Antioxidant activity and reduction potential of dietary herbs on acrylamide formation in asparagine-glucose reaction model

ABSTRACT Natural ingredients are increasingly being used to prevent the formation of acrylamide during food processing. This study investigated the antioxidant activities of Ocimum basilicum (basil), Lippia adoensis var. adoensis (kesse), Lippia adoensis var. koseret (koseret), Rosmarinus officinalis (rosemary), and Thymus schimperi (tosign) extracts and their acrylamide reduction effect in the asparagine-glucose model system. Folin-Ciocalteu and aluminum chloride methods were used to calculate the total phenolic content (TPC) and total flavonoid content (TFC). Antioxidant activities were determined using 2,2-Diphenyl-1-Picryl-Hydrazyl (DPPH) scavenging and phosphomolybdenum assay methods. Acrylamide contents of the samples were determined by a reversed phase high-performance liquid chromatography (RP-HPLC-DAD) method. Kesse had the highest concentrations of TPC (30.20 ± 1.23 mg gallic acid equivalent per gram of herb extract), TFC (15.87 ± 0.55 mg catechin equivalents per gram), and the strongest DPPH scavenging activity (IC50 = 108 μg/mL). The amount of acrylamide formed during the frying process was decreased by a factor of 26.8% to 65.81% with the addition of dietary herb extracts to the asparagine-glucose reaction model. The amount of acrylamide formed from an asparagine-glucose reaction model containing herb extracts was negatively correlated with the total phenolic and total flavonoid content with, R2 values of 0.920 and 0.930. This study suggests a potential application of these dietary herb extracts as a natural antioxidant for lowering acrylamide formation during the Maillard reaction in fried food products.


Introduction
Individuals are exposed to acrylamide to varying degrees through eating, smoking, drinking water, environmental exposure, and occupational exposure; nevertheless, there are growing worries about the toxicity of acrylamide and its possible health consequences in relation to fried and baked foods. [1]hemical reaction models are widely used to investigate food reaction mechanisms due to the complex chemicals and multiple reactions involved. [2]Acrylamide is a processing contaminant formed during the cooking process or by asparagine degradation in the presence of carbonyl compounds via Maillardreactions. [3]Reactive carbonyls heated with asparagine form a series of intermediates that eventually lead to the formation of acrylamide (Figure 1).Several antioxidants were found to be effective in retaining the Maillard reaction intermediates via the C 6 or C 8 position of the α-ring during this process. [5] variety of herbs and spices have been added to food since ancient times in order to enhance flavor and improve organoleptic properties. [6]The dried leaves of Lippia adoensis var.koseret, Lippia adoensis var.adoensis, and Thymus schimperi, locally known as koseret, kesse, and tosign, respectively, are endemic dietary herbs to Ethiopia.[9] The leaf of kesse is used to flavor milk and butter, as well as to prepare spiced red pepper in different parts of Ethiopia. [7]Tosign is a wild-growing species of thyme that is well known in Central, Eastern, and Northern Ethiopia. [10]It is used in the food and aroma industries; it is widely used as a culinary ingredient; and it serves as traditional medicine for diabetic patients. [11,12]osemary is a popular dietary herb, and its extract is widely used as an antimicrobial, antiinflammatory, and food preservative and has potential antioxidant activity. [13,14]Ocimum basilicum L. (basil) is one of the most frequently used culinary herbs known to possess strong antioxidant and antimicrobial activities due to its phenolic acids and aromatic compounds. [15]resh basil leaves are primarily used in Ethiopia for the preparation of spiced hot red pepper (berbere), "mitten shiro," paste of spiced chili pepper. [16]Amongst natural preservatives, rosemary, thyme, and basil have been widely applied and reported for their preservation abilities due to their antioxidant and antimicrobial activity as well as their higher total phenolic content. [17,18]any studies have reported the inhibitory potential of phytochemicals on acrylamide-induced toxicities due to their antioxidant properties and ability to regulate intracellular signaling pathways. [19,20][26] Furthermore, there has been little research done on the antioxidant activity and antimicrobial activity of koseret and tosign, [24,27,28] but to our knowledge, no research has been reported on the effect of endemic dietary herbs (kesse, koseret, and tosign) leaf extracts on acrylamide reduction in the asparagine-glucose reaction model.
Therefore, in light of many health-promoting benefits attributed to the consumption of these dietary herbs, the objective of the current study was to evaluate the antioxidant activity of these selected dietary herb extracts and their acrylamide reduction effect on an asparagine-glucose reaction model system.Furthermore, the correlation between the antioxidant activity of these dietary herb extracts and their effect on acrylamide reduction was evaluated.A propose reaction mechanism for formation of acrylamide.Source [4] .

