Application of DPPH assay for the evaluation of the antiradical activity of Creatine Lysinate

The aim of the current study was the estimation of the radical-scavenging activity of Creatine lysinate against 0.05 mM methanol solution of DPPH radical by measuring the decrease in the absorbance at λ = 516 in methanol. The antiradical effect of the compound examined is presented with the following parameters calculated: radical scavenging activity in [%], IC 50 value; antioxidant power 1/IC 50 , and Trolox equivalent activity. relative radical scavenging activity (RRSA, [%]), and a relative decrease in radical scavenging activity (RDRSA, [%]).From the experimental results, it was observed that the DPPH binding ability of Creatine lysinate (IC 50 = 73.75 mM) is lower compared to the standard Trolox (IC 50 = 0.001154 mM), which antioxidant power (1/IC 50 = 8.67) is higher in comparison with Creatine lysinate (1/IC 50 = 0.014). The experimental data show that Creatine lysinate (IC 50 = 73.75 mM) is more active compared to Creatine monohydrate (IC 50 = 102.48 mM) due to lower IC 50 and higher scavenging activity. In comparison with mono application of alone antioxidant, the beneficial effect of the combination of Creatine monohydrate and Creatine lysinate with other antioxidants in form of food supplements could be an important strategy for a synergistic effect in the reduction of free radicals in the treatment of disorders resulted from oxidative stress.


Introduction
Reactive oxygen species include charged and neutral species such as superoxide anion (O 2 -• ), singlet oxygen ( 1 O 2 ), hydroxyl (HO • ), alkoxyl (RO • ), and peroxyl (ROO • ) radicals [1]. Oxidative stress is as a result of the overrun of free radicals and the decreased activity of endogenous antioxidant protective enzymes [2]. The increased generation of reactive oxygen species leads to the disruption of multiple metabolic processes which results at the beginning of the development of the earlier pathological processes in neurodegenerative diseases such as Alzheimer, Parkinson, Huntington's [3], amyotrophic lateral sclerosis (Lou Gehrig disease) [4], cancer [5], rheumatic diseases [6], and aging [7]. Creatine as an antioxidant is important for human health and can provide benefits for different diseases [8]. Pre-exercise and postexercise Creatine supplementation prevents bone mineral content and density [9] in the aging population [10], and supports bone health in older women [11]. Creatine exerts a protective effect in pathological conditions of the brain and muscle [12]. Creatine supplementation shows effectiveness on aging muscle [13,14] as on muscle function in childhood myositis [14], muscle function in cancer [15], muscular atrophy [16], sarcopenia [17], myopathy [18], and in spinal and bulbar muscular atrophy [19].
Creatine or vitamin D supplementation can help in individuals with a spinal cord injury [20]. Creatine supplementation can provide a beneficial effect on the cognitive function of healthy individuals [21], traumatic brain injury [22], and the mental-associated decrease in visuomotor skills [23]. Dietary supplementation with Creatine is important for bipolar depression [24], pregnancy [25], vascular health [26], and cancer therapy [27][28][29]. Antioxidants from natural sources and foods [30] are important for protection against age-related diseases [31,32]. The investigation of antioxidant nutraceuticals [33] is an important therapeutic approach for the effective decrease of reactive oxygen species [34].
The aim of the current study was the comparative evaluation of the radical-scavenging activity (RSA) of Creatine lysinate against 0.05 mM methanol solution of DPPH (2,2-diphenyl-1picrylhydrazyl) radical by measuring the decrease in the absorbance at λ = 516 nm. Based on the eventually experimentally confirmed antiradical properties of Creatine lysinate, the study would help to justify the advantages of choosing both antiradical compounds in the more active combination over monotherapy, which would contribute to increasing the effectiveness against oxidative stress-related diseases.

I.
Determination of radical scavenging activity by DPPH assay.

Preparation of phosphate buffer solution pH = 7
For the preparation of phosphate buffer solution with pH = 7,accurate 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 a volumetric flask of 100.0 ml with distilled water. 7. DPPH assay procedure DPPH assay was performed according to the following procedure: 5 ml 0.1 mM methanol solution DPPH was mixed separately with 5 ml of Creatine lysinate in concentrations 20 mM, 40 mM, 60 mM, and 80 mM to obtain final concentrations respectively of 0.05 mM methanol solution DPPH and 10 mM, 20 mM, 30 mM, 40 mM of Creatine lysinate. As control recorded a mixture of 5 ml 0.1 mM methanolic DPPH solution and 5 mlof methanol.The mixtures were shaken vigorously and allowed to stand for incubation in dark for 1 h at temperature: 25 o C  27 o C After incubation, the absorbances were measured against blank methanol at λ= 516 nm using a UV-VIS spectrophotometer Hullett Packard N: 8452 A. All the tests were performed in triplicates and the results were averaged.

