Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524124EnglishN-0001November30HealthcareREVERSE PHASE - LIQUID CHROMATOGRAPHIC METHOD FOR THE ANALYSIS OF CLOPIDOGREL IN PHARMACEUTICAL DOSAGE FORM English0319Tarun Jain*English Anil BhandariEnglish Sanjay SharmaEnglish Veerma RamEnglish Laik AhmedEnglish Balaram DeoraEnglishA selective and sensitive reverse phase high-performance liquid chromatographic (RP-HPLC) method has been developed for the analysis of Clopidogrel in pharmaceutical dosage form. The elution of Clopidogrel and Diazepam (Internal Standard) was achieved using octadecyl C 18 column (4.6 i.d. / 5μ particle size/ 250 mm length) as stationary phase. The mobile phase consisted of Methanol: Water {pH adjusted to 3.5 with 0.1% Acetic Acid} (95:05, v/v) as eluents at a flow rate of 1.0 mL min-1. Detection was carried out at 220 nm. Quantitation was done by external standard calibration method. The retention time of Diazepam (IS) and Clopidogrel bisulphate was found to be 3.39 and 4.41 minutes respectively. The method has been validated for linearity, specificity, robustness, accuracy and precision. Linearity for Clopidogrel bisulphate was in the range of 10-100 μg mL-1. The total run time of analysis was 6 minutes and the lower limits of detection (LOD) and Quantification (LLQ) were 0.020 and 0.050 μg mL-1, respectively. This validated HPLC method using a simple mobile phase is sensitive enough for future monitoring Clopidogrel in biological samples. EnglishClopidogrel; Reverse Phase –High Performance Liquid Chromatography; Therapeutic Drug Monitoring; Bioanalytical estimation of Clopidogrel.1. Introduction: Clopidogrel bisulphate [methyl (s)-2- chlorophenyl (4, 5, 6, 7-tetrahydrothieno- [3,2-C]pyridin-5-yl) acetate bisulphate](Figure 1) is an oral antiplatelet agent (thienopyridine class) to inhibit blood clots in coronary artery disease, peripheral vascular disease, and cerebrovascular disease. Clopidogrel is a pro-drug whose action may be related to adenosine diphosphate (ADP) receptor on platelet cell membranes. The specific subtype of ADP receptor that clopidogrel irreversibly inhibits is P2Y12 and is important in platelet aggregation and the cross-linking of platelets by fibrin. 1 The blockade of this receptor inhibits platelet aggregation by blocking activation of the glycoprotein IIb/IIIa pathway. The IIb/IIIa complex functions as a receptor mainly for fibrinogen and vitronectin but also for fibronectin and von Willebrand factor. Activation of this receptor complex is the "final common pathway" for platelet aggregation, and is important in the cross-linking of platelets by fibrin. Platelet inhibition can be demonstrated two hours after a single dose of oral clopidogrel, but the onset of action is slow, so that a loading-dose of 300- 600mg is usually administered. Clopidogrel is a pro-drug activated in the liver by cytochrome P450 enzymes, including CYP2C192 . The active metabolite has an elimination half-life of about 8 hours and acts by forming a disulfide bridge with the platelet ADP receptor 3, 4, 5 . A few analytical methods including gas chromatography–mass spectrometry (GC–MS) 6 , liquid chromatography–mass spectrometry (LC–MS) 7, 8 and highperformance liquid chromatography (HPLC) with UV detection9, 10 have been published for determination of the clopidogrel in the biological fluids. In these methods however, complex two steps extraction methods6, 7 or long analytical run time (10 min7 , 16 min9 and 12 min10 ) are required. While LOQ of 0.1 µg mL-1 , 0.125µg mL-1 and 0.2 µg mL-1 have been reported in published papers8, 9, 10. In human single dose pharmacokinetic studies a LOQ of less than 0.03 µg mL-1 is required for measuring of the analyte up to three half lives post-dose. The present work describes simple and fast method for analysis of the Clopidogrel in pharmaceutical dosage formulation and biological fluids, with sensitivity of 0.05 µg mL-1 and analytical run time of 6.0 min. 2. Materials and Methods: 2.1. Chemicals and standard solutions: Clopidogrel [purity 99.9%] was gifted by IPCA Pharmaceutical PVT Ltd, Ratlam [MP] and Diazepam [IS] from Alembic Pharmaceuticals, Baddi, Himachal Pradesh. All reagents used were of HPLC grade except acetic acid which was of analytical grade. Water was glass tripledistilled and further purified with a 0.44 µ filtration membrane using vacuum pump. A stock solution of Clopidogrel (200µg mL-1 ) and IS (100µg mL-1 ) was prepared in methanol. Working standards of the Clopidogrel (0.5–100 µg mL-1 ) were prepared by serial dilution of the stock solution in methanol. Working standard solution of the IS (10 µg mL-1 ) was prepared in methanol. All solutions were stored at 4 °C and found to be stable for at least 4 weeks. 2.2. Equipments: The HPLC system used was Cecil ADEPT CE 4700 solvent delivery system, a system controller (CE 4900), a UV–PDA detector (CE 4200) operated at wavelength of 220 nm, a degasser and a data processor all from Cecil, England, United Kingdom. 2.3. Chromatographic conditions: The method was developed using Thermo C18-ODS column [250mm×4.6mm I.D., 5µm particle size] which was protected by a guard column [1 cm X 4.0mm I.D, 5 µm particle size]. A mixture of consist of methanol and 0.1% acetic acid (pH adjusted to 3.5) [95:05 v/v] used as the mobile phase. The detection wavelength was 220 nm. The solutions were filtered, degassed and pumped at a flow rate of 1.0 mL/min. The injection volume was 20µl. 2.4. Preparation of sample solution: Twenty Clopidogrel bisulfate (75 mg) tablets were weighed, transferred to a clean and dry mortar and ground into a fine powder. Tablet powder equivalent to 200mg drug was then transferred to a 100 ml volumetric flask, 100 ml of methanol was added, and the flask was attached to a vortex shaker for 10 min to disperse the material completely. Now the solution was centrifuged at 10,000 rpm for 10 min and supernatant was filtered through a 0.45µm pore size Nylon 66 membrane filter using vacuum pump. The filtrate was further diluted using mobile phase to give test solutions containing 1000 and 100µg mL-1 . The stock solution of pure Clopidogrel was prepared by dissolving 20 mg of pure drug in 100 ml of methanol to give stock solution of 200µg mL-1 . The solution was further diluted with mobile phase to prepare test solution of 100µg mL-1 . 2.5. Method validation: The proposed method was validated as per ICH guidelines. [11] 2.5.1. Linearity and range: A stock solution of the drug was prepared at strength of 200µg mL-1 . It was diluted to prepare solutions containing 10–100?