Ferulic acid as major antioxidant phenolic compound of the Tetragonisca angustula honey collected in Vera Cruz-Itaparica Island , Bahia , Brazil

This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract The antioxidant activity of Tetragonisca angustula honey (TAH) and its ethanolic extract (TAEE) were investigated. The total levels of phenolic (TPC) and flavonoids (TFC) were also evaluated. The results for TPC were 19.91 ± 0.38 and 29.37 ± 1.82 mg GAE g-1 and for TFC 0.20 ± 0.02 and 0.14 ± 0.01 mg QE g-1 of TAH and TAEE, respectively. Antioxidant activities were 73.29 ± 0.49% and 93.36 ± 0.27% in the DPPH● assay and 71.73 ± 4.07% and 97.86 ± 0.35% in ABTS●+ for TAH and TAEE, respectively. The total reducing activity was determined by the method of reducing power (PR) and iron ion (Fe III) and the results varied in PR from 151.7 ± 25.7 and 230.7 ± 25.2 mg GAE L-1, for TAH and TAEE respectively and for (Fe III) in EC50 0.284 in TAEE and 0.687 in TAH. Chemical analysis by HPLC-DAD of the ethanolic extract (TAEE) revealed the presence of ferulic acid as majority phenolic component in the extract. The 1H NMR analysis confirmed this structure and showed the also presence of glucose, citric acid, succinic acid, proline and hydrocarbon derivatives. In addition, the botanical origin was also investigated and showed a multifloral characteristic, having found 19 pollen types with a botanical predominance of the Anacardiaceae family, with Tapirira pollen occurring as predominant (42.6%) and Schinus as secondary (25.7%). The results showed that T. angustula honey is an interesting source of antioxidant phenolic compounds due to its floral origin and can act as a protector of human health when consumed.


Determination of antioxidant components 2.3. Total phenolic content (TPC)
Concentration of total phenolics was determined with the Folin-Ciocalteau phenol reagent (Synth ® ) according to the methodology described by Singleton et al. (1999). Aliquots (2.5 g) of TAH and TAEE were diluted in 25 mL distilled water (100 mg/mL concentration) of stock solution, in 0.5 ml of this solution 2.5 ml of 0.2 N Folin-Ciocalteu reagent was added for 5 min. Subsequently, they were added with 2 mL of sodium carbonate (Na 2 CO 3 ) solution at 7.5%. After the reaction mixture incubated for 1 hour at room temperature and protected from light, absorbance was measured using a WPA Lightwave II spectrophotometer (Biochrom) at 760 nm. The values were interpolated on a calibration curve made with gallic acid (Sigma ® ). The results were expressed in mg equivalent of gallic acid per gram (mg GAE g -1 ). The blank consisted of a solution of 0.5 mL of ethanol together with 2.5 mL of Folin-Ciocalteu reagent and 2.0 mL of sodium carbonate solution.

Total flavonoid content (TFC)
The total flavonoid content was determined by the colorimetric reaction with aluminum trichloride (Dinamic) performed according to the method described by Woisky and Salatino (1998). A 2.5-g aliquot of TAH and TAEE samples was diluted in distilled water (100 mg/mL concentration) and mixed 2.5 mL with 2.5 mL of a methanolic solution (2 g/100 mL) of aluminum trichloride (AlCl 3 ). The reading was performed on a spectrophotometer (Biochrom) at 420 nm after 1 hour at room temperature and in the dark.
The absorbance values were interpolated on a standard quercetin curve (Sigma ® ). The results were then expressed in mg equivalent of quercetin per gram (mg QE g -1 ). The blank contained a solution of 2.5 mL of ethanol together with 2.5 mL of AlCl 3 reagent without methanol.

