Sanjaykumar et al/RRBB 2(1&2), 2011, 31-39 ISSN 22297154

Research and Reviews in Biomedicine and Biotechnology [RRBB] Volume [2], Issue [1&2], 2011, 31-39 Available online at www.rrbb.in

Research paper Synthesis, Characterization and Antioxidant activity of Tetrazoloquinoline thiocarbohydrazide derivatives Sanjaykumar BD 1, Jayakumar Swamy BHM 1*, Pramod N 1, Patel Ashish Haribhai 1, Shivakumar B 2, Shivakumar Hugar 2
Post Graduate Studies, Paul Ehrlich Laboratory, Dept. of Pharmaceutical Chemistry, SCS College of Pharmacy, Harapanahalli-583131, Karnataka, India 2 BLDEA College of Pharmacy, Bijapur-586103, Karnataka, India *Corresponding author Email: drbhmjs@yahoo.co.in; Ph: +919448329098 Article history: Received on 18.01.2011; Accepted on 11.03.2011 Copyright: 2011 rrbb.in ABSTRACT A series of ten novel N-[tetrazolo[1,5-a]quinolin-4-ylmethylidene]thiocarbohydrazide derivatives were synthesized by the reaction of N-[tetrazolo[1,5-a]quinolin-4ylmethylidene]thiocarbohydrazide with various substituted aromatic aldehydes by refluxing with dimethyl formamide. The newly synthesized compounds were characterized by their IR, 1 HNMR and Mass spectral studies and have been evaluated for their anti-oxidant (In-vitro) activity by standard methods using ascorbic acid as reference standard drug. From the results it is concluded that, the compounds exhibited significant to moderate antioxidant activity. Key words: 2-Chloroquinoline-3-carbaldehyde, Schiff bases, Tetrazoloquinoline-4carbaldehyde, Thiocarbohydrazide, Anti-oxidant activity. INTRODUCTION Nitrogen containing heterocycles are one of the most extensively synthesized and screened compounds as they show diverse pharmacological activities. The development of tetrazole chemistry has been largely associated with wide scale of applications of these classes of compounds in medicine, biochemistry, agriculture and also large number of medicinally important tetrazole heterocyclic incorporated drugs approved by the FDA [1]. Quinolines and their derivatives are important constituents of pharmacologically active synthetic compounds. The quinoline nucleus also occurs in the structure of numerous naturally occurring alkaloids which have been associated with a broad spectrum of biological activities [2]. The fusion of quinoline to the tetrazole ring is known to increase the biological activity [3]. The tetrazole group, which is considered as a carboxylic group pharmacore, possesses a wide range of biological activities. Several substituted tetrazoles have been shown to possess antiinflammatory [4-5], antimalarial [6], anticancer [7], antifungal [8-11], anticonvulsant [12], antibacterial [13-14], vasorelaxing [15], antiviral [16] and CNS dispersant [17] activities. The tetrazole functionality plays an important role in medicinal chemistry, primarily due to its ability to serve as bioequivalent of the carboxylic acid group. Heterocyclic synthesis of tetrazole derivatives is obviously an important task in modern medicinal chemistry. They have been associated with a broad spectrum of various biological activities. The schiff bases derived from thiocarbohydrazide are known to exhibit diverse activities like antibacterial [18], www.rrbb.in 31
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Sanjaykumar et al/RRBB 2(1&2), 2011, 31-39 anticarcinogenic [19], antiviral [20], herbicidal [21] and antifungal [22] activities. As part of our continuous effort to find better antioxidant agents in this area, a series of new N[tetrazolo[1,5-a]quinolin-4-ylmethylidene]thiocarbohydrazide derivatives were synthesized as outlined in the scheme-1. MATERIALS AND METHODS Chemicals All chemicals used were of analytical grade, from SD Fine. Melting points of all the synthesized compounds were determined by open capillary tube method, these are uncorrected. The purity of all compounds was checked by TLC was run on Silica Gel G plates using Chloroform and Ethanol (9.5:0.5). Spots were visualized using iodine vapour chamber. IR spectra were recorded on Shimadzu IR spectrophotometer by using KBr pellets technique. 1HNMR was recorded on Bruker AMX 60 MHz spectrophotometer by using DMSO as solvent. Synthesis of N-[tetrazolo[1,5-a]quinolin-4-ylmethylidene]thiocarbohydrazide derivatives [6a-j] Synthesis of Tetrazolo[1,5-a]quinoline-4-carbaldehyde [2]: In a clean and dry round-bottom flask, 2-Chloroquinoline-3-carbaldehyde (0.001M, 0.191g) synthesized according to the literature [23] was taken in absolute ethanol (5ml), ptoluenesulphonic acid (0.001M, 0.190g) and sodium azide (0.0015M, 0.0975g) were added and the reaction mixture was refluxed for 65 hours at 125-1350C in oil bath. After completion of the reaction, the reaction mixture was poured into ice cold water and the resulting precipitate was filtered, dried, and recrystalized from Dimethyl formamide (DMF) as whitish light yellow needle shaped crystals (Yield 76 %, Melting point (MP) 240-242C). Synthesis of Thiocarbohydrazide [3] [24]: In a clean dry round-bottomed flask, a vigorously stirred solution of 100% hydrazine hydrate (1 mol, 50 ml) in water 30 ml, carbon disulphide (0.2 mol, 15 ml) was added drop wise. The temperature of solution raised to 60 C. The reaction mixture was refluxed for 30 minutes, then cooled in ice bath. The separated thiocarbohydrazide was filtered, washed with ethanol or ether and dried (Yield 12g, MP 170-172C). Synthesis of N-[tetrazolo[1,5-a]quinoline-4-ylmetylidene]thiocarbohydrazide [4] [25]: In a round bottom flask, containing the solution of Tetrazolo[1,5-a]quinoline-4-carbaldehyde (0.01 mol, 1.98 gm) in 1,4-Dioxane (25 ml) was added an equivalent amount of thiocarbohydrazide (0.01 mol, 1.06 gm). The reaction mixture was refluxed for 8 hours at 155-160C, partially concentrated and cooled. The separated solid product was filtered, dried and recrystalized from DMF (Yield 64.3 %, MP 224-226C). Synthesis of N-[tetrazolo[1,5-a]quinolin-4-ylmethylidene]thiocarbohydrazide derivatives [6a-j]: N-[tetrazolo[1,5-a]quinoline-4-ylmethylidene] thiocarbohydrazide (0.1M, 0.286g) in DMF (50ml) was taken in a round bottom flask and the various aromatic aldehydes (0.1M) were added. The reaction mixture was refluxed for 24-30 hours at 160-1700C and cooled, then the poured into ice cold water. The separated solid was filtered, recrystalized from aqueous DMF to give a pure light brown crystalline powder.

