal tract, resulting in a notable reduction

in the bioavailability of these toxins without interfering with the utilization of

vitamins and other micronutrients.

56

Moschini et al. (2008) suggested that the deleterious effects of AF could be

overcome or, at least, diminished by sorbents as if aluminosilicates that are by

themselves had no toxic effects.

Deng et al. (2010) found that bentonites could be used in aflatoxin-

contaminated feeds and diets to reduce bioavailability of these mycotoxins including

aflatoxins.

3.19 Biological Control of Aflatoxin Production

Dorner and Cole (2002) conducted an experiment to determine the potential

for biological control of peanuts aflatoxin contamination during storage with

nontoxigenic strains of A. flavus and A. parasiticus. Their result confirmed that

treatment of soil with specific, nontoxigenic strains of A. flavus and A. parasiticus

significantly reduces preharvest aflatoxin contamination in addition to reducing

aflatoxin contamination that occurred during storage.

Dorner et al. (2003) determined the efficacy of different formulations of

nontoxigenic strains of A. flavus and A. parasiticus in reducing aflatoxin

contamination of peanuts. The technology of incorporating conidia into granules such

as pesta and pregelatinized corn flour are viable options to consider for commercial

production of aflatoxin biocontrol formulations.

The ability of two non-aflatoxin producing strains of A. flavus to interfere with

aflatoxin production by a toxigenic A. flavus strain examined using a replacement

series with suspended disc culture method suggests that the substantial inhibition of

aflatoxin yield for inoculum mixtures results from the failure of spore germlings to

establish a cooperative mycelial network (Wicklow et al., 2003).

3.20 Fungal Growth and Toxin Inhibitory Activity of Common Medicinal

Plants

Buchanan et al. (1975) reported the antiaflatoxigenic activity of various

substances inhibiting the growth and AF biosynthesis. AF inhibitory compounds such

as aflastatins and blasticidin A, Walnut tannins-derived gallic acid, anticalmodulin,

trifluroperazine. Neem (Azadirachta indica), Thymus x-prolock and Thymus eriocalyx

57

were capable of inhibiting growth of A. parasiticus and its AF production in a dose

dependent manner by unknown mechanisms.

Neem leaf extract above 10% (v/v) effectively inhibited aflatoxin production

by A. parasiticus and A. flavus. About 0.9-1% ammonia inhibited fungal growth

together with aflatoxin production (Rasooli et al., 2004).

Rasooli and Abyaneh (2004) found that the oils from T. eriocalyx severely

inhibited fungal growth. The same observation was noted when the fungus was

exposed to undiluted oil of T.X-Porlock. The fungus was resistant to dilution 1/32 of

this oil. MIC and MFC techniques were employed to assess fungistatic and fungicidal

properties of the oils. It was found that both oils had static effect at 1/8 dilution and

fungicidal property at 1/4 dilution. Study of fungicidal kinetics of essential oils

revealed more than 50% death in 15 and 30 min at lower and higher spore populations

respectively. 90-100% lethal effects were observed within 2 hours of the exposure to

the oils. Aflatoxin production was significantly inhibited at fungistatic dilutions of

both oils.

Suberu (2004) carried out preliminary studies on A. flavus inhibition by

extracts of the lichens Hypogymnia physodes as well as Ramalina farinacea and

Bentex T fungicide. The inhibitory potentials of the extracts of the lichen are

established by the results on the mycelial growth, sporulation, and germination of

spores of A. flavus. The extracts of the lichens and Bentex-T suppressed mycelical

growth, sporulation and germination of the spores of A. flavus. Dry weight of the

mycelia mat of A. flavus inhibition was high with the extracts of H. physodes (75%)

followed by the extract of R. farinacea (70%) and modest with Bentex-T (60%). Both

the extracts of the lichens, and Bentex-T caused significant spore germination

inhibition.

Tulare walnut, a cultivar highly resistant to aflatoxin formation due to

endogenous phytochemical constituents capable of inhibiting aflatoxigenesis in

A. flavus was investigated. The activity, located entirely in the pellicle (seed coat),

was extractable to various degrees with polar solvents, although some activity

remained unextractable, indicating that the bioactivity resided in a complex of

hydrolyzable tannins. These tannins upon hydrolysis by fungal tannase present in

58

A. flavus, yielded potent aflatoxin biosynthesis inhibiting gallic acid and ellagic acid.

Comparison of the gallic and ellagic acid content in the pellicle of Tulare and Chico

cultivars showed that the gallic acid content increased rapidly with maturation of the

nut and was 1.5-2 times higher in Tulare than in Chico. Gallic acid content in the

pellicle at maturity of a series of commercial English walnut cultivars, and two black

walnut species, was determined as an indicator of potential for conventional breeding

or genetic manipulation has the potential to provide new cultivars with high resistance

to aflatoxigenesis (Mahoney and Molyneux, 2004).

