Spondias purpurea L. Stem Bark Extract: Antioxidant and in vitro Photoprotective Activities

Plant phenolic extracts are widely recognized as an important source of natural antioxidant substances and potential compounds for cosmetic formulations. This study aimed to evaluate the chemical profile, photoprotective and antioxidant activities of stem bark extract of Spondias purpurea L. (ciriguela) and its application in photoprotective formulations. Thirty phenolic constituents were annotated by ultra-performance liquid chromatography coupled with an electrospray ionization quadrupole time-of-flight mass spectrometry in mode negative (UPLC-QTOF-MS). The stem bark extract antioxidant and chelation potential, expressed in half maximal inhibitory concentration (IC50), showed 6.25 and 352.22 μg mL, respectively. The phenolic extract was used as an active ingredient in six sunscreen formulations, with concentrations ranging from 0.2 to 10%. The ultraviolet (UV) protection properties of the formulations were evaluated by sun protection factor (SPF) values obtained in 0.2 mg mL (0.495 to 2.27) and 2.0 mg mL (2.29 to 15.87). The SPF value for the extract (14.37 and 26.16) was high, but there was a reduction in the base formulation. However, these results suggested that the bioactive extracted of stem bark of Spondias purpurea L. has interesting potential to reduce the damage caused by UV radiation and may be utilized as an active ingredient in a sunscreen formulation.


Introduction
Skin neoplasms are one of the highest incidences worldwide form of cancer and globally represent 7.35% of all cancer cases in 2018, according to Ferlay et al. 1 Melanoma and non-melanoma type represented 1.04 million and 28.7 thousand of cases in all world, respectively. The main cause of skin cancer is prolonged exposure to solar ultraviolet (UV) radiation. 2 Prolonged exposure to UV radiation causes physical changes to the skin, at the tissue or cellular level. Besides, it has a high carcinogenic potential with direct action on both deoxyribonucleic acid (DNA) and proteins; and indirectly, it induces the formation of several reactive oxygen species (ROS). 3,4 ROS can induce lipid peroxidation, mitochondrial damage, and changes in the structure of nucleic acids, proteins, and genes. 5 Furthermore, skin exposed to UV radiation is susceptible to the appearance of photoallergies, erythema, accelerated skin photoaging, and the development of skin neoplasms. 2,4,6 The daily application of sunscreen products is highly recommended for photoaging prevention, sunburns, and the appearance of skin cancer. 7 Most photoprotection filter formulations are composed of chemical agents, organic or inorganic, which absorb, filter, disperse, and reflect the radiation UV. 6,8 However, these conventional formulations can be photounstable, reducing their effectiveness and generating by-products that can cause phototoxicity and photoallergic reactions. 9,10 Many studies 2,3 have demonstrated the photoprotective action of polyphenolic compounds due to their broad spectrum of UV absorption and antioxidant properties that protect cells from oxidative stress. Phenolic compounds are secondary metabolites that have relevant antioxidant properties related to their chemical structure, such as conjugation potential, the number of aromatic rings, and the position of hydroxyl and other functional groups. These compounds are multifunctional antioxidants that can break the chain reaction (free radical scavengers) and to chelate metal ions such as iron and copper, inhibiting the oxidation of low-density lipoproteins. 5,6,11 Research in cosmetic and phytocosmetic formulations, using phenolic extracts obtained from plants, have shown UV photoprotection potential, erythema suppression, powerful free radical removal effect, among other synergistic properties. 2,6,8 Anacardiaceae family is rich in secondary metabolites such as phenolic compounds with interesting biological activities. 12,13 Spondias purpurea L. is a medium-sized tree of the Anacardiaceae family, and their fruits (ciriguela) and other aerial parts generally have many polyphenolic compounds, such as flavonoids, tannins, anthocyanidins, catechins, epicatechins, as well as other non-volatile and volatile compounds. 14,15 Some studies have reported the chemical profile of fruits 12 and the photoprotective and antioxidant action of phenolic extracts of Spondias purpurea L. fruits peel. 4 However, little is known about the chemical compounds present in the stem bark of this species.
In the present study, innovative sunscreen formulations containing stem bark phenolic extract of Spondias purpurea L. were prepared, aiming to reduce the synthetic organic filter concentration without compromising the photoprotective efficacy. The hypothesis that this phenolic extract could act as constituents of photoprotective formulations was evaluated through their in vitro antioxidant and photoprotective action and the identification of the presence of polyphenol compounds. Besides, formulations containing different content of Spondias purpurea L. stem bark extract and formulation with just the commercial filter were investigated to assess their photoprotective activity and synergistic action. It is important to highlight that, at the best of our knowledge, there is no other work about the development of the sunscreen products containing Spondias purpurea L. stem bark extract.

