Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524137EnglishN-0001November30General SciencesISOLATION AND CHARACTERIZATION OF LOLDUVIN-S: A NOVEL ANTIMICROBIAL PROTEIN FROM THE INK OF INDIANS QUID LOLIGO DUVAUCELI
English0414Smiline GirijaEnglish J.Vijayshree PriyadharshiniEnglish Pandi Suba K.English Hariprasad G.English Raghuraman R.EnglishObjectives: Bioactive compounds from the marine habitat have been always represented as the greatest unexploited source of potentially active pharmaceutical agents. Squid ink, being reported for its various therapeutic applications has not been extensively studied for its bioactive molecules. Thus an attempt has been made in this study to isolate and characterize a novel antimicrobial protein from the ink of Loligo duvauceli.
Methods: The fresh squids were decapitated and the ink sac was dissected to collect the ink. Melanin free ink was obtained by ultracentrifugation. The ink was then subjected to SDSPAGE to analyze the proteins present in it and a new protein with a molecular weight of approximately 60 Kda was selected as the target protein. Native PAGE was then performed to isolate the protein in an original form and was eluted using passive gel elution method. The eluted protein was then subjected to an enzyme linked coupled assay for the L-amino oxidase
[LAO] activity. The Km and Vmax values for the L-amino acids utilized as substrates for the assay were determined by Lineweaver Burk Plots. The eluted protein was sterilized by syringe filtration after which the antimicrobial potential of the protein was checked by agar well diffusion method against the drug resistant pathogens such as ESBL, MRSA and the pathogenic yeast C.albicans. The MIC value of the protein was determined by Microbroth dilution method.
Results: A new protein of approximately 60 Kda was successfully isolated and was characterized to exhibit the L-amino acid oxidase activity. The protein was scored to possess a promising antibacterial and antifungal activity against the test pathogens. The MIC value was determined as 25 μg/ml for ESBL producing strains of E.coli and K.pneumoniae. MIC value for the methicillin resistant S.aureus and C.albicans was deduced as 12.5 μg/ml. This new protein exhibiting a LAO activity was further named as Lolduvin - S.
Conclusion: This study was concluded by stating that Lolduvin-S exhibits the LAO activity and had been reported to possess a promising antimicrobial activity against the dreadful drug resistant pathogens. Lolduvin-S could be employed in near future as a novel therapeutic agent to treat various systemic ailments.
EnglishAntimicrobial, drug resistant pathogens, Microbroth dilution method.INTRODUCTION
Natural bioactive substances have the least quantum of side effects when compared with the synthetic products. Although most antibiotics were derived from terrestrial life, it is the marine world that may provide the pharmaceutical industry with the next generation of medicines. At present, a number of marine natural products are in the market or in clinical trials. Due to the huge diversity of marine bioactive compounds with respect to their chemical structure, mode of action and applicability, the extreme concern has been increased towards the screening of marine natural products for their biomedical potential1 . In spite of incredible evolution in medicinal field, some deadly diseases are not curable. The emergence and spread of antimicrobial resistance is now threatening to undermine our ability to treat infections and save lives. Drug resistant bacteria such as extended spectrum beta lactamase (ESBL) producing strains of Escherichia coli, Klebsiella pneumoniae, methicillin resistant Staphylococcus aureus (MRSA) continue to cause a large number of infections and deaths particularly in developing countries2 . Thus a novel therapeutic compound from an alternate source with unique metabolic and physiological capabilities producing different kinds of metabolites must be employed for future therapeutic scenario globally. Squids under cephalopod family, is known to produce a black pigmented ink as a defensive ploy against its predators. This ink has already been reported to exhibit various therapeutic values3 . The crude ink extracts from various species of cephalopods have already been studied for its antimicrobial potential against biofilm bacteria, Staphylococcus aureus4 , clinical bacterial isolates and pathogenic yeasts5 , preservative property6 , antioxidant values7 and anti-retroviral activity8 . The ink has also been studied for its various bioactive proteins possessing cytolytic property9 , antitumour fractions10 and antimicrobial potential11. With these review background this study has been aimed at isolating and characterizing a new and novel protein from the ink of the Indian squid Loligo duvauceli and to study its antimicrobial potential against the drug resistant pathogens such as MRSA and ESBL producing strains of E.coli and K.pneumoniae.
METHODS
Collection and dissection of squids: Fresh squids were caught from the south west coast of Chennai and were decapitated by the fishermen. The squid was fixed in 4% formalin (4ml formalin in 96ml of water) and was identified as Loligo duvauceli by an eminent Zoologist based on the Zoological taxonomy. The dissection of the squids was performed as per the instructions of the Zoologist. After dissection of the ink sac the surface of the gland was sterilized with ethanol and was blotted with sterile cotton. The ink duct was cut with sterile scissors and the sac was gently squeezed and the excreted ink was collected in sterile brown bottles. The ink was stored at 4ºC until use.
Preparation of melanin free ink: The extracted fresh ink was subjected to centrifugation under cold condition at 15,000 rpm for 30 min to remove melanin. The supernatant was used as melanin free ink. The samples were frozen and kept at - 80°C until use.
Estimation of protein
The protein estimation was done by Lowry‘s method12 , a protein assay that is based on the reaction where the peptide bonds of proteins react with copper under alkaline conditions producing Cu+ ions that react with the Folin‘s reagent producing blue colour.
SDS Polyacrylamide gel electrophoresis:
SDS-PAGE was carried out using an acrylamide concentration of 7.5% in the resolving gel13. Stock solutions were prepared as recommended14 and stored in brown bottles at 4?C. The separating gel mix (12%) was prepared in a 25ml conical flask using acrylamide monomer 2.8ml, separating gel buffer (4x) 1.750 ml, 10 % SDS 0.070 ml, 10% APS 0.035 μl and distilled water 2.345 ml (Final volume – 7ml). The solution was de-gased for 5 min and 3 μl of TEMED was added with gentle mixing and was poured into the glass plates. 100 μl of N-butanol was added over the gel mix and the arrangement was left undisturbed for 20 min. The stacking gel mix (5%) was prepared by mixing acrylamide monomer 0.333 ml, 4x Stacking Gel Buffer 0.500 ml, 10 % SDS 0.020 ml, 10% APS 0.010 μl and distilled water 1.137 ml (Final volume – 2.0 ml). The electrophoretic run was performed as per standard protocols by adding the samples to the wells along with a molecular weight marker. Staining was done using Coomasie brilliant blue and destaining was done with ethanol and acetic acid. The gel was then analysed in the documentation unit. The molecular weight of the unknown peptides was thus determined by comparing it with the standard molecular weight marker bands.
Native Polyacrylamide gel electrophoresis:
Native – PAGE was performed without SDS using the same methodology as described above. Staining was not done. The unstained gel was then carefully removed and the target protein band portion corresponding to target protein band size was excised in sterile phosphate buffer and was eluted as follows.
Passive gel elution of the target protein:
Elution of the protein was performed by passively eluting the proteins from polyacrylamide gel pieces15. The excised gel pieces corresponding to the target protein band were placed in a clean microcentrifuge tubes. The elution buffer was prepared using Tris HCl 50 mM, NaCl 150 mM and EDTA 0.1 mM (pH 7.5). 0.5- 1ml of cold elution buffer is added so that the gel pieces were completely immersed. The gel pieces were crushed using a sterile scalpel and was incubated in a rotary shaker at 30ºC overnight. Centrifugation was done at 5000-10000g for 10 minutes and the supernatant was carefully pipetted into a new microcentrifuge tube. An aliquot of the supernatant might be tested for the presence of the same protein by subjecting it to SDS-PAGE
Enzymatic assay for L – amino acid oxidase [LAO] activity:
LAO activity of the protein was performed by an enzyme coupled assay16. Purified protein was prepared for the assay in 50 mM/L phosphate buffer and 150 mM/L KCl. The reaction mixture was prepared as follows: Tris HCl 0.1 mM/L (pH 7.6), Horseradish peroxide 10 μg, O-dianisidine 0.2 mM/L and L-amino acids 2 mM/L. The test sample was mixed with 100 μl of the reaction mixture. Reaction was performed at room temperature for 1-60 min. The activity was monitored by absorbance at 436 nm. The increase in absorbance was transformed into molar concentration of the end product o-dianisidine (8.31 x 103 mol/L). The Km and Vmax values of the Lamino acids were determined by Lineweaver-Burk plots.
Antimicrobial bioassay:
The antimicrobial assay was performed by Nathan‘s agar well diffusion method17 with the drug resistant strains that included the extended spectrum beta lactamase (ESBL) producing strains of E.coli and K.pneumoniae, methicillin resistant S.aureus (MRSA) and Amphotericin B resistant C.albicans isolated from various clinical specimens from the patients visiting the outpatient unit of Meenakshi General Hospital (Data not given). The control strains include E.coli (ATCC 25922), K.pneumoniae (ATCC 10031), S.aureus (ATCC 25923) and C.albicans (ATCC 10231).
A minimum of four colonies of the test organisms were touched with a sterile loop and transferred into Sterile Mueller Hinton broth and C.albicans into Saboraud‘s dextrose broth under aseptic conditions and was incubated for two hours at 37ºC. After incubation the density of each microbial suspension was adjusted equal to that of 106 c.f.u/ml (standardized by 0.5 MacFarland standards) and was used as the inoculum.
Agar well diffusion assay
Fifty microlitres (50μl) of inoculum of each test and control organism was spread as lawn cultures onto sterile Mueller Hinton agar plates using L-rods to achieve a confluent growth. The agar plates were allowed to dry and wells or cups of 8 mm were made with a sterile agar borer in the inoculated agar plates. The eluted protein was subjected to syringe membrane filtration prior to the bioassay and an aliquot of the same was streaked onto nutrient agar plates and was incubated at 37 C for 24 hrs for sterility checking. A 50 μl volume of the eluted protein in phosphate buffered saline was propelled directly into the wells of the inoculated specific media agar plates for each test organism. The plates were then allowed to stand for 10 minutes for diffusion of the protein to take place and incubated at 37ºC for 24 hrs. Sterile DMSO served as Negative control. Erythromycin 30 µg (for bacteria) and Amphotericin B 100 U (for yeast) were included as positive controls. After incubation the plates were observed for the zone of inhibition around the wells and the zone size was measured using an antibiotic sensitivity measuring scale (Himedia). The antimicrobial efficacy was graded based on the zone diameter as high activity (> 15 mm), moderately active (10- 14 mm), trace activity (5-9 mm) and no activity (< 4 mm)18 . Determination of MIC value for the protein MIC value for the purified protein was determined by Microbroth dilution method19. Serial dilutions of the eluted protein were done in a 96 well microtitre plate with sterile phosphate buffer. The dilution factor was 5, 2.5, 1.25, 0.625, 0.312 and 0.156 μg/100 μl. To each dilution 100 μl of the culture broths of the test strains and control strains were added in their respective wells and the plate was incubated at 37 C for 24 hrs. After incubation the spectrophotometric analysis was performed and the OD values were recorded. The MIC value was also confirmed with Microbial Spot Checker board method20 where 3 μl of each dilution was spotted onto Mueller Hinton agar plates and incubated at 37 C for 24 hrs. After incubation the spot showing the complete absence of microbial growth indicates the minimum inhibitory concentration value.
RESULTS
Centrifugation of the crude ink at 15,000 rpm for 15 min yielded melanin free ink. Using Lowry‘s method, a standard curve of absorbance was obtained as a function of initial protein concentration and it was used to determine the unknown protein concentration. Thus the total protein in the melanin free ink was determined as 65.5 mg/ml. SDS PAGE showed a major protein band with a molecular weight of 60 KDa. Two weak bands were also obtained and their molecular weights were determined as 100 KDa and 80 KDa (Figure 1). The 60 Kda band was the target protein of the study as it has not been reported earlier. The protein was successfully isolated by Native Gel Electrophoresis (Figure 2) and was eluted by passive gel elution technique. L-amino acid oxidase (LAO) assay performed showed that when the purified protein and 2 mM/L amino acids were incubated for 1 min, L-lysine and LArginine proved to serve as excellent LAO substrates. Km and Vmax values of lysine were 33-87 µmol and 1.93-2.71 µmol/sec respectively and Km and Vmax values for arginine is 25-125 µmol and 1.56-3.30 µmol/sec. The reactions were completed within 30 secs at room temperature for lysine and arginine at a concentration of 0.02-2 mM/L. Thus in this study we report a novel protein with LAO activity from the ink of Loligo duvauceli which has been named as Lolduvin-S. The protein after membrane filtration yielded no growth on nutrient agar plate; thus it was sterile and was used for bioactivity assays. Lolduvin-S had scored to possess high antibacterial activity against the drug resistant test strains (Table 1). The zone size ranges from a mean value of 20 mm for E.coli (ESBL), 21 mm for K.pneumoniae (ESBL), 22 mm for S.aureus (MRSA) and 20 mm for C.albicans. The zone size was compared with the control strains (Figure 3). The MIC value was determined as 25 µg/ml for E.coli and K.pneumoniae, 12.5 µg/ml for S.aureus and C.albicans. The previous dilution that showed the visible decrease in the number of colonies was determined as the bacteriostatic dose and was deduced as 12.5 µg/ml for E.coli and K.pneumoniae, 6.25 µg/ml for S.aureus and C.albicans. Thus a novel protein called Lolduvin-S has been isolated and characterized to possess a potent LAO activity from the ink of Loligo duvauceli. The protein had also a promising antimicrobial activity against the dreadful resistant pathogens like ESBL and MRSA organisms.
DISCUSSION
The marine ecosystem offers rich niche for marine flora and fauna. The protein structures, metabolic pathways, reproductive systems, sensory and defense mechanisms developed by marine microorganisms and other lifeforms are distinctive in nature. During the last few decades several novel compounds in point of view of potential drug development have been isolated from marine habitat with various therapeutic applications21. The antimicrobial property of the squid ink is already a known fact and the ink has been found to posses a promising antimicrobial effect in our previous studies. The proteins present in various species of gastropods and cephalopods have been characterized earlier22. This study is thus undertaken to characterize a novel antimicrobial protein from the ink of Loligo duvauceli. The melanin free ink has been used in various studies for the analysis of the protein and it holds suitable for various protein analysis The total protein estimated in this study from the squid ink suggests the availability of the bioactive protein for various pharmaceutical applications. Determination of the molecular weight of the proteins is best achieved by the bioinstrumentational tool such as the SDS PAGE. The electrophoretic separation of the proteins from other species of squid has been achieved with 2.5% and 7.5% gel resolution23. In this study we employed 12.5% gel that is suitable for the separation of the protein from Loligo duvauceli‘s ink. Apart from the major protein band at 60 Kda, two other weak bands at 100 Kda and 80 Kda have also been observed. This shows the presence of tyrosinase24 and peroxidase25 respectively which has been already reported from the ink of various cephalopods and squids. The 60 Kda protein in Loligo duvauceli has not been reported anywhere earlier and thus it has been considered as the target protein. Native PAGE separates the protein in a pure form without denaturation though effective and high amount of proteins can be obtained with gel filtration chromatography methods26. Due to high cost, gel filtration chromatography is not done for the isolation of the proteins: thus the passive gel elution protocol suggested by the Pierce technologies, USA is followed for the same. It is a simple and rapid protocol for the elution of target protein. The elution protocol is slightly modified by performing the elution under cold condition. The protein eluted is rechecked by subjecting the protein for SDS-PAGE and by obtaining the same band at 60 Kda. A similar type of protein called Escapin with a molecular weight of 60 Kda has been reported from the ink of a sea hare under the Phylum Mollusca earlier27 . Escapin is known for its L-amino oxidase activity which is a hydrogen peroxide induced destruction of the target cells (such as micro-organisms) in the presence of Lamino acids. With this view an attempt to check the LAO activity of Lolduvin-S protein is done by Enzyme linked coupled assay. Lolduvin-S has been found to possess an enzymatic L-amino acid oxidase activity. Hydrogen peroxide is sufficient to cause bacteriostasis and does so with a concentration dependency and threshold similar to that of amino acids as substrates for the enzymatic activity. Both lysine and arginine are substrates for LAO activity from the squid ink. It shows strong and rapid activity in this study when using arginine or lysine as substrates, with reactions being completed within 30 secs at room temperature. A similar protein has been reported to be bacteriostatic and bactericidal from the purple gland of the sea hare Aplysia dactylomela28 and the substrates determine the bactericidal or bacteriostatic effects. Another protein called achacin possesses a bacteriostatic effect that seems to be mediated through its oxidation of the L– amino acids lysine and/or arginine and the consequent subsequent production of hydrogen peroxide29. Another antimicrobial protein has been named as escapin from the Aplysia sp., which has been cloned and characterized. Thus this study reports the new protein Lolduvin-S with a potent LAO activity from melanin free ink of Loligo duvauceli. Lolduvin-S from the melanin free ink sample has been found to be more effective against the Gram positive bacteria, gram negative bacteria and the pathogenic yeast.
In an earlier study, a protein in low concentration with potent L-amino acid oxidase activity showing a broad antibacterial activity against Gram-positive and Gram-negative bacteria by H2O2 generation has been reported from the skin mucous of the rock fish30. In correlation with this, the MIC value is determined at low concentration range of 6.25µg/ml. A similar protein has also been isolated from two purple pigmented bacteria from Mediterranean sea whose enzymatic activity is potent in generating the hydrogen peroxide in the presence of Lamino acids31 . A BLAST search suggests that the cDNA encoded a novel antibacterial protein sharing identity with a number of L-amino acid oxidases is effective against MRSA strain32. In correlation with these reports, this study states that the new protein Lolduvin-S from Loligo duvauceli‘s ink possess a promising antibacterial property against the drug resistant pathogens. The bioactive protein Lolduvin-S also scores to possess a high antifungal activity against C.albicans with a MIC value of 12.5 µg/ml. The same property has been reported from novel proteins from snake venoms33. Four glycoproteins with similar activity have been studied from a shell less gastropod sea hare34. A similar protein called Aplysianin-A with antifungal activity has been isolated, cloned and characterized from the purple fluid released from a sea hare with LAO activity35. The antifungal activity of nine amphibian skin secretions has been reported to possess a similar protein with LAO activity36 . Several cell surface proteins with LAO activity have been isolated from marine phytoplanktons with potent antifungal activity37. In relation with these reports, Lolduvin-S from Loligo duvauceli‘s ink possesses a potent antifungal activity. In conclusion, Lolduvin–S, a new and novel protein from the ink of the Indian squid Loligo duvauceli with a potent antimicrobial and LAO activity, can be considered in near future as a valuable pharmaceutical antimicrobial agent from the marine habitat. However further characterization and structural analysis of the same is under progress to acquire a detailed understanding of this novel protein. Lolduvin-S being found to be bactericidal against the ESBL and MRSA pathogens, it can definitely aid in the control and emergence of drug resistant strains globally.
ACKNOWLEDGEMENT
We are thankful to Dr.Mehar Sultana.,M.Sc.,M.Phil.,Ph.D., Professor, Department of Zoology, Presidency College, Chennai, Tamilnadu for speciating the squid selected for our study.
Englishhttp://ijcrr.com/abstract.php?article_id=2142http://ijcrr.com/article_html.php?did=21421. McConnell O, Longley RE, Koehn FE, Gullo VP. The discovery of natural products with therapeutic potential. Butterworth-Heinemann Bost 1994; 109–174.
2. Schwaber MJ, Carmeli Y. Antimicrobial resistance and patient outcomes: the hazards of adjustment. Crit Care 2006; 10(5): 164.
3. Takai M, Kawai Y, Inoue N, Shinano H. Cepahalopod ink – bioactivity, Bull Japanese Soc Sci Fisheries 1992; 58: 2373 – 2378.
4. Atushi Mochizuki. An antiseptic effect of cuttlefish ink, Bull of the Jap Soc of Sci Fisheries 1979; 45(11): 1401-1403.
5. Smiline Girija AS, Hariprasad G, Vijayashree Priyadharsini J, Pandi Suba K, Raghuraman R, Gnanavendhan SG. Antimicrobial potential of Loligo duvauceli ink against the common clinical bacterial and yeast isolates. Biomed 2008; 28(3): 213-215.
6. Saloua Sadok, Abdelwahed, Amor. Combined effect of sepia soaking and temperature on the shelf life of peeled shrimp Peneaeus Kerathurus. Food Chem 2004; 88: 115-122.
7. Lei M, Wang JF, Pang L, Wang YM, Chen SG, Xue CH. Effects of sepia on the metabolization of blood lipid and antioxidant ability in hyperlipidemia rats. Chin J Mar Drugs 2007; 3: 30-33.
8. Rajaganapathy J, Thyagarajan SP, Edward JK. Study on the cephalopod‘s ink for anti-retroviral activity. Ind J Exp Biol 2000; 38(5): 519-20.
9. Gian Luigi, Elio Nisco, Gabriella, Paola and Anna Palumbo. Toxicity of melanin free ink of Sepia officinalis to transformed cell lines: identification of the active factor as tyrosinase. Biochem and Biophy Resear Comm 2003; 308: 293-299.
10. Takaya Y, Uchisawa H, Matsue H, Okusaki B. An investigation of the antitumour peptide form squid ink. Biol Pharm Bull 1994; 17: 846-849.
11. Charles Derby, Hsiuchin Yang, Paul Micah, Ko-Chun Ko and Phang. Cloning, characterization and expression of escapin, a broadly antimicrobial FAD – containing Lamino acid Oxidase from ink of the sea hare Aplysia californica. J of Expe Biol 2005; 208: 3609-3622.
12. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Estimation of proteins. J of Biol Chem1951; 193: 265-266.
13. Nagashima Y, Kikuchi N, Shimakura K, Shiomi K. Purification and characterization of an antibacterial protein in the skin secretion of rockfish. Comp Biochem Physiol 2003; 136: 63-71.
14. Anbalagan K. An introduction to electrophoresis. Laboratory Manual, Yercaud Biotech 1999; 1st Edition.
15. Pierce Laboratories. Technical Resource. Purify proteins from Polyacrylamide gels. Rockford, USA. www.piercenet.com.
16. MacHeroux P, Seth O, Bollschweiler C, Ghisla A. L-amino oxidase from the malayan pit Viper calloselasma rhodostoma comparative sequence analysis and characterization of active and inactive forms of the enzyme. Eur J Biochem 2001; 268: 1679-1686.
17. Nathan P, Law EJ, Murphy DF. A laboratory method for the selection of topical antimicrobial agents. Burns 1978; 4: 177 178.
18. Rios JL, Recio MC, Villar A. Screening methods for natural products with antimicrobial activity: a review of the literature. J Ethnopharma leaflets 1988; 23: 127-149.
19. Mcginnis, Ronaldi MG, Lorian V, Williams, Wilkins. Antibiotics in laboratory medicine (4th Edition), Baltimore 1996; 176-211.
