INTRODUCTION

Man and animals consume plant materials and plant products routinely as part of their

nutritional requirement. These materials comprise of many known and unknown organic

constituents. The hormones (phytohormones) and their derivatives are universally present in

plants (10-100g/kg of wet plant tissue) and are likely to be consumed by all herbivorous and

omnivorous animals including man. Phytohormone entry into the animal cells is expected to

result in either growth modulation or modulation of cell function in a temporal manner. Such

responses contributes to or results in metabolic changes within a cell. In recent years,

phytohormones have been considered as biologically significant compounds in animal health

and in agriculture. The use of naturally occurring substances (natural products) as

supplements in the diet has been under consideration as a possible factor for growth and

disease control. Further more, plant growth regulators (PGRs) have been reported to have

biological action in the animals too. Therefore, biologically active substances such as the

phytohormones as component of foods have received much attention recently (Maeda et al.,

1985; Wood et al., 1994; Hansawasdi et al., 2000). It has been recognized that endocrine

active substances are potentially present in food as natural compounds (Hughes et al., 1991;

Sharpe et al., 1993; McGarvey et al., 2001). Many varieties of chemicals have been

recognized for use in agriculture, and PGRs are among those most widely employed. The use

of PGRs in agriculture for enhancing plant food production capacity in a wide variety of

crops is also increasing at a steady rate. The need to increase the world food supply

substantially is one of the biggest challenges being encountered since the past few decades

(Hudson, 1976) and the use of plant growth regulators will have a major contribution towards

reaching the desired goal (Nickell, 1982). Further, the amount of these substances being

deposited into the environment may soon exceed those that of the insecticides (Mickel,
1978). Information on their in vivo influences on cellular metabolism in higher animals

remains very limited (Guarra, 1970). This subject has, therefore recently attracted the interest

of many researchers.

1.1 HISTORICAL PERSPECTIVE ON PHYTOHORMONES

The development of a plant from the stage of a seed is very complex, yet the process is

highly organized and integrated encompassing a cascade of sequential events. It is firmly

established that the integration and coordination of plant growth and development is

mediated through complex hormone functions. The presence of phytohormones in plants can

be traced back to 1880, when Julius Sachs suggested the existence of chemical messengers

responsible for plant growth. Further impetus to such a line of thinking arose from the

publication of Power of movements in Plants in 1981, wherein Charles Darwin

incorporated his observation on the phototropic movements in canary grass coleoptiles

(Darwin, 1881). The works of Boysen-Jensen (1913), Paal (1919) and Went (1926) on

phototropism culminated in the discovery of auxin as the first plant hormone. An intensive

search for the identification of the causative factor to the devasting bakanae disease in rice by

Japanese scientists (Kurosawa, 1926; Yabuta and Hayashi, 1939) resulted in the

identification of gibberellins, as phytohormones. A Tower of Babel situation developed in

1960s with the identification of abscission II in Cotton bolls (Ohkuma et al., 1963), dormin

from Acer (Wareing et al., 1964) and Lupin factor from Lupinus luteus (Rothwell and Wain,

1964). All the three substances were chemically alike and the substance abscisic acid was

given the status of phytohormone. Studies to identify the cell division inducing substances in

the young embryonic tissues brought later into light the existence of cytokinins (Letham,

1978).

Higher plants generated active substances, termed phytohormones, which had the

character of tissue hormones. Plant hormone is an organic compound synthesized in one part
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of a plant and translocated to another part, wherein at very low concentration it caused a

physiological response (Salisbury and Ross, 1992). Plant hormones are signaling molecules

produced within a plant, and occuring in extremely low concentration. Hormones regulated

cellular processes in targeted cells locally as well as in other locations of the plant. Hormones

also determined the formation of flowers, stems, leaves, the shedding of leaves, and the

development and ripening of fruit. Plant hormones shaped the plant, affected seed growth,

time of flowering, the sex of flowers and the senescence of leaves and fruits. They

determined which tissues grew upward or which grew downward, affected leaf formation and

stem growth, plant longevity and even plant death. They are naturally produced within plants,

though very similar chemicals are produced by fungi and bacteria which also affected plant

growth (Srivastava, 2002; Helgi, 2005). Until recently, plant growth and development was

thought to be regulated only by five groups of hormones, namely the auxins, gibberellins,

cytokinins, abscisic acid and ethylene. In addition to these several other classes of

compounds came to be known which regulate plant growth and development. They include

phenolics (Letham, 1978; Harborne, 1980), polyamines (Evans, 1989), methyl jasmonates

(Sembdner, 1993; Creelman, 1997) and brassinosteroids (Mandava,1988). Among these

substances, however, there is consensus among scientists to consider brassinosteroids as the

sixth class of phytohormones. Each class exhibited stimulatory as well as inhibitory

functions, and most often worked in tandem with each other, with varying ratios of one or

more interplaying to affect growth regulation (Rost, 1979).

