Chapter -1

I - INTRODUCTION

The use of plant as medicine is as old as human civilization. Medicinal and

aromatic plants have been used since ancient times in the traditional medicinal

practices for primary health care needs. The wide spread use of herbal remedies and

healthcare preparations from commonly used traditional herbs and medicinal plants. As

per the traditional medicinal programme of the World Health Organization (WHO)

nearly 80% of the world population used phytoproducts (Dubey et al., 2004).

Medicinal plants wealth is rapidly diminishing, day by day, due to genetic

erosion, loss of biodiversity, urbanisation, expansion of agriculture, increasing

deforestation and construction of dams. If it continues, mankind will forever, loss some

of the most important sources of drugs. Conservation of biodiversity has assumed

considerable importance in view of an increasing human population as well as due to

depleting natural resources.

1.1. MEDICINAL PLANTS IMPORTANCE AND MICROPROPAGATION

It is estimated that world market for plant derived drugs may accounts for about

Rs. 2,00,000 crores. Presently Indian contribution is less than Rs. 2000 crores, Indian

export of raw drugs has steadily grown. India is one of the world’s 12 biodiversity

centres with the presence of over 45,000 different plant species. Indians diversity is

unmatched due to the presence of 16 different agro-climatic zones, 10 vegetation

zones, 25 biotic provinces and 426 biomes (habitats of specific species) of these, about

15000 - 20000 plants have good medicinal values. However, only 7000 - 7500 species

are used for their medicinal values by traditional communities.

In India, forest cover is disappearing at an annual rate of 1.5 m ha/ year, what is

left at present is only 8% as against a mandatory 33% of the geographical area. Many

valuable medicinal plants are under the verge of extinctions. The Red Data Book of

India has 427 entries of endangered species of which 28 are considered extinct, 124

endangered, 81 vulnerable, 100 rare and 34 insufficiently known species (Thomas,

1997).

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Predicted growth in world population is posing a serious challenge to crop and

medicinal plant production, particularly in developing countries. The augmentation of

conventional breeding with the use of marker-assisted selection plants promises to

facilitate substantial increases in medicinal value plants and their by products.

However, knowledge of the physiology and biochemistry of plants is extremely

important for interpreting the information from molecular markers and deriving new

and more effective paradigms in plant breeding (Bourgaud et al., 2001).

1.1.1. Secondary metabolites

Secondary metabolites are chemicals produced by plants for which no role has

yet been found in growth, photosynthesis, reproduction, or other "primary" functions

(Hartmann, 1991). These chemicals are extremely diverse; many thousands have been

identified in several major classes. Each plant family, genus, and species produces a

characteristic mix of these chemicals, and they can sometimes be used as taxonomic

characters in classifying plants (Agosta and William, 1996). Humans use some of these

compounds as medicines, flavorings, or recreational drugs (Bidlack and Wayn, 2000).

1.1.2. Secondary metabolites Synthesis and purpose in plants

Secondary metabolites are produced within the plants besides the primary

biosynthetic and metabolic routes of compounds aimed at plant growth and

development, such as carbohydrates, amino acids, proteins and lipids. They can be

regarded as products of biochemical “side tracks” in the plant cells and not needed for

daily functioning of the plant. Phylogenetically, the secondary bioactive compounds in

plants appear to be randomly synthesised – but they are not useless junk. Several of

them are found to hold important functions in the living plants. For example,

flavonoids can protect against free radicals generated during photosynthesis.

Terpenoids may attract pollinators or seed dispersers, or inhibit competing plants.

Alkaloids usually ward off herbivore animals or insect attacks (phytoalexins). Other

secondary metabolites function as cellular signalling molecules or have other functions

in the plants. Those plants producing bioactive compounds seem to be the rule rather

than the exception. Thus, most plants even common food and feed plants are capable of

producing such compounds. However, the typical poisonous or medicinal plants

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contain higher concentrations of more potent bioactive compounds than food and feed

plants (Cooper and Johnson, 1998).

1.1.3. Importance of Secondary metabolites

Secondary metabolites can be classified on the basis of chemical structure (for

example, having rings, containing a sugar), composition (containing nitrogen or not),

their solubility in various solvents, or the pathway by which they are synthesized (e.g.,

phenylpropanoid, which produces tannins). A simple classification includes three main

groups: the terpenes (made from mevalonic acid, composed almost entirely of carbon

and hydrogen), phenolics (made from simple sugars, containing benzene rings,

hydrogen, and oxygen), and nitrogen-containing compounds (extremely diverse, may

also contain sulfur). High concentrations of secondary metabolites might result in a

more resistant plant. Their production is thought to be costly and reduces plant growth

and reproduction (Simms, 1992; Karban and Baldwin, 1997; Harvell and Tollrian,

1999; Stotz et al., 1999; Siemens et al., 2002).

Based on their biosynthetic origins, plant secondary metabolites can be

structurally divided into five major groups: polyketides, isoprenoids (e.g. terpenoids),

alkaloids, phenylpropanoids and flavonoids. The polyketides are produced via the

acetatemevalonate pathway; the isoprenoids (terpenoids and steroids) are derived from

the five-carbon precursor isopentenyl diphosphate (IPP), produced via the classical

mevalonate pathway or the novel non-mevalonate or Rohmer pathway; the alkaloids

are synthesized from various amino acids; phenylpropanoids having a C6–C3 unit are

derived from aromatic amino acids phenylalanine or tyrosine; and the flavonoids are

synthesized by the combination of phenylpropanoids and polyketides (Verpoorte,

2000).

Secondary metabolites are currently being obtained commercially by extraction

from whole plants. Large scale plant tissue culture is an attractive alternative to the

traditional methods of plantation, as it offers two advantages. Firstly, the controlled

supply of biochemical independent of plant availability (politics, climate, pests) and

secondly a well defined production systems which result in higher yields and more

consistent quality of the product.

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During the last 30 years, plant cell and tissue cultures have been

comprehensively studied for the production of secondary metabolites. However,

despite promising results, this technology has led to only a few realisations for the

production of commercial compounds, at the industrial scale. Lack of industrial success

can be attributed to severe bottlenecks that have been identified during the last decades.

Rational engineering of secondary metabolic pathways in plants requires a thorough

knowledge of the whole biosynthetic pathway and a detailed understanding of the

regulatory mechanisms controlling the onset and the flux of the pathways. Information

is not yet available for the vast majority of secondary metabolites, explaining why only

limited success has been obtained by metabolic engineering.

Today, only a few secondary plant metabolic pathways (e.g. flavonoids,

terpenoids, indole and isoquinoline alkaloids) in plants are well understood as a result

of many years’ classical biochemical research. Plants synthesize an extensive array of

secondary metabolites, often with highly complex structures. Currently, most

pharmaceutically important secondary metabolites are isolated from wild or cultivated

plants because their chemical synthesis is not economically feasible. Biotechnological

production in plant cell cultures is an attractive alternative, but to date this has had only

limited commercial success because of a lack of understanding of how these

metabolites are synthesized.

The biosynthetic pathways of secondary metabolites are often long, complex

multi-step events catalyzed by various enzymes, and still largely unknown. The best-

studied class of secondary metabolites is the alkaloids; more than 12000 structures are

known (Facchini et al., 2001). The production of specific alkaloids is often restricted to

certain plant families. By contrast, flavonoids are abundant in many plant species. Still

tissue culture plays an important role in synthetic chemical area. In our times around 30

percent of the prescriptions are based on plants or contain plant components.

Traditional medicinal systems utilise plant based medicines and now there is revival of

traditional medicinal systems all over the world putting great pressure on biodiversity

and its being destroyed in developing countries to fulfil the demands of global markets.

Tissue culture techniques could provide a better alternative. The products of market

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interest in the first place belong to glycosides and alkaloids. Besides steroids, enzymes

and pigments are of considerable interest.

Efforts are being made at the global level to preserve biodiversity and to achieve

genetic conservation. In vitro technology and cryopreservation have received attention

and significantly complement existing methods. Micropropagation is one of the most

convenient and beneficial way to propagate plants species. It has many advantages

such as rapid clonal propagation, establishment, easy maintenance and distribution of

pathogen-free clones and maintenance of true - to - type plant species, with an ever

increasing human population, decreasing in forest land area of medicinal plants and an

ever increasing demand at the national and international market, In vitro technology

appears a safe and sound alternative for the production of medicinal valuable plants.

1.1.4. In Vitro Culture Techniques

1.1.4.1. Micropropagation Technology

In 1902, Haberlandt noticed that the plant cells can be grown in synthetic media.

The discovery by Haberlandt that the plant cells have the capacity to grow in a nutrient

medium in presence of sufficient light made an impact in plant propagation and crop

improvement. This has become possible with the development of techniques to

regenerate whole plants from the tissue cultured cells. Micropropagation technology is

being widely utilized commercially in the ornamental plant industry and in other plant

production organization. This propagation method was widely used after the discovery

of plant growth regulators.

The discovery of Auxin and Cytokinin created the great opportunities for in

vitro propagation of higher plants (Ezeibekwe et al., 2009). There are four basic stages

for successful micropropagation of plantlets. The first stage, the preparative stage or

stated as phase zero, involved the correct pre-treatment of the starting plant material so

as to ensure that they are disease free as far as possible. The second phase is the

establishment of clean starting tissue for aseptic growth and development. It involves a

sterilization protocol for producing aseptic tissues.

These aseptic tissues will be used for the next stage of shoot multiplication

which can be carried out in a number of ways. Generally plant growth regulators are

used for shoot multiplication. The shoots obtained in phase two will be used for root

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induction at the third phase either in vitro or in vivo. Finally, at phase four, the in vitro

plantlets are acclimatized for better survival when transferred to greenhouse conditions

or to the soil.

1.1.4.2. Plasticity and Totipotency

Two concepts, plasticity and totipotency, are central to understanding plant cell

culture and regeneration. Plants, due to their sessile nature and long life span, have

developed a greater ability to endure extreme conditions and predation than have

animals. Many of the processes involved in plant growth and development related to

environmental conditions. This plasticity allows plants to alter their metabolism;

growth and development to best suit their environment. Particularly important aspects

of this adaptation, as far as plant tissue culture and regeneration are concerned, are the

abilities to initiate cell division from almost any tissue of the plant and to regenerate

lost organs or undergo different developmental pathways in response to particular

stimuli.

When plant cells and tissues are cultured in vitro they generally exhibit a very

high degree of plasticity, which allows one type of tissue or organ to be initiated from

another type. In this way, whole plants can be subsequently regenerated. This

regeneration of whole organisms depends upon the concept that all plant cells can,

given the correct stimuli, express the total genetic potential of the parent plant. This

maintenance of genetic potential is called ‘totipotency’. Plant cell culture and

regeneration do, in fact, provide the most compelling evidence for totipotency.

1.1.4.3. Establishment of Aseptic Explants

The plant tissues or explants collected from the wild or the green house are

usually contaminated with microorganisms and other contaminants. These

microorganisms such as bacteria or virus must be removed during the preparation of

aseptic explants otherwise they would kill the explants either due to their overgrowth or

due to the release of toxic substances into the medium. The potential sources of

contamination in the cultures are the plant tissues, instruments, culture medium, and

environment of the transfer area, technicians and incubation room.

1.1.4.4. Plant Growth Regulators

The most usual groups of plant growth regulators (PGR) used in tissue culture

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research are the Auxins and Cytokinins. The amount of PGR in the culture medium was

critical in controlling the growth and morphogenesis of the plant tissues (Skoog and

Miller, 1957). Generally a high concentration of Auxin and a low concentration of

Cytokinin supplemented into in the medium could promote cell proliferation with the

formation of callus. On the other hand, low Auxin and high Cytokinin concentration in

the medium resulted in the induction of shoot morphogenesis.

