Comparing anti-hyperglycemic activity and acute oral toxicity of three different trivalent chromium complexes in mice

Food and Chemical Toxicology 50 (2012) 1623–1631

Contents lists available at SciVerse ScienceDirect

Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox

Comparing anti-hyperglycemic activity and acute oral toxicity of three different trivalent chromium complexes in mice Fang Li a, Xiangyang Wu b,⇑, Yanmin Zou c, Ting Zhao a, Min Zhang b, Weiwei Feng c, Liuqing Yang b,⇑ a

School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Rd., 212013 Zhenjiang, Jiangsu, China School of Chemistry and Chemical Engineering, Jiangsu University, 301 Xuefu Rd., 212013 Zhenjiang, Jiangsu, China c School of Pharmacy, Jiangsu University, 301 Xuefu Rd., 212013 Zhenjiang, Jiangsu, China b

a r t i c l e

i n f o

Article history: Received 26 August 2011 Accepted 9 February 2012 Available online 18 February 2012 Keywords: Chromium complexes Coordinated ligands Diabetes Anti-hyperglycemic activity Acute oral toxicity

a b s t r a c t Three different ligands (rutin, folate and stachyose) of chromium(III) complexes were compared to examine whether they have similar effect on anti-hyperglycemic activity as well as the acute toxicity status. Anti-hyperglycemic activities of chromium rutin complex (CrRC), chromium folate complex (CrFC) and chromium stachyose complex (CrSC) were examined in alloxan-induced diabetic mice with daily oral gavage for a period of 2 weeks at the dose of 0.5–3.0 mg Cr/kg. Acute toxicities of CrRC and CrFC were tested using ICR mice at the dose of 1.0–5.0 g/kg with a single oral gavage and observed for a period of 2 weeks. Biological activities results indicated that only CrRC and CrFC could decrease blood glucose level, reduce the activities of aspartate transaminase, alanine transaminase, alkaline phosphatase, and increase liver glycogen level. In acute toxicity study, LD50 values for both CrRC and CrFC were above 5.0 g/kg. The minimum lethal dose for CrFC was above 5.0 g/kg, while that for CrRC was 1.0 g/kg. Anti-diabetic activity of those chromium complexes was not similar and their acute toxicities were also different. CrFC represent an optimal chromium supplement among those chromium complexes with potential therapeutic value to control blood glucose in diabetes and non-toxicity in acute toxicity. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Diabetes mellitus is a common disease caused by absolute or relative absence of insulin. The prevalence of diabetes is increasing due to dietary changes, aging, urbanization, and increasing prevalence of obesity and physical inactivity (Moreira et al., 2010). According to International Diabetes Federation (IDF), the world prevalence of diabetes among adults (aged 20–79 years) was 285 million representing 6.4% in 2010. This figure is estimated to increase up to 439 million (7.7%) by 2030 (Shaw et al., 2010). Diabetes mellitus is a disorder with abnormally high blood glucose levels caused by the failure of the body to produce enough insulin, or insulin is unable to make glucose accessible to cells. Poor glucose control brings about progressive complications in various body functions, and affects mineral status such as chromium (Krejpcio et al., 2011). Chromium has shown significantly beneficial affectivity in the insulin system (Anderson, 2003; Vincent et al., 2001). Research has shown that chromium facilitates insulin signaling and chromium supplementation improves systemic insulin sensitivity ⇑ Corresponding authors. Tel./fax: +86 511 88791800 (X. Wu), +86 511 88791180 (L. Yang). E-mail addresses: [email protected] (X. Wu), [email protected] (L. Yang). 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2012.02.012

and glycemic control in diabetic patients (Jain and Kannan, 2001; Sharma et al., 2011). Spectroscopic and crystallographic investigations indicate that molecular basis of chromium insulin interaction emphasized role of chromium in glucose metabolism (Sreekanth et al., 2008). Due to the low absorption rate of chromium salts, it has become necessary for designing and development of new organic chromium compounds (Yang et al., 2005). Some different organic chromium complexes have been reported, such as chromium picolinate (Trent and Tiedingcancel, 1995), chromium propionate (Król and Krejpcio, 2010), chromium histidinate (Dogukan et al., 2010), chromium nicotinate (Jennings et al., 1997), chromium complex of D-phenylalanine (Yang et al., 2006), chromium complex of seaweed polysaccharides (Zhang et al., 2002) and chromium complex of baicalein (Deng, 2007). However, the different coordinate ligands of these organic chromium complexes exhibited different bioactivities. Yang et al. (2006) found that chromium (D-phenylalanine)3 could decrease significantly blood glucose levels of insulin-challenge to obese ob/ob(+/+) mice. Supplementary chromium propionate given orally for a period of 8 weeks to high-fructose fed Wistar rats was able to ameliorate insulin resistance symptoms (Król and Krejpcio, 2010). It has been established that chromium picolinate is a muscle building agent and also been found to improve glucose metabolism and serum lipid metabolism (Kim et al., 2002). Studies by Deng (2007) and Zhang et al. (2002)

1624

F. Li et al. / Food and Chemical Toxicology 50 (2012) 1623–1631

using chromium complex of seaweed polysaccharides and chromium complex of baicalein in diabetes did not confirm any positive effect on lipid metabolism and carbohydrate metabolism. Chromium(III) ion-specific ligand environment could affect the biological activity of chromium (Preuss et al., 2008). There are little studies on the effects of the coordinated ligands on anti-hyperglycemic activity of the different organic chromium complexes. This study was therefore to evaluate and compare anti-hyperglycemic activity of the chromium rutin complex (CrRC), chromium folate complex (CrFC) and chromium stachyose complex (CrSC) in alloxan-induced diabetic mice and acute oral toxicity of CrRC and CrFC. It was also to provide an understanding of the potential biological activity of chromium as dietary supplementation in pharmaceuticals. 2. Materials and methods 2.1. Chemicals Stachyose (purity of 95.29%) and rutin (purity of 76.23%) were obtained from Xi’an Xiaocao Botanical Development Co., Ltd. (Xi’an, China). The standard sample of rutin was obtained from National Institute for Control of Pharmaceutical and Biological Products (China). The rutin was purified with water and ethanol re-crystallization till the purity of the rutin exceeded 95% (HPLC). Folic acid, with the purity of 98%, was obtained from Langrui fine chemicals Co., Ltd. (Shanghai, China). Alloxan was obtained from Sigma–Aldrich (Milwaukee, WI, USA). De-ionized water was used to prepare stock solutions of chromium complexes for all the experiments unless otherwise indicated. All the solvents and other chemicals were the analytical grades and were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The Cr contents of chromium rutin complex (CrRC, chemical formula [Cr3C54H100O53]Cl15), and chromium folate complex (CrFC, chemical formula [Cr2C57H87N21O36]) were determined using the atomic absorption spectrophotometry (TAS-986 atomic absorption spectrometer, Purkinje General Instrument Co., Ltd., Beijing, China). Cr content in CrRC and CrFC was 7.3% and 6.4%, respectively. 2.2. Synthesis and characterization of chromium(III) stachyose complex (CrSC) Aqueous solutions of CrCl36H2O (0.266 g, 1.0 mmol in 50 mL water) and stachyose (0.667 g, 1.0 mmol in 50 mL water) were mixed and adjusted to pH of 9 with aqueous sodium hydroxide (0.5 mol/L). The mixture was refluxed at 70 °C for 4 h and the resultant solution centrifuged at 4000g for 10 min. The supernatant was then concentrated to a volume of 10 mL. Fifty milliliters of ethanol was added and the mixture centrifuged once more. The precipitate was washed with acetone and ethanol for several times before lyophilization. A thin-layer chromatography was used to detect the product whilst stachyose was used as the control. The solvent system included n-butanol:acetone:water (ratio of 4:3:1). The product was characterized by elemental analysis (FLASH1112A analysis elemental analyzer, CE Instruments CO., Italy), atomic absorption spectrophotometry (TAS-986 atomic absorption spectrometer, Purkinje General Instrument Co., Ltd., Beijing, China), UV–visible spectrum (UV-2450 UV–visible spectrophotometer, Tosoh CO., Tokyo, Japan) and infrared spectrum (Nexus470 Fourier transform infrared spectrometer, Thermo Nicolet CO., USA). 2.3. Preparation of CrRC and CrFC 2.3.1. Preparation of CrRC CrRC was synthesized and its physicochemical characteristics determined the previous study method of Li et al. (2009). Ethanol solution of rutin (0.665 g, 1.0 mmol in 50 mL ethanol) was mixed with 50 mL aqueous solution of CrCl36H2O (0.399 g, 1.5 mmol in 50 mL water), the mixture was then warmed to temperature of 60 °C and titrated against aqueous sodium hydroxide (0.5 mol/L) to adjust the pH to 7.0. Immediately, the precipitate settled and filtered off. The filtrates were washed several times with hot ethanol and water and was allowed to finally dried under vacuo at 50 °C. UV–visible spectra (UV-2450 UV–visible spectrophotometer, Tosoh CO., Tokyo, Japan) indicated that the product did not contain the CrCl36H2O and rutin. A similar procedure for the physicochemical characterization of the product was carried out; as described for CrSC. 2.3.2. Preparation of CrFC CrFC was synthesized and its physicochemical characteristics determined using the method of the previous study (Yang et al., 2010). Folic acid (0.661 g, 1.5 mmol) was added to 30 mL water and the resultant titrated against aqueous sodium hydroxide (0.1 M) in order to adjust the pH to 7.0, then 10 mL aqueous solution of CrCl36H2O (0.266 g, 1.0 mmol) was added with continuously stirring. The mixture was then warmed at a temperature of 60 °C and then neutralization took place.

