ACTIVITY OF SUPEROXIDE DISMUTASE MIMIC OF [ Mn ( SALEN ) OAc ] COMPLEX COMPOUND NON-ENZYMATICALLY IN VITRO THROUGH RIBOFLAVIN PHOTOREDUCTION

The complex compound of [Mn(salen)OAc] can serve as mSOD and its activity has been determined nonenzymatically in vitro through riboflavin photoreduction. The complex was synthesized from Mn(OAc)2.4H2O and H2salen. Based on the elemental analysis, the C=56.69%; H=4.21%; and N=7.52% content are corresponding to the chemical formula of MnC18H17N2O4. The functional groups and ionic species in the complex have been analyzed by infrared spectroscopy and ESI-MS. SOD activity was determined by mixing complex at various concentrations with riboflavin and nitroblue tetrazolium (NBT), then the mixture was lighted with 20 watts tungsten lamp for 15 minutes in a closed box. Then the reduced NBT absorptions were measured at λ 560 nm. The difference of absorbance between standard and sample solutions (without and with riboflavin, respectively) was multiplied by 100% to obtain %inhibition of each various sample concentration against NBT. SOD activity was obtained from IC50 data defined as a 50% inhibition of the plot curve of % inhibition to the concentration of the complex. The result obtained for this compound is IC50 = 2.7 ± 0.1 μM as well as enzymatic method. Therefore, this method can be used to determine the SOD activity by giving more stability and accuracy of IC50 value.


INTRODUCTION
Superoxide radical (O2 •-) is one of the reactive oxygen species (ROS) which the most dangerous in comparison with other ROS (Vecchio & Lanza, 2009).The existence of excessive radicals in the body can cause tissue damage associated with the emergence of various diseases, such as rheumatoid arthritis, cancer, neurodegenerative disorders, complications of diabetes, stroke, inflammation and reperfusion injury.Superoxide reactivity could be overcome by a metalloenzyme known as superoxide dismutase (SOD), that catalyze the dismutation of O2 •-reactive species to O2 and H2O2 (Vecchio & Lanza, 2009).But the limited number of metalloenzyme in the body, the use of supplements containing SOD from plants extract or other living beings or synthetic SOD become an alternative.Synthetic SOD is known as SOD mimic or mSOD (Miriyala et al., 2012).
The in vitro indirect SOD test is an initial test conducted to determine a potential of a complex compound as an mSOD (Iranzo, 2011).The test principle is competition between mSOD and active redox indicator to react with O2 •-species.The activity of an mSOD of a complex compound shown by the ability of the complex compound to inhibit reduction process of the active redox indicator by O2 •-species.Through this indirect SOD test, SOD activity is obtained as IC50 value, that is a quantity used to express one unit of SOD activity.IC50 is a quantity that describes the amount of SOD to inhibit the reduction reaction of an active redox indicators by species O2 •-as much as 50%.
Based on the source of O2    Retnoningrum, 2014).However, there were only found 9 SOD mimics of manganese complex compound whose activities were determined by this rb-NBT non enzymatic method, those are: 1 complex of manganese(II)-bovine serum albumin (Yan et al., 2016) New in this research, the nonenzymatic method through riboflavin photoreduction using NBT indicator was used to determine SOD activity (IC50) of the [Mn(salen)OAc].The purpose of this study is to evaluate the ability of this rb-NBT nonenzymatically method in analyzing SOD activity of the [Mn(salen)OAc)] complex compound.

EXPERIMENTAL SECTION
In this research, the [Mn(salen)OAc] complex has been synthesized, characterized, and SOD activity tested by the in vitro nonenzymatic method through riboflavin photoreduction using NBT indicator (rb-NBT)

Instrumentation
Tools and instruments used in this study include glasswares, Perkin-Elmer 240 elemental analyzer, FTIR Nicolet Magna IR 750 series II spectrophotometer, ESI-MS Bruker Daltonics esquire3000 plus spectrometer, Sherwood Scientific magnetic susceptibility balance (MSB), and HANNA Instruments HI 8819W Professional ATC conductometer.While, equipment used in the SOD activity test include micropipette 1000 and 30-300 µL, 96-well microplate, light box (25 x 25 x 25 cm 3 ) coated by aluminum foil and using a 20 watts tungsten lamp, and Multiskan Go Thermofisher Scientific UV/vis double beam microplate reader spectrophotometer.

Synthesis of [Mn(salen)OAc] Complex
The [Mn(salen)OAc] complex was prepared using a published procedure (Boucher & Farrell, 1973) with using glass beakers instead of reflux and methanol as a solvent for recrystallization instead of petroleum.

SOD Assay: Riboflavin Photoreducion Test of [Mn(salen)OAc] Complex
The [Mn(salen)OAc] complex was tested for SOD activity using a previously described method (Kostyuk et al., 2007).The SOD activity measurement of the complex compound was based on the reduction of nitroblue tetrazolium by superoxide anions generated in riboflavin photoreduction, which was carried out at room temperature (25ºC) under fluorescent lighting (20 w, 20 cm).The sample solution was prepared in 12 variations of concentration and each 40 µL of them was added to a solution (200 µL) containing 6 µM of riboflavin, 0.8 mM of N,N,N',N'tetramethylethylenediamine (TEMED) in 16 mM phosphate buffer (pH 7.4), and 85 µM of nitroblue tetrazolium (NBT) in a microplate.The sample mixtures were subjected to light exposure as mentioned above for 15 min and the colorimetric activity assay by a microplate reader spectrophotometer follows the generation of NBT-diformazan, which absorbs at 560 nm (Janknegt, Rijstenbil, van de Poll, Gechev, & Buma, 2007).
The absorbance of sample blank mixture (blank 1), without riboflavin, was measured as well.In the standard mixture and its blank (blank 2), methanol solvent was added instead of the sample solution.The difference of absorbance between standard and samples (without and with riboflavin, respectively) was multiplied by 100% to obtain % inhibition of each various sample concentration against NBT (Retnoningrum et al., 2016).

