Photoprotective activity and HPLC-MS-ESI-IT profile of flavonoids from the barks of Hymenaea martiana Hayne (Fabaceae): development of topical formulations containing the hydroalcoholic extract

Abstract The development of photoprotective products has been highly focused on natural ingredients, and exposure to ultraviolet (UV) radiation is also related to the production of reactive oxygen species (ROS), which are extremely damaging to tissues. Hymenaea martiana Hayne has flavonoids related to its pharmacological activities, but a flavonoid profile of this species has not been reported yet. In this context, this work aimed to perform high-performance liquid chromatography-mass spectrometry-electrospray ionization-ion trap (HPLC-MS-ESI-IT) to characterize the flavonoids from Hymenaea martiana bark extract and develop a cosmetic formulation with photoprotective action. Total phenolic compounds, total flavonoids, sun protection factor (SPF) and antioxidant activity of the crude extract were determined. An HPLC-ESI-IT profile of flavonoids was developed. Photoprotective gel formulations were developed with the extract of H. martiana. The highest content of phenolic compounds was found in the crude extract, followed by the ethyl acetate fraction. The total flavonoid content in the samples demonstrated that these are the major phenolic compounds. The crude extract showed significant antioxidant and photoprotective activity. A flavonoid profile was developed, and bioactive flavonoids reported in the species were identified. In the development of the formulations, it was evidenced that the addition of the crude extract to the chemical filter increased the antioxidant activity and FPS, suggesting a synergistic effect in the photoprotection. Formulation F4 was considered a promising product. These results show the cosmetic potential of Hymenaea martiana, thus justifying the development of a cosmetic formulation with photoprotective activity.


Introduction
Medicinal plants have played an important role in therapeutics since antiquity, but their value as a clinical alternative has been gaining prominence in public policies in several countries around the world, including in Brazil. This country has standardized the methods of analysis, mainly in relation to some criteria, such as safety, efficacy and quality of drugs and cosmetics with active vegetable raw material.
The development and production of photoprotective products has been highly focused on natural active ingredients, and consumers have shown preference for using natural raw material, mainly vegetable derivatives [1]. Natural products with antioxidant activity have been highlighted, due to the fewer undesirable effects in relation to the synthetic additives [2]. Following this trend, the development of photoprotective formulations currently aims at the inclusion of natural products and plant extracts in the formulations [3], mainly raw materials with antioxidant activity [4].
Ultraviolet (UV) radiation is a range of the electromagnetic spectrum that lies between 100 and 400 nm and is divided into UVA (320-400 nm), UVB (290-320 nm) and UVC (100-290 nm) [5]. In addition to sunburn and skin cancer, exposure to UV radiation is also related to early cutaneous aging, which can be related to the production of Reactive Oxygen Species (ROS), which are extremely damaging to tissues [6].
As a result, natural chemical compounds with antioxidant and photoprotective activities, such as phenolic compounds and flavonoids, have shown important pharmaceutical and cosmetic potential, due to their biological actions already reported. the main pharmacological activities associated with flavonoidsinclude antioxidant activity [7], immunomodulatory, anti-inflammatory, bactericidal, antiviral, hepatoprotective and gastroprotective activity [8]. Other activities that have been studied are the antimicrobial activity [9] and photoprotective activity of flavonoids [10].
Among the medicinal plants found in the Caatinga biome, stands out the Hymenaea martiana Hayne, popularly known in the Brazilian Northeast as 'jatobá' . Jatobá has traditionally been used in the form of food, building material and traditional medicine, being used in the form of alcoholic extract, for the treatment of anemia, gastritis, inflammation, rheumatism, and also as antinociceptive and analgesic [11][12][13]. Some substances that have been related to the pharmacological activities are flavonoids, being astilbin the major component of the bark extract [7,11,[14][15][16][17]. Due to the presence of these chemical compounds whose absorption spectrum presents with two peaks maximum between 240-280 nm and another at 300-550 nm, Hymenaea martiana could then be associated with the development of photoprotective preparations [18].
In this context, this work aimed to perform a phytochemical study, with the characterization of flavonoids by high-performance liquid chromatography-mass spectrometry-electrospray ionization-ion trap (HPLC-MS-ESI-IT) from Hymenaea martiana bark extract, and to develop a cosmetic formulation with photoprotective action.