Boue et al. (2005) found that the volatiles generated from lipoxygenase (LOX)

normal and LOX deficient soybean (Glycine max) varieties with and without added

lipase inhibited A. flavus mycelial growth and aflatoxin production. The antifungal

aldehydes hexanal and (E)-2 hexenal were observed in both LOX normal and LOX

deficient lines and were detected at significantly higher amounts in soybean

homogenate with added lipase. These aldehydes may be formed through alternate

pathways, other than the LOX pathway, and may account for the inhibition of

A. flavus growth observed. Other volatiles detected, particularly the ketones and

alcohols, may contribute to the antifungal activity observed in both LOX normal and

LOX deficient soybean lines.

Guleria and Ashok kumar (2006) reported the antifungal activity of Himalayan

medicinal plants using direct bioautography. They established that among the twelve

plants tested, lipophilic extracts of Vitex negundo, Zanthoxylum alatum, Ipomea

carnea, Thuja orientalis and Cinnamomum camphora exhibited antifungal activity

against C. lunata or A. alternata by direct bio autography. The best antifungal activity

against C. lunata or A. alternata was shown by lipophilic leaf extract of T. orientalis.

Mohana and Raveesha (2007) evaluated the anti-fungal activity of eight

different plant extracts against some plant pathogenic field and storage fungi like

Fusarium solani and A. flavus by dry mycelial weight, spore germination and

poisoned food techniques. The phytopathogenic fungi were isolated from sorghum,

maize, and paddy seeds. Species of P. chrysogenum was completely inhibited at 10%

concentration. The antifungal activity of aqueous and petroleum ether extract of

D. hamiltonii showed better activity than tested synthetic fungicides. They concluded

59

that D. hamiltonii being an edible plant possessing significant broad spectrum

antifungal activity against important field and storage fungi would probably be an

important candidate plant for prevention of bio deterioration of grains during storage

and prevention of spoilage of processed food product.

Sandoss kumar et al. (2007) studied the inhibitory activity of medicinal plant

Zimmu on A. flavus growth and detoxification of its aflatoxin B1. Aqueous extract of

Zimmu leaves showed antifungal activity against A. flavus and recorded 73%

inhibition of A. flavus growth. They found that Zimmu is capable of significantly

reducing the population of A. flavus in soil and subsequent infection and aflatoxin

production in peanut kernels. It was also found that the population of beneficial

antagonistic microorganism viz., T. viridae was not affected.

Panda et al. (2010) observed the promising antifungal activity of crude,

petroleum ether, chloroform, ethanol, methanol and aqueous extracts of Cassia fistula

L. against Candida albicans, C. krusei, C. parapsilosis and C. tropicalis. Methanol

extract produced measurable antifungal activity against the filamentous A. niger,

A. flavus and A. fumigatus. They also found that for determining the antifungal

activity, the more accurate method of assessment is the broth dilution technique.

Velazhahan et al. (2007) gave an account on AFG1 detoxification ability of

leaves/seeds aqueous extracts of various medicinal plants. The seed extract of Ajowen

(Trachyspermum ammi (L.) Sprague ex Turrill) showed the maximum degradation of

AFG1 up to 65%. The dialyzed T. ammi extract was more effective than the crude

extract, capable of degrading >90% of the toxin. Significant levels of degradation of

other aflatoxin viz., AFB1 (61%), AFG2 (54%) and AFG2 (46%) by the dialyzed

T. ammi extract was also observed. Time course study of AFG1 detoxification by

dialyzed T. ammi extract showed that more than 78% degradation occurred within 6 h

and 91% degradation occurred 24 hours after incubation. AFG1 causes 2%

chromosomal aberration in corn at 40 mg/l concentration, after treatment with T.

ammi extract failed to induce chromosomal aberration demonstrating the degradation

of AFG1 by T. ammi extract. Hence, they suggested that the T. ammi extract might

provide a biologically safe method to protect poultry or livestock feeds and other

agricultural commodities from aflatoxins.

60

3.21 Culinary Herbs and Its Antifungal Activities

Moore and Atkins (1977) evaluated the fungicidal and fungi static effects of

an aqueous garlic extract on medically important yeast like fungi vaginitis Candida.

This study reported the consistent inhibitory and lethal effectiveness of garlic extract

against a wide variety of medically important opportunistic infections causing yeast-

like fungi.

Caporaso et al. (1983) have shown the garlic extracts growth inhibitory

activity on variety of fungi and bacteria. Antifungal activity in human urine and serum

after ingestion of garlic (A. sativum) was demonstrated. The maximum achievable

antifungal activity in these two body fluids after oral therapy, a maximum tolerable

dose of garlic extract administration to human volunteers and the antifungal activity in

urine along with serum were determined. All volunteers who ingested the fresh garlic

extract complained of burning sensations in the mouth, esophagus, and stomach,

which lasted for less than 10min. Volunteer who took a 25 ml dose of the extract,

additional symptoms of nausea, diaphoresis and light-headedness lasting for

30 minutes were noted. All urine and serum samples collected from the volunteers

before the ingestion of garlic failed to inhibit the growth of any of the fungal strains

tested. Detectable anticandidal and anticryptococcal activity was noted only in serum

collected 30 and 60 minutes after garlic extract ingestion. The antifungal activity of

garlic extract has been confirmed.