Evaluation of antioxidant properties 2.5.1. Free radical capture activity
The antioxidant activity of TAH and TAEE was determined by the methods of DPPH • (1,1-diphenyl-2-picrilhidrazil) according to a procedure described by Meda et al. (2005) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic) (ABTS •+ ) based on the methodology described by Lee et al. (2017), with some modifications. The samples were initially diluted to a concentration of 100 mg/mL in distilled water, to prepare the stock solution.
For the DPPH • assay, 0.3 mL of the stock solution was added to 2.7 mL of DPPH • 0.024% solution diluted in methanol. Distilled water (0.3 mL) in 2.7 mL of DPPH • solution (Sigma ® ) was used, serving as the blank sample. The mixtures were left for 1 h at room temperature in the dark and the absorbance was measured in an Evolution 220 UV-Vis spectrophotometer (Thermo Scientific) at 517 nm. Measurements were performed in triplicate. The reading of the optical density of the samples was correlated with the standard DPPH • control, establishing

Introduction
Jatai bee (Tetragonisca angustula) is an easily adaptable species of stingless bee that can be raised both in rural and urban areas (Grüter et al., 2011). Although they produce honey in a small amount, they provide a product that differs from Apis mellifera L. honey, especially in the peculiar flavor and aroma, what causes its prices to rise in the market. Honey produced by meliponini is a valuable product that has very different characteristics, being used by the old consumption tradition mainly for medicinal purposes (Almeida-Muradian et al., 2013). The honey is a natural source of health due to its therapeutic qualities, antimicrobial and anti-inflammatory activities, antioxidant properties and prebiotics (Sgariglia et al., 2010;Nishio et al., 2016).
Honey is the result of nectar and saccharin exudation of plants, collected, modified and stored by honey bees. It has a complex mixture composed mainly of water, carbohydrates, fatty acids, proteins, amino acids, vitamins, minerals, natural products and phenolic compounds. These phenolic compounds are largely responsible for the antioxidant activity of honey and can act as free radical reducing agents or as chelators of pro-oxidant metal ions, but they can also modulate several intracellular pathways (Wang and Lin, 2000;Alvarez-Suarez et al., 2012). Natural antioxidants are effective in reducing the risk of cardiovascular, metabolic, visual, immunological diseases and several other acute and chronic inflammatory processes (Rao et al., 2016). Phenolic compounds, comprising phenolic acids and flavonoids, are also considered as potential indicators of botanical origin (Alvarez-Suarez et al., 2012). Thus, honeys produced by distinct bee species or collected from different locations have different active compounds and, consequently, differ as for their biological properties.
Chemical and biological studies on Tetragonisca angustula honey are scarce in the literature, and it is known that these products have differences, especially in the phenolic profile, when compared to honey produced by Apis bees (Rodríguez-Malaver et al., 2009, Silva et al., 2013b. Recently, we described the identification of four hydroxycinnamic acid-spermidine amides from Tetragonisca angustula honey as anti-Neospora caninum (Lima et al., 2021). The aim of this study was to investigate the antioxidant activity and chemical profile of Tetragonisca angustula honey and its extract, as well as to identify its botanical origin.

Obtaining of the crude extract and fractions
The ethanol extract (TAEE) of the Tetragonisca angustula honey collected in the municipality of Vera Cruz on Itaparica Island (state of Bahia, NE Brazil) (12º 57' 32"S x 38º 36' 16"W), registered with SISGEN under N o . AA64E09, was prepared using Ethanol PA (1:3) according to the methodology of Siess et al. (1996), modified by Trusheva et al. (2007). Three hundred grams of TAH was added with 900 mL of ethanol and kept at room temperature, protected from light for a period of four days, when finally, the solvent was removed, pouring by hand. the percentage of discoloration of the DPPH • radical according to Equation 1: (1) Where: ADPPH = Absorbance DPPH and As = Sample Absorbance.
For the ABTS •+ assay a stock solution was prepared by dissolving ABTS •+ (Sigma ® ) (7 mM) and potassium persulfate (Dynamic) (2.45 mM) in ultrapure water and left protected from light at room temperature for 16h . The ABTS •+ solution was diluted with ethanol to reach an absorbance value of 1.0 (Di Castillo et al., 2015) at 753 nm (Dong et al., 2015) using a spectrophotometer (Thermo Scientific). Two hundred microliters of aqueous solutions of TAH and TAEE (100 mg/mL) were mixed with 2.8 mL of ABTS •+ diluted solution, the mixture was vortexed and left in the dark at room temperature for 30 minutes. The blank sample consisted of distilled water (200 µL) and ethanol (2.8 mL). The results were expressed as the radical scavenging capacity (% RSA) using Equation 1. The control sample was composed of ABTS •+ diluted solution and 200 µL of distilled water.