Plant materials
Stem bark samples of Spondias purpurea L. were collected in the municipality of Ararendá, Ceará, Brazil (4°45'18.0"S 40°49'41.3"W) and the use of these species was registered in the Brazilian National System of Management of Genetic Heritage and Associated Traditional Knowledge (SisGen) by registration number A53C3CE. The specimen was deposited in the Prisco Bezerra Herbarium (EAC) located at the Federal University of Ceará. Stem barks samples were dry at 20 °C for seven days and crushed into smaller sizes.

Stem bark extract (SBE)
The phenolics were extracted by macerating the dried material (each batch with 5 g of stem bark and 300 mL of 70% (v/v) ethanolic solution) under constant agitation (400 rpm for 48 h). The crude hydroethanolic extract was concentrated in a rotary evaporator at 45 °C and filtered through 0.28 μm filter paper and freeze-dried.

Total phenolic content (TPC)
The total phenolic was determined by the Folin-Ciocalteu method. 16 An aliquot (0.025 mL) of the hydroethanolic extract solution (1 mg mL −1 ) was diluted to 0.5 mL and mixed with 0.5 mL of Folin-Ciocalteu reagent, 1.0 mL of 20% m/v sodium carbonate (Na 2 CO 3 ), and 1.0 mL of distilled water. The mixture was vortexed and left to stand for 30 min at room temperature and out of the reach of light. Absorbance was then measured at 700 nm using the spectrophotometer (Genesys 10S, Thermo Scientific, USA). Gallic acid (10-50 µg mL −1 ) was used in the construction of the standard curve (y = 0.0156x + 0.0073, with coefficient of determination (r 2 ) equal to 0.9995). The values (triplicate) were expressed in terms of milligrams of gallic acid equivalents (mg GAE) per gram extract.

Total flavonoid content (TFC)
The total flavonoid content was determined according to the aluminum chloride colorimetric method described by Lin and Tang. 17 Briefly, aliquots of SBE solution (0.5 mL) was mixed with 1.5 mL of 95% ethanol, 0.1 mL of 10% m/v aluminum chloride hexahydrate (AlCl 3 .6H 2 O), 0.1 mL of 1 mol L −1 sodium acetate (CH 3 COONa), and 2.8 mL of deionized water. After 40 min at room temperature, the reaction mixture absorbance was measured at 415 nm against a deionized water blank on a spectrophotometer (Genesys 10S, Thermo Scientific, USA). Quercetin (0-100 µg mL −1 ) was used in the construction of the standard curve (y = 0.001x + 0.007, r 2 = 0.9988). The data (triplicate) were expressed as milligram quercetin equivalents (QE) 100 g −1 SBE.

DPPH• radical scavenging assay
The antioxidant activity of SBE was investigated by the DPPH • radical scavenging assay. The solution of SBE in ethanol was prepared at concentrations in the range of 1-250 µg mL −1 . 2.5 mL of each concentration were mixed with 1.0 mL of 0.3 mmol L −1 DPPH-ethanol solution, at room temperature, and in the dark for 30 min. Afterward, the absorbance was measured at 518 nm in a UV-Vis spectrophotometer (Genesys 10S, Thermo Scientific, USA). The blank solution was composed of ethanol. The SBE antioxidant activity was evaluated in comparison with ascorbic acid and expressed as half maximal inhibitory concentration (IC 50 ) value. The ability of the test sample to scavenge the DPPH • radical was calculated using the following equation 1: where A c was the absorbance of the control and A s was the absorbance of the sample with DPPH-ethanol solution.