The precipitate settled and filtered off. The filtrates were washed several times with minor amounts of dimethyl sulfoxide and hot ethanol dried under vacuo at 50 °C. UV–visible spectra (UV-2450 UV–visible spectrophotometer, Tosoh CO., Tokyo, Japan) indicated that the product did not contain the CrCl36H2O and folic acid. A similar procedure for the physicochemical characterization of the product used for CrSC was applied. 2.4. Experimental animals Three hundred and sixty ICR (Institute for Cancer Research) mice aged 4 weeks with 20 ± 1 g of body weight were obtained from Comparative Medicine Center in Yangzhou University (the license number SCXK (SU) 2007-0001). Two hundred and ten male mice were selected for the anti-hyperglycemic activity study whilst 150 female and male mice (female and male ratio; 50–50) were selected for the acute oral toxicity test. All the animals were kept and maintained under the standard guidelines and housed in the university-approved animal facility in rooms (license number: SYXK (SU) 2008-0024). The animals were kept and maintained at 24 ± 1 °C, humidity of 55–60% and 12-h photoperiod. The air exchange was about 18 times/h. The mice were allowed free access to food (standard pellet diet) and drinking water. They were treated in conformity with the animal ethical and animal welfare standards. The author of this study had obtained Jiangsu province post certificate of experimental animals (the license number 2091258) in China. The mice were housed 2 days for adjustment to the environment after arrival before use. 2.5. Anti-hyperglycemic activity study 2.5.1. Induction of diabetes in mice Diabetes in mice was induced according to the method of Nair et al. (2006). One hundred and seventy mice were treated with alloxan (185 mg/kg body weight) through intraperitoneal injection. The mice were then starved for 16 h prior to treatment. But they had access to drinking water. Ten mice for the control were injected with normal saline through intraperitoneal injection. Blood was collected from the tail vein of the mice. Blood glucose was determined by one touch glucometer (Johnson and Johnson Medical, Ltd., USA) after the injection for 72 h. The blood glucose level >11.1 mmol/L in the mice was taken as successful induction of diabetes. 2.5.2. Experiment design One hundred and ninety experimental animals were randomly divided into four normal groups (normal mice) and 15 diabetic groups (alloxan-induced diabetic mice) with 10 mice in each diabetic group. All the mice were allowed free access to standard solid diet for laboratory animals and drinking water. The grouping design and dose design of all drugs are shown in Table 1. The drugs were administered with oral gavage to both the normal and the alloxan-induced diabetic mice once daily for a period of 2 weeks. 2.5.3. Biochemical assays Individual body weights gain and blood glucose levels of the animal were determined prior to the administration of the test substances. This was on weekly base. Blood was obtained from the mice through retro-orbital puncture, and the mice were dissected in order to harvest the liver at the end of the treatment period; thus after 16 h starvation. The blood serum was separated by centrifuging at 5000g for 10 min. The activities of AST and ALT in serum were assessed by the IFCC method on Automatic Biochemical Analyser (Au2700, Olympus, Japan) using a commercial assay kit (Shanghai Rongsheng Biotech Co., Ltd.). The activity of ALP in serum was determined according to the method of Massion and Frankenfeld using a commercial assay kit (Beijing Leadman Co., Ltd.) (Buccolo and David, 1973). Liver glycogen level was determined by the method of Carroll et al. (1956). 2.6. Acute oral toxicity test 150 healthy ICR mice were randomly divided in to 15 groups (n = 10/group, male and female ratio was 50–50). The control group received normal saline by oral means whilst CrCl36H2O-treated groups received orally CrCl36H2O at the dose of 0.8, 1.6, 2.4, 3.2, 4.0 and 4.8 g/kg body weight, respectively. CrRC-treated and CrFC-treated groups were orally treated with CrRC and CrFC at the dose of 1.0, 2.0 and 5.0 g/kg body weight, respectively. Rutin-treated group and the group treated folic acid were treated with rutin and folic acid at doses of 5.0 g/kg body weight, respectively. All the mice were starved overnight before the treatment, but with access to drinking water. The animals were observed systematically and data were recorded at 1, 2, 4 and 6 h interval after a single administration of test substances and daily thereafter for a period of 2 weeks. Individual body weights of the animals were determined prior to the administration of the test substance weekly and thereafter. Blood was obtained from all the mice through retro-orbital puncture, and the mice were dissected in order to harvest internal organs including lung, heart, spleen, liver and kidney after 16-h starvation. The blood samples were collected in to two types of tubes; one with anticoagulant and the other without any additives. The anticoagulated blood was

1625

F. Li et al. / Food and Chemical Toxicology 50 (2012) 1623–1631

Table 1 The grouping design and doses of CrCl36H2O, chromium rutin complex (CrRC), chromium folate complex (CrFC), chromium stachyose complex (CrSC), metformin and carrier (control) on normal and alloxan-induced diabetic mice (ALX mice). Groups

Mice

Dose

Chromium content (mg/kg b.w.)

Normal control group Diabetic control group Positive control group CrCl36H2O-treated diabetic group CrRC-treated normal group Rutin-treated diabetic group CrRC-treated diabetic group (low dose group) CrRC-treated diabetic group (middle dose group) CrRC-treated diabetic group (high dose group) CrFC-treated normal group Folic acid-treated diabetic group CrFC-treated diabetic group (low dose group) CrFC-treated diabetic group (middle dose group) CrFC-treated diabetic group (high dose group) CrSC-treated normal group Stachyose-treated diabetic group CrSC-treated diabetic group (low dose group) CrSC-treated diabetic group (middle dose group) CrSC-treated diabetic group (high dose group)

Normal mice ALX mice ALX mice ALX mice Normal mice ALX mice ALX mice ALX mice ALX mice Normal mice ALX mice ALX mice ALX mice ALX mice Normal mice ALX mice ALX mice ALX mice ALX mice

Normal saline Normal saline 128 mg Metformin/kg b.w. 15.7 mg CrCl36H2O/kg b.w. 41.1 mg CrRC/kg b.w. 38.1 mg Rutin/kg b.w. 6.85 mg CrRC/kg b.w. 13.7 mg CrRC/kg b.w. 41.1 mg CrRC/kg b.w. 46.9 mg CrFC/kg b.w. 43.9 mg Folic acid/kg b.w. 7.81 mg CrFC/kg b.w. 15.6 mg CrFC/kg b.w. 46.9 mg CrFC/kg b.w. 46.5 mg CrSC/kg b.w. 43.5 mg Stachyose/kg b.w. 7.75 mg CrSC/kg b.w. 15.5 mg CrSC/kg b.w. 46.5 mg CrSC/kg b.w.

– – – 3 3 – 0.5 1 3 3 – 0.5 1 3 3 – 0.5 1 3

analyzed for blood morphology and haematology parameters using analytical hematology system Sysmex, XE-2100 (Sysmex Cooperation, Japan). The blood without any additives was for clinical biochemistry study. The serum was collected and transferred into sterile tubes for clinical biochemistry measurements using Olympus AU2700 equipment (Olympus Cooperation, Japan).

chromium complex containing 1:1 ratio of chromium to stachyose and its molecular formula, could be inferred as CrC24H48O27. Chen et al. (2003) and Liu et al. (2009) synthesized chromium complex of amorphophallus konjac oligosaccharide and chromium complex of chitooligosaccharide with similar method.

2.7. Statistical analysis The statistical values were presented as mean ± standard deviation of triplicate or 10 determinations. The one-way analysis of variance (ANOVA) for statistical analysis was used. The Student–Newman–Keuls (S–N–K) method or Dunnett test for the multiple comparisons among the groups was applied to determine the significant differences. A value of p < 0.05 denoted the presence of a statistically significant difference. Statistical analyses were carried out by SPSS version 16.0 (SPSS Inc., Chicago, USA).

3. Results 3.1. Synthesis and characterization of CrSC The complex was synthesized by addition of an aqueous chromium trichloride solution into an aqueous solution of stachyose in a molar ratio of 1:1. The complex could be isolated in the form of solid powders from organic solvents and the purity identified by the thin layer chromatography according to the different retention factor (Rf) of CrSC and stachyose (Rf of CrSC was 0.23, whilst the Rf of stachyose was 0.29). The product was poorly soluble in organic solvent such as methanol and ethanol but not soluble in water. CrCl3 in aqueous solution exhibited two major absorption bands (429 and 612 nm) in the visible region. In comparison with CrCl3 absorption spectrum, the complex in aqueous solution was shifted to the short-wavelength region (419 and 583 nm), which might be attributed to the spin allowed transitions 4T1g(F) 4A2g (v2) and 4 4 T2g A2g (v1) (Yang et al., 2005). IR spectra of the ligand and the complex present evidence of coordination between the chromium ion and stachyose. Stachyose exhibited an absorption band v (OH) at 3381 cm1, which shifted to 3394 cm1 in the infrared spectrum of the complex. This was an indication that the coordination of stachyose involves the oxygen atoms of the hydroxyl groups. The data of elemental analysis and atomic absorption spectrophotometry indicated that the content of C, H and Cr was 35.89%, 6.38% and 6.45% which were consistent with the stoichiometry of CrC24H48O27 requiring C 35.13%, H 5.90% and Cr 6.34%. On the basis of elemental analysis and spectrometry studies, the

3.2. Effect of chromium complexes on body weight level of mice The changes in body weight gain of the control and experimental mice during the experimental period are shown in Table 2. The day 1 in Table 2 was the first day in which all the drugs were administered with oral gavage for the mice. The day 1 in Table 2 also was 6 days after the beginning of the experiment (2 days for adjustment of mice and 4 days for induction of diabetes in mice), not the beginning day of the experiment. It was observed that the differences of body weight between the data given in Table 2 and data indicating that ICR mice with 20 g of body weight. In the fed state, the three chromium complexes (CrRC, CrFC, and CrSC) had no effect on body weight gain in the normal mice. The diabetic mice that were treated with the three chromium complexes exhibited no significantly increase for the average body weight gain when compared with the diabetic control group.