µg mL-1 of the drug. The solution was injected in triplicate into the HPLC column, keeping the injection volume constant 20 µL. 2.5.2. Specificity and selectivity: Specificity of the method towards the drug established through determination of purity for Clopidogrel peak after mixing and filtration with excipients. The resolution factor of the drug peak from the nearest other resolving peak was also studied. 2.5.3. Precision: Solutions in triplicate, of three different concentrations (80, 100 and 120µg mL-1 ), were analyzed on the same day and the values of relative standard deviation (R.S.D.) were calculated to determine intra-day precision. These studies were repeated on three different days to determine inter-day precision. 2.5.4. Accuracy: A known amount of standard drug was spiked in triplicate to the pre-analysed samples and the recovery of the drug was calculated. The accuracy of the method was calculated at three concentrations such as 80, 100 and 120 µg mL-1 . The assay concentrations of 80, 100 and 120 % were spiked to the pre-analysed samples. 2.5.5. Robustness: Robustness of method had been tested by varying the following parameters like Column temperature (± 5 °C), alteration in wavelength (± 5λ), flow rate (± 10%), pH (± 0.2%) and variation in mobile phase. 2.5.6. LOD and LOQ: To estimate the limits of detection (LOD) and quantitation (LLQ), diluted sample was injected six times and the signal-tonoise ratio (S/N) was determined. LOD and LLQ were regarded as the amounts for which S/N was 3:1 and 10:1 respectively. 2.6 Solution stability and system suitability parameters: The solution stability of Clopidogrel in the assay method had been carried out by keeping the test solution of sample and pure drug in air-tight volumetric flask at room temperature for 48 hrs. The same solution was assayed for every 8 hr interval up to the study period of 48 hrs. The overall system suitability was evaluated for the proposed method. The system suitability parameter studied were peak asymmetry (10%), capacity factor, theoretical plates, % Relative standard deviation of peak area, LOD (ng mL-1 ) and LLQ (ng mL-1 ). 2.7. Analysis of Dosage Form: Twenty Clopidogrel bisulfate [75 mg] tablets were weighed, transferred to a clean and dry mortar and ground into a fine powder. Tablet powder equivalent to 200mg drug was then transferred to a 100 ml volumetric flask. To this, 100 ml of methanol was added and vortexed for 10 min to disperse the material completely. This solution was then centrifuged at 10,000 rpm for 10 min and supernatant was filtered through a 0.45µm pore size Nylon 66 membrane filter and further diluted using mobile phase to give test solutions containing 100µg mL-1 . Six replicates of the final solution were analyzed and the observations were recorded. The above method was used for two different marketed formulations. 3. Results 3.1 Development and Optimization of the Stability Indicating Method: Satisfactory peaks were obtained with Methanol-Water (pH adjusted to 0.1% with acetic acid) at ratio 95:05, max 220 nm and flow rate 0f 1.0mL/min. The typical chromatogram under the optimum conditions is shown in figure 2 at Retention time 4.41 Minutes. The method has been optimized for future bioanalytical procedures using Diazepam as IS with retention time 3.39 Minutes as shown in figure 3. The overlay of Chromatograms shown in figure 4 indicates that the method succeeded in separation of drug and IS under similar set of conditions. A chromatogram of Clopidogrel with IS-Diazepam is shown in figure 5. 3.2 Analytical method validation: The present study aims at the analysis of Clopidogrel in dosage formulation and its future application in biomedical analysis. The stability indicating assay methods are gaining importance for the evaluation of active pharmaceutical substance. United State Food and Drug Administration (USFDA) emphasize the stability indicating assay methods for the estimation of active ingredients. The analytical method has been validated through the determination of linearity, range, specificity, accuracy, precision, detection limits, qauntitation limits, ruggedness, robustness and system suitability parameters. 3.2.1 Linearity: Linearity of the proposed method performed at concentration range of 10- 100µg mL-1 of Clopidogrel as shown in Table 1. The calibration curve drawn between concentration against the peak area counts shown in figure 6. The correlation coefficient (r2 ) found to be 0.9988. At 100±5 % of confidence limits, the regression equation is Y = 23.265X – 62.. 3.2.2 Specificity: It is a measure of the degree of interference from such things as other active ingredients, excipients, impurities, and degradation products, ensuring that a peak response is due to a single component only i.e. that no co-elution exist. The mobile phase injected prior to the standard solution and the chromatogram of the diluent revealed that at particular conditions, there was no peak in the retention time of Clopidogrel. It showed that the method was specific. The 3D chromatogram of the stability sample shown in figure 7 demonstrated that the retention of Clopidogrel was unaffected by the other impurities. 3.3.3 Precision: Precision of the proposed method was performed by intra and inter-day studies. Triplicates of three different concentrations (120, 100 and 80 µg mL-1 ) injected at 0, 2 and 4 Hours and on day 1, 2 and 3 for intra-day and inter-day precision respectively. The data obtained from precision experiments are shown in Table 2. The %R.S.D. values for intra-day and inter-day precision study were Englishhttp://ijcrr.com/abstract.php?article_id=2246http://ijcrr.com/article_html.php?did=22461. Savi, P.; Zachayus, J.L.; Delesque, N.T. Proceedings of the National Academy of Sciences of the USA. 2006, 103 (29), 11069–11074. 2. Pereillo, J.M.; Maftouh, M.; Andrieu, A.; Uzabiaga, M.F.; Fedeli, O.; Savi, P.; Pascal, M.; Herbert, J.M.; Maffrand, J.P.; Picard, C. Drug Metab. Dispos. 2002, 30 (11), 1288–1295. 4. Simon, T. New Engl. J. Med. 2009, 360, 363–75. 5. Collet, J. Lancet. 2009, 373, 309 . 6. Lagorce, P.; Perez, Y.; Ortiz, J.; Necciari, J.; Bressolle, F. J. Chromatogr. B. 1998, 720, 107. 7. Ksycinska, H.; Rudzki, P.; Kiliszek, M.B. J. Pharm. Biomed. Anal. 2006, 41, 533. 8. Mitakos, I.; Panderi, I. Anal. Chim. Acta. 2004, 505, 107. 9. Singh, S.S.; Sharma, K.; Barot,D.; Mohan, P.R.; Lohray, V.B. J. Chromatogr. B. 2005, 821, 173. 10. Souri, E.; Jalalizadeh, H.; Kebriaee-Zadeh, A.; Shekarchi, M.; Dalvandi, A. Biomed Chromatogr. 2006, 20, 1309. 11. ICH, Validation of Analytical Procedures: Text and Methodology (Q2 (R1)), International Conference on Harmonization. IFPMA, Geneva, 2005.3. Mega, J. L. New Engl. J. Med. 2009, 360, 354–362.

Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524124EnglishN-0001November30HealthcareFAST DISPERSING TABLETS OF DICLOFENAC SODIUM WITH NATURAL SUPERDISINTEGRANTS English2029Wyawahare N.SEnglish Mishra M.UEnglish Bhongade S.L.EnglishObjective: In the present study, fast dispersing tablets of Diclofenac Sodium were prepared by using natural super disintegrants obtained from husk and mucilage of seeds of plant Lepidium sativum. Fast dispersing or dissolving tablets are the dosage form which dissolve or disintegrate in oral cavity without the need of water or chewing. Diclofenac Sodium is a non steroidal anti-inflammatory drug taken to reduce inflammation and as an analgesic in conditions such as arthritis or acute injury with lower indication of gastrointestinal adverse effects. Method: The husk and mucilage were obtained from the seeds. The physicochemical properties like density, flow properties, particle size distribution, swelling index and water uptake studies of dried husk and mucilage were evaluated. It was observed that it shows rapid water uptake and swelling which makes it suitable candidate as disintegrants. Diclofenac Sodium fast dispersing tablets have been prepared by direct compression method by using mucilage and husk of the seeds of plant Lepidium sativum. Formulation was optimized on the basis of physical properties, swelling time, drug content, in vitro disintegration and in vitro drug release. The study revealed that husk and mucilage are effective at 4% concentration and showed rapid disintegration of the tablets (25 sec and 22 sec respectively). The husk as well as the mucilage did not interfere with the in vitro release studies. The tablets show almost complete release within 20 minutes. Conclusion: From this study, it can be concluded that natural disintegrants obtained from Lepidium sativum showed fast disintegrating property. EnglishLepidium sativum, husk and mucilage, fast disintegrationIntroduction Mouth dissolving tablets are the tablets which disperse rapidly in an oral cavity within a minute, without a need of water1 . For these formulations, the small volume of saliva is usually sufficient to result in tablet disintegration in the oral cavity.2 The gediatric and pediatric patients experience difficulty in swallowing tablets which leads to poor patient compliance. Similarly it is difficult to administer the drugs to mentally ill, bed ridden patients or to the patients having difficulty with swallowing the tablets.1 Fast dispersing tablets are a promising approach to overcome above problems. Mouth dissolving tablets breakdown rapidly in small particles without need of water and therefore it is convenient during travelling.2 Apart from patient compliance, rapid onset of action and increase bioavailability are the added advantage in designing of the tablets.3 Fast dissolving drug delivery (FDDTs,) can be achieved by various conventional methods like direct compression, wet granulation, moulding, spray drying, freeze drying and sublimation. In order to allow fast dissolving tablets to dissolve in the mouth, they are made of either very porous and soft- moulded matrices or compressed into tablets with very low compression force, which makes the tablets friable and/or brittle, which are difficult to handle, often requiring specialized peel-off blister packaging.4 Diclofenac is a non-steroidal antiinflammatory drug (NSAID) taken to reduce Inflammation and as an analgesic reducing pain in conditions such as arthritis or acute injury. It can also be used to reduce menstrual pain, dysmenorrhea. The name is derived from its chemical name: 2-(2,6- dichloranilino)phenylacetic acid. Mucilage and gum have been known since long time as a medicinal agent. Nowadays these gums and mucilage is gaining importance in pharmaceutical industries as thickener, suspending and emulsifying agents, binder and film former. As these are vegetative in origin hence the demand of these substances is increasing and therefore new sources been searched.5,6 Material and methods Diclofenac sodium (Panacea Biotech), microcrystalline cellulose PH 102 (NB Entrepreneurs), Sucrose (Loba chemicals), Talc (Burgoyne Lab and Co), magnesium stearate (Samar chemicals), Potassium dihydrogen orthophosphate (Krypton chemicals) Method Isolation of husk2 The seeds of the Lepidium sativum were powdered by automatic grinder and sieved through sieve #80. The seed powder was treated with chloroform. The husk was settled down in separating funnel remaining all the constituents of the seed floats at the top of the liquid. The husk was separated and dried at 400C till completely dried. The dried husk was pulverized by automatic grinder and passed through # 80 sieve. Isolation of mucilage5,6 The seed of Lepidium sativum contains the mucilage in the outer covering of the seed. The mucilage is enmeshed in the hard covering of the seed. The seeds of the Lepidium sativum swell rapidly but it did not separate from the surface of the seed and forms the thick covering around it which is difficult to remove by general mentioned methods. The seeds were soaked in water for 12 hrs in distilled water. The swollen material was transferred to the blender and blended for 10 min. The mass was then passed through eight fold of muslin cloth. The filtrate was collected. To the filtrate acetone was added in ratio 1:1, the mucilage was precipitated out. The precipitate was separated using separating funnel. The mucilage is dried in hot air oven at 400C till mucilage completely dried. The dried mass was pulverized using automatic blender and passed through # 80 seive. Characterization of Superdisintegrants2,5,6 The Superdisintegrants were evaluated for their physiochemical properties such as swelling index, loss on drying, flow properties, density and compatibility. Swelling index: The swelling index is the volume in ml occupied by 1 g of drug. The swelling index of Lepidium sativum seed powder and mucilage were determined according to BP method. 1g of each disintegrants was taken in separate graduated cylinder of 25 ml. To this 25 ml of water was added and shaken vigorously in every 10 min for 4 hrs. The volume occupied by swollen mucilage was measured. The test was carried out in duplicate and swelling index was calculated. The swelling index is tabulated in table no 1. Loss on drying: The moisture presents in the husk or mucilage influence the stability of the product. High moisture content contributes to the degradation of moisture sensitive drug as well as favor microbial contamination. The moisture content was determined by LOD method. 1g of sample was heated at 1050C in muffle furnace until constant weight was achieved. The %LOD was calculated and tabulated in table no 1. Density, compressibility and flow property: Bulk density was determined by taking accurate weight of sample in 100 ml graduated cylinder and volume was measured. For tapped density, cylinder was tapped until the powder bed volume achieves the constant reading. Compressibility index is a measure of ability of solid to get compressed. Flow property is determined in order to determine the effect of the disintegrant on the flow of the blend. Angle of repose is used to determine the flow property of the powder. It is determine by fixed height funnel method. The data is tabulated in table no 1. Compatibility studies: The drug and disintegrant were mixed properly in ratio 1:0.2. The physical mixtures were studies after 7 days. Preparation of the tablets Fast dispersing tablet of Diclofenac sodium 100 mg were prepared by direct compression method. The various ratios of drug and excipients were studied as shown in table no 2. The drug, disintegrant (husk or mucilage), microcrystalline cellulose and sucrose were pass through # 40 sieve. All the ingredients were mixed uniformly in a polybag. Talc and Magnesium stearate were passes through # 60 sieves and added to the polybag and mixed properly. The blend was compressed on a 12 station rotary punch tableting machine using 8 mm of concave punch set. Evaluation of tablets Tablets were evaluated for weight variation, hardness, friability, thickness and in-vitro disintegration time and in-vitro dissolution of the tablets. In weight variation test 20 tablets were randomly selected and average weight was determined using electronic balance. Tablets were weighed individually and compared with average weight and % variation was determined. The hardness of the tablet was determined using Monsanto type hardness tester and the applied pressure for crushing the tablets was determined. Roche frabilator was used to determine%friability of the tablets which was rotated at 25 rpm for 4 min. % friability = 100 x Wo-Wt/Wo. Drug content study of tablets was studied by method as specified in IP 1996. The data is tabulated in table no 3. Disintegration test: In vitro Disintegration time was determined using tablet disintegration