Study On The Synthesis, Anti-hyperglycemic Activity And Toxicity Of Novel Organic Chromium Complexes

Diabetes mellitus is a disorder with high abnormal blood glucose levels caused by the failure of the body to produce enough insulin, or the inability of the insulin to make glucose accessible to blood cells. Diabetes mellitus has been identified as one of the three diseases (cardiovascular diseases, caners and diabetes mellitus) difficult to cure. This has therefore triggered the research into secondary anti-diabetic functional foods or nutrient supplement as a measure to help cure the disease. Chromium(Ⅲ) as one of the important trace elements in the body has shown significantly anti-diabetic activity. The lack of chromium in the body system can result in the decline in insulin secretion and increases blood glucose levels. As a result of the low absorption rate of chromium salts, it has become necessary to find possible mechanisms such as the designing and development of new organic chromium complexes to help address the menace. Chromium picolinate is said to the most commonly applied dietary supplement. However, it has been reported that chromium picolinate exhibits genotoxicity and cytotoxicity which is likely caused by the ligand (picolinate). Hence the safety aspect of chromium picolinate in the body has been a matter of concern to the majority.In order to reduce the toxicity and improve the bioactivity of chromium, the organic ligand with less or no toxicity has drawn the attention of researchers.In this study, organic ligands including L-malic acid, stachyose, and taurine were selected. The aim of the study was to determine the effects of chromium malate complex, chromium stachyose complex and chromium taurine complex on alloxan-induced diabetic rats. The specific objectives of the study include; (a) to investigate the anti-hyperglycemic activity of five organic chromium complexes (chromium malate complex, chromium rutin complex, chromium folate complex, chromium stachyose complex and chromium taurine complex) administered to alloxan-induced diabetic rats, (b) to evaluate the acute and sub-acute oral toxicity of chromium malate complex, chromium rutin complex and chromium folate complex on the rabbits, and (c) to study the transportation and conversion of the chromium malate complex on alloxan-induced diabetic rats.Propolis, as the functional component of secondary anti-diabetic functional foods has been used over the world. In the domestic market for example, propolis accounts for 29.6% of the domestic functional foods; second to chromium compounds (45.3%). In order to investigate further the additional benefits of anti-diabetic activity and protective effects of liver injury on diabetes, this aspect of the study focused on evaluating the anti-diabetic effect of the combination of chromium malate complex and propolis in alloxan-induced diabetic mice and to test the acute oral toxicity of the combination.The stepwise experimental procedures and the results of the study is given below:(1) Chromium malate complex, chromium stachyose complex and chromium taurine complex were synthesized in a single step reaction in aqueous solution by chelating chromium (Ⅲ) with L-malic acid, stachyose, and taurine respectively. The structures of the chromium complexes were determined using infrared,UV-visible and atomic absorption spectroscopy and elemental analysis. The results of this study (1) indicated that the coordination sites of chromium malate complex, chromium stachyose complex and chromium taurine complex were hydroxyls in the carboxyl groups, hydroxyls and amino respectively. The molecular formulas of the three chromium complexes were inferred as Cr2C12H22O20, Crt24H48O27 and CrN6C12S6H54O24, respectively.In the case of second experiment (2), anti-hyperglycemic activities of chromium malate complex, chromium rutin complex, chromium folate complex, chromium stachyose complex and chromium taurine complex were examined in alloxan-induced diabetic mice with daily oral gavage for a period of two weeks at the dose of 0.5-3.0 mg Cr/kg. b.w. The results indicated that chromium malate complex, chromium rutin complex, chromium folate complex and anti-diabetic activity resulted in percentage reduction of blood glucose levels of 45.91%,29.1% and 34.7%, respectively. However, after a two-week period of administrating the mice with the chromium stachyose complex and chromium taurine complex, the effect on anti-diabetic activity of the rabbits were not significant. The significant increase in the glycogen levels of diabetic rabbits which were treated by chromium malate complex, chromium rutin complex, chromium folate complex were determined. This however, indicates that the ligand of the trivalent chromium compounds markedly affected its efficacy. Not all the trivalent chromium compounds possessed equal efficacy. Chromium malate complex, chromium rutin complex and chromium folate complex had hypoglycemic effect on alloxan-induced diabetic mice, while chromium stachyose complex and chromium taurine complex did not.In further investigation (3), ie the safety toxicological evaluation (the first step and the second step) of 3 chromium complexes,Acute toxicities of chromium malate complex, chromium rutin complex and chromium folate complex were tested using ICR mice with a single oral gavage and observed for a period of two weeks. CrCl3·6H2O was used as the positive control in this test. For the acute toxicities of chromium malate complex, chromium rutin complex and chromium folate complex tested using ICR mice with a single oral gavage (3), LD50 (median lethal dose) for CrCl3·6H2O