3.3. Effect of chromium complexes on blood glucose level of mice The changes in blood glucose levels of control and experimental mice during the experimental period are shown in Table 3. The normal control group was maintained in a normal range of blood glucose level. The blood glucose level did not change when compared with the control group that received a daily oral administration of the three different complexes at a dose of 3.0 mg Cr/kg for a period of 2 weeks. It was an indication that these complexes did not affect the blood glucose level of normal mice. The alloxan-induced diabetic mice exhibited significant hyperglycemia. After a 2-week administration with the CrRC and CrFC complexes, the blood glucose level decreased significantly when compared with the diabetic control group (p < 0.05). However, no significant differences in average blood glucose level between the CrSC-treated diabetic groups and diabetic control group were found. It was therefore deduced that CrRC and CrFC had hypoglycemic effect on alloxan-induced diabetic mice, while CrSC did not.

1626

F. Li et al. / Food and Chemical Toxicology 50 (2012) 1623–1631 Table 2 The changes in body weight (g) gain of the normal control, diabetic control mice and diabetic mice after 7 and 14 days of treatment. Groups

Normal control group Diabetic control group Positive control group ALX mice 15.7 mg CrCl36H2O/kg b.w. Normal mice 41.1 mg CrRC/kg b.w. ALX mice 38.1 mg rutin/kg b.w. ALX mice 6.85 mg CrRC/kg b.w. ALX mice 13.7 mg CrRC/kg b.w. ALX mice 41.1 mg CrRC/kg b.w. Normal mice 46.9 mg CrFC/kg b.w. ALX mice 43.9 mg folic acid/kg b.w. ALX mice 7.81 mg CrFC/kg b.w. ALX mice 15.6 mg CrFC/kg b.w. ALX mice 46.9 mg CrFC/kg b.w. Normal mice 46.5 mg CrSC/kg b.w. ALX mice 43.5 mg stachyose/kg b.w. ALX mice 7.75 mg CrSC/kg b.w. ALX mice 15.5 mg CrSC/kg b.w. ALX mice 46.5 mg CrSC/kg b.w.

Body weight (g) Day 1

Day 7

Day 14

28.7 ± 1.17a 25.24 ± 1.80 25.56 ± 1.12 24.18 ± 1.49 27.70 ± 0.56a 24.52 ± 1.78 24.85 ± 1.16 24.45 ± 1.42 24.63 ± 1.62 27.17 ± 1.33a 23.93 ± 1.45 24.60 ± 1.05 25.07 ± 1.59 25.52 ± 2.17 28.13 ± 1.99a 24.87 ± 1.70 25.20 ± 1.70 24.68 ± 1.42 24.82 ± 1.67

33.25 ± 0.90a 26.62 ± 1.70 26.68 ± 1.62 26.60 ± 2.84 31.75 ± 1.48a 26.56 ± 2.08 27.51 ± 1.42 26.70 ± 1.65 26.91 ± 1.62 31.75 ± 0.90a 26.53 ± 1.98 26.49 ± 1.51 27.08 ± 1.78 28.35 ± 1.67 32.05 ± 1.16a 27.40 ± 1.77 27.13 ± 1.55 26.90 ± 1.92 26.68 ± 1.62

35.01 ± 1.08a 28.59 ± 2.05 28.15 ± 1.65 28.10 ± 2.92 33.14 ± 1.65a 27.75 ± 1.85 30.10 ± 1.65 29.13 ± 1.82 28.17 ± 2.49 33.23 ± 0.93a 29.62 ± 1.57 28.15 ± 1.77 29.40 ± 1.86 30.45 ± 1.78 34.84 ± 1.84a 29.40 ± 1.51 28.96 ± 2.41 28.69 ± 2.19 29.23 ± 3.04

Each value is the mean ± SD of 10 separate experiments. p Values are shown as ap < 0.05 vs. diabetic control group in the same column. A value of p < 0.05 denoted the presence of a statistically significant difference according to Student–Newman–Keuls (S–N–K) method.

Table 3 The changes in blood glucose level (mmol/L) of the normal control, diabetic control mice and diabetic mice after 7 and 14 days treatment. Groups

Normal control group Diabetic control group Positive control group ALX mice 15.7 mg CrCl36H2O/kg b.w. Normal mice 41.1 mg CrRC/kg b.w. ALX mice 38.1 mg rutin/kg b.w. ALX mice 6.85 mg CrRC/kg b.w. ALX mice 13.7 mg CrRC/kg b.w. ALX mice 41.1 mg CrRC/kg b.w. Normal mice 46.9 mg CrFC/kg b.w. ALX mice 43.9 mg folic acid/kg b.w. ALX mice 7.81 mg CrFC/kg b.w. ALX mice 15.6 mg CrFC/kg b.w. ALX mice 46.9 mg CrFC/kg b.w. Normal mice 46.5 mg CrSC/kg b.w. ALX mice 43.5 mg stachyose/kg b.w. ALX mice 7.75 mg CrSC/kg b.w. ALX mice 15.5 mg CrSC/kg b.w. ALX mice 46.5 mg CrSC/kg b.w.

Blood glucose level (mmol/L) Day 1

Day 7

Day 14

6.25 ± 0.91b 25.56 ± 3.88a 25.44 ± 3.71a 25.13 ± 3.85a 5.60 ± 0.14b 24.86 ± 3.85a 25.00 ± 4.16a 25.43 ± 2.23a 26.55 ± 3.93a 6.06 ± 0.21b 24.45 ± 3.79a 24.88 ± 4.87a 24.99 ± 4.30a 24.53 ± 3.57a 7.40 ± 0.00 b 25.02 ± 4.25a 25.37 ± 4.03a 25.75 ± 3.64a 25.78 ± 5.30a

6.35 ± 0.49b 24.64 ± 4.01a 21.97 ± 4.31a 24.38 ± 3.19a 5.06 ± 0.44b 24.06 ± 4.00a 22.85 ± 3.83a 23.08 ± 3.43a 23.61 ± 4.83a 5.33 ± 0.40b 25.93 ± 4.54a 22.12 ± 3.94a 22.24 ± 2.93a 21.08 ± 3.91a 6.80 ± 0.75b 25.48 ± 1.32a 23.86 ± 1.73a 23.83 ± 1.53a 24.22 ± 1.11a

7.10 ± 0.99b 25.01 ± 2.71a 18.40 ± 2.86ab 23.16 ± 2.60a 6.27 ± 0.99b 25.15 ± 2.09a 20.09 ± 2.46ab 18.70 ± 2.33ab 18.83 ± 2.45ab 5.93 ± 0.25b 25.48 ± 3.77a 21.41 ± 2.01ab 18.36 ± 2.85ab 16.01 ± 4.70ab 6.23 ± 0.71b 24.17 ± 1.65a 23.26 ± 1.86a 22.83 ± 1.14a 22.14 ± 1.51a

Each value is the mean ± SD of 10 separate experiments. p Values are shown as ap < 0.05 vs. normal control group and bp < 0.05 vs. diabetic control group in the same column. A value of p < 0.05 denoted the presence of a statistically significant difference according to Student–Newman–Keuls (S–N–K) method.

3.4. Effects of chromium complexes on AST, ALT and ALP levels of mice

3.5. Effect of chromium complexes on liver glycogen level of mice

The activities of AST, ALT and ALP of the control and experimental mice are indicated in Table 4. When the normal mice were given a daily oral administration of the three complexes (CrRC, CrFC and CrSC) at a dose of 3.0 mg Cr/kg for a period of 2 weeks, the activities of AST, ALT and ALP did not change as compared with the control group indicating that the complexes did not affect the AST, ALT and ALP activities of the normal mice. When compared with control group, a significant increase in the AST, ALT and ALP activities were observed in diabetic control group. CrRC and CrFC administration had caused the decreases in the activities of serum AST, ALT and ALP in diabetic mice.

The effects of chromium complexes on liver glycogen level in the control and experimental mice are shown in Table 5. The treatment of CrRC, CrFC and CrSC did not produce any change of the liver glycogen level in the normal mice. CrRC and CrFC administration resulted in a significant increase in the level of liver glycogen in diabetic mice, while CrSC did not. 3.6. Acute oral toxicity The observation of control and experimental mice following administration of CrCl36H2O, CrRC and CrFC by a single oral ga-

1627

F. Li et al. / Food and Chemical Toxicology 50 (2012) 1623–1631 Table 4 The activities of serum aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP) of the control, diabetic mice and diabetic mice after 14 days treatment. Groups

AST

ALT

ALP

Normal control group Diabetic control group Positive control group ALX mice 15.7 mg CrCl36H2O/kg b.w. Normal mice 41.1 mg CrRC/kg b.w. ALX mice 38.1 mg rutin/kg b.w. ALX mice 6.85 mg CrRC/kg b.w. ALX mice 13.7 mg CrRC/kg b.w. ALX mice 41.1 mg CrRC/kg b.w. Normal mice 46.9 mg CrFC/kg b.w. ALX mice 43.9 mg folic acid/kg b.w. ALX mice 7.81 mg CrFC/kg b.w. ALX mice 15.6 mg CrFC/kg b.w. ALX mice 46.9 mg CrFC/kg b.w. Normal mice 46.5 mg CrSC/kg b.w. ALX mice 43.5 mg stachyose/kg b.w. ALX mice 7.75 mg CrSC/kg b.w. ALX mice 15.5 mg CrSC/kg b.w. ALX mice 46.5 mg CrSC/kg b.w.