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Sanjaykumar et al/RRBB 2(1&2), 2011, 31-39 Antioxidant activity of the synthesized compounds Reducing power assay and nitric oxide radical scavenging activity were used to assess antioxidant capacity of the test compounds [26-28]. Reducing power model: The standard drug and test compounds were dissolved in DMF so as to get different concentrations (25, 50, 75, 100 and 125g/ml). This was mixed with 2.5ml of (pH 6.6) 0.2 mol phosphate buffer and 2.5 ml of 1% potassium ferricyanide. The mixture was incubated at 50 0C for 20 minutes. 2.5 ml of 10 % trichloroacetic acid was added to the mixture, which was then centrifuged for 10 minutes at 1000 rpm. 2.5 ml upper layer of solution was mixed with 2.5ml of distilled water and 0.5 ml of 0.1% ferric chloride. The absorbance was measured at 700nm. The absorbance of the blank was also measured in similar manner. The results were compared with ascorbic acid, which was used as a reference standard antioxidant. The percentage increase in absorbance was calculated by using the formula: Percentage increase in absorbance = OD (Test) OD (Control) / OD (Test) x 100 Nitric oxide radical scavenging model: Nitric oxide (NO) radicals were generated from Sodium nitroprusside solution at physiological pH. 2ml (10mM) of Sodium nitroprusside were mixed with 0.5ml of phosphate buffer (0.01M, pH 7.4) and 0.5ml of different concentrations (25-125g/ml) of drug. The mixture was incubated at 25 0C for 150 min. Then 0.5ml of incubated solution was mixed with 1ml of Griesss reagent (1% Sulphanilamide, 5% O-Phosphoric acid) allowed to stand for 5 min at room temperature. Then 1ml Naphthyl ethylene diamine dihydrochloride (0.1 % w/v) was added to produce pink chromophore and allowed to stand for 30 min at 250C. Absorbance was read at 540 nm. The percentage inhibition of nitric oxide was calculated by using following formula: Percentage inhibition = Vc Vt / Vt X 100, where Vt is mean absorbance of test and Vc is mean absorbance of control group. RESULTS Physical data namely melting point, yield, molecular formula and molecular weight of the synthesized compounds is given in Table-1. Table 1: Physical data of synthesized compounds Compound Sl. No MP (C) Yield (%) Molecular formula Molecular weight Code 1 6a 110-112 57 C19H16N8OS 404.4 2 6b 164-166 26 C18H14N8OS 390.4 3 6c 100-102 57 C19H16N8S 388.4 4 6d 160-162 54 C18H13N8SCl 408.8 5 6e 136-138 66 C18H13N8SF 392.4 6 6f 84-86 67 C18H14N8S 374.4 7 6g 86-88 58 C19H16N8O2S 420.4 8 6h 78-80 67 C18H13N9O2S 419.4 9 6i 118-120 66 C20H19N9S 417.4 10 6j 142-144 56 C20H18N8O2S 434.4