20. Nkere CK, Iroegbu C. Antibacterial screening of the root, seed and stembark extracts of Picralima nitida. Afri J of Biotech 2005; 4(6): 522-526.
21. Epel D. Frontiers in squid production: prospecting for new antibiotics. Marine natural products. Pub No CSG-MP-02- 001. (Sea grant, California), 2002.
22. Kirsten and Benkendorff. 2005. Molluscan biological and chemical diversity: secondary metabolites and medicinal resources produced by marine molluscs. Bio Rev. 7: 917-921.
23. Anna Palumbo, Gabriella Fiore, Annarita Poli, Anna Di Cosmo, Marco d‘Ischia. Dopamine in the ink defence system of Sepia officinalis: biosynthesis, vesicular compartmentation in mature ink gland cells, nitric oxide (NO)/cGMP-induced depetion and fate in secreted ink. Biochem J 2004; 378: 785-791.
24. Gian Luigi, Elio Nisco, Gabriella, Paola, Anna Palumbo. Toxicity of melanin free ink of Sepia officinalis to transformed cell lines: identification of the active factor as tyrosinase. Biochem and Biophy Resear Comm 2003; 308: 293-299.
25. Ida Gesualdo, Franscesco, Margherita. Molecular cloning of a peroxidase mRNA specifically expressed in the ink gland of Sepia officinalis. BioChemica et Biophysica Acta 1997; 1353: 111-117.
26. Wang JF, Lei M, Wang YM, Pang L, Xu W, Xue CH. Study of the radioprotective effect of cuttlefish ink on hemopoietic injury. Asia Pac J Clin Nutr 2007; 16: 239-243.
27. Johnson PM, Yang H, Tai PC, Derby CD. Escapin: an antipredator protein in the defensive secretion of Aplysia californica. Chem Senses 2003; 28: A24 (abstract).
28. Melo V, Duarte A, Carvalho A, Siebra E , Carvalho. Purification of a novel antibacterial and haemagglutinating protein from the purple gland of the sea hare Aplysia dactylomela. Toxicon 2000; 38: 1415-1427.
29. Ehara T, Kitajima S, Kanzawa N, Tsuchiya T. Antimicrobial action of achacin is mediated by L-amino acid acid oxidase activity. FEBS Lett 2002; 531: 509-512.
30. Yoichiro, Nobuyo, GuoHua, Shoichiro, Kuniyoshi, Kazuo Shiomi, Yuji Nagashima. Antibacterial action of Lamino acid oxidase from the skin mucus of rockfish Sebastes schlegelii. Comparitive Biochem and Physiol 2008; 149(2): 394-400.
31. Daniel Gomez, Elena Espinosa, Marcelo Bertazzo, Patricia Lucas, Francisco Solano, Antonio Sanchez. The macromolecule with antimicrobial activity synthesized by Pseudoalteromonas luteoviolacea strains is an L-amino acid oxidase. Appl Microbio and Biotec 2007; 79(6): 925-930.
32. Kosuke Kasai, Takashi Ishikawa, Takafumi Komata, Kaori Fukuchi and Mitsuru Chiba. Novel L-amino acid oxidase with antibacterial activity against methicillin-resistant Staphylococcus aureus isolated from epidermal mucus of the flounder Platichthys stellatus. FEBS J 2009; 277(2) 453–465.
33. Gomes VM, Carvalho AO, Da Cunha M, Keller MN, Bloch C Jr, Deolindo P, Alves EW. Purification and characterization of a novel peptide with antifungal and antiviral activity from Bothrops jararaca venom. Toxicon 2005; 45(7): 817-827.
34. Ryosuke Iijima, Jun Kisugi and Masatoshi Yamazaki. A novel antimicrobial peptide from the sea hare Dolabella auricularia. Develop and Comp Immunol 2003; 27(4): 305-311.
35. Mitsuru Jimbo, Fumie Nakanishi, Ryuichi Sakai, Koji Muramoto, Hisao Kamiya. Characterization of L-amino acid oxidase and antimicrobial activity of Aplysianin-A, a sea hare-derived antitumor-antimicrobial protein. Fisheries Sci 2003; 69(6): 1240–1246.
36. Andre Gustavo, Marcia Souza, Carvalho Melhem, Frederico Oliveira Prado, Gabriela, Roberto Mitsuyoshi. Amphibian secretions for drug discovery studies: a search for new antiparasitic and antifungal compounds. Letters in Drug Design and Discovery 2007; 4: 67-73.
37. Brian Palenik, Francois MM. Morel Comparison of cell-surface L-amino acid oxidases from several marine phytoplanktons. Marine Eco Prog Series 2000; 59: 195-201.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524137EnglishN-0001November30HealthcareAN INSIGHT INTO METAL BASED ANTI-CANCER DRUGS
English1523Raj KaushalEnglish Nitesh KumarEnglish Rajeev KaushalEnglish Pamita AwasthiEnglishMetal complexes play critical role in the treatment of cancer. Cis-platin which is model anticancer drug has been used in the treatment of various types of cancer. But due to its side effects and resistance phenomenon efforts have been made to explore the possibility of synthesizing novel non-platinum based anti-cancer drugs. In addition to platinum based drugs, complexes of other transition metals like titanium, ruthenium, palladium and gold etc. also show pronounced anti-cancer activity. The Complexes with titanium and ruthenium have already been evaluated in phase I and phase II clinical trials. Some transition metal complexes show good anticancer activity against cis-platin resistant cell lines. This review will provide an insight into various platinum as well as non-platinum based anti-cancer drugs.
EnglishTransition metal, cis-platin, Nucleic acid, protein, platinum, titanium, ruthenium, palladium, gold, anti-cancer, drug, amines, biological activity.INTRODUCTION
Metals play an important role in our daily life due to their incorporation in our diet in varying quantities1,2. Due to potential pharmacological applications of transition metal based complexes such as antidiabetic, anti-neurological, anti-bacterial, anti-fungal, anti-cancer agents, metal complexes have been used in medicinal chemistry since sixteenth century. Transition metals have tendency to form variety of complexes due to the presence of vacant d-orbitals in their valence shell. They can take part in various biological processes which show their interaction with electron rich biological components like proteins and nucleic acid because metal centers are positively charged and favored to attack on negatively charged biomolecules3-6 . Various transition metals such as platinum(Pt), titanium(Ti), ruthenium(Ru), rhodium(Rh), iridium(Ir), molybdenum(Mo), copper(Cu) and gold(Au) in their complex form are effective against solid tumors in animals and human beings. The first metal complex discovered to exhibit anti-cancer activity was cis-platin (cisdiaminedichloroplatinum(II)). This drug is considered best for treatment of certain types of cancers but due to its toxicity, its utilization has been limited at broad range7- 9 . In coming era, interest has been growing in developing non-platinum based anticancer drugs due to their less toxicity.
Also, non-platinum compounds may provide different oxidation states, coordination geometries, and affinities for certain types of biological ligands10 . It has been established that ligands having O, N, S in their stem showed increased biological activity due to increase in coordination capacity11,12. It has been reported in literature that due to the symmetry of ligand(s) uptake of drug by cancerous cells has been increased13. The necessary conditions for a complex to have anti cancer activities are as i) Complex should be neutral so that it can diffuse through the hydrophobic cell membrane. ii) Complex should have square planer structure i.e. leaving group should be at cis-position. iii) Leaving groups should be labile, so that they can be easily substituted. iv) Groups which are not substituted should have low trans effect like NH3 , heterocyclic amines or diamines14. Amine ligands influence the anti-cancer property, because non leaving amine ligands are the reason for anti-cancer property15. Recently, some non metallocene titanium complexes having oxygen based ligands have been synthesized and it has been established that ligand lability is not essential to show anti-tumor activity16 .
Platinum Complexes as anti cancer agents:
The first metal based anticancer drug discovered was Cisdiaminedichloroplatinum(II) (cis-platin) by Rosenberg et al8,9.Cis-platin acts by interacting with DNA (Deoxy ribo nucleic acid) via cross linking with two adjacent guanine molecules, followed by the replacement of two chloride groups by water molecules and form aquated cisplatin which stops the replication of DNA and obstruct the cell growth which is the ultimate aim of anti- cancer drugs14. Cisplatin has been used in the treatment of various types of cancers such as testicular, ovarian, lung, neck, and head cancers. This metal complex used in the treatment of various cancerous malignancies and is one of the best-selling anti-cancer drug all over the world. Cis-platin has several disadvantages some of which may include that by treating the cells with cis-platin, necrotic and apoptic cell death occur simultanesly. Also, it has limited solubility in water hence it is given intravenously to reduce the harm to the kidney. Other side effects of using cis-platin are emesis, nausea, vomiting, nephrotoxicity, neurotoxicity, myelosuppression, ototoxicity14 . Also only a limited number of tumors can be treated with platinum based drugs. In addition to cis-platin many other platinum based drugs (Carboplatin, Oxaliplatin, nedaplatin and lobaplatin) passed for current tumor therapy1 . The new platinum complexes of the formula [Pt(2,2'- bipyridine)amino acid] n+ where n =1-2 and amino acid is an anion of L-histidine, LLysine, L-asparagine, L-tryptophan, or Ltyrosine, had been prepared by interaction of [Pt(2,2'-bipyridine)Cl2] and an appropriate amino acid (sodium salt) in water or water-methanol mixture which are highly negatively charged molecules. In case of Histidine the Platinum atom binds with –NH2 group of Histidine and in case of other amino acids Platinum binds by NH2 and COOgroups and these complexes were used against P-388 Lymphocytic leukemic cells18. An octahedral complex of Platinum(IV) with adamantylamine of composition bis(acetato)(1- adamantylamine)amminedichloro platinum(IV) had been prepared and showed resistance factor 2.84 fold lower than cis-platin because adamantylamine is a bulky hydrophobic ligand and the use increase the uptake of compound by the cancerous cells and able to overcome the acquired cis-platin resistance13. Pt(IV) complex with adamantylamine penetrate as a whole complex inside the cell membrane. It may be due to hydrophobicity of ligand. The symmetry of hydrophobic adamntylamine ligand lightens the penetration of whole complex inside the cell membrane. The penetration of Pt(IV) complex with adamantylamine had been improved and facilitates transport across cell membrane. This hydrophobic ligand enhances accumulation inside cancer cell and trigger rapid cell death in both cisplatin sensitive and cis-platin resistant cell lines15 . The Pt(II) Complexes bearing pyridine carboxyldimines containing bulky aromatic groups examined for their potential cytotoxicites against human ovarian carcinoma and cis-platin resistant cell line19. A series of trans-platinum(IV) complexes with functionalized aromatic carboxylate ligands cis, cis, transPt(NH3 ) 2Cl2 (CO2C6H4R)2 where R may be H, p-vinyl, p-methoxy, p-iodo, p-cyano, pcarboxyl had been synthesized and due to presence of aryl groups uptake of drugs had been improved and facilitate transport across cell membrane. These complexes were evaluated for cellular uptake and inhibition of cell proliferation against a panel of lung, colon and breast carcinoma cell lines20 .
Gold Compounds having anti-cancer activities:
The interaction of cytotoxic gold(III) compounds with DNA is weak than that of platinum analogues but gold(III) complexes have good interaction with model proteins and target proteins. The mode of action of gold(III) compounds is significantly different than that of cis-platin. Some compounds like [Au(en2)]Cl3 (en=ethylene diamine), [Au(dien)Cl]Cl2 (dien=diethylene diamine), [Au(cyclam)](ClO4) 2Cl, [Au(terpy)Cl]Cl2 (terpy=terpyridine), and [Au(phen)Cl2]Cl (phen=phenanthroline) were characterized in solid state and in solution. These compounds of gold were tested on human ovarian cell line A2780,which were either sensitive or resistance to cis-platin21. A new compound Chloro-glycylhistidinate gold(III) (GHAu) had good biological property and tested for cytotoxic properties in vitro against MOLT-4 (human leukemia) and C2C12 (mouse tumor) cell lines22,23 . Nowadays, gold(III) compounds are good cytotoxic agents. Plenty of gold(III) compounds were characterized and synthesized in the last fifteen years24. Gold complexes containing bipyridine ligands of general formula [Au(NΛN)Cl2]PF6 where (NΛN) = 2,2'-bipyridine, 4,4'-dimethyl bipyridine, 4,4'-dimethoxy-2,2'-bipyridine, 4,4'-diamino-2,2'-bipyridine showed moderate to good cytotoxicity in vitro towards human ovarian carcinoma cell line and cis-platin resistance variant25 .
RUTHENIUM COMPLEXES:
Ruthenium complexes have attracted much attention as building blocks for new transition metal based anti-tumor agents. Ruthenium compounds offer the potential over anti-tumor platinum(II) complexes currently used in the clinic because of reduced toxicity, a novel mechanism of action and the prospect of non cross resistance26-29. Organo ruthenium complexes due to presence of lipophilic arene can interact better than that of cisplatin inside the cell, by causing chlorine dissociation which is an important factor for cell death. Ruthenium(III) complexes of general formula [Ru(η 6 - arene)Cl2(NC5H4OOC-C5H4 FeC5H5)] where arene may be C6H6, C6H5Me, pPrC6H4Me, and C6Me6 and of formula [Ru(η 6 -arene)Cl2]2 (NC5H4OOC- RUTHENIUM COMPLEXES: Ruthenium complexes have attracted much attention as building blocks for new transition metal based anti-tumor agents. Ruthenium compounds offer the potential over anti-tumor platinum(II) complexes currently used in the clinic because of reduced toxicity, a novel mechanism of action and the prospect of non cross resistance26-29. Organo ruthenium complexes due to presence of lipophilic arene can interact better than that of cisplatin inside the cell, by causing chlorine dissociation which is an important factor for cell death. Ruthenium(III) complexes of general formula [Ru(η 6 - arene)Cl2(NC5H4OOC-C5H4 FeC5H5)] where arene may be C6H6, C6H5Me, pPrC6H4Me, and C6Me6 and of formula [Ru(η 6 -arene)Cl2]2 (NC5H4OOC-
Palladium Complexes:
The marginal anti-tumor activities of the palladium complexes were explained on the basis of fast reactivities of leaving groups as the reactivity of palladium(II) complexes is much higher as compared to platinum(II) complexes e.g. the reactivity of palladium(II) complexes having 1,2- Diaminocyclohexne and dicarboxylate ligands was 105 times more than that of platinum(II) complexes having similar ligands34 . The Pd(II) complexes of the form trans-PdCl2L2(where L=3- hydroxypyridine, 2-hydroxypyridine, 4- hydroxypyridine) had been synthesized and it had been found that the Solubility, reactivity, electronic and steric properties can be modified by varying the geometry and ligands around the metal center. Out of these the compound of 2-Hydroxy pyridine was found to be most active against A2780, A2780cisR and A2780ZD0473R ovarian cancer cell lines. It had been found that both metal and ligand take part in biological activity of the complex but due to the rapid hydrolysis of palladium complexes (105 faster that Platinum analogues), they dissociate easily before reaching their pharmacological target35 . The platinum(II) and palladium(II) complexes of 2,2'-bipyridine (bipy) with ethyl dithio carbamate (Et-dtc) in which the di thio carbamate ligand coordinate with pt(II) or pd(II) center as bidentate with two sulphur atoms were water soluble and were tested for their in vitro antitumor activity against chronic myllogenous leukemia cell line K562. But these complexes show cytotoxic concentration (Cc50) values lower than that of cis-platin. The mode of interaction of these complexes were investigated by circular-dichromism, ultraviolet difference and flouroscence spectroscopy. The interaction of Pd(II) complex with DNA and its anti- tumor activity against K562 is more than that of its Pt(II) analog36 . The series of palladium complexes with Salicyldamine thio semicarbazone having formula [pd(salt scR)PPh3], {H2Salt scR = Salicyldehyde thio semicarbazone R=H, 3-tert butyl, 3- methoxy, 5-chloro} were prepared by reaction of appropriate salicyldamine thiosemicarbazone with Pd(PPh3)2 in which thiosemicarbazone coordinate to the palladium in a tridentate manner that is through phenolic oxygen, imine nitrogen and thionate sulphur forming five or six membered chelate rings with in the structure and the fourth coordination site is occupied by PPh3. The biological activity of thiosemicarbazone ligand and palladium complexes were investigated towards WHC01 oesophageal cancer cell line and against two strains of malarial parasite plasmodium Falciparum W2 (Chloroquinone resistant)37 . Another palladium(II) compound with 5-methyl uracil of the general formula PdL2Cl2 where L= 5-methyl uracil was prepared by Anshu Srivastava et. al. and it had been found that this light brown compound was hygroscopic and had thermal stability up to 260?C with anti-tumor activity38 .
Titanium based anti-cancer drugs:
In the last few years there has been growing interest in developing nonplatinum based anti-cancer agents due to their pronounced biological activity39-51 . After the discovery of cis-platin, the first non-platinum anti-cancer drugs were budotitane and titanocene dichloride which are titanium based anti cancer drugs. Since titanium is second most abundant transition metal and ninth of all the elements on earth and pure titanium and titanium alloy are widely used for orthopedic and dental implants. Titanium is present in many biomaterials such as food in the form of whitening pigment. So it may incorporated in drugs and in to living systems with low toxicity10. Also Ti(IV) is an oxophillic metal and form strong bond with acidic DNA as well as other biological molecules. Titanium as a metal posseses a wide spectrum of anti-tumor properties. Titanium based compounds i.e. bis (β- diketonato)titanium(IV) [Budotitane] and titanocene derivatives offer an alterative for cancer chemotherapy. The anti-tumor activity of budotitane was reported in 1982.This was first non-platinum complex tested in clinical trials and used against ascites and solid tumor. This drug had maximum tolerable dose of 230 mg on two week schedule with side effects of cardiac arrhythmia. It had been reported that doses higher than maximum tolerable result in liver and kidney toxicity52 . Erich Dubler had synthesized and crystallized di-chloro derivatives of budotitane and found that anti-tumor activity appear due to unsubstituted phenyl rings, if phenyl rings get replaced by methyl groups, activity totally disappears53. The first metallocene i.e. titanocene dichloride show anti-tumor activity against colon, lung and breast cancer although the mechanism of cytotoxicity is not clear yet. This complex also exhibits antiproliferative activity against solid ascite tumor. Titanocene dichloride show anti-tumor activity against doxorubicin and cis-platin resistant ovarian carcinoma cells and also less toxic effect than cis-platin. The advantage of this complex is that no evidence of nephrotoxicity or myolotoxicity had been reported. It has been found in literature that studies on chemistry of titanium as anti tumor agent are more limited52. Titanocene dichloride are proved to be superior compounds of its derivatives since in addition to anti-tumor properties titanocene dichloride exhibits anti-viral54, antiarithmetic, and anti-inflammatory activities55. This compound exhibits higher toxicity than cis-platin, doxorubicin, mitoxantrone and vinblastine in human renal cell carcinoma. The other halides or pseudohalides of Cp2TiX2 (where X=F, Br, I, NCS, N3) were tested for ehrlich ascite tumor in mice and show anti proliferative activity similar to Cp2TiCl2. Budotitane and titanocene dichloride possess same limitation that they have low hydrolytic stability at physiological pH52 . In this respect titanium(IV) complexes offer a new outlook for chemotherapy. The novel titanocene compounds are better than cis-platin for apoptic effect in vitro and they can induce more apoptosis than cisplatin. TitanoceneY (bis-[(pmethoxybenzyl) cyclopentadienyl titanium dichloride) had better effect in prostate, pancreas, breast and ovarian cell lines and in uterine and renal cancer cells56 . Michael shavit et al studied Ti(IV) complexes of oxygen-based ligands. They had prepared the homoleptic complex of hydroxyamino 1,3,5 triazine ligands. These triazine ligands possess mild reactivity despite having no labile groups. This complex was effective against colon and ovarian cancer cells16 . Since titanocene dichloride is active against colorectal, lung and breast carcinomas, new derivatives may have antitumor activity profile. These complexes have advantage that they do not show common side effects such as emesis, alopecia, or bone marrow impairment52 . These features make titanium compounds interesting for combined therapy and further study16 . The novel achiral titanocene (Titanocene C and Titanocene Y) anti-cancer drug are almost ten times less toxic than cis-platin. The antiproliferative activity of titanocene Y had been studied in 36 human tumor cell lines and in explanted human tumors and albumin was the carrier protein to take titanocene at the target place inside the cell. Prostate, cervix and renal cell cancer were prime target of these titanocene57 . Titanocene dichloride react with trimethyl tin fluoride giving a new class of cytotoxic active substance in which Ti-F bond is 75 Kcal/mol more stable than Ti-Cl bond and due to fluorides ions product were not cytotoxic at concentration below 10-3M. But the drawback of this complex was that they show instability in water solution58 . Also titanocenyl amide complex having triflouromethoxy group on para position show more cytotoxicity than titanocene dichloride due to –OCF3 group on Para position and more stability in aqueous solution. Different compounds were synthesized by replacing –OCF3 by another groups and these were found active against breast cancer cell line MCF-7 59 . In addition to these, titanium alkoxide complex show toxicity in breast, colon and pancreatic cancer cell lines but molecular mechanism yet to be elucidated60 .
Englishhttp://ijcrr.com/abstract.php?article_id=2143http://ijcrr.com/article_html.php?did=21431. Hariprasath K , Deepthi B, udheer Babu I, Venkatesh P, Sharfudeen S, Soumya V. Metal complexes in drug research- Review. Journal of chemical and pharmaceutical research 2010;2(4):496-499.
2. Tripathi K. A Review- can metal ions be incorporated into drugs?. Asian J Research Chem 2009;2(1).
3. Sakurai H, Kojima Y, Kawabe K, Yasui H . Coordination chemistry review 2002;226:187-98.
4. Sadler PJ, LiH Sun H. Coordination Chem Rev 1999;185-186:689-709.
5. Ali H, Van Leir JE. Chem Rev 1999; 99: 2379-450.
6. Louie AY, Meade TJ. Chem Rev1999; 9: 2711-34.
7. Bruijnincx P C A and sadler P J. New trends for metal complexes with anticancer activity. Curr opin chem Biol 2008;12(2):197-206.
8. Wiltshaw B.E. Cis-Platin in the treatment of cancer. Platinum metals Rev 1979;23(3):90-98.
9. Reedijk J. Why does Cisplatin reach Guanine-N7 with competing S-donar ligands available in the cell?. Chem Rev 1999;99:2499-2510.
10. Tshuva EY and Peri D. Modern cytotoxic titanium(IV) complexes; insight on the enigmatic involvement of hydrolysis. Coordination chemistry reviews 2009;253:2098-2115.
11. Halder S, Peng S-M, Lee GH, Chatterjee T, Mukherjee A, Dutta S, Sanyal U, Bhattacharya S. New J Chem 2008;32:105-114.