Plant material
The barks of H. martiana were collected in the city of Petrolina, Pernambuco, Brazil, in July 2015, and were identified by a botanist of Herbarium Vale do São Francisco (HVASF), at the Federal University of São Francisco Valley, with voucher specimen n° 6444, coordinates 09"11'04.30° S, 040"18'05.40° W, 357 m high. The barks were dried at 40 °C for 72 h in air circulation oven (Ethiktechno®, Model TD 420), and pulverized using a mill (quimis®). All procedures for access to genetic patrimony and associated traditional knowledge were carried out and the project was registered in SisGen (Register #A3E4538).

Extract preparation
For the preparation of the crude extract, 300 g of the barks were submitted to extraction in the National Institute of Semiarid (INSA, Campina Grande, Paraíba, Brazil), using the Accelerated Solvent Extraction (ASE) Thermo Scientific Dionex® ASE 350, equipped with a stainless-steel cell extractor hermetic sealed. For this, the dried and powdered plant material had its granulometrically homogenized using sieves of mesh 14 and 35, then this material together with diatomaceous earth (2:1) was distributed in 15 extraction cells with 100 mL capacity for 20 g of vegetal material each. The extraction was done with 99.5% ethanol, with a temperature of 40 °C, for 15 min, flow of 5 mL/ min and average pressure of 1500 psi., with two extractions per cell.
After the extraction process, the solvent was concentrated in a Thermo Scientific Rocket Evaporator at 40 °C. The residual solvent was eliminated in an oven (Ethiktechno®) at 45 °C for 24 h, obtaining 67 g of the crude ethanolic extract (Hm-CEE).
The crude ethanolic extract (10 g) was solubilized in a mixture of methanol and water (3:7 v/v), which was submitted to a liquid-liquid partition in a separation flask, with manual shaking, extensively, with hexane, chloroform and ethyl acetate. After this process, the solutions were concentrated for solvent evaporation under vacuum at 50 °C, obtaining the 1.24 g of the hexane fraction, 0.18 g of the chloroform fraction, and 7.90 g of the ethyl acetate fraction.

Determination of total phenolic compounds
The total phenol content was measured by the colorimetric method using Folin-Ciocalteu reagent (Sigma®) and gallic acid (Sigma®) as standard, based on the method described by [19]. For this, an aliquot (40 μL) of the diluted extract and fractions was added to 3.16 mL of distilled water and 200 μL of the Folin-Ciocalteu reagent and mixed immediately. The mixture was allowed to stand for 6 min and then 600 μL of stock solution of Na 2 CO 3 (200 g/L) were added and mixed. The final solutions were left to stand at 20 °C for 2 h. At the end of the process, the absorbance of each solution was determined in a spectrophotometer (quimis®) at 756 nm against the blank (all components except the sample under analysis) and the results were plotted on a plot correlating the absorbance of the sample with concentration. The results were expressed as milligrams of gallic acid equivalent (mg GAE)/g of dry weight of plant extract. The standard curve was obtained using gallic acid as reference (50-1000 mg/L, R 2 = 0.9923). All assays were developed in triplicate.

Determination of total flavonoids
The total flavonoid content was measured for the samples with significant values for the total phenolic compounds, by the colorimetric method by metallic complexation [20]. A standard solution and the extract and fraction solutions (1 mg/mL) in ethanol 99% were prepared, and 0.2 mL of aluminum chloride 2.5% alcoholic solution and 3.80 mL of ethanol. The solutions could stand at room temperature for 30 min. The absorbance of each solution was obtained at 408 nm in a spectrophotometer (quimis®) against the blank (all solvents except the sample). The total flavonoids were expressed as milligrams of catechin equivalents per gram of sample (mg EC/g), using a standard curve with catechin as reference (2.5-20 μg/mL, R 2 = 0.9937). All assays were carried out in triplicate.