Gowda et al. (2004) evaluation revealed the best anti fungal compound of

clove oil which was at the 0.5-1% level showed 100% reduction in aflatoxin

production, followed by turmeric at 0.5-1% (77-85%), garlic at 0.5-1% (80-81%) and

onion at 0.5-1% (73-77%). The studied herbal compounds generally did not suppress

fungal growth, even though inhibition of toxin productions occurred, often to its

virtual exclusion. The outcome of this study suggests that the possible mode of action

of these compounds is by interfering the biosynthetic pathway of aflatoxin production

without actually affecting the growth of the fungus, thereby resulting in partial or

complete inhibition of aflatoxin production. However, the best anti-fungal compounds

are those that inhibit both fungal growth and toxin production.

61

The effect of daily oral administration of aqueous extract (600 mg/kg b.wt.)

and methanol extract (200 mg/kg b.wt.) of M. koenigii Spreng leaves on blood

glucose and plasma insulin level in alloxan induced diabetic rats for a period of eight

weeks was studied. Blood glucose levels of diabetic rats treated with aqueous and

methanol extracts of M. koenigii Spreng showed significant reduction (P<0.05) as

compared to diabetic control groups. Plasma insulin showed significantly high on 43rd

and 58th

days of treatment in aqueous and methanol extracts of M. koenigii treated

groups. The hypoglycemic effect might be mediated through stimulating insulin

synthesis and/or secretion from the beta cells of pancreatic islets of Langerhans

(Vinuthan et al., 2004).

Natural products may regulate the cellular effects of aflatoxins and evidence

suggests that aromatic organic compounds of spices can control the production of

aflatoxins. The use of spices in food products preservation has been traditional and

they are cultivated in many countries such as India, Japan and Russia. Spices occupy a

prominent place in the traditional culinary practices and are indispensable part of

daily diets of millions of people all over the world. They are essentially flavouring

agents used in small amounts and are reported to have both beneficial effect and

antimicrobial properties. Their antimicrobial properties have been found mostly due

to the presence of alkaloids, phenols, glycosides steroids, essential oils, coumarins

and tannins (Atanda et al., 2007)

Irkin and Korukluoglu (2007) investigated the A. niger control with fungicidal

concentration (MFC) doses of garlic (Allium sativam L.), Onion (A. cepa L.) and leek

(A. porrum L.) aqueous, acetone and ethyl alcohol extracts. They exposed that the

inhibitoriest plant was garlic, followed by onion and leek. They also found that

effectiveness of inhibition was related to the extraction solvent.

Atanda et al. (2007) study on essential oils of sweet basset (Ocimum

basiteceem), cassia (Cinnamomum cassia), coriander (Coriandrum sativum) and bay

leaf (Laurus nobites) against A. parasiticus CFR 223 revealed their protective effect.

Sweet basil oil at optimal protective dosage of 5% (v/v) was fungistatic on

A. parasiticus CFR 223 and aflatoxins produced in vitro and on fungal development

on sorghum grains (PÇ0.05) with a residual effect that lasted for 32 days. In contrast,

62

oils of cassia and bay leaf stimulated the mycelial growth of the fungus in vitro but

reduced the aflatoxin concentration (B1 and G1) of the fungus by 97.92% and 55.21%

respectively, while coriander oil did not have any effect on both the mycelia growth

and aflatoxin content of the fungus. The combination of cassia and sweet basil oils at

half of their optimal protective dosages (2.5% v/v) completely inhibited the growth of

the fungus. It was also uncovered that the addition of whole and ground basil leaves

markedly reduced aflatoxin contamination; however, 10% (w/w) of whole leaves was

more effective as the aflatoxin reduction was between 89.05% and 91%.

Rasooli et al. (2008) supposed that natural products may regulate the cellular

effects of aflatoxins and aromatic organic compounds of spices can control the

production of aflatoxins. They found the antifungal activity of hydrodistillated

essential oils of Rosmarinus officinalis and Trachyspermum copticum L. with special

reference to the inhibition of A. parasticus growth and aflatoxin production.

T. copticum L. oil showed a stronger inhibitory effect than R. officinalis on the growth

of A. parasiticus. Aflatoxin production was inhibited at 450 ppm of both oils with that

of R. officinalis being stronger inhibitor.

Skrinjar et al. (2009) established the antifungal property of spices and herbs

produced from botanically diverse plants grown in a wide variety of soils and

climates. Mint (Mentha piperita L.) and caraway (Carum carvi L.) have effect on the

growth of some toxigenic Aspergillus species and aflatoxin B1 production. All

concentrations of mint and caraway showed a high inhibitory effect against fungal

species tested (A. flavus, A. fumigatus, and A. ochraceus). Mint found to have stronger

effect than caraway.

Phytochemicals present in culinary plants found to have both health benefits

and antimicrobial activity. The antifungal properties of onion (A. cepa), ginger

(Z. officinale) and garlic (A. sativum) against A. flavus, A. niger and Cladosporium

herbarum were described. The blended, air dried, ethanol soaked and filtered plant

extracts of ginger, garlic and onions when tested showed no significant difference

between ethanol and plant extracts inhibitions in C. herbarum indicating an equivalent

antifungal property. The obtained results clearly confirmed the fact that soluble

extracts of ginger, garlic and onion have antifungal properties and are able to inhibit

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the growth of the fungi A. niger, A. flavus and C. herbarum albeit to different extents

(Tagoe et al., 2010).