Auxin alone or with the presence of a very low concentration of Cytokinin was

important in the induction of root primordial. There is a number of naturally occurring

Auxins, however, most of these are not generally available for routine use. Because of

their stability, synthetic Auxins are extensively employed. The most commonly used

are 2, 4- dichlorophenopxyacetic acid (2,4-D), 1-napthaleneacetic acid (NAA) and

indole-3-butyric acid (IBA). In some chemical compounds which are not strictly

Auxins, such as dicamba (3, 6-dichloro-o-anisic acid) or picloram (4-amino-3, 5, 6-

trichloropyridine-2-carboxylic acid), have been used as Auxin to substitute IBA.

Cytokinins of adenine derivatives are characterized by the ability to induce cell

division in tissue cultures usually in the presence of Auxin.

The most common type of Cytokinin found in plants is zeatin. Cytokinin also

occurs asribosides and ribotides. In tissue culture and crown gall culture, Cytokinins

promote shoot initiation. In moss, Cytokinins induce bud formation. Kinetin, the

prototype molecule for the synthetic adenyl cytokinins and zeatin which is about 10

times more potent and generally considered the prototype of the naturally occurring

Cytokinins, is widely used in tissue culture (Ezeibekwe et al., 2009).

1.1.4.5. Plant cell culture media

Culture media used for the in vitro cultivation of plant cells are composed of

three basic components: (1) Essential elements, or mineral ions, supplied as a complex

mixture of salts; (2) An organic supplement supplying vitamins and/or amino acids;

and (3) A source of fixed carbon; usually supplied as the sugar sucrose. For practical

purposes, the essential elements are further divided into the following categories: (1)

macroelements (or macronutrients); (2) microelements (or micronutrients); and (3) an

iron source.

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1.1.4.5.1. Macroelements

As is implied by the name, the stock solution supplies those elements required in

large amounts for plant growth and development. Nitrogen, Phosphorus, Potassium,

Magnesium, Calcium and Sulphur (and Carbon, which is added separately) are usually

regarded as macroelements. These elements usually comprise at least 0.1% of the dry

weight of plants.

1.1.4.5.2. Microelements

These elements are required in trace amounts for plant growth and development,

and have many and diverse roles. Manganese, Iodine, Copper, Cobalt, Boron,

Molybdenum, Iron and Zinc usually comprise the microelements, although other

elements such as Nickel and Aluminium are frequently found in some formulations.

Iron is usually added as iron sulphate, although iron citrate can also be used.

Ethylenediaminetetraacetic acid (EDTA) is usually used in conjunction with the iron

sulphate. The EDTA complexes with the iron so as to allow the slow and continuous

release of iron into the medium (Murashige and Skoog, 1962).

1.1.4.5.3. Organic supplements

Only two vitamins, thiamine (vitamin B1) and myoinositol (considered as B

vitamin) are considered essential for the culture of plant cells in vitro. However, other

vitamins are often added to plant cell culture media for historical reasons.

According to Berlin and Sasse (1985), different plants required different

nutrients, and callus could be derived from different plant parts and they required

different nutritional constituents and appropriate plant growth regulators. (Ravishankar

and Venkataraman, 1993; Ramachandra Rao, 2000). Plant growth regulators are

important and the balance between Auxin and Cytokinin concentration is crucial in

establishing callus cultures and maintaining them.

However, some callus becomes habituated and they no longer require the

addition of a particular plant growth regulator for their maintenance and growth.

Mostly callus formed from the same explants can normally be grown on the same

medium. A suitable medium for initiation and maintenance of callus can only obtained

by trial and error. The maintenance of cultures can determine whether a culture retains

its organogenic potential (Berlin and Sasse, 1985). The most important factor in

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maintaining morphogenic potential is the tolerance of chromosome stability. The

variation of chromosome number of plants in a long-term cell suspension culture has

been well documented. Polyploidy and aneuploidy are a major source of somaclonal

variation.

Sucrose concentration is one of the important factors in a plant cell culture. It is

utilized as a carbon source. It was found that an increase in the sucrose concentration in

a culture medium could result in an increase of secondary metabolite production

(Dicosmo and Misawa, 1995). The enhancing effect of sucrose was most impressively

shown in the case of rosmarinic acid formation in Coleus blumei cell suspension

cultures, where the rosmarinic acid content increased six fold in medium containing

5% sucrose compared with that in the 2% sucrose control medium (Ellis and Towers,

1970).

In plants, amino acids fulfil a wide variety of functions. Their common role is to

serve as building blocks of proteins, which exert manifold functions in plant

metabolism, and as metabolites and precursors they are involved in plant defence,

vitamin, nucleotide and hormone biosynthesis, and as precursors of a huge variety of

secondary compounds. One way or the other, as active catalysts or as precursors, amino

acids are essentially involved in all metabolic, regulatory, and physiological aspects of

plant metabolism (Miflin and Lea, 1977).

Optimization of medium nutrients is also important to increase the productivity

of particular secondary metabolites. There were a number of reports describing the

effects of medium nutrients on secondary metabolites production in plant cell cultures.

One of the investigations was the manipulation of cell growth inhibition medium

resulted an increase in the production of secondary metabolites, and the establishment

of two stage culture system for production of phytochemicals.

In this system, the cells were first cultured in a medium appropriate for

maximum cell biomass production and then transferred to the growth limiting medium

for maximum production of secondary metabolites. Physical factors such as light,

temperature, pH of the medium, aeration rate, can also affect secondary metabolite

synthesis in a cultured plant cells. The effect of light on secondary metabolite

biosynthesis was found to be quite varied. Light illumination usually induced

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chloroplast differentiation which sometimes resulted in elevation of secondary

metabolism production.

1.1.4.6. Callus culture

Theoretically all living cells are capable of giving rise to full plants and this

phenomenon is called cellular totipotency. In cultures, isolated plant cells/tissues may

be induced to form an actively growing mass of cells called callus which can be

multiplied for an indefinite period by routine sub culturing. It is an actively dividing

and more or less undifferentiated tissue. It can be obtained from isolating tissues,

organs and embryos in vitro; generally first undergo dedifferentiation before all

division starts.

It can be considered as a wound response from almost any part of the original

plant, both from plant organs (e.g. roots, leaves, petioles and stems) and from specific

tissue types or cells (e.g. pollen, endosperm, mesophyll). This wound response is

characterized by limited cell division and a rapid increase in metabolic activity, but

does not necessary lead to callus development. On the other hand, the growth response

resulted in continued cell division and is usually dependent on an exogenous supply of

Auxin.

Nobecourt were the first to induce callus culture from carrot (Daucus carota L.)

root tissues with the aid of IAA (indole-3-acetic acid) in 1939. A portion of the callus

tissue when transferred to the differentiation medium could result in shoot or bud

regeneration or the formation of somatic embryos (Pierik, 1988). The age and

physiological state of the mother plant could affect the formation of callus. Generally,

the explant material should be healthy and vigorous growing.

Allan (1991) reported that tissues from plants that were about to enter dormancy

were best for callus induction. The importance of plant age was obviously observed

from tree species, where callus usually could only be initiated from juvenile tissue, and

not explants from mature trees. The season of the year to collect the explants could also

affect callus initiation of explants derived especially from a field grown plant (George

and Sherrington, 1984).

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Cell culture of several plant species had been established for the production of

secondary metabolites such as podophyllotoxin and its derivatives, glucoside, 5-

methoxypodo phyllotoxin. Production cell culture of Podophyllum hexandrum was

increased by a factor of 6- to 30-fold after the addition of the precursor Coniferyl

alcohol, solubilized as a β- cyclodextrin complex or a glucoside from coniferin

(Woerdenbag et al., 1990). High producibility (0.2 % dry weight) of camptothecin was

reported for Catharanthus acuminata with the addition of yeast extracts and

jasmonates in the cell culture (Song and Byun, 1998).

1.2. GENERAL INTRODUCTION OF DIABETES MELLITUS

The disease diabetes mellitus (DM) has been recognized since antiquity, first

having been described around 1500 BC. in Egypt. The complete clinical description of

diabetes was given by the Ancient Greek physician Aretaeus of Cappadocia

(1st Century), who noted the excessive amount of urine which passed through the

kidneys and gave the disease the name “diabetes.” (Harper and Douglas, 2010; Dallas

and John, 2011). In Ayurveda, the oldest text book of Hindu medicine (Charak Samhita

and Sushruta Samhita of 6th century BC), diabetes is described as a urinary disorder, in

which the urine is sweet to taste and the disease is accordingly termed “Madhumeha”

(honey-urine) (Dwivedi et al., 2007).

Symptoms like lack of energy and tending to sleep, dryness of throat, sweet taste

in mouth, burning sensation in hands and swarming of ant on urine are discussed there.

Complaints like boils, carbuncles and gangrene are also mentioned. Instruction like

sugar, fats and oil restrictions and need for exercise too are given in them. The word

“Diabetes” is derived from the Greek word which means ‘Siphon’ a reference to the

copious urine, excretion that characterizes that affliction. Mellitus is the Latin word for

‘honey’. Thus the name “Diabetes mellitus” meaning passing of sweet urine was

introduced in Oxford English Dictionary (2011).

Diabetes mellitus is a metabolic disorder initially characterized by a loss of

glucose homeostasis with disturbances of carbohydrate, fat and protein metabolism

resulting from defects in insulin secretion, insulin action, or both (Barcelo and

Rajpathak, 2001). Without enough insulin, the cells of the body cannot absorb

sufficient glucose from the blood; hence blood glucose levels increase, which is termed

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as hyperglycemia. The disease crosses many boundaries, from physicians to geneticists,

and represents still a major health problem in the world (Pari and Saravanan, 2004).

1.2.1. Pathophysiology of diabetes mellitus

The pancreas plays a primary role in the metabolism of glucose by secreting the

hormones insulin and glucagon (Figure 1.1). The islets of Langerhans secrete insulin

and glucagon directly into the blood. Insulin is essential for proper regulation of

glucose and for maintenance of blood glucose levels (Worthley, 2003).

Glucagon is a hormone that opposes the action of insulin. It is secreted when

blood glucose level falls. It increases blood glucose concentration partly by breaking

down stored glycogen in the liver by a pathway known as glycogenolysis.

Gluconeogenesis is the production of glucose in the liver from non-carbohydrate

precursors such as glycogenic amino acids (Sowka et al., 2001).

Fig. 1.1. The role of pancreas in the human body

1.2.2. Types of diabetes mellitus

WHO classification of diabetes introduced in 1980 and revised in 1985 was based on

clinical characteristics. The two most common types of diabetes

1. Type I or Insulin-dependent diabetes mellitus (IDDM) and

2. Type II or Non-insulin-dependent diabetes mellitus (NIDDM).

(Holt, 2004; Tiwari and Rao, 2002).

3. Secondary diabetes

According to Cooke (2008), secondary diabetes is caused by the following

conditions. They are,

a. Pancreatic disease

Recurrent pancreatitis, malnutrition diabetes (calcific pancreatitis),

Haemochromatosis, pancreatectomy, islet- β-cell toxins.

b. Hormonal, Metabolic disturbances

Gestational diabetes, steroid excess, acromegaly, glucagonoma, hepatic

cirrhosis, catecholamine-induced diabetes.

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c. Drug induced diabetes

Thiazide diuretics

Genetic and other syndromes

Insulin receptor abnormalities

Insulin receptor antibodies

Glycogen storage diseases

DIDMOAD syndrome (Diabetes insipidus diabetes mellitus optic atrophy)

Prader- Willi syndrome, Lipodystrophy, Ataxia telangiectasia, diabetes

insipidus.

