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1. INTRODUCTION

1.1 General introduction

Nature always stands as a golden mark to exemplify the outstanding

phenomenon of symbiosis. Herbal medicine is the oldest form of healthcare known

to mankind. Primitive man observed and appreciated the great diversity of plants

available to him. Current estimates of the number of species of flowering plants

range between 200,000 and 250,000 in 300 families and 10,500 genera [Evans

2005]. More than 60% of approved and pre-new drug application (NDA) candidates

are either natural products or related to them, not including biologicals such as

vaccines and monoclonal antibodies [Snader KM et al.1997].

An amazing variety and number of products have been found in nature.

The total number of natural products produced by plants has been estimated to be

over 5, 00,000. One hundred sixty thousand natural products have been identified, a

value growing by 10,000 per year [Dictionary of Natural Products 2001]. About

100,000 secondary metabolites of molecular weight less than 2500 have been

characterized, half from microbes and the other half from plants [Henkel et al. 1999]

[Roessner CA et al.1996] Despite a rapidly expanding literature on phytochemistry,

only a small percentage of the total species has been examined chemically, and there

is a large field for future research. Although herbal medicines are effective in the

treatment of various ailments, very often these drugs are unscientifically exploited

and /or improperly used. Therefore, these plant drugs deserve detailed studies in the

light of modern science.

At the turn of the nineteenth century, methods became available for the

isolation of active principles from crude drugs. The development of chemistry made

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it possible to isolate and synthesize chemically pure compounds that would give

reproducible biological results. In 1806, Serturner (1783-1841) isolated the first pure

active principle when he purified morphine from the opium poppy. Many other

chemically pure active compounds were soon obtained from crude drug preparations

including emetine by Pelletier (1788-1844) from ipecacuanah root; quinine by

Carentou (1795-1877) from cinchona bark; strychnine by Magendie (1783-1855)

from nux vomica; and, in 1856, cocaine by Wohler (1800-1882) from coca [Robert

ES et al.1997] .

However, man did not require the modern methods of investigation to

collect for him a materia medica of plants which he often used in conjunction with

magical and other ritual practices. It is interesting to reflect that such collections of

herbal medicines compiled over centuries by trial and error, and presumably using

the patient as experimental animal throughout, must surely contain some material

worthy of further investigation and should not be too readily discarded. One obvious

line of approach is to start with folk medicines of the world on the assumption that

these materials have already been subjected to some human screening. This interest

in drugs of plant origin is due to several reasons, namely, conventional medicine can

be inefficient (e.g. side effects and ineffective therapy), abusive and or incorrect use

of synthetic drugs results in side effects and other problems, a large percentage of

the world‘s population does not have access to conventional pharmacological

treatment, and folk medicine and ecological awareness suggest that ―natural‖

products are harmless. It has been estimated that 56% of the lead compounds for the

medicines in the british national formulary are natural products or are derived from

natural products [Lixin Z et al.2005 ].

Isolation and characterization of pharmacologically active compounds

from medicinal plants continue today. In addition to this historical success in drug

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discovery, natural products are likely to continue to be sources of new commercially

viable drug leads. The history of the indigenous plants being used as anti fertility

agents have been emphasized by researchers way back in the 19th

century [Chaudhry

RR et al. 1986 ] [Farnsworth NR et al.1975]. of the 520 new pharmaceuticals

approved between 1983 and 1994, 39% were derived from natural products, the

proportion of antibacterial and anticancer agents of which was over 60 percentage

[Buss et al.2007] [Cragg et al. 1997].

The abundance of plant and microbial secondary metabolites and their

value in medicine are undisputed, but one question that is only partly answered

concerns the reasons for this abundance of complex chemical substances. In the past,

the production of what we would now call ―bioactive‖ substances was a mystery.

Taking morphine as an example of a secondary metabolite whose value to the plant

is not entirely obvious, fourteen steps are required from available amino acids,

including at least one step that is highly substrate specific [Gerardy et al.1993]. The

presence of morphine in the tissues of Papaver somniferum must therefore confer a

selectional advantage on the plant [Stone et al.1993]. Genetic code is required for

each of the enzymes involved in the biosynthesis, valuable amino acids are utilized

in forming the enzymes, and a relatively scarce nutrient (nitrogen) is locked up in

the compounds produced. If the morphine did not continue to have value for the

plant, mutants would have arisen with the advantage of not having a drain on their

metabolic resources. We can only guess the ecological functions of morphine.

