Research Journal of Chemical Sciences ______________________________________________ ISSN 2231-606X Vol. 3(12), 54-59, December (2013) Res. J. Chem. Sci. International Science Congress Association 54 Synthesis, Characterization of some Antidiabetic Copper Complexes with Ethylenediamine Tripathi I.P., Mishra Mahendra Kumar, Kamal Arti , Mishra Chinmayi , Tripathi Ruchita, Shastri Laxmi Kant and Pandeya Krishna Bihari 3 Department of Physical Sciences, Faculty of Science and Environment, Mahatama Gandhi Chitrakoot Gramodaya Vishvavidyalaya, Chitrakoot, Satna, MP, INDIA Department of Biotechnology, Mahatama Gandhi Chitrakoot Gramodaya Vishvavidyalaya, Chitrakoot, Satna, MP, INDIA Mahatama Gandhi Chitrakoot Gramodaya Vishvavidyalaya, Chitrakoot, Satna, MP, INDIA Available online at: www.isca.in, www.isca.me Received 27th November 2013, revised 4th December 2013, accepted 18th December 2013 Abstract Biological trace metals such as iron, zinc, copper, and manganese are essential for human health. Development of inorganic pharmaceutical agents is very interesting creation in field of medicinal inorganic chemistry and these agents are being used to treat various chronic diseases with the help of essential elements and other biological trace metals. Metallotherapy is an expanding of interest in the research field of treatment of diabetes mellitus. Especially, orally active anti-diabetic and anti- metabolic syndrome copper complexes have been developed and progressed since last decades, where several highly potent anti-diabetic copper complexes with different coordination structures have quite recently been disclosed, using experimental diabetic animals and enzyme inhibitarory action (-glucosidase inhibition) also . In the present study we have synthesized, characterized and evaluate % of - glucosidase of Cu (II) metal complexes and concluded that [Cu (en)] 2NO (complex ) shows lowest IC50 value 0.475 mg/ml while [Cu (en)] 2Cl (Complex ) shows moderate activity. Keywords: Chronic diseases, metallotherapy, anti-diabetic, anti-metabolic syndrome and -Glucosidase Inhibition. Introduction Diabetes mellitus (DM), a metabolic disorder related with multiple etiologies, is 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 2030 if successful strategies are not implemented for its prevention and control. Diabetes mellitus is a clinically and genetically heterogeneous group of disorders, characterized by hyperglycemia. Several pathogenic processes are involved in the development of diabetes. These are destruction of the -cells of the pancreas from inadequate insulin secretion and/or diminished tissue responses to insulin at one or more points in the complex pathways of hormone action. Deficient supply of insulin causes abnormalities in carbohydrate, fat, and protein metabolism and these results in disability and premature death 3, 2. Metallotherapy is a new therapeutic strategy to treat diabetes with metal complexes. It is first studied by Coulson and Dandona in 1980 that ZnCl stimulate lipogenesis in rat adipocytes similarly to the action of Insulin. In three decades there are many researchers reported insulin-mimetic activity, glucosidase and -amylase inhibition with different coordination of different ligand with transition metals. The development of modern chemotherapy is based on metals and metal complexes which play a key role in modifying the pharmacological properties of known drugs after coordinating to a metal. The resulting precursors allow these drugs to be released in a controlled fashion and at specific location. This approach may lead to the rescue of drugs that have failed because of poor pharmacology or high toxicity. For example, complexation of nonsteroidal anti-inflammatory drugs to copper overcomes some of the gastric side effects of these drugs. In the present work we are synthesizing the copper complexes to evaluate their -glucosidase activity. Material and MethodsChemicals: Water, DMSO, CuSO.5HO, CuCl, Cu(NO.HO, Potassium Chloride, Potassium Bromide, p-nitrophenyl--D-glucopyranoside were purchased from SRL, India and ethylenediamine 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[Cu (L) 32+ complex was prepared from three different salts of copper and ethylenediamine. 