Vol.50 Issue No.02 February 2013

INDIAN DRUGS 50(02) February 2013 1

2 INDIAN DRUGS 50(02) February 2013

Vol. 50 No. 02

February 2013

review article

- Protease Inhibitors: A Review Sapkale P. V., Jadhav S. B. and Sable P. N...... 5

original research articles

- Method Development and Validation of Olanzapine in Pure and Pharmaceutical Dosage form by Rp-Hplc Method Mastiholimath V.S., Dandagi P.M., Gadad A.P., Murali Krishna N.V. and Mannur V...... 20

- Quantitative Determination of Deferasirox in Bulk and Pharmaceutical Formulation by Uv Spectrophotometric Method Marathe G. M., Pande V. V., Patil P. H., Mutha R. E. and Bari S. B...... 27

- Preclinical evaluation of nootropic activity of Glabridin Rich Extract of Glycyrrhiza glabra using Passive Avoidance Paradigm in rats Desai S. K., Pandey C. H. and Mulgaonkar S.M...... 33

- Antihyperglycemic and In Vitro Antioxidant Activities of Punica Granatum Linn. in Alloxan Induced Diabetic Rats Patil U. S., Bandawane D. D., Bibave K. H. and Chaudhari P. D...... 39

short notes

- A Novel Method For Isolation of Mangiferin from Mangifera Indica L. Bark Gholkar M. S and Laddha K. S...... 47

- Anti-Inflammatory Effect of Seeds ofTamarindus Indica in Wistar Rats Hivrale M. G., Mali A. A. and Bandawane D. D...... 49

Indian Drug Manufacturers' Association 102-B, 'A-Wing', Poonam Chambers, Dr. A.B. Road, Worli, Mumbai - 400 018 Tel : 022-2494 4624 / 2497 4308 Fax: 022-2495 0723 E-mail: [email protected] / [email protected], Website: www.idma-assn.org

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Founder Editor *Dr. A. Patani, D.Sc. (Germany) Editorial Committee Editor Dr. Gopakumar G. Nair, Ph.D. Associate Editors Mr. J. L. Sipahimalani, B. Pharm. Hons. (London), MRCS, FRPharmS Dr. Nagaraj Rao, D.Sc. (Germany) Dr. George Patani, Ph.D. Consulting Editor Dr. S. G. Deshpande, M.Sc. (Tech.), Ph.D.

Editorial Board Editorial Advisory Board Prof. K. G. Akamanchi, Ph.D. (Tech.) Prof. Y. K. Agrawal, Ph.D., F.I.C., F.R.M.S. Dr. Evans Coutinho, Ph.D. (Tech.) Prof. H. L. Bhalla, Ph.D. Prof. Padma Devarajan, M.Pharm., Ph.D. (Tech.) Dr. B. N. Dhawan, M.D. Dr. Prashant M. Dikshit, Ph.D. Prof. S. S. Handa, Ph.D. Prof. A. K. Gadad, M.Pharm., Ph.D. Dr. C. I. Jolly, Ph.D. Dr. K. N. Ganesh, Ph.D. Dr. C. L. Kaul, Ph.D. Dr. (Mrs.) Gopa Ghosh, Ph.D. Dr. S. P. S. Khanuja, Ph.D. Dr. Parthajyoti Gogoi, Ph.D. Prof. J. K. Lalla, Ph.D. Dr. Nirmala D. Grampurohit, Ph.D. Dr. D. B. Anantha Narayana, Ph.D. Dr. (Mrs.) S. S. Mahajan, M.Sc. (Tech.), Ph.D. Dr. Nitya Anand, Ph.D. Prof. Bhushan Patwardhan, Ph.D. Dr. Harish Padh, Ph.D. Dr. Ashwinikumar Raut, M.D. Dr. M. K. Raina, Ph.D. Dr. Sanjay Singh, M.Pharm, Ph. D. Dr. A. V. Rama Rao, Ph.D. (Tech.), D.Sc. Prof. Saranjit Singh, M.Pharm., Ph.D. Dr. G. N. Singh, M.Pharm., Ph.D. Prof. N. Udupa, M.Pharm., Ph.D. Prof. R.T. Sane, Ph.D. Dr. K. Valliappan, M.Pharm., Ph.D. Prof. M. N. Saraf, M.Pharm., Ph.D. Dr. A. A. Natu, Ph.D. Dr. P. D. Sethi, Ph.D. Dr. N. G. N. Swamy, Ph.D. Dr. Ashok Vaidya, M.D., Ph.D., F.A.I.M. Dr. J. S. Yadav, Ph.D., FNA

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review article

Protease Inhibitors : A Review Sapkale P. V., Jadhav S. B*. and Sable P. N.

(Received 29 August 2012) (Accepted 19 December 2012)

Abstract

HIV protease inhibitors were first invented between 1989 and 1994 by researchers working forthe pharmaceutical companies of Hoffmann- La Roche Inc. (in Nutley, New Jersey), Abbott Laboratories and Merck & Co., Inc. HIV protease inhibitors are used in the treatment of patients with AIDS and were considered the first breakthrough in over a decade of AIDS research. Currently, there are fiveHIV protease inhibitors approved by FDA for the treatment of HIV infection. These drugs work at the final stage of viral replication and attempt to prevent HIV from making new copies of itself by interfering with the HIV protease enzyme. As a result, the new copies of HIV are not able to infect new cells. Occurrence of protease along with structural properties, classification of inhibitors like Saquinavir, Ritonavir, Indinavir, Nelfinavir etc and life cycle of virus confirm the role of protease inhibitor. Other parameters like adverse effect, application, structure activity relationship and dose regime shows need of medication for person suffering from HIV virus.

Keywords: Viral replication, enzyme, Lifecycle, • Metalloproteases Structural properties, Adverse reaction. • Glutamic acid proteases

INTRODUCTION The threonine and glutamic-acid proteases were not described until 1995 and 2004, respectively. The Protease mechanism used to cleave a peptide bond involves A protease (also termed peptidase or proteinase) making an amino acid residue that has the cysteine is any enzyme that conducts proteolysis, that is, begins and threonine (proteases) or a water molecule protein catabolism by hydrolysis of the peptide bonds (aspartic acid, metallo- and glutamic acid proteases) that link amino acids together in the polypeptide chain nucleophilic so that it can attack the peptide carboxyl forming the protein. group. One way to make a nucleophile is by a catalytic triad, where a histidine residue is used to activate Classification of Protease serine, cysteine, or threonine as a nucleophile. Within Proteases are currently classified into six broad each of the broad groups proteases have been groups: classified, by Rawlings and Barrett, into families of related proteases. For example, within the serine • Serine proteases proteases families are labelled Sx where S denotes • Threonine proteases the serine catalytic type and the ‘x’ denotes the number • Cysteine proteases of the family, for example S1 (chymotrypsins). An up to date classification of proteases into families is • Aspartate proteases found in the MEROPS database 1, 2.

*For correspondence Occurrence of protease Modern College of Pharmacy, Sector 21 Yamuna nagar, Nigdi, Pune-411044. Proteases occur naturally in all organisms. These E-mail: [email protected] enzymes are involved in a multitude of physiological

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reactions from simple digestion of food proteins to Proteases are divided into four major groups highly regulated cascades (e.g., the blood-clotting according to the character of their catalytic active cascade, the complement system, apoptosis site and conditions of action: serine proteinases, pathways, and the invertebrate prophenoloxidase- cysteine (thiol) proteinases, aspartic proteinases, and activating cascade). Proteases can either break metallo proteinases. Attachment of a protease to a specific peptide bonds limited( proteolysis), depending certain group depends on the structure of catalytic on the amino acid sequence of a protein, or break site and the amino acid (as one of the constituents) down a complete peptide to amino acids (unlimited essential for its activity. Proteases are used throughout proteolysis). The activity can be a destructive change, an organism for various metabolic processes. Acid abolishing a protein’s function or digesting it to its proteases secreted into the stomach (such as pepsin) principal components; it can be an activation of a and serine proteases present in duodenum (trypsin function, or it can be a signal in a signaling pathway. and chymotrypsin) enable us to digest the protein in Bacteria also secrete proteases to hydrolyze (digest) food; proteases present in blood serum (thrombin, the peptide bonds in proteins and therefore break plasmin, Hageman factor, etc.) play an important role the proteins down into their constituent monomers. in blood-clotting, as well as lysis of the clots, and the Bacterial and fungal proteases are particularly correct action of the immune system. Other proteases important to the global carbon and nitrogen cycles in are present in leukocytes (elastase, cathepsin G) the recycling of proteins, and such activity tends to be and play several different roles in metabolic control. regulated by nutritional signals in these organisms. Proteases determine the lifetime of other proteins The net impact of nutritional regulation of protease playing important physiological role like hormones, activity among the thousands of species present antibodies, or other enzymes; this is one of the in soil can be observed at the overall microbial fastest “switching on” and “switching off” regulatory community level as proteins are broken down in mechanisms in the physiology of an organism. By response to carbon, nitrogen, or sulfur limitation. complex cooperative action the proteases may A secreted bacterial protease may also act as an proceed as cascade reactions, which result in rapid exotoxin, and be an example of a virulence factor in and efficient amplification of an organism’s response bacterial pathogenesis. Bacterial exotoxic proteases to a physiological signal. Proteases are part of many destroy extracellular structures. Protease enzymes laundry detergents5. are also used extensively in the bread industry as bread improver3. Protease Inhibitors Protease inhibitors are a class of drugs used to Proteases, also known as proteinases or treat or prevent infection by viruses, including HIV proteolytic enzymes, are a large group of enzymes. and Hepatitis C. Protease inhibitors prevent viral Proteases belong to the class of enzymes known as replication by inhibiting the activity of proteases, hydrolases, which catalyze the reaction of hydrolysis e.g. HIV-1 protease, enzymes used by the viruses of various bonds with the participation of a water to cleave nascent proteins for final assembly of new molecule. Proteases are involved in digesting virions. Protease inhibitors have been developed or long protein chains into short fragments, splitting are presently undergoing testing for treating various the peptide bonds that link amino acid residues. viruses: HIV/AIDS antiretroviral protease inhibitors Some of them can detach the terminal amino (saquinavir, ritonavir, indinavir, nelfinavir, amprenavir acids from the protein chain (exopeptidases, such 6 etc.) . as aminopeptidases, carboxypeptidase A) while others attack internal peptide bonds of a protein HIV protease inhibitors were first invented (endopeptidases, such as trypsin, chymotrypsin, between 1989 and 1994 by researchers working for pepsin, papain, elastase)4. the pharmaceutical companies of Hoffmann- La Roche

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Inc. (in Nutley, New Jersey), Abbott Laboratories HIV PROTEASE and Merck & Co., Inc. HIV protease inhibitors are Human immunodeficiency virus (HIV) is a used in the treatment of patients with AIDS and were lentivirus that has two major species13, HIV-1 which considered the first breakthrough in over a decade causes the majority of the epidemic, and HIV-2, a of AIDS research. HIV protease inhibitors can lower close relative whose distribution is concentrated in the viral load carried by AIDS patents. There are five western Africa14. HIV infection was first described HIV protease inhibitors approved by US FDA for the in 1981 in San Francisco and New York City. In treatment of HIV infection. These medications work at 1985, HIV was identified as the causative agent of the final stage of viral replication and attempt to prevent acquired immune deficiency syndrome (AIDS) and HIV from making new copies of itself by interfering its complete genome was immediately available. with the HIV protease enzyme. As a result, the new This knowledge paved the way for the development of selective inhibitors15. HIV-2 carries a slightly lower copies of HIV are not able to infect new cells7. The risk of transmission than HIV-1 and infection tends United States Food and Drug Administration (US FDA) to progress more slowly to AIDS. In common usage approved the drug invirase in December 1995; and HIV usually implies HIV-1. HIV-1 protease is one of 8, 9, 10 norvir and crixivan in March, 1996 . Invention of the best known aspartic proteases, and an attractive 11, 12 protease inhibitors are summarized in (Table I) . target for the treatment of AIDS16.

Table I: Invention of protease inhibitors are summarized in tabulated form as below11, 12

Name Trade name Company Patents Notes Saquinavir Fortovase, Hoffmann–La U.S. Patent It was the first protease inhibitor approved by the Invirase Roche 5196438 FDA (December 6, 1995). Ritonavir Norvir Abbott U.S. Patent Laboratories 5541206 Indinavir Crixivan Merck & Co. U.S. Patent 5413999 Nelfinavir Viracept Agouron U.S. Patent Pharmaceuticals 5484926 Amprenavir Agenerase GlaxoSmithKline U.S. Patent The US FDA approved it April 15, 1999, making 5585397 it the sixteenth FDA-approved antiretroviral. It was the first protease inhibitor approved for twice-a-day dosing instead of needing to be taken every eight hours. The convenient dosing came at a price, as the dose required is 1,200 mg, delivered in eight very large gel capsules. Production was discontinued by the manufacturer December 31, 2004 as it has been superseded by fosamprenavir. Lopinavir Kaletra Abbott - Is only marketed as a combination, with ritonavir. Atazanavir Reyataz Bristol-Myers The FDA approved it on June 20, 2003. Atazanavir Squibb was the first PI approved for once- daily dosing. It appears to be less likely to cause lipodystrophy and elevated cholesterol as side effects. It may also not be cross-resistant with other PIs.

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After the discovery of HIV protease, it only took 10 years for its first inhibitor to reach the market17. The first reports of highly selective antagonists against the HIV protease were revealed in 1987. Phase I trials of saquinavir began in 1989 and it was the first HIV protease inhibitor to be approved for prescription use in 1995. Four months later, two other protease inhibitors, ritonavir and indinavir, were approved. In 2009, ten protease inhibitors had reached the market for treatment against HIV but one protease inhibitor, amprenavir, was withdrawn from the market in 2004 because it produced resistance and showed side effects18.

Life cycle of HIV HIV belongs to the class of viruses called retroviruses, which carry genetic information in the form of RNA. The mRNA is then translated into viral proteins and the third virally encoded enzyme, namely HIV protease, is required to cleave a viral polyprotein precursor into individual mature proteins. The viral RNA and viral proteins assemble at the surface of the cell into new virions. The virions bud from the cell and are released to infect other cells. All infected cells are eventually killed because of this extensive cell damage, from the destruction of the host’s genetic system to the budding and release of virions shown in (Fig. 1) 19, 20.

Mechanism of Action for Protease Inhibitors

There are several steps in the HIV life cycle that Fig. 1: Life Cycle of HIV19, 20 may be interfered with, thus stopping the replication of the virus. A very critical step is the proteolytic cleavage been proposed, for example inhibition of adipocyte of the polypeptide precursors into mature enzymes differentiation, triglyceride accumulation and and structural proteins catalyzed by HIV protease21. increased lipolysis. Theories considering the effect HIV protease inhibitors are peptide-like chemicals that of protease inhibitors on insulin-stimulated glucose competitively inhibit the action of the virus aspartyl uptake have also been linked to the lipodystrophic protease. These drugs prevent proteolytic cleavage syndrome. It is possible that protease inhibitors of HIV Gag and Pol polyproteins that include essential can cause a decrease in insulin-stimulated tyrosine structural and enzymatic components of the virus. phosphorylation of IRS-1, representing inhibition of This prevents the conversion of HIV particles into their early steps in insulin signaling. Decreased adiponectin mature infectious form22, 23. Protease inhibitors can secretion and induced expression of interleukin-6 alter adipocyte metabolism causing lipodystrophy, a associated with HIV protease inhibitors may also common side effect associated with the use of most contribute to inhibition of insulin-stimulated glucose 24, 25 HIV protease inhibitors. Many mechanisms have uptake .

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Design of Protease inhibitors non scissile junction, present in inhibitors other than those containing the reduced peptide bond isosteres, Protease inhibitors were designed to mimic the is positioned between the Asp25/Asp25´ carboxyls transition state of the protease’s actual substrates. A of the protease, within hydrogen-bonding distance to peptide linkage consisting of –NH-CO- is replaced by at least one carboxylate oxygen of each aspartate. A hydroxyethylene group (-CH2-CH (OH)-) which the feature common to almost all complexes of HIV-1 PR protease is unable to cleave. HIV protease inhibitors fit is a buried water molecule that bridges the P2 and the active site of the HIV aspartic protease and were P10 CO groups of the inhibitor and Ile50 and Ile150 rationally designed utilizing knowledge of the aspartyl NH groups of the flaps. This water is approximately protease’s mode of action. The most promising tetrahedrally coordinated and is completely separated transition state mimic was hydroxyethylamine, which from the bulk solvent33. The functional substitution lead to the discovery of the first protease inhibitor, of this water has led to the design of urea-based saquinavir. Following that discovery, other HIV inhibitors (Fig. 2). protease inhibitors were designed using the same principle26, 27, 28.

