Chapter 4

Results and Discussion

4.1 IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES AND THEIR IMPLICATIONS IN THE DISEASED
CONDITION

4.1.1 IDENTIFICATION OF DIFFERENTIAL EXPRESSED GENES FROM TRANSCRIPTOME

The comparison of 13 lesion and non-lesion skin samples of psoriasis patients resulted in identification of 244 up-regulated genes and 183 down-regulated
genes (Table 8).

Table 8 Differential gene expression results of 183 down regulated and 244 up regulated genes
Down regulated Up regulated
Genes LogF Genes LogF Genes LogF Genes LogF Genes LogF Genes LogF Genes LogF
C C C C C C C
IL37 -6.46 GPD1L -2.73 NAP1L3 -2.24 SPRR1A 2.01 LYN 2.25 DICER1 2.65 CHRNA9 3.54
MSMB -6.38 SORBS1 -2.71 NRN1 -2.23 CD24 2.01 PRC1 2.26 KIF20A 2.65 TMPRSS4 3.55
WIF1 -5.67 PLLP -2.68 CILP -2.23 FERMT1 2.01 LAMP1 2.27 IL13RA1 2.66 SPTLC2 3.55
CCL27 -4.85 CYP4B1 -2.68 ARHGEF -2.23 AURKA 2.02 DCTN5 2.28 NETO2 2.68 RAB31 3.56
40
RBP4 -4.73 PRR4 -2.68 UBE2I -2.23 RALBP1 2.02 SHC1 2.29 BIRC5 2.68 UPP1 3.59
FABP7 -4.55 PDLIM3 -2.67 FAM107 -2.22 CCNB2 2.02 IL19 2.30 WWTR1 2.68 CCNB1 3.65
A

36
CLDN8 -4.39 HMGCS2 -2.65 PPARG -2.22 SMC3 2.02 MKI67 2.30 SERPINB 2.69 OAS1 3.73
13
HSD11B -4.25 PIP -2.65 SYNM -2.21 SMOX 2.02 TGM3 2.30 RAB27A 2.69 LTF 3.74
1
RERGL -4.19 SSPN -2.65 ABHD6 -2.21 MAPK14 2.03 CCNA2 2.30 CCL18 2.72 IRF7 3.75
COBL -3.97 BTC -2.63 HSPB7 -2.20 PRRG4 2.03 RAB5A 2.31 OAS3 2.75 AMMECR 3.78
1
MMP28 -3.94 HPGDS -2.62 PTN -2.20 MELK 2.03 KIAA010 2.31 HSPA4 2.76 GALNT6 3.83
1
CMAHP -3.81 GPD1 -2.62 SAP18 -2.20 BCL2A1 2.03 LCK 2.32 ACTR2 2.76 STEAP4 3.84
COCH -3.79 LMOD1 -2.61 ITGA7 -2.19 MFHAS1 2.04 CRABP2 2.32 PTPRC 2.76 S100A9 3.89
MUC7 -3.75 ZSCAN18 -2.58 DBN1 -2.19 CDH1 2.04 LAMP3 2.33 MAPKAP 2.77 TMPRSS1 3.92
K2 1D
UST -3.72 LPL -2.56 ADCY2 -2.19 PRKCI 2.04 GBP1 2.34 ISG15 2.78 MMP1 3.98
GAL -3.70 ITPR2 -2.55 CRIP1 -2.19 BHLHE4 2.05 CFB 2.34 SEC24A 2.78 TYMP 4.05
0
SERHL2 -3.69 CAMK2N -2.52 RTN1 -2.18 MTF1 2.05 OSMR 2.36 GK 2.79 ATP1B1 4.05
1
LGR5 -3.66 PCP4 -2.52 DNAJB2 -2.18 KRT6A 2.05 TMOD3 2.36 LIPG 2.79 IFI44L 4.07
AQP9 -3.63 PARD3 -2.51 TPM2 -2.18 CLCA2 2.06 CLCN3 2.36 KCNJ15 2.83 CXCL2 4.07
FAM189 -3.61 LRRC17 -2.51 LRP4 -2.17 EPHA2 2.07 IFI27 2.36 UGCG 2.85 TTC39A 4.09

37
A2
CHP2 -3.55 AGTR1 -2.50 SEMA3G -2.17 TRIB2 2.07 TLE3 2.37 RGS20 2.85 TREX2 4.15
SCGB2A -3.45 APOC1 -2.49 KRT18 -2.17 ISG20 2.08 HIPK3 2.38 ATP6V1A 2.85 WNT5A 4.17
1
PLN -3.37 PLIN1 -2.48 ZNF107 -2.17 TRIM14 2.09 NEK2 2.40 SLC7A5 2.86 ASPM 4.21
ACSBG1 -3.30 DDAH1 -2.48 ELL3 -2.16 AURKB 2.11 EZR 2.40 RGS1 2.88 MCL1 4.22
LAMB4 -3.26 RNASE4 -2.48 GYG2 -2.13 FOXE1 2.11 DENND1 2.41 PBK 2.89 ARNTL2 4.27
B
CDHR1 -3.23 C1orf115 -2.47 NFATC1 -2.13 SQLE 2.11 FBXW11 2.41 LTB4R 2.89 PKP1 4.29
CHL1 -3.23 ADIPOQ -2.47 POSTN -2.13 GINS2 2.11 PNP 2.41 VNN1 2.89 DSC2 4.31
NR3C2 -3.21 FRZB -2.46 COX7A1 -2.12 GAREM 2.12 PTPN12 2.42 KIF11 2.90 OASL 4.32
FAM149 -3.18 BCL2 -2.46 KANK2 -2.12 LARP4 2.12 KTN1 2.43 CARHSP1 2.90 CDC20 4.34
A
FADS2 -3.16 FMO5 -2.45 SGCE -2.12 PRPF4B 2.12 MICALL1 2.44 TNFRSF2 2.91 GM2A 4.37
1
ATP1A2 -3.15 ATP2A3 -2.43 ASPN -2.11 PPARD 2.13 SAMSN1 2.44 SLC7A11 2.92 SERPINB3 4.50
BHLHE4 -3.14 LEPR -2.41 GIPC2 -2.11 PDZK1IP 2.13 TPBG 2.44 SERPINA 2.95 RSAD2 4.53
1 1 1
BCHE -3.14 ADH1B -2.41 COL21A1 -2.11 DHX9 2.14 IL36RN 2.45 IFI44 2.96 ALDH3B2 4.60
NCALD -3.13 CD34 -2.40 MEOX2 -2.11 MCFD2 2.14 DLGAP5 2.45 TGM1 2.98 CHAC1 4.61
OMD -3.13 EFHD1 -2.39 KIAA146 -2.09 IFIT1 2.14 PTBP1 2.46 DSG3 2.98 ZC3H12A 4.66

38
 7
FXYD6 -3.13 RCAN2 -2.39 ITM2A -2.08 INA 2.14 SLC38A1 2.46 HSD17B1 3.01 CHI3L2 4.73
LEP -3.08 NPTX2 -2.38 RAI14 -2.08 BCL3 2.15 PRSS53 2.46 GK3P 3.02 MMP12 4.81
TSPAN8 -3.08 ARHGAP -2.36 PPP1R3C -2.08 REL 2.16 ARSF 2.47 TWF1 3.02 OAS2 4.94
6
SCGB1D -3.07 ABAT -2.36 DCLK1 -2.08 HMMR 2.16 KIF2C 2.48 ALDH1A3 3.08 KRT16 4.95
2
PLCB4 -3.02 AOC3 -2.35 PAMR1 -2.07 FGFBP1 2.16 VSNL1 2.51 PKP3 3.10 HERC6 5.15
SGCG -3.02 CMA1 -2.35 PPARGC -2.07 PPIF 2.16 XAF1 2.51 IL1RN 3.12 SLC6A14 5.18
1A
GABRP -3.01 FA2H -2.35 DOCK4 -2.07 BUB1B 2.16 PTBP3 2.52 MTMR1 3.13 STAT1 5.29
CFH -3.00 OLFML2 -2.35 MUC1 -2.07 CXCR4 2.16 AGPS 2.52 RAB27B 3.15 CCL20 5.38
A
ID4 -2.99 PRUNE2 -2.35 SAMD4A -2.06 CYP2E1 2.18 CLEC7A 2.52 SAMD9 3.16 ADAMDE 5.45
C1
SERPIN -2.95 CD1A -2.34 NEK9 -2.06 CYP7B1 2.18 STAT3 2.52 ELL2 3.17 KLK13 5.49
A5
C14orf13 -2.94 EMX2 -2.33 PARVA -2.06 LDLR 2.18 APOL1 2.53 CXCL13 3.19 EHF 5.56
2
TPPP3 -2.94 OGN -2.32 DKK2 -2.05 NAMPT 2.18 FOSL1 2.54 SLC5A1 3.19 HPSE 5.67
SDC2 -2.91 ARHGEF -2.32 RAB2A -2.05 GLUL 2.19 SERPINB 2.55 IL1B 3.21 PI3 5.72

39
 10 1
RGS13 -2.91 SLC47A1 -2.30 MYH11 -2.05 KIF5B 2.19 PSENEN 2.55 IFI16 3.32 KYNU 5.74
CD207 -2.87 C1orf68 -2.29 SNX1 -2.05 RIT1 2.19 SP110 2.55 CXCL9 3.34 LCN2 5.98
F3 -2.87 ITGB5 -2.29 LYVE1 -2.05 UBA6 2.20 MPZL2 2.56 CKS2 3.35 KLK6 6.05
FAR2 -2.86 TMEM25 -2.27 KIAA036 -2.04 CDK1 2.20 NDC80 2.57 GDPD3 3.35 IL8 6.11
5A 8
TNMD -2.84 TIMP4 -2.27 DES -2.04 CDKN3 2.20 KLK10 2.58 HYAL4 3.39 CXCL1 6.41
ACACB -2.83 KRT19 -2.26 ADIRF -2.04 SHB 2.21 MPZL1 2.60 MX1 3.40 ATP12A 6.54
NAP1L2 -2.82 ECM2 -2.26 ACTG2 -2.03 TP63 2.21 CDH3 2.61 PLAT 3.43 IL36G 6.74
ADRB2 -2.80 FERMT2 -2.25 EPCAM -2.02 DNASE1 2.22 APOBEC 2.61 TCHH 3.44 SPRR2C 7.10
L3 3A
CIDEC -2.78 GHR -2.25 MAPT -2.02 YOD1 2.22 HMGCS1 2.62 ARG2 3.46 RHCG 8.18
PATZ1 -2.78 PPP1R1A -2.24 TMEM47 -2.01 TM9SF4 2.23 HSPA4L 2.63 DDX58 3.46 AKR1B10 8.22
PDGFD -2.77 ANG -2.24 CDO1 -2.01 PRSS2 2.23 ALOX12 2.63 RRM2 3.49 S100A12 8.85
B
LPHN3 -2.76 CNN1 -2.24 NACC2 -2.01 CXCR2 2.23 SYNCRIP 2.63 PTP4A1 3.52 SERPINB4 10.46
CA6 -2.73 PCOLCE2 -2.24 PDLIM4 -2.00 ADAP2 2.24 NUSAP1 2.64 CDK5R1 3.52 TCN1 11.48

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From these differentially expressed genes, we noticed many genes reported to be associated with
psoriasis. The ontology of the filtered genes were investigated and presented in Figure 5. Genes
up-regulated in psoriatic lesions compared to non-lesional psoriatic skin were enriched in
intracellular signaling, defence response, homeostatic process, response to wounding, regulation
of apoptosis, T cell activation, cell proliferation, protein kinase cascade, cytokine mediated
signalling and leukocyte activation, while genes down-regulated in the lesional skin were
enriched with cell adhesion, response to organic substance, hormone stimulus and endogenous
stimulus, cytoskeleton organization, blood circulation, lipid biosynthetic process and regulation
of growth. However, the top leads for overall gene patterns were involved in processes such as
immune response, intracellular signaling cascade, cell adhesion, cytoskeleton organization,
regulation of cell proliferation and apoptosis.

Figure 5 Gene ontology of differentially expressed genes

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4.1.1.1 INVOLVEMENT OF DIFFERENTIALLY EXPRESSED GENES IN IMMUNE
RESPONSE INTRACELLULAR SIGNALING CASCADE

We noted that majority of the genes in the immune response and intracellular signaling cascade
were up-regulated. There is a significant interest in the role of genes involved in intracellular
signaling cascades of inflammation. Transmigration of circulating leukocytes across the
endothelium is one of the key processes of inflammation. Neutrophils, the active participants of
inflammatory reactions accumulate in the epidermis marking a histological characteristic of
psoriasis. Various mediators implicated in neutrophil chemotaxis are expressed more in the
immune response and signaling cascade category. For instance genes involved in neutrophil
attraction such as CXCR2, CXCR4 and ligands CXCL1 and CXCL2 (Nickoloff et al, 2007).
CXCL8 (IL8) a potent chemoattractant for neutrophil (Duan et al, 2001; Futosi et al, 2013) to the
epidermis and cornified layer, leading to Munro abscesses, a characteristic feature of psoriasis is
found to be upregulated in our analysis. STAT1 (Hald et al, 2013) and STAT3 (Andres et al,
2013) involved in signal transduction mechanisms for many cytokine receptors are also
expressed in higher levels in the skin lesions of psoriasis. STAT3 has been shown to play a role
in psoriasis pathomechanism through IL23 signaling pathway. IL19 (Li et al, 2005), a member of
IL-10 cytokine family is an inducer of STAT3 and signaler for abnormal keratinocyte
differentiation and proliferation was also upregulated in lesions. Immune cascades channelized
through various second messengers, signal transmitters and integrators such as LYN, MAPK14
are expressed in higher amount in lesional psoriatic skin.

4.1.1.2 DIFFERENTIALLY EXPRESSED GENES RELATED TO CYTOSKELETON AND

CELL ADHESION

The end product of epidermal differentiation is the stratum corneum, which is composed of
tightly attached corneocytes but it is dominated by high keratin intermediate filaments. During
the keratinocyte differentiation, distinct morphological changes occur such as cell flattening and
stratification. The rearrangements in the cytoskeleton compartments give rise to these marked
morphological changes. Various genes involved in cytoskeleton formation and cell-adhesion
were differentially regulated in our analysis. Genes involved in epidermal differentiation
participating in cornification process such as small proline rich proteins - SPRR1A and SPRR2C,
Transglutaminase - TGM1 and TGM3 and trichohyalin (TCHH) expressions were up-regulated
in lesional psoriatic skin region compared to non-lesional regions. We also observed elevated
expression of cytoskeletal linker genes such as PKP1 and PKP3. Calcium dependent cell
adhesion molecules such as cadherins - CADH1, CADH3 and CDHR1 (Furukawa et al, 1997),

42
the key controllers of keratinocyte stratification were highly expressed in the lesional region. The
levels of genes concerned with epidermal integrity, involved in the formation of intracellular
adhesion structures such as desmocollins - DSC2 and DSG3, plakophilin - PKP1, PKP3 were
also up-regulated in the lesional patches. The integrins - ITGA7 and ITGB5 showed a markable
decrease correlating with disturbed epithelial cell proliferation and altered adhesive properties in
the lesions. The kallikreins- KLK6, KLK10 and KLK13 (Pampalakis et al, 2007), involved in
skin desquamation and keratinocyte activation showed increased expression levels in lesional
skin areas. These data suggest that an extensive change in cytoskeleton architecture occurs which
are closely related to keratinocyte terminal differentiation.

4.1.1.3 DIFFERENTIALLY EXPRESSED GENES LINKED TO REGULATION OF CELL

PROLIFERATION AND APOPTOSIS

One of the main histological features, the epidermal hyperproliferation mediated by suppression
of apoptosis mechanism is a hallmark of psoriasis. We observed several genes involved in cell
proliferation were differentially upregulated which includes cyclin- A2, B1, and B2 (CCNA2,
CCNB1, CCNB2) and cell-cycle genes CDC20. The nuclear peroxisome proliferator activated
receptors (PPAR) play an essential role in regulating cell differentiation. PPARδ is found to
regulate keratinocyte differentiation, block apoptosis and induce angiogenesis in normal
keratinocytes (Romanowska et al, 2008). The PPARδ’s expression is found to be increased in our
data analysis validating the hallmark of psoriasis. In contrast PPARγ and a transcriptional
cofactor PPARGC1A activated by it are upregulated in our analysis as reported by Sertzning et
al, 2011. Dysfunctional apoptosis has an important role in the pathogenesis of psoriasis. MCL-1
an anti-apoptotic BCL2 family member localized in mitochondria is known to exert its anti-
apoptotic activity by antagonizing BAX and BAK activation (Perciavalle et al, 2012). We noted
that there is a fourfold increase in the expression level of MCL-1 in the lesional patch of psoriatic
skin.

4.1.2 PATHWAYS ASSOCIATED WITH PSORIASIS

To identify the biological pathways of psoriasis, the associated genes were converted to their
corresponding proteins and they were mapped to the pathway data from MSigsDB. Only those
proteins whose pathways can be mapped were considered for further analysis. The pathways
associated with inflammation which is the first immune system response to infection or irritation
appeared in the pathway enrichment analysis as shown in Table 9.

43
 Table 9 Top five pathways enriched in the analysis
Pathways enriched Number of genes in the
enriched pathway
Immune system 44
Cytokine signaling in immune system 22
Cytokine-cytokine receptor interaction 18
Chemokine signaling pathway 17
PPAR signaling pathway 11

The interplay between various mediators and pathways provoke skin inflammation and epidermal
hyperproliferation. For instance NF-κB, MEK1/ERK (Hobbs et al, 2003) and STAT3 signaling
pathways are involved in the regulation of keratinocyte response to stress, infection and injury.
Furthermore, the integrin mediated activation of Erk MAPK not only contributes to perturbed
keratinocyte dysfunction and proliferation but also to inflammation in the skin. Similarly,
continuous activation of NFκB through RelA in keratinocytes is crucial for the initiation of
inflammation in the skin (Rebholz et al, 2007). The constant upregulation of STAT3 in the
psoriatic skin also leads to the development of lesions (Sano et al, 2005).
The cutaneous inflammation provokes epidermal hyperplasia through cytokine mediated signal
transductions. The interaction between various immune cells such as dentritic cells, T-cells and
keratinocytes orchestrated by numerous cytokines result in the development of psoriatic plaques.
Psoriatic kertinocytes report high expression of cytokines such as IL-1α, IL-1β, IL-6, IL-15, IL-
18 and IL-20. These members of cytokine family are implicated in various developmental
alterations and immune disturbances. IL-1 is a pro-inflammatory cytokine involved in the
activation of neutrophils, a key inflammatory infiltrate in psoriatic lesions. IL-15 and IL-18
exerts their activity on human defence system by mediating the inflammation reactions. IL-6 and
IL-20 are involved in the growth and differentiation of dermal and epidermal cells. Thus the
involvement of various cytokines composes the complex and intricate network in psoriasis
pathogenesis (O’Shea and Murray, 2008).
Chemokines are the carriers of different subsets of inflammatory cells resulting in the formation
of a pro-inflammatory circle (Lee et al, 2012). IL-8 (CXCL8) is a chemoattractant of intra-
epidermal neutrophil and plays a major in the development of subcorneal microabscesses, a
characteristic feature of psoriasis (Nickoloff and Turka, 1994). Other chemokines such as MCP-1
(CCL2), IP-10 (CCL10) and other CXC3 ligands act as chemo-attractants for predominantly
monocytes and Th1 cells.

44
Peroxisome proliferator-activated receptor (PPAR) pathway mediated through PPARγ, PPARβ/δ
is involved in the regulation of lipid metabolism and energy homoeostasis. The increased
expression of pro-inflammatory signal inducers in psoriasis leads to over-expression of
PPARβ/δ. The upregulated PPARβ/δ expression in lesional patches is accompanied by high
expression and accumulation of various lipid molecules such as lipoxygenase products, which are
efficient activators of PPARs in keratinocytes. The activation of PPARβ/δ in epidermis is known
to trigger numerous inflammatory cascades and gene dysregulation, characteristic of psoriasis
(Romanowska et al, 2008).

4.1.3 PROTEIN INTERACTION NETWORKS

The protein-protein interaction map formed by 159 proteins whose pathways were well
established produced 2044 connections. The 2044 connections were restricted to 1995
connections since the same interactions were reported by various researchers and through
different in-vitro techniques. Only a single instance of the connections was considered for the
construction of the PPIs. These connections were visualized as protein interaction networks with
1392 nodes and 1995 edges (Figure 6).

Figure 6 Protein-protein interaction network. The proteins - nodes and interactions - edges.
Red nodes -up regulated proteins; cyan nodes – down regulated proteins

45
We identified primary nodal proteins with minimum of 5 interactions in the networks. The
degree of distribution of proteins measures the number of interacting partners. The number of
interacting partners of each node decreases with the power-law proving that the networks have
scale free properties. The proteins in the network have an average of 2.88 interacting partners.
The presence of sparsely connected proteins as neighbours of densely connected proteins was
indicated by low average clustering coefficient value. Topological coefficient measures the
extent to which each protein shares its neighbour with others. The modular framework
organization of the network was confirmed by the decreased nature of the topological coefficient,
which proves that the neighbours of sparsely linked nodes were more connected than primary
nodal proteins. The shortest path length indicates that the proteins were closely inter-linked.
Figure 7 and Table 10 summarize the distribution of topological metrics.

Figure 7 Topological properties of central network (a) Average clustering coefficient (b)
Degree distribution of proteins (c) Shortest path length distribution (d) Topological
coefficients

46
 Table 10 Topological parameters of the network

Topological parameters Values

Number of nodes 1392
Number of edges 1995
Clustering coefficient 0.057
Characteristic path length 4.357
Network diameter 12
Network density 0.002
Average number of neighbours 2.88

4.1.4 HUB PROTEINS AND SUB-NETWORKS

A total of 15 hub proteins - SHC1, MAPK14, LYN, KRT18, STAT3, STAT1, PPARGC1A,
LCK, PTPRC, PRKCI, PPARG, CDH1, BCL2, EZR and MAPT were identified based on
different algorithms with high centrality statistics like MCC, closeness, degree and betweenness.
The top 20 proteins were identified based on these metrics and are listed in Table 11. These
interconnected hub proteins appeared almost in three categories proving their significant function
in maintaining the global network structure and communication. Most of the hub proteins play an
active role in enzyme binding, transcription activation, protein kinase activity, lipid binding and
signaling cascades.

Table 11 Top 20 hub proteins identified by different algorithms by Cytoscape hubba

MCC Closeness Degree Betweenness
MAPK14 SHC1 CDK1 SHC1
LYN LCK SHC1 MAPK14
KRT18 CDH1 LCK LYN
STAT3 STAT1 UBE2I KRT18
GHR MAPK14 LYN STAT3
PPARGC1A STAT3 STAT3 GHR
LCK LYN MAPK14 PPARGC1A
ADRB2 KRT18 STAT1 LCK
PTPRC UBE2I BCL2 ADRB2
PRKCI BCL2 EZR PTPRC
HSPA4 EGFR MAPT PRKCI

47
 PPARG EZR KRT18 HSPA4
CDH1 PRKCD PTPRC PPARG
SDC2 MAPT CDH1 CDH1
BCL2 PPARGC1A PPARG SDC2
UBE2I PTPRC PRKCI BCL2
MAPT PRKCI DHX9 UBE2I
CCNA2 PTK2 PARD3 EZR
STAT1 FYN CXCR4 CCNA2
SHC1 JAK2 PPARGC1A STAT1
*The highlighted genes appear in more than two columns

The three highly interconnected subnetworks (Figure 8) were raised from the hub proteins which
were considered as roots for clustering.

We also observed the presence of one hub-hub connected cluster. STAT1 formed the central hub
of the first cluster with a score of 1.067 containing 89 nodes and 97 edges. LYN forms the
central hub of second cluster with a score of 1.011 with 92 nodes and 95 edges and the last
cluster had two connected hub proteins PTPRC and MAPT with a cluster score of 0.981 with 53
nodes and 55 edges. The third cluster is connected through a sole node FYN.

48
Figure 8 Top three sub-networks. Proteins involved in immune mediated cascades are highlighted. (a) Cluster 1 (b) Cluster 2 (c) Cluster 3

49
4.1.5 SIGNIFICANT RESULTS OF SUBNETWORKS

The global view of the modules and their function are obtained by compiling the protein
ontologies and pathways enriched in the top three modules. The biological process (Figure 9)
and pathway annotation of the three modules are given in Table 12 and 13.