apparatus (USP apparatus). The test was performed using disintegration apparatus with distilled water as medium. The water was heated up to 370C. The tablets were dropped in each of 6 tubes of the apparatus. Time in second for complete disintegration of tablets was measured. In-vitro dissolution: In vitro dissolution drug release was carried out using a digital tablet dissolution test apparatus (Veego Model No DA- 6D) in 900 ml of pH 7.4 phosphate buffer at 37±0.50C at 75 rpm using USP type II apparatus Aliquot were withdrawn at 5, 10 15, 20, 30 and 45 min and were replaced immediately with the same volume of the fresh buffer. Aliquot were diluted suitably and assayed spectrometrically (Shimadzu model 1700) at 274nm. The data is tabulated in table no 3. Result and Discussion Preformulation characteristics of the mucilage contribute in the formulation of the efficient fast dispersing tablets with natural disintegrants. The super disintegrants obtained from Lepidium sativum posses good physiochemical properties. Swelling index of the mucilage and Husk obtained from Lepidium sativum were found to be 27 and 25 respectively. The swelling factor is related to the disintegration of tablets rapid water uptake and rapid swelling leads to rapid disintegration of tablets.7,8 The LOD value is within prescribe limit as specified in official. The compressibility index and angle of repose value indicates that the mucilage and the husk powder have good flow characteristics with moderate compressibility. Different batches were formulated using different ratio of drug, mucilage and husk of the seeds of Lepidium sativum by direct compression technique. Other excipients such as sucrose, microcrystalline cellulose, talc and magnesium stearate were incorporated in the formulation. Coarser grade of Microcrystalline cellulose (PH102) was selected as diluent as it facilitate the flow property of the blend from the hopper. Sucrose was selected as sweetener and talc and Magnesium stearate as lubricants. In the compatibility studies no physical change was observed after 7 days. The preliminary trial was designed with 2% of disintegrant and the tablets were evaluated. Further batches were designed with increase concentration of the disintegrant. Tablets were prepared having uniform weight (200 mg) due to uniform die fill and reveled less %variation with the average. Hardness of all the formulation was kept constant (4.0-4.5 kg/cm3 ).All the formulation passes the test for friability. The drug content was found in the range of 98.50 – 102.3% (acceptable limit) The in vitro disintegration time was found to be in range of 30 to 120 sec (official NMT 3 min). It was observed that the disintegration time decreases with increase in concentration of the disintegrants. Formulation with 4% of husk and mucilage as disintegrants shows disintegration time 25 sec and 22 sec. Mucilage of seeds of Lepidium sativum has a high swelling ability and may disintegrate tablets by mainly swelling pressure. As with 5% of the husk and mucilage the Disintegration time increase that the 4%. This was due to excessive amount of the mucilage lead to excessive swelling. There was no effect of mucilage on drug release from tablets as all the formulation showed more than 90% release at 20 min at 2 to 4% of disintegrants but release rate decreases with 5% of disintegrants. This was due to higher concentration of mucilage causing excessive swelling8 . The results are showed by Fig 1 and Fig 2 for mucilage and husk respectively. Conclusion From the present study, it can be concluded that natural super disintegrants like Lepidium sativum mucilage and husk showed fast disintegration of tablets and can be used as Superdisintegrants in place of synthetic disintegrants. Acknowledgement The Authors wish to thank Panacea Biotech, Punjab for the generous gift of the sample of Diclofenac Sodium. Englishhttp://ijcrr.com/abstract.php?article_id=2247http://ijcrr.com/article_html.php?did=22471. Sharma S, Bharadwaj S and Gupta GD, Fast dissolving tablets of Promethezine Theoclate by Using natural Superdisintegrants, Research Journal Pharmaceutics and Technology, July-Sept 2008, 218-7. 2. Mutasem M. Rawas-Qalaji, F. Estelle R. Simons, and Simons KS. Fast-disintegrating Sublingual Tablets: Effect of Epinephrine Load on Tablet Characteristics, AAPS PharmSciTech 2006; 7(2) Article 41. 3. Setty MC, Prasad DVK, Gupta RM and SA B. Development of Fast Dispersible Aceclofenac Tablets: Effect of Functionality of Superdisintegrants. Indian Journal of Pharmaceutical Sciences Mar-Apr 2008; 70 (2); 180-6. 4. Biradar SS, Bhagwati ST, Kuppasad IJ, Fast Dissolving Drug Delivery Systems: A Brief Overview, The Internet Journal of Pharmacology, 2006 Volume 4 Number 2. 5. Patel DM, Prajapati DG and Patel NM. Seed Mucilage from Ocimum americanum Linn. as disintegrants in Tablets : Separation and Evaluation. Indian Journal of Pharmaceutical Sciences May-Jun2007; 69 (3); 431- 5. 6. Ravikumar, Shirwaikar AA, Shirwaikar A, Prabhu SL, Rajendran MK, Studies of Disintegrant properties of seed mucilage of Ocimum gratissium, Indian Journal of Pharmaceutical Science, Nov-Dec 2007, 753-6. 7. H. Omidian1, K. Park*. Swelling agents and devices in oral drug delivery, J. Drug Del. Sci. Tech., 18 (2) 83-93 2008, 83-11. 8. Zhao Na, Augsburger LL. The influence of swelling capacity of Superdisintegrants in different pH media on the dissolution of Hydrochlorothiazide from directly compressed tablets, AAPS Pharm Sci Tech, 2005; 06(01) E120-E126. DOI:10, 1208. 9. Schiermeier S and Schmidt PC. Fast dispersible ibuprofen tablets, European Journal of Pharmaceutical Sciences Volume 15, Issue 3, April 2002, Pages 295-10

Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524124EnglishN-0001November30General SciencesISOLATION AND CHARACTERIZATION OF A BACTERIOPHAGE SPECIFIC TO DRUG RESISTANT KLEBSIELLA PNEUMONIAE DR1 English3043Wanpen ThamniamtonEnglish Varaporn BoonsarnEnglish Parichat PhumkhachornEnglish Pongsak RattanachaikunsoponEnglishA lytic bacteriophage isolated from sewage water was found to attack drugresistant Klebsiella pneumoniae DR1 isolated from a clinical specimen. The phage, designated kpdr1, had an isomeric head (60 nm in diameter) with a contractile tail (93 nm long and 13 nm wide). It was identified in Myoviridae family. From the host range test, the phage was highly specific to K. pneumoniae DR1. A onestep growth experiment of phage kpdr1 at 37 C after the phage infection showed that the latent period was 30 min, the burst period was 180 min, and the average burst size was about 52 phage particles/infected cell. The phage was able to survive after incubating at 37 C for 24 h in pH 5-12. The phage was also stable at the temperature of 60 C for at least 3 min. Various disinfectants could inactivate the phage after treating at 37 C for 24 h. Knowledge of the properties of K. pneumoniae DR1 bacteriophage kpdr1 may be important for the development of controlled infection. EnglishBacteriophage, Klebsiella pneumoniae, drug resistant bacteria, phage therapyIntroduction Klebsiella pneumoniae is a gramnegative bacterium which clinically the most important member of the Klebsiella genus of Enterobacteriaceae. It causes urinary tract infection (UTI), pneumonia, and abdominal infection. It normally affects persons with immunocompromised such as hospital patients, diabetes patients and people with chronic lung disease. Usually, alcoholics also suffer from K. pneumoniae infection. Thus, the infections are either hospital-acquired or community-acquired[1]. Treatment for K. pneumoniae infection is usually done by using antibiotics such as cephalosporins, quinolones, or aminoglycosides. However, there is a widespread development of antibiotic resistant strains of K. pneumoniae[2], so the need for new antibiotics and alternative approaches to control drug-resistant K. pneumoniae infection is receiving increased attention[3]. One such alternative is the possible therapeutic use of bacteriophage or bacteriophage therapy[4,5] Bacteriophages or phages are the viruses that infect bacteria. There are many of different phages, each of which may infect only one or several types of bacteria. Phages are common in all natural environments and are directly related to the numbers of bacteria present[6]. When a phage meets a suitable host bacterium, it binds to the particular molecules on the outermost part of the host and then injects its genetic material into the host cell. Lytic phages instruct the machinery in the host cell to make more phages. Fully viable progeny phages burst out and kill the bacteria. The released phages attack new bacteria. This process continues until all the bacteria are eliminated from the ecosystem[5] . ystem[5] . One of the advantages of phage therapy over antibiotics is that phages infect only specific bacteria, so they can kill only the harmful bacteria without affecting the