obtained was 2.39 g/kg body weight (95% confidence intervals:1.776-3.218 g/kg), that of chromium rutin complex and chromium folate complex were greater than 5.0 g/kg each. The minimum lethal dose for both chromium malate complex and chromium folate complex were over 5.0 g/kg b.w., while that of chromium rutin complex was 2.0 g/kg b.w.Three chromium (Ⅲ) complexes with different ligands (rutin, folic acid or L-malic acid) were compared to examine whether they have similar effect on sub-acute toxicity status. ICR mice were orally administered with chromium rutin complex, chromium folate complex and/or chromium malate complex at doses of 6 and 36 mg Cr/kg bodyweight/day for a period of 28 days. The results showed that oral administration of all these chromium complexes do not cause adverse effects on body weight, organ weights, hematology and serum biochemical parameters, hepatic and renal lipid peroxidation and cellular 8-OHdG levels. However, supplemental chromium folate complex or folic acid, not chromium malate complex or chromium rutin complex could induce abnormalities in metabolism of Zn. This can be inferred from the increase in renal Zn concentration and a decrease in hepatic Zn concentration.Collectively, the ligands affected the metal-toxicity of chromium in their complexes. In the acute toxicity study, the three chromium complexes were "non toxic". The minimum lethal dose for chromium malate complex and chromium folate complex was above 5.0 g/kg while that of chromium rutin complex was 2.0 g/kg b.w. Chromium malate complex and chromium rutin complex were safe in the sub-acute toxicity status while chromium folate complexes were not since chromium folate complex induced abnormalities in metabolism of Zn in the body.Following the investigation in (3), additional study (4) where pharmacokinetic study was performed using non-diabetic rats after intravenous administration at a single dose of 500 Crμg/kg b.w. Chromium levels in the blood stream, tissue samples, excreta (urine and faeces) were determined using ICP method. The pharmacokinetic parameters were obtained from this study. The results in this study indicated that chromium malate complex leaves the blood stream after intravenous administration for 30 min, and the elimination rate of chromium was around 85% in the blood stream. The complex leaves of chromium malate in the blood stream ends up appearing in the urine and entering tissue cells. For liver and kidney examined, the chromium content (livers and kidneys) is maximal 1 h after injection. After 36 h, however, no chromium was left in the livers and kidneys. Urine and fecal samples collected over time intervals of 30 min to 36 h after injection of the complex revealed that chromium loss of urine and fecal occurs rapidly for the first 2 h after injection and continues at a substantially lower rate over the next 34 h. The excretion rate of chromium in the urine and fecal was 82.4% after 36 injections.In order to determine the interaction between chromium (Ⅲ) malate complex with the high molecular mass components of the human serum albumin and apo-transferrin, a fifth (5) study was embarked upon. In this study, ultraviolet and fluorescence spectra as well as isothermal titration calorimetry was used. Cr3+ was binded with human serum albumin and apo-transferrin. The results showed that the interaction of Cr3+ with the human serum albumin and apo-transferrin obstructed atπ→π* transition of benzene rings (the bind sites of the tryptophan and tyrosine residues) cause the increase of the absorbance at 225 nm and decrease at 280 nm. The interaction also quenched the fluorescence of the two proteins. The interactions between Cr3+with the human serum albumin and apo-transferrin were mainly hydrogen bonds and van der Waals.To obtain the additional benefits of anti-diabetic activity and protective effects of liver injury on diabetes, anti-diabetic effect and acute oral toxicity of a combination of chromium (Ⅲ) malate complex and propolis were assessed (6). The anti-diabetic effect of the combination of chromium (Ⅲ) malate complex and propolis was determined and compared with chromium (III) malate complex and propolis only in alloxan-induced diabetic mice through daily oral gavage for 2 weeks. Acute oral toxicity of the combination of chromium (Ⅲ) malate complex and propolis was tested in ICR mice at the dose of 1.0-5.0 g/kg body weight with a single oral gavage and also observed for two weeks. The results therefore indicated that the increase in anti-diabetic efficacy and the protective effects of liver injury for diabetes of chromium malate complex combined with propolis was significant. In acute toxicity study, LD50 (median lethal dose) values for the combination was greater than 5.0 g/kg body weight. The combination might represent the nutritional supplement with potential therapeutic value to control blood glucose and exhibit protective effects of liver injury for diabetes and non-toxicity in acute toxicity.Taken together; three chromium complexes with natural active organic ligands have been synthesized. Chromium malate complex represent an optimal chromium supplement among those chromium complexes with potential therapeutic value to control blood glucose in diabetes and non-toxicity in acute and sub-acute toxicity. The fates of distribution and excretion, and transportation mechanism have been elaborated. The combination of chromium (Ⅲ) malate complex and propolis represent the nutritional supplement with potential therapeutic value to control blood glucose and exhibit protective effects of liver injury for diabetes and non-toxicity in acute toxicity.