Kinetics of DPPH• scavenging by bacterioruberin from Haloferax alexandrinus GUSF-1 (KF796625)

This is the first account of the kinetics of free radical scavenging by bacterioruberin obtained from cells of Haloferax alexandrinus GUSF-1(KF796625), grown at optimum conditions of 25% NaCl, pH 7, 42 °C, 150 rpm in NaCl Tryptone yeast extract medium and light. Bacterioruberin separated from methanolic extract displayed characteristics absorption peaks at 368, 386, 463, 492 and 525 nm and gave an m/z value of 740.4 (C50H76O4) in Liquid Chromatography-Mass Spectroscopy validating its purity. Bacterioruberin (13 µM) decolorized and decayed 0.2 mM 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH•) monitored at 517 nm and reached a steady state within 30 min. An EC50 of 6.50 µM ± 0.27 (4.81 µg/mL ± 0.2) was deduced for the 0.2 mM DPPH•-bacterioruberin reaction using the GraphPad Prism 9 statistical software and employing the right-angled triangle technique. The study also revealed a comprehensive information of the total kinetic activity of bacterioruberin with DPPH•: the antioxidant activity index was 16.38 ± 0.67; time needed to reach the steady state with the added EC50—30 min; the antiradical power 30.77 ± 1.27 and the antiradical efficiency of 54.7 × 10–3 ± 2.24, thus reflecting the strong antioxidant nature of bacterioruberin. Scavenging of DPPH• by bacterioruberin was a pseudo-first-order reaction with a rate constant k2 of 2.76 × 10–5 ± 0.001 µM−1 s−1 calculated at t = 0 or initial time and t = 30 min. The knowledge of the kinetics of bacterioruberin to scavenge DPPH• adds to its effective application as an antioxidant in medicinal use, pharmaceutical products and others. Additionally, the use of simple conventional method of DPPH• free radical scavenging, monitored using an easily available laboratory spectrophotometer, will certainly help in the effective use of any antioxidant compound.


Introduction
Oxidative stress in a biological system is the inability of the body to eliminate the free radical reactive species through the use of endogenous antioxidants (Yusuff et al. 2019). Natural antioxidants are gaining importance as nutraceuticals, dietary supplements (Guerin et al. 2003) and as therapeutic medicines (Firuzi et al. 2011). They are preferred over synthetic antioxidants because of their inability to accumulate in the human body and cause harm (Deng et al. 2011).
Free radical scavenging of 1,1-diphenyl-2-picrylhydrazyl (DPPH • ) at fixed endpoint has been widely used to evaluate the antioxidant properties of natural compounds capable of scavenging free radicals (Brand-Williams et al. 1995) and is preferred since it simulates reactive oxygen and nitrogen-free radical species, which affects the biological systems (Arnao 2000). The use of DPPH • provides an easy and rapid method to evaluate the potential of an antioxidant molecule scavenging free radicals which can be measured as a change in optical density/absorbance and referred to as the strength of the antioxidant. (Brand-Williams et al. 1995).
Knowledge of kinetic parameters such as antiradical efficiency and time required to reach steady-state provides additional information about antioxidant behavior. Hence, they are more informative than mere total antioxidant capacity determinations at fixed endpoint even if carried out by different methods (Sánchez-Moreno et al. 1998).
Recently, through separation and chemical profiling of the hexanolic cell extracts of Haloferax alexandrinus GUSF-1 (KF796625), the antioxidant property of bacterioruberin and other C 30 , C 40 , and C 50 isoprenoids has been confirmed . Although the authors Zalazar et al. 2019 investigated the antioxidant activity of bacterioruberin obtained from genetically modified Haloferax volcanii strain (HVLON3) and arrived at EC 50 value using the decolorization of DPPH • monitored through electron paramagnetic resonance analysis, they did not investigate other kinetic parameters or delve into the kinetics of the DPPH • -bacterioruberin interaction.
Therefore, the present study used bacterioruberin from cells of Haloferax alexandrinus GUSF-1 (KF796625), studied the kinetic parameters and behavior of this bacterioruberin to scavenge the 1,1-diphenyl-2-picrylhydrazyl (DPPH • ) free radicals using the conventional spectrophotometric method which is simple, reproducible, reliable, can be carried out routinely and does not require sophisticated instrumentation.