Co crystals of HIV-1 PR with a variety of inhibitors have been grown in a number of different crystal forms. The structures of these complexes have been used to investigate the binding of substrate- based modified oligopeptide inhibitors as well as of the inhibitors of non-peptidic nature. Binding of an inhibitor introduces substantial conformational changes to the enzyme. The overall movement of the subunits can be described as a rotation of up to about 2± around a hinge axis located in the subunit sheet interface. This motion, which slightly tightens the cavity of the active site, is also accompanied by a very large motion of the flap region as much as 7°A for the tips of the flaps29. However, the enzyme structure is well conserved among the different Fig. 2: Hydrogen binding between HIV-PIs and molded complexes, with rms deviations between the C atoms structure32 seldom exceeding 0.6A°. Such differences are well Binding site within the agreement range for protein structures refined independently or crystallized in different The HIV protease is a C2-symmetric homodimeric space groups30. Most of the inhibitors co-crystallized enzyme consisting of two 99 amino acid monomers. with HIV PR, including all peptidomimetic inhibitors, Each monomer contributes an aspartic acid residue 21 are bound in the enzyme active site in an extended that is essential for catalysis . Asp-25 and Asp-25´ 34 conformation so that when they are superimposed is shown in (Fig. 3) . The HIV protease has the upon one another, their functional elements align quite sequence Asp-Thr-Gly, which is conserved among well overall31. The contacts between the main chain other mammalian aspartic protease enzymes. An of the peptidomimetic inhibitors and the protease extended beta-sheet region on the monomers shows are almost uniform for all the complexes in (Fig. 2)32. by an arrow in (Fig. 3), known as the flap, constitutes Following a similar pattern, the hydrogen bonds are in part the substrate binding site with the two aspartyl made mostly between the main-chain atoms of both residues lying on the bottom of a hydrophobic the enzyme and the inhibitor. The hydroxyl group at the cavity35, 36, 37. Each flexible flap contains three

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characteristic regions: side chains that extend outward 2) Second-generation protease inhibitor therapy; (Met46, Phe53), hydrophobic chains extending inward boosting of protease inhibitors (Ile47, Ile54), and a glycine rich region (Gly48, 49, a) Amprenavir/fosamprenavir 51, 52). Ile50 remains at the tip of the turn and when b) Lopinavir the enzyme is unliganded a water molecule makes c) Atazanavir hydrogen bonds to the backbone of Ile50 on each monomer shows by an arrow in (Fig. 3). d) Tipranavir e) Darunavir

First generation protease inhibitor therapy Saquinavir The first approved protease drug. The discovery and development of the Hoffmann–La Roche drug Saquinavir, the first inhibitor of HIV-1 PR to be approved by the US FDA, proved to be a classic example of serendipity coupled with hard work. Initial inhibitors created in that study were peptide derivatives utilizing transition-state mimetic concepts42, 43. The basic design criterion relied on the Fig. 3: schematic diagram showing structure observation that HIV-1 PR, unlike other proteases, 34 of a HIV-1 protease. is able to cleave Tyr-Pro or Phe-Pro sequences in the viral polyprotein. Because the amide bonds of HIV proteases catalyze the hydrolysis of peptide proline residues are not susceptible to cleavage by bonds with high sequence selectivity and catalytic mammalian endopeptidases, the design of HIV-1 PR proficiency. The mechanism of the HIV protease shares many features with the rest of the aspartic inhibitors based on this criterion was expected to bring protease family although the full detailed mechanism potential advantages of higher selectivity. Reduced of this enzyme is not fully understood38. The water amides and hydroxyethylamine isosteres most readily molecule seems to play a role in the opening and accommodate the imino acid moiety characteristic closing of the flaps as well as increasing the affinity of a Phe-Pro or Tyr- Pro retroviral substrate and, between enzyme and substrate. The aspartyl therefore, were chosen for further studies. As residues are involved in the hydrolysis of the peptide was shown later, the reduced amide isosteres bonds39. The preferred cleavage site for this enzyme were relatively poor inhibitors but, in contrast, the is the N-terminal side of proline residues, especially compounds incorporating the hydroxyethylamine between phenylalanine and proline or tyrosine and moiety were very potent and highly selective inhibitors proline40, 41. of HIV PR. A minimum sequence required for potent inhibition, as well as the unexpected preference for Development of Protease Inhibitors R stereochemistry at the hydroxyl-bearing carbon, Classification of PIs was determined by enzymatic studies of a series of 1) First generation protease inhibitor therapy related compounds. The minimum length inhibitor included three residues on the N-terminal side of a) Saquinavir the isostere and two residues on the C-terminal side. b) Ritonavir Varying the side chains of the residues in all subsites c) Indinavir did not lead to dramatic improvement of the potency d) Nelfinavir of inhibitors. The most marked improvements in

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potency were achieved by varying the amino acid Ritonavir (Norvir) at subsite P10, via replacement of proline by (S, S, The development of the Abbott drug Ritonavir S)-decahydroisoquinoline- 3-carbonyl (DIQ). The shows the shift of the thinking of the designers from resulting compound (later designated as Ro 31-8959) the creation of symmetric inhibitors of this inherently had a Ki value of 0.12 at pH 5.5 against HIV-1 PR and symmetric enzyme to their ultimate conversion to an even better inhibition constant against HIV-2 PR asymmetric compounds. The concept was first tested (Ki < 0.1). It was also shown to be highly selective, with the synthesis and characterization of symmetric causing less than 50% inhibition of the human aspartic inhibitors of HIV-1 PR49, 50, 51, which were designed to proteases. The compound was subsequently used match the C2 symmetry of the homodimeric HIV PR. for further clinical trials and was finally approved Although symmetry was not thought to be an absolute by the US FDA in 1995 under the name Saquinavir requirement for the design of HIV PR inhibitors, it (Invirase). This design effort was accompanied by only was expected to be useful in tightly constraining the limited structural studies. The first crystal structure rather rigid ligands52. It was also expected that the to be solved was of a complex with another peptidic less peptidic nature of the inhibitors might enhance inhibitor, Ro 31-8588, which exhibited the expected their stability in vivo. Such symmetric inhibitors were S-configuration of the carbon bearing the central expected to confer higher specificity for retroviral hydroxyl group44, 45. The subsequent crystallographic proteases over the related mammalian proteases, study of Ro 31-8959 showed that, as predicted, this whose substrate binding sites are less symmetric. A inhibitor bound in an extended conformation, forming hypothetical tetrahedral intermediate of the cleaved a characteristic set of hydrogen bonds with the peptide divided by the C2 axis of the enzyme was enzyme46, 47. With the exception of the flap, the two taken as the template for the development of inhibitors. fold symmetry of the enzyme was preserved, allowing This hypothetical axis was placed on the carbonyl ‘O’ the S and S0 subsites to be essentially equivalent. A atom or between the C-N atoms of the cleaved peptide comparison of the HIV-1 PR/Ro 31-8959 structure with bond respectively, and one half of the template was the structure of HIV-1 PR complexed with a longer deleted. The C2 operation was then implemented inhibitor, JG-365, showed that the conformation of on the remaining template, generating symmetric the inhibitor in the binding cavity critically depended inhibitors in either the mono-ol or diol form. The first on the nature of the P10 residue and on the presence of these symmetric inhibitors showed good kinetic or absence of the extension beyond subsite S20 48. profiles, and the concept of symmetry seemed to It was also established that short inhibitors preferred work remarkably well in cell cultures. the R-configuration of the central carbon. Moreover, the carbonyl ‘O’ atom of the DIQ group was able to maintain the hydrogen bond between the water molecules connecting the inhibitor with the flap regions (Wat 301).

Ritonavir

Indinavir (Crixivan) The discovery and development of the Merck drug Indinavir was a long and complicated research project. Saquinavir Similarly to the approach taken at Roche, the original

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design strategy began with a compound based on the developed by Agouron, AG-1002 and AG-1004, had transition-state mimetic concept. This approach was a statine isostere instead of a normal peptide bond52. successfully implemented in an earlier design of renin These compounds were peptidomimetic inhibitors inhibitors, although no approved drugs resulted from with the flanking amino acid sequences naturally the project. One important structural feature present recognized by HIV-1 PR and were 272 WLODAWER & in most tight-binding aspartic protease inhibitors is VONDRASEK bound to the active site in an extended a critical hydroxyl group that hydrogen bonds to the conformation. They could form 16 and 18 hydrogen carboxyl groups of the catalytically active aspartic bonds, respectively, due to the presence of hydrophilic acids. Incorporation of a hydroxyethylene isostere as side chains. Despite the large number of hydrogen a dipeptide mimic resulted in compounds that were bonds, the inhibitors had relatively low potency, with potent and selective inhibitors of HIV PR. The available binding constants of 0.55 M for AG-1002 and 0.32 structure-activity data indicated that an S-hydroxyl ¹M for AG-1004. A possible explanation for this low was the preferred configuration. The constituent potency was the absence of a P10 group, as well as hydroxyethylene isostere was extensively examined the large free energy required for desolvating of the in a series of peptidomimetic inhibitors of different hydrophilic side chains53. length and with particular residues occupying subsites on both sides of the nonscissile isostere. Residues in these positions were systematically changed to establish the relationships between particular modifications and the efficiency of the inhibitors53. The important questions of bioavailability were also addressed by these modifications. Some of the useful directions of synthesis were based on the results of molecular modeling and calculations54.

Nelfinavir

Second-generation protease inhibitor therapy

Amprenavir/fosamprenavir Amprenavir reached the market in 199954. It is an N,N-disubstituded amino-sulfonamide nonpeptide Indinavir HIV protease inhibitor and shares some common features with previous protease inhibitors55. It has Nelfinavir (Viracept) a core similar to that of saquinavir but with different The pathway of the discovery and development functional groups on both ends. On one end it has of Viracept by Agouron Pharmaceuticals is the least a tetrahydrofuran carbamate group and on the completely described in the literature of the four other end is an isobutylphenyl sulfonamide with US FDA-approved protease inhibitor drugs. Little is an added amide. This structure results in fewer known about the basis of its synthesis, and no crystal chiral centers, that makes it easier to synthesize structure of the complex with HIV-1 PR has been and gives it enhanced aqueous solubility. That in released. This inhibitor is the only compound among turn gives better oral bioavailability56. However, these four that does not utilize a peptidomimetic amprenavir was withdrawn from the market in 2004 concept, although its development pathway may since fosamprenavir, its prodrug, proved superior have utilized some peptidomimetics. Two inhibitors in many aspects.

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protease inhibitors. It is unique among the other protease inhibitors as it can only be absorbed in an acidic environment61, 62.

fosamprenavir

Lopinavir Lopinavir was marketed in 200057. It was originally designed to diminish the interactions of the inhibitor Atazanavir with Val82 of the HIV-1 protease, a residue that is often mutated in the drug resistant strains of the Tipranavir virus58. It is a peptidomimetic HIV protease inhibitor Tipranavir is a nonpeptidic HIV-1 protease and its core is identical to that of ritonavir. Instead inhibitor and reached the market in 2005. Unlike other of the 5-thiazolyl end group in ritonavir, lopinavir has HIV protease inhibitors on the market, tipranavir was a phenoxyacetyl group and the 2-isopropylthiazolyl developed from a nonpeptidic coumarin template group in ritonavir was replaced by a modified valine in and its antiprotease activity was discovered by high- which the amino terminal had a six-membered cyclic throughput screening. This sulfonamide containing urea attached. Fosamprenavir was marketed in 2003, 5, 6-dihydro-4-hydroxy-2-pyrone had emerged and is a phosphoester prodrug that is rapidly and from screenings of 3-substituted coumarins and extensively metabolized to amprenavir. The solubility dihydropyrones. It possesses broad antiviral activity and bioavailability is better than of amprenavir which against multiple protease inhibitor resistant HIV-163, 64. results in reduced daily pill burden59.

Tipranavir

Darunavir Lopinavir Darunavir reached the market in 200665. It is a nonpeptidic analogue of amprenavir, with a critical Atazanavir change at the terminal tetrahydrofuran (THF) group. Atazanavir was marketed in 200360. It is an Instead of a single THF group, darunavir contains azapeptide protease inhibitor designed to fit the C2- two THF groups fused in the compound, to form symmetry of the enzyme binding site. Atazanavir a bis-THF moiety which makes it more effective showed better resistant profiles than previous HIV than amprenavir. With this structural change, the

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stereochemistry around the bis-THF moiety confers bis-THF moiety, instead of a single THF moiety like orientational changes, that allow for continued binding on amprenavir, it can form more hydrogen bonds and with the protease which has developed a resistance increase binding energy72. for amprenavir66. Development of Drug Resistance In common with other retroviral polymerases, HIV RT is unable to edit transcription errors during nucleic acid replication, and thus enhances the mutational rates of the virus73, 74. As one of the results, divergent viral populations are present during infection and the sequences of the proteases from these different strains differ, sometimes substantially. Many of these differences do not affect the activity of the enzyme, and the structures of fully active proteases that differ in a number of positions in their sequences, from strains such as NY5 or SF2, have been reported. Darunavir However, high rates of viral turnover in HIV infection and the inability of HIV reverse transcriptase to correct Structure-activity relationship transcriptional errors also mean that populations of resistant virus will eventually emerge during any All the HIV protease inhibitors on the market antiviral therapy as a result of drug selection of viral contain a central core motif consisting of a 75. It has previously been shown that the clinical hydroxyethylene scaffold, with the only exception strains being the central core of tipranavir, which is based on use of RT inhibitors has led to the rapid development a coumarin scaffold67. A very important group on the of resistance and cross-resistance against different HIV protease inhibitors is a hydroxyl group on the core RT inhibitors. Similarly, drug-induced mutations motif which forms a hydrogen bond with the carboxylic in HIV PR alter the susceptibility of the enzyme to acid on the Asp-25 and Asp-25´ residues in the binding inhibition by specific inhibitors. A recent survey of the site shows in (Fig. 4)68, 69. Hydrogen bonds between mutations associated with drug resistance has shown the water molecule, which is linked to Ile50 and Ile50’, the modifications of 45 residues in HIV-1 PR, almost and carbonyl groups of the peptidomimetic inhibitors half of the total, for enzyme isolated from samples seem to connect them with the flap regions70. On the obtained with the help of 21 drugs. An obvious other hand, on the nonpeptidic inhibitors, there is a and expected mechanism for the development of proton acceptor which replaces the tetracoordinated resistance to HIV PR inhibitors is caused by the water molecule and interacts directly with the two Ile50 changes of specificity-determining residues that can residues on the flap of the enzyme. Specific pockets directly interfere with the binding of the inhibitor to the in the binding site of the HIV protease, often referred enzyme. The mutations that do not directly change to as S1,S1’,S2 and S2’, recognize hydrophobic the shape or character of the binding cavity can amino acids on natural substrates shows in (Fig. 4). indirectly influence inhibitor binding via long range The potency of inhibitors bearing hydrophobic groups structural perturbations of the active site, or they can complementing these areas is therefore increased71. change the efficiency of catalysis and the stability of Some residues in the enzyme binding site are capable the enzyme. A characterization of resistant variants of forming hydrogen bonds with hydrophilic groups isolated from patients undergoing therapy with the on the inhibitor, for example with the THF moieties protease inhibitor MK-639 (Indinavir) was published on amprenavir and darunavir. Since darunavir has a in 199576.

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Some of these variants exhibited cross-resistance 8. Nephrolithiasis to all members of a panel of six structurally diverse 9. Photosensitivity protease inhibitors. This study provided the first evidence that inhibition of HIV-1 PR can also lead Current status to the emergence of drug-resistant mutants in vivo In November 2006 darunavir was still the most and that combination therapy with multiple protease recent HIV protease inhibitor78 to reach the market. inhibitors may not preclude the loss of antiviral activity In 2006, GlaxoSmithKline discontinued the phase II resulting from resistance selection. A similar study using clinical isolates was also reported by Winslow et al. Five noncontiguous regions of HIV-1 PR, as described originally by Fontenot et al, were found to be conserved across all examined isolates. These regions include residues 1–9 (N terminus), 21–32 (the sequence surrounding the catalytic aspartate found in the active site), 47–56 (flap region), 78–88 (substrate-binding region), and 94–99 (C terminus and the dimerization region). The mutational analyses have shown that diversity is allowed at some positions, where as mutations in these conserved regions often result in abnormal gag processing in vitro77.

Application of Protease Inhibitors 1) Tissue culture drug resistance analysis of a novel HIV-1 protease Inhibitor termed PL-100 in non-B HIV-1 sub types. Fig. 4: A simplified image of protease inhibitors binding to the active site of the HIV-168. 2) The new and less toxic protease inhibitor saquinavir now maintains anti-HIV-1 properties in vitro indistinguishable from those of the parental compound saquinavir. 3) Prediction of drug-resistance in HIV-1 subtype C based on protease sequences from ART naive and first-line treatment failures in North India using genotypic and docking analysis.

Adverse effect of Protease Inhibitors 1. Gastrointestinal intolerance 2. Asthenia 3. Headache and dizziness Fig. 5: Trends in annual rates of death due to 7 leading 4. limb and facial tingling causes among persons 25-44 years old in the United 5. Numbness and rashes States during period 1987-2011. Dramatic decrease in the rate of death due to AIDS coincides with the 6. Dyslipidaemia introduction of HIV protease inhibitors (source: 7. crystalises in urine increase risk of urinary National Vital Statistics, Centers for Disease Control and Prevention, Atlanta)81. calculi

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Table II: Inhibitors currently used in clinical practice81 Summarized data including antiviral activity, side-effects, and pharmaceutical boosting

Generic Commonly Most common side effects Position in the present name and recommended dosage and off-target activities therapeutic arsenal abbreviation Ritonavir 100–200 mg p.o. BID as nausea, diarrhea, practically only (RTV)a pharmacokinetic booster abdomenalgia, hyperlipidemia, pharmacoenhancing of of various PIs (original lipodystrophy syndrome, various PIs pharmacodynamic dose inhibition of the cytochrome was 600 mg p.o. BID) P450 3A4

Saquinavir 1000 mg + diarrhea, hyperlipidemia, second-line HAART therapy (SQV) RTV 100 mg p.o. BID lipodystrophy syndrome

Indinavir (IDV) 800 mg + RTV 100 mg nephrolithiasis, lipodystrophy second/third-line HAART p.o. BID syndrome, hyperlipideamia, therapy in case of resistance hepatotoxicity or intolerance Nelfinavir (NFV) 1250 mg p.o. BID diarrhea, hyperlipidemia, second/third-line HAART lipodystrophy syndrome therapy in case of resistance or intolerance, approved for therapy of children Lopinavir 400 mg + RTV 100 mg diarrhea, hyperlipideamia, first line option for PI based (LPV/r)b p.o. BID lipodystrophy syndrome HAART regimen Amprenavir 600 mg + RTV 100 mg diarrhea, toxoallergic rash, replaced by its prodrug (APV) p.o. BID hyperlipidemia, lipodystrophy fosamprenavir Fosamprenavir 700 mg p.o. + diarrhea, toxoallergic rash, first-line option for PI (FPV) RTV 100 mg p.o. BID hyperlipidemia, lipodystrophy based HAART syndrome regimen Atazanavir 300 mg + RTV 100 mg hyperbilirubinemia, first-line option for PI (ATV) p.o. q24h or ECG abnormalities based HAART 400 mg p.o. q24h (1° atrioventricular block) regimen Tipranavir 500 mg + toxoallergic rash, second-line HAART (TPV) RTV 200 mg p.o. BID hepatotoxicity, therapy in case of intracranial hemorrhage, resistance lipodystrophy syndrome, diarrhea Darunavir 600 mg + nausea, diarrhea, recently approved for (DRV) RTV 100 mg p.o. BID hyperlipidemia, first-line HAART or 800 mg + RTV 100 headache, toxoallergic rash mg p.o. q24h

Abbreviations: BID - twice per day, q24h - every 24 hours a Ritonavir is at present used in therapy of HIV infection practically only as a pharmacokinetic booster. b Lopinavir is marketed by the manufacturer only as (Kaletra), in co-formulation together with low doses of ritonavir which acts as a pharmacokinetic booster.