1.2. PHYTOHORMONES TYPE AND ITS ROLE IN PLANTS AND HUMAN

HEALTH

1.2.1. Abscisic acid (ABA)

Abscisic acid (ABA) is an isoprenoid plant hormone, that is synthesized in the

plastidal 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway (Nambara and Marion-Poll,

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2005). This class of PGR is normally produced in the leaves of plants, originating from

chloroplasts, especially when plants are under stress and also known to interfere with

defences against pathogen. ABA plays important roles in many cellular processes including

seed development, dormancy, germination, vegetative growth, and environmental stress

responses. These diverse functions of ABA involve complex regulatory mechanisms that

control its production, degradation, signal perception, and transduction (Xiong, 2003; Helgi

Opik, 2005).

1.2.2 Auxins

Auxins are a class of plant growth substances and morphogens located in shoot and

root meristematic tissue, young leaves and in mature root cells. It is positively influence cell

enlargement, bud formation and root initiation. Auxins promote the production of other

hormones and in conjunction with cytokinins, they control the growth of stems, roots and

fruits and are capable of converting stems into flowers. Auxins also induce sugar and mineral

accumulation at the site of application. The most common auxin found in plants is indole-3-

acetic acid (Daphne, 2005).

1.2.3. Cytokinins

Cytokinins are a group of chemicals that are involved in many plant processes,

including cell division, shoot and root morphogenesis, chloroplast maturation, cell

enlargement, auxiliary bud release and senescence (Kieber, 2002). Auxin is known to

regulate the biosynthesis of cytokinin (Nordstrom, 2004). They aid to delay senescence or the

aging of tissues, mediating auxin transport throughout the plant, and also affects internodal

length and leaf growth. They exhibit a highly-synergistic effect in concert with auxins and

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the ratios of these two groups of plant hormones affected most major growth periods during a

plant's life time. Cytokinins counter the apical dominance induced by auxins and they in

conjunction with ethylene promote the abscission of leaves, flower parts and fruits

(Deborah, 1983).

1.2.4. Ethylene

Ethylene is a gas formed through the Yang Cycle from the breakdown of methionine.

It has been shown that ethylene is produced from essentially all parts of higher plants,

including leaves, stems, roots, flowers, fruits, tubers, and seedlings. It exists as a gas and acts

at trace levels throughout the life of the plant by stimulating or regulating the ripening of

fruit, the opening of flowers, and the abscission (or shedding) of leaves. Ethylene has very

limited solubility in water and does not accumulate within the cell, but diffuses out of the cell

and escapes out of the plant. Ethylene is produced at a faster rate in rapidly growing and

dividing cells, especially in darkness. Ethylene affects cell growth, cell shape and fruit-

ripening (Crocker et al., 1935). Ethylene acts physiologically as a hormone in plants (De

Paepe and Van der Straeten, 2005; Chow and McCourt, 2006). In plant defense, ethylene is

mainly known for its positive role in resistance against necrotrophic pathogens in concert

with Jasmonic acid.

1.2.5. Gibberellins

Gibberellins include a large range spectrum of chemicals that are produced naturally

within plants and by fungi. They were first discovered by Japanese researcher, Eiichi

Kurosawa who noticed that a chemical produced by the fungus Gibberella fujikuroi led to

abnormal growth in rice plants (Grennan, 2006). The gibberellins were named GA1...GAn in

order of their discovery and Gibberellic acid (GA3) was the first gibberellins to be

structurally characterized. There are currently 136 gibberellins identified from plants, fungi

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and bacteria. GA3 is widespread and so far ubiquitous in both flowering (angiosperms) and

non-flowering (gymnosperms) plants as well as in ferns. Gibberellins are important in seed

germination and for effecting enzyme production which in turn mobilizes food production

used for growth of new cells. They promote flowering, cellular division, and seed growth

after germination. Gibberellins also reverse the inhibition of shoot growth and dormancy

induced by ABA (Tsai, 1997).