Chemicals and reagents
Analytical grade chemicals and reagents from Himedia laboratories-India, 1,1-diphenyl-2-picrylhydrazyl (DPPH • ) from Sigma, USA and silica gel GF254 plates from Merck, Darmstadt, Germany, were used for thinlayer chromatography (TLC). A 0.2 mM solution of (DPPH • ) was prepared in methanol (97% purity) and used in the study, further dilutions were done with methanol, as and when required.

Standard curve of DPPH • solution
The linearity range of the DPPH • solution was determined. Varying concentrations of DPPH • (0-0.2 mM) were prepared from 0.2 mM stock solution in methanol and incubated at room temperature (28 °C) in the dark for 30 min. A linear regression was obtained from the curve by plotting the concentrations v/s absorbance obtained at 517 nm (Additional file 1: Fig. S1).

Culturing of Haloferax alexandrinus GUSF-1 (KF796625) for bacterioruberin
Haloferax alexandrinus GUSF-1 with accession number KF796625, an isolate from a salt pan (Sequeira 1992), was cultured in Tryptone Yeast Extract containing 25% (w/v) NaCl (NTYE) (Raghavan and Furtado 2004) and as indicated in results, at different concentrations of NaCl, temperatures, pH and in the presence of light. Culture broth was withdrawn daily and cells separated out using the centrifuge (Biofuge-Heraeus Stratos; 8000 rpm, 4 °C, 10 min) were washed with 15% (w/v) NaCl. Growth was monitored as dry weight of washed cells. The cells were then extracted in methanol and clear methanolic extracts were obtained on centrifugation. The culture was also grown under conditions yielding optimum growth, and 100 mg of wet washed cells was used to prepare the methanolic extract which was stored at − 20 °C till further use. All experiments were carried out in triplicates.

Estimation of antioxidant potential of methanolic extracts
The methanolic extract was checked for DPPH • free radical scavenging, according to . Here, methanolic extract was mixed with 1 ml of 0.2 mM methanolic DPPH • (1:2 v/v) and incubated in the dark (28 °C) for 30 min; thereafter, the absorbance was read at 517 nm. The antioxidant capacity, expressed as the % DPPH • radical scavenging activity (%DPPH • RSA), was calculated using the following equation: Page 3 of 11 Alvares and Furtado J Anal Sci Technol (2021) 12:44 where A B is the absorbance of 1 mL of 0.2 mM DPPH • + 0.5 mL of methanol and A S is the absorbance of 1 mL of 0.2 mM DPPH • + 0.5 mL of methanolic extract after incubation for 30 min.

Preparation of bacterioruberin
The wet pellet obtained by growing culture under physicochemical parameters yielding maximum antioxidant was treated with methanol, and the cell debris were then separated by centrifuging to obtain a clear cell-free methanolic extract of cells. Components from this were then fractionated with distilled hexane and distilled water according to the method of Asker et al. (2002). The pigment was recovered in the hexane layer, washed several times with distilled water and dried over anhydrous sodium sulfate and other impurities were removed using acetone precipitation. The resulting pigmented residue was re-dissolved in hexane. The orange-colored hexanolic extract, containing bacterioruberin, was purified by preparative TLC using a developing system of methanol-chloroform (7: 93, v/v) in minimum light (Asker et al. 2002). Bacterioruberin was checked for purity by scanning between 300 and 800 nm using the UV-Vis dualbeam spectrophotometer (UV-1601, Shimadzu, Kyoto, Japan) in quartz cuvettes of 1 mL volume and 1 cm path length and methanol as the reference solution. The identity and purity were further re-confirmed and validated using Applied Biosystems API 2000 Liquid Chromatography-Mass Spectroscopy (LC-MS) system with Ion spray Voltage (IS) -45,000 V; Ion source gas (GS1) -20.0 psi, 1 ppm lowest sensitivity and analyst software Q1/MS (A1).

Free radical scavenging activity of bacterioruberin by steady-state measurement
Bacterioruberin in methanol corresponding to 13 µM and 1 mL of 0.2 mM DPPH • reagent was mixed in the spectrophotometric quartz cuvette and incubated in the dark at 28 °C. The decrease in absorbance of DPPH • radical was spectrophotometrically monitored at 517 nm, at initial 1 min and then every 5 min (1-35 min) until the reaction reached a plateau/steady state. The reaction mixture was also scanned at appropriate intervals using the UV-Probe 2.42 software. The percentage of DPPH • remaining at different intervals was determined according to Mishra et al. 2012 by using Eq. (2) (1) where A 0 is the absorbance of mixture at 517 nm containing 1 mL of 0.2 mM DPPH • + 0.5 mL of methanol and A f is the absorbance of the reaction mixture at 517 nm containing 1 mL of DPPH • + 0.5 mL of bacterioruberin.