129.67 ± 21.57b 452.25 ± 12.23a 239.16 ± 51.39ab 183.34 ± 18.19ab 117.00 ± 5.46b 421.67 ± 43.11a 227.33 ± 28.44ab 194.33 ± 15.82ab 159.67 ± 31.57b 121.23 ± 13.36b 417.26 ± 16.46a 313.35 ± 7.26ab 286.33 ± 12.25ab 174.67 ± 14.57ab 164.09 ± 21.28b 452.19 ± 4.03a 438.21 ± 24.57a 413.17 ± 6.00a 412.33 ± 20.52a

43.33 ± 6.66b 184.48 ± 5.79a 149.33 ± 12.53ab 101.50 ± 8.78ab 41.50 ± 2.52b 194.52 ± 9.50a 112.25 ± 6.5ab 104.17 ± 2.34ab 97.35 ± 5.25ab 37.25 ± 1.25b 192.17 ± 8.81a 93.25 ± 4.50ab 77.45 ± 0.51ab 66.05 ± 1.53ab 44.27 ± 10.0b 189.29 ± 3.15a 185.85 ± 9.25a 183.18 ± 3.77a 180.01 ± 3.32a

118.21 ± 20.03b 383.32 ± 24.00a 353.45 ± 35.25a 324.50 ± 31.5ab 122.50 ± 4.25b 300.31 ± 20.23a 306.35 ± 26.5ab 252.26 ± 24.5ab 172.25 ± 13.25ab 153.45 ± 32.05b 351.25 ± 36.25a 275.50 ± 34.52ab 250.07 ± 28.25ab 205.15 ± 55.81ab 151.34 ± 25.10b 335.06 ± 32.44a 340.43 ± 23.10a 319.29 ± 26.07a 313.51 ± 25.00a

Each value is the mean ± SD of 10 separate experiments. p Values are shown as ap < 0.05 vs. normal control group and bp < 0.05 vs. diabetic control group in the same column. A value of p < 0.05 denoted the presence of a statistically significant difference according to Student–Newman–Keuls (S–N–K) method.

Table 5 The liver glycogen (LG) level (lg/100 mg) in the control, diabetic mice and diabetic mice after 14 days treatment. Groups

LG (lg/100 mg)

Normal control group Diabetic control group Positive control group ALX mice 15.7 mg CrCl36H2O/kg b.w. Normal mice 41.1 mg CrRC/kg b.w. ALX mice 38.1 mg rutin/kg b.w. ALX mice 6.85 mg CrRC/kg b.w. ALX mice 13.7 mg CrRC/kg b.w. ALX mice 41.1 mg CrRC/kg b.w. Normal mice 46.9 mg CrFC/kg b.w. ALX mice 43.9 mg folic acid/kg b.w. ALX mice 7.81 mg CrFC/kg b.w. ALX mice 15.6 mg CrFC/kg b.w. ALX mice 46.9 mg CrFC/kg b.w. Normal mice 46.5 mg CrSC/kg b.w. ALX mice 43.5 mg stachyose/kg b.w. ALX mice 7.75 mg CrSC/kg b.w. ALX mice 15.5 mg CrSC/kg b.w. ALX mice 46.5 mg CrSC/kg b.w.

990.34 ± 35.17b 582.39 ± 72.80 666.67 ± 38.51a 644.42 ± 74.63a 982.25 ± 55.67b 563.00 ± 77.84a 686.06 ± 54.57ab 687.36 ± 56.20ab 812.88 ± 71.55ab 951.69 ± 41.77b 606.91 ± 42.67a 683.92 ± 40.48ab 753.94 ± 71.17ab 839.98 ± 37.09ab 956.05 ± 43.13b 525.03 ± 76.87a 586.66 ± 56.87a 626.94 ± 49.82a 665.46 ± 41.55a

Each value is the mean ± SD of 10 separate experiments. p Values are shown as a p < 0.05 vs. normal control group and bp < 0.05 vs. diabetic control group in the same column. A value of p < 0.05 denoted the presence of a statistically significant difference according to Student–Newman–Keuls (S–N–K) method.

vage during acute toxicity study are indicated in Table 6. The observed symptoms associated with ingestion of CrCl36H2O and CrRC included minor to more noticeable shakings, sleepy or/and bloody tears. The LD50 (median lethal dose) for CrCl36H2O was 2.39 g/kg body weight (95% confidence intervals: 1.776–3.218 g/ kg), and that of CrRC and CrFC were greater than 5.0 g/kg each. The minimum lethal dose for CrFC was over 5.0 g/kg, while that of CrRC was 1.0 g/kg. The body weight gain of CrCl36H2O-treated group at the dose of 2.4 g/kg body weight increased gradually throughout the study period, but was significantly lower as compared to the control group (Table 7). The increase in body weight of CrRC-treated and CrFC-treated group during 2-week period was not significantly different from the control group (Table 7). Comparing this with the control group, the organ weight (absolute and relative) of lung, heart, spleen, liver and kidney of the CrRC and

Table 6 The observation of mice following administration of CrCl36H2O, chromium rutin complex, chromium folate complex and carrier (control) by oral gavage during acute toxicity study. Groups

Normal control group 0.8 g CrCl36H2O/kg b.w. 1.6 g CrCl36H2O/kg b.w. 2.4 g CrCl36H2O/kg b.w. 3.2 g CrCl36H2O/kg b.w. 4.0 g CrCl36H2O/kg b.w. 4.8 g CrCl36H2O/kg b.w. 5.0 g 5.0 g 1.0 g 2.0 g 5.0 g 1.0 g 2.0 g 5.0 g

Rutin/kg b.w. Folic acid/kg b.w. CrRC/kg b.w. CrRC/kg b.w. CrRC/kg b.w. CrFC/kg b.w. CrFC/kg b.w. CrFC/kg b.w.

Mice

Effects

D/T

Mortality latency (h)

Symptoms of toxicity

0/10 0/10 0/10 5/10 10/10 10/10 10/10

– – – <72 <48 <48 <24

1/10 1/10 0/10 1/10 2/10 0/10 0/10 0/10

<24 <24 – <48 <48 – – –

None None None Shakings Shakings, sleepy Shakings, sleepy Bloody tears, shakings, sleepy None None None Sleepy Sleepy None None None

CrFC-treated mice indicated no significant changes (Table 8). However, the weight (absolute and relative) of the organs of CrCl36H2O-treated group at the dose of 2.4 g/kg body weight was significantly higher as compared to the control group (Table 8). From Tables 9 and 10, blood morphology, hematology and clinical biochemistry values of CrCl36H2O, CrRC and CrFC-treated mice were within the range of the control animals tested and indicated no significant changes. 4. Discussion It has been established that the ligand of the trivalent chromium compound affects its efficacy and toxicity (Chaudhary et al., 2005; Preuss et al., 2008; Shrivastava et al., 2005). Previous studies indicated that organic forms of chromium were more bioactive than chromium chloride. Recognizing the therapeutic potentials of trivalent chromium supplementation, many organic chromium complexes were synthesized and their bioactivities

1628

F. Li et al. / Food and Chemical Toxicology 50 (2012) 1623–1631

Table 7 The changes of body weight gain of the female and male ICR mice following administration CrCl36H2O, chromium rutin complex, chromium folate complex and carrier (control) by oral gavage during acute toxicity study. Days Day 0

Day 7

Day 14

Female groups Normal control group 0.8 g CrCl36H2O/kg b.w. 1.6 g CrCl36H2O/kg b.w. 2.4 g CrCl36H2O/kg b.w. 5.0 g Rutin/kg b.w. 5.0 g Folic acid/kg b.w. 1.0 g CrRC/kg b.w. 2.0 g CrRC/kg b.w. 5.0 g CrRC/kg b.w. 1.0 g CrFC/kg b.w. 2.0 g CrFC/kg b.w. 5.0 g CrFC/kg b.w.

20.30 ± 0.78 20.16 ± 0.76 19.83 ± 0.21 20.33 ± 0.58 20.26 ± 1.00 19.70 ± 0.75 20.07 ± 0.31 20.80 ± 0.72 20.50 ± 0.79 20.47 ± 0.5 20.03 ± 1.01 20.07 ± 0.12

25.36 ± 0.32 25.36 ± 1.61 24.10 ± 0.75 22.22 ± 0.55a 26.05 ± 0.64 23.95 ± 1.62 24.40 ± 0.5 26.85 ± 0.49 25.95 ± 1.48 25.17 ± 0.60 24.80 ± 2.00 25.16 ± 1.04

27.00 ± 1.00 26.43 ± 1.76 26.00 ± 0.17 25.00 ± 0.46a 27.90 ± 1.41 25.75 ± 2.47 28.15 ± 2.47 29.10 ± 4.38 27.80 ± 0.84 26.77 ± 0.49 27.27 ± 1.55 27.43 ± 1.27

Male groups Normal control group 0.8 g CrCl36H2O/kg b.w. 1.6 g CrCl36H2O/kg b.w. 2.4 g CrCl36H2O/kg b.w. 5.0 g Rutin/kg b.w. 5.0 g Folic acid/kg b.w. 1.0 g CrRC/kg b.w. 2.0 g CrRC/kg b.w. 5.0 g CrRC/kg b.w. 1.0 g CrFC/kg b.w. 2.0 g CrFC/kg b.w. 5.0 g CrFC/kg b.w.