12. Kovala-Demertzi D, Dermertzis M A, Miller JR, Papado-poulou C, Dodorou C, Filousis G. J Inorg Biochem 2001;86:555-563.
13. Kozubik A, Horvath V, S-Sindlerova L, Soucek K, Hofmanova J , Sova P , Kroutil A , Zak F , Mistr A , Turanek J. High effectiveness of Platinum(IV) complex with adamantylamine in overcoming resistance to cis-platin and suppressing proliferation of ovarian cancer cells in vitro. Biochemical pharmacology 2005;69:373-383.
14. Kostova I, Platinum complexes as anticancer agents. Recent trends on anti-cancer drug discovery 2006;1:1- 22.
15. Kozubik A, Vaculova A, Soucek K, Vondracek J, Turanek J , and Hofmanova J. Novel anticancer platinum(IV) complexes with adamantylamine:their efficacy and innovative chemotherapy strategies modifying lipid metabolism. Metal based drugs 2008;417:897.
16. Shavit M, Peri D, Melman A. Antitumor reactivity of non metallocene titanium complexes of oxygen based ligands:is ligand lability essential?. J Biol inorg Chem 2007;12:825-830.
17. Ott I. And Guest R. Non platinum metal complexes as anticancer drugs. Arch Pharm Chem Life Science 2007;340:117-126.
18. Kumar L, Kandasamy NR, Srivastava T S, Amonkar A J, Adwankar M K and Chitnis MP. Synthesis and spectroscopic studies of potential anticancer [Platinum(II)(2,2?Bipyridine)(amino Acid)n+ (n=1 or 2). Journal of inorganic biochemistry 1985;23:1-11.
19. Conard ML, Enman JE, Scales SJ, Zhang H, Vogels CM, SALEH MT, Decken A, Westcott SA, Synthesis, characterization and cytotoxicites of platinum(II) complexes bearing pyridine carboxaldimines containing bulky aromatic groups. Journal of inorganic biochemistry 1985;23:1-11.
20. Han Ang W, Pilet S, Scopelliti R, Bussy F, Juillerat-Jeanneret L and Dyson PJ. Synthesis and characterization of platinum(IV) anticancer drugs with functionalized aromatic carboxylate ligands:influence of the ligands on drug efficacies and uptake. J Med Chem 2005;48:8060- 8069.
21. Gabbiani C, CasiniA and Messori L. Gold(III) compounds as anticancer drugs. Gold Bulletin;2007:40/1 22.
22. IENKEN M W, IPPERT BL, Zangrando E, Randaccio L. Inor Chem, 1992;31:1983.
23. Carotti S, Marcon M, Marussicn M, Mazzei T, Messori L, Mini E, Orioli P. Chem Biol Interaction 2000;125:29.
24. Coronnello M, Mini E, caciagli B, Cinellu M A, Bindoli A, Gabbiani C, Messori L, J Med Chem 2005;48:6761.
25. Casim A. , Diawara M C, Scopelliti R, Zakeeruddin S H, Gratzel M, and Dyson p j. Synthesis, characterization and biological properties of gold(III) compounds with modified bipyridine and bipyridylamine ligands. Dalton transactions 2010;39:2239-2245.
26. Fruhauf S, Zeller WJ. New platinum, titanium and ruthenium complexes with different patterns of DNA damage in rat ovarian tumor cells. cancer Res 1991;51: 2943-2948.
27. Coluccia M, Sava G, Loseto F, Nassi A, Boccarelli A, Giordano D, Alessio E, Mestroni G. Antileukemic action of Rucl2(DMSO)4 isomers and prevention of brain involvement on p388 leukemia and on p388/DDP subline. Eur J Cancer 1993;29A:1873-1879.
28. Clarke MJ. Ruthenium metalopharmaceuticals. Coord Chem Rev 2003;236:209-233.
29. Alessio E, Mestroni G, Bergamo A, Sava G. Ruthenium anti-cancer drugs. In: Sigel, H(Eds) Metal complexes in tumor diagnosis and as anti-cancer agents 2004;42:323-351.
30. Auzias M, Therrien B, Suss-Fink G, Stepnicka P, Han Ang W, and Dyson P J. Ferrocenyl pyridine Arene Ruthenium complexes with anticancer properties: synthesis, structure, electrochemistry and cytotoxicity. Inog Chem 2008;47:578-583.
31. Beckford FA. Reaction of the anticancer organometallic ruthenium compound, [(η 6 -pcymene)Ru(ATSC)Cl)]PF6 with human serum albumin. International Journal of Inorganic Chemistry 2010;Article ID 975756:7 Pages.
32. Moreno V, Lorenzo J, Aviles FX, Garcia MH, Ribeiro JP, Morais Ts, Florindo P, and Robalo MP. Studies of the antiproliferative activity of Ruthenium(II) cyclopentadienyl derived complexes with nitrogen coordinated ligands. Bioinorganic Chemistry and applications 2010;Article ID 936834:11Pages.
33. Brabec V, Novakova O. DNA binding mode of Ruthenium complexes and relationship to tumor cell toxicity. Drug Resistance updates 2006;9:111- 122.
34. Kim J Y. Synthesis of some Palladium(II) complexes of 1,2- Diamino cyclohexane and dicarboxylates as cis-platin analogues of palladium series. Arch Pharm Res 1992;15(4): 336-342.
35. Abu-Surrah AS, Al-sa‘doni HH, Abdalla MY. Palladium based chemotherapeutic agents. Routes towards complexes with good antitumor activity.Cancer therapy 2008;6:1-10.
36. Islami-Moghaddam M, MansouriTorshizi H, Divsalar A and Saboury A. A Synthesis, characterization,cytotoxic and DNA binding studies of diimine platinum(II) and palladium(II) complexes of short hydrocarbon chain ethyl dithio carbamate ligand. J Iran Chem Soc Sep2009;63:552-569.
37. Chellan P, Shunmoogam-Gounden N, Hendricks D T, Gut J, Rosenthal P J, Lategan C, Smith PJ, Chibale K and Smith GS. Synthesis structure and invitro biological screening of palladium(II) complexes of functionalized salicyaldimine thiosemicarbazone as antimalarial and anticancer agents. Eur J Inor Chem 2010;3520-3528
38. Srivastava A, Gupta Dc. Synthesis and structural investigations of coordination compounds of palladium(II) with 5-methyl uracil. Int Journal of Applied Engineering research, Dindigul 2010;1:2.
39. PCA Bruijnincx, PJ Sadler. Curr Opin Chem Bio 2008;12:197.
40. Jakupec M A, Galanski M, Arion V B, Hartinger Cg, Keppler BK. Dalton Tran 2008:183.
41. Clarke MJ, Zhu f, Frasca DR. Chem. Rev 99;1999:2511.
42. Kratz F, Schutte MT. Cancer J 1998;11:176.
43. Kopf-Maier P. Eur J Clin Pharmcol 1994;47:1.
44. Desoize B. Anticancer Res 2004; 24:1529.
45. Xu G, Cui YB, Cui K, Gou SH. Prog Chem 2006;18:107.
46. Galanski M, Arion VB, Jakupec MA, Keppler BK. Curr Pharm Des 2003;9:2078.
47. Ott I, Gust R. Arch Pharm Chem Life Sci 2007;340:117.
48. katsaros N, Anagnostopoulou A. Crit Rev Onc Hemt 2002;42:249.
49. Evangelou AM. Crit Oncol Hemat 2002;42:249.
50. Kostova I. Curr Med Chem Rev 1987;87:1137.
51. Kopf-Maier P, Kopf H. Chem Rev 1987;8:1137.
52. Menendez E. Titanium complexes in cancer treatment. Critical reviews in oncology/Hematology 2002;42:309- 315.
53. Dubler E, Buschmann R, Schmalle H W. Isomer abundance of bis(β- diketonato complexes of titanium(IV) crystal structure of the antitumor compound budotitane[TiIV(bzac)2(OEt)2] and of its dichloro-derivative [TiIV(bzac)2Cl2]. Journal of inorganic Biochemistry 2003;95:97-104.
54. Tonew E, Tonew M, Heyn B, Schroer HP, Zentralo, Baktenal Parasitenskol Infeknonskr Bakt Hugg. Abt. Orig. Reahz A. 1981;250:425.Chem Abst 1982;96:82566.
55. Farlie DP, Whitencuse MW, Broomhead JA. Chem Biol Interact 1987;61:277.
56. O‘Connor K, Gill C, Tacke M, Rehmann FJK, Strohfeldt K, Sweeney N, Fitzpatrick JM, Watson RWG. Novel titanocene anticancer drugs and their effect on apoptosis and the apoptotic pathway in prostate cancer cells, Apoptosis 2006;11:1205-1214.
57. Pampillon C, Claffey J, Hogan M, Tacke M. Novel achiral titanocene anticancer drugs synthesized from bisN,N-dimrthylamino fulvene and lithiated heterocyclic compounds. Biometals 2008;21:197-204.
58. Eger S, Immel T A, Claffey J, MullerBunz H, Tracke M, Groth U and Huhn T. Titanocene diflourides with improved cytotoxic activity. Inog Chem 2010;49:1292-1294.
59. Gao LM and Melendez E. Cytotoxic properties of titanocenyl amides on breast cancer cell line MCF-7. Metal based drugs 2010; Article I.D.286298:6 pages.
60. Williamson EA, Boyle TJ, Raymond R, Farrington J, Verschraegen C, Shaheen M, Hromas R. Cytotoxic activity of the titanium alkoxide (OPy)2Ti (4AP)2 against cancer colony forming cells. Invest new drugs. DOI 10.1007/S:10637-010-9530-3.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524137EnglishN-0001November30General SciencesEFFECT OF THICKNESS AND ANNEALING TEMPERATURES ON STRUCTURAL AND MORPHOLOGICAL PROPERTIES OF (SB2S3)1-X (BI2S3)X THIN FILMS
English2436Jessy Mathew N.English Rachel OommenEnglish C. Sanjeeviraja English Usha Rajalakshmi PEnglish(Sb2S3)1-x (Bi2S3)x (with x= 0.03, 0.05, 0.1) thin films were prepared on chemically well cleaned glass substrates by thermal evaporation technique for two thickness. The orthorhombic crystal structure of the (Sb2S3)1-x (Bi2S3)x powder samples and thin films have been found out from XRD powder data and confirms the transition of films from amorphous to polycrystalline with thermal treatment at 523K. Also lattice parameters, crystallite sizes and interplanar spacings for the newly prepared ternary compounds (Sb-Bi-S) have been calculated. The XPS spectra were carried out to identify the surface compositions. The elemental percentage in each compound was verified by EDAX. Increase of crystallite size with thickness and temperature was proved by
XRD and the influences of thickness and annealing temperature on roughness and grain size was observed from AFM measurements.
EnglishThermal evaporation technique, Ternary compounds, Amorphous, Polycrystalline, Orthorhombic crystal structure1. INTRODUCTION
The ternary semiconductor compounds in thin film form have become the focus of attention because of their important physical and chemical properties since these properties could be changed by varying the annealing temperature and thickness. Thin films of semiconductor chalcogenides of the chemical formula m2 V-B - n3 VI-B basically have the structure of antimony sulphide which are most important materials for the applications in photosensitivity, photoconductivity and thermoelectric power1 . Antimony sulphide and Bismuth sulphide are layer structured direct band gap semiconductors with orthorhombic crystal structure have been received considerable interest because of their structural, morphological, optical and electrical properties which allow their wide use in many devices. A number of methods have been used for the preparation of V-VI compound thin films by the thin film investigators like, N.S. Yesugade et al.2 prepared Sb2S3 and Bi2S3 thin films by electrodeposition technique, Gang Xie et al.3 synthesized M2S3 (M = Sb, Bi) via a hydrothermal treatment, Pawan Kumar et al.4 prepared Bi2S3 thin films by spray pyrolysis technique, Siham Mahmoud et al.5 produced thin films of Bi2S3 by thermal evaporation technique, I.K. El Zawawi et al.6 prepared Sb2S3 thin films by thermal vacuum evaporation technique, I. Grozdanov et al.7 fabricated thin films of Sb2S3 by chemical deposition technique etc. Pawar et al.2 obtained Bi2S3, Sb2S3 and As2S3 films by a solution-gas interface technique. This paper reports the investigations on the effect of thickness and annealing temperature on the structural and morphological properties of thermally evaporated (Sb2S3)1-x (Bi2S3)x (with x= 0.03, 0.05, 0.1) thin films from newly prepared powder samples. Characterizations were done by means of Energy Dispersive X-ray Analysis (EDAX), X-ray Diffraction (XRD) technique, X-ray Photoelectron Spectroscopy (XPS) and Atomic Force Microscopic (AFM) analysis. To the best of our knowledge nobody has performed the work on the preparation and characterization of (Bi2S3)x doped (Sb2S3)1-x (with x= 0.03, 0.05, 0.1) thin films from the freshly prepared powder samples and thermal vacuum evaporation technique as the method of deposition.
2. MATERIALS AND METHODS
Bulk thin film samples of (Sb2S3)1-x (Bi2S3)x (with x= 0.03, 0.05, 0.1) were prepared from powder materials of Sb2S3 and Bi2S3 (99.999% purity, Sigma-aldrich) on chemically cleaned glass substrates by thermal vacuum evaporation technique. The conventional thermal evaporation method is being widely used for the growth of binary and ternary compounds because of its simplicity and this method supports high quality and pure films for thin film applications. For a particular composition, the constituent compounds Sb2S3 and Bi2S3 were weighed and grinded for several hours and sintered. The newly prepared powder samples were used as the source material. During deposition the precleaned substrates were placed in a rotative sample-holder to get uniformly coated films. Deposition rate and film thickness were controlled during deposition by quartz oscillator thickness monitor. The substrate temperature was maintained at 300K during the deposition. The deposition chamber was evacuated to a residual pressure of about 10-5 torr. The elemental compositions of the prepared powder and thin films were determined using an Energy Dispersive X-ray Analysis (EDAX) (model: INCA Oxford) based on SEM images. X-ray Diffractograms of the prepared material and the investigated films of as-deposited and annealed (473K, 523K) samples were carried out by using an X-ray Diffractometer (XRD) (model: D8 Advance XRD) with CuKα (λ=1.5406Å) radiation. To analyse the surface compositions and purity, the x-ray photoelectron spectroscopy (XPS) data were recorded with the specimen mounted on a specially designed sample holder8 using an AlKalpha laboratory x-ray source that was operated at 150 watts and an electron energy analyser with five channeltrons from Specs GmbH, Germany. The data were recorded with 20 eV pass energy with 1 eV energy resolution. The chamber base pressure was 6×10-11 mbar. Surface morphological studies were done before and after heat treatment by means of Atomic Force Microscope (AFM) and surface roughness and grain size were estimated.
3. RESULTS
3.1. Compositional analysis
Fig. 1 shows the Energy Dispersive X-ray Analysis (EDAX) spectra for the powder samples and the corresponding thin films of (Sb2S3)1-x(Bi2S3)x, with x= 0.03, 0.05, 0.1. The calculated and observed percentages of the elements in different compositions from EDAX analysis are shown in table 1 and are in good agreement each other. (Figure 1, Table 1)
3.2. Structural analysis
The X-ray diffractograms of the as-deposited, annealed (473K, 523K) thin films and powder samples of (Sb2S3)1-x(Bi2S3)x with x= 0.03, 0.05, 0.1 are shown in figures (2-13). It is clear from figs. (2-7) the as-deposited films and the films annealed at 473K are amorphous in nature9-10 . As reported by F. Perales et al.11, it was proved that the effect of annealing at 473K cannot change the amorphous nature. Thin films have been annealed for 1hr at 523K to investigate the effect of higher annealing temperature, and the growth of polycrystalline from amorphous phase. This observation is in agreement with the results of M. S. Droichi et al.12 for Sb2S3 and Mahmoud et al.10 for Bi2S3 thin films.
(Fig. 2 Fig. 3)
(Fig. 4 Fig. 5)
(Fig. 6 Fig. 7)
From figs. (8-10), it is clear that the increase of thermal treatment improves crystallinity of SbBi-S films with orthorhombic structure. The Xray diffractograms of (Sb2S3)1-x(Bi2S3)x with x= 0.03, 0.05, 0.1 for powder samples are shown in figures (11-13).
(Fig.8 Fig. 9)
The orthorhombic crystal structure of these compounds, both in powder and thin film forms, with cell parameters have been found out from XRD powder data. The position of peaks appeared in the diffractograms of the powder sample lie very near to those listed in the PDF for Sb2S3 (PDF 42-1393) and Bi2S3 (PDF 17- 0320). The estimated values of the lattice parameters lie close to the reported values of Sb2S3 and Bi2S3 11, 13 .
(Fig. 10 Fig.11)
(Fig.12 Fig.13)
The crystallite size D estimated using the Sherrer‘s formula14:
where K is the shape factor (0.94), β is the full width at half maximum (FWHM) of the diffraction expressed in radians. The estimated lattice parameters for both powder and thin films and crystallite size for the annealed film samples of (Sb2S3)1-x(Bi2S3)x with x= 0.03, 0.05, 0.1 of different thickness are recorded in table 2. Using Bragg‘s relation14, the interplanar spacing dhkl was also calculated from powder diffraction data:
where λ is the X-ray wavelength, n is the order number and θ is the Bragg‘s angle. Table 3 shows the calculated dhkl values of Sb-Bi-S compound in comparison with the standard powders of Sb2S3 (JCPDS card No. 42-1393) and Bi2S3 (JCPDS card No. 17-0320) and in agreement with the recorded values by A.A. ElShazly et al.15 and Siham Mahmoud et al.5 . The surface composition and purity of the asdeposited (Sb2S3)0.95 (Bi2S3)0.05 thin film were studied by XPS analysis and the core level spectra are given in figs. (14-16). The photoelectron spectra of the Sb(3d3/2), Bi(4d5/2) and S(2p) show the peaks at the binding energy 537.61eV, 440.01eV and 162. 21eV respectively.
(Fig. 14)
(Table 2)
3.3 Morphological analysis
The surface topographical images for Sb-Bi-S thin films, deposited at room temperature and annealed at 523K for 1 hr, of film thickness 900nm and 1200nm recorded from atomic force microscope (AFM) are shown in fig.15. This measurements have been taken for films from the compound with x=0.05. The measured grain size and RMS roughness from AFM images for thickness 900nm and 1200nm are given in table 4. It is observed from the AFM images that the average grain size and roughness increase with increasing thickness and temperature16 . (Fig. 15)
(Table 3)
(Table 4)
3. DISCUSSIONS
As the crystallization process proceeds with thickness at temperature 523K, the intensity of the (310) peak increases for x=0.03 compound, (120) peak increases for x=0.05 compound and (130) peak increases for x=0.1 compound, indicating further orientation of the film crystallites in these directions. And also, in all the three compounds, more peaks appeared in films of highest thickness (1200nm). Some peaks appeared, which are common to both Sb2S3 and Bi2S3 like, (120) and (250) for x=0.03, (120) and (240) for x=0.05, (120), (130), (240) and (250) for x=0.1. The calculated values of the lattice parameters for the three compounds lie close to the reported values of Sb2S3 and Bi2S3. The calculated interplanar spacing dhkl values of Sb-Bi-S compound lie close to the standard powders of Sb2S3 (JCPDS card No. 42-1393) and Bi2S3 (JCPDS card No. 17-0320). As K.Y. Rajpure et al.17 point out, annealing promotes fusion of small crystallites thus reducing the grain-boundary area, which leads to an increase in diffusion length due to decrease in scattering from boundaries. This leads to an increase in crystallite size of the particles. The crystallite size increases with: a) increasing film thickness and b) increasing annealing temperature16. The linear increase of surface area with the film thickness suggests that a porous film structure with its relatively large internal surface area accessible to the absorbing gas even in the lowest layaers of the film. 4.
CONCLUSIONS
The thermal evaporation technique has been used to prepare (Sb2S3)1-x(Bi2S3)x with x=0.03, 0.05, 0.1 thin films on glass substrate for two thickness. The measurements obtained from EDAX showed the elemental percentages of respective compounds. The orthorhombic crystal structure of all compounds with x=0.03, 0.05 and 0.1 and lattice parameters and the dependence of crystallite size on thickness were showed by X-ray Diffraction (XRD) measurements. The as-deposited amorphous thin films transformed to polycrystalline films during the thermal treatment at 523K. The XPS core level spectra of Sb(3d3/2), Bi(4d5/2) and S(2p) showed the peaks of corresponding binding energies. Dependence of grain size and RMS roughness on thickness and annealing temperature was proved by atomic force microscopic (AFM) photographs.
ACKNOWLEDGEMENT
The measurements have been taken in the UGCDAE Consortium for Scientific Research, Indore. The authors are much grateful to the supporting authorities, especially to Prof. Ajay Gupta- the centre Director of Indore. The corresponding author is also much grateful to the financial support given by DST- CURIE Project.
Fig.15 AFM images of the (Sb2S3)0.95(Bi2S3)0.5 thin films for thickness 900nm [as-deposited (a), annealed (b)] and for 1200nm [as-deposited (c), annealed (d)]
Englishhttp://ijcrr.com/abstract.php?article_id=2144http://ijcrr.com/article_html.php?did=21441. Lokhande C D, Sankapal B R, Sartale S D, Pathan H M, Giersig M, Ganesan V. A novel method for the deposition of nanocrystalline Bi2Se3, Sb2Se3 and Bi2Se3- Sb2Se3 thin films - SILAR. Appl Surf Sci 2001; 182:413-7.
2. Yesugade N S, Lokhande C D, Bhosale C H. Structural and optical properties of electrodeposited Bi2S3, Sb2S3 and As2S3 thin films. Thin Solid films 1995; 263:145-9.
3. Gang Xie, Zheng-Ping Qiao, Ming-Hua Zeng, Xiao-Ming Chen, Sheng-Li Gao. A single-source approach to Bi2S3 and Sb2S3 nanorods via a Hydrothermal treatment. Crystal Growth and Design 2004; 4:513-6.
4. Pawan Kumar, Nidhi Jain, Agarwal R K. Effect of substrate temperature on optical properties of Bi2S3 chalcogenide thin films. Chalcogenide Letters 2010; 7:89-94.
5. Siham Mahmoud, Fouad Sharaf. Optical and electrical properties of Bismuth sulphide (Bi2S3) thin films prepared by thermal evaporation. Fizika A5 1996; 4:205-13.
6. El Zawawi I K, Abdel-Moez A, Terra F S, Mounir M. Substrate temperature effect on the optical and electrical properties of antimony trisulfide thin films. Thin Solid Films 1998; 324: 300-4.
7. Grozdanov I, Ristov M, Sinadinovski Gj, Mitreski M. Fabrication of amorphous Sb2S3 films by chemical deposition. J NonCryst Solids 1994; 175: 77-83.