Total reducing activity
The total reducing activity of TAH and TAEE was evaluated using two methodologies: the modified Folin-Ciocalteu (PR) method as described by Berker et al. (2013), with some modifications, and the reducing power of the ferric ion (Fe III) determined by the method described in the literature by Berker et al. (2007).
The Folin-Ciocalteu reagent (Synth ® ) was diluted in isobutanol (Dynamic) at a retention of 1: 2 (v/v) and an aliquot (60 µL) was mixed with 40 µL of stock solution of TAH and TAEE, followed by adding 700 µL of sodium hydroxide solution (0.1 M) and 1.200 µL of distilled water. The tubes were vortexed and allowed to react in the dark, at room temperature, for 20 min. Ethanolic gallic acid solution (Sigma ® ) was used as a standard (100-600 µg/mL). The absorbance was read at 665 nm using a UV-Vis spectrophotometer (Thermo Scientific) and the results were interpolated in a standard curve of gallic acid and the data expressed in mg equivalent of gallic acid per liter (mg GAE L -1 ).
For the test of reducing power of the ferric ion (Fe III), in test tubes, 1 mL of the stock solution of each sample was added in 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of potassium ferricyanide (Dynamic) at 1%. The mixture was incubated in a water bath at 50 °C for 20 minutes. Subsequently, the tubes were kept at room temperature. 2.5 mL of 10% trichloroacetic acid (TCA) (Dynamic) was added and the mixture was stirred vigorously. Then, 2.5 mL of distilled water and 0.5 mL of 0.1% iron chloride (Dynamic) were added. The mixture was kept for 2 minutes. Absorbance was measured with a spectrophotometer (Thermo Scientific) at 700 nm. The concentration value that induced 50% inhibition of free radicals (EC 50 ) was calculated from the absorbance graph and measured in triplicate, depending on the concentrations of honey and extract [0.25; 0.5; 1.0; 1.5; 2.0; 2.5; 3.0].
2.6. Determination of chemical properties 2.6.1. NMR analysis 1 H NMR spectra were measured on a Bruker Advance 500 spectrometer operating at 500 MHz for 1 H and 125 MHz for 13 C, using deuterated chloroform (CDCl 3 ) as solvent (approximately 10-15 mg of samples were dissolved in 0.5 mL of solvent and transferred into a 5-mm NMR tube), internal lock and TMS as internal reference.

HPLC-DAD analysis
The extract (10 mg) was mixed with methanol until completely fluid, filtered on a 0.22-µm PTFE filter and placed in a vial for injection in a high-performance liquid chromatography apparatus with ultraviolet diode array detector (UV/DAD). The HPLC analysis was used with mobile phase reagents containing a gradient at 0.1% acetic acid (CH 3 COOH) in H 2 O (A) and methanol (B) in the following elutions: 0-30 min, 100-0% B; 31-37 min, 0-100% B; 38-40 min, 0-100% B. A DAD semiprep system was used for the isolation of pure compounds and comprised a LiChrospher ® 100 RP-18 column (250mm x 4mm x 5µm) with a 10-µL/min flow rate and column temperature adjusted to 30°C, with a binary gradient phase of the reagents under the same conditions. Suggestion for the identification of phenolic compounds was based on the retention times of authentic markers acquired in Sigma Aldrich ® as well as evaluation of the UV-Vis spectrum.