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Sanjaykumar et al/RRBB 2(1&2), 2011, 31-39

Synthesis of Tetrazoloquinoline thiocarbohydrazide derivatives: SCHEME

Compounds 6a-e 6f-j

R 4-OCH3 H

R 2-OH

R 4-CH3

R 4-Cl

R 4-F

4-(OH), 3-NO2 4-N(CH3)2 3,4(OCH3) 2 3-OCH3

Spectral data 6c: IR (KBr) cm-1: 2838 (C-H), 1619 (C=N), 1509 (C=C), 970 (N-N), 1252 (C=S), 3370 (NH). 1HNMR (DMSO) ppm: 2.37 (s, 3H, CH3), 7.320-8.134 (m, 9H, Ar-H), 9.295 (s, 1H, S=C-NH), 8.657 (s, 1H, -CH=N). ESIMS (m/z): 388 (M+). 6e: IR (KBr) cm-1: 2849 (C-H), 1632 (C=N), 1508 (C=C), 963 (N-N), 1227 (C=S), 3295 (NH). 1HNMR (DMSO) ppm: 7.228-8.368 (m, 9H, Ar-H), 9.127 (s, 1H, S=C-NH), 8.716 (s, www.rrbb.in 34

Sanjaykumar et al/RRBB 2(1&2), 2011, 31-39 1H, -CH=N). ESIMS (m/z): 392 (M+). 6f: IR (KBr) cm-1: 2848 (C-H), 1656 (C=N), 1493 (C=C), 958 (N-N), 1213 (C=S), 3370 (NH). 1HNMR (DMSO) ppm: 6.406-7.884 (m, 10H, Ar-H), 9.075 (s, 1H, S=C-NH), 8.719 (s, 1H, -CH=N). ESIMS (m/z): 374 (M+). 6h: IR (KBr) cm-1: 2850 (C-H), 1617 (C=N), 1528 (C=C), 960 (N-N), 1214 (C=S), 3271 (NH). 1HNMR (DMSO) ppm: 7.251-8.362 (m, 9H, Ar-H), 8.889 (s, 1H, S=C-NH), 8.686 (s, 1H, -CH=N). ESIMS (m/z): 419 (M+). 6j: IR (KBr) cm-1: 2837 (C-H), 1623 (C=N), 1509 (C=C), 962 (N-N), 1262 (C=S), 3372 (NH). 1HNMR (DMSO) ppm: 3.827 (s, 6H, OCH3), 7.063-8.414 (m, 8H, Ar-H), 9.147 (s, 1H, S=C-NH), 8.631 (s, 1H, -CH=N). ESIMS (m/z): 434 (M+). Reducing power assay: All the synthesized compounds (6a-j) were assessed for their in vitro antioxidant activities using the reducing power assay at various concentrations. Ascorbic acid was used as reference standard. The antioxidant activity of tested compounds 6a, 6b and 6i showed significant activity. The results reveal that 6c, 6f, 6g, 6h and 6j possesses moderate activity and 6d and 6e gives mild antioxidant activity when compared to control (Table 2). Nitric oxide Scavenging: All the synthesized compounds (6a-j) screened for their in vitro antioxidant activity using the nitric oxide scavenging method at various concentrations. Ascorbic acid was used as reference standard. The antioxidant activity of tested compounds 6a, 6b and 6i showed significant activity. The results reveal that 6c, 6f, 6g, 6h and 6j possesses moderate activity and 6d and 6e gives mild antioxidant activity when compared to control (Table 3). In both the model the same compounds have shown similar anti-oxidant activity. Compounds namely 6a, 6b and 6i which contain p-methoxy phenyl, o-hydroxyl phenyl and p-N-dimethyl amino phenyl rings have shown significant anti-oxidant activity. These compounds contain electron donating group at para, ortho and para position respectively. This shows that the presence of electron donating group at para and meta position of phenyl ring might have favored the activity. ACKNOWLEDGMENT The authors are thankful to shri. Sha. Bra. Chandramouleshwara Shivacharaya Swamiji President, T. M. A. E. Society, Harapanahalli. Sri. T.M. Chandrashekaraiah Secretary, T.M.A.E. Society, Harapanahalli through Principal S.C.S College of Pharmacy, Harapanahalli, for providing necessary facilities to carryout this work.