8. Dhaka R S, Shukla A K, Maniraj M, D‘Souza S W, Nayak J, Barman S R. An ultrahigh vacuum compatible sample holder for studying complex metal surfaces. Rev Sci Instrum 2010; 81: 043907.
9. Tigau N, Rusu G I, Gheorghies C J. On the structural and optical properties of antimony trisulfide thin films. Optoelect Adv Mater 2002; 4: 943-8.
10. Mahmoud S, Eid A H, Omar H. Optical characteristics of bismuth sulphide (Bi2S3) thin films. Fizika A6 1997; 3:111-20.
11. Perales F, Lifante G, Agullo-Rueda F, de las Heras C. Optical and structural properties in the amorphous to polycrystalline transition in Sb2S3 thin films. J Phys D Appl Phys 2007; 40: 2440-4.
12. Droichi M S, Vaillant F, Bustarret E, Jousse D. Study of localized states in amorphous chalcogenide Sb2S3 films. J Non-Cryst Solids 1988; 101: 151-5.
13. Sheng-Yue Wang, You-Wei Du. Preparation of nanocrystalline bismuth sulphide thin films by asynchronous-pulse ultrasonic spray pyrolysis technique. J Cryst Growth 2002; 236:627–34.
14. Nicolae Tigau. Structural characterization and optical properties of annealed Sb2S3 thin films. Rom Journ Phys 2008; 53: 209-15.
15. El-Shazly A A, Sayem M A M, ElSamanoudy M M, Ammar A H, Assim E M. The effect of deposition rate and heat treatment on conduction and charge carrier transport mechanisms in Sb2S3 films. Appl Surf Sci 2002; 189: 129-37.
16. Kasturi L Chopra. Thin Film Phenomena. 2 nd ed. New York: McGraw-Hill; 1969.p.188, 183.
17. Rajpure K Y, Bhosale C H. Effect of composition on the structural, optical and electrical properties of sprayed Sb2S3 thin films prepared from non-aqueous medium. J Phys and Chem Solids 2000; 61:561-8.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524137EnglishN-0001November30HealthcareEFFECT OF SOLANUM TRILOBATUM EXTRACT ON ERYTHROCYTE MEMBRANE SURFACE CHARGE
IN AGED RATS
English3751Amarnath Kanchana English Chinnakannu PanneerselavamEnglishObjectives: Oxidative stress an unavoidable consequence of oxygen metabolism in aerobic cells is postulated to be one of the most important causes of age related changes. The changes induced by free radicals are believed to be the major source of ageing, disease development, destruction of normal cell function and membrane rigidity. The present study was intended to determine the effect of chloroform extract of Solanum trilobatum (CST) on membrane surface charge density in aged rat erythrocytes upon oxidative stress.
Methods: The leaves of Solanum trilobatum were subjected to chloroform and the extract was prepared.
Chloroform extract of Solanum trilobatum (CST) (150 mg/day/kg body weight) was administered orally
for 90 days to young and aged rats. The erythrocyte membrane was isolated and was used to perform the
studies such as estimation of protein carbonyls, glycoproteins, surface charge and enzymatic and non
enzymatic levels. Key findings: A noteworthy decline in surface charge levels with simultaneous increase in protein carbonyls and shrink in glycoprotein and antioxidants status was prominent in erythrocytes of aged rats contrast to young rats. Administration of CST improved the erythrocyte surface charge density to near normalcy in aged rats. In addition a decrease in protein carbonyls level and increase in glycoproteins as well as antioxidant status was observed in aged rat erythrocytes on CST therapy.
Conclusions: Therefore the polyphenols and flavanoids are the phytochemical compounds in CST that might play a central role in defending the oxidative stress related loss of membrane surface charge in a way maintaining the erythrocyte membrane integrity and functions in aged individuals.
EnglishAging; Erythrocyte surface charge; Protein carbonyls; Glycoprotein; Antioxidants; Solanum trilobatum; Phytochemicals.INTRODUCTION
Oxidative damage apparently increases with age and thus may overwhelm the natural repair system in the organism1 leading to the onset of age-related diseases with a progressive increase in the chance of morbidity and mortality. At the cellular level, the reactive oxygen species inclination due to oxidative stress damages vital cell components like poly unsaturated fatty acids, protein and nucleic acids2 . Free radical attack on cellular proteins, damages the proteins3 leading to oxidation of side chains of lysine, arginine, proline and threonine thereby yield protein carbonyl derivatives the markers of protein oxidation4 . These oxidative modifications cause loss of structural and catalytic functions of the cell contributing to serious deleterious changes affecting cell survival5 . The cell membrane is a structural barrier that plays an essential role in protecting cellular integrity and underlying cause of ageing process6 . Changes in the macromolecules of membrane are one of the earliest signs of erythrocytes membrane alterations during ageing. Reactive oxygen species generated in the aqueous or lipid phase can attach to the erythrocyte membrane and can induce oxidation of lipids, proteins and carbohydrates, triggering disruption in membrane7 and further enhances the development of one or better-recognized age related modifications, such as alteration of intrinsic membrane properties (fluidity, ion transport, loss of enzyme activity, protein crosslinking), inhibition of protein synthesis and DNA damage4 , ultimately resulting in the cell death. Carbohydrates in erythrocytes have many important functions that are necessary for the proper functioning of the cell to survival. Carbohydrate is thought to be highly necessary for cell surface negative charges and have a crucial role in the clearance of senescent cells and essentially to prevent aggregation of erythrocytes from each other8 . Like most biological surfaces, erythrocytes exhibit a negative surface charge that is mainly attributed to sialic acid residues located on the glycoproteins in the membrane surface9 . Oxidative stress or other damaging effects to surface sialosaccharide may itself play role in aggregation of erythrocytes, increasing the adhesiveness to endothelial cells contributing for the development of various pathologies including diabetes mellitus, atherothrombotic complications10and sequestration of circulating erythrocytes by macrophages11 . Age associated oxidative changes in the glycomoieties of erythrocyte glycoprotein affects the surface charge including aggregation and binding of erythrocytes to macrophages12 and found to be associated with cardiovascular risk factors such as, hypertension, hyperlipoproteinemia and myocardial ischemia13and thus crucial for erythrocyte survival14 . During lifetime, an antioxidant network counteracts the deleterious action of free radicals and reactive species on macromolecules. Cells synthesize some of their antioxidant enzymes, as the superoxide dismutase, catalase, and glutathione peroxidase, as well as the peptides with thiol groups, as glutathione, while other non-enzymatic antioxidants from nature through nutrition, as vitamin C, vitamin E, and carotenoids. Several repair systems help antioxidant action by the recovery of damaged macromolecules15. Together, these systems play an important role in the ability of the body to respond to the oxidant challenge of using molecular oxygen to drive reactions that yield the necessary energy for life processes. However various anti ageing agents are being ushered into the field of medicine to conflict ageing and age-associated disorders. Flavonoids, the potential dietary antioxidant found ubiquitously in plants, attracted global interest in combating the devasting effects of oxidation in cells and tissues of an organism. Dietary antioxidants are considered beneficial because of their potential protective role against oxidative stress, which is involved in the pathogenesis of multiple diseases such as cancer and coronary heart disease16,17. The potential antioxidant effect in vivo of individual food polyphenols (PP) or concentrated extracts has been widely investigated in cultured cells18,19 live animals20 and men21,22,23. Herbal remedies as antioxidant supplement is thus globally growing up owing to broad spectrum of beneficial biological properties. Owing to the presence of phenolic compounds and the free radical scavenging properties24 of an anti cancer herabal extract Solanum trilobatum25 , the present study is carried out to study the effect of Chloroform extract of Solanum trilobatum (CST) on erythrocyte membrane surface charges during animal aging. The study also explores the effect of CST on erythrocyte membrane protein carbonyl levels and the antioxidant status in aged rats.
2. MATERIALS AND METHODS
2.1. Chemicals
Dextran 500 (500,000 molecular weight), Polyethylene glycol (PEG, 8000 molecular weight), Bovine serum albumin (BSA), and 2,4- dinitro-phenylhydrazine (DNPH) were purchased from Sigma Chemical and Company, Saint Louis, MO, USA. All other chemicals were of reagent grade .
2.2. Preparation of Chloroform extract of Solanum trilobatum (CST)
Extraction of plant materials
The leaves of Solanum trilobatum were collected from the local market and samples of the plant were identified and authenticated by Dr. Brindha, Botanist, Dept. of Pharmacognosy, Captain Srinivasa Murti Drug Research Institute for Ayurveda (CCRAS, New Delhi), Arumbakkam, Chennai, Tamil Nadu. The fresh leaves were shad dried, powdered and extracted successively with 1.2 L of chloroform, in a Soxhlet extractor for 18-20 h. The extracts were concentrated to dryness under reduced pressure and controlled temperature (40o -50oC). The chloroform, extract yielded a brown semisolid residue weighing 6.2 g (2.39% w/w). The extract was then filtered through Whatmann No.1 filter paper and concentrated using concentrator. Then it was frozen and subjected to lyophilization .
2.3. Animals, treatment and fixation of dosage and duration
Male albino rats of Wistar strain, weighing approximately 120–160 g (young) and 380–420 g (old) were used. The animals were obtained from King Institute of Preventive Medicine, Chennai. The animals were divided into two major groups namely, Group Ia: normal young rats (3–4 months old) and Group IIa: normal aged rats (about 24 months old) and two experimental group Id (Young rats) and group IId (Aged rats) on CST administration for 90 days. Each group consisted of six animals. The animals were maintained on commercial rat feed containing 5% fat, 21% protein, 55% nitrogen free extract, 4% fiber (w/w) with adequate mineral and vitamin contents and had access to water ad libitum. CST (Chloroform extract of Solanum trilobatum) was supplemented orally (oral gavage) at the dosage of 150 mg/kg body weight/day for 90 days, whereas young control and aged control rats received vehicle alone in a similar manner. On completion of 90 days of CST supplementation, the blood was collected with 3.8% sodium citrate from jugular vein and used for the isolation of erythrocytes and erythrocyte membranes. Fixation of dose and duration: Trial studies were carried out with different concentrations of Chloroform extract of Solanum trilobatum (50, 100, 150, 200 and 250 mg) dissolved in physiological saline and supplemented at various durations (30, 60, 90 and 120 days) orally to rats. The effectual dosage (150 mg/kg body weight/day) was selected on the basis of the concentration at which Chloroform extract of Solanum trilobatum was capable of inhibiting lipid peroxidation significantly above which there was (200 and 250 mg/kg body weight) no significant inhibition of lipid peroxidation. Similarly the effectual duration was found to be 90 days above which there was insignificant inhibition of lipid peroxidation. As per the results obtained, the forthcoming biochemical and molecular parameters were carried out with Chloroform extract of Solanum trilobatum supplementation for 90 days alone. The body weight of the animals were increased at the end of experiment as in group Ia (from 74.50 ± 5.6 to 80.40 ± 6.7), group Id (from 74.50 ± 5.6 to78.30 ± 3.3), group IIa (from 120.4 ± 4.8 to 155.9 ± 5.0) and group IId (from 126.2 ± 5.0 to 162.0 ± 5.3) in respect to their initial body weight.
2.4. Preparation of erythrocytes and erythrocyte membranes
Isolation of erythrocytes and erythrocyte membranes was done according to Dodge et al. (1963)26 with slight modifications. Briefly, blood collected from animals with 3.8% sodium citrate was subjected to centrifugation at 3000 rpm for 10 min at 4 0C. The plasma and buffy coat were removed by aspiration and the erythrocytes were washed three times with cold (4 0C) phosphate-buffered saline (PBS: 0.15 M NaCl, 1.9 mM NaH2PO4, 8.1 Mm Na2HPO4, pH 7.4). Washed erythrocytes were hemolyzed in 40 volumes of 5 mM sodium phosphate buffer (pH 8.0) (containing 1 mM EDTA and 0.5 mM phenylmethylsulfonyl fluoride, as protein inhibitor), and centrifuged for 20 min at 4 0C at 15,000 rpm. The supernatant (or hemolysate) was decanted carefully and saved, while the pellet were washed repeatedly and incubated for 45 min at 370C to reseal the membrane in the presence of 0.8 Mm MgCl2-ATP solutions, for hemoglobin free ghosts. Hemoglobin amount was estimated by Drabkin and Austin (1932)27 method and erythrocyte membrane protein content by Lowry et al. (1951)28 .
2.5. Determination of erythrocyte surface charge
Erythrocyte partition coefficients were determined at room temperature by a two-phase aqueous system containing 5% Dextran 500 and 4.3% PEG 8000 in isotonic phosphate buffer (pH 6.8) by the method of Walter (1985)29 . The two-phase system used is ?charge sensitive‘ whose partition coefficient ratio indicates the level of erythrocyte surface charge.
2.6. Determination of protein carbonyls
Protein carbonyls content was determined by the reliable method based on the reaction of carbonyl groups with 2, 4- dinitrophenylhydrazine to form 2, 4-dinitrophenylhydrazone as suggested by Levine et al. (1994)30 .
2.7. Determination of glycoproteins
Lipids were extracted from the erythrocyte membrane pellets according to the method of Folch et al. (1957)31using chloroform–methanol mixture (2:1 v/v). The resulting defatted residue was suspended in sodium acetate buffer (containing 2 mg cysteine HCl/ml, final pH 7.0) and deproteinized by 4–5 volumes of ethanol, evaporated to dryness in the cold under a vacuum and subjected to hydrolysis by heating with 2 ml of 4 N HCl for 4–6 h. The hydrolyzed material was neutralized with 4 N sodium hydroxide and used for estimating erythrocyte hexose (Niebes, 1972)32, hexosamine (Wagner, 1979)33 and sialic acid (Warren, 1959)34 .
2.8. Determination of antioxidants
Superoxide dismutase (SOD) in hemolysate was assayed by the method of Marklund and Marklund (1974)35, while erythrocyte membranes were used to estimate catalase (CAT) (Beers and Seizer, 1952)36. Hemolysate glutathione peroxidase (GPx) was determined by Rotruck et al. (1973)37 method, glutathione reductase (GR) by the procedure of Staal et al. (1969)38, glucose-6-phosphate dehydrogenase (G6PD) by Zinkham et al. (1958)39 and glutathione-S-transferase (GST) according to Habig et al. (1974)40. Hemolysate reduced glutathione (GSH) was estimated by Moron et al. (1979)41 and oxidized glutathione (GSSG) was assayed according to the method of Tietze (1969)42. Erythrocyte redox state was determined by the redox index: (GSH + 2 X GSSG) / (2 X GSSG / 100).
2.9. Statistical analysis
The results are expressed as mean standard deviation (SD) for six rats in each group. Differences between groups were assessed by one-way ANOVA using the SPSS version 7.5 software packages for Windows, USA. Post hoc testing was performed for inter-group comparisons using the least significance difference (LSD) test; statistical significance at p-valuesEnglishhttp://ijcrr.com/abstract.php?article_id=2145http://ijcrr.com/article_html.php?did=21451. Kowald A. Theoretical Gompertzian implications on life span variability among genotypically identical animals. Mech Ageing Dev. 1999; 110:101-7.
2. Halliwell B, The antioxidant paradox. Lancet 2000; 355:1179 – 1180.
3. Sinclair AJ, Barnett AH, Lunec J. Free radicals and antioxidant systems in health and disease. Br J Hosp Med. 1990; 43: 334- 44.
4. Bandyopadhyay U, Das D, Banerjee RK. Reactive oxygen species, oxidative stress and pathogenesis. Curr. Sci. 1999; 77:658– 666.
5. Levine RL, Williams JA, Stadtman ER, Shacter E. Carbonyl assays for determination of oxidatively modified proteins. Methods Enzymol. 1994; 233: 346-57.
6. Spitelle,r G. Are changes of the cell membrane structure causally involved in the aging process? Ann N Y Acad Sci. 2002; 959:30-44.
7. Grune T, Shringarpure R, Sitte N, Davies K. Age-related changes in protein oxidation and proteolysis in mammalian cells. J Gerontol A Biol Sci Med Sci. 2001; 11:459-67.
8. Jovtchev S, Djenev I, Stoeff S, Stoylov S. Role of electrical and mechanical properties of red blood cells for their aggregation. Colloids Surf. Physiochem. 2000; 164:95–104.
9. Aminoff D. Methods for the quantitative estimation of N-acetylneuraminic acid and their application to hydrolysates of sialomucoids.. Biochem J. 1961; 81:384- 92.
10. Wautier JL, Paton RC, Wautier MP, Pintigny D, Abadie E, Passa P, Caen J. Increased adhesion of erythrocytes to endothelial cells in diabetes mellitus and its relation to vascular complications. N Engl J Med. 1981; 305: 237-42.
11. Beppu M, Hayashi T, Hasegawa T, Kikugawa K. Recognition of sialosaccharide chain of glycophorin on damaged erythrocytes by macrophage scavenger receptors. Biochim. Biophys. Acta. 1995; 1268: 9-19.
12. Ando K, Kikugawa K, Beppu M. Binding of anti-band 3 autoantibody to sialylated poly-N-acetyllactosaminyl sugar chains of band 3 glycoprotein on polyvinylidene difluoride membrane and sepharose gel: further evidence for anti-band 3 autoantibody binding to the sugar chains of oxidized and senescent erythrocytes. J Biochem. 1996; 119:639-47.
13. Chien S, Weinburg R, Li S, Li Y. Endobeta-N-acetylglucosaminidase from fig latex. Biochem Biophys Res Commun. 1976; 76:317-23.
14. Sangeetha P, Balu M, Haripriya D, Panneerselvam C. Age associated changes in erythrocyte membrane surface charge: Modulatory role of grape seed proanthocyanidins. Exp Gerontol. 2005; 40: 820-8
. 15. Cutler RG, Rodriguez H. (Eds.). Critical Reviews of Oxidative Stress and Aging: Advances in Basic Science, Diagnostics and Intervention. World Scientific Publishing, Singapore. 2003; 1523.
16. Duthie GG, Duthie SJ, Kyle JAM. Plant polyphenols in cancer and heart disease: implications as nutritional antioxidants. Nutr Res Rev. 2000; 13:79–106.
17. Trichopoulou A, Vasilopoulou E. Mediterranean diet and longevity. Br J Nutr. 2000; 84:205–9.
18. Youdim KA, Martin A, Joseph JA. Incorporation of the elderberry anthocyanins by endothelial cells increases protection against oxidative stress. Free Rad Biol Med. 2000; 29:51–60.
19. Schroeter H, Spencer JPE. Rice-Evans C, Williams RJ. Flavonoids protect neurons from oxidized lowdensity-lipoproteininduced apoptosis involving c-Jun Nterminal kinase (JNK), c-Jun and caspase- 3. Biochem J. 2001; 358:547–57.
20. Hininger JF, Dragst LO, Daneshvar B, Lauridsen ST, Hansen, Sandstrom MB. The effect of grape-skin extract on oxidative status. Br J Nutr. 2000; 84:505–13.
21. Abu-Amsha Caccetta R, Burke V, Mori TA, Beilin LJ, Puddey IB, Croft KD. Red wine polyphenols, in the absence of alcohol, reduce lipid peroxidative stress in smoking subjects. Free Rad Biol Med. 2001; 30:636–42.
22. Natella F, Ghiselli A, Guidi A, Ursini F, Scaccini C. Red wine mitigates the postprandial increase of LDL susceptibility to oxidation. Free Rad Biol Med. 2001; 30:1036–44.
23. Breinholt V, Lauridsen ST, Dragsted LO. Differential effects of dietary flavonoids on drug metabolizing and antioxidant enzymes in female rat. Xenobiotica. 1999; 29:1227– 40.
24. Monavalli B, Raja Rajeswari A, Gowri V, Kanchana A. Invitro Antioxidant Activity of Methanolic Extract of Solanum trilobatum Leaves. J. of Natural Science and Technology - Life Sciences and Bioinformatics. 2010; 2:168-174.
25. Mohanan PV, Devi KS. Effect of Sobatum on radiation-induced toxicity in mice. Cancer Lett. 1998; 123:141-5.
26. Dodge JT, Mitchell C, Hanahan DJ. The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocyte. Arch. Biochem. Biophys. 1963; 100:119–130.
27. Drabkin DL, Austin JH. Spectrophotometric studies, spectrophotometric constants for common hemoglobin derivatives in human, dog and rabbit blood. J. Biol. Chem. 1932; 98:719– 733.
28. Walter H. Surface properties of cells reflected by partitioning red blood cells as a model. In: Walter H, Brooks, D.E., Fisher, D. (Eds.), Partitioning in Aqueous TwoPhase Systems. Academic, Orlando, FL, 1985; 327–376.
29. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951; 193:265– 275.
30. Levine RL, Williams JA, Stadtman ER, and Shacter E. Carbonyl assay for determination of oxidatively modified proteins. Methods Enzymol. 1994; 233:346—357.
31. Folch J, Less M, Sloane Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 1957; 226:497–509.
32. Niebes P. Determination of enzymes and degradation products of glycosaminoglycans metabolism in the serum of healthy and varicose subjects. Clin. Chim. Acta. 1972; 42:399–408.
33. Wagner WD. A more sensitive assay discriminating galactosamine and glucosamine in mixtures. Anal. Biochem. 1979; 94:394–396.
34. Warren L. The thiobarbituric acid assay of sialic acid. J. Biol. Chem. 1959; 234:1971– 1975.
35. Marklund S, Marklund G. Involvement of the superoxide anion radical in the autooxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. J. Biochem. 1974; 47:469–474.
36. Beers RF, Seizer IW. A spectrophotometric method for measuring breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 1952; 195:133–140.
37. Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG,. Selenium: biochemical role as a component of glutathione peroxidase purification and assay. Science 1973; 179:588–590.
38. Staal GE, Visser J, Veeger C. Purification and properties of glutathione reductase of human erythrocytes. Biochim. Biophys. Acta 1969; 185:39–48.
39. Moron MS, Depierre JW, Mannervik B. Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochim. Biophys. Acta. 1979; 582:67–78.
40. Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferases, the first enzymatic step in mercapturic acid formation. J. Biol. Chem. 1974; 249:7130– 7139.
41. Zinkham WH, Lenhard RE, Childs B. A deficiency of glucose-6-phosphate dehydrogenase activity in erythrocytes from patients with favism. Bull. Johns Hopkins Hosp. 1958; 102:169–175.
42. Tietze F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal. Biochem. 1969; 27:502–522.
43. Samoilov MV, Mishnev OD, Kudriavtser IV, Naumor AG, Daniltov AP. Morphofunctional characteristics of erythrocytes in chronic kidney failure and purulent intoxication. Klin. Lab. Diagn. 2002; 6:18–23.