(i) The effective concentration (EC 50 )
GraphPad Prism 9 (San Diego, CA) statistical program with the built-in equation for nonlinear regression, i.e. Asymmetric (five-parameter, 5P) was the statistical model used to derive the plot of scavenging of 0.2 mM DPPH • by bacterioruberin by plotting log concentration of bacterioruberin on the X-axis and % DPPH • RSA on the Y-axis (Chen et al. 2013). The EC 50 was then calculated from this log concentration-response curve by using a mathematical method based upon the principle of a right-angled triangle (Alexander et al. 1999).
The theoretical value was calculated by using the concentration-effect curve shown in Fig. 4a, wherein the maximum response chosen is a true representation of the E max and by selecting the two concentrations corresponding to two recorded points on either side of the 50% maximal response. From the raw data/curve, the values of A, B, C, and D are known, and the 50% maximal response is calculated from minimum (baseline) and maximum responses selected from the raw data. The EC 50 was derived according to Alexander et al. 1999, using Eq. (3) and compared with EC 50 of beta-carotene; Further, the time needed to reach the steady state (TEC 50 ) at which no further scavenging of DPPH • takes place at the added EC 50 concentration of bacterioruberin was also calculated graphically from the plot of % DPPH • remaining v/s time.
(ii) Antioxidant activity index (AAI) (Scherer and Godoy 2009) Furthermore, the antioxidant activity index (AAI) was calculated as: Final concentration of DPPH · µg/mL EC 50 µg/mL Page 4 of 11 Alvares and Furtado J Anal Sci Technol (2021) 12:44 Moreover, the antiradical efficiency (ARE) of bacterioruberin was deduced from EC 50 value converted to µg/mL and expressed as g BR/kg DPPH • and TEC 50 at 30 min, as under: (iv) Antiradical power (ARP) (Mishra et al. 2012) Additionally, antiradical power (ARP), the antioxidant action was calculated as reciprocal of ratio of EC 50 (µmoles/µmole of DPPH • ) (v) Reaction stoichiometry (Brand-Williams et al. 1995) This was expressed as the amount of EC 50 ratio (µmoles/µmole of DPPH • ) of bacterioruberin required to reduce 100% DPPH • radicals and was deduced and calculated from EC 50 value × 2. From this, the number of DPPH • molecules reduced by one mole of bacterioruberin was further calculated thereof. (Mishra et al. 2012). The formation of DPPH-H on addition of different µM concentrations of bacterioruberin to fixed 0.2 mM DPPH • was checked, and the rate constant was calculated from the slope of this plot. All reactions were carried out as five independent experiments and expressed as a mean ± standard deviation (n = 5) derived using Microsoft Excel 2019.

Large scale preparation and separation of bacterioruberin
Earlier, Alvares and Furtado (2018) demonstrated the decolorization of the deep purple color of DPPH • by colonies of Haloferax alexandrinus GUSF-1 and also of their methanolic extracts as a consequence of free radicals scavenging activity. In a recent report,  attributed this antioxidant activity to the several compounds present in the cells of Haloferax alexandrinus GUSF-1 among which, the C 50 isoprenoid bacterioruberin was most dominant.
As seen in Fig. 1a-d, maximum antioxidant produced by Haloferax alexandrinus GUSF-1 grown, separately at 150 rpm for over a period of 6 days in TYE medium with  (Fig. 2a, b) validated its purity. These features were consistent with those reported by Asker et al. (2002) and .