1.2.2.1. Type I diabetes mellitus

It is a result of cellular mediated autoimmune destruction of the insulin secreting

β-cells of the pancreas, which results in an absolute deficiency of insulin for the body.

Patients are more prone to ketoacidosis (Elizabeth et al., 2008). It occurs in children

and young, usually before 40 years of age, although disease onset can occur at any age.

The patient with type I diabetes must rely on insulin medication for survival. It may

account for 5 -10 % of all diagnosed cases of diabetes. Autoimmune, genetic and

environmental factors are the major risk factors for type I diabetes (Cavallerano and

Cooppan, 2002; Abebe et al., 2003; NDFS, 2005; Johns, 2007). Diabetic ketoacidosis

is caused by reduced insulin levels, decreased glucose use, and increased

gluconeogenesis from elevated counter regulatory hormones, including

catecholamines, glucagon and cortisol. Primarily it affects patients with type I diabetes,

but also may occur in patients with type 2 diabetes. Patients with diabetic ketoacidosis

usually present with polyuria, polydypsia, polyphagia, and weakness (Trachtenbarg,

2005).

1.2.2.2. Type II diabetes mellitus

Two key features in the pathogenesis of type II diabetes mellitus are a decreased

ability of insulin to stimulate glucose uptake in peripheral tissues, insulin resistance,

and the inability of the pancreatic β-cell to secrete insulin adequately, β-cell failure.

The major sites of insulin resistance in type 2 diabetes are the liver, skeletal muscle and

adipose tissue (Ostenson, 2001; White et al., 2003). Both defects, insulin resistance and

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β-cell failure, are caused by a combination of genetic and environmental factors.

Environmental factors such as lifestyle habits (i.e., physical inactivity and poor dietary

intake), obesity and toxins may act as initiating factors or progression factors for type

II diabetes. The genetic factors are still poorly understood (Uusitupa, 2002; Lindstrom

et al., 2003; Holt, 2004). Type II diabetes is increasingly being diagnosed at any age.

Nowadays and it accounts for 90-95% of all diagnosed cases of diabetes. It is

associated with old age, obesity, family history of diabetes, impaired glucose

metabolism, physical inactivity, and race /ethnicity (Holt, 2004; Li et al., 2004; NDFS,

2005).

1.2.3. Complications

All forms of diabetes increase the risk of long-term complications. These

typically develop after many years (10–20), but may be the first symptom in those who

have otherwise not received a diagnosis before that time. The major long-term

complications relate to damage to blood vessels.

Diabetes doubles the risk of cardiovascular disease. The main "macrovascular"

diseases (related to atherosclerosis of larger arteries) are ischemic heart disease (angina

and myocardial infarction), stroke and peripheral vascular disease (Boussageon et al.,

2011).

Diabetes also causes "microvascular" complications—damage to the small

blood vessels. Diabetic retinopathy, which affects blood vessel formation in the retina

of the eye, can lead to visual symptoms, reduced vision, and potentially blindness.

Diabetic nephropathy, the impact of diabetes on the kidneys, can lead to scarring

changes in the kidney tissue, loss of small or progressively larger amounts of protein in

the urine, and eventually chronic kidney disease requiring dialysis.

Diabetic neuropathy is the impact of diabetes on the nervous system, most

commonly causing numbness, tingling and pain in the feet and also increasing the risk

of skin damage due to altered sensation. Together with vascular disease in the legs,

neuropathy contributes to the risk of diabetes-related foot problems (such as diabetic

foot ulcers) that can be difficult to treat and occasionally require amputation

(Boussageon et al., 2011).

1.2.4. Clinical features and Aetiology of diabetes

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Genetic, autoimmune and environmental factors are involved in the aetiology of

diabetes. Genetic disorders associated with glucose intolerance sometimes causes

clinical diabetes. In NIDDM, diet and obesity has ascribed great significance. NIDDM

has strong genetic basis, being autosomal dominant in some isolated population, but

more usually polygenic in nature. Before the discovery of insulin, degeneration and

inflammatory reaction in and around the islet of Langerhans has been reported. IDDM

showed antipancreatic cellular hypersensitivity associated with certain HLA types often

found in disease of autoimmune character and the presence of islet cell antibodies.

In human, two chromosomal regions show association with, and linkage to type

I diabetes: the MHC (major histocompatibility complex) H2L region on chromosome

bp21, IDDM and the insulin gene region (Ins) on chromosome Hp15. Davies et al.

(1994) have found that, in addition to chromosome bp21 and Hp15, linkages to

chromosome h4 and b4 were also confirmed.

Autoimmune reaction against one or more components of pancreatic β-cell

results in the destruction of the β-cell. These observations suggest that destructive

process leading to IDDM is probably immune mediated (Riserus et al., 2009).

A viral infection causing human diabetes is evident from the studies of patients

with congenital rubella syndrome. Children infected congenitally may show rubella

virus in the pancreas with insulin it is and β-cell destruction. More convincing evidence

for viral involvement is derived from the study of a young boy who died from

overwhelming viral infection and diabetic ketoacidosis (Elizabeth et al., 2008).

Environmental agents may have a deleterious effect on β-cell. Number of

chemicals is known to have relatively specific cytotoxic effect on β-cell including

alloxan, streptozotocin and rodenticide racor. Encephalomyocarditis virus and

causative B virus can induce diabetes in animals by infecting pancreatic β-cell. Some

wild viruses exist which have specific β- cell tropism and diabetogenic potential in

animals and the patients with congenital rubella syndrome, particularly those with

HLA-DR3 (Riserus et al., 2009).

1.2.5. Clinical Manifestations

The manifestations of symptomatic diabetic mellitus vary from patients to

patient. Most often medical help is sought because of symptoms related to

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hyperglycemia (polyuria, polydypsia, polyphagia), but the first event may be acute

metabolic decomposition resulting in diabetic coma. A recent survey list shows more

than 60 distinct genetic disorders associated with glucose intolerance and some times,

clinical diabetes (Cooke, 2008).

1.2.6. Pathogenesis

The pathogenesis of complication of long standing diabetes, such as

macroangiopathy, retinopathy and nephropathy are currently the subject of a great deal

of research. Although it is felt that these complications are due to genetic disorders

untreated to the metabolic abnormalities, most of the available evidences suggest that

the complications of diabetes mellitus are a consequence of these metabolic

derangements (Cooke, 2008).

1.2.7. Epidemiology of diabetes mellitus

The prevalence of diabetes mellitus is increasing with ageing of the population

and lifestyle changes associated with rapid urbanization and westernization. The

disease is found in all parts of the world and is rapidly increasing in its coverage

(Sobngwi et al., 2001; Kamalakkanan and Prince, 2003).

1.2.8. Prevalence and incidence of diabetes mellitus

Globally, the prevalence of diabetes, without type distinction, was estimated to

be 4% in 1995. According to WHO, it is estimated that 3% of the world’s population

have diabetes and the prevalence is expected to double by the year 2025 to 6.3%

(Andrade-Cetto and Heinrich, 2005; Attele et al., 2002). There will be a 42% increase

from 51 to 72 million in the developed countries and 170% increase from 84 to 228

million, in the developing countries. Thus, by the year 2025, over 75% of all people

with diabetes will be in the developing countries, as compared to 62% in 1995

(Ramachandran et al., 2002).

There are many reasons behind this projected increase in prevalence such as,

increase in life expectancy at birth, physical inactivity and obesity and possibly a

genetic predisposition (Sobngwi et al., 2001; Wild et al., 2004). Age, ethnic, regional

and racial differences have also been found to play a role for the diabetic incidence in

heterogeneous populations within the same area (NDFS, 2005; Alberti et al., 2007).

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1.2.9. Diabetes in India

India has more diabetics than any other country in the world, according to the

International Diabetes Foundation, although more recent data suggest that China has

even more (BBC, 2010; Grens and Kerry, 2012). The disease affects more than 50

million Indians - 7.1% of the nation's adults - and kills about 1 million Indians a year.

The average age on onset is 42.5 years (Gale and Jason, 2010). The high incidence is

attributed to a combination of genetic susceptibility plus adoption of a high-calorie,

low-activity lifestyle by India's growing middle class (Kleinfield, 2006).

1.2.10. Risk factors

The predisposing factors associated with diabetes mellitus include modifiable

and non-modifiable factors. Among the modifiable risk factors, residence seems a

major determinant, since urban residents have a 1.5 to 4 fold higher prevalence of

diabetes compared to their rural counterparts. This is attributable to lifestyle changes

associated with urbanization and westernization, diet, obesity and physical inactivity,

Age, ethnicity, history of gestational diabetes and family history of type II diabetes are

the main non-modifiable determinants of diabetes prevalence (Colagiuri et al., 2006;

Libman and Arslanian, 2007).

1.2.11. Management of diabetes mellitus

Diet, exercise, modern drugs including insulin and oral administration of

hypoglycaemic drugs such as sulfonylureas and biguanides manage the pathogenesis of

diabetes mellitus. Insulin plays a key role in glucose homeostasis along the side of a

counter regulatory hormone, glucagon, which raises serum glucose. Carrier proteins

(GLUT 1- 5) are essential for glucose uptake into cells. In individuals with type II

diabetes, a common sequence of therapy starts with diet treatment and exercise

followed by oral antihyperglycemic agents. In general, insulin therapy has been

considered to be the last therapeutically option when diet, exercise and oral

antihyperglycemic agent therapies have failed.

Traditionally Medicinal plants are also used for the treatment of diabetes

throughout the world (Pari and Saravanan, 2004; Koski, 2006). Management of

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diabetes without any side effect is still a challenge for the medical system. This leads to

an increasing search for improved antidiabetic drugs.

1.2.12. Medicinal plants

1.2.12.1. The importance role of traditional medicinal plants in diabetes mellitus

treatment

As long as human beings have been living on Earth they have used remedies

from nature to improve their health or to cure illnesses. Documentation of this can be

found as far back in time as approximately 6000 years. It is interesting to note that

certain plants that are described in plates being that old are still in use both in what

today is called traditional medicine, but also the active ingredients from these plants are

used as single compounds in modern medicine.

Medicinal products had up to the beginning of the 20th century mainly been

produced by using extracts or powder of medicinal plants as the main active ingredient.

The chemical and biological achievements mentioned above, were amongst those that

were important for the development of more modern medicines based on pure

compounds isolated from plants or micro-organisms. In order to decide what plants to

use for isolation of active ingredient the scientific discipline ethnopharmacology

evolved. Ethnopharmacology involves studies of cultures still using traditional

medicines and can be defined as the observation, identification, description and

experimental investigation of the ingredients and the effects of such indigenous drugs

(Bruneton, 1999; Samuelson, 2004).

Medicinal plants, since time immemorial, have been used in virtually all

cultures as a source of medicine. It has been estimated that about 80-85% of population

both in developed and developing countries rely on traditional medicine for their

primarily health care needs and it is assumed that a major part of traditional therapy

involves the use of plant extracts or their active principles (Tomlinson and Akerele,

1998; Elujoba et al., 2005; Ignacimuthu et al., 2006).

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The active principles of many plant species are isolated for direct use as drugs,

lead compounds or pharmacological agents. Different species of medicinal plants are

used in the treatment of diabetes mellitus. For diabetes treatment, before the discovery

of insulin by Banting and Best in 1922, the only options were those based on traditional

practices (Ribnicky et al., 2006). The following table (Table-1.1.) shows various

medicinal plants identified to have the property of controlled blood glucose level

experimentally (Kavishankar et al., 2011).

