Bangladesh Journal of Pharmacology Volume: 12; Number 1; Year 2017 Cite this article as: Pandey AK, Kashyap PP, Kaur CD. Anti-inflammatory activity of novel Schiff bases by in vitro models. Bangladesh J Pharmacol. 2017; 12: 41-43. Anti-inflammatory activity of novel Schiff bases by in vitro models Sir, A comprehensive collection of enzyme activation, moderator release, cell relocation, tissue collapse and repair are involved in an inflammation reaction (Vane, 2000) for host defense and usually activated in most disease conditions. Currently research for new drugs or compounds which posses anti-inflammatory activity is going on which may lead to the discovery of new therapeutic agent which could be used to suppress the inflammation and in various disease conditions where the inflammation response in increase the disease process. An exhaustive investigation on different thia- diazole compounds have been done in recent years, many of which were found to posses wide spectrum of pharmacological actions. Moreover, various reports indicate that semicarbazides and thiadiazoles deriva- tives show anti-inflammatory (Labanauskas et al., 2001, Palaska et al., 2002) and analgesic (Amir and Shikha, 2004) activities. The present study was carried out to look into the anti-inflammatory activity of novel thiadiazole derivatives (7a-f) by different in vitro models for the first time. Test compounds were synthesized in laboratory and compounds were checked for purity by TLC, IR, 1H NMR and elemental analysis. Test compounds are as follows: 5-(2-Hydroxyphenyl)-2-{N-(4-methoxybenzylidene)-2- aminophenyl}-1,3,4-thiadiazole (7a), 5-(2-Hydroxyphe- nyl)-2-{N-(4-methoxybenzylidene)-2-aminophenyl}- 1,3,4-thiadiazole (7b), 5-(2-Hydroxyphenyl)-2-{N-(4-me- thoxybenzylidene)-3-aminophenyl}-1,3,4-thiadiazole (7c), 5-(2-Mercaptophenyl)-2-{N-(4-methoxybenzyl- idene)-3-aminophenyl}-1,3,4-thiadiazole (7d), 5-(2-Hy- droxyphenyl)-2-{N-(4-methoxybenzylidene) -4- aminophenyl}-1,3,4-thiadiazole (7e), 5-(2-Mercaptophe- nyl)-2-{N-(4-methoxybenzylidene) -4-aminophenyl}-1,3, 4-thiadiazole (7f). All the chemicals used were pur- chased from Ideal Chemicals, Raipur. All the chemicals were of Merck and Lobachem. The Instrument used was of Model UV-1800, Shimadzu, Japan and cuvette used was of 1 cm path length and made up of quartz. First model is the inhibition of protein denaturation which was evaluated by the methods with slight modification (Mizushima and Kobayashi, 1968; Sakat et al., 2010) with slight modification. 500 µL volume of 1% bovine serum albumin was added to 100 µL of test dilution. The mixture was kept at room temperature for 10 min, then heated for 15-20 min at 51°C. Then the solution was brought to normal room temperature and absorbance at 660 nm was recorded. Acetyl salicylic acid was used as standard. The process was carried out in triplicates and protein denaturation inhibition percentage was calculated using formula: % Inhibition = 100 - [(A1-A2) / A0] X 100 where, A1 is sample absorbance, A2 is product control absorbance and A0 is the absorbance of the positive control Second model is RBC membrane stabilization or Inhibition of RBC hemolysis. The blood was collected from healthy human volunteer, who had not taken any NSAIDS for last 2 weeks earlier to the experiment and mixed with equal volume of Alsever’s solution (2% dextrose, 0.7% sodium citrate, 0.5% citric acid and 0.4% NaCl). The mixture was centrifuged for 10 min at 3,000 rpm. The obtained solution was washed with saline three times. RBC layer was collected and diluted with phosphate buffer saline (PBS) to make 10% v/v solution (Sadique et al., 1989, Saket et al., 2010). 