Synthesis and Characterization of Some Selenium Nanometric Compounds: Spectroscopic, Biological and Antioxidant Assessments ()
1. Introduction
Selenium element was mentioned important due to its interesting role in the life construction of plants and biological systems, which absorb organoselenium compounds accumulated in the soils [1] [2] . It is an essential nutrient at trace level but toxic in excess [3] . Selenium is a trace mineral that is essential to good health but required only in small amounts [4] . Selenium is incorporated into proteins to make selenoproteins, which are important as antioxidant enzymes that help to prevent cellular damage from free radicals and to the development of chronic diseases such as cancer and heart disease [5] . Other selenoproteins help to regulate thyroid function and play a role in the immune system [6] . Because of its antioxidant role, selenium has been studied for its potential to protect the body from many degenerative diseases, including Parkinson’s and cancer [7] . Vitamin A (vit-OH; Figure 1) is an essential nutrient for human body. The vitamin A has biological activities [8] . Vitamin A deficiency was a major nutritional deficiency disorder in many developing countries, especially affects young children, in whom it can cause xerophthalmia and lead to blindness, and can also limit growth, weaken innate and acquired host defenses, exacerbate infection and increase the risk of death [9] . Generally, metal ions were required for many critical functions in humans [10] -[12] . The ability to recognize to the molecular level and to treat diseases caused by inadequate metal-ion function constitutes an important aspect of medicinal bioinorganic chemistry [10] [12] . Metal complexes have been playing a key role in the development of modern chemotherapy [13] , for example, complexation of non-steroidal anti-inflammatory drugs to copper overcomes some of the gastric side effects of these drugs [14] . The release of cytotoxins such as nitrogen mustards from redox-active metals such as cobalt in the hypoxic regions of solid tumors has the potential to improve drug activity and reduce toxicity [15] . The metal based drugs were also being used for the treatment of a variety of ailments viz. diabetes, rheumatoid arthritis, inflammatory and cardiovascular diseases as well as diagnostic [16] . A number of drugs and potential pharmaceutical agents also contain metal-binding and potentially influence their bioactivities [17] - [19] . Metal-organic frameworks were a burgeoning field in the last two decades, not only stems from their tremendous potential applications in areas such as catalysis, molecular adsorption, magnetism, nonlinear optics, and molecular sensing, but also from their novel topologies and intriguing structural diversities [20] [21] . On the other hand, many organic drugs, which possess modified pharmacological and toxicological properties administered in the form of metallic complexes [22] , have the potential to act as ligands and the resulting metal-drug complexes are particularly important both in coordination chemistry and biochemistry [23] [24] , however, the study of metal-drug complexes is still in its early stages, thus representing a great challenge in current synthetic chemistry and coordination chemistry. Recently, the trend of metal drug complexes has significant interest in order to achieve an enhanced therapeutic effect in combination with decreased toxicity. To the best of our knowledge, little attentions have been made to discuss the interaction between vitamin A and selenium (IV) metal ions and the literature is still poor in such spectroscopic characterizations. The interpretation is based on the ability of the cited drug to form complex associate with vital antioxidant metal ions like Se (IV). The spectral characteristic and the stability of the formed complex were also discussed.
2. Experimental@NolistTemp# All chemicals used throughout this study were Analar or extra pure grade and received from Aldrich chemical company.
Figure 1. 3, 7-Dimethyl-9-(2, 6, 6-trimethyl-cyclohex-1-enyl)- nona-2, 4, 6, 8-tetraen-1-ol ( Vitamin A; vit-OH).
2.1. Preparation of Selenium Nano Particles
The Se metal nano particles was prepared previously [25] by mixing equal volumes of aqueous solutions of 0.01 M of SeO2 with 0.1 M of acetamide as following equation.
The mixture was heated on a water bath to approximately 90˚C for about ~24 hrs. The precipitate filtered off, washed several times with bidistilled water and dried in vacuo over CaCl2. The selenium metal product was received in powder solid form. The elemental analysis for the obtained product shows the absence of carbon and nitrogen elements. The yield of selenium was ~70%.
2.2. Preparation of Solid Selenium (IV) vit-OH Complex
A mixture of solid powder of selenium metal (1 mmole) and the vit-OH (4 mmole) in toluene solvent (50 mL) was refluxed for 12 hr at 60˚C. The un-reacted of selenium metal powder was removed by filtration, and the resultant brownish red solution were reduced to ca. 1/3 of its volume and cooled to room temperature. The solid brown complex obtained was then collected by filtration, washed with little amount of toluene and dried in vacuo over anhydrous calcium (II) chloride. Elemental analysis, Calculated: %C = 78.45, %H = 9.88, %Se = 6.45; Found: %C = 78.32, %H = 9.56, %Se = 6.31.
