Α-glucosidase Inhibition by New Schiff Base Complexes of Zn(ii) We Prepared and Characterized the Zn(ii) Complexes with Four Different Schiff Bases (n-salicylidene-β-alanine (n- Sβ), N-n'-bis (salicylidene) Ethylenediamine (n-bse), N, N'-bis (salicylidene)-phenylenediamine (n- Bsp), and 1-[

There are many reports that divalent alkaline earth, first-row transition metals, and Zn(II) ions have α-glucosidase inhibitory effects. Cu(II) and Zn(II) ions, in particular, have strong α-glucosid-ase inhibitory effects. Several Schiff bases also display α-glucosidase inhibitory effects. In this study, we focused on safe and highly effective complexes including Zn(II) ion.(2-dimethylaminoethylimino) methyl]naphtholate (DMN)) and investigated their α-glucosidase inhibitory effects in vitro, using α-glycosidases from Saccharomyces sp. and rat small intestine, and in vivo, using a sucrose tolerance test. The Zn(II) complexes with DMN showed the highest in vitro and in vivo α-glucosidase inhibitory effects in this study.


Introduction
In 2006, the World Health Organization predicted that the number of patients with type 2 diabetes mellitus in the world could increase to 360 million by 2030 [1].Early detection and rapid cure are very important, because complications of diabetes such as nephropathy, retinopathy, and neuropathy are difficult to treat.Oral antidiabetic medicines have been used as one of the principle therapeutic methods to treat diabetes.α-Glucosidase inhibitors comprise one class of oral antidiabetic medicines.As of 2013, three types of α-glucosidase inhibitors, acarbose, voglibose, and miglitol, are used in medical practice (Figure 1).α-Glucosidase is an enzyme that metabolizes disaccharides into monosaccharides in the small intestine.Inhibiting this enzyme delays the digestion and absorption of carbohydrates, which results in suppression of both postprandial hyperglycemia and excessive insulin secretion.

Materials and Animals
All reagents and solvents used in this study were of the highest commercially available grade and were used as obtained.(CH 3 COO) 2 Zn•2H 2 O, ZnCl 2 , salicylaldehyde, β-alanine, ethylenediamine, 2-hydroxy-1-naphtaldehyde, LiOH•H 2 O, HEPES, NaOH, KH 2 PO 4 , dithiothreitol, α-glucosidase (from Saccharomyces sp.), maltose, d-(+)-glucose, powdered acacia, and a Glucose C-II Test kit were purchased from Wako Pure Chemical Industries (Osaka, Japan).O-phenylenediamine was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan).N, N-dimethylethylenediamine was purchased from Tokyo Chemical Industry Co. (Tokyo, Japan).EDTA•2Na•2H 2 O and Triton X-100 were purchased from Nacalai Tesque, (Kyoto, Japan).Rat small intestine acetone powder was obtained from Sigma Chemical Co.(St.Louis, MO, USA).Twelve-week-old ddYmice, which are non-inbred mice maintained in a closed colony, were purchased from Shimizu Laboratory Supplies Co. (Kyoto, Japan).All the mice were maintained on a 12 h light/dark cycle in our temperature-controlled central animal facility for breeding under fixed condition.The animal study was approved by the Experimental Animal Research Committee at the Kyoto Pharmaceutical University (KPU) and was performed according to the Guidelines for Animal Experimentation.

Synthesis of Four Ligands and Their Zn(II) Complexes
We synthesized four Schiff base ligands and their Zn(II) complexes (Schemes 2-5).The syntheses of these ligands were performed as described in previous reports [9] [21] [22].The intended Zn(II) complexes were readily

Preparation of α-Glucosidase from Rat Small Intestine Acetone Powder
The rat small intestine acetone powder was suspended in a 10 mM sodium phosphate buffer (pH 6.8).The suspension was homogenized, sonicated for 30 min and followed by the addition of 2% Triton X buffer (pH 7.0) containing 3 mM EDTA-2Na and 1 mM dithiothreitol (DTT) in the ice bath and centrifugation for 60 min at 20,000 g (4˚C).The supernatant was subjected to the ammonium sulfate precipitation.The precipitates were collected and dialyzed.The resulting solution was used for the assay.

Inhibition of α-Glucosidase in in Vitro
Solutions of ligands or Zn(II) complexes at various concentrations were prepared, and their α-glucosidase inhibitory effects were evaluated using a modified Dahlqvist method [23].The substrate and test solution were mixed and incubated at 37˚C for 5 min.A solution containing α-glucosidase enzyme (5 unit/ml) from Saccharomyces sp. or rat small intestine was added continuously and incubated at 37˚C for 1 h.After incubation, the reactions were terminated by heating at 90˚C for 5 min.The glucose concentration was determined by using a Glucose C-II Test kit.

Inhibition of α-Glucosidase in in Vivo
Twelve-week-old ddY mice were fasted for 6 h and were then orally administered one of the test solutions.After 30 min, a 5% acacia solution of maltose or glucose was orally administered [24].Blood samples were obtained from the tail vein at 0, 15, 30, 60, and 90 min.The blood glucose levels were measured using a glucose oxidase method (Glucocard; Arkray, Kyoto, Japan).
The substrate and test solution were mixed and incubated at 37˚C for 5 min.Next, an α-glucosidase enzyme solution was added and incubated at 37˚C for 30 min.After incubation, the reactions were terminated by heating at 90˚C for 5 min.The glucose concentration was determined by using a Glucose C-II Test kit.Substrate solutions containing sucrose at concentrations of 75, 150, and 300 mM (Saccharomyces sp.experiment) and of 15, 30, and 60 mM (rat small intestine experiment), and were prepared.[Zn 2 {(DMN) 2 Cl 2 }], which showed the highest activity in vitro, was used the IC50 value in in vitro study.