44. Stadtman ER. Protein oxidation in aging and age-related diseases. Ann N Y Acad Sci. 2001; 928, 22-38
45. Beal MF. Oxidatively modified proteins in aging and disease. Free Radic Biol Med. 2002; 32: 797-803.
46. Hyun DH, Lee M, Halliwell B, Jenner P. Proteasomal inhibition causes the formation of protein aggregates containinga wide range of proteins, including nitrated proteins. J Neurochem. 2003; 86:363-73
47. Arivazhagan P, Panneerselvam C. Favorable effect of L-Carnitine supplementation on glycoprotein status in aged rats. J. Clin. Biochem. Nutr. 1999; 26:193–200.
48. Carney JM, Starke-Reed PE, Oliver CN, Landum RW, Chen MS, Wu JF, Froemming GR, O‘Brien NM. U937 cells as a model to study the effect of phytochemicals on superoxide anion production. Nutr Res. 1997; 17:1091–1103.
49. Leb L, Snyder LM, Fortier NL, Anderson M. Antiglobulin serum mediated phagocytosis of normal senescent and oxidized RBC: role of anti-IgM immunoglobulins in phagocytic recognition. Br. J. Haematol. 1987; 66:565–570.
50. Vomel T. Properties of ATPases and energy-rich phosphate in erythrocyte of young and old individuals. Gerontology. 1984; 30:22–25.
51. Sato M, Maulik N, Das DK. Cardioprotection with alcohol: role of both alcohol and polyphenolic antioxidants. Ann N Y Acad Sci. 2002; 957:122-35.
52. Kalyan Reddy, Manda Craig Adams, Nuren Ercal. Biologically important thiols in aqueous extracts of spices and evaluation of their in vitro antioxidant properties. Food Chemistry. 2010; 118:589-593.
53. VanDuijn MM, Van den Zee J, Vansteveninck J, Van den Broek PJ. Ascorbate stimulates ferricyanide reduction in HL-60 cells through a mechanism distinct from the NADH-dependent plasma membrane reductase. J. Biol. Chem. 1998; 273:13415–13420.
54. Halliwell B. Use of desferrioxamine as a 'probe' for iron-dependent formation of hydroxyl radicals. Evidence for a direct reaction between desferal and the superoxide radical. Biochem Pharmacol. 1985; 342:229–233.
55. Davis TA, Anderson EC, Ginsburg AV, Goldberg AP. Weight loss mproves lipoprotein lipid profiles in patients with hypercholesterolemia. J Lab Clin Med. 1985; 106:447-54
56. Inal MG, Kanbak G, Sunal. Antioxidant enzume activities and malondialdehyde levels related to ageing. Clin. Chim. Actam. 2001; 305:75-80
57. Sini N, Devi KS. Pharmaceutical biology. Antioxidant activities of the chloroform extract of Solanum trilobatum. 2004; 959:30-44.
58. Pastore A, Federici G, Bertini E, Piemonte F. Analysis of glutathione: implication in redox and detoxification. Clin Chim Acta. 2003; 333:19-39.
59. John S, Kale M, Rathore N, Bhatnagar D. Protective effect of vitamin E in dimethoate and malathion induced oxidative stress in rat erythrocytes. J Nutr Biochem. 2001; 12:500-504.
60. Sastre J, Asensi M, Gasco E, Pallardo FV, Ferrero JA, Furukawa T, Vina J. Exhaustive physical exercise causes oxidation of glutathione status in blood: prevention by antioxidant administration. Am J Physiol. 1992; 263:992-5.
61. Sah NK, Taneja TK, Pathak N, Begum R, Athar M and Hasnain SE. The baculovirus antiapoptotic p35 gene also functions via an oxidant-dependent pathway. Proc. Natl. Acad. Sci. USA. 1999; 96: 4838–4843.
62. Choe M, Jackson C, Yu BP, 1995. Lipid peroxidation contributes to age-related membrane rigidity. Free Radic Biol Med. 18:977-84.
63. Ishige K, Schubert D, Sagara Y. Flavonoids protect neuronal cells from oxidative stress
by three distinct mechanisms. Free Radic Biol Med. 2001; 30:433-46.
64. Myhrstad MC, Carlsen H, Nordstrom O, Blomhoff R, Moskaug JO. Flavonoids increase the intracellular glutathione level by transactivation of the gammaglutamylcysteine synthetase catalytical subunit promoter. Free Radic Biol Med. 2002; 32:386-93
65. Nakagawa T, Yokozawa T, Satoh A, Kim HY. Attenuation of renal ischemiareperfusion injury by proanthocyanidin-rich extract from grape seeds. J Nutr Sci Vitaminol (Tokyo). 2005; 51:283-6.
66. Saija A, Scalese M, Lanza M, Marzullo D, Bonina F, Castelli F. Flavonoids as antioxidant agents: importance of their interaction with biomembranes. Free Radic Biol Med. 1995; 19:481-6.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524137EnglishN-0001November30HealthcareLYSOSOMAL ENZYMES
English5256Jayamathi GEnglish Vishnu Priya VEnglishLysosomal enzymes are implicated in tissue remodeling and in regulating the immune responses.Lysosomal enzymes can be incorporated into the explanation of mechanisms of development of various diseases and give scientific grounds for prevention of inflammatory disease. This review highlights synthesis, functions and regulation of lysosomal enzymes.
EnglishPhagocytosis, endocytosis, acid hydrolases, lysosomes, alpha1 –antitrypsinINTRODUCTION
Lysosomes are small intracellular organelle present in all animal cells. They destroy any foreign material which enters the cell such as bacteria or virus. Lysosomes also remove the worn out and poorly working cellular organelles by digesting them to make way for their new replacements. Since they remove cell debris, they are also known as scavengers, cellular housekeepers or demolition squads. Lysosomes form a kind of garbage disposal system of cell. During breakdown of cell structure, when the cell gets damaged, lysosomes burst and the enzymes eat up their own cells. So, lysosomes are also known as suicide bags of a cell. The major function of lysosomes and lysosomal proteases is not to kill the cell but to take care of cellular homeostasis and possibly differentiation by recycling cellular components. 1 The release of lysosomal enzymes are normally intended to degrade ingested microbes, could also lead to tissue destruction and amplification of inflammatory response with continued recruitment of new leukocytes. Altered lysosomal membrane stability leads to release lysosomal hydrolases, ensuing altered metabolism of different connective tissue constituents including collagen and also involved in the destruction of non - collagenous components of the extracellular matrix. Hence the present study is designed to give precise account on lysosomal enzymes, may open up new horizons in the research field. 2
Synthesis of lysosomal enzymes
More than 50 hydrolases involved in the lysosomal degradation of protein, carbohydrate, lipids and nucleic acids have been identified. The hydrolases are enclosed by a membrane containing a set of highly glycosylated lysosomal membrane proteins. The targeting of acid hydrolases depends on the presence of mannose-6-phosphate (M6P) residues that are recognized by specific receptors mediating the intracellular transport to an endosomal or prelysosomal compartment. The lysosomal apparatus is responsible for the intracellular digestion of externally and internally generated macromolecules. Coated vesicles internalize most extracellular macromolecules by endocytosis to form early endosomes, which move from the plasma membrane towards the cell nucleus. They become acidic and give rise to 'late' endosomes. This increasing acidity leads to the dissociation of lysosomal enzymes. Late endosomes also fuse with primary lysosomes (which contain lysosomal hydrolases and bud from the Golgi) to form secondary lysosomes. Secondary lysosomes might remain in the cell and become residual bodies, or be transported to the cell surface, where they fuse with the plasma membrane and exocytose their digested materials. 3 Lysosomal enzymes are synthesized with an Nterminal sequence of 20-25 aminoacids recognized by signal recognition particle which enable the nascent polypeptide to be transferred across the membrane of endoplasmic reticulum. Signal peptidase removes signal peptide. Preformed oligosaccharides undergo Nglycosylation with asparagine residue. Furthermore sulfatase family members are formed from sulfated mono and polysaccharides, glycolipid and hydroxyl steroids, and are modified in endoplasmic reticulum. Lysosomal enzymes are synthesized and are glycosylated in the rough endoplasmic reticulum. They are then transferred to the Golgi bodies, where they acquire mannose-6- phosphate (M6P) residues on their highmannose and hybrid-type oligosaccharide chains. This recognition marker is specific to lysosomal hydrolases and allows these hydrolases to be sorted from other proteins. Upon arrival of golgi the oligo saccharide chain of lysosomal enzymes are further trimmed and modified by the addition of complex sugar residues , sulfate groups and by the formation of M6P recognition marker. 4 Enzymology of lysosomes: Some important enzymes found within lysosomes include:
Lipase, which digests lipids
Amylase, which digests amylose, starch, and maltodextrins
Proteases, which digest proteins
Nucleases, which digest nucleic acids
Phosphoric acid monoesters
The proteolytic capacity of lysosomes comprises a mixture of endo- and exo-peptidases, called cathepsins, which act in concert to degrade proteins to a mixture of amino acids and dipeptides. 5and 6 Some cathepsins, for example, G and E, also function outside the lysosome. All of the proteases are active at an acidic pH, although this may not be their pH-optimum. They are synthesized in the form of inactive precursors, preproenzymes, which are transported to the lysosome by the mannose-6-phosphate pathway like other lysosomal hydrolases. Proteases are classified by the catalytic residue in the active site involved in the mechanism of peptide bond cleavage. Cathepsins with a serine (cathepsins A and G), cysteine (B, C, F, H, K, L, O, S, and W) or an aspartic acid (D and E) residue in the active site have been characterized. 6
Endopeptidases
Cysteine proteases: cathepsins B, C, H, K, L, O, S, and W
Aspartyl proteases: cathepsins D and E
Serine proteases: cathepsin G in azurophil granules of neutrophils
Exopeptidases
Carboxypeptidases: lysosomal carboxypeptidase (cathepsin A or protective protein)—serine protease; cathepsin B (dipeptidase); cathepsin X, mono- or dipeptidase; lysosomal carboxypeptidase B; prolylcaboxypeptidase; peptidyl dipeptidase B
Aminopeptidases: cathepsin H—true aminopeptidase; dipeptidyl peptidase I (cathepsin C); dipeptidyl peptidase II; tripeptidyl peptidase (TPP-I)
Lysosomal enzymes in various cells:
Lysosomes are subcellular organelles which perform many important cellular functions. For example, lysosomes digest foreign material and engulfed viruses and bacteria presenting in phagosomes during the process of phagocytosis. The influx of neutrophils and mononuclear phagocytes into tissues may be seen as the hallmark of inflammation and significantly contributes to both the injury and the subsequent repair seen in the normal tissues. Lysosomes are found in all eukaryotic cells, but are most numerous in disease-fighting cells, such as leukocytes 7 found that peritoneal macrophages in culture release lysosomal enzymes in response to phagocytic, but the mechanisms that regulate macrophage lysosomal enzyme secretion are not fully understood. Polymorphonuclear leukocytes also release lysosomal enzymes during phagocytosis 7 and the mechanisms that control this secretary process are well documented. Macrophage lysosomal enzyme release has many similarities to secretion from polymorphonuclear leukocytes and it is tempting to suggest that the processes might be regulated in the same way. Thus, macrophage lysosomal enzyme release is not controlled by the same regulatory mechanisms as the degranulation processes in polymorphonuclear leukocytes, platelets, and mast cells. Lysosomal enzyme release from macrophages is a much slower process than secretion from the other cell types and this may be functionally very important. Prolonged enzyme release from macrophages is consistent with the major role of this cell in chronic inflammation. In contrast, mast cells, granulocytes and platelets, which contribute primarily to acute inflammation, exhibit rapid degranulation processes. Platelets secrete lysosomal enzymes during the "platelet release reaction" early in clot formation. During the "platelet release reaction" induced by thrombin or collagen, mammalian blood platelets secrete lysosomal enzymes into the surrounding medium. 8and 9 Finally, inflammatory substances may leak from cells simply as a result of cell death due to plasma membrane injury . A number of animal, bacterial, and chemical toxins, as well as synthetic detergents may cause such lysis of the outer cell membrane.
FUNCTIONS OF LYSOSOMES 10
Cellular Digestion: Lysosomal enzymes degrade proteins into dipeptides and carbohydrates into monosaccharides. Sucrose and polysaccharides are not digested and remain in the lysosomal vacuoles.
Autophagy: By the process of autophagy, lysosomes constantly remove cellular components like mitochondria etc. Cytoplasmic organelles become surrounded by smooth endoplasmic reticulum and lysosomes attach with it and discharge their contents into autophagic vacuole and the organelle is digested. Autophagy is a general property of eukaryotic cells.
Exocytosis: Contents of the primary lysosome mat be released into the medium by exocytosis and it occurs during replacement of cartilage by bone during development where osteoclasts release lysosomal enzymes . It can also occur in bone remodeling under influence of parathyroid hormone. Crinophagy refers to the process by which secretary granules produced in excess are removed by lysosomes.
Endocytosis: Lysosomes may fuse with vesicles or vacuoles formed by endocytosis and release their enzymes into it for digestion. The material for digestion may be food (protozoa) or a foreign body like parasite .The products of digestion are absorbed and assimilated leaving undigested which are released outside by exocytosis. Role in germ cells and fertilization: The acrosome in spermatozoa may be considered as a special lysosome containing protease and hyaluronidase along with acid phosphatase .The lysosome in ova help in digestion of stored food Role of lysosomes in diseases: Lysosomes are involved in many diseases like rheumatoid arthritis, silicosis, acute inflammatory responses, anorexia, myocardial infarction, different storage diseases etc.
Lysosomal enzymes release
Although a series of studies have indicated that mechanism which account for release of lysosomal enzymes can provoke acute inflammation, may progressed to chronic state also. Regurgitation during feeding: Human neutrophils release lysosomal hydrolases during phagocytosis. Microtubules were more prominent in phagocytosing than in resting cells, and were observed near primary lysosomes and forming phagosomes. ?Regurgitation during feeding? resulted from degranulation of primary lysosomes into newly formed phagosomes which were still open to the extracellular space as well as from the ingestion of additional material directly into already loaded secondary lysosomes. 11 Phagocytosis: When cells engage in phagocytosis (leukocytes which engulf immune complexes in the synovial fluid of patients with rheumatoid arthritis) they release a portion of their lysosomal hydrolases into the surrounding medium . This effect appears due to extrusion of lysosomal materials from incompletely closed phagosomes open at their external border to tissue space while joined at their internal border with granules discharging acid hydrolases into the vacuole (phagolysosome). Under such circumstances lysosomal enzymes are selectively released to the outside of the cell without necessarily causing cytoplasmic damage. 12 Reverse Endocytosis: It and may be pertinent to the pathogenesis of tissue injury. When leukocytes encounter immune complexes which have been dispersed along a nonphagocytosable surface,there is similar, selective release of lysosomal enzymes directly to the outside of the cell. Perforation from within: Another mechanism for lysosomal enzyme release followed phagocytosis of crystals was due to "perforation from within" of the lysosomal membrane, rather than lysis by crystals of the plasma membrane. Enzyme release occurs when certain materials gain access to the vacuolar system wherein they interact with, and finally rupture, lysosomal membranes . A wave of membrane damage results with release of cytoplasmic and lysosomal enzymes followed by cell and tissue death.
Regulation of lysosomal proteases
Selective secretion of lysosomal enzymes from neutrophils during acute inflammation. Discharge of lysosomal content is requires extracellular calcium and can be modulated by several different classes of hormones, protease inhibitors such as α2 -macroglobulin and α1- antiprotease, drugs and other agents which results in the provocation of acute inflammation and connective tissue degradation. These antiproteases are present in serum and synovial fluids. They are thought to function by binding to and covering the active sites of proteases. Protease-antiprotease imbalance is probably important in the pathogenesis of emphysema. The most prominent protease inhibitor in human serum is α1-antitrypsin. It has the highest molar concentration of all inhibitors and is responsible for approximately 90% of the total trypsininhibiting activity of normal serum α1- antitrypsin is a glycoprotein with a carbohydrate portion of 12.4% containing galactose, mannose,fucose, acetyl hexosamine, and sialic acid. Its amino acid composition is unremarkable except perhaps for the content of only two cysteine residues 13and 14.
CONCLUSION
Concluding, the present study, the lysosomal enzymes are crucial for the degradation of numerous macromolecular substrates and have been involved in many inflammatory responses. Our understanding of the importance of lysosomal cysteine proteases has advanced considerably in recent years. It is now evident that they regulate biological processes such as matrix remodeling and the immune response. Although their exact roles in the pathobiology of various diseases are uncertain, continued research should clarify their roles in various grounds. Accurate knowledge of lysosomal enzyme is essential to expand our current understanding of intracellular proteolysis that plays important role in health and disease.
Englishhttp://ijcrr.com/abstract.php?article_id=2146http://ijcrr.com/article_html.php?did=21461. Turk B and Turk V. Lysosomes as Suicide Bags in cell death: Myth or reality? J Biol Chem 2009; 284: 21783-21787
2. Layik MN, Yamalik F, Caglayan K, Kilinc I, Etikan and Eratalay K. Analysis of human gingival tissue and gingival crevicular fluid beta-glucuronidase activity in specific periodontal diseases. J Periodontol. 2000; 71: 618-624.
3. Varki AP, Reitman Ml, Tabas I and S. Kornfeld,. Studies of the synthesis, structure and function of the phosphorylated oligosaccharides of lysosomal enzymes. J Bio sci. 1983; 5: 101–104.
4. Dell'Angelica EC, Mullins C, Caplan S and Bonifacino JS. Lysosomal related hydrolases. FASEB J. 2000;14: 1265-1278.
5. Barrett AJ, Rawlings ND and JF. Jr. Woessner, 1998. Handbook of Proteolytic Enzymes. Academic Press, London.
6. Mason RW, 1996. Lysosomal Metabolism of Proteins. In: Subcellular Biochemistry: Biology of Lysosome, Lloyd, J.B. and R.W. Mason (Eds.). Vol. 27, Plenum Press, New York, pp: 159–190.
7. Weissmann G, Zurier RB, Spieler PJ and Goldstein IM. Mechanisms of lysosomal enzyme release from leucocytes exposed to immune complexes and other particles. J Exp Med. 1971;134: 149s-165s
8. Davey MG and Luscher EF. Release reactions of human platelets induced by thrombin and other agents. Biochiini. Biophys. Acta. 1968;165: 490-506
9. Holmsen H and Day HJ. The selectivity of the thrombin-induced platelet release reaction: Subcellular localization of released and retained constituents. J Lab Clin Med; 1970;75: 840-855.
10. Luzio JP, Pryor PR and Bright NA. Lysosomes: Fusion and function. Nat Rev Mol Cell Bio. 2007; 8: 622-632. 11. Weissmann G. Lysosomes and joint disease. Arth Rheum.1966; 9: 834-840.
12. Becker EL,. Some interrelations of neutrophil chemotaxis, lysosomal enzyme secretion and phagocytosis as revealed by synthetic peptides. Am J Pathol 1976; 85: 385-394.
13. Parrott DP and Lewis DA. Protease and antiprotease levels in blood of arthritic rats. Ann Rheumatic Dis. 1977;36: 166-169.
14. Kueppers F. α1,-Antitrypsin. Am J Hum Genet 1973; 25: 677-686
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524137EnglishN-0001November30HealthcareEVALUATION OF ANTHELMINTIC ACTIVITY OF MOMORDICA CHARANTIA FRUIT EXTRACT
English5760Shripad M.BairagiEnglish Ritesh S. MantriEnglish Nitin NemaEnglishThe present study is an attempt to explore the anthelmintic activity of Methanolic extract of fruits of Momordica charantia. Various doses of methanolic extract were evaluated for their anthelmintic activity on adult Indian earthworms,Pheretima posthuma. All extract able to show anthelmintic activity at 25, 50 and 100 mg/ml. The activities are comparable with standard drugs, piperazine citrate and albendazole. All doses of momordica charantia showed dose dependent anthelmintic activity in comparison to standard drug. The data were found statistically significant. It is concluded that methanolic extract of M.charantia is having anthelmintic activity.
EnglishMomordica charantia, Cucurbitaceae, Anthelmintic, Piperazine citrate, Albendazole.
INTRODUCTION
Momordica charantia (Fam: Cucurbitaceae) grows in tropical areas. The herbaceous, tendrilbearing vine grows to 5 m. It bears simple, alternate leaves 4–12 cm across, with 3–7 deeply separated lobes. Each plant bears separate yellow male and female flowers. Fruit has a distinct warty looking exterior and an oblong shape, it is hollow in cross section, with a relatively thin layer of flesh surrounding central seed cavity filled with large flat seed and pith.[1] M.charantia contain novel and biologically active phytochemicals including triterpens, proteins and steroids. In numerous studies, at least three different group of constituent found in all parts of M.charantia have clinically demonstrated hypoglycemic properties. [2] The hypoglycemic chemical includes mixture of steroidal saponin known as charantins, insulin like peptide and alkaloid. M.charantia fruits and seeds has been shown to reduce total cholesterol and triglycerides in both the presence and absence of dietary cholesterol [3,4].Noval phytochemical in M.charantia demonstrated the ability to inhibit an enzyme named guanylate cyclase. This enzyme thought to be linked to pathogenesis and replication of not-only psoriasis but leukemia and cancer as well. [5- 7]The phytochemical momordin has clinically demonstrated cytotoxic activity against Hodgkin‘s lymphoma in vivo.[8
MATERIAL AND METHOD
The fruit of Momordica charantia were collected from M.P.K.V.Rahuri, Maharashtra, India. And was authenticated by botanical survey of India, Pune, Maharashtra.
Preparation of Extract:
The methanolic extract of air dried fruit powder (500 gm) was prepared by using soxhlet apparatus, concentrated and vaccum dried which give dark a brownish mass (62.20gm).
Anthelmintic Bioassay:
Healthy Indian earthworms (Pheretima posthuma) selected due to its anatomical and physiological resemblance with the intestinal round worm parasites of human being were used in the present study.[9-11].All the earthworms were of approximately equal size. They were procured from local supplier and maintained at MES College of Pharmacy, Sonai. The methanolic extract of Momordica charantia was tasted in various doses in each group. Normal saline water used as control. Piperazine citrate and albendazole were used as standard drug for comparative study with methanolic extract.The anthelmintic activity was assessed using earthworm by the Naragund [12] reported method. Earthworm divided into six groups. Each group containing five earthworms. First group (I) serve as normal control which receive saline water only. Second (II) group receive standard piperazine citrate 10 mg/ml, third group (III) receive standard albendazole 10 mg/ml.Group four (IV), group five (V) and group six (VI) receive dose of methanolic extract of 25 mg/ml, 50 mg/ml and 100 mg/ml respectively. Observations were made for the time taken to cause paralysis and death of individual worm for two hours. Paralysis was confirmed when the worm did not revive even in normal saline water. Death was concluded when worms lost their motility followed by fading away of their body colour. [13]
Statistical analysis:
The data expressed as mean ± standard deviation (n=5). For determining the statistical significance, standard error mean and analysis of variance (Anova) at 5% level significance was employed. P values < 0.05 were considered significant
. RESULTS
The methanolic extract of M.charantia produced a significant anthelmintic activity in dose dependent manner as shown in Table-1. Normal saline water used as control. The activity shown by methanolic extract is of considerable importance. All data were found to be statistically significant at 5% level of significance (PEnglishhttp://ijcrr.com/abstract.php?article_id=2147http://ijcrr.com/article_html.php?did=21471. Grover JK,Yadav SP,Pharmacological actions and potential uses of momordica charantia,J.Ethnopharmacol,93 (1),2004,123-32.
