Synthesis, Characterization and Effectiveness of Chelated Mineral as Aflatoxin Absorbents

The bioinorganic projects focus the synthesis of transition complexes using ligand – peptide conjugates that will impart coordination environments similar to those found in biological systems [1]. The design of new complexes plays an important role in bioinorganic chemistry [2-4]. A large number of metal-binding substances such as amino acids and proteins are present in biological systems. Low– molecular weight substances are involved in the absorption and transport of metal ions and metalloproteins play various roles, such as enzymatic catalysis, oxygen transport, and metal ion storage and transport [5,6]. Proteins such as metallothioneins protect organisms from the toxic effects of exogenous metal ions [7]. Most transition metal amino acid complexes have considerable biological activity, such as antitumor properties [8]. Amino acids usually increase the diffusibility of complexes and enhance their biological action inside the cell [9]. Such systems are widely used in the field of chemotherapy. Structural studies of many transition metal complexes have shown that amino acids coordinate in various ways, dependent upon the metal ion, its oxidation state and the primary structure of the amino acid [10]. Methionine is one of the nine essential amino acids needed by human beings and its copper (II) complex showed some antiulcer activity as revealed by animal model studies [11] and has also found some interest in veterinary medicine, for copper supplementation [12-14].

Preparation of the complexes

Solutions of 0.08 mole of Mn, Cr, Co, Cu and Zn sulfate were mixed with 0.08 mole of methionine. The reaction mixture was refluxed for two hours and then left overnight where the complexes were precipitated. Then filtered, washed with distilled water and dried in vacuum desiccators over P₄O₁₀. The melting points of the complexes are over 300ºC.

Analysis of the metal content

The complexes were digested and decomposed with aqua regia. The metal ion contents were determined by atomic absorption (Table 1) spectra.

Table 1: Analytical data, electronic spectra and μeff for methionine complexes

Carbon, hydrogen and nitrogen analysis

These were done at the Micro Analytical Laboratory (Table 1), Faculty of Science, Tanta University, Egypt.

Instruments and working procedures

IR spectra: The KBr IR spectra were recorded using Perkin –Elmer spectrophotometer model 1430 covering the frequency range 200-4000 cm^-1.

UV-Vis spectra: The spectral studies were measured using PYEUnicam spectrophotometer model 1750 covering the wavelength range 190-900 nm. The complexes were measured in nujol mull following the method described by Lee et al. [15].

Magnetic susceptibility measurements: Molar magnetic susceptibility corrected for diamagnetic using Pascal?s constant were determined at room temperature applying the Faraday’s method. The apparatus was calibrated with Hg [Co(SCN)₄] [16].

Thermal analysis: Differential thermal analyses (DTA) and thermo gravimetric analyses (TGA) were carried out using Shimadzu DTA-50. The rate of heating was 20ºC/min.

Effect complexes on aflatoxin concentration: One liter of yeast extract broth was divided into 10 flasks. Each flask’s content was mixed with one of the tested compounds to reach a final concentration of 2000 μg/ml. All flasks were artificially contaminated with 104 (c.f.u.) of toxigenic Aspergillus flavus strain and incubated at 25-27ºC for 21 days after which autoclaving of the media was done at 121ºC for 30 min. then mycotoxin concentration was determined using Fluorometer Vicam V 1.3 Series 4, according to [17].

To study physical and chemical properties of chelated mineral and its effect on aflatoxin.

Infrared spectra of amino acid complexes

Methionine complexes: The most important ir spectral bands for methionine and its complexes are given in (Figure 1). The bands a 3342-3269, 3409-3349-3277, 3413.9-3237, 3317 cm^-1 for Mn, Cr, Co, Cu and Zn methionine complexes respectively, arise the coordination of H₂O molecules and asymmetric stretching vibration of NH₂ [18]. The presence of new band at 3269, 3277, 3237 and 3317 cm^-1 in Mn, Co, Cu and Zn methionine complexes respectively, are due to γs NH₂. So, the nitrogen of the amino group is involved in coordination [19]. It is worthwhile to mention that the free methionine exist as zwitterions (NH₃. AA. COO-) due to the presence of characteristic γ (NH₃) and σ NH₃. The amino acid in complexes does not exist in zwitterions, NH₃ get deprotonated and binds to metals through the neutral NH₂ group. The transformation of NH₃to NH₂ must result in upward shift in γs NH₂ and δNH₂ [20,21]. On complexation, the two δNH₂ stretching vibration at 1695 and 1590 cm^-1 together with some deformational motions of this group could be observed. The position of these bands clearly supports the involvement of this group in bonding. Moreover, the γ (C-N) stretching mode at 1352 cm^-1 is affected to different extent in complexation [22]. The IR spectra showed strong evidence in support of the involvement of carboxylate group in coordination. The γ_as COO- and γ_s COO- at 1610 and 1410 cm^-1 record shifts in complexes. New ir bands appeared at 588-440 cm^-1 are due to γ M-O and γ M-N. So, methionine act as bidentate ligand.The band at 2370cm^-1 attributted to γ (SH) unaffected on complexation indicated that (S) atom is not involved in chelation.

Figure 1: Infrared spectra of methionine and its complexes (1) Methionine (2) Manganese methionine (3) Cromium methionine (4) Cobalt methionine (5) Copper methionine (6) Zinc methionine.