2.3. Instrumental Analyses
The elemental analyses of carbon, and hydrogen contents were performed using a Perkin Elmer CHN 2400 (USA). The molar conductivities of freshly prepared 1.0 × 10−3 mol/cm3 dimethylsulfoxide (DMSO) solutions were measured for the dissolved compounds using Jenway 4010 conductivity meter. The electronic absorption spectrum of Se (IV) complex was recorded in DMSO solvent within 600 - 200 nm range using a UV2 Unicam UV/Vis Spectrophotometer fitted with a quartz cell of 1.0 cm path length. The infrared spectra with KBr discs were recorded on a Bruker FT-IR Spectrophotometer (4000 - 400 cm−1). Raman laser of samples were measured on the Bruker FT Raman with laser 50 mW. The 1H-NMR spectra were recorded on Varian Mercury VX-300 NMR spectrometer. 1H spectra were run at 300 MHz spectra in deuterated dimethylsulphoxide (DMSO-d6). Chemical shifts are quoted in δ and were related to that of the solvents. The thermal studies TG/DTG-50H and DTA-50 H were carried out on a Shimadzu thermo-gravimetric analyzer under nitrogen till 800˚C. Scanning electron microscopy (SEM) images were taken in Quanta FEG 250 equipment. The X-ray diffraction patterns for the complexes were recorded on X ‘Pert PRO PANanalytical X-ray powder diffraction, target copper with secondary monochromate.
2.4. Antimicrobial Test
According to Gupta et al., 1995 [26] , the hole well method was applied. The investigated isolates of bacteria and fungi seeded in tubes with nutrient broth (NB) and Dox’s broth (DB), respectively. The seeded (NB) for bacteria and (DB) for fungi (1 ml) were homogenized in the tubes with 9 ml of melted (45˚C) nutrient agar (NA) for bacteria and (DA) for fungi. The homogenous suspensions poured into Petri dishes. The holes (diameter 0.5 cm) done in the cool medium. After cooling in these holes, about 100 ml of the investigated compounds applied using a micropipette. After incubation for 24 h in an incubator at 37˚C and 28˚C for bacteria and fungi, respectively, the inhibition zone diameter were measured and expressed in cm. The antimicrobial activities of the investigated compounds were tested against some kinds of bacteria as Escherichia coli (Gram − ve) and Staph albus (Gram + ve) as well as some kinds of fungi as Aspergillus flavus and Aspergillus niger. In the same time with the antimicrobial investigations of the compounds, the pure solvent also tested. The concentration of each solution was 1.0 × 10−3 mol/L. Commercial DMSO was employed to dissolve the tested samples.
3. Results and Discussion
3.1. Characterization of Selenium Nano Scale
The formation of brown colored selenium metal in nano scale size was prepared upon the heating of an aqueous mixture of SeO2 with acetamide at 90˚C. The isolated of selenium metal powder occurs during the decomposition of acetamide according to the important role of metal ion on the decomposition process via aqueous media. The infrared spectrum of the SeO2 with acetamide at 90˚C product is shown in Figure 2. The infrared spectrum of the obtained selenium metal product shows no bands due to characteristic groups of acetamide (carbonyl and amide groups). The XRD patterns of Se metal are shown in Figure 3. All definite peaks of Se metal are indexed as Se metal structure, which are matched and compared with the standard data. The SEM micrograph of the prepared Se metal is shown in Figure 4. The average grain size of selenium metalwere calculated and found in between 0.65 µm. From Figure 4 it is so difficult to observe inhomogeneitiy within the same micrograph due to that the solution route synthesis with acetamide precursor is very fine and the particle size must be in nano-range. The grain size for nano compounds were calculated according to Scherrer’s formula [27] .
where D is the crystalline grain size in nm, q, half of the diffraction angle in degree, l is the wavelength of X-ray source (Cu-Kµ) in nm, and B, degree of widening of diffraction peak which is equal to the difference of full width at half maximum (FWHM) of the peak at the same diffraction angle between the measured sample and standard one.
Figure 2. Infrared spectra of acetamide as a free ligand and Se metal.
Figure 3. XRD spectrum of the resulted Se metal.
The Raman laser spectrum of Se metal is reported in Figure 5. For the pure selenium sample the spectrum is characterized by a strong band at 237 cm−1 which is attributed to the vibrational mode of –Se-Se-Se-chains [28] [29] . Additional weaker features can be revealed at 141 cm−1 which are assigned to the presence of Se8 rings and to the bending modes of Se units.