1.3. GENERAL INTRODUCTION OF ANTIOXIDANT ACTIVITY

Antioxidants are often described as “free radical scavengers” meaning that they

neutralize the electrical charge and prevent the free radicals from taking electrons from

other molecules. They play a key role in the body defence system against reactive

oxygen species (ROS) which are known to be involved in the pathogenesis of aging

and many degenerative diseases such as cardiovascular diseases, diabetes mellitus,

cataract, cancers, atherosclerosis, hypertension, ischemia/reperfusion injury,

neurodegenerative diseases (Alzheimer's disease and Parkinson's disease), rheumatoid

arthritis and ageing (Hill et al., 1993; Beckman and Ames, 1998; McCord, 2000).

To protect the cells and organ systems of the body against reactive oxygen

species, humans have evolved a highly sophisticated and complex antioxidant

protection system. It involves a variety of components, both endogenous and

exogenous in origin, that function interactively and synergistically to neutralize free

radicals (Sies, 1997; Blomhoff, 2005).

There are two lines of antioxidant defence within the cell. The first line, found

in the fat soluble cellular membrane consists of vitamin E, beta-carotene, and

coenzyme Q. Of these, vitamin E is considered the most potent chain breaking

antioxidant within the membrane of the cell. Inside the cell, water soluble antioxidant

scavengers are present. These include vitamin C, glutathione peroxidase, superoxide

dismutase (SOD) and catalase (Halliwell, 1996; Gutteridge and Halliwell, 2000).

Plant cells are known to have both enzymatic and non-enzymatic defence

mechanisms to counteract the destructive effects of activated oxygen species. The

antioxidant defence system consists of low molecular weight antioxidants such as

ascorbate, glutathione, α-tocopherol and β-carotenoids, peptides, vitamins, flavonoids,

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phenolic acids, alkaloids as well as several antioxidant enzymes such as superoxide

dismutase (SOD), guaiacol peroxidase (POD), ascorbic acid peroxidase (APX) and

glutathione reductase (GR) (Astley and Lindsay, 2002; Manach et al., 2004; Manach et

al., 2005; Williamson and Manach, 2005).

1.3.1. Oxidative stress

Oxidative stress is a state characterized by an excess of reactive oxygen species

(ROS) in the body, which creates a potentially unstable cellular environment that is

associated with tissue damage accelerated aging and degenerative diseases. Free

radicals and reactive oxygen species (ROS) are species with incomplete electron shells

that make them move chemically reactive than those with complete electron shells.

They are by products of metabolic processes. Some of the reactive species which are of

particular interest from the point of view of oxidative stress are: Super oxide radical

(O(2)(-)); hydroxyl radical (HO(-)); peroxyl radical (ROO(-));

Hydrogen peroxide (H(2)O(2)); Alkoxyl radical; singlet oxygen ((1)O(2)); nitric oxide

((-)NO); peroxynitrite (ONOO(-)).

Oxidative stress is induced by a wide range of environmental factors including

UV stress, pathogen invasion (hypersensitive reaction), herbicide action, oxygen

shortage, cigarette smoke, automobile exhaust fumes, and air pollutants. There are

numerous types of free radicals that can be formed within the body. The most common

ROS include: the superoxide anion (O2-), the hydroxyl radical (OH-), singlet oxygen

(One O2), and hydrogen peroxide (H2O2). Superoxide anions are formed when oxygen

(O2) acquires an additional electron, leaving the molecule with only one unpaired

electron. Within the mitochondria O2- · is continuously being formed. The rate of

formation depends on the amount of oxygen flowing through the mitochondria at any

given time.

Hydroxyl radicals are short-lived, but are the most damaging radicals within the

body. This type of free radical can be formed from O2- and H2O2 via the Harber-Weiss

reaction. The interaction of copper or iron and H2O2 also produce OH· as first observed

by Fenton. These reactions are significant as the substrates are found within the body

and could easily interact. Hydrogen peroxide is produced in vivo by many reactions.

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Hydrogen peroxide is unique in that it can be converted to the highly damaging

hydroxyl radical or be catalyzed and excreted harmlessly as water.

Glutathione peroxidase is essential for the conversion of glutathione to oxidized

glutathione, during which H2O2 is converted to water. If H2O2 is not converted into

water, instead singlet oxygen (one O2) is formed. Singlet oxygen is not a free radical,

but can be formed during radical reactions and also cause further reactions. Singlet

oxygen violates Hund's rule of electron filling in that it has eight outer electrons

existing in pairs leaving one orbital of the same energy level empty. When oxygen is

energetically excited one of the electrons can jump to empty orbital creating unpaired

electrons. Singlet oxygen can then transfer the energy to a new molecule and act as a

catalyst for free radical formation. The molecule can also interact with other molecules

leading to the formation of a new free radical (Siwik et al., 2001).

O2 + e¬ - → O2-*

O2-* + H→ HO2*

2HO2*→ H2O2 + O2

2H2O2→ H2O + O2

H2O2 + ∟H2→ 2H2O +∟

O2-*+ H2O2 → O2+ HO- + HO* (Harber - Weiss reaction)

H2O2 +Fe2 + → Fe3+ + HO- + HO* (Fenton reaction)

1.3.2. Production of Free radicals in the Human body

Free radicals and other reactive oxygen species are derived either from normal

essential metabolic processes in the human body or from external sources such as

exposure to X-rays, ozone, cigarette smoking, air pollutants and industrial chemicals.

Free radical formation occurs continuously in the cells as a consequence of both

enzymatic and non-enzymatic reactions. Enzymatic reactions which serve as sources of

free radicals include those involved in the respiratory chain, in phagocytosis, in

prostaglandin synthesis and in the cytochrome P450 system. Free radicals also arise in

non-enzymatic reactions of oxygen with organic compounds as well as those initiated

by ionizing radiations.

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Some internally generated sources of free radicals are: Mitochondria;

Phagocytes; Xanthine oxidase; Reactions involving iron and other transition metals;

Arachidonate pathways; Peroxisomes; Exercise; Inflammation; Ischaemia/reperfusion.

Some externally generated sources of free radicals are: Cigarette smoke;

Environmental pollutants; Radiation; Ultraviolet light; certain drugs; Pesticides;

Anaesthetics and Industrial solvents; Ozone.

If free radicals are not inactivated, their chemical reactivity can damage all

cellular macromolecules including proteins, carbohydrates, lipids and nucleic acids.

Their destructive effects on proteins may play a role in the causation of cataracts. Free

radical damage to DNA is also implicated in the causation of cancer and its effect on

LDL cholesterol is very likely responsible for heart disease. In fact, the theory

associating free radicals with the aging process has also gained widespread acceptance

(Halliwell, 1996; Gutteridge and Halliwell, 2000).

1.3.3. Importance of Free radicals

Free radicals are naturally produced taken within the body and have beneficial

effects that cannot be overlooked. The immune system is the main body system that

utilizes free radicals. Foreign invaders or damaged tissue is marked with free radicals

by the immune system. This allows for determination of which tissue need to be

removed from the body. Because of this there is a need for antioxidant supplementation

(Ghosal, 2000).

1.3.4. Oxidative Stress in various diseases

A growing body of evidence suggests oxidative stress involvement in

neurodegenerative diseases; however, it remains to be determined whether oxidative

stress is a cause, result, or epiphenomenon of the pathological processes. ROS

contribute to oxidative stress, which is linked to numerous degenerative conditions

including cardiovascular disease, inflammation, Alzheimer’s disease, Parkinson’s

disease, diabetes, Aging etc. The other conditions associated with oxidative stress are

Atherosclerosis; Cancer; Pulmonary dysfunction; Cataracts; Arthritis and;

22

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inflammatory diseases; Diabetes; Shock, trauma and ischemia; Renal disease and

hemodialysis (Hunt et al., 1988; Baynes, 1991; Dean and Davies, 1993).

Mechanisms involved in the role of ROS and oxidative stress in disease

development may include alteration of important bio molecules causing oxidative

modifications in nucleic acids, modulation of gene expression through activation of

redox sensitive transcription factors and modulation of inflammatory responses through

signal transduction (Mantovani et al., 2008).

1.3.5. Mechanism of Antioxidants in the body

Antioxidants provide protection against oxidative attack by decreasing oxygen

concentration, intercepting singlet oxygen, preventing first chain initiation by

scavenging initial radicals, binding of metal ion catalysts, decomposing the primary

products of oxidation to non radical compounds and chain breaking to prevent

continuous hydrogen removal from substrates. Antioxidants react with radicals and

other reactive species faster than biological substrates, thus protecting biological targets

from oxidative damage. Furthermore the resulting anti oxidant radical possess high

stability that is the antioxidant radical interrupts (rather than propagate) a chain

reaction (Hunt et al., 1988).

1.3.6. Exogenous Antioxidant system

The body gets some of its antioxidants from the environment, more specifically

from the food that is consumed (exogenous antioxidants). While the enzymatic

antioxidants are intrinsic to the organism, the non-enzymatic components are both of

intrinsic and exogenous nature. The non-enzymatic antioxidants consist of nutrient and

non nutrient compounds.

1.3.6.1. Enzymatic Antioxidants

Among the enzymatic antioxidants mainly three groups of enzymes play

significant roles in protecting cells from oxidant stress:

Superoxide dismutases (SOD) are enzymes that catalyze the conversion of two

super oxides into hydrogen peroxide and oxygen. The benefit here is that hydrogen

peroxide is substantially less toxic that superoxide. SOD accelerates this detoxifying

reaction roughly 10,000-fold over the non-catalyzed reaction.

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SODs are metal-containing enzymes that depend on bound Manganese, Copper

or Zinc for their antioxidant activity. In mammals, the Manganese-containing enzyme

is most abundant in mitochondria, while the zinc or copper forms predominant in

cytoplasm. Interestingly, SODs are inducible enzymes - exposure of bacteria or

vertebrate cells to higher concentrations of oxygen results in rapid increases in the

concentration of SOD.

Catalase is found in peroxisomes in eukaryotic cells. It degrades hydrogen

peroxide to water and oxygen and hence finishes the detoxification reaction started by

SOD.

Glutathione peroxidase is a group of enzymes, the most abundant of which

contain Selenium. These enzymes, like catalase, degrade hydrogen peroxide. They also

reduce organic peroxides to alcohols, providing another route for eliminating toxic

oxidants. In addition to these enzymes, glutathione transferase, ceruloplasmin,

hemoxygenase and possibly several other enzymes may participate in enzymatic

control of oxygen radicals and their products.

1.3.6.2. Non- Enzymatic Antioxidants

1.3.6.2.1. Nutrient Compounds

1.3.6.2.1.1. Vitamin C

Ascorbate, an essential vitamin found in fruits and vegetables, has been

particularly well studied in its role as an antioxidant and is suggested to serve several

physiological functions including, the preventing free-radical-induced damage to DNA,

quenching oxidants which can lead to the development of cataracts, improving

endothelial cell dysfunction, and decreasing LDL induced leukocyte adhesion. Vitamin

C readily scavenges reactive oxygen and nitrogen species and may thereby prevent

oxidative damage to important biological macromolecules such as DNA, lipids, and

proteins. Vitamin C also reduces redox active transition metal ions in the active sites of

specific biosynthetic enzymes. Ascorbic acid, or vitamin C, has the potential to protect

both cytosolic and membrane components of cells from oxidant damage. In the cytosol,

ascorbate acts as a primary antioxidant to scavenge free radical species that are

generated as by-products of cellular metabolism. For cellular membranes, it may play

an indirect antioxidant role to reduce the a-tocopheroxyl radical to a-tocopherol. The

24

Chapter -1

erythrocyte results indicate that ascorbate can interact directly with the plasma

membrane as an antioxidant. Excellent sources of vitamin C include: parsley, broccoli,

bell pepper, strawberries, oranges, lemon juice, papaya, cauliflower, kale, mustard

greens (Food and Nutrition Board, 1998).