16 INDIAN DRUGS 50(02) February 2013

clinical development of brecanavir, an investigational aspartic proteases, Bioorg Med Chem Lett. 2009, 19:3945-3948. protease inhibitor for the treatment of HIV, due to 10. http:// www. google. com/ patents?vid=5196438 Accessed insurmountable issues regarding formulation. In on 23/06/2012. the summer of 2009, GlaxoSmithKline and Concert 11. http:// www. google. com/ patents?vid=5541206 Accessed Pharmaceuticals announced their collaboration to on 23/06/2012. develop and commercialize deuterium-containing 12. http:// www. google. com/ patents?vid=5413999 Accessed on 23/06/2012. medicines79. One of them is CTP-518, a protease 13. http:// www. google. com/ patents?vid=5484926 Accessed inhibitor for the treatment of HIV, expected to on 23/06/2012. enter phase I clinical trials in the second half of 14. http:// www. google. com/ patents?vid=5585397 Accessed 2009. CTP-518 is a novel HIV protease inhibitor on 23/06/2012. developed by replacing certain key hydrogen atoms 15. Davies D.R.: The structure and function of the aspartic of atazanavir with deuterium80. Pre-clinical studies proteinases, Ann Rev Bio Chem. 1990, 19 (1):189– 215. have demonstrated that this modification fully retains 16. brik A., Wong C.H.: HIV-1 protease: mechanism and the antiviral potency but can evidently slow hepatic drug discovery, Org Biomol Chem. 2003, 1(1):5-14. metabolism and thereby increase the half life and 17. Krausslich H.G., Ingraham R.H., Skoog M.T., Wimmer E., plasma trough levels. CTP-518, therefore, has the Pallai P.V., Carter C.A.: Activity of purified biosynthetic proteinase of human immunodeficiency virus on natural potential to be the first HIV protease inhibitor to substrates and synthetic peptides, J F U.S.A. 1989, eliminate the need to co-dose with a boosting agent, 86(3): 807–11. such as ritonavir. Protease Inhibitors currently used in 18. Kohl N.E., Emini E.A., Schlei W.A. et al: Active human clinical practice are shows in tabulated form in Table immunodeficiency virus protease is required for viral infectivity, Proc Natl Acad Sci. 1988, 8 (13):4686–90. II. Also due to the use of protease inhibitors it was 19. Seelmeier S., Schmidt H., Turk V.: Human immunodeficiency observed that dramatic decrease in the rate of death virus has an aspartic-type protease that can be due to AIDS occurs, as shown in Fig. 581. inhibited by pepstatin A, Proc Natl Acad Sci. 1988, 85 (18):6612–6. References 20. McPhee F., Good A.C., Kuntz I.D., Craik C.S.: Engineering 1. barrett A. J., Rawlings N.D., Woessner J.F.: The Handbook human immunodeficiency virus 1 protease heterodimers of Proteolytic Enzymes, Academic Press, 2003, 2nd edition. as macromolecular inhibitors of viral maturation, Proc 89. Natl Acad Sci. 1996, 93(21): 11477–81. 2. Hedstrom L.: Serine Protease Mechanism and Specificity, 21. http://en.wikipedia.org/wiki/HIV accessed on 23/5/2012. Chem Rev. 2002, 102: 4501-4523. 22. Wlodawer A., Vondrasek J.: Inhibitors of HIV-1 protease: 3. Hooper N.M.: Proteases in Biology and Medicine. London a major success of structure-assisteddrug design, Annu Portland Press, 2002, 90. Rev Biop Biomol Struct. 1998, 27:249-284. 4. Puente X.S., Sanchez L.M., Overall C.M., Lopez-Otin C.: 23. A. Wlodawer: Rational approach to AIDS drug design Human and Mouse Proteases: a Comparative Genomic through structural biology, Annu Rev Med. 2002, 5:595- Approach, Nat Rev Genet. 2003, 4:544-558. 614. 5. Rang H.P., Dale M.M., Ritter J.M., Flower, R.J.: Rang and 24. Prejdova J., Soucek M., Konvalinka J.: Determining and Dale’s Pharmacology Philadelphia Churchill Livingstone overcoming resistance to HIV protease inhibitors, Curr Elsevier, 2007, 6th edition, 234-236. Drug Targ Infect Disord. 2004, 4:137-152. 6. brunton L.L., Lazo J.S., Parker K.L.G., Gilmans´s.: The 25. De Clercq E.: New approaches toward anti-HIV Pharmacological Basis of Therapeutics, United States of chemotherapy, J Med Chem. 2005, 48:1297-1313. th America, McGraw-Hill, 2006, 11 edition, 458-460. 26. yin P.D., Das D., Mitsuya H.: Overcoming HIV drug 7. http:/ / emedicine. medscape. com/ article/ 211316- resistance through rational drug design based on overview, Accessed on 5/7/2012. molecular, biochemical, and structural profiles of 8. Kurup A., Mekapati S.B., Garg R., Hansch C.: HIV-1 HIV resistance, Cell Mol Life Sci. 2006, 63:1706- Protease Inhibitors: A Comparative QSAR Analysis, Cur 1724. Med Chem. 2003, 10:1679-1688. 27. De Clercq A.: The history of antiretrovirals: key discoveries 9. Shi H., Liu K., Leong S.Q.: Expedient solid-phase synthesis over the past 25 years, Rev Med Virol. 2009, 19:287- of both symmetric and asymmetric diol libraries targeting 299.

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57. Rose R.B., Rose J.R., Salto R., Craik C.S., Stroud. R.M.: 70. Dreyer G.B., Boehm J.C., Chenera B., Des Jarlais Structure of the protease from simian immunodeficiency R.L., Hassell A.M. et al.: A symmetric inhibitor binds virus complex with an irreversible nonpeptide inhibitor, HIV-1 protease asymmetrically, Biochem. 1993, Biochem. 1993, 32:12498–507. 32:937– 47. 58. Rich D.H., Sun C.Q., Vara Prasad J.V., Pathiasseril A., 71. Dreyer G.B., Lambert D.M., Meek T.D., Carr T.J., Toth M.V. et al: Effect of hydroxyl group configuration in Tomaszek T.A. et al: Hydroxyethylene isostere hydroxyethylamine dipeptide isosteres on HIV protease inhibitors of human immunodeficiency virus-1 protease inhibition evidence for multiple binding modes, J Med structure-activity analysis using enzyme kinetics, x-ray Chem. 1991, 34:1222– 25. crystallography, and infected T-cell assay, Biochem. 59. Greenlee W.J.: Renin inhibitors, 1990, Med Res Rev. 1992, 31:6646–59. 10:173–236. 72. collins J.R., Burr S.K., Erickson J.W.: Activated dynamics 60. Appelt K.: Crystal structures of HIV- 1 protease-inhibitor of flap opening in HIV-1 protease, Adv Exp Med Biol. complexes, Persp Drug Disc Des. 1993, 1:23–48. 1995, 362:455–60. 61. Swain A.L., Miller M.M., Green J., Rich D.H., Schneider J. et al: X-ray crystallographic structure of a complex between 73. erickson J.W., Anderson P.S., Kenyon G.L., Marshall a synthetic protease of human immunodeficiency virus 1 G.R.: Design and structure of symmetry-based inhibitors and a substrate-based hydroxyethylamine inhibitor, Proc of HIV-1 protease, In Perspectives in Drug Discovery and Natl Acad Sci USA. 1990, 87:8805–9. Design Leiden, The Netherlands: ESCOM Sci. 1993, 62. Greer J., Erickson J.W., Baldwin J.J., Varney M.D.: 109–128. Application of the three dimensional structures of protein 74. Roberts J.D., Bebenek K., Kunkel T.A.: The accuracy target molecules in structure-based drug design, J Med of reverse transcriptase from HIV-1, Sci. 1988, Chem. 1994, 37:1035–54. 242:1171–73. 63. Darke P.L., Nutt R., Brady S., Garsky V., Ciccarone T. et al: 75. Schinazi R.F., Larder B.A., Mellors J.W.: Mutations in HIV-1 protease specificity of peptide cleavage is sufficient retroviral genes associated with drug resistance, Antivir. for processing of Gag and Pol polyproteins. Biochem, 1997, 5(8):129–42. Biophys Res Commun. 1988, 156:297–303. 76. erickson J.W., Burt S.K.: Structural mechanisms of 64. Davies D.R.: The structure and function of the aspartic HIV drug resistance, 1996, proteinases, Annu Rev Biophys Chem. 1990, 19:189– Annu Rev Pharm Toxicol. 215. 36:545– 71. 65. DeSolms S.J., Giuliani E.A., Guare J.P., Vacca J.P., 77. Schonazi R.F., Larder B.A., Mellors J.W.: Mutations in Sanders W.M. et al: Design and synthesis of HIV protease retroviral genes associated with drug resistance, Antivir. inhibitors. Variations of the carboxy terminus of the HIV 1997, 5(8):129–42. protease inhibitor, J Med Chem. 1991, 34:2852–57. 78. Winslow D.L., Stack S., King R., Scarnati H., Bincsik 66. backbro K., Lowgren S., Osterlund K., Atepo J., Unge T. A., Otto M.J.: Limited sequence diversity of the HIV et al: Unexpected binding mode of a cyclic sulfamide HIV-1 type 1 protease gene from clinical isolates and in vitro protease inhibitor, J Med Chem. 1997, 40:898–902. susceptibility to HIV protease inhibitors, Res Hum 67. chen X., Tropsha A.: Relative binding free energies of Retrovir. 1995, 11:107–13. peptide inhibitors of HIV-1 protease: the influence of 79. Fontenot G., Johnston K., Cohen J.C., Cohen W.R., the active site protonation state, 1995, J Med Chem. Robinson J., Luftig R.B.: PCR amplification of HIV-1 38:42–48. proteinase sequences directly from lab isolates allows 68. DeVita V.T., Broder S., Fauci A.S., Kovacs J.A., Chabner determination of five conserved domains, Virol. 1992, B.A.: Developmental therapeutics and the acquired 190:1–10. immunodeficiency syndrome, Ann Intern Med. 1987, 106:568–81. 80. De Clercq E.: Anti-HIV drugs: 25 compounds approved within 25 years after the discovery of HIV, Int J Antimicro 69. Dorsey B.D., Levin R.B., McDaniel S.L., Vacca J.P., Agents. 2009, 33:307-320. Guare J.P. et al.: The design of a potent and orally bioavailable HIV protease inhibitor, J Med Chem. 1994, 81. http://www. gsk. com/ media/ press releases, Accessed 37:3443–51. on 21/7/2012.

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original research articles

METHOD DEVELOPMENT AND VALIDATION OF OLANZAPINE IN PURE AND PHARMACEUTICAL DOSAGE FORM BY RP-HPLC METHOD Mastiholimath V.S.*, Dandagi P.M., Gadad A.P., Murali Krishna N.V. and Mannur V.

(Received 11 April 2012) (Accepted 23 January 2013)

Abstract

A simple and reliable reverse phase high-performance liquid chromatography method was developed and validated for Olanzapine in pure and pharmaceutical dosage form. The method was developed on BDS Hypersil C18, (150 mm x 4.6 mm, 3μm) with a mobile phase of 0.01M tetra butyl ammonium hydrogen sulphate : methanol (80:20 v/v). The effluent was monitored by SPD-M20A, prominence UV-VIS detector at 234 nm. Calibration curve was linear over the concentration range of 10 –60μg/ml. For inter–day and intra–day precision % relative standard deviation values were found to be 0.18% and 0.24% respectively. Recovery of olanzapine was found to be in the range of 99.93 -100.00%. The limit of detection (LOD) and quantitation (LOQ) were 0.39275 and 1.1901μg/ml, respectively. The retention time and run time was very short; hence it is cost effective, making it more economical and rapid. Also this method can be used for the analysis of large number of samples.

Keywords: Olanzapine, RP-HPLC, Validation, estimation of olanzapine which includes HPLC3-9, Pharmaceutical dosage form. stability indicating method10, HPTLC11-13, HPLC- MS14,15 and spectroscopic method16-20. The present INTRODUCTION paper describes a new quantitative reversed-phase Olanzapine1, 2 is official in Indian Pharmacopoeia. high-performance liquid chromatographic method, It is chemically known as 2-methyl-4-(4-methyl-1- coupled with Shimadzu Prominence UV-VIS detector, piperazinyl)-10Hthieno [2,3-b][1,5]benzodiazepine as an alternative technique for quality control of OLN products. The purpose of this investigation was to (Fig.1). The molecular formula is C17H20N4S and molecular weight is 312.4. It is used as an develop and validate a method using a simple, rapid, antipsychotic drug due to antagonism at dopamine sensitive, precise, accurate and specific reversed and drugs serotonin type 2 receptors, with greater phase HPLC assay. activity at serotonin 5-HT2 receptors than at dopamine type 2 receptors. Antagonism at muscarinic receptors, H1-receptors, and alpha (1)-receptors also occurs with Olanzapine.

Extensive literature survey reveals that several analytical methods have been reported for the

*For correspondence Department of Quality Assurance KLE University’s College of Pharmacy Belgaum-590 010, Karnataka E-mail: [email protected] Fig. 1: Chemical structure of Olanzapine

20 INDIAN DRUGS 50(02) February 2013

MATERIALS AND METHODS RESULTS Chemicals and solvents Method development and optimization The reference sample of olanzapine was supplied A mixture of 0.01M tetra butyl ammonium hydrogen by Chandra Labs Pvt Ltd., Hyderabad. HPLC grade sulphate and methanol in the ratio of 80:20v/v was water and methanol were purchased from E. Merck found to be the most suitable mobile phase for ideal (India) Ltd., Mumbai. Tetra butyl ammonium hydrogen separation of olanzapine. The solvent mixture was sulphate of AR Grade was obtained from S.D. Fine filtered through a 0.45 μ membrane filter and sonicated Chemicals Ltd., Mumbai. Tablet formulations of before use. It was pumped through the column at a Oleanz® were procured from a local pharmacy with flow rate of 1ml/min. The column was maintained at labeled amount of 5 mg per tablet. ambient temperature. The pump pressure was set at 800 psi. The column was equilibrated by pumping the Instrument mobile phase through the column for at least 30 min The HPLC system is of Shimadzu, LC-20AT, prior to the injection of the drug solution. The detection a SPD-M20A prominence UV-VIS detector, of the drug was monitored at 234 nm. The run time was HPLC Pump LC-20AT pump and Injector loop set at 6 min. Under these optimized chromatographic rheodyne, model No. 2767, 20 µl volume loop. conditions the retention time obtained for the drug Data acquisition was performed by the Spinchrom was 2.61 min. A typical chromatogram showing the software. separation of the drug is given in Fig. 2.

Chromatography condition Validation procedures Chromatographic analysis was performed on The method validation was carried out according a BDS Hypersil reversed phase C-18 column with to the recommendations for analytical method 21,22 150 x 4.6mm internal diameter and 3μm particle validation . size. The mobile phase consisted of 0.01M tetra System suitability butyl ammonium hydrogen sulphate : methanol (80: 20 v/v) and that was set at a flow rate of 1 ml/ System-suitability tests are an integral part min. The mobile phase was degassed and filtered of method development and are used to ensure through 0.45 μ filter under vacuum before pumping adequate performance of the chromatographic into HPLC system. The effluent was monitored by system. Retention time (tR), number of theoretical UV detection at 234 nm. plates (N) and tailing factor (T) were evaluated for six replicate injections of the drug at a concentration Preparation of 0.01M tetra butyl ammonium of 50 μg/mL. The results which are given in Table I hydrogen sulphate were within acceptable limits. Dissolve 0.33954 g of tetra butyl ammonium hydrogen sulphate in small amount of distilled water transferred in to 100 mL volumetric flask and make up to final volume with water.

Preparation of mobile phase and diluents 800 mL of the 0.01M tetra butyl ammonium hydrogen sulphate was mixed with 200 mL of methanol. The solution was degassed in an ultrasonic water bath for 5 minutes and filtered through 0.45 μ filter under vacuum. Fig. 2: Chromatogram of Olanzapine at 234 nm

INDIAN DRUGS 50(02) February 2013 21

Linearity Several aliquots of standard stock solution(1mg/ ml) (0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 ml) of olanzapine drug were taken in different six standard 10.0 mL volumetric flasks and diluted up to mark with mobile phase. Evaluation was performed with UV detector at 234 nm. The peak area was recorded for all the peaks and calibration graph was obtained by plotting peak area versus concentration of olanzapine. The plot of peak area against concentration of Concentration µg/mL olanzapine was found to be linear in the range of 10 to 60μg/mL with correlation coefficient of Fig. 3: Calibration curve of Olanzapine 0.9996. The calibration data of olanzapine is given Inter-day precision in Table II and the calibration curve of olanzapine is shown in Fig. 3. In the inter-day variation studies, six injections of standard solution (50μg/mL) were injected at different Accuracy days. % RSD was calculated and is presented in Accuracy was performed in triplicate after spiking Table V. pure drug equivalent to 80, 100, and 120% of the Table I: Results from system suitability studies standard concentration of olanzapine (50 μg/mL). The Required results obtained (Table III) indicate that recovery was Property Values ±SD* %RSD excellent, not less than 100% ± 2. The results indicate limits the method is highly accurate for determination of Retention 2.55±0.0066 0.26 RSD ≤ 1% the olanzapine. time (tR) Theoretical Sensitivity 2672±14.88 0.56 N > 2000 plates (N) Limit of detection (LOD) and limit of quantitation Tailing (LOQ) were determined from standard deviation and 1.568±0.0223 1.421 T ≤ 2 factor(T) slope method as per ICH guideline, for olanzapine LOD was found to be 0.39275μg/mL and LOQ was *Average of six determinations. found to be 1.1μg/mL. Table II: Calibration data of Olanzapine by RP- Precision HPLC method The precision of the method was demonstrated S.No Concentration Peak Area by intra-day and inter-day variation studies. (μg/mL) (m V.s) Intra-day precision 1 10 729.418 In the intra-day studies, six injections of 2 20 1419.661 standard solution (50μg/mL) were injected into the 3 30 2205.335 chromatographic system in different time interval 4 40 2949.615 within a day. %RSD was calculated and is presented in Table IV. 5 50 3636.578 6 60 4314.348

22 INDIAN DRUGS 50(02) February 2013

Table III: Results from recovery studies

Sample Area Initial Amount Amount %Recovery ± SD* %RSD (m V.s) amount added recovered* (μg/mL) (μg/mL) (μg/mL) 80% 3329.88 5 40 44.97 99.93 ±1.98 0.06 100% 4072.52 5 50 55 100.00 ±3.97 0.097 120% 4811.37 5 60 64.98 99.97±3.93 0.081 *Average of three determinations.

Table IV: Intra-day precision results for Olanzapine

S. No Concentration (μg/mL) Retention time (min) Area (m V.s) 1 50 2.563 3713.551 2 50 2.550 3699.609 3 50 2.567 3696.849 4 50 2.563 3700.417 5 50 2.567 3703.568 6 50 2.54 3710.65 AVG 2.5583 3704.10 SD 0.0109 6.6153 %RSD 0.43 0.18

Table V: Inter-day precision results for Olanzapine

S. No Concentration (μg/mL) Retention time (min) Area (m V.s) 1 50 2.567 3674.252 2 50 2.557 3649.501 3 50 2.567 3664.827 4 50 2.553 3657.62 5 50 2.553 3668.155 6 50 2.555 3665.12 AVG 2.558667 3663.2458 SD 0.006623 8.6234408 %RSD 0.26 0.24

Robustness rate. It was observed that there were no marked Robustness of the method was determined changes in the chromatograms, which demonstrated by making slight changes in the chromatographic that the RP-HPLC method developed is robust. The conditions, such as change in wavelength and flow results are shown in Table VI.

INDIAN DRUGS 50(02) February 2013 23

Table VI: Robustness studies of Olanzapine by RP-HPLC method

Mean % RSD S.No Condition Modification Mean Peak area ± SD Mean Rt ± SD* (for AREA)

1 Flow rate 0.9 4195.7726±15.06 2.813±0.015 0.359 (ml/min) 1.1 3232.094±29.694 2.313±0.0057 0.249

2 Wavelength 232 4125.60567±44.5 2.81±0.01 1.079 (nm) 236 4323.51±8.727 2.54±0.0057 0.201

*Average of three determinations.