II.
Calculation methods

Calculation of radical scavenging activity(RSA, [%])
The results of DPPH-radical scavenging activity (RSA), and for not-scavenged radical (R, [%]), for a period of 1 h reaction of 0.05 mM methanol solution of DPPH with solutions of standard Trolox, and 10 mM ÷ 40 mM Creatine lysinate, were calculated by the equation: A DPPH control -absorbance of the solution of DPPH-radical before interaction with the compound investigated Asample -absorbance of the solution of DPPH-radical after reacting with the compound investigated the absorbance of the DPPH solution in control is measured against methanol.

Calculation of IC50 value (inhibitory concentration)
The IC 50 value is the concentration of the test samples at which the inhibition percentage reaches 50%. A lower IC 50 value corresponds to a higher antiradical activity of the tested sample. IC 50 values (mM), were calculated from the DPPH radicalscavenging curve of Creatine lysinate and standard Trolox at λ = 516 nm according to the following procedure: 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.
The higher TEAC value means a higher DPPH radical scavenging activity.
5. Calculation of relative radical scavenging activity (RRSA, [%]) and a relative decrease in radical scavenging activity (RDRSA, [%]) The relative radical scavenging activity (RRSA, [%]) and the relative decrease in radical scavenging activity (RDRSA, [%]) for Creatine lysinate with concentrations 10 ÷ 40 mM, were compared to the activity of standard Trolox with the same concentrations, and were calculated by the following equations: RSA sample -radical binding activity of Creatine lysinate RSA Trolox -radical binding activity of the standard Trolox.

Results and Discussion
Antioxidant methods are classified as hydrogen atom transfer (HAT)-based, and electron transfer (ET)-based assays. In spectrophotometric ET-based methods is measured the capacity of a redox-potential compound in the reduction of a colored oxidizing agent. The degree of color change in the reduction of an oxidant (either an increase or decrease of absorbance at a specific wavelength) is correlated to the concentration of the antiradical compound. In electron transfer-based methods, the increase in the radical-scavenging effect of the test compounds is directly proportional to the decrease of the absorbance of a solution of: 1. 1,1-diphenyl-2-(picrylhydrazyl) (λ = 516 nm): DPPH free radical scavenging assay 2. 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (λ = 734 nm): ABTS method [35]. The DPPH (1,1-diphenyl-2-picrylhydrazyl; α,α-diphenyl-βpicrylhydrazyl) is a stable free organic radical with dark purple color. The DPPH radical scavenging method is a decolorization assay, which mechanism is based on the reaction between the DPPH solution and compounds by measuring its capacity to directly scavenge the DPPH radicals. In this interaction, DPPH is reduced to its nonradical form 1,1′-diphenyl-2-(2,4,6trinitrophenol) hydrazine (DPPHH), and antioxidants are oxidized from DPPH. The determination of the capacity of compounds to directly scavenge the DPPH radicals is based on the monitoring of the decrease of absorbance of the DPPH radical most commonly at λ = 517 nm [36,37]. The method is applicable for the study of the radical scavenging activity of the test compounds because they do not possess a measurable absorbance at the absorption maximum λ = 517 nm, where the DPPH-test is carried out. DPPH method has been applied for the investigation of the antioxidant activity and of free radical scavenging effect of extracts from different plants such as Piper longum L. [38], Zingiber officinale var. rubrum [39], milk thistle [40].
Marinova et al. [41] have reported that the literature review described different modifications of the original methods of Blois [36] and Brand-Williams et al. [37] for the determination of the DPPH free radical scavenging activity. Following Blois for the DPPH method, are mixed 1 ml 0.1 mM methanol solution of DPPH solution is added with 3 ml of various concentrations of compounds in methanol or with 3 ml of reference standard Butylhydroxytoluene. After 30 min. at 25 ºC in dark, the absorbance is measured at λ = 517 nm [36]. The modification methods include the differences in the following parameters [41] The reducing ability of Creatine lysinate and standard Trolox presented as a percentage of antiradical activity (RSA %), was assessed by DPPH free radical scavenging assay. For the investigation of the radical scavenging activity was applied methodology described by the original DPPH methods of Blois [36] and Brand-Williams [37] with the following parameters modified in our previous work [42] Table 2 are summarized data for radical scavenging activity RSA (%), and for not scavenged radical (R). In our previous work, the following results forstandard Trolox have been obtained:  The scavenging activity percentages are subjected to a linear regression analysis against the respective concentrations. Figure  3. is illustrated the calibration curve which presents the linear relationship between the enhanced radical binding activity with the increase of concentration from 10 mM to 40 mM.    From the regression equations for the radical-binding effect, the values for the radical-scavenging activity of 0.01 mM, 0.02 mM, 0.03 mM, and 0.04 mM solutions of Creatine lysinate were calculated. The obtained data were used for the calculation of relative radical scavenging activity (RRSA, [%]) and a relative decrease in radical scavenging activity (RDRSA, [%]) shown in Table 3. The beneficial effect of combinations in the reduction of free radicals could be more effective in comparison with the mono application. Due to radical-scavenging activity, the application of Creatine monohydrate and Creatine lysinate in combination with other kinds of antioxidants such as Vitamin C or Coenzyme Q in form of food supplements could be an important strategy for obtaining of synergistic effect in an additive treatment of disorders resulting from oxidative stress-related diseases.