1.2.6. Brassinolides

These are plant steroids that are chemically similar to animal steroid hormones. They

were first isolated from pollen of the mustard family and extensively studied in Arabidopsis

plant. They promote cell elongation and cell division, differentiation of xylem tissues, and

they inhibit leaf abscission. Plants that are deficient in brassinolides suffer from dwarfism

(Nomura et al., 1997).

1.2.7. Salicylic acid

Salicylic acid (SA) is a phenolic phytohormone derived during the metabolism of

salicin and is found in plants. It helps in plant growth and development, photosynthesis,

transpiration, ion uptake and transport. SA induced specific changes in leaf anatomy, and

chloroplast structure, endogenous signaling and mediating plant defense against pathogens

(Hayat and Ahmad, 2007). It plays a role in the resistance to pathogens by inducing the

production of pathogenesis related proteins (Hooft Van Huijsduijnen, 2009). It is involved in

the systemic acquired resistance in plants in which a pathogenic attack on one part of the

plant induced resistance in other parts. The signal can move to nearby plants when salicyclic

acid gets converted to its volatile ester, methyl salicylate (Taiz and Zeiger, 2002). Salicylic

acid is used to treat acne, warts and other dermatological problems in humans. Sodium

salicylate has been found to suppress proliferation of human lymphoblastic leukemia,

prostate, breast, and melanoma human cancer cells.

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1.2.8. Jasmonates

The jasmonates (JAs) are a group of plant hormone which help to regulate plant

growth and development. Jasmonates include jasmonic acid and its esters, such as methyl

jasmonate. Like the related prostaglandin hormones found in mammals, the jasmonates are

cyclopentanone derivatives and are derived biosynthetically from linolenic acid by the

octadecanoid pathway. It seems to promote the production of defense proteins that are used

to ward off invading organisms. They also have a role in seed germination, storage of protein

in seeds, and root growth (Creelman and Mullet, 1997). Jasmonic acid, the plant stress

hormone, kills lymphoblastic leukemia cells. Methyl jasmonate has been found to induce cell

death in a number of cancer cell lines.

1.2.9. Polyamines

Polyamine is an organic compound having two or more primary amino groups.

Polyamines are strongly basic molecules with low molecular weight. They includes, many

substances that play important roles in both prokaryotic and eukaryotic cells, such as

putrescine H2N-(CH2)4-NH2, cadaverine H2N-(CH2)5-NH2, spermidine H2N-((CH2)4-NH-)2-

H, and spermine H2N-((CH2)4-NH-)3-H. They are essential for plant growth and development

and affect the process of mitosis and meiosis (Wang et al., 2003). In human, polyamines can

enhance the permeability of the blood-brain barrier (Zhang, 2009).

1.2.10. Nitric oxide (NO)

NO is one of the few gaseous signaling molecules known. In plants, nitric oxide can

be produced by any of four routes such as: (i) L-arginine-dependent nitric oxide synthase

(Corpas et al., 2004; Corpas et al., 2006; Valderrama et al., 2007). (ii) by plasma membrane-

bound nitrate reductase, (iii) by mitochondrial electron transport chain, or (iv) by non-

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enzymatic reactions. It is a signaling molecule, that acts mainly against oxidative stress and

also plays a role in plant pathogen interactions. Treating cut flowers and other plants with

nitric oxide has been shown to extend the wilting time. An important biological reaction of

nitric oxide is S-nitrosylation, the conversion of thiol groups, including cysteine residues in

proteins, to form S-nitrosothiols (SNOs). S-Nitrosylation is a mechanism for dynamic, post-

translational regulation of most or all the major classes of protein. It serves as signal in

hormonal and defense responses in animals too (Stryer and Lubert, 1995). Nitric oxide is

used as an anti-anginal drug in human, since it causes vasodilation, and helps in

atherosclerosis by improving blood flow to the heart (Hayward et al., 1990; Finer and

Barrington, 2006; Chotigeat et al., 2007). Impaired production in humans may lead to

various carcinomas and inflammatory conditions including juvenile diabetes, multiple

sclerosis, arthritis and ulcerative colitis (Tylor et al., 1997).

1.3. PHYTOHORMONE EFFECTS ON ANIMALS

Although phytohormones are quantitatively less important compounds than the

naturally or industrially produced hormones, their natural occurrence in plants must be taken

into consideration, while evaluating the contribution of chemicals having potential hormone

effects on humans. The effect on human health by chemicals mediated through the endocrine

system has generated a lot of interest and investment. Scientists have been concerned about

endocrine active substances and their possible negative impact on human health, as foods,

when fortified by phytohormones can have a major affect on humans (Verger and Leblanc,

2003). Hormones work with the immune and nervous systems to regulate growth,

reproduction, metabolism, immunity, cognition and behavior. Altering hormonal signals can,

therefore cause alteration in the body leading to health complications.