Ferrous ion chelation (FIC) assay
FIC assay was carried out by the method of Chew et al. 18 with some modifications. In this order, 1 mL of 0.1 mmol L −1 ferrous sulfate (FeSO 4 ) was mixed, 1 mL of the extract solution at concentrations 5, 10, 25, 50, 125, 250 μg mL −1 and 1 mL of 0.25 mmol L −1 ferrozine (3-(2-pyridyl) 5,6-diphenyl-1,2,4-triazine-p-p'-disulfonic acid). The tubes were vortexed for 1 min. After 10 min, readings were performed on a UV-Vis spectrophotometer (Genesys 10S, Thermo Scientific, USA) at 562 nm. The entire experiment was carried out in triplicate. The same method was performed with the EDTA as a positive control for further analysis and comparison of results. The results were expressed as ferrous ion chelation ability (%) (equation 2) and IC 50 . (2) where A c was the absorbance of the control and A s was the absorbance of the sample with ferrozine-FeSO 4 solution.
Analysis of chemical profile using ultra-performance liquid chromatography coupled with an electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-QTOF/ MS E ) A Waters Acquity ultra performance liquid chromatography (UPLC) equipment coupled to a quadrupole/TOF system (Waters, USA) was used to identify the chemical compounds present in SBE. The UPLC analysis conditions included the use of Waters Acquity UPLC BEH column (150 mm × 2.1 mm, 1.7 μm) at a fixed temperature of 40 °C. An exploratory gradient using water (A) and acetonitrile (B) (both with 0.1% formic acid) as mobile phases varying from 2 to 95% B (0-18 min), flow rate of 0.4 mL min −1 and injection volume of 5 μL was the method adopted. The chemical profiles were determined by coupling the Waters ACQUITY UPLC system to a QTOF mass spectrometer (Waters, Milford, USA) with an electrospray ionization interface (ESI) in negative ionization modes. The ESI (−) modes were in the range of 110-1200 Da, with a fixed temperature of 120 °C and a desolvation gas temperature of 350 °C. Leucine enkephalin was used as a lock mass. The mass spectrometry (MS) mode used Xevo G2-XS QTOF. The spectrometer operated with MS E centroid programming using a tension ramp from 20 to 40 V. 10 mg sample of hydroethanolic extract SBE were solubilized in 4 mL of 80% (v/v) solution of methanol and homogenized in a vortex. An aliquot of 1 mL was filtered in a 0.22 µm poly(tetrafluoroethene) (PTFE) filter and injected into the system. The acquisition and analysis of data were controlled using MassLynx 4.1 software (Milford, USA). 19 The compounds were annotated based on their exact mass and comparison with previously published data.
Determination of the spectrophotometric profile of aqueous extract of SBE and octyl methoxycinnamate Spectrophotometric profile was obtained according to a method adapted from Munhoz et al. 20 The dry extract was solubilized in water and the UVB filter in ethanol, both at a concentration of 0.2 and 2.0 mg mL −1 . Scanning between the wavelengths of 200 and 400 nm was carried out using a spectrophotometer (Genesys 10S, Thermo Scientific, USA) to verify the absorption in the ultraviolet regions A, B, and C. The blank solution was composed of water and ethanol, and the experiment was performed in triplicate.

Determination of in vitro sun protection factor (SPF)
The evaluation of the in vitro UVB photoprotection was carried out using the spectrophotometric method described by Mansur et al. 21 Mansur's method is simple and easily reproducible. The SPF determination is the correlation between the erythemogenic effect (EE) and the radiation intensity at each wavelength (I) ( Table 1). Subsequently, spectrophotometric scanning at wavelengths between 260-400 nm, with intervals of 5 nm was performed. The readings were performed using quartz cell (1 cm), and distilled water used as blank. Calculation of SPF was obtained according to the equation 3. (3) where CF: correction factor (equal to 10); EE (λ) : erythematous effect of radiation of wavelengths (λ); I (λ) : intensity of sunlight at wavelength (λ); Abs (λ) : spectrophotometric absorbance reading for preparing the solution at a wavelength (λ).

Statistical analysis
All experimental data were represented as mean ± standard deviation (SD). Statistical values were assessed by using the Minitab ® software 22 with a one-way analysis of variance (ANOVA), p < 0.05 expected to indicate statistical significance.