Melissopalynological analysis
The botanical origin of TAH was confirmed through melissopalinological analysis (Von der Ohe et al., 2004). TAH was submitted to the Erdtman acetolysis process (Erdtman, 1960) and the resulting pellet was mounted on slides with glycerinated gelatin for the identification and counting of the pollen grains that make up the pollen spectrum of the sample. The pollen grains were characterized using an Olympus ® optical microscope (CX41) and the images were captured with an Olympus ® digital camera (Evolt E-330) attached to the 100x objective. Pollen types were identified in double blind, using specialized literature (Online Palynological Database, 2018) and through consultation on the database and images of the Palinoteca of the Federal University of Reconcavo da Bahia. The frequency class of the pollen types was determined following the classification proposed by Louveaux et al. (1978).

Statistical analysis
The descriptive statistics of mean and standard deviation were determined for the variables TPC, TFC, DPPH • , ABTS •+ and PR and calculated by Student's t test at 1% significance. The normality of the data was verified by Shapiro-Wilks. Pearson's linear correlation coefficients, whose significance was calculated by the Student's t test under 5% probability. The analyzes were performed using the statistical program R (R Core Team, 2020).

Antioxidant properties
Due to the presence of phenolic compounds found in the sample and the antioxidant potential of these compounds was initially determined, the total phenolic contents (TPC) and it was observed that TAH and TAEE showed 19.91 ± 0.38 and 29.37 ± 1.82 mg GAEg -1 . Silva et al. (2013b) studying Jandaira honey (M. subnitida) collected in the Atlantic Forest region, reported values of total phenolic content of 1.2 to 1.3 mg GAE g -1 in pure honey and 125.90 to 391.9 mg GAE g -1 in the ethanolic fraction, values of the extract higher than that found in this study. Our results are within the average of that reported in the methanolic extract of stingless bees from the Brazilian Amazon region, with values from 17 to 66 mg GAE g -1 (Silva et al., 2013a).
Since phenolics and flavonoids are described in the literature as having antioxidant activity, we determined the antioxidant activity of this sample and the values found to inhibit antioxidant activity at a concentration of 100 mg/mL showed that the capture of DPPH • and ABTS •+ radicals by TAH were 73.29 ± 0.49% and 71.73 ± 4.70%, respectively. On the other hand, TAEE captured 93.36 ± 0.27% and 97.86 ± 0.35% of the radicals DPPH • and ABTS •+ , respectively. The percentages of radical scavenging obtained for TAH in this study were similar to results reported in the literature on honey from several countries (Almeida et al., 2016). Thus, natural honey has a very broad antioxidant activity, ranging from 23.89 to 100% inhibition (Dżugan et al., 2017).
Variations in the profile of phenolic compounds can be attributed to the botanical characteristics of honey and the chemical capacity it has to capture the analyzed radicals (Atoui et al., 2005). In honey, the antioxidant capacity may be the result of a combination of several compounds, including phenolics, peptides, organic acids, enzymes, minerals, products of the Maillard reaction and other minor compounds (Sant'ana et al., 2012).
The reducing power, which is an index of antioxidant activity, was evaluated using the Folin-Ciocalteu reagent and the reducing capacity of the ferric ion. The reducing power of TAH and TAEE using the Folin-Ciocalteu reagent was 156.63 ± 20.06 and 230.66 ± 25.23 mg GAE L -1 , respectively. The reducing power of Fe (III) was influenced by the concentration of honey, in all tested concentrations [0.25; 0.5; 1.0; 1.5; 2.0; 2.5; 3.0], it was indicated that TAEE had a significant reducing power (EC 50 0.284) and showed better activity than TAH (EC 50 0.687), due to the extraction process, it was expected that the extract would show results superior to raw honey.
According to Table 1, most of the correlations were not significant between the parameters evaluated in honey and extract. However, four significant correlations were observed, two positive and two negative. The significant positive correlations r=0.70 (*p=0.024) were between TPC/TFC of T. angustula honey, characteristics observed by Al et al. (2009) who reported a correlation similar to that found in this study, between total phenolics and total flavonoids ( =0.84), as well as a significant correlation between RSA and total phenolic content ( =0.94), as well as between RSA and total flavonoids ( =0.83).
Another positive correlation r=0.64 (*p=0.047) between PR/DPPH • of TAEE was found in this study, which can determine a high antioxidant activity by these methods related to TAEE. Chua et al. (2013) mentioned that both phenolic compounds and honey flavonoids play important roles in antioxidant activity. In the honey samples analyzed by these authors, the flavonoid content was correlated with DPPH • ( =0.9276) and with the antioxidant power of iron reduction ( =0.9910); while the total phenolic content was only correlated with the iron reducing power ( =0.9033).
The significant negative correlations found indicate an inversely proportional relationship r = -0.64 (*p=0.048) observed between PR/TFC both evaluated in the extract and r = -0.75 (*p=0.012) between the PR of the TAEE /TFC of the TAH. The inversely proportional correlation related to TFC and PR may be due to the fact that flavonoids can chelate metals preventing their iron reduction activity (Wang and Lin, 2000).
The reducing power (PR) correlated significantly with TFC and DPPH • , as TAH and TAEE interact with Fe, justifies the high efficiency of TAEE as an agent that can protect against oxidative damage induced by iron (Pardo-Andreu et al., 2006). Li et al. (2009) reported the existence of a similar linear correlation between the reducing power and the total phenolic content and Tuksitha et al. (2018), suggested that stingless honeys have useful amounts of phenolic compounds and flavonoids that are capable of acting as natural antioxidants.
Corroborating what was expected; some substances that make up the honey of Tetragonisca angustula are potentially responsible for its biological activity and can act simultaneously or in isolation. Considering the beneficial action of phenolic compounds for human health in the treatment of cardiovascular and chronic diseases (Moniruzzaman et al., 2014;Oršolić and Car, 2014), the use of T. angustula honey in the diet is a promising alternative as a source of phenolic compounds with antioxidant potential. Phenolic substances are important antioxidants, related to the stabilization of free radicals, acting as reducing agents, inhibitors of enzymes, as metal chelators, in addition to participating in the signaling pathway (Wang and Lin, 2000). Ayna et al. (2020) demonstrated that the use of antioxidants as nutritional supplements and, in particular, caffeic, ferulic and quercetin acids, protected cells and reduced apoptosis-induced cell death in the immune system, liver, heart and kidneys.