Figure 9 Biological process of the three clusters is shown as venn diagram. (a) Cluster 1 (b)
Cluster 2 (c) Cluster 3. Total number of genes in the module is enclosed within the parenthesis
following the GO ID. (GO:0007243: Protein kinase cascade, GO:0007166: Cell surface receptor
linked signal transduction, GO:0007242: Intracellular signaling cascade, GO:0016310:
Phosphorylation, GO:0007165: Signal transduction process, GO:0007242: Intracellular signaling
cascade and GO:0042981: Regulation of apoptosis)

50
 Table 12 The biological process annotation of the three modules

Biological Process
Module 1 Module 2 Module 3
4
GO:00072 GO:00071 GO:00072 GO:0007166 GO:00072425 GO:0016310 6
GO:00071 GO:00072 GO:00429
431 662 423 657 428 819
GNA1 MAPKAP STAT5
GNA13 GNA13 GNA13 GNA13 GRB2 3 K2 A PTPRC PTPRC ZFP36 CASP6
STAT5
NMI FGFR4 NMI IL27RA CCR1 A KIT SNCA FLT1 PHKG1 PTPRC PTPRC
STAT5 STAT5
FGFR3 FGFR3 FGFR3 A NEDD9 B SRC BTK BMX PKN1 PKN1 CASP3
PRKC MAPKAP MAPKAP
STAT5A IL27RA GNAI1 BCAR1 KIT SH VDR AKT1 STAT1 K2 K2 YWHAZ
STAT5 CSNK2 PTPN1
STAT5B GNAI1 STAT5A B IL7R BTK RAC1 A2 1 MARK4 PRKCSH TUBB
CXCL1
KIT STAT5A CCR1 2 STUB1 AKT1 SH2B2 HSF1 KDR MARK1 MARK1 APOE
CSNK2 RPS6K
SRC CCR1 STAT5B A2 SRC APOE INPP5D PAK2 A5 CSNK2A2 RPS6KA3 LGALS1
CXCR RPS6K
AKT1 STAT5B KIT AKT1 PTK2 4 TEC CXCR4 A3 RPS6KA3 MAPK12 PPP2CB
TNFRSF ANP32 MAPK1
CXCR4 KIT SRC FOS 1B A PTPRC INSR 2 MAPK12 FYN HSPB1
SYK CXCL12 AKT1 PRMT1 FCER1G INSR FLT1 SYK GSK3A FYN APOE AATF
EGFR SRC VDR GP6 SH2B2 SYK HCLS1 GHR FYN GSK3A TSC2 SEMA4D
FLT1 AKT1 CXCR4 APOE CD4 GHR SPHK1 MATK JAK1 PPP2CB ANP32A
SOCS3 FOS RAC1 CXCR4 CD24 ZFP36 BMX EGFR JAK2 INSR INSR
TNFRSF1
SOCS1 B SYK TRPV4 CSF1R EGFR STAT1 PIK3CG PPP5C
STAT1 PTK2 EGFR MS4A2 PTPN6 PIK3C VAV1 LYN KIT

51
 G
PTPN11 PRMT1 FLT1 INSR PTPRC LYN CISH PHKG1 AKT1
STAT2 CXCR4 SOCS3 PILRB FLT1 SOCS2 BRCA1 PKN1 FOS
TYK2 CD4 SOCS1 SYK SPHK2 SOCS3 STAT2 PRKCD PRMT1
RPS6KA5 SYK BMX GHR PTPN2 SOCS1 PTPN11 MARK4 SYK
IFNAR2 EGFR STAT1 EGFR SPHK1 PKN1 RPS6KA5 CDK2 FLT1
FYN PTPN6 VAV1 PIK3CG EVL IRS1 RPS6KA3 MARK1
PRKC
ADRA1B FLT1 PRKCD LYN GRIA3 D MAPK12 TYK2
MARK
JAK1 PTPN2 BRCA1 SOCS2 STAT1 1 FYN PRKCQ
JAK2 MYH9 PTPN11 MYH9 VAV1 TYK2 NPHS1 MAPK3
PRKC
PIAS1 STAT1 STAT2 MARK4 CISH Q TSC2 PDGFRA
IFNAR
VAV1 RPS6KA5 IRS1 KDR 2 ADRA1B PDGFRB
PTPN11 TYK2 IFNAR2 PTPN11 DOK2 JAK1 EIF2AK2
RPS6KA
KDR IFNAR2 DOK2 5 CRKL EPOR FGFR4
RPS6KA5 FYN GRB10 IRF9 CD36 JAK2 FGFR3
IRF9 ADRA1B CCR5 CBLC DOK3 CAMK2G
MAPK
IFNAR2 JAK1 ARRB2 CD19 3 ADRBK2
NCOA
CCR5 JAK2 PRAM1 FYN 6 PRKDC
ARRB2 PIAS1 JUN GSK3A PIAS1 MAP4K1
PDGFR
FYN A TSC2 FGFR3 MAPKAPK2
PDGFR ADRA1
JUN B B NMI KIT

52
 PDGFRA ITGAL JAK1 GNAI1 SRC
ADRA1B FGFR4 CD79B GRB2 PTK2
JAK1 FGFR3 JAK2 CCR1 CAMK2D
PDGFRB USP9Y CD79A MAP4K1 CSF1R
JAK2 GNAI1 TEC
1
Protein kinase cascade, Cell surface receptor linked signal transduction, Intracellular signaling cascade, 4Cell surface receptor linked signal
2 3

transduction, 5Intracellular signaling cascade, 6Phosphorylation, 7Signal transduction process, 8Intracellular signaling cascade, 9Regulation of
apoptosis

53
 Table 13 The biological pathway annotation of the three modules
Biological Pathways
Module 1 Module 2 Module 3
Cytokine signalling JAK STAT Interferon Cytokine signaling in JAK Kit Immune p38 MK2 Signaling
in immune system signaling pathway signaling immune system STAT pathway system pathway by
signaling NGF
pathway
STAT1 PTPN2 STAT1 SOCS3 STAT1 CISH SOCS1 CISH GRB2 MAPKAPK2 MAPKAPK2 MAPKAPK2
JAK2 CAMK2D JAK2 TYK2 JAK2 CRKL SOCS2 CSF2RA JAK2 TSC2 TSC2 TSC2
PTPN6 SOCS3 PTPN6 IFNAR2 PTPN6 CSF2RA SOCS3 GH2 PTPN6 YWHAZ YWHAZ RPS6KA3
STAT2 TYK2 STAT2 IL2RG JAK1 GH2 JAK2 GHR SOCS1 RPS6KA3 SRF MAPK12
STAT5B IFNAR2 STAT5B IL2RB PIAS1 GHR LYN GRB2 EPOR MAPK12 HSPB1 PPP2CB
STAT5A IL2RG STAT5A CSF2RB SOCS1 GRB2 MAPK3 IL7R CRKL PPP2CB TH GSK3A
VAV1 IL2RB JAK1 AKT1 IFNGR1 IL7R PTPN6 JAK2 LYN GSK3A ETV1 CASP3
FYN CSF2RB PIAS1 CREBBP IRF9 IRS1 TYK2 PTPN6 MAPK3 FYN LSP1 AATF
JAK1 IRF2 SOCS1 EP300 SOCS3 TYK2 GRB10 S100B
PIAS1 EIF2AK2 IFNGR1 PTPN11 PRKCD SOCS1 KIT PRKCSH
SOCS1 KPNA1 IRF9 IRF1 SOCS2 MATK MBL2
IFNGR1 SYK PTPN2 SOCS3 SH2B2 PTPRC

IRF9 RELA CAMK2D EPOR MAP4K1 STUB1

PRKCD IRF1 IRF2 PIK3CG ITGAL

CBLC

54
4.1.5.1 ENRICHMENT ANALYSIS OF MODULE1

Module 1 consisted of 89 members, 40 (44.94%) genes were annotated to the cell surface
receptor linked signal transduction. 33 (37.07%) genes were involved in intracellular signaling
and 25 (28.08%) members are implicated in protein kinase cascade. Our analysis uncovered a
coordinated functional pattern by the module1 members involved in cell surface receptor linked
signal transduction, intracellular signaling and protein cascade biological processes. The
synchronized activity of the members involved in the biological functions (BP) inferred above is
validated by the pathway annotation.
The type-1 (IL2, IL6 and GCSF) and type-2 cytokine mediated (IFNG) intracellular signaling
pathways (Gadina et al, 2001) and Jak-STAT signal transduction pathway (Yamaoka et al, 2004)
are engaged in the neutrophil recruitment and accumulation during injury and inflammation
acting via neutrophil cell surface receptors (Yamaoka et al, 2004) (Figure 10).

Figure 10 Signal transduction by cytokine receptors. JAK-STAT pathway is activated by type

I and II cytokine receptors while IRAK family members activate IL1 and IL18 receptors via
MyD88

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GCSF (Granulocyte-colony stimulating factor) are involved in neutrophil differentiation and
survival while IL-6 pathway is enriched with genes involved in neutrophil activation and
harmonization of various inflammatory responses. Members of IFNG (type-2 interferon)
pathway act by delaying the apoptosis of neutrophils a key attribute of psoriasis. The
synchronized function of all these biological processes and pathways in neutrophil trafficking has
a major implication in psoriasis.

4.1.5.2 ENRICHMENT ANALYSIS OF MODULE 2

Module 2 was enriched with the total of 92 members. The biological process ontology analysis of
module 2 members are also enriched with 79 (85.56%) genes in cell surface receptor linked
signal transduction, 68 (73.91%) in intracellular signaling cascade, but the third highest hit
corresponded to 53 genes involved in phosphorylation process (54.34%). Majority of the
members of top two hits of biological process were overlapping with the members of module1.
We investigated the role of phosphorylation process in the disease by the pathway annotation.
Apart from immune mediated pathways KEGG’s JAK-STAT signaling pathway, KEGG’s
insulin pathway and PID’s signaling events mediated by stem cell factor receptor (c-kit) scored
top in the list. Janus kinase (Jak) family is one of the ten families of non-receptor tyrosine kinase
recognized to have an important role in immune-inflammatory processes. Receptor attached Jaks
are activated by ligand attachment which in-turn switch on the STATs via phosphorylation of its
SH2 domain. The translocation of STAT’s mediated by its phosphorylation process and it is
crucial for the recruitment of inflammatory mediators such as interleukins IL6 and IL10 families
(Schindler et al, 2007). The Kit ligand Stem cell factor (SCF), is the ligand of the c-kit proto-
oncogene product and in addition they have an implication in the inflammatory process. Binding
of SCF homodimers to c-kit induces homodimerization and intermolecular tyrosine
phosphorylation of the SCF providing docking sites for signal transduction molecules (Reber et
al, 2006). c-Kit signal transduction covers many interconnected pathways which have major roles
in inflammatory mediator recruitment and activation of MAP kinase, PI-3 kinase, JAK/STAT,
Src and PLC-γ pathways (Figure 11a). The occurrence of insulin pathway in our annotation was
serendipity and its functional importance was investigated to understand its implication in the
disease pathogenesis. The proinflammatory cytokine IL-1β is concerned in the development of
insulin-resistance in keratinocytes, leading to the alterations in keratinocyte proliferation and
differentiation. The chronic dermal inflammation reports the presence of high IL-1β levels
thereby activating p38MAPK. This activation in turn induces insulin resistance via
phosphorylation of serine residues of IRS (Insulin Receptor Substrate) resulting in blockade of

56
differentiation but simultaneously IL-1β induced activation of p38MAPK and JNK leads to the
proliferation of keratinocytes, a trademark of psoriasis via PI3-K pathway (Buerger et al, 2012)
as observed in Figure 11b. Module2 emphasise the importance of phosphorylation and kinases
in the development of inflammation and keratinocyte proliferation.

Figure 11 Signaling cascades of module 2 members. (a) Signal transduction process of c-Kit;
(b) IL1β blocks insulin-mediated differentiation but induces proliferation of keratinocytes

4.1.5.3 ENRICHMENT ANALYSIS OF MODULE 3

Module 3 is enriched with 53 members with biological process clustered 20 (37.73 %) genes
under signal transduction process, 13 (24.52 %) genes in intracellular signaling cascade and 11
(20.75%) genes in the regulation of apoptosis. The biological process clustering identified
significant overlap between the members of signal transduction process and apoptosis process.
The pathway annotation of the clusters from module 3 produced similar results with that of our
previous modules 1 and 2. The Reactomes immune system was reported as the top hit in the
pathway annotation followed by PID’s p38 mediated signaling by MAPKAP kinases and
Reactome’s signaling by NGF (Nerve Growth Factor). Mitogen-activated protein kinase-
activated protein kinases (MAPKAPK or MKs) play an essential role in the inflammatory
response by cytokine biosynthesis (Gaestel, 2006). One of the unique features of psoriasis is the
presence of marked proliferation of terminal cutaneous nerves in the psoriatic plaques. NGF
influence many pathological events in psoriasis by being mitogenic to keratinocytes, promoting
angiogenesis and activates T cells (Raychaudhuri et al, 2008). NGF and Tyrosine Kinase

57
Receptors (TRKs) (Figure 12) play crucial role in the prevention of apoptosis and promoting
keratinocyte cell proliferation in psoriasis.

Figure 12 Signal transduction by NGF

On careful observation of the subnetworks, we found that the top clusters with high enrichment
scores was populated by the members of non-receptor tyrosine kinases (nRTKs) (Table 14).

Table 14 Gene functional clustering of sub-networks along with their enrichment scores
Clusters Genes Gene
Enrichment
score
1 ADRBK2, PDGFRA, FYN, FLT1, FGFR3, RPS6KA5, PRKCD, 10.45
EIF2AK2, JAK1, BMX, PDGFRB, EGFR, CAMK2D,
CAMK2G, KIT, SRC, TYK2, FGFR4, KDR, LCK, LYN
2 CSF1R, MATK, TYK2, MAP4K1, BTK, TEC,SYK 6.14
3 NPHS1, FCGR2B, CD19, MS4A1, FCAR, CD79B, PECAM1, 4.87
MME, CSF2RA, LIME1, FCGR2A, CTLA4, GP6,
TRAT1,CSF1R, PILRB, CD79A, CD22
4 CISH, SOCS2, SOCS3, SOCS1 4.29
5 IFNGR1,IFNAR2, IL2RG, IL27RA, CSF2RB, IL2RB 3.81
6 PKN1, CSNK2A2, MARK4, MARK1, MAPK12, GSK3A, 3.84
RPS6KA3, PHKG1, MAPKAPK2
7 KPNA1, CSE1L, XPO1, KPNA6 2.12
*Non-receptor kinases are highlighted

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The three prime module functions are coordinated by various nRTK families such as JAK, SRC,
TEC and SYK. Src-family tyrosine kinases (SFKs) are activated by the stimulation of growth-
factor receptors and chemokine receptors. The activation of SFK members regulates cell growth
and differentiation, in particular kertinocytes. The SFKs are implicated in various activities of
keratinocytes and cytokine production (Ayli et al, 2008). Similarly the JAKs, TECs and SYKs
are also involved in various signal transduction pathways of the inflammatory mechanisms
(Gaestel et al, 2009) highlighted in our module enrichment analysis. Our study supports the
hypothesis that these nRTKs can be key orchestrators of inflammation and epidermal
hyperproliferation evidenced by their high expression levels in psoriatic plaques.

4.2 INVESTIGATION OF THE CROSS TALKS BETWEEN PSORIASIS

VULGARIS AND THEIR ASSOCIATED COMORBIDITIES

4.2.1 LINKING PSORIASIS WITH ITS COMORBIDITIES

The differential expression analysis of psoriasis microarray data resulted in 507 differentially
expressed genes (DEG) (113-upregulated; 394-downregulated), on submitting the differentially
expressed genes of psoriasis transcriptome to DAVID server, around fourteen major disease
categories were enriched (Table 15).

Table 15 Disease categories associated with psoriasis based on the genes involved

Sl.No Disease class Disease Number Genes involved

of genes
1. Musculoskeletal Rheumatoid 16 PLAT, MMP9, TLR2,
Diseases arthritis GGH, PTPN22, CXCR2,
MMP1, MMP12, TYMS,
IL12RB1, CCR5, CD274,
IL1B, SERPINA1,
FCGR3B, SELE
2. Neoplasms Esophageal 4 TYMS, IL8, CXCR2,
cancer MMP1
Stomach 5 TYMS, IL8, CXCR2,
Neoplasms MMP1
Leiomyoma 3 IL12RB1, IL8, IL1B
Oral cancer 5 IL8, MMP9, TGFA,
CYP2E1, MMP1
3. Virus Diseases Hepatitis C 10 IFI27, IL12RB1, CCR5,
LDLR, IL19, IL1B, OAS1,
CXCR2, MX1, IL20
Hepatitis B 6 CCR5, OAS3, OAS1,
OAS2, MX1, STAT1
4. Otorhinolaryngologic hearing 5 PLAT, SLC26A4, GJB6,

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 Diseases loss/deafness ESPN, GJB2
5. Bacterial Infections Tuberculosis 9 IL12RB1, IL8, TLR2,
and Mycoses PTPN22, IL1B, CXCR2,
SERPINA1, CYP2E1,
STAT1
6. Nervous System Subarachnoid 5 MMP9, SERPINA3, IL1B,
Diseases hemorrhage MMP12, MMP1
Multiple sclerosis 15 IL8, MMP9, CCR1,
APOC1, PTPN22, CXCR2,
OAS1, IL7R, CXCL10,
CCR5, IL1B, CD24, MX1,
FCGR3B, SELE
Alzheimer 3 C3,CD14, DNM3
Disease
7. Digestive System Pancreatitis, 5 IL8, PRSS2, PRSS3,
Diseases chronic SERPINA3, IL1B
8. Stomatognathic Periodontitis 9 PLAT, CCR5, S100A8,
Diseases MMP9, TLR2, IL1B,
SERPINA1, FCGR3B,
MMP1
9. Respiratory Tract COPD 7 IL8, MMP9, SERPINA3,
Diseases IL1B, SERPINA1, MMP12,
MMP1

Asthma 15 CYP2J2, MMP9, TLR2,

CXCR2, EHF, IL12RB2,
CCR5, CXCR4, FCGR1B,
SERPINA3, FUT3, IL1B,
SERPINA1, FUT2, SELE
10. Cardiovascular Atherosclerosis, 13 PLAT, F12, CYP2J2,
Diseases coronary LDLR, CCR5, SELL,
MMP9, APOC1, TLR2,
IL1B, FCGR3B, SELE,
MMP1
Myocardial 3 JAK2, CD14, CCL1
Infarction
Coronary artery 7 PLAT, CCR5, LDLR,
disease MMP9, IL1B, SELE,
MMP12

11. Digestive System Cirrhosis; 3 IL8, IL1B, CYP2E1

Diseases pancreatitis
12. Skin and Connective systemic lupus 9 CCR5, IL8, CFB, TLR2,
Tissue Diseases erythematosus PTPN22, IL1B, CXCR2,
FCGR3B, SELE
13. Hemic and Lymphatic Sarcoidosis; 3 IL12RB2, IL12RB1, MMP1
Diseases tuberculosis
14. Nutritional and Diabetes, type 2 2 IL12RB2, IL12RB1
Metabolic Diseases
Obesity 5 CXCL1, DPN, NAIP,
PAPPA, IL24

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Each disease category had a minimum of one disease and a maximum of four disease classes.
Five diseases based on their common co-occurrence with psoriasis were selected to study their
relationship with psoriasis. The comorbidities associated with psoriasis chosen for the study were
T2DM, obesity, MI, AD and RA. The association between psoriasis and the five major
comorbidities were verified against the published literatures (Wolf et al, 2008; McGowan et al,
2005; Gelfand et al, 2006; Bechtel et al, 2009; Gribble, 1955)

Independent meta-analysis was carried out for psoriasis and its co-morbidity using their
respective microarray expression data. The aim of the meta-analysis was to identify the genes
which were significantly upregulated and down regulated in diseased condition when compared
with the normal samples (Sitras et al, 2015; Nazir et al, 2014). The number of genes differentially
expressed and associated with five psoriasis comorbidities ranged from 36 (MI) to 2222 (RA). A
psoriasis specific interactome was constructed and utilized the knowledge obtained from the
interactome to mine out the commonality between psoriasis and its comorbidities (Figure 13).
The psoriasis diseasome constructed using the knowledge obtained above revealed that the
comorbidities were connected with psoriasis through shared genes or proteins which interact to
form the cellular interactome.

Figure 13 Venn diagram showing the number of overlapping genes between psoriasis and its
comorbidities

The commonalities between the diseases were assessed based on their interaction patterns. The
number of shared genes or proteins in the psoriasis diseasome ranged from 31 (MI) to 312
(T2DM) (Figure 14-16).

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Figure 14 Psoriasis diseaseome with shared proteins of AD. The protein-protein interaction
network of psoriasis. The yellow nodes denote proteins involved in psoriasis. Red nodes
indicated common proteins shared by Alzhimer’s disease and psoriasis. Blue nodes indicate
indirect interactions

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Figure 15 Psoriasis diseaseome with shared proteins of MI. The protein-protein interaction
network of psoriasis. The violet nodes denote proteins involved in psoriasis. Red nodes indicated
common proteins shared by myocardial infarction disease and psoriasis. Blue nodes indicate
indirect interactions

63
Figure 16 Psoriasis diseaseome with shared proteins of T2DM. The protein-protein
interaction network of psoriasis. The green nodes denote proteins involvement in psoriasis. Red
nodes indicated common proteins shared by type II diabetes and psoriasis. Blue nodes indicate
indirect interactions

64
Figure 17 Psoriasis diseaseome with shared proteins of obesity. The protein-protein
interaction network of psoriasis. The pink nodes denote proteins involvement in psoriasis. Red
nodes indicated common proteins shared by obesity and psoriasis. Blue nodes indicate indirect
interactions

65
AD and psoriasis were directly linked through 13 proteins, while the association also occur
indirectly through the interaction of 84 proteins. In case of T2DM 26 proteins directly connected
both the diseases and 210 proteins contribute to their interaction via partnering with other
proteins. MI and psoriasis were connected directly through 7 proteins and indirectly through 49
proteins. Similarly obesity and psoriasis were connected directly through 24 proteins and
indirectly through 11 proteins. RA showed highest percentage of connections. They were
connected directly through 201 proteins and indirectly through 386 proteins (Figure 18).

Figure 18 Psoriasis diseaseome with shared proteins of RA. The protein-protein interaction
network of psoriasis. The brown nodes denote proteins involvement in psoriasis. Red nodes
indicated common proteins shared by rheumatoid arthritis and psoriasis. Blue nodes indicate
indirect interactions

66
The Molecular Comorbidity Index (MCI) which shows the strength of association between
psoriasis and its comorbidities was also studied for the better understanding. T2DM leaded the
MCI list followed by RA, AD, MI and obesity (Figure 19).

Figure 19 Molecular comorbidity index. Number of proteins shared between psoriasis and its
comorbidities

The proposed network protocol not only provides a global analysis of the proteins but also
presents a detailed view on specific proteins and their association with the comorbidity under the
study. We noted the appearance of apolipoprotein E (ApoE) in psoriasis and Alzheimer’s. Lipid
metabolism is the common term connecting both the disease. ApoE polymorphisms are shown to
be associated with both the diseases disrupting the lipid metabolism. When the direction of
regulation was considered the gene was downregulated in both the cases as established by the
previous reports (Kim et al, 2009; Al harthi et al, 2014). An elevated level of MCP-1 (CCL2) is
observed between psoriasis and myocardial infarction. The recruitment of inflammatory
mediators by MCP-1 forms a common link connecting both the diseases (Kölliker Frers et al,
2015). Leptin an important regulator of mass of adipose tissue is found to be downregulated in
obesity and psoriasis. Many polymorphisms are reported in leptin in both the disease. The gene
alters the endothelial cell morphology and causes epithelial hyperplasia in psoriatic patients
(Karpouzis et al, 2014) while in obesity the alteration in the gene lacks the control of metabolic
regulation of adepocytes (Paracchini et al, 2005). Early growth response-1 (Egr-1) is upregulated
in type II diabetes and psoriasis. Egr-1 mediated Egr-1/GGPPS/Erk1/2 pathway is one of the
pathways involved in insulin resistance in hyperinsulinism condition observed in type II diabetes
(Shen et al, 2011). In psoriasis the protein mediates the expression of psoriasin induced by IL-
17A via ERK-Egr-1 pathway. Egr-1 is a crucial modulator in Th17-mediated immune response
in epidermal keratinocytes (Jeong et al, 2014). Upregulation and downregulation of various
chemokines (CCL4, CCL18, CCL27, CCR1, CCR5, CCR7, CXCL1, CXCL9, CXCL10,

67
CXCL13 and CXCR4) involved in the recruitment of leukocytes and angiogenesis were observed
to occur in both psoriasis and RA which links immune mediated diseases (McInnes and Schett,
2007; Firestein, 2003). To assess the role of direct and indirect contributors to the diseases we
further studied the differential expression patterns of the linker proteins.

4.2.2 DIRECTION OF EXPRESSION OF THE COMMON GENES/PROTEINS AND LINKER

GENES/PROTEINS

The deregulation of common set of genes either in similar or opposite direction can be an
underlying cause for the comorbidities. To generate the molecular interpretation of the
comorbidities, we compared the direction of dysregulation of the genes shared by psoriasis and
its comorbidities with the help of meta-analysis (Figure 20).

Figure 20: Number of protein regulated in similar and opposite direction. (a) Proteins
upregulated (b) Proteins downregulated

The DEGs of the comorbidities were compared with DEGs of psoriasis individually. We noted
significant overlaps between the DEGs upregulated in psoriasis and those downregulated in its
comorbidities. Similarly DEGs downregulated in psoriasis were observed to be overlapping with
the DEGs upregulated with psoriasis associated comorbidities. Considerable overlap was also
observed between DEGs deregulated in same direction between psoriasis and its comorbidities.
The DEGs deregulated in the same direction can be treated as putative signatures of the
comorbidities while the DEGs deregulated in the opposite direction can contribute to inverse
comorbidities. We noted a maximal hit of DEGs deregulated in both the direction was from
T2DM followed by RA, obesity, AD and MI. Highest number of genes upregulated in the similar
direction was reported in T2DM followed by RA, AD, obesity and MI. Similarly the highest
number of down-regulated genes in the same direction was seen in RA followed by obesity, AD,
T2DM and MI. The highest number of DEG upregulated in opposite direction followed the same

68
pattern of DEGs upregulated in similar direction while the highest number of DEGs down-
regulated in opposite direction was observed in AD followed by obesity, T2DM, MI and RA.
When the overall DEG count was compared the genes expressed in similar directions were
highly contributing to the positive comorbidity.

4.2.3 EXPLORING THE BIOLOGICAL PROCESSES SHARED BY THE PROTEINS IN

PSORIASIS DISEASOME

To identify the biological functions shared by the psoriasis and its comorbidities, a functional
enrichment analysis was performed based on the BP. RA shared the highest number of
overlapping BP with psoriasis. The list followed the order of T2DM, obesity, AD and MI starting
from highest to lowest overlapping processes. Defence response, immune response and
inflammatory response were enriched in all the comorbidities. Response to wounding showed a
similar pattern of expression in almost all the comorbidities except T2DM. Proteins contributing
to cell-cell signaling were upregulated in AD, T2DM and RA. Around five biological processes
were shared by obesity and RA with connection to psoriasis. To investigate the degree of
similarity between psoriasis and its comorbidities, Jaccard coefficient was computed for all the
comorbidities (Figure 21). The highest JC value attributed to obesity followed by T2DM, RA,
MI and AD. These observations highlights the critically impaired BP between psoriasis and its
comorbidities were from immune mediated processes.

Figure 21 Similarity between psoriasis and its comorbidities. Each cell is colored according
to the Jaccard correlation coefficient which represents the similarity between the comorbidities
considering, (a) biological process and (b) biological pathways.

69
4.2.3.1 OVERLAP OF IDENTIFIED BIOLOGICAL PATHWAYS IN THE PSORIASIS
DISEASOME

We further investigated the pathways that were common between the psoriasis and its associated
comorbidities. Highest number of pathway overlaps was observed with RA followed by T2DM,
obesity, AD and MI. Similar to the BP, the degree of similarity in the pathways between
psoriasis and its comorbidities was also assessed using Jaccard coefficient (Figure 21b).
Psoriasis shared the highest number of pathways with obesity followed by RA, MI, T2DM and
AD. We noted eight pathways were overlapping with all the comorbidities. Angiogenesis, CCKR
signaling map, Gonadotrophin-realising hormone receptor pathway, heterotrimeric G-protein
signaling pathway-Gi alpha and Gs alpha mediated pathway, heterotrimeric G-protein signaling
pathway-Gq alpha and Go alpha mediated pathway, inflammation mediated by chemokines and
cytokine signaling pathway, integrin signaling pathway and Wnt signaling pathway. Toll like
receptor signaling pathway was shared by AD, T2DM, MI and RA. Seven pathways involved in
blood coagulation, Cadherin signaling, FGF signaling, Ionotropic glutamate receptor pathway,
Metabotropic glutamate receptor group III pathway, Muscarinic acetylcholine receptor 1 and 3
signaling pathway, and Nicotinic acetylcholine receptor signaling pathway were enriched in
AD,T2DM, obesity and RA. PDGF signaling pathway was found to be overlapping with T2DM,
MI, obesity and RA. It was observed that the biological pathways involved in the comorbid
diseases were not only shared by psoriasis but also by the other diseases which are almost
unrelated to each other except few. These observations shed lights over the existing associations
between the psoriasis with its multi-morbidities at the pathway level.