1.3.6.2.1.2. Vitamin E (α-tocopherol)

Vitamin E (tocopherol) is a fat-soluble vitamin which functions solely as a

membrane bound antioxidant that prevents cell membrane damage by inhibiting per

oxidation of membrane phospholipids and disrupting free radical chain reactions

induced by formation of lipid peroxides. Vitamin E also increases the bioavailability of

Vitamin A by inhibiting its intestinal oxidation. As the only membrane-bound lipid-

soluble antioxidant, Vitamin E plays a key role in preventing cellular injury from

oxidative stress associated with premature aging, cataracts, uncontrolled diabetes,

cardiovascular disease, inflammation, and infection. Exogenous supplementation of

functionally efficient antioxidants like vitamin E reactivates the enzymatic antioxidant

system and guards against the insult caused by ROS during the pathogenesis of the

diseases.

Increased production of reactive oxygen species secondary to phagocyte

respiratory burst occurs in pulmonary tuberculosis (TB) Vitamin E and Selenium

supplementation reduces oxidative stress and enhances total antioxidant status in

patients with pulmonary TB treated with standard chemotherapy. Vitamin E is found

only in foods of plant origin. Wheat germ is the richest source of the vitamin. Vegetable

oils and whole grains are additional rich sources of this nutrient. Nuts, peanut butter,

salad dressings and vegetable oils are also good sources of Vitamin E (Albanes et al.,

1996; Rapola et al., 1997; Food and nutrition Board, 1998).

1.3.6.2.1.3. β-Carotenoids

Carotenoids are nature’s most widespread pigments and have also received

substantial attention because of both their provitamin and antioxidant roles. More than

600 different carotenoids have been identified in nature. Carotenoids have a 40-carbon

skeleton of isoprene units. The structure may be cyclized at one or both ends, may

have various hydrogenation levels, or may possess oxygen-containing functional

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Chapter -1

groups. Lycopene and ß-carotene are examples of acyclized and cyclized carotenoids,

respectively. Carotenoid compounds most commonly occur in nature in the all-trans

form.

Mixtures of carotenoids or associations with others antioxidants (e.g. Vitamin E)

can increase their activity against free radicals. Carotenoids are found in colored fruits

and vegetables. Apricots, antaloupe, carrots, pumpkin and sweet potato are sources of

a-carotene and b-carotene; pink grapefruit, tomatoes and watermelon are sources of

lycopene, z-carotene, β -carotene, phytofluene and phytoene. Mango, papaya, peaches,

prunes, squash and oranges are sources of lutein, zeaxanthin, and β -cryptoxanthin, α,

β- and ω-carotene, phytofluene and phytoene, whereas green fruits and vegetables such

as green beans, broccoli, brussel sprouts, cabbage, kale, kiwi, lettuce, peas and spinach

are sources of lutein, zeaxanthin, α- and β-carotene. Carotenoid concentrations in fruits

and vegetables vary with plant variety, degree of ripeness, time of harvest, and growing

and storage conditions (Food and Nutrition Board, 1998; Williamson and Manach,

2005).

1.3.6.2.2. Non – Nutrient Compounds

1.3.6.2.2.1. Polyphenols - the potent antioxidants in plant foods

Phenolic antioxidants, a specific group of secondary metabolites play the

important role of protecting organism against harmful effects of oxygen radicals and

other highly ROS. Their formation in human organisms is closely connected with the

development of a wide range of degenerative diseases, mainly arteriosclerosis and

other associated complications, cancer and aging.

Among natural antioxidants plant poly phenols play a very important role.

Flavonoids are a class of poly phenolic compounds is widely and ubiquitously found in

fruits, vegetables, grains, nuts, and medicinal plants. High consumption of plant

phenolics in the daily diet has been found to provide their ability to low density lipo

proteins, platelet aggregation, growth of tumour cells and inflammation reactions. They

are one of the major groups of nonessential dietary components appearing in vegetable

foods. They are a wide chemical compounds group that are considered as secondary

plant metabolites, with different activity and chemical structure, including more than

8,000 different compounds (Astley and Lindsay, 2002; Williamson and Manach, 2005).

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Chapter -1

1.3.6.2.2.2. Flavonoids

Flavonoids are a group of phenolic compounds with antioxidant activity that

have been identified in fruits, vegetables, and other plant foods and that have been

linked to reducing the risk of major chronic diseases. More than 4000 distinct

flavonoids have been identified. Flavonols (quercetin, kaempferol, and myricetin),

flavones (luteolin and apigenin), flavanols (catechin, epicatechin, epigallocatechin,

epicatechin gallate, and epigallocatechin gallate), flavanones (naringenin),

anthocyanidins, and isoflavonoids (genistein) are common flavonoids in the diet.

Flavonoids are most frequently found in nature as conjugates in glycosylated or

esterified forms but can occur as aglycones, especially as a result of the effects of food

processing (Astley and Lindsay, 2002; Manach et al., 2004; Manach et al., 2005)

1.3.6.2.2.3. Phenolic acids

Phenolic acids can be subdivided into two major groups, hydroxybenzoic acids

and hydroxycinnamic acids. Hydroxybenzoic acid derivatives include p-

hydroxybenzoic, protocatechuic, vannilic, syringic, and gallic acids. They are

commonly present in the bound form and are typically a component of a complex

structure like lignins and hydrolyzable tannins. They can also be found in the form of

sugar derivatives and organic acids in plant foods. Food processing, such as thermal

processing, pasteurization, fermentation, and freezing, contributes to the release of

these bound phenolic acids Hydroxycinnamic acid derivatives include p-coumaric,

caffeic, ferulic, and sinapic acids.

They are mainly present in the bound form, linked to cell-wall structural

components, such as cellulose, lignin, and proteins through ester bonds. Ferulic acids

occur primarily in the seeds and leaves of plants, mainly covalently conjugated to

mono- and disaccharides, plant-cell-wall polysaccharides, glycoproteins, polyamines,

lignin, and insoluble carbohydrate biopolymers. Wheat bran is a good source of ferulic

acids, which are esterified to hemicellulose of the cell walls. Free, soluble-conjugated,

and bound ferulic acids in grains are present in the ratio of 0.1:1:100. Food processing,

such as thermal processing, pasteurization, fermentation, and freezing, contributes to

the release of these bound phenolic acids (Manach et al., 2004; Manach et al., 2005).

1.3.6.2.2.4. Alkaloids

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The alkaloids are heterocyclic, nitrogen containing compounds, usually with

potent activity and bitter taste. They are of limited distribution in the plant kingdom.

The various groups have diverse clinical properties. The first medically used alkaloid

was morphine, isolated in 1805 from the opium poppy Papaver somniferum.

Diterpenoid alkaloids, commonly isolated from the plants of the Ranunculaceae, or

buttercup family, are commonly found to have antimicrobial properties. Solamargine, a

glycoalkaloid from the berries of Solanum khasianum, and other alkaloids may be

useful against HIV infection as well as intestinal infections associated with AIDS.

While alkaloids have been found to have microbiocidal effects, the major anti diarrheal

effect is probably due to their effects on transit time in the small intestine (Sethi, 1979).

1.3.6.2.2.4.1. Berberine

Berberine is an important representative of the alkaloid group compound.

Berberine is a quaternary ammonium salt from the protoberberine group of

isoquinoline alkaloids. Berberine is strongly yellow colored, which is why in earlier

times Berberis species were used to dye wool, leather and wood. Wool is still today

dyed with Berberine in northern India. Under ultraviolet light, Berberine shows a

strong yellow fluorescence. Because of this it is used in histology for staining heparin

in mast cells. As a traditional medicine or dietary supplement, Berberine has shown

some activity against fungal infections, Candida albicans, yeast, parasites, and

bacterial/viral infections. It is potentially effective against trypanosomes and

plasmodia. The mechanism of action of highly aromatic planar quaternary alkaloid

Berberine is attributed to their ability to intercalate with DNA (Birdsall

and Kelly, 1997).

During the last few decades, many studies have shown Berberine has various

beneficial effects on the cardiovascular system and significant anti-inflammatory

activities (Kuo et al., 2004). Berberine has drawn extensive attention towards its

antineoplastic effects (Sun et al., 2009; Tang et al., 2009). It seems to suppress the

growth of a wide variety of tumour cells, including breast cancer, (Kim et al., 2009)

leukemia, melanoma, (Serafim et al., 2008) epidermoid carcinoma, hepatoma,

pancreatic cancer, (Pinto-Garcia et al., 2010) oral carcinoma, tongue carcinoma, (Ho et

al., 2009) glioblastoma, prostate carcinoma and gastric carcinoma. (Tang et al., 2009).

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Animal studies have shown that Berberine can suppress chemical-induced

carcinogenesis, clastogenesis, (Sindhu and Manoharan, 2010) tumour promotion,

tumour invasion, (Thirupurasundari et al., 2009) prostate cancer, (Wang et al., 2009)

neuroblastoma, (Choi et al., 2008) and leukemia (Lin et al., 2006). It is a

radiosensitizer of tumour cells but not of normal cells.

1.4. INTRODUCTION OF IN SILICO ANALYSIS

Life represents the highest manifestation of chemistry. Malfunctions in the

biochemical system lead to diseases and discomforts. Today, extensive efforts are being

made to understand the mechanisms at molecular levels. A so-called reductionist

approach toward health and disease, advocates selecting a bio-molecular target and

correcting its function by a molecular lead (ligand). This approach has been successful

as several very effective ligands have been discovered and used in clinical practices in

recent years.

1.4.1. Modern Medicine

The development of a new drug is called drug discovery. Modern medicine has

made tremendous leaps in the field of drug discovery. The drug design industry is now

one of the major players in the bioinformatics and biotechnology industries. The past

two decades have seen the development of numerous procedures to diagnose and treat

patients.

Drug research is comparatively less expensive than health care. The National

Institute of General Medical Sciences (NIGMS), United States of America (USA)

states that in 1990, research and developmental costs were only 3.7 % of health care

costs. Trying to cure a disease with insufficient research leads to unsuccessful treatment

and soaring health care costs resulting in severe economic impact. Research is the step

which reduces health care costs while potentially saving lives (Skyler, 2000).

Drug discovery and its approval cost a pharmaceutical biotech company

millions of dollars. In the year 1997, the top twenty pharmaceutical companies spent

$16 billion on drug development alone (Cherfils, 1993). Thousands of compounds

were screened and studied in order to find one drug. On a positive note, when a drug

is approved, the company gets billions of dollars in revenue and no other company

can duplicate it, as it is patented for at least 20 years. For example, in 2000,

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Prilosec, a drug commonly used to treat stomach ulcers, earned $4,102 billion in

sales (Kuntz, 1994).

The development of any prospective drug begins with years of strenuous

research to figure out the complexity of the medical problem. Often, it takes a

minimum of 5 years, to discover the drug, 2-5 years of preclinical testing and 3-10

years of clinical testing. A patent is granted for 20 years at the end of the preclinical

phase. The whole process takes 5-10 years after which the drug is forwarded to the

FDA (Food and Drug Administration) for final approval (Bohacek, 1994; Jones, 1995;

Peters, 1996; Klebe, 2000). Drug design is the first step in drug discovery research.