Table VII: Ruggedness studies of Olanzapine by RP-HPLC method

Injection S. No Analyst-1 Analyst-2 Number Area Retention Theoretical Area Retention Theoretical (m V.s) time (min) Plates(N) (m V.s) time (min) Plates(N) 1 1 3780.498 2.55 2647 3786.926 2.537 2619 2 2 3767.652 2.56 2633 3783.783 2.561 2625 AVG 3774.075 2.555 2640 3785.355 2.549 2622 SD 9.083 0.007 9.899 2.222 0.0169 4.242 %RSD 0.240 0.276 0.374 0.058 0.6657 0.161

Table VIII: Assay studies of Olanzapine by RP-HPLC method

Label claim Standard Area* Sample Area* Amount found (%)Recovery Sample (mg) (m V.s) (m V.s) (mg) Oleanz® 5 3789.1933 3665 4.99 99.73

*Average of three determinations

Ruggedness to the mark with the same solvent (Stock solution). It was checked by determining precision on the Further pipette out 0.5 mL of the above stock solution same instrument, but by a different analyst. Results into a 10 mL volumetric flask and dilute up to the mark of reproducibility are shown in Table VII. with the same solvent to obtain the concentration of 50 μg/mL. Mix well and filter through Whatman filter Assay paper (No. 41). Preparation of Standard Solution Preparation of Sample Solution Accurately weigh and transfer 25mg of Olanzapine working standard into a 25 mL volumetric Weigh 5 Olanzapine Tablets and calculate the flask, add about 15 mL of mobile phase and sonicate average weight. Accurately weigh and transfer the to dissolve it completely and make up the volume sample equivalent to 5 mg of Olanzapine into a 10

24 INDIAN DRUGS 50(02) February 2013

Table IX: Characteristic parameters of ACKNOWLEDGEMENT Olanzapine for the RP-HPLC method The authors wish to thank Chandra Labs Ltd, Parameters RP-HPLC Hyderabad for providing the necessary facilities to carry out work. Calibration range (µg / ml) 10-60 Detection wavelength 234 REFERENCES Mobile phase(Buffer : methanol) 1. Indian Pharmacopoeia. Ghaziabad: The Indian 80:20 (V/V) pharmacopoeia commission, 2007, 3, 857-858. Regression equation (Y) 72.455x+5.6381 2. http://en.wikipedia.org/wiki/Olanzapine 3. Patel S, Patel N. J.: Simultaneous RP-HPLC and HPTLC RT 2.61 Estimation of Fluoxetine Hydrochloride and Olanzapine Slope (b) 72.455 in Tablet Dosage Forms, Indian J Pharm Sci. 2009, 71(4), 477–480. Intercept (a) 5.6381 4. eswarudi M.M., Sushma M, Sushmitha M, Yamini K.: Correlation coefficient(r2) 0.9996 Validated spectroscopic method for the determination of salbutamol sulphate in bulk and pharmaceutical dosage LOD (µg / ml) 0.3927 forms, Int Res. J.Pharma. 2012, 3(4), 310-313. LOQ (µg / ml) 1.1901 5. Rao R.A., Delhi raj N, Jagadeesh.: RP-HPLC method for quantitative estimation of olanzapine in tablet dosage mL volumetric flask. Add about 7 mL of diluent and forms, Intl J. Pharma. Bio. Sci. 2012, 3(3), 267-272. sonicate it to dissolve completely and make up the 6. Karpinska J, Sokół Y, Bernatowicz A , Szulecka A, Kotowska U.: Studies on photodegradation of levomepromazine and volume up to the mark with diluent. Mix well and filter olanzapine under simulated environmental conditions, through Whatman filter paper (No.41). Further pipette Photochem. Photobiol. Sci., 2012, 11, 1575-1584. out 1 mL of the above stock solution into a 10mL 7. Rani P, Sekaran B.: Development of HPLC method for volumetric flask and dilute up to the mark with the the determination of Olanzapine in bulk and dosage forms, Inter J of Pharmtech research. 2009, 1(3), 654-657. same solvent. Mix well and filter through Whatman 8. Basavaiah K, Rangachar U.A., Tharpa K.: Quantitative filter paper (No.41).The results of assay are shown Determination of Olanzapine in Pharmaceutical in Table VIII. Preparations by HPLC, J. Mex. Chem. Soc. 2008, 52( 2), 120-124. DISCUSSION 9. Goyal S, Sharma D, Srinivas S.K.: Simultaneous estimation of risperidone, olanzapine and quetiapine A rapid and simple RP-HPLC method for and their degradation products by HPLC, Acta determination of olanzapine has been developed and Pharmaceutica Sciencia. 2010, 52, 345-352. validated and the results are shown in Table IX. The 10. Krishnaiah C.H., Murthy M.V., Kumar R.: Development of a stability-indicating UPLC method for determining RP-HPLC method involved a mobile phase consisting Olanzapine and its associated degradation products of 0.01M tetra butyl ammonium hydrogen sulphate: present in active pharmaceutical ingredients and methanol in the ratio of 80:20% (V/V) accomplished at pharmaceutical dosage forms, J of Pharm and Biomed 234 nm. The retention time was 2.61 min at a flow-rate Anal. 2011, 54, 667-673. 11. Patel R.B., Patel M.R..: Development and validation of of 1.0 ml/min and the injection volume was 5 μl. This an HPTLC method for determination of olanzapine in chromatographic assay fulfilled all the requirements formulations, JAOAC int. 2010, 3, 811-819. to be identified as a reliable and feasible method, 12. Shah C.R., Shah N.J., Patel D.R.: Stability-indicating including linearity, accuracy, sensitivity, precision, simultaneous HPTLC method for olanzapine and ruggedness and robustness. The chromatographic fluoxetine in combined tablet dosage form, 2008, 70(2), 251-255. system allows the analysis of a large number of 13. Raggi M.A., Sabbioni C, Mandrioli R, Albers L.: Rapid samples in a short period of time. Therefore, the analysis of Olanzapine and Desmethyl Olanzapine method is suitable for analysis of large samples during in human plasma using high-performance liquid routine analysis of formulations and raw materials. chromatography with coulometric detection, J of Analytica Chemica Acta. 2004, 51, 111-117.

INDIAN DRUGS 50(02) February 2013 25

14. Nirogi R.V.S., Kandikere V.N., Mauryaa S.: Development and Pharmaceutical Formulation by Using UV-Visible and validation of a sensitive liquid chromatography/ Spectroscopy Method, Intern. J. of Pharma. Sci. and electrospray tandem mass spectrometry assay for the Drug Research. 2011, 3(1), 52-55. quantification of Olanzapine in human plasma, J of 18. Basavaiah K, Tharpa K, Vinay K.B.: Spectrophotometric Pharm and Biomed Anal. 2006, 41, 935-942. Determination of Antipsychotic Drug Olanzapine in 15. Josefsson M, Roman M, Skogh E.: Liquid Pharmaceuticals, Jordan Journal of Chemistry. 2009, chromatography/tandem mass spectrometry method for 4(1), 65-76. determination of Olanzapine and N-desmethylOlanzapine 19. basavaiah K, Rajendraprasad N.: Determination in human serum and cerebrospinal fluid, J of Pharm of Olanzapine by spectrophotometry using and Biomed Anal. 2010, 53, 576-582. Permanganate, Brazillian J of Pharm Sci. 2009, 16. Gopinath S, Kumar R.S., Alexander S, Danabal P.: 45(3), 540-550. Development of a rapid and sensitive SPE-LC-MS/MS 20. ICH: Q2B, Analytical validation-methodology; 1996. method for the simultaneous estimation of fluoxetine and olanzapine in human plasma, Biomed chromatogr. 21. ICH: Q2A, Text on validation of analytical procedure; 2011. 1994. 17. Rubesh S, Kiran C.H., Duganath N.: Simultaneous 22. ICH: Q2 (R1), Validation of analytical procedures:text Estimation of Fluoxetine HCl and Olanzapine in Bulk Drug and methodology; 2005.

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QUANTITATIVE DETERMINATION OF DEFERASIROX IN BULK AND PHARMACEUTICAL FORMULATION BY UV SPECTROPHOTOMETRIC METHOD Marathe G. M., Pande V. V.*, Patil P. H., Mutha R. E. and Bari S. B.

(Received 25 October 2012) (Accepted 23 January 2013)

ABSTRACT

A simple, fast and reliable zero order spectrophotometric method was developed for determination of deferasirox in bulk and pharmaceutical dosage forms. Beer’s law was obeyed in concentration range of 2–12 µ/ml deferasirox at 245.6 nm wavelength. The correlation coefficient was found to be (r2 = 0.999), precision (repeatability % RSD 1.29), percentage recovery 100.054±0.271. The detection limit (DL) and quantitation limit (QL) were 0.247 µg/ml and 0.75 µg/ml respectively. The proposed method was found to be simple, accurate, precise, reproducible and gave an acceptable recovery of the analyte, which could be directly and easily applied to analysis of bulk and pharmaceutical tablet formulations of deferasirox.

Keywords: Deferasirox, UV spectrophotometry, who are receiving long term blood transfusions Area under curve for conditions such as beta-thalassemia and other chronic anemias. INTRODUCTION Literature survey revealed terbium-sensitized Deferasirox is 4-[3, 5-bis-(2-hydroxyphenyl)-[1, fluorescence method for the determination of deferasirox 2, 4]-triazol-1-yl]benzoic acid, molecular formula in biological fluids and tablet formulations3. It also C H N O molecular weight: 373.4, deferasirox) 21 15 3 4 revealed method development using UV spectroscopy4-5. (Fig. 1) is an orally active iron chelating agent. RP-HPLC methods have been developed for the Deferasirox is a white to slightly yellow powder and estimation of Deferasirox6-8. The aim of this study is a non-chiral compound. At the physiological pH of is to develop a simple, sensitive and validated, UV the intestine, the solubility is about 40 mg/L. In an spectrophotometric method for the determination of iron balance metabolic study in iron overloaded adult deferasirox as per ICH guidelines9-11. thalassaemic patients, deferasirox reduced chronic iron overload in adult and paediatric patients (aged 2 years and older) due to blood transfusions. The underlying conditions requiring transfusion include beta-thalassaemia, sickle cell disease, and other congenital and acquired anaemias (myelodysplastic syndromes, Diamond-Blackfan syndrome, aplastic anaemia and other very rare anaemias). Deferasirox (ICL670) is a novel once-daily, orally administered iron chelator to treat chronic iron overload in patients with transfusion-dependent anemias1-2. Its main use is to reduce chronic iron overload in patients

*For correspondence H.R. Patel Institute of Pharmaceutical Education & Research, Karwand Naka, Shirpur, Dist. Dhule (M.S.) 425405 E-mail: [email protected] Fig. 1: Structure of Deferasirox

INDIAN DRUGS 50(02) February 2013 27

EXPERIMENTAL Construction of calibration curve Instrument Aliquot of the standard stock solution (0.2, 0.4, Shimadzu UV-Visible double beam 0.6, 0.8, 1.0, 1.2 mL) was transferred into a series of Spectrophotometer (model-1700) was used for volumetric flasks (10 mL) and volume was adjusted spectral studies and validation. up to the mark with water to get desired concentration (2 – 12 µg/mL). The absorbance of the prepared Reagents and solutions solutions was measured at 245.6 nm

Methanol (HPLC grade) was procured from Assay Merck Fine Chemicals (Mumbai, India), the pure drug deferasirox was obtained as a gift sample from The assay of the proposed method was Hetero Drugs Limited, Hyderabad. ascertained by performing assay of the standard drug with reference to the sample drug and finding out the Standard solution absorbance. From the absorbance percentage purity was calculated (Table I). Stock Standard Solution: Accurately weighed quantity of deferasirox (10 mg) was dissolved in 10 Validation mL of methanol to get 1000 µg/mL, from that 1mL diluted upto 10 mL with methanol (concentration Linearity: To establish linearity of the proposed 100 µg/mL). methods, separate series of solutions of deferasirox (2-12 µg/mL) in water were prepared from the stock Preparation of working standard solution: solutions and analyzed. Least square regression 12µg /mL of deferasirox solution was prepared by analysis was performed on the obtained data. The diluting 1.2 mL of stock solution with water in 10 mL absorbance of the prepared solutions was measured volumetric flask up to the mark. Working standard at 245.6 nm (Fig. 3 and Table II, III). solution of deferasirox was scanned between 200-400 nm on Shimadzu double beam UV visible Precision spectrophotometer. The wavelength maximum Repeatability: Repeatability expresses the exhibited for deferasirox was at 245.6 nm (Fig. 2). precision under the same operating conditions over a short interval of time. Six determinations of same concentration were performed. Percentage RSD was found to 1.2 (Table IV).

Conc (µg/mL)

Fig. 3: Linearity of Deferasirox Fig. 2: Spectrum of Deferasirox

28 INDIAN DRUGS 50(02) February 2013

Table I: Assay of Deferasirox

Formulation Claim of tablet (mg/tablet) % Label claim [n=3] % RSD Desirox ® 250mg 99.49667 0.446356

Table II: Linearity of Deferasirox by Zero Order Spectroscopic Method

Sr. No. Conc. (µg/mL) Absorbance % RSD 1 2 0.111 1.37 2 4 0.234 0.65 3 6 0.349 0.72 4 8 0.469 0.56 5 10 0.591 0.33 6 12 0.715 0.42

Table III: Results of Linearity Study by Zero Order Spectroscopic Method

Beer’s law limit Correlation Regression Slope (m) Y-Intercept (c) (µg/mL) coefficient (R2) equation (y) 2-12 0.999 y=0.060x- 0.009 0.060 0.009

Table IV: Repeatability of Deferasirox by Zero Order Spectroscopic Method

Concentration (µg/mL) Mean of absorbance (n= 6) S.D % R.S.D 6 0.3475 0.0045 1.29

Table V: Results of Intra-Day and Inter-Day precision Study by Zero Order Spectroscopic Method

Concentration Intra-day [n=3] Inter-day [n=3] % RSD % RSD [µg/mL] Amount found [µg/mL] Amount found [µg/mL] 6 5.97 0.422 5.97 0.421 8 7.98 0.433 8.02 0.826 10 10.00 0.237 10.01 0.502

Table VI: Accuracy by Zero Order Spectroscopic Method

Percentage Percentage recovery [n=3] % RSD Mean ± SD 80% 100.3667 0.943869 100% 99.91667 0.641985 100.054±0.271 120% 99.88 0.820558

INDIAN DRUGS 50(02) February 2013 29

Table VII: Summary of validation parameter by Zero Order Spectroscopic Method

Parameter Result

Beer’s law limit (µg/mL) 2-12

Regression equation (y) y=0.060x- 0.009

Correlation coefficient (R2) 0.999

Repeatability (% R.S.D) 1.29

Accuracy (Mean±SD) 100.054±0.271

Assay (Mean± R.S.D) 99.49667±0.446

DL and QL (µg/mL) 0.247 and 0.75

Fig. 5: Second order derivative of Deferasirox

Fig. 4: First order derivative of Deferasirox Fig. 6: Area under curve of Deferasirox Intra-day and inter-day precision Accuracy Intra-day and inter-day precision were determined Standard addition and recovery experiments were by analyzing three different solutions of deferasirox conducted to determine the accuracy of the proposed within the same day and three different days over method (Table VI). a period of one week. Intra-day precision was estimated by analyzing 6 µg/mL, 8 µg/mL, 12 µg/mL Detection limit (DL) and quantitation limit (QL) for three times within same day. Inter-day precision The DL and QL of deferasirox by the proposed was estimated by analyzing above mentioned methods were determined using calibration standards. concentration of deferasirox for three different days DL and QL were calculated as 3.3r/S and 10r/S, over a period of one week (Table V). respectively, where S is the slope of the calibration

30 INDIAN DRUGS 50(02) February 2013

curve and r is the standard deviation of y-intercept of proposed method was found to simple, accurate, regression equation. DL and QL of deferasirox was precise, reproducible and gave an acceptable found to be 0.247 and 0.75 respectively. recovery of the analyte, which can be directly and easily applied to analysis of bulk and pharmaceutical First-Derivative spectrometry tablet formulations of deferasirox. The summary of The entire above obtained zero-order spectrums validation parameters by zero order spectroscopic were transformed to get first-order derivative spectra method is mentioned in Table VII. and the minima values of the derivative spectra were CONCLUSION recorded. First order derivative spectrum (D1) of (12 µg/ mL) at delta lambda 2 with scaling factor 1 (Fig. 4). The method described here is direct method for the analysis of deferasirox without any extraction Second order-Derivative spectrometry process to eliminate the excipients; it does not use The above obtained zero-order spectra were time consuming procedure such as standard addition transformed to get second-order derivative spectra method and also any expensive equipment. Due and the minima values of the derivative spectra were to the results of the present study the developed recorded. First order derivative spectrum (D2) of spectrophotometric method is accurate, sensitive, (12 µg/mL) at delta lambda 4 with scaling factor 1 is precise, reproducible and can be easily and directly shown in Fig. 5. applied to the bulk pharmaceutical preparations. Additionally, the short analysis time and low costs are Area Under Curve the other advantages of these methods for routine Area Under Curve (AUC) method involves the analysis. Its advantages over other existing methods calculation of integrated value of absorbance with are its simplicity, speed and low cost. respect to the wavelength between two selected wavelength 240.6nm and 250.6 nm. Area calculation ACKNOWLEDGMENT processing item calculates the area bound by the The authors are thankful to Hetero limited, curve and the horizontal axis. The horizontal axis is India for providing gift sample of deferasirox and selected by entering the wavelength range over which Department of Pharmaceutical Analysis, H. R. Patel the area has to be calculated. The wavelength range Institute of Pharmaceutical Education and Research, is selected on the basis of repeated observations so Shirpur, Dist. Dhule for support. as to get the linearity between area under curve and concentration. Area bound by the curve was 0.190 REFERENCES units (Fig. 6). 1. Waldmeier F., Bruin G.J., Glaenzel U., Hazell K., Sechaud R., Warrington S., Porter J.B.: Pharmacokinetics, RESULTS AND DISCUSSION metabolism, and disposition of deferasirox in beta- thalassemic patients with transfusion-dependent iron A simple, fast and reliable zero order overload who are at pharmacokinetic steady state, Drug spectrophotometric method was developed Metab Dispos. 2010, 38(5), 808-816. for determination of deferasirox in bulk and 2. Piga A., Galanello R., Forni G.L., Cappellini M.D., Origa R., Zappu A., Donato G., Bordone E., Lavagetto A., Zanaboni pharmaceutical dosage forms. Beer’s law was obeyed L., Sechaud R., Hewson N., Ford J.M, Opitz H., Alberti in concentration range of 2–12 µg/mL deferasirox at D.:Randomized phase II trial of deferasirox Exjade®, 245.6 nm wavelength. The correlation coefficient was ICL670), a once-daily, orally-administered iron chelator, found to be (r2 = 0.999), precision (repeatability% in comparison to deferoxamine in thalassemia patients with transfusional iron overload, Haematologica. 2006, RSD 1.29), percentage recovery 100.054±0.271. 91(7), 873-880. The detection limit (DL) and quantitation limit (QL) 3. Manzoori J.L., Abolghasem J., Mohammad A; Vahid P., were 0.247µg/mL and 0.75 µg/mL, respectively. The Elnaz T, Jalil V., Terbium-sensitized fluorescence method

INDIAN DRUGS 50(02) February 2013 31

for the determination of deferasirox in biological fluids estimation of deferasirox in pharmaceutical dosage and tablet formulation. Luminescence. 2011, 26(4), form, AJPI’s J of Analytical Chem.2011, 1(6), 244-250. 9-15. 4. Ananda kumar K., Ramu C., Subhash V., Jayamariappan 8. Chauzit E., Bouchet S., Micheau M., Mahon F.X., Moore M.:Development and validation of newer analytical N., Titier K., Molimard M.: A method to measure deferasirox methods for the estimation of deferasirox in bulk and in in plasma using HPLC coupled with MS/MS detection and tablet dosage form by UV spectroscopy and RP-HPLC, its potential application, Ther Drug Monit. 2010, 32(4), J Pharm Res.2011, 4(9), 2998-3000. 476-481. 5. Sambasivarao V., Ashok kumar G.: Spectrophotometric 9. beckett A.H., Stenlake J.B.:Practical Pharmaceutical determination of deferasirox in formulations using Folin- Chemistry. CBS Publishers and Distributors New Delhi, ciocalteu and ferric chloride reagents, IJRRPAS. 2011, 1986. 1(2), 62-71. 10. ICH Harmonized tripartite guidelines, Text on Validation of 6. chakravarthy V.K., GowriSankar D.: LC Determination Analytical Procedures Q2A, (ICH, Geneva, Switzerland, of Deferasirox in Pharmaceutical Formulation J Global 1994). Trends PharmaSci.2010, 1(1), 42-52. 11. ICH Harmonized tripartite guidelines, Validation of 7. Suman A., Naveen Babu K., RatnaPrasad G., Ramana Analytical Procedures: MethodologyQ2B, (ICH, Geneva, M.V.:Development and validation of LC method for the Switzerland, 1996).