Perhaps a mammalian herbivore that consumed too many poppies would become

drowsy and itself fall prey to a carnivore. It may be significant that the cannabinoids,

produced in greatest abundance in the nutritious growing tips of the plant, also

induce mental effects that could compromise an herbivore‘s ability to escape a

predator. Whatever their natural protective functions, natural products are a rich

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source of biologically active components that have arisen as the result of natural

selection, over perhaps 300 million years. Natural product medicine has come from

various source materials like plants, micro organisms, marine organisms, vertebrates

and invertebrates [Cragg GM et al.2000]. The challenge to the medicinal chemist is

to exploit this unique chemical diversity.

About 1500 plants with medicinal uses are mentioned in ancient texts

and around 800 plants have been used in traditional medicine. However, most of

these plants have not been screened systematically to prove their ethno medical uses,

to isolate the active ingredients and to develop them as drugs or lead molecules for

drug development.

1.2 Discovery of new medicines from plants

Plants have been utilized as medicines for thousands of years. These

medicines initially took the form of crude drugs such as tinctures, teas, poultices,

powders, and other herbal formulations [Balick et al.1997]. The specific plants to be

used and the methods of application for particular ailments were passed down

through oral history. Eventually information regarding medicinal plants was

recorded in herbals. In more recent history, the use of plants as medicines has

involved the isolation of active compounds, beginning with the isolation of

morphine from opium in the early 19th century [Kinghorn et al.2001]. Drug

discovery from medicinal plants led to the isolation of early drugs such as cocaine,

codeine, digitoxin, and quinine, in addition to morphine, of which some are still in

use [Butler et al. 2004] [Newman et al.2000]. Isolation and characterization of

pharmacologically active compounds from medicinal plants continue today. More

recently, drug discovery techniques have been applied to the standardization of

herbal medicines, to elucidate analytical marker compounds. The following provides

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a brief review of the importance of medicinal plants in drug discovery including

noteworthy compounds isolated from this source, our research involving anticancer

and cancer chemopreventive drug discovery using medicinal plants, and finally

current challenges in regard to medicinal plant drug discovery.

Drug discovery from medicinal plants has evolved to include numerous

fields of inquiry and various methods of analysis. The process typically begins with

a botanist, ethnobotanist, ethnopharmacologist, or plant ecologist who collects and

identifies the plant(s) of interest. Collection may involve species with known

biological activity for which active compound(s) have not been isolated (e.g.,

traditionally used herbal remedies) or may involve taxa collected randomly for a

large screening program. It is necessary to respect the intellectual property rights of

a given country where plant(s) of interest are collected [Baker et al.1995].

Phytochemists (natural product chemists) prepare extracts from the plant materials,

subject these extracts to biological screening in pharmacologically relevant assays,

and commence the process of isolation and characterization of the active

compound(s) through bioassay-guided fractionation. Molecular biology has become

essential to medicinal plant drug discovery through the determination and

implementation of appropriate screening assays directed towards physiologically

relevant molecular targets. Pharmacognosy encapsulates all of these fields into a

distinct interdisciplinary science.

The definition and practice of pharmacognosy have been evolving since

the term was first introduced about 200 years ago, as drug use from medicinal plants

has progressed from the formulation of crude drugs to the isolation of active

compounds in drug discovery. The American Society of Pharmacognosy refers to

pharmacognosy as ‗‗The study of the physical, chemical, biochemical and biological

properties of drugs, drug substances, or potential drugs or drug substances of natural

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origin as well as the search for new drugs from natural sources‘‘. As practiced today,

pharmacognosy involves the broad study of natural products from various sources

including plants, bacteria, fungi, and marine organisms. Pharmacognosy includes

both the study of botanical dietary supplements, including herbal remedies [Tyler,

1999] [Cardellina, 2002] as well as the search for single compound drug leads that

may proceed through further development into Food and Drug Administration

(FDA)-approved medicines. Drug discovery from medicinal plants is most

frequently associated with the second of these two endeavors. Colleagues in Sweden

have suggested a revised definition for pharmacognosy for these types of activities,

namely as ‗‗a molecular science that explores naturally occurring structure–activity

relationships with a drug potential‘‘ [Bruhn et al. 1997].

