2.0 review of literature m -...
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2.0 REVIEW OF LITERATURE
edicinal plants are of great interest in the field of biotechnology and are
considered as rich sources of ingredients in drug development. The
increase in demand for plant based therapeutics both in developed and
developing countries is largely due to the growing credits conferred to natural
products as being non-norcotic, causing minimal side effects and easily available at
affordable prices. The withania plants are tremendously known for their roots rich in
steroids and alkaloids and are a valuable constitute of traditional Ayurvedic drug
preparations against many diseases (Kiritikar and Basu, 1975; Williamson, 2002). The
major biochemical constituents of withania plants are a class of secondary metabolites
known as withanolides. These withanolides are known to possess diverse therapeutic
properties and has been used for centuries to treat various disorders. Several
withanolides have been isolated and characterized until date, but withaferin A is
considered to be multifunctional with strong anticancer and anti-inflammatory
properties (Berghe et al., 2012; Szic et al., 2014).
The ruthless collection of these plants by local herbalists and Ayurvedic drug
companies has lead this genus to the verge of extinction. To meet with the growing
demand for pharmaceutical industries, it is found to necessitate in vitro propagation of
such endangered plants. The present investigation was made to in vitro propagate
W. coagulans by optimizing the potential growth regulators and efforts were made to
explore the potent withanolides for their anti-inflammatory properties against LPS
activated Bone Marrow Derived Macrophages (BMDM’s) and the under lying
mechanism.
The literature relevant to the present study entitled “Computational
bioactivity analysis of major withanolides and their experimental validation in
in vitro cultures of Withania species” is reviewed in this chapter under the following
headings:
2.1 Withania as a medicinal plant
2.1.1 Withania somnifera
2.1.2 Withania coagulans
2.1.3 Withanolides of withania species
M
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2.2 Pharmacological properties of withanolides
2.2.1 Anti-microbial
2.2.2 Anti-inflammatory
2.2.3 Anti-cancer
2.2.4 Hypolipidemic and hypocholesterolemic activities
2.2.5 Hepatoprotective
2.2.6 Immunomodulatory
2.2.7 Antihyperglycaemic activity
2.2.8 CNS activity
2.2.9 Cardiovascular effect
2.3 Analytical tools employed to study plant secondary metabolites
2.3.1 Qualitative and quantitative analysis
2.3.2 High performance thin layer chromatography
2.4 In silico analysis
2.4.1 ADME predictions
2.4.2 Molecular docking
2.4.3 Scoring function
2.4.4 Target proteins
2.5 Toll-like receptors (TLRs) in inflammation
2.5.1 Toll-like receptor (TLR) 4 signalling
2.5.2 NF-kB and Mitogen-activated protein kinases activation
2.5.3 Chronic Inflammation
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2.1 Withania as a medicinal plant
Since the beginning of human civilization, medicinal plants have been used
for their therapeutic values by mankind. Nature has been a source of therapeutic
agents for centuries and an impressive number of modern drugs have been developed
from natural sources. As source of medicines, plants continue providing us with new
remedies and around 25% of today’s medical prescriptions are based on plant derived
substances or analogues. The efficacy and safety of herbal medicines have turned the
major pharmaceutical population towards medicinal plants research (Sara et al.,
2009).
The family Solanaceae comprises of 84 genera that includes 3,000 species
scattered throughout the world. The sixty five known withania species are densely
distributed in the drier parts of tropical and subtropical zones (Schonbeck-Temesy,
1972; Hepper, 1991; Warrier, 1996; Hunziker, 2001). In Ayurveda, withania is
claimed to have potent aphrodisiac, rejuvenative, sedative and life prolonging
properties. The plant has been traditionally used to promote youthful strength,
endurance and health, nurturing the timely elements of the body and increasing the
production of body fluids such as muscle fat, lymph, blood, cells and semen. It also
helps to counteract chronic fatigue syndrome, bone and body weakness associated
with dehydration, impotency, emaciation, premature ageing and muscle tension.
Bruised leaves and fruits are locally applied to glands, ulcers and tumours
(Williamson, 2002). Among the genera withania, Withania somnifera and
Withania coagulans are the two most esteemed species having high economical and
medicinal significance, being used and cultivated in several regions such as Pakistan,
Afghanistan, Egypt, Iran, Palestine, Spain, Jordan, Morocco, Canary Island, Eastern
Africa, South Africa, Congo, Madagascar and India (Dymock et al., 1981; Javanshir,
2000; Sharma, 2004; Panwar and Tarafdar, 2006). The botanical description and the
illustration of these two prevalent species of Withania are described in Table 2.1 and
Figure 2.1.
2.1.1 Withania somnifera
Withania somnifera (L) Dunal, (Solanaceae) popularly known as
Ashwagandha is the most commonly used herb in Ayurvedic and indigenous medical
system for more than 3000 years. Various parts of the plant have been used for
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centuries to treat a variety of ailments. Many pharmacological studies have been
carried out to illustrate multiple biological properties of W. somnifera (Mishra et al.,
2000). The similarity of therapeutic properties with those of Asian ginseng has led
Ashwagandha being called as Indian ginseng (Singh and Kumar, 1998). The species is
widely distributed in Africa, the Indian sub-continent and the Mediterranean. In India,
it grows well in the drier regions of tropical and sub-tropical areas of Punjab,
Haryana, Uttar Pradesh, Madhya Pradesh, Bihar, Jharkhand, Uttarkhand, Rajasthan
and Maharashtra and some parts of Jammu and Kashmir ascending upto 1,650 m in
the Himalayas (Kapoor, 2005).
Classification:
Kingdom : Plantae
Division : Angiosperm
Class : Dicotyledoneae
Order : Tubiflorae
Family : Solanaceae
Genus : Withania
Species : somnifera Dunal
(Singh et al.,2011)
Vernacular names:
Sanskrit : Asvagandha
Hindi : Asgandh
Kannada : Viremaddinagaddi
Malayalam : Amukkuram
Tamil : Amukkira
Telugu : Vajigandha
English : Winter cherry
2.1.2 Withania coagulans
Withania coagulans (L.) Dunal (Solanaceae) is commonly known as Indian
cheese maker, is well known for its ethnopharmacological activities. The fruit and
berries are used commercially for milk coagulation (Sanjay et al., 2007). Surveys of
existing literatures have shown that the plant is used in various traditional systems of
medicine like Ayurveda and Unani, and has been recommended for treating various
disorders including ulcers, rheumatism, bronchitis, and degenerative diseases (Maurya
et al., 2010; Khodaei et al., 2012). W. coagulans is a small ever green shrub that is
reputedly used for treating dyspepsia, flatulent colic and other intestinal disorders.
The plant is a native of the Asia-temperate (Western Asia- Afghanistan) and Asia-
tropical (Indian Subcontinent - India, Nepal) regions. This plant species is sparsely
distributed in the eastern Mediterranean region and extends to South Asia.
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Table 2.1: Botanical description of W. somnifera and W. coagulans(Jain et al., 2012)
S.No. Description Withania somnifera (L.)Dunal
Withania coagulans (stocks)Dunal
1 Habit Undershrub Herb
2 Leaves Alternate, broadly ovate,subacute, entire margins
Alternate, elliptic lanceolate-coriaceous, obtuse, entiremargins, glabrous, coated withminute stellate hairs on boththe surfaces
3 InflorescenceAxillary, umbellatecymes
Axillary
4 Flowers Monoecious Dioecious
5 CalyxAccrescent,gamosepalous with5sepals
Campanulate, gamosepalouswith 5 sepals clothed with finestellate grey tomentum
6 Corolla Campanulate, greenish-yellow with 5 petals
Campanulate, greenish-yellowwith 5 petals
7 Androecium Anthers 1.2 mm long,broadlyovate
Anthers long and filamentousin male flowers, smaller infemale flowers
8 GynoeciumOvary ovoid/globose,glabrous
Ovary ovoid/globose, withoutstyle or stigma
9 Style Filiform Glabrous
10 StigmaMushroom-shaped, 2-lamellate
Mushroom-shaped, 2-lamellate
11 Fruit (Berry)Globose, enclosed inthepersistent calyx, seedsyellow,reniform
Globose, smooth, closely girtby the enlarged membranouspersistent calyx
12 SeedsGlobose, enclosed in thepersistent calyx, yellow,reniform
Globose, ear shaped, glabrous,enclosed in the persistentcalyx yellow, reniform
13 Flowering Throughout the year November-March*
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Figure 2.1: Botanical descriptions of W. coagulans and W. somnifera (plantillustrations.org)
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Classification:
Kingdom : Plantae
Division : Magnoliophyta
Class : Magnolipsida
Order : Solanales
Family : Solanaceae
Genus : Withania
Species : coagulans
(Gupta, 2012)
Vernacular names:
Hindi : Puni-ke-bij, Akri
Persian : Tukhme- Kaknaje-hindi
Afgan : Spicebajja
Punjabi : Khamjira
Sindhi : Punir band, Punir-ja-fota
(Mathur et al., 2011).
2.1.3 Withanolides of withania species
The phytoconstituent profiles of withania have been of great interest to the
scientific and research community. Chemical characterization of withania started off
with Power and Salway (1911) who identified and isolated amorphous alkaloid
(C12H16N2) from South African strains of W. somnifera. Later in 1933, Majumdar and
Guha investigated W. somnifera plant from Bengal and confirmed the alkaloid
presence. Laboratory analysis till date has revealed over 35 chemical constituents in
the roots of W. somnifera (Rastogi and Mehrotra, 1998). Among these, the
biologically active constituents are steroidal lactones (withaferin A, withanolides
A-Y, withasomniferin A, withasomidienone, withasomniferols A-C, withanone, etc),
alkaloids (isopellertierine. Anferine), and saponins with additional acyl group
(sitoindoside) (Gupta and Rana, 2007; Maurya et al., 2010). Withanolides are
traditionally believed to account for the plants medicinal properties and they bear
resemblance to Ginsenosides both in their appearance and action. Withanolides from
the withania plants have been researched in a variety of clinical examinations for their
numerous therapeutic activities including cancer and immune functioning (Grandhi
et al., 1994).
Withanolides are a group of naturally occurring C28 steroidal lactones with an
intact or modified ergostane skeleton. They are mainly produced by the solanaceae
family, and in particular to the genera Withania, Physalis, Dunalia, Datura,
Tubocapsicum, Nicandra and Jaborosa (Glotter, 1991). Among these, plants,
Withania, Physalis and Datura have been widely distributed in the southern
peninsular regions of Tamil Nadu with rich content of withanolides (Gupta and Ray,
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1991; Ravikumar et al., 2010). Nearly 400 withanolides or closely related congeners
have been discovered in 58 Solanaceae species under 22 genera (Eich, 2008). Some of
the withanolides have been discovered in certain Ajuga species like Parviflora Benth
and Tacca species such as Taccaceae (Huang et al., 2002), as well as in certain
marine organisms. Nevertheless, their occurrence is by far predominant in Solanaceae
(Eich, 2008). Different withanolides such as withacoagin and coagulan from the roots
and fruits of W. coagulans and withanolide A and withaferin A from the roots and
leaves of W. somnifera has been reported (Khare, 2007). The basic skeleton of
withanolides is shown in the Figure: 2.2.
Figure 2.2 Basic structure of withanolides. C28 steroidal lactones with anintact ergostane skeleton
Withanolides are synthesized via mevalonate pathway during terpenoids
formation and arise during initial cyclization of 3S-squalene-2,3-epoxide (Kreis and
Muller-Uri, 2010). Synthesised withanolides generally contain polyoxygenated
ergostane skeleton. The most characteristic feature of withanolides is the ability to
introduce oxygen functions in almost every functional site of the carbocyclic skeleton
and compounds side chain (Naz, 2002). These withanolides were initially classified
based on the chemotypes of withania species and the regions of the collected plant.