3.2. Characterization of Selenium (IV) Vitamin A Complex
The reaction of vit-OH with selenium (0) metal in toluene solvent gave a brown colored solid complex. The found and calculated percentage of elemental analysis is in a well agreement with each other and proves the suggested molecular formula of the resulted selenium complex with molar ratio 1:4 (Se: vit-OH). The complexes have high melting points ~220˚C. The molar conductivity of selenium complex in DMSO has 15 Ω−1∙cm2∙mol−1, so that, it is non-electrolytes nature [30] [31] . Vitamin A ligand behaves as monodentate ligand and coordinates to selenium metal through the oxygen of hydroxyl group.
3.2.1. Electronic Absorption Spectra
The formation of the Se-vit-OH complex was also confirmed by UV-Vis spectroscopic technique. Figure 6, show the electronic absorption spectrum of the complex in DMSO in the 200 - 600 nm range. It can see that the free vitamin A has one distinct absorption band at 350 nm which may be attributed to n → π* intra-ligand transition. In the spectra of the selenium complex this band is blue shifted clearly to 310 nm, where the complex show two bands at 239 and 310 nm assigned to π → π* and n → π* respectively, suggesting the ligand is binding to selenium metal through the lone pair of electrons on oxygen atom of –OH group. The electronic absorption spectrum of the selenium complex in DMSO solution has significant band at 370 nm due to the charge transfer transition from metal-to-ligand.
3.2.2. Infrared Spectra
The infrared spectra of vit-OH free ligand and its selenium complex were recorded in Figure 7. The spectra are similar but there are some differences which could give indication on the type of coordination. The IR spectrum of vit-OH shows a very strong broad band at 3406 cm−1 which assigned to ν(O-H) stretching vibration of alcoholic OH group, ionization of the alcoholic OH group with subsequent ligation through oxygen atom seems a plausible explanation [32] . To ascertain the involvement of ν(OH) of alcoholic group of vit-OH in the coordination process to be followed the stretching vibration bands of ν(C-O) in selenium vit-OH complex, the examination of this complex show that the ν(C-O) is shifted to lower wavenumber from 1073 and 1176 cm−1 in case of free ligand to 1084 and 1143 cm−1 in the complex. This result indicates that the alcoholic group is participated in the complexation [33] and the vit-OH acts as monodentate ligand. The vit-OH complex show two weak bands observed at 2955 and 2929 cm−1 which assigned to the stretching vibration of ν(C-H) of CH3 and CH2 groups. Involvement of the oxygen atoms of hydroxyl group are also confirmed by the presence of new bands in the selenium complex at 520 and 488 cm−1 due to the ν(M-O) stretching vibrations respectively [34] .
Figure 5. Raman laser spectrum of Se metal.
Figure 6. UV-vis electronic spectrum of Se-vit-OH complex.
Figure 7. Infrared spectrum of Se-vit-OH complex.
3.2.3. 1H-NMR Spectra
The 1H-NMR data of vit-OH and its Se (0) complex is shown in Figure 8. 1H-NMR spectrum of vit-OH show the signal at δ = ~ 9 ppm, which assigned to the proton of alcoholic OH group that is disappear in the Se-vit-OH complex. The disappearance of the signal of the proton of the hydroxyl group in the 1H-NMR spectrum of the complex confirms that the hydroxyl group contribute in the complexation between vit-OH and Se (0) metal [35] . The proton NMR spectrum for Se (0) complex shows a singlet peaks at 1.30, 3.20 and 4.50 ppm. These peaks are assigned to protons of -CH3, -CH2, and =CH groups, supporting the complex formula as suggested in Figure 9.
3.2.4. Thermal Analysis
The obtained Se-vit-OH complex was studied by thermogravimetric TG/DTG and DTA differential thermal analysis from ambient temperature to 800˚C under N2 atmospheres. The TG curves were redrawn as mg mass loss versus temperature and DTG curve was redrawn as rate of loss of mass versus temperature (Figure 10 and Figure 11). The thermal decomposition of [Se(vit-OH)4] complex occurs at four decomposition steps. The first degradation step takes place within a temperature range of 50˚C - 120˚C at DTGmax = 80˚C and it correspond to the loss of three terminal methyl groups with an observed weight loss 3.30% (calcd. = 3.67%). The second decomposition step occur within temperature range 120˚C - 225˚C at DTGmax = 185˚C and DTA = 195˚C (exothermic) that assigned to the loss of another 14CH3 molecules with a weight loss (obs =17.50%, calcd. = 17.15%). The third step occur within temperature range 225˚C - 330˚C at DTGmax = 285˚C and DTA= 300˚C (exothermic) due to the loss of C22H22O4 organic molecule with mass loss (obs. = 28.65%, calcd. = 28.58%). The fourth step occur within a temperature range 330˚C - 435˚C at DTGmax = 365˚C and DTA = 370˚C (exo) due to the loss of C23H47 (organic moiety) with a mass loss (obs. = 26.60%, calcd. = 26.37%). The Se-metal contaminated with polluted carbon atoms remains as final residual till 800˚C.
