Prospective enzyme inhibitory activity of Bioactive molecules from

Withania somnifera, Phyllanthus amarus and Phyllanthus uirnaria – A

systematic analysis

Dhivya Dharshini S,
Roll No. 191706004
MSc. Bioinformatics
Manipal School of Life Sciences,
Manipal Academy of Higher Education, Manipal

Project Guide: Project Co-guide:

Dr. Annamalai Muthusamy Dr. Bobby Paul

Professor & Head, Assistant Professor & Head,
Department of Plant Sciences, Department of Bioinformatics,
MSLS, MAHE. MSLS, MAHE.
 Introduction - Medicinal Plants

• Rich resources of ingredients which can be used in drug development

pharmacopoeial, non-pharmacopoeial or synthetic drug

• Important potential source of therapeutics or curative aids.

• Plays vital roles in disease prevention and their promotion.

• Modern medicine utilizes uses 80% of active compounds.

• Affordability, accessibility and low cost

• Plant species contain active ingredients such as alkaloids, phenols, tannins,

cryogenics, glycocides, terpeniods.
(Garg et al., 2021; Oteng Mintah et al., 2019; Sofowora et al., 2013, SO et al., 2018)
 Withania somnifera
• Solanaceae family - commonly called Ashwagandha.

• 200 different formulations in Siddha, Ayurvedha and Unani medicine

• Contains over 35 phytochemicals and two active principles are

Sitoindosides VII-X and Withaferin-A

• Used to treat conditions – anxiety, arthritis, bronchitis and insomnia. 

• Withaferin-A, Withanone and TEG (Triethylene glycol) are biologically

active constituents of Ashwagandha leaves.

• Extract of leaves shows anti-anxiety, anti-inflammatory, anti-apoptotic

properties 

• Extract of roots shows anti-stress activity

(Kaushik et al., 2017; Palliyaguru et al., 2016; Singh, 2015; Zahiruddin et al., 2020) Fig 1. Withania somnifera.
 Phyllanthus amarus
 Phyllanthaceae family - commonly called Carry Me Seed

 Widely spread throughout the tropics and subtropics

 Primarily contains lignans (e.g., phyllanthine and

hypophyllanthine), geraniin and 5 flavonaoids.

 Contains minor compounds like hydrolysable tannins and

alkaloids.

 Used to treat urinogenital disorders, kidney problems, diabetes,

jaundice.

 Extracts isolated from Phyllanthus amarus display a broad range Fig 2. Phyllanthus amarus.
of pharmacological activities.
(Joseph & Raj, 2010; Meena, 2018)
 Phyllanthus urinaria

 Commonly referred to as chamber bitter is herb species

 More than 500 chemical compounds (phytochemicals) have been

isolated.

 It has a wide range of uses in its pharmacognostic properties. 

 The ethanolic and acetone extract has anti-inflammatory and antioxidant

activity

 Entire plant can be used to cure diabetes, jaundice, hypertension and

hepatitis B virus.

 Effective against peptic ulcer and gastric ulcer caused due to

Helicobacter pylori Fig 3. Phyllanthus urinaria.

(Geethangili & Ding, 2018a; Sarin et al., 2014)

 Aim and Objectives

To estimate the enzyme inhibitory activity of bioactive molecules from three

medicinal plants, Withania somnifera, Phyllanthus amarus and Phyllanthus
urinaria through in silico approaches. 

1. Identification of bioactive molecules reported from Withania

somnifera, Phyllanthus amarus and Phyllanthus urinaria

2. Inhibitory activity of selected bioactive molecules against

enzymes involved in different diseases

3. Molecular docking studies of bioactive molecules against selected

enzymes
 Methodology

Literature Resources Databases Autodock tools Visualization tools

PDB, PubChem AutoDock, 
PubMed, Scopus Pymol, Chimera,
and DrugBank
and Google Scholar AutoDock Vina BIOVIA

Target Structures Ligand Structures

• Deleting Water
• Adding charges Protein
Protein Preparations
Preparations Ligand
Ligand Preparations
Preparations • Making all bond rotatable
• Saving as .pdbqt • Saving as .pdbqt

• Setting up the grid Grid Preparations • MGLTools Autdock

box on entire target • Auto dock Vina

Virtual Screening/Docking

Evaluation of Results
 Protein and Ligand Preparation:
Bioactive molecules, 2,3-dihydrowithaferin-A, Geraniin, Withanoside IV are
obtained from PubChem database and saved in SDF or MOL format.

Protein structure was modified by adding hydrogen, removing atomic clashes and
optimizing hydrogen bonds using Discovery Studio Visualizer

Before the virtual screening, ligands were prepared to construct 3D geometry, to

establish accessible tautomer, assign correct bonding and ionization states and it
can be done using the MGL tool AutoDock version 4.2.6.

The amino acid in the receptor’s active site was determined according to the
proteins reported.

