STUDIES ON ANTIVIRAL ACTIVITY AND STUDIES ON ANTIVIRAL ACTIVITY AND CYTOTOXICITY OF MORINDA CITRIFOLIA NONICYTOTOXICITY OF MORINDA CITRIFOLIA NONI

P.SELVAM M.PHARM (PH.CHEM),(PH.D).,FISAR.,P.SELVAM M.PHARM (PH.CHEM),(PH.D).,FISAR.,

Assistant Professor,Assistant Professor,Dept.of Pharmaceutical ChemistryDept.of Pharmaceutical Chemistry

A.K. College of PharmacyA.K. College of PharmacyAnand nagar.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 combinationsfor 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-3H-indol-3-ylidene)amino]-N(4,6-dimethyl-2-pyrimidinyl)-benzene sulfonamide and its derivatives.

N

R1

O

N S

O

O

NH

N

N

CH3

CH3

R

Euro. J. Pharm. Sci. 14 (2001) 313-316.

• P. Selvam et al , synthesized cytostatic activity of some 3-[5-amino-6-(2,3-dichlorophenyl)-[1,2,4] Triazin-3-yl]-6,8-Dibromo-2-substituted-3H-Quinazolin-4-ones.

R=CH3; C2H5; C6H5

R1, R11=H; Br;

N

N

N

NN

Cl Cl

NH2R

R11

R1

O

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

N

N

O

SO2NH

R

R1

O

6 ,8 Disubt itut ed N-Benzoyl-4-(4-oxo-2-phenyl-

4H -quinazolin-3-yl)-benzenesulphonamide

W here R, R1 = B r

N

N

O

SO2NH

R

R1

O

6 ,8 Disubt itut ed N-Benzoyl-4-(4-oxo-2-phenyl-

4H -quinazolin-3-yl)-benzenesulphonamide

W here R, R1 = B r

N

N

O

SO2NH

R

R1

O

6 ,8 Disubt itut ed N-Benzoyl-4-(4-oxo-2-phenyl-

4H -quinazolin-3-yl)-benzenesulphonamide

W here R, R1 = B r

VIRUS DISEASE CELL LINE

HIV AIDS MT-4

HCV HEPATITIS HUH 5.2

VIRUSES, DISEASES AND CELL LINE

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 life-threatening liver disease.

Current standard therapy for chronic hepatitis C consists of the combination of pegylated interferon alpha (IFN-_) 2a in combination with ribavirin.

50% effective concentration (EC50) was de.ned as the

concentration of compound that reduced the luciferase

signal by 50%.

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 was removed and 3-fold serial dilutions in complete DMEM (without G418) of the test compounds were added in a total volume of 100 _L. After 4 days of incubation at 37°C, cell culture medium 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

*(% 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.

  Cell growth* Viral RNA*

Concentration  

50 µg/ml 78 13

10 105 91

2.0 100 97

0.40 99 78

0.08 92 100

Results CC50 EC50

  > 50 23

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  

N

N

N

CH2 NH CO

N

N

CH2 HN NH

AntiHCV Activity of Methanolic extract of wrightia tinctoria on HCV sub genomic replicon replication in huh-5-2-cells

CONCENTRATIONµg/ml

MWT

Cell growth* Viral RNA*

50 83 0

10 102 67

2.0 110 100

0.40 115 100

0.08 111 100

Results CC50> 50 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-IIIBCELL LINE: MT-4 CELLSASSAY: MTT METHOD

EC50,CC50MAXIMUM PROTECTION

Anti HIV activity and cytotoxicity of the compounds in MT-4

cells.

Anti-HIV Activity and Cytotoxicity of Morinda citrifolia 

 EC50and CC50 value are expressed in g/ml

 

COMPOUND STRAIN

EC50 CC50 MAXIMUM PROTECTION

MC IIIB >0.1920 0.1920 19

MC IIB >0.1930 0.1930 17.3

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 EC90 CC50 Maximum Protection

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 >0.00256 0.00256 2

EC50, EC90 and CC50 value are expressed in g/ml

FUTURE PLAN

• ANTIVIRAL ACTIVITY AGAINST SARS-CoV in VERO CELLS

• ANTIVIRAL ACTIVITY AGAINST AVIAN FLU(H5N1) IN MDCK CELLS

ACKNOWLEDGEMENT

Dr. MYRIAM.W MOLECULAR MEDICINEBELGIUM

Dr.JOHAN NEYTS REGA INSTITUTE FOR MEDICAL RESEARCHBELGIUM

THANK YOU…BY

P.SELVAM P.SELVAM Asst.Prof Asst.Prof

Dept.of Pharm.Chemistry Dept.of Pharm.Chemistry A.K. College of Pharmacy A.K. College of Pharmacy

Anand nagar. Anand nagar.