Determination of total antioxidant capacity (TAC)
The Total Antioxidant Capacity was determined by the phosphomolybdenum method [21]. A volume of 0.1 mL of sample solutions (1 mg/mL) were added to 1 mL of reagent solution (sulfuric acid 600 mmol/L, sodium phosphate 28 mmol/L and ammonium molybdate 4 mmol/L). The tubes were incubated at 95 °C for 90 min, and then the absorbance was measured at 695 nm against the blank. Ascorbic acid was used as reference and the Total Antioxidant Capacity was expressed as equivalents of ascorbic acid. The TAC (%) was calculated using the following formula: where A s is the absorbance of the sample, A b is the absorbance of the blank, and A aa is the absorbance of ascorbic acid. All assays were carried out in triplicate.

Inhibition of 2,2-azino-bis-(3 ethylbenzothiazoline)-6-sulfonic acid (ABTS + ) radical
The inhibition of ABTS + radical (Sigma®) by the samples was assayed according to a method previously described [22]. The ABTS + solution 7 mmol/L was prepared by adding potassium persulfate 140 mmol/L and incubating the mixture away from light at room temperature for 12-16 h (time required for radical formation) before its use. The ABTS + solution was standardized with the dilution with ethanol to an absorbance of 0.7 (± 0.02) at 732 nm. The absorbance of the samples was measured using 30 µL of the sample solutions (1 mg/mL), added to 3 mL of the standardized ABTS + solution 7 mmol/L, at different time points (6,15,30,45,60 and 120 min) at 734 nm. The inhibition of the oxidation was calculated and plotted as a function of reference antioxidant concentration (Trolox) and expressed as Trolox Equivalent Antioxidant Capacity (TEAC, µmol/L).

DPPH free radical scavenging assay
The free radical scavenging activity was measured using the 2,2-diphenyl-1-picrylhydrazil (DPPH) assay with adaptations [23]. Samples were diluted in methanol to an initial concentration of 5 mg/mL, followed by serial dilution to 0.078 mg/mL. Then, 40 µL of each dilution was transferred to the 96 well plate and 250 µL of the 0.008% DPPH methanolic solution was added. The absorbance of each sample was measured at 517 nm in a spectrophotometer, 25 min after the addition of DPPH solution to the sample solutions. The Antioxidant Activity (AA) was calculated using the following formula: AA% = [(A c − A s )/A c ] × 100, where A c is the absorbance of the control, and A s is absorbance of the sample. All assays were carried out in triplicate.

In vitro photoprotective activity
The photoprotective activity was evaluated spectrophotometrically by the method of diluted solutions. Samples were dried in an oven at 40 °C for 60 min, and then dilutions were prepared (100 mg/L). A spectrophotometer (quimis®) was used, with quartz cuvettes with 1 cm optical path for the acquisition of the spectra and ethanol as blank. For maximum absorption wavelength (λmax) determination, spectrophotometric scanning of the crude extract was performed at wavelengths between 260 and 400 nm, with intervals of 5 nm. Calculations of the Sun Protection Factor (SPF) were made considering the intervals λ determined using the following formula: SPF = CF × AA × EE × SA, where CF is Correction Factor, AA is Amount of absorbance at 290-320 nm, Erythemogenic Effect of Radiation (λ), and SA is Spectrophotometric reading of sample absorbance (λ). The values of EE×I are constant and previously determined [24,25]. The synthetic filter benzophenone-3 (10 mg/L) and the standard flavonoid quercetin (10 mg/L) were used as positive controls.
The chromatograph was coupled to an Amazon SL ion trap mass spectrometer (Bruker Daltonics®), equipped with an electrospray ionizer and ion trap analyzer, under the following conditions: 3500 V capillary; 500 V end plate; nebulizer 60 psi; gas flow 10.0 L/ min and gas temperature 330 °C.
The results were analyzed using the GNPS website on-line database (Global Natural Product Social Molecular Networking) (GNPS, 2017). The data obtained in the chromatograph coupled to the mass spectrometer were converted to the mzXML format directly into the software Data Analysis 4.2 (Bruker Daltonics®), and submitted to dereplication analysis at GNPS website, and the substances were considered as identified in the sample if the mass spectra obtained at least six combining ions and cosine score above 0.5 [26]. Molecular formulas and classifications of substances were obtained from PubChem [27].