Electronic spectra and magnetic moments of methionine complexes

For the chromium complex (Figure 2), three spin allowed transition have been observed at 249,286 and 428 nm assigned to charge transfer, but for 428nm is due to ⁴A_2g (F) ? ⁴T_1g(F) respectively [23]. The observed magnetic moment is 3.88 B.M due to the existence of octahedral geometry [23]. Mn methionine complex exhibit absorption bands at 245, 248, 251, 261, 263, 267and 444 nm are due to d-d transitions. Its magnetic moments is 5.88 typified the existence of octahedral structure [24]. The nujol mull spectra of cobalt methionine showed two bands at 249 and 434 nm, the band at 249 nm is due to charge transfer but the band at 434 nm is due to⁴T_1g(F) ? ⁴T_1g(P) transition. The U_eff= 5.8 B.M is consistent with octahedral geometry [25].Cu methionine complex exhibit absorption bands at 252,255,265,270 and 274 due to charge transfer type. The U_{eff =}1.5 B.M is consistent with tetrahedral structure [26].

Figure 2: Electronic spectra of: (1) copper methionine (2) manganes emethionine (3) cobalt methionine (4) chromium methionine

Thermal analysis of complexes

The DTA of Co methionine Co (L)3 (HL).4H₂O, (Figure 3) gave one endothermic peak at 205.3°C and two exothermic peaks at 305.7°C, 509.2°C, the order of reactions is 2.2, 0.95 and 1.56 respectively, i.e., the first and second types [27]. The first broad DTA peak at 205.3°C (56.3-236.3)°C is due to dehydration of lattice and coordination water molecules [27]. The broad features of DTA peak may be due to strong thermal agitation accompanying the water elimination and loss of water molecules occurs in more than one step. This is confirmed by appearance of TGA peak at (53-233.2)°C [28] .The two strong exothermic peaks at 305.7°C and 509.2°C are due thermal agitation and decomposition steps of metal complex as evident from TGA. The latter peak at 509.2 and the associated TGA (415.2-560°C) and (560-700)°C is assigned to the decomposition of the complex with weight loss 18.8 and 10.2% ended with the formation of CoO.

Figure 3: DTA curve for cobalt methionine complex.

The DTA and TGA of manganese methionine Mn(L)₃. 2H₂O, (Figure 4) gave one endothermic peak at 99.3 and exothermic peaks at 247.8°C and 513°C.The first peak is assigned to dehydration process of the lattice and coordinated water molecules with activation energy 353.3 KJ/mole, the larger the E_a values is may be due to the thermal agitation accompanying the water elimination [27].The two exothermic peaks at 247 and 513°C with activation energies 79.2 and 220.15 KJ/ mole are corresponding to the thermal agitation and decomposition of complexes ended with the formation of MnO 13.4% [28].

Figure 4: DTA and TGA curves for Manganese methionine complex.

The TG and DTA curves of copper methionine complex Cu (L).3H2O, (Figure 5), pointed to that the dehydration process of water molecules takes places in one step in the temperature range (29.6-240)°C with activation energy 160.4 KJ/mole [28]. The strong exothermic peak at 250°C is due thermal agitation and decomposition steps of metal complexes as evident from TG. The latter broad band peak with maximum at 350°C and the associated TGA peaks with the temperature range (240-500)°C showed weight loss of 49.33% is assigned to the decomposition of complex ended with the formation of Cu O as final product [29].

Figure 5: DTA and TGA curves for Copper methionine complex.

The TG and DTA curves of zinc methionine complex Zn (L)₂ (HL). H₂O, (Figure 6) gave four exothermic peaks at 69.5, 175, 334 and 563.9°C with activation energies of 93, 42.6,101.6 and 240.5 KJ/mole, their order of decomposition is 1.34 ,1.12 , 0.19 and 0.93. The first band at 69.5°C (40.8-94)°C is due to dehydration of H₂O molecule this is confirmed by TGA peaks in the range (26.9-85)°C. The three strong exothermic peaks are due to thermal agitation and decomposition which occurs in three steps with weight loss 27.9, 24.7 and 27.9 ended with the formation of ZnO 15.2% as evident from TG [30,31].

Figure 6: DTA and TGA curves for zinc methionine complex.

Biological activity against aflatoxin

Table 2 show that the addition of methionine complexes results in reduction of aflatoxin percentage to different degree. The complexes have the ability to absorb mycotoxin with high affinity. In vitro results suggested that the complexes are a strong binder for aflatoxin. An inert, stable and insoluble complex between chelated mineral and aflatoxin was assumed to be responsible for toxin absorption [32]. Absorptions by all metal complexes increase with increasing their levels. The activity order of complexes are copper methionine>zinc methionine>cobalt methionine. The geometric structure of the complexes plays an important role for adsorption of aflatoxin. So, the tetrahedral structure has the great ability to bind aflatoxin than octahedral structure [33-37].

Table 2: The effect of complexes on the concentration (%) of aflatoxin.

Transition metalamino acid complex (copper, zinc and cobalt methionine) have considerable biological activity against aflatoxin and the geometry structure of complex play an important role for adsorption of aflatoxin.