In vitro evaluation of the antioxidant potential of derivatives eugenol via ABTS radical capture assay

Eugenol is a natural product present in different plant species, especially in the flower buds of Syzygiu Aromatum . This secondary metabolite exhibits antioxidant action, in addition to acting as an anti-inflammatory, antitumor, antibacterial, antifungal, antipyretic, anesthetic and analgesic agent. Given the biological potential of eugenol, the antioxidant activity of eugenol derivatives was evaluated. Thus, eugenol isolated from flower buds of Syzygiu aromaticum was subjected to different types of reactions such as O -alkylation. The eugenol derivatives were characterized by 1 H and 13 C NMR techniques. The evaluation of antioxidante activity was carried out using the ABTS radical capture assay. Six derivatives were synthesized with yields ranging from 75-92%, which exhibited low antioxidant potential compared to radical capture assays, with EC 50 values from 0.085 to 0.327 mg/mL. These results motivate new studies, since the search for new antioxidant agents is extremely necessary in view of the numerous pathologies that are associated with oxidative stress.


Introduction
In recent decades, numerous studies have suggested that oxidative stress is associated with several pathological conditions involving chronic inflammation, which contributes to aging and chronic diseases, to mention cancer.Oxidative stress refers to the imbalance between the levels of reactive oxygen species (ROS) and their antioxidants.These species are generated in various processes involving biochemical and physiological activities in the form of by-products (Tan, Norhaizan, Liew, & Rahman, 2018).Phenolic compounds are examples of natural antioxidants.Reports indicate that they are more potent than vitamins E, C and carotenoids.Its action is closely related to the ability to donate hydrogen atoms, suppressing the formation of ROS, acting as metal chelators (which catalyze the Fenton reaction) or also as enzyme inhibitors/activators (Silva, Monte, Lemos, Nascimento, Costa, & Paiva, 2018).Eugenol, is a naturally occurring phenolic compound, found in the essential oil of clove Syzygium aromaticum (L.) Merr.& amp; LM Perry, Myrtaceae which has widespread antioxidant action.In addition, eugenol has a range of biological activities, to mention, acts as anti-inflammatory, antitumor, antibacterial, antifungal, antipyretic, anesthetic and analgesic (Batiha, Alkazmi, Wasef, Beshbishy, Nadwa, & Rashwan, 2020).In view of the constant search for new effective therapeutic substances to prevent diseases caused by oxidative stress, combined with the potential of eugenol, the synthesis and evaluation of the in vitro antioxidant activity of different eugenol derivatives was carried out.

Reagents and equipment
The reagents and solvents used were obtained in commercial form.The reactions were monitored by thin-layer chromatography (TLC) using silica gel plates (with fluorescent indicator F254).TLC plates were visualized using UV light.The purification step was performed by liquid chromatography on a glass column using silica gel 60 (70-230 mesh) as the stationary phase, and the hexane and ethyl acetate solvents in different proportions as mobile phase.Spectral analyzes of the synthesized compounds were obtained using the Fourier transform infrared spectrophotometer (Spectrum 400 FT-IR / FT-NIR Spectrometer model PerkinElmer), the 1 H NMR and 13 C NMR spectra were obtained on a Varian Unity Plus spectrometer 300 and 400 MHz with the chloroform-d solvent and tetramethylsilane was used as an internal standard.The carbon and hydrogen contents of the compound 2c was determined by the Dynamic Flash Combustion technique, in a CHNS-O elementary analyzer, CE Instruments, model EA 1110.

Isolation procedure of the essential oil
Syzygium aromaticum flower bud (Clove) was purchased from a market in Recife, Brazil.The essential oils from clove (100 g) were separately isolated using a modified Clevengertype apparatus and hydrodistillation for 2h.The oil layers were separated and dried over anhydrous sodium sulfate, stored in hermetically sealed glass containers, and kept at low temperature (-5ºC) until analysis.Total oil yields were expressed as percentages (g/100 g of fresh plant material).All experiments were carried out in triplicate.

Isolation of the eugenol
The essential oil obtained as described above (0.8 g) was chromatographed on a column of silica gel and eluted with a gradient of hexane / ethyl acetate (95:5, 85:15 and 60:40) to 100% dichloromethane.Twenty-five fractions each of 30 mL were collected and analysed by TLC eluting with hexane / ethyl acetate (7:3), fractions were grouped to yield five groups (G:1-5).Group G-2 was purified by chromatography using a mixture of hexane / ethyl acetate (80:20) as eluent, resulting in yellow oil 0.68 g (eugenol).