Preparation of photoprotective formulations
The base gel used was composed of Carbopol® 940 (Table 1). This base was prepared by dispersing the gelling agent (Carbopol®) in water with methylparaben preservative, along with a humidifying agent (propylene glycol). Thereafter, it could stand for 24 h to facilitate preparation of the gel in the dispersion of Carbopol® in water. Subsequently, the alkalizing agent (triethanolamine) was added and mixed until a pH range of 5.0-5.5 was obtained obtaining a clear gel.
From the prepared gel base, the chemical filter and Hm-CEE were incorporated in different concentrations ( Table 2). The chemical filter used was a water-soluble UVA/B filter composed of benzophenone-3 (Fagron®). The formulations were evaluated for photoprotective activities with adaptations in the concentration of the sample solution to 1 mg/mL, using the gel base as white, and antioxidant activity.
The SPF of the prepared formulations was determined using the previously described methodology, with adjustments in concentration of the sample solution to 1 mg/mL [24].
The formulations were submitted to physicochemical quality control tests, as appearance, color, odor, pH, consistency by extensibility and centrifugation resistance test.
The appearance, homogeneity, and organoleptic characteristics were evaluated by macroscopic analyses. The pH value (MS Tecnopon®, model mPA-210, Brazil) was determined by inserting the electrode directly into the aqueous dilution 1:10 (w/v) of the samples. The determination of consistency by extensibility was performed as proposed by [28], using 0.3 g of the sample placed between two glass plates, 10 × 20 cm and 0.5 cm thick, laid on a graph paper. The diameters of the formulations were measured after the addition of weights, every three minutes, on the top plate. The average extensibility was calculated in cm 2 by multiplying the square of the diameter by π/4. Centrifugation test was performed 24 h after preparation of the formulations at 3000 rpm (Fanen, model 206 BL, Brazil) for 30 min at room temperature.
For the preliminary stability study, the freezing/ defrosting cycle method was used [29]. The formulations were submitted to physicochemical tests (appearance, color, odor, pH, consistency by extensibility and centrifugation resistance test), before (T0) and after

Total phenolic compounds and total flavonoids
The total phenolic content of the crude extract and fractions is presented in Table 3. The highest content of phenolic compounds was found in Hm-CEE, followed by the ethyl acetate fraction and the values found in the present study is lower to that found in another study with the bark of this species [31]. The content of flavonoid compounds found were significant in this study, and these results can indicate that flavonoids are the major phenolic compounds present in the extract and ethyl acetate fraction of H. martiana. The difference between the results found in this study and the previous values reported in the literature can be justified by the different extraction methods, and the volumes and dilutions in the methods for the determination of phenolic compounds and flavonoids used [31].
Flavonoids were described as the major compounds of the crude extract and the ethyl acetate fraction of H. martiana previously [14,32]. Flavonoids have, among other biological activities, a characteristic antioxidant activity, which is related to their capacity to eliminate free radicals donating electrons or hydrogen atoms or cations of metal chelate compounds [33]. The complex chemical structure of flavonoids, as well as the diversity of their molecules, makes the structure-activity relationship more complicated than that of phenolic acids. Some of the structural characteristics and the nature of the substitutions in rings B and C can determine the antioxidant and photoprotective activity of flavonoids. The degree of hydroxylation and the positions of the hydroxyl groups on ring B, in particular a ring B ortho-dihydroxy structure in the positions 3' and 4' (known as the 'catechol' group), results in increased activity as greater radical stability is conferred by relocation [33]. Dihydroxyflavonoids that present the catechol group in ring B have already been described in H. martiana. Astilbin and taxifolin were previously described as substances related to the bioactivities of the species [7,14,32,34]. Other important compounds found in the species include engelitin, eucrifin and daucosterol [14,34]. Table 4 presents the antioxidant activity of the crude extract and fractions of H. martiana in vitro. These data demonstrate the high antioxidant capacity (TEAC), analyzed by the ABTS + radical inhibition method, with 83.75 ± 11.96% and 98.98 ± 1.06% for crude extract and the ethyl acetate fraction, respectively. The ethyl acetate fraction had the highest total antioxidant capacity (phosphomolybdenum method), with 85.97 ± 6.30%. The crude extract showed the highest scavenging activity in vitro in the the DPPH radical assay, with 91.4%. It was observed that the fractionation potentiated the antioxidant power in the ABTS + assay and the phosphomolybdenum method and was supported  by the DPPH radical scavenging assay. The significant antioxidant activity of the crude extract and the ethyl acetate fraction points to the antioxidant potential of the species being studied.