21.50 ± 0.88 21.63 ± 1.21 21.93 ± 0.55 20.63 ± 1.18 21.07 ± 0.31 20.50 ± 0.50 21.40 ± 0.72 20.90 ± 1.45 19.93 ± 1.00 21.57 ± 0.81 20.67 ± 0.57 20.8 ± 1.47

30.70 ± 0.85 29.70 ± 1.61 28.53 ± 0.81 26.15 ± 3.18a 29.20 ± 0.35 28.53 ± 1.58 31.20 ± 0.61 28.83 ± 2.29 31.20 ± 1.13 30.63 ± 0.46 30.60 ± 1.51 30.13 ± 2.47

33.37 ± 1.27 31.90 ± 2.87 31.83 ± 1.29 30.60 ± 2.26a 32.57 ± 0.67 31.10 ± 2.06 33.67 ± 0.58 31.37 ± 2.14 33.65 ± 0.35 32.00 ± 1.24 32.40 ± 1.24 31.90 ± 1.00

Each value is the mean ± SD of 10 separate experiments. p Values are shown as p < 0.05 vs. normal control group in the same column. A value of p < 0.05 denoted the presence of a statistically significant difference according to Dunnett test. a

demonstrated. In this study, three different chromium complexes (CrRC, CrFC and CrSC) were chosen to examine whether their anti-hyperglycemic activities were influenced by ligands.

When the normal groups were administered with CrRC, CrFC or CrSC, a non-significant difference in body weight over 1–14 days period was observed when compared with the control group. Studies by Lukaski et al. (2007) indicated that chromium picolinate supplementation of women do not independently influence body weight under conditions of controlled energy intake. The decrease in body weight observed in uncontrolled diabetics might be the result of protein wasting due to the unavailability of carbohydrate for utilization as an energy source (Yanardag et al., 2009). When alloxan-induced diabetic mice were administered with CrRC, CrFC or CrSC, a non-significant difference in body weight gain over 1–14 days period was observed when compared with the diabetic control groups as well. Study conducted by Wu et al. (2011) has shown that the treatment of chromium chloride and chromium(III) malate complex does not cause significant body weight gain in diabetic rats. In alloxan-induced mice, after a 2-week period of the administration of CrRC and CrFC at the dose of 3 mg Cr/kg body weight, CrRC and CrFC resulted significant anti-diabetic activity, resulting in percentage reduction of blood glucose levels of 29.1% and 34.7%, respectively. However, after a 2-week period of administrating the mice with the CrSC, the effect on anti-diabetic activity was not significant. It might suggest that not every chromium complex is necessarily hypoglycemic under insulin-deficient conditions. Similar conclusion had been drawn by Machalin´ski et al. (2006) who found that 3 weeks of the treatment with chromium ‘‘454’’ and chromium picolinate in insulin-deficient streptozocin-treated diabetic rats results in a 38% and 11% reduction of blood glucose levels, respectively. The appreciation of the anti-hyperglycemic effect follows the order: CrSC < CrRC < CrFC. CrFC exhibited the highest anti-diabetic property, whilst the effect of CrRC was comparable to that produced by the standard drug metformin. Preuss et al. (2008) compared metabolic effects of five different commercial trivalent chromium compounds (citrate, amino acid chelate, chelavite, polynicotinate, and nicotinate). They found that only three en-

Table 8 Organ weight (absolute and relative) of ICR mice following administration CrCl36H2O, chromium rutin complex (CrRC), chromium folate complex (CrFC) and carrier (control) by oral gavage during acute toxicity study. Group

Normal control group 0.8 g CrCl36H2O/kg b.w. 1.6 g CrCl36H2O/kg b.w. 2.4 g CrCl36H2O/kg b.w. 5.0 g Rutin/kg b.w. 5.0 g Folic acid/kg b.w. 1.0 g CrRC/kg b.w. 2.0 g CrRC/kg b.w. 5.0 g CrRC/kg b.w. 1.0 g CrFC/kg b.w. 2.0 g CrFC/kg b.w. 5.0 g CrFC/kg b.w.

Normal control group 0.8 g CrCl36H2O/kg b.w. 1.6 g CrCl36H2O/kg b.w. 2.4 g CrCl36H2O/kg b.w. 5.0 g Rutin/kg b.w. 5.0 g Folic acid/kg b.w. 1.0 g CrRC/kg b.w. 2.0 g CrRC/kg b.w. 5.0 g CrRC/kg b.w. 1.0 g CrFC/kg b.w. 2.0 g CrFC/kg b.w. 5.0 g CrFC/kg b.w.

Parameter Lung

Heart

Spleen

Liver

Kidney

0.155 ± 0.018 0.165 ± 0.025 0.167 ± 0.011 0.189 ± 0.018 0.149 ± 0.013 0.161 ± 0.026 0.165 ± 0.010 0.167 ± 0.013 0.175 ± 0.009 0.159 ± 0.012 0.163 ± 0.031 0.164 ± 0.042

0.120 ± 0.017 0.124 ± 0.016 0.121 ± 0.021 0.123 ± 0.034 0.127 ± 0.016 0.119 ± 0.022 0.139 ± 0.041 0.134 ± 0.037 0.141 ± 0.038 0.124 ± 0.018 0.135 ± 0.022 0.144 ± 0.036

0.085 ± 0.015 0.096 ± 0.031 0.116 ± 0.023 0.133 ± 0.017a 0.099 ± 0.024 0.092 ± 0.038 0.100 ± 0.017 0.101 ± 0.010 0.089 ± 0.005 0.092 ± 0.016 0.104 ± 0.017 0.080 ± 0.010

1.407 ± 0.300 1.640 ± 0.327 1.681 ± 0.394 1.825 ± 0.201a 1.494 ± 0.381 1.495 ± 0.415 1.657 ± 0.383 1.578 ± 0.094 1.607 ± 0.299 1.340 ± 0.203 1.560 ± 0.399 1.494 ± 0.315

0.128 ± 0.037 0.166 ± 0.482 0.177 ± 0.042 0.233 ± 0.014a 0.174 ± 0.033 0.175 ± 0.050 0.200 ± 0.048 0.160 ± 0.032 0.188 ± 0.044 0.195 ± 0.066 0.183 ± 0.046 0.157 ± 0.048

Relative lung weight

Relative heart weight

Relative spleen weight

Relative liver weight

Relative kidney weight

0.506 ± 0.054 0.572 ± 0.096 0.582 ± 0.064 0.660 ± 0.042 0.487 ± 0.052 0.550 ± 0.031 0.520 ± 0.035 0.532 ± 0.023 0.587 ± 0.081 0.556 ± 0.060 0.546 ± 0.089 0.587 ± 0.026

0.399 ± 0.050 0.429 ± 0.055 0.417 ± 0.044 0.422 ± 0.085 0.411 ± 0.025 0.407 ± 0.054 0.424 ± 0.084 0.410 ± 0.089 0.441 ± 0.064 0.409 ± 0.097 0.443 ± 0.082 0.466 ± 0.096

0.285 ± 0.048 0.334 ± 0.086 0.407 ± 0.093 0.474 ± 0.097 0.323 ± 0.070 0.309 ± 0.095 0.312 ± 0.029 0.323 ± 0.038 0.298 ± 0.041 0.310 ± 0.095 0.351 ± 0.063 0.276 ± 0.043

4.627 ± 0.525 5.653 ± 0.845 5.757 ± 0.834 6.379 ± 0.599a 4.813 ± 0.894 5.040 ± 0.323 5.150 ± 0.749 5.066 ± 0.487 5.309 ± 0.323 4.657 ± 0.314 5.185 ± 0.963 5.091 ± 0.795

0.424 ± 0.069 0.564 ± 0.099 0.607 ± 0.099 0.719 ± 0.102a 0.441 ± 0.033 0.589 ± 0.017 0.620 ± 0.092 0.512 ± 0.105 0.616 ± 0.071 0.591 ± 0.031 0.607 ± 0.102 0.532 ± 0.034

a

Each value is the mean ± SD of 10 separate experiments. p Values are shown as ap < 0.05 vs. normal control group in the same column. A value of p < 0.05 denoted the presence of a statistically significant difference according to Dunnett test.

1629

F. Li et al. / Food and Chemical Toxicology 50 (2012) 1623–1631

Table 9 The results of blood morphology and hematology of ICR mice following administration CrCl36H2O, chromium rutin complex (CrRC), chromium folate complex (CrFC) and carrier (control) by oral gavage during acute toxicity study. Group

Normal control group 0.8 g CrCl36H2O/kg b.w. 1.6 g CrCl36H2O/kg b.w. 2.4 g CrCl36H2O/kg b.w. 5.0 g Rutin/kg b.w. 5.0 g Folic acid/kg b.w. 1.0 g CrRC/kg b.w. 2.0 g CrRC/kg b.w. 5.0 g CrRC/kg b.w. 1.0 g CrFC/kg b.w. 2.0 g CrFC/kg b.w. 5.0 g CrFC/kg b.w.