3.22 Culinary Plant Extracts and Their Other Health Benefits

M. koenigii leaves (Rutaceae) are used traditionally in Indian ayurvedic

system to treat diabetes. The effect of mahanimbine (carbazole alkaloid from

M. koenigii leaves) on blood glucose and serum lipid profiles on streptozotocin –

induced diabetic rats was investigated. Mahanimbine possess anti-hyperglycemic and

anti-lipidemic effects. Diabetes was induced in adult male Wister rats by intra-

peritoneal injection of streptozotocin (45 mg/kg). Mahaimbine (50 and 100 mg/kg)

were administrated as a single dose per week to the diabetic rats for 30 days. The

control group received 0.3% w/v sodium carboxy methyl cellulose for the same

duration. Fasting blood sugar and serum lipid profiles were measured in the diabetic

and non-diabetic rats. In addition, in vitro alpha amylase and alpha glycosidase

inhibitory effects of mahanimbine were performed. In the diabetic rats, the elevated

fasting blood sugar, triglycerides, low density lipoprotein levels, sugar, triglycerides

was increased by mahanimbine at a dose of 50 and 100 mg/kg. In addition,

mahanimbine showed appreciable alpha amylase inhibitory effect, weak alpha

glycosidase inhibitory effects when compared with acarbose. Mahanimbine has

beneficial effect in the management of diabetes associated with abnormal lipid profile

and related cardiovascular complications (Dinesh Kumar et al., 2010).

M. koenigii is used as an analgesic, febrifuge, stomachic, carminative and for

the treatment of diarrhoea, dysentery and skin eruption (Manvi and Sarin, 2010).

The use of spices in food not only improves the flavour and taste of food, but

they also show potential human health benefits with their important preservative and

antioxidant properties. These are commonly related to the content of vitamins,

flavonoids, terpenoids, carotenoids, coumarines, curcumins, etc., rendering them as

preservative agents in foods. Antimicrobial properties are attributed to their essential

oils as well (Polovka and Suhaj, 2010).

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3.23 Bioactive Compounds of Selected Culinary Plants

Martin and Moody (1988) demonstrated the chemical synthesis of bioactive

component of culinary plant M. koenigii (L) spreng. 1-Oxygenated carbazoles are

prepared in 4 steps from indole-2-carboxylates by condensation with y-butyrolactones

to give the lactones, followed by hydrolysis with concomitant decarboxylation to the

alcohols and oxidation to the aldehydes. The aldehydes are found to cyclise to

1-methoxy-carbazoles on treatment with boron trifluoride-methanol or with

methanolic hydrogen chloride. The methoxycarbazoles were converted into the

corresponding carbazolequinones by demethylation and oxidation. The carbazole

alkaloids murrayafoline-A and murrayaquinone-A also were prepared.

Sukari et al. (2001) reported the isolation of carbazole alkaloids from roots of

M. koenigii (Rutaceae). Two carbazole alkaloids were isolated from the petroleum

ether root extract for the first time from this plant. They also reported the the structure

elucidations of the compounds using spectroscopic methods.

Rahman and Gray (2005) reported the isolation of a benzoisofuranone

derivative, 3Ç-(1Ç-hydroxyethyl)-7-hydroxy-1-isobenzofuranone, and a dimeric

carbazole alkaloid, 3,3‟-[oxybis(methylene)]bis(9-methoxy-9H-carbazole), along with

six known carbazole alkaloids and three known steroids from the stem bark of

M. koenigii. They ascertained the structures of these compounds unambiguously by

UV, IR, MS and a series of 1D and 2D NMR analyses.

Cardenas-Ortega et al. (2005) characterized seventeen compounds from the

essential oil of Chysactinia mexicana using gas chromatography-mass spectrometry

analysis. Eucalyptol (41.3%), Piperitone (37.7%) and linalyl acetate (9.1%) were

found as the major components. They also found that the essential oil of leaves and

piperitone completely inhibited A. flavus growth at even relatively low concentrations.

Noor Haslizawati Abu Bakar et al. (2007) reported the isolation of four

carbazole alkaloids, identified as mahanimbine, girinimbine, murrayanine,

murrayafoline-A and one triterpene from stem, bark and roots of M. koenigii.

Previously they established the structures of these compounds by infrared (IR), mass

65

spectrometry (MS) and nuclear magnetic resonance (1H NMR,

13C NMR, HMQC and

HMBC) spectroscopy.

Chowdhury et al. (2008) analyzed the chemical composition of the leaf oils of

M. koenigii (L.) Spreng and M. paniculata (L.) Jack. They used gas chromatography

mass spectroscopy (GC-MS) for analyzing the leaves essential oil of them. They

revealed that M. koenigii oil contains 39 compounds of which the major is 3-carene

(54.22%) followed by caryophyllene (9.49%) where as oil of M. paniculata contains

58 compounds of which the major are caryophyllene oxide (16.63%), þ-caryophyllene

(11.81 %) spathulenol (10.21%), þ-elemene (8.94%), germacrene D (6.95%) and

cyclooctene, 4-methylene-6 (6.37%).