Chemical properties
Honey is a very complex matrix with very specific physical-defined properties and this complexity makes the analysis of honey difficult in terms of its different properties.
The 1 H NMR spectra (see Figure 1) of TAEE showed three characteristic absorption regions of honey: aliphatic hydrogen (0.8 -3.0 ppm) (see Figure 1A), carbohydrate (3.0 -5.6 ppm) and aromatic (6.5 -8.5 ppm) (see Figure 1B). Individual signs were assigned based on data from the literature (Kosaka et al., 2015;Kortesniemi et al., 2016;Salgueiro et al., 2016;Consonni et al., 2019). The signal of citric acid, succinic acid, proline and hydrocarbon derivatives were observed in the aliphatic region (Spiteri et al., 2015), while the aromatic hydrogen signal confirms the presence of phenolic acid and flavonoids in the honey investigated here.
There are studies that show that the content of protons in aromatic groups is positively related to the antioxidant capacity of food products (Polak and Bartoszek, 2015;Bartoszek et al., 2017). This fact may suggest that these  compounds found in the extract are mainly responsible for the antioxidant properties of this honey. Then, the samples were analyzed by HPLC-DAD, below the chromatogram obtained at the wavelength of 360 nm (see Figure 2) and as shown in Table 2 shows the class of compounds characterized in the TAEE chromatogram.
The class of major compounds featured in the TAEE's 360 nm chromatogram demonstrated a characteristic region of phenolic acids and flavonols. As can be seen in Figure 2 and Table 2, the HPLC-DAD analysis of TAEE in comparison with authentic standards suggests the major peak 5 [(λmax) 299 (sh) 323 and 211 nm] as ferulic acid.
Ferulic acid is a phenolic acid widely distributed in the plant kingdom. It presents a wide range of potential therapeutic effects useful in the treatment of cancer, diabetes, pulmonary and cardiovascular diseases, as well as hepatic, neuro and photoprotective effects and inflammatory antimicrobial and anti-inflammatory activities (Kampa et al., 2004;Bobany et al., 2010;Paiva et al., 2013).
Phenolic compounds, aromatic acids, alcohols, terpenes and sugars have been observed in other studies with stingless honey in Brazil (Silva et al., 2013a;Silva et al., 2013b;Costa et al., 2018;Ciappini, 2019). In samples of stingless  honey from Australia, Portugal and Malaysia, ferulic, gallic, caffeic, chlorogenic, vanillic and rosmarinic acids were found, as well as myricetin, quercetin and kaempferol (Tomas-Barberan et al., 2001;Moniruzzaman et al., 2014). A wide variety of chemical constituents in small amounts are part of honey and many are known for their antioxidant properties that include phenolic acids and flavonoids.