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 The use of plant growth regulators (PGRs) in agriculture for rising plant food

production and employing a wide variety of crops has been increasing at a steady rate. These

crops are used in preparing functional foods whose numbers have increase in recent years.

Hence, it is essential to understand the effect of these hormonal molecules on humans and

animals (Verger and Leblanc, 2003). The presence of abscisic acid and some of its

conjugates have been reported in the brain, heart, lung, liver and kidney of pig and

remarkable quantity in the brain of rat. Concern has, therefore been raised for its

biosynthesis, localization and physiological function in rat brain (Le Page-Degivry, 1986). It

has been reported that fecundity, longevity and egg vitality in insects were affected by PGR

treatment (Guarra., 1970; Alanso, 1971; Visscher, 1980; Visscher, 1983; De Man et al.,

1991). PGRs caused increase in the number of splenic plaque forming cells, circulating

WBCs, hematocrit value and thymus weight in young deer mice (Olson, 1981). The

molecular mechanism behind PGR induced toxicity seems to involve oxidative stress

resulting in the generation of free radicals causing lipid peroxidation (Candeias et al., 1995;

Tuluce et al., 2006).

Administration of subacute ABA and GA3 promotes lipid peroxidation and alters the

antioxidative system in rat tissues due to an increase in superoxide radicals generated under

stress (Celik et al., 2007). The administration of subacute naphthalene acetic acid (NAA),

2,4-dichlorofenoxyacetic acid (2,4-D), and 2,3,5-triiodobenzoic acid (TIBA) promotes

malondialdehyde (MDA) content in the tissues, inhibits the antioxidative defense mechanism

and activates or inhibits immune potential enzymes involved in the development of rat spleen

and lung tissues (Ismail Celik and Yasin Tuluce, 2007). Abscissic acid and GA3 have also

been found to affect sexual differentiation and other physiological phenomenon in mice

(Ozmen et al., 1995).

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 It has been reported that IAA in combination with horse radish peroxidase (HRP) kills

human cancer cells, and this has been suggested as a new form of anticancer treatment

(Wardman, 2002; Folkes and Wardman, 2003). Studies by Furukawa et al., (2004), indicated

that indole acetic acid (IAA) induced neuronal apoptosis in S phase and led to

microencephaly in rat fetuses. Incubation of rat neutrophils and lymphocytes for 24 hours in

the presence of IAA (1mM) showed an increase in the activities of SOD, CAT and GPx (de

Melo et al., 2004). IAA was found to prevent the loss of cell membrane integrity by inducing

the activities of SOD, CAT and GPx in rats (de Melo et al., 2004). The IAA administration to

animals has a good potential for increasing the phagocyte capacity with no prooxidant effect

(Patricia et al., 2006)

IAA was found to be teratogenic in mice and rats at 500mg/kg/day and cleft palate was

induced in both species at this dose. In mice, other malformations including exencephaly,

ablepharia, dilated cerebral ventricles, and crooked tail were also observed (John et al.,

1979). On the other hand, some PGRs have been shown to affect the antioxidant defense

systems and MDA content (Celik et al., 1997). IAA was found to inhibit AST and activate

amylase, CPK and LDH in human serum (Celik et al., 1997). The level of LDH and CPK

was increased significantly by IBA (indole butyric acid) and AST by IAA (Celik et al.,

2002). IAA causes death and marked ultra structural changes in cultured neutrophils by

increasing the production of O2 and H2O2 radicals (Mariza Pires de Melo et al., 1998). IAA

and Kinetin produced substantial systemic organ toxicity in the erythrocyte, liver, brain,

heart, lungs, spleen, and kidney during the period of a 21-day sub acute exposure, by

affecting the antioxidant and immune potential enzymes (Celik, et al., 2006) and MDA

content in rats at sub chronic levels (Celik and Tuluce, 2006). IAA increased the activities of

bovine carbonic anhydrase and human carbonic anhydrase-II of erythrocytes (in vitro), while

10
the kinetin was found to have no effect on either bovine or human carbonic anhydrase-I or

human carbonic anhydrase-II isozymes in vitro (Celik et al., 2007). The administrations of

IBA at sub acute and sub chronic levels affected the antioxidant defence system (Nuray

Topalca et al., 2009), and decreased AChE, BChE and ADA activities, whereas it increased

the MPO activity in various tissues of rats (Zeycan Yilmaz and Ismail Celik, 2009).