Figure 8. 1HNMR spectrum of Se-vit-OH complex.
Figure 9. Suggested structure of Se-vit-OH complex.
Figure 10. TG/DTG curves of Se-vit-OH complex.
3.2.5. Kinetic Studies
Most commonly used methods are the differential method of Freeman and Carroll [36] integral method of Coat and Redfern [37] and the approximation method of Horowitz and Metzger [38] . In the present investigation, the general thermal behaviors of the vitamin A comple in terms of stability ranges, peak temperatures and values of kinetic parameters are discussed. The kinetic parameters have been evaluated using the Coats-Redfern equation:
(1)
This equation on integration gives:
(2)
where 
is the linear heating rate, 
is the gas constant, T is the DTG temperature peak, 
, is the fraction of the sample decomposed at time t, A is the pre-exponential factor. A plot of left-hand side against 1/T was drawn. E* is the energy of activation in J∙mol−1 and calculated from the slop and A in (s−1) from the intercept value. The entropy of activation ΔS* in (JK−1∙mol−1) was calculated by using the equation:
(3)
where kB is the Boltzmann constant, h is the Plank’s constant and Ts is the DTG peak temperature [39] .
The Horowitz-Metzger equation is an illustrative of the approximation methods.
(4)
When n = 1, the LHS of equation 4 would be log[−log(1 − α)]. For a first-order kinetic process the HorowitzMetzger equation may be written in the form:
where θ = T − Ts, wγ = wα − w, wα = mass loss at the completion of the reaction. The plot of log[log(wα/wγ)] vs θ was drawn and found to be linear from the slope of which E* was calculated. The pre-exponential factor, A, was calculated from the equation:
The entropy of activation, ΔS*, was calculated from Equation (3). The enthalpy activation, ΔH*, and Gibbs free energy, ΔG*, were calculated from; ΔH* = E* − RT and ΔG* = ΔH* − TΔS*, respectively. The kinetic parameters such as activation energy (ΔE*), enthalpy (ΔH*), entropy (ΔS*) and free energy change of the decomposition (ΔG*) have been evaluated graphically as shown in Figure 12 and Figure 13 by employing the Coats-Redfern
Figure 12. Coats-Redfern plots of the first, second, third and fourth thermal decomposition steps of Se-vit-OH complex.
Figure 13. Horowitz-Metzger plots of the first, second, third and fourth thermal decomposition steps of Se-vit-OH complex.
Figure 14. Biological evaluation of vit-OH, Se-vit-OH complex and DMSO solvent.
and Horwitz-Mitzger relations. The most significant result is the considerable thermal stability of the Se-vit-OH complex reflected from the high values of the activation energy of the decomposition. The entropy change ΔS* of the formation activated complex from the starting reactants is in most cases of negative values. The negative sign of the ΔS* suggests that the thermodynamic behavior is non-spontaneous (more ordered) reactions and the degree of structural “complexity” (arrangement, “organization”) of the activated complex was lower than that of the starting reactants, also the thermodynamic behavior of selenium complex is endothermic reactions (ΔH > 0) and endergonic (ΔG > 0), during the reactions. The thermodynamic data obtained with the two methods are in harmony with each other. The correlation coefficients of the Arrhenius plots of the thermal decomposition steps were found to lie in the range 0.97 to 0.99, showing a good fit with linear function.
3.2.6. Antimicrobial Assessment of Se-vit-OH Complex
Peng et al. [40] reported that 36 nm of Se metal has lower toxicity than selenite or selenomethionine with higher particle sizes, but all of these forms of (Se) possess similar ability to increase selenoenzyme levels. They deduced also that the size of nanoparticles plays an important role in their biological activity: as expected, from range 5 - 200 nm for Se which can be directly scavenge free radicals in vitro in a size-dependent fashion. Accordingly the present techniques of synthesizing selenium metal within the range of (15 - 35 nm) is a unique and pioneer to increase the validity for selenium in biological reaction and leading new trends to decrease toxicity of selenium of higher particle size as reported in literature [40] . Antibacterial and antifungal activities of Sevit-OH complex are carried out against some kinds of bacteria as Escherichia coli (Gram − ve) and Staphalbus (Gram + ve) as well as some kinds of fungi as Aspergillus niger and Aspergillus flavus. The antimicrobial activity was estimated based on the size of inhibition zone around dishes. The free vitamin A was found to have the lowest activity against four types of bacteria and fungi, while the selenium complex was found to be more potent than the original chelate in their inhibition properties (Figure 14). This has been explained in terms of the greater lipid solubility and cellular penetration of the complexes [41] [42] .
Acknowledgements
This work was funded By Deanship of Scientific Research at the University of Dammam, Kingdom Saudi Arabia under Project Grants No. 2013173.
NOTES
*Corresponding author.