1.3 Medicinal Plants role in Human History

Over the centuries humans have relied on plants for basic needs such as

food, clothing, and shelter, all produced or manufactured from plant matrices

(leaves, woods, fibers) and storage parts (fruits, tubers). Plants have also been

utilized for additional purposes, namely as arrow and dart poisons for hunting,

poisons for murder, hallucinogens used for ritualistic purposes, stimulants for

endurance, and hunger suppression, as well as inebriants and medicines. The plant

chemicals used for these latter purposes are largely the secondary metabolites, which

are derived biosynthetically from plant primary metabolites (e.g., carbohydrates,

amino acids, and lipids) and are not directly involved in the growth, development, or

reproduction of plants. These secondary metabolites can be classified into several

groups according to their chemical classes, such as alkaloids, glycosides, saponins,

terpenoids, and phenolics [Harborne 1984].

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The use of hallucinogens in the past was usually associated with magic

and ritual. However, these hallucinogens have been exploited as recreational drugs

and accordingly may lead to habituation problems. Several well-recognized plants

that contain hallucinogenic or psychoactive substances (the compound names are

given in parentheses) include Banisteriopsis caapi (Spruce ex Griseb.) Morton (N,

N-dimethyltryptamine), Cannabis sativa L. (9-trans-tetrahydrocannabinol), Datura

species (scopolamine), Erythroxylum coca Lam. (cocaine), Lophophora williamsii

(Salm-Dyck) J.M. Coult. (Mescaline), Papaver somniferum L. (Morphine), and

Salvia divinorum Epling & Jativa (Salvinorin A) [McCurdy 2005] [Farnsworth et al.

1985]. Several of these plants are also used as drugs due to their desired

pharmacological activities, and some of the constituents of these plants have been

developed into modern medicines, either in the natural form or as lead compounds

subjected to optimization by synthetic organic chemistry.

Nowadays, plants are still important sources of medicines, especially in

developing countries that still use plant-based TM for their healthcare. In 1985, it

was estimated in the bulletin of the World Health Organization (WHO) that around

80 % of the world‘s population relied on medicinal plants as their primary healthcare

source [Anon 2003]. Even though a more recent figure is not available, the WHO

has estimated that up to 80 % of the population in Africa and the majority of the

populations in Asia and Latin America still use traditional medicines for their

primary healthcare needs [Blumenthal et al. 2006]. In industrialized countries, plant-

based traditional medicines or phytotherapeuticals are often termed complementary

or alternative medicine (CAM), and their use has increased steadily over the last 10

years. In the USA alone, the total estimated ―herbal‖ sale for 2005 was $4.4 billion,

a significant increase from $2.5 billion in 1995.

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1.4 Medicinal Plant Derived Compounds in Drug Development

Despite the recent interest in drug discovery by molecular modeling,

combinatorial chemistry, and other synthetic chemistry methods, natural-product-

derived compounds are still proving to be an invaluable source of medicines for

humans. The importance of plants in modern medicine has been discussed in recent

reviews and reports [Mukherjee PK 2002] [Mandal et al. 2008]. Other than the

direct usage of plant secondary metabolites in their original forms as drugs, these

compounds can also be used as drug precursors, templates for synthetic

modification, and pharmacological probes, all of which will be discussed briefly in

turn in this section.