Rasooli et al. (2008) showed that the major components of R. officinalis and

T. copticum L. oils were piperitone (23.65%), a-pinene (14.94%), limonene (14.89%),

1,8-cineole (7.43%) and thymol (37.2%), p-Cymene (32.3%), y-Terpinene (27.3%)

respectively. They also suggested that these essential oils could be safely used as

preservative materials on some kinds of foods to protect them from toxigenic fungal

infections.

Pande et al. (2009) carried out the pharmacognostic and phytochemical studies

on the leaves of M. koenigii (L) spreng. Phytochemically leaves volatile oil found to

contain alkaloid, volatile oil, xanthotoxin and sesquiterpine. The powder of dried

leaves was subjected to continuous soxhlet extraction with various organic solvents

such as petroleum ether (60-800C) chloroform, acetone, benzene, methanol and

ethanol. The phytochemical studies with help of thin layer chromatography (TLC)

method revealed that petroleum ether fraction contains volatile oil. The benzene

fraction showed presence of fixed oil and alkaloid. The chloroform and methanol

fraction showed alkaloid. Acetone fraction has confirmed presence of alkaloid. The

alkaloid was prominently found in benzene, chloroform, acetone and methanol

extract. The petroleum extract was found to contain volatile oil and fixed oil in

benzene extract.

Manvi and Sarin (2010) carried out the chemical characterization and

antimicrobial screening of volatile components of the Indian aromatic tree M. koenigii

(L) spreng. The essential oil of M. koenigii leaves exhibited potent antimicrobial

66

properties that explain the basis for their use in traditional medicines. The essential oil

so obtained on gas chromatography and mass spectroscopy analysis exposed

identification of thirty components. They also found that chemically, the essential oils

of M. koenigii are primarily composed of mono and sesquiterpenes as well as

aromatic polypropanoids synthesized via the mevalonic pathway for terpenes and the

shikmic acid pathway for aromatic poly-propanoids.

3.24 Role of Culinary Herbs in the Chemoprevention of Aflatoxin Toxicity

Among the various antimutagens/ inhibitors reviewed, chlorophyllin (CHL), a

water-soluble derivative of chlorophyll was identified as almost uniformly protective

against a broad range of direct- and indirect-acting mutagens, including aflatoxins.

Initial work with trout and rat liver enzymes in the Salmonella assay showed that

CHL was a potent antimutagen towards heterocyclic amines, polycyclic aromatic

hydrocarbons, aflatoxins and other classes of mutagen. Antimutagenic activity was

further demonstrated using the corresponding direct-acting mutagens in the absence of

an exogenous metabolizing system. Mutagen-inhibitor interaction molecular complex

formation was identified in spectrophotometry studies, suggesting that CHL acts as an

„interceptor molecule‟. In in vivo, CHL reduced hepatic AFB1-DNA adducts and

hepatocarcinogenesis when the inhibitor and carcinogen were co-administered in the

diet. Finally, co-injection of inhibitor and AFB1 into trout embryos established that

CHL was more effective than chlorophyll a in reducing AFB1-DNA adducts 2 weeks

after injection and liver tumors after 1 year (Dashwood et al., 1998).

3.25 Mode of Application Culinary Herbs and Their Activity

Schilter et al. (2003) point out: “Interactions between constituents will be

extremely important when data for highly purified preparations are used to assess

relatively unpurified material”. Already Yang et al. (2004) have discussed the

possibility of using in silico methods for the investigation of simple chemical

mixtures. Most of these interaction data are the derivatives of in vitro studies.

Naturally, such studies should be followed up with in vivo analysis. This is not at all

times practical due to the wide complexity of herbs and their extracts phytochemicals

(Rietjens et al., 2008) and is limited by the complex combinations of plant chemicals

(Jordan et al., 2010). Hence assessment of potential interactions between classes of

67

compounds, or the major compounds involved may give answer to the dearth of

information on potential matrix effects in medicinal herbs (Rietjens et al., 2008), and

the use of omics technologies to determine potential global effects of interactions in

mixtures (Jordan et al. 2010). Herbs in entirety and their extracts contain an

innumerable of phytochemicals. At least 50,000 compounds have been isolated from

plants, and one approximation notes the total number of plant metabolites likely

exceeds 200,000. Interactions between phytochemicals, and even between different

plants used in combination, form the basis of therapeutic use in traditional healing

paradigms such as traditional Chinese medicine (TCM) and Ayurveda. The failure of

reckoning true synergy between specific isolated phytochemicals within a herb or

herbal extract owing to the plenty of uncharacterized phytochemicals in whole plants

and many herb extracts, and the possibility of many complex interactions, provides

the explanation for the net activity, as other unknown constituents may have a

contributory role (Jordan et al., 2010).

3.26 Effect of Heat Treatment on Spices

It is well known that many antioxidant compounds are inactivated by heat

treatment, thereby reducing the final antioxidant status. However, some processing

steps may actually enhance antioxidant status due to the transformation of

antioxidants into more active compounds, such as the deglycosylation of onion

quercetin. The increase of antioxidant activity resulting from the inhibition of

enzymes has also been observed. The formation of certain antioxidants during thermal

treatment, considered to be the primary effect of Millard browning reactions, can

positively influence the total antioxidant status of food as well (Nicoli et al., 1997).