Results and Discussion
Phenolic, flavonoid content and antioxidant and chelating activity Polyphenols are secondary plant metabolites and have several potentially beneficial bioactive functions for humans, such as antimutagenic, anticarcinogenic, and antioxidant activity, already reported in the literature. 11 Several studies 4,23-26 have shown the high phenolic content in leaves, fruits, fruit peel, and stem bark of species of the Anacardiaceae family.
In this study, the total phenolics content determined in SBE was 523.26 ± 18.11 mg GAE g −1 (Table 3). This result suggests a higher content of phenolic compounds present in stem bark compared to other aerial parts of the Spondias purpurea L. such as fruit pulp and epicarp. 4,26 Silva et al. 4 found a lower total phenolic value, 28.68 ± 0.05 mg GAE g −1 , in the methanolic extract from the peel of Spondias purpurea L. fruit. Santos et al. 24 also found a high value for stem bark hydroethanolic extracts (403.26 mg GAE g −1 ) for Schinopsis brasiliensis bark extract, which also belongs to the Anacardiaceae family.
TFC of SBE is shown in Table 3. In general, the content of total flavonoids was low in SBE sample. This result suggests that flavonoids do not contribute strongly to antioxidant capacity of SBE. Villa-Hernández et al. 26 evaluated the flavonoid content in S. purpurea fruit (epicarp and pulp) and also found a lower value (2.4-4.7 µg g −1 ) when compared to the total phenolic content.
This study analyzed the antioxidant and chelating activity of Spondias purpurea L. stem bark hydroethanolic extract using the DPPH • and ferrous ion chelation method (Table 3 and Figure S1, Supplementary Information (SI) section). The SBE exhibited a scavenging activity of the DPPH • radical very efficient when compared with ascorbic acid, used in this work as standard. The results of inhibition of the radical can be seen in Table 3. One way to measure the inhibition capacity of the DPPH • radical is by calculating the IC 50 . The IC 50 is the necessary concentration of the antioxidant to reduce the DPPH • radical by 50% and decreasing values indicate higher antioxidant activity. The SBE test resulted in an IC 50 of 6.25 ± 0.52 µg mL −1 (Table 3), which is close to that found for ascorbic acid with an IC 50 of 9.85 ± 1.65 µg mL −1 (positive control). Also, the value of IC 50 for SBE was higher than that found by Silva et al. 4 with IC 50 of 27.11 µg mL −1 for peels of Spondias purpurea L. The high antioxidant activity presented can be linked to the high content of phenolic compounds present in SBE.
Chelating agents have a significant action as secondary antioxidants because they reduce the redox potential, thereby stabilizing the metal ion's oxidized form. 27 The chelating activity measured to the phenolic extract against Fe 2+ ions showed a lower chelation ability than EDTA (considered a powerful chelating agent). The results showed a chelation ability of 43.57 ± 0.95% and an IC 50 of 352.22 ± 15.01 µg mL −1 for SBE of Spondias purpurea L. stem bark. In general, lower metal chelations are observed for phenolic compounds than EDTA. 18,27 However, phenolic compounds are shown to be potential sources of natural chelating agents of metallic oxidants. Thus, SBE of Spondias purpurea L. may well be used as a source of the secondary antioxidant agent.

Characterization of phytochemical profiles
UPLC-QTOF-MS 2 analysis was carried out to annotated and compare the major chemical components present in the stem bark extract of Spondias purpurea L. 28 Table 4 shows 30 phytochemical compounds have been annotated, mainly phenolic compounds: phenolic acids, flavonoids (flavonols, flavan-3-ols, and flavanonols), tannins, and their derivatives, benzophenones, and others. The base peak chromatography in negative ionization mode is shown in Figure 1, and the structural proposed of each compound was inferred according to the detected m/z, error (ppm), calculated molecular formula, and obtained MS 2 fragment ions ( Table 4). The compounds were annotated and characterized by comparing MS and MS 2 spectra, together with the fragmentation mechanism, reference data related to family (Anacardiaceae), genus (Spondias), and species (S. purpurea L.), and the SciFinder, ScienceDirect, ChemSpider, PubChem databases, and Human Metabolome databases.

Simple phenolic acids and derivatives
In this study, five simple phenolic acids were annotated.