Chemically, these compounds may be classified as ergostane derivatives of their
structural pattern which are broadly divided into seven groups represented in Figure
2.3 (Glotter, 1991)
1. 5β, 6β –epoxides
2. 6α, 7α –epoxides
3. 5-enes
4. Intermediary compounds
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5. 5α,6α –epoxides
6. 6β,7β –epoxides
7. Phenolic withanolides
Figure 2.3 Structural classifications of withanolides
The diverse structural analogues of withanolides provide a great opportunity
to study structure activity relationship, lead optimization and target identification.
Therefore, withanolides represent a promising lead compounds in development of
new drugs. The isolated withanolides from W. somnifera and W. coagulans are listed
in Table 2.2 and 2.3.
Table 2.2: Withanolides identified in W. somnifera
S.No. Chemical constituent References
1 Withaferin A
Lavie et al. (1965), Kirson et al.(1970), Devi (1996), Gupta et al.(1996), Anjaneyulu and Rao(1997), Ali et al. (1997), Mohanet al. (2004), Oh et al. (2008a)
2 Withanolide D, 2, 24-dienolide Lavie et al. (1968), Kirson et al.(1970)
3 27-deoxywithaferin A Kirson et al. (1970)
4 Withanone, Trienolide Kirson et al. (1971)
5 5, 20α ®-dihydroxy-6α, 7α-epoxy-1- Menben-Von and Stapel (1973)
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oxo-(5α) - With a-2, 24-dienolide(steroidallactone) 2, 3-dihydrowithaferinA-3beta-O-sulfate
6Withanolide - WS 1 (aliphatic ketone),Withanolide - WS 2 (aliphatic ester)
Kundu et al. (1976), Khan et al.(1993)
7 Withanolide E Glotter et al. (1977)
8 Sitoindosides VII, VIII Bhattacharya et al. (1987)
9 Sitoindosides IX, X Ghosal et al. (1988)
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Withanosides I, II, III, IV, V, VI, VII,5a,20aF(R)-dihydroxy-6a,7a-epoxy-1-oxowitha-2,24-dienolide, coagulin Qandphysagulin D
Matsuda et al. (2001)
11 withanolide A, withanoside VIII, IX, XI Zhao et al. (2002)
12Withanolide A,withanoside IV and VI
Kuboyama et al. (2002),Tohda et al. (2005)
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Physagulin D (1,6)β-D-glycopyranosyl-(1-4)-β-D-glycopyranoside, 27-O-β-Dglycopyranosylphysagulin D, 27-O-β-Dglycopyranosylviscosalactone B, 4, 16dihydroxy-5β, 6β epoxyphysagulin D, 4-(1-hydroxy-2,3-dihydrowithaferin A,viscosalactone B, 27-desoxy-24,25-dihydrowithaferin A
Jayaprakasam and Nair (2003)
14Withanone, 27-hydroxy withanolide A,iso-withanoneand 6α, 7β-epoxy-1β,3β,5α-trihydroxywitha-24-enolide
Lal et al. (2006)
15 Ashwagandhanolide Subaraju et al. (2006), Mirjaliliet al. (2009a)
16 Withanolide B and Z, 7- hydroxylwithanolide Pramanick et al. (2008)
17 Withanoside IV, VI, physagulin D andwithastraronolide Ahuja et al. (2009)
18 Withanolidesulfoxide Mulabagal et al. (2009)
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Table 2.3: Withanolides identified in W. coagulans
S.No. Chemical constituent References
1 Withaferin A Subramanian andSethi, 1969
2 Withanolide G, I, J, K Gottlieb andKirson, 1981
3 3β-hydroxy- 2,3-dihydrowithanolide F, Ergosta-5,25-diene-3β,24 ε -diol
Budhiraja et al.,1983
4 Withanolide D, Δ3 isowithanolide F Velde et al., 1983
5Withanolide H: 14α, 20αF, 27-trihydroxy-1-oxo-20R,22R-with a-2,5,24- trienolide, 3β,14α,20αF,27-tetrahydroxy-1-oxo-20R,22R-witha-5,24-dienolide
Ramaiah et al.,1984
6 (20S, 22R) 6α, 7α- epoxy- 5α-hydroxy- 1- oxo- witha-2 ,24- dienolide, Withacoagin Neogi et al., 1988
7 17β, 27 dihydroxy-14, 20- epoxy -1- oxo- 22R- witha-3,5, 24- trienolide
Rahman et al.,1993
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14, 15β- epoxywithanolide I: [(20S, 22R) 17β, 20β-dihyroxy -14β, 15β- epoxy- 1- oxo- witha-3,5,24-trienolide], 17β- hydroxywithanolide K: [(20S, 22R) 14α,17β, 20β-trihydroxy 1- oxo- with a-2, 5, 24- trienolide],17β,20β- dihydroxy- 1- oxo- witha- 2,5,24- trienolide
Choudhary et al.,1995
9 Coagulin B, C, D, E, F, G, H, I, J, K, L, M, N, O Rahman et al.,1998(a, b, c, d)
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Withahejarin: [ 20 β-hydroxy-1-oxo-(22R) – witha-2,5,24 trienolide, Withapakistanin: [ 17β, 20 β-dihydroxy- 14, 15β- epoxy-1-oxo-(22R)- with a-3,5,24trienolide], Withasomniferine-A: [ 17β, hydroxyl- 6α, 7α-epoxide-1-oxo-(22R)-witha-4,24-dienolide], Coagulin A
Shahwar, 1999
11 Coagulin P, Q, R Rahman et al.,1999
12(22R), 20β-hydroxy- 1-oxowitha- 2,5,24- trienolide,(22R)-14,20-epoxy-17ß-hydroxy-1-oxowitha-3,5,25-trienolide, Coagulin U
Naz, 2002
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17β-hydroxy-14α,20α-epoxy-1-oxo-(22R)-witha-3,5,24-trienolide, 20β, hydroxy -1- oxo- (22R) – witha – 2, 5.24- trienolide, Withacoagulin: 20β,27-Dihydroxy-1-oxo-(22R)-witha-2,5,24-tetraenolide
Rahman et al.,2003
14 Coagulanolide: (17S,20S,22R)-14α,15α,17β,20β-tetrahydroxy-1-oxowitha-2,5,24-trienolide Maurya et al., 2008
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15
(20R,22R)-14,20a,27-trihydroxy-1-oxowitha-3,5,24-trienolide, (22R)-14a,15a,17b,20b-tetrahydroxy-1-oxowitha-2,5,24-trien-26,22-olide, Withacoagulin A, B,C, D, E, F, Withanolide F, L
Huang et al., 2009
16 Coagulansins A, Coagulansins B Jahan et al., 2010
17 Withacoagulin D, Withanolide K and J Kuroyanagi et al.,2012
2.2 Pharmacological properties of withanolides
The integral constituents of withania genera have always been a great interest
to the research community. The bioactive components are alkaloids, steroidal
lactones with ergostane skeletons such as withanolide A-Y, withaferin A,
withasomniferols A-C, withanone, withasomniferin A, etc (Gupta and Rana, 2007;
Maurya et al.,, 2010). Withanolide A (5α,20α-dihydroxy- 6α,7α -epoxy-1-oxowitha-
2,24-dienolide) and withaferin A (4β,27-dihydroxy-5β,6β-epoxy-1-oxowitha-2,24-
dienolide) are the key active withanolidal principles responsible for a diverse array of
pharmacological activities. They have chemically similar back bone but differ in their
side chain constituents (Sanghwan et al., 2007; Hemalatha et al., 2008).
2.2.1 Antimicrobial activity
Antibacterial and anti-fungal properties have been demonstrated in isolated
withanolides from the extracts of various parts of withania (Khan et al., 1993;
Choudhary et al., 1995). The methanolic extract of W. somnifera possessed maximum
inhibitory activity against a wide range of bacteria. Oral administration of fruit
extracts of W. somnifera successfully obliterated Salmonella infection in mice
subjects as revealed by increased survival rate as well as less bacterial load in vital
organs of the treated animals (Owais et al., 2005). The methanol, diethyl ether and
hexane extracts from leaves and roots of W. somnifera were evaluated for their
synergistic antibacterial activity by agar disc diffusion assay against Escherichia coli
and Salmonella typhimurium (Arora et al., 2004; Jain et al., 2012). Lalsare et al.
(2010) demonstrated antimicrobial and antioxidant activities from various extracts of
W. coagulans fruits. The methanolic, dichloromethane and pertroleum extract of
W. coagulans were treated against a wide array of fungal infections caused by
Trichoderma viridis, Aspergillus flavus, Fusarium laterifum, Aspergillus fumigatus,
Trichophyton mentogrophytes, Microsporum canis and Candida albicans (Maurya
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et al., 2010; Mughal et al., 2011). Also volatile oil from the fruits of W. coagulans has
antibacterial activity against Vibrio cholera and Staphylococcus aureus (Khare,
2007).
These anti-fungal and anti-bacterial properties have been demonstrated in
isolated withanolides from extracts of both W. somnifera and W. coagulans
respectively. Two withanolides (14, 15β-epoxywithanolideI[(20S,22R) 17β,20β-
dihydroxy-14β, 15β-epoxy-1-oxo-witha-3,5,24-trienolide] and 17β-hydroxy
withanolide K (20S,22R) 14α,17β,20β-trihydroxy- 1-oxo-witha-2,5,24-trien-olide])
were isolated from W. coagulans. The withanolides 17β- hydroxywithanolide K
(20S,22R) 14α,17β,20β-trihydroxy- 1-oxo-witha-2,5,24-trien-olide was found to be
active against a number of potentially pathogenic fungi Nigrospora oryzae,
Aspergillus niger, Curvularia lunata, Stachybotry satra, Allescheria boydii,
Drechsleraro strata, Microsporum canis and Epidermo phytonfloccosum and plant
pathogen Pleurotus ostreatus (Choudhary et al., 1995). The compound also showed
activity against gram positive bacteria (S. aureus) (Rahman and Choudhary, 1998a).
Withaferin A potentially exhibited significant antibacterial activity against Gram-
positive bacteria’s but were inactive against Gram-negative microorganisms and non-
filamentous fungi. Also another significant compound, withanolide D are proved with
antifungal cytotoxic activity against thirteen fungal species responsible for various
human infections (Roumy et al., 2010).
2.2.2 Anti-inflammatory activities
The anti-inflammatory potential of W. somnifera and W. coagulans has been
studied by several workers. Anbalagan and Sadique (1981) started off with
preliminary experiments and reported W. somnifera to possess efficient anti-
inflammatory activity compared to a common anti-inflammatory drug,
hydrocortisone. Budhiraja et al. (1984 and 1986) showed aqueous extracts of
W. coagulans fruits had significant anti-inflammatory activities in subacute models of
formalin-induced arthritis in rats. 3-β-Hydroxy-2, 3-dihydrowithanolide F was
isolated from W. coagulans extracts and produced the same pattern of anti-
inflammatory activity in formalin induced rat arthritis (Maurya et al., 2010). The
effect of W. somnifera on synthesis of glycosaminoglycans in the tissue granulation of
carrageenan-induced air pouch granuloma was studied by Begum and Sadique (1987).
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It was found out that W. somnifera root powder decreased the glycosaminoglycans
content by 92%, much higher than the drugs phynylbutazone and hydrocortisone. The
same team again proved the efficiency of root powders of W. somnifera in comparison
to hydrocortisone succinate in rheumatoid rats (Begum and Sadique, 1988).