4.2.4 BIOLOGICAL PROCESS AND PATHWAYS LINKING PSORIASIS WITH ITS

COMORBIDITIES

The findings of our work reveal that the psoriasis comorbidities are related at the molecular level
that may contribute to their co-occurrences. In this context, the biological process annotation
identified several process of inflammation such as defence response and immune response are
enriched in all the comorbidities. The pathway annotation also revealed the presence of
inflammatory pathways which includes inflammation mediated by chemokines and cytokine
signaling pathway, integrin signaling pathway and Wnt signaling pathway. The search for
inflammatory links was carried out in for all the diseases against published literatures.

The production of amyloid-β peptides can activate the innate immune response and evoke
Alzheimer’s disease. Pattern recognition receptor (PRR) such as C1q is involved in complement
cascade activation in the brain. The PPRs are also involved in the induction of proinflammatory

70
signaling pathways in Alzheimer’s disease (Salminen et al, 2009). Various immune related
proteins are produced by adipocytes. The peroxisome proliferator activated receptor (PPARγ) is
highly expressed in adipose tissue and they are found to be involved in macrophage function
(Marti et al, 2001). Furthermore an adipocyte protein leptin upregulated in obesity and type II
diabetes can stimulate the activity of macrophage and neutrophil colony-forming cells. The leptin
deficiency also imparts impaired lymphoid tissue development. PRRs play major role in
inflammation in obesity and type II diabetes (Pickup, 2004). The ischemic myocardial infarctions
activate pleiotropic inflammatory mediators. IL-8 and C5s are released during the injury playing
a major role in neutrophil recruitment. Neutrophil infiltrations, activation of complement and
cytokine cascades were reported by many researchers as inflammatory reactions in myocardial
infarctions (Kölliker Frers, 2015). Local production of cytokines accounts for systemic clinical
manifestation of rheumatoid arthritis. The cytokines are the products of IL-1, IL-6, colony
stimulating factor (CSF-1), GM-CSF and MCP-1. MCP-1 is chemotactic and plays a role in the
recruitment of inflammatory leukocytes into the inflamed joints (McInnes and Schett, 2007;
Koch et al, 1994). The occurrence of biological processes and pathways related to inflammation
might form a basic molecular link connecting psoriasis and its comorbidities.

4.3 IDENTIFICATION OF BIOMARKERS CHARACTERISTIC TO

PSORIASIS VULGARIS

4.3.1 WEIGHTED CORRELATION NETWORK ANALYSIS ORGANIZES EXPRESSION

TO MODULES

Weighted gene coexpression network analysis builds gene networks utilizing the coexpression
nature of the genes, hence facilitating the identification of genes which are tightly correlated
across different datasets. WGCNA provides a global view of microarray data analysis specific to
diseases, tissues and species. The modulePreservation function evaluates the module preservation
by implementing various network based statistics. Z-summary is one such statistic measure
summarizing the composite preservation. For each module in the primary dataset, the function
computes module preservation through the Z-summary statistics in the secondary datasets. The
Z-summary > 10 indicates evidence of strong preservation of the module across all the dataset
but Z-summary < 2 indicated no evidence of module preservation. Based on the Z-summary
statistics cutoff, five modules enriched across all the modules were chosen (Table 16).

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 Table 16 Z summary statistics of the conserved modules preserved across the datasets
Modules GSE6710 GSE34248 GSE41662
Turquoise 23.017 25.08 19
Black 13.14 7.76 10.40
Yellow 7.7 12.93 14.44
Pink 7.58 7.61 7.02
Red 6.67 17.15 9.39
Green - 7.83 5.88
Purple - 7.50 -
Brown - 5.05 9.28
Gold 5.46 8.35 -

The module conservation across all the datasets is shown as dendogram in Figure 22. Based on
WGCNA convention the top five enriched modules were named as turquoise, yellow, red, pink
and black. Since hub genes have been found to play important roles in many networks, highly
connected hub genes were expected to play an important role in biological systems.

72
Figure 22 Dendograms produced by average linkage hierarchical clustering of genes based on topological overlaps. (a) GSE13355, (b) GSE6710, (c)
GSE34248 and (d) GSE41662. The extent of gene conservation in the datasets are represented by the same module colours

Based on the kin values (kin > 0.9) ten highly connected hub genes were identified in each module. Figure 23 represents the modules and their respective
hub genes. The hub genes identified in each module is validated by the cytohubba plugin of the Cytoscape. Degree, the topological parameter which
determines the connectedness between the nodes is chosen as the parameter for hub gene selection. The top ten genes with high kin identified by WGCNA
in all the modules were reported as hubs by cytohubba.

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Figure 23 Network visualization of the top modules identifies psoriasis specific hub genes. (a) Turquoise module with 122 nodes and 622 edges. (b)
Yellow module with 107 nodes and 647 edges (c) Red module with 178 nodes and 546 edges (d) Black module with 96 nodes and 534 edges and (e) Pink
module with 113 nodes and 622 edges. The psoriasis specific hub genes in each module are labelled

4.3.2 COEXPRESSION LINK CONFIRMATION

The primary motivation of constructing the disease interactome network is to gain insights into different aspects and association of the hub genes with the
disease pathology. The association between the highly enriched ontological terms with the diseases in the five modules is investigated (Figure24).

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Figure 24 Enriched gene ontologies of the modules. (a) biological process and (b) cellular components. The bars are coloured based on the module
colours

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4.3.2.1 ASSOCIATION OF IMMUNE RESPONSE WITH TURQUOISE MODULE

The Z-score summary ranked turquoise module as the top most module with high conservation
pattern. The underlying psoriasis pathogenesis involves three predominant and independent
biologic processes: inflammation, epidermal hyperproliferation and altered differentiation with
parakeratosis (Gaspari, 2006). The highest number of genes in the module is found to be
associated with four biological processes: intracellular signaling cascade (p=0.066396),
keratinocyte differentiation (p = 0.0015), immune response (p = 3.85E-07) and epidermis
development (p = 1.73E-04). The cellular component analysis of turquoise module revealed the
presence of genes in cornified layer (p = 0.0026) and melanosome (p = 6.81E-04). Psoriasis, the
hyper proliferative immune mediated disease is reported to have elevated levels of melanosome.
Melanosome play a key role in protecting the skin cells from UV damage. The uptake of
melanosome is dynamically connected with the differentiation and proliferation of keratinocytes
(Choi et al, 2014). The changes associated with the keratinocyte differentiation suggest the
impairment of cornification process (Ligterink, 1995).

4.3.2.2 ASSOCIATION OF CELL ADHESION AND WOUND HEALING WITH RED

MODULE

The biological process annotation of red module reveals the enrichment of genes involved in cell
adhesion (p = 4.64E-06), wound healing (p = 2.04E-04) and regulation of cell proliferation (p =
0.0042). The lesional psoriatic skin exhibits elevated levels of endothelial cell activation markers
facilitating the adhesion of lymphocytes, monocytes and polymorphonuclear leukocytes to
endothelial cells and antigen presenting cells located in the dermal and epidermal layers of
psoriatic skin. The proliferation and lateral migration of keratinocytes are stimulated in wound
healing process (Morhenn et al, 2013). Psoriasis presents itself with many features of wound
healing. The genes located in extracellular matrix (p = 6.58E-07) and cytoskeleton (p = 0.0895)
were enriched in cellular component analysis. Activation of T cells is liked with pathogenesis of
psoriasis. The trafficking of T cells into the lesional zone is achieved by the cells in contact with
extracellular matrix (ECM) proteins. The ECM proteins provide co-stimulatory signals in CD3
triggered T cell activation (Glinski et al, 1993). Excessive actin cytoskeleton organization may
lead to cytoskeletal plasticity in the hyperthickened epidermis of the psoriatic skin. Red module
is dominated by the presence of genes involved in ECM and cytoskeleton organization.

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4.3.2.3 ASSOCIATION OF CELL CYCLE AND CELL DEATH WITH YELLOW MODULE

The yellow module encloses genes involved in biological processes related to cell cycle (p =
0.0043), mitotic cell cycle (p = 8.31E-05), regulation of apoptosis (p=0.0034) and regulation of
programmed cell death (p = 0.0038). Doubling of proliferative cell population accompanied by
increased germinal cells and shortening of individual cell cycle in psoriatic skin are reported by
several research groups worldwide (Weinstein et al, 1985). Dysfunctional apoptosis accompanies
psoriasis which is marked by the hyperproliferation and incomplete differentiation of epidermal
keratinocytes (Kastelan et al, 2009). The cellular component analysis revealed the presence of
organelles mediating the enriched biological processes such as mitochondria (p = 6.29E-04), golgi
apparatus (p = 0.061) along with cytoplasmic membrane-bounded vesicles (p = 9.89E-04). The
organelles are observed to be involved in the programmed cell death and cell cycle.

4.3.2.4 ASSOCIATION OF RNA EDITING AND SPLICING WITH PINK MODULE

The biological process annotation of pink module includes genes related to RNA processing (p =
0.00154), mRNA processing (p = 0.0018), RNA splicing (p = 0.003826) and RNA, via trans-
esterification reactions (p = 9.92E-05). During the malignant transformation of skin in psoriasis
the epidermal keratinocytes acquire the capacity to by-pass the cellular senescence leading to
their unrestricted proliferations. Various genes involved in RNA splicing and processing by their
altered and impaired splicing and processing mechanisms contribute to biological switches such
as senescence (Chaturvedi et al, 2003). We noted the involvement of organelles involved in RNA
processing and splicing such as membrane-enclosed lumen (p = 4.14E-06), intracellular organelle
lumen (p = 1.66E-05) and spliceosome (p = 0.013) were enriched in cellular component analysis.

4.3.2.5 ASSOCIATION OF ENERGY PRODUCTION WITH BLACK MODULE

The final black modules showed genes involved in electron transport chain (p = 7.34E-16),
oxidative phosphorylation (p = 1.23E-15) and oxidative reduction (p = 2.23E-07). Mitochondria
levels and ATP production were reported to be elevated in the lesional zones of psoriatic skin
(Hammar, 1975). The majority of the ATP produced in the epidermis is from the electron
transport chain of the mitochondrial system. Apart from playing key roles in cellular metabolism
and programmed cell death, mitochondrion is positioned as central player in innate immune
response. Cellular damage and stress are elevated in psoriasis which triggers the innate immunity
by generating the reactive oxygen group mediated by the power house of the cell. We noted the
presence of mitochondrion related components which includes mitochondrion itself (p = 4.71E-

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23), mitochondrial part (p = 1.98) and mitochondrial membrane (p = 1.55) leaded the cellular
component list of black modules.

4.3.3 PSORIASIS SPECIFIC HUB SIGNATURES AS BIOMARKERS - VALIDATION FOR

MODEL BUILDING

15 out of 50 hub genes were previously shown to be associated with psoriasis (Figure 25). The
involvement of hub signatures in the disease pathogenesis was strongly validated by the presence
of three hub genes S100A12, SNX7 and SPRR2C positioned in the epidermal differentiation
complex (EDC) located in the 1q21 linked/associated psoriasis locus.

Figure 25 Role and chromosomal location of PSHS genes proved to be previously associated
with psoriasis

Further, the hub genes from each module (Table 17) are analysed for its involvement in the
disease through the functional analysis using PESCADOR. The roles of genes contributing to the
major anomalies of psoriasis were analysed.

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 Table 17 The psoriasis specific hub signatures
Module Gene Description Location
Turquoise SPRR2C Small Proline-Rich Protein 2C  1q21.3

Turquoise TGM1 Transglutaminase 1  14q12

Turquoise ZC3H12A Zinc Finger CCCH-Type Containing 12A  1p34.3

Turquoise S100A12 S100 Calcium Binding Protein A12 1q21.3

Turquoise AKR1B10 Aldo-Keto Reductase Family 1, Member B10  7q33

Turquoise RHCG Rh Family, C Glycoprotein  15q26.1

Turquoise HPSE Heparanase  4q21.23

Turquoise IL36G Interleukin 36, Gamma  2q14.1

Turquoise TPBG Trophoblast Glycoprotein 6q14.1

Turquoise ADAP2 ArfGAP With Dual PH Domains 2 17q11.2

Red APLP2 Amyloid Beta (A4) Precursor-Like Protein 2  11q24.3

Red LAMA4 Laminin, Alpha 4  6q21

Red OLFML3 Olfactomedin-Like 3  1p13.2

Red PMP22 Peripheral Myelin Protein 22  17p12

Red FYN Proto-Oncogene, Src Family Tyrosine Kinase  6q21

Red AEBP1 AE Binding Protein 1  7p13

Red PCOLCE Procollagen C-Endopeptidase Enhancer  7q22.1

Red ZHX2 Zinc Fingers And Homeoboxes 2  8q24.13

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Red CDK14 Cyclin-Dependent Kinase 14  7q21.13

Red SNX7 Sorting Nexin 7  1p21.3

Pink RCOR1 REST Corepressor 1  14q32.31

Pink XPOT Exportin, TRNA  12q14.2

Pink UBE2D2 Ubiquitin-Conjugating Enzyme E2D 2  5q31.2

Pink MRPL13 Mitochondrial Ribosomal Protein L13  8q24.12

Pink STAM Signal Transducing Adaptor Molecule (SH3

 10p12.33

Domain And ITAM Motif) 1

Pink WSB2 WD Repeat And SOCS Box Containing 2  12q24.23

Pink G3BP1 GTPase Activating Protein (SH3 Domain) Binding

 5q33.1

Protein 1
Pink TXNDC9 Thioredoxin Domain Containing 9  2q11.2

Pink CPSF6 Cleavage And Polyadenylation Specific Factor 6  12q15

Pink PLK2 Polo-Like Kinase 2  5q11.2

Yellow CNOT7 CCR4-NOT Transcription Complex, Subunit 7  8p22

Yellow RAB1A Member RAS Oncogene Family  2p14

Yellow MRPL42 Mitochondrial Ribosomal Protein L42  12q22

Yellow CCT2 Chaperonin Containing TCP1, Subunit 2 (Beta)  12q15

Yellow CAPRIN1 Cell Cycle Associated Protein 1  11p13

Yellow TMX1 Thioredoxin-Related Transmembrane Protein 1  14q22.1

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Yellow NPEPPS Aminopeptidase Puromycin Sensitive  17q21.32

Yellow BZW1 Basic Leucine Zipper And W2 Domains 1  2q33.1

Yellow EIF5 Eukaryotic Translation Initiation Factor 5  14q32.32

Yellow PWP1 Endonuclein  12q23.3

Black SNRPG Small Nuclear Ribonucleoprotein Polypeptide G  2p13.3

Black RPL26L1 Ribosomal Protein L26-Like 1  5q35.1

Black COX7B Cytochrome C Oxidase Subunit VIIb  Xq21.1

Black PSMB1 Proteasome (Prosome, Macropain) Subunit, Beta

 6q27

Type, 1
Black PSMB3 Proteasome (Prosome, Macropain) Subunit, Beta
 17q12

Type, 3
Black PSMB6 Proteasome (Prosome, Macropain) Subunit, Beta
 17p13.2

Type, 6
Black UQCRQ Ubiquinol-Cytochrome C Reductase, Complex III
 5q31.1

Subunit VII
Black NDUFAB1 NADH Dehydrogenase (Ubiquinone) 1, Alpha/Beta
 16p12.2

Subcomplex, 1
Black SLIRP SRA Stem-Loop Interacting RNA Binding Protein
 14q24.3

Black TIMM8B Translocase Of Inner Mitochondrial Membrane 8 11q23.1

Homolog B

4.3.3.1 GENES REGULATING CELL CYCLE AND CELL PROLIFERATION

Psoriasis manifests itself with epidermal hyperplasia leading to excessive scale formation. Large
turnover of germinative cells and mitosis are reported in psoriatic lesions when compared to
normal skin. We noted the elevated levels of genes involved in cell survival and proliferation.
BZW1 (BZAP45) acts as a co-regulator of subset of genes controlled at the G1/S transition phase

81
of the cell cycle (Mitra et al, 2001). AKR1B10 promotes cell survival by modulating lipid
synthesis, mitochondrial function and oxidative stress, CDK14 (PFTK1) the cyclin dependent
kinase is involved in cell cycle progression and cell proliferation, CAPRIN1 and PMP22 are
reported to have tight correlation with cell proliferation, CNOT7 represses the genes which are
associated with the inhibition of cell proliferation (Aslam et al, 2009) and NPEPPS (PSA) the
aminopepetidase is found to associate itself with the spindle apparatus during mitosis. Microarray
analysis of cell cycle regulated genes identified periodic expression of G3BP1 along with other
cell cycle regulated genes throughout the cell cycle validating the involvement of G3BP1 in cell
cycle regulation as attributed by several studies (Irvine et al, 2004). The genes acting as
checkpoints were enriched in the PSHS list.

4.3.3.2 GENES CONTRIBUTING TO CYTOSKELETON REARRANGEMENT

Cytosketal proteins contribute to various biological processes such as cell adhesion, cell
proliferation and cell migration which play crucial role in psoriasis pathogenesis. Cytoskeletons
are also involved in the organization of immunological synapses and cellular activation. ADAP2
(centaurin-α2, 17q11) by partnering with ARF6 is involved in actin cytoskeleton rearrangements
such as exocytosis and cell motility (Venkateswarlu et al, 2007). PCOLCE is a positive regulator
of collagen deposition which is predominantly expressed in ECM. TXNDC9 (PHLP3) is a
phosducin which is required for the maintenance of β-tubulin level and actin-based cystoskeletal
remodelling (Stirling et al, 2006). CCT2 is a mammalian cytosolic chaperonin required for
appropriate folding of proteins involved in cytoskeletal formation and contractility activity.
RAB1A is implicated in microtubule dependent anterograde transport of melanosome.

4.3.3.3 GENES INVOLVED IN IMMUNE RESPONSE

Psoriasis is often termed as immune mediated disease. Chronic inflammation is considered as a

perpetrator of psoriasis by disrupting the normal signaling mechanisms in the skin. NF-κB
orchestrates inflammation and complex biological processes. Psoriasis is marked by elevated
levels of phosphorylated NF-κB. AEBP1 expressed abundantly in macrophages is a novel
proinflammatory mediator that induces NF-κB activity (Holloway et al, 2012). The elevated
expression of ZC3H12A (MCPIP1) significantly attenuated lipopolysaccharide induced
inflammatory cytokine network indicating their novel role in regulation of macrophage activation
(Liang et al, 2008). Migration of transepidermal leukocytes result in the accumulation of
neutrophils in Munro’s microabscesses in the stratum corneum which is the characteristic feature
of psoriasis. Heparanase (HPSE) involved in degradation of heparin sulphate aids the leukocytes
to pass through the barrier of the basement membrane (Tao et al, 2013). The overexpression of

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proinflammatory cytokines is considered to be crucial for initiation, maintenance and
reoccurrence of psoriatic skin lesions. IL-36γ, an IL-1F expressed in elevated levels in psoriasis
acts as an alarmin and expressed in response to the innate immune system. IL-36γ also induces
the production of several pro-inflammatory cytokines that contribute to inflammation. The
signal-transducing adaptor molecules (STAMs) were implicated in cytokine signaling via IL2
mediated C-Myc induction. S100A12 a member of S100 calcium binding protein is implicated in
variety of functions which includes cell proliferation and differentiation, dynamics of
cytoskeletal rearrangements and inflammation. The S100A12 is constantly expressed in
neutrophils validating its involvement in inflammation (Yang et al, 2001).

4.3.3.4 GENES RELATED WITH KERATINOCYTE PROLIFERATION AND

ANGIOGENESIS

Hyperproliferation of keratinocytes is one of the anomalies contributing to severity of psoriasis.

Several biochemical mechanisms have been implicated in the overproduction of keratinocytes in
psoriatic lesions. Amyloid precursor like protein (APLP2) expressed in mammalian epidermis
acts as an epidermal growth factor promoting keratinocyte proliferation and migration (Siemes et
al, 2006). Impaired signalling pathways involving kinases and their activation and transcription
have been hypothesized to be involved in the pathophysiology of psoriasis. Tyrosine
phosphorylation in response to calcium and TPA exposure occurs during early keratinocyte
differentiation. FYN, a non-receptor tyrosine kinase’s activity was induced in keratinocyte
differentiation (Calautti et al, 1995). Similarly, PLK2, a polo-like kinase has been associated
with the proliferation of keratinocytes (Kristl et al, 2008). The fast phase of keratinisation
contributes to the generous scales seen in psoriasis. The changes associated with the keratinocyte
differentiation suggest the associated cornification process would be incomplete. SPRR2C, a
small proline-rich family member and TGM1, a transglutaminase connecting cornified envelope
of keratinocytes were also observed to be a part of PSHS list (Cabral, 2001; Phillips et al, 2004).
One of the major cofactor of psoriasis development is angiogenesis which results due to the
changes in the superficial microvasculature in the skin. We noted the presence OLFML3 in the
PSHS list which is a proangiogenic factor that interacts with BMP modulators in order to
promote angiogenesis.

4.3.4 CLASSIFICATION OF THE DISEASE PHENOTYPE BASED ON PSORIATIC HUB

GENE EXPRESSION

To validate the efficiency of the hub signatures in distinguishing the psoriatic samples from the
normal samples, a classifier was built using the top ten hub genes from five enriched modules.

83
The expression values of each signature gene was retrieved from the training and test dataset and
visualized as heatmap. The heatmaps were clustered into diseased and normal categories based
on the PSHS expression values. The heatmaps indicate a good clustering of samples which were
validated by the dendograms on the top (Figure 26-27).

Figure 26 Heatmap of differential gene expression of PSHS in training data-set. Expression

levelswere colored orange for lowintensities and yellowfor high intensities (see scale at the top
left corner). The hierarchical clustering of PSHS in the training data-set clustering the diseased
(D) and normal (N) samples was also observed

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Figure 27 Heatmap representing the gene expressions of PSHS in the test datasets. The hierarchical clustering of PSHS in diseased (D) and normal
(N) samples in all the three test data-sets was also shown

The random forest algorithm is selected to build the classifier as it chooses subsets of genes and iteratively builds multiple parallel decision trees. The
random sampling selection and iteration reduces inefficiencies caused by noise and outliers during classifier training. The mean error rate of the classifier is
0.027%. The prediction ability of the classifier is validated using three datasets a, b and c which was not used in training steps of classifier building. The
performance for each of the dataset is assessed using sensitivity, specificity, precision, recall and other accuracy measures such as F1 score, MCC and
kappa statistics (Table 18). The sensitivity values ranged from 0.96 to 1 (mean 0.96) and specificity value was 1 for all the data sets. The F1 score, which
considers the precision and recall for computing the classifier’s accuracy ranged from 0.95 to 1, the MCC value which assesses the overall quality of the
binary classifier was predicted to be positive for all the three validation data sets and ranges from 0.86 to 1 and the kappa statistics which determines the accuracy of the classifier

85
by eliminating the prediction by chances ranges from 0.85 to 0.95. The overall measures of the
accuracy validate the ability of the classifier to distinguish the disease phenotype.

Table 18 Accuracy scores of PSHS based classifier in distinguishing diseased from normal state
using RF method
Measures Dataset a* Dataset b** Dataset c***
Sensitivity 0.9655 1 0.9176
Specificity 1 1 1
Precision 1 1 1
Recall 0.9655 1 0.9176
F1 score 0.9825 1 0.9571
MCC score 0.9533 1 0.8678
Kappa statistics 0.9522 1 0.8591
*GSE13355 (both diseased and normal (first 32 samples)), **GSE34248 (diseased), GSE41662
(diseased), GSE16161 (normal) and GSE7553 (normal), ***GSE30999 (diseased) and GSE13355
(normal, next 32 samples)

4.4 DOCUMENTATION OF TRADITIONAL SIDDHA MEDICINES FOR SKIN

DISEASES FROM KATPADI TALUK, VELLORE DISTRICT, TAMIL NADU,
INDIA

4.4.1 HEALERS’ SOCIO-DEMOGRAPHIC INFORMATION

We noted that the healers were mostly male. Since many of the healers we visited were located
inside villages they were uneducated or poorly educated except few. Most of the practitioners aged
between 66-75 years. The practitioners had a minimum of three years of experience and maximum
of 55 years of experience. 75% of the practitioners were practising it as their full time job. Some
practitioners subject their patients to basic biomedical tests such as blood glucose levels to validate
their diagnosis and treatment. The consultation charges ranged between INR 50-300. Accessibility
to the resources plays an important role to determine the knowledge over its usage. We noted that
the healers agreed with the usage of same plants for various disease categories. The agreement is
because majority of the healers rely on the local drug store and nearby forest for their medicinal
preparations.

4.4.2 DATA ANALYSIS

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The data obtained from the survey were entered in Microsoft excel spreadsheets for further analysis.
The documented plant species were sorted according to the disease categories. The scientific names,
common names or local names, plant parts used were documented based on the prescription by the
healers. Majority of the healers we interviewed acquired their knowledge from their family heads
and the doctors with recognized degree were also found to have a family background of Siddha
medicine. Only few healers reported their specialization category which included psoriasis,
leucoderma and eczema. Though, the practioners were willing to share the names of the plants and
few general medicines, the other information such as the time for procuring herbs, compositions,
mode of preparation and applications were not disclosed, since the knowledge about the medicines
were transferred only between the family members and not to others. Similar rules were adhered by
majority of the practioners participating in the interview and who were practising Siddha for many
generations. The herbs obtained from the local forest or local drug stores were processed in the
backyard or on the rooftop of the healer’s house. The herbs were shade dried or sun dried (based on
the preparatory protocol) and powdered using traditional instruments such as mortar and pestle made
from stone. Separate spaces were utilized by the healers for pre-processing of herbs and for the
production of complete drugs or formulations. A separate furnace section was observed in the
backyard for heating purpose in few healers’ homes. In many cases we observed that the healer’s
family and students were completely involved in the production of drugs. The prepared drugs were
stored in glass jars or mud pots.

4.4.3 HEALTH PROBLEMS AND THEIR TREATMENTS

The survey covered around 19 major illness categories pertaining to various skin diseases. The
medications included medicines from single plant, combinations of plants, animal products and
minerals. Around 102 species from 55 families of medicinally important plants were reported. The
formulations were also prescribed based on the severity of the disease. The compositions of the
formulations were given in Table 19.

