Application of ABTS method for assessment of radical-binding effect of Creatine monohydrate

Due to its antioxidant properties Creatine exhibits benefits on muscle, bone, and brain function, and can be of great importance for the prevention of the oxidative stress-related diseases. The aim of current study was the investigation of antiradical effect of Creatine monohydrate by the application of ABTS method. The radical-scavenging activity of Creatine monohydrate against methanol solution of ABTS radical was evaluated by measuring the decrease in the absorbance at λ = 744 nm. For the estimation of antiradical effect of the compound examined the following parameters were calculated: radical scavenging activity in [%], IC 50 value; antioxidant power 1/IC 50 , Trolox equivalent activity. Linear relationship between the enhanced radical scavenging activity and decrease of absorbances and of not-bound ABTS-radical with the increase of concentration of Trolox (0.002 mM ÷ 0.75 mM) and Creatine monohydrate (20 mM ÷ 200 mM) has been established. Linearity was characterized by coefficients of linear regression, which were proven to be higher than 0.97. From the experimental results it was observed that Creatine monohydrate (IC 50 = 100.98 mM) exerts antiradical effect, but is less active compared to Trolox (IC 50 = 0.2 mM) due to higher IC 50 value and lower antioxidant power (1/IC 50 , = 0.01) than Trolox (1/IC 50 = 5).


Introduction
The disballance between the decreased activity of endogenous antioxidant protective enzymes and the overproduction of free radicals [1] leads to oxidative stress. Brain neurons are particularly sensitive to the effect of oxidative stress, caused by glutamate and ß-amyloid peptide, due to the increased oxygen consumption [2]. Low brain Creatine content has been associated with cognitive and movement disorders, epilepsy, and muscle myopathies [3]. In regulating the contractile ability of vascular smooth muscle, endothelial cells produce superoxide radicals [4]. Oxidative stress is associated with the development of neurodegenerative diseases such as Alzheimer, Parkinson, Huntington [5], Lou Gehrig disease (amiotrophic lateral sclerosis) [6]. Oxidative stress is a factor in pathogenesis of cancer [7], rheumatic diseases [8], and aging [4]. The consumption of antioxidants in form of dietary supplements [9] is of great importance for the prevention of the oxidative stress-related diseases [10]. Multiingredient nutritional supplements improve cognitive function [11]. The insufficient biosynthesis is a factor for Creatine deficiency syndromes which leads to the developmental of mental disorders [12]. Creatine supplementation has been investigated to be effective against different diseases [13]. The potential therapeutic role of Creatine in health has been proven as a result from its antioxidant properties [14].
It has been reported that in adults Creatine exhibits beneficial properties on muscle, bone [15], and brain function [15,16]. It has been investigated that dietary Creatine is essential for brain health [17] due to improves cognition [18] and enhances hippocampal-dependent spatial memory, and bioenergetics [19].
In several studies have been investigated therapeutic benefits of Creatine supplementation against neurodegenerative diseases such as Parkinson [20] Huntington [21], amyotrophic lateral sclerosis [22], and encephalomyopathies [23]. In different studies has been reported that Creatine supplementation are effective in muscular atrophy and sarcopenia [24], can improve reproductive perinatal outcomes [25,26], and during pregnancy prevents acute deficits in skeletal muscle after birth asphyxia [27]. It has been described that in chronic heart failure Creatine supplementation are effective in combination with with Coenzyme Q 10 (Ubiquinone, Vitamin Q 10 ) which is one of the most important lipid antioxidants, and prevents the generation of free radicals and modifications of proteins, lipids and DNA. The main biochemical action of Coenzyme Q 10 is as a cofactor in the electron transport chain, in the series of redox reactions involved in the synthesis of adenosine triphosphate. In many diseases associated with increased generation and action of reactive oxygen species, the concentration of coenzyme Q 10 in the body decreases and its deficiency leads to dysfunction of the respiratory chain. The potential use of Coenzyme Q 10 in combination with Creatine may help prevent: cardiovascular disease, mitochondrial disorders, Parkinson's disease, muscular dystrophy and aging [28]. The aim of current study was the evaluation of the radicalscavenging activity of Creatine monohydrate against methanol solution of ABTS radical by measuring the decrease in the absorbance at λ = 744 nm. III. ABTS-radical-scavenging method for invitro study of radical-scavenging activity of Creatine monohydrate.

Preparation of 7 mM ABTS stock solution
An accurarely measured amount of 0.3841 g ABTS diammonium salt (M = 548.7) was dissolved in phosphate buffer solution pH = 6.8 and diluted in volumetric flask of 100.0 ml with phosphate buffer solution pH = 6.8 to obtain a solution of concentration 7 mM.

Preparation of 2.45 mM potassium persulfate stock solution
An accurarely measured quantity of 0.