beneficial bacteria present in the body. On the other hand, antibiotics usually target both pathogenic and normal flora. Thus, phage therapy is considered safer for therapeutic use[5]. The application of phages to treat bacterial infections has been successfully used both in infected human and experimentally infected animal[7] . In this study, we isolated a phage, kpdr1, against drug-resistant K. pneumoniae DR1 from sewage water and also characterized the phage in some aspects. The phage from this study may be useful as a potential therapeutic agent for controlling K. pneumoniae DR1 infection which usually occurring in our communities. Materials and Methods Bacterial Strains and Culture Conditions K. pneumoniae strain was isolated from a patient hospitalized at the Sappasitiprasong Hospital, Ubon Ratchathani, Thailand and was designated as DR1 in our nomenclature. The strain was grown in Brain Heart Infusion (BHI) medium (HiMedia, Mumbai, India) at 37 C and confirmed as K. pneumoniae by the standard biochemical tests. Antibiotic susceptibility testing by Kirby-Bauer?s method revealed that the K. pneumoniae DR1 strain was resistant to most of the commonly used drugs. Staphylococcus aureus ATCC25923 and Escherichia coli ATCC25922 were used as control strains in antibiotic susceptibility testing. To test the host range of phage, kpdr1, several bacterial strains were used (Table 1). All of the bacterial strains used in this study were grown in BHI medium at 37 C. The bacterial cultures were stored as stock cultures in BHI broth supplemented with 20% (v/v) glycerol at -70 C. Phage Isolation Sewage water samples for phage isolation were obtained from the sewage treatment tank located at the Sappasitiprasong Hospital. The samples were stored at 4 C overnight to allow larger suspended sediments to settle out. These crudely clarified samples were centrifuged (4,500 g for 10 min) to remove bacterial cells and debris. The supernatant was passed through a 0.22 m membrane filter (SartoriusAG, Goettingen, Germany). For phage enrichment, the filtrate was added to equal volume of double strength BHI broth and inoculated with an early log phase host culture, K. pneumoniae DR1. After incubation at 37 C overnight, the culture was centrifuged (4,500 g for 10 min) and the supernatant was passed through 0.22 m membrane filter. The filtrate was stored at 4 C and subjected to the following phage detection. Phage Detection and Host Range Determination Phage detection was performed by using a spot test method[8]. The test was used as an initial step for the presence of phage by observing lytic activity of phages. Soft agar in 5 ml (BHI broth with 0.7% agar) was seeded with 0.1 ml of a log-phase host culture, mixed thoroughly, and poured onto a BHI agar plate. After solidification, 10 l of phage filtrate was spotted onto the top agar layer. After drying, the plate was incubated at 37 C overnight. A clear zone in the plate, resulting from the lysis of host cells, indicated the presence of phage. Spot tests were also used for host range studies. The procedures were as described above but using the other bacterial hosts listed in the table 1. Phage Propagation Phage was purified by single plaque isolation[8] using K. pneumoniae DR1 as a propagating strain. A single plaque was picked from the lawn of the bacterial host, and propagated in 10 ml of an early log phase K. pneumoniae DR1 culture in BHI broth. After incubated at 37 C overnight, phage lysate was centrifuged at 4,500 g for 10 min. The supernatant was filtered through 0.22 m membrane filter. Phage stock was stored at 4 C, and an aliquot was frozen at -70 C. Phage titer was counted as plaque-forming unit (pfu/ml) using the double-layer agar plaque method[9] . One-Step Growth Curve For one-step growth experiments, a method of Capra et al.[10] was used. A mid-exponential phase culture of K. pneumoniae DR1 (optical density at 600nm = 0.5) was harvested and suspended in one-fifth of the initial volume of fresh BHI broth. Phages were added at a multiplicity of infection (MOI) of 0.5, and allowed to adsorb for 30 min at 37 C. Cells were harvested by centrifugation (10,000 g for 5 min), and resuspended in BHI broth. Decimal dilutions were made, incubated at 37 C, and at intervals, aliquots from each dilution were collected for phage counts. Latent period, burst time and burst size were calculated from the one-step growth curve. Phage Stability Resistance to physical and biocides was determined according to the methods described by Lu et al.[8] and Capra et al.[10] with minor modifications. Thermal inactivation was examined at 50, 60, 70, and 80 C. A tube containing sterile, deionized water was preheated to a desirable temperature. Phage solution was added into the tube at final concentration of 106 pfu/ml. After heating at intervals between 30 s to 3 min, phage samples were taken for phage counts. Results were expressed as the concentration (pfu/ml) of active viral particles and its log plotted against time. Phage stability was also examined at pH values ranging from 2 to 13, after incubation for 24 h at 37 C. Phages were added into each pH value at final concentration of 106 pfu/ml. The surviving phages were immediately counted and the results were expressed as the concentration (pfu/ml) of active viral particles and its log plotted against pH values. Resistance to disinfectants was also determined using commercial ethanol (10, 50, 75, and 95% v/v), isopropanol (10, 50, 75 and 95% v/v), and sodium hypochlorite (200, 400 and 600 ppm). After treating at different concentrations of each disinfectant for 24 h at 37 C, the surviving phages were counted and the results were expressed as a percentage of the initial viral counts. Phage Preparation for Electron Microscopy Phage preparation for direct visualization by electron microscopy was carried out as described by Watanabe et al.[11] with some modifications. Briefly outlined, five hundred milliliters of BHI medium was inoculated with a bacterial host, K. pneumoniae DR1, and grown to an optical density at 600 nm of 0.5. The bacterial host was then infected with 5 ml of a phage suspension (106 pfu/ml) and incubated overnight at 37 C. After centrifugation at 4,500 g for 20 min, the supernatant was collected, and was centrifuged at 4 C with a 70.1Ti rotor at 28,500 rpm for 1 h in a Beckman L- 80 ultracentrifuge (Beckman, CA, USA). The resulting pellets were resuspended in 5 ml of phage buffer (20 mM Tris-HCl [pH 7.4], 100 mM NaCl, 10mM MgSO4). A purified phage was recovered after centrifugation (4,500 g for 20 min) and the supernatant was passed through a 0.22 m membrane filter. The purified phage was stored at 4 C for electron microscopy. Electron microscopy A drop of the purified phage suspension was applied to a carboncoated grid for 5 min, then removed with a pipette and immediately replaced with a solution of 2% (w/v) uranyl acetate. After 1 min, the liquid was removed with a filter paper. The grids were examined in a JEOL JEM- 1230 transmission electron microscope (JEOL, Tokyo, Japan) at 80 kV accelerating voltage. Results Isolation and Morphology of Phage Five sewage water samples were collected for phage isolation. One sample was obtained a phage specific to the drug resistant K. pneumoniae DR1, designated kpdr1. The phage was found to produce small central clear plaques (average diameter, 1.0 mm) surrounding with a large halo (Fig. 1). The phage had an isometric head that was 60 nm in diameter and a long contractile tail (13 93 nm) under the electron microscope (Fig. 1). The virion had a baseplate at the distal end of its tail but did not show other additional structures such as collars or fibers. The phage was classified as a member of the Myoviridae family[6] . Host Range Determination The host range of phage kpdr1 was determined by testing the formation of clear zones at 37 C after overnight incubation on lawns of 17 bacterial strains. The results, summarized in Table 1, showed that the phage had a highly specific host range. The phage was able to infect only K. pneumoniae DR1 strain. All the tested strains were insensitive to this phage. One-Step Growth Curve Multiplication parameters of the lytic cycle of phage kpdr1 were determined from the one-step growth curve (Fig. 2). The latent and burst periods were 30 and 180 min, respectively, and the burst size was estimated at 52 phage particles per infected cell. Phage Stability Phage kpdr1 was able to survive at 50 and 60 C. There was not much difference of phage counts between 50 and 60 C. Heating at 60 C for 180 s was insufficient for complete inactivation but was reduced the phage titer about 2 log (Fig. 3). However, the phage was totally inactivated at 70 and 80 C after only 30 s (data not shown). Phage kpdr1 maintained its infectivity when incubated at 37 C for 24 h in a pH range between 5 and 12 (Fig. 4). An obvious decrease in phage particle