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Sanjaykumar et al/RRBB 2(1&2), 2011, 31-39 REFERENCES 1. Myznikov LV, Hrabalek A, Koldobskii GI. Drugs in tetrazole series. Chem. Het. Comp. 2007; 43: 1-9. 2. El-Subbagh HI, Abu-Zaid SM, Mahran MA, Badria FA, Alofaid AM. J. Med.Chem. 2000; 43: 2915-2921. 3. Gupta R, Gupta AK, Paul S. Synthesis of new 4-(4,5-diphenyl-1h-imidazol-2-yl) tetrazolo[1,5-a]quinolines from tetrazolo[1,5-a]quinolines. Ind. J. Chem. 2000; 39B: 847. 4. Farghaly AM, Bekhit AA, Park JY. Arch. Pharm. Pharm. Med. Chem. 2000; 333: 5357. 5. Bekhit AA, El-Sayed OA, Aboulmagd E, Park JY. Tetrazolo[1,5-a]quinoline as a potential promising new scaffold for the synthesis of novel anti-inflammatory and antibacterial agents. Eur. J. Med. Chem. 2004; 39: 249-55. 6. Vlabhov R, St Parushev, Vlahov J, Nickel P, Snatzke G. Synthesis of some new quinoline derivatives-potential antimalarial drugs. Pure. Appl. Chem. 1990; 62(7): 1303-06. 7. Dalla Via L, Gia O, Gasparotto V, Ferlin MG. Discovery of a new anilino-3Hpyrrolo[3,2-f] quinoline derivative as potential anti-cancer agent. Eur. J. Med. Chem. 2007; XX: 1-6. 8. Sharma K, Fernandes PS. A facile route to the synthesis of substituted 2hydrazinoquinolines and its ring closer reaction and their biological activity. Ind. J. Het. Chem. Oct-Dec 2005; 15: 161-168. 9. Rajendra SP, Karvembu R. Synthesis and antifungal activities of schiff bases derived from 3-amino-2H-pyrano[2,3-b]quinoline-2-ones. Ind. J. Chem. Jan 2002; 41B: 222-224. 10. Kategaonkar AH, Pokalwar RU, Sonar SS, Gawali VU. Synthesis, in vitro antibacterial and antifungal evaluations of new a-hydroxyphosphonate and new a-acetoxyphosphonate derivatives of tetrazolo [1,5-a] quinoline. Eur. J. Med. Chem. 2010; 45: 11281132. 11. Gupta M, Paul S, Gupta R. The one pot synthesis of antifungal active 9-substituted-3aryl-5H,13aH-quinolino[3,2-f][1,2,4]triazolo[4,3-b], [1,2,4]triazepines. Ind. J. Chem. Apr 2010; 42B: 475-481. 12. Zie Z, Chai K, Piao H, Kwak K, Quan Z. Synthesis and anticonvulsant activity of 7alkoxyl-4,5-dihydro-[1,2,4]triazolo [4,3-a]quinolines. Bioorg. Med. Chem. Lett. 2005; 15: 4803-05. 13. Gupta R, Gupta AK, Paul S. Microwave-assisted synthesis and biological activities of some 7/9-substituted-4-(3-alkyl/aryl-5,6-dihydro-s-triazolo[3,4-b][1,3,4] thiadiazole-6yl)-tetrazolo[1,5-]quinolines. Ind. J. Chem. Nov 2000; 39B: 847-852. 14. Bawa S, Kumar S. Synthesis of schiffs bases of 8-methyl-tetrazolo[1,5-a]quinoline as potential anti-inflammatory and antimicrobial agents. Ind. J. Chem. Jan 2009; 48B: 142145. 15. Ferlin MG, Chiarelotto G, Antonucci F, Caparrotta L, Froldi G. Mannich bases of 3Hpyrrolo[3,2-f]quinoline having vasorelaxing activity. Eur. J. Med. Chem. 2002; 37: 427434. 16. Benard C, Zouhiri F, Normand-Bayle M, Danet M, Desmaele D, Leh H. Linker-modified quinoline derivatives targeting HIV-1 integrase: synthesis and biological activity. Bioorg. Med. Chem. Lett. 2004; 14: 2473-76. 17. Shukla JS, Saxena S. Indian Drugs. 1980; 18:15. 18. Baseer MA, Jadhav VD, Phule RM, Archana YV, Vibhute YB. Orient. J. Chem. 2000; 16: 553. 19. Moubaraki B, Murray KS, Ranford JD, Xu XY. Chem. Commun., 1998; 353. 20. Blumenkopf TA, Harrington JA, Koble CS, Bankston DD, Morrsion RW, Bigham EC, Styles VL, Spector T. J. Med. Chem, 1992; 16: 2306. 21. Pathak P, Jolly VS, Sharma KP. Orient. J. Chem. 2000; 16: 161.

