STUDIES ON ANTIVIRAL ACTIVITY AND
CYTOTOXICITY OF MORINDA CITRIFOLIA NONI
P.SELVAM M.PHARM (PH.CHEM),(PH.D).,FISAR.,
Assistant Professor,
Dept.of Pharmaceutical Chemistry
A.K. College of Pharmacy
Anand nagar.
Morinda citrifolia L (Noni) has been used
in folk remedies by Polynesians for over
2000 years, and is reported to have a
broad range of therapeutic effects.
The fruit juice is in high demand in alternative medicine for
different kinds of illnesses
The Polynesians utilized the whole
Noni plant in various combinations
for herbal remedies.
Herbal and natural products of folk
medicine have been used for centuries in
every culture throughout the world.
Morinda citrifolia L (Noni) is one of the traditional folk
medicinal plants that has been used for broad range of
therapeutic and nutritionalvalue.
Morinda citrifolia L (Noni) has been used in
folk remedies by Polynesians for over 2000
years, and is reported have a broad range of
therapeutic effects, including
Antibacterial,
Antiviral,
Antifungal,
Antitumor,
Antihelmin, analgesic,
Hypotensive, anti-inflammatory,
Immune enhancing effects.
Medicinal use of Noni :
The fruit juice is in high demand in alternative
medicine for different kinds of illnesses such as
Arthritis, Diabetes,
high blood pressure,muscle aches
menstrual difficulties,Headaches,
Heart disease, AIDS, Cancers,
Gastric ulcers, Sprains, Mental depression,
Poor digestion, Atherosclerosis,
• P. Selvam et al , Synthesized anti-HIV
activity of 4-[(1,2-dihydro-2-oxo-3Hindol-3-ylidene)amino]-N(4,6-dimethyl2-pyrimidinyl)-benzene
sulfonamide
and its derivatives.
R1
R
CH3
O
N
N
S
O
O
Euro. J. Pharm. Sci. 14 (2001) 313-316.
N
NH
N
CH3
• P. Selvam et al , synthesized cytostatic
activity of some 3-[5-amino-6-(2,3dichlorophenyl)-[1,2,4] Triazin-3-yl]6,8-Dibromo-2-substituted-3HQuinazolin-4-ones.
Cl
O
N
R1
Cl
N
N
N
N
R
NH2
R11
R=CH3; C2H5; C6H5
R1, R11=H; Br;
Indian J. Heterocyclic chem. Vol.14, Jan-Mar, 2005, 255-256.
DETAILS OF ANTIVIRAL ASSAY
• ANTIHCV ACTIVITY IN HUH 5.2 CELLS
• ANTIHIV ACTIVITY IN MT-4 CELLS
MTT ASSAY PRINCIPLE
96 MICROTITER PLATE ASSAY
O
S O2 NH
O
R
N
O
N
R1
O
S O2 NH
R
O
N
6,8 Disubt itut ed N-Ben zo yl-4 -(4 -o xo -2 -ph eny l4H -qu in azolin -3-yl)-ben zenes ulph on ami de
W h ere R, R 1 = B r
S O2 NH
O
N
R
N
N
R1
6,8 Disubt itut ed N-Ben zo yl-4 -(4 -o xo -2 -ph eny l4H -qu in azolin -3-yl)-ben zenes ulph on ami de
W h ere R, R 1 = B r
R1
6,8 Disubt itut ed N-Ben zo yl-4 -(4 -o xo -2 -ph eny l4H -qu in azolin -3-yl)-ben zenes ulph on ami de
W h ere R, R 1 = B r
VIRUSES, DISEASES AND CELL LINE
VIRUS
DISEASE
CELL LINE
HIV
AIDS
MT-4
HCV
HEPATITIS
HUH 5.2
Anti HCV activity of compound on
HCV Subgenomic replicon assay in
human hepatoblastoma cells
Hepatitis C virus (HCV) is an enveloped singlestranded(-) RNA virus that
belongs to the separate genus Hepacivirus of the family Flaviviridae.
HCV causes acute and chronic liver disease, including chronic
hepatitis, cirrhosis, and hepatocellular carcinoma.
Worldwide more than 170 million people are chronically infected with
HCV and are thus at increased risk of developing serious lifethreatening liver disease.
Current standard therapy for chronic hepatitis C consists of the
combination of pegylated interferon alpha (IFN-_) 2a in combination
with ribavirin.
Anti-HCV Assay in Huh 5-2 Cells.
Huh 5-2 cells were seeded at a density of 5 x 103 per well
in a tissue culture–treated white 96-well view plate in
complete DMEM supplemented with 250 _g/mL G418.
