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INTERNATIONAL JOURNAL OF

PHARMACEUTICAL INNOVATIONS

Official publication of

Nova college of Pharmaceutical Education and Research

Affiliated to Jawaharlal Nehru Technological University Kakinada&

Approved by Pharmacy Council of India and AICTE New Delhi.

Jupudi, Ibrahimpatnam- 521456,Krishna district, Andhra Pradesh.

Email id: [email protected]

mailto:[email protected]

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INTERNATIONAL JOURNAL OF PHARMACEUTICAL INNOVATIONS

Editorial Board

Editor in chief

Dr. P. Selvam M.Pharm, Ph.D., FNABS, (NCPER)

Associate editor

M. Tejaswi M. Pharm.(NCPER)

Editorial Board Members

Dr. G. Narasimhan M.Pharm, Ph.D (Harvard University, USA)

Dr. K.Senthil Kumar.,M.Pharm., Ph.D,(New York University Medical Center, New York, USA)

Dr. Solomon Arulraj David., Ph.D., (University of Johannesburg, South Africa)

Dr. D. Selvakumar , M.Pharm., Ph.D (Wayne StateUniversity, USA).

Dr. M. Chandramohan ,M.D ,Ph.D (Madurai)

Dr. N. Murugesh B Sc, MBBS., M.Sc., Ph.D (Madurai)

Dr. P. Vasanthraj, M.Pharm, Ph.D (AIMST, Malaysia)

Dr. R. Govindharajan, M.Pharm., Ph.D (Head Dabur R&D centre, Dubai)

Dr. K. Prabhu., M.Pharm, Ph.D (Nova College of Pharmacy, Jangareddygudem)

Dr. Kasthur Reddy, PhD (Mako Research Labs, Hyderabad)

Dr. P. Venkatesh, M.Pharm ,Ph.D ( Senior Principal Scientist, Orchid R&D centre, Chennai)

Dr. Alagasamy, M.Pharm, Ph.D (Hyderabad)

Dr. D. Sriram., M.Pharm., Ph.D., (BITS-Hyderabad)

Dr. Subashini., M.Pharm, Ph.D, (MSU University, Kuala Lumbur, Malaysia)

Dr. Anand Kumar., M.Pharm Ph.D, (Astrazeneca R&D Centre, Banglore)

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INSTRUCTIONS TO AUTHORS

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established by the first identification in the text of the particular table or figure.

Journal Articles

Shashi A, Jain SK and Pandey M: In-vitro evaluation of antilthiatic activity of seeds of Dolichos

biflorus and roots of Asparagus racemosus . International Journal of Plant Sciences 2008; 1:67-71.

A Book

Kalia AN: A Text Book of Industrial Pharmacognosy. CBS Publishers & Distributors, First Edition

2005.

A Chapter in a Book

Nadkarni KM: Indian Materia Medica. Popular Prakashan, Mumbai, Edition 3, Vol. I, 2000:

242-246.

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Phytochemical Analysis of successive extract of............

Chemical Constituent Aqueous Extract Ethanolic Extract

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requirements are same as those applicable to full papers. Submission of Manuscript All manuscripts (must be in English and in MS Word format) and should be submitted via our online

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human studies should also send a notary verified letter of approval from the Ethics Committee or the

Institutional Review Board. Authors are requested to send their research articles strictly according to the given format

mentioned in the guidelines to the authors.


http://[email protected]/

http://[email protected]/

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Contents

Invited article:

Novel Protocol Treatment of Dengue Fever & Swine Flu-A (H1N1) M. Chandramohan S.C. Vivekananthan, D. Sivakumar P. Selvam------- Pg no: 7-12

Research article:

Design of Novel Quinazolin-4(3H)-ones as Novel inhibitors of HIV Integrase/LEDGF.

Periyasamy Selvam1*

, Dr. Guoping Hu2, Prof. Yun Tang

2, Dr. Xi Li

2, Prof. Jin Huang

2-----Pg no : 13-20

Comparative antioxidant evaluation of three Indian cardio-protective medicinal plants in in-vitro studies

U.Subasini, S. Thenmozhi, Mohamed shabi, C. Sasikala, NinaVarghese, G.V. Rajamanickam,G.P.Dubey

Short communications :

Investigation on Cytotoxicity of Morinda citrifolia L Noni fruit Extracts

P. Selvam1*

, S.K. Ammaji1, V.Jyothirmai

1, M.Prathima

1, T. Paul Pandi

1, K. S. Praveen

2, Hitesh Jagani

2,

and J. Venkata Rao---Pg no: 21-28

Colorimetric Method Development and validation for the Estimation of Milnacipran

and Amlodipine Drugs In Bulk And Formulation

Mubarakunnisa.Md*, P. Jaya Kumar, P.Kalvari., Giri Kumari, R.Sandhya Rani, S. Ramesh

S. Udaya Bhanu, ---Pg no: 29-35

Colorimetric Method Development and Validation for the Estimation of Nebivolol in Bulk

and Formulation

P.J.V. Sagar*, M. Tejaswi, A. Jeevan kumar, A. Bhargavi, B. Adina Helga, CH. Harsha,

J. Roopu Vemula, J. Eliazar---Pg no: 36-41

Colorimetric Method Development and Validation for the Estimation of Valsartan and

Ramipril drugs in Bulk and Formulation.

A.Swathi*, M.Tejaswi N. Varun Kumar, N. Swapna Priya, N. Ravi Teja, N. Subramanyam, N. Nirmal

Babu, S. Vema Reddy. , ---Pg no: 42-48 INTERNATIONAL JOURNAL OF PHARMACEUTICAL INNOVATIONS

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Invited Article

Novel Protocol Treatment of Dengue Fever & Swine Flu-A (H1N1)

M. Chandramohan S.C. Vivekananthan, D. Sivakumar P. Selvam,

Nova College of Pharmaceutical Education and Research,

Jupudi, Ibrahimpatnam, Krishna district-521456.

Introduction:

Amidst the gloomy situation of declaration of India as “HYPER ENDEMIC” for dengue fever with yearly

average incident of 19775 with mortality of 111.5 in India with worldwide incidence mounting to 100 million and

mortality is abort 12% with endemicity occupying in more than 100 countries; we see literature reports as late as

13th march 2013 stating that there is no specific drugs available for dengue fever; consonance with the same mood

the entire world’s Medical Fraternity is lamenting the same.

With anguish we wondered “How this is happening in this NEW MILLENNIUM” which has ushered in most

advanced scientific methods in diagnosis and treatment. We wanted to conquer this dreaded infection and initiated

methodical analysis of pathophysiology of the disease and causes of severe manifestations of the disease namely

Dengue haemorrhagic fever [DHF] and Dengue Shock Syndrome [DSS] and propensity for mortality and

morbidity.

This flavivirus, dengue encompasses four serotype namely D1, D2, D3 & D4. If the infection is from one serotype

with low viral load the manifestation will be mild fever and ends up as self liming disease or undetected sub

clinical form. Trouble starts with one serotype infection followed by 2nd

type with high viral load and high level of

non structural antigen NS1 along with higher than physiological level of secreted cytokines; Tumor necrosis factor

alpha (TNF-alpha) Interleukin 1B [IL-B] and Interleukin-6 [IL-6] may cause severe manifestation of the disease

DHF; when the cytokines level goes up to exponential level they precipitate “Cytokine Storm” and cause profound

damage to microcirculatory systems of all the organs of the body and reach a point of no return causing DFS, with

profound capillary leak, haemorrhage, coagulopathy, refractory thrombocytopenia and death becomes inevitable .

One other malady in all viral infections especially in susceptible children [congenital]or other age group can

progress from cytokine storm to catastrophic “Haemophagocytic Lympho Histiocytosis” manifesting as

uncontrolled indiscriminate phagocytosis of all haemopoitic cell ending in pancytopenia. Without targeting the

above said five precipitating factors, we are at present tackling the disease with symptomatic and supportive

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measures along with trying in futile to control the vector mosquitoes Aedes Aegypti and Aedes Albopictus. We sat

down to search world literature through the internet targeting the virus for antivirals and cytokines for anti-

cytokines. As in Biblical verses; “Ask Seek and knock; the door will open” and for us the door opened and rich

haul of details of our choice poured out and we had wide choice to pick and choose. We had chosen two already in

use drugs for long time and had shown to have functional metamorphosis as antiviral agents; namely Chloroquine

[CQ] and S-Adenosyl L-Methionine. We tagged on to these Ribavirin; well known broad spectrum antiviral

agent. While picking anti-cytokines, we chose again two drugs already in use for other indications and shown to

have functional metamorphosis as anti-cytokines namely CQ again and Doxycycline; to these two anti-cytokines

we selected and added Zileuton, Monteleukast and Zaltoprofen.

Modes of Anti Dengue virus actions:

1. Chloroquine: Chloroquine [CQ] had been shown to have 13 modes of antiviral actions as per literature

survey against 13 human pathogenic viruses from 1990 and our valuable contribution is Anti Hepatitis –C

virus [HCV] action in Huh 5-2 cell line studies; Chandramohan et al [2006] and by 3D-Crystallography

assay at NS5B polymerase, Periyasamy Selvam et al [2000]. As for anti dengue virus [DV] action of CQ

we gathered four good scientific reports as early as in 1990 by Randolph VD et al spelled out that CQ

inhibits proteolytic processing of Flavivirus PrM proteins to as late as 2011; CQ blockade of NS3 of DV as

shown by the Novartis institute of Tropical Medicine, Singapore; in addition two more cell line studies by

Navarro-Sanchez 2003 and Johan Nytes.

2. S-Adenosyl L.Methionine [SALM]

Lim SV et al 2011 had shown that SALM binds at NS5 Methyl Trasferace and blocks the D.V attachment

and viral entry into the target cells and terminates viral replication. They have spotted the compound by

virtual screening based on the matching of structural configuration in 5 million chemicals

3. Ribavirin [RV]

Thakampuniya et al 2011 had shown that broad spectrum antiviral agent Ribavirin blocks DV replication

in MK2 cell line at 50.9 Mµ concentrations

Anti-Cytokines Actions

1. Chloroquine again hold another useful action of anti-cytokine activity as shown by (Jang CH et al 2006,

Weber SM, 2000) So CQ had Dual Anti DFV action, that is antiviral and anti-cytokine action to our advantage.

Another plus point is; it is available as injectable form which can be used in patients with vomiting and

diarrhea. Dose 5mg /kg oraly and 2mg/kg IM o.d

2. The popular antibiotic Doxycyline had been shown by (Castro et al [2011]) that it blocks TNF-α, IL B1 and

IL6. Dose 100mg b.d

3. Zileuton; anti asthmatic agent is having good anti-cytokine action namely blockage of synthesis of cystienyl

leukotrine and leukotrine B4 and can be used beneficial for DFV infection. Dose: 600mg b.d

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Monteleukast

Cysteinyl L Receptor – Antagonist

Zaltoprofen

Preferential Cox-2 blocker acting at the site of inflammation only

We launched a two pronged attack on DV infection and cytokine excess state with a scientific and judicious

combination of three antiviral agents acting at different sites of replication with additive action, along with five anti

cytokines and anti inflammatory agents again acting at different levels with additive actions has formed a

formidable therapeutic combipack. We formulated a strategy to start the whole set of antivirals and anticytokines

from “DAYONE” of disease of DV infection so that both viral load and cytokine level will not escalate and we

will be able to win the battle against DV with ease and certainty.

Anti-Swine Flu A-H1N1 Programme:

In the same spree we culled out again two already in use drugs Chloroquine [CQ] antimalarial and

Nitazoxanide [NTZ] Antiprotozoal which are shown to have functional metamorphosis as Anti-A[H1N1]

Anti-A H1N1 action:

Chlroquine blocks the M2 channels of A[N1H1] and prevents attachment and cell entry of the targeted host cells.