Sample preparation and extraction
Koseret, basil, and rosemary were collected from the Wondo Genet Agriculture Research Center, 25 km northeast of Hawassa, Ethiopia, while kesse and tosign were collected from Dinsho, Bale National Park, 370 km southeast of Addis Ababa, Ethiopia.The herbs were validated by the Herbarium, Department of Botany, Hawassa University.Approximately 25 g of powdered dried herbs were mixed into 250 mL of 96% (v/v) ethanol for 12 h in an enclosed beaker with constant shaking. [29]Following that, each extract was filtered and concentrated under a vacuum at 40°C in a rotary evaporator (Buchi, 3000 series, Switzerland).The extracts were sealed in a polyethylene bag and stored at 4°C until the experiment was performed.

Total phenolic content
The total phenolic content of basil, koseret, kesse, rosemary, and tosign ethanolic extracts (1 mg/mL) was determined using the Folin-Ciocalteu colorimetric reaction method as described by Ghani et al. [30] with some modifications.An aliquot (0.3 mL) of each herb extract was treated with 1.5 mL of 10-fold diluted Folin-Ciocalteu.After adding 1.5 mL of 7.5% Na 2 CO 3 to the solution, it was allowed to stand in the dark at room temperature for 90 min.The estimation of the phenolic content was carried out in triplicate.The absorbance of the blue color solution was measured in a UV-visible spectrophotometer (JENWAY, 6300, UK) 765 nm against the blank.The total phenolic content was estimated from the gallic acid (20-160 µg/mL) calibration curve: y = 0.0053× + 0.2566 R 2 = 0.9954; the results were expressed as milligrams of gallic acid equivalent per gram of herb extract (mg GAE/g).

Total flavonoid content
One of the widely followed methods for the determination of total flavonoid content in plant extracts is the aluminum chloride colorimetric assay, where Al(III) is utilized as a complexing agent. [31]The total flavonoid content was determined using the aluminum chloride method, [32] with minor modifications.The analysis was based on the formation of a pink color of the flavonoid aluminum complex.Briefly, 1 mL of each dietary herb extract (1 mg/mL) was added to a test tube and diluted with 1.25 mL of distilled water, followed by 75 μL of 5% NaNO 2 .After 5 min, 150 μL AlCl 3 solutions (10%) was added.After another 5 min, 1 mL of 1 M NaOH solution was added to the mixture.A blank was prepared by adding distilled water instead of aluminum chloride solution.The solution was thoroughly mixed, and the absorbance was measured against the blank at 510 nm using a UV-visible spectrophotometer.All the experiments were repeated three times for precision, and the total flavonoid content of the extracts was calculated from the regression equation of the catechin calibration curve (y = 0.0058× + 0.2361; R 2 = 0.9821) and expressed as milligram catechin equivalents per gram of herb extracts (mg CE/g).

DPPH radical scavenging activity
The stable organic radical 2,2-Diphenyl-1-picrylhydrazyl (DPPH) has been widely used in antioxidant activity studies of plant extracts. [33]The DPPH radical scavenging activity of dietary herb extracts was determined using the method of Sub et al. [34] with some modifications.Two milliliters of DPPH reagent (0.06%, w/v) in methanol was mixed with 1 mL of dietary herb extracts with concentrations ranging from 50 to 750 μg/mL.For the blank solution, the extracts were substituted with methanol.The mixture was vigorously shaken (using a vortex mixer) and incubated at room temperature in the dark for 30 min.The absorbance of the reaction mixture and ascorbic acid was measured at 520 nm in a UV-vis spectrophotometer (JENWAY, 6300, UK).The DPPH scavenging ability of the extracts and standard was expressed by the equation below.
where A c is the absorbance of the control and A s is the absorbance of the test samples.All the tests were performed in triplicate, and the graph was plotted with the average of three observations.The IC 50 value for herb extracts was measured from a plotted graph of scavenging activity against different concentrations of extracts, where IC 50 represents the amount of extract needed to scavenge 50% of the DPPH radical.

