J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2

ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2

'Nexus' - Alumni Association SJCOPS, Chalakudy 1 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ISSN : 2350-1332

2 'Nexus' - Alumni Association SJCOPS, Chalakudy ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2

Contents Page No.

• Isolation of a Triterpenoid from Methanolic Extract of Leaves of Bischofia Javanica Blume from Sikkim Himalaya 1 - 5 L.Sutharson, L.K.Nath, J Rajan Vedasiromoni, G.Mariappan

• Development and Validation of Spectrophotometric Method for Simultaneous Determination of Montelukast Sodium and Levocetirizine Hydrochloride 6 - 10 N.Tamilselvi, Abdel Salam Hamid idris Mohamed, Basheer C, Nidhin Louis L

• Formulation and In-Vitro Evaluation of pH Sensitive Mebeverine Hydrochloride Nanoparticles for Colon Targeting 11 - 19 Bhaktha Pragalathan G, Ayyappan T, Vetrichelvan T

• A Retrospective Study to Determine the Prevalence of Left Ventricular Systolic Dysfunction 20 - 23 Jayakumar K.S, Anitha L, Harikrishnan S, Perumal P

• An Insight into Chemometrics Concepts in Pharmaceutical Analysis 24 - 33 N. Satheeshkumar, S. Shantikumar, David Paul

• A Review on Clinical Trials of Natural Polyphenol Nutraceuticals in the Management of Diabetes 34 - 42 K. Umaa, V. Abinaya, T. Gokul, G. Saipriya, A. Nagarajan

• Microchip: New Era in Novel Drug Delivery Systems - A Review 43 - 55 P. Manikandan, R.Nethaji, N.S.Surenderan, G.Babu

• In-vitro Techniques for Cytotoxic Evaluation 56 - 69 Rajasree R.S, Greeshma P, Sibi P.I.

• A review on analytical tools used for controlling drug expenses 70 - 74 Siraj Sundaran, Sanjay Sreekumar, Babu G.

• Green Coconut Water-Alginate Hydrogel: Anti-microbial Studies 75 - 80 Anish John, Dineshkumar B, Krishnakumar K, Amulya K. Mohan, Jaison T. James, Sincy Sunny P.

'Nexus' - Alumni Association SJCOPS, Chalakudy J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ISSN : 2350-1332

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Published by “NEXUS”-Alumni Association SJCOPS St James College of Pharmaceutical Sciences (SJCOPS) Chalakudy-680307, Kerala

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'Nexus' - Alumni Association SJCOPS, Chalakudy ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2

Journal of Pharma Innovative Research (JPIR)

Published by (Registration No: KERENG02482, Govt. of India) 'NEXUS' - Alumni Association SJCOPS St James College of Pharmaceutical Sciences, Chalakudy - 680 307

Approved by Government of India Ministry of Information and Broadcasting, New Delhi- 110066

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Journal of Pharma Innovative Research (JPIR) is intended primarily to promote prompt publication (original research articles and review articles) in all fields of pharmaceutical science. Specific scientific topics of interest to the journal include: Pharmaceutics, Pharmaceutical Chemistry, Industrial Pharmacy, Pharmacology, Pharmacognosy, Phytochemistry, Pharmacodynamics, Pharmacokinetics, Pharmacogenomics, Biopharmaceutics, Physical Pharmacy, Drug Design, Pharmaceutical Analysis, Drug Stability, Quality Control and Assurance, Pharmaceutical Engineering, Hospital and Clinical Pharmacy.

Chief Editor Dr. K. Krishnakumar M.Pharm., Ph.D. The Chairman 'NEXUS' - Alumni Association SJCOPS St. James College of Pharmaceutical Sciences Chalakudy-680307, Kerala

'Nexus' - Alumni Association SJCOPS, Chalakudy J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ISSN : 2350-1332

Editorial - Advisory Board 1. Dr. (Prof). Ramadas medical Superintendent Regional Cancer Centre, Trivandrum 2. Dr. N. Udupa Research Director, manipal University, Manipal 3. Dr. K.B. Sudhikumar dean Student Affairs Kerala University of Health Sciences, thrissur 4. Dr. C. I. Jolly Research Adviser CARe KERALAM LTD, Koratty, Kerala 5. Dr. T.K. Ravi the Principal College of Pharmacy Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore 6. Dr. S. Gopalakrishnan Professor of Pharmaceutics College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore 7. Dr. Jalaja S. Menon Assistant Professor Kerala Agricultural University, Thrissur 8. Dr. Seethalakshmi K.K. Research Programme Coordinator Kerala Forest Research Institute Peechi, Thrissur 9. Dr. Sheela Karalam B R&D Special Officer oushadhi, Thrissur 10. Dr. M. Kannapiran Professor RVS Dental College and Hospital, Coimbatore 'Nexus' - Alumni Association SJCOPS, Chalakudy ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2

Journal of Pharma Innovative Research (JPIR)

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'Nexus' - Alumni Association SJCOPS, Chalakudy J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ISSN : 2350-1332 . Example: Mandell GL, Petri WA; Antimicrobial agents: Penicillins, Cephalosporins and other beta lactam antibiotics. In Goodman and Gillman’s: The Pharmacological basis of Therapeutics. 9th Edition, Hardman JG, Limbard LE editors, McGraw-Hill, New York, 1996:1073-1101. For Thesis

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'Nexus' - Alumni Association SJCOPS, Chalakudy ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2

Sutharson et.al.,/ Journal of Pharma Innovative Research, 2015; 2(1): 1-5 Isolation of a Triterpenoid from Methanolic Extract of Leaves of Bischofia Javanica Blume from Sikkim Himalaya L.Sutharson*1, L.K.Nath2, J Rajan Vedasiromoni3, G.Mariappan4

1K.T.N College of Pharmacy, Chalavara, Palakkad,Kerala - 679505 2Department of Pharmaceutical Sciences, Dibrugargh University,Assam-786004 3National Institute of Pharmaceutical Education and Research, Kolkata- 700032 4Department of Pharmaceutical chemistry, Himalayan Pharmacy Institute,Sikkim-737136 Corresponding Author Email:[email protected]

ABSTRACT Sikkim is the state abundant with medicinal plants. The methanolic extract of leaves of Bischofiaja- vanicaBlume (BJ) had shown significant anti-inflammatory and anticancer activities in our previous research work. Hence in this research work leaves ofBJwas undergone for isolation of triterpenids. Methnolic extract of BJ has been isolated by column chromatography followed by TLC using n-hexane-chloroform-ethylacetate as mobile phase in different ratio.The sub fraction (SF-7-10) of F-18 to F-22 in the eluting solvent system of n-hexane-chloroform-ethyl acetate in the ratio of 7:2:1 gave the pure triterpenoid.The sample was further studied by IR, 1H and 13C NMR and Mass spectra and confirmed that Friedelin-3α-acetate with molecular formula C32 H54O2. Key words: Triterpenoid;Column chromatography

INTRODUCTION Sikkim is a very small hilly state in the Eastern Himalayas with a total geographical area of 7096. sq. kms. Sikkim Himalaya is abundant with medicinal plants, herbs, shrubs, and bamboo. More than 242 species of medicinal plants are reported to be found in the state[1].BischofiajavanicaBlume (Euphorbi- aceae), known as kainjal in Nepali a evergreen tree widely distributed over the Southeast Asia, Japan and Australia. In India it is distributed over the Sub-Himalayan region, Orissa and south west coast from Konkan to Nilgiris. [2] This plant has been utilized significantly for various ailments like topical treatment for ulcer, sores and boils. [3] It possesses antiulcer, antimicrobial, anthelmintic and antidysen- teric activities. Tribes of Chhattisgarh and Sikkim use the leaf juice of the plant for the treatment of cancerous Wound. [4]B. javanica have been reported to contains the phytoconstituents like tannin, β-amyrins, betulinic acid, chrysoeriol, ellagic acid, fiestin, friedelan-3-alpha-ol, epifriedelinol, friedelin, luteolin and glucoside, quercetin, quercitrin, β-sitosterol, stigmosterol, ursolic acid, triacontane, fatty acids including linoleic acid, stearic acid, iscchofianin, corigalin, furosin, geranin, punicalagin[5-6]Anti- inflammatory and analgesic potency of BJ were studied by the authors in a previous research work. [7] Methanolic extract of leaves of BJ has shown significant anticancer activities on human leukemic cell

'Nexus' - Alumni Association SJCOPS, Chalakudy 1 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ISSN : 2350-1332 lines and the presence of triterpenoids could be the reason behind the activities [8]. In this research work we have made an attempt to isolate triterpenoids from BJ.

MATERIALS AND METHODS Extraction of Phytoconstituents from Bischofiajavanica Collection of Plant Material The leaves of Bischofiajavanica were collected from Melli region of Sikkim (India) in the month of June. The plant material was identified and authenticated at Botanical Survey of India (BSI), Sikkim branch. A herbarium was also kept in the parent institute for future reference (LS/BJ/03/RPS). The collected leaf of B.javanica was shade dried for 15 days and size reduced using laboratory grinder in to coarse powder. It was stored in a well closed container free from environmental changes till usage.

Extraction Procedure The powdered leaves (5 Kg) were extracted with methanol in soxhlet apparatus at 50-60°C. After exhaus- tive extraction the extract was concentrated by distilling the solvent for further use. The concentrated extract was kept in the desiccator. Yields of the prepared extract were 5.0 %w/w (250g) of the dried leaf powder. The extract was used for the study of preliminary chemical analysis[9-10].The methanol extract was concentrated, suspended in distilled water and partitioned with petroleum ether, chloroform. [11- 12] The chloroform soluble fraction was subjected to thin layer chromatographic analysis. The aqueous and petroleum ether fraction did not show any positive pharmacological activities under perview of this investigation and was discarded. Dried powdered leaves of Bischofiajavanwas extracted with methanol. Methanol extract shown positive test for triterpenoid. Methanol extract was Partitioned- pet ether, chloroform and water. Pet-ether fraction showed absence of triterpenoid. Chloroform fraction showed presence of triterpenoid and aqueous fraction showed absence of triterpenoid). Then the Chloroform fraction was subjected to chromatographic separation.

Isolation and Purification of Phytoconstituents from the Leaf of B.javanica The dried solid chloroform fraction of the leaf extract and silica gel60-120 (E.Merck) was layered on the top of the glass column, 80 cm in length, 3.5 cm diameter. The bottom of the column was plugged with glass wool. A thin layer of cotton was placed over it. The solvent was allowed to flow down slowly till the mixture was adsorbed on the top of the column. Gradient elution was carried out using n-hexane and ethyl acetate as solvent by increasing the polarity of n-hexane by adding increments of ethylacetate. [13-14] The fractions obtained from the column chromatography using different ratios of n-hexane: ethyl acetate were numbered as F-1 to F-7 for the ratio 10:0, F-8 to F-17 for the ratio 9:1, F-18 to F-23 for 8:2, F-24 to F-35 for 7:3 and F-36 to F-49 for 6:4. TLC study was performed on the fractions and the fraction F-18 to 22 revealed the elution of the mixture of triterpenoid amongst all the fractions. So the fractions were combined together and was further purified in column chromatography using n-hexane- chloroform-ethyl acetate (8:1:1, 7:2:1, 6:3:1) as solvent system. The sub fraction (SF-7-10) of F-18 to F-22 in the eluting solvent system of n-hexane-chloroform-ethyl acetate in the ratio of 7:2:1 gave the pure triterpenoid, which was confirmed by TLC studies. The isolated compound LS-1 was further examined by different physico-chemical techniques for its structure elucidation. The infrared (IR) absorption spectra of the isolated compound were taken with Perkin Elmer FTIR-1750

2 'Nexus' - Alumni Association SJCOPS, Chalakudy ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 spectrophotometer, in potassium bromide discs. The spectra were recorded in the region of 4000 cm-1 to 400 cm-1. The spectra of the isolated compound with absorption bands were recorded and tabulated. The 1H and 13C NMR spectra of the isolated compound from chloroform fraction of B.javanica leaves were undertaken in Brucker DRX 600 Spectrometer in CDCl3 (Duterated chloroform) solution. The spectra are recorded and tabulated.Mass spectrum (ES-MS) of the isolated compound from the leaves of B.javanicawas recorded on a Micromass Quattro II instrument.

RESULTS AND DISCUSSION The concentrated methanol extract obtained from the shade-dried leaf of B.javanicawas fractionated successively with petroleum ether, chloroform and water. It was observed that only the Chloroform fraction exhibited significant pharmacological activities under purview of this investigation. So this fraction was subjected to phytochemical analysis. The preliminary phytochemical group tests indicated the presence of steroids, flavonoids, proteins, tannins, saponins and reducing sugar (Table 1). The thin layer chromatographic study of the chloroform fraction of methanol extract of B.javanica showed the presence of about five components with selected mobile phases and spraying reagent. This fraction was subjected to coloumn chromatographic separation on Silica gel coloumn in which forty-nine fractions were collected. The fractions having identical Rf values (F-18-22) from TLC studies were mixed together. They were again purified with a silica gel (60-120) coloumn, which yielded a pure compound (LS-1) which was again confirmed by TLC studies. The compound is a white crystalline powder,melting point is 292-294°C, insoluble in water, soluble in organic solvents. It was further subjected to chemical tests and TLC studies to confirm the chemical nature of the compound LS-1, which are presented in. The chemical nature of the isolated compound was further characterized from its physical parameters and spectral (IR, Mass, 13C and 1H NMR) data [15- 16]. The IR spectrum shows the presence of absorption bands at 2931, 1738 and 1245 cm-1. Other prominent peaks were 2870, 1461, 1386, 1025 and 526 cm-1.The IR spectrum of the compound LS-1 revealed the presence of carbonyl group (1738 cm-1), C-H stretching and bending were confirmed by absorption at 2870 and 1461 cm -1. The 1H NMR data revealed the eight singlet methyl protons in the compound showing the signals at 1.16, 1.18, 1.13, 1.04, 0.99, 0.82, 0.76 and 0.98 ppm. The signal at 4.36 ppm revealed the acetyl substitution at C-3 and the singlet at 2.03 ppm confirmed the acetyl group. The methylene link in the skeleton showed α and β configuration and the signalbetween 1.24-1.59 ppm.13C NMR data revealed the 32 carbons in the skeleton with eight methyl groups. The acetyl group at C-3 showed the chemical shift at 75.17 and the C=O group has provided the δC at 171. Further DEPT (Dis- tortionless Enhancement by Polarization Transfer)-90and DEPT-135 studies confirmed singlet, doublet, triplet and Quadralet carbons of 13C NMR studies and inferred the pentacyclictriterpenoid skeleton for the test compound LS-1 [17-19](Table 2). The Mass spectra (ES-MS) showed the m/z (%) 494{M+Na+} (40), 472{M+1} (20) and confirmed the compound with molecular weight 470.77. From the available literature on Friedelin compounds and the 1H NMR, and 13C NMR spectral data, [17-20] the structure of the compound LS-1 was found to be Friedelin-3α-acetate with molecular formula C32 H54O2 (Fig- ure1).

CONCLUSION The study was concluded that the triterpenoid (Friedelin-3α-acetate) was isolate from methanolic extract of BischofiaJavanicaBlume leaves and its structure was characterized.

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Table1:Preliminary phytochemical test of methanol extract of B. javanicaleaf

Phytoconstituents B.javanica Methanol Extract Chloroform Fraction Alkaloids + + Amino acids _ _ Proteins + _ Reducing sugar + _ Steroids and triterpenoids + + Flavanoids + + Tannins + _ Saponins + + Anthraquinones _ _ + veindicates presence and – ve indicates absence of the phytoconstituents

Table2:Hydrogen and carbon chemical shifts of compound LS-1 isolated from the leaves of Bischofiajavanica

Position δH Position δC Position δH Position δC H-1 1.59(α) C-1 17.83 H-16 1.46(α) C-18 42.77 1.36(β) C-2 32.68 1.54(β) C-19 35.55 H-2 1.58(α) C-3 75.17 H-17 1.47 C-20 28.16 1.38(β) C-4 49.99 H-18 1.51 C-21 32.77 H-3 4.63 C-5 38.27 H-19 1.78(α) C-22 39.25 H-4 1.55 C-6 41.3 1.29(β) C-23 9.92 H-6 1.25(α) C-7 19.31 H-21 1.76(α) C-24 14.48 1.48(β) C-8 52.95 1.28(β) C-25 18.11 H-7 1.35(α) C-9 36.97 H-22 1.58(α) C-26 18.21 1.49(β) C-10 59.85 1.25(β) C-27 20.15 H-9 1.51 C-11 35.3 CH3-23 0.98 C-28 31.78 H-10 1.53 C-12 30.55 CH3-24 0.76 C-29 35.01 H-11 1.34(α) C-13 39.65 CH3-25 0.82 C-30 32.08 1.42(β) C-14 38.36 CH3-26 0.99 C-31 171.0 H-12 1.37(α) C-15 32.32 CH3-27 1.04 C-32 21.37 1.27(β) C-16 36.02 CH3-28 1.13 H-15 1.50 C-17 39.99 CH3-29 1.18 CH3-30 1.16 COOCH3 2.03

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Figure 1Structure of Friedelin-3α-acetate (Compound LS-1)

REFERENCES 1. Sikkim forest gov [home page on internet][cited 21/07/2004].Available from http://www.sik- kimforest.gov.in/medicine_main.htm. (Accessed on 15.1.15) 2. Nayar C, Chopra K. Glossary of Indian Medicinal Plants. New Delhi: CSIR Publications; 1970.p.37. 3. Cambie RC, Ash J. Fijian Medicinal Plants. Australia: CSIRO Publications; 1984.p.1441- 45. 4. Mishra SS, Tewrai RS .Chemical investigation of Bischofiajavanica. Current Science 1971;40:11-12. 5. Perry LM, Metzger J. Medicinal plants of East and Southeast Asia. London: MIT Press; 1980.p.32-139. 6. Khan MR, Kihara M, Omoloso AD. Anti-microbial activity ofBidenspilosa, Bischofiajavani- ca, Elmerilliapapuanaand Sigesbekiaorientalis. Fitoterapia 2001;72:662-665. 7. Sutharson L, Nath LK, Kar PK, Shila EB, Rajan JV. Anti-inflammatory and antinociceptive activities of methanolic extract of leaves of BischofiajavanicaBlume on experimental animals. Asian J Chem 2007;7:5150-6. 8. SutharsonLingadurai, Soma Roy, RajanVedasiromoniJoseph,Lila Kant Nath. antileukemic activity of leaf extract of BischofiajavanicaBlume on human leukemic cell lines. Indian Jour- nal of Pharmacology 2011;43(2):143-149. 9. tyler VE, Brady LR. Pharmacognosy. Philadelphia: Lea and Febiger Publishers;1988.p.123- 134. 10. Evans WC. Trease and Evans Pharmacognosy. London: W.B.SaundersCompany Limited; 2002.p.171-393. 11. Su NB, Kang YH, Pinos RE, Santarsiero BD, Mesecar AD, Soejarto DD, Fong HS, Pezzuto JM, Kinghorn AD. Isolation and absolute stereochemistry of coussaric acid, a new bio-active triterpenoid from the stems of Coussareabrevicaulis. Phytochemistry 2002;64:293-302. 12. Lieu X, Cui Y, Yu Q, Yu B. Triterpenoids from Sanguisorbaofficinalis. Phytochemistry 2005;66:1671-1679. 13. Pathak A, Kulshreshta DK, Maurya R. Coumaryltriterpene lactone, phenolic and naphthalene glycoside from stem bark of Diospyrosangustifolia. Phytochemistry 2004;65:2153-2158.

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Tamilselvi et.al.,/ Journal of Pharma Innovative Research, 2015; 2(1): 6-10 Development and Validation of Spectro- photometric Method for Simultaneous Determination of Montelukast Sodium and Levocetirizine Hydrochloride N.Tamilselvi*, Abdel Salam Hamid idris Mohamed, Basheer C, Nidhin Louis L KMCH College of pharmacy, Coimbatore Corresponding Author Email: [email protected]

ABSTRACT A simple and sensitive spectrophotometric method has been developed for simultaneous determination of Montalukast sodium and Levocetirizine hydrochloride in a binary mixture. In the proposed method, the absorbances were measured at 229.0 nm and 284.0 nm corresponding to the absorbance maxima of Montalukast sodium and Levocetirizinein methanol.Linearity range observed was 4 to 20 µg/ml for Montalukast sodium and 2 to 10 µg/ml for levocetirizine hydrochloride. LOD and LOQ were 1.1 and 0.7 µg/ml for Montalukast sodium and 3.3 and 2.1 µg/ml for Levocetirizine hydrochloride. Developed method was applied to laboratory mixture and its marketed formulation. The method was validated statistically and recovery study was performed to confirm the accuracy of the method. The method was found to be rapid, simple, accurate and precise. Key-words: Levocetirizine hydrochloride; simultaneous-equation

INTRODUCTION Levocetirizine, the (R) enantiomer of cetirizine, is a potent and selective antagonist of peripheral H1- receptors. Montelukast is a Leukotriene receptor antagonist (LTRA) used for the maintenance treatment of asthma and to relieve symptoms of seasonal allergies.1 Literature survey reveals HPLC[,2,3] and Spectrophotometric [4,5] methods for simultaneous estimation of Levocetirizine Hydrochloride and Montelukast Sodium in combined dosage form. The present work is aimed at the development of simple, rapid, accurate, precise and sensitive spectrophotometric method based on simultaneous equations for simultaneous estimation of Levocetirizine Hydrochloride and Montelukast Sodium in tablet dosage form. Developed method is validated with various parameters as per ICH guidelines like accuracy, precision, linearity. MATERIALS & INSTRUMENTS UV-VIS spectrophotometer 1700(SHIMADZU), Electronic balance AY220(SHIMADZU) were the instruments used for analysis.Methanol Reagent grade was used as the solvent.