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Table 19 Formulations prescribed by the Siddha healers
Sl.No Formulation Main Ingredients
1. Punga thailam Millettia pinnata
Cocos nucifera
Edible camphor
2. Chirattai Cocos nucifera
thailam
3. Vetpalai Wrightia tinctoria
thailam Cocos nucifera
4. Milagai thailam -
5. Mathan thailam Datura metal
Cocos nucifera
Curcuma aromatica
Copper sulphate

6. Arkunpul -
thailam
7. Karappan Mercurous Chloride
thailam Eletteria cardamomum
Copper sulphate
Zinc sulphate
Cinnamomum camphora
Elemental mercury
Psoralea corylifolia
Lead sulphide
Curcuma aromatica
Hydnocarpus kurzii
Vemonia anthelmintica
Elemental Sulphur
Cocos nucifera
8. Sivanar vembu Indigofera aspalathoides
kuzi thailam Corallocarpus epigeous
Celastrus paniculatus
9. Nuna thailam Morinda Pubescens

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10. Elathi Elettaria cardamomum
chooranam Syzygium aromaticum
Mesua ferrea
Zizyphus jujube
Oryza sativa
Callicarpa macrophylla
Cyperus rotundus
Santalum album
Piper longum
11. Parangipattai Smilax china
chooranam Saccharum officinarum

12. Rasaganthi Elemental Mercury

mezhuku Elementary Sulphur
Mercurous chloride
Arsenic trisulphide
Magnetite ore of Iron
Copper sulphate
Zinc carbonate with traces
of Zinc sulphate
Lead monoxide
Zingiber officinale Roscoe
Trachyspermum ammi L.
Curcuma longa L.
Embelia ribes Burm.f.
Acorus calamus L.
Cinnamomum
zeylanicum(Bl.)
Cucurbita pepo L.
Semecarpus anarcadium
L.
Terminalia chebula Retz.
Nigella sativa L.
Centratherum

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anthelminticum L.
Premna herbacea Roxb.
Taxus baccata L.
Vitis vinifera L.
Piper longum L.
Alpinia speciosa L
Saussurea lappa Clarke
Celastrus paniculatus
Willd
Foeniculum vulgare
Miller
Elataria cardamomum L.
Myristica fragrans Houtt.
Piper nigrum L.
Cuminum cyminum L
Psoralea corylifolia L.
Quercus infectoria oliver
Piper longum L.
Calamus rotang L
Strychnos nux –vomica L.
Strychnos potatorum L.
Asteracantha longifolia
L.Nees
Sesamum indicum L.
Cocos nucifera L.
Dolichos biflorus L
Acalypha fruticosa Forsk
Azima tetracantha Lam
Withania somnifera L
Corallocarpus epigaeus
Rotrl
Plumbago rosea L.
Gallus domesticus L
Borassus flabellifer L

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13. Idivallathy Semecarpus anacardium
mezhugu Sesamum indicum
Cocos nucifera
Smilx china
Withania somnifera
Plumbago indica
Curcuma aromatica
Nigella sativa
Celastrus paniculatus
Hyoscyamus niger
Piper betle
Terminalia chebula
Piper longum
Saussurea lappa
Mercurous chloride
Borassus flabellifer
14. Kilinjal Lime-shell
mezhugu Emblica officinalis
15. Nandi mezhugu -
16. Iru nelli karpam Elemental sulphur
Emblica officinalis
17. Triphala Terminalia chebula
karpam Terminalia belerica
Emblica officinalis
Acacia catechu
Pterocarpus marsupium
Aya Chendooram
Silasathu Parpam
Sangu Parpam
18. Karisalai Wedalia calendulacea
karpam Eclipta prostrata
Indigofera tinctoria
Acalypa indica
Centella asiatica

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 Ghee
Eclipta prostrata
Acacia nilotica
19. Neeradimuthu Semicarpus anacardium
vallathy lehyam Hydnocarpus kurzii
Smilax china
Calamus rotang
Nigella sativa
Cuminum cyminum
Acorns calamus
Indicofera aspalathoides
Azima tetracantha
Corallocarpus epigaeus
Withania somnifera
Enicostemma littorale
Calotropis gigantea
Ficus racemosa
Boerhaavia diffusa
Toddalia asiatica
Azadirachta indica
Wrightia tinctoria
Hydragyrum
Elemental Sulphur
Copper sulphate
Zinc sulphate
Sesamum indicum
Borassus flabellifer
Indigofera tinctoria
Ghee
20. Agasthiar Asafoetida
kuzhambu Mustard
Rock salt
Mercury
Borax

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 Aconitum napellus
Arsenic disulphide
Trachyspermum ammi
Nigella sativa
Arsenic trisulphide
Croton tiglium
21. Parangipattai Smilax china
rasayanam Plumbago zeylanica
Clitoria ternatea
Withania somnifera
Curculigo orchiodes
Piper longum
Abies spectabilis
Cinnamomum tamala
Piper nigrum
Carum capticum
Hyoscyamus niger
Nigella sativa
Alpinia officinarum
Alpinia galangal
Terminalia chebula
Terminalia bellarica
Phyllanthus emblica
Piper cubeba
Costus specius
Papaver somniferum
Coriandrum sativum
Nardostachys grandiflora
Syzygium aromaticum
Myristica fragrans
Cinnamomum verum
Zingiber officinale
Ghee
Sugar
Cow’s milk
Honey
22. Mega virana -

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 kalimbu
23. Amirtha vennai Mercuric chloride and
butter

Based on the informant consensus factor (Fic) we found that the highest agreement among the
informants was found for the category fungal infection (Fic=0.98) followed by acne vulgaris
(Fic=0.96) and psoriasis (Fic=0.95). Patients with eczema, pruritus, fungal infections frequently
visit the Siddha healers (Table 20). The predominant and prominent skin diseases and their
prescribed herbal medications are discussed below.

Table 20 Informant consensus factor and number of patients visiting the Siddha healers

Disease category Fic Average number of

patients/week*
Fungal infection 0.98 5-15
Acne 0.96 5-10
Psoriasis 0.95 2-5
Pruritis 0.83 2-5
Scabies 0.80 2-5
Eczema 0.78 5-7
Hair loss 0.74 5-7
Lice in hair 0.73 2-5
Heel cracks 0.67 2-3
Alopecia 0.67 2-3
Warts 0.65 5-7
Impetigo 0.53 2-3
Wounds and cuts 0.51 2-3
Leucoderma 0.45 2-5
Seborrheic dermatitis 0.45 2-5
*Includes new and follow-up patients

4.4.3.1 PRURITUS

Pruritus is commonly termed as ooral or chori by the Siddha healers. The symptoms include itching
of the superficial skin and provoking scratching sensation. Around 15 plants were recorded for this
disease category (Table 21).

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 Table 21 Plants used in the treatment of pruritus

Scientific name Common name Family Parts used UV IAR

Acacia catechu Karunkaali Fabaceae Root 0.23 0.89

Aristolochia indica Isvaramuli Aristolochiaceae Root 0.64 0.92

Calamus rotang Linn Bettam, pirampu Palmae Leaves and 0.68 0.92
Arecaceae roots

Calotropis gigantean Irrukam Apocynaceae Leaves and 0.95 1

roots
Desmodium triflorum Sirupullati Fabaceae Whole plant 0.41 1

Diospyros montana Vakanai Ebenaceae Bark and root 0.23 0.78

Roxb
Eupatorium Ayapanai Asteraceae Leaves 0.45 0.68
triplinerve vahl
Flacourtia indica Katukalli Salicaceae Whole plant 0.27 1
Jasminum Kattumullai Oleaceae Root 0.59 0.48
angustifolium wild
Piper nigrum Melagu Piperaceae Fruit 0.35 0.92
Prunus amygdallus Vatamkottai Rosaceae Kernel 0.23 0.55
batsch
Syzygium aromaticum Graambu Myrtaceae Flowerbuds 0.59 0.83
Tragia involucrata Chenthatti, kancori, Euphorbiaceae Root 0.82 0.88
Linn. kandudi

Trichosanthes dioica Peyupadal Cucurbitaceae Leaves 0.45 1

Roxb
Vernonia Kattu shiragam Asteraceae Seed 0.73 0.86
anthelmintica

Calotropis gigantea (irrukam) was the most highly cited plant in the disease category with a UV of
0.95 and IAR value of 1. The roots and leaves of C. gigantea were crushed and applied on the
affected areas of the skin (Kadiyala et al, 2013). Thailam (~ medicated oil) such as Punga thailam
and Chirattai thailam were also prescribed for external applications. The healers also advised their

95
patients to follow a strict diet regimen with certain diet restriction such as avoiding chicken, brinjal
and other spicy food items.

4.4.3.2 IMPETIGO

Impetigo is termed as sirangu and classified under the category of karappan. It is commonly
observed in children. The appearance of red boils which later breaks down to become irregular
ulcers (pun) were the noted symptoms of the disease. The ulcers break to release pus leading to itchy
skin. The rhizomes of Curcuma longa (Shawket et al, 2013) and leaves of Ocimum americanum
were crushed and applied over the boils. Punga thailam was administrated orally and Vetpalai
thailam was prescribed for external application.

4.4.3.3 SCABIES

Scabies termed as chori sirangu in Siddha medicinal terms was one of the highly frequent skin
disease. Leaves of Acalypha indica L. was the highly prescribed medicine for scabies based on the
UV and IAR values of 1 and 0.99, respectively. The juice from crushed leaves of A. indica was
applied over the affected areas (Jagatheeswari et al, 2013). Milakai thailam and Mathan thailam
were prescribed for external use and Iru nelli karpam (~ herbo-mineral medicine) and chooranam (~
powder) were recommended for oral consumption. Table 22 cites the plants used for treating
scabies.

Table 22 Plants cited for curing scabies

Scientific name Common name Family Parts used UV IAR

Acalypha indica L. Kuppaimeni Euphorbiaceae Leaves 1 0.99

Aegle marmelos Vilvam Rutaceae Fruit 0.68 1

Azadirachta indica Vembu Meliaceae Leaves and bark 0.98 0.95
Biophytum sensitivum Tintanali Oxalidaceae Whole plant 0.68 0.92
Calotropis gigantean Irrukam Apocynaceae Leaves and roots 1 0.93
Cassia fistula Konrai Fabaceae Ariel part 1 0.94
Clerodendrum Perukilai Lamiaceae Leaves 0.41 0.875
infortunatum
Coccinia grandis Kovai Cucurbitaceae Leaves 0.77 0.92
Diospyros montana Roxb Vakanai Ebenaceae Bark and root 0.29 0.86
Ficus exasperata Maramthinniatti Moraceae Bark, root and 0.82 0.94
leaves

96
Melia azedarach Malaivembu Meliaceae Bark 0.31 0.95
Syzygium aromaticum Graambu Myrtaceae Flower buds 0.59 0.83
Thespesia populnea Poovarasu Malvaceae Ariel part 0.95 0.95
correa
Vernonia anthelmintica Kattu shiragam Asteraceae Seed 0.73 0.86

4.4.3.4 ECZEMA

Eczema is called padai and few healers classify them under karappan. Two types of eczema were
defined by the healers which includes wet and dry eczema. The dry eczema starts with itching and
leads to hyper-pigmentation while in wet eczema itching is followed by blister development further
leading to hyper-pigmentation at later stages. Based on the quantitative metrics C. longa has the
highest UV (0.98) and IAR index (1.00). The immediate remedy prescribed by many healers was the
application of rhizome powder of C. longa on the affected skin (Jurenka, 2009). The fresh rhizomes
of C. longa and Cyperus rotundus (Nagulendran et al, 2007) were grounded without water and
applied externally. In adults Rasaganthi mezhugu (~ wax) was prescribed along with parangipattai
chooranam for oral consumption and Arkunpul thailam for external application. Few healers
prescribed Karappan thailam for external application. In children Vetpalai thailam was prescribed
for external application and Punga thailam for oral intake. Plants prescribed by various healers for
eczema were reported in Table 23. The plants used for treating skin disorders associated with
eczema which includes boils, skin eruptions and itch were provided in Table 24.

Table 23 Plants used in the treatment of eczema

Scientific name Common Family Parts UV IAR

name used
Achyranthes aspera Nayuruvi Amaranthaceae Leaves 0.55 0.96
Acorus calamus Vasambu Acoraceae Rhizome 0.82 1
Aerva lanata Sirrupulayvayr Amaranthaceae Whole 0.45 0.77
plant
Amaranthus spinosus Mullikkirai Amaranthaceae Leaves 0.32 0.96
Callicarpa tomentosa Vettilaipattai Verbenaceae Leaves 0.95 0.95
Calotropis gigantea Irrukam Apocynaceae Leaves 0.85 0.93
and
roots

97
 Capparis zeylanica Suduthorati, Capparaceae Leaves 0.68 0.92
Kathotti
Cassia alata Seemaiagathi Caesalpiniaceae Leaves 0.59 0.96
Cassia fistula Konrai Fabaceae Ariel 0.98 0.94
part
Cassia tora Thagarai Cesalpinaceae Leaves 0.32 0.92
Centella asiatica Vallarai Apiaceae Leaves 0.18 0.92
Cleome viscosa Naai kadugu Cleomaceae Leaves 0.55 0.875
Cryptolepis Paal kodi, Apocynaceae Root 0.23 1
buchanani Maattaan kodi and bark
Diospyros montana Vakanai Ebenaceae Bark 0.39 0.86
Roxb and root
Elephantopus scaber Anashovadi Asteraceae Leaves 0.09 1
(L)
Cyperus rotundus Korai Cyperaceae Rhizome 0.91 1
kizhangu
Curcuma longa Manjal Zingiberaceae Rhizome 0.98 1
Vernonia Kattu Asteraceae Seed 0.73 0.86
anthalmintica shiragam

Table 24 Plants cited for treatment of boils, skin eruption and itch

Disease Scientific name Common name Family Parts UV IAR

category used

Boils Achyranthes Nayuruvi Amaranthaceae Leaves 0.45 0.96

aspera

Basella alba Kodip pasali Basellaceae Leaves 0.91 1

Butea Porasu Fabaceae Bark 1 1.27

monosperma
(Lam.) Taubert
Callicarpa Vettilai-pattai Lamiaceae Leaves 0.15 0.95
tomentosa

98
 Capparis Suduthorati, Capparaceae Leaves 0.68 0.92
zeylanica Kathotti

Cinnamomum Karruwa Lauraceae Bark 0.41 1

verum
Citrus Champalam Rutaceae Flowers 0.14 1
aurantifolia

Clerodendrum Perukilai Lamiaceae Leaves 0.41 0.875

infortunatum
Coccinia grandis Kovai Cucurbitaceae Leaves 0.77 0.92

Heliotropium Thel kodukku Boraginaceae Leaves 0.51 0.93

indicum
Piper nigram Melagu Piperaceae Fruit 0.91 0.92
Schrebera Kattupparutticceti Oleaceae Whole 0.27 1
swietenioides plant
Skin Cassia tora Thagarai Cesalpinaceae Ariel 0.32 0.92
eruption part
Datura metel Karoo Omatay Solanaceae Leaves 0.23 1

Desmostachya Dharbapul Poaceae Culms 1 0.95

bipinnata
Eupatorium Ayapanai Asteraceae Leaves 0.45 0.88
triplinerve vahl
Leucas aspera Thumbai Lamiaceae Leaves 0.55 0.9
Spreng

Ocimum Tulsi Lamiaceae Leaves 0.95 0.95

tenuiflorum

Pongamia Punkamaram Fabaceae Bark 0.23 1

pinnata

Terminalia Tanrikkai Combretaceae Seeds 0.32 1

bellerica
Tragia Chenthatti, kancori, Euphorbiaceae Root 0.82 0.88
involucrata Linn kandudi

99
 Biophytum Tintanali Oxalidaceae Seeds 0.68 0.92
sensitivum
Clerodendrum Parugilai Lamiaceae Roots 0.23 1
viscosum vent and
leaves
Coccinia grandis Kovai Cucurbitaceae Leaves 0.77 0.92

Desmodium Sirupullati Fabaceae Whole 0.41 0.93

triflorum plant
Putranjiva Irukolli, Karupala Putranjivaceae Seed 0.77 0.93
roxburghii
Thespesia Poovarasu Malvaceae Latex 0.95 0.95
populnea correa
Itch Acalypha indica Kuppaimeni Euphorbiaceae Leaves 0.28 0.92
L.
Andrographis Nilavembu Acanthaceae Leaves 0.4 0.96
paniculata
Ardisia solanacea Manipudbam Primulaceae Root 0.41 1
Roxb

Calophyllum Punnai Calophyllaceae Seed and 0.73 1

inophyllum leaves

Cassia auriculata Aavaram Fabaceae Leaves 0.82 1

Cassia fistula Konrai Fabaceae Ariel 1 0.94

part
Cynodon Arugumpul Poaceae Leaves 0.73 0.97
dactylon
Gymnema Cherukurinja Apocynaceae Leaves 0.91 1
sylvestre
Melia azedarach Malaivembu Meliaceae Stem 0.31 0.95
Piper betle Vetrili Piperaceae Leaves 0.23 1

Sesamum indicum El Pedaliaceae Seed 0.55 0.9

100
 Sphaeranthus Kottakaranthai Asteraceae Whole 0.41 1
indicus plant

Thespesia Poovarasu Malvaceae Bark 0.95 0.95

populnea correa

4.4.3.5 PSORIASIS

Different types of psoriasis were well documented in Siddha medicinal system. The most common
type of psoriasis, the plaque psoriasis was designated as Kanda karappan and Kutta karappan by the
Siddha healers. Similarly scalp psoriasis was termed as Manda karappan and psoriatic arthritis was
named as Thimir vata karappan. Few healers label psoriasis as Kalanchaka padai. The highly
prescribed treatment for psoriasis was Vetpalai thailam, prepared from the leaves of Wrightia
tinctoria (Dhanapal et al, 2012; Jose and Joji, 2014). Chirattai thailam prepared by destructive
distillation of dry coconut shells was also suggested by few healers. Parangipattai chooranam, a
formulation made from various plant components was prescribed for oral intake by majority of the
healers. The other medicines for oral consumptions include Neeradimuthu vallathy lehyam (~
confections), Idivallathy mezhugu and Sivanar vembu kuzi thailam.

4.4.3.6 LEUCODERMA

Vitiligo or leucoderma was termed as venpulli noi or venpadai by the Siddha healers. Leucoderma
was characterized by de-pigmentation of the skin leading to white patches. Vitiligo was divided into
four categories; vatha venpadai, pitha venpadai, kaba venpadai and mega venpadai. Leaves of
Cassia senna was the highly used species with the UV of 0.85 and IAR values of 0.88. The
preparations from Morinda pubescens were prescribed by many healers which include Nuna kadugu
for oral consumption and Nuna thailam for external application. The above two medications were
prepared from the leaves of M. pubescens (Ramaswamy et al, 2012). The plants cited by other
Siddha healers were listed in Table 25.

Table 25 Plants prescribed for the treatment for leucoderma

Scientific name Common name Family Parts used UV IAR

Cassia senna Nelavakai Fabaceae Leaves 0.85 0.88
Ixora arborea Roxb Irruvatchi Rubiaceae Root and Bark 0.45 1
Lannea coromandelica Anaikarai Anacardiaceae Bark 0.74 0.85
Rhinacanthus nasutus Nagamalli Acanthaceae Leaves 0.50 0.9

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4.4.3.7 WARTS AND ULCERS

Wart is called unni in Siddha terminology. Local healers prescribed the application of Coccinia
grandis leaf paste over the warts. Majority of the healers prescribed the usage of Unni marunthu, a
mineral based ointment to remove warts and corns. Lannea coromandelica was also prescribed by
few healers for the removal of warts. Pungal (~ulcers) were divided into inguinal bubo, ulcers
induced by diabetes and genital ulcers. Agasthiar kuzhambu and Parangipattai rasayanam were
commonly used to treat inguinal bubo and genital ulcers. Mega virana kalimbu was also prescribed
by few healers. The application of turmeric powder mixed with coconut oil was the first measure
prescribed by many practitioners to control the inflammatory response of the disease.

4.4.3.8 WOUNDS AND CUTS

The gel obtained from the leaves of cactus Aloe vera was the highly reported medicine for the
category with the UV of 0.95 and IAR value of 1. The fresh leaves were cut and the gel from the
leaves was topically applied over the skin (Dorai, 2012). The healers also suggested other remedies
which include juice from the leaves of Cynodon dactylon, rhizome paste of C. longa and bark paste
of Santalum album in combination. Siddha kalimbu, a polyherbal formulation made from 12 herbal
ingredients was also prescribed by few healers (Devi and Sampath Kumar, 2011). The plants cited
by the Siddha practitioners for treating wounds and cuts were reported in Table 26.

Table 26 Plants prescribed for treating wounds and cuts

Scientific name Common name Family Parts UV IAR

used
Aloe vera Katrazhi Asphodelaceae 0.95 1
Abutilon indicum Thuthi Malvaceae Leaves 0.64 0.92
Acacia caesia (L.) Indu, seengaikodi Fabaceae Bark 0.68 1
Willd
Acacia dealbata Seegai Fabaceae Leaves 0.52 0.27
Acacia leucophloea Velvelam Fabaceae Bark 0.59 1
(Roxb.) Willd
Achyranthes aspera Nayuruvi Amaranthaceae Whole 0.55 0.96
plant
Aerva lanata Sirru pulayvayr Amaranthaceae Ariel part 0.45 0.77
Anacardium Munthiri Anacardiaceae Bark 0.41 0.75
occidentale

102
Argemone Mexicana Kudiyotti, bhramathandu Papaveraceae Leaves 0.23 1
Azima tetracantha Mulsangu Salvadoraceae Roots 0.36 1
Lam
Blepharis Naalilai naagam Acanthaceae Leaves 0.41 0.85
maderaspatensis
Calotropis gigantean Irrukam Apocynaceae Latex 0.82 0.93
Cleome viscosa Naai kadugu Cleomaceae Leaves 0.55 0.90
Cynodon dactylon Arugumpul Poaceae Leaves 0.73 0.97
Desmodium triflorum Sirupullati Fabaceae Whole 0.41 0.933
plant
Euphorbia hirta L. Chithirapaladai Euphorbiaceae Leaves 0.27 1
Euphorbia thymifolia Chinamampatchaiarisi, Euphorbiaceae Leaves 0.55 0.90
sittirapaladi
Gloriosa superba L. Sengandhal Colchicaceae Stem 0.80 0.94
Helicteres isora Valampuri Malvaceae Leaves 0.36 1
Homonoia riparia Kattualari Euphorbiaceae Leaves 0.64 1
Lour.
Lannea Anaikarai Anacardiaceae Leaves 1 0.85
coromandelica and bark
Rhinacanthus nasutus Nagamalli Acanthaceae Leaves 0.50 0.9
Tridax procumbens Thalaivettukaay Asteraceae Leaves 0.36 1

4.4.3.9 FUNGAL INFECTIONS – DERMATOPHYTOSIS

The treatment option for various fungal infections such as tinea corporis and tinea versicolor were
given by various Siddha practitioners. Azadirachta indica with high UV of 0.94 and IAR value of
0.95 were used by many healers. The lesions were covered with a preparation made from the
crushed seeds of Cassia occidentalis (Vipul and Anjana, 2011) soaked in leaf juice of Euphorbia
ligularia mixed with cow’s urine. Similarly the lesions were also covered with the medicine made
by mixing root bark of Alangium salvifolium (Wuthi-udomlert et al, 2002) and seeds of Randia
dumentorum (Kumar et al, 2006) with lime juice. The above medication was also given for oral
consumption. For tinea interdigitale, Amirtha vennai, a mineral based ointment was prescribed for
external application. For tinea versicularis the leaves of Cassia alata and Ocimum tenuiflorum were
prescribed by few practitioners. The plants prescribed for the treatment of fungal infections were
shown in Table 27.

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 Table 27 The plants prescribed for the treatment of fungal infections

Scientific name Common name Family Parts used UV IAR

Abutilon indicum Thuthi Malvaceae Leaves 0.64 0.92
Acalypha indica L. Kuppaimeni Euphorbiaceae Leaves 0.28 0.92
Amaranthus tricolor Thandukeerai Amaranthaceae Leaves and 0.55 0.90
seeds
Azadirachta indica Vembu Meliaceae Leaves 0.94 0.95
Carica papaya Pappali Caricaceae Leaves 0.55 1
Cassia alata Seemaiagathi Caesalpiniaceae Leaves 0.59 0.96
Cassia fistula Konrai Fabaceae Ariel part 0.18 0.54
Cassia tora Thagarai Cesalpinaceae Leaves 0.32 0.62
Centella asiatica Vallarai Apiaceae Whole 0.48 0.52
plant
Crinum asisticum Visamumgil Amaryllidaceae Whole 0.56 0.81
plant
Euphorbia Chinamampatchaiarisi, Euphorbiaceae Leaves 0.55 0.90
thymifolia sittirapaladi
Ficus exasperata Maramthinniatti Moraceae Bark, root, 0.82 0.94
leaves
Jasminum Kattumullai Oleaceae Root 0.59 0.94
angustifolium willd
Mimosa pudica Thotta siningi Fabaceae Whole 0.69 0.93
plant
Tragia involucrata Chenthatti, kancori, Euphorbiaceae Root 0.82 0.88
Linn kandudi
Trichosanthes pudalangai Cucurbitaceae Leaves 0.45 0.68
cucumerina Linn

4.4.3.10 ALOPECIA AREATA AND HAIR LOSS

Baldness (Alopecia) was termed as vazhukai and puzhu vettu by the healers. The leaf paste of
Indigofera tinctoria (Saraswathi et al, 2012) mixed with castor oil was heated, filtered and cooled.
The oil was prescribed for the external application on the affected regions of the scalp. Latex of
Nerium oleander was also prescribed by the healers. Juice of the Colocasia esculenta corns were

104
used by few practioners to treat baldness (Prajapati et al, 2011). Leaf paste of Trichosanthes dioica
Roxb was also prescribed by few healers for alopecia. Oil made by heating flowers of Hibiscus
rosasinensis, leaves of Eclipta prostrata and coconut oil were filtered and cooled. The oil was
applied on the scalp on regular basis to prevent hair loss (Thorat et al, 2009). Leaves of
Indoneesiella echioides and seeds of Tectonal grandis L. were also recommended by very few
healers to control hair loss. Triphala karpam, Karisalai karpam and Rasaghendhi mezhugu were
advised for oral consumption.

4.4.3.11 ACNE VULGARIS AND FOOT CRACKS

The paste from Santalum album (sandalwood) with a UV of 0.91 and IAR index of 1, was the highly
prescribed treatment for acnes and pimples. Kunkiliya vennai was also advised by few healers for
external application. Leaves of Abutilon indicum, Acorus calamus and Allium sativum (garlic) were
grounded and boiled in sesame oil. The filtered and cooled oil was adviced for external application.
Fresh rhizomes of Curcuma longa were prescribed for external application as an immediate remedy.
The leaf paste of Lawsonia inermis was applied on the foot cracks (kal vedippu) (Badoni Semwal et
al, 2014). Kilinjal mezhugu was prescribed by the healers for the external application to heal the foot
cracks. In some cases healers prescribe Nandi mezhugu for oral intake depending on the severity of
the disease. Table 28 lists few plants prescribed for the treatment of acne, pimple and foot cracks.