Drugs are usually designed in a manner to bind to, interact with and affect the activity

of the key molecule under study.

Generally to design a drug based on certain theoretical concepts but the

effectiveness of this aspect is far from optimal. There are two main types of research in

drug design. The first method, called High Throughput Screening (HTS), identifies

active compounds in collections or libraries as quickly as possible and with high

statistical accuracy. Success in HTS depends on uncontrollable factors. For instance,

HTS hits (successfully bound compounds) eventually decompose and some HTS hits

have poor physico-chemical properties for use as potential drugs. Many compounds on

the order of thousands or more must be tested to find a hit.

The other method, structure-based drug design, involves detailed knowledge of

the binding sites of targets (such as proteins) associated with the disease. A drug's

effectiveness depends on structural interaction with the receptor or target molecule. The

most common model is the one in which the drug molecule fits itself into the crevice of

the target protein similar to a key in a lock. This strategy results in inhibition of the

protein's function, and ultimately halts the progress of the disease.

Structure-based drug design offers tremendous potential and a powerful

alternative to traditional screening techniques. This drug design method involves basic

knowledge of bioinformatics, proteomics, biochemistry and computer modeling of

three dimensional protein structures.

The aim of anti diabetic therapy, both in insulin-dependent diabetes mellitus and

non-insulin-dependent diabetes mellitus patients, is to achieve normoglycemia (normal

30

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blood glucose level). However, this goal has been only partly achieved. Better

understanding of the various complex biochemical processes involved in the onset and

progress of diabetes is now driving the anti – diabetic drug development.

Protein interactions with ligands, other proteins, or surfaces are controlled by a

complex array of intermolecular interactions. Such interactions depend both on the

specific interactions in the binding site as well as the non-specific forces outside the

binding pocket. This interplay of specific and non-specific forces controls all protein

interactions ranging from bimolecular collisions in solutions to adhesion between cells.

The complexity of interactions between proteins and flexible target molecules,

including other proteins, nucleic acids and small molecules, is often determined by the

considerable flexibility of the protein binding sites and by the structural rearrangements

that occur upon binding of the associated molecule.

A goal of many biophysical studies is to determine the molecular forces that

control biological interactions and to use this information to rationally manipulate

protein function by modifying the protein, the interacting ligand, or both. The forces

that control protein behaviour and their physico chemical origins are inferred from

equilibrium binding kinetic measurements or are calculated with molecular models.

Calculated energies are used to identify the role of the physical and chemical

interactions in protein function and behaviour. Although detailed calculations are

feasible for small molecules, such calculations become prohibitive as the size and

complexity of the biological macromolecules increase.

Virtual ligand screening using in silico methods can provide prospective leads

and is a practical alternative to high-throughput screening of large compound libraries

provided the binding modes and affinities of the distinct ligands can be predicted

correctly. The docking and scoring problems countered in this endeavour are central to

the theory of bimolecular interactions and are ultimately determined by the nature of

the underlying binding energy landscape. However, the desired synergy of adequate

conformational sampling combined with accurate evaluation of energetic has been

difficult to achieve with any computational model.

1.4.2. Current trends in ligand-protein docking

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Computer aided methods for the identification and characterization of ligand–

protein interactions have undergone considerable advances in the past decade. Ligand

docking and screening algorithms are now frequently used in the drug-design process,

and have additional application in the elucidation of fundamental biochemical

processes. There are several well-established docking algorithms which have been

previously reviewed (Cherfils, 1993; Kuntz, 1994) as well as many more recently

introduced methods.

Protein–protein docking methodology has considerable overlap with that of

ligand–protein docking; however, differences between the two docking tasks have

resulted in most algorithms being intended for either one purpose or the other. The

issues faced in designing docking algorithms have been reviewed (Jones, 1995;

Lengaeur, 1996; Verkhiker, 2000). Various workers tested the performance of several

docking algorithms.

Most docking algorithms rely on the binding site being predefined, so that the

search space is limited to a comparatively small region of the protein. As the location

of the binding region of the protein can frequently be inferred from comparison with

other known protein structures or biochemical constraints. The binding site may be

identified by comparison with the protein co-crystallized with a different ligand, or

comparison with proteins of similar function.

In the absence of prior information, putative docking sites may be identified by

cavity detection programs (Peters, 1996; Brady, 2000). The search algorithm must

effectively sample the search space of the ligand–protein complex, i.e. the translation,

rotation, and conformation space of the ligand relative to the protein.

However, for even moderate sampling increments, a search of all possible

combinations within the search space results in the number of combinations, being

frequently in the billions. For example, given a ligand with ten rotating bonds, sampled

in 60° increments, there are over 107 possible conformations in the rotation space

alone. Thus a search algorithm must be able to effectively sample regions of the search

space in the vicinity of the correct solution, without an exhaustive search of all

conformations and orientations.

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The scoring function used within docking algorithms has two roles – as a target

function for the search algorithm (a quantity to be optimized), and to give a ranking to

the set of final solutions generated by the search. Ideally, the combination of the search

algorithm and the scoring function should result in a single solution close to the actual

ligand position. In practice, docking algorithms are tested for their ability to reproduce

known ligand conformations in an X-ray crystal structure within a given margin, and to

recognize one of the conformations closest to the experimental structure as the best

solution combine aspects from two or more search algorithms.

1.4.3. Drug – Like Compounds

Drug-like compounds are molecules which contain functional groups and/or

have physical properties similar to the majority of known drugs (Walters, 2002). Since

a drug-like compound is a potential starting point to develop a new drug, researchers

have made more efforts to extract sets of ‘rules’, i.e. ‘filters’ or ‘criteria’, to identify if a

compound is a ‘drug-like’ compound or not.

The pioneering study on ‘rules’ for drug-like compounds was carried out by

Lipinski in the 1980s and 1990s. As a result of the (Walters, 2002) analysis, Lipinski

proposed a very simple set of physicochemical parameter ranges named ‘Rule of Five’

(RO5). The set of rules was associated with 90% of orally active drugs that have

achieved phase II clinical stages (Lipinski, 2004). Compounds that fail to survive the

phase I human toleration studies or the preclinical stage do not receive an International

Non-Proprietary Name (INN name) and a United States Adopted Name (USAN name).

INN names and USAN names are assigned when a compound enters phase II

clinical states. A compound entering into phase II is a real drug in the sense that it has

all the properties of a real drug. The name of RO5 comes from the parameter cut-off

values all containing 5’s (Lipinski, 1997). There are actually only four simple

physicochemical parameter ranges to be drug like according to the RO5

(Lipinski, 2003):

Molecular weight (M.Wt.) ≤ 500 Lipophilicity Clog P ≤ 5 H-bond donors ≤ 5 (sum of OH and NH) H-bond acceptors ≤ 10 (sum of O and N atoms)

1.4.4. ADME Properties

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ADME drug properties, namely, Absorption, Disposition, Metabolism,

Elimination and toxicity, are important and critical for clinical success. Selection of

drug candidates that meet with the best ADME properties should increase the

likelihood of clinical success. The important drug-like properties can be elucidated by

examining an orally available drug. The following events would occur after taking a

drug into the body (Li, 2005).

Dissolution: Separation of the active components of a drug from its matrix. Absorption: Movement of the drug through the intestinal epithelium into the

systemic circulation. Disposition: Entrance of the drug into the system circulation and distribution

to various organs and tissues. Metabolism: Biotransformation of the drug by the drug metabolizing

enzymes. Biological interaction: Interaction of the drug and its metabolites with

intended and unintended targets. Drug-drug interaction: Interaction of the drug along with other drugs. Elimination: Removal of the drug from the body.Therefore, the desirable drug should have the following properties which are

preferred results of the above events.

High solubility and absorption, ready distribution to intended target tissues,

appropriate metabolic stability and minimal formation of toxic metabolites, extensive

interactions with intended targets and minimal interactions with unintended targets,

minimal interaction with co-administered drugs, minimal toxicity and an appropriate

rate of elimination (Li, 2005), a drug candidate with these desirable drug-like

properties will be probably successful in clinical trials.

1.4.5. In Silico Drug Designing

In the last decade the number of protein structures published in the RCSB

Protein Data Bank have risen from approximately 4000 (1995) to 32000 (2005)

(www.rcsb.org). This increase in available 3D protein structures marked the beginning

of the structure-based inhibitor design era. Computer-aided ligand design is concerned

with the prediction of molecules that are expected to bind with high affinity to key

regions of pharmacologically relevant enzymes, to inhibit or alter their function.

There are two major problems that the in silico design process needs to deal

with. Firstly, the prediction of the optimum binding orientation a ligand will have in

34

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the active site of the receptor. Secondly, the estimation of binding affinity of the ligand-

receptor complex. Various docking programs can be used to perform conformational

sampling of specific inhibitors addressing the binding mode prediction problem.

Scoring functions can be used to estimate the binding affinity between the target

protein and different conformations of the ligand or different ligands (Wolf and

Dormeyer, 2003; Ferrari et al., 2004). Thirdly, the biological results have to support

the in silico predictions to validate the strategy for future use in drug design.

1.4.6. Protein surface

The protein surface is the outer or the topmost boundary of a protein. The

topology of the surface of a protein is intimately related to its function; parts of the

surface are directly involved in interactions with other molecules; the solvent protein

interface is almost certainly related to the structure of the native molecule; and the

chemical reactivity of the various functional groups will depend on their relation to this

interface (Lee and Richards, 1971).

There are three types of protein surfaces: Van der Waals surface, solvent

excluded surface (Connolly surface), and solvent accessible surface. Van der Waals

surface corresponds to the envelope containing the atomic spheres of Van der Waals

radius. The shape of the Van der Waals surface of a molecule may be misleading,

especially for macromolecules, since it frequently contains small gaps, pockets and

clefts which are sometimes too small to be penetrated even by a solvent molecule like

water.

For all practical purposes, the van der Waals surface of these oddments cannot

enter into contact with a solvent or a drug molecule and therefore is not truly an

accessible surface. To "smooth" the roughness of the van der Waals surface, (Lee and

Richards, 1971) the concept of a contact surface and a solvent accessible surface was

introduced. These surfaces are obtained by rolling a spherical probe of a diameter

corresponding to the size of a solvent molecule (usually water) on the original van der

Waals surface. As a result, the area where the probe touches the van der Waals surface

is called the contact surface, the centre of the spherical probe traces a surface called the

solvent accessible surface and the patches over narrow gaps and clefts traced by the

35

Chapter -1

surface of the probe are called re-entrant surfaces. The Connolly surface is composed

of contact surface and re-entrant surface (Connolly, 1983).

1.4.7. Molecular Docking

The computational process of searching for a ligand that is able to fit both

geometrically and energetically to the binding site of a protein is called molecular

docking. The molecular docking finds best orientation of two molecules to each other.

The interaction can be modeled by a scoring function that includes terms describing

inter and intra-molecular energies.

1.4.8. Mechanics of DockingTo perform docking, the first requirement is the structure of the protein. Usually

the structure has been determined in the lab using a biophysical technique called as x-

ray crystallography or NMR spectroscopy. This protein structure and a database of

potential ligands serve as inputs to a docking program. The success of a docking

program depends on two components namely the search algorithm and the scoring

function.

1.4.9. The Search AlgorithmThe search space consists of all possible orientations and

conformations of the protein paired with the ligand. With the present

computing resources, it is impossible to exhaustively explore the search space. This

would involve enumerating all possible distortions of each molecule since molecules

are dynamic and exist in an ensemble of conformational states and all possible

rotational and translational orientations of the ligand relative to the protein at a given

level of granularity. Most docking programs in use account for a flexible ligand, and

several are attempting to model a flexible protein receptor. Each snapshot of the pair is

referred to as a pose.