Granuloma tissue formation was inhibited when subcutaneous cotton-pellet implanted
rats were treated with extracts of aerial parts of W. somnifera and produced a high
anti-inflammatory activity compared to the drug hydrocortisone sodium succinate
(Al-Hindawi et al., 1992; Singh et al., 2010). Bhattacharya et al. (2000) reported the
synthesis of a live plasma protein, alpha-2-macroglobulin increased greatly upon
inflammation and this was decreased effectively upon treatment with W. somnifera
compared to other standard anti-inflammatory drugs. The hydroalcoholic extracts of
W. coagulans fruits showed significant anti-inflammatory activity against carrageenan
induced paw oedema rat models (Rajurkar et al., 2001; Gupta et al., 2013) followed
by, Lalsare and Chutervedi (2010) proved it with various solvent extracts of W.
coagulans fruits.
These activities are mainly attributed due to their high bioactive steroids,
especially the major withaferin A which is shown to have similar structure and
function to glucocorticoids and has a complex influence on inflammatory system
(Davis and Kuttan, 2000). Withaferin A is known to play as potent inhibitors of pro-
inflammatory mediators and a promising treatment against inflammatory cascades of
various disorders (Kaileh et al., 2007). Preliminary studies on withanolides anti-
inflammatory activity started with Subramanian and Sethi (1972) who assessed the
activity of withaferin A, withanone and other new withanolides on acute and subacute
models of inflammation and found significant variation in biological activities of
withanolides. Withanolide fraction from aerial parts of W. somnifera from Iraq had
antigranuloma activity and reduced the weight of inflammation induced adrenal
glands. Withaferin A was also found to have adrenal and granuloma inhibiting
activity without affecting the spleen and body weight (Al-Hindawi et al., 1992; Patel
et al., 2013). Other reports have also indicated withanolides as an inhibitor of NfkB
mediated inflammations (Bargagna-Mohan et al., 2006; Oh et al., 2008b; Kour et al.,
2009; Oh and Kwon, 2009; Maitra et al., 2009).
2.2.3 Anti-cancer activity
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The anticancer property of withania species has been extensively studied by
several researchers and was identified as an effective agent in preventing cancer
through reduction in tumor size (Davis and Kuttan, 2000; Prakash et al., 2002;
Winters, 2006; Senthilnathan et al., 2006; Widodo et al., 2007; Singh et al., 2011;
Khazal et al., 2013; Szic et al., 2014). Initial studies started with oral administration
of W. somnifera extracts simultaneously with urethane for seven months and reduced
tumor incidence was found (Singh et al., 1986). Treatment with W. somnifera root
extracts on mice with induced skin cancer exhibited significant decrease in the
number of skin lesions compared to control group (Prakash et al., 2002). Also a
number of studies have reported the anti-cancer activities in the leaf extracts of
W. somnifera (Diwanay et al., 2004; Christina et al., 2004; Leyon and Kuttan, 2004;
Malik et al., 2007; Aalinkeel et al., 2010; Malik et al., 2009). Ichikawa et al. (2006)
demonstrated that the anti-carcinogenic effect of W. somniferais mainly mediated
through withanolides activity of anti-proliferation, antiangiogenic, antimetastatic,
anti-invasive and proapoptosis which then results in suppression of NF-kB regulated
gene products. Additionally, the extracts of W. coagulans were demonstrated to
inhibit the incorporation of thymidine and hence proliferation of carcinomas.
Withaferin A was identified as the responsible component in extracts of
withania with potent tumor inhibiting activity by inhibiting more than 50% of RNA
synthesis and acting as mitotic poison, resulting in the cell cycle arrest in various
human derived carcinomas (Jayaprakasam et al., 2003; Choudhary et al., 2010;
Maurya et al., 2010; Chen et al., 2014). Earlier studies on withaferin A proved their
radio-sensitising and growth inhibitory effects on experimental mouse tumors and
increase the tumor free survival in a dose dependent manner (Devi et al., 1995;
Sharada et al., 1996; Ganasoundary et al., 1997). Similarly withaferin A in tumor
cultures decreased the expression of nuclear factor-kappa β and tumor necrosis factor
resulting in the arresting of apoptotic signalling (Choudhary et al., 2010). Apart from
this, studies on withanolide A also showed growth inhibitory and radio sensitizing
effects on mouse carcinomas and resulted in the mitotic arrest of the chicken
fibroblast cells (Gupta and Keshari, 2013). These withanolides are also said to induce
apoptosis via mitochondria by cytochrome C release and caspase activation (Senthil
et al., 2007). The presence of unsaturated lactone as a side chain upon which allelic
primary alcohol groups are attached and highly oxygenated rings on the other end of
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the molecule has been suggested as the chemical system with carcinostatic properties
(Rastogi et al., 1998; Khare, 2007).
2.2.4 Hypolipidemic and hypocholesterolemic activities
The aqueous extract of W. somnifera root and W. coagulans fruit have been
reported to decrease total amount of lipid, cholesterol and triglycerides in
cholesterolemic animals (Andallu and Radhika, 2000; Hemalatha et al., 2006).
W. somnifera root powder was effective in decreasing lipid profiles of normal
subjects. Visavadiya and Narasimhacharya (2007) investigated hypocholesterolemic
activity of W. somnifera in male swiss albino rats and suggested that the activity is
mediated through increased the HMG-CoA reductase activity and bile content of
liver. In another study, hypoglycemic and hypocholesterolemic effect of W. somnifera
roots were assessed on human subjects and suitable parameters were analysed in
blood and urine samples of the subjects after 30 days treatment. Significant increase
in urine sodium, urine volume and decrease in serum cholesterol levels and decreased
LDL and VLDL were observed indicating the roots of withania to be a potential
source of hypocholesterolemic agents (Andallu and Radhika, 2000; Gupta and Rana,
2007; Singh et al., 2010). Further, a significant decrease in lipid-peroxidation was
found after administration of W. somnifera extracts to hypercholesteremic animals
when compared to the control groups.
The hypolipidemic and hypocholesterolemic activities of W. coagulans were
also reported by Hemalatha et al. (2006) and Datta et al. (2013). Administration of
aqueous extracts of W. coagulans fruits to high fat diet-induced rats for 7 weeks
significantly reduced serum cholesterol, lipoprotein and triglyceride levels. The
extract also showed hypolipidemic property in triton induced hypercholesterolemia
(Jain et al., 2012). The histopathological examination of liver tissues of withania
extract treated rats showed comparatively lesser degenerative changes compared to
hyperlipidemic controls. The hypolipidemic effect of
W. coagulans fruits was much comparable to Ayurvedic product containing
“Commiphoramukkul” to treat high cholesterol levels (Hemalatha et al., 2006). The
hydroalcoholic extract of W. coagulans fruits were also effective and comparable to
drug “atorvastatin” in controlling lipid levels in high cholesterol diet induced rats.
Hoda et al. (2010) showed aqueous and chloroform extracts of W. coagulans fruits to
21
effectively decrease total cholesterol, triglyceride, LDL and VLDL levels. The
extracted coagulin L from the fruits of W. coagulans was found to be the responsible
element with effective anti-dyslipidemic effect on mice (Maurya et al., 2008).
2.2.5 Hepatoprotective activity
The W. somnifera root powder influenced the levels of lipid peroxidation
thereby provided hepatoprotection (Mohanty et al., 2008). In an in depth study
conducted to examine the effect of these extracts on hepatic cells of
Clarias batrachus reported flavonoids as the responsible molecules in the
W. somnifera extract, stimulating the neuroendocrine system resulting in hyper-
activity of the liver cells endomembrane and exit of molecules through surface
exocytosis (Verma et al., 2009). Studies on pharmacological properties of
W. coagulans by Budhiraja et al. (1986) and Rajurkar et al. (2001) reported the
hepatoprotective activity of extracts of W. coagulans fruits. 3β-hydroxy-2,3-dihydro
withanolide F isolated from W. coagulans was screened for its hepatoprotective effect
against CCl4 induced hepatotoxicity, and the compound was found capable with
marked protective effect after histopathological examinations (Maurya et al., 2010;
Gupta, 2012). Similarly, withaferin A at dose 10 mg/kg, significantly protected CCl4
induced hepatotoxicity as effective as hydrocortisone in rat models (Rastogi
et al., 1998; Khare, 2007).
2.2.6 Immunomodulatory activity:
Administration of W. somnifera extract was found to significantly reduce
leucopenia induced by cyclophosphamide (CTX) and sub-lethal dose of gamma
radiation by means of its effective immune regulation and chemoprotection activity
(Kuttan, 1996; Davis and Kuttan, 1998). W. somnifera treatment significantly
increased RBC count, platelet count and Hb concentration (Ziauddin et al., 1996). In
another study, administering powdered root extract from W. somnifera inhibited
cyclophosphamide–induced delayed type hypersensitivity (DTH) reactions and
enhanced the phagocytic activity of macrophages compared to control group
(Agarwal et al., 1999; Davis and Kuttan, 2000). Extracts of W. somnifera root are also
capable of inhibiting the mitogen induced DTH reactions and lymphocyte
proliferation in rats (Rasool and Varalakshmi, 2006). The extracts of withania are also
known to enhance total white blood cell count, haemoglobin concentration, red blood
22
cell count, platelet count and enhanced phagocytic activity of macrophages (Davis
and Kuttan, 2002).
Withacoagulins A-F along with ten other withanolides isolated from aerial
parts of W. coagulans exhibited strong inhibitory activity on excess proliferation of T
and B-cells and helped in immune modulation (Huang et al., 2009). Coagulin H
significantly inhibited IL-2 production by 80% and docking study predicted coagulin
H bound to IL-2 receptor binding site more effectively than the drug prednisolone.
Based on these computational and experimental results, coagulin H was identified as a
potent immunosuppressive candidate (Mesaik et al., 2006). Similarly withaferin A has
been reported to possess both immune suppressive and immune activating properties
and implement specific and selective effects on human B and T lymphocytes via
antigen recognition (Bahr and Hansel, 1982; Rastogi et al., 1998; Aggarwal et al.,
1999; Davis and Kuttan, 2000; Gautam et al., 2004; Rasool and Varalakshmi, 2006;
Narinderpal et al., 2013).
2.2.7 Antihyperglycaemic activity
W. coagulans has been used since time immemorial in Ayurvedic medicines
to treat diabetes (Gurson and Saner, 1971; Budhiraja et al., 1977; Huang et al., 2009;
Ojha et al., 2014). Administration of aqueous fruit extracts of W. coagulans was
found to significantly lower the blood glucose level (Hemalatha et al., 2004; Saxena,
2010). Long term study on diabetic rats showed a reduction of 54.1% and 52.9% in
post prandial glucose levels and fasting blood glucose levels after 30 days treatment
(Hoda et al., 2010). The ethanol extracts of W. coagulans fruits also reduced blood
glucose level by 52.6% and decrease of 75% sugar level in urine (Jaiswal et al.,
2010). Even W. somnifera has been evaluated for its hypoglycemic effects in human
subjects during clinical studies. Six type 2 diabetes subjects were treated with extract
powder for 30 days and a significant decrease in blood glucose levels were identified
upon comparison with control (Singh et al., 2010). Significant improvement in signs
and symptoms were observed and attained euglycemis in type 2 diabetes mellitus by
Lopez-Ridaura et al. (2004) and Jaiswal et al. (2009). Alam et al. (2009) reported the
combined effect of W. coagulans and Trigonellafoenum graecum in controlling Type
2 diabetes.