6 'Nexus' - Alumni Association SJCOPS, Chalakudy ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 Reference Standards Levocetrizine Hydrochloride and Montelukast Sodium standard were procured Hetero pharmaceuticals,Hyderabad, which was found to be 99.56% pure. Sample used was Montek LC, Sun pharma.with a composition of Montelukast Sodium-10mg and Levocetirizine Hydrochloride-5mg

Method:

Spectral characteristics of Montelukast Sodium and Levocetirizine Hydrochloride in Methanol SHIMADZU 1700 UV-VIS spectrophotometer was used for scanning Montelukast Sodium and Levo- cetirizine Hydrochloride in Methanol (solution C) from 200-300nm after enabling a blank correction in the above region. An absorption band ranging from 200-300nm was observed with maximum absorption at 229nm AND 284nm for Levocetirizine Hydrochloride and Montelukast Sodium respectively. The spectrum obtained with solution C. Standard mixture of Levocetirizine Hydrochloride and Montelukast Sodium (each of 10µg/ml) is shown in figure4. Overlay spectrum of LevocetirizineHydrochloride (6µg/ ml) and MontelukastSodium (12µg/ml). Linearity Linearity range observed was 4 to 20 µg/ml for Montalukast sodium and 2 to 10 µg/ml for levocetirizine hydrochloride in methanol at 229 and 284nm for both of the drugs. Precision The precision of an analytical procedure expresses the closeness of agreement between a series of measurements from multiple sampling of the same homogenous sample under prescribed conditions. Preci- sion may be considered at two levels: repeatability and reproducibility. Repeatability expresses the precision under the same operating conditions over a short interval of time. The precision of an analytical procedure is usually expressed as the standard deviation of a series of measurements. The repeatability and reproducibility of the method was studied using concentrations of 8µg/ml Montelukast Sodium & 4µg/ml Levocetirizine Hydrochloride and 12µg/ml Montelukast Sodium & 6µg/ml Levocetirizine Hy- drochloride respectively. Which were prepared from stock solution B. The absorbance was measured at 229 and 284nm against Methanol as blank. The absorbance was measured two more times for each concentration and their mean values were calculated and the data is given in table no.5. The intra-day and inter-day precision study was carried for Montelukast Sodium and Levocetirizine Hydrochloride by estimating the corresponding responses three times on the same day and on three different days (1st, 2nd and 3rd day) for 12gµ/ml Montelukast Sodium and 6µg/ml Levocetirizine Hydrochloride. The results are reported in terms of relative standard deviation in Table 1,2 and 3. Accuracy The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted as reference value and the value found. Accuracy was evaluated by carrying out a recovery study and the method was found to be accurate. Recovery Studies Twenty tablets were accurately weighed and finely powdered in a mortar. The power equivalent to 100mg of Montelukast Sodium was accurately weighed out and transferred into a 100ml volumetric

'Nexus' - Alumni Association SJCOPS, Chalakudy 7 Tamilselvi et al., 2015/ JOURNAL OF PHARMA INNOVATIVE RESEARCH, 2015; 2(1): 7-11

was adjusted upto the mark with Methanol In this simultaneous equation method, the to acquire a final concentration of overlain spectra of drugs showed the Ȝmax 1000µg/ml of Montelukast Sodium & of 229.0 nm and 284.0 nm for Montelukast J. Pharm.500µg/ml Inn. Res. of | JuneLevocetirizine 2015 | Vol - 2 Hydrochloride. | Issue - 2 Sodium & Levocetirizine HydrochlorideISSN : 2350-1332 flask. Methanol (50ml) was added to it for dissolving and the solution was filtered through Whatman- nfilterpaper No 41 and the volume was adjusted upto the respectively.mark with MethanolBoth the to drugs acquire obeyed a final linearity concen - trationMeasurement of 1000µg/ml of of Absorbance Montelukast Sodium & 500µg/mlrange of Levocetirizine 4-20 ȝg/ml Hydrochloride. and 2-10 ȝg/ml a. Accurately pipetted out 1ml of the above respectively and correlation coefficient (r2) Measurementsolution and of transferredAbsorbance into a 10ml standard were found to be <1 in both cases. The a. Accuratelyflask and pipetted made out up 1ml to of the the volumeabove solution with and transferredpercentage into purity a 10ml of drugs standard in binary flask mixtureand made up to the volume with Methanol to acquire a final concentration of 100µg/ml of Montelukast Sodium & 50µg/mlMethanol of Levocetirizine to acquire a Hydrochloride.final concentration of was found to be 99.08 to 99.87 % for b. Accurately100µg/ml pipetted of Montelukast out 0.8ml of the Sodium above solution & andmontelucast transferred to sodium a 10ml and standard 98.5 toflask 101.1 and % added for known50µg/ml amount of of Levocetiriz standard solutionsine Hydrochloride. at 50, 100, and 150%Levcetrizine level and made Hydrochloride. up to the volume The with accuracy Metha - nol. The absorbance of each solution was measured at 229 and 284nm using Methanol as blank. Data obtainedb. Accurately is furnished pipettedin Table 4. out 0.8ml of the of the method was determined by RESULTSabove AND solution DISCUSSION and transferred to a 10ml performing recovery study by standard standard flask and added known amount of addition method. The % recoveries were In this simultaneous equation method, the overlain spectra of drugs showed the λmax of 229.0 nm and 284.0standard nm for M solutionsontelukast at Sodium 50, 100, & Levocetirizine and 150% Hydrochloridefound near respectively. to 100 Both % for the Montelukastdrugs obeyed linearitylevel range and 4-20 made μg/ml up and to the2-10 volume μg/ml respectively with andSodium correlation Levocetirizine coefficient Hydrochloride. (r2) were found The to be <1 in both cases. The percentage purity of drugs in binary mixture was found to be 99.08 to 99.87 % forMethanol. montelucast The sodium absorbance and 98.5 of each to 101.1 solution % for Levcetrizineexperiment Hydrochloride. was repeated T he three accuracy times of in the a methodwas was measured determined at by 229 performing and 284nm recovery using study byday standard for intra-day addition and method. on three The different % recoveries days wereMethanol found near asto 100 blank. % for DataMontelukast obtained Sodium is Levocetirizinefor inter-day Hydrochloride. precision. T Thehe experiment method was was repeated three times in a day for intra-day and on three different days for inter-day precision. The method was foundfurnished to be in precise Table 4.as % RSD for intra-day and inter-dayfound precision to be precise were

Table no: 1 Intraday study of MontelukastSodium Table no: 1 Intraday study of MontelukastSodium

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Tamilselvi et al., 2015/ JOURNAL OF PHARMA INNOVATIVE RESEARCH, 2015; 2(1): 7-11

Table no: 2 Intraday study of Levocetirizine Hydrochloride

Table no: 3 Interday studies Of Montelukast Sodium and LevocetirizineHydrochloride

Table no: 4 Accuracy (Recovery studies)

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CONCLUSION The proposed method is simple, precise, and accurate for the rapid for simultaneous determination of Montelukast Sodium Levocetirizine Hydrochloride in combined tablet dosage forms and this method may be successfully applied in control laboratories for their determination in combined dosage form.

REFERENCES 1. ArindamBasu, KrishnenduBasak, MithunChakraborty, Inder Singh Raw at Jan-Mar2011 Vol.3, No.1, 405-410. 2. Atul S. Rathore, L. Sathiyanarayanan and K.R. Mahadik 1:106. doi:10.4172/2153- 2435.1000106. 3. SushmaSomkuwar, A.K. Pathak, Vol I, Issue 3, June 2012. 4. Choudhari A, Kale S, Abnawe B, Kuchekar V, Patil GN. Vol.2,No.1, 04-09, 5. Patel Nilam K. and Pancholi S. S. 2011, 3 (5): 135-140.

10 'Nexus' - Alumni Association SJCOPS, Chalakudy ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2

Bhaktha et.al.,/ Journal of Pharma Innovative Research, 2015; 2(1): 11-19 Formulation and In-Vitro Evaluation of pH Sensitive Mebeverine Hydrochloride Nanoparticles for Colon Targeting

Bhaktha Pragalathan G*, Ayyappan T, Vetrichelvan T

Dept.of Pharmaceutics, Adhiparasakthi College of Pharmacy, Melmaruvathur,Tamilnadu Corresponding Author Email:[email protected]

ABSTRACT Mebeverine Hydrochloride (MBH) is having poor oral bioavailability and has some adverse effects, even though it shows direct action on the smooth muscle of the GIT (especially colon), because of this reason the MBH is used as model drug for site-specific drug delivery system. The purpose of research work was to develop Eudragit® EPO based nanoparticle suspension of MBH in order to increase its solubility and efficacy. The nanoparticle suspension was prepared by nanoprecipitation technique. The 32 factorial designswere used to study the effect of Eudragit® EPO and Pluronic® F-68 on characteristics of nanoparticle suspension of MBH. The optimized formulation was subjected to lyophilization. The lyophilized nanoparticle suspension and the redispersed lyophilized nanoparticle suspension were characterized by particle size, drug content, entrapment efficiency, DSC, SEM, and in-vitro drug release. Formulation F9 subjected to SEM analysis to know the size and its surface morphology. The optimized batch (Formulation F9) showed particle size 178.3 nm with entrapment efficiency 88.74±5.20%. The highest cumulative percent of drug release with formulation F9 was found to be 96.85±0.37% in 12 hours. The results of the present investigation conclude that the formulation F9 was considered as best among various formulations with respect to particle size, entrapment efficiency and in-vitro drug release.

Key-words:Nanoparticle suspension, 32factorialdesigns; Irritable bowel syndrome.

INTRODUCTION

Irritable bowel syndrome (IBS) is a disorder that leads to abdominal pain and cramping, change in bowel movements, and other symptoms. IBS is not the same as inflammatory bowel disease (IBS),

'Nexus' - Alumni Association SJCOPS, Chalakudy 11 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ISSN : 2350-1332 which includes crohn’sdisease and ulcerative colitis. In IBS, the structure of the bowel is not abnormal. IBS can be classified as either diarrhea-predominant (IBS-D), constipation-predominant (IBS-C) or IBS with alternating stool pattern (IBS-A or pain-predominant). In some individuals, IBS may have an acute onset and develop after an infectious illness characterized syndrome has consequently been termed “post-infectious IBS”(IBS-PI).

The primary symptoms of IBS are abdominal pain or discomfort in association with frequent diarrhea or constipation, a change in bowel habits. There may also be urgency for bowel movements, a feeling of incomplete evacuation (tenesmus), bloating or abdominal distention. In some cases, the symptoms are relieved by bowel movements, people with IBS, more commonly than others. Have gastro esophageal reflux, symptoms relating to the genitourinary systems, chronic fatigue, fibromyalgia, head- ache, backache and psychiatric symptoms such as depression and anxiety. Some studies indicate that up to 60% of persons with IBS also have a psychological disorder, typically anxiety or depression.

The main problem with the therapeutic effectiveness of MBH is its absorption which is highly variable and dose dependent thus reducing the bioavailability to 65–75% . MBH is soluble in acidic pH and is predominantly absorbed from upper gastro intestinal tract (GIT) to duodenum to jejunum regions. There are indications of its active absorption from the duodenum and jejunum regions of GIT 1.

The inherent shortcomings of conventional drug delivery and the potential of nanoparticle as drug delivery systems have offered tremendous scope for researchers in this field and are fast moving from concept to reality. Nanoparticle may be used for oral administration of gut-labile drugs or those with low aqueous solubility. These colloidal carriers have the ability to cross the mucosal barrier as such. In addition to the potential for enhancing drug bioavailability via particle uptake mechanisms, nanoparticulate oral delivery systems also have slower transit times than larger dosage forms increasing the local concentration gradient across absorptive cells, thereby enhancing local and systemic delivery of both free and bound drugs across the gut. These colloidal carriers are expected to develop adhesive interactions within the mucosa and remain in the gastrointestinal tract, while protecting the entrapped drug from enzymatic degradation, until the release of the loaded drug or their absorption in an intact particulate form2, 3.

Rectal dosage forms such as suppositories and enemas are not always effective since a high variability in the distribution of these dosage forms is observed. Suppositories are only effective in the rectum because of the confined spread and enema solutions can only offer topical treatment to the sigmoid and descend- ing colon. Thus, an effective therapy of the colonic disorders can be effectively achieved by using site- specific pH-sensitive drug delivery system. The present studies are aimed to targeting the colon by using pH-sensitive system and protect the active drug against first pass effect and gastrointestinal disturbances. Advantages of targeting drug to the diseased organ (colon) includes, delivery of drug in its intact form as close as possible to the target site, ability to reduce the conventional dose, reduce incidence of adverse side effects and avoidance of mucosal metabolism. MBH is known to suffer from extensive first pass effect. In an attempt to improve its oral bioavailability and possibility to restrict its absorption only to the colon, Mebeverine hydrochloride nanoparticle was prepared by nanoprecipitation method 1.

12 'Nexus' - Alumni Association SJCOPS, Chalakudy ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 MATERIALS AND METHODS:

Preparation of pH sensitive nanoparticle:

Nanoparticle suspension of Mebeverine hydrochloride (MBH) was prepared by nanoprecipitation method. MebeverineHCl 200 mg and specific amount of Eudragit®-EPO were dissolved in 15 ml of methanol. The organic solution was quickly injected to30 ml aqueous solution containing pluronic® F-68 under stirring at 2000 rpm. Stirring was continued for 2 hours at 400C for complete evaporation of methanol. The volume was adjusted up to 40 ml with aqueous solution of 200 mg of HPMC K-15 to obtain a nanoparticle suspension. The optimized nanoparticle suspension was lyophilized at – 420C for 72 hours and which was also redispersed in water to get aqueous nanoparticle suspension 4.A prior knowledge and understanding of the process and the variable under investigation led to preliminary experiments. Based on the preliminary data, the 32 factorial design was adopted to optimize the amount of Eudragit®-EPO (x1) and pluronic® F-68 (x2) identify the independent variable affecting the drug content and the percentage drug encapsulation efficiency (dependent variable). The response surfaces of the obtained result were also plot-ted. The coded and the actual values of the experimental design are given in table 1. The data analysis of values obtained from various batches for drug content and encapsulation efficiency were subjected to multiple regression analysis using PCP dissolution software the equation fitted was

Y: ß0+ ß1 X1+ ß2 X2+ ß11 X12+ ß22 X22+ ß12 X 1X2

Where y is the measured response; X is the level of factors; ß is the co-efficient computed from the responses of the formulations 4, 8.

In-vitro Characterization of pH Sensitive Nanoparticle

I. Particle size analysis:

Particle size analysis of nanoparticle was performed by photon correlation spectroscopy (PCS). This technique yields the mean particle diameter and particle size distribution. Samples were analyzed using Mastersizer 2000 (Malvern Instruments, Malvern, UK), which allows sample measurement in the range of 0.020 – 2000.00 µm 5.

Polydispersity was determined according to the equation:

D (0.9) – D (0.1)Polydispersity = ———————

D (0.5)

Where D (0.9) corresponds to particle size immediately above 90% of the sample, D (0.5) corresponds to particle size immediately above 5% of the sample, D (0.1) corresponds to particle size immediately above 10% of the sample.

II.Estimation of drug content:

The nanoparticle suspension was weighed accurately (equivalent to 10 mg) of the MBH nanoparticle suspension was diluted with phosphate buffer pH 7.4 and the drug content were estimated by using UV -Visible spectrophotometer(Shimadzu UV-1700) at 263 nm against blank solvent system 6.

'Nexus' - Alumni Association SJCOPS, Chalakudy 13 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ISSN : 2350-1332 III.Estimation of entrapment efficiency:

The entrapment efficiency of the prepared formulation was determined by measuring the concentration of free drug in the dispersion medium. The amount of entrapped drug was determined by taking 10 ml of nanoparticle suspension and it was centrifuged for 2 hours at 14000 rpm. The supernatant was separated and filtered through 0.45µm Millipore (Millipore filter). The filtrate was diluted with phosphate buffer pH 7.4 and measured specrophotometrically (Shimadzu UV-1700) the amount of free drug was determined in filtered drug was determined. Initial drug–free drug the entrapment efficiency was calculated using the following equation 7.

IV.Statistical Analysis:

A prior knowledge and understanding of the process and the variable underinvestigation led to preliminary experiments. Based on the preliminary data, the 32 factorial design was adopted to optimize the amount of Eudragit®-EPO (x1) and pluronic® F-68 (x2) identify the independent variable affecting the drug content and the percentage drug encapsulation efficiency (dependent variable). The response surfaces of the obtained result were also plot-ted. The coded and the actual values of the experimental design are given in table 1. The data analysis of values obtained from various batches for drug content and encapsulation efficiency were subjected to multiple regression analysis using PCP dissolution software the equation fitted was

Y: ß0+ ß1 X1+ ß2 X2+ ß11 X12+ ß22 X22+ ß12 X 1X2

Where y is the measured response; X is the level of factors; ß is the coefficient computed from the responses of the formulations 4, 8.

V.In-vitro drug release study:

The in-vitro drug release of the nanoparticle suspension was studies by using dialysis method the formulation equivalent to 10 mg of MBH were placed in a dialysis bag. The dialysis bag was suspended in a beaker containing 100 ml of phosphate buffer pH 7.4 on a magnetic stirrer at 100 rpm, with temperature adjusted to 37±0.5°C at selected time interval sample was removed and replaced with fresh medium. The sample was filtered through 0.45µm Millipore (Millipore filter) the samples were analyzed for drug release by measuring absorbance 263 nm using UV -visible spectrophotometer (Shimadzu UV- 1700) 11.

VI.Differential Scanning Calorimetric Analysis (DSC):

Differential scanning calorimetry (DSC) are one of the most powerful analysis technique which offer- ing the possible of detecting chemical interaction between drug and polymer. The differential scanning calorimetry of thermograms of MBH, Eudragit®-EPO, and lyophilized nanoparticle suspension were obtained using DSC-Shimadzu 60 with TDA trend line software 9.

VII.Scanning electron microscopy (SEM) Analysis:

The morphology of nanoparticle was examined by using scanning electron microscopy (SEM, JSM- 6360LV scanning microscope Tokyo, Japan).The nanoparticle formulation was mounted on metal stubs using double-sided tape and coated with a 150 A0 layer of gold under vacuum. Stubs were visualized

14 'Nexus' - Alumni Association SJCOPS, Chalakudy ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 under scanning electron microscope. SEM has been used to determine particle size distribution, surface topography, texture and examine the morphology of fractured or sectioned surface 10.

RESULTS AND DISCUSSION:

I.Particle size analysis:

Theparticle size ofthenanoparticle suspensionwas found to be 178.3 nm and polydispersityindex 0.810. Increasein particlesizeof nanoparticle suspension with decrease inthe polydispersityindexwasobserved- withincreaseinthe polymer content.The smallerparticlesizeobtained atlowpolymercontentmay be due to highdistribution efficiencyof theinternalpolymer-solvent phase into the external phase.Increase inthe viscosity of internal phase with increased amount of polymer also provides resistance for mass transfer in turn diffusion of polymer – solvent phase into the external phase leading to particle enlargement.

II.Drug content:

The results of drug content are shown in the table 1. The drug content of the nanoparticle were in the range from 43.16±1.84 to 93.14±2.77% depends on the concentration of polymeradded. The maximum drug content was found in formulation F9 with 93.14±2.77%. Drug content was increases with increasing in concentration of polymer added to the formulation.

III.Drug entrapment efficiency:

The entrapment efficiency of the MBH pH sensitive nanoparticle was exhibit maximum in formulation F9 with 88.74±5.20%. Drug entrapment efficiency of the nanoparticle was in the range from 44.64±3.89 to 88.74±5.20%. The results were also in the table 1. As the Eudragit®-EPO concentration increased from 100-200 mg the encapsulation efficiency was increased. When the Eudragit®-EPO concentration was maintained constant of 100 mg, the pluronic® F-68 concentration was varied as 25 mg, 50 mg, and 100 mg the encapsulation efficiency was also increased. The results indicates the Eudragit®-EPO concentration plays a major role in drug entrapment efficiency equal to the pluronic® F-68 concentration.

It can be explained on the basis of hydrophilic - lipophilic interaction between MBH and Eudragit®- EPO. Consequently with increase in the Eudragit®-EPO amount, MBH gets preferentially dispersed in the internal organic phase. Pluronic® F-68, also displayed similar trend and increase in encapsulation efficiency which can be due to the formation of interpenetrated network chain between the hydrophobic portion of pluronic® F-68 with Eudragit®-EPO during precipitation.

IV. In-Vitro drug release studies:

The release rate of nanoparticle depends upon

I) desorption of the surface-bound/adsorbed drug; ii) diffusion through the nanopaticle matrix; iii) diffusion (in case of nanocapsule) through the polymer wall; iv) nanoparticle matrix erosion: and v) a combined erosion/diffusion process. Thus, diffusion and biodegradation govern the process of drug release. The mechanism of drug release from nanoparticle is determined by different physical–chemical phenomena. The exponent n has been proposed as indicative of the release mechanism. In this context, n = 0.5 indicates Fickian release and n = 1 indicates a purely relaxation controlled delivery. Intermedi- ate values 0.5 < n < 1 indicate an anomalous behavior (non-Fickian kinetics corresponding to coupled

'Nexus' - Alumni Association SJCOPS, Chalakudy 15 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ISSN : 2350-1332 diffusion/polymer relaxation. The average percentage release was fitted into different release models: zero order, first order, Higuchi’s square root plot and Korsmeyer-Peppas models. The models giving a correlation coefficient close to unity were taken asthe order of release In vitro drug release data of all selected factorial formulations was subjected to goodness of fit test by linear regression analysis according to zero order and first order kinetic equations,Higuchi’s,andKorsmeyer-Peppas models to ascertain the mechanism of drug release. It is evident that values of ‘r’of factorial formulation F9 was found to be0.9975. This data reveals that drug release follows peppas release kinetics with fickian diffusion mechanism Drug release for selected factorial formulation F9 are 96.85±0.37% at the end of 12 hours. Kinetic exponent ‘n’ for these formulations indicate diffusion through the nanoparticle. Finally, it shows that the different drug release rates may be attributed to different sizes of the nanoparticle. It is expected as theparticle size of Eudragit®-EPO nanoparticle is smaller, their surface area will be more and the drug release is faster. The results of in-vitro drug release studyare shown in the table 2.

V.Differential Scanning Calorimetric (DSC) Analysis:

MBH exhibits a sharp melting endothermic peak at 130ºC whereasEudragit®-EPO showed a broad melting endothermic peak at 129.5º. The thermogram of lyophilized nanoparticle suspension displayed sharp endotherm at 129.750C, corresponding to Eudragit®-EPO but no drug peak it explains monotet- icbehavior of the system, where drug gets completely dissolve in below it temperature. It is evident that there was no chemical interaction between drug and polymer.

VI.Scanning Electron Microscopy (SEM) Analysis:

MBH nanoparticle has shown smooth and spherical shape. The results is depends on the ratio of surfac- tant and polymer used in the optimized formulationF9.