Total antioxidant activity
Total antioxidant activity of dietary herb extracts was determined using the phosphomolybdenum assay method according to Zengin et al. [33] with a minor modification.In an acidic medium, Mo (VI) is reduced to Mo (V) by antioxidant compounds or extracts, followed by the formation of green Mo (V) complexes with maximum absorption at 695 nm.An aliquot (0.3 mL) of 0.5 and 1 mg/mL of the test extracts were mixed with 3 mL of the reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate) taken in test tubes.The tubes were incubated in a boiling water bath at 95°C for 90 min.The reaction mixture was allowed to cool to room temperature, and the absorbance of the solution was measured against a blank containing 3 mL of reagent solution.The ascorbic acid calibration curve (y = 0.9405 + 0.235, R 2 = 0.997) was used to calculate the total antioxidant activity, which was expressed as milligram ascorbic acid equivalent per gram of herb extract (mg AAE/g).

Asparagine-glucose model preparation
Chemical reaction models are widely used to investigate the reaction mechanism because the food reaction system contains complex chemicals and numerous simultaneous reactions. [2]To study the function of dietary herb extracts for acrylamide reduction, chemical model systems were developed, following the method of Zhu et al. [35] with some modifications.L-asparagine monohydrate powder (1.32 g) and D-glucose anhydrous powder (1.80 g) were combined with 10 mL of phosphate buffer solution (0.2 M, pH 6.80) in a 25 mL flask and vortexed for at least 5 min.The flask was filled with prepared dietary herb extracts or BHT (0.3 mL, 2 mg/mL), while the control system received the same concentration and volume of phosphate buffer, which was then heated in a thermostat silicone oil bath at 175°C for 20 min.After heating, the reaction mixtures were immediately cooled in an ice bath to stop further reactions.

Extraction of acrylamide
The extraction of acrylamide was performed according to the method described by Miao et al. [36] Acetone (20 mL) and 100 μL of water were added to the asparagine-glucose reaction model sample, and then the flask was placed in an ultrasonic bath at 40°C for about 20 min.The resulting solution was filtered through a Whatman No. 1 filter paper and the filtrate was evaporated under vacuum to achieve dryness.Then, 3 mL of water was added and shaken thoroughly to dissolve the residue.

Analysis of acrylamide by HPLC
A reversed-phase high-performance liquid chromatography system (RP-HPLC-DAD) was used to determine acrylamide.The Agilent 1260 Infinity II Quaternary System (Hewlett-Packard-Strasse 8, 76337 Waldbronn, Germany).The HPLC system included a quaternary pump, degasser, auto-sampler, and diode array detector.The column was an Agilent rapid resolution column (Agilent Zorbax Eclipse C 18 , for Agilent Technologies), with (4.6 × 75 mm, 3.5 μm) made in the USA.HPLC-grade water and acetonitrile were used for solvents 'A' and 'B', respectively.The aqueous solution was filtered through a 0.45 μm syringe filter, and the acrylamide sample was injected into the column using an auto sampler injection loop.The flow rate was set to 1 mL/min at 30°C, and the injection volume for each sample was 20 μL.Analyses were performed in triplicate.

Preparation of standard solutions and method validation
A standard acrylamide stock solution (1 mg/mL) was made by dissolving 100 mg of acrylamide in 100 mL of double-distilled water.The stock solutions were also diluted with water to yield a series of standards (0.05-2.5 mg/L).The calibration curve for acrylamide analysis was created by plotting the ratio of acrylamide peak area versus acrylamide concentration levels (Figure 2).The peak area is proportional to the acrylamide concentration, and the calibration curve was plotted using the regression equation, y = 85.71× + 10.828, R 2 = 0.9892, where y is the concentration of acrylamide (mg/L) and x is the peak area.The peak area is linearly related to the acrylamide concentration (0.05 to 2.5 mg/L).

Statistical analysis
All statistical analyses were performed in triplicate, and the results were expressed as mean ± standard deviation (SD) for n = 3. Statistical analysis was performed with JMP Pro 13 software, and comparisons were made with one-way ANOVA followed by Duncan's new multiple-range tests.The level of significance was set at p < .05.The IC 50 value was calculated using Origin software 2021.The correlation analysis was conducted using Pearson's correlation method.