Little work was carried out by the pharmaceutical industry during 1950-

1980; however, during the 1980-1990 massive growth has occurred. This has

resulted in new developments in the area of combinational chemistry, new advances

in the analysis and assaying of potential plant materials as drug leads by

conservationists. New plant drug development programs are traditionally undertaken

by either random screening or an ethno botanical approach, a method based on the

historical medicinal/ food use of the plant. One reason why there has been

resurgence in this area is that conservationists especially in USA have argued that by

finding new drug leads from the rainforest, the value of the rainforests to society is

proven and that this would prevent these areas being cut down for unsustainable

timber use. However, tropical forests have produced only 47 major pharmaceutical

drugs of worldwide importance. It is estimated that a lot more say about 300

potential drugs major importance may need to be discovered. These new drugs

would be worth $ 147 billion. It is thought that 125000 flowering plant species are

of pharmacological relevance in the tropical forests. It takes 50000 to one million

screening tests to discover ONE profitable drug. Even in developed countries there

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is a huge potential for the development of nutraceuticals and pharmaceuticals from

herbal materials. For example the UK herbal materia medica contains around 300

species, where as the Chinese herbal materia medica contains around 7000 species,

one can imagine what lies in store in the flora- rich India.

Indigenous systems of medicine like Ayurveda, Siddha and Unani mainly

used medicinal plants for treatment of various ailments of the human beings and

animals. Plants are symbols of growth, rejuvenation, prosperity and general well

being. Medicinal plants in particular have added advantage of possessing

tremendous natural healing power. Even as we commence the new century with its

exciting prospect of gene therapy, herbal medicine remains one of the common

forms of therapy available to most of world‘s population [Anatas et al. 1998].

Plant constitutes a major percentage of naturally occurring materials used

as drugs by the people all over the world. The use of plants as food and medicine has

started ever since man was born in this universe. Even in this modern world people

realize the value of plants as drugs as serious side effects of modern medicine are

increasingly felt by them. The practice of wide use of plant drugs is prevalent among

the people in some countries like India, China and Greece, etc., which can boast of

ancient civilizations and culture.

The inherited skill of using plants for medicinal purposes is highly in

vogue amongst the native people especially the Gypsis, Bedowin-Arabs, American-

Mexican Indians and practically every ancient race. The importance of plant

medicine popularly known as herbal medicine and their powers to cure diseases of

human being as well as animals are well documented in ancient literature. In the Rig

Veda which is considered to be one of the oldest repositories of human knowledge,

written between 4500 and 1600 BC, the medicinal use of plants is emphasized. In

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the Atharva Veda which is known as the fourth Veda, the use of plants is

documented in greater detail. In the Ayurveda which is considered as the Upaveda to

the Atharva Veda, definite properties of plant remedies and their uses are given in

detail.

In fact Ayurveda is the very foundation of the ancient medical science in

India followed by the monumental treaties of Charaka and Sushruta. The realization

that there is something interesting in the properties of medicinal plants dawned with

advent of Chemistry in the late 18th

century. Chemists gradually started isolating

pure substances from various anatomical parts of medicinal plants and concluded

that certain active molecules are responsible for therapeutic actions of the plants.

The technological advancements and expanding knowledge in the fields

of chemistry, botany and biology have helped in substantiating the value of the

medicinal plants as whatever are the glories of our ancient remedies; the scientific

mind will not be satisfied by mere claims no matter from whatever source they

originate, unless corroborated by experimental and clinical evidences.

The organized research based on traditional and folklore claims has

resulted in the discovery of many therapeutically useful products.Isolation of

salicylic acid from the bark of willow tree and Salix alba leads to the synthesis of

aspirin in 1899 by the German company Bayer was used world wide in folk

medicine for the relief of aches, fever and rheumatic pain. Since then many

compounds were introduced as a result of laboratory research for drugs with anti-

inflammatory activity (AIA); though many of them produced a dramatic

symptomatic improvement in rheumatic process, did not arrest the progress of the

disease process and all of them shared the common side effect. In traditional system

of medicine the practitioners use various indigenous plants for the treatment of

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different types of arthritic conditions. One such plant drug used by siddha

practitioners is Glycosmis pentaphylla commonly called bala or atibala is claimed by

folklore for various ailments like rheumatism, seminal weakness, and diarrhoea.