Table 1: Comparisonofdrugcontentwithentrapmentefficiencyofformulation

S.No Batchcode *Drugcontent(%) *Drugentrapmentefficiency(%)

1 F1 43.16±1.84 44.64±3.89

2 F2 53.43±1.84 52.91±3.23

3 F3 63.71±1.22 57.87±0.72

4 F4 58.67±1.09 53.51±2.96

5 F5 69.05±2.57 63.69±2.77

6 F6 79.13±1.71 74.34±6.33

7 F7 73.29±1.55 71.75±7.61

8 F8 82.86±3.48 80.88±3.70

9 F9 93.14±2.77 88.74±5.20

16 'Nexus' - Alumni Association SJCOPS, Chalakudy ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 F 9 0.00 15.97±0.17 21.77±0.30 30.56±0.35 38.52±0.93 45.05±0.28 51.74±0.47 58.39±0.53 65.41±0.36 74.10±1.04 84.89±0.36 91.55±0.71 96.85±0.37 F 8 0.00 15.97±1.25 26.51±0.34 31.81±0.34 40.40±0.46 44.93±1.52 52.72±0.18 59.38±0.32 66.21±0.19 73.39±0.73 81.15±0.44 89.69±0.46 - F 7 0.00 16.07±1.14 21.37±0.17 29.24±0.81 36.29±0.32 43.50±0.79 50.07±0.47 54.89±0.89 61.98±1.40 68.92±1.75 77.24±1.28 - - 0.00 17.28±0.17 21.18±0.30 29.25±0.52 36.90±0.75 43.11±0.17 50.08±0.45 54.90±0.87 62.09±1.49 68.93±1.74 76.45±2.95 85.24±1.81 92.40±0.55 F 6 F 5 0.00 15.06±0.46 25.90±0.34 30.99±0.34 39.19±0.35 43.88±0.61 50.16±0.47 54.17±0.50 60.94±0.68 66.87±0.34 74.56±0.46 - - F ormulation code 0.00 12.74±0.35 20.43±0.46 29.20±0.52 36.65±0.75 43.06±0.17 50.03±0.45 54.85±0.86 60.52±0.62 67.35±1.25 72.84±0.35 - - F 4 F 3 0.00 16.57±1.14 21.68±0.63 28.75±0.16 36.29±0.33 43.20±0.30 50.07±0.46 53.68±0.61 61.96±1.39 68.91±1.73 77.23±1.17 82.91±0.31 - able 2: In-vitrodrug release data of M BH pH sensitive nanoparticle formulation T F 2 0.00 15.16±0.46 21.26±0.17 27.82±0.69 36.26±0.34 43.17±0.30 49.74±0.17 53.65±0.62 61.93±1.09 68.88±1.72 77.20±1.16 - - F 1 0.00 15.26±0.23 20.96±0.46 27.82±0.69 35.45±0.86 43.16±0.29 50.04±0.45 54.55±1.52 60.52±1.60 65.84±0.35 70.71±0.65 - - ime in T hours 0 1 2 3 4 5 6 7 8 9 10 11 12

'Nexus' - Alumni Association SJCOPS, Chalakudy 17 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ISSN : 2350-1332 CONCLUSION

MBH nanoparticle suspensions were prepared by the nano precipitation technique. The application of factorial design gave a statistically systematic approach for the formulation of nanoparticles with desired particle size, high drug content and high entrapment efficiency. Drug: polymer ratio and concentration of stabilizer were found to influence the particle size, drug content and entrapment efficiency of MBHEudragit®-EPO nanoparticle formulation F9. The maximum drug release was found in the formulation F9.The results of the present investigation conclude that the formulation F9 was considered as best among various formulations with respect to particle size, entrapment efficiency and in-vitro drug release.

ACKNOWLEDGEMENT

We express our sincere thanks to our management, Adhiparasakthi College of Pharmacy, Melmaru- vathur, Kanchipuram for their support to providing the facilities to complete the work. We also extend our thanks to Shasun Pharmaceutical Pvt. Ltd., Puducherry, Cipla Pharmaceuticals, Mumbai, Indiafor providing us gift samples of drug and polymers.

REFERENCES

1. dandagi PM, Mastiholimath VS., Gadad AP, Kulkarni AR, Konnur BK; pHsensitive mebeverine hydrochloride microspheres for colon delivery. Indian Journal of Pharmaceutical Sciences, 2009; 71(4): 461-464.

2. Allemann E,JCLeroux, and R Gurny; Polymeric nano and microparticulate for the oral delivery of Peptides and peptidomimetics. Advances Drug Delivery-Review. 34; 1998: 171-189.

3. ArbosP.Campanero MA, Arangoa MA, Renedo MJ, and Irache JM; Influence of the surface characteristics of PVM/MA Nanoparticles on their bioadhesiveproperties, J.Control Release 89; 2003: 193.

4. BothirajaC, AtmarampawarP, SheikhS, PraveenSher; Eudragit®-EPO based nanoparticle suspension of Andrographolideinvitroand invivo. Nanoscienceand Nanotechnology Letters, 2009; 1:156-154.

5. YadavAV, Selvakumar,Kalimuth; Formulation and evaluation of Carvedilolloadedeudragit100 nanoparticle.InternationalJournalof Pharmaceutical Technology and Research, 2009;1(2): 179- 183.

6. Sowjanya M, Ayyappan T, Vetrichelvan T; Formulation and In-Vitro evaluation of Mebeverine Hydrochloride colon targeted micropellets for the treatments of irritable bowel syndrome. Inter- national Journal of Chemical and Phamaceutical Science, 2013; 4(1).

7. ParsiSwathi, Velrajan G; Formulation and evaluation of targeted and controlled release Mebev- erine Hydrochloride microspheres. International Journal of Pharma and Bio Sciences, 2013; 4 (2): 11-20.

8. AtmaramPandurangPawar, BothirajaChellampillai;Improved bioavailabilityof orallyadministere-

18 'Nexus' - Alumni Association SJCOPS, Chalakudy ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 dAndrographolidefrompH-sensititivenanoparticle. European Journal of Metabolism and Pharma- cokinetics, 2011; 35:123-129.

9. vikramMPandya, JayvadanK. Patel, Dhaval JPatel; Formulation optimationandcharacterization of Simvastatinnanosuspension preparedby nanoprecipitationtechnique. DerPharmacia Lettre, 2001; 3(2) : 129-140.

10. AnilkumarJ, ShindeHarinathN More;Designandevaluationofpolylactic-co- glycolic acidnanopar- ticles containing Simvastatin. International Journal of Drug Development and Research, 2011; 3(2): 133-137.

11. PanchaxariM Dandagi,PravinPatil, PunitDPatel,VinayakS Mastiholimath,AnandPGadad; Devel- opment andcharacterization ofa particulate drugdelievery system forEtoposide. Indian Journal Of Novel Drug Delivery, 2011; (30): 43-51.

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Jayakumar et.al.,/ Journal of Pharma Innovative Research, 2015; 2(1): 20-23 A Retrospective Study to Determine the Prevalence of Left Ventricular Systolic Dysfunction

Jayakumar K.S*1, Anitha L2, Harikrishnan S2, Perumal P*1

1Nirmala College of Pharmacy, Muvattupuzha P.O, Ernakulam District 2College of Pharmaceutical Science, Medical College, Trivandrum Corresponding Author E-mail: [email protected]; [email protected]

ABSTRACT Retrospective study carried out in the department of cardiology of SreeChitraTirunal Institute for Medical Sciences and Technology (SCTIMST). The data of patients admitted to SCTIMST from 2004 january onwards with Left Ventricular Systolic Dysfunction (LVSDF) were collected by retrospective methods from the records library. The collected data where analysed statistically by using SPSS version 11.The total sample size was about 150 which has been calculated by using the formula16s2/d2+1 The objective of the study was to determine whether males or females are more prone to LVSDF, which age class is more affected, to determine the most common co- morbidities among these patients, also to determine among the LVSDF which is more prevalent. We were able to find out that the popular type of LVSDF was Coronary Artery Disease (CAD) and dilated cardiomyopathy (DCM), males are more effected with LVSDF than females, the age group of 50-64 suffered mostly from LVSDF and finally the most common co morbidity was Dyslipidaemia followed by Diabetes mellitus and Hypertension

Key words:LVSDF; Prevalence; Hypertension

INTRODUCTION Left ventricular systolic dysfunction is one of the leading causes of death globally. In India, the incidence of left ventricular systolic dysfunction is expected to rise in parallel with increase in life. In left ventricular systolic dysfunction, the heart is unable to pump sufficient blood to meet the body’s metabolic needs at normal filling pressure.1 Ventricular systolic dysfunction is characterized by loss of contractile strength of the myocardium accompanied by ventricular dilation. In an attempt to stabilize compromised pump performance, the failing heart is subjected to increased adrenergic stimulation.2Although this compensatory mechanism may initially support and maintain cardiac output, the continuous adrenergic stimulation may be harmful. The most compelling evidence that chronic adrenergic stimulation is

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harmful to the failing heart derives from the experience with beta adrenergic blocking agents which consistently improve left ventricular function in subjects with chronic heart failure by reversing intrinsic systolic dysfunction.3,4,5Therefore, a retrospective study was designed to determine the prevalence of left ventricular systolic dysfunction. CASE REPORT

The data of patients admitted to SCTIMST from 2004 january onwards with LVSDF were collected by retrospective methods from the records library. The duration of study is six months ie the patients who have completed a minimum treatment period of six months are chosen. The sample size was calculated by using the formula 16s2/d2+1. According to this sample size the total number of cases was 150.The collected data where analyzed statistically by using SPSS version 11.The statistical significance was as- sessed by

 fisher’s exact test.

 Paired t test.

 Z test for proportion.

 Comparison by ANCOVA

The study area was Records library, Department of Cardiology Of SreeChitraTirunal Institute for Medi- cal Science and Technology, ThiruvanathapuramEthical committee clearance had been obtained from both medical college Thiruvanathapuram (No: C13/EC07/07/MCT and from SCTIMST, Thiruvana- thapuram (No:IEC/174)

RESULTS& DISCUSSION

The sex distribution of the patients were analysed in the study group. For this study we collected data of 150 patients admitted to SCTIMST from January 2004 to December 2006 diagnosed with left ventricular systolic dysfunction. The study group consists of males (80.7%) and females (19.3%) (Figure 1).The age group distribution of the patients was studied. The total population was divided in to four groups and included all patients in this group. Here 72 patients (48%) were of age group 50-64, followed by 46 patients (30.7%) from age group 35-49, only 18 patients (12%) from the group 65-80 and 14 patients (9.3%) from the group 20-34 (Figure 2).Figure 3 showed that distribution of sample population according to diagnosis. The study population consists of 150 patients among whom 100 patients were diagnosed with coronary artery disease with left ventricular systolic dysfunction and 50 patients with dilated cardiomyopathy. Figure 4indicated that the prevalence of co-morbidities among the sample population. Among the total 150 population, 32% of the population were having no co-morbidities, 39.3% of the population having diabetes mellitus, 38% of the population with hypertension, 1.3% of the population with renal dysfunction and about more than half of the population about 52% possessdyslipidaemia.

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Figure 1: Distribution of the sample according to sex Figure 2: Age group of patients

Figure 3: Distribution of sample according to diagnosis

Figure 4: Prevalence of co-morbidities among the sample

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CONCLUSION The study concluded that the most common type of LVSDF was CAD and DCM. The patients with age group between 50-64 are more effected with LVSDF. The male to female ratio was 80.7: 19.3. 3. Males were more affected with coronary artery disease and dilated cardiomyopathy.The co-morbidities were present included diabetes mellitus, hypertension and dyslipidemia. Among this most common co- morbidity was dyslipidemia. REFERENCES

1. Pollock SG, Lystash J, Tedesco C, Craddock G, Smucker ML. Usefulness of bucindolol in congestive heart failure. Am J Cardiol.1990;66:603-607.

2. olsen SL, Gilbert EM, Renlund DG, Mealey PC, Taylor DO, Yanowitz FD, Bristow MR. Carve- dilol improves left ventricular function and symptoms in heart failure, a double-blind randomized study. J Am CollCardiol. 1995;25:1225-1231.

3. valentin Fuster, R. Wayene Alexander et al. Hurst’s THE HEART, volume 1,11th edition.

4. Yusoff K. Vitamin E in cardiovascular disease. Asia Pac J ClinNutr 2002;11:S443-S447.

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Satheeshkumar et.al.,/ Journal of Pharma Innovative Research, 2015; 2(1): 24-33 An Insight into Chemometrics Concepts in Pharmaceutical Analysis N. Satheeshkumar*, S. Shantikumar, David Paul

Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research (NIPER-Hyderabad), Hyderabad, Telangana, India Corresponding Author E-mail: [email protected]

ABSTRACT Chemometrics involves the application of statistical and mathematical methods to analytical data to permit collection and extraction of maximum useful information. The developments over the past years revolutionized different fields of pharmaceutical sciences. Chemometrics has advanced in conjunction with state of the art analytical instrumentation and computation- al capability. The future of the automated laboratory depends largely on the chemometrical concepts and its applications. As instruments acquire data more rapidly, there will be the need to optimize the instrumental and interpretative functions. This review highlights the concepts with applications of chemometrics in pharmaceutical analysis and to enlighten the importance of Quality by design (QbD) approach employed in the pharmaceutical sciences.

Keywords: Chemometrics; Quality by design; Design of experiments.

INTRODUCTION Automated systems are becoming increasingly important tools for appropriate monitoring and controlling of the analytical process. The technological developments in the past years had revolutionized the instrumental analysis and made instruments to become smaller, faster and cheaper while continuing to increase accuracy, precision and availability. Data analysis methods have also benefitted in several ways from advances in technical computing; commercially available programming packages allow scientists to undergo complex calculations in a simple manner. Furthermore, software sold with many commercial instruments contains automatic data processing algorithms. The advances in computing allow researchers to obtain chemically relevant information and optimizing experimental programing.1 Chemometrics is applied to resolve both descriptive and predictive problems in experimental sciences, especially in chemistry. In descriptive applications characteristics of chemical systems are modeled with the intent of understanding the relationships and structure of the system (i.e., model understanding and identification). In predictive applications, characteristics of chemical systems are modeled with the

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intent of forecasting new properties of interest. In both cases, the smaller datasets with large and highly complex properties, involving several variables and hundreds to thousands of cases or observations.2 Chemometrics methods are versatile and it involves high level of abstraction and application of the statistical and mathematical methods, mainly the multivariate analytical methods. These tools involve various algorithms and analogous ways for processing and evaluating the data. It can be easily implemented to various fields namely medicine, pharmacy, food control and environmental monitoring.3 Different chemometric arenas for the pharmacy and medical fields are shown in Figure 1.

Figure 1: Applications of Chemometrics in Pharmaceutical sciences

Chemometrics tools are found to be a useful tool in estimating the quality of drugs, metabolites identification, impurity profiling studies and various other analytical studies 6, 7. Chemometrics involves the application of statistical and mathematical methods to analytical data to permit maximum collection and extraction of relevant information’s 1. To perform the quality of complex analytical samples, novel methods have to be explored.4 Nowadays HPLC is extensively employed for quality control of drugs due to its sensitivity, superior precision, high resolution and extensive applicability. LC–MS, GC–MS, and LC– NMR have been increasingly used in complex analytical and chemical exploration of drugs 10. This advancement in instrumentation in every sort of analysis has generated enormous amounts of data which record even minute differences between samples and this enables us to provide large implications for the discrimination of complex analytical information’s. To obtain accurate quantitative results from the fingerprint, pretreat- ment of chromatographic data is necessary.5 To eliminate or reduce unwanted sources of variations due to instrumental responses from hyphenated techniques and to obtain more efficient results, data preprocessing techniques are applied. The advances

'Nexus' - Alumni Association SJCOPS, Chalakudy 25 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ISSN : 2350-1332 in computing allows researchers to obtain chemically relevant information from their analytical data; however, this is not possible to be achieved by using simple data processing techniques.6 The purpose of this review is to put an insight into the various multivariate calibration chemometrics methods with applications and to enlighten the importance of Quality by design (QbD) approach employed in the pharmaceutical sciences. Preliminary treatment of Data The quantity of data generated by chromatographic and mass spectrometric characterization is more and during the analytical process, measured data points (variables) will be associated to each sample (observation). This data is normally assembled and stored in data tables called data matrices, where each observation is represented by one row in the table and each of the descriptor variables is represented by one column.7 Normalization Variation in sample concentration might affect the multivariate analysis of the entire chromatographic profile. So normalization of data is examined before carrying out multivariate analysis, the total area of all peaks can be calculated 11. No normalization of data is examined before carrying out multivariate analysis in the following way: each chromatogram consists of N peaks with each peak area Ci of the ith component being utilized. The total area of all peaks can be calculated by

(1) So the peak area after normalization is as follows

Ci= (2) Then, each peak is expressed as percentage of the sum of peak areas.8 Local Least Square (LLS) method: Major problem in analyzing samples from complex matrixes is occurrence of signal shift. It causes a significant influence in chromatographic profile. To eliminate the chromatographic shift, the LLS method is used to correct retention time shift. When we are using pattern approach, then it is necessary to correct retention time in all chromatograms unless it causes serious problem. To match all chromatographic profiles with their retention times, all common constituents will be selected and utilized for chromatographic alignment.9 Methods for extracting chemical information The aim of these methods is to detect similarities or to extract useful information from the data obtained from analytical instrument. These methods are important for finding out the interested analyte in complex mixture and Similarity Analysis (SA), SCC and IT are commonly used tools. Spectral Correlative Chromatography (SCC) It is a technique used to identify the chemical component present in different chromatograms as acquired

26 'Nexus' - Alumni Association SJCOPS, Chalakudy ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 from hyphenated instrument. It is based on the fact that the same chemical components should have the same spectra no matter what or how they are eluted through diverse chromatographic columns. The spectral information is utilized to pick up the targeted component from the other two-way chromatograms. The procedure for carrying out SCC is given in the following steps: 1. Assess peak purity of targeted component and obtain its UV or MS spectrum from the chromatogram. 2. Identify this component in the chromatogram of interest through comparison by a series of spectrum at each scan point of other chromatogram by their correlation coefficients. 3. Get a curve of the correlation coefficient versus scan point in the direction of retention time and further validate. Step 2 with consideration of the information in the local chromatographic cluster where the target exists. The result obtained by SCC is highly accurate in the case of MS because the mass spectrum provides specificity compared to the chromatogram10. Information Theory (IT) method The value of information data depends on the separation degree and concentration distribution of each chemical component in a chromatogram. The more they resolute with uniform concentrations, the higher the value of information contents, i.e., more chemical information can be obtained from this chromatogram. Information content of a chromatogram is calculated by using mathematical equation. By correlating the magnitude of information content, maximal chemical information under certain chromatographic conditions that include all extraction and detection parameters can be resolved 11. Methods for resolution of mixtures The main goal in the analysis of any multicomponent system is to get useful information from the raw experimental data and knowledge of the number of chemical components in the analyzed sample. The common aim of all resolution methods is to provide the linear model of individual component contribu- tions using solely the raw experimental measurements. HELP and OPA are two important and commonly used multivariate tools for the resolution of mixtures.12 Heuristic Evolving Latent Projections (HELP) HELP is a multivariate resolution method used to resolve two-way bilinear data into spectra and chromatograms of the pure constituents13. It employs the feature of visual interface from latent variable projection graph and also provides information of the local rank of the data matrix. This method employs the following steps: 1. To find out the total number of components by employing Singular Value Decomposition (SVD). 2. further, Eigen value plot is constructed which is plotted between logarithms of Eigen values against retention time. 3. then zero concentration regions and selective concentration region of the compound are determined from Eigen value plot. Selective concentration region is defined as the region in which only a single component elutes out i.e., having rank equal to one and zero concentration region in which no component elutes out. 4. then Evolving Latent Projection (ELP) graph gives us information about the selective region of the individual component in wavelength as well as retention time space. Such region can be identified

'Nexus' - Alumni Association SJCOPS, Chalakudy 27 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ISSN : 2350-1332 by passing straight lines in ELP graphs. 5. In the end spectra and chromatogram of individual components can be resolved through simple mathematic computation. S= (CTC-1)CTX (3) Represents data matrix generated by hyphenated system with total rows representing spectral profile S and columns showing chromatograms C.13 Orthogonal Projection Analysis (OPA) OPA compares each spectrum with one or more than one reference spectra and searches for the least correlated spectrum. OPA is a stepwise process and selects one key variable in each step. Then calculates dissimilarity based on the mathematical concept of orthogonalisation. The first dissimilar plot represents a comparison of each spectrum with the average spectrum.14 Methods for displaying data These methods are useful to resolve net analytical signal by discovering the dominant factors while excluding the relevant interference, thus giving a more accurate estimate. These include various methods such as LDA, Hierarchical Clustering Analysis (HCA), SIMCA and PCA, but among these PCA and LDA are found to be most commonly used. Principal Component Analysis (PCA) Principal component analysis generates a new set of orthogonal variables that contains information similar to the original set. PCA is an unsupervised multivariate projection method, commonly used to obtain an overview of complex datasets. PCA reduces the dimensionality of the data by means of latent variables, which allows for the data to be represented in a meaningful manner. In PCA the central idea is to minimize the dimensionality of a data set consisting of a large number of interrelated variables, while keeping maximum variation in the data set. This is done by transforming to latent variables, the principal components (PCs) that are uncorrelated and ordered so that the first few PCs retain most of the variation in all of the original variables.15 PCA a multivariate method used to explore the main source of variability present in the data sets. It is used to detect cluster formatting and to establish relationship between object and variable.16 The sample variation in data is given in terms of latent variable as given below: X=UP = (3) where X is a given data matrix, Ui and Pi are score and loading vector, respectively and orthogonal to each other. Generally score matrix (U) gives the relationship between samples while loading matrix (P) shows the importance of each variable (Figure 2).

Figure 2: Flow of complicated data and chemometric extraction of information’s.