100 µL of 10% RBC solution was added to 100 µL of test dilution. The resulting solution was heated at 56°C for 30 min followed by centrifugation at 2,000 rpm for 8-10 min at room temperature. Clear supernatant was collected, and absorbance was recorded at 560 nm. Membrane stabilization percentage was calculated by the method as described elsewhere (Shinde et al., 1999, Sakat et al., 2010). Third model is proteinase inhibition assay. Trypsin inhibition was evaluated by the method described elsewhere (Oyedepo and Femurewas, 1965; Sakat et al., 2010). 100 µL of 1% bovine serum albumin was added to 100 µl of test dilution. The mixture was incubated at room temperature for 5-6 min. Reaction was inhibited by adding 250 µL of trypsin followed by centrifugation. The supernatant was separated and absorbance was recorded at 210 nm. Acetylsalicylic acid was used as standard and proteinase inhibition percentage was calculated Inhibition of albumin denaturation by test compounds was studied. Selected test compounds were effective in A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2017; 12: 41-43 Journal homepage: www.banglajol.info Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088; DOI: 10.3329/bjp.v12i1.29675 Letter to the Editor http://www.bioxbio.com/if/html/BANGL-J-PHARMACOL.html inhibiting heat induced albumin denaturation. IC50 of most potent 7f was observed as 147.6 µg/mL. Results are shown in Table I. Acetylsalicylic acid was used as standard anti-inflammation drug. Inhibition of RBC hemolysis or RBC membrane stabilization effect by test compounds was studied. The compounds inhibited the heat induced hemolysis of RBCs. IC50 of 7f was observed as 167.1 µg/mL. Proteinase inhibition activity of test compounds was studied. The test compounds exhibited significant anti-proteinase activity. Maximum inhibition was observed in 7f and IC50 were observed as 186.3 µg/mL. The main cause of inflammation is denaturation of protein. Anti-inflammatory drugs like phenybutazone have been found to posses ability to thermally induce protein denaturation (Mizushima and Kobayashi, 1968). The ability of the test compound to inhibit protein denaturation was studied as a part of study on the mechanism of the anti-inflammatory activity. The test compounds showed relationship as increase in % inhibition with increase in concentration. To further prove the mechanism of anti-inflammatory action of test compounds, Stabilization of RBC membrane was studied. All compounds effectively inhibited the heat induced hemolysis. The results provide evidences for membrane stabilization effect of the test compounds as an additional mechanism for their anti-inflammatory effect. Due to the resemblance of RBC membrane with lysosomal membrane, this effect may possibly inhibit the release of neutrophils’ lysosomal content at the location of inflammation. The compounds suppressed the RBC haemolysis. Even though specific mechanism of membrane stabilization is yet to be known, it may be possible that the test compounds show the effect by maintaining the surface area/volume ratio of the cell. This could be done by growth of membrane and or contraction of the cell and interaction with membrane proteins (Shinde et al., 1999). Proteinase has been implicated in arthritics. Neutrophils are a wealthy