2. Raza H,Moduation of xenobiotic metabolism and oxidative stress in chronic streptozotocin induced diabetic rat fed with momordica charantia fruit extract,J.Biochem Mol.Toxicol.14(3),2000,131-39
3. Jayasooyiya,AP, Effect of momordica charantia powder on serum glucose level and various lipid parameters in rats fed with cholesterol free and cholesterol rich diets,J.Ethnopharmacol,72(1-2),2000,331-36
4. Ahmed I,Hypotriglyceridemic and hypocholesterolemic effect of anti-diabetic momordica charantia (karela) fruit extract in streptozotocin induced diabetic rat, J.Diabetis Res.Clin.Pract,51(3),2001,155-61
5. Takemoto,DJ,Partial purification and characterization of a guanylate cyclase inhibitor with cytotoxic properties from the bitter melon(momordica charantia),J.Biochem.Biophys.Res.Commun ,94(1),1980,332-39.
6. Claflin AJ,Inhibition of growth and guanylate cyclase activity of an undifferentiated prostate adenocarcinoma by an extract of the balsam pear (momordica charantia abbreviata),J. Proc Nati.Acad. Sci.,75(2),1978,989-93.
7. Vesely DL, Isolation of a guanylate cyclase inhibitor from the balsam pear(momordica charantia abbreviate)J.Biochem.Res.Commun.77(4),1 977,1294-99.
8. Terenzi A, Anti-CD30 (BER=H2) immunotoxins containing the type-1 ribosome-inactivating proteins momordin and PAP-S (pokeweed antiviral protein from seeds) display powerful anitumour activity against CD30+tumor cells in vitro and in SCID mice.Br.J.Haematol,92(4),1996,872- 79.
9. Vidyaethi RD,A textbook of zoology,Chand and Co.Press,New delhi, 14th edn , 1977,329.
10. Thron GW,Harrison‘s principles of internal medicine, MC Grew Hill,New York,1977,1088.
11. Vigear Z, Atlas of medical parasitology, Publishing House,Singapore,2nd edn ,1984,216.
12. Jayachandran E,Bhatia K,Naraguand V and Ray A,Anthelmintic activity of 2[3-amino,5- 8 methyl-6-carboxamidepyrazol-1-yl] 6- fluro-7-substituted (1,3) benzothiazoles on pheritima postuma, Indian drugs,40(7),2003,408.
13. Mali RG,Hundiwale JC,Sonawane RS,Patil RN,Indian J.Nat.Prod.,20(4),2004,10.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524137EnglishN-0001November30HealthcareHEALTH SEEKING BEHAVIOR OF HIV POSITIVE PATIENTS ATTENDING VOLUNTARY COUNSELING AND TESTING CENTER - A GENDER PERSPECTIVE
English6169Shaikh MohsinEnglish Patil RajkumarEnglish Pathan SameerEnglishThe epidemic of Acquired Immuno Deficiency Syndrome (AIDS) has emerged as a serious public healthproblem in many parts of the world and a gender based difference in the health seeking behavior has significantly precipitated to it. Aims: To study the health seeking behavior of HIV positive cases and impact of gender discrimination over it. Settings and Design: It is a cross sectional study conducted in Voluntary Counseling and Testing Center (VCTC) – Baroda. Methods: A semi-structured and pretested proforma was used to interview HIV positive patients attending VCTC located at Sayaji Hospital, Vadodara. With the help of VCTC counselors, In-depth interview of all patients were arranged to collect the detailed information on health seeking behavior. Prior verbal and written consent was taken before starting each interview. This study included 100 HIV positive cases (>13years) attending VCTC during April-December 2007. Results: The present study included 100 individuals with equal ratio of male and female, 73 % were in age group 21-40 years, 92% were literate and 60 % were married. 54 % patients consulted private clinic for their health problem while 30 % went to government hospital of which
majority were females (70 %). None of the female patients contacted VCTC initially for counseling purpose, while 13 % patients didn‘t consult any health care providers before reaching VCTC. 54 % patients consulted private GP initially of which 21 % didn‘t satisfied and visited Government hospital later. Over all 48 % patients reached SSG hospital and were referred to skin, TB, urology and general medicine before reaching VCTC.
EnglishStudy of gender based health behavior is the vital link to control the spread of HIV. The need of the hour is to strengthen the health services with the focus of gender.INTRODUCTION
The epidemic of Human Immunodeficiency Virus (HIV) infection that causes Acquired Immuno-Deficiency Syndrome (AIDS) has emerged as a serious public health problem in many parts of the world. Estimates at the end of 2006 suggest that 39.5 million men, women and children are living with HIV/AIDS worldwide and almost 22 million have already lost their lives1 . In sub-Saharan Africa, young women (aged 15-24 years) are infected more frequently than young men. In 2001, the estimated infection rates for young women were 6-11% compared to 3- 6% for young men1 Focus of Gender in HIV/AIDS: Of the 37.2 million adults living with HIV/AIDS at the end of 2006, 17.7 million or nearly 50 percent were women2 . Rather than representing equity between the sexes, the one-to-one ratio of male to female HIV infections demonstrates the way in which gender inequalities affect HIV/AIDS incidence rates. Since the HIV/AIDS pandemic began more than 20 years ago, infection rates among women have accelerated. Traditional gender roles held by many of the world‘s societies definitely affects the health seeking behavior of women, which continued spread of HIV, particularly from men to women. In most societies, women also carry a disproportionate amount of the burden of caring for family members living with HIV/AIDS, and experience the brunt of the stigma associated with HIV infection. Correcting the gender imbalance that contributes to and is exacerbated by the HIV/AIDS pandemic will depend upon improving women‘s social and economic status, and increasing men's responsibility for HIV prevention and care, so as to provide better health care access. Unequal social roles and vulnerability to men‘s demands prevent women to have better health care access. Number of facts associated with HIV transmission like illiteracy, employment, gender discrimination poor health infrastructure etc., stimulates already prevalent stigma related to HIV/AIDS. Finally all these factors such as gender roles, economic, cultural and social factors and stigma related to HIV/AIDS are likely to influence the motivation of HIV infected people to practice safer sex3 . United Nations for HIV/AIDS has reported 5.7 million people in India with HIV/AIDS this year, which is almost 15 % of the global burden of HIV/AIDS, and 37 % of all the infected people in India are women4 . Nash Ojanuga and Gilbert (1992) systematized the obstacles which women face into four categories5 : 1.Institutional barriers: unequal treatment by health providers, 2. Economic barriers: different access to resources, 3.Cultural barriers: social status of women which situates them in socially inferior positions, 4. Education barriers: women having less access to education.
Objective: To study the health seeking behavior of HIV positive cases and impact of gender discrimination over it.
Need of the study
Women are most vulnerable and likely to get discriminated with HIV infection, having so many reasons in the background than men who plays dominant bread earner role in the society, there was a need of gender based research to go through the health seeking behavior of the HIV positive patients to understand the discrimination. It would also help in policy designing for national AIDS control Program.
METHODOLOGY
This is VCTC based cross sectional study consisting of qualitative data collection of HIV positive patients conducted between April ‘07 to December ‘07. Total number of 50 male and 50 female HIV positive patients who visited VCTC, Vadodara during the study period in age group >13 years were purposively selected for this research. The following selected criteria was adopted for inclusion of HIV positive patients attending VCTC in this research, 1) 50 adult male and 50 adult female >13 years of age. 2) HIV positive patients diagnosed minimum 2 months back. 3) Patient who voluntarily took part in the research and visited VCTC – SSGH after HIV infection. 4) Patients with HIV antibody test positive according to NACO guidelines6 . (ELISA and Rapid test). Information on Health seeking behavior and access to health care of HIV positive patient was collected through semi-structured, pre-designed and pre-tested proforma following in-depth. interview technique with the help of health seeking behavior pathway (Annexure). In-depth interview of each and every HIV positive patient was done with the help of expert VCTC counselors, through previously prepared questionnaire at VCTC in a private room with prior informed, written and verbal consent. Proper care has been taken to maintain the confidentiality. Prior permission from the Ethical committee of the university has been taken to carry out whole research. Analysis of the study was done using Epi Info 6.04 d Statistical Package and Chi square test was applied wherever needed. Following steps were involved in documentation and data management after data collection.
Limitations of study:
We could have been covered VCTCs of Gujarat but we didn‘t get the permission to do so and also could have consulted the other high risk groups like male and female sex workers, homosexual group, or truck drivers to understand their health seeking behavior.
RESULTS
The study included 50 male and 50 female HIV positive (diagnosed minimum 2 months back) cases who visited VCTC. Over two third of the patients (73%) were within 21-40 years of age group among both the genders. Almost 60 % of HIV positive patients were married with significantly higher percentage of males (58 %) as compared to females (42 %). Whereas total number of widow/widower found is 23 % and more than two third of them were females as compared to males. Majority of female patients (80 %) were housewives while majority of males were involved in high risk occupations like truck driving, auto driving, call center job and sales work. Of total patients, 8 % were illiterate, 43 % patients were educated upto 7th standard, 30 % untill 10th standard, 14 % untill 12th standard and only 5 % had studied untill graduation or higher; without much gender difference. Of the total economically self dependant patients (49%), more than two third were males. Out of total 66 % of patients, 42 % had TB infection in the past while 30 % had STD (with equal gender distribution) history. All HIV positive patients had undergone CD4 count testing of whom 49 % were females and 51 % were males (Table – 1). Out of 37 patients on ART, 78 % (n=29) cases experienced at least one ADR related to ART use. 86 % cases had history of ADR in the form of nausea, vomiting and in 31 % cases, in the form of skin reaction with no gender difference. Out of 100 patients, 8 directly went to VCTC without getting referred by anyone and all of them were males, while 62 % patients were referred to VCTC by Doctors ( Private and Government). 7 % patients were referred by the family members, 6 % by their friends, 5 % by NGOs and 9 % of patients by their relatives other than their family members. There was no statistically significant gender difference (Table 2). Table 2, shows the distribution of HIV positive patients by appearance of first sign/symptoms noticed by them when asked. HIV positive patients had history of single or multiple symptoms during initial health problems. Majority of patients observed weight loss (26 %) followed by fever (23 %), cough (22 %), skin problems (17 %), diarrhea (10 %), burning micturition (6 %) and genital ulcer (5 %). There is no statistically significant gender difference seen. 54 % patients initially contacted private clinic/hospital for their symptomatic problems of whom 59 % were males as compared to 41 % females. 30 % patients contacted government hospital initially and 70 % of them were females as compared to 30 % males, which is statistically significant (P3 health care providers before reaching VCTC. Out of 100 patients studied, 8 directly contacted VCTC voluntary bases immediately. 79 % patients had first health care contact within 6 months of observing first sign/symptom by themselves, while 13 % patients took >6 months to contact health care providers. There is no significant gender difference seen (Table-2). 13 % visited VCTC because of Tuberculosis infection, 10 % due to skin problems, 9 % cases visited VCTC because of their high risk behavior, 7 % gave a reason of their spouse being HIV positive, 4 % due to penile ulcer and same percentage of patients visited VCTC as a part of pre operative investigation. 2 % had pneumonia and were consulted VCTC for HIV testing while 2 % were consulted during visa procedure for HIV testing and others during ANC check-ups, for insurance purpose and some MSM behavior. 46 % patients gave a reason that they visited VCTC for other health problems like low grade fever, chronic cough, diarrhea and weight loss. According to health care seeking pathway, Initially 3 patients directly went to Quack with the hope of getting treated – but finally consulted GP/SSGH for their treatment. 8 Patients directly went to VCTC - Baroda for testing and for counseling purpose. 54 Patients directly went to Private General Practitioner out of which 9 patients changed GP for the 3rd time and 2 patients changed GP treatment for the 4th time and finally went to VCTC. 21 Patients who consulted private GP for the first time - were not satisfied and moved to SSG Hospital/Govt. CHC/PHC for further testing and treatment purpose. 38 Patients directly consulted SSGH/Govt. CHC/PHC without going to Private GP. 5 Patients went to NGO directly with their suspicion of having HIV infection and after counseling they were sent to VCTC. 16 patients consulted Govt. CHC/PHC of which 6 patients went to private GP for the first and then to Govt. CHC/PHC. 8 patients out of those 16 went to SSGH because of their unsatisfactory treatment and counseling, finally all were guided to VCTC. Over all 48 patients reached SSGH after consulting one or more GP of whom 15 patients reached OPD-1 (Skin and VD OPD) for skin lesions over body parts and private parts while 16 patients reached OPD-18 (General Medicine OPD) for their generalized health problems and 8 patients reached OPD-17 (TB and Chest diseases OPD) for their suspected koch‘s infection. 8 Patients consulted OPD-25 (Urology OPD) for suspected UTI and 2 patients visited OPD -4 (Orthopedic OPD) for their orthopedic problems.
CONCLUSION
Majority of male patients (59 %) had contacted private clinic/hospital initially for their health problems as compared to 41 % females because of fear of social stigma related to HIV/AIDS and belief of more privacy at private setup. Nearer to two third (70 %) female patients contacted government hospital initially for their problems as compared to 30 % males (PEnglishhttp://ijcrr.com/abstract.php?article_id=2148http://ijcrr.com/article_html.php?did=21481. Joint United Nations Programme on HIV/AIDS (UNAIDS) 2006, AIDS Epidemic Update (English original). Available at :http://www.unaids.org/ globalreport/documents/20101123_GlobalR eport_full_en.pdf. , Accessed on 1st January 2011.
2. Joint Unied Nations Programme on HIV/AIDS (UNAIDS) 2000. Men and AIDS: A Gendered Approach. Geneva: UNAIDS. Available at: www.thebody.com/ unaids/men/contents.html. Accessed on 1st August 2007.
3. Population Council (2007), ?Reducing HIV risk behaviors among key populations by Increasing community involvement and building social capital?. Available at: www.popcouncil.org, Accessed on 1st November 2007.
4. United Nations Program on HIV/AIDS (2007). www.unaids.org.in . Accessed on 10th January 2007
5. Ojanuga, D Nash, Gilbert., (1992). Women‘s access to health care in Developing countries. Social Science and Medicine Vl.35, August 1992:613.
6. National AIDS Control Organization (NACO), (2002), Specialist‘s Training and Reference Module. New Delhi.
7. Matlin, S. and Spence, N. (2001) "The Gender Aspects of the HIV/AIDS Epidemic." Presentation at the Expert Group Meeting on the HIV/AIDS Pandemic and Its Gender Implications, Windhoek, Namibia. Available at:www.un.org/womenwatch/daw/csw/hivai ds/matlinspence.html. Accessed on 10th January 2009.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524137EnglishN-0001November30HealthcareA COMPARATIVE STUDY ON EXTENDED TIMED GET UP AND GO (ETGUG) TEST BETWEEN RIGHT AND LEFT HEMIPLEGICS
English7077Sankar Sahayaraj MuthukaruppanEnglish RavindraSubbannaEnglish Harilal BapurajapanickerEnglishObjective: The purpose of the study is to enlighten the role of limb dominance in gait training and for tailoring the gait training programme in physical therapy treatment between the categorical (temporal parameters) and the representational (visuo-spatiial parameters) hemispheres.
Methods: between 45-55 years old left hemisphere dominant sixty males with hemiplegic (30 right side and 30 left side), satisfying the inclusion criteria were chosen for the study. Extended Timed Get Up and Go (ETGUG) test was used to assess the duration of sit to stand, gait initiation, walking, turn around, sitting down, and speed of the walk. Independent?t‘ test was used to compare the components of both the groups.
Results: The findings showed that there were significant durational changes in the various components of
ETGUG test between right side and left side hemiplegic subjects.
Conclusion: The gait rehabilitation should be emphasized on standing up, turning and sitting down for left sided hemiplegics and gait initiation, walking and speed for right sided hemiplegics. Temporal and the spatial parameters should be considered during gait training and gait training programme should be given for right and left side hemiplegic subjects differently.
EnglishHemiplegic patient, ETGUG test, limb dominance, gait training.INTRODUCTION
Hemispheric specialization is related to handedness. Handedness appears to be genetically determined. 96% of right-handers had left cerebral dominance, and the remainder had right cerebral dominance. Left dominance was observed in 70% of left-handers, bilaterality in 15% and right-dominance in 15%1 . There are anatomic differences between the two hemispheres that may correlate with the functional differences. During gait the left lower limb contributed mainly to body weight transfer during walking; whereas the right lower limb was responsible for propulsion.2 The leading limb mainly contributes to forward progression, where as the trailing limb provides control and contributes to propulsion to a lesser extent.3 The concept of cerebral dominance and a dominant and non dominant hemisphere has been replaced by a concept of complementary specialization of the hemispheres, One for sequential analytic processes (the categorical) and the other one for visuo-spatiial relations (the representational hemisphere).4 In India the incidence of cerebrovascular disease was found to be 13/1,00,000 population/year in a study conducted in 1969 - 71 at Vellore5 , 31/1,00,000 population/year in a study conducted at Rohtak6 and 124/1,00,000 population / year in a study conducted at Baruipur, West Bengal7 . A WHO study in 1990 quoted incidence of mortality due to stroke in India to be 73/1,00,000 per year. 8 Hand dominance affects an individual‘s functional ability after a stroke; yet current constrained induced movement therapy studies have not sufficiently addressed dominance and its role in functional recovery9 . People with non dominant could possibly returns to a previous level of functioning at a quicker phase compared with an individual with a dominant side stroke. Gait asymmetry in temporal and kinematics variables challenging the assumption of that right and left limbs are functioning symmetrically10. Gait asymmetry in people without impairments can be explained in terms of actions taken by the lower limb to propel the body segments and to control their forward progression.11 Subjects with right hemisphere lesions had greater troubles than those with left hemisphere lesions in regaining the ability to stay seated unassisted.12 Walking speed, step length, and double support ratio, have been varied more in cognitive impairments than in healthy older adults. The objectives of the study were to investigate the duration of various components of ETGUG Test between right side hemiplegic subjects, and left side hemiplegic subjects to modify the treatment protocol for the right and left side stroke subjects.
Need of the Study
Changing life styles, and habitual intake of junk food and the added stress increase the incidence of stroke in developing countries like India. Over the past few decades, demographic shift caused by increasing life expectancy resulted in a burgeoning aging population in India. The previous urban community-based studies had documented an age adjusted incidence rate of stroke from 13 in 1970 to 105 per 100 000 persons per year in 2001.5,13 Stroke rehabilitation is a long drawn process and the knowledge about the contributing factors might help in tailoring the treatment and make it cost effective by reducing treatment duration. The ability to walk is the prime factor that determines whether a patient will go home or to a nursing home and whether he or she will return to the previous level of productivity after a stroke. 14,15,16,17 This study aimed to provide an opportunity for Neuro - physical therapist as they would be able to scientifically understand and assess the role of dominant hemispheres during the gait training programme, the role of parametric screening tool utilizing in physical therapy assessment, and the role of visuo-spatial, cognitive, psychosomotor and limb dominance in gait rehabilitation programme in stroke.
MATERIALS AND METHODS
It was an observational study. 30 right side affected and 30 left side affected hemiplegic subjects (Middle Cerebral Artery infarcts and Brunstorm stage V), taking treatment in the physiotherapy department were taken for this study. Subjects included in the study were 45-55 years 8 and having the ability to walk 20 meters independently without walking aids. Subjects were excluded if they had deteriorating medical conditions, structural and functional orthopedic problems of lower limb, and neurological problems like cerebellar lesions, basal ganglion lesions and previous history of head injury. Also subjects under sedative therapy, visually impaired subjects and non co-operative subjects were excluded.
The Extended Timed Get Up and Go (ETGUG) test is a potential, objective, assessment tool that can be used in almost any clinical setting with minimal equipment, professional expertise or training. It better isolates functional deficits thereby aiding the therapist in devising prevention strategies and guiding both treatment and further testing
Multi-memory stopwatch is the basics of ETGUG and measures the total time to complete a task, as well as the time intervals for components within that task. The laps and the phases of test with which they correspond are as follows: Lap task: Sit to stand, Gait initiation, Walk 1, Turn around, Walk 2, Slow down, stop, turn around and sit down. Since the distance for walk 1 and walk 2 are to know walking speeds, can be calculated from the times taken for these components of the ETGUG test. This is important objective measure of functional ability in the stroke subjects. In order to avoid errors; subjects were oriented about the study prior to the conduct of the test. Standard protocol for ETGUG test was followed. 18
Statistical Methods
Data were statistically analyzed by using Independent t test for comparing each components of ETGUF between right and left sided hemiplegics by SPSS version 16 for windows.
RESULTS
Stand up, Turning, Sit down and speed, were increased in left hemiplegic subjects is showed Figure 2. It showed they took maximum time to complete the tasks than right hemiplegic subjects. So these components are difficult for left hemiplegic subjects.
Stand up
Group I right hemiplegics has the mean of 1.5540 with SD of 0.12227 and Group II left hemiplegics has the mean of 1.6230 with a SD of 0.11114. The mean difference is 0.690 and the t calculated value of 2.287 df= 58 at 5%. P value is 0.026. Thus there is a significant difference between the two groups and result indicates that right group is able to stand up easily than left group
Turning
Group I right hemiplegics has the mean of 7.6170 with SD of 0.43560 and left hemiplegics group has the mean of 8.7253 with a SD of 0.95102. The mean difference is 1.1083 and the t calculated value of 5.803, df= 58 at 5%. P value is 0.000. Thus there is a significant difference between the two groups and result indicates that right group is able to turn easily than left group.
Sitting down
Group I right hemiplegics has the mean of 3.3410 with SD of 0.15103 and left hemiplegics group has the mean of 3.6940 with a SD of 0.13361. The mean difference is 0.3530 and the t calculated value of 9.588, df= 58 at 5%. P value is 0.000. Thus there is a significant difference between the two groups and result indicates that right group is able to sit in the chair easily than left group.
Speed
Group I right hemiplegics has the mean of 0.5200 with SD of 0.2387 and left hemiplegics group has the mean of 0.5724 with a SD of 0.3101. The mean difference is .0525 and the t calculated value of 7.343, df= 58 at 5%. P value is 0.000. Thus there is a significant difference between the two groups and result indicates that right group is able to walk faster than left group. Gait initiation, Walk I and Walk II took longer duration to complete the task in right hemiplegic subjects than left side hemiplegic subjects are shown in Figure 3. So these components are difficult for right hemiplegics than left hemiplegics.
