Research Journal of Chemical Sciences ______________________________________________ ISSN 2231-606X Vol. 4(6), 13-17, June (2014) Res. J. Chem. Sci. International Science Congress Association 13 Synthesis, Spectral, Electrochemical analysis and Screening for - Glucosidase inhibition of some complexes of Cobalt (II) and Ethylenediamine Tripath I.P., Mishra Mahendra Kumar, Tripathi Ruchita, Mishra Chinmayi, Kamal Arti, Shastri Laxmikant, Dwivedi Atul, Shukla Umesh Kumar and Pandeya Krishna Bhihari Mahatma Gandhi Chitrakoot Gramodaya Vishwavidyalaya, Chitrakoot, Satna, MP, INDIA Available online at: www.isca.in, www.isca.me Received 13th March 2014, revised 19th Apirl 2014, accepted 28th May 2014May Abstract Schiff bases, having amine group and their metal complexes are widely used for industrial purposes and also reveal a wide range of biological applications. This work describes the most promising biological activities. Cobalt metal complexes, the [Co(en)]2NO has most potent inhibitory activity among three complexes, having 784.12 µg/ml IC50 value while [Co(en)]2Cl shows mild inhibition i.e. 47.39±0.83 %. Keywords: Cobalt metal, diabetes mellitus, metallo-therapeutics, cyclic voltameter, -Glucosidase Inhibition. Introduction Diabetes is chronic metabolic and non communicable disease with multiple etiologies, is considered as one of the five leading causes of death in the world. The total number of people with diabetes is projected to rise from 171 million in 2000 to 366 million in 20301,2. Diabetes mellitus clinically are of types (Type I and Type II) and genetically heterogeneous groups of disorders characterized by hyperglycemia. The main cause of Type I diabetes mellitus is autoimmune destruction of -cells3,4. However etiopathogenesis of Diabetes type II is complex in nature but it is believed that due to impaired insulin secretion, resistance of insulin, genetic and environmental factors are responsible for causing this type of diabetes4-6. Metallo-therapeutics is an area of growing interest as is evident through the clinical trials that are being conducted worldwide for the usages of metals in therapeutics. Metallotherapy is a new therapeutic strategy to treat diabetes with metal complexes. Coulson and Dandona in 1980 that ZnCl stimulate lipogenesis in rat adipocytes similarly to the action of Insulin. Since many metal complexes have been synthesized, also being with different metals with different coordination modes7,10. The aim of present work is to synthesize, characterize and observe the electrochemical behavior of cobalt complexes in sodium perchlorate solution to access further -Glucosidase inhibitory activity of cobalt (II) complex with ethylenediamine. Material and Methods Chemicals: Water, CoSO.7HO, CoCl2.6HO, Co (NO.6HO, Potassium Bromide, p-nitrophenyl--D-glucopyranoside, were purchased from SRL, India and ethylenediamine, sodium perchlorate monohydrate from Alfa Acear, Great Britain. Acarbose, –glucosidase rat intestinal acetone powder was procured from Sigma Aldrich, USA. All solvent were HPLC grade, chemicals were A.R grade and used further any purification. Synthesis of Complex: The [Co (L)2+complex was prepared from three different salts of cobalt and ethylenediamine. 2 mM aqueous solution of metal salts was stirred in a beaker and 6mM of ethylenediamine were added drop by drop. With continuous string, a orange red colored solution were obtained, which were transferred in a petri dish to remove solvent in incubator at 45 °C. After few days a orange red colored complex [Co (en)] SO, [Co (en)] 2Cl and [Co (en)] 2NO (1, 2 and 3respective) solid obtained11. Infra Red Spectroscopy: Infrared (IR) spectra were obtained by the KBr method using a Bruker Alfa-T model Fourier transform (FTIR) spectrometer (Bruker Instrument, Germany).The spectrometer was equipped with a Globar IR source, KBr beam splitter, and detector. For each spectrum, 16 scans were obtained with the resolution of 4 cm-1. The obtained IR spectra were processed by means of the program OPUS 7.0. Cyclic Volta metric: The cyclic voltammetric measurements were carried out with a Metrohm Instrument (Germany) having an electrochemical cell with a three-electrode system. The reference electrode was an Ag/ AgCl. Platinum wire was used as a working electrode, while a platinum wire electrode used as an auxiliary electrode. The 3 mg of complex were dissolved in 25 ml supporting electrolyte (0.1 M Sodium perchlorate) solution. The volatammogram, peak position and area