Table 28 Plants used in the treatment of acne vulgaris and foot cracks

Disease Scientific Common name Family Parts UV IAR

name used
Pimples Heliotropium Thel kodukku Boraginaceae Leaves 0.50 0.93
indicum
Mimosa pudica Thotta siningi Fabaceae Whole 0.69 0.93
plant
Santalum Santhanam Santalaceae Stem 0.91 1
album
Symplocos Vellilathi Symplocaceae Stem 0.14 0.95
racemosa bark
Roxb.
Foot Anacardium Munthiri Anacardiaceae Seed 0.41 0.77
cracks occidentale coat
Lawsonia Maruthani Lythraceae Leaves 0.41 1

105
 inermis L.
Tribulus Nerunchi Zygophyllaceae Whole
terrestris plant
Acne Curcuma Kasturimanjal Zingiberaceae Rhizome 0.68 0.97
aromatica
Euphorbia Chinamampatchaiarisi, Euphorbiaceae Leaves 0.55 0.90
thymifolia Sittirapaladi
Symplocos Vellilathi Symplocaceae Stem 0.54 0.95
racemosa bark
Roxb.

4.4.3.12 PEDICULOSIS AND SEBORRHEIC DERMATITIS

Siddha healers prescribe many medications to control lice. Oil prepared by heating seed kernel
powder of Anamirtha cocculus (cantiropam) with sesame oil was highly prescribed to control lice.
Andrographis paniculata leaf paste or rhizome paste of Acorus calamus was applied on the scalp for
15 minutes and washed. Since they were common in every household, they were highly cited and
used as control measures. Few practitioners prescribed the usage of Gloriosa superb and Ocimum
amaericanum for controlling the pediculosis. The oil made from Phyla nodiflora was used to control
seborrheic dermatitis (Sharma and Singh, 2013). The fresh leaves of P. nodiflora were also
prescribed to control Seborrhea and it has high UV and IAR index value of 0.92 and 1, respectively.
Table 29 reports the plants prescribed for seborrheic dermatitis.

Table 29 Plants prescribed to control seborrheic dermatitis

Scientific name Common name Family Parts UV IAR

used
Amaranthus Thandukeerai Amaranthaceae Seeds 0.55 0.90
tricolor
Aristolochia Aadutheendaapaalai Aristolochiaceae Leaves 0.36 0.96
bracteolate
Azadirachta indica Vembu Meliaceae Leaves 0.34 0.95
Centella asiatica Vallarai Apiaceae Whole 0.53 0.92
plant
Crinum asiaticum Visamumgil Amaryllidaceae Whole 0.86 1

106
 plant
Eclipta prostrate Karisalaankanni Asteraceae Leaves 0.54 0.95
Euphorbia Chinamampatchaiarisi, Euphorbiaceae Leaves 0.55 0.92
thymifolia sittirapaladi
Heliotropium Thel kodukku Boraginaceae Leaves 0.50 0.93
indicum
Ocimum Nai thulasi Lamiaceae Leaves 0.82 0.94
americanum
Phyla nodiflora Podutalai Verbenaceae Leaves 0.92 1
juice
Piper nigram Melagu Piperaceae Fruit 0.91 0.92
Sesamum indicum El Pedaliaceae Seed 0.55 0.90
Syzygium Graambu Myrtaceae Flower 0.59 0.83
aromaticum buds
Tectona grandis L. Tekkumaram Lamiaceae Seed 0.82 0.94

4.4.3.13 PREMATURE GREYING

Premature greying (ilam narai) is one of the top causes for many to visit the healers. Oil made from
Indigofera tinctoria (Dorai, 2012) and oil from Eclipta alba (Datta et al, 2009) prepared by the
healers themselves was prescribed to many patients. The intake of fresh leaves of Murraya koenigii
(curry leaves) paste with butter milk in the morning was also suggested by few practitioners.

4.4.4 ROLE OF DIET IN SIDDHA MEDICATION

Diet plays a major role in Siddha medicine. The cure for the diseases is not only governed by the
medications but also by observing proper diet. The restrictions were suggested to avoid drug-drug
and food-drug interactions. In clinical practice, along with the medications prescribed by the healers
the patients tend to add various over-the-counter drugs. When all the drugs are digested together
they may interact. Combined usage of herbs with drugs may mimic, increase or decrease the effects
of either components leading to herb-drug interactions (Fugh-Berman, 2000). To avoid these
negative outcomes pathiyam (~ diet restrictions) were observed while taking these medications. For
instance when the patient is treated for anaemia using Annabedhi chenduram, the clinical findings
suggest the improvement of haemoglobin levels within a short time; however the patients with the
habit of chewing betal nuts showed negligible increase in the Hb level. The tanic acid present in the
betal nut has a tendency to precipitate iron and prevents absorption; hence specifications to avoid

107
certain dietary articles were suggested (Veluchamy and Thayagarajan, 1983). Vegetables such as
brinjal, tomato, tubers, bitter gourd were restricted. Beef, salted dry fish, squid and crabs were also
avoided. Pickles and other oily items were also restrained from consumption. Patients with skin
diseases were advised to avoid salt, tamarind and vinegar. Though several restrictions were advised,
the healers also ask their patients to take up few items regularly. Drum stick (Moringa oleifera)
leaves, leafy vegetables, onions, rice gruel without salt, pepper, green gram, parched rice flour and
non-salted fish were allowed for consumption while taking the medicines.

4.4.5 BATH POWDERS

The traditional Siddha healers prescribe the usage of certain herbal bathing powders to scrub their
body depending upon the disease condition. People with pruritus were advised to use oil cake of
Madhuca longifolia for taking bath instead of soaps. Most of the healers prescribed the usage of
bengal gram powder to everyone. The dried fruit remains of Luffa acutangula (akasaveni) was
prescribed as a scrub. Powder made from Albizia amara (arappu) and powder made from the flower
and leaves of Hibiscus rosasinensis were used for bathing purposes in patients with skin diseases.
Application of rhizome paste of C. longa was advised because it avoids spreading of the disease and
prevents excessive sweating.

4.5 INVESTIGATION OF THE MOLECULAR MECHANISM OF ACTION OF

HIGHLY PRESCRIBED SIDDHA TOPICAL THERAPY FOR PSORIASIS
VULGARIS

4.5.1 COMPOUND PROFILING

The GC-MS analysis identified a total of 235 and 176 compounds from the methanol, and petroleum
ether extract, respectively (Figure 28-29). Since only trace elements were identified from the
chloroform extract we eliminated the results obtained from the extract.

108
Figure 28 GC-MS Chromatogram of methanol extract of W. tinctoria

109
 Figure 29 GC-MS Chromatogram of petroleum ether extract of W. tinctoria

After removing the compounds without structures a total of 316 structures were obtained. The
redundancy check was carried out with the remaining dataset. Finally 312 compounds were selected
for further analysis. The distributions of molecular properties of the compounds are shown in Figure
30.

110
Figure 30 The distribution of molecular properties of 312 compounds obtained after first phase of selection

111
4.5.2 DRUG-LIKENESS AND ORAL-BIOAVAILABILITY ANALYSIS

Quality measure, DL associates the pharmacokinetic and pharmaceutical properties of the

compounds. DL minimizes the time and cost of drug discovery process along with the development
of potent drugs. Hence DL is considered as a highly fruitful filter. OB another pharmacokinetic
parameter which computes the percent of oral dose reaching the systemic circulation to exhibit the
desired pharmacology action was also considered as a screening measure, since poor orally
bioavailable drugs fail to produce the required action. The compounds were screened for the
possession of desired ADMET properties. 126 compounds possessing required ADMET properties
along with satisfying DL, OB and skin permeability were finally retained for the further analysis.

4.5.3 TARGET FISHING

Due to the presence of numerous bio-active components in W. tinctoria, a major challenge lies in the
identification of molecular targets contributing to its pharmacological nature. The detection of the
targets aiding the bioactive components to perform the desired action will help in deciphering the
mechanism of action of the herb under molecular level. Among 126 compounds 67 compounds
(Table 30) were mapped to the 238 targets (Table 31) by STITCH database. 59 compounds without
any relevant targets were eliminated from further analysis. Minimum 1 and maximum 10 proteins
were targeted by the compounds. An average of 5 protein hits was maintained by the majority of the
compounds in the list revealing the promiscuous roles of few compounds.

112
 Table 30 Compounds with desirable molecular properties used in the study

Compound name ID Bioavail MW1 AlogP nHBA nHBD TPSA2 nRotBt nAR wQED Toxicity Skin
ability permi-
ability3
2,3-Dihydro-benzofuran C1 1 111.99 0.874 1 0 9.23 0 1 0.505 Neutral -1.91
1,2,3-Benzenetriol C2 1 119.98 -0.286 3 0 0 0 1 0.513 Neutral -1.50
3-O-Methyl-d-glucose C3 1 179.97 -2.382 6 0 26.3 6 0 0.514 Neutral -2.80
1,1,2,2-Tetramethyl-3- C4 1 375.99 3.595 1 0 0 0 0 0.514 Neutral -2.22
[(methyl)methylene]-8-
oxobicyclo[4.3.0]non-4(5)-ene
Phenol, 2,4-bis(1,1- C5 1 183.99 3.561 1 0 0 2 1 0.519 Neutral -0.73
dimethylethyl)
3-(1-Cyclopentyl)-1-propanol C6 1 111.99 -0.852 1 0 0 3 0 0.52 Neutral -2.18
4-Amino-1,5-pentandioic acid C7 1 169.99 -1.469 4 0 17.07 7 0 0.524 Neutral -2.35
3- C8 1 175.99 0.571 2 0 26.3 0 1 0.527 Neutral -2.53
Spirotetramethyleneisobenzofu
ran-1(3H)-one
1-Dodecanol, 3,7,11-trimethyl C9 1 195.99 0.549 1 0 0 10 0 0.529 Neutral -0.64
1-Methyl-2-cyano-3- C10 1 136.01 -0.57 2 0 27.03 1 0 0.539 Neutral -1.99
ethylpiperidine
3á,6-Dichloro--5-cholestene C11 1 392.34 3.706 0 0 0 5 0 0.546 Neutral -0.54
4,8,12-Trimethyltridecan-4- C12 1 223.99 1.507 2 0 26.3 8 0 0.558 Neutral -0.70

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olide
Ursa-9(11),12-dien-3-one C13 1 375.99 3.276 1 0 17.07 0 0 0.56 Neutral -1.62
Methane, oxybis[dichloro- C14 1 181.89 2.481 1 0 9.23 2 0 0.574 Neutral -1.62
Cyclopropaneoctanoic acid C15 1 184.15 -1.184 2 1 37.3 8 0 0.582 Neutral -1.36
Olean-12-en-3-ol, (3á)- C16 1 375.99 3.012 1 0 0 0 0 0.583 Neutral -1.22
Lupeol C17 1 426.39 3.231 1 1 20.23 1 0 0.584 Neutral -1.97
2,3-Di-O-methyl-D- C18 1 163.97 -1.186 5 0 27.69 2 0 0.586 Neutral -2.50
xylopyranose
3,4-Di-O-methyl-L- C19 1 163.97 -1.186 5 0 27.69 2 0 0.586 Neutral -2.60
arabinopyranose
9,19-Cyclolanost-24-en-3-ol, C20 1 363.99 3.487 1 0 0 3 0 0.591 Neutral -2.01
(3á)-
3-Keto-urs-12-ene C21 1 378.01 2.935 1 0 17.07 0 0 0.599 Neutral -1.91
4-Vinylphenol C22 1 120.06 1.352 1 1 20.23 1 1 0.6 Neutral -1.27
2(1H)Naphthalenone,3,5,6,7,8, C23 1 195.99 2.284 1 0 17.07 1 0 0.605 Neutral -0.93
8a-hexahydro-4,8a-dimethyl-
6-(1-methylethenyl)-
Benzoic acid, 4-ethoxy-, ethyl C24 1 179.98 1.221 3 0 35.53 5 1 0.608 Neutral -1.69
ester
Benzoic acid, ethoxy-, ethyl C25 1 179.98 1.221 3 0 35.53 5 1 0.608 Neutral -1.69
ester
Norolean-12-ene C26 1 428.37 2.477 2 2 40.46 0 0 0.613 Neutral -2.49

114
Benzoic acid, 3-hydroxy-, 1- C27 1 179.98 0.318 3 0 26.3 4 1 0.615 Neutral -1.52
methylpropyl ester
4-Vinyl-2-methoxy-phenol C28 1 139.99 0.854 2 0 9.23 2 1 0.616 Neutral -1.43
2-Methoxy-3-vinylphenol C29 1 139.99 0.854 2 0 9.23 2 1 0.616 Neutral -1.43
Isosteviol methyl ester C30 1 332.24 0.682 3 1 46.53 2 0 0.617 Neutral -2.32
2-Propenoic acid, 3-(2- C31 1 155.98 0.766 3 0 17.07 2 1 0.624 Neutral -1.68
hydroxyphenyl)-, (E)-
Bicyclo[3.2.2]nonane-1,5- C32 1 219.98 -0.631 4 0 43.37 4 0 0.624 Neutral -2.03
dicarboxylic acid, 5-ethyl ester
4-Hydroxy-2- C33 1 150.07 -0.072 1 1 37.3 1 1 0.626 Neutral -1.71
methylacetophenone
α-Amyrin C34 1 426.39 2.336 1 1 20.23 0 0 0.629 Neutral -2.10
Viminalol C35 1 426.39 2.366 1 1 20.23 0 0 0.629 Neutral -2.10
3,4-Dimethoxybenzene-1,2- C36 1 159.98 -0.72 4 0 18.46 2 1 0.637 Neutral -2.10
diol
Methyl commate B C37 1 375.99 2.366 1 0 0 0 0 0.642 Neutral -2.10
Urs-12-en-3-ol, (3á)- C38 1 375.99 2.366 1 0 0 0 0 0.642 Neutral -2.10
3-Isopropyl-6,7- C39 1 211.99 -0.406 2 0 0 1 0 0.644 Neutral -2.31
dimethyltricyclo[4.4.0.0(2,8)]d
ecane-9,10-diol
Stigmasta-3,5-dien-7-one C40 1 410.35 2.537 1 0 17.07 6 0 0.645 Neutral -0.64
Ethyl iso-allocholate C41 1 391.97 0.035 5 0 26.3 6 0 0.647 Neutral -2.55

115
2-Allyl-5-t-butylhydroquinone C42 1 206.13 2.471 2 2 40.46 3 1 0.648 Neutral -0.83
1,3,5-Triazine-2,4-diamine, 6- C43 1 164.98 -0.464 5 0 37.08 2 1 0.658 Neutral -3.08
chloro-N-ethyl- (CAS)
1,4-naphthoquinone, 5- C44 1 179.98 0.24 3 0 43.37 1 1 0.664 Neutral -2.14
methoxy-
Dicinnamamide, (E)- C45 1 147.07 0.934 2 1 43.09 2 1 0.667 Neutral -1.83
15- C46 1 187.99 1.475 2 0 18.46 1 1 0.667 Neutral -1.57
methyltricyclo[6.5.2(13,14).0(
7,15)]pentadeca-9,11,13-
heptene1,3,5,7,
1-methoxymethylthymine C47 1 170.07 -0.404 5 1 58.64 2 0 0.678 Neutral -2.45
Oleanolic acid C48 1 411.01 213 3 0 17.07 1 0 0.687 Neutral -2.35
Katonic acid C49 1 407.98 2.13 3 0 17.07 1 0 0.69 Neutral -2.34
Betulin C50 1 442.38 2.141 2 2 40.46 2 0 0.691 Neutral -2.5
8-amino-6-methoxy-2- C51 1 188.09 -0.783 2 1 48.14 1 2 0.692 Neutral -2.03
methylquinoline
2-Allyl-3,4- C52 1 191.98 0.851 3 0 35.53 5 1 0.728 Neutral -1.69
dimethoxybenzaldehyde
Desmosterol C53 1 384.34 2.826 1 1 20.23 4 0 0.73 Neutral -0.66
Pramipexole C54 1 193.98 -0.266 3 0 37.66 3 1 0.733 Neutral -2.48
Cycloeucalenol C55 1 426.39 1.949 1 1 20.23 5 0 0.736 Neutral -1.69
Indolo[2,1-b]quinazolin-6,12- C56 1 252 1.105 4 0 49.74 0 2 0.741 Neutral -2.45

116
dione, 8-methyl-
Stigmast-5-en-3-ol, (3á)- C57 1 414.39 1.3 1 1 20.23 6 0 0.742 Neutral -0.73
(CAS)
Á-Sitosterol C58 1 413.39 1.3 1 1 20.23 6 0 0.742 Neutral -0.59
Ethyl5,6,7,8-tetrahydro-2,7,7- C59 1 263.15 1.394 4 1 55.4 3 0 0.749 Neutral -2.48
trimethyl-5-oxo-3-
quinolinecarboxylate
Indigo C60 1 262.07 -0.272 4 2 58.2 0 2 0.751 Neutral -2.48
Stigmasterol C61 1 412.37 1.257 1 1 20.23 5 0 0.768 Neutral -0.71
Ergost-5-en-3-ol, (3á) C62 1 351.99 1.976 1 0 0 5 0 0.768 Neutral -0.62
Campesterol C63 1 400.37 1.976 1 1 20.23 5 0 0.769 Neutral -0.62
25-hydroxycholesterol C64 1 402.35 1.116 2 2 40.46 5 0 0.775 Neutral -1.22
3à,5-CYCLO-ERGOSTA- C65 1 351.99 2.25 1 0 17.07 4 0 0.778 Neutral -1.02
7,22-DIEN-6-ONE
Cholesterol C66 1 386.35 1.555 1 1 20.23 5 0 0.796 Neutral -0.64
Quercetin 7,3',4'-trimethoxy C67 1 327.96 -1.05 7 0 53.99 4 2 0.828 Neutral -2.24
1
Daltons; 2Å2, 3cm/h

Table 31 The information of 238 potential proteins targeted by the bioactive compounds

Target ID Gene symbol Gene name

T1 SEPT12 Septin-12
T2 ABCA1 ATP-binding cassette sub-family A member 1

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T3 ABCB1 Multidrug resistance protein 1
T4 ABCG5 ATP-binding cassette sub-family G member 5
T5 ABCG8 ATP-binding cassette sub-family G member 8
T6 ACP1 Low molecular weight phosphotyrosine protein phosphatase
T7 ADCK1 Uncharacterized aarF domain-containing protein kinase 1
T8 ADCK2 Uncharacterized aarF domain-containing protein kinase 2
T9 ADCK4 AarF domain-containing protein kinase 4
T10 ADCK5 Uncharacterized aarF domain-containing protein kinase 5
T11 AHR Aryl hydrocarbon receptor
T12 AKR1B10 Aldo-keto reductase family 1 member B10
T13 ALB Serum albumin
T14 ALLC Probable allantoicase
T15 ALOX15 Arachidonate 15-lipoxygenase
T16 ALOX15B Arachidonate 15-lipoxygenase B
T17 ALOX5 Arachidonate 5-lipoxygenase
T18 ANXA5 Annexin A5
T19 APOA1 Apolipoprotein A-I
T20 APOB Apolipoprotein B-100
T21 APOE Apolipoprotein E
T22 AR Androgen receptor
T23 ASAP1 Arf-GAP with SH3 domain, ANK repeat and PH domain-containing protein 1
T24 ASB6 Ankyrin repeat and SOCS box protein 6

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T25 ATP5C1 ATP synthase subunit gamma, mitochondrial
T26 ATP5H ATP synthase subunit d, mitochondrial
T27 ATP5O ATP synthase subunit O, mitochondrial
T28 ATP6V0A2 V-type proton ATPase 116 kDa subunit a isoform 2
T29 ATXN2 Ataxin-2
T30 ATXN2L Ataxin-2-like protein
T31 BAX Apoptosis regulator BAX
T32 BCL2 Apoptosis regulator Bcl-2
T33 BIRC2 Baculoviral IAP repeat-containing protein 2
T34 C10ORF2 Chromosome 10 Open Reading Frame 2
T35 C17orf88 Chromosome 17 Open Reading Frame 88
T36 C19ORF26 Chromosome 19 Open Reading Frame 26
T37 CA10 Carbonic anhydrase-related protein 10
T38 CA11 Carbonic anhydrase-related protein 11
T39 CA14 Carbonic anhydrase 14
T40 CA2 Carbonic anhydrase 2
T41 CA6 Carbonic anhydrase 6
T42 CABC1 Chaperone Activity Of Bc1 Complex-Like, Mitochondrial
T43 CASP3 Ataxin-2
T44 CASP8 Caspase-8
T45 CASP9 Caspase-9
T46 CAT Catalase

119
T47 CDA Cytidine deaminase
T48 CDC25A M-phase inducer phosphatase 1
T49 CENPH Centromere protein H
T50 CH25H Cholesterol 25-hydroxylase
T51 CHRM1 Muscarinic acetylcholine receptor M1
T52 CHST5 Carbohydrate sulfotransferase 5
T53 CHST6 Carbohydrate sulfotransferase 6
T54 CHST7 Carbohydrate sulfotransferase 7
T55 CLDN4 Claudin-4
T56 CNP 2',3'-cyclic-nucleotide 3'-phosphodiesterase
T57 COMT Catechol O-methyltransferase
T58 COMTD1 Catechol O-methyltransferase domain-containing protein 1
T59 CRH Corticoliberin
T60 CSPG5 Chondroitin Sulfate Proteoglycan 5
T61 CTSE Cathepsin E
T62 CYP11A1 Cholesterol side-chain cleavage enzyme, mitochondrial
T63 CYP17A1 Steroid 17-alpha-hydroxylase/17,20 lyase
T64 CYP19A1 Aromatase
T65 CYP1A1 Cytochrome P450 1A1
T66 CYP1A2 Cytochrome P450 1A2
T67 CYP21A2 Steroid 21-hydroxylase
T68 CYP27A1 Sterol 26-hydroxylase, mitochondrial

120
T69 CYP2B6 Cytochrome P450 2B6
T70 CYP2F1 Cytochrome P450 2F1
T71 CYP7A1 Cholesterol 7-alpha-monooxygenase
T72 CYP7B1 25-hydroxycholesterol 7-alpha-hydroxylase
T73 DHCR24 Delta(24)-sterol reductase
T74 DHCR7 7-dehydrocholesterol reductase
T75 DHRS2 Dehydrogenase/reductase SDR family member 2, mitochondrial
T76 DNAJC10 DnaJ homolog subfamily C member 10
T77 DRD1 D(1A) dopamine receptor
T78 DRD2 D(2) dopamine receptor
T79 DRD3 D(3) dopamine receptor
T80 DYNLT1 Dynein light chain Tctex-type 1
T81 EBP 3-beta-hydroxysteroid-Delta(8),Delta(7)-isomerase
T82 EBPL Emopamil-binding protein-like
T83 EGFR Epidermal growth factor receptor
T84 EN1 Homeobox protein engrailed-1
T85 ENSG000000232055 -
T86 ENSG000000232938 -
T87 ENSG00000184154 -
T88 ENSG00000197149 -
T89 ENSG00000198555 -
T90 ENSG00000214255 -

121
T91 ENSG0000022448 -
T92 ENSG00000228981 -
T93 ENSG00000233525 -
T94 ENSG00000234851 -
T95 EPM3 Epithelial Membrane Protein 3
T96 ERP27 Endoplasmic reticulum resident protein 27
T97 ESR1 Estrogen receptor
T98 ESR2 Estrogen receptor beta
T99 ETV3 ETS translocation variant 3
T100 FABP6 Gastrotropin
T101 FAM102A Protein FAM102A
T102 FAM123B APC Membrane Recruitment Protein 1
T103 FAM20C Extracellular serine/threonine protein kinase
T104 FDFT1 Farnesyl-Diphosphate Farnesyltransferase 1
T105 FDPS Farnesyl pyrophosphate synthase
T106 FECH Ferrochelatase, mitochondrial
T107 FOXF1 Forkhead box protein F1
T108 FRMPD2 FERM and PDZ domain-containing protein 2
T109 FYN Tyrosine-protein kinase Fyn
T110 GGPS1 Geranylgeranyl pyrophosphate synthase
T111 GNB2L1 Guanine nucleotide-binding protein subunit beta-2-like 1
T112 GP1BA Platelet glycoprotein Ib alpha chain

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T113 GPBAR1 G-protein coupled bile acid receptor 1
T114 GPR109A G Protein-Coupled Receptor 109A
T115 GRHL1 Grainyhead-like protein 1 homolog
T116 HEPH Hephaestin
T117 HMGCS1 Hydroxymethylglutaryl-CoA synthase, cytoplasmic
T118 HMGCS2 Hydroxymethylglutaryl-CoA synthase, mitochondrial
T119 HRH2 Histamine H2 receptor
T120 hs6M1-27 Olfactory receptor OR6-27
T121 HSD3B1 3 beta-hydroxysteroid dehydrogenase/Delta 5--4-isomerase type 1
T122 IAH1 Isoamyl acetate-hydrolyzing esterase 1 homolog
T123 IL12B Interleukin-12 subunit beta
T124 IPO7 Importin-7
T125 IPO8 Importin-8
T126 IQCJ IQ domain-containing protein J
T127 IRAKA4 Interleukin-1 Receptor-Associated Kinase 4
T128 KPNB1 Importin subunit beta-1
T129 KRT33B Keratin 33B, Type I
T130 KRT34 Keratin, type I cuticular Ha4
T131 KRT35 Keratin, type I cuticular Ha5
T132 KRT36 Keratin, type I cuticular Ha6
T133 KRT38 Keratin, type I cuticular Ha3-II
T134 LBR Lamin-B receptor

123
T135 LCAT Phosphatidylcholine-sterol acyltransferase
T136 LDLCQ3 3-Hydroxy-3-Methylglutaryl-CoA Reductase
T137 LDLR Low-density lipoprotein receptor
T138 LIAS Lipoyl synthase, mitochondrial
T139 LPL Lipoprotein lipase
T140 LSR Lipolysis-stimulated lipoprotein receptor
T141 LSS Lanosterol synthase
T142 MAF Transcription factor Maf
T143 MAPK14 Mitogen-activated protein kinase 14
T144 MARS Methionine--tRNA ligase, cytoplasmic
T145 MCCC1 Methylcrotonoyl-CoA carboxylase subunit alpha, mitochondrial
T146 MLN Promotilin
T147 MVK Mevalonate kinase
T148 MYBL1 Myb-related protein A
T149 NFE2L2 Nuclear factor erythroid 2-related factor 2
T150 NPC1 Niemann-Pick C1 protein
T151 NPC1L1 Niemann-Pick C1-like protein 1
T152 NR1H2 Oxysterols receptor LXR-beta
T153 NR1H3 Oxysterols receptor LXR-alpha
T154 NR3C2 Mineralocorticoid receptor
T155 OAS1 2'-5'-oligoadenylate synthase 1
T156 OR12D1P Olfactory Receptor, Family 12, Subfamily D