Since no scientific validation on the anti rheumatic activity is carried on the roots,

the roots are selected for the study [Hwang et al.2001]

1.5 Free radicals in human diseases

Free radicals are fundamental to any biochemical process and represent

an essential part of aerobic life and metabolism (In living systems, free-radicals are

generated as part of the body‘s normal metabolic process, and the free radical chain

reactions are usually produced in the mitochondrial respiratory chain, liver mixed

function oxidizes, by bacterial leucocytes, through xanthine oxidase activity,

atmospheric pollutants, and from transitional metal catalysts, drugs and xenobiotics.

Free radicals or oxidative injury now appears the fundamental mechanism

underlying a number of human neurological and other disorders. Oxidative stress,

the consequence of an imbalance of prooxidants and antioxidants in the organism, is

rapidly gaining recognition as a key phenomenon in chronic diseases [Rindfleisch et

al. 2005].

Reactive Species

Reactive Nitrogen Species Reactive Oxygen Species

●Nitric Oxide (NO˙) Oxygen centered radical Oxygen Centered non-radical

●Nitric Dioxide (NO2˙) ●Superoxide anion (˙O2). ●Hydrogen peroxide (H2O2)

●Peroxy nitrite (OONO-) ●Hydroxyl radical (˙OH) ● Singlet oxygen (O2)

●alkoxyl radical (RO˙)

●peroxyl radical (ROO˙)

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1.6 Antioxidants

Antioxidants play an important role as a health protecting factor. Science

evidence suggests that antioxidants reduce the risk of chronic diseases including

cancer, cardiovascular disease, cataracts, atherosclerosis, diabetes, arthritis, immune

deficiency diseases and aging. Most of the antioxidant compounds are derived from

plant sources. The main characteristic of an anti oxidant is its ability to trap free

radicals. Highly reactive free radicals and oxygen species are present in biological

system from a wide variety of sources. Antioxidants are important in the prevention

of human diseases. Antioxidant compounds may function as free radical scavengers,

complexes of pro-oxidant metals, reducing agents and quenchers of singlet oxygen

formation With this background, in the present study an attempt was made to

evaluate the isolated phytoconstituents compounds for their biological properties.

Carotenoids have been reported to act as radical scavengers due to the extensive

system of conjugated double bonds in their molecule that makes them very

susceptible to radical addition.

1.7 Arthritis

Arthritis (from Greek arthro-, joint + -itis, inflammation; plural:

arthritides) is a form of joint disorder that involves inflammation of one or more

joints. There are over 100 different forms of arthritis. The most common form,

osteoarthritis (degenerative joint disease), is a result of trauma to the joint, infection

of the joint, or age. Other arthritis forms are rheumatoid arthritis, psoriatic arthritis,

and related autoimmune diseases. Septic arthritis is caused by joint infection.The

major complaint by individuals who have arthritis is joint pain. Pain is often a

constant and may be localized to the joint affected. The pain from arthritis is due to

inflammation that occurs around the joint, damage to the joint from disease, daily

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wear and tear of joint, muscle strains caused by forceful movements against stiff

painful joints and fatigue.

There are several diseases where joint pain is primary, and is considered

the main feature. Generally when a person has "arthritis" it means that they have one

of these diseases, which include

Osteoarthritis

Rheumatoid arthritis

Gout and pseudo-gout

Septic arthritis

Ankylosing spondylitis

Juvenile idiopathic arthritis

Still's disease

Symptoms include

Inability to use the hand or walk

Malaise and a feeling of tiredness

Weight loss

Poor sleep

Muscle aches and pains

Tenderness

Difficulty in moving the joint

Diagnosis is made by clinical examination from an appropriate health

professional, and may be supported by other tests such as radiology and blood tests,

depending on the type of suspected arthritis. All arthritides potentially feature pain.

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Pain patterns may differ depending on the arthritides and the location. Rheumatoid

arthritis is generally worse in the morning and associated with stiffness; in the early

stages, patients often have no symptoms after a morning shower. Osteoarthritis, on

the other hand, tends to be worse after exercise. In the aged and children, pain might

not be the main presenting feature; the aged patient simply moves less, the infantile

patient refuses to use the affected limb.

Elements of the history of the disorder guide diagnosis. Important

features are speed and time of onset, pattern of joint involvement, symmetry of

symptoms, early morning stiffness, tenderness, gelling or locking with inactivity,

aggravating and relieving factors, and other systemic symptoms. Physical

examination may confirm the diagnosis, or may indicate systemic disease.