were calculated using NOVA 1.9 software. -Glucosidase Inhibition: -glucosidase inhibitory activity was performed following the modified method of Pistia Brueggeman and Hollingsworth12, 13. In brief, Rat-intestinal acetone powder was dissolved in 4 ml of 50 mM ice cold phosphate buffer and Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(6), 13-17, June (2014) Res. J. Chem. Sci. International Science Congress Association 14 sonicated for 6 minutes at 4°C. After vortexing for 20 minutes, the suspension was centrifuged (10,000 rpm, 4°C, 30 minutes) and the resulting supernatant was used for the assay. A reaction mixture containing 50 l of phosphate buffer (50 mM; pH 6.8), 20 l of rat -glucosidase (1 U/ml) and 25 l of sample of varying concentrations was pre-incubated for 5 min at 37°C, and then 25 l of 3 mM PNPG was added to the mixture as a substrate. After incubation at 37°C for 30 min, enzymatic activity was quantified by measuring the absorbance at 405 nm in a micro titer plate reader (Bio-TEK, USA). Acarbose was used as a positive control and water as negative control. Experiments were done in triplicates. IC50 value was quantified using formula, Y = 24.935ln(x) - 37.049, R˛ = 0.9883. The percentage of enzyme inhibition by the sample was calculated by the following formula: % Inhibition = {[(AC - AS)/AC] ×100} Where, AC is the absorbance of the control and AS is the absorbance of the tested sample. The concentration of an inhibitor required to inhibit 50% of enzyme activity under the mentioned assay conditions is defined as the IC50 value. Results and Discussion Infra Red Spectroscopy: In the IR spectrum of compound 1, the characteristic N-H bending vibration is observed as a strong bond at 1584 cm-1, which is a commonly observed fact for chelated ethylenediamine complex. The -NH stretching vibrations are found in the range 3110-3304 cm-1, the -CH stretching vibration 3050 and 2885 cm-1 and -CN stretching vibration are found in the range of 1190, 1140, 800 and 590 cm. IR spectra of complexes show a peak in the range of 3500-3400 cm-1 which denotes presence of -OH group. IR assignments and spectra of the complex 1, 2 and 3 are given in table 1 and figure 1, 2 and 3 respectively14. Table-1 Representing IR spectra of Complexes S.No Complexes Group Bond cm - 1 1 [Co(en)]SO-OH 3500-3400 -CH (Stretching Vibration) 3099 -NH (Bending vibration bonded with metal) 1616 ( merged) -NH (Stretching vibration) 3210 -CN (Stretching Vibration) 1056 2 [Co(en)]2Cl -OH 3500-3400 -CH (Stretching Vibration) 3089 (merged) -NH (Bending vibration bonded with metal) 1584 -NH (Stretching vibration) 3169 -CN (Stretching Vibration) 1055 3 [Co(en)]2NO-OH 3500-3400 -CH (Stretching Vibration) 3208 -NH (Bending vibration bonded with metal) 1581 -NH (Stretching vibration) 3169 -CN (Stretching Vibration) 1056 Figure-1 Representing IR Spectraof [Co (en)] SO Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(6), 13-17, June (2014) Res. J. Chem. Sci. International Science Congress Association 15 Figure-2 Representing IR Spectra of[Co (en)] 2Cl Figure-3 Representing IR Spectra of[Co (en)] 2NO3 Figure-4 Represents the voltamogram of [Co(en)2+ system Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(6), 13-17, June (2014) Res. J. Chem. Sci. International Science Congress Association 16 Figure 4 showes cylic voltamogram (CV) scaned three times cathodically in the potential region 0.00 to -0.750V using Ag/AgCl electrode in 0.1 M NaClO solution [Co(en)2+system. In this scan voltamogram showes only single reduction peak at -541.99 mV. Peak width half of complex were 0.14887 and peak (1/2) were 80.138 mV. -Glucosidase Inhibition: There are several cobalt (II) complex has been reported to have antidiabetic activity15,16. The inhibitory effects of acarbose (positive control), [Co(en)]SO, [Co(en)Cl, [Co(en)NO on -glucosidase activity were concentration dependent. From the result, the apparent IC50values (table 4) of the test compounds with respect to the glucosidase activity were estimated (figure 5 and 6). In comparison with acarbose, all test compounds except for [Co(en)]2Cl showed mild -GI effect (table 2 and 3). Table-2 Represents the Absorbance of Nitrophenol S.N Conc. in µg/ml Acarbose Error±SD Conc. of Complexes µg/ml Complex1 Error±SD Complex2 Error±SD Complex3 Error±SD 1 10 0.312 0.001 100 0.344 0.002 0.322 0.002 0.353 0.003 2 20 0.248 0.003 200 0.307 0.002 0.312 0.003 0.293 0.003 3 40 0.167 0.002 400 0.290 0.001 0.265 0.003 0.255 0.002 4 60 0.123 0.003 600 0.241 