After incubation for 24 hours at 37°C (5% CO2) medium
50% effective concentration (EC50) was
was removedde.ned
and 3-fold
as the serial dilutions in complete
DMEM (without
G418) of the test compounds were added
concentration of compound that reduced
in a total volume
of 100 _L.
the luciferase
After 4 days of
incubation
at 37°C, cell culture medium
signal
by 50%.
was removed and luciferase activity was determined
using the Steady-Glo luciferase assay system the
luciferase signal was measured using a Luminoskan
Ascent.
The 50% effective concentration (EC50) was de.ned as the
concentration of compound that reduced the luciferase
signal by 50%.
Cytostatic Assay.
Huh 5-2, monocells were seeded at a density of
5 x103 cells per well of a 96-well plate in complete DMEM with
the appropriate concentrations of G418 or hygromycin.
Serial dilutions of the test compounds in complete DMEM
without or G418 or hygromycin were added 24 hours after
seeding.Cells were allowed to proliferate for 3 days at 37°C,
after which the cell number was determined by means of
the(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)2-(4-sulfophenyl)-2H-tetrazolium) /phenazine methosulfate
method (Promega).
The 50% cytostatic concentration(CC50) was de.ned as the
concentration that inhibited the proliferation of exponentially
growing cells by 50%.
Activity of the compounds on HCV subgenomic replicon replication
in huh-5-2 cells
Cell growth*
Viral RNA*
Concentration
Results
50 µg/ml
78
13
10
105
91
2.0
100
97
0.40
99
78
0.08
92
100
CC50
EC50
> 50
23
*(% of untreated control)
Interferon alfa-2b at 10,000 units/well reduced the signal in the viral RNA (luciferase )
assay to background levels; without any cytostatic activity.
invitro antiviral activity against HCV
The compound reduced the viral RNA below
25% and promoted cell growth more than 85 %
with respect to the untreated control,
considered as positive antiviral activity.
RESULTS AND DISCUSSION
The 50% effective concentration for inhibition
of HCV subgenomic replicon replication in
Huh 5-2 cells (luciferase assay) by noni was
23 µg/mL.
The concentration that reduced the growth of
exponentially proliferating Huh 5-2 cells by
50% was greater than 50 µg/mL
AntiHCV Activity of Methanolic extract of wrightia tinctoria on
HCV sub genomic replicon
replication in huh-5-2-cells
N
CONCENTRATION
µg/ml
N
MWT
N
CH2
NH Cell
CO
growth*
Viral RNA*
50
83
0
10
102
67
110
100
0.40
115
100
0.08
111
100
Results
CC50> 50
N
N
CH2
2.0
HN
NH
EC50=15
 Percentage of untreated control
 Interferon α-2b at 10,000 units/well reduced the signal in the
viral RNA (Luciferase) assay to background level without any
cytostatic activity
In vitro antiHIV activity and cytotoxicity studies
VIRUS : HIV-1 HTLV-IIIB
CELL LINE: MT-4 CELLS
ASSAY: MTT METHOD
EC50,CC50
MAXIMUM PROTECTION
Anti HIV activity and cytotoxicity
of the compounds in MT-4 cells.
Anti-HIV Activity and Cytotoxicity of Morinda citrifolia
COMPOUND
STRAI
N
EC50
CC50
MAXIMUM
PROTECTION
MC
IIIB
>0.1920
0.1920
19
MC
IIB
>0.1930
0.1930
17.3
EC50and CC50 value are expressed in g/ml
RESULTS
Morinda citrifolia has been evaluated for its anti-HIV
activity and cytotoxicity against HIV-1(IIIB) replication in
acutely infected MT-4 cells.
Morinda citrifolia exhibited a maximum protection of 18
against HIV-1 (IIIB) strains in acutly infected MT-4 cells.
Morinda citrifolia displayed distinct cytostatic activity against
(MT-4) cells-Adult T Cell leukemia with (CC50 =0.1930
g/ml).
Anti-HIV Activity and Cytotoxicity Of Wrightia tinctoria
Compound
Strain
EC50
CWT
III B
> 7.1
> 7.1
7.1
48
MWT
IIIB
>44.8
>44.8
44.8
2
EWT
IIIB
>60.2138
>60.2138
60.2138
1
ETWT
IIIB
>0.00256
EC90
>0.00256
EC50, EC90 and CC50 value are expressed in g/ml
CC50
0.00256
Maximum
Protection
2
FUTURE PLAN
• ANTIVIRAL ACTIVITY AGAINST
SARS-CoV in VERO CELLS
• ANTIVIRAL ACTIVITY AGAINST
AVIAN FLU(H5N1) IN MDCK
CELLS
ACKNOWLEDGEMENT
Dr. MYRIAM.W
MOLECULAR MEDICINE
BELGIUM
Dr.JOHAN NEYTS
REGA INSTITUTE FOR MEDICAL RESEARCH
BELGIUM
THANK YOU…
BY
P.SELVAM
Asst.Prof
Dept.of Pharm.Chemistry
A.K. College of Pharmacy
Anand nagar.