With the help of AutoDock Vina, the 3D grid was set up to the whole structure of
the protein (blind docking) as there is no information on the target pocket.
 Results:

 As per the literature source, the bio active compounds of three medicinial plants
Withania somnifera, Phyllanthus amarus, Phyllanthus urinaria are listed in the table

 The 3D structures of obtained bioactive compounds were retrieved from PDB (

https://www.rcsb.org/)

Withania somnifera Phyllanthus amarus Phyllanthus

urinaria

Bioactive 66 94 77
Compounds

3D Structure 61 40 70
 Results

 We have screened 23 bioactive molecules obtained from three medicinal plants for
potential interaction with human proteins.

 We have determined the docking or binding-free energy (kcal/mol)

 Analysis of the ligand-protein interaction as a structural complex was done using Autodock
Tools v1.5.6, Pymol, and UCSF Chimera v 1.15.

 2,3-dihydrowithaferin-A showed the highest binding affinity with Aldose Reductase (-

16.7).

 Geraniin has shown good interaction ability with top-six receptors namely,
Acetylcholinesterase, ACE, Pancreatic α-amylase, 5-LOX, COX-2, COX-1 and Pancreatic
lipase
 Results: Bioactive molecules of WS, PA & PU
showing enzyme inhibitory activity
Table 1. Binding affinity value between selected Receptor and Ligand molecules

Molecule Name PDB ID Compound Name PubChem ID Binding Affinity

Aldose Reductase 2R24 2,3-dihydrowithaferin-A 418033 -16.7
Acetylcholinesterase 4PQE Geraniin 3001497 -16.5
Angiotensin-Converting
Enzyme (ACE) 1O8A Geraniin 3001497 -16.0
Pancreatic α-amylase 3BAW Geraniin 3001497 -16.0
5-lipoxygenase (5-LOX) 3V98 Geraniin 3001497 -15.2
Cyclooxygenase-2 or COX-2 5KIR Withanoside IV 71312551 -14.5
Cyclooxygenase-1 or COX-1 6Y3C Geraniin 3001497 -14.4
Acetylcholinesterase 4PQE 2,3-dihydrowithaferin-A 418033 -14.3
Pancreatic Lipase 2PPL Geraniin 3001497 -14.2
Acetylcholinesterase 4PQE Withanoside IV 71312551 -14.1
 Results: Bioactive molecules of WS, PA & PU showing
enzyme inhibitory activity
Table 2. Docking results showing amino acid residues after the interaction of binding molecule with the
respective receptor.

Molecule Name Compounds Amino Acid residues

TYR48, TRP20, CYS298, PHE122, TRP79, CYS80,
Aldose Reductase 2,3-dihydrowithaferin-A
PHE115, CYS303, TYR309, THR113, LEU300.
SER293, VAL294, PHE295, TYR341, TYR124, LEU76,
Acetylcholinesterase Geraniin
TYR72

Angiotensin-Converting HIS353, HIS513, VAL380, GLU376, ASP453, LYS454

Geraniin
Enzyme (ACE)
PHE348, ASP353, ARG303, GLY304, ILE312, ASN301,
Pancreatic α-amylase Geraniin
ALA310
GLN549, ALA453, SER447, ARG370, ARG246, ILE365,
5-lipoxygenase (5-LOX) Geraniin
ASP442

Pancreatic Lipase Geraniin GLU310, PHE404, ASN346, TYR387, GLU349

 Results: Compounds (W. somnifera) inhibiting various
enzymes
Alpha glucosidase Alpha amylase Aldose reductase
0 0 0
-2 -2 -2

-4 -4
-4
-6
-6 -6
-8
-8 -8 -10

-10 -12
-10
-14
-12 -12
-16
-14 -14 -18

5-LOX
Acetylcholinesterase 0

i th f e D

D
ith fe IV
W no in-A

Ph ano in A
ul S
W ha ide-

ag e
in
0

W itha ide

ys lid
r
a r
-2

l
s
ow o
dr an

t
-2

i
hy ith
3- W
-4
-4

di
2,
-6
-6
-8
-8

-10 -10

-12 -12

-14 -14

-16
 Ol
e an
Re o

-9
-8
-7
-6
-5
-4
-3
-2
-1
pa M Ur l ic
nd a so a c
lic id

-12
-10
-8
-6
-4
-2
0
us llo Ta
in ti ur L acid
M ic a nin oc up
ac c Ta ho e
at id ur
an A od O lic ol
n eo xa ac
Co in B xy zo id
ch l o
Ga rilag 9, o n
l l i in 12 An lic a e
cA ,1 ah ci
Tr cid 5- Ph yg d
op Oc yl rin