Total phenolic contents
The appearance of a blue-colored solution due to the reduction of phospho-molybdic and phospho-tungstic acids contained in the Folin-ciocalteu reagent indicates the presence of phenolic compounds in the samples [37] and the result expressed in milligram gallic acid equivalent per gram (mg GAE/g).The total phenolic content of five dietary herb extracts ranged from 19.28 ± 1.18 to 30.20 ± 1.23 mg GAE/g and decreased in the order of kesse > tosign > rosemary > koseret > basil (Table 1).The total phenolic content of basil extract was significantly (p < .05)differed from each other; however, kesse with tosign extract, and koseret with rosemary extract had shown no significant difference (p > .05).The total phenolic content of koseret, tosign, and rosemary extracts of the current study was higher than the previous report presented by Belachew et al. and Engida et al. [8,16] while, rosemary extract was lower than the previous finding of Turan et al. [38] The total phenolic contents of Ocimum basilicum and Ocimum gratissimum extracts ranged from 9.09 to 27.41 mg GAE/g, as reported by Uyoh et al., [39] which agrees with the results of the current study.

Total flavonoid content
The aluminum chloride colorimetric assay, which uses Al (III) as a complexing agent, is one of the most widely used methods for determining total flavonoid content in plant extracts. [31]The total flavonoid contents varied from 5.13 ± 0.0.53 to 15.87 ± 1.06 mg CE/g and decreased in the order of kesse > tosign > koseret > rosemary > basil (Table 1).The results of this investigation showed that the total flavonoid content of kesse, koseret, rosemary, and tosign extracts differed significantly (p < .05)from that of bail, while there was no difference between kesse and tosign, koseret, and rosemary extracts.The total flavonoid content of the current study was lower than that of the previous reports on different extracts of tosign and koseret. [16,40]

DPPH scavenging activity
Substances capable of donating hydrogen atoms or electrons are able to convert the DPPH (purple) free radical into the non-radical form. [41,42]Results have shown that at 750 μg/mL, DPPH scavenging activity of dietary herb extracts ranged from 86.09% to 55.07%.Kesse extracts showed the strongest antioxidant activity with an inhibitory percentage of 86.09% which was closest to the value of ascorbic acid (91.16%) used as a positive control (Figure 3).The current results are also consistent with the Where x and y are total phenolic and total flavonoids expressed as gallic acid and catechin equivalents, respectively.Values are expressed as mean ± SD (n = 3) from triplicate experiments.Means with different letters within the same column were significantly different at the level of p < .05.
findings of Aburigal et al., [43] who found that the DPPH scavenging activity of basil leaves from different locations varied from 89.22% to 69.33%.The IC 50 value of DPPH scavenging activity was calculated from the percentage of inhibition against the concentration of the extracts (Table 1).Basil had the lowest DPPH scavenging activity with an IC 50 value of 406.98 ± 3.43 μg/mL, whereas kesse had the highest DPPH scavenging activity with an EC 50 value of 107.80 ± 2.37 μg/mL.The IC 50 values of koseret, rosemary, and tosign extracts were not different, whereas they were significantly different (p < .05)with kesse and basil extracts.The IC 50 values of koseret, rosemary, and tosign extracts were not different, but the extracts of kesse and basil had significantly (p < .05)different IC 50 values from one another.The strongest values of free radical scavenging activity of kesse and tosign extracts could be due to their higher total phenolic content and total flavonoid content among the three herb extracts obtained from this investigation.Engida et al. [16] reported that extract of tosign showed stronger DPPH scavenging activity than the present study.The difference in the DPPH radical scavenging activity might result from the extraction method, extraction solvent, and geographical location. [44]tal antioxidant activity Kesse extract showed the strongest total antioxidant activity, ranging from 37.32 ± 0.45 mg AAE/g at 0.5 mg/mL to 65.81 ± 1.50 mg AAE/g at 1 mg/mL, whereas basil extract had the weakest total antioxidant activity (9.09 ± 0.225 mg AAE/g at 0.5 mg/mL to 20.10 ± 0.451 mg AAE/g at 1 mg/mL, respectively, Figure 4).The results also indicated that the total antioxidant activity of the other three dietary herb extracts increased with increasing concentration.At a concentration of 1 mg/mL, there was no difference between rosemary and koseret extracts, but these values revealed a significant difference (p < .05) between kesse, tosign and basil extracts.