Nitazoxanide –NTZ had been shown to block maturation of Haemagglutinin surface antigen of the virus, 500 in

number and wholesomely prevents virus attachment on to the targeted cell’s receptor sites, endocytosis, trafficking

inside the Nucleus of the cell, collecting essential materials for assembly of the virus, maturation of the virus and

final process of exocytosis.

These two Anti-A[H1N1] drugs have additive and complementary actions and when added with the five

anti-cytokines combination will work in consonance and will have lethal blow on the virus and prevent cytokine

excess or cytokine storm and will cure the patients radically. I have put up this proposal to W.H.O in 2009 itself for

trial and implementation and got a pleasant reply that “Clinical Task Force considering with attention to your

proposal; thank you sharing your ideas with us”; no further communication followed.

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New protocol treatment for Dengue Viral Fever

Material and Method:

Recruited 3 male 3 female patients; age ranged between 16 and 36, who had fever, with conjunctival

injection, body pain and retrobulbar pain who were positive for NS1 antigen by rapid card test and the same

remaining sera tested for anti dengue anti bodies IgM and IgG by ELISA assays proved positive denoting that all

the patients had been suffering from secondary dengue infection.

All the seven drugs were given from the day one of the diagnosis in the following oral dose schedule 1.

CQ 300mg o.d 2.SALM 400 TDS 3) RV 200mg QID 4) Doxycycline 100mg b.d 5) Zileuton 600mg b.d 6)

Monteleukast 10mg o.d 7) Zaltoprofen 80 tds

We found the fever and other symptomatologies abated in 5 to 14 days while dengue IgM and IgG ELISA

assay had been repeated on every 4 to 6 days. To our surprise; an eye opener result noticed in these six patients and

the Dengue Anti IgM and IgG rendered negative in 16 to 30 days by ELISA assay while the antibodies usually

persist in the blood for 90 days and lifelong respectively.

Conclusion:

These results denote that prompt two prong attack on dengue virus and the illness with multimodal action might

have drastically restricted the DV load and yielded minimal anti bodies production which were cleared very early

and interpreted as “RADICAL CURE” It is heartening to note that among the pessimistic attitude and lamenting

medical fraternity that there is no specific anti viral agent available we have picked up effective and economical

anti viral agents against DV and very effective anti cytokine combination tackling the Dengue disease

manifestations successfully. In both the programmes the antivirals should be used as prophylaxis for all the

inmates of the premises of the patients. In case of severe refractory thrombocytopenia patient we have given

intravenous Anti-D immunoglobulin 50µg/kg for 2 days and shown remarkable recovery (Gaurav Kharya et al

2011).

These two programmes exemplifies “BENCH TO BED SIDE” motto of utilizing the laboratory proved

benefits to be made available to sick patients in the hospital beds as early as possible.

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References:

Castro JE et al “Modulation of cytokine and cytokine receptor/antagonist by treatment with Doxycycline and

Tetracycline in patients with dengue fever” Clin Dev Immunol. 2011;2011:370872

Chandramohan M, Vivekananthan SC, Sivakumar D, Selvam P, Neyts J, Katrien G, De Clercq E “Preliminary

report of anti – hepatitis C virus activity of chloroquine and hydroxychloroquine in Huh – 5 – 2 cell line” Indian J.

Pharm.Sci. 2006, 68(4): 538 – 540.

Chandramohan, M et al “Chloroquine therapy in viral hepatitis” Pro. Southern regn conference of Association of

Physicians of India. Madurai PP. 22 – 1980

Jang CH et al., “Chloroquine Inhibits Production of TNF-alpha, Il-1beta and Il-6 from lipopolysaccharide-

stimulated human monocytes/macrophages by different modes” Rheumatology [Oxford] 2006; 45(6) 703-10

Jean Franc¸ois Rossignol “Thiazolides, a New Class of Anti-influenza Molecules Targeting Viral Hemagglutinin at

the Post-translational Level” 2009 284: 43 23, PP29789 – 808 J. Biol. Chem

Kharya G et al “Management of severe refractory thrombocytopenia in dengue hemorrhagic fever with intravenous

anti-D immune globulin” Pediatr Hematol Oncol. 2011;28(8):727-32

Lango D.L., D.L. Kasper, et al “Harrison’s Principles of Internal Medicine” 18th Edition 2012 pp 1621 publisher

M.C. Graw-Hill Company Inc. Newyork U.S.A.

Lim SV, Rahman MB, Tejo BA “Structure-based and ligand-based virtual screening of novel methyltransferase

inhibitors of the dengue virus” BMC Bioinformatics. 2011 Nov 30;12 Suppl 13:S24

Navarro-Sanchez E, Altmeyer R, Amara A, Schwartz O, Fieschi F, et al. (2003) Dendritic-cell-specific ICAM3-

grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived

dengue viruses. EMBO Rep 4: 723–728.

P. Selvam, Chandramohan,M. Suresh kumar et al “Design, Molecular Modelling Studies on Chloroquine and Its

analogues as Inhibitors of HCV NS5B RNA Polymerase ” 22nd

International Conference - Antiviral Research

82(2009) A52

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Periyasamy Selvam, Priya Srinivasan, Tanvi Khot, R. Padmanaban, M. Chandramohan “Studies of Dengue NS3

Protease Inhibitory Activity of Novel Isatin Derivatives” 25th International Conference on Antiviral Research –

Abstract 99-2012

Randolph VB, Winkler G, Stollar V (1990) Acidotropic amines inhibit proteolytic processing of flavivirus prM

protein. Virology 174: 450–458.

Ratree Takhampunya et al “Inhibition of dengue virus replication by mycophenolic acid and ribavirin” Journal of

General Virology (2006), 87, 1947–1952

Weber SM, Levitz SM (2000) Chloroquine interferes with lipopolysaccharide induced TNF-alpha gene expression

by a nonlysosomotropic mechanism. J Immunol 165: 1534–1540.

WHO: Technical Guides for Diagnosis Treatment, Surveillance, Prevention and Control of Dengue Hemorrhagic

Fever, WHO Geneva, 1975.

Dr. M. Chandramohan MD., Ph.D., F.I.C.C

Distinguished Professor TN Dr. MGR Medical University/ Stem Cell Therapist

Chairman & Prof. of Hepatology, Bharat Ratna Kamarajar Jaundice, Liver Hospital and

Research Centre, Retd. Addl.Prof. & HOD of Internal medicine Research Wing, Madurai Medical College

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Research Article

Quinazolin-4(3H)-ones as Novel inhibitors of HIV Integrase/LEDGF interaction

Periyasamy Selvam1*

, Dr. Guoping Hu2, Prof. Yun Tang

2, Dr. Xi Li

2, Prof. Jin Huang

2

1Nova College of Pharmaceutical Education and Research, Ibrahimpatnam, Krishna Dt, India

2School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China

E mail: [email protected]

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Abstract

Novel Quinazoline derivatives were investigated for inhibition of HIV Integrase/ Lens epithelium derived

growth factor (LEDGF) protein-protein interaction by using ALPHA screen technique. Hypothetical binding

modes of the selected compound in HIV integrase were generated using GLIDE docking tool. Quinazoline

derivatives (SPS-I and SPS-II) inhibits HIV IN/LEDGF interaction and SPS-I more potent compound

(9.23±0.4 µM) in this series. Molecular modeling studies indicate that the SPS-I and II can bind within the

active site of HIV integrase (DDE) and thus interrupt the binding of HIV integrase with LEDGF.

Keywords: Quinazoline, HIV Integrase, LEDGF, Protein-Protein interaction

Introduction:

AIDS is a fatal pathogenic disease caused by retrovirus Human Immunodeficiency Virus (HIV). No

effective vaccine is available till now to combat HIV/AIDS. The only available option is chemotherapy

(HAART) that can reduce the viral load and improve the quality of life of HIV/AIDS patients. Present

therapeutic agents are suffering with emergence of resistance and thus demanding novel molecular targets,

newer therapeutic agents to sustain the treatment and enhance the life span of the infected population

(Frauke Christ, et al., 2013). HIV integrase (IN) plays important roles at several steps, including reverse

transcription, viral DNA nuclear import, targeting viral DNA to host chromatin and integration (Engelman

et al., 2013). Review of literature have demonstrated that HIV-1 Integrase interacts with a cellular lens

epithelium-derived growth factor (LEDGF/p75) and that this viral/cellular interaction plays an important

role for tethering HIV-1 preintegration complexes (PICs) to transcriptionally active units of host chromatin

(Serrao et al., 2013; Christ et al., 2013; Hu et al., 2012 and Peat et al., 2012). Small molecule inhibitors of

HIV IN/LEDGF have emerged as promising new class of antiviral agents for the treatment of HIV/AIDS

(Taltynov et al., 2008 and Hou et al., 2008). Present study is to investigate the novel quinazoline

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derivatives as potential inhibitor of HIV Integrase-LEDGF/P75 protein-protein interaction.

Materials and Methods

Compounds: Quinazoline derivatives were synthesized by reaction between 2-phenyl-benzoxine with

sulphanamide in presence of glacial acetic acid (Selvam et al., 2008)

Molecular modeling :

The crystal structure of the dimeric CCD of HIV-1 IN in complex with Compound 3 (PDB code 3LPT) was

used in this study. Compounds 3 were used to induce the receptor. The IFD workflow of Maestro 9.0 was

utilized with Prime 2.1 and Glide 5.5 to adjust the receptor structure, especially in the binding pocket. This

workflow set up a sequence of tasks in which ligands were first docked with Glide, Prime refinement was

implemented to allow the receptor to relax, and finally the ligands were redocked into the relaxed receptor

with Glide. All docking calculations were run in the “Standard Precision” mode of Glide, the center of the

grid box was set to that of the ligand, and the box size was set to auto. All other parameters were left at

default settings.

B) Ligand preparation: Ligprep 2.3 was used to prepare these compounds (SPS-I and II) before docking.

During this process, the OPLS_2005 force field was chosen and possible ionization states at the pH range of

5.0–9.0 were generated.

HIV IN /LEDGF assay: The HIV-1 IN CCD was expressed and purified as described (Jenkins et al.,

1996)The IBD of p75 containing a GST tag was prepared as previously reported (Du et al., 2008) The

LEDGF/p75–IN AlphaScreen assay was developed as described elsewhere. (Frauke Christ, et al., 2010 )

Reactions were performed in a 25 μL final volume in 384-well ProxiPlates. The buffer was composed of 25

mM HEPES, pH 7.3, 150 mM NaCl, 2 mM MgCl2, 1 mM DTT, and 0.1% BSA. The His6-tagged HIV IN

16

CCD was added to a final concentration of 40 nM and incubated with test compounds at varying

concentrations (0.1–100 μM) and room temperature for 30 min. Subsequently, the remaining components

containing a GST-tagged LEDGF/p75 IBD (final concentration, 40 nM), nickel chelate acceptor beads (final

concentration, 8 μg/mL), and glutathione donor beads (final concentration, 8 μg/mL) were added to the well.

Proteins and beads were incubated at room temperature for 2 h. Incubation was performed in the dark to

avoid direct light exposure. The plates were measured with an EnVision Multilabel Plate Reader, with the

final emission ranging from 520 to 620 nm.

Results:

Quinazoline derivatives (SPS-I and SPS-II) were investigated for inhibition of HIV Integrase/LEDGF

interaction (Fig 1, Table 2) and compounds SPS-I and SPS-II inhibits HIV IN/LEDGF interaction at the

concentration of 10.08 and 9.23 µM, respectively. Whereas standard quinoline derivatives13

(compound 3)

reported in nature was found to be 11.91 µM. SPS-I more potent compound (IC50: 9.23 µM) in this series.