Significant losses (if any) would raise a question as to whether the spices retain their

beneficial health effects after heat processing (Polovka and Suhaj, 2010). An

overview on effect of heat treatment on culinary herbs and spices were given

elsewhere (Polovka and Suhaj, 2010).

3.27 Bioanalytical Studies on Culinary Oil

Hartman et al. (1987) described a method for the rapid determination of the

hydroxyl value of oils, fats and related products. Their method used toluene–p-

sulphonic acid as a catalyst and this allows the sample, dissolved in toluene, to be

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acetylated in 10 minutes at room temperature. The fatty acid anhydrides formed

during the acetylation are decomposed with aqueous sodium hydroxide and tert-

butanol, and are then titrated with hydrochloric acid. The addition of an aliquot of the

sample to the blank after the decomposition of the acetic anhydride reduces the

number of titrations required to two.

Examination of the oils for their physico-chemical characteristics revealed that

their moisture contents were generally below 1%. Palm oil had the highest

biochemical oxygen demand (BOD, 2576.88 ± 12.37 mg/l), while palm kernel oil had

the lowest (450.25 ± 7.42 mg/l). Palm oil had the highest iodine value (40.32 ± 0.27)

and coconut oil the lowest (13.18 ± 0.22) (Okpokwasili and Molokwu, 1996).

A combination of 1H NMR and

31P NMR spectroscopy and multivariate

statistical analysis was used to classify 192 samples from 13 types of vegetable oils,

namely, hazelnut, sunflower, corn, soybean, sesame, walnut, rapeseed, almond, palm,

groundnut, safflower, coconut, and virgin olive oils from various regions of Greece.

Different artificial mixtures of olive-hazelnut, olive-corn, olive-sunflower, and olive-

soybean oils were prepared and analyzed by 1H NMR and

31P NMR spectroscopy.

Subsequent discriminate analysis of the data allowed detection of adulteration as low

as 5% w/w, provided that fresh virgin olive oil samples were used, as reflected by

their high 1,2-diglycerides to total diglycerides ratio (DÇ0.90) (Vigli et al., 2003).

Peanuts have been reported to contain bioactive phytochemicals, particularly

isoflavones (genistein, daidzein, and biochanin A) and trans-resveratrol. Four methods

of extraction (stirring, sonication, soxtec, and microwave-assisted sonication (MAS))

for runner peanuts was compared. Quantification of the selected compounds was

conducted by revere-phase high-performance liquid chromatography (RP-HPLC). The

MAS and soxtec methods extracted significantly higher amounts of the

phytochemicals. The defatted peanuts gave significantly higher amounts of the

phytochemicals compared to the nondefatted peanuts. The high levels of isoflavones

may be attributed to heat-induced conversion of conjugate glycosides to aglycons

(Chukwumah et al., 2007).

Anyasor et al. (2009) investigated the chemical analysis of groundnut

(A. hypogaea) oil. Seeds of six varieties of A. hypogaea, boro light, boro red, mokwa,

69

ela, campala and guta as well as oil from three geographical zones in Nigeria;

northern, eastern and western were investigated. Gas chromatograph analysis showed

high concentrations of oleic and linoeic acids in the oil samples. Lauric acid was

highest (8.1%) in the mokwa while capric acid was totally absent. The high iodine

values denote high degree of unsaturation of the oil caused by the extent of oxidation

and degree of heat treatment during oil processing. The peroxide value of local and

refined oils was less than the standard peroxide value (10 mEqkg-1

) for vegetable oil

deterioration. Fresh oils have value less than 10 mEq kg-1

and values between 20 and

40 mEq kg-1

results in rancid taste.

Phenolic compunds in oil palm fruit (E. guineensis) were extracted in soluble

free (SFP), insoluble bound (ISBP) and esterified (EFP) forms. The total phenolic

content (TPC) of the oil palm fruit extracts was determined using the Folin-Ciocalteu

method and found to range from 5.03 to 9.04 g/L per gram of dried weight (DW). The

antioxidant activities of oil palm phenolic extracts were analyzed using free radical

scavenging assays and showed that oil palm phenolic extracts contained antioxidant

activities in the order of ISBP > EFP > SFP. Eight different phenolic acids were

identified and quantified using a simple reversed-phase high performance liquid

chromatography (HPLC) with a diode array detector (DAD) and liquid

chromatography/tandem mass spectrometry (LC/MS/MS). Ferulic, p-hydroxybenzoic

and p-coumaric acid were the dominant phenolic acids found in oil palm fruit extracts

and ranged from 55 to 376 µg/g of DW (Neo et al., 2010).