In vitro SPF determination
The in vitro photoprotective effect of the crude extract was determined by the Mansur method [24] and a significant absorption in the UVB/UVA regions was observed, in the spectrophotometric scanning for the crude extract and the positive controls, quercetin and the synthetic filter, suggesting a possible photoprotective activity (Figure 1). Aiming the total protection of the skin, which is not achieved by several photoprotective formulations containing only chemical synthetic sunscreens, plant extracts rich in antioxidant compounds are being widely used. This can be explained by the presence of phenolic compounds, which can provide protection against UV rays and neutralize free radicals after sun exposure. For this reason, studies have investigated antioxidant substances that absorb ultraviolet radiation in the UVA range (320-400 nm) and UVB range (290-320 nm), and thus can be used as natural sunscreen. The application of the photoprotective potential of natural products in sunscreens formulations is a trend [3,35,36], including polyphenols, especially flavonoids [10,35,37]. Some studies have presented the photoprotective activity of this class of chemical compounds, pointing to the beneficial potential of natural substances in photoprotective and cosmetic formulations [35][36][37][38][39][40][41].
Due to their photochemical properties, phenolic compounds such as flavonoids are great candidates for natural photoprotective and antioxidant substances. As Figure 1 presents, the photoprotective potential of the H. martiana extracts showed relevant values. When compared to the positive control (quercetin and ben-zophenone3), the UV spectra of the crude extract of H. martiana evidenced an important absorption in the UVB/UVA regions, suggesting a possible photoprotective activity.
Probably due to the high concentrations of phenolic compounds and expressive antioxidant capacity, the crude extract presented significant values for SPF, with 12.43 ± 1.25 in the concentration of 100 mg/L, value like the SPF presented for the flavonoid standard quercetin, which showed an SPF of 12.12 ± 0.02 in the concentration of 10 mg/L, while the synthetic filter benzophenone-3 obtained 8.45 ± 0.06. These values can show the photochemical potential for the  extract, and these results were above the levels required for sunscreens in Brazil, according to the Brazilian National Health Surveillance Agency [42], which requires that the minimum SPF value of a photoprotective formulation should be 6.0. These values were also higher than that recommended by the United States Food and Drug Administration (FDA), which considers as a sunscreen a formulation with an FPS value greater than 2.0 [43]. Therefore, these samples presented adequate values for the future development of photoprotectors, with an important antioxidant activity. The content of phenolic compounds was correlated to antioxidant activity (DPPH radical sequestration) and SPF, and the Pearson (ρ) and R 2 coefficients were calculated. The results indicate the strong correlation between the antioxidant activity and the total phenolic compounds content of the analyzed samples (R 2 = 0.995; ρ = 0.997). Several studies point to a strong relationship between the presence of phenolic compounds and antioxidant activity in medicinal plants and fruits [44,45]. A strong correlation between the phenolic compounds content and the photoprotective activity (SPF) was also presented in the analyzed samples (R 2 = 0.954; ρ = 0.977), important data for the future development of photoprotectors from plant extracts that present phenolic compounds as chemical markers, as is the case of the species in study.