Parameter WBC (109/L)

RBC (1012/L)

PLT (109/L)

MPV (fL)

PDW (fL)

Hb (g/L)

HCT (%)

MCV (fL)

MCHC (g/L)

RDW (%)

7.70 ± 1.13 8.65 ± 1.91 8.40 ± 0.71 8.50 ± 0.57 8.85 ± 0.35 7.60 ± 1.56 7.55 ± 0.67 7.60 ± 0.42 7.25 ± 1.25 7.55 ± 1.48 7.90 ± 1.13 7.65 ± 0.49

8.30 ± 0.30 8.24 ± 0.11 7.61 ± 1.01 7.42 ± 0.74 7.64 ± 1.67 8.34 ± 1.06 8.60 ± 0.29 8.32 ± 0.08 8.55 ± 0.39 8.21 ± 0.87 8.85 ± 0.35 7.74 ± 0.82

790 ± 125 760 ± 96 832 ± 79 864 ± 125 665 ± 13 591 ± 56 740 ± 50 831 ± 81 633 ± 140 577 ± 147 753 ± 67 706 ± 96

5.42 ± 0.21 5.31 ± 0.22 5.42 ± 0.57 5.06 ± 0.07 5.30 ± 0.42 5.53 ± 0.07 5.40 ± 0.42 5.67 ± 0.41 5.20 ± 0.28 5.25 ± 0.35 5.62 ± 0.08 5.70 ± 0.12

8.30 ± 0.56 8.22 ± 0.71 7.31 ± 0.85 8.40 ± 0.71 8.17 ± 1.06 8.80 ± 0.14 8.96 ± 0.07 8.34 ± 0.89 7.75 ± 0.50 8.06 ± 0.92 8.91 ± 0.05 7.70 ± 1.41

150.5 ± 7.8 141.5 ± 14.8 154.0 ± 2.8 142.5 ± 13.4 142.5 ± 12.0 145.5 ± 14.7 146.1 ± 8.5 143.7 ± 17.7 148.9 ± 3.5 143.1 ± 11.3 154.4 ± 2.8 146.8 ± 21.9

44.6 ± 4.9 45.5 ± 4.8 49.2 ± 1.4 46.2 ± 2.8 41.2 ± 9.9 46.3 ± 5.7 47.6 ± 4.2 47.0 ± 1.4 46.5 ± 2.1 44.9 ± 4.3 48.8 ± 1.4 46.7 ± 4.2

55.1 ± 2.3 54.9 ± 3.5 55.4 ± 2.8 56.3 ± 1.6 53.4 ± 2.1 55.1 ± 0.8 53.6 ± 2.2 52.9 ± 0.7 54.6 ± 0.4 55.3 ± 0.6 54.9 ± 0.5 51.9 ± 0.7

319.1 ± 7.1 312.5 ± 17.7 316.4 ± 10.6 306.7 ± 3.5 356.6 ± 61.5 317.1 ± 2.8 314.2 ± 8.5 312.4 ± 17.0 318.9 ± 8.5 316.4 ± 4.5 317.8 ± 3.5 314.7 ± 21.2

15.7 ± 1.9 14.4 ± 1.5 16.6 ± 1.4 15.9 ± 2.5 14.8 ± 1.8 15.1 ± 2.9 16.2 ± 1.0 15.6 ± 0.1 14.8 ± 0.9 14.5 ± 2.1 16.3 ± 1.1 15.9 ± 0.7

WBC = white blood cell, RBC = red blood cell, PLT = platelet count, MPV = platelet mean volume, PDW = platelet distribution width, Hb = hemoglobin, HCT = hemataocrit, MCV = mean corpuscular volume, MCHC = mean corpuscular hemoglobin concentration, RDW = Red cell distribution width.

hanced insulin sensitivity (polynicotinate, chelavite, and picolinate) decreased systolic blood pressure (polynicotinate and picolinate). The result in this study is in line with previous studies which indicate that the ligand of the trivalent chromium compound markedly affects its efficacy, not all the trivalent chromium compounds possess equal efficacy. Alloxan produces oxygen radicals in the body which cause pancreatic injury that is responsible for increased blood glucose level in animals (Halliwell and Gutteridge, 1985). However, it is has been established that action that is not specific to pancreas as other organs such as liver can also be affected by alloxan administration as seen from the elevation of marker enzymes (Hamden et al., 2009). Common biochemical markers of liver damage are known to increase the activities of some enzymes such as AST, ALT and ALP in the blood. The increase in the activities of AST, ALT and ALP in serum might be due to the leakage of these enzymes from the liver cytosol into the blood stream (Ghosh et al., 2004). In the present study, these values decreased by the administration of CrRC and CrFC. The decrease in the increasing enzyme activities indicated that CrRC and CrFC prevented damage in the liver. Similar observation was made by Machalin´ski et al. (2006) who reported that ALT and AST are significantly reduced by chromium 454 and chromium picolinate in STZ-treated diabetic rats; thus an indication that the functioning of the liver is improved to some extent. Although Jain et al. (2007a) reported that ALT, AST and ALP values with large variation are not significantly reduced by chromium niacinate or chromium picolinate in STZ-treated diabetic rats, but they stated that Cr3+ supplementation can lower the risk of vascular inflammation in aspects of proinflammatory cytokines tumor necrosis factor-alpha (TNF-a), interleukin-6 (IL-6), interleukin-8 (IL-8), C-reactive protein and MCPI (monocyte chemoattractant protein-1), oxidative stress, lipid peroxidation and lipids levels in diabetic rats (Jain et al., 2007a,b). The liver is one of the most important organs in the metabolism of drugs and other substances. Short-term diabetes acutely impairs the ability of the liver to synthesize glycogen (Ferrannini et al., 1990), since alloxan or streptozotocin causes selective destruction of B cells of islets resulting in marked decrease in insulin levels. It is rational that glycogen levels in liver decrease since they depend on insulin for influx of glucose (Hems et al., 1975). The significant increase in the glycogen levels of CrRC and CrFC-treated diabetic animals was found. It was reported by a previous study that chromium supplementation significantly increased the liver glycogen content in fish that fed the glucose diet (Shiau and Lin, 1993). Human studies have mostly used 1000 lg Cr3+ per day (Martin et al., 2006; Vrtovec et al., 2005). Assuming an average 60 kg body

weight, this would relate to an intake of nearly 17 lg Cr3+/kg body weight. The dose of CrSC, CrRC or CrFC used in this study is 0.5– 3.0 mg Cr3+/kg body weight/day of mice. This dose was similar to that used in the previous studies which was administered with oral gavage chromium(III) propionate at the dose of 1 and 5 mg Cr3+/kg body weight/day for a period of 5 weeks to enable the study into the carbohydrate metabolism in insulin-resistance rat model and to also evaluate the anti-diabetic potential of chromium(III) propionate complex in STZ injected rats fed with high diets (Król and Krejpcio, 2010, 2011), but much higher than that used for human supplementation (Jain et al., 2007a). From the point of pharmacology, this quantity was chosen to increase chances in order to obtain the anti-diabetic activity of Cr3+ because of its low absorptions (2–5%). Anderson (1997) fed rats with diets containing 100 mg of Cr as [Cr(pic)3] per kg diet for a period of 24 weeks without observing any acute toxic effects. Staniek et al. (2010a) also fed rats with diets containing 1000 mg Cr(III)/kg diet (given as [Cr3O(O2CCH2CH3)6(H2O)3]NO3), equivalent of 100 mg Cr/kg body weight/day for a period of 4 weeks without observing any genotoxic effects in the rats. The dose in the current study was not expected to have acute toxic effects. No ill effects were found in the administration of all drugs by oral gavage with the normal mice once daily for the 2 weeks period. Some manifestations of genotoxicity of chromium picolinate have been reported, including skeletal and neurological defects in the offspring of mice receiving chromium picolinate during pregnancy or lactation (Bailey et al., 2006, 2007). Chromium(III) complexes with organic ligands other than picolinate generally showed low toxicities, except for study by Shrivastava et al. (2005) which reported the presence of significant cytotoxicities of a wide range of chromium(III) complexes with common O/N-donor ligands. The nature of the ligand was one of the crucial factors that affected the toxicity of chromium(III) nutritional supplements. In the present study, two chromium complexes (CrRC and CrFC) were chosen in order to compare their acute oral toxicities. The analyzed haematological parameters including total white blood cell (WBC), total red blood cell (RBC), platelet count (PLT), platelet mean volume (MPV), platelet distribution width (PDW), hemoglobin (Hb), hemataocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC) and Red cell distribution width (RDW) for mice received the CrRC or CrFC by oral means were not significantly different when compared with the control mice. The biochemical analyses which included aspartate transaminase (AST), blood urea nitrogen (BUN), blood glucose (GLU), total protein (TP), alanine transaminase (ALT), triglycerides (TG), total cholesterol (CHOL), high density

1630

F. Li et al. / Food and Chemical Toxicology 50 (2012) 1623–1631

Table 10 Serum biochemical indices of mice following administration CrCl36H2O, chromium rutin complex (CrRC), chromium folate complex (CrFC) and carrier (control) by oral gavage during acute toxicity study. Group

Normal control group 0.8 g CrCl36H2O/kg b.w. 1.6 g CrCl36H2O/kg b.w. 2.4 g CrCl36H2O/kg b.w. 5.0 g Rutin/kg b.w. 5.0 g Folic acid/kg b.w. 1.0 g CrRC/kg b.w. 2.0 g CrRC/kg b.w. 5.0 g CrRC/kg b.w. 1.0 g CrFC/kg b.w. 2.0 g CrFC/kg b.w. 5.0 g CrFC/kg b.w.