23
This hypoglycemic activity of withania is reported mainly due to their high
content of Ca and Mg which plays significant role in diabetes management (Giugliano
et al., 2000; Rai et al., 2007; Kumar et al., 2009). Jaiswal et al. (2010) reported
antidiabetic and hypoglycemic activity of aqueous fruit extracts of W. coagulans by
Laser Induced Breakdown Spectroscopy for detection of glycaemic elements present
in the extract and reported the trace elements like Ca and Mg responsible for
antidiabetic potential of this indigenous shrub. Ca2+ ion mediates insulin gene
activation and expression via CREB (Calcium Responsive Element Binding Protein)
and is responsible for release and breakdown of stored insulin (Veiga et al., 2006).
Alkaloidal and steroidal content of this plant has been the major factor
responsible for hypoglycemic activity (Adebajo et al., 2006). Coagulin L from fruits
of W. coagulans has been reported to have antihyperglycemic activity in rats (Maurya
et al., 2008). The median effective dose of coagulanolide isolated from the fruits of
W. coagulans was determined in streptozotocin-induced diabetic rats and was
compared to the antidiabetic drug metformin (Maurya et al., 2008). Also treatment
with W. coagulans dried fruit extract for 4 weeks significantly reversed the
hyperglycemic levels in streptozotocin-induced diabetic rats comparable to the drug
glipizide (Datta et al., 2013).
2.2.8 CNS activity
The extracts from various parts of both the plants of withania have the
capacity to modulate neurotransmitters. Bhatnagar et al. (2009) observed that the
extracts of withania work as a suppressor of corticosterone secretion and activates
choline acetyltransferase, which in turn increases the levels of serotonin in
hippocampus. The bioactive metabolites isolated from withania was found to be
effective in alleviating disorders associated with central nervous system such as
anxiety, depression, epilepsy and catalepsy (Subramanian and Sethi, 1971; Budhiraja
et al., 1977; Bhattacharya et al., 1997; Jain et al., 2001; Dhuley, 2001; Naidu et al.,
2006). Withanolide VI and withanolideA isolated from W. somnifera induces neurite
outgrowth in cultured neurons and in rodents injected with Aβ 25-35 (Kuboyama et
al., 2002). Recently Sehgal et al. (2012) revealed that the semi purified root extracts
of W. somnifera reversed behavioural deficits, accumulation of β-amyloid peptides
(Aβ), plaque pathology and oligomers in the brains of Alzheimer’s diseased mice
24
subjects by enhancing low density lipoprotein receptor proteins in the brain
microvessels.
2.2.9 Cardiovascular effect
W. somnifera when tested in experimental rats, was reported to exert a strong
cardioprotective effect on isoprenaline-induced myonecrosis (Mohanty et al., 2004).
Maintenance of myocardial antioxidant status and augmentation of endogenous
antioxidants contributes to its cardioprotective effect of W. somnifera at 50 mg/kg
dose. Also a new withanolide (3β-hydroxy-2, 3-dihydrowithanolide F) with its unique
chemical structure similar to aglycones of cardiac glycosides has been isolated from
W. coagulans fruits. This withanolides upon administration to dogs produced a
moderate fall of blood pressure which was induced by atropine (Budhiraja et al.,
1983).
2.3 Analytical tools employed to study plant secondary metabolites
Plants synthesize a wide range of phytochemicals that are useful in the
maintenance of health and vitality of humans. These include primary and secondary
metabolites with their unique functions and metabolic activities. The herbal extracts,
singularly and in combinations, contain myriad of such compounds in which no single
active constituent is responsible for the overall efficacy. The quality assurance and
quality control still remains a challenge because of the high variability of chemical
constituents involved. Due to natural variability in plant material, chemical analysis is
a great challenge and requires special approaches.
2.3.1 Qualitative and quantitative analysis
Secondary metabolite analysis can either be qualitative or quantitative in
nature. Qualitative analysis is used to identify a characteristic compound or a
metabolite species present in the mixture. Qualitative analysis is less instructive and
has to be done as a routine. The classical qualitative analysis scheme for identification
purpose has been around for well over 100 years, but still continues to be an important
part of any analytical training. This is because it offers an effective means for
presenting descriptive nature and illustrates important compositions.
25
In quantitative analysis, we are interested in the relative amount of certain
secondary metabolite present in the mixture. Knowing the composition of a sample is
very important and there are several ways that have been done to make it possible.
Also it is necessary to develop methods in estimations of active constituents or marker
compounds as the quantitative target to assess the inherent and authenticity of quality.
Planar chromatography is the most versatile option required for the identification tests
for the quality control of the herbal drug. In its traditional form, thin layer
chromatography is frequently used for analysis of crude plant extractions and has a
long record in almost all pharmacopeia for its use in analysis of plant extracts. There
are other analytical techniques such as HPLC and HPTLC that can ascertain the
presence of certain compounds in plants and also quantify them more precisely
(Sreekumar and Ravi, 2007).
2.3.2 High performance thin layer chromatography
High-performance-thin-layer-chromatography (HPTLC) is an advanced form
of instrumental TLC which not only include the use of high performance adsorbent
layers (silica material with refined uniform particles of approximately 5 µm in
diameter compared to 12 µm in TLC), but also adopts instrumentation such as
advanced development chambers. It usually implies a standardized methodology for
development, optimization and documentation after a proper validation of methods.
The HPTLC technique is applied in qualitative and quantitative separation of
compounds of a mixture, where the quantitative mode operates in a more optimized
way and hence applicable in the assay of compounds in a sample.
There are several advantages of using HPTLC for analysis of compounds
compared to HPLC and spectrometric titrations (Sethi, 1996; Kalasz and Bathori,
2001). They are (Shewiyo et al., 2012)
The process of separation is easy to follow: especially with coloured
compounds.
Several samples can be separated in parallel on the same silica plate resulting in
high through-put and a rapid low cost analysis.
Specific and sensitive colouring agents can be applied to detect the separated
spots.
Two-dimensional separations can be performed easily.
26
HPTLC can combine and consequently use varied modes of evaluation,
allowing compounds identification with different colours or light-absorption
characteristics.
Contact detection allows microbial activity in the spots to be assessed and
radiolabelled compounds to be monitored.
The TLC plates are disposable and therefore regeneration and clean-ups is not
required.
The development and detection of the separated spots on a plate are different
processes in time and therefore, after separation, the plates can be stored for a
long time and detection can be performed at a later stage.
Literature search reveals a huge number of published papers describing
various usage of the HPTLC technique in many analytical fields. To emphasize on the
importance, opportunities and challenges of the HPTLC technique, older review
papers are available in the literature (Kalasz and Bathori, 2001; Nyiredy, 2002; Poole,
2003). Kaale et al. (2011) recently reported the availability of a large number of
studies on HPTLC methods that have been developed, validated and applied in
pharmaceutical analysis of an active ingredient. HPTLC serves a convenient tool in
analysing the distribution pattern of phytoconstituents, which is unique for each plant.
HPTLC fingerprinting can be applied for authentication of herbal extracts and
formulations. Various workers developed HPTLC protocol for analysis of
phytoconstituents in crude drugs such as bergenin, gallic acid and catechine in
Bergenia lingulata and Bergeniacilliata (Dhalwal et al., 2008). HPTLC technique is
widely employed in pharmaceutical companies in process development, detection and
identification of adulterants in herbal products. It is also used in the quality control of
biological matrices such as whole plant, leaves, flowers and health foods and herbal
formulations (Soni and Naveed, 2010).
HPTLC method has been developed by Sharma et al. (2007) for estimation
of withanolide A and withaferin A in different plant parts such as root, stem, leaf and
fruit of two morphotype of W. somnifera. Patel et al. (2009) developed fingerprint
profile and analysed the withaferin A in old and young roots after complete ethyl
acetate extraction. Distinct HPTLC fingerprint profile was developed for standard
drug and for young and old root extracts by using toluene:ethyl acetate:acetone (2:3:3)
27
as mobile phase. The fingerprinting of hydroalcoholic extracts of withania
formulations has been done using HPTLC in an attempt to understand the heavy metal
content and confirmed its prescription to be safer (Chandran et al., 2010). Recently,
Jirge et al. (2011) developed and validated a HPTLC protocol to determine variations
by herbal manufacturers of Ashwagandha formulations. They employed HPTLC
standardization of four Ayurvedic formulations for their withaferin A and β-sitosterol-
D-glucoside content and their variation in the same plant product. A selective HPTLC
analytical method has also been developed for fingerprinting and determination of
constituents in extracts of Ashwagandha root and their polyherbal formulation (Alam
et al., 2012). Natural populations of W. somnifera and W. coagulans from Iran were
analysed for their withaferin A content by means of HPTLC finger print and was
found out their accumulation were higher in aerial parts than in root extracts. The
result also showed a high level of variations in the Iranian natural populations of
withania, which can be utilised for conservation and breeding programs of a selective
morphotype to improve withaferinA production (Mirjalili et al., 2009b). Similarly,
HPTLC method standardization and withanolide A quantification has been carried out
in field roots of W. coagulans obtained from different geographical locations of Iran.
A morphotype has been identified with increased phytoconstituents and withanolides
content for their in vitro propagation (Preethi et al., 2014).
2.4 In silico analysis
2.4.1 ADME predictions
Computational approach is one of the fastest and newest developing
technique in pharmacokinetics, ADME (absorption, distribution, metabolism and
elimination) predictions, drug discovery and toxicity analysis (Boobis et al., 2002).
In silico analysis of pharmacokinetic and phytochemical parameters are primarily
based on ADMET (absorption, distribution, metabolism, excretion and toxicity) and
Lipinski’s rule of five. ADME describes the potential utility of compounds as drug
leads. Unlike in vivo and in vitro ADME assays, in silico ADME prediction is
particularly cheap and efficient in search of a great number of compounds by
screening libraries and virtual molecules prior to their synthesis. Studies have
demonstrated that initial screening of drug candidates for their ADME properties is a
successful approach to enhance the drug quality (Di and Kerns, 2008). Thus, the
in silico ADME prediction plays a crucial role in facilitating pharmaceutical
28
companies to wisely select drug candidate’s prior expensive clinical trials. The
ADME data can also be used to identify potential liabilities, structural modifications,
select compounds for experimental studies, prioritize lead compounds, and diagnose
in vivo assay results (Di and Kerns, 2008).
ADME-Tox properties can be classified into two categories namely, the
“physiological” and “physicochemical” categories. The physiological ADME-Tox
properties can be further grouped into in vitro ADME-Tox properties (such as MDCK
permeability and Caco-2 permeability, liver microsomes, etc.) and in vivo
pharmacokinetic properties (such as human intestinal absorption – HIA, plasma
protein binding – PPPB, oral bioavailability –F, urinary excretion, area under the
plasma concentration –AUC, total body clearance –CI, elimination half time –t1/2 and
volume of distribution) are governed by many factors. On the other hand, the
physicochemical property which includes logarithm of octanol-water partition
coefficient –logP, aqueous solubility, and logarithm of octanol-water distribution
coefficient (logD) are governed by simple physicochemical laws (Wang and Hou,
2009). In the last few decades, several new ADME-Tox models have been published
and many new software packages and databases have emerged to theoretically
estimate these parameters for a given chemical structure. Also many in silico
approaches for predicting ADME properties of drug compounds for their chemical
structure have been initiated and developed, ranging from database approaches such
as QSAR and 3-dimensional QSAR to structure based methods such as pharmacore
modelling and ligand-protein docking (Yamashite and Hashida, 2004). Schrödinger’s
QikProp is an extremely fast ADME predicting program with following benefits:
Wide range of prediction – QikProp predicts the widest varieties of
pharcological properties such as octanol/water and water/gas coefficient, logPs,
logS, logKhsa, logBB, Caco-2 and MDCK cells permeabilities, overall CNS
activity, oral absorption level, log IC50 for HERG K+ channel blockage.