Characterization of flavonoids by HPLC-MS-ESI-IT
For a more in-depth study on the chemical composition of H. martiana, a metabolomic study by an HPLC-MS-ESI-IT analysis was performed to identify the main flavonoids of the species. Metabolomic studies are characterized by the separation of complex matrix components, using chromatographic and spectroscopic methods, as well as bioactivity studies. Hyphenated techniques stand out for this type of study, based on the comparison of the information obtained with databases, resulting in the detection of compounds already known or even in the structural elucidation of new compounds. Several natural compounds have been identified using the metabolomic study as flavonoid glycosides, isoflavonoids and flavonoid derivatives [26,34,46,47].
The flavonoid profiling was developed by HPLC-MS method using the GNPS website as an online database, and 18 known flavonoids were detected (Figure 2, Table 5), among two flavanonols, six flavonols, four isoflavones, five flavones and one flavan-3-ol.
All compounds identified showed biological activities in the literature, including antioxidant and photoprotective activities. The dihydroflavonoid astilbin was previously detected, which was previously identified as the major component of Hymenaea martiana [14,34]. This flavonoid has several pharmacological activities already reported, as anti-inflammatory [14], anti-ischemic [48], antinociceptive and antiedematogenic [32] and antioxidant [49].
Among the flavonoids identified in the ethyl acetate fraction, only quercetin has photoprotective activity reported in the literature [10]. It also has antioxidant activity and anti-inflammatory activity reported [50]. This flavonoid was tested in this study and presented important absorption in the UVA/UVB region, and significant photoprotection (SPF of 12.12 ± 0.02) in a low concentration (10 mg/L). These data show the importance of the identification of this flavonoid in H. martiana.
From the flavonoids identified in this study, only astilbin, taxifolin [14,34] and quercitrin [34] have been previously described in the species. Therefore, this is the first report of the identification of all other flavonoids described in Table 5, evidencing the contribution of this work for the phytochemical study of Hymenaea martiana.