Parameter AST (U/L)

BUN (mmol/ L)

GLU (mmol/ L)

TP (g/L)

ALT (U/L)

TG (mmol/ L)

CHOL (mmol/ L)

HDL (mmol/ L)

LDL (mmol/ L)

121.0 ± 16.6 135.0 ± 26.9

6.28 ± 1.30 6.17 ± 0.25

7.94 ± 1.98 7.92 ± 0.76

51.5 ± 3.53 50.0 ± 1.41

145.0 ± 38.2 167.0 ± 25.5

1.79 ± 0.09 2.41 ± 1.18

2.63 ± 0.62 2.99 ± 0.30

1.15 ± 0.11 1.44 ± 0.18

1.40 ± 0.43 1.48 ± 0.13

128.5 ± 23.1

7.25 ± 1.94

7.36 ± 0.49

52.5 ± 0.71

155.5 ± 37.5

1.99 ± 0.35

2.72 ± 0.93

1.35 ± 0.50

1.35 ± 0.46

117.0 ± 29.7

6.71 ± 0.69

8.28 ± 0.90

53.5 ± 0.71

139.5 ± 33.2

2.00 ± 0.79

3.05 ± 1.30

1.50 ± 0.64

1.85 ± 0.13

120.0 ± 11.0 138.5 ± 20.1 135.0 ± 19.8 122.5 ± 18.9 146.5 ± 25.9 128.0 ± 5.7 131.0 ± 26.7 144.5 ± 14.8

7.07 ± 1.32 6.82 ± 1.22 7.91 ± 0.46 7.55 ± 1.73 6.75 ± 0.97 6.93 ± 0.10 6.67 ± 1.17 6.28 ± 0.64

7.79 ± 2.17 8.23 ± 0.44 8.0 ± 1.72 7.87 ± 0.90 6.54 ± 2.80 6.86 ± 1.06 6.36 ± 1.08 6.28 ± 2.44

50.5 ± 4.95 54.0 ± 0.01 51.5 ± 6.36 51.0 ± 1.41 61.0 ± 5.66 60.5 ± 4.95 58.5 ± 3.54 52.5 ± 2.12

161.0 ± 29.8 190.5 ± 24.7 173.0 ± 22.6 137.0 ± 36.8 220.5 ± 27.6 202.5 ± 14.8 203.5 ± 31.8 177.5 ± 2.12

2.00 ± 1.05 2.13 ± 0.60 2.03 ± 0.66 2.62 ± 0.26 2.72 ± 0.19 1.84 ± 0.81 2.68 ± 0.37 2.48 ± 0.70

2.70 ± 0.16 3.20 ± 0.51 3.42 ± 0.52 3.18 ± 0.74 2.76 ± 0.66 3.18 ± 0.45 2.9 ± 0.79 2.73 ± 0.62

1.20 ± 0.24 1.52 ± 0.44 1.55 ± 0.49 1.49 ± 0.23 1.17 ± 0.49 1.50 ± 0.52 1.42 ± 0.55 1.53 ± 0.74

1.47 ± 0.71 1.62 ± 0.39 1.70 ± 0.13 1.66 ± 0.49 1.69 ± 0.10 1.77 ± 0.57 1.50 ± 0.45 1.57 ± 0.54

AST = aspartate transaminase, BUN = blood urea nitrogen, GLU = blood glucose, TP = total protein, ALT = alanine transaminase, TG = triglycerides, CHOL = total cholesterol, HDL = high density lipoprotein cholesterol, LDL = low density lipoprotein cholesterol.

lipoprotein cholesterol (HDL) and low density lipoprotein cholesterol (LDL) indicated that no significant differences for any of the parameters among the control mice and the mice that received CrRC or CrFC orally. Similar observation was made by Staniek and Krejpcio (2009) and Staniek et al. (2010b) who reported that haematological parameters and biochemical analyses in rats were not significantly changed by chromium propionate in the acute toxicity test in studies on the effects of chromium propionate supplementation on pregnancy outcome. According to Loomis and Hayes classification (Loomis and Hayes, 1996), chemical substance with a LD50 within the range of 5.0–15.0 g/kg is considered as practically non-toxic. The LD50 values of CrRC and CrFC found in this range indicated that they could be regarded as practically non-toxic in acute ingestion. Wu et al. (2011) and Staniek et al. (2010b) reported that the potential acute toxicity of chromium malate complex and chromium propionate complex, were evaluated as ‘‘non-toxic’’ for the rats. Contrast to CrRC and CrFC, acute oral toxicity of CrCl36H2O was evaluated as relatively higher than that of CrRC and CrFC. Yu et al. (1999) also reported that CrCl3 increased cancer rates in the progeny of male mice that received a single intraperitoneal injection of aqueous CrCl3 at 2 weeks before mating. 5. Conclusion The anti-diabetic activity of CrRC, CrFC and CrSC was examined and the results compared using alloxan-diabetic mice with daily oral gavage for a period of 2 weeks at the dose of 0.5–3.0 mg Cr/ kg body weight. The acute oral toxicities of CrRC and CrFC was tested in the dose of 1.0–5.0 g/kg body weight and observed for a period of 2 weeks. The biological activity results indicated that CrRC and CrFC at dose of 0.5–3.0 mg Cr/kg body weight could decrease the levels of blood glucose, reduce the activities of AST, ALT and ALP, and increase liver glycogen level in alloxan-diabetic mice. In the acute oral toxicity test, LD50 values for both CrRC and CrFC were above 5.0 g/kg. The minimum lethal dose for CrFC was above 5.0 g/kg, while that for CrRC was 1.0 g/kg. Collectively, the anti-diabetic activity of trivalent chromium complexes was not equally effective and their acute oral toxicity was also different. It is therefore recommended that CrFC should represent the optimal chromium supplement among the three chromium complexes with potential therapeutic value to control blood glucose in diabetes and non-toxic in acute oral study.

Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgments This work was supported financially by Specialized Research Fund for the Doctoral Program of Higher Education of China (20103227110004) and Graduate Innovative Projects in Jiangsu University (CX10B_019X). We also appreciate Dr. Mohammed Takase for language polishing. References Anderson, R.A., 1997. Lack of toxicity of chromium chloride and chromium picolinate in rats. J. Am. Coll. Nutr. 16, 273–279. Anderson, R.A., 2003. Chromium and insulin resistance. Nutr. Res. Rev. 16, 267–275. Bailey, M.M., Boohaker, J.G., Sawyer, R.D., Behling, J.E., Rasco, J.F., Jernigan, J.J., Hood, R.D., Vincent, J.B., 2006. Exposure of pregnant mice to chromium picolinate results in skeletal defects in their offspring. Birth Defects Res. Part B 77, 244– 249. Bailey, M.M., Townsend, M.B., Jernigan, P.L., Sturdivant, J., Rasco, J.F., Vincent, J.B., Hood, R.D., 2007. Prenatal and lactational exposure to chromium picolinate and picolinic acid – the effects on the neurological development of mice. Neurotoxicol. Teratol. 29, 408-408. Buccolo, G., David, M., 1973. Quantitative determination of serum triglycerides by use of enzyme. Clin. Chem. 19, 476–482. Carroll, N.V., Longly, R.W., Joseph, H.R., 1956. Determination of glycogen in liver and muscle by use of anthrone reagent. J. Biol. Chem. 220, 583–593. Chaudhary, S., Pinkston, J., Rabile, M.M., Van Horn, J.D., 2005. Unusual reactivity in a commercial chromium supplement compared to baseline DNA cleavage with synthetic chromium complexes. J. Inorg. Biochem. 99, 787–794. Chen, X.-M., Fu, D.-X., Ou, Y.-F., 2003. Preparation of Cr(III)-amorphophallus konjac oligosaccharide complex and study on its effect on blood glucose in mice. Chin. J. Biochem., 24. Deng, Y., 2007. Study of chromium complexes with bacalin bacalein and mdg-1 polysaccharides for anti-hyperglycemia. A Dissertation of Shanghai Jiao Tong University for the Degree of Master. Dogukan, A., Tuzcu, M., Juturu, V., Cikim, G., Ozercan, I., Komorowski, J., Sahin, K., 2010. Effects of chromium histidinate on renal function, oxidative stress, and heat-shock proteins in fat-fed and streptozotocin-treated rats. J. Renal Nutr. 20, 112–120. Ferrannini, E., Lanfranchi, A., Rohner-Jeanrenaud, F., Manfredini, G., Van de Werve, G., 1990. Influence of long-term diabetes on liver glycogen metabolism in the rat. Metabolism 39, 1082–1088. Ghosh, R., Sharatchandra, K., Rita, S., Thokchom, I., 2004. Hypoglycemic activity of Ficus hispida (bark) in normal and diabetic albino rats. Indian J. Phamacol. 36, 222–225. Halliwell, B., Gutteridge, J., 1985. Free Radicals in Biology and Medicine. Oxford, London. Hamden, K., Boujbiha, M.A., Masmoudi, H., Ayadi, F.M., Jamoussi, K., Elfeki, A., 2009. Combined vitamins (C and E) and insulin improve oxidative stress and