Statistical Analysis
All experimental data are expressed as the mean ± standard derivation (SD).Statistical analyses of the in vitro data were performed using Student's t-test.Statistical analyses of the in vivo data were performed using one-way analysis of variance (ANOVA), followed by Dunnett multiple comparison post-hoc tests.Differences were considered to be statistically significant when p values were < 0.01 or < 0.05, as noted.

Structural Characteristics
Four Schiff base ligands and their Zn(II) complexes were characterized by several physicochemical methods.These data are shown in Table 1 and Table 2.For elemental analysis, both calculated and measured values of the percent concentration of C, H, and N were identical and within the estimated range of experimental error.In the IR spectra, we observed the frequencies due to the ν C=N of Zn(II) complexes with four Schiff bases.And we observed the parent peak of these Zn(II) complexes in the Mass Spectra.When we measured NMR spectrometry, the Zn(II) ion induced the 13 C NMR chemical shift changes of the N-sβ, as shown in Table 2.

Inhibition of the α-Glucosidase from Saccharomyces sp. in in Vitro
In

Inhibition of the α-Glucosidase from Rat Small Intestine in in Vitro
In in vitro experiments involving rat intestinal α-glucosidase, the Zn(II) complexes exhibited α-glucosidase inhibitory effects in the following order:

Discussion
There have been many reports in recent years ascribing various biological activities to many metal ions or their complexes [27] [28].The study of divalent alkaline earth, first-row transition metal, and Zn(II) ions have become particularly popular [5] [29].In 2014, Kumar et al. reported that α-glucosidase inhibitory activity of Schiff base complexes containing Mn, Co, Ni, Cu, Sr, and Cd were more effective than that of the free Schiff base ligand [30].Schiff base metal complexes have received a lot of attention from many researchers.In this study, we synthesized four Schiff base ligands and their Zn(II) complexes (Schemes 2-5).We chose Zn(II) from among many metals for this study because the Zn(II) ion was an essential trace element and Zn(II) compounds possessed a wide margin of safety.Figure 2 and Figure 3 showed that Zn(II) complexes derived from Schiff bases inhibited the α-glucoidases from yeast and rat small intestine in in vitro study; however, the Schiff base ligands did not show activity by themselves.From these results, we could say that the α-GI effect might be mainly caused by the Zn(II) in the complexes.In addition, the compounds that belonged to the "Schiff bases" did not necessarily Zinc gluconate 1.70 [31] [N-sβ-Zn] 4.50 [32] [N-bsE-Zn] 9.05 [33] [N-bsP-Zn] 13.3 [34] Zn-His 12.0 [35] Zn-Cys 18.2 [35] showed an α-GI effect.The C-N double bond is considered an important factor for the α-GI effect of the Schiff bases; however, we think that the α-GI effect of several Schiff bases may be caused by factors other than their C-N double bond.[N-bsP-Zn] shows weaker effect than other Zn(II) complexes (Figure 2(a) and Figure 3(a)).
As the reason for this result, we consider the possible involvement of the stability constant (Table 3).Moreover, cysteine and histidine residues play an important role in the active center of α-glucosidase [36].Thus, we expect that ligands which bind more strongly with Zn(II) ion than cysteine and histidine may prevent the α-GI effect of the Zn(II) ion.In Figure 4(a) and Figure 5(a), DMN shows a α-GI effect equivalent to the effect of zinc acetate against maltase in the in vivo study.Therefore, we think that the α-GI effect of [Zn 2 {(DMN) 2 Cl 2 }] against maltase may be caused by a synergic effect from the combination of the Zn(II) ion and this ligand.
In conclusion, Zn(II) ions and their complexes exhibited α-GI effects in in vitro and in vivo studies.Additionally, although Schiff base ligands did not show a α-GI effect in in vitro, DMN showed an anti-hyperglycemic effect in the glucose-loading test during the in vivo study.We considered that changing the ligand structures of Zn(II) complexes might result in synergic action between the metal ion and the ligand in producing a α-GI effect.Finally, when searching for candidate compounds, we should look at their physical properties such as their stability constants, molecular weights, or lipid solubilities.

Figure 2 .
Figure 2. Inhibition of the α-glucosidase activity ((a) yeast and (b) rat small intestine) by Zn(II) complexes and Schiff base ligands.Maltose (0.1 M) was used as the substrate.Data are presented as the mean ± SD (n = 3 − 4).

[
Zn 2 {(DMN) 2 Cl 2 }], which showed a particularly high inhibitory effect against rat intestinal α-glucosidase, and its Schiff base ligand, DMN, were selected for oral maltose and glucose tolerance testing.In the maltose tolerance test, the postprandial blood glucose levels in the [Zn 2 {(DMN) 2 Cl 2 }] group were significantly lower than those in control groups.In the glucose tolerance test, the postprandial blood glucose levels in the DMN group and the (CH 3 COO) 2 Zn•2H 2 O group were lower than those in the control groups.The values of the area under the curve (AUC) also showed similar transitions in each test (Figures4(a)-(b) and Figures 5(a)-(b)).

Table 1 .
in vitro experiments involving yeast α-glucosidase, the Zn(II) complexes exhibited α-glucosidase inhibitory effects in the following order: (CH 3 COO) 2 Zn•2H Analytical data of four schiff base ligands and their Zn(II) compounds.

Table 3 .
Stability constants of the Zn(II) complexes.