Preparation of photoprotective formulations
The significance of these results supports the antioxidant and photoprotective potential of H. martiana. Since no patents were found with photoprotective formulations with the genus Hymenaea or with the species studied in the available data, the results obtained in this study support the development of a novel formulation with photoprotective action from the barks of Hymenaea martiana.
Four test formulations were obtained using the Carbopol® gel base, the UVAB chemical filter and the Hm-EEB. The formulations presented visual aspects and homogeneity suitable for cosmetic formulations before and after the freezing/defrosting cycles.
The formulations had pH values between 5.5 and 6.0 (Figure 3) after the manipulation and after the thermal stress, values compatible with cutaneous pH (Isaac et al. 2008). In addition, the formulations did not present any changes or phase separations after the centrifugation test over the entire period of this study (T0 and T12). The extensibility of the formulations was calculated (Figure 3), showing important data about the efficacy and sensory aspects. A photoprotective gel should have a suitable extensibility, contributing for the formation of a film on the skin. In this way, the formulation can guarantee the specified Sun Protection Factor (SPF). The results presented by the proposed formulations are above the values found for emollients used in commercial emulsions [28], demonstrating that the gels developed with the extract of H. martiana achieved an adequate extensibility for photoprotective formulations.
Thus, the results for the quality control and preliminary stability suggest that the formulations showed adequate quality control properties, even after thermal stress.
The antioxidant and photoprotective activities of the formulations were evaluated ( Table 6). The results showed that the formulation F1, which contains only the chemical UVAB filter, did not present significant antioxidant activity. The formulation F2, composed of 5% of Hm-BSE, presented 77.55 ± 0.02% AS (DPPH method), 26.60 ± 1.90% TEAC (ABTS method) and 15.00 ± 0.55% TAA (Phosphomolybdenum method), demonstrating that the replacement of the chemical filter by crude extract interfered with the antioxidant activity, causing a significant increase. The F3 formulation, composed of 5% of the UVAB chemical filter and 5% of the Hm-BSE, presented 29.52 ± 0.11% AS, 4.60 ± 0.10% TEAC and 6.55 ± 0.09% TAA, demonstrating lower values in relation to the formulation containing only the crude extract. The F4 formulation, composed  of 10% of the UVAB chemical filter and 10% of the crude extract, presented 57.88 ± 0.10% AS, 34.60 ± 1.20% TEAC and 12.92 ± 0.12% TAA, evidencing that the increase in the concentration of the sunscreens increased the antioxidant activity of the formulation F4 in relation to the formulation F3. The results show that the gel with photoprotective activity containing only the chemical filter UVAB (formulation F1), which is already commercialized, did not present antioxidant activity in the tested methods, and that the addition of the crude extract under study greatly increased this activity. The addition of the crude extract, therefore, may provide an advantage for the formulations. However, it can be observed that the addition of the UVAB chemical filters and the crude extract at 5% concentration (F3 formulation) caused a dropin the antioxidant activity. This result may suggest a chemical interaction between the chemical filter and the crude extract, which could be justified by the pH of the crude extract of H. martiana bark, which was considered mildly acidic in a previous study [63]. The acidic pH could interfere with the electrons in the chemical structure of the solar filters, causing the product to absorb UV radiation at different wavelengths [63]. It is noteworthy that this interference was not found in photoprotective activity.
On the other hand, increasing the amount of the crude extract to 10% resulted in an increase in both antioxidant activity and photoprotective activity, indicating that this concentration positively affected the activities evaluated. Thus, pH monitoring in preliminary stability studies is more important for monitoring this possible interaction [30,64]. Therefore, the results presented for the antioxidant activity of the formulations tested may add greater value to the development of the photoprotective formulation, since it may provide a decrease in the use of synthetic additives with antioxidant action in the formulation, which could bring fewer undesirable effects [2,64].
The evaluation of the photoprotective activity of the formulations tested is shown in Figure 4. The formulation containing only the crude extract presented an FPS value greater than 6.0, demonstrating the photoprotective potential of the extract under study. The formulations containing the chemical filter and the crude extract (formulations F3 and F4) showed a considerable increase in the FPS values, in relation to the formulations containing the filters tested alone (formulations F1 and F2), which may suggest a synergistic effect between the chemical filter and the crude extract in the photoprotective activity. . evaluation of ph (a) and extensibility (b) of the photoprotective formulations (F1-F4) before (t0) and after (t12) the preliminary stability study. Results are expressed as mean values ± standard deviation, where a (p < 0.05 t0 versus t12) and B (p < 0.05 t0 versus t12), according to Student's t-test. The formulation F4 presented the best result for the photoprotective activity, with significant antioxidant activity. It can be considered a promising product, since the addition of natural sunscreens to the chemical filters is considered an alternative to increase the safety and efficacy of photoprotectors [2,10,41].

Conclusions
The results presented in this study showed that the crude extract of H. martiana contains phenolic compounds, mainly the flavonoids, major class of the chemical composition of the extract. Several flavonoids were identified in the species, and the flavonoids astilbin, taxifolin and quercitrin were identified in the extract, corroborating previous studies. The knowledge about the antioxidant and photoprotective activity can add therapeutic and cosmetic value to this species, with a strongly positive correlation between the phenolic compounds and the evaluated activities. Formulations containing the crude extract were prepared, presenting relevant antioxidant and photoprotective activity. The results showed a synergistic effect between the crude extract and benzophenone-3, bringing promising results for the development of a formulation with photoprotective action. The results obtained in this study up to the present moment show the relevant antioxidant and photoprotective activity of the crude extract, demonstrating the cosmetic potential of Hymenaea martiana, thus justifying the development of medicinal or cosmetic formulations associated with oxidative stress and skin care.