Gait initiation
Group I right hemiplegics has the mean of 3.2377with SD of 0.66638 and left hemiplegics group has the mean of 2.6910 with a SD of 0.18894. The mean difference is 0.5467 and the t calculated value of 4.323, df= 58 at 5%. P value is 0.000. Thus there is a significant difference between the two groups and result indicates that right group is easily able to perform gait initiation than left group
Walk I
Group I right hemiplegics has the mean of 11.547 with SD of 0.5708 and left hemiplegics group has the mean of 10.503 with a SD of 0.5425. The mean difference is 1.045and the t calculated value of 7.266, df= 58 at 5%. P value is 0.000. . Thus there is a significant difference between the two groups and result indicates that right group is walk faster than the left group.
Walk II
Group I right hemiplegics has the mean of 11.5540 with SD of 0.48604 and left hemiplegics group has the mean of 10.4987 with a SD of 0.58557. The mean difference is 1.0553 and the t calculated value of 7.596 df= 58 at 5%. P value is 0.000. Thus there is a significant difference between the two groups and result indicates that right group is more faster walk than the left group. Figure 3 showed the time duration to complete the task is longer in right hemiplegic subjects than left hemiplegic subjects. Also the left hemiplegic subjects were able to finish the task faster than in right hemiplegic subjects.
Total time
Group I right hemiplegics has the mean of 39.0800 with SD of 2.08339 and left hemiplegics group has the mean of 37.9000 with a SD of 1.52131. The mean difference 1.1800 and the t calculated value of 2.505, df= 58 at 5%. P value is 0.015. Thus there is a significant difference between the two groups and result indicates that right group requires maximum time to finish the task than left group.
DISCUSSIONS
The absence of well recognized measures to evaluate functional recovery after a stroke and the large variety of rehabilitation techniques and protocols add credence to the questioning of the value of rehabilitation in terms of costs and psychosocial benefits.19, 20, 21 The maximum weight-bearing difference between the 2 lower extremities during the Sit To Stand task was highest for the subjects with stroke who had the lowest scores on the Functional Independence Measure.22
The present study analyzed various components of functional mobility in right and left side hemiplegic subjects. In our study, Standing up (Sit to stand), turning and sitting down require maximum balance, which is significantly reduced in trailing limb (left limb) affected subjects. Leading limb (right limb) affected subjects took longer duration for gait initiation and finishing the task (walking) than the trailing limb affected subjects. This implies right side affected stroke subjects (right handed) had more difficulties with gait initiation and walking and left side affected subjects had more difficulties in sit to stand, turning and sitting downGait performance in subjects with stroke is characterized by slower gait velocity and residual spatial and temporal left-right asymmetry, compared with that in healthy adults. The average walking speed of stroke subjects is lower than that of healthy controls but the values vary depending on the severity of the stroke, the time post-stroke and the age of the subjects23 . In this study these variables were controlled. Previous studies suggested that the gait velocity of patients with stroke of varying severity ranges from approximately 0.18 to 1.03m/s, 24,25,26,27 whereas that of healthy adults of similar ages averages about 1.4m/s28 in our present study also suggested the same. The speed was less than the normal and for the Right side hemiplegics it was 0.5200 ± 0.2387 m/s and left side hemiplegics group it was 0.5724 ± 0.3101. The right side affected group was able to walk faster than left side affected group. The ability to complete the task is faster in left sided hemiplegic subjects. But both the groups took more than 30 seconds to complete the task. So they were under the risk category, since the falling criteria is more than 30 sec for ETGUG test.
CONCLUSIONS
ETGUG test components were not in similar. In stroke patient‘s conventional rehabilitation program will not provide additional benefit in terms of lower extremity motor recovery and mobility. So the training of functional mobility and gait training should be concentrated differently for right side affected hemiplegics and left side affected hemiplegics in physiotherapy. Training should not be concentrated only on temporal parameters. The temporal and spatial parameters as well as hemisphere dominance should be considered for gait training protocols.
ACKNOWLEDGEMENT
The author expressing his sincere thanks to Professor James C. Wall, PhD Department of Physical Therapy, University of South Alabama for his advices in ETGUT Test.
Englishhttp://ijcrr.com/abstract.php?article_id=2149http://ijcrr.com/article_html.php?did=21491. Annett, M. (2002). Handedness and brain asymmetry the right shift theory. East Sussex: Psychology Press.
2. Hirasawa Y. Left leg supporting human stright (bipedal) standing. saiensu. 1981; 6:32-44.
3. Sadegli H, Allard1 P, Duhaime M. Contribution of lower limb muscle perser in gait of people with not impairments Phys Ther 2000, 8: 12: 188-1196.
4. William F. Ganong, Review of medical physiology. 18th ed. San fransisco: PrenticeHall international, Inc; 1997. p.257.
5. Abraham J, Rao PSS, Inbaraj SG, Shetty G, Jose CJ, An epidemiological study of hemiplegia due to stroke in South India. Stroke 1970; 1: 477-81
. 6. Bansal BC, Parkash C, Jain AC, Brahmanandan KRV. Cerebrovascular disease in young individuals below the age of 40 years. Neurol India 1973; 21: 11-8.
7. Bhattacharya S, Saha SP, Basu A, Das SK. A 5-year prospective study of incidence, morbidity and mortality profile of stroke in a rural community of Eastern India. J Indian Med Assoc 2005; 103: 655-9
8. Prasad K: Epidemology of cerebrovasular disorders in India. In: Recent concepts in stroke by Bansal BC (ed) Indian college of physicians, New Delhi, 199; 4-19.
9. Hellige J. Hemispheric Asymmetry: What‘s Right and What‘s Left? Cambridge, Mass: Harvard University Press; 1993.
10. SadeghiH, Allard P, Duhaime M. Functional gait asymmetry in able bodied subjects. Human Movement science. 1997; 16:243- 258.
11. Gunderson LA, Valle DR, Barr AE, et al, Bilateral analysis of the knee and ankle during gait: an examination of the relationship between lateral dominance and symmetry. Phys Ther 1989; 69:640-650 (medlins).
12. Wade DT, Hewer C, Wood VA, Stroke; Influence of patient‘s sex and side of weakness on outcome, Arch Phys Med Rehabil 1984; 65:513-6 13. Banerjee TK, Mukherjee CS, Sarkhel A. Stroke in the urban population of Calcutta: an epidemiological study. Neuroepidemiology 2001;20: 201–207
14. Dimitrijevic, M. R.; Faganel, J.; Sherwood, A.M.; and Mckay, W. B.: Activation of paralysed leg flexors and extensors during gait in patients after stroke. Scandinavian J. Rehab. Med. 1981; 13:109-115
15. Shiavi, R.; Bugle, H. J.; and Limbird, T.: Electromyographic gait assessment, part 1: adult EMG profiles and walking speed. J. Rehab. Res. and Devel 1987; 24:13-23
16. Shiavi, R.; Bugle, H. J.; and Limbird, T.: Electromyographic gait assessment, part2: preliminary assessment of hemiparetic synergy patterns. J. Rehab. Res. and Devel. 1987; 24:24-30
17. Wagenaar, R. C.; Meijer, O. G.; van Wieringen, P. C. W.; Kuik, D. J.; Hazenberg, G. J.; Lindeboom, J.; Wichers, F.; and Rijswijk, H.: The functional recovery of stroke: a comparison between neurodevelopmental treatment and the Brunnstrom method. Scandinavian J. Rehab. Med. 1990;22:1-8
18. Wall C. James, Bell Churan, Campbell Stewart; Davis Jennifer the Timed get-up and-go Test Revisited: measurement of the component tasks. J Rehabil Res January/February 2000; 37(1): 109-114.
19. Brunnstrom, S.: Movement Therapy in Hemiplegia: a Neurophysiological Approach. Hagerstown, Maryland, Harper and Row, 1970
20. Kaplan, P. E.: Hemiplegic: rehabilitation of the lower extremity. In Stroke Rehabilitation, pp. 119-146. Edited by P. E. Kaplan and L. J. Cerullo. Boston, Butterworth, 1986
21. Stevens, R. S.; Ambler, N. R.; and Warren, M. D.: A randomized controlled trial of a stroke rehabilitation ward. Age and Ageing 1984; 13:65-75
22. Lee MY, Wong MK, Tang FT, et al. Comparison of balance responses and motor patterns during sit-to-stand task with functional mobility in stroke patients. Am J Phys Med Rehabil 1997; 76: 401– 410.
23. Olney SJ, Richards C. Hemiparetic gait following stroke. Part I: Characteristics. Gait Posture 1996; 4: 136-148.
24. Brandstater ME, de Bruin H, Gowland C, Clark BM. Hemiplegic gait: analysis of temporal variables. Arch Phys Med Rehabil 1983; 64: 583-7.
25. Knutsson E, Richards C. Different types of disturbed motor control in gait of hemiplegic patients. Brain 1979; 102: 405-30.
26. Olney SJ, Griffin MP, McBride ID. Temporal, kinematic, and kinetic variables related to gait speed in subjects with hemiplegia: a regression approach. Phys Ther 1994; 74: 872-85.
27. Wade DT, Heller WA, Maggs J, Hewer RL. Walking after stroke. Scand J Rehabil Med 1987;19:25-30
28. Murray MP, Kory RC, Clarkson SB. Walking patterns in healthy old men. J Gerontol 1969; 24: 164-78
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524137EnglishN-0001November30HealthcareSIX MINUTE WALK DISTANCE IN HEALTHY ADULTS AGED BETWEEN 20-30 YEARS
English7883Prem.VEnglish R.D. ChakravartyEnglish Karvannan H.English RimmiEnglish Vinita AryaEnglish ManikankannaEnglishOBJECTIVE: To find the normal range of distance covered during six minute walk distance (6MWD) in
healthy adults aged between 20-30 years.
METHODS & RESULTS: Six minute walk distance was performed in a 100 feet hallway by 50 males and 50 females healthy adults ranging in age from 20-30 years. Each subject underwent a thorough physical examination including weight, standing height and BMI. Any subject with underlying cardiopulmonary, neurological or musculoskeletal pathology or conditions that could interfere with the walk test was excluded. All subjects underwent 6MWT and pre and post measurements such as distance, heart rate were recorded. Males walked 45 m more than females. Height of females is significantly correlated with 6MWD. There was significant increase in heart rate and respiratory rate following six minute walk test.
CONCLUSION: Six minute walk distance covered in healthy individuals of 20-30 years is 560±61 in males and 514±32 in females.
EnglishSix minute walk distance, Heart rate, Healthy adults .INTRODUCTION
An individual‘s response to exercise is an important clinical assessment tool, since it provides a composite assessment of their respiratory, cardiac and metabolic system. The current goal standard for assessing a person‘s aerobic response is the maximum incremental cardiopulmonary exercise test. However, most daily activities are performed at submaximal levels of exertion and therefore it has been proposed that submaximal functional tests are better reflection of physical capabilities. The ability to work a set distance is a quick, safe, easy and inexpensive way to assess physical function. It is an important component of quality of life, since it reflects the capacity to undertake day to day activities. According to the American Thoracic Society (ATS), the most precise indication for the performance of the six minute walk test (6MWT) is mild or moderate lung or heart disease, in which it is used in order to measure treatment response, as well as to predict morbidity and mortality. Balke developed a simple test for examining functional capacity, measuring the distance walked during a definite period of time. A twelve minute performance test was then developed to evaluate the physical fitness of healthy individuals. This test was subsequently modified for use in patients with chronic bronchitis. In order to allow the test to be used in patients with respiratory diseases, for whom twelve minute walking was too demanding, a shortened version six minute walk test was developed and found to perform equally as well.1-3 Twelve minute walk test (12MWT) is a practical guide to everyday disability nevertheless it is both time consuming for investigator and exhausting for patients, therefore the possibility of using walking test of shorter duration to assess exercise tolerance was explored.4 Compared to traditional laboratory index of exercise capacity such as cycle, treadmill and step ergo meter; walk test require less technical expertise and equipment making them inexpensive and easy to administer. More importantly, they employ an activity that individuals perform on a daily basis that is walking. 6MWT has been frequently used to measure outcomes before and after treatment, in patients with moderate to severe heart and lung diseases and in their prognosis. It has also been used to measure functional status and for epidemiological research purposes. The distance covered in 6MWD has been showed to accurately predict morbidity and mortality from cardiopulmonary diseases. In healthy elderly subjects, 6MWT represents submaximal exercise, but at almost 80% of the VO2 max. A recent review of functional walking test concluded that 6MWT is easier to carry out, more acceptable and provides a better reflection of activities of daily living than other walk test.5 The normal six minute walk distance has been reported in western countries 1, 5-7 , but there is no normative data for Indian population. Hence the present study aims to generate a normative data for Indian population.
METHODOLOGY
Subjects
The study was carried out at Kasturba Medical College, Mangalore. There were 100 healthy volunteered subjects comprising 50 males and 50 females aged 20-30 years. Each subject underwent a thorough physical examination including weight, standing height and BMI( Appendix I). Any subject with underlying cardiopulmonary, neurological or musculoskeletal pathology or conditions that could interfere with the walk test was excluded. All subjects underwent 6MWT and pre and post measurements such as distance, heart rate were recorded. The healthy adults included were age group between 20-30 years and adults excluded were, Underlying cardiopulmonary, neurological, musculoskeletal pathology, Smoker for more than one year, Alcoholic for more than one year (occasional alcoholic can be included), Cognitively impaired subject, Uncooperative subjects.
PROCEDURE
Baseline measurements such as pulse rate, respiratory rate and dyspnoea level using Modified Borg 0-10 scale were taken. The materials required were 100 Ft. hallway, Sphygmomanometer, Stethoscope, Timer, Watch, Pen and relevant paper work, Cones, Chair, Inch tape. We instructed the subject that objective of this test is to walk as far as possible for six minutes and were told to ?walk back and forth in this hallway as quickly as you can so that you cover as much ground as possible?. They were informed that they could slow down or rest if necessary. We demonstrated way of walking and turning around the cones placed at the two ends of the hallway. They were also instructed not to run and jog. We positioned the subject at the starting line. As soon as subject started walking, timer was started. At the end of each minute subject were given feedback on the elapsed time and standardized encouragement in the form of statements such as ?you are doing well, keep up the good work? and ?do your best?. Test was terminated at the end of six minutes.1
Post test measurement
Heart rate was recorded using three finger palpation method at rest and at the end of test. Respiratory rate was recorded by observation method prior to and upon completion of 6MWT. At the end of the test distance was measured and dyspnoea was rated using modified Borg 0-10 scale.
DATA ANALYSIS
Test used for the study is student unpaired t-test. Result has been analyzed by SPSS.vers.14.0
RESULTS
The baseline characteristics of males and females are demonstrated in table 1.Mean distance covered in males is 45m greater than females table 2.There was a significant increase between pre test and post test heart rate table 3. There was a significant increase between pre test and post test respiratory rate table 4. There was no difference in pre test and post test Borg Scale table 5.
DISCUSSION
The present study showed considerable variability in the 6MWD of the healthy subjects aged 20-30 years; ranging 455 to 600m.On average the 6MWD was 560±61m in males and 514±32m in females. An important part of the variability is 6MWD was explained by height, sex, age and weight as independent variables. Distance walked in males and females is directly proportional to height and age and it is significant in females. Study done by T.Troosters et al. showed considerable variability in 6MWD of healthy subjects aged 50-85years,ranging 383-820m on an average 6MWD was 631±93m and it was 84m greater in males as compared to female subjects. An important part of the variability in 6MWD was explained by height, sex, age and weight as independent variable.8 The greatest 6MWD from among several repetition in a wide age range of healthy volunteers showed that the distance walked after the first walk was the best and in 86% individuals an average increase of 43m was observed from first to best 6MWD.Best 6MWD average 698±96m and was inversely related to age, directly to height and greater in male then female.9 The present study shows the correlation between height of the subject and the distance walked which is directly proportional to each other. Because taller individuals have greater stride length and so the distance covered is more. When compared between males and females, males cover longer distance because of greater functional capacity and more muscle mass. Moreover, distance walked is also directly proportional to age of the subject because the present study covers young population in a narrow range. In this age group as the as the age increases muscle mass increases. Study done in Chinese population shows marked similarity with the independent variables – height and sex. Height is another important determinant of 6MWD. This is not surprising as taller people have, in theory, a larger stride length and, thus, greater 6MWD.Young males are also found to have greater exercise capacity and 6MWD than young females, probably as a result of their greater muscle mass.10 In the present study, subjects were able to reach 50% HRmax which was 101 for males and 100 for females. The less distance walked during 6MWD could be due to different persons administering the test and variability in understanding the instructions by the subjects. Though there is not much difference in mean height of the subjects as compared to study done by Bernadine Camarri et al., the lesser distance walked by subjects may be because of lower HRmax as compared to 80% of HRmax in their study.11 The functional status and capacity can be effectively measured by functional walk test, the best being 6MWT.The measurement properties of the 6MWT have been the most extensively researched and established. In addition 6MWT is easy to administer, better tolerated and more reflective of activities of daily living than the other walk tests. Therefore the 6MWT is currently the test of choice when using a functional walk test for clinical or research purposes.
Limitations of the Study
There are certain limitations to present study. only 100 subjects were recruited, the sample size was inadequate for establishment of more accurate value of distance walked in Indian population.
Future Studies
Future studies need to be done with larger sample size with more trials. Studies are also needed for varying age groups for eg.30-70 years.
CONCLUSION
Six minute walk distance covered in healthy individuals of 20-30 years is 560±61 in males and 514±32 in females.
Englishhttp://ijcrr.com/abstract.php?article_id=2150http://ijcrr.com/article_html.php?did=21501. ATS Statement Guidelines for Six Minute Walk Test American Journal of Respiratory and Critical Care Medicine vol 166, pp 111- 117 .
2 Sherra Solway, Lina Brooks, Yves Lacasse, Scott Thomas A Qualitative Systematic Overview Of The Measurement Properties Of Functional Walk Test used in the Cardiorespiratory Domain chest 2001;119:256-270
3. Kervio, Gaelle, Carre, Francois,Ville, Nathlie Reliability and Intensity of the 6MWT in healthy Elderly subjects Med. Sci. Sports Exerc, vol 35, no 1,pp. 169-174,2003
4. CR Mcgavin, S P Gupta 12 Minute walking test for assessing disability in chronic bronchitis. British Medical Journal 1976,1,822-823
5. Two, Six, Twelve min walking test in respiratory diseases British Medical Journal,vol284,29 may 1982
6. Fryderyk Prochaczek, Hanna Winiarska1 et al. Six-minute walk test on a special treadmill: Primary results healthy volunteers. Cardiology Journal 2007, Vol. 14, No. 5, pp. 447–452.
7. Paul L. Enright and Duane L. Sherrill Reference Equations for The Six Minute walk in Healthy Adults. AMJ Respir J 1999;14:270-274
8. T.Troosters, R.Gooselink, M.Decramer Six Minute Walking Test in healthy elderly subjects. Eur Respir J 1999;14:270-274
9. Gibbons, William J, Fruchter, Nadine, Sloan, Sherry et al Reference Values for a multiple repetition Six Minute Walk Test in healthy adults older than 20 years. Journal of Cardiorespiratory Rehabilitation 21(2):87-93 March/April 2001.
10. A.M. Li, J.Yin, C.C.W.Yu, T.Tsang, H.K.So, E.Wong et al The 6 MWT in healthy children; reliability and validity Eur Respir J 2005;25:1057-1060
11. Bernadine Camarri, Peter R. Eastwood, Nola M.Cecins, Philip J. Thompson, Sue Jenkins Six minute walk distance in healthy subjects aged 55-75 years. Respiratory Medicine (2006)100,658-665
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524137EnglishN-0001November30TechnologySEA WATER ABSORPTION PHENOMENON IN BANANA FIBRE REINFORCED VINYL ESTER COMPOSITES
English8488Rajesh GhoshEnglish A RamakrishnaEnglish G.ReenaEnglishThe ageing response of banana fibre reinforced vinyl ester composites in sea water environment is investigated. The main objective was to evaluate the effects of sea water on the mechanical properties. Fibre mats were reinforced into vinyl ester matrix and composite laminates were made. These were then subjected to sea water ageing and water absorption and mechanical properties were tested. It is observed that during the initial stages there was increased rate of water absorption following the Fickian law. On prolonged time of immersion, moisture absorption led to plasticization of the matrix and also reduced the mechanical properties of the specimen.
EnglishSea water, absorption, banana fibre, vinyl ester.INTRODUCTION
Fibre reinforced polymers are increasingly shown interest from the engineering and structural view point. A lot of work is done and investigated on glass fibre reinforced composites. But glass fibre has a detrimental effect on the environment and hence there is a growing interest on bio fibres on the possibility of replacing glass fibres. These composites are being used for marine applications such as water storage vessels, pipelines, small boats etc. Researchers found that prolonged exposure of glass fibre composites caused degradation in flexural strength and modulus [1, 2]. It is observed that the polymer matrix gets degraded by a hydrolysis reaction of the unsaturated groups within the resin [3]. Vinyl ester composites show superior chemical stability in sea water atmosphere [4, 5]. The absorption of water into the macromolecular network of a thermoset matrix causes swelling and plasticization of the matrix [6- 8]. Researchers have reported that absorption of water (distilled or sea water) causes changes in the thermophysical and mechanical properties by plasticization and hydrolysis [9, 10]. It is reported that degree of degradation depends on the degree of crosslinking of the polymers [11]. Moisture may also affect the fibers. Experimental work is done on glass fibre reinforced in epoxy or vinyl ester resin. But much work is not done on banana fiber reinforced in polymer matrix and the composite exposed to sea water environment. In the present work banana fibre reinforced vinyl ester composite specimens were immersed in sea water and the moisture gain is noted and compared with distilled water moisture gain.
EXPERIMENTAL
Banana fibers were procured from Tamil Nadu - India. The fibers were knitted in the form of mats. The mats were alkali treated. Vinyl ester is procured from ECMAS India pvt ltd. Composites were made by hand layup process in an MS die with inner cavity of dimensions 200mm x 200mm x 10mm. These laminates were later oven cured. Composite specimens were later cut to size as per ASTM standards. The cut edges of the specimens were coated with a thin layer of adhesive.
Sea water absorption tests: These specimens were first weighed and then immersed in sea water taken from the coast of Visakhapatnam – India (Bay of Bengal). Specimens were periodically taken out of the water; the surface is wiped with a tissue paper and weighed in an electronic balance. The water uptake was plotted against square root of immersion time.
The moisture absorbed M (in %) is calculated using
The moisture absorbed M (in %) is calculated using where Wt is the measured weight of the specimen at time t and Wo is the initial dry weight of the specimen.