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T157 OR51A1P olfactory receptor, family 51, subfamily A, member 1 pseudogene
T158 OR51V1 Olfactory receptor 51V1
T159 OR52A4 Putative olfactory receptor 52A4
T160 OR52A5 Olfactory receptor 52A5
T161 OR7D4 Olfactory receptor 7D4
T162 OXSM 3-oxoacyl-[acyl-carrier-protein] synthase, mitochondrial
T163 PAPD7 Non-canonical poly(A) RNA polymerase PAPD7
T164 PARP1 Poly [ADP-ribose] polymerase 1
T165 PCCA Propionyl-CoA carboxylase alpha chain, mitochondrial
T166 PDE4A cAMP-specific 3',5'-cyclic phosphodiesterase 4A
T167 PDGFB Platelet-derived growth factor subunit B
T168 PDIA5 Protein disulfide-isomerase A5
T169 PDIA6 Protein disulfide-isomerase A6
T170 PDILT Protein disulfide-isomerase-like protein of the testis
T171 PDSS1 Decaprenyl-diphosphate synthase subunit 1
T172 PDSS2 Decaprenyl-diphosphate synthase subunit 2
T173 PGR Progesterone receptor
T174 POR NADPH--cytochrome P450 reductase
T175 PPARA Peroxisome proliferator-activated receptor alpha
T176 PPWD1 Peptidylprolyl isomerase domain and WD repeat-containing protein 1
T177 PRPS2 Phosphoribosyl Pyrophosphate Synthetase 2
T178 PTGER2 Prostaglandin E2 receptor EP2 subtype

125
T179 PTGIR Prostacyclin receptor
T180 PTGIS Prostacyclin synthase
T181 PTGS2 Prostaglandin G/H synthase 2
T182 PTGES Prostaglandin E Synthase
T183 PTPN13 Tyrosine-protein phosphatase non-receptor type 13
T184 PTPN2 Tyrosine-protein phosphatase non-receptor type 2
T185 PYGB Glycogen phosphorylase, brain form
T186 PYGL Glycogen phosphorylase, liver form
T187 PYGM Glycogen phosphorylase, muscle form
T188 RBM24 RNA-binding protein 24
T189 RHOG Rho-related GTP-binding protein RhoG
T190 RNASEL 2-5A-dependent ribonuclease
T191 ROBO4 Roundabout homolog 4
T192 RORC Nuclear receptor ROR-gamma
T193 RPL23A 60S ribosomal protein L23a
T194 RPL8 60S ribosomal protein L8
T195 RPS2P4 Ribosomal Protein S2 Pseudogene 4
T196 SART1 U4/U6.U5 tri-snRNP-associated protein 1
T197 SC5DL Sterol-C5-Desaturase
T198 SCAP Sterol regulatory element-binding protein cleavage-activating protein
T199 SCARB1 Scavenger receptor class B member 1
T200 SKP1 S-phase kinase-associated protein 1

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T201 SLC19A1 olute Carrier Family 19 (Folate Transporter), Member 1
T202 SLC22A15 Solute carrier family 22 member 15
T203 SLC47A1 Multidrug and toxin extrusion protein 1
T204 SLC6A1 Sodium- and chloride-dependent GABA transporter 1
T205 SLC6A11 Sodium- and chloride-dependent GABA transporter 3
T206 SLC6A12 Sodium- and chloride-dependent betaine transporter
T207 SLC6A13 Sodium- and chloride-dependent GABA transporter 2
T208 SLC6A6 Sodium- and chloride-dependent taurine transporter
T209 SLC6A8 Sodium- and chloride-dependent creatine transporter 1
T210 SLCO1B1 Solute carrier organic anion transporter family member 1B1
T211 SLCO1C1 Solute carrier organic anion transporter family member 1C1
T212 SNCA Alpha-synuclein
T213 SOAT1 Sterol O-acyltransferase 1
T214 SOAT2 Sterol O-acyltransferase 2
T215 SQLE Squalene monooxygenase
T216 SREBF2 Sterol regulatory element-binding protein 2
T217 SRR Serine racemase
T218 SULT1A1 Sulfotransferase family 1A member 1
T219 TADA2A Transcriptional adapter 2-alpha
T220 TBX22 T-box transcription factor TBX22
T221 TBXA2R Thromboxane A2 receptor
T222 TM7SF2 Delta(14)-sterol reductase

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T223 TMCO3 Transmembrane and coiled-coil domain-containing protein 3
T224 TMEM19 Transmembrane Protein 19
T225 TNPO1 Transportin-1
T226 TNPO2 Transportin-2
T227 TOP2A DNA topoisomerase 2-alpha
T228 TP53 Cellular tumor antigen p53
T229 TPMT Thiopurine S-methyltransferase
T230 TPSD1 Tryptase delta
T231 TRPA1 Transient receptor potential cation channel subfamily A member 1
T232 TSPO Translocator protein
T233 TXNDC16 Thioredoxin domain-containing protein 16
T234 TXNDC5 Thioredoxin domain-containing protein 5
T235 TXNIP Thioredoxin-interacting protein
T236 TYMP Thymidine phosphorylase
T237 VSX1 Visual system homeobox 1
T238 WDR18 WD repeat-containing protein 18

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4.5.4 COMPOUND TARGET NETWORK ANALYSIS

67 compounds along with their 238 potential target hits were mapped to generate a compound-target
bipartite graph (Figure 31). The network contained a total of 305 nodes (67- compounds and 238 -
proteins) and 328 edges. The connectivity distribution of the network measured by the centralization
parameter is found to be 0.026 and the network heterogeneity which reflects the tendency of a
network to incorporate various hub nodes was found to be 1.127. These parameters together indicate
that few compounds and proteins may be biased and act as key players in the C-T network.
Lanosterol synthase (LSS) (T141) has the highest number of compound connections while many
protein hits maintained a minimum of one connection with the active components in the compound
list. The degree of few nodes had a huge number of C-T interactions while many nodes had few or
smaller interactions. The result was consistent with the fore-mentioned centralization and
heterogeneity measure. In the compound list, 32 compounds possessed degree connectedness more
than 5. The average degree connectedness for the compounds is found to be 4 with 18 compounds
(C4, C12, C16, C26, C27, C29, C36, C42, C43, C48, C53, C56, C58, C61, C63, C64, C65 and C66)
possessing a maximum of 10 protein connections. When the interacting protein partners were
investigated we observed LSS (T141) established the maximum of 8 connections followed by
LDLCQ3 (T136) with 6 connections and AR (T22) with 5 connections. These highly connected
nodes were attributed as hubs of the network which were considered to play a significant role in
treating the disease or its associated effects.

Nodes with high betweenness score indicates that the node in certain paths is crucial in maintaining
the node connectivity. A node is considered significant if it incorporates many paths which link pairs
of nodes. 15 compounds (C7, C8, C14, C23, C30, C31, C36, C40, C42, C43, C44, C52, C60, C62
and C67) and 3 protein targets (T14, T116 and T141) were observed to have high betweenness value
of 1. When the network parameters were analyzed we noted that the degree and betweenness were
correlating with each other. The top three compounds (C36, C42 and C43) with high degree were
also observed to possess large betweenness.

129
Figure 31 A global perspective of compound – Target (C-T) network. The circular nodes and diamonds nodes denote the candidate compound
and targets for W. tinctoria respectively

130
4.5.5 C-T NETWORK: MECHANISM OF ACTION OF W. TINCTORIA

The investigation of network parameters revealed that the nodes in the network have multiple
connections establishing a synergistic path to exhibit its activity. The multi-compound, multi-target
mechanism acts as a background for governing the therapeutic efficiency of W. tinctoria.

Among the potential protein target hits, four proteins such as APOE (T21), CAT (T46), IL12B
(T123) and TP53 (T228) which were previously associated with psoriasis emerged as direct targets
for five compounds. Disruption of lipid metabolism is an important characteristic feature in the
pathogenesis of psoriasis. The continuous loss of lipids through the psoriatic lesions disrupts the
lipid homeostasis (Pietrzak et al, 2010). Lipid metabolism in the epidermis is tightly regulated by the
expression of the apolipoprotein E (APOE). Normal healthy skin secretes 85 mg of cholesterol
within a time period of 24 hours; on the contrary the psoriatic skin loses 1-2 g of cholesterol with the
scales during the same period (Al Harthi et al, 2014). The compounds C58 and C66 are the direct
controllers of APOE (T21) from the herb. α-sitosterol (C58) a structural homolog of cholesterol and
cholesterol (C66) itself were supplemented by the herb in order to compensate the lost component.
The plasma and erythrocytes of the psoriatic patients were reported to have increased levels of
catalase, CAT (T46). The anti-oxidant enzyme is involved in the reduction of hydrogen peroxide.
Pyrogallol (1, 2, 3 benzenetriol) (C2) is a generator of free radical and is involved in the suppression
of mouse lymphocytes in concentration dependent manner. The elevated expressions of CAT
enzyme in the psoriatic skin neutralize the free radicals through its scavenging activity (Willsteed
and Regan, 1985). Since psoriasis is a immune mediated disorder, the compound is found to
suppress the humoral immune response at high doses; hence prescribed as a topical remedy for
psoriasis. Pyrogallol is found to be the direct hit for CAT enzyme. The transcriptional factor p53
(T228) is an important regulator of cell cycle. The protein inhibits the cell division in response to
DNA damage and making time for DNA repair. The protein can also trigger apoptosis. An elevated
expression of p53 has been reported in both the lesional and un-involved skin parts of psoriasis
patients (Hannuksela-Svahn et al, 1999). In spite of elevated expression of pro-apoptotic p53,
apoptosis in psoriatic lesions remains impaired. Tryptanthrin derivative (C56) was detected as an
inhibitor of p53 protein in our analysis. Tryptanthrin has been reported to decrease the amount of
mutant p53 in MCF-7 cell line (Yu et al, 2007) . Psoriasis is a TH1-mediated disease with enhanced
expression of IL-12 in the psoriatic lesions. IL-12 stimulates the pathogenic inflammatory T cells
leading to the induction of psoriaform lesions in mice (Barrie and Plevy, 2005). The compound
benzoic acid, 3-hydroxy,1-methylpropyl ester (C27) is identified as a direct inhibitor for the IL-12
(T123). Pyrogallol (C2) has been proven to induce the activity of Caspase 3 (T43) and Caspase 8
(T44) thus leading to the activation of apoptosis via the mitochondrial pathway (Park et al, 2007).

131
The compounds arrested the apoptosis induction via p53 but induced the same process via Caspase 3
(T43) and Caspase 8 (T44).

4.5.6 INVOLVEMENT OF TARGETS IN THE IMPAIRED BIOLOGICAL PROCESS IN

PSORIASIS

The careful assessment of the network revealed that the number of targets interacting with the herbal
compounds was found to be involved in major biological processes which were impaired in
psoriasis. The epidermal keratinocytes in psoriatic lesions undergo hyperproliferation with
incomplete differentiation and decreased apoptosis along with the formation of Munro’s
microabscesses. Proteins related to keratinocyte proliferation such as FYN (T109), SART1 (T196)
and Top2A (T227) were observed in the target list. TOP2A (topo-isomerase 2α) implicated in DNA
strand repair mechanism is found to be elevated in rapidly proliferating cells. The inhibitors of the
enzyme arrest the protein-DNA cleavable complex leading to DNA strand breaks finally to cell
death (Syrovets et al, 2000). SART1 encoding SART1(800) expression is elevated in highly
proliferating cells. The silencing of SART1 has been reported to induce apoptosis in Caspase 8
dependent manner (Allen et al, 2012). Fyn, a non-receptor tyrosine kinase plays a major role in
epidermal keratinocyte differentiation and transformation. Several studies suggests that knockdown
of Fyn disrupts the T cell receptor-induced activation of mature T-cells (Appleby et al, 1992).
Proteins promoting apoptosis such as CASP3 (T43) and CASP8 (T44) also appeared in the target
list. The hyperproliferating keratinocytes is supplied with rich blood vessels contributed by proteins
involved in angiogenesis such as Platelet-derived growth factor receptor (PDGF-β) (T167) which is
validated by the enhanced expression of PDGF-β in the psoriatic lesions (Krane et al, 1991). Another
protein involved in angiogenesis is Thymidine phosphorylase (TYMP) (T236) a key factor in
controlling the vascular growth. The protein is attributed to be identical to platelet derived
endothelial cell growth factor (PD-ECGF), however the mechanism by which it induces the
angiogenesis has not been elucidated completely (Mitselou et al, 2012).

4.6 IDENTIFICATION OF MOLECULAR MECHANISM OF SIDDHA ORAL

MEDICATIONS FOR PSORIASIS VULGARIS

Herbal concoctions contain various active components which hit on multiple biological targets
concerned in the pathogenesis of the disease. The presence of numerous chemical constituents
provides a challenge to analytical chemist and pharmacologist. The current work uses a systems
approach to study the Siddha formulation to understand the mechanism of action of the bioactive
constituents.

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4.6.1 HERBAL INGREDIENTS COMPOSITION

The ingredients of the PC were screened to mine out the bioactive components present in them. A
total of 155 compounds were obtained after structural verification and duplicate elimination. The
rhizome of S. china enclosed around 46 compounds, I. aspalathoides contained 35 compounds, E.
littorale enclosed 19 compounds, root bulb of A. tetracantha enclosed 7 compounds, E. antiquorum
and E. tirucalli contributed 24 compounds independently (Table 32).

Table 32 155 compounds from the Parangichakkai chooranam formulation and their corresponding
predicted oral bioavailability (OB) class and drug-likeness (DL) scores

ID Molecule name OB DL
Class
SC1 (2R,3S)-2-(3,5-dihydroxyphenyl)chroman-3,5,7-triol 1 0.661
SC2 (2S,3R)-3,5,7-trihydroxy-2-(4-hydroxyphenyl)chroman-4-one 1 0.638
SC3 3,4,5-trihydroxybenzoic acid 1 0.544
SC4 5-[(Z)-2-(3,4-dihydroxyphenyl)vinyl]resorcinol 4 0.655
SC5 Astilbin 4 0.205
SC6 β-sitosterol 1 0.617
SC7 Caffeic acid 6 0.655
SC8 Cinchonine 10 0.926
SC9 Cis-Dihydroquercetin 7 0.488
SC10 Cis-resveratrol 5 0.526
SC11 Coumarin 3 0.548
SC12 Cudranin 10 0.768
SC13 Dihydrokaempferol 3 0.638
SC14 Diosgenin 8 0.729
SC15 Engeletin 1 0.289
SC16 Ergosterol 1 0.628
SC17 Gramin 7 0.687
SC18 Isoengelitin 1 0.589
SC19 Moracin M 6 0.656
SC20 Oleanolic acid 3 0.701
SC21 Oxyresveratrol 10 0.974
SC22 Palmitic acid 2 0.36
SC23 Piceid 6 0.319

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SC24 Protocatechuic acid 3 0.531
SC25 Resveratrol 3 0.744
SC26 Sitogluside 3 0.523
SC27 Taxifolin 6 0.488
SC28 Vanillic acid 1 0.759
SC29 Yamogenin 2 0.729
SC30 Epicatechin 5 0.545
SC31 2',6'-triacetyl-3,6-diferuloylsucrose 2 0.063
SC32 6-[(1R)-3-methoxy-3-oxo-1-(2,4,5- 1 0.123
trihydroxyphenyl)propyl]catechin
SC33 6-[(1S)-3-methoxy-3-oxo-1-(2,4,5- 1 0.123
trihydroxyphenyl)propyl]catechin
SC34 8-[(1R)-1-(3,4-dihydroxyphenyl)-3-methoxy-3-oxopropyl]-3- 1 0.182
epicatechin
SC35 8-[(1R)-1-(3,4-dihydroxyphenyl)-3-methoxy-3- 1 0.182
oxopropyl]catechin
SC36 Catechin-(7,8-bc)-4beta-(3,4-dihydroxyphenyl)dihydropyran- 1 0.241
2(3H)-one
SC37 Cinchonain 1a 4 0.289
SC38 Cinchonain 1c 4 0.289
SC39 Cinchonian 1b 4 0.289
SC40 Helonioside B 2 0.0062
SC41 Malic acid 7 0.567
SC42 Neotigogenin-3-O-α-L-rhamnopyranosyl(1→6)-β-D- 2 0.146
glucopyranoside
SC43 Neotigogenin-3-O-β-D-glucopyranosyl(1-4)-O-[α-L- 3 0.068
rhamnopyranosyl(1-6)]-β-D-galactopyranoside
SC44 Smiglaside E 1 0.047
SC45 γ-Methylene glutamic acid 2 0.589
SC46 Chlorogenic acid 8 0.267
IA1 (-)-Spathulenol 3 0.555
IA2 3-Acetoxy-bisnor-5-cholenic acid 1 0.694
IA3 Alloaromadendren 6 0.355
IA4 α-amorphene 2 0.365

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IA5 α-amyrin 1 0.537
IA6 α-caryophyllene 3 0.241
IA7 β-Bisabolene 3 1
IA8 Caffeic acid 6 0.655
IA9 Caryophyllene oxide 5 0.465
IA10 Catechin 5 0.545
IA11 Copaene 3 0.382
IA12 Dehydroisoandrosterone acetate 3 0.685
IA13 Dodecanoic acid 3 0.467
IA14 Ellagic acid 5 0.416
IA15 Erythroxydiol X 1 0.825
IA16 Ethyl iso-allocholate 10 0.63
IA17 Ferulic acid 4 0.802
IA18 3,4,5-trihydroxybenzoic acid 1 0.544
IA19 Palmitic acid 2 0.36
IA20 Mucronulatol 1 0.925
IA21 Stearic acid 2 0.32
IA22 Phyllocladene 4 0.39
IA23 Phytol 4 0.496
IA24 Prasterone 1 0.748
IA25 Pregnanetrial 9 0.747
IA26 Quercetin 5 0.467
IA27 Resorcinol 3 0.601
IA28 Rimuene 4 0.396
IA29 Rutin 1 0.08
IA30 Salicylic acid 4 0.699
IA31 Stigmasterol 5 0.633
IA32 Tetradecanoic acid 3 0.409
IA33 Tau-cadinol 4 0.59
IA34 Vanillin 6 0.738
IA35 Beta-sitosterol 1 0.617
EL1 Gentianine 7 0.652
EL2 Apigenin 3 0.755
EL3 Genkwanin 4 0.88

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EL4 Isovitexin 4 0.229
EL5 Swertisin 4 0.255
EL6 Myristic acid 3 0.409
EL7 Stearic acid 2 0.32
EL8 Oleic acid 4 0.426
EL9 Betulin 2 0.707
EL10 Vanillic acid 5 0.759
EL11 Syringic acid 5 0.791
EL12 P-hydroxy benzoic acid 10 0.699
EL13 Protocathechuic acid 3 0.632
EL14 P-coumaric acid 5 0.749
EL15 Ferulic acid 4 0.802
EL16 Myricetin 2 0.35
EL17 3,3’-methylenebis (4-hydroxycoumarin) 10 0.426
EL18 Catechin 5 0.545
EL19 Erythrocentaurin 5 0.594
AT1 3-Indolylmethylglucosinolate 4 0.184
AT2 Myricetin 2 0.35
AT3 Quercetin 5 0.467
AT4 Rutin 1 0.08
AT5 Isorhamnetin 6 0.602
AT6 Rhamnazin 5 0.712
AT7 1-Methoxy-3-indolylmethyl-glucosinolate 0 0.212
EA1 24-Methylene-4,4,14-trimethyl-Delta8-sten-3beta-ol 1 0.501
EA2 3β-taraxerol 1 0.437
EA3 Antiquol B 1 0.442
EA4 Antiquol C 1 0.408
EA5 β-sitosterol 1 0.617
EA6 Boeticol 1 0.442
EA7 Camelliol C 1 0.261
EA8 Cycloartanol 4 0.565
EA9 Cycloeucalenol 5 0.61
EA10 Euphadienol 1 0.443
EA11 Euphol 5 0.443

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EA12 Euphorbol 1 0.501
EA13 Euphorbyl-acetate 1 0.4
EA14 Friedelanol 2 0.538
EA15 Fumaric acid 2 0.626
EA16 Lemmaphylla-7,21-dien-3β-ol 1 0.419
EA17 O-Acetyl-euphorbol 1 0.384
EA18 O-acetyl-tirucallol 1 0.349
EA19 Olean-12-ene-3β-ol acetate 1 0.429
EA20 Taraxerol 4 0.437
EA21 Taraxerone 1 0.395
EA22 Tirucallol 1 0.443
EA23 Tirucallol acetate 1 0.349
EA24 β-Amyrin 2 0.484
ET1 Quercitrin 1 0.187
ET2 Lanosterol 5 0.443
ET3 Cyanin 1 0.065
ET4 12-O-2Z,4E-Octadienoyl-4-deoxyphorbol-13-acetate 1 0.35
ET5 Cycloeuphordenol 4 0.613
ET6 Cyclotirucanenol 4 0.567
ET7 Casuarinin 1 0.037
ET8 Euphadienon-3 1 0.365
ET9 Euphorbia factor Ti2 1 0.322
ET10 Euphorbia factor Ti5 1 0.281
ET11 Euphorbia factor Ti6 1 0.26
ET12 Euphorbia factor Ti7 1 0.22
ET13 Euphorbia factor Ti8 1 0.2
ET14 Euphorbia factor Ti9 1 0.22
ET15 Euphorbinol 1 0.457
ET16 Euphorginol 1 0.457
ET17 Geranin 1 0.53
ET18 Maculaniol 1 0.538
ET19 Neochlorogenic acid 2 0.267
ET20 Taraxerone 1 0.354
ET21 Tellimagrandin-II 1 0.039

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ET22 Tirucallol 1 0.443
ET23 Tinyatoxin 1 0.344
ET24 Euphorbol 1 0.457

4.6.2 MOLECULAR PROPERTY COMPARISON – ADMET EVALUATIONS

RO5 provides a group of guidelines which identifies the crucial properties favouring oral absorption
of drugs. The rule also emphasizes potential bioavailability issues when two or more rules are
violated. The cut-off scheme was determined for all the components (Figure 32). The size of the
molecule determines its molecular weight. An increased size of the molecule requires a large cavity
to be formed in water to promote its solubilisation leading to decreased solubility. The average MW
of the compounds ranged between 626.36 and 293.85 Da. Majority of the bioactive components
possessed favourable MW (< 500). An increased logP decreases aqueous solubility of the compound
which in turn reduces the absorption. The bioactive compounds from all the plants had favourable
AlogP (< 5). The average AlogP ranged between -3.17 and 2.70. Only three compounds 9-
Nonacosene, Camelliol C and Euphol 3-O-cinnamate showed AlogP above the cutoff. The hydrogen
bonds increase the aqueous solubility of the compounds. The Hbonds in the compounds should be
broken down in order to permeate the lipid membrane. The average nHBDon ranged between 1 and
5. The average nHBAcc ranged between 2 and 11. The molecular flexibility of the compounds was
assessed using nRotB. As suggested by Veber et al, nRotB influence the uptake of drugs by lumen.
The average nRotB ranged between 3.33 and 6.52. Veber also suggested TPSA as an indicator for
permeation. Crossing the lumen and uptake by lipid bilayer requires smaller TPSA (< 140 Å2). The
average TPSA values ranged between 35.60 and 189.01 Å2.

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 Figure 32 Distribution of molecular properties of the PC compounds

Acute toxicity studies categorize the compounds with respective to its relative toxicity values. The toxicity of the individual compounds was assessed
based on the LD50 value predicted by the pkCSM webserver. The average LD50 values of the compounds were found to be 2.14 mol/kg (SC), 2.25
mol/kg (IA), 2.11 mol/kg (EL), 2.41 mol/kg (AT), 2.60 mol/kg (EA) and 2.36 mol/kg (ET). The toxicity prediction by the server indicates that the
compounds possess nil/less oral toxicity.

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4.6.3 ORAL BIOAVAILABILITY AND DRUG LIKENESS EVALUATION

The micro-average and macro-average F1 scores of the classifier were 0.77 and 0.64, respectively
proving the efficiency of the multi-label classifier. To further validate the efficiency of the classifier
we compared the predicted OB percentage from our dataset with few known compounds from
TCMSP. For instance quercetin had 46.43% of OB value in TCMSP database the same compound
was predicted to fall in the 5th category by our classifier. Similarly for resveratrol, the database value
is 19.07% and but our classifier assigned to 3rd category. Oleic acid has an OB of 33.13% in TCMSP
and our classifier categorized the compound to 4th class. Similarly the other available compounds
were also verified. Majority of the compounds fell in category 1 revealing that the compounds with
less oral bioavailability dominate the list. Though majority of the compounds had poor oral
bioavailability, the compounds such as quercetin, salicylic acid, swertisin were identified to have
favourable OB which aid in the mediation of desired functions.

The drug-likeliness of the compounds assessed by QED scores revealed that the compounds with
high scores (≥ 0.5) showed less violations and good OB%. Majority of the compounds in the plants
showed QED score ≥ 0.5. When the individual molecular properties were compared two key
properties MW and AlogP were found to control the QED scores.

4.6.4 FUNCTIONAL PRIORITIZATION OF TARGETS

To identify the functional targets of the compounds, a target retrieval strategy which utilizes mining
of targets from databases (HIT, PCIDB, UNPD and TCMSP), experimental interactions along with
structural similarity (STITCH and BindingDB) and broad literature search was employed. A total of
155 compounds were mapped to 583 targets. The above scheme mapped 46 compounds from SC to
331 targets, 35 compounds from IA mapped to 294 proteins, 19 compounds from EL mapped 225
proteins, 7 compounds from AT were mapped to 193 protein targets, 24 compounds from EA
mapped 98 protein targets and 24 compound from ET mapped 55 protein targets.

When the family classes of the target proteins were investigated using PANTHER (Thomas et al,
2003), the receptor class (PC00197) which includes G-protein coupled receptors, cytokine receptors,
ligand-gated receptors, nuclear hormone receptors and protein kinase receptors dominated the list in
SC, IA, AT and EA. The proteins which are grouped under receptor were known to be implicated in
inflammation, keratinocyte migration and differentiation such as G-protein coupled receptors -

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CCR2, C5AR1 and androgenic receptor α/β along with nuclear hormone receptors - PPAR α/γ/δ.
While transferases, comprising acetyl-transferase, acyl-transferase, glycosyl-transferase, kinase,
methyl-transferase, nucleotidyl-transferase, phosphorylase, transaldolase, transaminase and
transketolase occupied the top protein class in EL and ET. Mediators involved in transmission of
extra cellular immune signals located on the cell surface and cytoplasm of innate immune cells such
as JAK1, MAPKs, SRC kinase family members and PKCs were enriched in transferase class.

