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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
10
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
15
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
18
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
19
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
20
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:
21
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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