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Sanjaykumar et al/RRBB 2(1&2), 2011, 31-39 22. Chohan ZH, Pervez H, Khan KM, Supuran CT. J. Enzyme Inhib. Med. Chem. 2005; 20: 81. 23. Meth-Cohn O, Narine B and Rarnowski B. A versatile new synthesis of quinolines and related fused pyridines part. The synthesis of 2-chloroquinoline-3-carbaldehydes. J.C.S. Perkin I, 1520-1529. 24. Audrieth LF, Scott ES, Kippur PS. J. Org. Chem. 1954; 19: 733. 25. Bekhit AA, El-Sayed OA, Aboulmagd E, Park JY. Eur. J Med. Chem. 2004; 39: 249-255. 26. Krishnaraju AV, Rao CV, Rao TVN, Reddy KN and Trimurtulu G. In vitro and In vivo Antioxidant Activity of Aphanamixis polystachya Bark. Ame. J. Infe. Dis. 2009; 5 (2): 60-67. 27. Hossain MA, Shaha SK and Aziz F. Antioxidant potential study of some synthesized Nheterocycles. Bangla. Med. Res. Counc. Bull. 2009; 35: 49-52. 28. Antolovich M, Prenzler PD, Patsalides E, McDonald S and Robards K. Methods for testing antioxidant Activity. Analyst 2002; 127: 183198.

Sources of support: None declared Conflict of interest: None declared

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Table 2: Antioxidant activity of synthesized compounds by reducing power assay

Absorbance (Mean SEM) of triplicates Sl. No. 01 02 03 04 05 06 07 08 09 10 11 12 Compound Code 25 g/ml 50 g/ml 75 g/ml 0.180 + 0.001 0.213+ 0.001*** 0.202+ 0.003** 0.199+ 0.002** 0.186+ 0.003** 0.189+ 0.002* 0.211+ 0.004* 0.196+ 0.002** 0.194+ 0.004** 0.209+ 0.008** 0.192+ 0.003*** 0.210+ 0.001*** 0.234+ 0.002*** 0.217+ 0.002*** 0.221+ 0.002*** 0.215+ 0.003** 0.207+ 0.003* 0.213+ 0.004* 0.209+ 0.003** 0.225+ 0.004** 0.217+ 0.005** 0.203+ 0.001*** 0.203+ 0.010* 0.274+ 0.001*** 0.243+ 0.004*** 0.255+ 0.003*** 0.216+ 0.004** 0.241+ 0.003* 0.246+ 0.004* 0.220+ 0.002** 0.253+ 0.001** 0.240+ 0.001** 0.261+ 0.004*** 0.234+ 0.001** 0.318+ 0.016*** 0.306+ 0.006*** 0.304+ 0.005*** 0.288+ 0.006** 0.272+ 0.004* 0.277+ 0.004* 0.269+ 0.003** 0.281+ 0.006** 0.309+ 0.003** 0.297+ 0.006*** 0.263+ 0.003** 0.349+ 0.001*** 0.326+ 0.004*** 0.330+ 0.001*** 0.309+ 0.002** 0.301+ 0.004* 0.296+ 0.003* 0.311+ 0.004** 0.312+ 0.003** 0.323+ 0.004** 0.339+ 0.010*** 0.326+ 0.004** 17.22 12.22 10.55 3.33 5.00 17.22 8.88 7.78 16.12 6.67 16.66 30.00 20.56 22.78 19.44 15.00 18.33 16.11 25.00 20.56 12.78 12.78 100 g/ml 125 g/ml 25 g/ml %Increase in Absorbance 50 g/ml 75 g/ml -------100 g/ml 125 g/ml