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Preclinical evaluation of nootropic activity of Glabridin Rich Extract of Glycyrrhiza glabra using Passive Avoidance Paradigm in rats Desai S. K.*, Pandey C. H. and Mulgaonkar S.M. (Received 18 September 2012) (Accepted 23 January 2013)

ABSTRACT

The study was aimed at establishing the nootropic potential of Glycyrrhiza glabra using appropriate experimental model. Nootropic activity evaluation of Glabridin Rich Extract (GRE) of roots and stems of Glycyrrhiza glabra was carried out in rats using passive avoidance paradigm. In this model, scopolamine induced imbalance in the level of biochemical parameters like increase of lipid peroxidation and acetylcholinesterase levels and decrease of superoxide dismutase, reduced glutathione and dopamine levels leading to impairment in cognitive functions were normalized following administration of GRE. An enhancement in step down latency was also seen in scopolamine induced memory impairment following GRE treatment. The mechanism of such protection of Glycyrrhiza glabra may be due to reduction in anticholinesterase levels, monoamino oxidase inhibition and augmentation of cellular antioxidants. Our data suggests that Glycyrrhiza glabra is a screened candidate that deserves to be investigated further as an herbal alternative for treatment of Alzheimer’s disease (AD).

Keywords: Glycyrrhiza glabra, Glabridin Rich Extract, inflammatory drugs and facilitation of brain cholinergic Passive avoidance paradigm, Acetylcholinesterase, neurotransmission with anti-cholinesterases are some Dopamine, Antioxidant parameters, Alzheimer's positive approaches for management of AD4. disease. Nootropic agents such as piracetam are used INTRODUCTION primarily for improving memory, mood and behaviour. However, the side-effects like mental confusion, Normal ageing is known to deteriorate memory muscle tremors, shivering, headaches that are in human beings. Oxygen free radicals, the harmful associated with these agents have limited their use5. by-products of oxidative metabolism, are known Since the cholinesterase inhibitors confer only modest to cause organic damage to the living system benefits, additional non-cholinergic AD therapies are that may be responsible for the development of urgently needed. Multipotent agents aiming at diverse Alzheimer’s disease (AD) in the elderly 1, 2. AD targets are expected to act better than the single target is a neurodegenerative disorder characterized aiming counterparts in the fight against AD3. by a progressive loss of memory and cognition. Pathology of AD includes defective beta-amyloid Glycyrrhiza glabra Linn. of the family Leguminosae, β (A ) protein metabolism, abnormalities of adrenergic, is a genus of perennial herbs and shrubs distributed dopaminergic neurotransmission and the potential in the subtropical and warm temperate regions of 3 involvement of inflammatory and oxidative pathways . the world, chiefly in the Mediterranean countries and Reducing oxidative stress by anti-oxidants, China6. In the traditional system of medicine, the protecting brain inflammatory lesions using anti- roots and rhizomes of Glycyrrhiza glabra have been *For correspondence used clinically for centuries. Roots have demulcent, Department of Pharmacology & Toxicology antacid, antiulcer, antiinflammatory, expectorant, Prin. K. M. Kundnani College of Pharmacy, 23-Jote Joy tonic, diuretic, laxative and sedative properties. They Building, Rambhau Salgaonkar Marg, Cuffe Parade, also possess antipyretic, antimicrobial, antiherpes, Colaba, Mumbai- 400 005. anxiolytic and memory enhancing activity7. E-mail: [email protected]

INDIAN DRUGS 50(02) February 2013 33

Liquorice roots contain many bioactive phytochemical screening by the methods previously components, such as glycyrrhizin, liquiritin, liquiritenin described by Kokate 13. and glabridin. Among these functional components, glabridin, a major polyphenolic flavonoid, is found in Column Chromatography and Characterization the cork layer and decayed part of its thickening roots8. The crude extract, dissolved in 15mL of ethyl Glabridin is an isoflavone which has a wide variety of acetate-hexane (1:4 v/v), was subjected to column pharmacological activities, such as cytotoxic activity chromatography using various concentrations of silica and antimicrobial activity against Helicobacter pylori gel of 230-400 mesh size (30g) and ethyl acetate- and has shown beneficial effects in the treatment hexane (1:4, 1:3, 1:2 & 1:1 v/v respectively). Fractions of cardiovascular and central nervous system showing UV spectrum similar to standard Glabridin disorders9, 10. It has shown promising memory were combined to obtain Glabridin Rich Extract (GRE). enhancing activity in various behavioural models of GRE (100 ug/mL) in ethanol was scanned in UV range memory11. The present investigation was undertaken of 200-400nm and compared with standard Glabridin to evaluate the nootropic activity of Glabridin rich (20 ug/mL) by using UV/VIS spectrophotometer extract from Glycyrrhiza glabra. (Jasco - UV-550 spectrophotometer).

MATERIALS AND METHODS Animals Drugs and Chemicals Albino wistar rats of either sex weighing between Scopolamine hydrobromide, dopamine hydro- 120-170g were used. Animals were housed in chloride, serotonin hydrochloride, acetylthiocholine polypropylene cages under standard conditions of iodide were purchased from Sigma Aldrich Chemicals, humidity (50± 5%), temperature (25± 2ºC) and light U.S.A., Glabridin was gifted by Pravinchandra & Co, (12 h light/ 12 h dark cycle) and fed with standard Mumbai and piracetam was gifted by IPCA Labora- pellet diet and water ad libitum. Experimental protocol tory, Mumbai, India. All the other chemicals were was reviewed and approved by the Institutional Animal obtained from local sources and were of analytical Ethics Committee and care of laboratory animals was grade. Volume of oral administration and intraperi- taken as per CPCSEA guidelines (Protocol no: 1011 toneal injection was 1 mL/100g of rat. 02, Reg. No. 25/1999/CPCSEA).

Plant Materials Passive avoidance paradigm 14, 15 Fresh roots and stems of Glycyrrhiza glabra L. Albino wistar rats of either sex were divided into were collected from Sheetal Herbal Products. The 5 groups (n=6), Group I: normal control (negative plant was authenticated at Agharkar Research control), Group II: toxicant control (Scopolamine Institute, Pune, India (Certificate reference no: hydrochloride, 0.4mg/kg, i.p.), Group III: standard 47083). treatment group (piracetam 200mg/kg), Group IV: GRE5 treatment group (5mg/kg) and Group V: GRE10 Preparation of Glabridin Rich Extract (GRE) treatment group (10mg/kg). The standard drug and The method of Jiao et al. 12 was followed with the test drugs were administered to the respective necessary modification. groups (p.o.) once daily for 8 days. Selection of test treatment doses was done based on the data In brief, 30g dried powder of liquorice roots obtained from acute toxicity studies and preliminary and stems were sonicated with 80mL ethyl acetate dose range finding studies. The apparatus consisted for 30min. The extract was dried over water bath of a box (27 cm×27cm×27 cm) having three walls of maintained at 40°C and vacuum dried to obtain a plastic and front wall of plexiglass, featuring a grid crude extract. The extract was subjected to preliminary floor (made up of 3 mm stainless steel rods set 8

34 INDIAN DRUGS 50(02) February 2013

mm apart), with a wooden platform (10 cm×7 cm×1.7 Inc., San Diego, USA was the software used for cm) in the centre of the grid floor. Electric shock statistical analysis. (6 mA) was delivered to the grid floor. Training (on 08th day of drug treatment) was carried out in two similar RESULTS sessions. Each rat was gently placed on the wooden GRE Characterization platform set in the centre of the grid floor. When the rat stepped-down placing all its paws on the grid floor, UV scanning in the range of 200-400nm showed shocks were delivered for 15sec and the step-down an absorbance peak at 228nm that coincided with the 22 latency (SDL) was recorded. SDL was defined as peak record for standard Glabridin , confirming the the time (in seconds) taken by the mouse to step presence of glabridin in GRE. The yield of GRE was down from the wooden platform to grid floor with found to be 0.25%w/w. Preliminary phytochemical all its paws on the grid floor. Animals showing SDL screening of GRE has shown presence of saponin in the range of 2– 15 seconds during the first test glycosides, flavonoids and phenolic compounds. were used for the second session and the retention Passive avoidance paradigm test. The second session was carried out 90min after the first test. During second session, if the animals Scopolamine significantly decreased SDL as stepped down before 60sec, electric shocks were compared to control group indicating impairment delivered once again for 15sec. During the second of memory (amnesia). GRE administration at both test, animals were removed from shock free zone, doses reversed the amnesia induced by scopolamine if they did not step down for a period of 60sec and (Table I). were subjected to retention test. Retention memory was tested after 24 hr (i.e., 09th day, 24 hr after MDA, the lipid peroxidation marker was last dose) in similar manner, except that the electric significantly elevated in toxicant group when compared shocks were not applied to the grid floor observing with the normal group. Treatment with GRE5 and an upper cut-off time of 300sec and immediately after GRE10 to scopolamine intoxicated rats depleted rats were humanely sacrificed using ether. significantly (P<0.01 and P<0.001 respectively) the increased levels of MDA. Significant decline in brain The whole brain was removed aseptically and GSH and SOD levels was observed in the toxicant divided in two equal parts. 5% (w/v) homogenate scopolamine treated group when compared with was prepared in HCl-butanol solution from the first normal control group of rats. Treatments with GRE5 half of the brain which was utilized for estimation of and GRE10 significantly restored the GSH and SOD 16 dopamine . A 10% (w/v) homogenate was prepared levels depleted by scopolamine (Table I). in phosphate buffer solution (pH 7.4) from the second half of the brain which was further utilized The AChE activity in brains of toxicant scopolamine for estimation of various biochemical parameters group was found to be significantly higher as compared 17 like acetylcholinesterase (AChE), superoxide to the control group (P<0.001). GRE10 treatment 18 19 dismutase (SOD), reduced glutathione (GSH), decreased the AChE activity that was comparable 20 21 lipid peroxidation (LPO) and total protein . to the standard treatment (Fig. 1).

STATISTICAL ANALYSIS A significant decrease in dopamine level was The results are expressed as mean ± SEM from observed in the toxicant scopolamine group when 6 animals in each group. Results were statistically compared with the normal control group of rats analyzed using one-way ANOVA followed by Tukey (Fig. 2). Treatments with GRE5 and GRE10 restored Kramer test; P < 0.05 was considered significant. significantly the dopamine levels depleted by GraphPad InStat version 4.00 of Graph Pad Software scopolamine (P<0.05 and P<0.001).

INDIAN DRUGS 50(02) February 2013 35

Fig. 1: Effect of GRE on brain AChE level in passive Fig. 2: Effect of GRE on brain dopamine level in avoidance paradigm passive avoidance paradigm

All values expressed as mean ± SEM, n = 6. One-way All values expressed as mean ± SEM, n = 6. One-way ANOVA followed by Tukey-Kramer test is applied for statistical ANOVA followed by Tukey-Kramer test is applied for statistical x z analysis, P values: < 0.001, < 0.05 where treatment groups analysis, P values: x < 0.001, z < 0.05 where treatment groups a compared with toxicant control and < 0.001 where toxicant compared with toxicant control and a < 0.001 where toxicant control compared with normal control control compared with normal control.

DISCUSSION AND CONCLUSIONS disease are demonstrated. Moreover, strong evidence supporting the involvement of oxidative damage in In the present study, passive avoidance behaviour neurodegenerative disease has been suggested by based on negative reinforcement was used to examine various clinical studies24. The drugs with antioxidant the long-term memory using piracetam as a standard effects might be beneficial for preserving brain for comparison. Increase in SDL following treatment function. Antioxidant enzymes such as superoxide with GRE5 and GRE10 is suggestive of animal dismutase (SOD), glutathione peroxidase (GPx) retaining the memory of the shock once it entered and catalase as well as glutathione reductase and the shock free zone. So, long term treatment with ascorbate are involved in the reduction of oxidative GRE exhibited pronounced effect in reversal of the stress. Antioxidant enzymes display reduced activities scopolamine induced amnesia. in the affected brain region of patients of Alzheimer’s disease. Augmentation of endogenous antioxidants Many clinical studies have reported strong by therapeutic substances has recently evoked evidence that oxidative stress is involved in the scientific interest because any such property ofa pathogenesis of Alzheimer’s disease. Oxygen-free therapeutic agent can be expected to cause significant radicals, implicated in the process of age related decline improvement in the endogenous defense against in the cognitive performance may be responsible for oxidative stress25. These agents also reduce the the development of Alzheimer’s disease in elderly oxidative damage and promote a functional recovery 23 persons . Brain tissue homogenates of toxicant in degenerative disorders. control group showed a depletion in the level of SOD, GSH, dopamine and serotonin and a marked rise Our results are in accordance with El-Sherbiny in LPO and AChE levels. An increased oxidation of who has reported increased oxidative stress within lipids, proteins and deoxyribonucleic acid, alterations rat brain with altered brain levels of SOD and in mitochondrial function and a possible role of GSH26. Administration of scopolamine significantly amyloid beta and its precursor protein in oxidative increased the MDA level, an important marker for reaction in experimental models of Alzheimer’s LPO and reduced both GSH and SOD activities in rat

36 INDIAN DRUGS 50(02) February 2013

Table I: Effect of GRE on various biochemical parameters in passive avoidance paradigm

Parameters / Groups SDL (sec) LPO GSH SOD (nmol MDA/g tissue) (nmol/mg protein) (U/mg protein) Normal Control (Group I) 178.72 ± 3.04 348.10 ± 14.93 3.64 ± 0.04 12.91 ± 0.34 Scopolamine (Group II) 143.72 ± 4.94a 443.72 ± 3.99 a 1.75 ± 0.05 a 6.45 ± 0.45 a Piracetam (Group III) 210.35 ± 6.17 x 333.03 ± 10.05 x 4.66 ± 0.26 x 16.24 ± 0.60 x GRE5 (Group IV) 161.20 ± 1.62 z 401.12 ± 4.80 y 2.32±0.1269 z 9.35 ± 0.52 y GRE10 (Group V) 210.43 ± 4.59 x 338.87 ± 1.42 x 4.33± 0.07 x 14.06 ± 0.49 x

All values expressed as mean ± SEM, n=6. One-way ANOVA followed by Tukey-Kramer test is applied for statistical analysis. P values : x<0.001, y<0.01, Z<0.05 where treatment groups compared with toxicant control and a<0.001 where toxicant control compared with normal control. brain. Treatment with GRE10 for 8 days produced a 2. Nagy I.Z.: On the True Role of Oxygen Free Radicals in significant fall in MDA and restored the GSH and SOD the Living State, Ageing and Degenerative Disorders, Ann N Y Acad Sci. 2001, 928, 187-199. activities in rat brain. GRE may exert a protective effect 3. Kang S.Y., Lee K.Y., Koo K.A., Yoon J.S., Lima S.W., against oxidative damage induced by scopolamine Kima Y.C., Sung S.H.: ESP-102, a standardized by augmenting the activities of GSH and SOD in rat combined extract of Angelica gigas, Saururus chinensis brain. GRE treatment groups have shown a reduction and Schizandra chinensis, significantly improved scopolamine-induced memory impairment in mice, Life in AChE and increase in dopamine levels in brain. Sci. 2005, 76 (15), 1691–1705. 4. Adnaik R.S., Sapakal V.D., Patil V.M., Naikwade N.S., The presence of flavonoids and phenolic Magdum C.S.: Prasham: A promising memory enhancer compounds in GRE as seen in preliminary in mice, J Pharmacol Sci. 2008, 2, 55-60. phytochemical analysis might have been responsible 5. blazer D.G., Federspeil C.F., Ray W.A., Schaffner W.: for the antioxidant effects. The present study The risk of anticholinergic toxicity in the elderly: a study of prescribing practices in two populations, J Gerontol. demonstrates that GRE has therapeutic potential 1983, 38(1), 31-35. in improving memory as seen in passive avoidance 6. Muralidharan P., Balamurugan G., Babu V.: paradigm model that may be attributed to reduced Cerebroprotective effect of Glycyrrhiza glabra Linn. levels of brain AChE, increased levels of dopamine root extract on hypoxic rats, Bangladesh J Pharmacol. 2009, 4, 60-64. and supplemented by its antioxidant properties. As 7. Ambawade, S.D., Kasture, V.S., Kasture, S.B.: a result, the susceptible brain cells might have been Anticonvulsant activity of roots and rhizomes of exposed to less oxidative stress resulting in reduced Glycyrrhiza glabra, Indian J Pharmacol. 2002, 34, 251- brain damage and improved neuronal function 255. probably due to acetylcholinesterase and monoamine 8. Hayashi H., Hiraoka N., Ikeshiro Y., Yamamoto H.: Organ specific localization of flavonoids in Glycyrrhiza glabra oxidase inhibitory activity translating into improved L., Plant Sci. 1996, 116 (2), 233–238. memory function 25. 9. Fukai T., Sakagami H., Toguchi M., Takayama F., Iwakura I., Atsumi T.: Cytotoxic activity of low molecular REFERENCES weight polyphenols against human oral tumor cell lines, Anticancer Res. 2000, 20(4), 2525-2536. 1. Smith J.V., Luo Y.: Elevation of oxidative free radicals in Alzheimer’s disease models can be attenuated by Ginkgo 10. Fukai T., Marumo A., Kaitou K., Kanda T., Terada S., biloba extract EGb 761, J. Alzheimers Dis. 2003, 5 (4), Nomura T.: Anti-Helicobacter pylori flavonoids from 287-300. licorice extract, Life Sci. 2002, 71(12), 1449-1463.