Molecular modeling studies (Fig 2 ) indicate that the SPS-I and SPS-II can bind within the active site of HIV

integrase (DDE) and thus interrupt the binding of HIV integrase with LEDGF. This compounds already

demonstrated for inhibition of HIV replication in MT-4 cells (Selvam et al., 2008). Quinazolines are the

novel class of inhibitors of HIV IN/LEDGF interaction (protein-protein) and this lead molecule along with

the residues identified through modeling stuides is suitable for further molecular modifications

References

Christ F, Voet A, Marchand A, Nicolet S, Desimmie BA, Marchand D, Bardiot D, Van der Veken NJ, Van

Remoortel B, Strelkov SV, De Maeyer M, Chaltin P, Debyser Z. Rational design of small-molecule inhibitors

of the LEDGF/p75-integrase interaction and HIV replication. Nat Chem Biol. 2010 :6(6):442-8.

http://www.ncbi.nlm.nih.gov/pubmed?term=Christ%20F%5BAuthor%5D&cauthor=true&cauthor_uid=20473303

http://www.ncbi.nlm.nih.gov/pubmed?term=Voet%20A%5BAuthor%5D&cauthor=true&cauthor_uid=20473303

http://www.ncbi.nlm.nih.gov/pubmed?term=Marchand%20A%5BAuthor%5D&cauthor=true&cauthor_uid=20473303

http://www.ncbi.nlm.nih.gov/pubmed?term=Nicolet%20S%5BAuthor%5D&cauthor=true&cauthor_uid=20473303

http://www.ncbi.nlm.nih.gov/pubmed?term=Desimmie%20BA%5BAuthor%5D&cauthor=true&cauthor_uid=20473303

http://www.ncbi.nlm.nih.gov/pubmed?term=Marchand%20D%5BAuthor%5D&cauthor=true&cauthor_uid=20473303

http://www.ncbi.nlm.nih.gov/pubmed?term=Bardiot%20D%5BAuthor%5D&cauthor=true&cauthor_uid=20473303

http://www.ncbi.nlm.nih.gov/pubmed?term=Van%20der%20Veken%20NJ%5BAuthor%5D&cauthor=true&cauthor_uid=20473303

http://www.ncbi.nlm.nih.gov/pubmed?term=Van%20Remoortel%20B%5BAuthor%5D&cauthor=true&cauthor_uid=20473303

http://www.ncbi.nlm.nih.gov/pubmed?term=Van%20Remoortel%20B%5BAuthor%5D&cauthor=true&cauthor_uid=20473303

http://www.ncbi.nlm.nih.gov/pubmed?term=Strelkov%20SV%5BAuthor%5D&cauthor=true&cauthor_uid=20473303

http://www.ncbi.nlm.nih.gov/pubmed?term=De%20Maeyer%20M%5BAuthor%5D&cauthor=true&cauthor_uid=20473303

http://www.ncbi.nlm.nih.gov/pubmed?term=Chaltin%20P%5BAuthor%5D&cauthor=true&cauthor_uid=20473303

http://www.ncbi.nlm.nih.gov/pubmed?term=Debyser%20Z%5BAuthor%5D&cauthor=true&cauthor_uid=20473303

http://www.ncbi.nlm.nih.gov/pubmed/?term=Nat.+Chem.+Bio.%2C+2010%2C6%2C442

17

Frauke Christ, ZegerDebyser., The LEDGF/p75 integrase interaction,a novel target for anti-HIV therapy.,

Virology 435, 2013; 102–109

Du L, Zhao Y, Chen J, Yang L, Zheng Y, Tang Y, Shen X, Jiang H. D77, one benzoic acid derivative,

functions as a novel anti- HIV- 1 inhibitor targeting the interaction between integrase and cellular LEDGF/p

75. Biochem Biophys Res Commun. 2008; 10;375(1): 139-44.

Engleman A, Kessl JJ, Kvaratskhelia M. Allosteric inhibition of HIV-1 integrase activity.

Curr Opin Chem Biol. 2013:339-45.

Hou Y, McGuinness DE, Prongay AJ, Feld B, Ingravallo P, Lunn CA, Howe JA Screening for antiviral

inhibitors of the HIV integrase - LEDGF/p75 interaction using the AlphaScreen (TM) luminescent proximity

assay. J Biomol Screening 2008;13:406–414.

Hu G, Li X, Zhang X, Li Y, Ma L, Yang LM, Liu G, Li W, Huang J, Shen X, Hu L, Zheng YT, Tang Y,

Discovery of inhibitors to block interactions of HIV-1 integrase with human LEDGF/p75 via structure based

virtual screening and bioassays. J Med Chem. 2012;55(22):10108-17.

Jenkins TM, Engelman A, Ghirlando R, Craigie R. A soluble active mutant of HIV-1 integrase: involvement

of both the core and carbozyl-terminal domains in multimerizatio. J Biol Chem. 1996;271(13):7712-8.

Peat TS, Rhodes DI, Vandegraaff , Le G, Smith JA, Clark LJ, Jones ED, Coates JA, Thienthong N, Newman

J, Dolezal O, Mulder R, Ryan JH, Savage GP, Francis CL, Deadman JJ. Small molecule inhibitors of the

LEDGF site of human immunodeficiency virus integrase identified by fragment screening and structure based

design. PLos One. 2012;7(7):e40147.

18

Selvam P, Murgesh N, Chandramohan M, De Clercq E, Synthesis, antiviral and cytotoxic activities of 6-

bromo/6,8-dibromo-4-(4-oxo-2-phenyl-4H-quinazolin-3-yl)-benzene sulphonamide, Journal of Applied

Chemistry 7,18-23,2008

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hydroxy qunioline inhibitors of the HIV- Integrase- lens epithelium – derived growth factor/p75 (IN-

LEDGF/p75) interaction. J. Med. Chem. 2013; 56(6) :2311-22

Taltynov O, Desimmie BA, Demeulemeester J, Christ F, Debyser Z. Cellular co factors of lentiviral integrase

from target validation to drug discovery. Mol Biol Int. 2012 :863405

19

Fig 1. Structure of Quinazoline derivatives

Table 1. HIV Integrase/LEDGF inhibitory activity of QUINAZOLIN-4(3H)-ONES

**Nat. Chem. Bio., 2010,6,442

The results are IC50 ±S.D, n = 3 for HIV-1 IN/LEDGF inhibitory activity

cConcentration required to inhibits HIV IN/LEDGF interaction.

Compounds MW Inhibitory (%) (@10µM) LEDGF-IN (IC50 µM)

SPS-I 377.42 49.96 10.08 ±3.11

SPS-II 456.31 52.52 9.23±0.47

Compound 3** 313.74 44.07 11.91±0.61

20

Fig 2. Molecular Modelling of Quinazolin-3(4H)-one derivatives

SPS-I SPS-II

21


Comparative antioxidant evaluation of three Indian cardio-protective medicinal

plants in in-vitro studies

U.Subasini1*, S. Thenmozhi

2, Mohamed shabi

3, C. Sasikala

3, Nina Varghese

3,

G.Victor Rajamanickam4, and G.P.Dubey

5

1Management and Science University, IMS, Shah Alam, Malaysia 40100

2Swamy Vivekanandha College of Pharmacy, Thiruchengode – 637 205 Namakkal Dt,

3Masterskill University, Medical campus, Johor Bharu, Malaysia - 81750

4

Sri Sairam Groups of Institutions, Medical Campus, West Tambaram, Chennai – 44 5

Banaras Hindu University, Varanasi

* - Corresponding author,

Dr. Subasini Uthirapathy

Associate Professor

Faculty of Medicine

MSU University, International Medical School

Shah Alam

Malaysia - 637 205

Email – [email protected]

[email protected]



22

Abstract:

A number of Indian medicinal plants have been used for thousands of years in the management of cardiovascular

diseases such as atherosclerosis, hypertension. Oxidative damage remains as the important factor for the

development of cardiovascular diseases. A comparative antioxidant evaluation of three cardio-protective plants

pays the better way to fix the proportion of individual plants in the development of new formulation. With this

aim, the antioxidant activity of three medicinal plants is evaluated in the in-vitro condition. 70 % ethanolic extract

of Nelumbo nucifera (NN), Withania somnifera (WS) and Terminalia arjuna (TA) were tested for their antioxidant

activity and IC50 values were determined. To confirm the potent antioxidant plant their phenol, tannin, Vitamin C,

Vitamin E and carbohydrate content were also estimated. HPTLC profile of all the three extracts were also taken.

The results from the ABTS and nitric oxide radical scavenging assay showed that IC50 value of all the three extracts

were similar. Whereas in antilipid peroxidation assay NN has lower IC50 value than other two extracts. Tannin,

Vitamin C, Vitamin E and carbohydrate content of TA was found to be high. In NN, Total phenolic content was

found to be high. In conclusion, NN is more potent in exhibiting antioxidant activity than the other two extracts.

Whereas free radical scavenging activity is exhibited by all the three extracts equally. Along with NN, TA is more

potent in phytoconstituents content. Thus increasing the content of NN or TA in the formulation can increase the

potency in cardiovascular disorder.

Keywords: ABTS, nitric oxide radical, tannin, HPTLC, Vitamin C

INTRODUCTION:

Free radicals are highly responsible for the development of various diseases including cardiovascular

disease (Halliwell and Gutteridge, 1984). Various phenolic compounds which are widely distributed in food and

medicinal plants are versatile antioxidants against reactive oxygen species that cause cell damage and are

implicated in many diseases (Pietta, 2000). Polyphenol compounds are reported to be a good source of natural

antioxidants (Hagerman et al. 1998). Besides the traditional resources used for antioxidants, many plant species

have been investigated in the search for natural antioxidants (Banias et al. 1992).

23

Nelumbo nucifera (Geartn) (NN) belongs to the family Nelumbonaceae is a handsome aquatic herb, with

stout, creeping rhizome is found through out India. Ethanolic extract from seeds exhibits antioxidative and

hepatoprotective effects (Sohn et al., 2004). Ethanolic extract of rhizome reduces blood sugar level

(Pulok et al.,

1997). Ethanolic extract of rhizome exhibits various psycho-pharmacological effects like reduction in spontaneous

activity, decrease in exploratory behavioral pattern by the head dip and Y-maze test, reduction in muscle relaxant

activity by rotarod, 30o inclined screen and traction test and potentiated the pentobarbitone induced sleeping time in

mice (Pulok et al., 1996). It causes reduction of triglyceride and cholesterol level

(http://www.pfaf.org/database/search_use.php?K%5B%5D=Cardiotonic&START=100).

Withania somnifera (L) (WS) belongs to the family Solanaceae, is an erect, branched, unarmed shrub grows

throughout the drier parts and sub-tropical India. It exhibits adaptogenic and cardioprotective activity in rats and

frogs (Jayant, 2000)9. The hypotensive effect of WS might be due to autonomic ganglion blocking action and a

depressant action on the higher cerebral centers (Malhotra et al., 1981)

Terminalia arjuna (TA) (belongs to the family Combretaceae is a large tree with smooth and thick bark found

throughout the greater parts of India. It exhibits antioxidant and hypocholesteremic effects (Gupta et al., 2001).

Early physiological studies carried on the isolated frog and rabbit heart revealed that the bark of TA had

cardiotonic and stimulant actions (Ghoshal, 1909). It was subsequently found that intravenous administration of the

glycoside, obtained from the bark of TA resulted in rise in blood pressure (Ghosh, 1926). Interestingly the aqueous

extract of the bark in isolated rat atria demonstrated positive inotropic activity. This was again confirmed in

subsequent work where aqueous extract of the bark in isolated rat atria produced inotropic action which was

abolished by propanolol and cocaine (Karamsetty et al., 1995).