28 'Nexus' - Alumni Association SJCOPS, Chalakudy ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 Linear Discriminate Analysis (LDA) LDA is a supervised pattern recognition method and seeks a linear function of the variable in multivariate space which maximizes the ratio between both variances compared to the within-group variance. Since multi-dimensional data arise when the number of variables larger than the number of observations, then we cannot use LDA directly. In that case PCA is employed to compress data and to transform the original data set comprising of a large number of inter-correlated variables into a reduced new set of variables. LDA makes a discriminate function for each group according to this equation:

F(Gi) = ki + where i is number of groups(G), ki is the constant of each group, n is number of parameters used to classify a set of data into a given group and Wj is weight coefficient, assigned by LDA to a selected parameter (Pi).17 Soft Independent Modelling of Class Analogy (SIMCA) In order to test the robustness, a popular classification method SIMCA was used to assign unknown samples to its existing classes. SIMCA is a clustering method that separates PCA models for a prior determined class of data that operates on the residual matrix 5. There are five steps involved to obtain the classification model: 1. Construction of separate PCA models for each class. 2. determine the optimal number of PCs by validation. 3. Fitting the unknown samples to each predefine model, provided that the class are distinct enough. 4. deciding whether the samples belong to the corresponding class by referring to the object-to-model distance and leverage (distance of the sample to the model center). 5. Validate the classification results with statistical test called significance test.18 Quality by Design (QbD) QbD is a concept in which Quality by Design approach have been used in the advancement of product and process quality especially by the pharmaceutical industry. Quality by Design (QbD) has become an important concept for the pharmaceutical industry that is further defined in the International Conference on Harmonization (ICH) guidance on pharmaceutical development as "a systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management” 25. Quality is built into product and process by design based on scientific understanding. It includes knowledge rich submission which shows product knowledge and process understanding. The quality of method developed has become increasingly important in a QbD environment and for this quality attributes like robustness and rugged- ness should be verified early in the method development stage to ensure method performance over the lifetime of the product. Otherwise, if a non- robust or non- rugged method is adapted, significant time and resource may be required to redevelop, revalidate and retransfer the developed analytical methods. The developed method will be more robust which gives greater level of confidence in case of variations in conditions and this approach gives greater transfer success when method is transferred from research level to quality control department. It provides a space for invention of new techniques by continuous improvement throughout method development cycle. The main reason behind the adoption of QbD ap-

'Nexus' - Alumni Association SJCOPS, Chalakudy 29 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ISSN : 2350-1332 proach is to provide greater compliance with regulatory authorities 19, 20. QbD can be applied for various analytical methods which include, chromatographic techniques like HPLC (For stability studies, method development, and determination of impurities in pharmaceuticals), hyphenated technique like LC–MS, analysis of genotoxic impurity, dissolution studies etc. shown in Figure 3.

Figure 3: Applications of QbD in Pharmaceutical sciences Design of experiments (DOE) and statistical analysis have been applied widely to analytical sciences. The major advantage of using DOE to pharmaceutical analysis is that it allows all the potential factors to be evaluated simultaneously and systematically. Using DOE, one can evaluate and identify the critical factors based on statistical analysis. Once the critical factors have been identified, the optimal analytical method can be performed by using proper DOE with optimization all critical factors.21,22 HPLC (High-Performance Liquid Chromatography) HPLC is used for the analysis of multicomponent pharmaceutical formulation. Various kinds of injection and selective treatment are required for the analysis of samples by HPLC. Optimization of the different chromatographic conditions like selection of the column, selection of various mobile phases with various compositions, temperature of the column and selection of one specific wavelength has to be done for accurate analysis of results. HPLC is a very sensitive analytical technique for determination in which some factors like error in linear regression, error in chromatographic area, fluctuation during single wavelength detector response etc. could affect the outcome of chromatographic analysis. For the statistical comparison, various statistical tests are being involved, like the t-test, ANOVA test, F test and so forth. Various chemometric methods can also be used along with the combination of detectors. Evalu- ation of the response from detector is based on the function of the peak area. The results obtained from HPLC chemometric calibrations were compared with those obtained by instrumental analysis using HPLC.23,24 Application of DOE, to study the robustness of a HPLC method for sitagliptin and piogli- tazone was carried out for six selected factors the percentage of acetonitrile in the mobile phase, the pH of mobile phase, the detector wavelength, the column temperature, the flow rate and the strength of the buffer. These factors are examined in 2-level screening experimental designs created using JMP@ (SAS

30 'Nexus' - Alumni Association SJCOPS, Chalakudy ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 Institute) software. The system suitability parameters the capacity factor, tailing factor and resolution were calculated. It was analyzed using the ANOVA method by least squares fitting.25 Software and tools Number of software packages and other tools for multivariate analysis, including: High-D, JMP (Sta- tistical software), MiniTab, Calc, PSPP, SAS (software), SciPy for Python, SPSS, TMVA - Toolkit for Multivariate Data Analysis in ROOT, The Unscrambler, MATLAB, Eviews, Prosensus ProMV and Um- etrics SIMCA. CONCLUSION Chemometrics is used in optimizing experimental procedures, extracting useful information from the chromatographic data and resolution of the mixture into linear components. It is an application driven discipline, chemometric techniques are widely used in pharmaceutical analysis and improved chemometric methods of analysis will makes to utilize fully all the benefits of novel analytical methodologies. Advanced analytical instrumentation and data processing, led to a need in advanced methods for design experiments, calibration of instruments and evaluate the resulting data. Various chemometric methods are applied for the analysis of data with an aim to achieve maximum accuracy, precision and robustness. The implementation of chemometric techniques with a view of ensuring overall production process control necessitate the use of analytical techniques capable of providing accurate results in a simple and rapid manner.

ACKNOWLEDGMENTS The authors would like to thank Dr. Ahmed Kamal, Project Director, National Institute of Pharmaceuti- cal Education & Research, Hyderabad for his encouragement and support.

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25. International Conference on Harmonisation Q8 (R2).

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Umaa et.al.,/ Journal of Pharma Innovative Research, 2015; 2(1): 34-42 A Review on Clinical Trials of Natural Polyphenol Nutraceuticals in the Management of Diabetes

K. Umaa*1, V. Abinaya2, T. Gokul2, G. Saipriya2, A. Nagarajan*2

1Dept. of Pharmaceutical Chemistry and 2Dept. of Pharmacognosy, PSG College of Pharmacy, Coimbatore-04 Corresponding Author Email: [email protected], [email protected]

Abstract

Polyphenols are potential nutraceuticals used in recent years for their unique biological properties. This natural plant component is being given as food supplement in case of type 2 diabetes mellitus. In this review, all the potential efficacies of poly phenols that include flavonoids, lignans, polymeric lignans, stilbenes and phenolic acids evaluated through clinical trials are being discussed. Based on several reports on survey, it is evident that if taken as diet polyphenols have been found to modulate the carbohydrate and fat metabolism, attenuate hyperglycemia and insulin resistance, improve adipose tissue metabolism and reduce stress and inflammatory processes to a great extent. Due to this, the development of long-term diabetes complications like cardiovascular disease, neuropathy, nephropathy and retinopathy also seem to be prevented. Hence, in order to standardize the dose and duration of polyphenol dietary supplement in diabetics, it is obvious that an investigation on human clinical studies is the definite need of the day.

Key words: Polyphenols, Flavonoids, Type 2 diabetes mellitus (T2DM), Diabetic complications

INTRODUCTION

Diabetes mellitus (DM), a metabolic disorder in the endocrine system has become a life-threatening disease with India fast becoming a diabetic capital of the World. Diabetes treatment involves usage of drugs including oral hypoglycemic agents such as sulphonylureas, metformin, gliclazide, DPP4 inhibitors and insulin therapy. In spite of the invention of several drugs for diabetes that help intervene at many stages of the disease, they are associated with adverse effects such as glitazone causing cardiac and renal toxicities demanding alternate medicine devoid of toxicities. The widespread use of drugs derived from natural sources for diabetes and other diseases is seen as a promising option that has emerged as a new paradigm in therapy popularly known as Complimentary and Alternate Medicine (CAM). The uncertain-

34 'Nexus' - Alumni Association SJCOPS, Chalakudy ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ties in the drug formulation in terms of authenticity of the herb used, method of extraction or preparation of the formula and more importantly contamination with heavy metals and other impurities limit the use of herbal products for therapy. Among the various types of compounds present in natural products, polyphenols, terpenes, alkaloids, flavanoids etc. have found use in diabetes treatment. We have chosen polyphenols for the present review since there are numerous, such as tea polyphenols, resveratrol and others reported to be useful in the ailment of diabetes.

DIETERY SUPPLEMENTS FROM THE PLANT SOURCE

More than 800 plants found to have anti-diabetic properties. Ethano-pharmacological surveys shows that more than 1200 plants were used in traditional medicines for the hypoglycemic activity. Some of the herbal extracts have been shown hypoglycemic activity in human and animal models and can be used for the treatment of type 2 diabetes mellitus. For diabetes, many conventional drugs have been derived from the active molecule of medicinal plants like Momodicacharantia, Gymnemasylvester, Azardichtaindica etc. Many plant constituents are also usedas anti-diabetic agentslikepolyphenols,isoflavones,resveratrol ,apigenin,epigallocatechingallate,hespiridine,cyanidine,myricetin,quercetin,Catechinetc.

POLYPHENOLS

Polyphenols are present in many dietary ingredients andhave gained importance owing to their perceived health benefits. Apart from exerting its antioxidant benefit, polyphenols are also known to modulate -es sential signaling molecules involved in carbohydrate and lipid metabolism eliciting protective action against metabolic disease and the harmful effects of insulin resistance thus slowing down progression towards T2DM.Polyphenols are a structural class of organic chemicals characterized by the presence of large multiples of phenol structural units that are present in plant based foods such as fruits, vegetables, whole grains, cereals, tea, coffee and cocoa. Anti-diabetic action of polyphenols may be considered to be operating through different mechanisms: inhibition of glucose absorption in the gut or its uptake by peripheral tissues. Several other polyphenolic components such as Caffeic acid, black and green tea extracts, EGCG, isoferulic acid have been studied for their glucose uptake in adipocytesin-vitro on cultured cells. Even though polyphenol rich supplementation had no effect on glycemia, it significantly reduced the level of glycatedhemoglobin (HbA1c) indicating that polyphenols could help reduce the risk of diabetic complications such as the beneficial effect of curcumin in reducing renal damage. Literature reports indicate the utility of polyphenols in the management of diabetes and the list of components used are: Citrus flavonoids inhibit Glucose-6-phosphatase, Quercetin and Resveratrol inhibit GLUT4 and AMPK, Isoflavones like genisteinhelp in revival of pancreatic β-cells. Other than this, high polyphenolic dark chocolate helps in reducing oxidative stress in T2DM, Cyanidine’s role in adipose tissue metabolism, Grape seed proanthocynidine function in reducing neuropathy, nephropathy and retinopathy are important.

'Nexus' - Alumni Association SJCOPS, Chalakudy 35 JOURNAL OF PHARMA INNOVATIVE RESEARCH (ISSN: 2350-1332) Universal Pharmaceutical Research Publication. All rights reserved J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ISSN : 2350-1332 Umaa et al., 2015/JOURNAL OF PHARMA INNOVATIVE RESEARCH, 2015; 2(1): 36-44

Figure1: Chemical structures of a few common dietary polyphenols Figure1: Chemical structures of a few common dietary polyphenols Table 1:MECHANISMS OF ACTION INVOLVED IN INVITRO STUDIES CLINICALCLINI TRIALSCAL TRIALS

Several clinical trials involving Genistein POLYPHENOLS SOURCE MECHANISM Several clinical trials involving Genistein supplementation,Isoflavonesat a dose/day of 90mg, pome- supplementation,Isoflavonesat a dose/day of Flavanoids Citrus G6 phosphatase granate polyphenols at a dose of 2 capsules per day (equivalent to 753mg of polyphenols) and fenugreek 90mg, pomegranate polyphenols at a dose of inhibition seeds were reported in the literature indicating beneficial effects of polyphenol in the management of 2 capsules per day (equivalent to 753mg of Tea polyphenols Green tea AMPK inhibition diabetespolyphe purportedlynols) acting and fenugreek through various seeds mechanisms were independently evaluated through in-vitro methods. These results are summarized in Table 1. Quercetin Apple GLUT4 & AMPK reported in the literature indicating inhibitors beneficial effects of polyphenol in the Table 1:MECHANISMS OF ACTION INVOLVED IN INVITROGenistein STUDIES Soya beans Revival of pancreatic management of diabetes purportedly acting ȕ cell through various mechanisms independently Resveratrol Red grapes Attenuates ȕ cell POLYevaluatePHENOLSd through inSOURCE-vitro met hods. These MECHANISM mass Flavanoidsresults are summarizedCitrus in Table 1. G6 phosphatase inhibition Procynidin Apple Decrease Tea polyphenols Green tea AMPK inhibition apolipoproteinȕ synthesis Quercetin Apple GLUT4 & AMPK inhibitors Genistein Soya beans Revival Aofnthocyanines pancreatic β cell Strawberry Adipose tissue metabolism Resveratrol Red grapes Attenuates β cell mass Catechin Green tea Decrease the synthesis Procynidin Apple Decrease apolipoproteinβ synthesis of thromboxane A2

Anthocyanines Strawberry Adipose tissue metabolism

Catechin Green tea Decrease the synthesis of thromboxane A2

Through a clinical study (NCT01245270), eight male volunteers upon ingestion of a concentrated bil- berry extract showed reduced postprandial glycaemia and insulin probably through reduced rates of carbohydrate digestion and/or absorption. In another study,olive leafpolyphenols (51.1 mg of oleuro38- pein, 9.7 mg of hydroxytyrosol per day) on insulin action and cardiovascular risk factors in middle-aged overweight men elicitedimprovement in insulin sensitivity and pancreatic β-cell secretory capacity.

36 'Nexus' - Alumni Association SJCOPS, Chalakudy ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 Table 2: Clinical trial involving polyphenols and the administered doses

Substance Principle / Dose/Day Days Number of Biomarkers Significantly Given Polyphenols of Isofla- Groups Affected vones Geinstein geinstein 54mg 365 30 women Reduced excretion of pyridinium supplement of age group crosslinks and increase in bone between (47- mineral density in femur and 57) lumbar spine Isoflavone isoflavones 1,332mg 84 32 post Lowers fasting insulin,insulin supplement menopausal resistance, LDL & cholesterol women with diet control type 2 diabetes mellitus Isoflavone isoflavones 90mg 365 15 young No changes in bone mineral den- enriched soy healthy sity or other bone parameters protein women

Moreover, type 2 diabetes also significantly influences the benefits of dietary polyphenols, although only limited studies have been conducted so far. An understanding of type 2 diabetes-mediated changes in pharmacokinetics and bioactivity of dietary polyphenols will augment the benefits of these phytochemicals. Ostertaget. al., described the health benefits of polyphenols and remarked that the poor biological activities may be due to the low bioavailability. This was supported by the finding that only nano to micromolar concentrations of polyphenols detected in samples in-vivo as against the required high milli molar levels for defence against free radicals. Polyphenols from popular dietary components such as fenugreek, bitter melon, cinnamon, pomegranate, apple, curcumin, tea are widely used in the management of diabetes. However the clinical trial information using polyphenols from these sources are not comprehensive. There were as many as 39 clinical trial records found in clinical trial database that disclose various polyphenol rich dietary sources highlighting the doses, frequency and duration of administration. The data recorded in these trials can be summarized as follows.

CLINICAL TRIAL DATA ON THE USE OF POLYPHENOLS IN THE MANAGEMENT OF DIABETES

Olive oil polyphenolcould promote HDL anti-atherogenic and cholesterol efflux function, grape seed powder reduces plasma concentrations of large LDL and large LDL-cholesterol particles in obese humans.Short-term supplementation of polyphenol, viz.,soyisoflavones could play lipolytic role and that epigallocatechin-gallate combined with resveratrol may improve metabolic health and body weight regulation.In view of the complications including calcium loss observed with habitual coffee and tea consumption, it was suggested that supplementation with adequate dietary calcium may be necessary for diabetic patients.Polyphenol-rich functional foods and nutraceuticals help activate intestinal T regula-

'Nexus' - Alumni Association SJCOPS, Chalakudy 37 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ISSN : 2350-1332 tory cells and homeostatic regulation of the gut microbiotathat could help reduce low-grade inflamma- tion. Supplementation of olive leaf polyphenols for 12 weeks has resulted in asignificant improvement of insulin sensitivity and pancreatic β-cell secretory power. Green tea seems to reduce fasting blood glucose levels in a small interventiontrial; although no improvement in HbA1c levels were seen. Human nutrigenomic studies with Mediterranean diet (MedDiet) and olive oil (OO) interventions, revealed that gene expression changes manifested by improvement in systemic markers for oxidation and inflamma- tion toward a protective action.Effectiveness of Hibiscus sabdariffa L. (HS) in addressing the risk factors associated with cardiovascular disease has been reported. Adding a spice mix to hamburger meat, prior to cooking helped in reducing the urinary malondialdehydeand improving postprandial endothelial dysfunction in men with Type 2 diabetes.A natural mixture of grape polyphenols at nutritional doses was demonstrated to efficiently prevent fructose-induced oxidative stress and insulinresistance. A test drink enriched in pomegranate polyphenols, consumed with a high-fat meal, was shown to reduce postprandial lipaemia and improve vascular function and blood pressure compared to placebo.A high-polyphenol chocolate administration was effective in protecting oral glucose challenge.Red wine administeredat 30 g alcohol/d for 4 weeks, has resulted in a beneficial effect on insulin resistance presumably through the non-alcoholic fraction of red wine (mainly polyphenols), that emphasizes the greater protective effects of red wine on cardiovascular disease than other alcoholic beverages. The role of nutritional supplements, e.g. L-carnitine and polyunsaturated fatty acids (PUFAs), exert lipid-lowering effects in overweight hyperlipidemic subjects highlightingthelipid-lowering effects of polyphenols and carnitine (PPC).Metabolic response to sweetened dried cranberries (SDC), raw cranberries (RC), and white bread (WB) in humans with type 2 diabetes were compared that showed the use of polydextrose as a bulking agent has a potential toreducecalorie content.Role of polyphenol rich chocolate in subjects with chronic fatigue syndrome (CFS) has suggested that high cocoa liquor/polyphenol rich chocolate (HCL/PR) may benefit these patients with improved symptoms.

Among the polyphenols that were evaluated through clinical trials for the management of diabetes, the following summary can be made. Olive oil polyphenols, soy isoflavone and pomegranate polyphenols exert beneficial cardiovascular effects. Olive oil polyphenol exclusively improved pancreatic beta cell secretory power. Few combinations such as epigallocatechin-gallate with resveratrol and cranberry with white-bread exhibited favorable energy expenditure to maintain a healthy metabolic process. Anti-inflammatory and protective effects are observed with respect to olive oil polyphenols. Insulin resistance can be addressed with grape polyphenols. At the outset, after the elaborate study, it is being observed that olive oil polyphenols greatly enhance the overall health condition of diabetic patients through improving insulin resistance and beta cell secretory power.

Based on prior findings and reports it is inferred that polyphenols without glycosidic linkage, viz., (Epi) catechin, Caffeic acid, Anthocyanins and Proanthocyanidin suffer from rapid onset and faster elimination necessitating frequent dose administration. However polyphenols with glycosidic linkage such as Hesperidin, Rutin, Genistin, Daidzin have higher Tmax, higher AUC and longer elimination half lives indicating that they possess a sustained duration of action which might be beneficial to the patient in overcoming frequent administration of the drug and combating non-compliance in diabetes management.

38 'Nexus' - Alumni Association SJCOPS, Chalakudy ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 CONCLUSION Although the trials reported above indicate a potential benefit to diabetic patients, it still remains to be unclear about the evidence, for using these foods in treating type 2 diabetes. Further, the bioavailability, dose, frequency of administration and toxicity profiles along with the pure active polyphenols from each of these dietary sources need to be understood before embarking on recommendation for a clinical dose. The dietary polyphenols are characterized by a low intrinsic activity and poor absorption from the intestine in addition to their rapid elimination. The present review can be used for further exploring the pharmacokinetic pitfalls of polyphenols in management of Diabetes.