3.28 Molecular Mechanism and Role of Ver-l Gene in Aflatoxin Biosynthesis

Liang et al. (1997) believed that aflatoxin contamination would be prevented

logically by blocking aflatoxin synthesis by toxigenic fungi in the field prior to

harvest or during storage. A thorough understanding of aflatoxin biosynthesis at the

molecular level might aid for this approach. A. flavus has eight chromosomes with an

estimated genome size of about 33-36 Mbp harboring an estimated 12,000 functional

genes (Jiujiang yu et al., 2007). The aflatoxin biosynthetic pathway has been

extensively studied both biochemically and molecular biologically in A. flavus. The

biosynthesis mechanism and the enzymes involved in their production have been well

characterized (Juvvadi and Chivukula, 2006). This complex biosynthesis process

70

involves at least 18 enzyme activities (Lee et al., 2004). The genes directly involved

in aflatoxin formation comprise an aflatoxin pathway gene cluster (25 genes) which

includes those encoding polyketide synthases, fatty acid syntheses, carboxylases,

dehydrogenases, reductases, oxidases, oxidoreductases (Jiujiang yu et al., 2004).

Buchannan and Stahl (1984) reported that the level of AF in the lactose

containing culture was over 8 fold higher than previous record. Over expression of

CA747470, gene resulted in lower AF production, suggesting this gene may play a

role in regulating the AF pathway CA747470 appears to be involved in vegetative

growth, which may explain its negative effect on AF production. They suggested that

the AF production by Aspergillus species is a highly regulated process, involving

multiple layers of transcriptional and post transcriptional regulation. Prieto et al.

(1996) identified the aflatoxin biosynthesis genes by genetic complementation in an

A. flavus mutant lacking the aflatoxin gene cluster.

Woloshuk and Prieto (1998) found that most of the genes involved in the

biosynthesis of aflatoxin are contained within a single cluster in the genome of these

filamentous fungi. In addition, gene expression is coordinated during aflatoxin

production and is under the control of a positive regulatory gene belonging to a family

of fungal transcriptional activators associated with various metabolic pathways in

fungi. A detailed note on new gene associations with aflatoxin production under

aflatoxin conductive and non-conductive growth conditions was given by Micheli. Yu

et al. (2007) identified gene expression profiles in aflatoxin supportive media versus

non-aflatoxin supportive media to identify A. flavus and A. parasiticus potential

regulatory networks controlling aflatoxin contamination in food and feeds. They

reported consistent expression of genes in several aflatoxin-supportive media.

Duran et al. (2007) showed that a gene called veA controls aflatoxin and

sclerotial production in A. parasiticus. Their study in A. flavus showed that the veA

homolog in A. flavus is not only necessary for the production of aflatoxins B1, B2, and

sclerotia, but also regulates the synthesis of the mycotoxins cyclopiazonic acid and

aflatrem. Molecular studies on regulation of fungal sexual development or formation

of resistant structures are still limited, and only a few genes involved have been

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identified, mainly in A. nidulans, a model system for fungal development genetic

regulation studies. One of these genes is called velvet, or veA. Kato et al. (2003)

studies on A. nidulans showed that veA not only regulates morphogenesis but also is

necessary for the production of ST, the penultimate precursor in the AF biosynthetic

pathway. Calvo et al. (2004) found a veA homolog in A. parasiticus and generated a

veA deletion mutant unable to produce AF intermediates or sclerotia. The fact that

veA controls the production of AF, CPA, and aflatrem adds to the importance of veA

as a potential target to control Aspergillus mycotoxin contamination (Duran et al.,

2007). The ver1 is a middle gene in the aflatoxin biosynthetic pathway (Roze et al.,

2011). Ver1 of A. parasiticus was the first gene found to be involved in aflatoxin

biosynthesis and was isolated based on its ability to complement the genetic block in a

VA-accumulating mutant of A. parasiticus. The nucleotide sequence of ver1 suggests

that it encodes a ketoreductase responsible for deoxygenation (Yabe and Nakajima,

2004). The number of studies (Anderson and Green, 1994; Cleveland et al., 1987; Lin

and Anderson, 1992) conducted previously demonstrated that the appearance of

several aflatoxin metabolic enzymes and the accumulation of nor-1, ver1, and omtA

transcripts (Liang et al., 1997) coincide with the cessation of exponential growth of

the fungus and the onset of aflatoxin biosynthesis (Fig 3.1). Previous studies

suggested that aflatoxin synthesis is regulated in part at the transcriptional level. The

þ-glucuronidase reporter gene construct for monitoring aflatoxin biosynthesis in

A. flavus the ver1–GUS construct was utilized to identify plant substances, which

affect aflatoxin biosynthesis in A. flavus (Flaherty et al., 1995). It was observed that

GUS activity paralleled AFB1 accumulation under toxin-inducing growth conditions,

suggesting that the rate of transcription plays a role in determining AFB1 levels in the

aflatoxin producing organism (Liang et al., 1997).

3.29 Interceptor Molecule Hypothesis

Mutagen-inhibitor interaction (molecular complex formation) studies helps in

many ways to understand their mechanism of action and there by the prevention of

toxic effects. The various studies conducted on the carcinogenesis prevention can give

a clue for utilizing plant compounds for preventing aflatoxin synthesis. Hartman and

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Shankel (1990) reviewed the literature for inhibitors that interact directly with

mutagens and carcinogens and serve in effect to „sequester‟ these compounds from

harm‟s way. By reducing the bioavailability of many genotoxins, these putative

„interceptor molecules‟ might represent an important first line of defense, perhaps

rivaling such mechanisms as induction of detoxification enzymes or inhibition of

carcinogen activating enzymes. In the period since the review by Hartman and

Shankel published, a number of papers has described the ability of CHL, chlorophylls,

and other porphyrins to form molecular complexes with various carcinogens and

mutagens in a manner consistent with the interceptor molecule hypothesis (Dashwood

et al., 1998).