Putrescine decreased and abscisic acid increased the enzymatic activity of CAT and

G6PD in rat erythrocytes (Ciftci et al., 2003). Kinetin inhibited muscle creatine kinase

activity (CK-MB), while it activated AST and ALT activities (Celik and Kara., 1997). The

activities of LDH, CPK and AST were increased significantly by kinetin (Celik et al., 2002).

Kinetin increased DNA in the nuclei of fibroblasts in cell culture at low doses, but at higher

doses it caused foamy and vacuolized cytoplasm in these cells (El-Mofty., 1988). Kinetin

exhibited effective free radical scavenging activity in vitro and anti thrombotic activity in

vivo (Hsiao et al., 2004). Isopentenyladenine and its related compounds enhanced the

proliferation of animal cells in culture (Galli, 1984). The use of exogenous

isopentenyladenine, a trace cytokinin plant hormone reportedly affected DNA and protein

synthesis in cultured myoblasts (Kazumi Yagasaki et al., 1986). Researchers have found that

some plant stress hormones shared the ability to adversely affect human cancer cells also.

Sodium salicylate has been found to suppress proliferation of lymphoblastic leukemia,

prostate, breast, and melanoma in human cancer cells.

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1.4. CURRENT KNOWLEDGE ON GIBBERELLIC ACID

Gibberellic acid (GA3 )-C19 H22 O6

O
H

CO
HO OH
H

COOH CH2
CH3

(3S, 3aS, 4S, 4aS, 7S, 9aR, 12S)-7, 12-dihydroxy-3-methyl-6-methylene-2-oxoperhydro-4a,

7-Methano-9b, 3-propeno (1, 2-b) furan 4 -carboxylic acid (IUPAC).
Gibberellic acids are a group of plant growth regulators that have been identified in

different plants (Macmillian et al., 1961). Gibberellic acid is a white crystal powder, easily

soluble in alcohol, acetone, ether acetate, and in pH 6.2 phosphoric acid buffer solution. This

compound rapidly breaks down in alkaline solution and by heat. Gibberellic acid is stable in

dry or acid solution. The oral LD50 value is reported as 6300mg/kg for rat. It is a most

extensively used phytohormone in agriculture. It is a diterpene hormone derived from acetyl

Co-A, through the mevalonic acid pathway. It plays an important role in many cellular

processes, such as promotion of cell elongation, breaking seed and bud dormancy, stimulates

fruit setting and growing, parthenocarpic fruit development, flowering, mobilization of food

reserves in grass, seed germination, juvenility and sex expression in plants (Salisbury and

Ross, 1992). GA3 is used in Egypt to increase the growth of some fruits and vegetables

(Weaver, 1961). The World Health Organization listed gibberellins A3 (GA3) as a plant

growth regulator related to pesticides (WHO, 1990).

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1.5. EFFECT OF GIBBERELLIC ACID ON ANIMALS

1.5.1. General effects

Experiments on the effect of GA3 on rat indicate that the GA3 has biological actions in

animals too. GA3 was reported to have positive influence on body weights, food conversion

rate and fecundity on rats, poultry, pigs and calves (Tesh and Tesh, 1971; Madacsi et al.,

1988; Abd-Elhamid et al., 1994; Elkomy, 2003; Elkomy et al., 2007). It was also found to

increase the content of protein, glucose, cholesterol, hemoglobin and platelet numbers and

the GGT enzyme activity in the different tissues of rat, 2 hr after administration

(Pugazhendhi et al., 2003). EI-Okazy, (2008) found that the treatment of mice with GA3 for

11 weeks increased, the weight of liver, kidney and spleen, WBC number and the activity of

liver AST with a record of decreased RBC and serum creatinine in a dose dependent manner.

GA3 caused enhancement of lipid peroxidation and reduction of antioxidant defense in

different tissues of rat (Muthuraman and Srikumar, 2009). Administration of GA3 to rat

caused reduction in liver carbohydrates and total protein content, and in the serum level of

AST, ALT and ALP in a dose dependent manner (Saber et al., 2003).