Radiographs are often used to follow progression or help assess severity.

1.7.1 Rheumatoid arthritis

Rheumatoid arthritis is a systemic disease and it involve rheumatoid

nodules, vasculitis, eye inflammation, cardio pulmonary disease are manifestation of

the disease. Rheumatoid arthritis is not an inherited disease. Researchers believe that

some people have genes that make them susceptible to the disease. People with these

genes will not automatically develop rheumatoid arthritis. There is usually a

"trigger," such as an infection or environmental factor, which activates the genes.

When the body is exposed to this trigger, the immune system responds

inappropriately. Instead of protecting the joint, the immune system begins to

produce substances that attack the joint. This is what may lead to the development of

rheumatoid arthritis. It is autoimmune disease which means the body‘s immune

system mistakenly attack on healthy tissues. The normal joint lining is very thin and

it has very few blood vessels in it but in the rheumatoid arthritis joints the lining is

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very thick and crowded with the white blood cells. The white blood cells secrete

chemical substances like interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-

alpha) that produce pain, joint swelling and joint damage. Recent discoveries show

the presence of novel cytokines like IL-17, IL-18 and RANK ligand in the

pathogenesis of chronic arthritis.

Figure 1.1 Rheumatoid arthritis joint

Figure 1.2 Difference between normal joint lining and rheumatoid joint lining

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Rheumatoid arthritis is diagnosed by rheumatoid factor. These are

abnormal antibodies (IgG) which are present in blood. These are reacted with

antigen and form antigen-antibody complex that leads to pain and inflammation of

synovial membrane. The American College of Rheumatology requires at least four

of the following seven criteria to confirm the diagnosis [American Rheumatism

Association 1959] [Jorgensen 1991].

• Morning stiffness around the joint that lasts at least 1 hour

• Arthritis of three or more joints for at least 6 weeks

• Arthritis of hand joints for at least 6 weeks

• Arthritis on both sides of the body for at least 6 weeks

• Rheumatoid nodules under the skin

• Rheumatoid factor present in blood testing

1.7.2 Lupus

Lupus is a common collagen vascular disorder that can be present with

severe arthritis. Other features of lupus include a skin rash, extreme photosensitivity,

hair loss, kidney problems, lung fibrosis and constant joint pain.

1.7.3 Gout

Gout is caused by deposition of uric acid crystals in the joint, causing

inflammation. There is also an uncommon form of gouty arthritis caused by the

formation of rhomboid crystals of calcium pyrophosphate known as pseudogout. In

the early stages, the gouty arthritis usually occurs in one joint, but with time, it can

occur in many joints and be quite crippling. The joints in gout can often become

swollen and lose function. Gouty arthritis can become particularly painful and

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potentially debilitating when gout cannot successfully be treated. When uric acid

levels and gout symptoms cannot be controlled with standard gout medicines that

decrease the production of uric acid (e.g., allopurinol, febuxostat) or increase uric

acid elimination from the body through the kidneys (e.g., probenecid), this can be

referred to as refractory chronic gout or RCG.

Arthritis is predominantly a disease of the elderly, but children can also

be affected by the disease. More than 70% of individuals in North America affected

by arthritis are over the age of 65. Arthritis is more common in women than men at

all ages and affects all races, ethnic groups and cultures. In the United States a CDC

survey based on data from 2007–2009 showed 22.2% (49.9 million) of adults aged

≥18 years had self-reported doctor-diagnosed arthritis, and 9.4% (21.1 million or

42.4% of those with arthritis) had arthritis-attributable activity limitation (AAAL).

With an aging population this number is expected to increase.

1.7.4 Treatment

There is no cure for either rheumatoid or osteoarthritis. Treatment

options vary depending on the type of arthritis and include physical therapy, lifestyle

changes (including exercise and weight control), orthopedic bracing, and

medications. Joint replacement surgery may be required in eroding forms of arthritis.

Medications can help reduce inflammation in the joint which decreases pain.

Moreover, by decreasing inflammation, the joint damage may be slowed.