Nowadays, pharmaceutical companies are seeking for polyherbal formulations mainly for their less toxic and

high synergestic effect. A study should be undertaken, to find out the more potent herb in invitro condition and

then the result might be useful to the Pharmaceutical industry to fix the concentration of individual herb in the

formulation. With this aim three Indian medicinal plants which are reported and traditionally used for the

cardiovascular diseases. (NN, TA, WS) are selected for the present study. The In vitro antioxidant activity of the

70 % ethanolic extract of all the three extracts were carried out. In order to find out the more potent extract their

HPTLC profile along with the phytoconstituents content were estimated.

http://www.pfaf.org/database/search_use.php?K%5B%5D=Cardiotonic&START=100

24

MATERIALS AND METHODS

Chemicals and reagents:

2,2’-Azinobis-3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) was purchased from Sigma, MO, USA. The

other chemicals used were of analytical grade.

Collection and identification of herbs:

Earlier references reveal that flower and seed of NN, root of WS and bark of TA were cardioprotective in

nature (http://www.pfaf.org/database/search_use.php?K%5B%5D=Cardiotonic&START=1008, Lakshmi et al.,

(2000), Caius et al., 1930). So that the same parts of NN, WS and TA were collected respectively from Nagercoil,

Madurai and Thirunelveli, Tamilnadu, India. They were identified and authentified and stored in Centre for

Advanced Research in Indian System of Medicine (CARISM), SASTRA University, Tamilnadu, India. The

voucher number is 0092 for NN, 0064 for WS and 0094 for TA.

Preparation of extracts:

The collected plant materials were cleaned, shade dried for 15 days and coarsely powdered in grinding

mill. 70 % ethanolic extracts (70:30 Ethanol: water) of all the three medicinal plants were prepared as follows. 100

gm each of the dried plant was soaked in 70 % ethanol for 3 days in room temperature (26-/28°C). The solvent was

evaporated under reduced pressure and then lyophilized. The yield of extract for NN, WS and TA was calculated

as 8.49 %, 3.83 % and 27.7 % respectively. The samples were dissolved in 70 % ethanol and used for further in-

vitro antioxidant activity.

ABTS+·

radical cation decolorization assay:

ABTS radical cation decolorisation assay was estimation by following the method of Re et al., (1999)17

.

ABTS radical cation (ABTS+·) was produced by reacting ABTS solution (7 mM) with 2.45 mM ammonium

persulfate and the mixture was allowed to stand in dark at room temperature for 24 h before use. After 24 h the

ABTS solution was diluted with 70 % ethanol, to an absorbance of 0.70 (±/0.02) at 745 nm. One milliliter of

diluted ABTS solutions were mixed with 1.0 ml of extract containing different concentration of extract and the

percentage inhibition was calculated against control. The control tubes contain 1.0 ml of ABTS and 1.0 ml of 70 %

ethanol. Water was used as blank.

% Inhibition = (Control O.D. – Test O.D./ Control O.D.)X100

IC50 value is 50 % inhibition of ABTS decolorisation and this was calculated using GraphPad Prism version 5.0.

Triplicate was carried out and the mean value was used for IC50 determination.

http://www.pfaf.org/database/search_use.php?K%5B%5D=Cardiotonic&START=1008

25

Lipid peroxidation:

Preparation of rat Heart homogenate:

Rats weighing 200-250 g were sacrificed by decapitation and whole heart was dissected out and

homogenized (100 mg/ml) in ice-cold phosphate buffer (0.1 M, pH 7.4) medium and used as a source of

polyunsaturated fatty acids for determining the extent of lipid peroxidation. The tissue homogenate was

centrifuged at 800/g for 15 min at 4°C and the supernatant was used for further study. Control tube contains 25 μl

of heart homogenate along with 25 μl of ferrous sulphate (0.15 M). Different concentration of extracts were mixed

with 25 μl of heart homogenate along with 25 μl of ferrous sulphate and incubated in a shaking water bath at 37°C

for 40 min in open test tubes. Stock solutions of different concentrations of the extracts were diluted to 1.0 ml with

buffer and were added to the homogenate at the beginning of the incubation period. In case of control experiments,

volume adjustments were done with phosphate buffer. Incubations were terminated by the rapid addition of 3.5 ml

20 % (v/v) acetic acid. All the tubes were subjected for Thiobarbituric acid Reactive substances assay.

Thiobarbituric acid reacting substances (TBARS) assay:

After incubation was terminated, 600 μl of 0.5 % Thiobarbituric acid (TBA) in 20 % (v/v) acetic acid (pH

3.5) was added to each sample. The tubes were then incubated at 85°C for 60 min. After cooling 50 μl of 10 %

sodium dodecyl sulfate (SDS) was added and centrifuged at 5000 rpm for 10 min and the absorbance of the

supernatant was determined at 532 nm (Okhawa et al., 1979). The absorbance was recorded against blanks

prepared in the same way as the experimental samples but without homogenate. Percentage inhibition and IC50

values were determined as mentioned in ABTS radical cation decolorization assay. The experiment was repeated

thrice.

Determination of Nitric Oxide Radical Scavenging Activity:

Nitric oxide radical scavenging activity was determined by following the method of Govindharajan et al.,

(2003)19

with slight modification. Sodium nitroprusside (5 mM) in phosphate-buffered saline (PBS) was mixed

with 3.0ml of different concentrations (10 – 320 µg/ml) of the drugs dissolved in the suitable solvent systems and

incubated at 25 °C for 300 min. The samples from the above were reacted with Greiss reagent (1% sulphanilamide,

2 % H3PO4 and 0.1 % napthylethylenediamine dihydrochloride). The absorbance of the chromophore formed

during the diazotization of nitrite with sulphanilamide and subsequent coupling with napthylethylenediamine was

read at 546 nm

26

(Acont—Atest)

NO scavenged (%) = __________ X 100

Acont

where Acont is the absorbance of the control reaction and Atest is the absorbance in the presence of the sample of

the extracts

Determination of total polyphenol content:

Total polyphenols were determined by Folin–Ciocalteu procedure (Slinkard and Singleton, 1977). 70 %

ethanolic extract of NN was dissolved in the same solvent. 0.5 ml of sample was mixed with 0.25 ml of Folin–

Ciocalteu reagent and 1.25 ml 20 % aqueous sodium carbonate solution, samples were vortexed and absorbance of

blue colored mixtures recorded after 40 min at 725 nm against a blank containing 0.5 ml of 70 % ethanol, 0.25 ml

of Folin–Ciocalteu reagent and 1.25 ml 20 % aqueous sodium carbonate solution. The calibration curve was

prepared from Phenol. The amount of total polyphenols was mentioned as mg of phenol/g of extract.

Measurements were done in triplicate.

Quantification of tannins:

Tannin content of 70 % ethanolic extract of NN was determined by following the method of Okwu

(2005)21

. 500 mg of 70 % ethanolic extract was weighed in to 100 ml bottle. 50 ml of the same solvent was added

and shaken for 1 hr in a mechanical shaker. This was filtered in to a 50 ml volumetric flask and made up to the

mark. Then 5 ml of the filtrate was pipette out in to a tube and mixed with 3 ml of 0.1 M FeCl3 in 0.1 N HCl and

0.008 M Potassium ferrocyanide. The absorbance was measured in a spectrophotometer at 630 nm wavelength. A

blank sample was prepared and the color developed was read at the same wavelength. Different concentration of

tannic acid was used as standard. The final concentration of tannins was represented as mg/gm of extract.

Measurements were done in triplicate.

27

Estimation of Total Carbohydrate:

Total carbohydrate content of plant material was estimated by following the method of Dubois (Dubois et

al., 1954)22

. 50 mg of sample was ground well with 2-3 ml of 5 % TCA. To the de-proteinized supernatant 10 ml of

45 % ethanol was added to precipitate the polysaccharides. After setting it to stand overnight in cold, the tube was

centrifuged for 10 minutes at 4000 rpm. The dried precipitate was analyzed for total carbohydrate by dissolving in

2 ml of 1N NaOH. The sample was made up to 1ml with water. 1 ml of 5 % Phenol and 5ml of concentrated

sulphuric acid were added. The mixture was mixed thoroughly with a glass rod. The solution was allowed to stand

for 10 minutes at room temperature and its optical density was read at 490 nm in a spectrophotometer and standard

graph was prepared by using different concentration of D-Glucose ranging from 10 to 100 µg/ml.

Estimation of Vitamin C:

Vitamin C in the plant was estimated according to the method of Sarojini and Nittala (1999) with slight

modifications. 1.0 gm of plant material was soaked in 75 % ethanol for 24 hours. The extract was filtered. The

filtrate was used as sample for the estimation of vitamin C. 0.1 ml of sample was made up to 1.0 ml with water.

Different concentrations of ascorbic acid from 4 – 20 µg/ml were used as the standard. Blank contains 1.0 ml of

water. 0.5 ml of dinitro phenyl hydrazine (0.2 %) was added to all the tubes including blank, test and standard test

tubes. Incubate all the tubes at room temperature for 3 hours. 2.5 ml of 85 % Sulphuric acid was added to all the

tubes. The absorbance was measured at 520nm

Estimation of Vitamin E by Dipyridyl Method:

Vitamin E in the plant was estimated according to the method of Jayashree et al., (1985)

with slight

modifications. 1.0 gm of plant material was soaked in 100 ml of ethanol: petroleum ether (1.5:2.0) for 24 hours.

The petroleum ether fraction was separated and evaporates to dry. The precipitate formed was mixed with 5.0 ml

of ethanol. 0.2 ml of extract was made up to 1 ml with ethanol. To this, 0.2 ml of 2,2’, - dipyridyl reagent (0.2

%), 0.2 ml of ferric chloride (0.5 %) solution and 2.0 ml of butanol were added. For blank, 0.2 ml of ethanol, 0.2

ml of 2,2’- dipyridyl reagent, 0.2 ml of ferric chloride and 2.0 ml of butanol were added The red colour developed

was read exactly after 15 min. at 520 nm. Different concentrations of α-tocopherol were used as standard. The

calculated tocopherol was mentioned as µg/100 g of plant material.

28

HPTLC fingerprinting of all the three extracts:

Chromatography was performed on preactivated (1100C) silica gel HPTLC plate silica 60GF254. 70 %

hydroalcoholic extract of all the three extracts dissolved in methanol at the concentration of 10 mg/ml was applied

on the silica gel coated plate with an automatic applicator Linomat 5 with N2 flow (CAMAG, Switzerland). The

number of bands for each sample is 2. Initially the TLC chamber was saturated with Butanol:acetic acid: water

(4:1:1) for NN, Chloroform : methanol (8:2) for WS and TA for 1 hour. The sample applied plate was developed

using the mobile phase upto 80 mm of the plate. The HPTLC runs were in laboratory conditions of 25 ± 50C and

50 % relative humidity. After development the plate was withdrawn and air dried and spots were visualized in UV

light (UV cabinet, CAMAG, Switzerland). The plate was photodocumented at 254 and 366 nm. The plate was

scanned in the scanner, CAMAG, Swizerland. Scanning conditions for NN are Position of first track – 10.3 mm,

distance between tracks – 10.0 mm, scan start position Y- 10.0 mm, scan end position – 84.0 mm, wavelength –

366 nm, for WS are Position of first track – 10.8 mm, distance between tracks – 11.4 mm, scan start position Y-

10.0 mm, scan end position – 82.0 mm, wavelength – 366 nm and for TA are Position of first track – 9.6 mm,

distance between tracks – 11.4 mm, scan start position Y- 6.0 mm, scan end position – 85.0 mm, wavelength – 366

nm,. Other scanning conditions like slit dimensions – 6.0X0.45 mm, scanning speed – 20.0 mm/s, data resolution –

100 μm/step, lamp – D2 & W are same for all the three extracts.