Free radical scavenging activity of bacterioruberin
The two characteristics absorption peaks of 0.2 mM DPPH • in methanol were observed at 517 nm and 325 nm, while purified bacterioruberin displayed peaks at 368, 386, 463, 492 and 525 nm. Spectrophotometric monitoring was employed to decipher the interaction of bacterioruberin with DPPH • at 517 nm. The addition of bacterioruberin in methanol with one of the absorption peaks at 525 nm when in contact with the chromogen DPPH • in a 1:2 ratio instantly reduced the absorption peak of DPPH • at 517 nm as displayed in Fig. 3a(i-v).
Further the peaks at 463, 492 and 525 nm corresponding to bacterioruberin were completely abolished thus indicating that the interaction of bacterioruberin had no interference in the measurement of DPPH • at 517 nm. The incremental decrease of the DPPH • peak at 517 nm on the addition of bacterioruberin reflected receipt of H + by DPPH • from the added bacterioruberin and highlighted the free radical scavenging property of bacterioruberin carried out using similar method of DPPH • , as reported by Biswas et al. (2016), Squillaci et al. (2017) and Hou and Chi (2018). The extent of change in color corresponded to the H + received from the donor bacterioruberin molecule. Free radical scavenging activity of bacterioruberin was monitored as a function of time. The addition of 13 µM of bacterioruberin to 0.2 mM DPPH • decreased its absorbance at 517 nm, in the dark at 28 °C. In contrast, the absorbance of control DPPH • at 517 nm remained unchanged for over 30 min (Fig. 3b) and indicated that it did not undergo auto-oxidation nor was affected by the environment in which the reaction was carried out. The purple color of DPPH • changed to yellow with time, as DPPH • received an electron or a H + from bacterioruberin, the donor molecule. The simultaneous decrease in % DPPH • remaining was noted; 81% on addition of bacterioruberin (13 µM/0.013 mM) within 1 min, 66% at 5 min, 54% at 10 min, 45% at 15 min, 38% at 20 min, 32% at 25 min and remained steady at 31% from 30 min onwards (Fig. 3b).
Further, as seen in Fig. 3c, the DPPH • was converted to DPPH-H molecule inversely proportional to the decay of radical till steady state. At 30 min, an amount of 0.073 mM DPPH • was converted to DPPH-H.
The subsequent slower rate pointed to the role of slow secondary reactions, possibly involving dimerization of phenol-derived radicals. The dependence of absorbance of A B (517 nm) and A S (517 nm) (t=x) is therefore exponential and a power function, obtained by nonlinear regression analysis. The reaction occurs via the HAT radical mechanism wherein H + is donated by bacterioruberin as in ArOH + DPPH • = ArO − + DPPH-H and is possibly similar to the reactivity of the antioxidant present in Corchorus olitorius (C.olitorius) and Vernonia amygdalina (V. amygdalina) towards DPPH • (Yusuff et al. 2019).