Control Standard (Ascorbic acid) 6a 6b 6c 6d 6e 6f 6g 6h 6i 6j

52.22 35.00 41.66 20.00 33.89 36.67 22.21 40.56 33.33 45.00 30.00

76.67 77.78 68.89 60.00 51.11 53.89 49.44 56.11 71.67 65.00 46.10

93.88 81.12 83.33 71.67 67.22 64.45 72.78 73.32 79.44 88.83 81.11

The values are MEAN+., n=3, *p<0.01 **p<0.001 ***p<0.0001 vs control.

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Table 3: Antioxidant activity of synthesized compounds by Nitric oxide scavenging determination

Absorbance 75 g/ml 0.093+0.003 0.062+ 0.002*** 0.064+ 0.001*** 0.063+ 0.001*** 0.063+ 0.003** 0.076+ 0.001* 0.075+ 0.004* 0.068+ 0.003** 0.073+ 0.001* 0.067+ 0.003** 0.064+ 0.001*** 0.066+ 0.003** % Inhibition 100 g/ml 0.058+ 0.001*** 0.060+ 0.002*** 0.059+ 0.005*** 0.060+ 0.002** 0.069+ 0.002* 0.067+ 0.001* 0.062+ 0.002** 0.070+ 0.004* 0.066+ 0.001** 0.061+ 0.001*** 0.065+ 0.002** 125 g/ml 0.053+ 0.001*** 0.054+ 0.001*** 0.055+ 0.003*** 0.059+ 0.002** 0.063+ 0.004* 0.061+ 0.001* 0.057+ 0.001** 0.066+ 0.002* 0.058+ 0.004** 0.055+ 0.001*** 0.059+ 0.002** 25 g/ml 25.67 22.36 20.77 13.41 4.49 3.33 14.81 1.08 19.23 20.77 20.77 50 g/ml 75 g/ml -------38.80 34.78 32.85 30.98 12.04 17.72 27.39 13.41 27.39 30.98 24.00 50.00 45.31 47.61 47.61 22.36 24.00 36.76 27.39 38.80 45.31 40.90 60.34 55.00 57.62 55.00 34.78 38.80 50.00 32.85 40.90 52.45 43.07 75.41 72.22 69.09 57.62 47.61 52.45 63.15 40.90 60.34 69.09 57.62 100 g/ml 125 g/ml

Sl. No. 01 02 03 04 05 06 07 08 09 10 11 12

Compound Code Control Standard (Ascorbic acid) 6a 6b 6c 6d 6e 6f 6g 6h 6i 6j

25 g/ml 0.074+ 0.003*** 0.076+ 0.001*** 0.077+ 0.001*** 0.082+ 0.002** 0.089+ 0.003* 0.090+ 0.005* 0.081+ 0.002** 0.092+ 0.006* 0.078+ 0.002** 0.077+ 0.002*** 0.077+ 0.002**

50 g/ml 0.067+ 0.002*** 0.069+ 0.001*** 0.070+ 0.005*** 0.071+ 0.004** 0.083+ 0.002* 0.079+ 0.004* 0.073+ 0.003** 0.082+ 0.006* 0.073+ 0.005** 0.071+ 0.001*** 0.075+ 0.001**

The values are MEAN+ n=3, *p< 0.01 **p< 0.001 ***p< 0.0001 vs control.

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