0.001 0.240 0.002 0.213 0.002 5 80 0.104 0.004 800 0.217 0.003 0.222 0.003 0.192 0.000 6 100 0.098 0.004 1000 0.186 0.002 0.205 0.003 0.168 0.003 Table-3 Represents the % inhibition of -Glucosidase S.N Conc. in µg/ml Acarbose Error±SD Conc. of Complexes µg/ml Complex1 Error±SD Complex2 Error±SD Complex3 Error±SD 1 10 19.96 0.29 100 11.48 0.535 17.22 0.51 9.17 0.65 2 20 36.16 0.64 200 21.08 0.514 19.71 0.65 24.59 0.78 3 40 56.72 0.39 400 25.36 0.392 31.96 0.65 34.53 0.59 4 60 68.03 0.82 600 37.96 0.392 38.22 0.39 45.24 0.44 5 80 73.01 0.92 800 44.22 0.680 42.93 0.68 50.64 0.00 6 100 74.12 0.97 1000 52.18 0.000 47.39 0.83 56.73 0.64 Figure-5 Represents the % Inhibition of - Glucosidase Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(6), 13-17, June (2014) Res. J. Chem. Sci. International Science Congress Association 17 Figure-6 Represents the % Inhibition of - Glucosidase by Acarbose Table-4 Represents the IC50 Value S.No Compounds IC 50 Value µg/ml 1 Acarbose 25.60 2 Complex1 927.89 3 Complex2 784.12 Conclusion The variety and extent of metal ions involvement has been recently appreciated but it has a very long history in toxicology, medicine and pharmacology. For instance, Cr, V, Mn, Cu, Ni, Co, Fe, Zn and Mo among the transition metals are very essential to life. In the present work we have synthesized cobalt complexes with ethylenediamine. The intense peak of IR at 1581 cm-1 support the synthesis of cobalt complex and cylic voltamogram represents only reduction peak at -541.99 mV. IC50 values represents the [Co(en)NO have good inhibitory activity among these three complexes References 1.Wild S., Roglic K., Green A., Sicree R. and King H., Global prevalence of diabetes, Estimation for the year 2000 and projections for 2030, Diabetes Care, 27, 1047–1053 (2004) 2.Eman A. Alam, Initiation of Pharmaceutical Factories depending on more Application of Biotechnology on some Medicinal Plants Review Article (In Vitro Production of some Antioxidant, Analgesic, Antibacterial, Antidiabetic agents), Research Journal of Recent Sciences, 1(ISC-2011),398-404 (2012)3.Thomas H.E, Kay T.W., Beta cell destruction in the development of autoimmune diabetes in the non-obese diabetic (NOD) mouse, Diabetes Metab. Res. Rev.16(4), 251-261 (2000)4.Iwahashi H., Itoh N., Yamagata K., Imagawa A., Nakajima H., Tomita K., Moriwaki M., Waguri M., Yamamoto K., Miyagawa J., Namba M., Hanafusa T., Matsuzawa Y., Molecular mechanisms of pancreatic beta-cell destruction in autoimmune diabetes: potential targets for preventive therapy, Cytokines Cell Mol. Ther.,4(1), 45-51 (1998)5.Leahy J.L., Pathogenesis of type 2 diabetes mellitus, Arch. Med. Res., 36(3), 197-209 (2005)6.Kumari M. and Jain S, Tannins: An Antinutrient with Positive Effect to Manage Diabetes, Research Journal of Recent Sciences, 1(12), 70-73, (2012)7.Kaku K., Pathophysiology of Type 2 Diabetes and Its Treatment Policy, J. M. A. J., 53(1), 41–46 (2010) 8.Coulston L., Dandona P., Insulin-like effect of zinc on adipocytes, Diabetes, 29(8), 665-667 (1980)9.Pandeya K.B., Tripathi I.P., Mishra M.K., Dwivedi N., Pardhi Y., Kamal A., Gupta P., Dwivedi N. and Mishra C., A Critical Review on Traditional Herbal Drugs: An Emerging Alternative Drug for Diabetes, I.J.O.C.,3(1), 1-22 (2013) 10.Tripathi I.P., Mishra M. K., Kamal A., Mishra C., Tripathi R., Shastri L. K., Pandeya K. B., Synthesis, Characterization of some Antidiabetic Copper Complexes with Ethylenediamine, Res. Jrn. of Chem. Sci.,3(12), 54-59 (2013)11.Beattie J.K., Conformational Analysis of Tris (ethylenediamine) Complexes, Acc. Chem. Res, 253-259 (1971)12.Pistia-Brueggeman G. and Hollingsworth R.I., A preparation and screening strategy for glycosidase inhibitors, Tetrahedron, 57, 8773-8778 (2001)13.Shinde J., Taldone T., Barletta M., Kunaparaju N., Hu B., Kumar S., Placido J., Zito S.W., Alpha-glucosidase inhibitory activity of Syzygium cumini (Linn.) Skeels seed kernel in vitro and in Goto-Kakizaki (GK) rats, Carbohydr. Res., 343, 1278-1281 (2008)14.http://people.cedarville.edu/student/s1209916/co_en.pdf (2014)15.Talba T., Shui X. W., Cheng Q., Tian X., Antidiabetic effect of glucosaminic acid-cobalt (II ) chelate in streptozotocin-induced diabetes in mice, Diabetes Metabolic Syndrome and Obesity: Targets and Therapy, , 137–140 (2011)16.Saker F., Ybarra J., Leahy P., Hanson R.W., Kalhan S.C., Ismail-Beigi F., Glycemia-lowering effect of cobalt chloride in the diabetic rat: role of decreased gluconeogenesis, Am. J. Physiol.,274(6 Pt 1), E984-91 (1998)