Flavonoid derivatives
Flavonoids and their glycosides are one of the major classes of compounds annotated in Spondias purpurea L. 12 A total of 11 flavonoid derivatives were detected in SBE sample.  38 Therefore, this compound with the molecular formula C 20 H 17 O 11 was annotated proposed to be quercetin pentoside. 12,35 Ampelopsin and its glycosylated derivatives were  Figure S2, SI section) related to aglycone fragmentation of ampelopsin. 29 Compound 28 (C 21 H 22 O 13 ) has been annotated assigned as ampelopsin glucoside. 29 MS 2 fragment of this compound has shown the characteristic aglycone ion at m/z 319.0241 (Figure 2). These substances have numerous pharmacological properties reported as antimicrobial, anti-inflammatory, antioxidant, and anticarcinogenic. 39 A total of six flavan-3-ols (flavonoid subgroup) and derivatives were annotated at peaks 9, 11, 19, 20 31 Correlating this information with data from the literature, it was possible to identify this compound digalloyl benzophenone derivative known as maclurin-3-C-(2,3-di-O-galloyl)-β-D-glucoside. 31,43 The benzophenones are intermediates in the biosynthetic pathway of xanthones, and their synthetic derivatives have been used in the composition of sunscreens due to their high capacity to absorb sunlight in the UVB and UVA spectrum region. These compounds are rarely reported in species outside the Clusiaceae and Moraceae family. 31,44 However, glycosylated benzophenones derivatives were annotated in leaves extracts, 45 peel and seed 31 of Mangifera indica L.
(Anacardiaceae family). This is the first study to report these compounds in the specie Spondias purpurea L. The various groups of polyphenols annotated corroborate with the antioxidant action observed for SBE, since many of these compounds are widely reported in the literature due to their antioxidant potential, which is strongly related to the chemical structure of these metabolites.
Determination of the spectrophotometric profile of SBE and octyl methoxycinnamate Figure S3 (SI section) shows the scanning profile of the dry extract and octyl methoxycinnamate (OM). OM is one of the most widely used organic UV filter, better known by its commercial name, octinoxate and has been used as positive control in previous studies. 49,50 It was possible to observe that the extract has a greater absorption in the UVC (100-280 nm) region and the synthetic filter in the UVB region (280-320 nm). However, SBE showed a peak around 280 nm, also demonstrating the energy absorption by the chromophores in the UVB region. According to Wróblewska et al. 10 extracts containing polyphenols such as flavonoids generally show peaks of absorbing ultraviolet between 240-280 nm. Silva et al. 4 correlated the photoprotection action in peels extracts from Spondias purpurea L. with its significant content of polyphenolic compounds. Thus, the absorption profile of the extracts of S. purpurea L. aerial parts demonstrated the possibility of its use as a sun protection agent.
In vitro UVB photoprotection of the SBE Table 5 shows the sun protection of the SBE and the octyl methoxycinnamate. The method described by Mansur et al. 21 is widely used to measure the in vitro SPF, what proves good correlation with in vivo tests because it relates the absorbance of the substance with the erythematous effect of radiation and the intensity of light at a wavelength from UVB region. 51 According to Yang et al. 52 the spectrophotometric screening method described by Mansur et al. 21 demonstrated a high level of reproducibility and reliability compared to the US FDA-guided in vivo SPF testing method. According to Resolution RDC 30/2012 53 published by ANVISA (National Health Surveillance Agency), the minimum value of sun protection factor (SPF) for products used by the Brazilian population is 6. The test showed a satisfactory photoprotective activity for the SBE (Table 5). It was possible to observe a better result of SPF than other works in the literature by spectrophotometric method (in vitro): Medina et al. 54 obtained a SPF of 1.44 for Byrsonima crassifolia leaf extract; de Carvalho et al. 55 showed SPF equal to 8 ± 0.31 for hydroethanolic extract of Pterodon emarginatus, fruit; and Lima-Saraiva et al. 56 found SPF of 6.27 ± 0.69 at 100 mg L −1 for hydroethanolic extract (aerial parts) of Schinopsis brasiliensis Engl. Therefore, S. purpurea L. stem bark extract could be considered a promising active ingredient, as it presented high sun protection factors at low dilution.