Accurate ADME predictions – QikProp predicts the ADME properties based on
3D molecular structure; unlike fragment based methods. Therefore, they provide
equally accurate results in predicting molecular properties from a molecules
scaffold structures compared to the novel scaffolds of a well-known drug
analogue.
29
Jorgensen rule of three and Lipinski rule of five – QikProp has ability to
accurately check for Jorgensen rule of three and Lipinski rule of five violations
to provide an at-a-glance measure of compounds drug characteristics.
Similarity check – QikProp automatically identifies molecular similarity
between a submitted ligand and compounds of 1700 molecular databases and
user-specified libraries.
Lead generation – QikProp rapidly screens compound libraries for hits and
identifies molecules with user defined properties that fall inside the normal
range of known drugs. Thus making it simpler to filter the candidates with
suitable and unsuitable ADME properties.
Lead optimization – QikProp plays important roles in lead optimization by
analysing within defined classes of compounds as well as by identifying the
compounds to eliminate because they exhibit extreme values of predicted
properties.
Improving the accuracy level – QikProp computes around twenty physical
descriptors thereby improving the predictions by fitting to additional or
proprietary experimental data and generating alternate QSAR models.
Easy-to-use-interphase – QikProp accepts a wide variety of input formats,
including MDL SD files, Maestro files and PDB files. Calculations are easily set
up and results can be plotted and analysed using Maestro interface.
ADME screening provided peer analysis for final selection of potential drug
candidates from the compound library generated for nevirapine and 47 nevirapine
structural derivatives (Sengupta et al., 2008). In a study of Das et al. (2011), the best
fit ligands benzoxazinone were subjected to in silico ADME screening and was
concluded that the series has the significant potential for type 2 diabetes based on
ADME screening and docking analysis and thus benzoxazionepharmacore could be
used for further development. Withanolides were also screened for their ADME
properties before their molecular docking against the bacterial and viral components
to study their novelty and recommendation for future drug candidature against the
infections caused by these components (Santhi and Aishwarya, 2011; Regon et al.,
2014). In another study, ADME analysis was done using QikProp software before
molecular dock analysis of withaferin A and withanone to prove their efficiency as
small molecule drugs (Vaishnavi et al., 2012).
30
2.4.2 Molecular docking
In recent years, the search for novel drugs has evolved from a trial and error
process into computer based approaches. In structure-based design, the structures of
known target proteins are used in order to discover the novel compounds with
therapeutic relevance. This approach can be accomplished by molecular docking and
involves the formation of protein-ligand complexes (Holger et al., 2001). Docking is
frequently used to predict the binding orientation of small drug candidates into their
appropriate protein targets in order to predict the activity and affinity of the drug
molecule. Hence docking plays an important role in rational designing of drugs. The
development of docking methods is also concerned with making the right assumptions
of target proteins and drug candidates and therefore finding the acceptable
simplification by providing sufficient accuracy and predictive model for protein-
ligand interactions (Abraham et al., 1998). Given the structure of a protein and ligand,
the task is to predict the structure of the resulting complex from their interactions.
During the course of this process, the protein and the ligand adjust their conformation
to achieve an overall “best-fit” and this kind of conformation adjustment in their
overall binding is referred to as “induced-fit” (Wei et al., 2004). In their bound
conformation, the ligand exhibits chemical and geometric complementarily, both of
which are essential for successful drug activity.
The molecular docking tool has been generated to obtain a preferred
geometry of interaction of the receptor-ligand complexes having minimum interaction
energies and based on different scoring functions viz. dock score, sum of steric and
electrostatic fields. Some of the important protein-ligand docking tools used are listed
in Table 2.4. Accuracy and efficiency of the geometric modelling of the dock,
depends on the scoring function. The scoring functions used in the molecular docking
have been adapted from salvation and entropy of the dock complex. The challenge of
the lead-generation phase of the protein-ligand docking is to quickly screen millions
of possible compounds that fit particular receptors with high specificity and affinity.
The set of ligands thus selected can be screened further using more involved
computational technique such as free-energy perturbation theory or in experimental
assays.
31
Table 2.4: Characteristics of current open access protein-ligand docking tools
S.No. Program Designer/Company
License terms Docking approach Supportedplatforms
Scoring function Reference
1 AutoDock D.S. Goodsell andA.J. OlsonThe ScrippsResearch Institute
Free foracademic use
Genetic algorithmLamarckian geneticalgorithmSimulated annealing
Unix, Mac, OSX,Linux, SGI
AutoDock (force-field methods)
Good sell andOlson, 1990
2 DOCK I. KuntzUniversity ofCalifornia, SanFrancisco
Free foracademic use
Shape fitting (spheresets)
Unix, Linux, Sun,IBM AIX,, OSX,Mac, Windows
ChemScore, GB/SAsolvation scoring
Kuntz et al.,1982
3 FRED OpenEyeScientificSoftware
Free foracademic use
Shape fitting(Gaussian)
Unix, Linux, SGI,Mac, IBM AIX,OSX, Windows
ScreenScore, PLP,Gaussian shapescore, user defined
Schulz-Gaschand Stahl, 2003
4 FlexX T. Lengauer andM. RareyBioSolveIT
Commercialfree evaluation(6 weeks)
Instrumentalconstruction
Unix, Linux, Sun,SGI, Windows
FlexXScore, PLP,ScreenScore,DrugScore
Rarey et al.,1997
5 GOLD CambridgeCrystalographicData Centre
Commercialfree evaluation(2months)
Genetic algorithm Linux, SGI, IBM,Sun, Windows
GoldScore.ChemScore userdefined
Jones et al.,1997
6 Glide Schrodinger Inc. Commercial Monte Carlo sampling Unix, Linux, IBM,AIX, SGI,
GlideComp,GlideScore
Friesner et al.,2004
7 LigandFit Accelrys Inc. Commercial Monte Carlo sampling Linux, SGI, IBMAIX
LigScore, PMF, PLP Venkatachalamet al., 2003
32
2.4.3 Scoring function
Scoring functions are fast and approximate mathematical methods used to
predict the strength of the interactions between two molecules after docking. Most
commonly, one of the molecules is small and organic drug compound and the second
is a biological target such as protein receptor (Jain, 2006). Scoring function is also
been developed to predict the strength of other types of interactions such as protein-
protein or protein-DNA (Robertson and Varani, 2007). The scoring function takes a
docked pose as input and returns the strength in numbers indicating the likelihood that
the pose represents favourable binding interactions. Most of the scoring functions are
physics based molecular mechanics estimating the force fields. Scoring methods
ranges from molecular mechanics force fields such as OPLS, AMBER or CHARMM
through empirical free energy scoring function or knowledge based functions. The
docking methods utilize the scoring functions in one of two ways. The first approach
utilizes the full scoring function to rank the protein ligand complex formed. The
system is then modified by the search algorithm and the same scoring function is
again implemented to rank the new structure (Taylor et al., 2002).
2.4.4 Target proteins
Proteins involved in cell cycle
Cell cycle deregulation is a distinguished hallmark of tumour cells (Stewart
et al., 2003). Normal cells possess the ability to arrest cell cycle after DNA damages
to maintain genome integrity whereas tumour progressing cells are characterised of
deregulation of cell cycle whereby the damaged DNA possessing cells proceed to
undergo DNA replication and cell division, resulting in an unrestrained cell
proliferation. Cancers such as breast, lung and gastric cancer are known to exhibit
uncontrolled cell growth and division. The best parameter to judge the efficacy of
anti-cancer therapies is through their ability to arrest cell cycle. It is essential to
identify and eliminate cells proliferating inappropriately and therefore cell cycle
regulators play a vital role in tight check of cell cycle (Meikrantz and Schlegel, 1995;
King et al., 1996).
The timing and order of events involved in cell cycle are monitored during
cell cycle check points that occur at G1/S phase boundary and during G2/M phase
33
transitions (Murray and Hunt, 1993). These check points ensures critical events after a
particular phase of a cell cycle is completed and before a new phase is been initiated,
thereby preventing the formation of abnormal cells. It is at these checkpoints that the
cell determines and chooses the machinery for its own beneficial. The cell cycle
control system depends on three protein families: the cell division cycle 25 protein
(Cdc25), the cyclins and the cyclin dependent protein kinases (CDKs). Arresting the
cell cycle involves down regulation of these proteins. In cancer, mutations are
observed in genes encoding CDK, CDK-activating enzymes, cyclins, and other check
point proteins (Sherr, 1996; Mc Donald and el Deiry, 2000). The cell cycle phases and
the mediators involved in them are presented in Figure 2.4.
Figure 2.4: Cell cycle phases and components. Upon proliferative stimuli, the D-cyclin level increases and forms complexes with CDK4/6, leading to phosphorylationof Rb. Cyclin E along with CDK2 further phosphorylates and results in transitionfrom G1 to S phase. Cyclin A binds with CDK2 in S phase and CDK1 in G2/M phase.In M phase, CDK1 is in complex with cyclin B (Bjorner, 2013).
The cell division cycle 25 proteins (Cdc25) are phosphatases that activate
the CDKs and cyclins which in turn regulate the progression of cell cycle.
Mammalian cells express three Cdc25 - Cdc24A, Cdc25B, Cdc25C of which Cdc25A
mainly controls G1/S transitions and, Cdc25B and Cdc25C predominantly activates
G2/M progression (Molinari et al., 2000; Mailand et al., 2000; Donazelli and Draetta,
2003; Santamaria et al., 2007). It is now evident that all the three Cdc25 isoforms
cooperate to play essential roles in spatial and temporal regulation of the CDKs
during various stages of the cell cycle (Boutros et al., 2006; Boutros et al., 2007;
Rudolph, 2007; Lavecchia et al., 2009). Cdc25s have been associated to undergo
34
oncogenic transformation and elevated expression of Cdc24A and Cdc25B at both
mRNA and protein level has been reported in a wide variety of primary human
cancers with poor prognosis such as breast cancer (Galaktionov et al., 1995), prostate
(Ngan et al., 2003), colon cancer (Takemara et al., 2000; Hernandez et al., 2001) and
lung cancers (Wu et al., 1998; Sasaki et al., 2001). Enhanced expression of Cdc25s in
tumours correlates with specific clinical and pathological features resulting in more
aggressive tumours and a short disease free survival (Boutros et al., 2006; Boutros
et al., 2007). The inhibition of Cdc25 phosphatases may thus contribute as the novel
approach in the development of anticancer therapeutics.
Selective inhibitors of Cdc25 with potent and reversible potential should
facilitate the elucidation of biological functions for these enzymes. Furthermore,
potential inhibitors prove to be useful in targeted cancer therapies due to oncogenic
nature of Cdc25A and Cdc25B in cancer cell lines (Brisson et al., 2004). Although
structures of the catalytic domains of these Cdc25 have been available (Fauman et al.,
1998; Reynolds et al., 1999), very little studies have been carried out of its interaction
with the small molecule inhibitors. Synthetic compounds based on phosphatase
inhibitors of Cdc25 are either not potent or fail to enter the cells effectively. Inhibitors
derived from natural sources such as menadione, dysidiolide, dnacin B1 and
coscinosulfate were found to form irreversible adducts with Cdc25 (Lyon et al.,
2002). Withanolides being a natural compound with proven track record of
therapeutic properties can be used as a one such potential inhibitor.