Chemical analysis of essential oils
Quantitative GC analysis were carried out using a PerkinElmer Clarus 500 GC apparatus equipped with a flame ionization detector (FID) and a non-polar DB-5 fused silica capillary column (30 m x 0.25 mm x 0.25 μm film thickness) (J & W Scientific).The oven temperature was programmed from 60 to 240 °C at a rate 3 °C.min - .Injector and detector temperatures were 260 °C.Hydrogen was used as the carrier gas at a flow rate of 1 mL.min -1 in split mode (1:30).The injection volume was 0.5 µL of diluted solution (1/100) of oil in n-hexane.The amount of each compound was calculated from GC-FID peak areas in the order of DB-5 column elution and expressed as a relative percentage of the total area of the chromatograms.Analyses were carried out in triplicate.The qualitative Gas Chromatography-Mass Spectrometry (GC-MS) analysis were carried out using a Varian 220-MS IT GC system with a mass selective detector, mass spectrometer in EI 70 eV with a scan interval of 0.5 s and fragments from 40 to 550 Da. fitted with the same column and temperature program as that for the GC-FID experiments, with the following parameters: carrier gas = helium; flow rate = 1 mL.min -1 ; split mode (1:30); injected volume = 1 µL of diluted solution (1/100) of oil in n-hexane.

Identification and quantification
Identification of the components was based on GC-MS retention indices with reference to a homologous series of C8-C40 n-alkanes calculated using the Van der Dool and Kratz equation (Van, Dool & Kratz 1963) and by computer matching against the mass spectral library of the GC-MS data system (NIST 14 and WILEY 11th) and co-injection with authentic standards as well as other published mass spectra (Adams, 2007).Area percentages were obtained from the GC-FID response without the use of an internal standard or correction factors.

Evaluation of the in vitro antioxidant activity
The evaluation of antioxidant activity was performed according to methodology which are based on the capture of ABTS radicals (2,2'-azino-bis(3-ethylbenzothiazoline-6sulphonic acid).The ABTS free radical scavenging test was carried out according to the methodology described by Gupta, Kumar, Ganguly, Singh, Rana and Pandey, (2021) was used, in which (+)-6-hydroxy-2,5,7,8-tetramethylchroman-2carboxylic acid (Trolox) as a positive control.Compounds and essential oil were tested at concentrations of 0.010 to 0.500 mg/mL and all tests were performed in triplicate.The results were expressed using the EC50 value, which represents the concentration of the sample necessary to sequester 50% of the ABTS radical.
Antiradical efficiency was established using linear regression analysis in the 95% confidence interval (P < 0.05) obtained by the GraphPad Prism 5.0 statistical program.

Results and Discussion
The GC-MS analysis of the essential oil of S. aromaticum enabled the identification of 98.70% of the chemical composition (Table 1).Eugenol (86.01%) was the major component identified in the oil.The eugenol was purified by column chromatography and used in synthesis.The eugenol derivatives (2a-f) were obtained through reactions of Oalkylation, oxidative hydroboration and hetero coupling of the eugenol as shown in Figure 1.
The eugenol derivatives (2a-f) were synthesized with yield values that varied from 70 to 92% (Table 2), these values being equal or higher when compared to those described in the literature.In addition, the spectroscopic data obtained from compounds 2a-f are in accordance with the values reported in the literature cited in Table 2.The new compound 2c showed 75% yield in 24 h.
The values of the effective concentrations of the compounds 2a-f are shown in Table 3.The synthesized eugenol derivatives exhibited the following order of antioxidant activity 2e >2f> 2c >2d>2a>2b.TROLOX was used as positive control, which had the following EC50 values 0.004 mg/mL.The EC50 values of the compounds 2a-f demonstrate a low antioxidant action when compared to the EC50 of eugenol (0.050 mg/mL).The compounds 2e and 2f had the lowest EC50 values from 0.085 to 0.088 mg/mL, respectively, allowing the conjecture that the incorporation of the propargyl and acetyl groups favors the capture of free radicals (ABTS) when compared to the other groups inserted in the eugenol structure (e.g.compounds 2a-d).Compound 2b showed highest EC 50 value, allowing conjecture that the removal of the establishment of the allyl group, through oxidative hydroboration, drastically reduced the ability of this compound to capture ABTS free radicals.These results corroborate the data described in the literature, which describe that the phenolic hydroxyl and the allylic unit, present in eugenol, effectively contribute to the antioxidant activity of this natural product.Specifically, unsaturation's are considered to be very valuable for eugenol activity in relation to free radicals (Gulçin, 2011;Farias, Oliveira, Dutra, Fernandes, Pereira, Oliveira, Stefanello, Lencina and Barschaka, 2013), justifying the unsatisfactory result of compound 2b.Additionally, this study marks the first steps towards the development of new antioxidant agents, however, more studies are needed to measure the antioxidant properties of these eugenol derivatives.

Conclusion
Different eugenol derivatives were synthesized, which were obtained in good yields that varied from 75 to 92%.The evaluation of the antioxidant capacity of the eugenol derivatives provided effective concentration values in the range of 0.085 to 0.327 mg/mL, which implies a low antioxidant capacity when compared to the ABTS radical elimination test.Thus, the results obtained in this study are the first steps towards the development of new therapeutic substances that come to play an antioxidant action.
RI a = Retention indices calculated from retention times in relation to those of a series C8-C40 of n-alkanes on a 30m DB-5 capillary column.RI b = Retention indices from the literature.RI = retention índice, MS = mass spectroscopy and CI = Co-injection with authentic compounds.

Table 1 .
Percentage composition of essential oils from flower buds of Syzygium aromaticum.

Table 2 .
Information on the time and yield of the synthesized compounds 2a-f.
a Yield of isolated product.