Free radical scavenging kinetics at different concentrations of bacterioruberin
Data at column 1 under experiment no in Table 1 was used to construct a concentration-effect curve (Fig. 4a) using the GraphPad Prism 9 statistical model with the built-in equation; Asymmetric (five-parameter, 5P), as also employed by Chen et al. (2013). From the minimum and maximum response, the amount required to scavenge 50% of the original concentration or 50% maximal response was calculated. The 50% maximal response was interpolated to the x-axis. Also, A and B were selected on the y-axis as two closest points on either side of the 50% maximal response from the raw data. These points were then interpolated as D and C values on the x-axis. From Fig. 4a, b and Table 1, the following values were obtained: 100% response calculated from the baseline (minimum) and maximum data was 87.37% and hence the 50% maximal response was fixed at 43. Equation (3) was applied to experimental data from column 1 of Table 1, and the following values were calculated: Response interval (y) between responses A and B = 21.66%; the interval between 50% maximal response and the next highest concentration (y′) = 14.65%; x = 5.25 − 4.97 = 0.28 The effectiveness of EC 50 of bacterioruberin in scavenging 50% of 0.2 mM DPPH • free radicals, calculated using the right-angled triangle method of Alexander et al. 1999, was therefore taken as 6.50 µM ± 0.27 (4.81 µg/mL ± 0.2) ( Fig. 4a; Table 1) and that reported for beta-carotene was 18 µM ± 0.2 (10 µg/mL ± 0.2) which was three times lower than bacterioruberin. This is a very simple, precise and rapid method for the calculation of the EC 50 , (Ralevic et al. 1995) and does not require expensive computational aids, thus making the technique particularly useful for laboratory calculations (Alexander et al. 1999).
The antiradical efficiency is the reflection of a combination of kinetic and static approaches to characterize the antioxidant efficiency of a molecule (Huang et al. 2005). The antiradical efficiency of bacterioruberin deduced according to Sánchez-Moreno et al. 1998 (EC 50 calculated as: g Bacterioruberin/Kg DPPH • = 61.04 ± 2.58) was 54.7 × 10 -3 ± 2.24, which with TEC 50 of 30 min, fitted with an intermediate decay similar to that reported for gallic acid, tannic acid and α-tocopherol and showed a very high efficiency as characterized by Sánchez-Moreno et al. 1998.
Bacterioruberin had an antiradical power (ARP) of 30.77 ± 1.27 calculated from the EC 50 (µmoles of bacterioruberin/µmole of DPPH • = 0.03 ± 0.017) using the equation according to Mishra et al. 2012, higher than that reported for all compounds tested in their study. Further  (Table 1) for calculation of the EC 50 of bacterioruberin using the right-angled triangle method; c time to reach steady state TEC 50 with the EC 50 concentration of bacterioruberin. Data are expressed as mean ± SD of five independent experiments Page 9 of 11 Alvares and Furtado J Anal Sci Technol (2021)  Many attempts to explain the structure-activity relationship of some polyphenols are reported in the literature. It is known that the monophenols are less efficient than the polyphenols and that the number of hydroxyl groups is an important factor that enhances activity (Cuvelier et al. 1992;Shahidi et al. 1992;Salah et al. 1995). The accessibility of the radical center of DPPH • to each polyphenol also contributes to the antioxidant power obtained (Yoshida et al. 1989). Another important parameter of antioxidant action is the stoichiometry of reactants which is the amount of antioxidant required theoretically to reduce 100% of DPPH • radicals. Bacterioruberin has an extensive system of 13 conjugated double bond, two acyclic phi φ end groups, four OH at positions C-1, C-1′, C-3′′, C-3′′′. The length, conjugated double bonds and functional groups of a molecule are reported to contribute to the antioxidant capacity (Mandelli et al. 2012) and this explains the lower EC 50 values and corresponding kinetic parameters.
The conversion of DPPH • to DPPH-H was carried out at fixed 0.2 mM concentration of DPPH • and different concentrations of bacterioruberin, and hence represented by the equation is the concentration of radical at the t = 0; (DPPH • C ) t=30 is the concentration of the radical at steady time t = 30, k obs is therefore the pseudo-firstorder rate constant obtained for the fixed reaction time of 30 min. The second-order rate constant k 2 deduced from slope of the plot of k obs v/s concentration of DPPH • (Fig. 5) was 2.76 × 10 -5 ± 0.001 µM −1 s −1 for the bacterioruberin-DPPH • interaction at a steady state of 30 min. In the absence of studies reporting rate constant for bacterioruberin, the rate constant value of 2.76 × 10 -5 ± 0.001 µM −1 s −1 obtained in this study was found to be greater than that of curcumin (2 × 10 −5 ± 0.12 µM −1 s −1 ) but lower than the value reported for gallic acid 4 × 10 -5 ± 0.4 µM −1 s −1 by Mishra et al. 2012, although both of these molecules being unrelated to bacterioruberin in molecular weight.
Expression of results in terms of the kinetic approach does not take into account only the activity of an antioxidant but also provides information on how quickly the antioxidant acts (Squillaci et al. 2017). Hence, it is pertinent to note that results of antioxidant activity based on kinetic data and on the measurement at a fixed endpoint should be combined so as to provide comprehensive information of the total antioxidant activity of a compound. This is the first study reporting the kinetics of free radical scavenging by bacterioruberin from a genetically unmodified Haloferax alexandrinus GUSF-1 investigated by the simple, conventional colorimetric assay for DPPH • chromogen and monitored using an easily available spectrophotometer and highlighting its unique antioxidant potential.

Conclusions
In summary, this study on the kinetic behavior of antioxidant, bacterioruberin, from cells of Haloferax alexandrinus GUSF-1(KF796625) on decay of 0.2 mM DPPH • enabled us to fix EC 50 at 6.50 µM ± 0.27 (4.81 µg/ mL ± 0.2) and TEC 50 of 30 min and also the other kinetic parameters such as AAI at 16.38 ± 0.67, ARP at 30.77 ± 1.27 and the ARE of bacterioruberin was 54.7 × 10 -3 ± 2.24, showed a stoichiometric value of 0.06 ± 0.002 and reduced 15.38 ± 0.63 molecules of DPPH • . These kinetic parameters are not yet reported for bacterioruberin interacting with DPPH • . Further, the scavenging reaction of DPPH • by bacterioruberin was pseudo-first order with a second-order rate constant Fig. 5 Plot of pseudo-first-order rate constant (k obs ) versus bacterioruberin concentration. All measurements were carried out in methanol medium. Data are expressed as mean ± SD of five independent experiments. The second-order constant k 2 was calculated from the slope of this plot