Cyclin is an essential cell cycle regulator in all eukaryotes. Cyclin binds to
the cyclin-dependent kinase (CDKs) to form Cyclin/CDK complex which then
activates and phosphorylates a critical set of proteins to set the succeeding events into
motion that defines mitosis (Smits and Medema, 2001). These events include nuclear
membrane breakdown, assembly of spindle apparatus, chromosomal condensation and
segregation of sister chromatids (Sanchez and Dynlacht, 2005). Given the central role
of cyclins in cell division, its deregulation not surprisingly contributes to cancer.
Cyclins are overexpressed in several human carcinomas and level of expression of
cyclins correlates with the aggressiveness of the tumour (Ikuerowo et al., 2006). Thus
cyclin B serves as effective targeted cancer therapeutics (Yuan et al., 2004).
Inhibition of cyclin expression either by antisense methodology or by antibody
35
microinjection lengthens the G1 phase duration and causes reduction in proliferation
as demonstrated in nude mice (Arber et al., 1997).
The Cyclin Dependent Kinases (CDKs) holoenzymes are a family of
serine/threonine kinases which play a major role in regulating the cell cycle
progression in all eukaryotic organisms (Nilsson and Hoffman, 2000). As their name
suggest, their activity is controlled in part by regulatory subunits called Cyclins that
bind and activate and provides substrate specificity for their catalytic partners (Sherr
and Roberts, 1999). Inhibitory phosphorylation on N-terminal tyrosine and threonine
residues maintains CDK complexes in an inactive state (Parker and Worms, 1992;
Squire et al., 2005). The structure-function relationship of cyclin-CDK complexes
have been extended further by mutational analysis in the N-terminal helix of the
mitotic cyclin by Goda et al. (2001). This active site is highly conserved in all known
mitotic cyclins.
In order to keep the cells in a controlled the cell cycle progression in a
controlled state, the cyclins and CDKs are tightly regulated through inhibition,
subcellular localization and degradation. The CDK inhibitors (CKI) bind CDKs alone
or in combination with cyclins thereby regulates the activity of CDKs. These
inhibitors are in turn regulated by both intercellular and extracellular signals. For
example, p21 is transcriptionally regulated by p53 tumor suppressor and induced upon
DNA damage (el-Deiry et al., 1993). Genes such as c-myc, are known to inhibit or
activate cell proliferation by affecting the formation of CDK complexes.
Proteins involved in apoptosis
Apoptosis or the programmed cell death is a physiological homeostatic
mechanism and is critically important for the survival of multicellular organisms
(Lockshin and Zakeri, 2007). As a result of this process, unwanted cells are
eliminated in a well-organised sequential process, characterised by various
morphological and biochemical changes such as pyknosis, mitochondrial membrane
permeability, plasma membrane blebbing, and activation of caspase cascades
(Elmore, 2007). Activation of apoptosis is mainly mediated through intrinsic
mitochondrial pathway and extrinsic death receptor pathway which involve a variety
of caspase family members (Hengartner, 2000; Fulda and Debatin, 2006). The
regulation of Bcl-2 family members dissipates the mitochondrial membrane potential
36
resulting in the release of proapoptotic proteins such as cytochrome C and apoptosis
inducing factor from the intermembrane space into the cytosol. Following this, the
apoptosome, a complex that turns out from the interacting cytochrome C and
apoptosis protease-activating factor, results in activation of caspases. Activation of
various caspases subsequently cleaves poly-(ADP-ribose) polymerase (PARP),
ultimately leading to apoptosis mechanism.
A rational approach to treat cancer is to selectively eliminate the proliferating
tumor cells via apoptosis and spare quiescent and terminally differentiated cells
(Schwartz and Shah, 2005; Petrelli and Giordano, 2008). The failure to control cancer
cells associated with apoptosis induction has been considered to be a critical cause of
resistance against cancer therapies (Fulda and Debatin, 2006). The entire apoptosis
mechanism is discussed in detail in Figure 2.5.
Figure 2.5: Apoptosis pathways. The extrinsic cell death pathway mediated by TNFreceptor superfamily is initiated by the recruitment of adapter proteins, like FADD(Fas associated death domain), via DD (death domain), which in turn binds to deatheffector domain containing caspases. Formation of this death inducing signallingcomplex (DISC) leads to activation of caspase-8/10 which then activates caspase-3. Inintrinsic mitochondrial pathway, proapoptotic Bcl-2 family members, Bak and Baxtranslocates to the mitochondria where it forms an oligomeric pore in the outermitochondrial membrane. This releases cytochrome c and other pro-apoptotic factorsinto the cytosol. This mechanism triggers the assembly of apoptosome from Apaf-1,caspase-9 and ATP as a third component. Subsequently apoptosome activatescaspase -3, leading to cell death (Kalimuthu and Kwon, 2013).
37
In mammalian cells, five antiapoptotic proteins Bcl-2, Bcl-XL, Bcl-w, MCL1
and A1 antogonises the proapoptotic functions of BAX and BAK (Youle and Strasser,
2008). Overexpression of Bcl-2 and Bcl-XL enhances the cell survival by suppression
of apoptosis (Yang and Korsmeyer, 1996; Boise et al., 1995; Reed, 1995; White,
1996). Unlike Bcl-2 and Bcl-XL, over expression of BAK and BAX accelerates cell
death (Oltvai et al., 1993). Both groups of proteins are proposed to regulate apoptosis
by means of homo and hetero dimerization (Sedlak et al., 1995; Yang et al., 1995).
The structures of pro and antiapoptotic proteins are illustrated in Figure 2.6.
Figure 2.6: Pro and anti-apoptotic proteins involved in the apoptosis mechanism.The hypothesis is that upon engagement of BH3-only proteins termed as “activators”,notably Bim and Bid at the trigger site of BAX leads to major conformational changeincluding allosteric release of its C-terminal helix and exposure of its BH3 domain foractivation and oligomerization within the mitochondrial membrane (Shamas-Dinet al., 2013).
The B-cell lymphoma 2 (Bcl-2) family proteins is been discovered in many
types of cancer cells and promotes cell survival (Vaux et al., 1988) leading to
impaired apoptosis, a critical step in tumor developments (Hanahan and Weinberg,
2000). The pro- and anti-apoptotic family members heterodimerize each other and
seemingly titrate their functions (Oltvai et al., 1993). Mutagenesis studies established
that BH1, BH2 and BH3 domains strongly influences these homo and
heterodimerizations (Yin et al., 1994; Chittenden et al., 1995). Proapoptotic proteins
allow its insertion into the groove of the BH3 domains of the anti-apoptotic proteins
(Muchmore et al., 1996). The fourth “BH4 site” is the N-terminal helix is conserved
38
for inhibition of apoptosis and binding of drug molecules at this site results in
suppressing their inhibitory profile and supports the function of other pro-apoptotic
proteins under tumerogenesis. The BH4 region of the Bcl-2 binds with regulatory
domain of the inositol 1,4,5 triphosphate (IP3) receptor which in turn controls the
calcium efflux from the endoplasmic reticulum, thereby inhibiting the initiation phase
of calcium mediated apoptosis (Rong et al., 2009).
Bcl-2 and Bcl-XLare the best model to be screened under the category of
anti-apoptotic proteins since it has a higher global flexibility with pliable binding
pockets within the BH3 domain compared to the deeper hydrophobic pockets of other
anti-apoptotic proteins that restrict the binding to specific BH3 domain containing
proteins (Lee et al., 2009). In fact, the Bcl-2 coding nucleotides are currently being
tested in clinical trials for various cancer treatments (Rudin et al., 2002). In addition,
by utilising the structures of Protein-Protein complexes, small molecule inhibitors of
Bcl-2 have been designed (Petros et al., 2004). Wang et al. (2000) were the first to
report small molecule inhibitors of Bcl-2 by building the modelled Bcl-2 in the form
of peptide complex. Subsequently they employed a computational docking strategy to
screen around 193,833 compounds from the available compound libraries. In another
study using computer-based screening, around 206,876 organic compounds were
searched from National Cancer Institute 3D database to identify the potential binders
of Bcl-2 (Enyedy, 2001).
BAK and BAX are the two main pro-apoptotic proteins considered to be
essential gateway for apoptosis mechanism (Wei et al., 2001) and when deleted, give
rise to severe developmental defects (Lindsten et al., 2000). In normal cells, BAX is
largely cytosolic and translocate to mitochondrial membrane only upon receiving
apoptotic stimulus (Wolter et al., 1997; Edlich et al., 2011). Cytosolic BAX
comprises a globular bundle of nine helices and the last helix is assumed to regulate
the BAX activity as it neither anchors the BAX to the mitochondrial membrane nor
resides in a hydrophobic groove on the surface of the cytosolic BAX (Suzuki et al.,
2000). Defining how a BAX metamorphoses from an inert cytosolic monomer to
cytotoxic mitochondrial membrane perforating oligomer has been deemed the “holy
grail” of apoptosis research (Youle and Strasser, 2008). This pivotal mechanism is
poorly understood because no structure of any activated form of BAX is available.
39
However, the mechanism by which BAX are activated in the first place has been the
theme of much debate (Leshchiner et al., 2013).
Poly (ADP-ribose) polymerases (PARPs) are nuclear protein enzymes
involved in synthesis of poly (ADP ribose) in DNA damages and repairing of single-
strand breaks (SSBs) through base excisional repair (BER) (Do and Chen, 2013).
Escalating the DNA damages and to deteriorate the DNA repair system are the other
important features leading to genomic instability which provides valuable clues in the
rational development and exploitation of inhibitor drugs in clinical setting and
designing of a new therapeutic approach in cancer. The human genome integrity is
constantly under stress from both endogenous insults such as reactive oxygen species
and exogenous insults such as chemotherapeutic agents. Cellular response depends
upon the magnitude of insult and if the damage is extensive and irreparable, cell death
occurs.
Catalytic domain of PARP is highly conserved and forms the active site
(Ame et al., 2004; Otto et al., 2005). Under normal condition, the inactive PARP
resides in the nucleoplasm and once the DNA breaks are introduced, synthesis of poly
(ADP-ribose) takes place at the sites of breakage. In vitro studies indicates that
PARP-1 binds tightly to DNA nick which activates its catalytic domain inducing poly
(ADP-ribosyl)ation. This allows for the recruitment of DNA repair proteins such as
DNA polymerase, DNA ligase and scaffolding proteins (El-Khamisy et al., 2003;
Houtgraaf et al., 2006). More recent studies by Helleday et al. (2011) proposed that
PARP inhibitors results in trapping of PARP from DNA repairing and stalling of
replication forks. A considerable effort is centred in order to manipulate DNA damage
responses to selectively induce death in tumor cells (Helleday et al., 2008).
Accordingly, PARP inhibitors that compete with β -NAD+ at their active site are
arisen as a new potential therapeutic strategies as chemo-and radio-potentiation for
cancer treatments (Rouleau et al., 2010).
Proteins involved in Inflammation
Inflammation is an adaptive response triggered by noxious conditions and
stimuli such as tissue injury and infection (Kumar and Cotran, 2003; Majno and Joris,
2004). Inflammation is a complex process mediated by immune cells such as
macrophages and monocytes (Chen et al., 2005) which results in restoration of
40
damaged tissue structure and function (Lawrence et al., 2002). It is generally thought
that a controlled inflammatory response is beneficial by providing protection against
infections and tissue damage, but become detrimental if deregulated to cause septic
shocks. Acute inflammatory response involves the coordinated delivery of blood
components to the site of infection, causing increased swelling, temperature, redness
and pain (Kumar and Cotran, 2003; Koh et al., 2010). It is well known that,
macrophages along with neutrophils and dendritic cells play important roles in innate
immune reactions (Carralot et al., 2009). The key inflammatory mediators such as
cyclooxygenase-2 (COX-2), nitric oxide (NO), nitric oxide synthase (iNOS) and
prostaglandins (PGE2) and proinflammatory cytokines such as interleukins (IL) and
tumor necrosis factor (TNFα) are released from activated macrophages (Nakagawa
et al., 2012). Lipopolysaccharide (LPS), a cell wall component of Gram-negative
bacteria is reported to activate these macrophages and triggers a series of signalling
cascades which leads to activation of MAPKs and NF-kB pathways (Zhang and
Dong, 2005). Herein, LPS induced macrophages is a well-established model to study
innate immune responses (Aderem and Ulevitch, 2000).