RESULTS:

Data of Table 1 shows the antioxidant activity of NN, WS and TA in different methods. Along with the

concentration of extracts the percentage inhibition of TBARS formation, NO radical scavenging and ABTS+·

radical cation decolorization were found to be increased (Table 1). The IC50 value calculated using Graphpad

software version 5.0 reveals that there is no difference between all the three extracts in ABTS and NO radical

scavenging assays. Whereas, NN prevent the TBARS formation at the lower concentration itself followed by WS

and TA (Fig 1).

29

Table 1: Effect of NN (Nelumbo nucifera), WS (Withania somnifera) and TA

(Terminalia arjuna ) on In-vitro antioxidant assay

Parameters Extract % Inhibition

Anti-lipid

peroxidation

Concentration

(μg/ml)

100 200 300 400 500

NN 37.4 ± 3.0 50.88 ± 5.0 65.09 ± 6.2 76.5 ± 6.5 86.7 ± 7.3

WS 37.7 ± 3.0 46.6 ±4.0 59.2 ± 5.0 77.4 ± 6.2 89.3 ± 7.3

TA 26.2 ± 2.3 36.6 ± 2.5 55.9 ± 3.6 68.2 ± 5.6 87.9 ± 6.8

Nitric oxide

radical

scavenging

% Inhibition

Concentration

(μg/ml)

100 200 300 400 500

NN 31.5 ± 2.8 40.1 ± 3.5 53.0 ± 4.2 60.4 ± 5.2 68.1 ± 5.9

WS 24.4 ± 1.9 35.7 ± 3.2 52.9 ± 4.0 63.6 ± 5.2 76.8 ± 6.3

TA 28.5 ± 2.1 40.7 ± 3.5 55.4 ± 4.5 70.5 ± 5.9 85.7 ± 6.8

ABTS+·

radical

cation

decolorization

% Inhibition

Concentration

(μg/ml)

20 40 60 80 100

NN 21.4 ±2.3 35.6 ± 5.9 48.5 ± 9.8 56.6 ± 8.7 72.6 ± 9.6

WS 11.2 ± 2.5 35.6 ± 6.5 45.6 ± 8.9 68.9 ± 5.8 79.8 ± 9.8

TA 33.5 ± 2.5 45.6 ± 6.5 54.2 ± 8.7 65.6 ± 9.8 75.8 ± 8.5

Values Are Mean ± SD

30

IC50 value

LPONO

ABTS

0

100

200

300

400NN

WS

TA

195

226

270275

280

270

63.2

59.858.9

IC5

0

Fig. 1: IC50 Value of NN (Nelumbo nucifera), WS (Withania somnifera) and TA (Terminalia arjuna) in Anti-lipid

peroxidation assay, Nitric oxide radical scavenging assay and ABTS+·

radical cation decolorisation assay

Table 2 reveals that Phenolic content of NN is high followed by TA and WS. Moreover, TA exhibits the

presence of higher concentration of all the other phyto-constituents like tannin, carbohydrate, Vitamin C and

Vitamin E.

31

Table 2: Total Phenolic, tannin, Vitamin C, Vitamin E and carbohydrate content of

NN (Nelumbo nucifera),WS (Withania somnifera) and TA (Terminalia arjuna)

S.No. Plants Total Phenolics

(mg/100 gm)

Total Tannins

(mg /100 gm)

Vitamin C

(mg/100 g)

Vitamin E

(mg/100 g)

Total

Carbohydrate

(mg/100g m)

1 NN 10.20 ± 0.1 4.30 ± 0.3 0.36 ± 0.2 0.42 ± 0.2 672.0 ± 2.3

2 WS 2.86 ± 0.3 3.20 ± 0.2 0.26 ± 0.2 0.46 ± 0.6 748.0 ± 3.2

3 TA 8.05 ± 0.2 5.10 ± 0.1 1.47 ± 0.1 0.58 ± 0.3 1154.0 ± 5.3

HPTLC profile of WS showed the presence of 8 peaks followed by NN with 7 peaks. The % Area of the

peak with maximum Rf value in all the three extracts was found to be high (Table 3, Fig 2, 3 and 4). The HPTLC

profile of TA showed the presence of only 3 major peaks (Table 3, Fig 4).

Fig. 2: HPTLC profile of NN. A – Photodocumentation of HPTLC plate at 254 nm. B-

Photodocumentation of HPTLC plate at 366 nm. C - Peak display of second track

A B C

32

Fig. 3: HPTLC profile of WS. A – Photodocumentation of HPTLC plate at 254 nm. B-

Photodocumentation of HPTLC plate at 366 nm. C - Peak display of second track

A B C

Fig. 4: HPTLC profile of TA. A – Photodocumentation of HPTLC plate at 254 nm. B-

Photodocumentation of HPTLC plate at 366 nm. C - Peak display of track

A B C

33

Table 3: Rf value and % Area of HPTLC plate for all the three extracts

Extract Track number Total number

of peaks

Peak

number/Rf

Peak number/% area

NN 1 7 1/0.33, 2/0.42,

3/0.51, 4/0.63,

5/0.67, 6/0.74,

7/0.89

½.08, 2/3.46,

3/18.21, 4/8.12,

5/6.28, 6/3.86,

7/57.99

2 8 1/0.32, 2/0.41,

3/0.46, 4/0.53,

5/0.61, 6/0.65,

7/0.74,

8/0.88

1/1.3, 2/4.94, 3/3.99,

4/20.83, 5/8.97,

6/7.29, 7/6.03,

8/46.65

WS 1 6 1/0.34, 2/0.64,

3/0.65, 4/0.74,

5/0.85,

6/0.89

1/0.61, 2/3.02,

3/2.67, 4/7.14,

5/30.40, 6/56.15

2 8 1/0.34, 2/0.46,

3/0.54, 4/0.63,

5/0.72, 6/0.76,

7/0.84,

8/0.88

1/0.65, 2/1.35,

3/1.98, 4/5.83,

5/10.56, 6/8.03,

7/20.77, 8/50.83

TA 1 3 1/0.51, 2/0.69,

3/0.79

1/12.87, 2/11.54,

3/29.77

2 3 1/0.50, 2/0.68,

3/0.78

1/12.38, 2/16.06,

3/40.57

34

DISCUSSION:

Antioxidant may be defined as “a substance when present at lower concentrations compared with

those of an oxidizable substrate such as fats, proteins, carbohydrates or DNA, significantly delays or

prevent oxidation of substrate. Consequently, organisms contain a complex network of antioxidant

metabolites and enzymes that work together to prevent oxidative damage to cellular components such as

DNA, proteins and lipids (Burits and Bucar, 2000)25

. In general, antioxidant systems either prevent these

reactive species from being formed, or remove them before they can damage vital components of the cell

(Vertuani et al., 2004).

Various extracts were tested in this study for their radical scavenging activities on two different

radicals like NO, ABTS. The ABTS systems provide information on the reactivity of a test compound

with a stable free radical. The ABTS radical cation decolorisation assay is a widely used method to

measure the total anti-oxidative status of various biological specimens because of the good

reproducibility and quality control (Brand Williams et al., 1995)27

. This method apply de-colorisation

assay to monitor the decrease in their absorbance during the reaction. For example the ABTS radical

absorb at 734 nm. When an antioxidant is added to the radical, there is a degree of de-colorisation owing

to the presence of antioxidant which reserves the formation of the ABTS radical cation. The scavenging

activity of cation is shown by increasing the concentration of extracts and decreases the color of ABTS.

NO a vasodilator, reacts with superoxide radical results in the formation of peroxynitrite. NOS

inhibitors are important drugs for treating various diseases related with inflammation (Miller et al., 1992).

In our present experiment, Nitric oxide was generated from sodium nitroprusside and measured by the

Greiss reaction. Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates

nitric oxide (Green et al, 1982) which interacts with oxygen to produce nitrite ions that can be estimated

by use of Greiss reagent. Scavengers of nitric oxide compete with oxygen leading to reduced production

of nitric oxide (Marcocci et al., 1994). All the three extracts were found to scavenge the nitric oxide with

increasing the concentration. Similar IC50 value of all the three extracts reveals that, all extracts are

similar in their NO radical scavenging activity.

Anti-lipid peroxidation activity of extracts was found to be increased with increasing the

concentration. The inhibition could be caused by the absence of ferryl–perferryl complex or by

scavenging of OH radical or superoxide radicals or by changing the ratio of Fe3+

/Fe2+

or by reducing the

rate of conversions of ferrous to ferric or by chelating the iron itself (Braughler et al., 1986). NN extract

35

exhibits lower IC50 value than other extracts which reveal that NN is more potent in the antioxidant

activity. This effect might be due to the presence of higher concentration of phenol (Table 2).

Total phenolic content of all the three extracts were calculated in the present study and their

values are mentioned in the Table 2. Polyphenolics is a highly inclusive term that covers many different

subgroups of phenolic acids and flavonoids. More than 5000 polyphenolics, including over 2000

flavonoids have been identified, and the number is still growing. Many of the phenolics are often

associated with sugar moieties that further complicate the phenolic profiles of plants. Polyphenols are

especially important antioxidants, because of their high redox potentials, which allow them to act as

reducing agents, hydrogen donors and singlet oxygen quenchers. In addition, they have a metal chelating

potential. The antioxidant activity of the dietary polyphenolics is considered to be much greater than that

of the essential Vitamins, therefore contributing significantly to the health benefits of fruits (Tsao &

Deng, 2004).

Total carbohydrate and glycosides play an important role in immunomodulatory reactions and

their free radical scavenging activity has been reported earlier by Morelli

et al., (2003)33

. This made us to estimate the level of Total carbohydrate in different extracts. TA has

shown to consist of higher concentration of glycosides. Presence of carbohydrate in all the three extracts

might prevent the formation of ferryl-perferryl complex.

The tannin concentration of TA was found to be high (Table 2). The high tannin content could be

partly responsible for bitter principle associated raw plant. Flavonoids and tannins are reported to inhibit

superoxide radical generation (Priyadarsini et al., 2002). That NO promotes IL-1β induced cartilage

destruction through the activation of MMP-3 and -9 (Kato et al., 2003). No significant difference

between the IC50 value of NN, TA and WS might be due to the presence of tannin in all the three extracts.

Vitamin C concentration of TA was found to be higher among all the three extracts (Table 2).

Vitamin C is one of the most potent reducing agents for biological systems. When Vitamin E turns into

tocopheroxyl radical this reacts with Vitamin C that restores tocopherol (Bohm et al., 1997). Anti-lipid

peroxidation activity of all the three extracts might be due to the presence of Vitamin C.

Vitamin E concentration of TA was found to be high (Table 2, Fig 1). Cherubini et al. (2001)

suggested that maintaining proper Vitamin E status is important to avoid increased risk for

36

atherosclerosis with advanced age. The antioxidant activity of all the extracts might be due to the

presence of Vitamin E.

SUMMARY AND CONCLUSION:

The antioxidant activity of NN, WS and TA was reported to exhibits the antioxidant activity. The

free radical scavenging activity of all the three extracts was similar. Whereas, NN exhibits more potent

anti-lipid peroxidation assay than other two extracts. Since all the three extracts are exhibiting potent

antioxidant activity, a poly herbal formulation can be produced by mixing the three extracts with NN

alone in higher amount to potent the biological activity. The presence of phenol, tannin, carbohydrate,

Vitamin C and Vitamin E in NN, WS and TA shows that these plants are potent neutraceutical agent.

37

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on the activity of plant extracts in lard. J. Am. Oil Chem. Soc., 69:520–524.

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antioxidant efficiency. J Am Chem Soc., 119: 621–2.

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Indian species of genus Terminalia. Indian Medical Research Memoirs., 16: 51–75.