4.6.5 NETWORK ANALYSIS

With increasing knowledge of the patho-mechanisms of complex diseases, the “one-target, one-
drug” paradigm has been shifted to “multi-target, multi-drug” theory. The application of multi-
component hypothesis has been implemented with the help of network theory. The network
medicine approach aids in understanding of the target-drug interaction through the visualization and
parametric analysis of the networks.

4.6.5.1 COMPOUND-TARGET NETWORK (C-T)

To understand the relationship between the compounds of PC and their respective targets a
compound-target network was constructed. After redundancy removal, the compound-target network
was constructed by connecting 155 compounds with 577 protein targets. The network enclosed a
total of 732 nodes and 2034 edges with an average degree of 3.52 nodes per target and 13.12 edges
per compound (Figure 33). The global network architecture is maintained by the highly connected
nodes namely the hubs. When the compound list was investigated to locate the hubs, resveratrol
from SC ranked first establishing 155 connections with its protein targets followed by quercetin from
IA and AT establishing 140 connections, apigenin from EL establishing 90 connections and caffeic
acid from SC establishing 62 connections with the targets. The target list was also investigated to
identify the highest number of targets shared by the compounds. The highest number of targets
(n=169) were shared by SC and IA. While the least number of targets (n=11) were shared by the
assistant drugs EA and ET. COX2 was targeted by 67 compounds in the list followed by COX1, AR
and NCOA2 with 49, 31 and 30 associated compounds respectively. Thus, indicating that many
protein targets in psoriasis might be inter-connected and show similar binding pattern with their
respective compounds. The above C-T network analysis reveals that various compounds can interact

141
with the same target and many targets interact with more than one compound composing multi-
compound targets and multi-targeted compounds respectively.

The systemic drugs prescribed for the treatment of psoriasis are summarized along with their targets
from Drugbank (Wishart et al, 2008). The drug targets were compared to understand the
commonality between the C-T and D-T. A total of 43 drugs and 125 targets were obtained. Around
34 targets were shared by the compounds and systemic drugs suggesting that the compounds may
also act through similar mechanism as the systemic therapeutics.

Figure 33 A C-T network connecting 155 compounds and 577 protein targets. Pink – SC, green
– IA, indigo – EL, yellow – AT, orange – EA and blue – ET. The cyan nodes denote the protein
targets.

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4.6.6 ROLE OF PHYTOCOMPOUNDS IN CONTROLLING DISEASE MEDIATORS

The major players which maintain the network integrity by controlling the key targets of the disease
were investigated. Resveratrol which establishes highest number of connections possess potent
antioxidant, anti-inflammatory and anti-proliferative properties. The anti-proliferative effect of
resveratrol was investigated by many groups. Resveratrol is found to inhibit the proliferation of
keratinocytes even at submolar level. Previous investigations suggest that the compound controls
proliferation by acting through SIRT1/ARNT/ERK dependent pathway. Resveratrol binding
activates SIRT1 and induces a conformational change in the enzyme. The compound antagonizes
AhR activity by binding to it, however the resveratrol/AhR complex translocates to nucleus and
dimerizes with ARNT. A cross talk between ARNT and ERK phosphorylation has been reported by
the scientific community (Figure 34). ARNT influence AREG expression and the downstream
EGFR–ERK pathway in keratinocytes thereby controlling the expression of epidermal differentiation
genes (Wu et al, 2014).

Figure 34 A working model of the mechanism of resveratol inhibition

Inflammation is a prime feature associated with psoriasis which includes the expression of
inflammatory cytokines, chemokines and other immune mediators. Activated MAPK phosphorylate
downstream protein kinases and transcription factors triggering the production of pro-inflammatory
cytokines. Genkwanin a non-glycosylated flavonoid is reported to decrease the LPS-induced
phosphorylation of p-38 and JNK conversely increase the MKP-1 expression at the post-translational
level. Reverse activation of MAPK requires phosphorylation of p38, JNK and ERK1/2. MKP-1, a
negative regulator of macrophage signaling responds to inflammatory stimuli by dephosphorylating

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activated p38, JNK and ERK1/2 thereby turning off the production of proinflammatory cytokines by
AP-1 signaling pathway (Figure 35) (Gao et al, 2014).

Figure 35 A working model of the mechanism of inhibition of genkwanin. The components

marked with the star symbol indicates the targets of genkwanin

Apigenin is reported to possess inhibitory effects against adhesion molecules (ICAM, VCAM)
prostaglandin (PG) E2, cyclooxygenase (COX2) and proinflammatory cytokines (IL6). The
inflammatory mechanism is arrested by apigenin mediated through non-canonical pathway. LPS-
induced activation of the downstream signallers was inhibited by apigenin by blocking the IKK
kinase activity and inhibiting the phosphorylation of p65 subunit, hence switching off the NF-κB
expression (Yin et al, 2001).

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Dendritic cell (DC) and T cell interactions play a crucial role in mediating the inflammation in
psoriasis. Quercetin, the polyphenolic flavonoid suppresses the DC activation through the inhibition
of phosphorylation of ERK and JNK. The LPS induced DCs were degraded by the blockage of Akt
and I-κB. The compound also inhibits macrophage activation by disrupting STAT1 expression,
blocking the IFNβ signaling involving TBK1 and disturbing the lipid rafts accumulation (Huang et
al, 2010; Li et al, 2016).

4.6.7 DISEASE-TARGET NETWORK (D-T)

The disease target network was constructed on the basis of targets mined from the databases and
microarray studies. The network enclosed psoriasis related proteins and their related PPIs. The D-T
network was annotated with the gene expression data. The network enclosed 1891 nodes and 2905
edges (Figure 36).

The targets mined out from C-T network were mapped to the D-T network to check their
involvement in the disease. Around 17 targets showed direct interactions which were proven targets
of psoriasis while 200 targets were found to act via indirect interactions mediated by the partner
proteins (Table 33).

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Figure 36 A D-T network bridging 1891 nodes and 2905 edges. The circular nodes indicate proteins
and triangular nodes indicate shared protein targets from mined from various approaches. Pink –
targets from microarray studies, green – common targets from disgenet and microarray studies,
orange – common between compound and drugbank targets, red- common between compound and
disgenet targets and yellow - common between compound and microarray studies targets

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 Table 33 The direct and indirect controllers of the disease identified from D-T network

Direct interactions Indirect interactions

CA6, CDKN2A, IFNG, ABCB1, ABCC2, ACPP, ADA, ADRA1B, ADRA2A, ADRA2B, ADRA2C,
IL10, IL13, IL6, ITGB2, AGTR1, AHR, AKR1C1, AKT1, ALOX5, AOX1, APC, AR, BAD, BAK1,
LYZ, MAPK1, MCL1, BAX, BBC3, BCL2, BCL2L1, BCL2L11, BDNF, BMP2, BRCA1, BRCA2,
MGST1, NFKB1, BSG, BTK, CALM1, CASP1, CASP3, CASP8, CASP9, CCNA2, CCNB1,
NFKBIA, STAT1, TGFB1, CCND1, CCNE1, CCR2, CD28, CD40, CD80, CD86, CDA, CDC25A,
TNF, TYRP1 CDK1, CDK2, CDK4, CDK6, CDK7, CDKN1A, CFLAR, CHEK2, CHUK,
CLDN4, COL1A1, COL2A1, CREB1, CRP, CRTC2, CTNNB1, CTSD,
CXCL11, CYP19A1, CYP1A1, CYP1A2, CYP2C9, CYP3A4, DDIT3,
DNMT1, E2F1, EGFR, EIF2S1, ELK1, ERBB2, ESR1, ESR2, F10, F2, F7,
FCER2, FGF2, FOS, FOXO1, FYN, GABBR1, GJA1, GSK3B, HCK,
HEY2, HIF1A, HSF1, HSP90AA1, HSPA5, HTR2A, ICAM1, IGF1R, IGF2,
IGFBP3, IGHG1, IKBKB, IKBKG, IL2, INS, INSR, IPO7, IRF1, IRS1,
ITGB1, JAK1, JUN, LCK, LRP1, LYN, MAP2, MAP2K1, MAPK11,
MAPK14, MDM2, MMP13, MMP2, MMP3, MMP9, MPO, MYC, NCF1,
NCOA1, NCOA2, NFE2L2, NOS1, NOS2, NOS3, NR1H3, NR1I2, NR1I3,
NR3C1, NR3C2, PCYT1A, PGR, PIK3CG, PLA2G4A, PLAT, PLG,
PMAIP1, PON1, POR, PPARA, PPARD, PPARG, PRKCA, PRKCB,
PRKCD, PRKCE, PRKCG, PRKCH, PRKCZ, PRKD1, PROC, PRSS1,
PTPN1, RAC1, RAF1, RASA1, RASSF1, RB1, RELA, RUNX1T1, RXRA,
SELP, SERPINE1, SHBG, SIRT1, SIRT2, SLC22A6, SLC2A4, SLC6A4,
SMAD1, SORD, SP1, SPARC, SPP1, SRC, SREBF1, SREBF2, STAT3,
SYK, TBXA2R, TGFB2, TP53, TPI1, TPM1, TPO, TRAF2, TYR, UCP2,
UCP3, VAV1, VCAM1, VEGFA, XRCC6

A total of 217 overlapping targets were submitted to the CTD server (Davis et al, 2015) to check
their alliance with psoriasis associated comorbidities. 15 comorbidities shared their proteins with our
compounds targets (Table 34). The highest number of targets was shared by diabetes mellitus (49),
followed by hypertension (38) and obesity (24).

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 Table 34 Proteins shared between psoriasis and its associated comorbidities
Sl. Comorbidities Proteins involved
No
1 Alzheimer's BAX, BCL2, BDNF, CALM1, CASP3, EIF2S1, ESR1, F2,
Disease GSK3B, IGF1R, IGF2, INS, MPO, NOS3, PPARG, TNF, TPI1,
VEGFA
2 Atherosclerosis AHR, ALOX5, CRP, ESR1, ICAM1, IFNG, IGF2, IL6, NOS2,
NOS3, PLAT, PON1, PPARG, SERPINE1, SIRT1, STAT3, TNF,
TNF, VCAM1, VEGFA
3 Cardiovascular CRP, ICAM1, MPO, NOS3, PON1, VCAM1
Diseases
4 Myocardial BAX, BCL2, BCL2L1, BRCA1, CREB1, ESR1, F2, GSK3B,
Infarction ICAM1, IL10, IL6, MMP2, MMP9, NOS2, NOS3, NR3C2, PLAT,
PRKCE, TGFB1, TNF
5 Autoimmune CD28, CD40, IFNG, IL6, SIRT1, STAT3
Diseases
6 Crohn’s Disease CRP, IFNG, IL10, IL6, PPARA, PPARG, TNF
7 Depressive BDNF, FGF2, GSK3B, IL6, NOS1, NR3C1, PON1, SLC6A4,
Disorder HTR2A, IL2, SLC6A4
8 Dermatitis BCL2, CASP8, CCR2, CYP1A1, FYN, IFNG, IL10, IL13, IL2,
IL6, ITGB2, NFE2L2, PLA2G4A, PLAT, PPARA, TNF
9 Diabetes Mellitus AR, BAX, BCL2, BCL2L1, CASP3, CASP8, CRP, CYP19A1,
CYP1A1, CYP1A2, DDIT3, EGFR, FGF2, ICAM1, IFNG, IL10,
IL6, INS, INSR, IRS1, MMP2, MMP9, MPO, NCF1, NFKB1,
NOS1, NOS2, NOS3, NR1I2, NR1I3, PON1, PPARA, PPARG,
PRKCA, PRKCB, PRKCD, PRKCE, PTPN1, RELA, SERPINE1,
SHBG, SIRT1, SLC2A4, SREBF1, STAT3, TGFB1, TNF, UCP2,
VEGFA
10 Fatty Liver CYP19A1, F2, INS, MYC, NR1H3, NR1I2, PPARA, PPARD,
SERPINE1, SIRT1, SREBF1, TNF, UCP2, PPARA
11 Hypertension ADRA2A, AGTR1, AHR, AR, BCL2, COL1A1, CRP, CYP1A1,

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 FOS, GJA1, HIF1A, ICAM1, INS, MMP2, NCF1, NOS2, NOS3,
NR3C1, PLAT, PLG, PPARA, PPARG, PRKCD, PROC, RELA,
SELP, SERPINE1, TGFB1, TNF, TP53, TPM1, UCP2, VCAM1,
NR3C2, VEGFA, ALOX5, CD40, SLC6A4
12 Leishmaniasis CRP, IFNG, IL10, IL6, TNF, IFNG, IL10, MMP2, TNF, IFNG,
CRP, IFNG, IL10, IL6, TNF
13 Obesity AKT1, CASP1, ESR1, F2, HTR2A, ICAM1, IGF2, IL6, INS, IRS1,
MMP9, NR1I2, NR1I3, PPARA, PPARD, PPARG, PRKCH,
SERPINE1, SIRT1, SREBF1, TNF, UCP2, UCP3, BDNF
14 Parkinson Disease ABCB1, BDNF, IGF1R, IGF2, IL6, INS, INSR, TNF, PRKCD
15 Pulmonary CYP1A1, CYP1A2, HTR2A, MMP9, NOS2, NOS3, TGFB1, TNF,
Disease, Chronic TP53
Obstructive

4.6.8 COMORBIDITIES LINKS – TARGETS OF THE PHYTOCOMPOUNDS

Various epidemiologic studies have demonstrated that psoriasis and its associated comorbidities
share common etiological linkage. Inflammatory mediators play a crucial role in connecting the
comorbidities together. Few common threads connecting psoriasis with its comorbidities and revival
by the compounds of PC were investigated.

Psoriatic skin and blood shows higher expression of Th-1 inflammatory cytokine TNF-α implicated
in the recruitment of T cells. Insulin resistance, a common thread connecting psoriasis, obesity and
diabetes mellitus may be mediated through TNF. TNF can promote insulin resistance through
various pathways such as impairing insulin signaling through the inhibition of insulin receptor
phosphorylation, activation of proliferator-activated receptor δ (PPARδ) leading to the promotion of
epidermal proliferation and glucose metabolism. The inflammation in psoriasis also leads to
increased expression of IGF-II in psoriasis. The protein is implicated in epidermal proliferation and
lipid metabolism in diabetes and hyperlipidemia (Romanowska et al, 2008).

Over expression of intracellular adhesion molecules such as ICAM-1 and VCAM-1 were observed in
the pro-inflammatory conditions such as psoriasis and atherosclerosis. The expressions of these

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endothelial molecules were triggered by NFκB which in turn is activated by the cytokine signaller
TNF. The inflammatory events which include migration, extravasation and infiltration of leukocytes
are mediated by these cell-cell/ cell-extracellular matrix adhesion molecules. These molecules also
mediate the transfer of leukocytes via endothelial barrier which were upregulated in psoriatic and
atherosclerotic lesions (Flammer and Ruschitzka, 2012).

The neurotrophin BDNF (Brain derived neurotrophic factor) is associated with regulation of
neuronal activity and synaptic actions related to neuronal plasticity. A cross sectional study with 94
psoriatic patients reported significantly lower levels of BDNF (Brunoni et al, 2015). The
psychological stress in terms of depression in psoriatic patients activates the neuroinflammatory
cytokines hence decreasing the levels of BDNF. A significant decrease of BDNF protein levels was
reported in the hippocampus and parietal cortex of the brain in Alzheimer disease. Decreased
production of BDNF leads to the reduction of neurotrophic support of cholinergic and 5-
hydroxytryptamine-containing neurons in process of neurodegeneration of Alzheimer’s (Hock et al,
2000). The neurotrophin BDNF levels are altered by dietary intake of omega-3 fatty acids such as
myristic acid, palmitic acid, stearic acid and oleic acid (Beltz et al, 2007).

4.6.9 PATHWAY ENRICHMENT ANALYSIS

To reflect the global architecture of the interactions between the targets and psoriasis pathways the
targets from each herb were mapped to canonical pathways from KEGG, BIOCARTA and Reactome
using MsigDB. The mapped pathways of each plant were investigated for their involvement in the
disease (Figure 37). Apart from pathways involved in immune system which are enriched in the
pathway analysis we also analyzed the pathways mediating secondary complications in psoriasis.

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Figure 37 P-T network. The orange diamond nodes indicate the compounds and the circular nodes
indicate the pathway in which they are involved

We noted the presence of immune pathways enriched in the pathway analysis of targets from SC.
The disrupted immune system shows a strong association in the pathogenesis of psoriasis. Two
transcription factors AP-1 and ATF-2 pathways triggering the immune signaling cascades were
observed in the list. AP-1 initiates cellular response to growth factors, cytokines, and various
intracellular signalling molecules. AP-1 modulates the decision of cells to proliferate or differentiate
or undergo apoptosis (Turpaev, 2006). Similarly ATF-2, the transcription factor of leucine zipper
family is implicated in inflammation along with cell proliferation and apoptosis. Previous reports
suggest that ATF2 is upregulated in infiltrating macrophages (Figure 38). ATF2 is also implicated to
play an active role in inflammatory pain (Yu et al, 2014).

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 Figure 38 The inflammatory response signaling pathway regulated by ATF-2

In IA, besides targets from immune system, we also noted targets involved in platelet and skin
homeostasis mediated by PPARα along with toll-like receptor signaling pathway. Platelets play a
major role in immune and inflammatory conditions. Earlier reports suggest that platelet aggregations
are enhanced in psoriatic patients and they remain in activated state under the diseased condition.
The psoriatic plaques enclose various chemokines that are produced by platelets once activated. The
platelet derived inflammatory mediators may be involved in leukocyte recruitment to the psoriatic
lesions. The activated platelets may induce abnormal levels of arachidonic acid lipoxygenase
products in lesional areas of psoriatic skin which may induce infiltration of neutrophils into the
pustules and microabscesses in psoriasis (Tamagawa-Mineoka et al, 2010). Peroxisome proliferator
active receptors are involved in linking innate immune system with lipid metabolic disorders.
Reduced levels of PPARα were noted in the skin of psoriatic patients. Ligands of PPARα such as
oleic acid were proved to improve the development of skin barriers along with the restoration of skin

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homeostasis, promotion of differentiation and regulation of epidermal apoptosis (Lima Ede et al,
2013). Activation of Toll like receptors (TLRs) by infiltrating primary dendritic cells (pDCs) has
been proposed to mediate psoriatic pathogenecity (Jariwala, 2007). Dermal DCs were found to
express TLR2 and 4 but not 9, however epidermal DCs express TLR4 and not TLR2 and 9
(Santegoets et al, 2011). The activation of TLR7 in the pDCs leads to increased productions of IFNs
and triggering the IFNs mediated downstream inflammatory cascades. The psoriatic lesions also
show elevated expression of TLR8 which leads to the activation of NFκB responsive pathways
(Suárez-Fariñas et al, 2013).

In AT we noted pathways related to wound healing mediated by glucocorticoid receptor (GR)

signaling and pathways associated with pruritis mediated by IL-2 and Thromboxane A2 receptor
signalling. Elevated expression of GR reported in psoriasis hinders cutaneous wound healing,
decreases migration and increase differentiation of keratinocytes (Man and Zheng, 2015). Besides its
role in inflammation, IL-2 containing lymphocytes serve as a pruritogenic mediator (Nakamura et al,
2003). Thromboxane A2 (TXA2) is an itch mediator of skin (Figure 39). Keratinocytes in the
epidermis and platelets were known to release TXA2. TXA2 triggers the itch signals to CNS and
result in sensitization of primary afferents to pruritogenic mediators and release of transmitters in the
epidermis. The transmitters and TXA2 in turn act on keratinocytes leading to the release of itch
mediators and enhancers (Andoh et al, 2007).

Figure 39 Mechanism of thromboxane A2 (TXA2), mediated itch process.Thromboxane

synthase is expressed in the epidermal keratinocytes (KC) and TP receptor (TP-R) is expressed in the
KC and primary sensory neurons. KCs produce and release TXA2, which in-turn act on primary
afferents to trigger the itch signals. TXA2 act on KCs to release itch mediators including TXA2.

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Calcium signaling pathway, neuro-active ligand receptor interaction pathway and metabolism of
lipids and lipoproteins were observed in EA. The psoriatic lesions exhibit low extracellular Ca2+
levels and high nuclear and cytosolic Ca2+ levels in epidermal basal and spinous cells. Elevated
expression of desmosomes may relate to impaired desquamation of the skin a key feature of psoriasis
(Menon and Elias, 1991). Impaired fat metabolism is considered as an important factor of
pathogenesis of psoriasis. Continuous loss of psoriatic scales leads to loss of lipids affecting the
serum lipid metabolism. Lipid deposition in reticular-endothelial system in psoriatic lesions results
in parakeratosis, congestion and inflammation (Pietrzak et al, 2010).

P53 mediated signaling pathway involved in apoptosis was observed in EL. The major role of
apoptosis in the pathogenesis of psoriasis is its mechanism of eliminating excess keratinocytes.
Apoptosis machinery is also implicated in delayed wound healing process in psoriatic plaques. The
marked thickening of epidermis suggests the imbalance of homeostasis relating to impairment of
apoptotic process. P53 and Bcl-2 are the central regulators of apoptotic process. It has been reported
that the expression of anti-apoptotic protein P53 was enhanced while the pro-apoptotic Bcl-2 was
decreased in psoriatic keratinocytes. Over expression of P53 in lymphocytes prolongs its survival
resulting in relapse and chronic characteristics of the disease (El-Domyat et al, 2007).

Pathways involved in signaling by GPCR and regulation of hemostasis were enriched in ET. We also
noted NFAT3 and keratinocyte differentiation pathway mediated by calcineurin. Calcineurin/NFAT
signalling pathway triggered by enhanced expression of Ca2+ levels regulate a milieu of cytokines
which includes IL2, IL3, IL4, IL13, IFNγ, TNFα and GMCSF and other cell surface receptors.
Calcineurin also significantly controls the keratinocyte differentiation in addition to cell/cell
adhesion formation (Baksh and Burakoff, 2000).

4.7 THEORETICAL STUDIES OF ANTI-PSORIATIC COMPOUNDS AND

EXPERIMENTAL EVALUATIONS OF THE LEAD COMPOUND

When the C-T network of PC was investigated we noted an enrichment of non-receptor tyrosine
kinase family members in particular five SRC kinase family members (SFK); Fyn, Src, Lck, Lyn and
Hck. These kinases were known to play crucial roles in cell-cycle control, adhesion, migration,
proliferation, survival and differentiation (Roskoski, 2015; Ayli et al, 2008). The SFKs are essential
for triggering various intracellular signalling cascades mainly the immune mediated pathways which

154
are activated in psoriasis. Since in psoriasis the immune signals are up regulated, targeting these
SFKs can arrest the propagation of the disease. The compounds targeting these kinases were mined
out from the C-T network from our previous objective. Interaction of these SFKs with the
compounds fumaric acid, genkwanin, quercetin, sitogluside, apigenin, myricetin and caffeic acid
were investigated.

From the C-T network we noted Src interacted with fumaric acid, genkwanin, quercetin and
sitogluside, Hck interacted with quercetin and sitogluside, Lck interacted with apigenin, genkwanin,
myricetin, and quercetin, Lyn interacted with genkwanin and quercetin and Fyn interacted with
apigenin, genkwanin, myricetin, quercetin and caffeic acid.

4.7.1 PROTEIN MODEL BUILDING AND VALIDATION

The activation of SFKs requires release of intra-molecular constraints between the SH2 and SH3
domains and phosphorylation of particular tyrosine residue in the activation loop. The protein
structures were modelled in the active state by phosphorylating the unique tyrosine residue required
for activation. The templates were chosen to mimic the physiological state of these kinases. The
sequence alignment of the kinases revealed high conservation of the functionally important sites
(Figure 40). The residues were numbered based on the human SRC terminology.

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Figure 40 Multiple sequence alignment of the SFKs highlighting the conservation of crucial
residues along with their secondary structures

Based on sequence identity and query coverage three different templates (PDB ID: 1Y57, 2DQ7 and
1QPE) were chosen for model construction. To further validate the protein models the secondary
structure of the templates were compared with predicted secondary structure of the SFKs using
PSIPRED (McGuffin and Jones, 2000) and PDBsum (De Beer et al, 2014). The secondary structure
comparison revealed good agreement between the template and target sequences across the sequence
length (Figure 41-45). The stereochemical parameters of the models obtained from various servers
further validate accuracy of the predicted models which are considered for further studies (Table
35).

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Figure 41 Structural insights of Src kinase (a) Cartoon representation of predicted Src kinase. (b)
Secondary structure prediction by PDBsum. Helices are labelled as H1, H2 and strands by their
sheets as A, B, C.., Motifs: β-beta turn, γ- gamma turn and beta hairpins (c) PSIpred results of
secondary structure prediction. The residues highlighted in yellow indicate sheets and pink indicate
sheets

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Figure 42 Structural insights of Fyn kinase (a) Cartoon representation of predicted Fyn kinase. (b)
Secondary structure prediction by PDBsum. Helices are labelled as H1, H2 and strands by their
sheets as A, B, C.., Motifs: β-beta turn, γ- gamma turn and beta hairpins (c) PSIpred results of
secondary structure prediction. The residues highlighted in yellow indicate sheets and pink indicate
helices.

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Figure 43 Structural insights of Lck kinase (a) Cartoon representation of predicted Lck kinase. (b)
Secondary structure prediction by PDBsum. Helices are labelled as H1, H2 and strands by their
sheets as A, B, C.., Motifs: β-beta turn, γ- gamma turn and beta hairpins (c) PSIpred results of
secondary structure prediction. The residues highlighted in yellow indicate sheets and pink indicate
helices.

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Figure 44 Structural insights of Lyn kinase (a) Cartoon representation of predicted Lyn kinase. (b)
Secondary structure prediction by PDBsum. Helices are labelled as H1, H2 and strands by their
sheets as A, B, C.., Motifs: β-beta turn, γ- gamma turn and beta hairpins (c) PSIpred results of
secondary structure prediction. The residues highlighted in yellow indicate sheets and pink indicate
helices.

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Figure 45 Structural insights of Hck kinase (a) Cartoon representation of predicted Hck kinase.
(b) Secondary structure prediction by PDBsum. Helices are labelled as H1, H2 and strands by their
sheets as A, B, C.., Motifs: β-beta turn, γ- gamma turn and beta hairpins (c) PSIpred results of
secondary structure prediction. The residues highlighted in yellow indicate sheets and pink indicate
helices.