Preparation of phosphate buffer solution pH = 6.8
For the preparation of phosphate buffer solution with pH = 6.8, an accurarely measured quantities of 0.1 g KH 2 PO 4 , 0.2 g K 2 HPO 4 , and 0.85 g NaCl were dissolved in distilled water and diluted in volumetric flask of 100.0 ml with distilled water.

Preparation of stock solution of Creatine monohydrate
An accurarely measured amount of 1. The results of ABTS-radical scavenging activity (RSA), and for not-scavenged radical (R, [%]), for a period of 10 min. reaction of methanol solution of ABTS with solutions of standard Trolox, and Creatine monohydrate, were calculated by the equation: A ABTS control -absorbance of the solution of ABTS-radical before interaction with the compound investigated Asample -absorbance of the solution of ABTS-radical after reacting with the compound investigated Absorbance of ABTS solution in control is measured against methanol.

Calculation of IC50 value (inhibitory concentration)
The concentration of the compound, at which the inhibition ot of ABTS-radical reaches 50 % is presented as IC 50 value. From the ABTS radical-scavenging curves of standard Trolox and Creatine monohydrate at λ = 744 nm were calculated IC 50 values (mM). The inhibition ratios (y) were plotted against the sample concentrations (x), and the respective regression line (y = a.x + b) was drawn. The sample concentration (x), was calculated by substituting the value of (y) with 50 in the regression equation.
Higher radical-scavenging activity of the compounds investigated corresponds to a lower IC 50 value.

Calculation of Trolox equivalent antioxidant capacity
The ABTS radical scavenging activity of sample was expressed as Trolox equivalent antioxidant capacity (TEAC) calculated as follows: The higher TEAC value means the higher ABTS radical scavenging activity.
The oxidation of ABTS with potassium persulfate generates a green ABTS + -radical which reduction in the presence of hydrogen donating antioxidants is measured [29]. DPPH and ABTS methods has been applied for the investigation of free radical scavenging effect of extracts from different plants as follows: 1. DPPH for Curcuma xanthorrhiza Roxb [30] 2. ABTS for Thymelaea hirsute [31].

Results for ABTS radical-scavenging activity
In spectra of methanol solutions of ABTS at λ = 744 nm the absorbance of control is 0.99425. Spectra of ABTS methanol solutions at λ = 744 nm after 10 min. interraction with Creatine monohydrate solutions (10 mM ÷ 100 mM) .are illustrated on Figure 1.  (Table 1) and with Creatine monohydrate solutions (10 mM ÷ 100 mM) ( Table 2) are presented. The results for the absorbance values of ABTS methanol solution at λ = 744 nm after 10 min. interraction with methanol solutions of standard Trolox (0.001 mM ÷ 0.375 mM) ( Figure 2) and with Creatine monohydrate solutions (10 mM ÷ 100 mM) (Figure 3) were putted against the corresponding concentrations into linear regression analysis and the linear dependence between the decrease of absorbances with an increase of concentration in the investigated ranges was observed. In calibration curves linearity is characterized by coefficient of linear regression, which is R 2 = 0.974 for Trolox and R 2 = 0.991 for Creatine monohydrate.  The high values for regression coefficients obtained from calibration curve after linear regression analysis prove the linear dependence between the increase of radical-scavenging activity with increase of concentration of standard Trolox (Figure 4). The data for ABTS-radical scavenging effect and for notscavenged ABTS-radical from Creatine monohydrate (10 mM ÷ 100 mM) are subjected to a linear regression analysis. On Figure 5 is shown calibration curve for linear relationship between the enhanced radical-binding activity and the decreased not-scavenged ABTS-radical with the increase of concentration of Creatine monohydrate from 10 mM to 100 mM. The results are expressed as: IC 50 and antioxidant power: 1/IC 50 . High radical-scavenging activity is associated with low IC 50 value, which defines that at less concentration the compounds exert high antiradical effect. The regression analysis method was employed and the obtained regression equations for standard Trolox and Creatine monohydrate were used to calculate the IC 50 value which determines the amount of antioxidant needed for decreasing the radical concentration by 50 %. Due to the lower IC 50 = 0.2 mM standard Trolox possess the higher antioxidant activity than Creatine monohydrate (IC 50 = 100.98 mM)

Calculation of Trolox equivalent antioxidant capacity
The ABTS-radical scavenging activity of Creatine monohydrate, expressed as Trolox equivalent antioxidant capacity is: TEAC = 0.002.

Conclusion
From the experimental results it was observed that the ABTSradical scavenging effect of Creatine monohydrate (IC 50 = 100.98 mM) is lower compared to the standard Trolox (IC 50 = 0.2 mM), which antioxidant power (1/IC 50 = 5) is higher in comparison with Creatine monohydrate (1/IC 50 = 0.01).