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lipoprotein oxidation are associated with the absence of atherosclerosis in octogenarians. J Am Geriatr

Soc., 49: 651-54.

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determination of sugar and related substances. Anal chem., 28; 350-356.

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biological fluids. Anal Biochem., 126: 131-138.

Gupta, R, S. Singhal, A. Goyle, V.N. Sharma, 2001. Antioxidant and hypocholesterolaemic effects of

Terminalia arjuna tree bark powder randomized placebo controlled trial. The journal of The Association

of Physicians of India., 49: 233–235.

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Carmichel Hospital for Tropical Diseases, Calcutta, India

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1998. High molecular weight plant phenolics (tannis) as biological antioxidants. J. Agric. Food Chem.,

46:1887–1892.

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from Goa, West coast of India. Indian J, Mar. Scie., 14; 228 – 229.

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39

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40

Short communications

Investigation on Cytotoxicity of Morinda citrifolia L Noni fruit Extracts

P. Selvam1*

, Sk. Ammaji1, V.Jyothirmai

1, M.Prathima

1, T. Paul Pandi

1,

K. S. Praveen2, Hitesh Jagani

2, J. Venkata Rao

1Nova College of Pharmaceutical Education and Research, Jupudi, Ibrahimpatnam, AP

2Manipal College of Pharmaceutical Sciences, Manipal University, Manipal 576104, Karnataka, India

Email: [email protected]

41

Abstract

Morinda citrifolia L Noni fruit Extracts were investigated cytotoxic effect on HepG2 (Human Liver Cancer) cell

culture by MTT assay. Methanol, ethanol and acetone extracts (MCF-Me and MCF Me ETOAC) Noni fruits

showed cytotoxicity against HepG2 cells with CTC50 (cytotoxicity 50%) values of 142, 234 and 90 µg/ml, where

as standard Cis platin was found to be 11.09 µg/ml. Acetone extract (MCF-AC) showed significant cytotoxicity

against HepG2 cells and MCF-AC merits for further investigation to screen its anti-cancer activity using in vivo

models.

Keywords: Morinda citrifolia, HepG2 cells, MTT.

Introduction

Morinda citrifolia L Noni is a versatile medicinal plant and reported to possess hepatoprotective (Wang et al., 2008

a,b), anticancer (Akihisa et al., 2008), immunomodulatory , anti-inflammatory (Palu et al., 2007), wound healing

(Nayak et al., 2007), antioxidant (Su et al., 2005), anti-tubercular (Saludes et al., 2002), wide spectrum of

biological activities (Pawlus et al., 2007) and anti-HIV (Umezawa et al., 1992). Recently much attention been

devoted for searching potential safe herbal medicines from natural products for the treatment of various diseases

and Morinda citrifolia used for the treatment of a variety of diseases and safe herbal drug (West et al., 2006).

Different extracts of Morinda citrifolia leaf exhibited maximum protection against replication of HIV-1 in MT-4

cells (Selvam et al., 2008).The present work is to study the cytotoxicity activity of various extracts prepared from

Morinda citrifolia fruit against HepG2 (Human liver cancer) cells by MTT assay method.

42

Materials and Methods

Extraction and Isolation

Morinda citrifolia fruits were collected from Noni Research cum demonstration centre, Vadakkancherry, Thirussur

District, Kerala, India, fresh fruits were cut into small pieces, shade dried at room temperature and then powdered.

Dried fruit powder (400gm) was subjected to hot continuous extraction in Soxhlet extractor with methanol, ethanol

and acetone for 72 hours. After extraction the solvent was distilled out and syrupy extract was concentrate under

the vaccuo and kept in a desicator. Dried extracts were studied on cytotoxicity against HepG2 (Human liver

cancer) cells by using MTT assay methods.

Preparation of suspensions

Methanolic extract and isolated compounds of Noni fruits (Morinda citrifolia L) dissolved in DMSO and the

volume was made up to 10 ml with DMEM/MEM to obtain a stock solution of 1mg/ml concentration and stored at

-20°C prior to use. Further dilutions were made to obtain different concentrations ranging from 1000–62.5 µg/ml

with respective media and used for in vitro investigations.

Cell lines and growth media

HepG2 (Human liver cancer) cells were cultured in MEM (Minimum Essential Medium) and DMEM (Dulbecco’s

Modified Eagles Medium) respectively. The medium also contains 10% fetal calf serum, penicillin (100 IU) and

streptomycin (100 µg).

In vitro cytotoxicity screening

The ability of the cells to survive a toxic insult is the basis of most cytotoxicity assays (Francis et al., 1986; Philip

et al., 1990). The monolayer cell culture was trypsinized and the cell count was adjusted to 1.0x105 cells/ml using

medium containing 10% new born calf serum. To each well of the 96 well microtiter plate, 0.1ml of the diluted cell

suspension (approximately 10,000 cells) was added. After 24 hours partial monolayer was formed, the supernatant

43

liquid was flicked off washed the monolayer once and 100l of different drug concentrations were added to the

cells in microtiter plates. The plates were then incubated at 37oC for 3 days in 5% CO2 atmosphere, and

microscopic examination was carried out and observations recorded every 24 hours. After 72 hours, the drug

solutions in the wells were discarded and MTT assay performed.

Results

Morinda citrifolia L Noni fruit extracts were investigated for cytotoxicity against human liver cancer cell.

Methanol, ethanol and acetone extracts (MCF-Me and MCF Me ETOAC) Noni fruits showed cytotoxicity against

HepG2 cells with CTC50 (cytotoxicity 50%) values of 142, 234 and 90 µg/ml, where as standard Cis-platin was

found to be 11.09 µg/ml (Table 1). Acetone extract (MCF-AC) showed significant cytotoxicity against human

cancer cells from liver origin. This in vitro study has proved the selective cytotoxicity Morinda citrifolia against

liver cancer cells.

Discussion

The Polynesians utilized the whole Noni plant (Morinda citrifolia) in various combinations for herbal remedies

and reported to possess wide spectrum biological activities such as arthritis, diabetes, high blood pressure, muscle

aches and pains, menstrual difficulties, headaches, heart disease, AIDS, cancers, gastric ulcers, sprains, mental

depression, senility, poor digestion, atherosclerosis, blood vessel problems and drug addiction (Wang et al., 2002

and McClatchey et al., 2002). The different extracts of stem and fruits of Morinda citrifolia L were screened for

their anti-HIV activity and cytotoxicity, some of this extracts exhibited marked cytotoxicity in MT-4 cells (Selvam

et al., 2007 and 2009). Acetone extract of Noni fruit showed cytotoxicity against HepG2 (human cancer cells)

from liver origin. This extracts merits further investigation to screen its anti-cancer activity using in vivo models.

44

References

Akihisa T, Matsumoto K, Tokuda H, Yasukawa K, Seino KI, Nakamoto K, H. Kuninaga H, Suzuki T and Kimura

Y.2008. Anti-Inflammatory and Potential Cancer Chemopreventive Constituents of the Fruits of Morinda

Citrifolia. Journal of Natural Product, 71:1322-25.

Bina S Siddiqui, Fouzia A Sattar, Sabira Begum, Tahsin Gulzar, and Fayaz Ahmad. 2007. Chemical Constituents

from the Stems of Morinda citrifolia Linn. Achieve Pharmaceutical Research, 30 (7): 793-798,

Francis D and Rita L. 1986. Rapid colorimetric assay for cell growth and survival modifications to the tetrazolium

dye procedure giving improved sensitivity and reliability. Journal of Immunological Methods, 89: 271-277.

Masakazu Kamata, Raymond P Wu, Dong Sung Anc, Jonathan P Saxe, Robert Damoiseauxd, Michael E Phelps,

Jing Huang and Irvin S.Y. Chena.2006. Cell-Based Chemical Genetic Screen Identifies Damnacanthal as an

Inhibitor of HIV-1 Vpr Induced Cell Death. Biochemical Biophysics Research Communication, 348 (3): 1101–

1106.

McClatchey W. 2002. From Polynesian healers to health food stores changing perspectives of Morinda citrifolia

(Rubiaceae). Integrated Cancer Therapy, 1 :110-20.

Nayak BS, Isitor GN, Maxwell NA, Bhogadi V and Ramdath D. 2007. Wound-Healing Activity of Morinda

Citrifolia Fruit Juice on Diabetes-Induced Rats. Journal Wound Care, 16 (2) :83-7.

Palu AK, Kim AH, West BJ, Deng S, Jensen J and White L. 2008. The Effects of Morinda Citrifolia L. (Noni) on

the Immune System, Its Molecular Mechanisms of Action. Journal of Ethnopharmacology, 115 (3): 502-7.

Palu AK, Kim AH, West BJ, Deng S, Jensen J and White L.2007. Anti-Inflammatory and Potential Cancer

Chemopreventive Constituents of the Fruits of Morinda Citrifolia (Noni). Journal of Natural Product, 70 (5): 754-

60.

http://www.ncbi.nlm.nih.gov/pubmed/14664736?ordinalpos=113&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

45

Pawlus AD and Kinghorn D. 2007. A Review of the Ethnobotany, Chemistry, Biological Activity and Safety of the

Botanical Dietary Supplement Morinda Citrifolia (Noni). Journal of Pharmacy and Pharmacology, 59 (12): 1587-

92.

Periyasamy Selvam, Christophe pannecuoque, Erik De Clercq. 2009. Studies on anti-HIV activity and cytotoxicity

of stem extracts of Morinda citrifolia L. (Noni). Noni clinical Research Journal, 3 (1-2): 47-50.

Periyasamy Selvam, Christophe pannecuoque. 2008. Studies on anti-HIV and cytotoxicity of leaf extract of

Morinda citrifolia L. Noni clinical Research Journal, 2 (1-2): 25-28.

Periyasamy Selvam, Narayanan Murugesh, Myriam witvrouw. 2007. Studies of Comparative Anti-HIV activity

and cytotoxicity of Morinda citrifolia L. Noni clinical Research Journal, 1 (1-2): 25-27.

Philip S, Rista S, Dominic S, Anne M, James, David V, Jonathan T.W, Heidi B, Susan K. and Michale R.B. 1990.

New colorimetric cytotoxic assay for anti cancer drug screening. Journal of National Cancer Institute, 82: 1107-

1112.

Saludes J, Garson MJ and Aguinaldo AM. 2002. Antitubercular Constituents from the Hexane Fraction of

Morinda Citrifolia Linn. (Rubiaceae). Phytotheraphy Research, 16 (7): 683-8.

Su BN, Pawlus AD, Jung HA, Keller WJ, McLaughlin JL and Kinghorn AD. 2005. Chemical Constituents of the

Fruits of Morinda Citrifolia (Noni) and their Antioxidant Activity. Journal of Natural Product, 68 (4): 592-8.

Umezawa K.1992. Isolation of 1-methoxy-2-foremyl-3-hydroxyanthraquinone from Morinda citrifolia and

neoplasm inhibitors containing the same. Japan Kokai Tokyo Koho JP 06 87, 736 (94-87, 736) Appl; 92/264, 311

07.

Wang MY, Anderson G, Nowicki D and Jensen J. 2008a. Hepatic Protection by Noni Fruit Juice against CCl4-

Induced Chronic Liver Damage in Female SD Rats. Plant Foods Human Nutrition, (3): 141-47.

46

Wang MY, Nowicki D, Anderson G, Jensen J and West B. 2008b. Liver Protective Effects of Morinda Citrifolia

(Noni). Plant Foods Human Nutrition, 63 (2): 59-63.

Wang MY, West BJ, Jensen CJ, Nowicki D, Su C, Palu AK and Anderson G. 2002 Morinda citrifolia (Noni) a

literature review and recent advances in Noni research. Acta Pharmacologica Sinica, 23: 1127-41.