The main effect of these mediators is to elicit inflammatory exudate locally
and prevent its access to extravascular tissues through post capillary venules. The
activated blood vessel tissue allows selective extravasation of neutrophils and
prevents the exit of erythrocytes. This selective permeability is afforded by ligation of
endothelial cell integrins with selectins and chemokine receptors on leukocytes (Pober
and Sessa, 2007). When the neutrophils becomes activated upon reaching the afflicted
tissue site either by direct contact with the pathogens or through actions of cytokines
secreted by infected cells, they releases toxic components of their granules such as
reactive oxygen species (ROS) and reactive nitrogen species (RNS) and attempts to
kill the invading agents. These highly potent effectors released from the neutrophils
do not discriminate between pathogens and host targets, leading to unavoidable
collateral damage to the host tissues (Nathan, 2002). A successful acute inflammatory
response results in the expulsion of infectious agents followed by a repair phase
mediated mainly by macrophages recruitment (Serhan and Savil, 2005). The switch in
lipid mediators from pro-inflammatory prostaglandins to lipoxins with anti-
inflammatory potential is crucial for the transition to resolution from inflammation.
Lipoxins inhibits the neutrophil recruitment and engage monocytes to remove dead
41
cells and initiate tissue remodelling (Serhan and Savil, 2005). Protectins and resolvins
contribute another class of lipid mediators, as well as growth factor produced by
macrophages and transforming growth factor β possess crucial role in resolution of
inflammation and initiation of tissue repair (Serhan, 2007).
The MAP kinases (MAPK) comprises of large enzyme family in both
prokaryotes and eukaryotes. These are serine/threonine kinases that act by modulating
the activity of cellular proteins such as cell surface receptors, other kinases enzymes,
transcription factors and structural proteins (Davis, 1993). They catalyse the transfer
of the terminal phosphoryl group of ATP to their appropriate protein substrates. It has
been recognised for many years that protein phosphorylation regulates many cellular
functions such as metabolism, survival, movement, division and death. Therefore, any
process that disrupts normal phosphorylation disrupts the function of cell and results
in diseases (Cohen, 2002). Several individual MAPK signal transduction pathways
have been characterised of which one pathway leads to activation of the extracellular
signal regulated protein kinases (ERK) which plays important roles in regulating
cellular responses and growth factors (Blumer and Johnson, 1994). The ERK MAP
kinases are activated in a wide variety of cell types by varying stimuli (Cobb et al.,
1991; Pearson et al., 2001). Boulton et al. (1991) coined the acronym ERK for
extracellular signal regulated protein kinase and cloned the cDNA of rat ERK.
ERK1/2 have small amino-terminal lobe and large carboxyterminal lobe with several
conserved helices and strands as described by Knighton et al. (1991). The small lobe
consists of five antiparallel sheets with conserved glycine rich (GxGxxG) ATP-
phosphate binding loop. The large C-terminal lobe is mainly helical with four short
conserved strands that contain most of the catalytic residues associated with
phosphoryl transfer from ATP to ERK substrate (Taylor and Kornev, 2011). Early
studies indicated that these enzymes are activated upon cellular stimulation by
bradykinin, fibroblast growth factors, epidermal growth factors, platelet derived
growth factors and nerve growth factors (Cobb et al., 1991). Then ERK1/2 are also
known to be activated by cytokines, osmotic stress and transmembrane receptors
proteins (Raman et al., 2007). The Ras-Raf-MEK-ERK signalling cascade is
dysregulated in a variety of diseases such as brain injury, cancer, inflammations,
cardiac hypertrophy and diabetes (Muslin, 2008; Tidyman and Rauen, 2009; Tanti
and Jager, 2009; Montagut and Settleman, 2009; Chico et al., 2009; Kim and Choi,
42
2010). Owing to the importance of ERK1/2 signalling cascade, they are known to
represent bonafide drug targets with considerable attention from large cadre of
biomedical scientists (Cohen, 2002).
A second MAPK pathway is regulated by changes in extracellular stimuli’s
induced by physical or chemical changes or by proinflammatory cytokines leading to
activation of transcription factors of c-Jun (Derijard et al., 1994; Kallunki et al.,
1994). The JNKs are a larger group of protein kinases and can be expressed in 10
different isoforms. They are activated by dual phosphorylation by MAPK kinases
MKK4 and MKK7 on specific threonine and tyrosine within their
activation/phosphorylation loop (Davis, 2000). Activation of JNK by extracellular
stimuli such as cytokines or stress leads to the phosphorylation of several transcription
factors and cellular substrates implicated in cell survival and proliferation and mRNA
stabilization. Since these pathways are implicated in a variety of disease states, JNK
constitutes valuable targets for drug discovery and development (Manning and Davis,
2003; Weston and Davis, 2007). JNK interacting protein 1 (JIP1), enhances JNK
signalling by creating proximity effect between the JNK and upstream kinases. An
oligopeptide corresponding to an 11 amino sketch (153-163) is known to inhibit JNK
activity by competing with JIP protein (Whitmarsh et al., 1998; Barr et al., 2002).
Recently, small molecules which mimic JIP serves as substrate-competitive inhibitors
have been reported and inhibit the phosphorylation of JNK substrates both in vitro
and in vivo in a dose dependent manner (Stebbins et al., 2008).
A third MAPK pathway leads to p38 pathway activation after varying stimuli
such as UV light, proinflammatory cytokines, microbial pathogens and increased
extracellular osmolarity (Han et al., 1994; Rouse et al., 1994; Freshney et al., 1994;
Raingeaud et al., 1995). p38 in mammals are in four forms: α, β, γ and δ and among
all p38α is best characterised and expressed in most cell types. p38 was initially
identified as a 38 kDa polypeptide with tyrosine phosphorylation in response to
endotoxins and shocks (Han et al., 1994). The amino-acid sequence identity shows
that p38α and p38β are 75% identical whereas p38δ and p38γ are 61% and 62%
identical, respectively. MAPK family members have marked sequence homologies
with around >40% sequence identity. Nonetheless, there are specificities found
around upstream activators, the MAPK kinases (Crews et al., 1992; Yan et al., 1994;
Derijard et al., 1995; Han et al., 1996; Raingeaud et al., 1996; Cuenda et al., 1996)
43
and the substrates activated by the active MAPKs (Derijard et al., 1994; Kallunki et
al., 1994; Sanchez et al., 1994; Raingeaud et al., 1995; Wang and Ron, 1996).
The nuclear factor-kB (NF-kB) family proteins are essential for immunity,
inflammation, cell proliferation and cell death (Baeuerle and Henkel, 1994; Barnes
and Karin, 1997; Bours et al., 2000; Karin et al., 2002). NF-kB exists in a latent state
in the cytoplasm and requires activation of signalling pathways. Such NF-kB
activating pathways are triggered by diverse extracellular stimuli leading to
phosphorylation and subsequent proteasome mediated degradation of inhibitory
mediators, the inhibitor of NF-kB (IkB) proteins (Karin and Benneriah, 2000).
Therefore, a key step for controlling NF-kB activity is by regulating IkB-NF-kB
interaction. Almost all signals leading to NF-kB activation converge on the activation
of a high molecular weight complex containing a serine specific IkB kinase (IKK).
IKK targets IkBα for ubiquitination and degradation. This IkB protein masks the
nuclear localization signals (NLS) located on each subunits of NF-kB to prevent its
translocalization. The IkBα protein is divided into three parts: an N-terminal domain
that integrates activating signals together, a central part with ankyrin repeats involved
in contact and inhibition of NF-kB subunits, and a C-terminal PEST region, rich in
proline, glutamic acid, serine, and threonine that regulates the half-life of the
molecule. IkB not only interferes with nuclear translocation of NF-kB but also has the
ability to displace NF-kB bound DNA (Ghosh et al., 1995). In response to diverse
stimuli, the inhibitory subunit IkB is phosphorylated and then undergoes degradation
to free NF-kB. Most of the stimuli such as LPS, pro-inflammatory cytokines and
ionizing radiations converge to IKK protein complexes in activating signal
transduction (Karin, 1999). After phosphorylation and degradation of IkB by means
of IKKs, leads to translocation of NF-kB to the nucleus to activate transcription of
target genes (Pahl, 1999). The gene coding for IkB is among the first to be transcribed
by NF-kB and newly synthesized IkB molecules migrates to the nucleus to turn off
NF-kB dependent transcription (Sachdev et al., 1998). Then, with the help of nuclear
export sequences, the newly formed NF-kB/IkB complexes returns back to cytoplasm
(Gilmore, 1999). The UV irradiation induces the activation of NF-kB after
phosphorylation and degradation of IkB apparently without the involvement of IKK
complexes (Li and Karin, 1998). Cell reoxygenation and oxidative stress also leads to
NF-kB activations (Imbert et al., 1996).
44
2.5 Toll-like receptors (TLRs) in inflammation
Deciphering the signalling pathways under normal and disease states remains
a major challenge. Toll-like receptors (TLRs) sense the invading pathogens and play
crucial roles in activation of innate and adaptive immunity. Recent studies of Toll-like
receptors (TLRs) signalling during bacterial infections revealed the molecular
mechanisms by which bacterial components induce innate defence and
proinflammatory responses (Jo et al., 2007; Basu et al., 2012). TLR mediated innate
immune responses are mainly regulated by mitogen activated protein kinase (MAPK)
and nuclear factor NF-kB pathways (Jo et al., 2007; Yuk and Jo, 2011). Both these
signalling pathways play major roles in the activation of antimicrobial responses and
in the generation of effector molecules during infections (Schorey and Cooper, 2003).
Moreover, reactive oxygen species (ROS), primarily derived from NADPH oxidases
(NOX), are important in controlling and shaping the key signalling network systems
during inflammatory responses (Bae et al., 2011; Brune et al., 2013).
Figure 2.7 illustrates different TLR’s involved in signaling cascades. Some
TLRs share common ligands, and heterodimerization between the ligands is common
(Huang et al., 2011). Therefore, it is necessary to have a sound knowledge of how this
excessive TLR activation disrupts immune homeostasis and results in the
development of inflammatory and autoimmune diseases. The list of endogenous and
exogenous ligands of different TLRs is represented in Table 2.5. TLRs are found to
be conserved evolutionarily from wide array of plants to mammals. Toll means
“great” in German and Anderson et al. (1985) coined it for a protein which played a
critical role in the early development of Drosophila embryos. Later it was found that
this protein also played an essential role in host innate immunity against fungal
infections in adult flies (Anderson et al., 1985; Lemaitre et al., 1996). Since then,
research interest has been focused on TLRs activation and functioning. All TLRs
show similar domain architecture comprising with an extracellular leucine-rich
domain sensing the pathogens, a Toll-interleukin 1 receptor (TIR) domain which
mediated the downstream signal transduction and a single transmembrane helix
(Basith et al., 2011). On the whole, 13 TLRs have been discovered until date and is
found to have common role in immunity. These TLRs differ in their ligand
specificity, cell localization, usage of adaptor proteins and cellular responses (Iwasaki
and Medzhitov, 2004). However, TLRs are not the only group of receptors for
45
invading pathogens, the system also possess certain non-TLRs such as cyctosolic
DNA receptors (Lee and Kim, 2007), nucleotide-binding domain and leucine-rich
repeat containing gene family (NOD receptors) (Martinon et al., 2007) and retinoic-
acid-inducible gene-I- (RIG-I) like receptors (RLRs) (Yoneyama and Fujita, 2009).