1.5.2. On animal histology

GA3 induced liver neoplasm in Egyptian toads and the tumor was diagnosed as

hepatocellular carcinoma (El-mofty et al., 1988). Gibberellic acid also was found to induce

chromosomal aberrations in human (zalinian, et al., 1990) and in mice lymphocytes (bakr, et

al., 1999). Exposure to GA3 has been reported to induce micro abscesses and hydropic

degeneration in the liver, while causing nuclear inflammatory infiltration in the mice kidney

(Ustan, 1992) and liver of rat (Saber et al., 2003). Moreover, gibberellin A3 was also reported

to induce breast and lung adenoma in mice (El-Mofty et al., 1994). Feeding of chicken with

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GA3 led to numerous histological lesions in different organs including liver, but returned to

normal following withdrawal (Abdelhamid et al., 1994).

1.5.3. Role in male Reproduction

Gibberellic acid also reported to have an effect on the reproductive structure and its

physiology in animals. The feeding of GA3 to Mus musculus doubled the proportion of

females producing litters without increase in litter size or number (Olson, 1981). GA3 also

has number of estrogenic hormone-like actions in mammals (Gawienowski et al., 1977a;

Gawienowski and Chatterijee, 1980). It is worthy noting that, in castrated male rats, GA3

partially restored the weight of the prostate, but did not significantly change the epididymis

and seminal vesicles (Bouton, 1969). GA3 exhibits synergistic uteropic effect with estradiol

in the immature female mouse and androgenic properties in male chicks (Gawienowski et al.,

1977a; Gawienowski 1977b). Treatment of mature cockerels with GA3 improved semen

quality traits, such as sperm concentration, sperm motility, quantity of the sperm and

decreased abnormal sperm (Elkomy, 2003; Kamel et al., 2009). Elkomy et al., (2007) had

mentioned that, gibberellic acid can have testosteronic biological effects on male chicks, by

inducing effects on chick comb and testes weights that was similar to testosterone effects.

Gibberellic acid was reported to have a close relationship between increased fertility and

testosterone hormone level and libido in male rabbits (Elkomy et al., 2003; Elkomy et al.,

2008) Administration with low, medium and high doses of GA3 increased seminal plasma

HDL, cholesterol and total lipids concentration (Kamel et al., 2009). Seminal plasma total

lipids plays an important role in the membrane structure of spermatozoa, sperm metabolism,

sperm capacitation and fertilization of the female gamete (Hafez, 1987). GA3 has a direct

androgenic-like action on testes in male rabbits and it has a positive effect on semen quality

and quantity (Kamel et al., 2009). Gibberellic acid was found to produce changes in the

activities of hexokinase, acid phosphatase and alkaline phosphatase enzymes in rat testicular
14
tissue (Ravikumar and Srikumar, 2005). The long term treatment of male rat with gibberellic

acid was reported to cause derangement and loss of germ cells, reduction in the size of the

seminiferous tubules and dystrophy of Leydig cells with reduced sperm count in the lumen of

testis. A dysregulatory and inhibitor role was thus established for GBA in rat testicular cell

function. (Malini, 1991; Ravikumar and Srikumar, 2005).

1.5.4. Growth effects on other organs

GA3 exhibited a significant increase in the body weight, feed conversion ratio,

percentage weight of carcass, liver, pancreas, intestine, thyroid and adrenal glands, packed

cell volume (PCV), hemoglobin, RBC, total blood protein, and a significant decrease in

serum total lipids, triglycerides and cholesterol levels in rabbits. Furthermore, an increase in

the activity of Glutathione peroxidase with a corresponding reduction in malondialdehyde

(MDA) content was also observed due to GA3, by maintaining the serum T3, T4, AST, GGT,

uric acid and creatinine level as normal in rabbits In female quails, GA3 induced significant

increase in liver and body weight, feed conversion, percentage of egg production, ovary and

oviduct relative weights, hatchability and also the hematological parameters, such as RBC,

hemoglobin, PCV and total protein. In contrast, GA3 administration resulted in a reduction in

egg weight, blood total lipids, triglycerides and GGT activity in female quills. Gibberellic

acid has been shown to decrease feed consumption with lower weights of the carcass, organs

(liver, gizzard and heart) and glands (adrenal, thyroid and pituitary) significantly in chick

(Abdelhamid et al., 1994). It was also reported to decrease the activities of transaminase in

serum, and the content of muscular protein and bone mineral density (calcium, magnesium

and phosphorus) in chicks (Abdelhamid et al., 1994).