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11. Dayananda B., Raghavan A.K., Khanum F., Singh B.A.: 19. ellman G.L.: Tissue sulphydryl groups, Arch Biochem In vitro antioxidant and free radical scavenging activity Biophys. 1959, 82(1), 70-77. of Glycyrrhiza glabra root extracts, Journal of Herbal 20. Ohkawa H., Ohishi N., Yagi K.: Assay of lipid peroxides Medicine and Toxicology. 2010, 4, 97-102. in animal tissues by thiobarbituric acid reaction, Anal 12. Jiao L.V., Hao L., Qipeng Y., Yan X., Tianxin W.: Preparative Biochem. 1979, 95(2), 351-358. Purification of the Major Flavonoid Glabridin from Licorice 21. lowry O. H., Rosebrough N. J., Farr A. L., Randall R. Roots by Solid Phase Extraction and Preparative High J.: Protein measurement with the folin phenol reagent, Performance Liquid Chromatography, Separation J Biol Chem. 1951, 193(1), 265-275. . 2010, 45, 1104-1111. Science and Technology 22. Vaya J., Belinky P.A., Aviram M.: Antioxidant Constituents 13. Kokate C.K., Practical Pharmacognosy, Delhi.India, From Licorice Roots: Isolation, Structure Elucidation And Vallabh Prakashan, 1989, 111. Antioxidative Capacity Toward LDL Oxidation. Free Radic 14. Joshi H., Parle M.: Zingibar officinale: Evaluation of its Biol Med. 1997, 23(2), 302-313. nootropic effect in mice, Afr. J.Trad.CAM. 2006, 3(1), 23. Joshi H., Parle M.: Pharmacological evidences for 64-74. antiamnesic potentials of Phyllanthus amarus in mice, 15. Parle M., Dhingra D.: Ascorbic Acid: A Promising Memory- Afr. J. Biomed. Res. 2007, 10, 165 – 173. Enhancer in Mice, J Pharmacol Sci. 2003, 93(2), 129- 24. Jeong E.J., Lee K.Y., Kim S.H., Sung S.H., Kim Y.C.: 135. Cognitive-enhancing and antioxidant activities of 16. Schlumpf M., Lichtensteiger W., Langemann H., Waster iridoid glycoside from Scrophularia buergeriana in P.G., Hefti F.: A fluorometric micromethod for the scopolamine treated mice, Eur J Pharmacol. 2008, simultaneous determination of serotonin, noradrenaline 588(1), 78-84. and dopamine in milligram amounts of brain tissue, 25. Dhingra D., Parle M., Kulkarni S.K.: Memory enhancing Biochem Pharmacol. 1974, 23(17), 2437-2446. activity of Glycyrrhiza glabra in mice, J Ethnopharmacol. 17. ellman G.L., Courtney K.D., Andres V.Jr., Featherstone 2004, 91(2-3), 361-365. R.M.: A new and rapid colorimetric determination of 26. el-Sherbiny D.A., Khalifa A.E., Attia A.S., Eldenshary acetylcholinesterase activity, Biochem Pharmacol. Eel-D.: Hypericum perforatum extract demonstrates 1961, 7, 88-95. antioxidant properties against elevated rat brain oxidative 18. Sun M., Zigman S.: An improved spectrophotometric status induced by amnestic dose of scopolamine, assay for superoxide dismutase based on epinephrine Pharmacol Biochem Behav. 2003, 76(3-4), 525- auto-oxidation, J Indian Med Assoc. 2006, 104(7), 396- 533. 397.

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ANTIHYPERGLYCEMIC AND IN VITRO ANTIOXIDANT ACTIVITIES OF PUNICA GRANATUM LINN. IN ALLOXAN INDUCED DIABETIC RATS Patil U. S., Bandawane D. D.*, Bibave K. H. and Chaudhari P. D.

(Received 10 September 2012) (Accepted 23 January 2013)

ABSTRACT

The present study was undertaken to evaluate antihyperglycemic and antioxidant activities of aqueous and methanolic extracts of leaves of Punica granatum. The aqueous and methanolic extracts (200 and 400 mg/kg) of the leaves were tested for their efficacy in alloxan induced diabetic rats. Glibenclamide (4 mg/kg), p.o. and insulin (5 unit/kg s.c.) were used as standard drugs. The maximum reduction in fasting blood glucose in diabetic rats was observed with aqueous extract as compared to methanolic extract. Oral glucose tolerance test in normal rats showed reduction in fasting blood glucose level at 60 min of extract administration. Aqueous and methanolic extracts of leaves of Punica granatum exhibited scavenging effect in concentration dependent manner on 2, 2- diphenyl-1-picrylhydrazyl (DPPH), nitric oxide and reducing power assay. Ascorbic acid was used as a standard. The findings of the present study suggested that Punica granatum possess significant antihyperglycemic and antioxidant activity.

Keywords: Punica granatum, Alloxan monohydrate, million people by the year 20252. The prevalence Antihyperglycemic, Antioxidant. of diabetes mellitus is increasing with ageing of the population and lifestyle changes associated with rapid INTRODUCTION urbanization and westernization3,4. Currently available Diabetes mellitus is a common endocrine pharmacotherapy for the treatment of diabetes disorder characterized by chronic hyperglycemia mellitus includes oral hypoglycemic agents and due to either insufficient insulin production by insulin. However these current drugs do not restore pancreatic beta cells or by cellular resistance to normal glucose homeostasis and they are not free insulin. It leads to disturbances of carbohydrate, from side effects. It is therefore become necessary to fat and protein metabolism1. Diabetes is now one make use of vast reserves of plant origins for medical of the most common non-communicable diseases purposes which will help to search effective as well 5 globally and is the fifth leading cause of death in as safer drug remedy for diabetes mellitus . most of the developed countries. Complications Oxidative stress and oxidative damage to from diabetes such as coronary artery, peripheral the tissue are common end points of chronic vascular disease, diabetic neuropathy, nephropathy diseases, such as atherosclerosis, diabetes and and retinopathy result in increasing disability, reduced rheumatoid arthritis6. Oxidative stress is currently life expectancy and enormous health costs virtually suggested as mechanism underlying diabetes and in every strata of society. Currently over 150 million diabetic complications7. During diabetes, persistent people are diabetic worldwide and it is estimated hyperglycemia causes increased production of that diabetes mellitus will affect more than 300 free radicals, especially reactive oxygen species (ROS), for all tissues from glucose auto-oxidation *For correspondence and protein glycosylation. Oxidants are generated Department of Pharmacology, as a result of normal intracellular metabolism in Progressive Education Society’s Modern College of Pharmacy,Sector No. 21, Yamunanagar mitochondria and peroxisomes, as well as from a 8 Nigdi, Pune- 411044, Maharashtra variety of cytosolic enzyme systems . It has been E-mail : [email protected] reported that the natural antioxidants present in

INDIAN DRUGS 50(02) February 2013 39

many plants reduce such damage and help to were identified by the botanist of Botanical Survey of prevent mutagenesis, carcinogenesis and aging due India, Pune and a voucher specimen (V.No. USP-1) to their radical scavenging activities9. These natural was deposited for future reference. antioxidants have been isolated from various fruits10, vegetables and medicinal plants11,12, and the identified Preparation of plant extracts compounds cover a wide range of phytochemicals Freshly collected Punica granatum leaves were including phenolics (e.g., cinnamic acid derivatives dried in shade and coarse powder was extracted with and flavonoids) and carotenoids (e.g., lycopene). water and methanol by maceration17. The extracted mixture was filtered through muslin cloth and Punica granatum Linn. is commonly known as evaporated at 60ºC up to one third of initial volume. pomegranate in English and Anar in Hindi belonging Remaining solvent was completely evaporated at 40ºC to family Punicaceae. It is a fruit-bearing deciduous and concentrated to dryness. The yield (13.6% w/w) shrub or small tree, native to Asia13. Pomegranates are drought-tolerant, and can be grown in dry areas of the powdered plant material was collected dried º with either a mediterranean winter rainfall climate and stored at 5 C in air tight container. or in summer rainfall climates. Today, it is widely Animals distributed throughout the world. P. granatum has been extensively used as a traditional medicine in India Male albino Wistar rats weighing 150-200 g for the treatment of dysentery, diabetes, diarrhea, were used for present study. The animals were helminthiasis, acidosis, hemorrhage and respiratory maintained under standard environmental conditions diseases. The leaves are used in hepatoprotection, and were fed with standard pellet diet and water microbial infection, inflammation, lipid regulation, ad libitum. The study was approved by Institutional gastroprotection, helminthes14 and diarrhea15. The Animal Ethics Committee (Reg. no. 884/OP/05/ac/ leaves are reported to contain apigenin, flavones, CPCSEA). CPCSEA guidelines were adhered during apigenin 4’-O-β glucopyranoside, luteolin 4’-O-β the maintenance and experiment. glucopyranoside, luteolin 3’-O-β glucopyranoside, Preliminary phytochemical screening luteolin 3’-O-β xylopyranoside flavones glycoside, and tannins like punicalin and punicafolin13. In The aqueous and methanolic extract of Punica Unani system of medicine flower extract of P. granatum were screened for the presence of granatum is used for the treatment of diabetes16. To various phytoconstituents like alkaloids, glycosides, our knowledge, there are no available reports on flavonoids, tannins, carbohydrates, amino acids and antihyperglycemic effect of the leaves of this plant. proteins18. Hence, the present study was carried out to evaluate Thin layer chromatomatography acute antihyperglycemic activity of aqueous and methanolic extract of P. granatum leaves in alloxan The aqueous and methanolic extracts of Punica induced diabetes in Wistar albino rats and its in granatum were subjected to thin layer chromatography vitro antioxidant activity through various analytical using plates of 250 µm thickness (TLC Silica gel 60 methods. F254 Merck, Germany).The spots were developed in two different solvent systems as follows: MATERIALS AND METHODS Solvent system A- Ethyl acetate: glacial acetic Collection of plant material acid: formic acid: water (100:11:11:27) (V/V). Fresh leaves of Punica granatum were collected from regions of Nasik district in the months of Solvent system B- Ethyl acetate: methanolic: September and October. The leaves of the plant water (200:33:7) (V/V)19.

40 INDIAN DRUGS 50(02) February 2013

Acute toxicity study glucose level of above 200 mg/dl were considered to be diabetic and used for the studies. Control rats Healthy adult Wistar albino rats of either sex, received similar volume of vehicle, normal saline starved overnight were divided into two groups (2ml/kg body weight) alone. consisting of five rats and were orally fed with the extract in increasing dose levels of 300, 500, 2000 Acute antihyperglycemic activity and 5000 mg/kg body weight. The acute toxicity study was carried out according to OECD guidelines no. Animals were divided into seven groups, each 423. The animals were observed continuously for consisting of six rats. The extracts were administered 2 h under the following profiles. orally in single dose. Group I: Normal control rats, administered saline Behavioural profile: Alertness, restlessness, (0.9% w/v), fearfulness and irritability. Group II: Diabetic control rats administered saline Neurological profile: Spontaneous activities, (0.9% w/v), reactivity, touch response, pain and gait. Group III: Diabetic rats administered glibenclamide (4 mg/kg), Autonomic profile: Defecation and urination. Group IV: Diabetic rats administered AEPG After period of 24 h, 72 h and 14 days they were (200 mg/kg), 20 observed for any lethality or death . Group V: Diabetic rats administered AEPG (400 mg/kg), Oral glucose tolerance test (OGTT) Group VI: Diabetic rats administered MEPG The oral glucose tolerance test was performed in (200 mg/kg), overnight fasted (18 h) normal rats. Rats divided into six groups, each consisting of six rats were administered Group VII: Diabetic rats administered MEPG 0.9% (w/v) saline, glibenclamide 4 mg/kg, Punica (400 mg/kg). granatum aqueous extracts (AEPG) 200 and 400 The effects of administration of AEPG and MEPG mg/kg and Punica granatum methanolic extracts in diabetic rats were observed by measuring fasting (MEPG) 200 and 400 mg/kg, respectively. Glucose blood glucose level. Fasting blood glucose levels (3 g/kg) was fed 30 min after the administration of were obtained from the tail tip of rats estimated by extracts. Blood was withdrawn from the tip of the tail using glucometer (Accu-check sensor-comfort). vein under ether inhalation at 0, 30, 60, 90 and 120 Blood glucose levels were determined before (0 hr) min of glucose administration and glucose levels and then at 2, 4, 6 and 8 hrs. after administration of were estimated using glucose oxidase–peroxidase plant extract. reactive strips and a glucometer21. In vitro antioxidant activity Induction of diabetes Determination of reducing power The animals were fasted for 12 h prior to the induction of diabetes as described by Prince and The reducing power was determined according Kamalakkannan (2004) with slight modification. to the method of Oyaizu. Various concentrations of Alloxan freshly prepared in saline was administered AEPG and MEPG (100- 1000 µg/ml) were mixed with intraperitoneally (i.p.) at single dose of 150 mg/ 1ml of 200 mmol/l sodium phosphate buffer (pH 6.6) kg. Development of diabetes was confirmed by and 1ml of 1% potassium ferricyanide. The mixture measuring blood glucose concentration at 3 days was incubated at 50ºC for 20 min. After incubation after the administration of alloxan. Rats with blood 1ml of 10% trichloroacetic acid (w/v) was added and

INDIAN DRUGS 50(02) February 2013 41

Fig.1: Hypoglycemic effect of AEPG and MEPG on fasting Fig.2 : Reducing power of aqueous and methanolic blood glucose level (FBG) of normal rats during OGTT. extract of Punica granatum leaves. Each value shown in mean±S.D. n (number of animals in each AEPG: Aqueous extract of Punica granatum leaves, MEPG: group) = 6. AEPG: Aqueous extract of Punica granatum leaves, Methanolic extract of Punica granatum leaves, EAECA: Ethyl MEPG: Methanolic extract of Punica granatum leaves, *p < acetate extract of Punica granatum leaves. 0.05 vs. glucose control,**p < 0.01 vs. glucose control.

Fig. 3 : DPPH radical scavenging activity of AEPG Fig. 4 : Nitric oxide scavenging activity of AEPG and MEPG. and MEPG. AEPG: Aqueous extract of Punica granatum leaves, MEPG: AEPG: Aqueous extract of Punica granatum leaves, MEPG: Methanolic extract of Punica granatum leaves Methanolic extract of Punica granatum leaves. the mixture was centrifuged at 2000 rpm for 10min. radical DPPH. AEPG and MEPG of concentrations The upper layer solution (2.5 ml) was mixed with 2.5 200-1000 µg/ml (0.1 ml) were added to 3 ml of a ml of deionised water and 0.5 ml of ferric chloride 0.004% methanolic solution of DPPH. Absorbance at (0.1%) solution. The absorbance was measured at 517 nm was determined after 30 minutes. Ascorbic 700 nm. A higher absorbance indicates a higher reducing acid was used as the reference compound. Lower power. Ascorbic acid used as a standard22. absorbance of the reaction mixture indicated higher free radical scavenging activity. Radical scavenging Determination of DPPH (1-1-diphenyl 2-picryl activity was expressed as the inhibition percentage of hydrazyl) radical scavenging activity free radical by the sample and was calculated using 22 The free radical scavenging activity of Punica the following formula granatum leaves were measured in terms of hydrogen % inhibition = (A - A ) /A X 100) donating or radical scavenging ability using the stable 0 t 0

42 INDIAN DRUGS 50(02) February 2013

Table 1. Effect of Aqueous (AEPG) and methanolic (MEPG) extract of leaves of Punica granatum on fasting blood glucose level in alloxan induced diabetic rats.

Experimental Fasting blood glucose level (mg/dl) groups (n=6) 0 h 2 h 4 h 6 h 8 h Normal Control 77.33 ± 2.29 75.66 ± 2.98 77.16 ± 2.54 77.66 ± 2.15 75.5 ± 2.487 (NC) Diabetic Control 490.83 ± 44.49## 488 ± 43.25## 493 ± 27.06## 506.84 ± 20.96## 504.84 ± 18.30## (DC) DC+AEPG 376.00 ± 32.27 349.16 ± 65.04 281 ± 60.78** 248.5 ± 77.83* 187 ± 65.20** (200 mg/kg) DC+AEPG 436.00 ± 16.15 311.5 ± 44.26* 251.34 ± 43.37** 275.67 ± 49.65* 159.34 ± 55.21** (400 mg/kg) DC+MEPG 476.16 ± 67.83 405.17 ± 60.48 380 ± 70.12* 340.83 ± 50.50* 318.34 ± 41.25* (200 mg/kg) DC+MEPG 438.00 ± 41.46 456.5 ± 38.50 398.17 ± 54.42* 326.84 ± 67.98* 302.67 ± 76.32* (400 mg/kg) DC+GL (4 mg/kg) 481.16 ± 41.23 470.33 ± 25.37 265 ± 29.15** 283.16 ± 31.96* 313.34 ± 11.66* DC+Insulin 499.29 ± 45.32 139.66 ± 41.24** 90.34 ± 6.3** 161.5 ± 8.9** 243 ± 23.18** (5 units/kg) n=6, Values are mean ± S.E.M., #p<0.05, ##p<0.01 as compared to normal control group; *p<0.05, **p<0.01 as compared to diabetic control group. AEPG: Aqueous extract of Punica granatum leaves; MEPG: Methanolic extract of Punica granatum leaves; GL: Glibenclamide, Data analysed by one way Analysis of Variance (ANOVA) followed by Dunnet’s multiple test for comparison.

where A0 was the absorbance of the control was mixed with 3.0 ml of different concentrations (10–100 µg/ml) of AEPG and MEPG and incubated (blank, without extract) and At was the absorbance in the presence of the extract. All the tests were at 250C for 150 min. The samples were added to performed in triplicate and the graph was plotted with Greiss reagent (1% sulphanilamide, 2% H3PO4 and the mean values. 0.1% napthylethylenediamine dihydrochloride). The absorbance of the chromaphore formed during Determination of nitric oxide radical scavenging the diazotization of nitrite with sulphanilamide and activity subsequent coupling with napthylethylenediamine was measured at 546 nm and referred to the Nitric oxide was generated from sodium absorbance of standard solutions of ascorbic acid nitroprusside and measured by the Greiss reaction. treated in the same way with Greiss reagent as a Sodium nitroprusside in aqueous solution at positive control. The percentage of inhibition was physiological pH spontaneously generates nitric measured by the following formula23. oxide, which interacts with oxygen to produce nitric ions that can be estimated by using Greiss reagent. % inhibition = (A - A ) /A X 100) Scavengers of nitric oxide compete with oxygen 0 t 0 leading to reduce production of nitric oxide. Sodium where A0 was the absorbance of the control nitroprusside (5 mM) in phosphate buffer saline (PBS) (blank, without extract) and At was the absorbance

INDIAN DRUGS 50(02) February 2013 43

in the presence of the extract. All the tests were compared to normal group. The blood glucose levels performed in triplicate and the graph was plotted with of diabetic untreated rats were significantly higher the mean values. than those of normal rats. Alloxan induced diabetic rats treated with AEPG (200 and 400 mg/kg) showed Statistical analysis reduction in blood glucose level by 50.26% and Values were represented as mean ± S.D. for six 63.45% at 8 h respectively. Diabetic rats treated with animals in each group. Data were analyzed using MEPG (200 and 400 mg/kg) showed reduction in blood one-way analysis of variance (ANOVA) followed by glucose level by 33.28% and 28.42% at 8 h. Alloxan Dunnet’s multiple test for comparison.The values induced diabetic rats treated with glibenclamide (4 mg/kg) showed reduction in blood glucose level were considered significant when p < 0.05. by 44.90 % at 4 h respectively. RESULTS Reducing power Acute toxicity study Fig. 2 depicts total reducing power of AEPG Acute toxicity study revealed the non-toxic nature and MEPG. Reducing power increased with an of the extracts. There was no lethality or any toxic increase in extracts concentration as evident by the reactions found among the tested animals when concentration dependant increase in optical density administered as a single dose. at 700 nm. The highest reducing power of AEPG, MEPG was shown at a concentration of 1000 μg/ Thin layer chromatography ml in the following order: ascorbic acid (2.1097) The results of thin layer chromatography showed > AEPG (1.8689) > MEPG (1.4561). The results the presence of three spot in the solvent system A obtained were comparable with standard antioxidant which is for flavonoid glycosides. Brownish-yellow ascorbic acid. coloured single spot in solvent system B indicated the presence of flavone and/or flavonol in AEPG DPPH (1-1-diphenyl 2-picryl hydrazyl) radical and MEPG. scavenging activity Fig. 3 depicts DPPH radical scavenging activity Oral glucose tolerance test (OGTT) of AEPG and MEPG. AEPG, MEPG and EAEPG Fig. 1 depicts the hypoglycemic effect of single showed a concentration dependent increase oral administration of AEPG (200 and 400 mg/kg) in scavenging of DPPH. The highest DPPH and MEPG (200 and 400 mg/kg) on OGTT of normal scavenging activity of AEPG, MEPG was shown rats. The aqueous extract at the dose of 200 and at a concentration of 1000 μg/ml in the following 400 mg/kg produced maximum fall in blood glucose order: ascorbic acid (94.2± 0.02) > AEPG (90.02± level at 30 min while methanolic extract at the dose 0.02) > MEPG (66.75± 0.005). The results obtained of 200 and 400 mg/kg produced maximum fall at 90 were comparable with standard antioxidant ascorbic min after glucose administration. acid.