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Manikandan et.al.,/ Journal of Pharma Innovative Research, 2015; 2(1): 43-55 Microchip: New Era in Novel Drug Delivery Systems - A Review

P. Manikandan*1, R.Nethaji1, N.S.Surenderan1, G.Babu2

1Department of Pharmaceutics, Devaki Amma Memorial College of Pharmacy, Chelembra, Malappuram 2Department of Pharmaceutical chemistry, Devaki Amma Memorial College of Pharmacy, Chelembra, Malappuram Corresponding Author Email: [email protected]

ABSTRACT Over the past 30 years, greater attention has been focused on development of sustained or controlled-release drug delivery system. The ideal drug-delivery system two pre requisites would require. First, it would be a single dose for the duration of treatment, whether it is for days or weeks or for the lifetime of the patient, as in diabetes or hypertension. Second, it should deliver the active entity directly to the site of action, thereby minimizing or eliminating side effects. Microchip is a new approach in the novel drug delivery system. It is a device act as an implant and delivers required quantity of drug on the site of action at specific time. This device having so many advantages like it stores the drug for long period of time and also protects the drug. Effective concentration of drug can be maintained in blood for long period of time. This system is more beneficial to treat chronic diseases. Chronic diseases require therapy for long period of time which improves patient non-compliance. Chronic diseases like diabetes, cancer, and osteoporosis can be treated by using microchip. There are some limitations in this type of drug delivery system which can be overcome in future research study. This type of drug delivery system can take new era in the world of medicine and technology. Key words: Controlled Release, Microchip, Cancer, Diabetes, Hypertension

INTRODUCTION Drug delivery refers to approaches, formulations, technologies, and systems for transporting a pharmaceutical compound in the body as needed to safely achieve its desired therapeutic effect. It may involve scientific site-targeting within the body, or it might involve facilitating systemic pharmacokinetics; in any case, it is typically concerned with both quantity and duration of drug presence. Drug delivery is often approached via a drug's chemical formulation, but it may also involve medical devices or drug- device combination products. Drug delivery is a concept heavily integrated with dosage form and route of administration, the latter sometimes even being considered part of the definition. Drug delivery technologies modify drug release profile, absorption, distribution and elimination for the benefit of improv-

'Nexus' - Alumni Association SJCOPS, Chalakudy 43 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ISSN : 2350-1332 ing product efficacy and safety, as well as patient convenience and compliance.1 Drug release is from diffusion, degradation, swelling and affinity based mechanism. Most common routes of administration include the preferred non-invasiveperoral (through the mouth), topical (skin), transmu- cosal, nasal,buccal/sublingual,vaginal,ocular and rectal)and inhalationsroutes. Many medications such as peptide and protein, antibody, vaccine and gene based drugs, in general may not be delivered using these routes because they might be susceptible to enzymatic degradation or cannot be absorbed into the systemic circulation efficiently due to molecular size and charge issues to be therapeutically effective. For this reason many protein and peptide drugs have to be delivered by injection or a Nano-needle array. For example, many immunizations are based on the delivery of protein drugs and are often done by injection.2Current efforts in the area of drug delivery include the development of targeted delivery in which the drug is only active in the target area of the body (for example, in cancerous tissues) and sustained release formulations in which the drug is released over a period of time in a controlled manner from a formulation. In order to achieve efficient targeted delivery, the designed system must avoid the host's defense mechanisms and circulate to its intended site of action. Types of sustained release formulations include liposomes, drug loaded biodegradable microspheres and drug polymer conjugates.3 IMPLANTABLE CONTROLLED DRUG DELIVERY SYSTEM The controlled drug delivery systems are used to release therapeutic agent at predictable rate in vivo. Most of controlled drug delivery systems are designed for implantable devices, transdermal, subcutane- ous, or intramuscular uses. Implantable devices can deliver drug which cannot be delivered via the oral route or are irregularly absorbed via GIT. These devices deliver drug in to blood stream. This replaces daily injections and repeated insertion of IV catheters. This is beneficial to the treatment of chronic diseases.Manikandanet al., 2015/ JOURNAL OF PHARMA INNOVATIVE RESEARCH, 2015; 2(1): 44-58 This system is particularly suitable for drug delivery requirements of insulin, steroids, chemothera- peutics, antibiotics, analgesics, heparin and contraceptives. These devices are placed under skin which reduces side effects causedJOUR by traditionalNAL administrationOF PHAR techniques.MA INN ImplantableOVATI VdevicesE RES canE controlARCH release by electrical power source to achieve zero order or manipulated non-zero release profile. Figure (ISSN: 2350-1332) 1 shows drug levels in the blood withUniversal (a) traditional Pharmaceutical drug dosing Research and (b)Pub controlled-deliverylication. All rights reserv dosing.ed

Currently available drug delivery implants Microchip & Microchip Device Design can be divided into two main categories, according to their way of delivery of drug in The microchip delivery system consists of a Currentlya passi availableve or drug acti vedelivery manne implantsr. Polyme can ber dividedsub intostrate two contmainaining categories, multiple according rese rtovoirs their wayimplants of delivery are ofthe drug mo sint common a passive p orassive active dru manner.g Polymercapable implants of holding are chemithe mostcals common in the spassiveolid, delivery systems. They are designed to liquid or gel form. Each reservoir is capped (i.e. with a conductive membrane) and wired maintain a constant diffusion rate of drug 'Nexus' - Alumni Association SJCOPS, Chalakudy 44out of the polymer or they degrade in the with the final circuitry controlled by a body at a particular rate, thereby releasing microprocessor. This central processor drug at that rate. A desirable drug delivery should be able to actively control electrically implant would be small in size, have the the exact time of release and the amounts of ability to protect the drug or biosensor from drugs dispersed by controlling the the body until needed, allow continuous and dissolution of the gold membrane. The pulsatile delivery of both liquid and solid system should be reasonable to manufacture drug formulations.4 by standard microfabrication techniques and still be cost-effective.Microchip allow Therefore, it is necessary to design a drug localized delivery of drug by direct delivery device that has the following placement of the device at the treatment site, characteristics: delivery on demand, programmable dosing cycles and automated delivery of multiple 1) It must be simple to use and manufacture. drugs. The microchip delivery system consists of a substrate containing multiple 2) It must be multi-welled so that drugs and reservoirs capable of holding chemicals in other molecules can be delivered for weeks the solid, liquid, or gel form. Each reservoir or years at a time, is capped (i.e. with a conductive membrane) 3) It must hold many different drugs or other and wired with the final circuitry controlled molecules of varying dosages and can by a microprocessor. This central processor release these substances in a controlled should be able to actively control electrically dependable manner, and the exact time of release and the amounts of drugs dispersed by controlling the 4) It must be biocompatible and small dissolution of the gold membrane. The enough to be implantable in the human body system should be reasonable to manufacture (i.e. a microchip).

ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 drug delivery systems. They are designed to maintain a constant diffusion rate of drug out of the polymer or they degrade in the body at a particular rate, thereby releasing drug at that rate. A desirable drug delivery implant would be small in size, have the ability to protect the drug or biosensor from the body until needed, allow continuous and pulsatile delivery of both liquid and solid drug formulations.4 Therefore, it is necessary to design a drug delivery device that has the following characteristics:

1) It must be simple to use and manufacture.

2) It must be multi-welled so that drugs and other molecules can be delivered for weeks or years at a time,

3) It must hold many different drugs or other molecules of varying dosages and can release these substances in a controlled dependable manner, and

4) It must be biocompatible and small enough to be implantable in the human body (i.e. a microchip). Microchip & Microchip Device Design The microchip delivery system consists of a substrate containing multiple reservoirs capable of holding chemicals in the solid, liquid or gel form. Each reservoir is capped (i.e. with a conductive membrane) and wired with the final circuitry controlled by a microprocessor. This central processor should be able to actively control electrically the exact time of release and the amounts of drugs dispersed by controlling the dissolution of the gold membrane. The system should be reasonable to manufacture by standard microfabrication techniques and still be cost-effective.Microchip allow localized delivery of drug by direct placement of the device at the treatment site, delivery on demand, programmable dosing cycles and automated delivery of multiple drugs. The microchip delivery system consists of a substrate containing multiple reservoirs capable of holding chemicals in the solid, liquid, or gel form. Each reservoir is capped (i.e. with a conductive membrane) and wired with the final circuitry controlled by a microprocessor. This central processor should be able to actively control electrically the exact time of release and the amounts of drugs dispersed by controlling the dissolution of the gold membrane. The system should be reasonable to manufacture by standard micro fabrication techniques and still be cost-effective.5

The Design Approach—an Overview6 1. The Substrate According to system design, the reservoirs will be patterned into the substrate. This can easily be done by standard etching techniques of microfabrication. Any material that can serve as a support, is suitable for etching, and is impermeable to the molecules to be delivered and to the surrounding fluids may be used as a substrate. For this in vivo application, biocompatibility should be considered. Non-biocompatible materials, however, can also be enclosed within biocompatible materials like poly (ethylene glycol). One example of a strong, nondegradable, easily etched substrate that is impermeable to the delivered chemicals and nondegradable to the surrounding environment within the body is Silicon. It should be noted that for some applications a material degradable over time might be preferred. For example, brain implants make the

'Nexus' - Alumni Association SJCOPS, Chalakudy 45 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ISSN : 2350-1332 removal of a device difficult or too endangering to the patient and therefore this device would not be applicable. 2. Release System The design of a release system depends on the treatment required by the patient whether it is a continuous or pulsed release. Drug delivery can be achieved by a passive or active release system. In the passive system, the drugs diffuse through a membrane or enter the body by the degradation of the substrate. Active systems are triggered by a microprocessor and are preferred due to a more predictable release profile. The exact time release and amounts of drugs can then be controlled. The chip can be placed stra- tegically as well for drugs that are too potent for a continuous release. The device being described will be employing an active system. 3. Reservoir Caps In the active timed-release devices, the reservoir caps consist of thin films of conductive material patterned in the shape of anodes surrounded by cathodes. Any conductive material that can oxidize and dissolve in solution upon application of an electric potential can be used for the fabrication of the anodes and cathodes. The anode is defined as the electrode where oxidation occurs. The portion of the anode directlyManikand aboveanet theal., 2015 reservoir/ JOURNAL oxidizes OF PHARMA and dissolves INNOVATIVE into RE solutionSEARCH, 2015 upon; 2(1): the 44-58 application of a potential between the cathode and anode. This exposes the release system to the surrounding fluids and results in the release of the molecules or drugs. Gold is chosen as the model membrane material because it is easily deposited and patterned, has a low reactivity with other substances and resists spontaneous corrosion in many solutions overJOUR the entireNAL pH range2.OF PHAR However,M theA PresenceINNOVATI of a smallV amountE RES ofE chlorideARCH ion creates an electric potential region which favors the formation(ISSN: 2350-13 of soluble32) gold chloride complexes (Frankenthal et al, 1982). Holding theUniversal anode Phpotentialarmaceutical in this Research corrosion Pu regionblication. enables All rights reproducible reserved gold dissolution. Potentials below this region are too low to cause appreciable corrosion, whereas potentials above this region result in gas evolution and formation of a passivating gold oxide layer that causes cor- rosioncorrosio to slown to or slowstop (Santinior stop et (Sal,a ntini1999). et Gold al, has alsosou beenrce. shownThe mi tocroprocesso be a biocompatibler will contr material.ol the 1999). Gold has also been shown to be a desired reservoir to be activated so that a 4. biocomp Controlatible Circuitry material. and Power Source variety of drugs may be contained in each specific reservoir. The input source can The control circuitry consists of a timer, demultiplexer, microprocessor or an input source. The micro- 4. Control Circuitry and Power either be a memory source, remote control processor will control the desired reservoir to be activated so that a variety of drugs may be contained Source device or a biosensor. A thin-film micro in each specific reservoir. The input source can either be a memory source, remote control device or a battery can be used as a power source. All of The control circuitry consists of a timer, biosensor. A thin-film micro battery can be used as a powerthes esource. can beAll pofatterned these can d irectlybe patterned onto directly the demultiplexer, microprocessor or an input onto the device. device.

Schematic diagram of general circuit desiign Schematic diagram of general circuit design 5. Reservoir filling known concentration into each reservoir 46 (Santini'Nexu ets ' - al, Alumni 1999).The Association volume SJCOPS, oChalakudyf the Three-dimensional printing is capable of reservoirs can be controlled by specifying fabricating complex structures by ink-jet the appropriate print head to deposit a pre- printing liquid binder onto loose, fine determined amount of binder. The drug is powder (Wu et al, 1999). The printing pushed out of the nozzle as the vapor bubble pattern can be obtained from a computer- within the nozzle expands upon heating. The aided-design model (CAD). Inkjet printing relationship between the amounts expanded in combination with a computeer-controlled by the vapor bubble to the heat added alignment apparatus is capable of depositing follows the ideal gas law relationship. as little as 0.2 nl of a liquid or gel solution of

6. Microfabrication

Manikandanet al., 2015/ JOURNAL OF PHARMA INNOVATIVE RESEARCH, 2015; 2(1): 44-58

corrosion to slow or stop (Santini et al, source. The microprocessor will control the 1999). Gold has also been shown to be a desired reservoir to be activated so that a biocompatible material. variety of drugs may be contained in each specific reservoir. The input source can 4. Control Circuitry and Power either be a memory source, remote control Source device or a biosensor. A thin-film micro battery can be used as a power source. All of The control circuitry consists of a timer, these can be patterned directly onto the demultiplexer, microprocessor or an input device.

ISSN : 2350-1332 Schematic diagram of general circuitJ. Pharm. des iigInn.n Res. | June 2015 | Vol - 2 | Issue - 2 5. Reservoir filling 5. Reservoir filling known concentration into each reservoir Three-dimensional printing is capable of fabricating complex(Santini structures et al, 1999).The by ink-jet printingvolume liquid of the binder ontoThree-di loose,m fineensional powder printing (Wu et isal, capable1999). The of printingrese patternrvoirs can can be b eobtained controll fromed bya computer-aided- specifying designfabricati modelng comple(CAD).x Inkjet structures printing by in ink-jecombinationt thewith app a computer-controlledropriate print head t oalignment deposit aapparatus pre- is printingcapable ofliquid depositing binder as little onto asloose, 0.2 nl of fin ae liquid ordet gelermined solution am ofount known of bin concentrationder. The dr uintog is each reservoirpowder (Santini(Wu et et al, al, 1999). 1999).The T volumehe printin of theg reservoirspush caned out be controlledof the nozzle by specifyingas the vapor the b uappropribble - atepattern print headcan to be deposit obtained a pre-determined from a compute amountr- of binder.within T thehe nozzdrug lise expandspushed out upon of heatinthe nozzleg. The as the vaporaided-de bubblesign within model the (CAD). nozzle Inexpandskjet printin upong heating. rela Thetionship relationship betw eenbetween the a themounts amounts exp aexpandednded byin the comb vaporination bubble wit toh the a computheat addedeer-controlle followsd the idealb ygas the law vaporrelationship. bubble to the heat added alignment apparatus is capable of depositing follows the ideal gas law relationship. as little as 0.2 nl of a liquid or gel solution of

6. Microfabrication

6. Microfabrication Microfabrication can be generally defined as the production of micro scale features in or on a material by techniques such as deposition, etching, micro molding along with patterning techniques such as photolithography or micro contact printing (Jonh et al, 2000).Fabrication of these microchips begins by depositing ~0.12 mm of low stress, silicon-rich nitride on both sides of prime grade, silicon wafers using a vertical tube reactor (Santini et al, 1999). The silicon nitride layer on one side of the wafer is patterned by photolithography and electron cyclotron resonance (ECR) enhanced reactive ion etching (RIE) to give a square device containing square reservoirs. The silicon nitride serves as an etch mask for potassium hydroxide solution at 85°C, which anisotropically etches square pyramidal reservoirs into the silicon along the crystal planes until the silicon nitride film on the opposite side of the water is reached. The newly fabricated silicon nitride membranes completely cover the square openings of the reservoir. Gold electrodes (0.3-0.5 mm thick) are deposited and patterned over the silicon nitride membranes by electron beam evaporation and lift- Off. Some portions of the electrodes must be protected from unwanted corrosion by an adherent, non-po- rous coating that isolates the electrode materials from the surrounding electrolyte. Silicon dioxide is used as a model protective coating because its physical properties can be tailored to a particular application by selecting the appropriate processing conditions2. A layer of plasma enhanced chemical vapordeposition silicon dioxide is deposited over the entire electrode containing surface. The silicon dioxide located over portions of the anode, cathode, and bonding pads are etched with ECR-

'Nexus' - Alumni Association SJCOPS, Chalakudy 47 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ISSN : 2350-1332 enhanced RIE to expose the underlying gold film. This technique is also used to remove the thin silicon nitride and chromium membranes located in the reservoir underneath the gold anode. The reservoirs are then filled with the molecules or drugs to be delivered by the aforementioned reservoir filling methods and subsequently sealed. 1.) Deposit layer of insulating material,silicon nitride (0.12 mm), onto the substrate by PECVD 2.) Pattern by photolithography and square reservoirs are etched by ECR-enhanced RIE 3.) With potassium hydroxide solution at 85°C, anisotropically etch square pyramida reservoirs into the silicon along the (111) crystal 4.) Invert and deposit gold electrodes (0.3-0.5 mm thick). Pattern by E-beam evaporation and lift off. Manikandanet al., 2015/ JOURNAL OF PHARMA INNOVATIVE RESEARCH, 2015; 2(1): 44-58 5) Deposit electrode protective coating,silicon dioxide, by PECVD. Silicon dioxide over anode, cathode and bonding pads are etched with ECR-enhanced RIE to expose gold film. 6.) Remove SiN layer in theJOUR insideN of ALreservoir OF byPHAR RIE to M exposeA IN goldN OVATImembrane.V E RESEARCH 7.) Fill reservoirs by inkjet printing through opening (500 (ImmSSN: x 2350-13500 mm)32) Universal Pharmaceutical Research Publication. All rights reserved

7. Device testing: MicrochipMicroch hasip hasdemonstrated demonstrated in vivo in vivo and in and vitro in releasecoll of drugsected usingfracti othens technology.are analyzed. D eviceBoth hasblood been vitro release of drugs using the technology. and urine are monitored to evaluate release. tested by releasing radio-labeled compounds and therapeutic drugs and detecting release by scintillation Device has been tested by releasing radio- Incremental and cumulative release profiles counting and liquid chromatography, respectably. In vitro testing is performed with flow cell configura- labeled compounds and therapeutic drugs measured from urine in a rate are shown in tion, in which the chip is mounted in a chamber of phosphate-buffer saline (PBS). Periodically the PBS and detecting release by scintillation Figure No. 11. These experiments have is replacedcounting via andinlet and liq uidoutlet chrotubesm andatography the collected, fractionsshown thatare analyzed. drug release Both bloodis reliable and urine and are monitoredrespecta tobly. evaluate In vitr release.o testing Incremental is performe and dcumulative rep releaseeatable. profiles Scientists measured are designing from urine in vivoin a rate arewith shown flo inw F celligure con No.fif guration,11. These iexperimentsn which the have shownexpe rimentsthat drug o nrelease the 100 is reliable –dose and IDDS. repeatable. The Scientistschip is aremounted designing in a inchamber vivo experiments of phosphate on -the 100 res–doseult fromIDD S.thes Thee experime result fromnts thesewill be experiments used to willb ufferbe used saline to establish (PBS). Periodicallthe long-termy the stability PBS i sof the deviceestab lishand the the efficacy long-term of released stability drugs. of the replaced via inlet and outlet tubes and the device and the efficacy of released drugs.

In-vivo release proofile (urine measurements)

48 'Nexus' - Alumni Association SJCOPS, Chalakudy 8. Delivery schedule Theory of operation The drug delivery schedule is heavily ¾ Each reservoir is sealed at one end dependent on patient need. However, the by a thin membrane of material that 400 reservoirs add flexibility to patient serves as an anode in an treatment. The multiple reservoirs can hold electrochemical reaction and multiple drugs and can release them in dissolves when an electric potential varying amounts. For example, with the is applied to it in an electrolyte. battery capabilities, the patient can be ¾ There must be at least one other administered 25 ml (one reservoir) per day. electrode on the device surface to At this rate, the drugs can be delivered serve as a cathode in the everyday for over a year. electrochemical reaction.

ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 8. Delivery schedule

The drug delivery schedule is heavily dependent on patient need. However, the 400 reservoirs add flex- ibilityManikand to patientanet al., 2015treatment./ JOURNAL The OmultipleF PHARMA reservoirs INNOVATIVE can RE SholdEARCH, multiple 2015; 2(1): drugs 44-58 and can release them in varying amounts. For example, with the battery capabilities, the patient can be administered 25 ml (one reservoir) per day. At this rate, the drugs can be delivered everyday for over a year. Theory of operation JOURNAL OF PHARMA INNOVATIVE RESEARCH - Each reservoir is sealed at one end by a thin membrane(ISSN: 2350-13 of material32) that serves as an anode in an Universal Pharmaceutical Research Publication. All rights reserved electrochemical reaction and dissolves when an electric potential is applied to it in an electrolyte. - There must be at least one other electrode on the device surface to serve as a cathode in the electrochemical¾ The reaction. cathode must be made of any ¾ An electric potential is applied to an - Theconductive cathode mustmaterial be made but of is any usuall conductivey materialanode but is musuallyembrane made when of therelease same from material made of the same material as the corresponding reservoir is desired. as the anodes to simplify fabrication procedures. anodes to simplify fabrication This causes oxidation of the anode - Inp addition,rocedures. any number of cathodes can be designed onmateria a microchip,l and and fo theyrmation can be of of differentthe shape¾ or sizeIn addition, to suit the any electrode number designof cathode desireds for a particularsoluble application. complex with the electrolyte - Thecan reservoirs be designed are onfilled a mi throughcrochip, the an dopen end with theions. drug/molecule to be released. they can be of different shape or ¾ The complex then dissolves in the - The open ends of the reservoirs are then sealed with a waterproof material. size to suit the electrode design electrolyte and the membrane - Thedesired device for is a submerged particular ainpplication. an electrolyte containing ionsdisappe thatars. form a soluble complex with the anode¾ material The reservo in its ionicirs are form. fille d through the ¾ The chemical in the newly opened - Ano penelectric end potentialwith the drugis applied/molecule to an t oanode membranereservo when ireleaser is now from exposedcorresponding to the reser - voir is desired.be released. This causes oxidation of the anode material andsurrou formationnding. of the soluble complex with ¾ ¾ the electrolyte The open ions. ends of the reservoirs are Electrolyte and is free to dissolve in then sealed with a waterproof the electrolyte and diffuse out of the - Thematerial. complex then dissolves in the electrolyte and the membranereservoir. disappears. - ¾T he The chemical devic ein isthe submnewlyerged opened in reservoir an is now exposed to the surrounding. electrolyte containing ions that form - Electrolyte and is free to dissolve in the electrolyte and diffuse out of the reservoir. a soluble complex with the anode material in its ionic form.