2 mM aqueous solution of metal salts was stirred in a beaker and 6 mM of ethelenediamine were added drop by drop. With Research Journal of Chemical Sciences ___________________________________________________________ISSN 2231-606X Vol. 3(12), 54-59, December (2013) Res. J. Chem. Sci. International Science Congress Association 55 continuous string, 2-3 ml of ethyl alcohol was added for precipitation. A deep blue colored solution were obtained, which were transferred in a petri dish to remove solvent in incubator at 45\rC. After few days a deep blue colored complex [Cu (en) 3] SO, [Cu (en) 3] 2Cl and [Cu (en) 3] 2NO (1, 2 and 3 respective) solid obtained. 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 supporting electrolyte 25 ml of 0.01 M solution of KCl solution. The volatammogram, peak position and area were calculated using NOVA 1.9 software.–Glucosidase Inhibition : Method for determination of Glucosidase was adopted from Misra S. et al.Rat intestinal acetone powder (Sigma chemicals,USA) was sonicated properly in normal saline (100:1 w/v) and after centrifugation at 3000 rpm × 30 mins the supernatant was treated as crude intestinal Glucosidase. 50 l various dilutions in DMSO (0.1mg /ml solution) were mixed and incubated with 50 l of enzyme in a 96-well microplate for 5 mins. Reaction mixture was further incubated for another 10 mins with 50 l substrate (5 mM, p-nitrophenyl--D-glucopyranoside) prepared in 100 mM phosphate buffer (pH~ 6.8) and release of nitrophenol was read at, 405 nm spectrophotometerically (MultimodeSynergyH4 micro plate reader, BioTek instrument, inc. Winoosci, VT, USA). All the samples were run in triplicate and acarbose was taken as standard reference compound. Several dilutions of primary solution (5mg/ml DMSO) were made and assayed accordingly to obtain concentration of the test sample required to inhibit 50 % activity (IC50) of the enzyme. Quantification was performed with respect to the standard curve of acarabose (Y = 26.63X + 46.26, R² = 0.958) results were expressed as milligram of acarbose equivalent per ml of extract. Percent -Glucosidase inhibition was calculated using following equation.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 1581 cm-1, which is a commonly observed fact for chelated ethylenediamine complex. The N-H stretching vibrations are found in the range 3110-3304 cm-1 and the C-H stretching vibration 2962 and 2885 cm-1. IR assignments and spectra of the complex 1, 2 and 3 are given in table 1 and figure 1, 2 and 3 respectively. Electrochemical studies of Complexes: Figure 4 shows cyclic voltammogram (CV) scanned cathodically in the potential region between +0.00 and -1.00 V vs Ag/AgCl in 0.1 M KClsolution [Cu(II)en2+ system. In this scan range, the CVs show a single reduction peak at -512.7 mV (B1) in the forward sweep and three overlapping oxidation waves A, A and Arespectively at -285.6, -175.8 and - 65.9 mV at 2 mV/s (table 2 and figure 4). It must be mentioned that extension of negative potential limit to – 0.8 V did not result in any other reduction peak except BTable-1 Representing the band assignment for complex 2 and 3 S. No. Complex Group Band (cm - 1 ) 1 [Cu(en)] SO N-H (bending) bounded with metal 1581 N-H (stretching) 3110-3304 C-H (stretching) 2962 and 2885 2 [Cu (en) 3] 2Cl N-H (bending) bounded with metal 1582 N-H (stretching) 3110-3304 C-H (stretching) 2963 and 2885 3 [Cu (en) 3] 2NON-H (bending) bounded with metal 1583 N-H (stretching) 3110-3304 C-H (stretching) 2962 and 2887 Research Journal of Chemical Sciences ___________________________________________________________ISSN 2231-606X Vol. 3(12), 54-59, December (2013) Res. J. Chem. Sci. International Science Congress Association 56 Figure-1 