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Chapter -1

1.4.10. The Scoring Function

The scoring function takes a pose as input and returns a number indicating the

likelihood that the pose represents a favourable binding interaction. Most scoring

functions are physics-based molecular mechanics force fields that estimate the energy

of the pose where a low or negative energy indicates a stable system and thus a likely

binding interaction. An alternative approach is to derive a statistical potential for

interactions from a large database of protein-ligand complexes, such as the Protein

Data Bank, and evaluate the fit of the pose according to this inferred potential.

1.4.11. Evaluation

One of the major uses of docking is in the ranking of ligands in order of their

relative binding affinities. Evaluation of the results may be done by analyzing drugs

based on these scores. Due to uncertainty in the approximation in the scoring function

it is better to consider a range of top scoring docking prediction. There can be

minimum energy positions that are redundantly reported, resulting in many nearly

identical orientations with slight differences in their scores. These redundant

orientations can overwhelm the ranked list of all the results, reducing their spatial

diversity. Clustering provides a useful tool for pruning redundant results from this top

scoring list. The most appropriate method for analyzing the dock results would be

correlating the result obtained by docking with the experimentally validated results.

1.4.12. Packages

There are many software packages available for docking and molecular

visualizations. Some of them are free and some are commercial. Here some of the tools

which used for my research purpose mentioned below.

1.4.12.1. Maestro

It is a powerful, all-purpose molecular modeling environment; Schrödinger

develops state-of-the-art chemical simulation software for use in pharmaceutical,

biotechnology, and materials science research. Since its founding in 1990, Schrödinger

has earned a reputation for its leadership in scientific development. Schrödinger's

products range from general molecular modeling programs to a complete suite of drug

design software including both ligand and structure based methods.

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Maestro is the linchpin of Schrödinger's computational technology. Far more

than just a visualization program, Maestro also helps researchers organize and analyze

data. Maestro's intuitive interface makes setting up calculations easy and

straightforward.

Computed results are automatically returned and incorporated into projects for

further study. Maestro's vast array of visualization options makes it possible to glean

insight into molecular properties as well as detailed intermolecular interactions.

Maestro is a powerful and versatile molecular modeling environment, and the portal to

the most advanced science in computational chemistry.

1.4.12.2. GLIDE (GRID BASED LIGAND DOCKING WITH ENERGETICS)

GLIDE uses a hierarchical series of filters to search for possible locations of the

ligand in the active site region of the receptor. The shape and properties of the receptor

are represented on a grid by several different set of fields that provide progressively

more accurate scoring of the ligand poses. Conformational flexibility is handled in

GLIDE by an extensive conformational search, augmented by a heuristic screen that

rapidly eliminates unsuitable conformations, such as conformations that have long

range internal hydrogen bonds.

1.4.12.3. High throughput Virtual Screening

High-throughput-Virtual screening (HTVS) has become a cornerstone

technology of pharmaceutical research. Investments into HTVS have been, and

continue to be, substantial. A current estimate is that biological screening and

preclinical pharmacological testing alone account for ~14% of the total research and

development (R&D) expenditures of the pharmaceutical industry.

1.4.12.4. Qikprop

QikProp efficiently evaluates pharmaceutically relevant properties for over half

a million compounds per hour, making it an indispensable lead generation and lead

optimization tool. It plays an important role during lead optimization by analyzing

similarity within a class of compounds as well as by identifying compounds to avoid

because they exhibit extreme values of predicted properties. To minimize the drug

candidates failure in clinical trials due to poor ADME (absorption, distribution,

metabolism, and excretion) properties. These late-stage failures contribute significantly

38

Chapter -1

to the rapidly escalating cost of new drug development. The ability to detect

problematic candidates early can dramatically reduce the amount of wasted time and

resources, and streamline the overall development process.

1.4.12.5. Ligprep

LigPrep goes far beyond simple 2D to 3D structure conversions by including

tautomeric, stereochemical, and ionization variations, as well as energy minimization

and flexible filters to generate fully customized ligand libraries that are optimized for

further computational analyses. Efficient and accurate 2D to 3D conversion is therefore

a key precursor to computational analyses. Beyond simple one-to-one structural

conversion, it is equally important to generate scientifically sound molecular models

that enumerate the different structural and chemical possibilities a ligand could sample,

as these variations could lead to dramatically different results in subsequent

computations. A versatile conversion program that can be configured to generate ligand

libraries with the desired structural and chemical features can significantly streamline

the entire in silico drug discovery process.

1.4.12.6. PyMOL

PyMOL is one of a few open source visualization tools available for use in

structural biology. The Py portion of the software's name refers to the fact that it

extends, and is extensible by the Python programming language. It can produce high

quality 3D images of small molecules and biological macromolecules, such as proteins.

Almost a quarter of all published images of 3D protein structures in the scientific

literature were made using PyMOL.

1.4.13. Image Representations

The data can be represented in nearly 20 different ways. Spheres provides a

CPK-like view, surface and mesh provide more volumetric views, lines and sticks put

the emphasis on bond connectivity, and ribbon and cartoon are popular representations

for identifying secondary structure and topology. PyMOL's quick demo, accessible

through the built-in Wizard menu, gets users started with all of the standard

representations.

1.4.14. Chimera

39

Chapter -1

UCSF Chimera is a highly extensible program for interactive visualization and

analysis of molecular structures and related data, including density maps,

supramolecular assemblies, sequence alignments, docking results, trajectories, and

conformational ensembles. High-quality images and animations can be generated.

Chimera includes complete documentation and several tutorials. Chimera is developed

by the Resource for Bio computing, Visualization, and Informatics, funded by the

National Institutes of Health National Center for Research Resources and National

Institute of General Medical Sciences.

1.4.15. Properties of Macromolecules

If a protein structure is known then designing inhibitors which will block

activity in the test-tube should be a relatively straightforward problem. More spice to

such a challenge is added if we at the same time attempt to make the ligand bio-

reductive.

40

Chapter -1

Diabetes mellitus is recognized as a leading cause of new cases of blindness,

and is associated with increased risk for painful neuropathy, heart disease and kidney

failure. Many theories have been advanced to explain mechanisms leading to diabetic

complications, including stimulation of glucose metabolism by the polyol pathway.

Additionally, the enzyme is located in the eye (cornea, retina, lens), kidney, and

the myelin sheath–tissues that are often involved in diabetic complications (Schrijvers

et al., 2004). Under normal glycemic conditions, only a small fraction of glucose is

metabolized through the polyol pathway, as the majority is phosphorylated by

hexokinase, and the resulting product, glucose-6-phosphate, is utilized as a substrate

for glycolysis or pentose phosphate metabolism (Lindstad et al., 1993; Gabbay et al.,

1996). However, in response to the chronic hyperglycemia found in diabetics, glucose

flux through the polyol pathway is significantly increased. Up to 33 % of total glucose

utilization in some tissues can be through the polyol pathway (Cheng, 1986).

Insulin stimulates numerous intracellular signalling pathways that regulate

cellular metabolism and growth (White and Kahn, 1994). The physiological effects of

insulin are mediated by its cell surface receptor, transmembrane glycoprotein with

intrinsic protein tyrosine kinase activity (Ebina et al., 1985; Ullrich et al., 1985).

Binding of insulin to the extracellular α-Chains results in autophosphorylation of

specific tyrosine residues in the cytoplasmic portion of the chains: two in the

juxtamembrane region, three in the kinase (catalytic) domain, and two in the C-

terminal tail (Tornqvist et al., 1987; Tavare et al., 1988; White et al., 1988; Feener et

al., 1993; Kohanski, 1993). Autophosphorylation of Tyr1158, Tyr1162 and Tyr1163 in

the activation loop (A-loop) of the kinase domain is critical for stimulation of kinase

activity and biological function (Rosen et al., 1983).

1.4.16. Dipeptidyl peptidase-4

Dipeptidyl peptidase-4 (DPP4), also known as adenosine deaminase complexing

protein 2 or CD26 (cluster of differentiation 26) is a protein that, in humans, is encoded

by the DPP4 gene (Kameoka et al., 1993).

The protein encoded by the DPP4 gene is an antigenic enzyme expressed on the

surface of most cell types and is associated with immune regulation, signal

transduction and apoptosis. It is an intrinsic membrane glycoprotein and a serine

41

Chapter -1

exopeptidase that cleaves X-proline dipeptides from the N-terminus of polypeptides.

DPP4 plays a major role in glucose metabolism. It is responsible for the degradation of

incretins such as GLP-1 (Barnett, 2006). Furthermore, it appears to work as a

suppressor in the development of cancer and tumours (Pro and Dang, 2004; Wesley et

al., 2005; Masur et al., 2006). A new class of oral hypoglycemics called dipeptidyl

peptidase-4 inhibitors work by inhibiting the action of this enzyme, thereby prolonging

incretin effect in vivo (Rosenstock and Zinman, 2007).

1.4.17. Glucagon-like peptide-1

Glucagon-like peptide-1 (GLP-1) is derived from the transcription product of

the proglucagon gene. The major source of GLP-1 in the body is the intestinal L cell

that secretes GLP-1 as a gut hormone. The biologically active forms of GLP-1 are:

GLP-1-(7-37) and GLP-1-(7-36) NH2. Those peptides result from selective cleavage of

the proglucagon molecule.

GLP-1 secretion by ileal L cells is dependent on the presence of nutrients in the

lumen of the small intestine. The secretagogues (agents that cause or stimulate

secretion) of this hormone include major nutrients like carbohydrate, protein and lipid.

Once in the circulation, GLP-1 has a half-life of less than 2 minutes, due to rapid

degradation by the enzyme dipeptidyl peptidase-4. It is a potent antihyperglycemic

hormone, inducing glucose-dependent stimulation of insulin secretion while

suppressing glucagon secretion. Such glucose-dependent action is particularly

attractive because, when the plasma glucose concentration is in the normal fasting

range, GLP-1 no longer stimulates insulin to cause hypoglycemia. GLP-1 appears to

restore the glucose sensitivity of pancreatic β-cells, with the mechanism possibly

involving the increased expression of GLUT2 and glucokinase. GLP-1 is also known to

inhibit pancreatic β-cell apoptosis and stimulate the proliferation and differentiation of

insulin-secreting β-cells. In addition, GLP-1 inhibits gastric secretion and motility. This

delays and protracts carbohydrate absorption and contributes to a satiating effect.

GLP-1 possesses several physiological properties that make it (and its analogs) a

subject of intensive investigation as a potential treatment of diabetes mellitus (Toft-

Nielsen et al., 2001; Meier et al., 2004).

• Increases insulin secretion from the pancreas in a glucose-dependent manner.

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• Decreases glucagon secretion from the pancreas by engagement of a specific G

protein-coupled receptor.

• Increases insulin-sensitivity in both alpha cells and beta cells

• Increases beta cells mass and insulin gene expression, post-translational

processing and incretion.

• Inhibits acid secretion and gastric emptying in the stomach.

• Decreases food intake by increasing satiety in brain.

• Promotes insulin sensitivity.

The physiological role of GLP-1 in post- prandial insulin secretion, it has been

shown that an oral dose of glucose triggers a much higher peak in plasma insulin

concentration compared to an intravenous dose.