counts was observed at pH higher than 10 and at pH lower than 6. At pH 2, 3, 4, and 13, phage could not be detected. After phage kpdr1 suspensions were treated with various concentrations of different disinfectants at 37 C for 24 h, phage particles mostly became inactivated (Table 2). Discussion For more than half a century, the human society has been relying primarily on antibiotics to treat infection disease caused by pathogenic bacteria. However, the worldwide spread of pathogenic bacteria that are resistant to a variety of antibiotics has made antibiotics less and less effective[3]. Drug-resistant K. pneumoniae is a common nosocomial pathogen and a very important cause of morbidity and mortality in several countries[12-14]. This clearly highlights the need for new antibacterial agents with fundamentally different modes of action than that of traditional antibiotics to control drug-resistant K. pneumoniae infection. Bacteriophages or phages represent the new class of antibacterial agents. Obviously, lytic phages are the best candidates for bacteriophage therapy as they undergo rapid growth, disrupt bacterial metabolism and reproduction, and lyse the bacterial cells. In this study, we found a lytic phage, kpdr1, against drug-resistant K. pneumoniae DR1 in sewage water from the local hospital where the host strain was isolated. Not surprisingly, phages can be found in places where their hosts exist[9,15]. Because phage kpdr1 use K. pneumoniae DR1 as a host organism, it can be found in places where its host is plentiful. Transmission electron micrograph showed that phage kpdr1 consisted of an isometric head and a long contractile tail. According to its morphology, this phage was classified as a member of the Myoviridae family. This virion morphology was similar to that of phage Kpp55 of K. pneumoniae which identified as the T-even like phage group[16]. However, the morphology of phage against K. pneumoniae found in this study was different from other previous reports such as phage K 1, KPO1K2, SS, and Kpn phage group all of which belonged to the Podoviridae family having an icosahedral head with a short noncontractile tail[17-19]. It is possible that further analysis of phage kpdr1 will indicate that it represents new virus family or similar to the previous families of viruses found in K. pneumoniae. The diversity of phage types against a single species is truly remarkable. On the basis of spot test to check the host range, phage kpdr1 was found to infect only drug-resistant K. pneumoniae DR1 strain. None of the ther bacterial hosts used in the present study showed susceptibility to this phage. Interestingly, the phage was not even able to infect the two other strains of K. pneumoniae. Thus, the host range of phage kpdr1 seems to be extremely narrow. The narrow host range of phage should be advantageous, in principle, as phage therapy results in less harm to the normal flora than commonly used antibiotics. Moreover, the high specificity of phage and its host could be applicable for phage typing in epidemiological studies as well[20] . Several studies documented that thermal and pH stability and chemical resistance of phages varied depending on types of phage[8,10,18]. Therefore, it is of interest to investigate the stability of phage kpdr1 in a wide range of temperature and pH and in some common used biocides. The results revealed that phage kpdr1 was quite stable in a broad pH range (5-12) and could be survived at 60 C for at least 3 min. However, phage kpdr1 was hardly able to survive in all disinfectants used in this experiment. This information may be useful for broad applications of the phage in phage therapy or in other applications. In conclusion, this study finds a lytic phage, kpdr1, infecting a drugresistant K. pneumoniae DR1 strain that will lead to a better controlling infection especially in the outbreak area. Further research is needed to evaluate the potential therapeutic use of the phage to control the drug-resistant K. pneumoniae DR1 infection in animal models. Acknowledgement The authors gratefully acknowledge Nual-anong Narkong, the Instrumental Center, Faculty of Science, Mahasarakham University for her advice and assistance with electron microscopy. Englishhttp://ijcrr.com/abstract.php?article_id=2248http://ijcrr.com/article_html.php?did=22481. Tsay RW, Siu LK, Fung CP, Chang FY. Characteristics of bacteremia between community-acquired and nosocomial Klebsiella pneumoniae infection: risk factor for mortality and the impact of capsular serotypes as a herald for community-acquired infection. Arch Intern Med 2002; 162: 1021- 27. 2. DeRyke CD, Pharm D, Wallace MR. Antimicrobial resistance update: Klebsiella pneumoniae carbapenemases. Drug Benefit Trends 2009; 21: 238-40. 3. Parisien A, Allain B, Zhang J, Mandeville R, Lan CQ. Novel alternatives to antibiotics: bacteriophages, bacterial cell wall hydrolases, and antimicrobial peptides. J Appl Microbiol 2008; 104: 1-13. 4. Sulakvelidze A, Alavidze Z, Morris JGJr. Bacteriophage therapy. Antimicrobe Agents Chemother 2001; 45: 649-59 . 5. Sandeep K. Bacteriophage precision drug against bacterial infections. Curr Sci 2006; 90: 631- 33. 6. Ackermann HW. Bacteriophage observations and evolution. Res Microbiol 2003; 154: 245-51. 7. Matsuzaki S, Rashel M, Uchiyama J, Sakurai S, Ujihara T, Kuroda M, et al. Bacteriophage therapy: a revitalized therapy against bacterial infectious disease. J Infect Chemother 2005; 11: 211-19. 8. Lu Z, Breidt JrF, Fleming HP, Altermann E, Klaenhammer TR. Isolation and characterization of a Lactobacillus plantarum bacteriophage, JL-1, from a cucumber fermentation. Int J Food Microbiol 2003; 84: 225-35. 9. McLaughlin MR, Balaa MF, Sims J, King R. Isolation of Salmonella bacteriophages from swine effluent lagoons. J Environ Qual 2006; 35: 522-8. 10. Capra ML, Quiberoni AL, Ackermann HW, Moineau S, Reinheimer JA. Characterization of a new virulent phage (MLC-A) of Lactobacillus paracasei. J Dairy Sci 2006; 89: 2414-23. 11. Watanabe K, Takesue S, Jin-Nai K, Yoshikawa T. Bacteriophage active against the lactic acid beverageproducing bacterium Lactobacillus casei. Appl Microbiol 1970; 20: 409-15. 12. Podschun R, Ullmann U. Klebsiella spp. As nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 1998; 11: 589- 603. 13. Yan JJ, Ko WC, Tsai SH, Wu HM, Wu JJ. Outbreak of infection with multidrug-resistant Klebsiella pneumoniae carrying blaIMP-8 in a university medical center in Taiwan. J Clin Microbiol 2001; 39: 4433-9 . 14. Kang CI, Kim SH, Bang JW, Kim HB, Kim NJ, Kim EC, et al. Community-acquired versus nosocomial Klebsiella pneumoniae bacteremia: clinical features, treatment outcomes, and clinical implication of antimicrobial resistance. J Korean Med Sci 2006; 21: 816-22. 15. Sundar MM, Nagananda GS, Das A, Bhattacharya S, Suryan S. Isolation of host-specific bacteriophages from sewage against human pathogens. Asain J Biotechnol 2009, 1: 163-70. 16. Wu LT, Chang SY, Yen MR, Yang TC, Tseng YH. Characterization of extended-host-range pseudo-T-even bacteriophage Kpp95 isolated on Klebsiella pneumoniae. Appl Environ Microbiol 2007; 73: 2532- 40. 17. Malik R, Chhibber S. Protection with bacteriophage K 1 against fatal Klebsiella pneumoniae induced burn wound infection in mice. J Microbiol Immunol Infect 2009; 42: 134-40. 18. Verma V, Harjai K, Chhibber S. Characterization of a T7-like lytic bacteriophage of Klebsiella pneumoniae B5055: a potential therapeutic agent. Curr Microbiol 2009; 59: 274-81. 19. Kumari S., Harjai K, Chhibber S. Efficacy of bacteriophage treatment in murine burn wound infection induced by Klebsiella pneumoniae. J Microbiol Biotechnol 2009; 19: 622-8. 20. Hagens S, Loessner MJ. Application of bacteriophages for detection and control of foodborne pathogens. Appl Microbiol Biotechnol 2007; 76: 513-9

Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524124EnglishN-0001November30HealthcareREDEFINING NUTRACEUTICALS English4451Ajay PiseEnglish Shilpa PiseEnglish D. SreedharEnglish Manthan J.English Virendra LigadeEnglish N. UdupaEnglishEnglishIntroduction Today, healthcare market is flooded with different new terminologies like Nutraceuticals, Cosmeceuticals, Biopharmaceuticals, Herbaceuticals, Ayuraceuticals, Skinceuticals, Dermaceuticals, Nutri-cosmetics and many more. Among all these new terminologies, nutraceuticals has gained more importance for the industry in India and abroad. Nowadays, nutraceuticals is a buzzword in global healthcare market. There is a growing craze for using nutraceutical products to improve quality of life. Interpretation of definitions The concept of nutraceuticals was defined and explained by different scholars and government regulatory bodies of different countries. Though the definitions slightly differs, but essence of all definitions remain same i.e. „food as medicine? The commonly adopted definition of a nutraceutical by food marketing industry is, “any food or food ingredient which is considered to have a beneficial effect on health”. Such description of nutraceutical is broadly used and can refer to anything from a vitamin supplement pill, to an energy enhancing drink, and more recently to foods which are claimed to have physiological effects. Term Nutraceutical is coined by Dr. Stephen DeFelice in 1989; he fused two words Nutrition and Pharmaceutical to design the term. He has defined nutraceuticals as, “Food, or parts of food, that provide medical or health benefits, including the prevention and treatment of disease is called as Nutraceuticals”1 According to this definition food parts such as garlic, soybean are considered as Nutraceuticals. This definition does not specify that crude or processed food (part of food) would be used as nutraceuticals. As per this definition whatever food we take in any form (crude or processed) would be considered as nutraceuticals. Such products may range from isolated nutrients, dietary supplements and specific diets to genetically engineered designer foods, herbal products, and processed foods such as cereals, soups and beverages. Also this definition does not differentiate between nutraceuticals, functional food, and dietary supplement. Health Canada has adopted different definition saying “A product isolated or purified from foods, and generally sold in medicinal forms not usually associated with food and demonstrated to have a physiological benefit or provide protection against chronic disease”9 Unlike other definitions this definition has clearly specified that nutraceuticals are the isolated or purified products of food. This definition does not include the word „treatment? of disease; it reflects that the concept of nutraceuticals is confined to „health benefits? and „prevention of disease?. As per this definition, if we are using isolated active ingredient of garlic in medicinal form for health benefits then it is categorised as Nutraceuticals. Alike previous definition, this definition also does not differentiate between functional food, dietary supplement, and designer food. Another widely adopted definition of nutraceuticals is that of Zeisel (1999) who described them as “Diet supplements that deliver a concentrated form of a presumed bioactive agent from a food, presented in a nonfood matrix, and used to enhance health in dosages that exceed those that could be obtained from normal foods.” Though different scholars have attempted to frame different definitions of Nutraceuticals, essence of all remains same. Several attempts have been made to define Functional food, Designer food, and Dietary supplements but ambiguity exist in interpretating clear differences between Nutraceuticals and these terminologies. Nutraceuticals, Dietary supplement, Functional food Nutraceuticals are also been called medical foods, designer foods, phytochemicals, functional foods and nutritional supplements. Many different terms and definitions are used in different countries, which can result in confusion. Nutraceuticals can refer to foods, dietary supplements, medical foods, and functional foods that may provide prevention and treatment of illness or disease. Some of the researchers have classified dietary supplement, and functional food under the term „Nutraceuticals?. Whereas some researchers are of opinion that nutraceuticals, functional food, and dietary supplements are different terminologies. These terminologies are further discussed as- Dietary Supplements including botanicals Vitamins, minerals, coenzyme Q, carnitine Gingsing, Gingko Biloba, Saint John's Wort, Saw Palmetto Functional Foods Oats, bran, psyllium and lignin's for heart disease and colon cancer Prebiotics - oligofructose for control of intestinal flora Omega-3 milk in prevention of heart disease Canola oil with lowered triglycerides for cholesterol reduction Stanols (Benecol) in reduction of cholesterol adsorption Medicinal Foods Transgenic cows and lactoferrin for immune enhancement Transgenic plants for oral vaccination against infectious diseases Health bars with added medications The term “Functional Foods” was first introduced in Japan in the mid 1980s. It refers to processed foods containing ingredients that aid specific body functions, in addition to being nutritious14 . The term nutraceuticals and functional food has no legal significance in the United States. In the United States this industry falls under dietary supplement. The DSHEA formally defined "dietary supplement" using several criteria. A dietary supplement2,10 - is a product (other than tobacco) that is intended to supplement the diet that bears or contains one or more of the following dietary ingredients: a vitamin, a mineral, an herb or other botanical, an amino acid, a dietary substance for use by man to supplement the diet by increasing the total daily intake, or a concentrate, metabolite, constituent, extract, or combinations of these ingredients. is intended for ingestion in pill, capsule, tablet, or liquid form. is not represented for use as a conventional food or as the sole item of a meal or diet. is labeled as a "dietary supplement." includes products such as an approved new drug, certified antibiotic, or licensed biologic that was marketed as a dietary supplement or food before approval, certification, or license (unless the Secretary of Health and Human Services waives this provision). In Korea, functional foods are defined as dietary supplement whose purpose is to supplement the normal diet and have to be marketed in measured doses, such as in pill, tablets14 . Health Canada has defined functional food as “A functional food is similar in appearance to, or may be, a conventional food that is consumed as part of a usual diet, and is demonstrated to have physiological benefits and/or reduce the risk of chronic disease beyond basic nutritional functions, i.e. they contain bioactive compound”. Presently, there is no universally accepted term for functional foods14 Health Canada has defined „Novel Food? as “Products that have never been used as food; foods that result from a process that has not previously been used for food; or, foods that have been modified by genetic manipulation. This category of foods are also refered as genetically modified foods”. In the opinion of Ekta K. Kalra, nutraceuticals differ from dietary supplements in the following aspects: Nutraceuticals must not only supplement the diet but should also aid in the prevention and/or treatment of disease and/or disorder. Nutraceuticals are represented for use as a conventional food or as the sole item of meal or diet. Nutraceuticals and Drugs Dictionary definition of drug says “Drug is any substance that, when absorbed into the body of a living organism, alters normal bodily function12”. Drug can also be defined as, “A substance used in the diagnosis, treatment, or prevention of a disease or as a component of a medication13” . As per the definition of nutraceuticals given by Dr. Stephen DeFelice, nutraceuticals are food or part of food which is used for health benefits including the prevention and treatment of disease. As per the definition of nutraceuticals given by Health Canada nutraceuticals are “Product isolated or purified from foods, and generally sold in medicinal forms not usually associated with food and demonstrated to have a physiological benefit or provide protection against chronic disease” Comparative analysis of above definitions suggest that nutraceuticals are not drugs but their effect is like drug. As per above discussed definitions nutraceuticals cannot be used in the treatment of the disease but it can be used to prevent the disease and for other health benefits. Therefore nutraceuticals are „Quasi Drugs? i.e. „Like Drugs?. Need of harmonized definition Today there is a craze for being healthy and maintaining health (baby-boomers), increasing awareness about preventive medicines under the concept of “prevention is better than cure”, heavy cost of allopathic treatment and attractive promises of nutraceuticals, greater acceptance by healthcare community and general perception of “natural is always good and safe” are a few factors responsible for rapid emergence of the Nutraceutical concept. Therefore nutraceutical products are becoming more popular in healthcare market around the globe. As demand is increasing for nutraceutical products, many pharmaceutical companies started launching the nutraceutical products in the market. As a result, market is flooded with several nutraceutical products. Present regulations for Pharmaceuticals are not applicable for the manufacturing and sale of the nutraceuticals. This has triggered manufacturing activities of nutraceuticals by many companies without considering the regulatory aspects. As the term Nutraceuticals is not recognised and defined officially by many countries, it remains as a market term. Health Canada