West BJ, Jensen CJ and Westendorf J. 2006. Noni Juice is Not Hepatotoxic. World Journal of Gastroenterology,

12 (22): 3616-20.

http://www.ncbi.nlm.nih.gov/pubmed/12466051?ordinalpos=126&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

47

Table 1. Determination of CTC50 by using MTT assay in HepG2 cells

Extract CTC50* value (in µg/ml)

MCF-ME 142 ± 11.71

MCF- ET 234± 20.23

MCF-AC 90.83 ± 5.89

Cis Platin (STD) 11.09 ±0.59

*Cytotoxic 50% concentration,

*Average of four independent determinations, values is mean ± S.E.M.

48


COLORIMETRIC METHOD DEVELOPMENT AND VALIDATION FOR THE

ESTIMATION OF MILNACIPRAN AND AMLODIPINE DRUGS IN BULK

AND FORMULATION

Mubarakunnisa.Md*, P. Jaya kumar, P.Kalvari giri kumari, R.Sandhya rani, S. Ramesh,

S. Udaya bhanu

* Nova College of Pharmaceutical Education and Research, Jupudi, Ibrahimpatnam, AP

Email: [email protected]

49

Abstract:

Colorimetric methods were developed and validated to the determination of milnacipran and amlodipine

in bulk and formulation. The reagents used (1, 10- phenanthroline and woll faster blue-black reagent)

were simple, economical and easily available. The good values and low relative standard deviation

obtained for the two methods shows that the development methods are precise and accurate, the

validation parameters like sensitivity, robustness were checked and the values are obtained within the

limits.the absorbance of milnacipran was measured at 485nm and Amlodipine at 620nm.

Key words: 1, 10-phenanthroline for Milnacipran and woll faster blue-black reagent for Amlodipine

Introduction:

Milnacipran hydrochloride used for the treatment of fibromyalgia and having antidepressant activity

under the class of serotonin-reuptake inhibitor1. Molecular formula C16H23N2O Chemical name is N, N-

dimethyl –2-(amino methyl) 1-phenyl cyclopropane caboxamide which is soluble in methanol and water.

Amlodipine having anti-hypertensive activity under the class of calcium channel blockers, molecular

formula C21H25N2O5I and chemical name is Methyl ethyl 2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-

6-methyl-1,4-dihydropyridine-3,5-dicarboxylate which is soluble in ethanol 4,5

. Literature survey

revealed that one RP-HPLC method have been reported for individual drugs. Few spectrophotometric

methods for Milnacipran were reported. Present study made to develop a simple economical, reliable,

colorimetric method for the estimation of two drugs simultaneously in pure and bulk formulation. The

developed method was validated and they are under ICH guidelines.

Materials and reagents:

A systronic (Schimadzu Corporation, Japan) model visican-167 digital spectrophotometer.

The cells used for absorbance measurements were 1cm matched quartz cells.

A schimadzu electronic weighing balance (Tokyo, Japan) BL-22OH Model

A magnetic stirrer (REMI Electrotechnic LTD., INDIA) MS-500 model1, 10- phenanthroline and woll

faster blue-black reagent.

50

Experimental method:

Preparation of 1, 10- phenanthroline reagent:

Weigh accurately 200mg of O-phenanthroline (Merck Specialities pvt. Ltd., Mumbai) was

dissolved in 100ml of distilled water with warming.

Preparation of Woll faster blue-black (WFBBL) reagent:

Weigh 200mg of WFBBL (Merck Specialities pvt. Ltd., Mumbai.) was dissolved in 100ml of

distilled water.

Preparation of standard stock solution of Milnacipran:

Milnacipran(1mg/ml) were prepared by dissolving 100mg of drug content in 40 ml distilled water

and make upto 100ml with distilled water. From the above stock solution standards were prepared by

pippetting 10ml, 40ml into each 100ml volumetric flask made upto mark with distlled water, the

concentrated solutions obtained were 100µg/ml, 400µg/ml respectively.

Preparation of standard stock solution of Amlodipine:

Amlodipine(1mg/ml) were prepared by dissolving 100mg of drug content in 40 ml distilled water

and make upto 100ml with distilled water. From the above stock solution standards were prepared by

pippetting 10ml, 40ml into each 100ml volumetric flask made upto mark with distlled water, the

concentrated solutions obtained were 100µg/ml, 400µg/ml respectively 7.

Procedure:

Method 1:

Aliquots of 0.5-3ml working standard solution (100µg/ml) were transferred into 100ml of

calibrated flask. To each flask add 0.5ml of ferric chloride solution and 2ml of 1, 10 phenanthroline was

added. The tube was heated in water bath upto 30min, after cooling the tube 2ml of acid was added and

make upto 50ml distilled water. The absorbance of formed color was measured after 5min at 485nm

against a reagent blank.

51

Method 2:

Different Aliquots of 0.5-3ml working standard solution (100µg/ml) were transferred into 100ml

of calibrated flask. To this 6ml of HCl solution and 2ml of WFBBL solution were added successively.

This mixture was transferred into separating funnel and then 5ml of chloroform was added then mix well

the contents. Keep aside for 5min, then two layers were separated, organic layer was separated was

collected. The absorbance measured at 620nm against the reagent.

Validation:

Linearity:

The linearity ranges for Amlodipine taken was 8-48µg/ml. the slope was found to be 0.0168, the

correlation coefficient was found to be 0.998

The linearity ranges for Milnacipran taken was 5-30µg/ml. the slope was found to be 0.0321, the

correlation coefficient was found to be 0.998

Both drugs accepted the Beer Lambert’s law.

Accuracy:

It expressed as percentage recovery by the assay of known amount in linearity ranges found at 3

different levels 50%, 100%, 150%.

Precision:

The intra and inter day was carried out for both drugs. The concentration range for Milnacipran is

5, 15, 30 µg/ml and for Amlodipine is 8, 24, 48 µg/ml. The percentage recovery for both the drugs

satisfies the criteria. The percentage recovery for both the drugs found to be 100%, 99% respectively.

52

References:

Wolff, M.E., 1981, Burger’s Medicinal Chemistry, part IV, Edn. Wiley interscience, NewYork.

Skoog, W., 1992, Fundamental of Analytical Chemistry, Saunders College, Publishing, 7th

Edn, pg no: 1-

3.

Coprners, K. A., 1967, Textbook of Pharmaceutical Analysis, a wiley intersciences publication, 1st Edn

pg no: 475-78.

Chatwal, G. R., 2005, Instrumental Methods of Chemical Analysis, Himalaya publishing house, pg no:

634-638.

Indian Pharmacopoeia, 2007, vol. 2, pg no: 634-638.

Sai Parveen, P., Sahiba, M., Rasool, Sk., 2011, Spectrometric Method for the Determination of

Milnacipran, Asian Journal of Pharmacy and Technology, vol: 4, pg no: 114-115.

Kiran Kumar, V., Srivani, V., 2011, Spectrophotometric Method for the Determination of Milnaciprin in

the Formulation, Research Journal of Pharmacy and Technology, vol: 4, pg no: 1250.

Priti J. Menta, Deepak M. Khatri, 2010, Development and Validation of RP-HPLC Formulation,

International Journal of Pharmacy and Pharmaceutical Sciences, vol: 2, pg no: 137-141.

Nilesh Jain, Arthi Jain, 2010, Spetrophotometric Method Development and Validation for Qualitative

Estimation of Amlodipine Besylate Using Hydrotropic Agent, Eurasian Journal of Analytical

Chemistry, Vol 5, No 3.

Topliss, J.G., 1983, Qualitative Structure Activity Reletionships of Drugs, Vol: 19, Academic Press,

London, pg no: 35.

53

Results and Discussion:

Table 1: Optical parameters of Milnacipran and Amlodipine drugs

Parameters Milnacipran Amlodipine

λmax(nm) 485 620

Beer’s limit (nm) 5-30 8-48

Molar absorbitivity (Lmole-1

cm-1

) 10.068×104 11.3×10

4

Sandell’s sensitivity

(µg cmcm-2

/0.001 absorbance unit)

0.00055 0.0024

Stability of colored products (mint) 100 90

Regression equation (Y= mx+c) 0.032x+0.02 0.016x+0.001

Correlation coefficient (R2) 0.998 0.998

Table 2: Validation parameters of Milnacipran and Amlodipine.

Validation parameters Range Results

Linearity MNC AML Correlation coefficient 0.998

5-30µg/ml 8-48 µg/ml

Precision

Intraday

Interday

5-30µg/ml

5-30µg/ml

8-48µg/ml

8-48µg/ml

%RSD=1.09

%RSD=1.56

Assay

(%Recovery)

50%

100%

150%

50%

100%

150%

99.49

100.61

99.02

Ruggedness Analyst-1

Analyst-2

%RSD=0.66

%RSD=0.002

54

Fig: 1 Linearity graph of Milnaciprin-phenanthroline

Fig: 2 Linearity graph of Amolidipine-woll faster blue black reagent

y = 0.0331x

0

0.2

0.4

0.6

0.8

1

1.2

0 10 20 30 40

AB

SO

RB

AN

CE

CONCENTRATION

ABSOR

Linear (ABSOR)

y = 0.0136x

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 20 40 60 80

AB

SO

RB

AN

CE

CONCENTRATION

abs

Linear (abs)

55


COLORIMETRIC METHOD DEVELOPMENT AND VALIDATION FOR THE

ESTIMATION OF NEBIVOLOL IN BULK AND FORMULATION

P.J.V. Sagar*, M. Tejaswi, A. Jeevan kumar, A. Bhargavi, B. Adina Helga, CH. Harsha,

J. Roopu Vemula, J. Eliazar




56

Abstract:

Present investigation was done on estimation of Nebivolol in bulk and formulation, a method

wasdeveloped using Brucine solution. Initially stock solution of Nebivolol 1mg/ml was prepared

bydissolving 10mg of drug in 10 ml of methanol. A concentration of 250 μg/ml was prepared and used

asstock solution. Effect of different parameters was calculated at 530nm and absorbance spectra

werederived. Validation parameters like linearity, sandall’s sensitivity, molar absorptivity, LOD & LOQ

werecalculated and checked whether they reached the ICH guidelines. The results were tabulated and

theysatisfied the ICH guidelines with % assay of 98.6 from formulation.

Keywords: Nebivolol bulk drug, Brucine solution, NaIO4, Methanol, Digital spectrophotometer

Introduction:

Nebivolol is a β1 receptor blocker with nitric oxide-potentiating vasodilatory effect used in treatment

of hypertension and, in Europe, also for left ventricular failure. It is highly cardioselective under certain

circumstances. Molecular Formula C22H25F2NO4 IUPAC NAME 1-(6-fluorochroman-2-yl)-{[2-(6-

fluorochroman-2-yl)-2-hydroxy-ethyl] amino} ethanol. A survey of literature showed some official

methods have been mentioned in pharmacopoeias. Very few colorimetric methods have been reported. In

this study an attempt is made to develop a simple colorimetric method for the estimation of present drug

in pure as well as in pharmaceutical dosage form i.e. tablet. The developed method was validated as per

ICH guidelines.

Materials and Reagents:

All the chemicals used were of analytical reagent grade and distilled water.

Nebivolol bulk drug was obtained from Matrix formulations (Hyderabad), their formulations Nebilol

2.5mg tablets (Intas pharmaceuticals, Dehradun, India), was bought from the nearby hetero pharmacy.

A Systronics (SCHIMADZU CORPORATION, Japan) model Visiscan-1137 digital spectrophotometer.

The cells used for absorbance measurements were 1-cm matched quartz cells.

A Schimadzu electronic weighing balance (Tokyo, Japan) BL 220H model.

http://en.wikipedia.org/wiki/Beta_blocker

http://en.wikipedia.org/wiki/Nitric_oxide

http://en.wikipedia.org/wiki/Hypertension

http://en.wikipedia.org/wiki/Left_ventricular_failure

57


Preparation of Stock solution:

Standard stock solution of nebivolol (1mg/ml) was prepared by dissolving 10mg of the drug in 10ml of Methanol.