Acute antihyperglycemic activity of AEPG and Nitric oxide radical scavenging activity MEPG in alloxan induced diabetic rats Fig. 4 depicts nitric radical scavenging activity of The effect of AEPG and MEPG on the blood AEPG and MEPG. The highest nitric oxide radical glucose levels of normal and diabetic rats is given in scavenging of AEPG and MEPG was found to be Table I. After oral administration of alloxan, there is 56.24 ± 0.02 and 51.02± 0.02 percent respectively a significant elevation of the plasma glucose levels at concentration of 100 μg/ml. Ascorbic acid was by 2-3 folds during experimental time periods when used as standard compound.

44 INDIAN DRUGS 50(02) February 2013

DISCUSSION extract such as phenolic groups such as flavonoids and tannins. Excess levels of nitric oxide are found The present study is the preliminary assessment in several diseases including diabetes. AEPG of the antihyperglycemic activity of the aqueous and showed maximum nitric oxide scavenging activity methanolic extracts of Punica granatum leaves. The extract shows dose dependant fall in fasting blood than MEPG which may be the greater presence of glucose level in alloxan induced diabetic rats. The flavonoids in the particular extract. AEPG showed diabetogenic agent alloxan is a hydrophilic and more potent antioxidant activity compared to MEPG chemically unstable pyrimidine derivative which is extracts. Ascorbic acid was used for comparing the toxic to pancreatic β - cells because it can generate antioxidant activity. toxic free oxygen radicals during redox cycling in the presence of reducing agents such as glutathione The present study showed that aqueous and and cysteine24. methanolic extract of Punica granatum leaves showed significant antihyperglycemic activity When AEPG and MEPG were administered to in alloxan induced diabetic rats. The possible glucose loaded normal rats (OGTT) fasted for 18 h, mechanisms by which Punica granatum brings about reduction in blood glucose levels was observed after its antihyperglycemic action may be through the 60 and 90 min respectively. The decline reached its insulinomimetic action of phytoconstituents or may maximum at 2 h. In the present study, the difference be through potentiation of pancreatic secretion of observed between the initial and final fasting blood insulin from the intact β-cells of islets28,29. It could be glucose levels of different groups under investigation due to free radical scavenging activity of plant extract revealed a significant elevation in blood glucose level as evident from in vitro antioxidant study30. in diabetic control group. Administration of AEPG and MEPG to diabetic rats showed a significant CONCLUSION decrease in fasting blood glucose level in alloxan It is concluded from the data that Punica granatum induced diabetic rats. leaves possesses significant antihyperglycemic and in vitro antioxidant activity than and it may prove to In our present experiment, we studied the in vitro be effective for the treatment of diabetes mellitus. scavenging activity of different concentrations of However, longer duration studies on chronic models AEPG and MEPG. Reactive oxygen species (ROS) are capable of damaging biological macromolecules are necessary to elucidate the exact mechanism of such as DNA, carbohydrates or proteins25. The action so as to develop it as a potent antihyperglycemic reducing power assay measures the electron-donating and antioxidant drug. ability of antioxidants using potassium ferricyanide reduction method. The reducing properties are ACKNOWLEDGEMENT generally associated with the presence of reductones, The author would like to thank Dr. Diwakar, which have been shown to exert antioxidant action by Director, Botanical Survey of India, Pune for breaking the free radical chain by donating a hydrogen authentication of plant material and Mr. Bhushan atom26. 2, 2-dipheny-l-picrylhydrazyl (DPPH) is Pimple for his help during the project work. a stable free radical at room temperature, which produces a violet solution in ethanol. The decrease REFERENCES of DPPH molecules by the action of test extracts can 1. Juvekar A. R. and Bandawane D. D.: Antihyperglycaemic be correlated with the number of available hydroxyl and antihyperlipidemic activity of Aegle marmelos Linn. leaf extract in streptozotocin-induced diabetes rats, Indian 27 groups . DPPH scavenging activity of AEPG and Drugs. 2009, 46 (7), 43-49. MEPG may be probably due to the presence of 2. King H, Aubert R. E. and Herman W. H.: Global substance with an available hydroxyl group in the burden of diabetes 1995-2025 prevalence, numerical

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estimates and projections, Diab. Care. 1998, 21, normal and streptozotocin induced diabetic rats, Adv. 1414-31. Pharmacol.Toxicol. 2009, 10 (3), 89-92. 3. Kamalakkanan N and Prince P. S. M.: Hypoglycaemic 18. Khandelwal K. R.: Practical Pharmacognosy: Techniques effect of water extracts of Aegle marmelos fruits in and Experiments, Nirali Prakashan, Pune 2006, 10th ed., streptozotocin-diabetic rats, J of Ethnopharmacol. 2003, 149-150. 87, 207-210. 19. Koua F. H. M., Babiker H. A., Halfawi A., Ibrahim R. 4. Sobngwi E, Mauvais-jarvis F, Vexiau P, Mbanya J. C. and O., Abbas F. M., Elgaali E. I. and Khlafallah M. M.: Gautier J. F.: Diabetes in Africans Part 1: epidemiology Phytochemical and biological study of Striga hermonthica and clinical specificities, Diabetes Metabolism. 2001, (Del.) Benth callus and intact plant, Res. Pharm. Biotech. 27, 628-634. 2011, 3, 85-92. 5. bandawane D, Juvekar A and Juvekar M.: Antidiabetic 20. ecobichon D. J.: The basis of toxicology testing, Newyork and Antihyperlipidemic Effect of Alstonia scholaris Linn CRC press. 1997, 43-86. Bark in Streptozotocin Induced Diabetic Rats, Ind J 21. Maithili V., Dhanabal S. P., Mahendran S. and Vadivelan Pharm Edu Res. 2011; 45 (2): 114-120. R.: Antidiabetic activity of ethanolic extract of tubers 6. baynes J. W. and Thorpe S. R.: Role of Oxidative Stress of Dioscorea alata in alloxan induced diabetic rats, Indian in Diabetic Complications. Diabetes. 1999, 48, 1-9. J Pharmacol. 2011, 43 (4), 455-459. 7. Moussa S. A.: Oxidative stress in Diabetes Mellitus, 22. Kumaran A. and Karunakaran R. J.: In vitro antioxidant Romanian J Biophys. 2008, 18 (3), 225-236. activities of methanol extracts of five phyllanthus species 8. Kangralkar V. A., Patil D. S. and Bandivadekar R. M.: from India, Elsevier J. LWT. 200, 40, 344–352. Oxidative stress and diabetes: a review, Int J Pharm 23. Saha M. R., Jahangir R. and Biva I. J.: In vitro nitric Appl. 2010, 1(1), 38-45. oxide scavenging activity of ethanol leaf extracts of four 9. cook N. C. and Saman S.: Flavonoids-chemistry, Bangladeshi medicinal plants, S. J. Pharm. Sci. 2008, metabolism, cardioprotective effect and dietary sources, 1, 57-62. J Nutr Biochem. 1996, 7, 66–76. 24. Szkudelski T.: The mechanism of alloxan and 10. luo J, Borgens R. B. and Shi R.: Polyethylene glycol streptozotocine action in B-cells of the Rat pancreas, immediately repairs neuronal membranes and inhibits Physiol Res. 2001, 50, 536. free radical production after acute spinal cord injury, J. 25. bagchi D., Garg A. and Krohn R. L.: Protective Neurochem. 2002, 83, 471–480. effects of grape seed proanthocyanidins and selected 11. lu Y and Foo L. Y.: Unusual anthocyanin reactions antioxidants against TPA-induced hepatic and brain lipid with acetone leading to pyranoanthocyanin formation, peroxidation and DNA fragmentation, and peritoneal Tetrahedron Letters. 2001, 42, 1371–1373. macrophage activation in mice, Gen Pharmac. 1998, 30, 771–776. 12. badami S., Moorkoth S., Rai S. R., Kannan E. and Bhojraj S.: Antioxidant activity of Caesalpinia sappan heartwood, 26. Gordon M. H.: The mechanism of antioxidant action in Biol. Pharm. Bull. 2003, 26, 1534-1537. vitro. In: BJF Hudson (Ed.), Food antioxidants, Elsevier London. 1990, 1–18. 13. Jurenka J.: Therapeutic applications of pomegranate Applied Sci., (Punica granatum L.): A review, Altern Med Rev. 2008, 27. Annie S., Rajagopal P. L. and Malini S.: Effect of Cassia 13, 128-144. auriculata linn. root extract on cisplatin and gentamicin- induced renal injury, 2005, 12, 555- 14. lansky E. P. and Newman R. A.: Punica Phytomedicine. 560. granatum (pomegranate) and its potential for the prevention and treatment of cancer and inflammation, J. 28. Prince P. S. M., Kamalakkannan N. and Menon V. Ethnopharmacol. 2007, 109, 177-206. P.: Antidiabetic and antihyperlipidaemic effect of alcoholic Syzigium cumini seeds in alloxan induced 15. bandawane D. D., Juvekar A. R., Jadhav S. B. and diabetic albino rats, 2004, 91, Dhole S. N.: Antidiarrhoeal activity of pomegranate fruit J. Ethnopharmacol. 209-213. rind- a comparative study, Indian Drugs. 2006, 43 (2), 102-105. 29. Ohno T., Horio F., Tanaka S., Terada M., Namikawa T. and Kitoh J.: Fatty liver and hyperlipidemia in IDDM of 16. Wang R., Ding Y., Liu R. and Du L.: Pomegranate: streptozotocin treated shrews. Life Sci. 2000, 66, 125- constituents, bioactives and pharmacokinetics. Fruits, 131. Vegetables, Cereal Sci. and Biotech. 2010, 4 (2), 77- 87. 30. Grover J. K., Yadav S. and Vats V.: Medicinal plants of India with anti diabetic potential. 17. Juvekar A. R. and Bandawane D. D.: Preliminary study J Ethanopharmacol. 2002, 81, 81. on hypoglycemic effect of Alstonia scholaris Linn. in

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Short Notes

A NOVEL METHOD FOR ISOLATION OF MANGIFERIN FROM MANGIFERA INDICA L. BARK

Abstract A simple and practical method for the isolation of mangiferin from Mangifera indica Linn. (Anacardiaceae) (Mango) bark has been developed. The method involves solvent extraction of the dried bark powder using a single solvent and further purification by recrystallization to yield pure mangiferin. Purity of the compound was ascertained using HPLC. The structure of the compound was elucidated by IR, MS and NMR spectral analysis.

Key words: Mangiferin, s olvent extraction, Mangifera dried at a temperature of 60°C and powdered. The indica. chemicals used were of analytical grade. HPLC- grade Acetonitrile was sourced from Merck (India). Mangifera is a large genus of evergreen trees, Distilled water was filtered through 0.45 µm filter. distributed in tropical and sub-tropical parts of South- Mass spectrum was recorded on Micromass Q-TOF East Asia. Three to four species are found in India, MS Mass Spectrometer. All 1H and 13C NMR spectra of which Mangifera indica is cultivated extensively were recorded on a JOEL 400-MHz instrument with for its fruit1. an internal standard of tetramethylsilane (TMS). UV/Vis Spectrum was recorded on a Jasco V-530 M. indica bark majorly contains tannins Spectrophotometer. (16-20%) such as protocatechuic acid and catechin.

Other constituents present are mangiferin, alanine, TLC was performed on 0.20 mm silica gel G60 F254 glycine, α-aminobutyric acid, kinic and shikimic (E. Merck) aluminium-supported plates. Compound acid2. Mangiferin (2-β-D-glucopyranosyl-1,3,6,7- was visualized either under UV light at 254 nm or with tetrahydroxy-9H-xanthen-9-one, fig. 1 found in the Ferric chloride reagent; reagent grade solvents were bark and leaves of Mangifera indica, is also found in used for extraction. HPLC analysis was performed leaves of Bombax ceiba, Canscora decussata and with a Jasco (Hachioji, Tokyo, Japan) system, Swertia chirata. It is also found in many angiosperms using 250 mm × 4.6 mm i.d., RP-18 (5-μm particle and ferns3. Mangiferin is currently of interest because size) column, with a flow rate of 1.00 ml/min, Injection of its activities such as antidiabetic, hypolipidemic, volume loop: 20 μl., monitoring at 257 nm (UV-1575), gastroprotective, antibacterial, chemopreventive, and elution program: 15 min isocratic, acetonitrile : antiallergic, antiviral, anti-inflammatory and is also water containing 0.1% v/v of o-phosphoric acid a potent antioxidant 3,4,5,6,7. Mangiferin isolation has (15 : 85). Chromatographic data were processed with earlier been reported to be done using the column Borwin Software. chromatography and hydroalcoholic solvent extraction techniques. 50 g of powdered M. indica bark was subjected to solvent extraction with methanol for 26 h using Soxhlet The bark of M. indica were collected from the apparatus. The methanolic extract was reduced to campus of the Institute of Chemical Technology, 1/5th of its original volume. Crude mangiferin was Matunga, Mumbai and authenticated by Dr. H. M. obtained as a yellow precipitate. Yield of crude Pandit, Khalsa College, Mumbai; a voucher specimen mangiferin been 8.1 % w/w of dry bark powder. was deposited in Medicinal Natural Products Filtered precipitate was further recrystallized using Research Laboratory, ICT, Mumbai. The barks were methanol as solvent. For 1 g of crude mangiferin

INDIAN DRUGS 50(02) February 2013 47

Based on the chemical and spectral studies, the isolated compound was identified as mangiferin.

The method described above employing solvent extraction method and subsequent purification by recrystallization was found to be efficient on laboratory scale for isolation of mangiferin. However, further optimization of this method is necessary to make it suitable for commercial extraction. Fig. 1: Mangiferin (2-β-D-glucopyranosyl-1,3,6,7- tetrahydroxy-9H-xanthen-9-one) REFERENCES 600ml of methanol was used. Crude mangiferin was 1. The Wealth of India, Council of Scientific & Industrial refluxed with methanol, solution filtered, concentrated Research, New Delhi, 2005, Vol VI, 265-285 to 1/5th the volume and left overnight for crystallization. 2. The Ayurvedic Pharmacopoeia of India, Government of India, Ministry of Health and Family Welfare, Department Light yellow coloured, needle-shaped crystals of pure of ISM&H, New Delhi, 2004, Part-I, Vol-III, 9-10 mangiferin were obtained. Yield of pure mangiferin 3. Dineshkumar B., Mitra A. and Manjunatha M.; Studies on from 50g of bark powder was found to be 3.30g the anti-diabetic and hypolipidemic potentials of mangiferin (6.6%w/w). The quantity of mangiferin in the dried (Xanthone Glucoside) in streptozotocin-induced Type 1 bark of M. indica was found to be 10.2% w/w using and Type 2 diabetic model rats, International Journal of Advances in Pharmaceutical Sciences, 2010, 1, 75-85 the HPLC method. 4. Saveri J. A., Lima Z. P., Kushima H., Brito A. R., Santos L. C., Viegas W. and Hirumalima C. A.; Polyphenols Thin layer chromatography was performed on with antiulcerogenic action from aqueous decoction of pre-coated silica gel G60 F254 (E. Merck) using ethyl mango leaves (Mangifera indica L.), Molecules, 2009, acetate: methanol: water: formic acid (10:1:1:0.1) 14, 1098-1110 5. Rasool M., Sabina E. P., Mahinda P. S. and Gnanaselvi B. as mobile phase, a single band was seen at Rf 0.41 which gave a green colour with ferric chloride reagent C.; Mangiferin, a natural polyphenol protects the hepatic damage in mice caused by CCl4 intoxication, Comp Clin (5% alcoholic Ferric chloride). Infra red (IR) spectrum Pathol, 2012, 21, 865-872 of the isolated compound showed a broad peak 6. yoshimi N., Matsunaga K., Katayama M., Yamada at 3407 cm-1(hydroxyl), 1618 cm-1 (aryl aldehyde), Y., Kuno T., Qiao Z. et al; The inhibitory effects of 1094 cm-1 (C-O stretching of ethers) and 1350 cm-1 mangiferin, a naturally occurring glucosylxanthone, in (C-O stretching of phenols). Molecular ion peak at bowel carcinogenesis of male F 344 rats, Cancer Lett., 2001, 163(2), 163-170 423 m/e gave the molecular weight of the compound. 7. Garrido G., Gonzalez D., Lemus Y., Garcia D., Lodeiro L., The UV/Vis maxima in methanol were found to be at Quintero G., et al; In vivo and in vitro anti-inflammatory 208, 241, 257, 315, 360 and 365 nm. The 1H NMR activity of Mangifera indica L. extract (Vimang), and 13C NMR was performed and the compound Pharmacological Research, 2004, 50(2), 143-149 was confirmed to be mangiferin by comparing with 8. Shahat A. A., Hassan A. A., Nazif N.M., Van Miert S., the published data8. The isolated compound melts Pieters L., Hammuda F. M., et al; Isolation of mangiferin from Bombax malabaricum and structure revision of at 269°C. Mangiferin showed 94% purity by HPLC. shamimin, Planta Medica, 2003, 69, 1068-1070.