Botanical origin
The sample collected in the municipality of Vera Cruz, Bahia, was evaluated for botanical origin through pollen analysis compared to the literature, having identified 19 pollen types distributed in 14 botanical families (as shown in Table 3), with the pollen type Tapirira occurring as pollen predominant (42.60%) and Schinus as secondary pollen (25.70%). These results confirm the influence of the diversity of flora in the Atlantic Forest region on the composition of honey (see Figure 3). The pollen types Borreria, Eucalyptus, Serjania, Tapirira and Waltheria represent genera that include botanical species considered nectariferous and important for the production of honey (Silva et al., 2013b;Nascimento et al., 2015). Predominant pollen from the Anacardiaceae family was found in Trigona honey (Frieseomelitta), Nigra paupera and Scaptotrigona ochrotica. Corroborating our results, the pollen type Tapirira guianensis was identified in samples of T. angustula in the north of the state of Pará (Novais et al., 2014) and in the northeast of Brazil; and Schinus was also considered frequent in the Northeast of Brazil (Novais et al., 2013).
The species of the Anacardiaceae family have shown promise in the search for bioactive compounds, involving the largest number of investigations, mainly regarding their metabolic constituents. Among these constituents are phenolic and catecholic compounds, or the mixture of them, called phenolic lipids, or terpenes and flavonoids, among other metabolites (Correia et al., 2006). The popular medicinal use aroused interest in the study of the chemical composition and biological activity of its extracts and metabolites and in the study of many species of these genera it was possible to detect flavonoids (mainly biflavonoids), terpenes, steroids, xanthones, phenolic lipids and derivatives (Correia et al., 2006;Ono et al., 2008, Chaves et al., 2010, thus corroborating the results presented in this work. The present study shows that T. angustula honey has high levels of phenolics and flavonoids and that the distribution of these compounds is influenced by the floral origin of the analyzed honey. Analysis by HPLC-DAD showed that the extracts are rich in phenolic acids, such as ferulic acid. NMR analysis showed signs of citric acid, succinic acid, proline and hydrocarbon derivatives and the presence of phenolic acid and flavonoids, in addition to carbohydrates. Finally, the total antioxidant capacity of honey is probably the result of the combined activities and interactions of a wide range of compounds, including amino acids, enzymes and possibly other minor components. However, Data represent analyses of 1.000 pollen grains (n = 1.000) counted in three different samples; standard deviation of means was ≤ 0.5. PP = predominant pollen (> 45% of total grains); SP = secondary pollen (16 to 45%); IMP = important minor pollen (3 to 15%); MP = minor pollen (<3%) (Louveaux et al., 1978).
phenolic compounds are well known for being the main contributors to this property, enriching the quality of honey as a functional food. Therefore, Tetragonisca angustula honey can be considered a natural source of antioxidants and can be added to the daily diet.