has adopted the term and framed certain regulatory guidelines. India Government and US FDA have not yet recognised the term. Absence of regulatory guidelines for nutraceuticals in a country like India results in heavy mushrooming of nutraceutical manufacturers and market may be flooded with several products with lofty claims without sufficient scientific evidences. In order to survive in competition and to reduce the price, manufacturers may compromise with quality of product, which may lead to disaster of health of society. Analysis of all these factors suggests that there is a need of harmonized definition of nutraceuticals which would be adopted globally. Such definition should clearly differentiate between the term nutraceuticals, dietary supplement and functional food. Proposed definition Still there is an ambiguity in distinguishing dietary supplements, nutrient supplements and drugs from nutraceuticals. We have attempted to frame a comprehensive definition of nutraceuticals. “Any natural product in its crude or processed form if taken for expected health benefits based on nutrient supplement and its physiological action can be explained scientifically in animals and human being is referred as nutraceutical product” Explaination of terms used in the definition Natural product: Product from natural sources including plant, mineral and animal sources Expected health benefits: For expected betterment of health including treatment, cure, mitigation or prevention of disease Scientifically explained physiological action: Scientifically explained mechanism of action of the product which supports the claims of beneficial effects in animals or human being Conclusion Though Nutraceuticals is considered as a bright landscape in healthcare market, it is in nascent stage. Many scholars around the world have proposed different definitions but no definition is considered as harmonised definition. This lack of harmonised definition is a major challenge. Absence of regulatory guidelines and lack of general awareness are also considered as challenges in development of the concept of Nutraceuticals. The proposed definition of nutraceutical would help in differentiating the concept of drug, functional food, dietary supplement and novel food. There is a need of accepting one harmonised definition and framing proper regulatory guidelines to control the excessive mushrooming of nutraceutical manufacturers. Englishhttp://ijcrr.com/abstract.php?article_id=2249http://ijcrr.com/article_html.php?did=22491. Ekta K. Kalra, “Nutraceutical - Definition and Introduction” cited from http://www.aapspharmsci.org/view.a sp?art=ps050325. Accessed on 12 Apr 2006 2. P A Francis, “A Regulation for Nutraceuticals” cited from http://www.pharmabiz.com/article/de tnews.asp?articleid=12303§ioni d=47. Accessed on 20th Apr 2006 3. R. K. Rishi, “Nutraceuticals: borderline between food and drugs” published in „The Pharma Review? vol 4 no. 20 (Feb. 06 ) 4. “Market status of nutraceuticals” cited from http://www.bccresearch.com/food/G A085R.html.Accessed on 12th Apr 2006 5. V. D. Deshmukh, “Nutraceuticals: dietary supplements a legal dilemma” IDMA 30th June 2005 no. 24, pg 38-39 6. Raja Prasanna, “Nutraceuticals to gain ground globally” published in „The Pharma Review? vol 4 no. 20 (Feb. 06) 7. „Nutraceuticals Market Review? cited at http://www.teknoscienze. com/agro/pdf/nov_dec03/bioceutical s.pdf. Accessed on 12th Apr 2006 8. “Regulatory status” cited from http://www.globalregulatory.com/lab class/nutraceutical_consultants.cfm. Accessed on 25 Apr 2006 9. “Introduction of Nutraceuticals” cited at http://www.anajana.org/nut_info_details.cfm?NutInf oID=4. Accessed on 25th Apr 2006 10. Presentation for FDA public hearing, June 8, 1999- Washington, DC by Gary L. Huber published in American Nutraceuticals Association newsletter. 11. „Nutraceuticals? cited at http://chemistry.about.com/od/chemi stryglossary/a/nutraceuticaldf.htm 12. „Definition of Drug? cited at http://en.wikipedia.org/wiki/Drug 13. „Definition of Drug? cited at http://www.answers.com/topic/drug 14. “Industry Insights-Nutraceuticals” report published by Cygnus Business Consulting and Research, Hyderabad

Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524124EnglishN-0001November30General SciencesANTI BACTERIAL ACTIVITY OF BARK EXTRACTS OF PTEROCARPUS MARSUPIUM ROXB. English5255H.R.AmbujakshiEnglishAnti bacterial activity of ethanol and water extracts of barks of Pterocrpus marsupium were tested by cup-plate agar diffusion method against Staphylococcus aureus, Bacillus sterothermophilus (Gram +ve) and Eschericia coli , Klebsiella pneumoniae (Gram -ve) bacteria. Ciprofloxacin was used as the standard. It was observed that all extracts have dose dependent inhibitory effect, ethanol extract being most effective. EnglishAntibacterial, Pterocrpus marsupium, cup-plate.Pterocarpus marsupium Roxb.(Leguminosae) is also known as Indian Kino tree or Bijasar, is common in the hilly regions of central and peninsular india1 .The extracts of leaves, flowers and gum obtained from the stem of this tree have been used medicinally in the treatment of diarrhea, toothache, fever, urinary tract and skin infections2 .The extract of the bark has been regarded as useful in the therapy of diabetes3 .The aqueous extract of the stem bark was found to reduce the blood glucose level in alloxan - induced diabetic rats4 . The ethnic uses includes the use of stem bark of this species in treating wounds, fever, stomachache, diabetes and jaundice5 .The current study was undertaken to evaluate the antibacterial activity of ethanol and water extracts of Pterocarpus marsupium Ethanol and water extracts were tested against Staphylococcus aureus, Bacillus sterothermophilus (Gram +ve) and Eschericia coli , Klebsiella pneumoniae (Gram -ve). Ciprofloxacin was used as the standard Materials and Methods Plant material The fresh barks of Pterocarpus marsupium Roxb were collected from the Jeddu Ayurveda vanaspathi abivriddi parishat, Bantwal Taluk, D.K. District, Karnataka. Extraction of plant material The barks were dried under sunny condition at ambient temperature and then ground by using grinder, to coarse powder and the powder was packed in soxhlet column and extracted with ethanol for 24 hrs. Successively drug is boiled for 30min using distilled water. The extracts were concentrated under reduced pressure at 50° C using rotary evaporator to a powder mass, which was then stored at 40° C until used. Crude extract was subjected to preliminary phytochemical screening to characterize the phyto constituents present. Screening for antibacterial activity Antibacterial activity was tested by cup-plate agar diffusion method to determine the zone of inhibition of two extracts. The extracts were freshly reconstituted with suitable solvents (H2O / DMSO) tested at concentration of 800 and 400 µg/ml against all the microorganisms. Ciprofloxacin (Himedia laboratories, Mumbai,) were separately dissolved in DMSO to give a concentration of Ciprofloxin 20 µg/ml. Sterile nutrient agar plates were prepared and 0.1 ml of 4he inoculums from standardized culture of test organism was spread uniformly. Wells were prepared by using a sterile borer of diameter 10mm and 100µl of the test substance, standard antibiotic and the solvent control were added in each well separately. The plates were placed at 4 0C for 1 h to allow the diffusion of test solution into the medium and plates were incubated at 370C for 24 h and the zone of inhibition of microbial growth around the well was measured in mm. Result and discussion: Preliminary phytochemical studies revealed the presence of tannins, flavonoids and glycosides. From the zone of inhibition produced by the extracts, it was observed that a concentration dependent antibacterial activity was observed with both alcoholic and water extracts. The alcohol extract was more potent than water extract. The antimicrobial activity may be due to the presence of tannins. Other constituents like flavonoids may also contribute to the activity. Acknowledgement: Authors are thankful to Mr. Premnath Reddy, Chairman, Acharya & B.M. Reddy College of Pharmacy, Bangalore for providing laboratory facilities to carry out this project. Englishhttp://ijcrr.com/abstract.php?article_id=2250http://ijcrr.com/article_html.php?did=22501. Jain, S. K., Medicinal plants, National Book Trust, New Delhi, 1968, p.116 2. Chopra R N, Nayar SL, Chopra IC, Glossary of Indian medicinal plants. New Delhi: CSIR: 1956. 3. Kirtikar, K.R and Basu, B.D., Indian Medicinal plants, 2nd Ed., Delhi, India, 1975,272 4. Vats V. Grover Jk, Rathi SS., J. Ethnopharmacol.2002, 79, 95-100. 5. Mankni K.L, Krishna V, Manjunath B.K, Vidya S.M, Jagadeesh Singh S.D, Manohara Y.N, AneesurRaheman, Avinash k.R, Indian J Pharmacol. June 2005, 37, 3, 165-168 6. Ali Ms, Ahmad VU, Usmanghani K, Azhar I and Amtul Z.,Fitopteropia,1999, 70; 299-304