Alpha glucosidase
in ta
e de Ph tetr e
ca yl ali
Alpha glucosidase

tri lan n
en th
oi in
ca
cid

Ge

-8
-6
-4
-2
0

-14
-12
-10
Re r Ol
pa M ani
nd al in e an
us loti ol
n ic

-9
-8
-7
-6
-5
-4
-3
-2
-1
0

-10
in ac
M i c a in
ac ci Ta Ta Ur Lup id
at d A ur ur so e
an od oc lic ol
ni eo ho ac
Co n B xy lic id
ril ch a
G a ag ol ci
l l i in Ox ic a d
cA 9,
12 a c
c ,1 An zol id
Tr id
op
Alpha amylase 5-
Oc
ah on
Ph yg e
in ta y ri
e de P ltet ne
Alpha amylase

ca hyl ral
tri la in
en nth
oi in
ca
cid

Re
-10
-9
-8
-7
-6
-5
-4
-3
-2
-1
0

pa Ur Lup
-8
nd G e
-6
-4
-2
0

-16
-14
-12
-10
us era Ol soli ol
in n ea c a
i c ii n Ta no ci
a ur lic d
M ci d oc
M al l A ho aci
ac oti li d
at n Ox c ac
an in
n a z id
Co in B An olo
ril Ta ah ne
Ga ag ur
lli in 9, od Ph ygri
cA 12 eo ylt ne
,1 xy etr
Aldose reductase

Tr cid 5- ch al
op Oc ol in
in ta i
e de Ph c ac
Aldose reductase

ca ylla id
tri n
en t h
oi in
ca
cid

Re Ur
pa Ol sol
nd G
-9
-8
-7
-6
-5
-4
-3
-2
-1
0

-10

ea i c
-8
-6
-4
-2
0

-18
-16
-14
-12
-10

us era no aci
in n lic d
i c i in
a T a Ta a
M ci d
M al l A u
ur r L cid
ac oti od oc up
a n eo ho eo
xy lic l
Ph tann in ch ac
ys in ol id
ag B i
u Ox c ac
Co lin D a z id
9, Ph olo
Ga rilag 12 yl ne
ll i i n ,1
cA 5- Ph lant
Acetylcholinesterase

Oc ylt hi
cid ta n
de An etra
ca a h l i n
Acetylcholinesterase

tri yg
en rin
oi e
ca
cid
inhibiting various enzymes

Ta
ur
oc
Re ho
l
-9
-8
-7
-6
-5
-4
-3
-2
-1

-10
0

pa Ox ic a
nd G Ol a ci
0

-16
-14
-12
-10
-8
-6
-4
-2

us era ea z o d
in n no lon
i c ii n
a Ta Ur li c e
M ci d ur s o ac
M al l A od li c id
ac oti
at n eo a
an in xy Lu cid
n ch p
Co in B 9, o e
r 12 Ph lic ol
5-LOX
5-LOX

,1
Results: Compounds (P. amarus & P. urinaria)

G a ilag ylt aci

5- Ph etr d
ll i in Oc
cA ta y al
Tr cid de An llan in
op ca ah thi
in tri yg n
e en ri
oi ne
ca
cid
 Interactions of potential binding molecule with their
respective receptor

Aldose Reductase + 2,3-dihydrowithaferin-A Acetylcholinesterase + Geraniin Angiotensin-Converting Enzyme + Geraniin

Binding Affinity : -16.7 Binding Affinity : -16.5 Binding Affinity : -16.0
 Interactions of potential binding molecule with their
respective receptor

Pancreatic α-amylase + Geraniin 5-lipoxygenase (5-LOX) + Geraniin Cyclooxygenase-2 or COX-2 + Withanoside IV

Binding Affinity : -16.0 Binding Affinity : -15.2 Binding Affinity : -14.5
 Interactions of potential binding molecule with their
respective receptor

Cyclooxygenase-1 or COX-1 + Geraniin Acetylcholinesterase + 2,3-dihydrowithaferin-A Pancreatic Lipase + Geraniin

Binding Affinity : -14.4 Binding Affinity : -14.3 Binding Affinity : -14.2
 Interactions of potential binding molecule with their
respective receptor

Acetylcholinesterase + Withanoside IV
Binding Affinity : -14.1
 Graphical Abstract of the Review Article
Selected medicinal plants

WS PU PA

Bioactive molecules from literature

Bioactive molecules

Click to add text

Protein is derived from PDB and

ligand from PubChem database
 Conclusion

 Through insilico approaches, a study has done to design drug targets, screening phytochemicals
and established ligand-protein interaction of bioactive compounds.

 Natural source of enzyme inhibitors exhibits great potential for new drug discovery

 Natural biomolecules could be effective in the treatment of various human diseases and illness

 Advantage of using natural biomolecules, is that well tolerated by people

 Interaction of secondary metabolites and inhibition of enzyme activity is a very troublesome

process to study in the laboratory.

 But due to development of recent technologies which hold the possibility for studying interaction
and generating new drugs through in-silico approaches.
 References
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 Acknowledgements

I would like to extend my sincere gratitude to,

Dr. K. Satyamoorthy
Director and Professor, MSLS, MAHE, Manipal

Dr. A. Muthusamy,
Dept. of Plant Sciences, MSLS, MAHE, Manipal

Dr. Bobby Paul & Faculty

Dept. of Bioinformatics, MSLS, MAHE, Manipal

Mr. Akshay, Mr. Sachin & Ms. Arya PhD Research Scholar
MSLS, MAHE, Manipal

All the Faculty members , MSLS, Manipal

THANK YOU