1.6. CURRENT KNOWLEDGE ON BRASSINOSTEROIDS

Sterols are an important class of organic molecules. They occur naturally in plants,

animals and fungi. Sterols of plants are called phytosterols, which include campesterol,
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sitosterol and stigmasterol. Phytosterols are C28 or C29 sterols, structurally very similar to

cholesterol (C27), but differing in their nucleus and/or side chain configurations or polar

groups. Among the 250 naturally occurring phytosterols, the -sitosterol, campestanol,

stigmasterol and dihydrobrassicasterol were the most frequently occurring phytosterols in

nature and thus in human diets (Oka et al., 1972; Oka et al., 1973(a); Oka et al., 1973; Oka et

al., 1974). Sterols and related compounds play an important role in the physiology of

eukaryotic organisms. Five food categories namely edible oils, vegetables, fruits, cereals and

pulses are known to contain these compounds. Besides, those naturally occurring phytosterol

fortified foods are also considered as a potential way by which humans are exposed to

phytosterol (Weihrauch and Gardner, 1978). Like phytoestrogens, the phytosterols also have

beneficial effects in animals including humans. Diets containing phytosterols at a level of 2-3

g/day can reduce total and LDL cholesterol level to about 10% and 20%, respectively.

Phytosterols are metabolized in the liver to C21 bile acids. Phytosterols are present in all

plants and in food products of plant origin. Phytosterols regulate the fluidity and permeability

of membranes and play an important role in adaptation of membranes to temperature

(Piironen et al., 2000). The use of high levels of phytosterols yielded estrogenic activity in

fish (MacLatchy and Van Der Kraak, 1995; Mellanen et al., 1996). A significant decrease in

testicular weight and sperm concentration was reported after long-term treatment with

phytosterols in albino rats (Malini and Vanithakumari, 1991). Phytosterol rich diets induce

elevation of plasma phytosterol five to 15-folds in infants above that observed in adults. In

countries, like Japan and United States and the European Union infants and young children

consuming 35-50mg of phytosterols/day/person, which was 7-11 times higher than that

required for their respective body weights, and an equivalent to that quantity was used during

post menopausal substitution (Setchell et al., 1998).

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 Mandava and Mitchell 1971, proposed that the pollen of rape plant contained a new

group of lipoidal hormone, termed brassins. Brassinosteroids are a new group of naturally

occurring polyhydroxy steroid hormones (phytosterols). In 1982, Gregory and Mandava

demonstrated that brassins could enhance crop yield, crop efficiency and seed vigour. This

biologically active plant growth promoter was named as brassinolide and was found to be a

steroidal lactones with an empirical formula of C28H48O6 (MW=480). It is insoluble or

poorly soluble in water, but freely soluble in organic solvents. Brassinolide was the first

brassinosteroid isolated in crystalline form (Grove et al., 1979). In 1982, the second

naturally occurring brassinosteroid termed castasterone was isolated from the insect galls of

chestnut (Castenea crenata). Up to now, seventy brassinosteroids and four brassinosteroid

conjugates have been characterized (Fujioka and Sakurai, 1997). Among natural

brassinosteroids, brassinolide is the most widely distributed biologically active

brassinosteroid. Natural brassinosteroids so far identified have a common 5-cholestane

skeleton, and their structural variations come from the kinds and orientation of functionalities

on the skeleton (Fujioka and Sakurai, 1997). The compounds can be classified as C27, C28

or C29 brassinosteroids depending on the alkyl substitution pattern of the side chain (Yokota,

1997). Brassinosteroids have been characterized from 44 plant species including 37

angiosperms (9 monocots and 28 dicots), 5 Gymnosperms, 1 Pteridophyte and 1 alga

(Fujioka, 1997). As brassinosteroids are reported in all plants tested so far, Sasse (1997)

suggested that brassinosteroids are ubiquitous in plant kingdom. It is present in almost every

tissue or organ such as pollen, seeds, flowers, fruits, shoots and leaves in variable levels.

Among them, reproductive organs (pollen and immature seeds) showed higher levels than

vegetative tissues (Sakurai and Fujioka, 1993).

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 Structure of 28-Homobrassinolide

Brassinosteroids existed endogenously in plant tissues at extremely low concentrations

(nanograms level). The level of endogenous brassinosteroids however, varies among plant

tissues. Pollen, the original source of brassinolide, and immature seeds are the richest

sources with a range of 1-100 ng per gram fresh weight, while shoots and leaves usually

have lower amounts of 0.01-0.1ng per gram fresh weight (Yokota and takahashi, 1986;

Takatsuto, 1994). In general, young growing tissues contained higher levels of

brassinosteroids than the mature tissues (Takatsuto, 1994). Brassinosteroids are plant growth

promoting hormones (Grove et al., 1979). In addition to growth, brassinosteroids also

influenced various other physiological processes like seed germination, rhizogenesis,

flowering, senescence, abscission, maturation and were also involved in conferring resistance

to plants against environmental stress (Sharma and Bhardwaj, 2007; Sharma and Bhardwaj,

2008). Due to multiple effects, Sasse (1997) considered brassinosteroids as plant hormones

with pleotropic effects.