1.8 In silico Docking Model

Molecular docking can be thought of as a problem of “lock-and-key”,

where one is interested in finding the correct relative orientation of the “key” which

will open up the “lock”. Here, the protein can be thought of as the ―lock‖ and the

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ligand can be thought of as a ―key‖. Molecular docking may be defined as an

optimization problem, which would describe the ―best-fit‖ orientation of a ligand

that binds to a particular protein of interest. However, since both the ligand and the

protein are flexible, a “hand-in-glove” analogy is more appropriate than “lock-and-

key’ [Wei et al. 2004]. During the course of the process, the ligand and the protein

adjust their conformation to achieve an overall ―best-fit‖ and this kind of

conformational adjustments resulting in the overall binding is referred to as

―induced-fit‖ [Goldman et al. 2000].

The focus of molecular docking is to computationally simulate the

molecular recognition process. The aim of molecular docking is to achieve an

optimized conformation for both the protein and ligand and relative orientation

between protein and ligand such that the free energy of the overall system is

minimized.

Two approaches are particularly popular within the molecular docking

community. One approach uses a matching technique that describes the protein and

the ligand as complementary surfaces [Meng et al. 2004] [Morris et al. 1998]. The

second approach simulates the actual docking process in which the ligand-protein

pair wise interaction energies are calculated [Feig et al. 2004]. Both approaches

have significant advantages as well as some limitations. These are outlined below.

1.8.1 Shape complementarities

Geometric matching / shape complementary methods describe the protein

and ligand as a set of features that make them dock able [Shoichet et al. 2004].

These features may include molecular surface / complementary surface descriptors.

In this case, the receptor‘s molecular surface is described in terms of its solvent-

accessible surface area and the ligand‘s molecular surface is described in terms of its

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matching surface description. The complementary between the two surfaces

amounts to the shape matching description that may help finding the complementary

pose of docking the target and the ligand molecules. Another approach is to describe

the hydrophobic features of the protein using turns in the main-chain atoms. Yet

another approach is to use a Fourier shape descriptor technique [Cai et al.2002]

[Kahraman et al. 2007]. Whereas the shape complementarity based approaches are

typically fast and robust, they cannot usually model the movements or dynamic

changes in the ligand/ protein conformations accurately, although recent

developments allow these methods to investigate ligand flexibility. Shape

complementarity methods can quickly scan through several thousand ligands in a

matter of seconds and actually figure out whether they can bind at the protein‘s

active site, and are usually scalable to even protein-protein interactions. They are

also much more amenable to pharmacophore based approaches, since they use

geometric descriptions of the ligands to find optimal binding.

1.8.2 Simulation

Simulating the docking process as such is much more complicated. In

this approach, the protein and the ligand are separated by some physical distance,

and the ligand finds its position into the protein‘s active site after a certain number

of ―moves‖ in its conformational space. The moves incorporate rigid body

transformations such as translations and rotations, as well as internal changes to the

ligand‘s structure including torsion angle rotations. Each of these moves in the

conformation space of the ligand induces a total energetic cost of the system. Hence,

the system's total energy is calculated after every move.

The obvious advantage of docking simulation is that ligand flexibility is

easily incorporated, whereas shape complementarity techniques must use ingenious

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methods to incorporate flexibility in ligands. Also, it more accurately models reality,

whereas shape complimentary techniques are more of an abstraction. Clearly,

simulation is computationally expensive, having to explore a large energy landscape.

Grid-based techniques, optimization methods, and increased computer speed have

made docking simulation more realistic.

1.8.3 Mechanism of docking

To perform a docking screen, the first requirement is a structure of the

protein of interest. Usually the structure has been determined using a biophysical

technique such 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: the search algorithm

and the scoring function.

1.8.4 Search algorithm

The search space in theory consists of all possible orientations and

conformations of the protein paired with the ligand. However in practice with

current computational resources, it is impossible to exhaustively explore the search

space—this would involve enumerating all possible distortions of each molecule

(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 attempt to model a flexible protein receptor. Each "snapshot" of

the pair is referred to as a pose.