RESULTS AND DISCUSSION
The water absorption into the composite specimen may be considered to be following three different modes. The principal mode being the diffusion of water molecules into the microgaps of the resin, while the other processes being capillary action through the interfacial gap between the fibre and the resin and also through the fiber, and transport of water through the microcracks in the matrix. The diffusion, in most of the cases, follows the equation
where Mt is the moisture content at time t, Mmax is the maximum moisture content at saturation and k and n are constants. The diffusion coefficient is an important parameter in Fick‘law. This can be found out from the following equation
where Mt , Mmax and t are as denoted above, and h is the specimen thickness. The diffusion coefficient can be found out by considering the slope of the first portion of the curve between moisture gain and square root of time by the following equation.
where k is the initial slope of the plot. Fig.1 represents the percentage moisture gain plotted against square root of time. The analysis of diffusion mechanism and kinetics can be performed by modifying eqn.(1) as shown below
Fig.2 shows the graph plotted against log(Mt / Mmax) against log(t).
The straight line in Fig.2 shows the fitting of the experimental data to eqn. (3). The values of k and n resulting from the graph in fig.3 is found to be 0.001254 and 0.59 respectively. The value of ?n‘ suggests that the initial diffusion follows Fick‘s law. The diffusion coefficient was found to be 2.67 x 10-6 mm2 /sec.
Effect of sea water on mechanical properties:
Tensile properties: Tensile test is done according to ASTM D638. The tests were carried out in a Hounsfield tensometer model H20KW. The cross head speed is 1mm/min.
Flexural properties: The flexural test is done according to ASTM D790 in a universal testing machine by UNITED calibration corp. with a cross head speed of 0.5mm/min. Fig.4 shows the values of tensile strength, tensile modulus, flexural strength and flexural modulus of the composite specimens before and after immersion in sea water. Sea water diffuses into the composite specimen following the Fick‘s law of diffusion initially. With elapsing time, the salts get deposited into the microcracks preventing further seepage. Hence the rate of water absorption slows down with time. With the ingress of water, the mechanical properties decrease as understood from the graphs.
CONCLUSION
The water uptake increases rapidly during the initial stages. The absorption process follows the Fickian diffusion process during the initial stages but later it follows non-fickian diffusion process due to the deposition of the salts present in the water. Absorption of sea water causes the deterioration of mechanical properties. The tensile strength is found to decrease by 6.46 % and the flexural strength is found to decrease by 12.19 %. There is a decrease in the tensile modulus and flexural modulus of the specimen by 12.32% and 10.09%.
Englishhttp://ijcrr.com/abstract.php?article_id=2151http://ijcrr.com/article_html.php?did=21511. Kootsokoos A, Mouritz AP, ?Sea water durability of glass and carbon polymer composites. Compos. Sci. Technol. 64, 1503-1511 (2004)
2. Gellert EP, Turley DM, ?Seawater immersion ageing of glass fibre reinforced polymer laminates for marine applications.? Composites 30A, 1259 (1999)
3. Srinivas MV, Dvorak GJ and Prochazka P, ?Design and fabrication of submerged cylindrical laminates – II, Effects of fibre pre-stress. International journal of solids and structures. 36, 3945 – 3976 (1999).
4. Apicella A, Migliaresi C, Nicholais L and Roccotelli S, ?The water ageing of unsaturated polyester based composites: Influence of resin chemical structure, Composites. 14, 4, 387 – 392 (1983).
5. Dvorak GJ, Prochazka P, and Srinivas MV, ?Design and fabrication of submerged cylindrical laminates – I, International journal of solids and structures. 36, 3917 – 3943 (1999).
6. Bradley WL, (1995). J. Mater. Sci 30:5537. doi:10.1007/BF00351570.
7. Pomies F, Carlsson LA, Gillespie JW Jr (1995) ASTM STP 1230. philadelphia.
8. Springer GS (ed) (1981) Environmental effects on composite materials, Technomic, CA.
9. Ellyin F, Rohrbacher C, ?Effect of aqueous environment and temperature on glass fibre epoxy resin composites?. J. Reinf. Plast. Compos. 19(17), (2000)
10. Wood C, Bradley WL, ?Determination of the effect of sea water on the interfacial strength of an interlayer eglass/graphite/epoxy composite by in situ observation of transverse cracking in an environmental SEM. Compos. Sci. Technol. 57, 1033 – 1043, (1997)
11. Rutowska M, Krasowska K, Heimwoska A, Steinka E, Janik H, ?Degradation of polyurethanes in sea water?. Polym. Degrad. Stab. 76, 233 – 239 (2002)
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524137EnglishN2011July20General SciencesPERIOD-DOUBLING PHENOMENA IN A SIMPLE-3D CHAOTIC OSCILLATOR WITH A DIODE PAIR
English8995G. KandibanEnglish V. BalachandranEnglish S. ManimaranEnglishIn this paper, in order to show some interesting phenomena of third-order chaotic oscillator circuit with a
smooth cubic nonlinearity, different kinds of attractors, time waveforms and corresponding power spectra
of systems are presented, respectively. The perturbation transforms an unpredictable chaotic behavior into
a predictable chaotic or periodic motion via stabilization of unstable, aperiodic, or periodic orbits of the
strange chaotic attractor. One advantage of the method is its robustness against noise. A theoretical
analysis of the circuit equations is presented, along with experimental and numerical results.
EnglishAutonomous third-order chaotic circuit; Smooth cubic nonlinearity; chaos.http://ijcrr.com/abstract.php?article_id=2152http://ijcrr.com/article_html.php?did=2152Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524137EnglishN-0001November30General SciencesHOURLY OZONE CONCENTRATION PREDICTION USING NEURAL NETWORK MODEL
English9699G.GeethaEnglishThe aim of the present work is to provide a methodological procedure to forecast hourly Ozone concentrations using Artificial Neural Networks (ANNs). The study area is the urban center of Chennai, and the results are presented here. The model can predict the mean surface ozone based on the parameters like concentration of Nitrogen-di-oxide, temperature, relative humidity, sun spot number, wind direction and wind speed. The model can perform well both in training and independent periods. The achieved results were satisfactory.
EnglishArtificial Neural Networks surface ozone Air pollutionINTRODUCTION
Ground-level ozone air pollution is of great concern because of its adverse effects on human health and ecosystems (Poupkou et al.2008, Cristofanelli and Bonasoni 2009). Ground-level ozone is not emitted directly into the atmosphere. It results from photochemical reactions between oxides of nitrogen (NOx) and volatile organic compounds (VOCs) in the presence of sunlight (Pudasaine etal2006, Vingarzan and Taylor 2003, Clapp and Lenkin 2001, Sillman 1999). Present paper aims to develop a simple model using neural network technique based on the data which are easily available. The performance of the model is satisfactory both in training and independent period.
Area of Study and data
Ground level ozone concentration and nitrogen dioxide measurements were carried out in the urban site Chennai, Tamil Nadu, India. India is a tropical country urbanized district. Its irregular shape covers about 174 Km2 (M.Pulikesi et al., 2005). It is geographically positioned between 12º9´ and 13º9´ of the Northern latitude and 80º12´ and 80º19´of the eastern longitude.
MATERIALS AND METHODS
A portable Aeroqual series S200 ozone monitor was used. An Aeroqual series S200 ozone monitor is constructed to measure low and high ozone levels. Its ultra low concentration ozone head measures the ozone concentration from 0.000 to 0.500 ppm, and a high concentration ozone head measures the ozone Concentration from 0.50 to 20.00 ppm. Accuracy of a low concentration ozone head is ± 0.001 ppm (from 0 to 0.100 ppm); ± 10% (0.100 to 0.500 ppm), while that of a high concentration ozone head is ± 10% (from 0.20 to 2.00 ppm); ± 15% (from 2.00 to 20.00 ppm), the measurement unit being either ppm or μg/m3. The operating Temperature range is from – 5 o C to 50o C, relative humidity limits are 5% and 95%. Similar kind of NO2 sensor has been used for nitrogen dioxide measurement. A gas sensitive semiconductor (GSS) type is used to measure the ozone and nitrogen dioxide values. Gas sensitive sensor head is interchangeable and replaceable. LCD type display. The temperature range is from -400C to 1240C humidity range 0 to 100%. Wind speed is calculated using a wind vane. Wind velocity is measured using AM- 4201digital Anemometer. Measurement in range 0.4 – 3 m\s has resolution 0.1 m\s of accuracy ± (2% + 0.2 m\s). The ambient temperature and humidity are measured by Thermo Hydrometer. Temperature accuracy ± 0.1oCand humidity accuracy ± 5%. Sampling was carried out for five days from 13- 11-2010 to 17-11-2010 for every 1 hour interval.
Brief review of neural network technique
Neural Networks are signal processing systems that attempt to emulate the behavior of biological nervous systems by providing a mathematical model of combination of numerous basic blocks called neurons connected in a network. It is remotely analogous to living nervous system and hence its name. As input to the model, a historical set of significant meteorological data is used, whereas the output, ozone concentration is predicted by the model. The network is trained with the past data. By the proper choice of training sets, after the learning process, the trained network is capable of predicting the ozone concentrations as an output according to the inputs and internal structure of the network established during the learning period. The transfer function used here is the sigmoidal function. The ANN‘s are product of the artificial intelligence,which miming the neurons networks, allow expert systems and learning skills..(Benvenuto et al,2000) The process of learning the training set of patterns means the determination of the optimum weights which minimize the mean square error between the outputs in the output layer and the desired values. Most commonly used ?back-propagation learning algorithm? [Rumbelhart et al., (1986)] is used for the training. Initially random weights between ±0.5 are assigned to each weight as initial guesses. The weights are learned through an iterative process. During learning the weights are updated. When the network learns the training set of patterns well enough it can be used for determining the output values for the pattern with unknown outputs (Test period or prediction period).
RESULTS AND DISCUSSION
The data is separated for training of the network and the network was trained. The weight values were fixed. Remaining data was used for testing of the network. The results suggest that the Sunspot number is one of the best predictors for ozone prediction. Table 1 gives the correlation coefficient values of the set of values. The correlation coefficient values give the relationship among the various predictors. The result obtained is shown in the Fig. 1.Surface ozone - predicted versus actual values. As indicated by the results it provided the highest performance. This was due to enabling of the ozone by Sunspot number and the input parameters, resulting in improved training and thus improved prediction. Root Mean Square Error (RMSE) training data set 0.176988 % Root means Square Error (RSME) for testing data 21.168520 % The above results validate the proposed model.
CONCLUSION
Our results suggest that neural network technique is promising tools for modeling and prediction of ozone. Results can be summarized as follows;
(i) The ANN constructed models suggests high feasibility of the application of ANN technique for the prediction of hourly ozone within reasonable error bounds.
(ii) The predictive analysis suggest a considerable link among solar/ Sunspot cycle, related variability and ozone formation.
(iii) It is concluded that the above model can be used for Predicting surface ozone concentration with nitrogen dioxide, temperature, % relative humidity, Sunspot number, wind direction and wind speed as predictors.
Englishhttp://ijcrr.com/abstract.php?article_id=2153http://ijcrr.com/article_html.php?did=21531. Chudzynski S., Czyzewski A., Ernst K., Pietruczuk A., Skubiszak W., Stacewicz T., Stelmaszczk K., Szymanski A., Sowka I., Zwozdziak A., Zwozdziak J., Observation of ozone concentration during the solar eclipse. Atmospheric Research, 2001, Vol. 57, p. 43-49. ELSEVIER . ISSN 0169- 8095.
2. Clapp I., J., Jenkin M., E., Analysis of the relationship between ambient levels of O3 . NO2 and NOx as a function of NOx in the UK Atmospheric Environment. 2001. Vol. 35 p.0401-0405 ELSEVIER ISSN 1352- 2310.
3. Cristofanelli P., and Bonasoni P., Background ozone in the southern Europe and Mediterranean area: Influence of the transport and processes. Environmental Pollution, 2009, Vol. 157, p. 1399-1406. ELSEVIER. ISSN 0269-7491.
4. Poupko A., Symeonidis P., Lisaridis I., Melas D., Ziomas I., Yay O.D., Blis D. Effects of anthropogenic emission sources on maximum ozone concentration over Greece. Atmospheric Research, 2008, Vol. 89, p. 374-381. ELSEVIER.ISSN 1352- 2310.
5. Pudasainee D., Sapkota B., Manohar L.S., Kaga A., Kondo A., Inoue Y. Ground levels ozone concentration and its association with NOx and meteorological parameters in Katmandu valley, Nepal. Atmospheric Environment, 2006, Vol. 40, p. 8081-8087 ELSEVIER.ISSN 1352-2310.
6. Pulikesi M, P.Baskaralingam, D.Ilango, V.N.Raidu, V.Ramamoorthy, S.Sivanesan, Air quality monitoring in Chennai, India, in the summer of 2005. J.Hazard.Mater. 136(2006)589-596.
7. Rumbelhart, D.; Hinton, G.E.; Wiliams, R.J. (1986): Learning internal representation by error propagation, In parallel distributed processing Exploration in the Microstructure of Cognition, Vol I, Cambridge.
8. Sillman S., The relation between ozone, NOx and hydrocarbons in urban and polluted rural environments. Atmospheric Environment, 1999, Vol. 33, P. 1821-1845. ELSEVIER.ISSN 1352-2310.
9. Vingarzan R., Taylor B. Trend analysis of ground levels ozone in the greater Vancouver/Fraser Valley area of British Columbia. Atmospheric Environment, 2003, Vol. 37, p. 2159-2171. ELSEVIER.ISSN 1352-2310
10. Westmoreland E. J., carslaw N., Carslaw D., Gillah A., Bates E Analysis of air pollution within a street canyon using statistical and dispersion modeling techniques. Atmospheric Enviroment, 2007, Vol. 41, p. 9195-9205. ELSEVIER.ISSN 1352-2310.
11. www.aeroqual.com/gas sensitive sensor/14-10-2010
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524137EnglishN-0001November30TechnologyA STUDY ON THE DESIGN OF MICRO-LATHE FOR EDUCATION AND APPLICATION
English100114S.Syath AbuthakeerEnglish P.V. MohanramEnglish G. Mohan KumarEnglishAs the factory automation progresses, the number of specialized product is increasing rapidly. Previous years have been characterized by the growth of 3-D micro-components production. Now-a-days, machined parts are becoming progressively smaller. So, production of machinery which remains in a conventional size is often inappropriate for such products. The term ?micro factory‘‘ represents an entirely new approach to design and manufacture which minimizes production systems to match the siz of the parts they produce. The micro-lathe was one of key components in "Micro-factories" claiming "small machine tools for small mechanical parts?. There is an alternative to manufacture microcomponents by micro-machine tools and micro-manipulators using conventional mechanical techniques. In India, Robot, Micro-factory, several prototypes of micro-machine tools (MMTs) and micromanipulators (MMs) have been developed. Furthermore, there is an increased need for engineers trained
in micro-machine tool design and operation. This development necessitates thorough and systematic education in both industry and education institution. Inexpensive educational micro-machine tools will facilitate the required education in India. In this study we design and manufacture a prototype of an inexpensive LabVIEW controlled micro-lathe. This will facilitate the micro-machine tool education in India as these activities become active.
EnglishMicro-machine, micro-lathe, LabVIEW1 INTRODUCTION
1.1.RATIONALE AND STIPULATION OF THE MICRO-MACHINE TOOL
There is a great effort towards the miniaturization in the last few decades. We can see the effects of that trend in every aspect of our lives. From the laptops to the cellular phones, we always prefer the smallest one since the idea of ?the smaller the better? has penetrated into our minds and one can be equipped with more gadgets as miniaturization goes further. Miniaturization process of mechanical components started with micro fabricated sensors and was followed by micro fabricated parts and micro actuators. In recent years integration of micro components such as precision mechanisms, sensors, actuators and embedded electronic circuits into micro systems has become one of the most prominent research areas all over the world. When the micro components were first introduced, they were simple and could be naturally integrated directly into the product. However, developments in micro system technology resulted in a large variety of micro components made from dissimilar materials and technologies. These miniaturized products use even smaller components, and in more and more cases they are micro components with sizes of components less than one millimeter. A new bid to manufacture pieces with overall sizes smaller than 1mm using conventional mechanical technology was made in[1]. This proposal was based on the development of micro-factories (composed of micro-machine tools, micro-manipulators (MMs), assembly devices, etc.) to manufacture and assemble 3-D micro-devices employing conventional mechanical techniques. The micro-factories can help to reduce the consumption of resources (energy, materials and space); and can help to increase the productivity[2]. The micro-machine tools (MMTs) in such micro-factories must be sufficiently precise to produce components according to industrial and research demands. The conservation of energy has been the slogan for the past decade in order to reduce energy consumption. The conservation of energy by reducing the machine tool size for machining micro components is attaining popularity as micro-factory. Micro-factory can be said that it is a small manufacturing system for achieving higher throughput with less space and reduced consumption of both resource and energy via downsizing of production processes. The energy-saving effect factoriesareminiaturizedto1/2size.Theenergysaving effect is large when the size of the processing and assembly Equipment is extremely large compared to the dimensions of the products. As for watch manufacturing, the amount of Energy consumption may be reduced to approximately 30 Percent of the conventional factory by the half-miniaturization of the production systems [3].The term micro-factory represents an entirely new approach to design and manufacture that minimize production systems to match the size of the parts they produce. In the earliest attempt to turn the concept of micro-factory [4] in to a reality a micro-lathe smaller than a human palm was developed in 1996[5]. And it was the first big success for the further step into the concentration on process physics of micromachining including materials and micro structural effects, machine tools, tooling and sensing, workpiece and design issues, software and simulation tools, and other issues [6] micro factory. The first micro press was developed in the year 2000[7]. In the micro-world, the error sources of MMTs can be reduced by reducing their sizes [8],[9],[10] and [11]. Some prototypes were made in order to demonstrate the advantages of this proposal. Countries such as Japan, Ukraine and Mexico have developed micro-machines tools with overall sizes from 130 × 160 × 85 mm to 32 × 28 × 30 mm[12],[13], and [14]. In Mexico, the research in this area began in 1999 and the main goal was to develop micromechanical technology for automated production systems based on low-cost and high efficiency equipment and instrumentation [15].In the low-cost micro-equipment development, the principal challenge is to obtain high precision employing low-cost components. Advanced countries like Japan, Taiwan, Korea, Europe, Ukraine ,Mexico, Gemany and USA not only manufacture production micromachine tool but also educational micromachine tool in a balanced manner. As the economic development of India progresses toward becoming an advanced nation there is a need for an effective policy for science, engineering and technology and their education. Particularly, that for the machinery, automobile and electrical industries is more urgent. Because the resources, fund and technology for the domestic industry are not sufficient, and also technology protection policy in the level of advanced nation is nonexistent, there is a great deal of problems associated with the Indian Machine tool Industry. In particular, Micro-machine tool systems which are based on an industry are less manufactured in India. Therefore, it is an urgent problem that the Indian industry develops capability to manufacture integrated special tool system, such as Micro-factory ,Micro-machine tool, micromanipulators, Robots; Educational high level technicians for the special fields. Sufficient equipment and other necessary materials are needed for experiments essential for effective education. It is more suitable to use specially built micro machine tool for educational uses. In our country, certain educational institutes recognize this problem and utilize educational micro-machine tool, but this machinery depend on total import. Therefore it is expected that this research contributes towards micro-equipment education by domestic production of educational MICRO LATHE so it can reduce the import and foreign exchange and eventually manufacture of Micro-lathe for production. The necessity of educational Micro-machine tool is considered by this method: and an economical prototype micro-lathe is designed, manufactured and studied. In the low-cost micro-equipment development, the principal challenge is to obtain high precision employing low-cost components. For this reason, we have proposed to use the labVIEW control systems to increase the micromachine tools accuracy without increasing significantly the total device cost.
1.2 DEVELOPMENT OF MICRO-LATHE An interest to produce mechanical parts with sizes less than 1 mm arose worldwide in the 80‘s. Some methods based on micro-electronic technology were proposed; nowadays, these developments are called Micro Electro Mechanical Systems (MEMS) [8]. The microelectronic technology allows developing micromechanical components with simple shapes (two and a half dimensions), and the materials employed in this technology are silicon, silicon oxide, metallic films (mainly aluminum), and piezoelectric materials like quartz. These microdevices applications are encountered in many industries such as the automotive, the biomedical, electronics, computer, etc. The manufacture of micro-components employing micro-mechanical systems (microfactories) was proposed in the 90‘s as a new alternative to cover some of the micro-world applications where MEMS could not be applied. A research group from Japan proposed the development of tools that allow generating other kind of application which can be made from different materials and can have 3-D geometry shapes. The main goal was to create MMTs, micro-manipulators (MMs), etc. at a scale comparable with the size of the produced microcomponents. Their proposal consisted of transferring the conventional mechanical methods to the micro-world and to develop micro-factories able to produce micro-devices. A micro-factory contains several systems: a manufacturing system, an assembly system, a quality control system, a transport system, a maintenance system, and others. The microfactories allow a decrease in the consumption of energy, space, and resources[16]. The produced micro-components can be used in the watch industry, the automotive industry, medical facilities, biology investigations, etc. [16] and [17]. For example, in the medical field, the micro-equipment demands are: microscopy, diagnosis, non-invasive surgery, etc. in the industrial field for the development of microrobots to inspect inaccessible or dangerous places, pipe inspection, transportation machinery, archeological research, etc. Another interesting application field is the development of micro-actuators, for example: micro-grippers. for manipulation with living cells, microgenerators, micro-motors, etc.[13]. The first micro-machine tool was developed in the National Institute of Advance Industrial Science and Technology of Japan in 1996 [12]. Nowadays, there are many groups in different countries around the world such as Germany, Korea, Switzerland, Mexico, USA, etc. interested in this field [18], [19] and [20]. Researchers from the National Institute of Advance Industrial Science and Technology of Japan developed an automated micro-factory to produce components for micro-bearings in 2000 [21]. The assembly of micro-bearings was made in the same micro-factory with a semi automated process. Particularly, in Mexico, investigation in micromechanics began in 1999. The main goal is to create technology for automated micromechanical devices for production based on low-cost and high efficiency equipment and instrumentation. To achieve this goal, it was proposed to work out the micro-equipment as sequence of generations where the first generation of micro-equipment is produced by conventional machine tools. The microequipment of this generation will be able to produce the second generation of microequipment having smaller overall sizes than the previous one. Employing the second generation, it would be possible to produce the third generation of micro-equipment, and so on. The sizes of each new generation devices are smaller than the sizes of the prec++edent ones. This process can be repeated until the micromachines with overall sizes of some micrometers have been obtained [14]. Based on this study, the prototype of LabVIEW controlled Micro-lathe is developed for production maximized education efficiency, it can be learned easily and a disjointing and assembling are possible.
Englishhttp://ijcrr.com/abstract.php?article_id=2154http://ijcrr.com/article_html.php?did=2154