Operation of microchip

Microchip Technologyo in Drug Delivery7 drug delivery focused on achieving 'Nexus' - Alumni Association SJCOPS, Chalakudy sustained drug release rates over time, while 49 The realization that the therapeutic efficacy a more recent trend is to make devices that of certain drugs can be affected dramatically allow the release rate to be varied over time. by the way in which they are delivered has Advances in microfabrication technology created immense interest in controlled drug have made an entirely new type of drug delivery systems. Much previous work in 51

J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ISSN : 2350-1332 Microchip Technology in Drug Delivery7 The realization that the therapeutic efficacy of certain drugs can be affected dramatically by the way in which they are delivered has created immense interest in controlled drug delivery systems. Much previous work in drug delivery focused on achieving sustained drug release rates over time, while a more recent trend is to make devices that allow the release rate to be varied over time. Advances in microfabrication technology have made an entirely new type of drug delivery device possible. Proof-of- principle experiments have shown that silicon microchips have the ability to store and release multiple chemicals on demand. Future integration of active control electronics, such as microprocessors, remote control units, or biosensors, could lead to the development of a 'pharmacy on a chip,' ie 'smart' microchip implants or tablets that release drugs into the body automatically when needed From pacemakers to artificial hips, people are getting more familiar with new technology placed in their bodies, but im- bedding microchips still raises many privacy concerns. People who are weary of daily needle injections for their medical issues could soon have new options: researchers have been developing an implantable microchip that will replace needles. In 2011, human trials began in Denmark and elsewhere. According to Plastics Today, researchers developed wireless and implantable microchips. The future of drug delivery may be a programmable, wirelessly controlled microchip implanted in a patient's body. A Massachusetts Institute of Technology (MIT) research team envisioned the implantable microchip idea 15 years ago. The team created a Micro-CHIPS company to explore commercialization. Recently, the researchers announced a successful in-body test results. Human clinical trials began in Denmark during 2011 with successful results. The Micro-CHIPS company plans to develop implants with the capability to transport hundreds of drug doses per chip. Dosages will be scheduled in advance or triggered remotely by radio communication over a special frequency called Medical Implant Com- munication Service. Implantable microchip devices will provide real-time dose schedule tracking, and as part of a network, physicians can remotely adjust treatment schedules as necessary. Micro-CHIPS expect that it will take a few years for its first product to reach the market. During this time, the company plans on refining the technology design for a microchip implant so it will deliver drugs for either one or two years. The company should complete the clinical studies for approval by regulatory authorities within the next few years, according to Plastics Today. Reuters reported that an implantable, wireless microchip successfully delivered osteoporosis medicine to a small group of Danish women. This accomplishment raises hope for a new kind of drug delivery system that will allow patients to skip regular injections. The device, currently being developed by privately held Microchips Incorporated, has a wireless receiver that signals the microchip to release the drug, according to Reuters. According to the Los Angeles Times, science researchers say they have devised a modern technique for giving patients their medicine. This new method involves a microchip embedded in the body where doctors can control drug release into their patients’ body remotely by a wireless connection. The drug chip was employed for delivering bone-strengthening hormones to women suffering with advanced osteoporosis who otherwise would have needed daily injections. After four months, the chips were safely removed from the patients' bodies, according to science researchers. According to Radio Frequency Identification Technology News (RFIDNews), Positive-ID declared that it has received an order for its Veri-Chip microchip to be employed for disaster preparation and crisis management by the Israeli Military. The Veri-Chip microchip was approved by the U.S. Food and Drug Administration for patient identification in 2004. The company’s integration partner intends to provide the microchips to the Israeli military. Veri-Chip will assist crisis situations and disaster recovery in com-

50 'Nexus' - Alumni Association SJCOPS, Chalakudy ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 bination with cameras capable of wirelessly receiving of both RFID scanned data and GPS data. A Web- enabled database will support the gathering and storing of information and images captured during disaster response operations. Positive-ID stated that it has developed its RFID glucose-sensing microchip, called the Gluco-Chip. This microchip will precisely calculate glucose levels in any diabetic’s body. According to the 2011 National Diabetes Fact Sheet, more than 25 million children and adults in the U.S. have diabetes, which is roughly 8 percent of U.S. population. The Gluco-Chip is FDA cleared and based on Positive-ID’s Veri-Chip microchip employed for patient identification. The company believes the measurement of glucose levels through this system will allow individuals with diabetes to monitor glucose levels in a less invasive manner, according to RFIDNews. Nano-microchip8 It's almost surreal, like something out of a sci-fi flick, but nano-microchips invisible to the naked eye are a reality that are already being hosted in wide-range of applications. Nanotechnology deals with structures smaller than one micrometer (less than 1/30th the width of a human hair), and involves developing materials or devices within that size. To put the size of a nanometer in perspective, it is 100,000 times smaller than the width of a human hair. More than ten years ago simple low-cost techniques improved the design and manufacture of nano-microchips. That unlocked a multitude of methodologies for their manufacture in a wide-range of applications including optical, biological, and electronic devices. The joint use of nano-electronics, photolithography, and new biomaterials, have enabled the required manufacturing technology towards nano-robots for common medical applications, such as surgical instrumentation, diagnosis and drug delivery.Japan's Hitachi says it has developed the world's smallest and thinnest microchip, that can be embedded in paper to track down parcels or prove the authenticity of a document. The integrated circuit (IC) chip is as minute as a speck of dust.Nano-electrodes implanted in the brain are increasingly being used to manage neurological disorders. Micro-Chip Applications9-11 Chemicals to be released Multiple chemicals can be stored inside and released from the microchip. Each reservoir can be filled with different chemicals or combination of chemicals. Chemicals in any form (solid, liquid, gel) can be delivered by microchip. Micro fluidic device such as pumps are limited to delivering liquids. The controlled release microchip consists of reservoir covered by a thin membrane of material that can be dissolved on demand. The form of the chemical or drug in the reservoir and the presence or absence of other materials such as polymer matrices or excipient has little or no effect on the electrochemical behavior of the membrane. Therefore, controlled release microchip has the potential for a high degree of flexibility in the type of chemicals they can store and release. Simplicity of release mechanism The microchip has no moving parts .A thin barrier membrane covers the each reservoir filled with one or more chemicals. The release of chemicals from the microchip is initiated by disintegration of the membrane. The membrane is removed by the application of an electric potential, which cause the membrane to dissolve by simple electrochemical reaction. The absence of moving parts potentially increases device reliability by decreasing the possibility of mechanical breakdown. (Jonh et al, 2000) Accuracy

'Nexus' - Alumni Association SJCOPS, Chalakudy 51 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ISSN : 2350-1332 A Variety of highly potent drug can potentially be delivered from the microchip in a safe manner. It is important that the amount of drug delivered to a patient matches the amount prescribed, especially for highly potent compounds. Each reservoir of microchip can be accurately filled with a small amount of the drug by using microinjection or ink-jet printing techniques (J.Yoo et al 1997).The amount of the drug administered from a microchip filled by this printing methods can be tightly controlled, and accidental overdose id unlikely because release from active devices can only occur when an electric potential is applied to an anode. Larger doses can be administered by simply opening several reservoirs simultaneously. Complex release patterns Complex release patterns (such as simultaneous constant and pulsatile release) can be achieved from the microchip. Any complex chemical or drug release pattern can be broken down into a combination of two parameters: Release time and Release rate. A unique feature of the controlled release microchip is the potential to control both of these parameters. The time at which release begins from any reservoir is determined by the time at which the anode membrane covering that reservoir is removed. Spontaneous release from reservoir will not occur if the anode membrane material is stable in the electrolyte solution. Therefore, an anode membrane material is selected that will not dissolve and open until the correct electric potential is applied. Potential for local delivery The microchip can be made small enough to make local chemical delivery possible. An advantage of local drug delivery is that high concentration of drug can be achieved at the site where it is needed, while keeping the systemic concentration of the drug at a low level. This technique is particularly useful if the drug has adverse side effect if administered systemically in high doses. Stability enhancement Some new protein based drugs have limited stability (i.e., shelf life ).Water penetration into this protein drug formulation is one of the most frequent causes of their instability. The membrane covering the filled reservoir of a microchip will prevent penetration of water into these reservoirs.Thus,the stability of protein drug is theoretically enhanced first, because the drug can be isolated from the outside environment (hermetically sealed) and second, because they can be stored in the microchip in their most stable form (solid, liquid, gel). In cancer therapy Measuring proteins in the blood can help doctors determine patients' cancer risk and monitor the health of the elderly and people with chronic diseases. But current methods for testing these proteins are too expensive and require too much blood to be performed regularly. A micro fluidic chip in clinical trials does on a single chip in 10 minutes what normally takes multiple technicians’ hours to do and with just a single drop of blood. Researchers hope to make bedside diagnostics based on blood proteins a reality by bringing down the cost of such tests by at least an order of magnitude. The diagnostic chip is being developed by Caltech chemistry professor James Heath and by Leroy Hood, the president and founder of the Institute for Systems Biology, in Seattle. Heath and Hood have founded a company called integrated .Diagnostics to commercialize the blood chip.

52 'Nexus' - Alumni Association SJCOPS, Chalakudy ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 Microchip for Antidepressants Depression is the fourth most important cause of disability in the world. In Britain, most depressed patients are managed in primary care and antidepressant drugs represent the mainstay of treatment. To-date, tricyclic antidepressants have been the most widely used group of drugs and still account for approximately 50% of all new prescriptions. Almost all previous studies have relied on indirect methods of assessment including self-reporting of tablet consumption and the counting of left-over tablets. More recently, mechanical devices such as the microprocessor-based Medication Event Monitoring System (MEMS) have been developed. The assay of blood for drug and its metabolites has also been used for dothiepin a ratio of nordothiepin: dothiepin of greater than 1.1 indicates noncompliance for a period of 48 h or longer. The MEMS system allowed us to identify the precise times at which opening of the container occurred. As a consequence it was possible to detect when patients ceased to take their medication, the occurrence of drug holidays, apparent increases in tablet consumption prior to review by research nurses and variability in the timing of drug taking during the study. Implantable technology for psycho- tropic medications may have its historical beginnings in the use of haloperidol or fluphenazine depot injection formulations, which represented a crude delivery system that delayed the delivery of the drug to the circulatory system by its slow dissolution from a lipophilic matrix. Additional applications and variations of reservoir-based microchip technology include Alleviation of osteonecrosis, using an implant that locally administered drug to bone in the affected joint. Application of active release from reservoirs from orthopedic implants to address a wide range of bone health issues. Laser-based photolytic opening of selected reservoirs as an enabler for reservoir opening in ophthalmic applications; ● Light-based actuation using a photosensitive gel that changes volume and acts as a valve to a reservoir; and Alternative circuit design to decrease the number of connections required for independently controlled reservoirs.This technology can be useful in diabetes, Parkinson’s disease, congestive heart failure, anti-coagulation. CURRENT DEVELOPMENTS 12 Microchip technology Electronic identification or radio frequency identification technology has been tested for identification purposes for over twenty-five years. Three types of devices can be categorized, as follows:  Implantable microchips for permanent application, which are injected or surgically implanted.  Microchips deposited in body cavities or orally ingested for temporary application.  Electronic devices that can be attached to the exterior of an animal.  A well-known company with the name Microchips has done research on microchip based drug delivery which is as following. (Santini et al, 1999)  microchips’ development of a long-term implant designed to provide 100% compliant delivery of parathyroid hormone for people who suffer from severe osteoporosis. Parathyroid hormone (PTH) is the only drug therapy available in the US that has an anabolic effect on bone, resulting in marked bone growth.  In November, Microchips’ was awarded the 2008 AAPS Drug Delivery Technology Award

'Nexus' - Alumni Association SJCOPS, Chalakudy 53 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ISSN : 2350-1332 for its osteoporosis research. The award is given by the American Association of Pharma- ceutical Scientists to recognize outstanding research pertaining to novel drug delivery technologies. Microchips’ device is being developed to conveniently deliver human parathyroid hormone (hPTH 1-34) to help build bone, prevent new fractures, and improve the quality of life for patients with osteoporosis. CONCLUSION It can be concluded that, the future trend requires advanced drug delivery system like individualized therapy and the capability to automate delivery system. Microchip based implantable drug delivery devices allow localized delivery by direct placement of the device at the treatment site, delivery on demand (pulsatile, adjustable continuous dosing, and emergency administration, programmable dosing cycle) automated delivery of multiple drug and dosing in response to physiological response. The designed microchip for drug delivery allows for storage and dependable controlled release of multiple drugs. This device is less complex and much more dependable than the aforementioned devices that attempt to control drug release rate (i.e. electro-mechanical or polymer systems). The microchip can be created by general microfabrication techniques and can also be self-contained, which eliminates the need for patient or doctor intervention. The proposed device described (assuming one dose per day) can last over a year; however, the delivery abilities do depend on patient need. Today, internal drug delivery devices that sense, stimulate, deliver to, and record from biological systems are being developed by application of the growing fields of micro technology and nanotechnology. Some of these devices are programmable, i.e., drugs can be stored and released on predetermined or real-time demand. A silicon microchip with the ability to provide on-demand controlled release of single or multiple drugs. The future may also hold the development of a biodegradable microchip that, once implanted, would not require removal. Like the available delayed-release antidepressants, implantable drug delivery systems such as microchips will enhance drug safety, tolerability, and efficacy because of the ability to maintain a more constant plasma drug level. These encouraging results for support the feasibil- ity of applying microchip-based implant technology to deliver other therapeutic peptides and proteins.

REFERENCES

1. A Controlled Release Microchip. MIT NewsJanuary20, 1999. http://web.mit.edu/newsoffice/ nr/1999/microchipcom.html. (Accessed on 15.02.15)

2. Santini JT, Cima MJ, Langer R. A Controlled release Microchip. Nature 1999; 397: 335-338.

3. Ramille M. Capito,Leah A. Lucas. Microchip for drug delivery, June 4, 2000

4. John TS Jr, Amy CR, Rebecca S, Michael JC, Robert L. Microchips as Controlled Drug Delivery Devices. AngewandteChemie International Edition 2000; 39: 2396-2407.

5. Sood A and Panchagnula R. Peroral Route: An Opportunity for Protein and Peptide Drug Deliv- ery”, Chemical Reviews 2000, 101: 3275-3303.

6. Soppimath KS, Aminabhavi TM, Kulkarni AR, Rudzinski WE. Biodegradable polymeric nanoparticles as drug delivery devices. Journal of Controlled Release 2001: 70, 1-20.

54 'Nexus' - Alumni Association SJCOPS, Chalakudy ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 7. madou MJ. Fundamentals of Microfabrication. 2nd Edition, FL: CRC Press, Boca Raton, 2002.

8. Kopecek J. Smart and genetically engineered biomaterials and drug delivery systems. European Journal of Pharmaceutical Sciences 2003, 20, 1-16.

9. John MM, Scott AU, Benjamin FP, Norman FS Jr, Christina MP, John TS Jr. Electro thermally activated microchips for implantable drug delivery and biosensing. Journal of Controlled Release 2005, 109:2444-2455. Journal of Pharmaceutical Sciences 2003, 20, 1-16

10. mark S. Microchip and Controlled-release drug reservoirs. WIREs’Nanomed Nan biotechnology 2010; 2:400-417.

11. maloney JM. An implantable microfabricated drug delivery system. Am. Soc. Mech. Eng. 2003, 70, 115–116.

12. Santini JT Jr, Hutchinson CE, Proos ER. Medical and dental implant devices for controlled drug delivery, US20070016163A1, 2007. Bhowmik D. Microchip drug delivery new era of drug delivery system. Elixir Pharmacy 2013, 57: 13925-13929.

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Rajasree et.al.,/ Journal of Pharma Innovative Research, 2015; 2(1): 56-69 In-vitro Techniques for Cytotoxic Evaluation Rajasree R.S.*1, Greeshma P.1, Sibi P.I.2

1College of Pharmaceutical Sciences, Government Medical College,Kottayam 2Dept. of Pharmaceutical Sciences, Mahatma Gandhi University, Ettumanoor ,Kerala Corresponding Author Email: [email protected]

ABSTRACT Analyzing the effect on cell growth and /or cell death has been an important component of many researches, especially in cancer treatment development. So in-vitro cytotoxicity testing is a challenging task.Many methods are involved for this purpose. Most of them are cell viability Assays. Here we briefly describe the currently used cytotoxic tests and their advantages and disadvantages. Key-words:In-vitro assay, cell viability

INTRODUCTION In vitro cytotoxicity testing is the scientific analysis of the effects of toxic chemical substance on cultured bacteria or mammalian cells. In vitro (literally glass) testing methods are primarily used to evaluate the toxicity of potential anti-cancer drugs. A significant part of drug discovery in the last forty years has been focused on agents to prevent or treat cancer. This is not surprising because, in most developed countries and, to an increasing extent, in developing countries, cancer is amongst the three most common causes of death and morbidity. Treatments for cancer may involve surgery, radiotherapy and chemotherapy and often a combination of two or all three is employed.Natural compounds from flowering plants play a significant role in cancer chemotherapy. Anticancer drugs in wide clinical use include vincristine and vinblastine from Catharanthusroseus, palitaxel (Taxol) and taxotere from species of yew (Taxus), etopo- side derived from lignans of Podophyllum spp. and camptothecin analogues, such astopotecan, from Camptothecaacuminata.[1] All of these are fundamentally cytotoxic and act principally by inhibiting cell proliferation, but by different mechanisms. Their cytotoxic effects are evaluated by using various invitro testing methods. Assessing cell membrane integrity is one of the most common ways to measure cell viability and cytotoxic effect. Cytotoxicity testing is based on one or more mammalian cell lines being grown under conditions, where they are actively growing and undergoing mitotic division. Cells are cultured in a microtitre well plate and the rate of multiplication and growth is measured indirectly by formation of a colour, the intensity of which is directly proportional to the number of cells present. A variety of experiments can be used and the most basic is to compare the rate of proliferation of a cancer cell line in the presence and absence of 56 'Nexus' - Alumni Association SJCOPS, Chalakudy ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 the test substance, usually after a specified time. [2] The major invitro cytotoxic evaluation techniques are MTT assay, MTS assay, SRB assay, Resazurin reduction assay, Protease viability marker assay, ATP assay, Neutral red uptake assay, Clonogenic assay, WST assay, Agar overlay diffusion method.The present review deals withthe study of various invitro cytotoxicity test, their advantages and disadvantages. MTT TETRAZOLIUM ASSAY PRINCIPLE The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay was the first homogeneous cell viability assay developed for a 96-well format that was suitable for high through put screening (HTS)[3] .The MTT substrate is prepared in a physiologically balanced solution, added to cells in culture, usually at a final concentration of 0.2 -0.5mg/ml, and incubated for 1 to 4 hours. The quantity of formazan (presumably directly proportional to the number of viable cells) is measured by recording changes in absorbance at 570 nm using a plate reading spectrophotometer. A reference wavelength of 630 nm is sometimes used, but not necessary for most assay conditions.Viable cell with active metabolism convert MTT into a purple colored formazan product with an absorbance maximum near 570 nm. When cells die, they lose the ability to convert MTT into formazan, thus color formation serves as a useful and convenient marker of only the viable cells.[4]The formazan product of the MTT tetrazolium accumulates as an insoluble precipitate inside cells as well as being deposited near the cell surface and in the culture medium. The formazan must be solubilized prior to recording absorbance readings. A variety of methods have been used to solubilize the formazan product, stabilize the color, avoid evaporation, and reduce interference by phenol red and other culture medium components.[5,6] Various solubilization methods include using: acidified isopropanol, DMSO, dimethylformamide, SDS, and combinations of detergent and organic solvent. [1,5,6] Acidification of the solubilizing solution has the benefit of changing the color of phenol red to yellow color that may have less interference with absorbance readings.[7] The amount of signal generated is dependent on several parameters including: the concentration of MTT, the length of the incubation period, the number of viable cells and their and metabolic rate etc. Reagent preparation: MTT Solution

1. dissolve MTT in Dulbecco’s Phosphate Buffered Saline, pH=7.4 (DPBS) to 5 mg/ml.

2. Filter-sterilize the MTT solution through a 0.2 µM filter into a sterile, light protected container.

3. Store the MTT solution, protected from light, at 4°C for frequent use or at -20°C for long term storage. Solubilization Solution

1. Choose appropriate solvent resistant container and work in a ventilated fume hood.

2. P repare 40% (vol/vol) dimethylformamide (DMF) in 2% (vol/vol) glacial acetic acid.. Add 16% (wt/vol) sodium dodecyl sulfate (SDS) and dissolve.Adjust to pH = 4.7

3. Store at room temperature to avoid precipitation of SDS. If a precipitate forms, warm to 37°C and mix to solubilize SDS.

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1. Prepare cells and test compounds in 96-well plates containing a final volume of 100 µl/well.Incu- bate for desired period of exposure.

2. Add 10 µl MTT Solution per well to achieve a final concentration of 0.45 mg/ml..Incubate 1 to 4 hours at 37°C.

3. Add 100 µl Solubilization solutions to each well to dissolve formazan crystals.

4. mix to ensure complete solubilization.

5. Record absorbance at 570 nm. MTS TETRAZOLIUM ASSAY PRINCIPLE More recently developed tetrazolium reagents can be reduced by viable cells to generate formazan products that are directly soluble in cell culture medium.[8-13] These improved tetrazolium reagents eliminate a liquid handling step during the assay procedure because a second addition of reagent to the assay plate is not needed to solubilize formazan precipitates, thus making the protocols more convenient. The negative charge of the formazan products that contribute to solubility in cell culture medium are thought to limit cell permeability of the tetrazolium.[14] This set of tetrazolium reagents is used in combination with intermediate electron acceptor reagents such as phenazine methyl sulfate (PMS) or phenazine ethyl sulfate (PES) which can penetrate viable cells, become reduced in the cytoplasm or at the cell surface and exit the cells where they can convert the tetrazolium to the soluble formazan product.[15] Reagent preparation MTS Solution (containing PES)

1. dissolve MTS powder in DPBS to 2 mg/ml to produce a clear golden-yellow solution.

2. dissolve PES powder in MTS solution to 0.21 mg/ml.

3. Adjust to pH 6.0 to 6.5 using 1N HCl.

4. Filter-sterilize through a 0.2 μm filter into a sterile, light protected container.

5. Store the MTS solution containing PES protected from light at 4°C for frequent use or at -20°C for long term storage. PROCEDURE

1. Prepare cells and test compounds in 96-well plates containing a final volume of 100 µl/well. An optional set of wells can be prepared with medium only for background subtraction.

2. Incubate for desired period of exposure.

58 'Nexus' - Alumni Association SJCOPS, Chalakudy ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 3. Add 20 µl MTS solution containing PES to each well (final concentration of MTS will be 0.33 mg/ml).

4. Incubate 1 to 4 hours at 37°C.

5. Record absorbance at 490 nm. One of the advantages of the tetrazolium assays that produce an aqueous soluble formazan is that absorbance can be recorded form the assay plates periodically during early stages of incubation. Multiple readings may assist during assay development; but caution should be taken to return the plates to the incubator between readings to maintain a nearly constant environment. Extended incubations with the tetrazolium reagent beyond four hours should be avoided. RESAZURIN REDUCTION ASSAY PRINCIPLE Resazurin is a cell permeable redox indicator that can be used to monitor viable cell number with protocols similar to those utilizing the tetrazolium compounds.[16]Resazurin can be dissolved in physiological buffers (resulting in a deep blue colored solution) and added directly to cells in culture in a homogeneous format. Viable cells with active metabolism can reduce resazurin into the resorufin product which is pink and fluorescent.The quantity of resorufin produced is proportional to the number of viable cells which can be quantified using a microplatefluorometer equipped with a 560 nm excitation / 590 nm emission filter set. Resorufin also can be quantified by measuring a change in absorbance; however, absorbance detection is not often used because it is far less sensitive than measuring fluorescence. The resazurin reduction assay is slightly more sensitive than tetrazolium reduction assays. [17]The major advantages of the resazurin reduction assay are that it is relatively inexpensive, it uses a homogeneous format, and it is more sensitive that tetrazolium assays. In addition, resazurinassays can be multiplexed with other methods such as measuring caspase activity to gather more information about the mechanism leading to cytotoxicity. [18] The disadvantages of the resazurin include the possibility of fluorescent interference from compounds being tested and the often overlooked direct toxic effects on the cells. [19] Reagent Preparation

1. dissolve high purity resazurin in DPBS (pH 7.4) to 0.15 mg/ml.

2. Filter-sterilize the resazurin solution through a 0.2 μm filter into a sterile, light protected container.

3. Store the resazurin solution protected from light at 4°C for frequent use or at -20°C for long term storage. PROCEDURE

1. Prepare cells and test compounds in opaque-walled 96-well plates containing a final volume of 100 µl/well. An optional set of wells can be prepared with medium only for background subtraction and instrument gain adjustment.