A variety of conformational search strategies have been applied to the

ligand and to the receptor. These include:

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Systematic or stochastic torsional searches about rotatable bonds

Molecular Dynamics simulations

Genetic Algorithms to "evolve" new low energy conformations

1.8.5 Ligand flexibility

Conformations of the ligand may be generated in the absence of the

receptor and subsequently docked or conformations may be generated on-the-fly in

the presence of the receptor binding cavity, or with full rotational flexibility of every

dihedral angle using fragment based docking. Force field energy evaluation are

most often used to select energetically reasonable conformations but knowledge-

based methods have also been used [Kearsley et al. 1994] [Klebe et al. 1994].

1.8.6 Receptor flexibility

Computational capacity has increased dramatically over the last decade

making possible the use of more sophisticated and computationally intensive

methods in computer-assisted drug design. However, dealing with receptor

flexibility in docking methodologies is still a thorny issue. The main reason behind

this difficulty is the large number of degrees of freedom that have to be considered

in this kind of calculations. Neglecting it, however, leads to poor docking results in

terms of binding pose prediction [Cerqueira et al. 2009].

Multiple static structures experimentally determined for the same protein

in different conformations are often used to emulate receptor flexibility.

Alternatively rotamer libraries of amino acid side chains that surround the binding

cavity may be searched to generate alternate but energetically reasonable protein

conformations [Taylor RD et al. 2003].

22

1.8.7 Scoring function

The scoring function takes a pose as input and returns a number

indicating the likelihood that the pose represents a favorable binding interaction.

Most scoring functions are physics-based molecular mechanics force

fields that estimate the energy of the pose; a low (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.

There are a large number of structures from X-ray crystallography for

complexes between proteins and high affinity ligands, but comparatively fewer for

low affinity ligands as the later complexes tend to be less stable and therefore more

difficult to crystallize. Scoring functions trained with this data can dock high affinity

ligands correctly, but they will also give plausible docked conformations for ligands

that do not bind. This gives a large number of false positive hits, i.e., ligands

predicted to bind to the protein that actually doesn‘t when placed together in a test

tube.One way to reduce the number of false positives is to recalculate the energy of

the top scoring poses using (potentially) more accurate but computationally more

intensive techniques such as Generalized Born or Poisson-Boltzmann methods.

Nowadays drug design is an important tool in the field of medicinal

chemistry where new compounds are synthesized by molecular or chemical

manipulation of the lead moiety in order to produce highly active compounds with

minimum steric effect [Cavasotto et al. 2004]. Nowadays, the use of computers to

predict the binding of libraries of small molecules to known target structures is an

increasingly important component in the drug discovery process [Koppen 2009] [

23

Schoichet 2004]. There is a wide range of software packages available for the

conduct of molecular docking simulations like, AutoDock, GOLD, and FlexX.

AutoDock 4.2 is the most recent version which has been widely used for virtual

screening, due to its enhanced docking speed [Collignon et al. 2011]. Its default

search function is based on Lamarckian Genetic Algorithm (LGA), a hybrid genetic

algorithm with local optimization that uses a parameterized free-energy scoring

function to estimate the binding energy. Each docking is comprised of multiple

independent executions of LGA and a potential way to increase its performance is to

parallelize the aspects for execution [Schames et al. 2004]. Docking of small

molecules in the receptor binding site and estimation of binding affinity of the

complex is a vital part of structure based drug design.

Inflammation is a process involved in the pathogenesis of several

disorders like arthritis and cardiovascular disease [Vane et al. 1998]. Human

dihydrofolate reductase and Cyclooxygenase (COX) is an endogenous enzyme

which catalyses the conversion of arachidonic acid into Prostaglandins and

thromboxanes. The enzyme exists in atleast two isoforms, COX-1 and COX-2.

Although both the isoforms catalyze the same biochemical transformation, the two

isoforms are subject to a different expression regulation. COX-1 is a constitutive

enzyme and is responsible for the supply of prostaglandins which maintain the

integrity of the gastric mucosa and provide adequate vascular homeostasis whereas

COX-2 is an inducible enzyme and is expressed only after an inflammatory stimulus

[Kurumbail et al. 1996] [Herschman et al.1996].

24

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