source of proteinase which carries many serine proteinases in their lysosomal granules. Formerly it was reported that proteinase of leukocytes play an important role the formation of tissue break in inflammation reactions and considerable level of protection was brought by proteinase inhibitors (Das and Chatterjee, 1995). The test compounds exhibited significant antiproteinase activity. Ajit Kumar Pandey, Pranita P. Kashyap and Chanchal Deep Kaur Shri Rawatpura Sarkar Institute of Pharmacy, Kumhari, Durg, Chhattisgarh 490042, India. Corresponding author: email: ajitpandey588@gmail.com References Amir M, Shikha K. Synthesis and anti-inflammatory, analgesic, ulcerogenic and lipid peroxidation activities of some new 2- [(2,6-dichloroanilino)phenyl]acetic acid derivatives. Eur J Med Chem. 2004; 39: 535-45. Das SN, Chatterjee S. Long term toxicity study of ART-400. Indian Indg Med. 1995; 16: 117-23. Labanauskas L, Kalcas V, Gaidelis P, Brukstus A, Dauksas V. Synthesis of 3-(3,4-dimethoxyphenyl)-1H-1,2,4-triazole-5- thiol and 2-amino-5-(3,4-dimethoxyphenyl)-1,3,4-thiadiazole derivatives exhibiting anti-inflammatory activity. Pharmazie 2001; 56: 617. Mizushima Y, Kobayashi M. Interaction of anti-inflammatory drugs with serum proteins, especially with some biologi- cally active proteins. J Pharm Pharmacol. 1968; 20: 169-73. Oyedepo OO, Femurewas AJ. Anti-protease and membrane stabilizing activities of extracts of Fagra santhoxiloides, Olax subscorpioides and Tetrapluera tetraptera. Ln. J Pharm. 1995; 33: 65-69. Palaska E, Sahin G, Kelicen P, Durlu NT, Altinok G. Synthesis and anti-inflammatory activity of 1-acylthiosemicarbazides, 1,3,4-oxadiazoles, 1,3,4-thiadiazoles and 1,2,4-triazole-3- thiones. Farmaco. 2002; 57: 101-07. 42 Bangladesh J Pharmacol 2017; 12: 41-43 Table I Anti-inflammatory activity of novel Schiff bases by in vitro models Concentration (µg/mL) % Inhibition In vitro models 7a 7b 7c 7d 7e 7f 50 Albumin denaturation 23.4 ± 1.0 24.8 ± 0.4 25.8 ± 0.6 25.9 ± 0.7 28.1 ± 0.3 29.8 ± 0.4 Membrane stabilization 17.8 ± 0.6 17.3 ± 0.6 21.6 ± 0.4 29.1 ± 0.7 32.3 ± 1.3 33.5 ± 1.1 Proteinase inhibition 17.4 ± 0.5 19.5 ± 0.4 20.9 ± 0.9 21.5 ± 0.4 23.8 ± 0.8 25.9 ± 0.6 100 Albumin denaturation 38.9 ± 0.5 38.6 ± 0.3 40.6 ± 0.7 40.9 ± 0.3 42.9 ± 0.1 45.4 ± 1.0 Membrane stabilization 22.3 ± 0.7 29.8 ± 0.4 33.7 ± 0.5 37.1 ± 0.7 42.6 ± 0.7 45.0 ± 1.1 Proteinase inhibition 33.5 ± 0.7 32.8 ± 0.8 33.9 ± 0.2 34.7 ± 0.6 38.0 ± 0.2 40.9 ± 0.4 200 Albumin denaturation 47.8 ± 0.3 49.9 ± 0.3 51.7 ± 0.3 50.9 ± 0.3 53.7 ± 0.6 58.1 ± 0.5 Membrane stabilization 38.2 ± 0.9 32.6 ± 0.5 42.8 ± 0.9 44.3 ± 0.6 47.8 ± 0.8 53.2 ± 0.3 Proteinase inhibition 36.2 ± 0.4 38.6 ± 0.8 41.5 ± 0.6 40.6 ± 0.4 47.9 ± 0.8 50.8 ± 0.6 Results are expressed as Mean ± SEM membrane system. Fitoterapia 1989; 60: 525-32. Sakat S, Juvekar AR, Gambhire MN. In vitro antioxidant and anti-inflammatory activity of methanol extract of Oxalic corniculata Linn. Int J Pharm Sci. 2010; 2: 146-56. Shinde UA, Phadke AS, Nari AM, Mungantiwar AA, Dikshit VJ, Saraf MN. Membrane stabilization activity: A possible mechanism of action for the anti-inflammatory activity of Cedrus deodara wood oil. Fitoterapia 1999; 70: 251-57. Vane JR. The mechanism of action of anti-inflammatory drugs. In: Advances in Eicosanoid Research. Serhan CN, Perez HD (eds). Vol 31. Berlin, Springer-Verlag, 2000, pp 1-23. Bangladesh J Pharmacol 2017; 12: 41-43 43 DatePrinted: This article was downloaded by you on: Mar 02, 2017