Figure 2.7: Various TLRs and their locations. The cell membrane bound TLRs useMyD88 as adaptor protein for signal transduction. TLR4 alone uses three or moreadaptors apart from MyD88 to elicit antiviral responses. Endosome located TLRs alsouse MyD88 except, TLR3, which uses TRIF, the universal adaptor protein recruitedby TLR3 and TLR4. All these pathways converge on TRAF6 (Tumor necrosis factorreceptor associated factor 6), and activates NF-κB, which in turn translocate tonucleus for expression of various genes involved immune responses (Krishnan et al.,2012).
Toll-like receptors (TLRs) were identified by means of genetic analysis as
the sensors that detect the released ligands. First LPS (Poltoraket al., 1998), then
DNA, RNA, flagellin and lipopeptides of microbes were shown to be ligands of
TLRs. Since TLRs are capable of recognizing molecules derived from bacteria,
viruses, fungi and protozoa, they are able to sense most of the infections which we
might ever encounter and their sensing role is indispensable. Other sensors are
inadequate to cope with wide array of infections in the absence of TLR signaling.
Experimental mice lacking MyD88 and TRIF cannot generate signals via any of the
TLRs were found to rarely survive the weaning age without supplementation of
46
proper antibiotic (Hoebe et al., 2003). Even mutations of individual TLRs such as
TLR2 (Takeuchi et al., 2000), TLR4 (Poltorak et al., 1998) and TLR9 (Tabeta et al.,
2004) cause quite obvious susceptibility to infectious agents and redundancy in
system to detect pathogenic microbes. Conventional adjuvants lead to strong adaptive
immune responses in the absence of TLR signaling (Gavin et al., 2006). This
redundancy is also seen in initiation of adaptive immune responses witnessed in germ
free mice when encountered with enteric microbes, which occurs if TLR signaling is
abolished by mutations (Slack et al., 2009). Thus TLRs although mediate adjuvant
effects, adaptive immune responses can none the less be perfect in their absence.
Table 2.5: Endogenous and exogenous ligands for TLRs
TLRLigands
Endogenous Exogenous
1 - Triacyl lipopeptide
2 Necrotic cells, HSPs (HSP-60, HSP-70,Gp-96), Biglycans
Peptidoglycans andlipoproteins
3 Self-messenger RNA Double-stranded RNA
4
Extra domain A-containing fibronectin,Fibrinogen, Polysaccharide fragments ofheparin sulphate, Oligosaccharides ofHyaluronic acid, Β-Defensin 2, Oxidized low-density lipoprotein, HGPs, Neutrophilelastase, High mobility group box 1 protein,Biglycans
LPS and Taxol
5 - Flagellin
6 - Diacyl lipopeptide
7 - Single-stranded RNA
8 - Single-stranded RNA
9 Chromatin-IgG complex Unmethylated CpG DNA
10 - Unknown
11 - Urapathogenic E. coli
47
2.5.1 Toll-like receptor (TLR) 4 signalling
LPS is a key component of gram negative bacterial cell wall and is composed
of three structural elements: a core oligosaccharide, a lipid component and an
O-specific chain with repeating sequences polysaccharides, which are responsible for
the proinflammatory properties of LPS (Alexander and Rietschel, 2001). The LPS
binding to the endothelial cells surface results in endothelial activation (Hack and
Zeerleder, 2001). LPS also activates macrophages to stimulate the produce
proinflammatory components which in turn modulates the endothelial function.
Collectively, these processes initiate a parallel cascade of events contributing to the
clinical manifestations of sepsis. The TLRs are pattern recognition receptors classified
based on the homology of the cytoplasmic domain (Slack et al., 2000). To date, there
have been 10 TLRs identified in humans (Chuang and Ulevitch, 2000; Du et al.,
2000; Chuang and Ulevitch, 2001) and TLR4 was established as the LPS signalling
receptor. The first host protein involved in the LPS recognition is LPS-binding protein
(LBP) (Schumann et al., 1990). LBP is an acute-phase protein which recruits LPS to
the cell surface by binding to LPS and forms a ternary complex with LPS receptor
molecule, CD14 (Schumann et al., 1990). Formation of LPS and CD14 complex
facilitates LPS transfer to LPS receptor complex composed of TLR4 and MD2
(da Silva Correia et al., 2001). MD2 is a secreted glycoprotein and functions as an
indispensable extracellular adaptor molecule in LPS initiated signalling events by
aiding ligand recognition (Nagai et al., 2002; Visintin et al., 2003). The TLR4
signalling cascades following LPS binding is enhanced by homodimerization of the
receptor and subsequent recruitment of Toll/IL-1 receptor (TIR) domain-containing
adaptor molecules (TIRAP) with cytoplasmic domains of the receptor (Zhang et al.,
2002; Lee et al., 2004). These adaptors include myeloid differentiation factor 88
(MyD88), TIR-containing adaptor inducing IFNb (TRIF) also called TIRAP-1 and
TRIF-related adaptor molecule (TRAM) also called TIRAP-2 (Akira and Takeda,
2004). Activation of TLR4 stimulated MyD88-dependent and independent pathway
which involves activation of NF-kB and mitogen-activated protein kinases (MAPKs)
(Figure 2.8). These pathways and activation regulates the balance between cell
viability and inflammation.
48
Figure 2.8: MyD88 dependent signalling pathway and activation of NF-kB andMAPKs. LPS binds to TLR4 receptor consisting of soluble CD14 and MD2 and as aresult, recruitment of adaptor proteins MyD88 and TIRAP takes place. Then IRAKproteins interacts with the receptor complex. IRAK1 recruits and activates TRAF6leading to downstream activation of MAPKs and IKKs. Activation of IKK complexresults in phosphorylation and degradation of IkB, permitting translocation of NFkBand expression of pro-inflammatory cytokines.
2.5.2 NF-kB and Mitogen-activated protein kinases activation
MyD88 originally cloned as an adaptor molecule possess a C-terminal TIR
domain and an N-terminal death domain (DD) (Wesche et al., 1997). During MyD88-
dependent signalling, MyD88 is recruited to the TLR4 through interaction with the
TIR domain of TLR4 (O’Neill et al., 2003). This multiplex in turn facilitates the
recruitment of IRAK1 and IRAK4 (Wesche et al., 1997; Li et al., 2002). The binding
of IRAK4 with the receptor complex facilitates the IRAK1 to transphosphorylate,
inducing IRAK1 kinase activity (Burns et al., 2003). The autophosphorylation of
IRAK1 results in the binding of TNF receptor-associated factor-6 (TRAF6) (Cao
et al., 1996). TRAF6 then becomes activated and associates with TAB2 which
activates the TAK1 (transforming growth factor-b-activated kinase) which is
constitutively associated with its adaptor protein TAB1 (Shirakabe et al., 1997;
49
Ninomiya-Tsuji et al., 1999; Yamaguchi et al., 2008). TAK1 acts as a common
activator of both NF-kB and MAPKs consisting of extracellular signal-regulated
kinase (ERK), c-jun NH2-terminal kinase (JNK) and p38 (Wang et al., 2001). NF-kB
activation starts with the assembly of high molecular weight protein complex known
as signalosome. This complex is made up of inhibitory-binding protein kB kinases,
IKKa and IKKb, along with a scaffolding protein known as NF-Kb essential
modulator (NEMO) (Wang et al., 2001). IKK activation involves TRAF interacting
protein with a forkhead-associated (FHA) domain (TIFA) protein (Ea et al., 2004).
TIFA then promotes oligomerization of TRAF6 and facilitates downstream activation
of NF-Kb (Ea et al., 2004). Activation of IKKs leads to phosphorylation of inhibitors
of NF-Kb (IkB) family, resulting in ubiquitin proteasome mediated degradation of
IkB members, thus permitting the release and translocation of activated NF-kB (Chen
et al., 1995). In addition to the activation of NF-kB, activation of TAK1 also leads to
activation of MAPKs (O’Neill et al., 2003).
2.5.3 Chronic Inflammation
Inflammation is the physiologic response to tissue injury caused by infection,
wounding and chemical damage (Philipa et al., 2004). Acute inflammation is the base
line of defence response, but chronic inflammation has been found to mediate a wide
array of inflammatory diseases (Kao et al., 2009). The host system resists infection in
many different ways and in one way or the other, must discriminate from self and
nonself molecular components. One example is downregulation of class I MHC
molecules during viral infections, a change that is perceived by NK cells.
Discrimination of self-nonself may also depend upon concomitants of infection. This
includes changes in expression of molecules indicative of ‘stresses’. Microbial
protease initiates proteolytic cascades that culminate innate immune response
(Ferrandon et al., 2007). But the most basic and broadly applied system for self-
nonself discrimination depends upon receptors susceptible to molecular signatures
unique to microbes.
Activated immune cells (macrophages, eosinophils, neutrophils, monocytes
and phagocytes) secrete increased amounts of inflammatory mediators such as
reactive oxygen species (ROS), nitric oxide (NO) and pro-inflammatory cytokines
(Block and Hong, 2005; Kim and de Vellis, 2005; Yoon et al., 2009; Tremblay et al.,
50
2011). Although macrophage activation is essential for host defence mechanisms,
aberrant activation of macrophages can lead to disastrous outcomes of inflammatory
diseases such as sepsis, inflammatory bowel and autoimmune diseases (Wyss-Coray
and Mucke, 2002; Block et al., 2007). Normal inflammatory responses are self-
limited by a process involving down-regulation of pro-inflammatory mediators and
increase of anti-inflammatory medators (Kim et al., 2008). However, chronic
inflammations leads to excess release of pro-inflammatory mediators including
COX-2, iNOS and cytokines such as IL-1, IL-6 and TNF-α. During this process, the
activation of immune cells upregulates inflammation (Rietchel et al., 1994; Tao et al.,
2009).
Figure 2.9: Roles of TLRs in inflammation and anti-inflammation. TLRactivation has both negative and positive effects. Over activation of TLRs leads tovarious inflammatory diseases such as sepsis and inflammatory bowel diseases.Positive effects of TLRs are essential in bridging the connection of innate andadaptive immune responses (Krishnan et al., 2012).
Receptors of innate immune system are activated by lipopolysaccharide
(LPS), a microbial component and a key molecule involved in the initiation of sepsis
syndrome (Medzhitov, 2001). However, activation of TLRs at excessive levels can
disrupt immune homeostasis and results in chronic inflammations. Figure 2.9
presents the positive and negative effects of TLR activations and their subsequent
inflammatory cascades. Chronic inflammation is the continued presence of
pro-inflammatory factors at higher levels than baseline and many folds lower than
acute inflammation. Inflamed tissues at chronic levels are characterised by the
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presence of infiltrating macrophages and lymphocytes, fibrosis, abundant blood
vessels and often tissue necrosis (Nathan, 2002; Sarkar and Fisher, 2006). Sepsis is
the leading cause of mortality in critically ill patients suffering from chronic
inflammation (Angus et al., 2001). The sepsis develops as a result of systemic
inflammatory response due to severe bacterial infection. In sepsis condition, immune
responses gets hyper activated and leads to excessive production of proinflammatory
cytokines leading to cellular injury (Pinsky, 2004). Thus, regulation of macrophage
activation may be a valuable therapeutic target for various inflammatory diseases.