IR Spectra of [Cu(en)]SO4 Figure-2 IR Spectra of [Cu (en)]2Cl Figure-3 IR Spectra of [Cu(en)]2NO3 Research Journal of Chemical Sciences ___________________________________________________________ISSN 2231-606X Vol. 3(12), 54-59, December (2013) Res. J. Chem. Sci. International Science Congress Association 57 Table-2 Oxidation and reduction peaks S.No. Name of Peak Peak (1/2) Peak position V 1 B -0.054234 -0.5127 2 A 0.040929 -0.28564 3 A 0.027677 -0.17578 4 A 0.025151 -0.065918 Figure-4 Cyclic volatammogram of [Cu (en)] species –Glucosidase Inhibition: A lot of literatures were reviewed and found that there are several complexes have been prepared to evaluate their anti diabetic activity. The development of a clinically useful metallopharmaceutics, the research of copper complexes on the long-term toxicity including side effects, clear-cut evidence of target molecule for the in vivo as well as in vitro pharmacological action and good pharmacokinetic property are essential9-11. Many copper complexes have been prepared to examine for their -glucosidase inhibitory activity. We have examined the three complexes of ethylenediamine with copper (II) for their % -glucosidase inhibition Table 3 demonstrate the IC50 value of Acarbose and metal complexes. Table 4 shows the absorbance of nitrophenol and Table 5 represents the % -glucosidase inhibition and fogire-5 and figure-6 were plotted according these values respectively. Table-3 IC50 value of Complexes S.No. Name of Complex IC50 value in mg/ml 1 Acarbose 0.1403 2 [Cu (en)] SO (Complex ) 0.6085 3 [Cu (en)] 2Cl (Complex ) 0.801 4 [Cu (en)] 2NO (Complex ) 0.4755 Research Journal of Chemical Sciences ___________________________________________________________ISSN 2231-606X Vol. 3(12), 54-59, December (2013) Res. J. Chem. Sci. International Science Congress Association 58 Table-4 The Absorbance of Nitrophenol S.No. Conc. in mg/ml Absorbance of Nitrophenol Acarbose Error±SD Complex1 Error±SD Complex2 Error±SD Complex3 Error±SD 1 0.1 0.155 00 0.249 0.001 0.269 0.001 0.257 0.0006 2 0.2 0.132 0.01 0.224 0.003 0.244 0.001 0.213 0.002 3 0.4 0.118 0.001 0.208 0.004 0.187 0.002 0.162 0.003 4 0.6 0.108 0.002 0.166 0.004 0.183 0.001 0.138 0.001 5 0.8 0.094 0.004 0.145 0.005 0.177 0.002 0.126 0.005 6 1.0 0.079 0.004 0.129 0.004 0.168 0.002 0.114 0.004 Table-5 % of - glucosidase inhibition S.No. Conc. in mg/ml % of - glucosidase inhibition Acarbose Error±SD Complex1 Error±SD Complex2 Error±SD Complex3 Error±SD 1 0.1 45.80 00 28.65 0.001 22.92 0.001 26.36 0.0006 2 0.2 53.85 0.01 35.82 0.003 30.08 0.001 38.96 0.002 3 0.4 58.74 0.001 40.40 0.004 46.42 0.002 53.58 0.003 4 0.6 62.24 0.002 52.43 0.004 47.56 0.001 60.46 0.001 5 0.8 67.13 0.004 58.45 0.005 49.28 0.002 63.90 0.005 6 1.0 72.38 0.004 63.04 0.004 51.86 0.002 67.33 0.004 Figure-5 Representing graph between concentration (mg/ml) and absorbance Research Journal of Chemical Sciences ___________________________________________________________ISSN 2231-606X Vol. 3(12), 54-59, December (2013) Res. J. Chem. Sci. International Science Congress Association 59 Figure-6 % inhibition curve of -Glucosidase v/s Concentration of sampleConclusion Sharp and intense peak at 1581 cm-1 in IR spectrum shows the formation of [Cu (en) ] complexes. The electrochemical behavior of Cu (II)-(en) system in 0.01 M KCl solution complex as it comprises electroxidation and electroreduction of copper (0), copper (I) and copper (II) species. All three complexes possess -glucosidase inhibition activity, among them [Cu (en)] 2NO have the highest -glucosidase inhibition, having IC50 value 0.4755 mg/ml. In this complex the % inhibition of -glucosidase may be due to the presence of NO2-species. Acknowledgments The authors (Prof. I. P. Tripathi and Mahendra Kumar Mishra) express their gratefulness to the MPCST, Bhopal, India, for the financial support in the forms of a project (6053/CST/RandD/2011) and a Junior Research Fellow. 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