Analysis of acrylamide content in asparagine-glucose model
The effect of dietary herb on acrylamide formation was evaluated in the current study using an asparagineglucose model, and the results of acrylamide analysis revealed the presence of a significant amount of acrylamide in all samples with varying ranges (Figure 5).The reaction model treated with kesse and tosign extracts resulted in the lowest concentration of acrylamide (1.245 ± 0.042 and 1.13 ± 0.032 mg/L), indicating a reduction of 64.42% and 63.6%, respectively.These results were significantly (p < .05)different from those in the control, BHT (1.952 mg/L), which showed a reduction of 37.01%.On the other hand, the reaction model treated with basil, koseret, and rosemary extracts had acrylamide contents of 2.27, 1.52, and 1.48, respectively, with acrylamide reduction percentages of (26.8, 51.0, and 52.1) % at p < .05 in comparison with the model without the herb extracts.The  acrylamide content in all dietary herb treatment groups was significantly different from that of the control group (p < .05).There are different reports about the effects of some plants and herbal extracts as additives on acrylamide mitigation.The effects might be, carbonyl trapping activity (Schiff base a key intermediate), asparagine precipitation effect, and other supposed mechanisms of action of the phytochemicals present in the extracts [5,19,45] Moreover, Mousa et al. [46] found that garlic powder was reduced acrylamide formation in an asparagine/glucose model system and similar observations were also made with our result that adding thyme and rosemary extracts to wheat dough decreased the amount of acrylamide in the bread model. [21,23,47]

Correlation analysis
The ability of phenolic and flavonoid molecules to donate hydrogen atoms to free radicals or reduce the free radicals is what causes them to act as potent antioxidants and deactivate free radicals. [40]The amount of acrylamide formed from an asparagine-glucose reaction model containing herb extracts was negatively correlated with the total phenolic and total flavonoid content with, R 2 values of 0.920 and 0.930, respectively (Figure 6).Accordingly, the asparagine-glucose model treated with kesse (the highest in total phenolic content and total flavonoid content) revealed the significant reduction of acrylamide (p < .05).A strong correlation could also be seen between DPPH-scavenging activity and acrylamide (R 2 = 0.959), and between total antioxidant activity and acrylamide (R 2 = 0.999) suggesting that the antioxidant activity may be due to the phenolic compounds and flavonoids present in herb extracts.
Jing et al. [2] reported that the acrylamide content was strongly correlated with the total phenolic content and DPPH scavenging activity with R 2 values of 0.983 and 0.966, respectively, and these results are in line with our findings.Some other researches demonstrated that the correlation between the reduction rate of acrylamide and antioxidant activity of plant extracts were a bit different from pure compounds. [28]According to Ciesarová et al. [48] the DPPH scavenging activity of the used spices was correlated (R 2 = 0.996) with the effect of the extracts on the decrease in acrylamide contents.

Conclusion
In the current study, dietary herbs significantly reduced the formation of acrylamide in asparagineglucose models.Among the studied herb extracts, kesse and tosign extracts were found to be most effective at mitigating acrylamide.In addition, the total phenolic content and total flavonoid content of the herbs and their antioxidant activities were strongly correlated with the reduction of acrylamide content in the asparagine-glucose model treated with herbal extracts, which suggests that antioxidants significantly reduce the formation of acrylamide by removing reactive free electrons from free radical intermediates formed by the Maillard reaction.Furthermore, our results suggest that these dietary herb extracts could be good candidates for the mitigation of acrylamide during the Maillard reaction.

Figure 3 .
Figure 3. DPPH radical scavenging activity of different dietary herb extracts and ascorbic acid as the control.The values are in three replicates (mean ± SD).

Figure 4 .
Figure 4. Total antioxidant activity of herb extracts.Each value represents the mean ± SD of triplicate experiments, and the mean values of the same concentration with different letters were significantly different at the level of p < .05.

Figure 5 .
Figure 5. Effects of dietary herb extract on acrylamide content in an asparagine-glucose reaction model heated at 175°C for 20 min.Data represent the mean ± SD of three experiments.Values of different letters are significantly different (P < .05).

Figure 6 .
Figure 6.Correlation between acrylamide contents formed by an asparagine-glucose model containing herb extracts at 175°C for 20 min and the total phenolic content (a), total flavonoid content (b), DPPH-scavenging activity (c), and total antioxidant activity (d) of the herb extracts.

Table 1 .
Total phenolic content, total flavonoid content, and IC 50 value (μg/mL) of DPPH scavenging activity for dietary herb extracts.