Preparation of Standard solution:

Different standard solutions of concentrations ranging from 0.5-3.0μg/ml were prepared from 1mg/ml stock

solution.

Method development:

Aliquots of the drug (0.5-3ml) were transferred into a series of 25 mL volumetric flasks. Then 3.0 mL of

Brucine solution and 1.5ml of NaIO4 solution and 2ml of acid solution was added successively. Then all

the solutions were kept aside for 15 min. Later the solution in each tube was made up to 25ml with

distilled water. It was boiled on a water bath for 15min. then it was cooled to room temperature and the

absorbance was measured at 530nm against the reagent blank.

Validation:

Linearity: The Linearity was observed in the range of (5-30µg/ml) for brucine solution, with nebivolol.

A calibration graph was plotted in the linearity range, an equation (Y=a+bx) and regression coefficient

(R2) was found to be 0.999.

Accuracy: It expressed as percentage recovery by the assay of known amount in linearity ranges found

at 3 different levels 50%, 100%, 150%.

Precision: The intra and inter day was carried out for the drug. The concentration range is 5, 15, 30

µg/ml .The percentage recovery for drug satisfies the criteria. The percentage recovery for both the drugs

found to be 100%, 99% respectively.

58

REFERENCES:

Deorge, R.F., Edt., Wilson and Gisvold’s Text book of organic Medicinal and pharmaceutical chemistry,

8th

Edn., Lippincott Company, 1982.

Goodman, L.S and Gilman, A.G., The pharmacological basis of therapeutics, 9th

Edn. By Hardman, J.G.,

Limbard, L.E., Editors in chief, MC Graw – Hill, 1996.

Korolkovas, A., Essentials of Medicinal chemistry, 2nd

Edn., Wiley Interscience, New York 1988.

L.M.Atherden, Edr, Bentley and Drivers Text Book of pharmaceutical chemistry, 8th

Edition,

oxford Universitry press, 1966, 4th

Impression.

Melentyeva, G., Antonova, L., pharmaceutical chemistry, Mir pulishers, Moscow, 1988.

Pub. Med. Health, US National Library of Medicine www.ncbi.n/m.nih.gov/ pubmedhealth/PMHdd1463

Rao, S.G., Bennett, R.M. “Pharmacologic Therapies in Fibromyalagia”, Best practice and Research

compendions 2007.

Topliss, J.G., Edt., Quantitative structure – activity relationships of Drugs, Vol 19, Academic Press,

London, 1983.

William O. Foye, Edt., Principles of Medicinal Chemistry, 3rd

Edn., Varghese, Bombay, 1989.

Wolff, M.E., Burger’s Medicinal chemistry, part IV, 4 th

Edn., Wiley interscience, New York,1981.

http://www.ncbi.n/m.nih.gov/

59


Table1. Optical parameters of nebivolol drug

Parameters

Result

λmax (nm) 530nm

Beer’s Limit (µg mL-1

) 5-30µg/ml

Molar Absorbitivity (L mole-1

cm-1

) 8.379×104

Sandell’s sensitivity 0.00005

Regression equation (Y= mx + c)$$

0.0178x+0.02

Slope (m) 0.0178

Intercept (c) 0.02

Correlation coefficient (R2) 0.999

LOD (µg mL-1

) 0.37

LOQ (µg mL-1

) 1.25

Table2. Validation parameters of nebivolol drug

Parameter Range Result

Linearity 5-30μg/ml Slope 0.0178

Intercept 0.0020

Correlation coefficient 0.999

Precision

Intraday precision

Interday precision

5-30 μg/ml

5-30 μg/ml

S.D 0.002

%R.S.D 1.24

S.D 0.0015

%R.S.D 1.35

Recovery 50%

100%

150%

99.56

99.76

100.1

LOD 5-30μg/ml 0.37

LOQ 5-30μg/ml 1.25

60

Fig 1: Linearity graph showing effect of different solutions on nebivolol drug

Fig 2: Graph showing absorption spectra of Nebivolol in Brucine solution

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1 2 3 4 5 6 7 8

abso

rban

ce

concentration

Volume of Brucinesolution

Volume of sodiumiodate

Volume of sluphuricacid

61


COLORIMETRIC METHOD DEVELOPMENTAND VALIDATION FOR THE

ESTIMATION OF VALSARTAN AND RAMIPRIL DRUGS IN BULK AND

FORMULATION

A. Swathi*, M.Tejaswi N. Varun kumar, N. Swapna Priya, N. Ravi Teja,

N. Subramanyam, N. Nirmal Babu, S. Vema Reddy



62

Abstract:

Colorimetric methods were developed and validation to the determination of Valsatran and Ramipril

inbulk and formulation. The reagents used are alizarin red S, Follens reagent were simple and

easilyavailable. The good values and low relative standard deviation obtained for the two methods

shows that development methods are precise and accurate, the validation parameters like sensitivity,

robustness werechecked and the values are obtained within the limits.

Key words: Alizarin Red S for Valsartan and Follens reagent for Ramipril.

Introduction: Valsartan used for the treatment of high blood pressure, congestive heart failure and

post myocardial infarction. Molecular formula C24H29N5O3. The chemical name 3-methyl-2[pentanoyl-

[4-[2-(2H-tetrazol-5-yl) phenyl] phenyl] methyl] amino-butanoic acid.

Ramipril used for the treatment of hypertension, congestive heart failure and diabetic nephropathy.

Molecular formula C23H32N2O5. Chemical name (2s, 3as, 6as)-1[(S)-N-(S)-1-carboxy-3-phenyl propyl]

alanyl) octa hydro cyclopenta[b]pyrrole-2-carboxylic acid-1-ethyl ester. Few HPLC methods were

reported for Ramipril and spectrophotometric methods for Valsatran have been reported. No colorimetric

method has been reported so far for these two drugs simultaneously. Present study involves the

colorimetric estimation of both drugs simultaneously in pure and in bulk formulation which is more

economical and sophisticated. The developed method was validated according to ICH guidelines.

Materials and reagents:

A systronic (schimadzu corporation, Japan) model visican-167 digital spectrophotometer.

The cells used for absorbance measurements were 1cm matched quartz cells.

A Schimadzu electronic weighing balance (Tokyo, Japan) BL-220H model

A magnetic stirrer (REMI Electro-technique Ltd., India) MS-500 model Alizarin Red S and Follens

reagent.

63


Preparation of Alizarin Red S:

Alizarin Red S (ARS) solution: Weigh 200mg of ARS and is dissolved in 100ml of distilled

water.

Preparation of Follens reagent: 4-amino phenazone solution:

Weigh accurately 500mg of 4-amino phenazo and is dissolved in 100ml of methanol containing

concentrated HCl.

Preparation of standard stock solution:

Standard stock solution of Valsartan (1mg/1ml) were prepared by dissolving 100mg of drug

content in 40 ml distilled water and make up to 100ml with distilled water. From the above stock solution

standards were prepared by pippetting 10ml, 40ml into each 100ml volumetric flask made up to mark

with distilled water, the concentrated solutions obtained were 200µg/ml, 300µg/ml respectively.

Procedure:

Method 1:

In a series of 125ml separating funnels, Aliquots of 0.5-3ml working standard solution

(100µg/ml) was taken. To this add 6ml of HCl solution and 2ml of ARS was added. The total volume of

the solution in each separating funnel was added and the contents were shaken for 2min. the two phases

were allowed to separate and the absorbance of the separated chloroform layer was measured at 470nm

against a similar reagent blank.

Method 2:

Aliquots of the drug (0.5-3ml) were transferred into a series of 10ml of calibrated tubes and then

3.0ml of 4-amino phenazone solution was added to each tube and kept aside for 15min. later the solution

in each tube was made upto 10ml with methanol. The absorbance measured at 485nm against the reagent

blank.

64

Validation:

Linearity:

The linearity ranges for Valsatran taken was 6-36µg/ml. The slope was found to be 0.0231, the

correlation coefficient was found to be 0.9990.

The linearity ranges for Ramipril taken was 6-60µg/ml. The slope was found to be 0.010, the

correlation coefficient was found to be 0.9996.

Both drugs accepted the Beer Lambert’s law

Accuracy:

It expressed as percentage recovery by the assay of known amount in linearity ranges found at 3

different levels 50%, 100%, 150%.

Precision:

The intra and inter day was carried out for both drugs. The concentration range for Valsatran is 6,

18, 36 µg/ml and Ramipril for is 10, 40, 60 µg/ml. The percentage recovery for both the drugs satisfies

the criteria. The percentage recovery for both the drugs found to be 100%, 99% respectively.

65

References:

Bilal Yilmaz, 2010, the determination of Ramipril in Pharmaceutical preparation by HPLC, Inter. J. of

Pharm. Sci. Review and Res., vol. 1(1):39-42.

Chatwal GR., Anand Sk., 2005, Instrumental methods of chemical Analysis, Himalaya publishing house,

pg no: 634-638.

Indian Pharmacopoeia, 2007, vol. 2, pg no: 634-638.

Motofumi, I., Takeo, k., Junichi, G., Toshio, N., 1990, Separation Ramipril optical isomers by HPLC, J.

Liquid Chromatogr., vol. 13(5):991-1000.

Sai Praveen, p., Sahiba, m., Rasool, Sk., 2011, Spectrometric method for the determination of Valsartan,

Asian Journal of pharmacy & Technology, 4(1), 114-115.

Satana, E., 2001, Simultaneous determination of Valsatran and Hydrochlorthiazide in tablets by first-

derivative ultra violet spectrophotometry and LC, J pharm. Biomed. Anal., 25, 1009-1013.

Skoog, W., 1992, Fundamental of Analytical Chemistry, Saunders College, Publishing, 7th

Edn, pg no: 1-

3.

Wolff, M.E., 1981, Burger’s Medicinal Chemistry, part IV, Edn. Wiley interscience, New York.

Zarapakar, SS., and Rane, SH., 2000, RP-HPLC determination of Ramipril and Hyrochlorthiazide in

tablets. Indian drugs, vol. 37: 589-593.

66


Table 1: Optical parameters of Valsatran and Ramipril.

Parameters Method-A Method-B

λmax(nm) 470 485

Beer’s limit (µg/ml) 6-36 10-60

Molar absorbitivity (Lmole-1

cm-1

) 5.26×104 4.82×10

4

Sandell’s sensitivity

(µg cmcm-2

/0.001 absorbance unit)

0.00082

0.003

Stability of colored products (mint) 80 120

Regression equation (Y= mx+c) 0.0239x+0.01 0.0108x+0.2

Correlation coefficient (R2) 0.999 0.999

Table 2: validation parameters of Valsartan and Ramipril

Validation parameters Range Results

Linearity Valsartan Ramipril Correlation coefficient 0.998

6-36 µg/ml 6-60 µg/ml

Precision

Intraday

Interday

6-36µg/ml

6-36µg/ml

6-60µg/ml

6-60µg/ml

%RSD=1.09

%RSD=1.56

Assay

(%Recovery)

50%

100%

150%

50%

100%

150%

99.11

100.61

99.63

Ruggedness Analyst-1

Analyst-2

%RSD=0.203

%RSD=0.352

67

Fig: 1 Linearity graph of Valsatran-Alizarin Red S

Fig: 2 Linearity graph of Ramipril- Follens reagent

y = 0.0226x + 0.0335

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 10 20 30 40

ab

sorb

an

ce

concentration

abs

Linear (abs)

y = 0.0104x + 0.0137

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 20 40 60 80

ab

sorb

an

ce

concentration

abs

Linear (abs)

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