2. Incubate for desired period of exposure.

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3. Add 20 µl resazurinsolution to each well.

4. Incubate 1 to 4 hours at 37°C.

5. Record fluorescence using a 560 nm excitation / 590 nm emission filter set. A general disadvantage of both the tetrazolium and resazurin reduction assay protocols is the requirement to incubate the substrate with viable cells at 37°C for an adequate period of time to generate a signal. Incubation of the tetrazolium or resazurin reagents with viable cells increases the possibility of artifacts resulting from chemical interactions among the assay chemistry, the compounds being tested, and the biochemistry of the cell. PROTEASE VIABILITY MARKER ASSAY PRINCIPLE Measurement of a conserved and constitutive protease activity within live cells has been shown to serve as a marker of cell viability. A cell permeable fluorogenic protease substrate (glycyl phenyl alanyl- amino fluorocoumarin; GF-AFC) has recently been developed to selectively detect protease activity that is restricted to viable cells. [20]The GF-AFC substrate can penetrate live cells where cytoplasmic aminopeptidase activity removes the glycine and phenylalanine amino acids to release aminofluorocoumarin (AFC) and generate a fluorescent signal proportional to the number of viable cells. Cell permeable glycylphenylalanyl-aminofluoroumarin (GF-AFC) substrate is converted by cytoplasmic aminopeptidase activity to generate fluorescent aminofluorocoumarin (AFC).As soon as the cells die; this protease activity rapidly disappears, thus making this protease activity a selective marker of the viable cell population. [21] Reagent Preparation GF-AFCsolution

1. thaw the GF-AFC substrate and Assay Buffer components from the Cell Titer-Fluor™ Cell Vi- ability Assay kit. [21]

2. transfer 10 µl of the GF-AFC Substrate into 10 ml of the Assay Buffer to prepare a 2X Reagent. Note: For multiplexing applications where total sample volume is a concern, a 10X Reagent can be prepared by adding 10 µl GF-AFC Substrate to 2 ml of Assay Buffer.

3. mix by vortexing the contents until the GF-AFC substrate is thoroughly dissolved.

Storage: Store the CellTiter-Fluor™ Cell Viability Assay components at –20°C. The diluted Cell- Titer-Fluor™ Viability Reagent should be used within 24 hours if stored at room temperature. Unused GF-AFC Substrate and Assay Buffer can be stored at 4°C for up to 7 days with no appreciable loss of activity. PROCEDURE

1. Set up opaque-walled 96-well assay plates containing cells in culture medium at desired density.

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An optional set of wells can be prepared with medium only for background subtraction and instrument gain adjustment.

2. Add test compounds and vehicle controls to appropriate wells so that the final volume is 100 μl in each well (25 μl for a 384-well plate).

3. Culture cells for the desired test exposure period.

4. Add CellTiter-Fluor™ Reagent in an equal volume (100 μl per well) to all wells, mix briefly by orbital shaking, then incubate for at least 30 minutes at 37°C. Note: Longer incubations may improve assay sensitivity and dynamic range. However, do not incubate more than 3 hours, and be sure to shield plates from ambient light. ATP ASSAY PRINCIPLE The measurement of ATP using firefly luciferase is the most commonly applied method for estimating the number of viable cells. ATP has been widely accepted as a valid marker of viable cells. When cells lose membrane integrity, they lose the ability to synthesize ATP and endogenous ATPases rapidly deplete any remaining ATP from the cytoplasm. Although luciferase has been used to measure ATP for decades, recent advances in assay design have resulted in a single reagent addition homogeneous protocol that results in a luminescent signal that glows for hours. [22]The ATP detection reagent contains detergent to lyses the cell, ATPase inhibitors to stabilize the ATP that is released from the lysed cells, luciferin as a substrate, and the stable form of luciferase to catalyze the reaction that generates photons of light. [23] The ATP assay is the fastest cell viability assay to use, the most sensitive, and is less prone to artifacts than other viability assay methods. The luminescent signal reaches a steady state and stabilizes within 10 minutes after addition of reagent and typically glows with a half-life greater than 5 hours. The ATP assay has the advantage that you do not have to rely on an incubation step with a population of viable cells to convert a substrate (such a tetrazolium or resazurin) into a colored compound. This also eliminates a plate handling step because you do not have to return cells to the incubator to generate signal. The ATP assay chemistry can typically detect fewer than 10 cells per well and has been used widely in 1536-well format. The ATP assay sensitivity is usually limited by reproducibility of pipetting replicate samples rather than a result of the assay chemistry. [23] ATP Assay Reagent Preparation

1. thaw the CellTiter-Glo® Buffer and CellTiter-Glo® Substrate and equilibrate to room temperature prior to use. For convenience the CellTiter-Glo® Buffer may be thawed and stored at room temperature for up to 48 hours prior to use.

2. transfer the appropriate volume (10ml ) of CellTiter-Glo® Buffer into the amber bottle containing CellTiter-Glo® Substrate to reconstitute the lyophilized enzyme/substrate mixture. This forms the CellTiter-Glo® Reagent.

3. mix by gently vortexing, swirling or inverting the contents to obtain a homogeneous solution.

'Nexus' - Alumni Association SJCOPS, Chalakudy 61 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ISSN : 2350-1332 The CellTiter-Glo® Substrate should go into solution easily in less than 1 minute. PROCEDURE

1. Set up white opaque walled microwell assay plates containing cells in culture medium at desired density.

2. Add test compounds and vehicle controls to appropriate wells so that the final volume is 100 μl in each well for 96-well plate (25 μl for a 384-well plate).

3. Culture cells for the desired test exposure period.

4. Equilibrate plates to ambient temperature for 30 min to ensure uniform temperature across plate during luminescent assay.

5. Add CellTiter-Glo® Reagent in an equal volume (100 μl per well for 96-well plates or 25 μl per well for 384-well plates) to all wells.

6. mix contents for 2 minutes on an orbital shaker to induce cell lysis.

7. Allow the plate to incubate at room temperature for 10 minutes to stabilize luminescent signal and Record luminescence. WST ASSAY PRINCIPLE AND PROCEDURE WST-1 Cell Proliferation Assay is a sensitive and accurate assay for cell proliferation and cytotoxicity. The assay is highly convenient as it is performed in a single cell tissue culture well and requires no washing, harvesting or solubilization of cells. Adherent or suspension cells are cultured in a microplate and then incubated with WST-1 and the assay is monitored with a spectrophotometer. The assay principle is based upon the reduction of the tetrazolium salt WST-1 to formazan by cellular dehydrogenase. The generation of the dark yellow colored formazan is measured at 420-480nm (optimal at 440nm) and is directly correlated to cell number. FEATURES

1. Uses WST-1, a high sensitivity tetrazolium salt.

2. Colorimetric assay.

3. Adherent or suspension cells.

4. No washing, harvesting or solubilization required. APPLICATIONS

1. for the detection of cell toxicity, death, viability or proliferation.

2. Ideal for high throughput screening. [24]

62 'Nexus' - Alumni Association SJCOPS, Chalakudy ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 NEUTRAL RED UPTAKE ASSAY PRINCIPLE The neutral red uptake assay provides a quantitative estimation of the number of viable cells in a culture. It is one of the most used cytotoxicity tests with many biomedical and environmental applications. It is based on the ability of viable cells to incorporate and bind the supravital dye neutral red in the lyso- somes. Most primary cells and cell lines from diverse origin may be successfully used.[25] PROCEDURE Cells are seeded in 96-well tissue culture plates and are treated for the appropriate period. The plates are then incubated for 2 h with a medium containing neutral red. The cells are subsequently washed, the dye is extracted in each well and the absorbance is read using a spectrophotometer. The procedure is cheaper and more sensitive than other cytotoxicity tests (tetrazolium salts, enzyme leakage or protein content). Once the cells have been treated, the assay can be complein<3 h. CLONOGENIC ASSAY PRINCIPLE Clonogenic assay or colony formation assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony. The colony is defined to consist of at least 50 cells. The assay essentially tests every cell in the population for its ability to undergo ‘‘unlimited’’ division. Clonogenic assay is the method of choice to determine cell reproductive death after treatment with ionizing radiation, but can also be used to determine the effectiveness of other cytotoxic agents. Only a fraction of seeded cells retains the capacity to produce colonies. Before or after treatment, cells are seeded out in appropriate dilutions to form colonies in 1–3 weeks. Colonies are fixed with glutaraldehyde (6.0% v/v), stained with crystal violet (0.5% w/v) and counted using a stereomicroscope.The assays detect all cells that have retained the capacity for producing a large number of progeny after treatments that can cause cell reproductive death as a result of damage to chromosomes, apoptosis, etc. [26] MATERIALS-REAGENTS

1. Appropriate culture medium containing serum

2. Phosphate-buffered saline (PBS) pH 7.2

3. trypsin .The trypsin solution is freshly prepared before the experiment from a stock solution containing 0.5 g trypsin, 0.2 g EDTA and 0.85 g NaCl

4. Isotonic buffer for cell counting when using a Coulter counter

5. Colony fixation-staining solution, glutaraldehyde 6.0% (vol/vol), crystalviolet 0.5% (wt/vol) in H2O. [26] EQUIPMENT

1. Pipettes

'Nexus' - Alumni Association SJCOPS, Chalakudy 63 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ISSN : 2350-1332 2. Culture dishes or six-well plates

3. tubes for dilution

4. Coulter counter or hemocytometer

5. Stereomicroscope

6. Colony counting pen

7. Statistical analysis software (SPSS) PROCEDURE Initial handling of cells

1. Before any experiment, ensure that all required materials from sterile pipettes, sterile test tubes, culture dishes and six well plates are at hand. Warm the medium, PBS and trypsin to 37 1C. Work out the cell dilutions and label the dishes or plates. The experiment has to continue smoothly to limit the total time, preventing adverse effects of pH and temperature changes.

2. Harvesting cells from a donor culture is performed using trypsinization. To detach cells from the plastic, the overlying medium is removed and cells are washed with PBS. PBS is removed and replaced by a solution containing trypsin.

3. Remove the medium above the cells.

4. Wash the cells with PBS.

5. trypsinize cells to produce a single-cell suspension. The trypsin solution should be left over the cells until they round up; this may be inspected under the microscope.

6. When cells start to round up indicating detachment from the culture dishes, resuspend the cells in medium to inhibit trypsinization. Adding sufficient volume of medium supplemented with serum neutralizes the trypsin solution. Detach the cells by pipetting up and down the medium with the cells.

7. Count the cells. The accurate number of cells that are plated is required to obtain the correct data for plating efficiency (PE) for unirradiated controls or after a certain treatment for a proper survival calculation.

8. Dilute the cell suspension into the desired seeding concentration and seed into flasks or plates as desired. The dilutions have to be performed accurately to seed the correct number of cells. Clonogenic assay setup

9. there are two essentially different ways to perform studies using this assay: In option (A), cells are plated before treatment. Cells are harvested from a stock culture and plated at appropriate dilutions into (cluster) dishes. After attachment of the cells to the dishes, which generally takes 2 h or more, the cells are treated. The treatment has to be performed before cells start replicating;

64 'Nexus' - Alumni Association SJCOPS, Chalakudy ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 otherwise, the numbers of cells per dish will increase, yielding more colonies. After treatment, the dishes are placed in an incubator and left there for a time equivalent to at least six potential cell divisions. This method is often used for a quick screening of the sensitivity of cells to different treatments. In option (B), cells are treated in dishes and subsequently re-plated in appropriate dilutions to assess clonogenic ability. The replating may be performed immediately after treatment or it may be delayed to allow repair processes. This method is used especially in radiologi- cal research to determine potentially lethal- and sublethal damage repair. [33] Fixation and staining of colonies

10. Remove the medium above the cells.

11. Rinse carefully with PBS.

12. Remove the PBS and add 2–3 ml of a mixture of 6.0% glutaraldehyde and 0.5% crystal violet.

13. Leave this for at least 30 min.

14. Remove the glutaraldehyde crystal violet mixture carefully and rinse with tap water. Do not place the dishes or plates under the running tap, but fill the sink with water and immerse the dishes or plates carefully.

15. Leave the dishes or plates with colonies to dry in normal air at room temperature (20 1C).Colo- nies can be counted up to at least 50 weeks after staining. Counting the colonies

16. the standard procedure is to count using a stereomicroscope and an automatic counting ‘‘colony counter pen. When for a certain drug a dose-modifying factor is calculated, the ratio of the radiation dose at a certain survival level after radiation alone to that for the combined treatment should then be calculated and not the ratio of the surviving fractions at ascertain radiation dose. SRB ASSAY PRINCIPLE The sulforhodamine B (SRB) assay is used for cell density determination, based on the measurement of cellular protein content. The method not only allows a large number of samples to be tested within a few days, but also requires only simple equipment and inexpensive reagents. The SRB assay is therefore an efficient and highly cost-effective method for screening. CytoScan™-SRB Cytotoxicity Assay is an accurate and reproducible assay based upon the quantitative staining of cellular proteins by sulforhodamine B (SRB). Sulforhodamine B is an anionic aminoxanthene dye that forms an electrostatic complex with the basic amino acid residues of proteins under moderately acid conditions, which provides a sensitive linear response. The color development is rapid and stable and is readily measured at absorbances between 560 and 580nm. [28] PROCEDURE The method described here has been optimized for the toxicity screening of compounds to adherent cells

'Nexus' - Alumni Association SJCOPS, Chalakudy 65 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ISSN : 2350-1332 in a 96-well format. After an incubation period, cell monolayers are fixed with 10% (wt/vol) trichloroa- cetic acid and stained for 30 min, after which the excess dye is removed by washing repeatedly with 1% (vol/vol) acetic acid. The protein-bound dye is dissolved in 10 mMTris base solution for optical density (OD) determination at 510 nm using a microplate reader. The results are linear over a 20-fold range of cell numbers and the sensitivity is comparable those of flourimetric method. FEATURES

1. measures total biomass by staining cellular proteins.

2. Linear response.

3. Simple, accurate and reproducible assay. [34-38] APPLICATIONS

1. for the detection of cell toxicity, death, viability or proliferation.

2. Ideal for high throughput screening. AGAR OVERLAY DIFFUSION METHOD PRINCIPLE The agar overlay was designed to determine the cytotoxicity of diffusible components from material or solution. A layer of agar was added over a cell monolayer to act as cushion to protect cell from mechanical damage while allowing the diffusion of leachable materials. The test articles were then placed on top of agar layer and incubated. The cell monolayer were examined and scored based on the degree of cellular destruction. PROCEDURE Six well culture seeded with verified quatity of cell and incubated 37 degree with 1% co2, until approxi- mate 18% confluent. The agar overlay consisted of equal mixture of 1N AGAR 1% and 10% bovin calf serum. Solid test article were placed directly on the solidified agar overlay. Liquid or gel test article were applied to sterile filter discs, testing Not less than 0.1ml per well. Powders resin or irregular materials were placed directly onto the solidified agar, testing not less than 100mg per well. Positive and negative references were included with each assay. All tests were performed using three test wells per test article. After the addition of test articles, the cell culture plates are incubated as described above for 24 to26 hr. Following incubation, cells were evaluated microscopically. [29] CONCLUSION There are a variety of assay technologies available that use standard plate readers to measure metabolic markers to estimate the number of viable cells in culture. Each cell viability assay has its own set of advantages and disadvantages. The ATP detection assay is by far the most sensitive, has fewer steps, is the fastest to perform, and has the least amount of interference whereas the tetrazolium or resazurin reduction assays offer less expensive alternatives that may achieve adequate performance depending on experimental design. The fluorogenic cell permeable protease sub-

66 'Nexus' - Alumni Association SJCOPS, Chalakudy ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 strate is far less cytotoxic than the tetrazolium and resazurin compounds while enabling many possi- bilities for multiplexing other assays to serve as orthogonal or confirmatory methods. Other than these methods, cytotoxicity can also be measured by using Sulphordamine B (SRB) assay, WST assay, Clo- nogenic assay and Agar over lay diffusion method. Regardless of the assay method chosen, the major factors critical for reproducibility and success include, using a tightly controlled and consistent source of cells to set up experiments and performing appropriate characterization of reagent concentration and incubation time for each experimental model system.Development of these in vitro techniques has made the effort to develop new drug molecules with anticancer property easy and we can use this as a way to proceed towards in vivo techniques to evaluate cytotoxicity. Due to ethical reasons also animal models may be used only when we confirm the activity of a new chemical entity, either natural or synthetic by in vitro cytotoxicity test. This can ultimately lead to the development of new drug molecules which possess least toxicity as well as increased efficacy to treat one of the most feared diseases by mankind. REFERENCES

1. da-Rocha AB, Lopes RM, Schwartsmann G.Natural products in anticancer therapy. Curr. Opin. Pharmacol. 1 (2001) 364–369.

2. Suffness M., Pezzuto J.M. In: K. Hostettmann (Ed.), Methods inPlant Biochemoistry, Assays for Bioactivity, Academic Press, London, 1991, vol.6.p.125.

3. mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Meth. 1983;65:55-63.

4. marshall NJ, Goodwin CJ, Holt SJ. A critical assessment of the use of microculturetetrazolium assays to measure cell growth and function. Growth Regul. 1995;5(2):69–84.

5. tada H, Shiho O, Kuroshima K. An improved colorimetric assay for interleukin 2. J. Immunol. Methods.1986;93:157–65.

6. Denizot F, Lang R. Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J. Immunol. Meth. 1986;89:271–277.

7. Plumb JA, Milroy R, Kaye SB. 1989. Effects of the pH dependence of 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyl-tetrazolium bromide-formazan absorption on chemosensitivity determined by a novel tetrazolium-based assay. Cancer Res 15;49(16):4435-40.

8. Cory A, Owen T, Barltrop J, Cory JG. Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture. Cancer Commun. 1991;3(7):207–212.

9. Barltrop J, Owen T. 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazoly)-3-(4-sulfophenyl) tetrazolium, inner salt (MTS) and related analogs of 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) reducing to purple water-soluble formazans as cell-viability indicators. Bioorg Med ChemLett. 1991;1:611–614.

'Nexus' - Alumni Association SJCOPS, Chalakudy 67 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ISSN : 2350-1332 10. Paull KD, Shoemaker RH, Boyd MR. et al. The synthesis of XTT: A new tetrazolium reagent that is bioreducible to a water-soluble formazan. J Heterocyclic Chem. 1988;25:911–914.

11. Ishiyama M, Shiga M, Sasamoto K, Mizoguchi M, He P. A new sulfonatedtetrazolium salt that produces a highly water-soluble formazan dye. Chem Pharm Bull (Tokyo). 1993;41:1118–1122.

12. tominaga H, Ishiyama M, Ohseto F. A water-soluble tetrazolium salt useful for colorimetric cell viability assay.AnalCommun. 1999;36:47-50.

13. Goodwin CJ, Holt SJ, Downes S, Marshall NJ. 1995. Microculturetetrazolium assays: a comparison between two new tetrazolium salts, XTT and MTS. J Immunol Methods 13;179(1):95-103.

14. Scudiero DA, Shoemaker RH, Paull KD, Monks A, Tierney S, Nofziger TH, Currens MJ, Seniff D, Boyd MR. Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res. 1988;48(17):4827–33.

15. Berridge MV, Herst PM, Tan AS. Tetrazolium dyes as tools in cell biology: New insights into their cellular reduction.Biotechnology Annual Review. 2005;11:127–152.

16. Ahmed SA, Gogal RM, Walsh JE. A new rapid and simple nonradioactive assay to monitor and determine the proliferation of lymphocytes: An alternative to [3H]thymidine incorporation assays. J Immunol Meth. 1994;170:211–224.

17. Shum D, Radu C, Kim E, Cajuste M, Shao Y, Seshan VE, Djaballah H. A high density assay format for the detection of novel cytotoxic agents in large chemical libraries. J EnzInhib Med Chem. 2008;23(6):931–945.

18. Wesierska-Gadek J, Gueorguieva M, Ranftler C, Zerza-Schnitzhofer G. A new multiplex assay allowing simultaneous detection of the inhibition of cell proliferation and induction of cell death. J Cell Biochem.2005;96(1):1–7.

19. InvitrogenalamarBlue Assay manual. Online at [http://tools.invitrogen.com/content/sfs/manuals/ PI-DAL1025 1100_TI%20alamarBlue%20Rev%201.1.pdf].

20. Niles AL, Moravec RA, Hesselberth PE, Scurria MA, Daily WJ, Riss TL. A homogeneous assay to measure live and dead cells in the same sample by detecting different protease markers. Anal Biochem. 2007;366:197–206.

21. CellTiter-Fluor Technical Bulletin. Online at http://www.promega.com/~/media/Files/Resources/ Protocols/Technical%20Bulletins/101/CellTiter-Fluor%20Cell%20Viability%20Assay%20Pro- tocol.pdf .

22. fan F, Wood KV. Bioluminescent Assays for High-Throughput Screening.ASSAY and Drug De- velopment Technologies 2007, 5(1): 127-136.

23. thompson JF, Geoghegan KF, Lloyd DB, Lanzetti AJ, Magyar RA. Mutation of a protease-sensitive region in firefly luciferase alters light emission properties. J BiolChem 1997, 272: 18766- 18771.

68 'Nexus' - Alumni Association SJCOPS, Chalakudy ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 24. Kim-Choi E, Danilo C, Kelly J, Carroll R, Shonnard D. Kinetic characterization and in vitro toxicity evaluation of a luciferase less susceptible to HPV chemical inhibition. Toxicolin Vitro 2006, 20: 1537-1547.

25. Eriksson J, Nordstrom T, Nyren P. Method enabling firefly luciferase-based bioluminometric -as says at elevated temperatures. Anal Biochem 2003, 314: 158–161.

26. Lei-Miao Y, Wei Y, Wang Y, Yu-Dong X, Yong-Qing Y. Long Term and Standard Incubations of WST-1 Reagent Reflect the Same Inhibitory Trend of Cell Viability in Rat Airway Smooth Muscle Cells. Int J Med Sci. 2013; 10(1): 68–72.

27. Rafehi H, Orlowski C, Georgiadis GT, Ververis K, El-Osta A, Karagiannis TC.Clonogenic Assay: Adherent Cells. J Vis Exp. 2011; (49): 2573.

28. Houghton P, Fang R, Techatanawat I, Steventon G, Hylands PJ, Lee CC. The sulphorhodamine (SRB) assay and other approaches to testing plant extracts and derived compounds for activities related to reputed anticancer activity.Methods. 2007, 42(4):377-87.