F. Li et al. / Food and Chemical Toxicology 50 (2012) 1623–1631 pancreatic and hepatic injury in alloxan diabetic rats. Biomed. Pharmacother. 63, 95–99. Hems, D.A., Harmon, C.S., Whitton, P.D., 1975. Inhibition by parathyroid hormone of glycogen synthesis in the perfused rat liver. FEBS Lett. 58, 167–169. Jain, S.K., Kannan, K., 2001. Chromium chloride inhibits oxidative stress and TNF-a secretion caused by exposure to high glucose in cultured U937 monocytes. Biochem. Biophys. Res. Commun. 289, 687–691. Jain, S.K., Rains, J.L., Croad, J.L., 2007a. Effect of chromium niacinate and chromium picolinate supplementation on lipid peroxidation, TNF-a, IL-6, CRP, glycated hemoglobin, triglycerides, and cholesterol levels in blood of streptozotocintreated diabetic rats. Free Radical Biol. Med. 43, 1124–1131. Jain, S.K., Rains, J.L., Croad, J.L., 2007b. High glucose and ketosis (acetoacetate) increases, and chromium niacinate decreases, IL-6, IL-8, and MCP-1 secretion and oxidative stress in U937 monocytes. Antioxid. Redox Signal. 9, 1581–1590. Jennings, D.S., Brevard, P.B., Flohr, J.A., Gloeckner, J.W., 1997. Chromium nicotinate supplementation: effects on body composition and strength in female colleigate athletes participating in off-season training. J. Am. Diet. Assoc. 97, A65. Kim, D.-S., Kim, T.-W., Park, I.-K., Kang, J.-S., Om, A.-S., 2002. Effects of chromium picolinate supplementation on insulin sensitivity, serum lipids, and body weight in dexamethasone-treated rats. Metabolism 51, 589–594. Krejpcio, Z., Wojciak, R.W., Krol, E., 2011. The effect of hyperglycemia on zinc, copper and chromium status in STZ-induced diabetic rats. Trace Elem. Electrolytes 28, 156–161. Król, E., Krejpcio, Z., 2010. Chromium(III) propionate complex supplementation improves carbohydrate metabolism in insulin-resistance rat model. Food Chem. Toxicol. 48, 2791–2796. Król, E., Krejpcio, Z., 2011. Evaluation of anti-diabetic potential of chromium(III) propionate complex in STZ injected rats fed high diets. Food Chem. Toxicol.. doi:10.1016/j.fct.2011.09.006. Li, F., Wu, X., Yang, L., Zou, Y., Zhao, J., Zhang, R., 2009. Synthesis and characterization of the chromium(III)complexes with rutin and quercetin. Chem. Res. Appl. 21, 899–902. Liu, B., Qin, Z.-K., Lin, X.-M., Mei, L., Liu, W.-S., Han, B.-Q., 2009. Promotion effect of chitooligosaccharides and its derivatives on pancreatic islet cells proliferation and insulin secretion. J. Clin. Rehab. Tissue Eng. Res. 13, 513–516. Loomis, T.A., Hayes, A.W., 1996. Loomis’s Essentials of Toxicology. Academic Press, San Diego, California. Lukaski, H.C., Siders, W.A., Penland, J.G., 2007. Chromium picolinate supplementation in women: effects on body weight, composition, and iron status. Nutrition 23, 187–195. Machalin´ski, B., Walczak, M., Syrenicz, A., Machalin´ska, A., Grymuła, K., Stecewicz, I., Wiszniewska, B., Da˛bkowska, E., 2006. Hypoglycemic potency of novel trivalent chromium in hyperglycemic insulin-deficient rats. J. Trace Elem. Med. Biol. 20, 33–39. Martin, J., Wang, Z.Q., Zhang, X.H., Wachtel, D., Volaufova, J., Matthews, D.E., Cefalu, W.T., 2006. Chromium picolinate supplementation attenuates body weight gain and increases insulin sensitivity in subjects with type 2 diabetes. Diabetes Care 29, 1826–1832. Moreira Jr., E.D., Neves, R.C.S., Nunes, Z.O., de Almeida, M.C.C., Mendes, A.B.V., Fittipaldi, J.A.S., Ablan, F., 2010. Glycemic control and its correlates in patients with diabetes in Venezuela: results from a nationwide survey. Diabetes Res. Clin. Pract. 87, 407–414. Nair, S.A., Shylesh, B.S., Gopakumar, B., Subramoniam, A., 2006. Anti-diabetes and hypoglycaemic properties of Hemionitis arifolia (Burm.) Moore in rats. J. Ethnopharmacol. 106, 192–197.

1631

Preuss, H.G., Echard, B., Perricone, N.V., Bagchi, D., Yasmin, T., Stohs, S.J., 2008. Comparing metabolic effects of six different commercial trivalent chromium compounds. J. Inorg. Biochem. 102, 1986–1990. Sharma, S., Agrawal, R.P., Choudhary, M., Jain, S., Goyal, S., Agarwal, V., 2011. Beneficial effect of chromium supplementation on glucose, HbA1C and lipid variables in individuals with newly onset type-2 diabetes. J. Trace Elem. Med. Biol. doi:10.1016/j.jtemb.2011.03.003. Shaw, J.E., Sicree, R.A., Zimmet, P.Z., 2010. Global estimates of the prevalence of diabetes for 2010 and 2030. Diabetes Res. Clin. Pract. 87, 4–14. Shiau, S.-Y., Lin, S.-F., 1993. Effect of supplemental dietary chromium and vanadium on the utilization of different carbohydrates in tilapia, Oreochromis niloticus  O. aureus. Aquaculture 110, 321–330. Shrivastava, H.Y., Ravikumar, T., Shanmugasundaram, N., Babu, M., Unni Nair, B., 2005. Cytotoxicity studies of chromium(III) complexes on human dermal fibroblasts. Free Radical Biol. Med. 38, 58–69. Sreekanth, R., Pattabhi, V., Rajan, S.S., 2008. Molecular basis of chromium insulin interactions. Biochem. Biophys. Res. Commun. 369, 725–729. Staniek, H., Krejpcio, Z., 2009. The effects of tricentric chromium(III) propionate complex supplementation on pregnancy outcome and maternal and foetal mineral status in rat. Food Chem. Toxicol. 47, 2673–2678. Staniek, H., Kostrzewska-Poczekaj, M., Arndt, M., Szyfter, K., Krejpcio, Z., 2010a. Genotoxicity assessment of chromium(III) propionate complex in the rat model using the comet assay. Food Chem. Toxicol. 48, 89–92. Staniek, H., Krejpcio, Z., Iwanik, K., 2010b. Evaluation of the acute oral toxicity class of tricentric chromium(III) propionate complex in rat. Food Chem. Toxicol. 48, 859–864. Trent, L.K., Tiedingcancel, D., 1995. Effects of chromium picolinate on body composition. J. Sport Med. Phys. Fit. 35, 273–280. Vincent, J.B., Sun, Y.J., Clodfelder, B.J., 2001. Biomimetic chromium complex enhances insulin sensitivity in healthy and type II diabetic model rats. Diabetes 50, A508. Vrtovec, M., Vrtovec, B., Briski, A., Kocijancic, A., Anderson, R.A., Radovancevic, B., 2005. Chromium supplementation shortens QTc interval duration in patients with type 2 diabetes mellitus. Am. Heart J. 149, 632–636. Wu, X.-Y., Li, F., Xu, W.-D., Zhao, J.-L., Zhao, T., Liang, L.-H., Yang, L.-Q., 2011. Antihyperglycemic activity of chromium(III) malate complex in alloxan-induced diabetic rats. Biol. Trace Elem. Res. 143, 1031–1043. Yanardag, R., Demirci, T.B., Ülküseven, B., Bolkent, S., Tunali, S., Bolkent, S., 2009. Synthesis, characterization and antidiabetic properties of N1-2,4dihydroxybenzylidene-N4-2-hydroxybenzylidene-S-methylthiosemicarbazidato-oxovanadium(IV). Eur. J. Med. Chem. 44, 818–826. Yang, X., Palanichamy, K., Ontko, A.C., Rao, M.N.A., Fang, C.X., Ren, J., Sreejayan, N., 2005. A newly synthetic chromium complex – chromium(phenylalanine)3 improves insulin responsiveness and reduces whole body glucose tolerance. FEBS Lett. 579, 1458–1464. Yang, X.-P., Li, S.-Y., Dong, F., Ren, J., Sreejayan, N., 2006. Insulin-sensitizing and cholesterol-lowering effects of chromium (D-phenylalanine)3. J. Inorg. Biochem. 100, 1187–1193. Yang, L., Li, F., Wu, X., Zhang, M., Zhang, R., Yan, S., 2010. A folic acid chromium(III) complex and its preparation method. China Patent, CN101691369A. Yu, W., Sipowicz, M.A., Haines, D.C., Birely, L., Diwan, B.A., Riggs, C.W., Kasprzak, K.S., Anderson, L.M., 1999. Preconception urethane or chromium(III) treatment of male mice: multiple neoplastic and non-neoplastic changes in offspring. Toxicol. Appl. Pharmacol. 158, 161–176. Zhang, L., Cao, Y., Pen, L.-L., Wen, Z.-J., Yang, Y.-S., 2002. Experimental study on reducing serum glucose of seaweed polysaccharides–Cr  (3+) complex. Sichuan J. Phys. Sci. 24, 69–71.