Medicinal Natural Products Research Laboratory, Gholkar M. S * and Laddha K. S Department of Pharmaceutical Sciences and Technology Institute of Chemical Technology, Nathalal Parikh Marg Matunga, Mumbai – 400019 E-mail: [email protected]

*For correspondence

(Received 29 November 2012) (Accepted 11 January 2013)

48 INDIAN DRUGS 50(02) February 2013

ANTI-INFLAMMATORY EFFECT OF SEEDS OF TAMARINDUS INDICA IN WISTAR RATS

Abstract

Tamarindus indica L. is highly valued in Indian medicine for management of painful inflammation, diabetes and constipation. In the present study, the anti-inflammatory potential was evaluated using carrageenan induced rat paw edema model. The coarse seed powder was extracted with distilled water, ethyl acetate and methanol and submitted to chemical tests which revealed the presence of alkaloids, tannins, flavonoids, glycosides, phenolic compounds and steroids. All the three extracts i.e. aqueous, ethyl acetate and methanolic extract of Tamarindus indica seeds at 500 mg/kg showed significant anti-inflammatory activity in the second phase of inflammation. For comparison purpose, weused indomethacin (10 mg/kg) as a reference drug. The results showed that methanolic extract exhibited more promising anti-inflammatory activity compared to aqueous and ethyl acetate extracts.

Keywords: Tamarindus indica seeds, Anti- Tamarindus indica L. (family: Leguminosae), inflammatory, Carrageenan. commonly known as tamarind, grows naturally in the tropical and sub-tropical regions of India3. It is INTRODUCTION widely found throughout the tropical belt, from Africa Inflammation is a part of the complex biological to south Asia, northern Australia, Southeast Asia, 4 response of vascular tissues to harmful stimuli, Taiwan and China . The healing power of tamarind is 5 such as pathogens, damaged cells or irritants. first mentioned in the traditional Sanskrit literatures . It is the defence reaction to limit the spread of In the Indian system of medicine, tamarind has wide 6 these injurious agents. This initiates the healing therapeutic application including inflammation , 7 8 process. Inflammation is considered as a protective diabetes , constipation, indigestion and flatulency . mechanism and the progressive destruction of the The seeds of Tamarindus indica are reported tissue would compromise the survival of the organism. to possess pharmacological activities such as However, chronic inflammation can also lead to a antidiabetic and hypoglycemic, antioxidant, anti-ulcer, host of diseases, such as hay fever, periodontitis, anti-venom, hepatoprotective, antibacterial, inhibition atherosclerosis, rheumatoid arthritis and even cancer of nitric oxide production and serin proteinase (e.g. gallbladder carcinoma). Therefore inflammation inhibitor9. Fruits and leaves of Tamarindus indica are is normally closely regulated by the body1. reported with antiasthamtic, hepatoprotective and antimicrobial activities10. Currently, synthetic drugs like non-steroidal anti-inflammatory agents (indomethacin, aspirin, Several studies have demonstrated that various diclofenac, etc.) and steroids are used in the treatment flavonoids like quercetin, luteolin, apigenin and tannins of inflammation. All these drugs possess immense are responsible for analgesic and anti-inflammatory side effects; also they are very expensive to develop. activity11, 12. The seeds of Tamarindus indica are Moreover in developing countries like India, most of reported to contain polymeric tannins and flavonoids the rural population relies on traditional remedies, like (+) catechin, procyanidin B2, (-) epicatechin, being cost effective and easily available2. It is procyanidin trimer, procyanidin tetramer, procyanidin therefore essential to study the traditionally used plant pentamer, procyanidin hexamer, taxifolin, apigenin, materials in order to provide a scientific base to the eriodictyol, luteolin and naringenin13. Thus it may be plant remedies by the people especially in the rural possible that seeds of T. indica may possess anti- area, where cost effectiveness and easy availability inflammatory potential due to the presence of above is of utmost importance. stated phytoconstituents. This forms the basis of the

INDIAN DRUGS 50(02) February 2013 49

current study which was designed to evaluate the alkaline test for flavonoid glycosides; Dragendorff’s, anti-inflammatory effect of different extracts of seeds Mayer’s, Hagger’s and Wagner’s test for alkaloids; of Tamarindus indica in Wistar rats. ferric chloride, lead acetate and potassium dichromate test for tannins and phenolics15. MATERIAL AND METHODS Acute Toxicity Study Experimental Animals The aqueous, ethyl acetate and methanolic Wistar rats (180-200 g) of either sex were extracts of Tamarindus indica seeds were tested used for the study. The animals were procured from for their acute toxicity in rats. Acute toxicity studies the National Institute of Biosciences (NIB), Pune. were performed according to OECD (Organization The animals were kept under standardized animal for Economic Co-operation and Development) house conditions with free access to standard pellet guidelines OECD 423. To determine short term diet and water ad libitum. The study was approved toxicity, the adult female Wistar rats were starved by Institutional Animal Ethical Committee (IAEC) overnight and were administered with aqueous, ethyl and experimental procedures were conducted in acetate and methanolic extract of Tamarindus indica accordance with the regulations of CPCSEA (884/ seeds orally in increasing dose levels of 100, 300, PO/ac/05/CPCSEA). 500, 2000, 5000 and 8000 mg/kg body weight. The Plant Materials mortality and general behavior of the animals were observed periodically for 48 h. The animals were The seeds of Tamarindus indica were purchased observed individually after dosing periodically for from the local market in the Pune region of Maharashtra 24 h with special attention during first two hours and in India in the month of September 2011 and identified then intermittently thereafter, for a total period of 14 and authenticated from Botanical Survey of India, days. The animals were observed for the signs of Pune. Voucher specimen (TAMIMAG6) has been toxicity which include changes in eyes and mucous deposited in the herbarium of BSI, Pune. membrane, skin and fur and behavior pattern. Attention was given to parameters like grooming, Preparation of Extract convulsions, tremors, salivation, lethargy, diarrhea, All the extracts were prepared by maceration loss of righting, reflex, sleep and coma16. technique14. The coarse powder of tamarind seeds (100 g) was extracted separately with 500 mL distilled Anti-inflammatory Study water, ethyl acetate and methanol for 72 h at room Wistar rats of either sex were selected for the temperature. The extracts were filtered and the study. The animals were randomly divided into 8 solvents were evaporated under vacuum to obtain groups. Animals from group I received distilled water reddish-brown powdered residues. (1 mL) and group II received indomethacin (10 mg/ kg) orally. Group II, IV and V received aqueous, ethyl Preliminary Phytochemical Screenings acetate and methanolic extract of Tamarindus indica The preliminary phytochemical analysis of seeds at 500 mg/kg body weight, per oral. aqueous, ethyl acetate and methanolic extract of Tamarindus indica seeds was performed as per Groups I : control the standard methods. A series of chemical tests Groups II : Standard were carried out viz. Molisch’s, Fehling’s, Benedict’s test for carbohydrates; Biuret and Million’s test for Groups III : Aqueous Extract proteins; Salkowski and Liebermann-Buchard’s test Groups IV : ethyl acetate Extract for steroids; Borntrager’s test for anthraquinone glycosides; foam test for saponins; Shinoda and Groups VI : Methanolic Extract

50 INDIAN DRUGS 50(02) February 2013

Table I. Anti-inflammatory activity of different extracts of Tamarindus indica seeds

Treatment Dose Increase in paw volume in mL 1 h 2 h 3 h 4 h 6 h Control 1 mL/rat 1.65±0.10 1.95±0.07 2.46±0.20 2.66±0.20 2.96±0.25 Indomethacin 10 mg/kg 1.41±0.09* 1.64±0.08* 1.48±0.15** 1.79±0.17** 1.70±0.15** (14.54) (15.89) (39.83) (32.70) (42.56) Aqueous 500 mg/kg 1.38±0.04* 1.61±0.17* 1.92±0.30* 2.05±0.21* 2.39±0.22* Extract (16.36) (17.43) (21.95) (22.93) (18.98) Ethyl acetate 500 mg/kg 1.55±0.05ns 1.86±0.13* 1.98±0.14* 2.07±0.07* 2.35±0.18* Extract (6.06) (15.38) (19.51) (15.85) (20.60) Methanolic 500 mg/kg 1.57±0.03ns 1.51±0.04** 2.03±0.04** 1.87±0.09** 2.10±0.12** Extract (4.84) (22.56) (23.10) (29.69) (29.05) n=6,*p<0.05 and **p<0.01, considered significant, by Dunnet Multiple Comparison Test (One way analysis of variance, ANOVA). Value expressed as mean±S.E.M.

0.1 mL of 0.1 % w/v of carrageenan suspension Variance (ANOVA) followed by Dunnet Multiple in 0.9% normal saline was injected subcutaneously comparison test. into the sub-planter of the left hind paw. All the test groups were treated with their respective RESULTS treatments, 1 h before the carrageenan injection. The 100 g powder of Tamarindus indica seeds The paw volumes were measured immediately after gave a percent yield of 24.1 % w/w for aqueous carrageenan injection and then at 1, 2, 3, 4 and 6 h extract, 10.8% w/w for ethyl acetate extract and using plethysmometer. 17.9 % w/w for methanolic extract. The aqueous and methanolic extracts were found to be crystalline, In the above model, the degree of edema formation powdered reddish-brown residue, whereas ethyl was expressed as an increase in paw thickness. The acetate extract was found to be brown in colour with % inhibition in paw volume was calculated by using sticky consistency. The preliminary phytochemical following formula, analysis of these extracts revealed the presence of secondary metabolites like alkaloids, anthraquinone % inhibition in paw volume = 100 × (1 – Vt/Vc) glycosides, tannins, flavonoids, phenolic compounds, steroids and saponins. where, As suggested by OECD guidelines, the test Vt = mean paw volume in the drug treated group. animals were observed individually, after dosing at Vc = mean paw volume in control group17. once, during the first 24 h periodically with special attention during first 2 h. The test animals did not Statistical Analysis exhibit any visible change and survived beyond The results are expressed as the mean ± S.E.M recommended duration of observation i.e., 48 h with of six animals. p<0.05 was considered as statistically 8000 mg/kg. Hence, all the three extracts were found significant when all groups were compared with the to be safe up to 8000 mg/kg. Therefore, dose of 500 control gruop. The difference between experimental mg/kg less than submaximal dose (800 mg/kg) was groups was compared by One-way Analysis of selected for the study.

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Table 1 indicates the anti-inflammatory activity or bradykinins which is due to presence of alkaloid, of aqueous, ethyl acetate and methanolic extract tannins and flavonoids. Since methanolic extract was of Tamarindus indica seeds. The aqueous extract found to be more potent that other two extracts, we at 500 mg/kg shows significant activity at 4 h with can say that the active constituents responsible for percentage inhibition of 22.93, while the ethyl anti-inflammatory effect have been extracted to more acetate extract showed significant activity at 6 h with extent in methanol. However, isolation of chemical percentage inhibition of 20.60. Similarly, methanolic constituents and mechanism responsible for the extract showed inhibition of paw edema in second anti-inflammatory activity ofTamarindus indica seeds phase (at 4 h) with percentage inhibition of 29.69 remain to be investigated. at 500 mg/kg. The standard indomethacin showed significant activity from 3 h onwards, maximum at ACKNOWLEDGEMENT 6 h with inhibition of 42.56%. The authors are thankful to the Management and the Principal, Modern College of Pharmacy, Nigdi, DISCUSSION Pune, for providing the necessary facilities to carry In the present study, the anti-inflammatory activity out research work. The authors are also thankful to was evaluated using carrageenan induced paw Mr. P. G. Diwakar, Joint Director, Botanical Survey edema model. This model has been widely used of India, Pune, for authentification of plant and Mrs. S.S. Nipate and Mr. B. P. Pimple for their valuable to screen natural products with anti-inflammatory suggestions. potentials18,19. Inflammation induced by carrageenan involves the release of platelet activation factor and REFERENCES pro-inflammatory mediators such as prostaglandins, 1. Mohan H.: Textbook of pathology: Jaypee Brothers kinins, tumor necrosis factor and nitric oxide20. Sub- Medical Publishers (P) Ltd., New Delhi, 2010, 6th ed., 130-173. planter injection of carrageenan in the rat hind paw 2. Juvekar A. R. and Bandawane D. D.: Antihyperglycemic induces inflammation in two distinct phases: the first and antihyperlipidemic activity of Aegle marmelose (L.) phase (0-2 h) which involves release of histamine and leaf extract in streptozotocin induced diabetic rats. Indian 5-hydroxytryptamine21,22 and second phase (2-6 h) Drugs. 2009, 46 (7), 43-49. which involves release of the inflammatory mediators 3. Siddhuraju P.: Antioxidant activity of polyphenolic compounds extracted from defatted raw and dry heated like prostaglandins, leukotrienes, polymorphonuclear Tamarindus indica seed coat. LWT. 2007, 40, 982- cells and bradykinins. These two phases are 990. linked with kinin release23. The knowledge of these 4. Adeola A. A.: Adeola O. O and Dosumu O. O.: Comparative analysis of Phytochemicals and antimicrobial properties mediators is important for interpreting the mode of of extracts of wild Tamarindus indica pulps. African J. drug action. Microbiol. Res. 2010, 4, 2769-2779. 5. Khan R. A., Siddiqui S. A., Azhar I. and Ahmed S. P.: It can be concluded that aqueous, ethyl acetate Preliminary screening of methanol and butanolextracts and methanolic seed extract have moderate to of Tamarindus indica for anti-emetic activity. J. Basic significant anti-inflammatory activity, among which and App. Sci. 2005, 2. the methanolic extract is much more efficient than 6. Paula F. S., Kabeya L. M., Kanashiro A., Figueiredo A., Azzolini A. E., Uyemura S. A. and Lucisano-Valim Y. M.: aqueous and ethyl acetate extract. Tannins and Modulation of human neutrophil oxidative metabolism flavonoids are reported to inhibit prostaglandin and degranulation by extract of Tamarindus indica L. synthesis12. As the anti-inflammatory effect of the fruit pulp. Food Chem Toxicol. 2009, 47: 163–170. seed extracts was more significant during later phase 7. Maiti, R., Jana D., Das U. K. and Ghosh D.: Antidiabetic of inflammation, perhaps there might be inhibition of effect of aqueous extract of seed of Tamarindus indica in streptozotocin-induced diabetic rats. Journal of prostaglandins, leukotrienes, polymorphonuclear cells Ethnopharmacology, 2004, 92: 85-91.

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8. Ghosh Roy M., Rahman A. S., Fatema R. A., Munmun M., bark in streptozotocine induced diabetic rats. Indian J. Sharmin N., Mamun A. A., Khatun A. and Rahmatullah M.: Pharm. Edu. Res. 2011, 45 (2), 114-120. Evaluation of anti-hyperglycemic potential of Tamarindus 17. bandawane, D. D., Juvekar A. R. and Juvekar M. R.: indica L. (Fabaceae) fruits and seeds in glucose- induced Antihyperglycaemic and antihyperlipidemic activity of Aegle hyperglycemic, mice. Adv. Natural Appl. Sci. 2010, 4, marmelose (L.) leaf extract in streptozotocine induced 159-162. diabetic rats. Indian Drugs. 2010, 46 (7), 43-49. 9. bhadoria S. S., Ganeshpurkar A., Narwaria J., Rai G. 18. Biswas M., Biswas K. Karan T. K., Bhattacharya S., and Jain A. P.: Tamarindus indica: Extent of explored Ghosh A. K. and Haldar P. K.: Evaluation of analgesic potential. Phco. Rev. 2011, 5, 73-81. and anti-inflammatory activities ofTerminalia arjuna leaf. 10. Fabiyi J. P, Kela S. L., Tal K. M. and Istifanus W. A.: J. Phytology. 2011, 3, 33-38. Traditional therapy of dracunculiasis in the state of Bauchi, 19. Nonato F. R., Barros T. A. A., Lucchese A. M., Oliveira Nigeria. Dakar Med. 1993, 38, 193–195. C. E. C., Santos R. R., Soares M. B. P. and Villarreal 11. Oyvind M. and Andersen K. R., 2006. FLAVONOIDS- C. F.: Anti-inflammatory and antinociceptive activities of Chemistry, Biochemistry and Applications, CRC Press, Blechnum occidentale L. extract. J. Ethnopharmacol. Taylor & Francis Group, New York, 458. 2009, 125, 102-107. 12. Mule S. N., Ghadge R. V., Chopade A. R., Bagul B. A., Patil 20. Tan-No K., Nakajima T., Shoji T., Nakagawasai O. and S. B. and Naikwade N. S.: Evaluation of antinociceptive Njima F.: Anti-inflammatory effect of propils through and anti-inflammatory activity of leaves ofGynandropsis inhibition of nitric oxide production on carrageenan- penthaphylla. J. Herb. Med. Toxicol. 2008, 2 (1), induced mouse paw oedema. Biol. Pharm. Bull. 2006, 41-44. 29, 96-99. 13. Sudjaroen Y., Haubner R., Wurtele G., Hull W. E., Erban 21. cuman R. K. N., Bersani-Amadio C. A. and Fortes Zb.: G., Spiegelhalder B., Changbumrung S., Bartsch H. and Influence of type-2 diabetes on the inflammatory response Owen R. W.: Isolation and structure elucidation of phenolic in rats. Inflame. Res. 2001, 50, 460-465. antioxidants from Tamarind (Tamarindus indica L.) seeds 22. Neto A. G., Costa J. M. L. C., Belati C. C., Vinholis A. and pericarp. 2005, 43, Food and Chemical Toxicol. H. C., Possebom L. S., Da Silva Filho A. A., Cunha W, 1673-1682. R., Carvalho J. T. C., Bastos J, K. and DaSilva M. L. 14. benni J. M., Jayanthi A. R. and Suresh R. N.: Evaluation A., Analgesic and anti-inflammatory activity of a crude of the anti-inflammatory activity ofAegle marmelos (Bilwa) root extract of Pfaffia glomerata (Sperng) Pedersen. J. root. Indian J. Pharmacol. 2011, 43, 393-397. Ethnopharmacol. 2005, 96, 87-91. th 15. Khandelwal, K.R., 2003. Practical Pharmacognosy, 6 23. Kuashik M. L. and Jalalpure S. S.: Evaluation of anti- Edn., Nirali Prakashan, Pune,159-162. inflammatory effect of ethanolic and aqueous extracts of 16. bandawane, D., Juvekar A. and M. Juvekar.: Antidiabetic Curcuma zedoaria Rosc root. Int. J. Drug Dev. & Res. and antihyperglycemic effect of Alstonia scholaris Linn. 2011, 3(1), 360-365.

Department of Pharmacology, Hivrale M. G., Mali A. A. and Bandawane D. D.* PES’s Modern College of Pharmacy Nigdi, Pune, Maharashtra 411044 E-mail : [email protected]

*For correspondence

(Received 22 August 2012) (Accepted 11 January 2013)

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