Characterization of H2Salen Ligand and [Mn(salen)OAc] Complex
Elemental analysis of the H2salen (C16H16N2O2) and [Mn(salen)OAc] complex compound (MnC18H17N2O4) resulted from synthesis show that each compound has C, H, and N elements percentage which fit the theoretical calculations (Table 1).These results indicate that H2salen and [Mn(salen)OAc] complex compound have been successfully synthesized.In the infrared spectrum of the [Mn(salen)OAc] complex (Figure 1 (below)), several different wave numbers are observed compared with the H2salen spectrum (Figure 1 (above)).The differences are observed in the vibration of O-H, C-H, C-O-H, Mn-N, and Mn-O functional groups.In the complex compounds, the vibration of O-H and C-H functional groups occurs successively in the wave numbers of 3426-3775 and 2611-3084 cm -1 , while the ligands occurred successively in the wave numbers of 3425-3789 and 2610-3052.The vibration of C-O-H which appeared at the wave numbers of 1717-1942 cm -1 in the H2salen spectrum, did not appear in the spectrum of [Mn(salen)OAc] complex.This fact indicates that after H2salen bound to Mn(III), the vibration of C-O-H did not happen again due to the release of the H atom of the -OH group of H2salen.Metal-ligand bond in the [Mn(salen)OAc] complex can be observed in the fingerprint region at the wave number of 593-624 cm -1 (Mn-N) and 452 cm -1 (Mn-O).
Measurement of the ESI-MS mass spectrometry of [Mn(salen)OAc] complex was carried out in methanol-acetonitrile.In the positive ionization spectrum (Figure 2 (left)), a cationic species with a high-intensity peak was observed at m/z 320.9 which corresponding to the four-coordinated cation [Mn(salen)] + .This suggests that the [Mn(salen)] + cation is the parent species of the [Mn(salen)OAc] complex compound.Besides, a moderate intensity peak also observed at m/z 269.0, which corresponding to a cationic free ligand species (salen + H) + .On the other side, in the negative ionization spectrum (Figure 2 (right)), a peak indicating two acetate ions which coordinated to the Mn(III) ion was observed, although the intensity was very low, that is at m/z 441, corresponding to the anion species of [Mn(salen)(OAc)2] -.In addition, the negative ionization spectrum is also observed three peaks at m/z 255.0, 265.0, and 283.1, which corresponding to the (salen -N) -, (salen -H) - and ( salen + N) -.Meanwhile, a peak at m/z 319 which corresponding to the [Mn (salen) -H) -species showed that the acetate ion binding to the Mn(III) ion could be broken).Based on the results of the ESI-MS, the [Mn(salen)OAc] complex compound have been successfully synthesized.
The conductometry measurement results of the [Mn(salen)OAc] complex compound can be seen in Table 2.The methanolic solution of the complex was provided a molar conductivity of 0.17 mS.This molar conductivity is smaller than the value of the smallest molar conductivity of NaCl ionic compound.These results indicate that the complex is a neutral [Mn(salen)OAc] complex compound, where the acetate bound as ligands to Mn(III) or in other words it is not as a free anion or a counter ion.The results obtained from this molar conductivity value measurements in accordance with the molecular formula of the ionic species that appear on the ESI-MS spectrum and the content of C, H, N on elemental analysis.
Based on the result of magnetic measurement, the [Mn(salen)OAc] complex compound is paramagnetic with the experimental magnetic susceptibility of 4.93 BM.This value indicates that the solution reaction of Mn(II) with H2salen caused oxidation of Mn(II) to Mn(III) ion.This result are consistent with the theoretical magnetic susceptibility of Mn 3+ , which has four unpaired electrons (μtheory = 4.90 BM).The potential as mSOD of the [Mn(salen)OAc] complex compound was determined by IC50 value, that is the concentration of the complex which is able to inhibit the reduction of NBT by superoxide anion as well as 50%.The SOD activity was tested in vitro non-enzymatically through riboflavin photoreduction and NBT is used as an indicator that can be reduced to NBTdiformazan when it reacts with superoxide anions generated from photoreduction of riboflavin.This indirect method includes several reactions: the photochemically excited riboflavin was first reduced by TEMED into semiquinone, which donated an electron to oxygen to form superoxide anion source.The superoxide readily converted a yellow NBT into a purple formazan product.The SOD activity was inversely related to the amount of formazan formed The measurement results of the inhibition of the [Mn(salen)OAc] complex against NBT reaction with superoxide in the form of absorption values before and after irradiation are shown in Table 3.An empty microplate before being filled by any mixture gives the same absorption, about 0,060, either before or after lighting.

Figure 2 .
Figure 2. The ESI-MS spectrum of [Mn(salen)OAc] complex compound in a solution of methanolacetonitrile for: positive ionization region (left) and negative ionization region (right) •-species source, while in the nonenzymatic method, O2

Table 1 .
Elemental analysis of C, H, and N for H2salen and [Mn(salen)OAc] complex

Table 2 .
The results of the molar conductivity measurements

Table 3 .
The absorbance data of SOD activity test of [Mn(salen)OAc] complex