1.4.18. Incretins

Incretins are a group of gastrointestinal hormones that cause an increase in the

amount of insulin released from the beta cells of the islets of Langerhans after eating,

even before blood glucose levels become elevated. They also slow the rate of

absorption of nutrients into the blood stream by reducing gastric emptying and may

directly reduce food intake. As expected, they also inhibit glucagon release from the

alpha cells of the Islets of Langerhans. The two main candidate molecules that fulfil

criteria for an incretin are glucagon-like peptide-1 (GLP-1) and gastric inhibitory

peptide (also known as: glucose-dependent insulinotropic polypeptide or GIP). Both

GLP-1 and GIP are rapidly inactivated by the enzyme DPP-4.

Another approach is to inhibit the enzyme that inactivates GLP-1 and GIP, DPP-

4. Several DPP-4 inhibitors that can be taken orally as a tablet have been developed.

In 1970, GIP was isolated and sequenced from intestinal mucosa (Brown).

Originally named gastric inhibitory peptide, GIP was renamed glucose-dependent

insulinotropic peptide in 1973 after Brown and Dupre, showed GIP stimulates insulin

secretion. However, initial research could not establish its utility as a treatment for

diabetes.

1.4.19. Dipeptidyl peptidase 4 inhibitors

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Inhibitors of dipeptidyl peptidase 4, also DPP-4 inhibitors or gliptins, are a class

of oral hypoglycemics that block DPP-4. They can be used to treat diabetes mellitus.

1.4.19.1. Risks and side effects of gliptin class Drugs

The first agent of the class - sitagliptin - was approved by the FDA in 2006

(FDA, 2006).

Glucagon increases blood glucose levels, and DPP-4 inhibitors reduce glucagon

and blood glucose levels. The mechanism of DPP-4 inhibitors is to increase incretin

levels (GLP-1 and GIP) (Dupre et al., 1995; Behme et al., 2003; McIntosh et al.,

2005), which inhibit glucagon release, which in turn increases insulin secretion,

decreases gastric emptying, and decreases blood glucose levels.

Long-term effects of DPP-4 inhibitors on mortality and morbidity are so far

inconclusive, although adverse effects, including nasopharyngitis (the common cold),

headache, nausea, hypersensitivity and skin reactions, have been observed in clinical

studies. Consistent with this FDA approval of Novartis' DPP-4 inhibitor vildagliptin

was delayed because of skin lesions with blistering observed in nonhuman primate

toxicology studies (The FDA's Decision on Galvus 2007). Other possible adverse

effects, including hypersensitivity reactions and pancreatitis, have been reported. These

effects may relate to DPP-4's function in restricting the inflammatory actions of the

chemokine CCL11/eotaxin, so that inhibiting DPP-4 might unleash the recruitment of

inflammatory cells (Forssmann et al., 2008).

Although one in vitro study found that DPP-4 inhibitors, together with GLP-

2, increased proliferation and migration of colon cancer cells, which might encourage

cancer cells to metastasize, (Masur et al., 2006) carcinogenicity has not been confirmed

in long-term, preclinical studies of the major DPP-4 inhibitors.

Based on this DPP4 protein controlling diabetes mellitus and its mechanism of

action in our body (Fig.1.2 & 1.3), the present study was aimed to assess the

bioactivity of Berberine in various antidiabetic investigations in silico approach with

potential for drug- development.

1.5. INTRODUCTION ABOUT TINOSPORA CORDIFOLIA

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Botanical synonym: Tinospora cordifolia (Willd.) Miers

Family – Menispermaceae (Willd.) Miers.

Synonyms – Guduchi, Tinospora

Vernacular names

English: Heartleaf moonseed

Tamil: Shindil kodi (º¢ó¾£ø ¦¸¡Ê), Kunali (ககககக)

Sanskrit: Guduchi, Amrita

Hindi: Giloya

Telugu: Amirtavalli (ககககககககககக), Teppatige

Bengali: Giloe, Gulancha

Gujarati: Gado, Galo

Urdu: Tippatige.

1.5.1. Scientific Classification

Kingdom: Plantae

Division: Magnoliophyta

Class: Magnoliopsida

Order: Ranunculales

Family: Menisoermaceae (Willd.) Miers

Genus: Tinospora

Species: cordifolia (Willd.) Miers

1.5.2. Habitat

Tinospora cordifolia (willd.) Miers Ex Hook.F. & Thoms. It is a large, glabrous,

succulent, perennial deciduous twiner with succulent stems and papery bark, climbing

shrub belonging to the family menispermaceae. It is distributed throughout tropical

Indian subcontinent, Sri Lanka and China, ascending to an altitude of 1200m.

45

Chapter -1

1.5.3. Habit

It thrives easily in the tropical region, often attains a great height, and seems to

be particularly fond of climbing up the trunks of large trees.

1.5.4. Plant botanical descriptions

Tinospora cordifolia is large extensively spreading glabrous, perennial woody

climber. The stem is rather succulent with long filiform fleshy aerial roots from the

branches. The bark is creamy white to grey, deeply left spirally, the space in between

being spotted with large rosette like lenticels. The wood is white, soft and porous and

the fleshy cut surface quickly assumes a yellow tint on exposure to air. The branches

bear smooth heart- shaped leaves, unisexual greenish flowers and red berries. In

auxiliary and terminal racemes or racemose panicles, the male flowers are clustered

and female are usually solitary.

Fig.1.4. Climbed Tinospora cordifolia plants in trunks of large trees

Fig.1.5. Tinospora cordifolia plant – Leaves with fruits

Fig.1.6. Tinospora cordifolia plants- Stem with flowers

Fig.1.7. Tinospora cordifolia plant – fruits

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The leaves are simple, alternate, entire, 7-9 nerved, long petiole and possess a

characteristic heart shaped, giving the name cordifolia to the plant. The drupes are

ovoid, glossy, succulent, red and pea – sized. The seeds are curved. Fruits are fleshy

and single seeded, fruits drupes, red when ripe. Flowers grow during the summer and

fruits during the winter. The odour is not characteristic, but the taste is bitter (Kirtikar

and Basu, 1975; Anonymous, 1976).

1.5.5. Plant Parts used

The parts used for medicinal purposes are the leave, bark, stem and root.

1.5.6. Chemical constituents

A variety of compounds have been isolated from aerial parts and roots of

Tinospora cordifolia. They belong to different classes such as alkaloids, diterpenoids,

lactones, glycosides, steroids, sesquiterpenoid, phenols, aliphatic compounds and

polysaccharides. Leaves of this plant are rich in protein and are fairly rich in calcium

and phosphorous (Sarma et al., 1998; Chintalwar et al., 1999).

1.5.7. Traditional uses

Tinospora cordifolia (Guduchi) is Indian medicinal plant and has been used in

Ayurvedic preparation for the treatment of various ailments throughout the centuries.

Ancient Hindu physicians prescribed it for gonorrhoea. The plant is used in Ayurvedic,

“Rasayanas” to improve the immune system and the resistance against infections. The

whole plant is used medicinally; however, the stem is approved for use in medicine as

listed by the Ayurvedic Pharmacopoeia of India (Ayurvedic Pharmacopoeia of India,

2004; Sinha et al., 2004).

In folk and tribal medicine the whole plant, powdered root and stem bark,

decoction of root and stem, juice of the root and paste or juice of leaves or stem of

Tinospora cordifolia are used to treat various ailments such as fever, jaundice,

diarrhoea, dysentery, general debility, cough, asthma, leucorrhoea, skin diseases,

fracture, bits of poisonous insects and venomous snakes and eye disorders (Singh

et al., 2003).

1.5.8. Modern therapeutic uses

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Today the drug and a tincture prepared from Tinospora cordifolia are approved

for use in the Indian pharmacopoeia. They are used for the treatment of general

weakness, fever, dyspepsia, dysentery, gonorrhoea, secondary syphilis, urinary

diseases, impotency, gout, viral hepatitis, skin diseases, and anaemia. In compound

formulations guduchi is clinically used to treat jaundice and rheumatoid arthritis. The

root is considered to be a powerful emetic and is used for bowel obstruction. A

decoction leaves are used for the treatment of gout and water extract is used in leprosy.

The starch from the roots and stems are nutrients used in chronic diarrhoea and chronic

dysentery. The fresh plant is more efficacious than the dried plant, its watery extract,

known as Indian quinine, is very effective in fevers due to cold or indigestion. The

plant is commonly used in rheumatism, urinary diseases, dyspepsia, general debility,

syphilis, skin diseases, bronchitis and impotence. In gonorrhoea, cough and chronic

fever the juice of the fresh plant is administered in doses of 56 to 112 ml with long

pepper and honey. It has been used as an antidote for snakebite (Kapoor). Tinospora

cordifolia is also used in malaria, environmental illness, asthma, upper respiratory tract

infection, urinary tract infection, general debility and amelioration of symptoms from

chemo/radiotherapy.

1.6. Aim and objectives

Tinospora cordifolia (Willd.) Miers ex Hook. F. & Thoms is a large deciduous

climbing shrub found throughout India, especially tropical part of India. The plant is

mainly known for its medicinal properties in Ayurvedic medicine. The dry stem, with

bark intact, constitute the drug. The bitter principle present in the stem is used in the

treatment of debility, dyspepsia, fever and urinary disease and the decoction of the

leaves is used for the treatment of Gout. The root is a powerful emetic and used for

visceral obstructions; its water extract used in treatment of leprosy.

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The extracts of stem, leaves, barks and roots show strong antioxidant activities.

The pharmaceutical significance of this plant is mainly because of various bioactive

compounds such as glucoside, alkaloidal constituents including Berberine etc., found in

this plant. The various reports on its multiple uses attracted attention for utilization of

the plant for medicinal purposes. Tinospora cordifolia natural strands are fast

disappearing and are threatened with extinction due to its indiscriminate collection and

over exploitation of natural resources for commercial purposes to meet the

requirements of the pharmaceutical industry. Tinospora cordifolia grows wild in forest

and other areas and there is no organized propagation information available so far.

Conventional vegetative propagation of this plant has limited potential for large

scale propagation of elite plants. At the present time, products are derived from

conventional field grown plants. In spite of the commercial success of these products,

there are many challenges associated with the growth and production. For instance, as

with many other field-grown medicinal plants, uncontrollable environmental effects

such as rainfall or drought can lead to plant-to-plant or year-to-year variation in yield

and amount of secondary metabolites produced by the plant species in question.

The main aim of this research is to develop and optimize efficient

micropropagation systems for the phytomedicinal species of Tinospora cordifolia.

These systems will provide a means to obtain large amounts of sterile, consistent plant

material, superior in their levels of active ingredients over a reduced time frame when

compared to conventional practices.

The developmental route and frequency of regeneration in any tissue culture

system is dependent on several factors the first one selection of appropriate explants,

second the preparation of the explants, third supplementation of plant tissue culture

media with the optimal combination of growth regulating compounds and amendments,

and final is optimization of environmental conditions for the development of

regenerate.

Based on this background, the present study was aimed to explore the possible

methods to enhance secondary metabolites through in vitro conservation

(micropropagation and callus) and assessment of its bioactivity of hypoglycaemic and

antioxidant activity of Tinospora cordifolia medicinal plant.

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Based on these facts the present study has been attempted with the following objectives.

1. Collection of Tinospora cordifolia medicinal plant from the south India. And

selection of plant based on in-vitro free radical scavenging activity.

2. To standardize wild Tinospora cordifolia plant crude drug.

3. To study of in-vitro conservation through micropropagation and callus.

4. To analyse the secondary metabolite content of Tinospora cordifolia wild,

micropropagated plants and callus through HPLC.

5. To evaluate the hypoglycaemic and antioxidant activity of Tinospora cordifolia

wild, micropropagted plants and callus in alloxan induced diabetic rats.

6. To study the interaction of Berberine and proteins involved in the control of

blood glucose level by using in silico method its in vivo evaluation.

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