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 Table 35 Template and structure validation statistics for the modeled proteins

Temp Query Identi MolProbity

Protein
late coverage ty Procheck (%) Verify ERRAT ProSA (%)
ID (%) 3D (F) (G) (H)
A B C D E I J
Src 1Y57 84 100 -
1QPE 50 92.9 5.9 1.0 0.3 0.0 98.22 79.04 -11.27 0.11 1.04
67
1
Fyn 1Y57 84 78 -
2DQ7 51 94.4 4.8 0.8 0.0 0.0 93.99 78.33 -9.47 0.05 1.08
96
1
Lck 1Y57 86 61 -
1QPE 54 92.5 6.2 1.3 0.0 0.0 94.70 80.93 -10.72 0.17 1.45
99
1
Lyn 1Y57 86 60 -
2DQ7 53 91.6 7.1 1.3 0.0 0.0 94.85 77.94 -9.66 0.11 1.17
70
3
Hck 1Y57 83 63 -
2DQ7 51 91.9 6.3 1.3 0.0 85.84 81.79 -10.76 0.11 1.30
70 0.5
4
A
Most favored regions, Additionally allowed regions, Generously allowed regions, DDisallowed
B C

regions, EOverall G-factor, FAveraged 3D-1D score 4 0.2, GOverall quality, HZ-score, IResidues with
bad bonds, JResidues with bad angles

4.7.2 EXTRA PRECISION DOCKING STUDIES

The interactions of phyto-compounds with their respective SFKs were investigated based on their
Glide XP dock score, Glide XP Emodel energy, Glide XP energy, mmgbsa binding free energy and
binding patterns independently (Table 36).

Table 36 Docking score and binding energy of the compounds binding to SFks
Protein Compound Glide Glide XP Glide XP MM-GBSA
Docking Emodel energy Energy ΔGBind
Score (kcal/mol) (kcal/mol) (kcal/mol)
Src Fumaric acid -4.11 -24.22 -21.19 -7.95
Genkwanin -6.47 -34.02 -30.63 -59.56
Quercetin -8.69 -49.41 -44.91 -68.38
Sitogluside -4.99 -56.12 -37.59 -65.96

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 Hck Quercetin -7.88 -40.81 -42.76 -52.20
Genkwanin -5.26 -33.16 -29.25 -56.82
Lck Apigenin -4.85 -38.55 -34.60 -40.05
Genkwanin -4.47 -20.58 -22.49 -41.63
Myricetin -8.22 -46.21 -36.01 -37.99
Quercetin -6.53 -48.42 -36.67 -55.49
Lyn Genkwanin -4.84 -23.54 -26.00 -61.84
Quercetin -6.33 -44.03 -41.34 -64.74
Fyn Apigenin -5.18 -37.74 -30.54 -43.53
Genkwanin -3.84 -30.32 -24.65 -36.06
Myricetin -5.16 -39.21 -34.30 -45.93
Quercetin -8.13 -17.56 -35.96 -48.66
Caffeic acid -3.43 -17.56 -19.67 -23.18

4.7.2.1 BINDING MODE ANALYSIS OF PHYTOCOMPOUNDS WITH SRC KINASE

Based on the C-T network analysis the compounds; fumaric acid, genkwanin, quercetin and
sitogluside were found to directly interact with SRC kinase. The Glide XP Dockscore of the ligands
ranged between -8.69 and -4.11. The Glide XP Emodel energy ranged between -56.12 kcal/mol and
-24.22 kcal/mol. The Glide XP energy value ranged between -44.91 kcal/mol and -21.18 kcal/mol.
The binding free energy calculated by prime MMGBSA method ranged between -68.386 kcal/mol
and -7.95 kcal/mol. When the energies were compared we noted quercetin lead the table with
favourable scores. Based on the multiple sequence alignment, the functional sites were analysed.

The binding pattern analysis revealed that the ligands preferentially formed hydrogen bonds with the
residues located in the protein kinase domain (Figure 46-49). Fumaric acid, genkwanin, quercetin
and sitogluside were found to interact with PTR419 by the formation of hydrogen bond. Fumaric
acid forms hydrogen bonds with the residues in the αE-activation segment loop and activation
segment HΦ interaction of the C lobe (485-413) which includes Arg388, Leu410 and Ala411.
Genkwanin forms hydrogen bond in the same region involving Tyr385, Gly 409, Ala411 and
hydrophobic interaction with Arg412. Quercetin forms hydrogen bonds with the residues located in
the αC-helix β5-strand involved in H Φ interactions (312-316) of N-lobe which includes Gln312,

163
Gln315 and Val316 and hydrophobic interaction with Leu 413. The compound also forms hydrogen
bonds with C-lobe residues Ala411 and Tyr385. Sitogluside forms hydrogen bonds with the C-lobe
αE-activation segment loop and activation segment HΦ residues Arg388, Leu410 and Ala411
(Roskoski, 2015).

Figure 46 Binding of fumaric acid with Src kinase (a) Binding of fumaric acid (CPK model) with
Src kinase (cartoon and mesh). (b) Interacting residues of Src kinase (sticks) with fumaric acid (ball
and stick)

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Figure 47 Binding of genkwanin with Src kinase (a) Binding of genkwanin (CPK model) with Src
kinase (cartoon and mesh). (b) Interacting residues of Src kinase (sticks) with genkwanin (ball and
stick)

165
Figure 48 Binding of quercetin with Src kinase (a) Binding of quercetin (CPK model) with Src
kinase (cartoon and mesh). (b) Interacting residues of Src kinase (sticks) with quercetin (ball and
stick)

166
Figure 49 Binding of sitogluside with Src kinase (a) Binding of sitogluside (CPK model) with Src
kinase (cartoon and mesh). (b) Interacting residues of Src kinase (sticks) with sitogluside (ball and
stick)

4.7.2.2 BINDING MODE ANALYSIS OF PHYTOCOMPOUNDS WITH HCK KINASE

Two ligands quercetin and genkwanin were found to interact with Hck kinase. The glide XP Dock
score were -7.88 and -5.26 kcal/mol for quercetin and genkwanin respectively. The Glide XP
Emodel energy were -40.81 kcal/mol and -33.16 kcal/mol and Glide XP energy were -42.76
kcal/mol and -29.25 kcal/mol for quercetin and genkwanin. The mmgbsa binding energy for
quercetin is -56.82 kcal/mol and -52.20 kcal/mol for genkwanin.

The interacting residues of the proteins with the ligands were investigated (Figure 50-51). The
binding pattern analysis reveals that quercetin forms a hydrogen bond with the phosphorylated
tyrosine residue PTR411. Quercetin interacts by hydrogen bonding with Phe302 and hydrophobic

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interaction with Ala301 located in αC-helix 5β-strand of N-lobe. The compound also forms
hydrogen bond with Gly401 and Arg414 located in the C-lobe αE-activation segment loop and
activation segment HΦ. Genkwanin interacts by forming hydrogen bonds with the residues Glu300,
Asn307 and hydrophobic interaction with Ala304 located in the N-lobes αC-helix β5-strand.
Genkwanin also forms hydrogen bond with Ala403 located in the C-lobes αE-activation segment
loop and activation segment HΦ (Sicheri et al, 1997).

Figure 50 Binding of genkawanin with Hck kinase (a) Binding of genkwanin (CPK model) with
Hck kinase (cartoon and mesh). (b) Interacting residues of Hck kinase (sticks) with genkwanin (ball
and stick)

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Figure 51 Binding of quercetin with Hck kinase (a) Binding of quercetin (CPK model) with Hck
kinase (cartoon and mesh). (b) Interacting residues of Hck kinase (sticks) with quercetin (ball and
stick)

4.7.2.3 BINDING MODE ANALYSIS OF PHYTOCOMPOUNDS WITH LCK KINASE

Lck expressed in T cells plays a major role in T cell receptor signalling. Apigenin, genkwanin,
myricetin and quercetin interacts with Lck. The Glide XP Dockscore of the ligands ranged between -
8.22 and -4.47. The Glide XP Emodel energy ranged between -48.42 kcal/mol and -20.58 kcal/mol.
The Glide XP energy value ranged between -36.67 kcal/mol and -22.49 kcal/mol. The prime’s
mmgbsa free energy of binding ranged between -55.49 kcal/mol and -37.990 kcal/mol.

The interaction pattern analysis of the ligands (Figure 52-55) revealed that all the ligands except
genkwanin formed hydrogen bond with PTR394, the phosphorylated tyrosine residue. Apigenin
forms hydrogen bonds with Glu390, Asp391, Thr395, Arg397 and Leu388 located in the C- lobes

169
activation loop (363-394). Similarly genkwanin is also involved in hydrogen bonding with Ala386,
Arg387 and hydrophobic interactions with Arg387 and Leu388 located in the C- lobes activation
loop. The compound also forms hydrogen bond with Ala287 located in the N-lobes αC-helix β5-
strand HΦ interaction region. Myricetin and quercetin forms hydrogen bonds with Gly384 and
Arg387 located in the C- lobe’s activation loop (Zhu et al, 1999).

Figure 52 Binding of apigenein with Lck kinase (a) Binding of apigenin (CPK model) with Lck
kinase (cartoon and mesh). (b) Interacting residues of Lck kinase (sticks) with apigenin (ball and
stick)

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Figure 53 Binding of genkwanin with Lck kinase (a) Binding of genkwanin (CPK model) with
Lck kinase (cartoon and mesh). (b) Interacting residues of Lck kinase (sticks) with genkwanin (ball
and stick)

171
Figure 54 Binding of myricetin with Lck kinase (a) Binding of myricetin (CPK model) with Lck
kinase (cartoon and mesh). (b) Interacting residues of Lck kinase (sticks) with myricetin (ball and
stick)

172
Figure 55 Binding of quercetin with Lck kinase (a) Binding of quercetin (CPK model) with Lck
kinase (cartoon and mesh). (b) Interacting residues of Lck kinase (sticks) with quercetin (ball and
stick)

4.7.2.4 BINDING MODE ANALYSIS OF PHYTOCOMPOUNDS WITH LYN KINASE

Genkwanin and quercetin interacts with Lyn. The Glide XP Dockscore of the compounds are -4.84
kcal/mol and -6.33 kcal/mol. The Glide XP Emodel energy for genkwanin and quercetin are -23.54
kcal/mol and -44.03 kcal/mol. The Glide XP energy for genkwanin and quercetin are -26.00
kcal/mol and -41.34 kcal/mol. The prime mmgbsa binding free energy for genkwanin and quercetin
are -61.74 kcal/mol and -64.74 kcal/mol, respectively.

The interacting residues of the ligands with Lyn were investigated (Figure 56-57). The binding
pattern analysis found that genkwanin formed hydrogen bonds with residues Glu289, Asn292,
Thr296 located in the N-lobes αC-helix β5-strand HΦ interaction region. The compound also formed
hydrogen bonds with Tyr363 and hydrophobic interaction with Val 391 in the C-lobes αE-activation
segment loop and activation segment HΦ interaction region. Quercetin formed hydrogen bond with
the phosphorylated tyrosine residue PTR397. The compound also formed hydrogen bonds with

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Glu289 in the N-lobes αC-helix β5-strand HΦ interaction region and Arg366, Gly387 and Arg390
located in the C-lobes αE-activation segment loop and activation segment HΦ interaction region
(Miyano et al, 2009).

Figure 56 Binding of genkwanin with Lyn kinase (a) Binding of genkanin (CPK model) with Lyn
kinase (cartoon and mesh). (b) Interacting residues of Lyn kinase (sticks) with genkwanin (ball and
stick)

174
Figure 57 Binding of quercetin with Lyn kinase (a) Binding of quercetin (CPK model) with Lyn
kinase (cartoon and mesh). (b) Interacting residues of Lyn kinase (sticks) with quercetin (ball and
stick)

4.7.2.5 BINDING MODE ANALYSIS OF PHYTOCOMPOUNDS WITH FYN KINASE

Fyn kinase plays a major role in immune mediated cascade in particular T cell activation. Apigenin,
genkwanin, myricetin, quercetin and caffeic acid interacts with Fyn kinase. The Glide XP Dockscore
of the ligands ranged between -8.13 and -3.84. The Glide XP Emodel energy ranged between -39.21
kcal/mol and -17.56 kcal/mol. The Glide XP energy value ranged between -35.96 kcal/mol and -
19.67 kcal/mol. The binding free energy calculated by prime mmgbsa ranged between -48.66
kcal/mol and -23.19 kcal/mol.

The interacting residues of ligands were investigated (Figure 58-62). The binding pattern analysis
revealed apigenin formed hydrogen bonds with Leu274 in the SH1 domain, Ser346 in the N-lobes
αC-β4 loop and αE helix HΦ interaction region, Asp349 and Ala391 and hydrophobic interaction
with Leu394 in the C-lobe’s activation segment. Genkwanin and myricetin formed hydrogen bond

175
with Ala391 located in the C-lobe’s activation segment. Myricetin also formed hydrogen bond with
Ala394 located in the same region. Quercetin formed hydrogen bonding with Thr97 and Glu94 in the
SH3 domains along with the Leu411 in the C-lobe’s activation segment. Similarly, caffeic acid also
formed hydrogen bonds with Glu94 and Leu411 (Kinoshita et al, 2006).

Figure 58 Binding of apigenin with Fyn kinase (a) Binding of apigenin (CPK model) with Fyn
kinase (cartoon and mesh). (b) Interacting residues of Fyn kinase (sticks) with apigenin (ball and
stick)

176
Figure 59 Binding of genkwanin with Fyn kinase (a) Binding of genkwanin (CPK model) with
Fyn kinase (cartoon and mesh). (b) Interacting residues of Fyn kinase (sticks) with genkwanin (ball
and stick)

177
Figure 60 Binding of myricetin with Fyn kinase (a) Binding of myricetin (CPK model) with Fyn
kinase (cartoon and mesh). (b) Interacting residues of Fyn kinase (sticks) with myricetin (ball and
stick)

178
Figure 61 Binding of quercetin with Fyn kinase (a) Binding of quercetin (CPK model) with Fyn
kinase (cartoon and mesh). (b) Interacting residues of Fyn kinase (sticks) with quercetin (ball and
stick)

179
Figure 62 Binding of caffeic acid with Fyn kinase (a) Binding of caffeic acid (CPK model) with
Fyn kinase (cartoon and mesh). (b) Interacting residues of Fyn kinase (sticks) with caffeic acid (ball
and stick)

4.7.3 OVERALL BINDING MODE ANALYSIS

When the overall binding energies were compared quercetin lead the list showing favourable binding
with all the SFKs of our study. The binding pattern analysis revealed that the ligands preferentially
bind to the protein kinase domain and their interactions restrict key functional residues of the N and
C lobes. In general, quercetin with high binding energy interacted with various functionally
important residues and key phosphorylated tyrosine residues of the SFKs except Fyn kinase. In
addition, the inhibitory modes of quercetin with the SFKs were validated by the molecular dynamics
and simulation analysis.

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4.7.4 MOLECULAR DYNAMICS AND SIMULATION ANALYSIS

A 25 ns production MD runs were carried out for the apo proteins and quercetin bound protein
complexes. Each SFK system was investigated independently to analyse the influence of quercetin
binding.

4.7.4.1 MOLECULAR DYNAMICS AND SIMULATION ANALYSIS OF SRC KINASE

Dynamic stability of apo-Src kinase and quercetin bound Src kinase was analysed using the
backbone root mean square deviation (RMSD) of Cα atoms. The comparison of the RMSD plots
indicated that the apo protein RMSD converged prior to the protein-ligand complex and became
stable after initial deviation at 15 ns. The apo protein maintained its stability throughout the entire
simulation time period and converging at 0.75 nm at the end of the simulation time. The ligand
induced disturbances in the protein is evident from the complex RMSD. The conformational change
associated with the binding of quercetin is validated by the increased RMSD of the complex and
occurrence of drifts in the plot.

To assess the flexibility of the residues in the apo and bound state root mean square fluctuation
(RMSF) of the backbone atoms were calculated for both the system. Comparable to the RMSD the
residual fluctuations were high in the complex system when compared to the apo state. To further
inspect the fluctuations in the systems PCA was performed for the backbone carbon atoms of the apo
protein and Src-quercetin complex system using the last 5 ns trajectory. The first and the second
motion modes were used for plotting. The comparison of the projections of the two eigen vectors of
the apo protein and protein-ligand complex system revealed that the protein-ligand complex explores
a large area of conformational space when compared to the apo protein. The overall compactness of
the systems was analysed by calculation of radius of gyration (Rg). In apo state after the initial
equilibration phase the apo proteins Rg remained relatively stable with a Rg of 2.6 nm. In contrast,
the protein-ligand complex system showed increased Rg value when compared to the apo state and
finally dropping to 2.7 nm at the end of the simulation time. The hydrogen bond analysis indicated
that the complex system maintained a minimum of 1 hydrogen bond throughout the simulation time.
Maximum of 5 hydrogen bonds and a minimum of 1 hydrogen bond were observed the Src-quercetin
complex system (Figure 63).

181
 (a)

(b)

(c)

182
 (d)

(e)

Figure 63 Conformational analysis of apo Src kinase and Src kinase-quercetin complex. (a)
RMSD, (b) RMSF, (c) Rg (d) Hydrogen bonds (e) Prevalent motions of the apo protein and protein-
ligand complex analysed by PCA

4.7.4.2 MOLECULAR DYNAMICS AND SIMULATION ANALYSIS OF HCK KINASE

The comparison of RMSD of the apo Hck and Hck-quercetin complex revealed that the apo protein
converged after 15 ns reaching an rmsd of 0.75 nm at the end of the simulation time period. The

183
Hck-quercetin complex rmsd was observed to be parallel with the apo protein before 7 ns during the
equilibration phase but the deviations increased above the apo protein before reaching the
convergence at 15 ns. The rmsd value of the protein-ligand complex is found to be 1 nm at the end of
the simulation time period. Similar to the rmsd the residual mobility was higher in the protein-ligand
complex system when compared to the apo protein. The PCA analysis of the apo protein indicated a
restricted population, exploring small conformational space with low degree of conformational
changes when compared with protein-ligand complex. The binding of quercetin to Hck kinase has
minimized the internal motions of the protein restricting the conformational changes of the protein
which is evident from the low Rg value (2.4 nm) when compared to the apo-protein (2.8 nm). The
hydrogen bond analysis indicated that the Hck-quercetin complex formed stable hydrogen bonds
over the simulation time by maintaining a minimum of 1 bond and maximum of 4 hydrogen bonds
(Figure 64).

(a)

(b)

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 (c)

(d)

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 (e)

Figure 64 Conformational analysis of apo Hck kinase and Hck kinase-quercetin complex. (a)
RMSD, (b) RMSF, (c) Rg (d) Hydrogen bonds (e) Prevalent motions of the apo protein and protein-
ligand complex analysed by PCA

4.7.4.3 MOLECULAR DYNAMICS AND SIMULATION ANALYSIS OF LCK KINASE

The comparison of RMSD values between the apo Lck and Lck-quercetin complex indicated that the
apo protein deviation peaked at 12 ns to 1.5 nm before reaching the convergence at 25 ns, in contrary
the protein-ligand complex converged before 5 ns and was stable throughout the simulation time.
High residual mobility in the apo protein was restricted by the ligand binding which is evident from
the protein-ligand complex RMSF plot. The PCA analysis shows the high conformational search
space exploration of the apo protein in contrast to the restricted motions of the protein-ligand
complex system. The binding of quercetin to Lck drastically decreased the conformational motions
of the protein and induced high compactness which is validated by the comparison of Rg between
the apo protein and the bound complex. Further the hydrogen bond analysis indicated that a
minimum of 2 and maximum of 7 hydrogen bonds were maintained between Lck and quercetin
throughout the simulation time period (Figure 65).

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(a)

(b)

(c)

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 (d)

(e)

Figure 65 Conformational analysis of apo Lck kinase and Lck kinase-quercetin complex. (a) RMSD,
(b) RMSF, (c) Rg (d) Hydrogen bonds (e) Prevalent motions of the apo protein and protein-ligand
complex analysed by PCA

4.7.4.4 MOLECULAR DYNAMICS AND SIMULATION ANALYSIS OF LYN KINASE

The RMSD of the apo Lyn and Lyn-quercetin complex after equilibration converged after 7.5 ns.
The rmsd of the apo protein was higher when compared to the protein-ligand complex. At the end of

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the simulation time period the 0.25 nm rmsd difference was observed between the systems. The
residual mobility of the apo protein was higher when compared to the protein-ligand complex. High
fluctuations were observed in the region lying between 175 and 250 in the apo protein which is
richly populated by loops. The PCA analysis revealed the presence of restricted motions in the
protein-ligand complex systems when compared to the apo-protein. The comparison of Rg between
the systems indicated that the binding of ligand decreased internal motions of the protein, however
the apo-protein is found to be highly compact as its Rg was low and stable throughout the simulation
time period when compared to the protein-ligand complex. The hydrogen bond analysis revealed that
a minimum of 1 and maximum of 5 hydrogen bonds were maintained between Lyn and quercetin
throughout the simulation time period (Figure 66).

(a)

(b)

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 (c)

(d)

(e)

Figure 66 Conformational analysis of apo Lyn kinase and Lyn kinase-quercetin complex. (a)
RMSD, (b) RMSF, (c) Rg (d) Hydrogen bonds (e) Prevalent motions of the apo protein and protein-
ligand complex analysed by PCA

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4.7.4.5 MOLECULAR DYNAMICS AND SIMULATION ANALYSIS OF FYN KINASE

The RMSD of the apo-Fyn was higher when compared to the Fyn-quercetin complex. A difference
of 0.5 nm RMSD was observed between the systems at the end of the simulation time period.
Similar to the RMSD the residual flexibility of the apo-protein was higher when compared to the
protein-ligand complex. The PCA analysis revealed that the motions of the protein in the protein-
ligand complex were restricted to limited conformational search space when compared to the apo-
protein. The restriction in the conformational search space was further validated by the low and
stable protein-ligand Rg. The hydrogen bond analysis indicated that a minimum of 1 and maximum
of 5 hydrogen bonds were maintained between Fyn and quercetin throughout the 25 ns simulation
time period (Figure 67).

(a)

(b)

(c)
191
(d)

192
 (e)

Figure 67 Conformational analysis of apo Fyn kinase and Fyn kinase-quercetin complex. (a)
RMSD, (b) RMSF, (c) Rg (d) Hydrogen bonds (e) Prevalent motions of the apo protein and protein-
ligand complex analysed by PCA

The binding of quercetin to the SFKs has impaired the tightly regulated protein structure by
restricting or inducing the conformational changes which is evident from the above analysis of
various parameters. To further assess the inhibition of Src, Fyn and Lyn which are implicated in T
cell signalling in psoriasis experimental verifications were carried out.

4.7.5 EFFECT OF QUERCETIN ON THE HACAT CELL VIABILITY

The cytotoxicity of quercetin on HaCaT cells was measured using MTT assay. Ponatinib, a potent
tyrosine kinase inhibitor was used as standard drug for comparison. As shown in Figure 68 (b),
quercetin at lower concentrations (5-25 µM) induced no significant cell death however at higher
concentrations 50 µM, 75 µM and 100 µM the cell viability was decreased to 53.61%, 37.48% and
29.36%, respectively. These results suggest the effectiveness of quercetin to induce cell death in
HaCaT cells.

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Figure 68 Effect of quercetin on HaCaT cells. (a) Cells treated with varying concentration of
Ponatinib (0.5-4.5 µM) for 24 hours. (b) Cells treated with varying concentration of Quercetin
(5-100 µM) for 24 hours.

4.7.6 QUERCETIN INHIBITS ACTIVATED SRC, FYN AND LYN

To investigate the expression levels of p-Src, p-Fyn and p-Lyn, the HaCaT cells were treated with
quercetin (51.65 µM) and ponatinib (1.43 µM) for 24 h. The flow cytometry analysis revealed that
the expression of these three SFKs were upregulated in untreated HaCaT cells. However, there was
a significant downregulation in the expression of these SFKs after the treatment with quercetin
through the induction of apoptosis (Figure 69). Furthermore, quercetin suppressed the expression
level of Src kinase similar to that of the reference compound Ponatinib (1.43 µM).

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Figure 69 Effect of quercetin on the expression levels of Src, Lyn and Fyn in HaCaT Cells. The HaCat cells were treated with Quercetin (51.65µM)
and Ponitinib (1.43µM) for 24 h. After the treatment, the cells were stained with FITC coupled monoclonal antibodies against Fyn, Lyn and Src and
analyzed by flow cytometry. The histograms showing the expression of (a) Src (b) Lyn and (C) Fyn

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The order of suppression (Src > Lyn > Fyn) correlated with the order of binding energy of quercetin
with these SFKs (Src > Lyn > Fyn) validating its role in disrupting the signaling mechanism
mediated by these kinases in the immune singnaling cascades.

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 Conclusion

Psoriasis is the chronic immune mediated disease of the skin. The identification of various genes
impaired in the disease condition can provide insight about the pathogenesis of the disease. The
current work mined out crucial genes implicated in psoriasis from microarray gene expression data
by utilizing system biology approach. The sub-networks derived by cluster analysis of the central
network identified rich kinome as putative targets implicated in T cell activation. The network based
analysis incorporating gene ontology and pathway-level information revealed the presence of novel
genes of non-receptor tyrosine kinases that are not previously studied in psoriasis. As these genes
are signal carriers in defense response they may act as a starting point in the drug discovery pipeline
for the development of new psoriatic drugs.

The current analysis of psoriasis and its associated comorbidities highlight psoriasis as a
paradigmatic disorder. The comprehensive network based approach revealed the existence of shared
component hypothesis between psoriasis and its associated co-morbidities. The findings suggest that
the most prevalent psoriasis comorbidities were interlinked through common molecular connections
which were evidenced by their shared biological functions and pathways. Significant overlaps was
observed in the biological process and pathways involved in inflammation in most of the
comorbidities establishing a link between them.

The hub signatures identified by WGCNA were found to be implicated in crucial cascades of
psoriasis which includes immune response, keratinisation and cytoskeleton remodelling. Hence, the
signature genes may act as promising biomarkers in the disease diagnosis and can be targeted for
therapeutic development.

Traditional system of medicines offer promising cure by utilizing holistic methods. The current
work integrating Siddha medicine with the network medicine approach is to provide scientific
validation for the traditional medicinal system. The network pharmacology approach was utilized to
decipher the mechanism of action of the highly prescribed topical and oral therapeutics for the
treatment of psoriasis. The constituents of the therapeutics were found to act in a synergistic way by
suppressing the immune system and inducing or reviving the disrupted mechanisms such as
apoptosis.

The PC’s C-T network analysis has identified compounds as direct inhibitors of non-receptor
tyrosine kinase in particular members of SRC kinase which are enriched in the diseased condition.
The docking analysis of the phyto-compounds with SFKs revealed that the compound quercetin

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interacted with all the members with favourable binding energy. The molecular dynamics and
simulation studies revealed that the flexible motions of the proteins were arrested by the binding of
quercetin in all the complexes except Src kinase. The in-vitro validation of quercetin binding with
Fyn, Lyn and Src showed that the expression levels of these kinases are markedly decreased in the
HaCaT cells.

The current work is an effort to promote Siddha medications (Tamil maruthuvam) which is at the
verge of extinction and a small endeavour to prevent it from disappearance. This work not only
highlights the pharmacological action of the herbal remedies for psoriasis but also emphasis on safe
herbal remedies offered by the Siddha medicinal system.

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