The in vitro interactions between free DNA and anticancer drugs, mutagens,

and carcinogens have been widely explained. However, in eukaryotic cells, the

majority of nuclear DNA is complexed with histone proteins to form chromatin.

Structural and dynamical properties alternation takes place due to their incorporation

and may therefore influence reactivity as well as repair. The relatively modest

differences in the attack of free and nucleosomal DNA by this wide variety of agents

like intercalators (ethidium bromide, actinomycin D), Minor-groove hinders

(Benzo[a]pyrene), major groove binders (Dimethylnitrosamine), cross-linkers

(nitrosoureas, mitomycin), strand breakers (Bleomycins), are perhaps surprising given

the considerable conformational restrictions placed on nucleosomal DNA. Also latest

evidence suggests that nucleosomes are in fact dynamic structures that transiently

expose DNA, providing access to regulatory proteins. Therefore, only agents that

react quickly with DNA, such as hydroxyl radical and UV light, would produce a

„snap shot‟ of nucleosomal structure. Other reagents may react more slowly on the

time scale of site exposure, resulting in a time-averaged picture of nucleosomal

structure and an overall foot printing effect within the core (Millard, 1996).

Cancer chemopreventive property compounds both natural and synthetic have

been subdivided into blocking agents and suppressing agents based on their

carcinogenesis stage activity. Blocking agents prevent carcinogens from modifying

DNA and causing mutations. This increases the expression of detoxification and

73

antioxidant genes. They are a form of cellular adaptation. Whereas the suppressing

agents inhibit the later promotion and progression stages of neoplastic disease. Their

actions include antagonism of oncogenes, activation of tumour suppressor proteins,

inhibition of angiogenesis, stimulation of apoptosis or terminal differentiation, and

modulation of arachidonic acid cascades (Hayes and McMahon, 2001). But in fungi

because many secondary metabolites are synthesized at highest levels at specific

times during its life cycle, they cells have evolved a complex „„molecular switch” that

activates the genes involved in secondary metabolism and controls the flow of

primary metabolites (carbon and nitrogen) through these pathways. To date, many

details of the „„molecular switch” have been discovered and it is clear that the switch

helps explain, at least in part, the mechanisms by which cells control when (temporal

switch) and to what extent pathway genes in secondary metabolism are activated

(Roze et al., 2011). Number of similar earlier studies conducted on the important

carcinogen aflatoxin producing organism suggested that aflatoxin synthesis is

regulated in part at the transcriptional level. Plant substances which affect aflatoxin

biosynthesis in A. flavus (Fig 3.2) would be identified using þ-glucuronidase reporter

gene construct (Flaherty et al., 1995).

74

Fig 3.1 Showing the involvement of genes and their corresponding enzymes in

aflatoxin biosynthesis (Courtesy: Cleveland et al., 2009)

75

3.30 Molecular Docking Studies on Aflatoxin and Its Gene Regulation

The whole genome of A. flavus is known, the whole genome microarray of

A. flavus is available and hence has been used to study the regulation of aflatoxin

biosynthesis genes (Schmidt-Heydt et al., 2009). Dashwood et al. (1998) used

computer generated molecular model of the AFB1-CHL complex to study the

interaction between chlorophilin and aflatoxin. They obtained energy-minimized

molecular models of the interaction between AFB1 and CHL using HyperChem based

on the previously described studies detailing the complexes between chlorophylls and

heterocyclic amines or polycyclic aromatic hydrocarbons. Amadasi et al. (2007) taken

the structures ß-CD, c-CD, AFB1, and OTA from the Cambridge Structural Database

(CSD) and performed their docking using GOLD and AutoDock to explain

interactions of aflatoxin B1 and ochratoxin A with ß - and c-cyclodextrins.

Yen et al. (2009) used LGA to elucidate interaction sites between AFB1 and

various forms of glycine N-methyltransferase (GNMT). Autodock 3.0 software was

used to identify the most favorable ligand binding interactions.

According to Amadasi et al. (2007) an agreed approach, combining different

docking tools and scoring functions based on different concepts, should allow a more

reliable analysis of the inclusion mechanism, overcoming errors and approximations

of each single molecular modeling tool. Identification of the exact nature of

regulatory proteins with regard to their anticalmodulin (CaM) binding character will

be useful not only to advance our knowledge of signal transduction pathways

regulating secondary metabolism but also to design suitable antiaflatoxigenic

compounds (Juvvadi and Chivukula, 2006). The current knowledge on the regulation

of aflatoxin biosynthesis in relation to external factors was available in the works of

Georgianna and Payne (2009).

Schmidt-Heydt et al. (2009) also reported the influence of various

combinations of the most important physical parameters, temperature and aw, on the

regulation of the aflatoxin biosynthesis genes of A. flavus by systematic analysis.

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Fig 3.2 : A model explaining aflatoxin biosynthesis in Aspergillus (Courtesy Roze

et al., 2011)