In animal tissues brassinosteroid was present in the form of brassicasterol. It is a

component of animals along with other sterols in shell fish such as clams, oysters and

scallops and available to human through these foods (Bergmann, 1949). In crab the major

sterols are cholesterol (57%) and brassicasterol (37%) and in slugs brassicasterol accounts for

about 4-13% of the total sterols (David kritchevsky, 2006). Unlike in animal systems, where

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receptors for steroid hormones are intracellular, the brassinosteroid receptor 1(BRL1) in

plants is located in the plasma membrane, functions at the cell surface and transduces extra-

cellular signals (Li, 1997).

1.6.1. Chemistry of Brassinosteroids

The metabolism of brassinosteroids in mammals has not yet been investigated. It may

be speculated, however that a normal catabolism of the steroidal skeleton takes place. Being

normal constituents of practically all plants, brassinosteroids have been, and are, consumed

by mammals. However, confirmation of their safety can be obtained from toxicological

studies. The acute toxicity data obtained at the Sanitary-Hygienic Institute of Belarus for 24-

epibrassinolide is: LD50 (orally) in mice (female) is more than 1000 mg/kg/body wt; LD50

(orally and dermally) in rats (male/female) is more than 2000 mg/kg/body wt., dermal

toxicity in rats (male/female) is more than 2000 mg/kg body wt. In micro-nuclear or

chromosomal aberration tests (mice CBAB1/6), 24-epibrassinolide was found to cause

spontaneous mutations. In acute, sub acute, and chronic experiments, 24-epibrassinolide

showed low toxicity and very little cumulative effect. In prolonged experiments, 24-

epibrassinolide showed no toxicity, but a pronounced adaptogenic effect (increasing adaptive

ability of the population). Studies on fish toxicity showed no negative effects, but

pronounced stimulative and toxico-protective properties (Vitvitskaya et al., 1997a and b).

1.6.2. Effect of 28-HB on animals

An ubiquitous constituent in all plant species, brassinoisteroids were, and are,

consumed by mammals in their diet through evolution and exhibit in them, some regulatory

role that is not recognised till date. Not much work has been reported in animals employing

this phytohormone. Recently, it was reported that 24-epibrassinolide, an isoform of

brassinosteroid that was structurally related to the insect hormone ecdysone, caused an

increase in food consumption, growth rate, and an egg laying capacity, life expectancy period
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and protection against environmental stress in bees. 24-epibrassinolide was also proved to be

an effective anti-hypercholesterolemic agent in humans and possessed inhibitory activity

towards measles, HIV, herpes simplex and arena viruses. Sub chronic treatment of rats with

homobrassinolide reduced lipid peroxidation rate and elevated antioxidant defense system

(Muthuraman and Srikumar, 2009). The activity of LDH in different tissues and

hematological parameters of rat were modified by homobrassinolide treatment (Vikram et

al., 2009). Further, a decrease in glucose and an increase in cholesterol were also observed

following homobrassinolide treatment in this study.

Though a number of works were conducted on animals with GA3 and few with 28-

homobrassinolide, unfortunately no immediate/short term extensive systematic trials on any

mammalian species has been done so for. Hence, the present study aims to investigate the

metabolic impact of 28-HB and GA3 in rats following sub acute treatment (sub lethal) and for

the duration (within a period of 10 hr post administration) within which peak levels of the

compound is reached. In humans and rats the isoflavones were reported to be absorbed from

the intestinal tract and the peak plasma concentration was reached within 5-10 hr (Setchell et

al., 2001) after which it excreted in the urine and in the feces. Investigation was therefore

initiated to understand the mechanism of action of these phytohormones in the tissues using

rat as an experimental model. To observe time dependent changes in the tissues and to

understand the mechanism underlying the early response to phytohormones, the rats were

sacrificed using anesthetic ether within hours of exposure to each phytohormone. The time

points were selected taking in relation to the rime at which maximum serum level of

isoflavonoids was observable in rats, that is 10 hr prier to being excreted in the urine and

feces. This study therefore, is unique, extensive in character and significantly relevant from

environmental, nutritional, biochemical, medicinal and biological points of view.

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