29. Barry AL, Badal RE. Quality Control Limits for the Agar Overlay Disk Diffusion Antimicrobial Susceptibility Test. J ClinMicrobiol. 1982, 16(6): 1145–1147.

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Siraj et.al.,/ Journal of Pharma Innovative Research, 2015; 2(1): 70-74 A review on analytical tools used for controlling drug expenses

Siraj Sundaran*, Sanjay Sreekumar, Babu G.

Dept. of Pharmacy Practice, Devaki Amma Memorial College of Pharmacy, Chelembra, Malappuram Corresponding Author Email:[email protected]

ABSTRACT Several analytical tools are present that help to quantify costs of drugs and identify areas where costs can be reduced. The major tools include VEN system, ABC analysis, therapeutic category analysis, price comparison analysis, total variable cost analysis, lead-time analysis, expiry date analysis, and hidden cost analysis. VEN system categorizes drugs by their relative public health value. ABC analysis examines the annual consumption of drugs and expenditures for procurement. Therapeutic category analysis considers the utilization and financial impact of various therapeutic categories of drugs and then compares cost and therapeutics benefit to select the most cost-effective drugs in each major therapeutic category. Price comparison analysis compares drug prices paid by different supply systems, as one measure of procurement efficiency. Total variable cost analysis compiles information on variable costs associated with purchasing and inventory management, which help in considering the options for change in terms of their impact on total variable costs. Lead-time analysis is as systematic approach to track procurement lead times, determining the points at which lead time can be reduced and adjusting safety stock. Expiry date analysis examines levels of stock on hand and their expiry dates and compares this information with average rates of consumption to assess the likelihood of wastage. Hidden cost analysis examines supplier performance to identify any hidden costs incurred because of problems such as late deliveries and short shipments. The inventory control management can show improvementin patient care and in the optimal use of resources.

Key-words: Drugs, Expense

INTRODUCTION A medical store is an extensively used facility in any hospital where a large amount of money is spent on purchases on a frequent basis. It is estimated that nearly 35% that is nearly one third of annual hospitals budget is spent on buying materials and supplies, including medicines [1]. There has always been

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a requirement of a very effective and efficient management of the medical stores to avoid unnecessary purchase and prevent under purchase that leads to shortage of drugs in hospital. Inventory management is defined as the continuing process of planning, organizing and controlling inventory which aims at minimizing the investment in inventory while balancing supply and demand. Drug inventory management helps in designing appropriate corrective measures.Inventory control is a scientific system which indicates as to what, when, and how much to order, and how much to stock so that purchasing costs and storing costs are kept as low as possible. Efficient priority setting, decision making in purchase and distribution of specific drugs, close supervision on drugs belonging to important categories, etc., depends on the drug and inventory management. Since the mid-1980s, the strategic benefits of inventory management and production planning and scheduling have become obvious [2]. Analytical tools for the inventory management 1. VEN categorization:The VEN system categorizes pharmaceuticals based on their relative public health value. The categories are vital (V), essential (E), and nonessential (N) (also known as D for desirable which makes it VED categorization). At times the health systems find a two category system more useful than the three-tiered VEN; for example, the categories might be V and N, differentiating between those medicines that must always be in stock and other medicines. V – Vital drugs potentially involve lifesaving drugs. Drugs that are crucial to providing basic health services are included in this group as well. E – Essential drugs are less severe in effectiveness when compared to vital drugs but they are significant for illnesses. N – Nonessential drugs are used for minor or self–limited illness. Drugs that are still questionable about their efficacy also belong to this class. To identify the drugs according to VEN system, a panel of pharmacists, doctors with different specializations should be set up since opin- ions can differ. The degree of importance of procurement and control procedures reduces from vital to nonessential drugs. 2. ABC analysis:The ABC analysis also known Pareto’s law or Always, Better and Controlexam- ines the annual consumption of medicines and expenditures for procurement by dividing the medicines consumed into three categories. Class A includes 10 to 20 percent of items, which account for 75 to 80 percent of expenditures. Class B items represent 10 to 20 percent of items and 15 to 20 percent of expenditures. Class C items are 60 to 80 percent of items but only about 5 to 10 percent of expenditures. ABC analysis can be used to measure the degree to which actual consumption reflects public health needs and morbidity, reduce inventory levels and costs by arranging for more frequent purchase or delivery of smaller quantities of class A items, seek major cost reductions by finding lower prices on class A items, where savings will be more noticeable. According to ABC classification, it suggests that the more analysis should be applied to materials with high inventory value. Class A should be most extensively handled and Class C is analyzed little. Advantage of ABC classification is that controlling small numbers of items amounting to 10-20 % will result in the control of 75-80% of the monetary value of the inventory held. Very strict control procedures should be used with A items and the controller should have great authority. Class B can be controlled by middle management. Low safety stock policy is applied to this class with quarterly or monthly orders. Class C items do not need to be highly controlled. Since the items have the lowest value compared to the class A and B, orders can be placed at a greater volume to take advantage of quantity discount. Rough estimates are sufficient to manage class C materials. 3. Therapeutic category analysis: This analysis considers the use and financial impact of various

'Nexus' - Alumni Association SJCOPS, Chalakudy 71 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ISSN : 2350-1332 therapeutic categories of medicines and then compares cost and therapeutic benefit to select the most cost-effective medicines in each major therapeutic category. The analysis reviews the volume of use of various therapeutic categories and subcategories of drugs. This technique builds on ABC analysis, i.e., produce a list of drugs with the volume and value of use calculated for each drug. Further sorts the ABC list into therapeutic categories, where coding systems followed in WHO essential drug list and American hospital formulary service pharmacologic and therapeutic classification system. Therapeutic category analysis helps to select medicines for a formulary or procurement list. 4. Price comparison analysis: This analysis compares pharmaceutical prices paid by different supply systems as one measure of procurement efficiency. Acquisition price comparison analysis states whether the system is getting the maximum benefit from available procurement funds, and in case not how much might be saved with alternative procurement practices. It can also compare supply system acquisition and selling prices with local private-sector prices to gauge the cost-effectiveness of in-house pharmaceutical services and to assess price elasticity for cost recovery. 5. Total variable cost analysis: Total cost analysis compiles information on variable costs associated with purchasing and inventory management to help managers consider options for change in terms of their impact on total costs. It is the sum of four components drug acquisition costs, inventory holding costs, purchasing cost, and shortage cost (emergency purchase, client outside purchase leading to loss of revenue, increased morbidity or mortality, etc.,). The compiled data are then reviewed to look for options to reduce the total cost and to perform what if analysis i.e., what happens to inventory holding cost if ordering costs are reduced and so forth. This analysis help in finding opportunities for cost reduction and modeling the cost impact of potential changes in the supply system. 6. Lead-time analysis:Lead time is defined as the interval between submitting an order and receiving the goods. Lead-time analysis is a systematic approach to tracking procurement lead times, determining the points at which lead time can be reduced, and adjusting safety stock appropriately. Pay- ment time should also be analyzed (when delayed payment to suppliers is feasible) which is the interval between receipt of goods at the warehouse and payment to the supplier. 7. Expiry-date analysis:Analysis of expiry dates versus inventory levels is useful for determining how much stock is at risk of wastage. This analysis examines levels of stock on hand and their expiry dates and compares this information with average rates of consumption to assess the likelihood of wastage (and to develop appropriate countermeasures). The analysis is done for each drug by determining the average monthly consumption during the past year and dividing the quantity in stock by the average monthly consumption to determine the stock position in months. Then the number of months remaining until expiry is calculated. If the months until expiry are greater than the stock position in months, there should be limited risk of wastage. But if the stock position is higher that the months until expiry; there is some risk. 8. Hidden-cost analysis: Hidden costs are those costs that occur due to poor supplier performance and are not obvious in the invoice price. The procurement officers should consider the expected hidden costs for each supplier based on the past performance. The hidden costs may include viz., commissions for local agents, cost of late deliveries, cost of delivery errors, loss due to poor packaging, replacement cost of unusable drugs, etc. Hidden costs may make one supplier considerably more expensive than a competitor that offers a higher unit price but better performance.

72 'Nexus' - Alumni Association SJCOPS, Chalakudy ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 CONCLUSION Among the inventory control techniques mentioned in this review the most commonly used are ABC analysis and VEN categorization methods. It is a usual practice of coupling ABC and VEN whereby the vital and essential drugs are not under - purchased just by considering the cost factor. Further the selection of techniques to be used depends on the need of institution. The inventory control management can show improvement in patient care and in the optimal use of resources. REFERENCES

1. SarbjeetKhurana, NeelamChhillar, Vinod Kumar Singh Gautam;Inventory control techniques in medical stores of a tertiary care neuropsychiatry hospital in Delhi. Health, 2013; 5(1): 8-13.

2. Silver EA, Pyke DF, Peterson R; Inventory management and production planning and scheduling. 3rd edition, John Wiley & Sons, USA, 1998: 9.

3. JD. Quick, J. Rankin, R. Laing, R. O’Connor, HV. Hogerzeil, MNG. Dukes et al; Managing drug supply. 2nd edition, Kumarian press, USA, 1997.

4. West D. Purchasing and Inventory management. In Pharmacy management: essentials for all practice settings. 2nd edition, Desselle SP, Zgarrick DP editors. McGraw-Hill,New York, 2009: 383.

5. dr. PoorwaWandalkar, Dr.PT. Pandit, AR. Zite; ABC and VED analysis of the drug store of a tertiary care teaching hospital. Indian Journal of Basic and Applied Medical Research, 2013;3(1): 126-131.

6. fS Vaz, AM Ferreira, I Pereira-Antao, MS Kulkarni, DD Motghare; Application of inventory control techniques for drug management at a rural health centre. Indian J. Prev. Soc. Med, 2008; 39(3& 4): 120-123.

7. Gupta R., Gupta KK., Jain BR., Garg RK; ABC and VED analysis in medical stores inventory control. Medical Journal Armed Forces India, 2007; 63: 325-327.

8. mcCaffrey DJ, Smith MC, Banahan BF, Frate DA, Gilbert FW; A Continued Look into the Finan- cial Implications of Initial Noncompliance in Community Pharmacies: An Unclaimed Prescrip- tion Audit Pilot. J Res Pharm Econ, 1998; 9(2):33-57.

9. Ayad K. Ali; Inventory Management in Pharmacy Practice: A Review of Literature. Archives of Pharmacy Practice, 2011; 2(4): 151-156.

10. SurabhiDwivedi, Arun Kumar, PreetiKothiyal; Inventory management: a tool of identifying items that need greater attention for control . The Pharma Journal, 2012; 1(7): 125–129.

11. Kant S, Pandaw CS, Nath LM; A management technique for effective management of medical store in hospitals. J AcadHospAdm, 1997; 89: 41-47.

12. Adeyemi SL, Salami AO; Inventory management:a tool of optimizing resources in a manufacturing industry. J SocSci, 2010; 23(2): 135-142.

'Nexus' - Alumni Association SJCOPS, Chalakudy 73 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ISSN : 2350-1332 13. vinita;To study hospital formulary management in tertiarycare hospital. Int J Pharm PharmSci, 2012; 4(2): 536-539.

14. Anand T, Ingle GK, Kishore J, Kumar R; ABC-VED analysis of a drug store in the department of community medicine of a Medical College in Delhi. Indian J Pharm Sci, 2013; 75(1):113-117.

15. viboonsunti C, Kumprakorb U, Sirisaard P; Stock management of drug inventory control in the community pharmacylaboratory. Thai J Pharm Sci, 2003; 27(3-4): 139-148.

16. duclos LK; Hospital inventory management for emergency demand. Journal of Supply Chain Management, 1993; 29: 29-38.

17. vR Thawani, AV Turankar, SD Sontakke, SV Pimpalkhute, GN Dakhale, KS Jaiswal, et al; Eco- nomic analysis of drug expenditure in Government medical college hospital Nagpur. Indian J of Pharma, 2004; 36:15-19.

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Anish et.al.,/ Journal of Pharma Innovative Research, 2015; 2(1): 75-80 Green Coconut Water-Alginate Hydrogel: Anti-microbial Studies

Anish John*, Dineshkumar B, Krishnakumar K, Amulya K. Mohan, Jaison T. James, Sincy Sunny P.

St. James Hospital Trust Pharmaceutical Research Centre, Chalakudy, Kerala Dept. of Pharmaceutics, St. James College of Pharmaceutical Sciences, Chalakudy, Kerala Corresponding Author Email: [email protected]

ABSTRACT Coconut water is the juice in the interior or Endosperm of young coconut. It is one of the natures most refreshing drinks, consumed for its nutritious and health benefiting properties. Coconut water easily forms hydro gel with the sodium alginate, which is a polysaccharide. Coconut water contains Monolausin, an antiviral, antibacterial and antiprotozalmanoglyceride that is used to kill the lipid coated viruses such as HIV, Herpes, Cytomegalovirus, flu and various Patho- genic bacteria. Based on literature survey, there is no report for biological activity of Green Coconut Water scientifically. Therefore, the present project work was designed to develop the Green Coconut Water – Alginate hydrogel formulation and to investigate its antimicrobial activity using well diffusion methods. Different concentrations of (4%, 3.5%, 3%, 2.5%) alginate is used to formulate the hydrogel with coconut water (1ml). The compatibility of coconut water and alginate is determined by FT-IR method. The FT-IR spectra showed that coconut water and alginate will mix without any interaction. PH of the preparation lies in the range between 6.5 to 7.1. This shows that the preparation is suitable for topical application. The sample D (2.5%) was exhibited considerable antimicrobial potential (zone of inhibition 2.5 mm) with standard Erythromycin (zone of inhibition 1mm). Key-words: gel; coconut water; microbial study

INTRODUCTION Coconut water is the juice in the interior or Endosperm of young coconut. It is one of the nature most refreshing drinks, consumed for its nutritious and health benefiting properties.Coconut water easily forms hydro gel with the sodium alginate, which is a polysaccharide. The formed hydro gel produces antimicrobial actions.1

'Nexus' - Alumni Association SJCOPS, Chalakudy 75 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ISSN : 2350-1332 Alginate is a linear unbranched polymers containing B(1,4) linked D-Mannuronic acid and α (1,4) linked L-glucoronic acid. It’s average molecular weight contains 50-10,00,000 residues. They are thermally stable, cold setting gelling agents. Alginates solubility and water holding capacity depends on PH(Precipitating below PH 3.5), molecular weight (low molecular weight Ca2+ alginate chains with less than 500 residues shows increased water binding with increasing size ), lonic strength ( low lonic strength increases extended nature of chains ) & nature of ions present.Alginate is used to stabilize phase separation in low-fat, fat- substitutes. Example is alginate / casein blends in starch 3-phase systems.2So further prospects are excellent as recombinant epimerizes with different specificities may be used to produce novel designer alginates. Alginate products were prepared as dressings, bandages or implants, has varying rates of disintegration in tissues depending upon the purpose for which they are to be used. Alginate has haemostatic property. The absorbable alginate fibredressing is used in surgery.3 The present investigation utilizes alginates which are precipitated in a solution of polyvalent ions like Ca2+, Zinc resulting in an aqueous insoluble product that could have utility in the fields of Medicine, dentistry, surgery etc. Alginate is a polysaccharide extracted from certain sea weeds. Coconut water as Antimicrobial Coconut water contains Monolausin, an antiviral, antibacterial and antiprotozalmanoglyceride that is used to kill the lipid coated viruses such as HIV, Herpes, Cytomegalovirus, flu and various Pathogenic bacteria.To develop green coconut water – Alginate hydrogel formulation and to investigate its antimicrobial activity using well diffusion method. Based on literature survey, there is no report for biological activity of Green Coconut Water scientifically. Therefore, the present project work was designed to develop the Green Coconut Water – Alginate hydrogel formulation and to investigate its antimicrobial activity using well diffusion methods. 1. Formulation of Green Coconut Water – Alginate hydrogel. 2. Characterisation of Green Coconut Water – Alginate hydrogel

• Colour

• PH

• FTIR 3. Antimicrobial Studies of Green Coconut Water- Alginate hydrogel MATERIAL & METHOD Tender Coconut Water, Sodium Alginate, Potassium Bromide, E-Coli Bacteria, Erythromycin Tablet, Weighing Balance, Stirrer, Incubator, Refrigerator, FTIR Spectrometer, EUTECH Instrument.

Anish et al., 2015/ JOURNAL OF PHARMA INNOVATIVE RESEARCH, 2015; 2(1): 76-80

COCONUT 3. Well was made in each nutrient Agar WATER plate using metal borer. SAMPLE 1ml 4% 4. Wells are filled with the different A concentrations of Green Coconut SAMPLE 1ml 3.5% Water and the standard (Erythromycin) B 5. If Substantial antimicrobial activity is ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 FormulationSAMPL of GreenE Coconut Water- Alginate 1ml Hydrogel4 3% present for std. & samples, zone of C GREEN COCONUT WATER ALGINATE inhibition appears around test product. SAMPLE A 1ml 4% SAMSAMPLPLE B E 1ml 1ml 3.5% 2.5% The zone of inhibition is simply the SAMPLE C 1ml 3% area on agar plate that remains free SAMPLE D D 1ml 2.5% from microbial growth. 6. The diameter of zone of inhibition is usually related to the level of antimicrobial activity present in the sample and standard which was compared. 7. If diameter of zone of inhibition is larger the antimicrobial action is

greater and was highly potent. FigureFigure 1:1: Different Differ concentrationse ntof Green Coconut concen water-Alginatetrations hydrogel of 8. The Kirby-Bauer zone of inhibition CHARACTERISATIONGreen Coconut OF GREEN waCOCONUTter-Al WATER-ALGINATEginate hy HdroYDROGELgel test method is designed to qualitatively 1.Colour and appearance – Detected by physical test. 2.PH – Detected by PH Meter, EUTECH Instrument, PH Tutor. PH/Co meter test the ability of antimicrobial agents 3.FT-IRCHAR - Detected Aby CTERISAFourier transform infraredTION spectrophotometer OF was taken GREE with coconutN water, to inhibit the growth of micro- alginateCOCO and preparedN mixtureUT of green coconut waterWATER- and alginate ALGINATE organisms over an 18-24 hour period ANTIMICROBIALHYDRO STUDIESGEL OF GREEN COCONUT WATER-ALGINATE HYDROGEL5 1. the nutrient Agar plate was prepared. of contract.

2. 1.Colou the Microbial suspensionr and or organisms app e(E-Coli)arance is spread over – theD nutrientetected agar plate b withy a ster- physicalile swab. test. H H 3. 2.PWell was made – inDetected each nutrient Agar plate by using P metal Mete borer. r, EUTECH 4. Wells are filled with the differentH concentrationsH of Greeno Coconut Water and the standard (Eryth- Instrumeromycin) nt, P Tutor. P /C meteer 5. 3.FT-IRIf Substantial antimicrobial - Detecte activity is presentd by for std. Fouri & samples,er zone transfor of inhibitionm appears 'Nexusinfrared' - Alumni Association spectropho SJCOPS, Chalakudy tometer was taken with 77 coconut water, alginate and prepared mixture of green coconut water and alginate RESULT AND DISCUSSION ANTIMICROBIAL STUDIES OF GREEN COCONUT WATER- II) Evaluation studies ALGINATE HYDROGEL5 1. The nutrient Agar plate was prepared. 1. Colour and appearance 2. The Microbial suspension or organisms (E-Coli) is spread over the nutrient agar Sam Sample Sample Sam plate with a sterile swab. 78

J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 ISSN : 2350-1332 around test product. The zone of inhibition is simply the area on agar plate that remains free from microbial growth.

6. the diameter of zone of inhibition is usually related to the level of antimicrobial activity present in the sample and standard which was compared.

7. If diameter of zone of inhibition is larger the antimicrobial action is greater and was highly potent.

8. the Kirby-Bauer zone of inhibition test method is designed to qualitatively test the ability of antimicrobial agents to inhibit the growth of micro-organisms over an 18-24 hour period of contract. RESULT AND DISCUSSION II) Evaluation studies 1. Colour and appearance

Sample A Sample B Sampl C Sample D

Colour Slight Yellow Yellowish Brown Yellowish brown Light Brown

Appearance Gel Gel Gel Gel

2.PH The PH of each sample is detected by PH Meter (EUTECH Instrument

Sample PH

A 6.5

B 6.9

C 7.0

D 7.1

This PH is equivalent to skin PH so these preparations are suitable for topical applications. 3 FTIR Studies FITR – SPECTRUM OF ALGINATE

78 'Nexus' - Alumni Association SJCOPS, Chalakudy ISSN : 2350-1332 J. Pharm. Inn. Res. | June 2015 | Vol - 2 | Issue - 2 FTIR-SPECTRUM OF COCONUT WATER

FTIR SPECTRUM OF MIXTURE OF COCONUT WATER AND ALGINATE

ANTIMICROBIAL STUDIES OF GREEN COCONUT WATER-ALGINATE HYDROGEL The efficiency of the mixture of Green Tender Coconut Water – Alginate Hydrogel can be studied from the diameter of their zone of inhibition formed.

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Sample Diamter Erythromycin 1 mm Sample A (4%) 1.8mm Sample B (3.5%) 1.7mm Sample C (3%) 1.6 mm Sample D (2.5%) 2.5mm

CONCLUSION This study indicated that formulation sample D (2.5% of Green Coconut Water-Alginate) Exhibited considerable antimicrobial activity conformed with Standard (Erythromycin) against E.coli usingWell diffusion method REFERENCES

1. Bhagya D, Prema L, Rajmohan T. Beneficial effects of tender coconut water on blood pressure and lipid profile level in experimental hypertension. Journal of cell and tissue Research 2010, 10(1): 2139-2144.

2. Peppas NA, Bures P, Leobandung W, Ichikawa H. Hydrogels in pharmaceutical formulations. Eur J Pharm Biopharm. 2000, 50(1):27-46.

3. donati I, Holtan S, Mørch YA, Borgogna M, Dentini M, Skjak-Braek G. New hypothesis on the role of alternating sequences in calcium-alginate gels. Biomacromolecules. 2005, 6(2): 1031- 40.

4. Wu J, Hou S, Ren D, Mather PT. Antimicrobial Properties of Nanostructured Hydrogel Webs Containing Silver. Biomacromolecules 2009, 10, 2686–2693.

5. venkatesan J, Jayakumar R, Mohandas A., Bhatnagar I , Kim SK. Antimicrobial activity of chitosan-carbon nanotube hydrogels. Materials 2014, 7(5): 3946-3955.

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