According to the chemical profile of SBE (Table 4) the main compounds that can be associated with the highest SPF values are flavonoids, benzophenones, and acid gallic derivatives. The flavonoids such as quercetin, myricetin, catechin, epicatechin, and their derivatives, have the potent antioxidant capacity, widely reported in the literature. 8,[57][58][59] These substances have conjugated bonds and chemical groups that absorb UV radiation at different wavelengths. 60 Adding to this, we still have benzophenones, which are classes of metabolites that have powerful antioxidant and photoprotective action. 31 Hwang et al. 61 showed the protective effects of acid gallic against photoaging caused by UVB radiation in vitro and in vivo. Besides, when associated with synthetic filters, these substances can help stabilize the characteristics of conventional UV filters during exposure to solar radiation. 60 Preparation of a cosmetic formulation and in vitro SPF determination SPF is one of the universally accepted parameters used to evaluate the efficacy of a sunscreen. 62 In the present study, formulations with SBE were produced in the concentrations 0.2, and 2.0 mg mL −1 , and SPF values of the formulations were described in Table 5. The test allowed verifying that the photoprotection activity is proportional to the amount of extract added to the formulation. The results obtained for the 0.2 mg mL −1 concentration was less significant, showing the dependence of the sun protection factor with the increase of the photoprotective agent concentration. Formulations containing only extract showed an increase in the SPF value as the incorporation of phenolic compounds of SBE of S. purpurea L. increased. Therefore, it was possible to observe that the concentration of 2.0 mg mL −1 extract was able to absorb the UVB radiation. The formulations FBE 2.5 , FBE 5 , and FBE 10 were found to possess SPF values of 6.89 ± 0.006, 10.15 ± 0.005, 10.84 ± 0.012, respectively. Silva et al. 4 in a study with Spondias purpurea L. fruits peels, obtained SPF of 0.70 and 0.79, respectively, in a formulation with 10% of hydroethanolic crude extract at a dilution of 2.0 mg mL −1 , demonstrating a superior photoprotective activity of the SBE at the same concentrations (Table 5).
However, the SPF values for formulations with extract were lower than the control (FBOM 7.5 ) and the analysis of the synergistic effect (FBE 10 OM 7.5 ). Thus, no synergistic effect was observed between the extract and the sunscreen. The SPF obtained was attributed to the absorptive and photoprotective characteristics of octyl methoxycinnamate. Although not contributing with the in vitro SPF, SBE presents phenolic compounds that could act preventing UV-induced damage by other mechanisms, e.g., capturing and inactivating ROS. 63 Many factors may interfere with the determination of SPF, as the type of emulsion, the concentration of the sunscreen, interactions of the formulation components, pH, rheological properties, or other factors that may increase or decrease the UVB absorption of the photoprotective substance. 64 Thus, the phenolic antioxidants of S. purpurea stem bark can be added in solar filters to complement UV filter photoprotection, reducing free radical damage generated after sun exposure and also to stabilize UV chemical filters. 10,50,62 The results of this research are in accordance with the increased interest in the use of plants to treat or prevent diseases and may contribute to the development and diversification of new sunscreen products by two different mechanisms: absorption of UV radiation and antioxidant activity. 60

Conclusions
In this study, the stem bark extract of Spondias purpurea L., showed high potential as antioxidant and photoprotective agents, corroborating with the elevated value of phenolic content and chemical profile obtained by UPLC-QTOF-MS 2 . A total of thirty compounds, including five simple phenolic acids, five hydrolysable tannins, eleven flavonoids and derivatives, two benzophenones, and four simple acids glycosylated were annotated. However, three compounds could not be annotated. These results led to the preparation of sunscreen formulations, with the same plant extract, and in combination with commercial UV filter. The SBE individually showed higher SPF values. However, these formulations, containing a sunscreen base and SBE, showed slightly lower SPF values compared to the commercial UV filter formulation. These results demonstrate that there was no synergic action between components of formulations. Due to its rich composition in phenolic substances and its positive outcomes for antioxidant activity, SBE of Spondias purpurea L. can provide a promising source of photoprotective agents for new phytocosmetic formulations.

Supplementary Information
Supplementary information (Figures S1-S3) is available free of charge at http://jbcs.sbq.org.br as PDF file.