Abstract
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-MS2). The stem bark extract antioxidant and chelation potential, expressed in half maximal inhibitory concentration (IC50), showed 6.25 and 352.22 µg mL−1, 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−1 (0.495 to 2.27) and 2.0 mg mL−1 (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.
Keywords:
ciriguela; UPLC-QTOF-MS2 profile; sunscreen formulation; stem bark extract
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 Ferlayet al.11 Ferlay, J.; Colombet, M.; Soerjomataram, I.; Mathers, C.; Parkin, D. M.; Piñeros, M.; Znaor, A.; Bray, F.; Int. J. Cancer 2019, 144, 1941. 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.22 Lacatusu, I.; Arsenie, L. V.; Badea, G.; Popa, O.; Oprea, O.; Badea, N.; Ind. Crops Prod. 2018, 123, 424.
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.55 Bazylko, A.; Borzym, J.; Parzonko, A.; J. Photochem. Photobiol. , B 2015, 149, 189. Furthermore, skin exposed to UV radiation is susceptible to the appearance of photoallergies, erythema, accelerated skin photoaging, and the development of skin neoplasms.22 Lacatusu, I.; Arsenie, L. V.; Badea, G.; Popa, O.; Oprea, O.; Badea, N.; Ind. Crops Prod. 2018, 123, 424. ,44 Silva, R. V.; Costa, S. C. C.; Branco, C. R. C.; Branco, A.; Ind. Crops Prod. 2016, 83, 509. ,66 Duque, L.; Bravo, K.; Osorio, E.; Ind. Crops Prod. 2017, 97, 431.
The daily application of sunscreen products is highly recommended for photoaging prevention, sunburns, and the appearance of skin cancer.77 Lin, C.; Lee, M.; Chi, M.; Chen, C.; Lin, H.; ACS Omega 2019, 4, 1801. Most photoprotection filter formulations are composed of chemical agents, organic or inorganic, which absorb, filter, disperse, and reflect the radiation UV.66 Duque, L.; Bravo, K.; Osorio, E.; Ind. Crops Prod. 2017, 97, 431. ,88 Santhanam, R. K.; Akhtar, M. T.; Ahmad, S.; Abas, F.; Ismail, I. S.; Rukayadi, Y.; Shaari, K.; Ind. Crops Prod. 2017, 96, 165. However, these conventional formulations can be photounstable, reducing their effectiveness and generating by-products that can cause phototoxicity and photoallergic reactions.99 Silva, A. C. P.; Paiva, J. P.; Diniz, R. R.; Anjos, V. M.; Silva, A. B. S. M.; Pinto, A. V.; Santos, E. P.; Leitão, A. C.; Cabral, L. M.; Rodrigues, C. R.; Pádula, M.; Santos, B. A. M. C.; J. Photochem. Photobiol. , B 2015, 193, 162.,1010 Wróblewska, K. B.; Baby, A. R.; Guaratini, M. T. G.; Moreno, P. R. H.; Ind. Crops Prod. 2019, 130, 208.
Many studies22 Lacatusu, I.; Arsenie, L. V.; Badea, G.; Popa, O.; Oprea, O.; Badea, N.; Ind. Crops Prod. 2018, 123, 424. ,33 Dario, M. F.; Oliveira, F. F.; Marins, D. S. S.; Baby, A. R.; Velasco, M. V. R.; Löbenberg, R.; Bou-Chacra, N. A.; Ind. Crops Prod. 2018, 112, 305. 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.55 Bazylko, A.; Borzym, J.; Parzonko, A.; J. Photochem. Photobiol. , B 2015, 149, 189.,66 Duque, L.; Bravo, K.; Osorio, E.; Ind. Crops Prod. 2017, 97, 431. ,1111 Rasera, G. B.; Hilkner, M. H.; Alencar, S. M.; Castro, R. J. S.; Ind. Crops Prod. 2019, 135, 294.
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.22 Lacatusu, I.; Arsenie, L. V.; Badea, G.; Popa, O.; Oprea, O.; Badea, N.; Ind. Crops Prod. 2018, 123, 424. ,66 Duque, L.; Bravo, K.; Osorio, E.; Ind. Crops Prod. 2017, 97, 431. ,88 Santhanam, R. K.; Akhtar, M. T.; Ahmad, S.; Abas, F.; Ismail, I. S.; Rukayadi, Y.; Shaari, K.; Ind. Crops Prod. 2017, 96, 165. Anacardiaceae family is rich in secondary metabolites such as phenolic compounds with interesting biological activities.1212 Engels, C.; Gräter, D.; Esquivel, P.; Jiménez, V. M.; Gänzle, M. G.; Schieber, A.; Food Res. Int. 2012, 46, 557. ,1313 Muñoz-Ramírez, A.; Torrent-Farías, C.; Mascayano-Collado, C.; Urzúa-Moll, A.; Phytochemistry 2020, 174, 112359. Spondias purpureaL. 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.1414 Bicas, J. L.; Molina, G.; Dionísio, A. P.; Barros, F. F. C.; Wagner, R.; Maróstica, M. R.; Pastore, G. M.; Food Res. Int. 2011, 44, 1843.,1515 Vargas-Simón, G. In Exotic Fruits, 1st ed.; Rodrigues, S.; Silva, E. O.; Brito, E. S., eds.; Academic Press: Cambridge, USA, 2018. Some studies have reported the chemical profile of fruits1212 Engels, C.; Gräter, D.; Esquivel, P.; Jiménez, V. M.; Gänzle, M. G.; Schieber, A.; Food Res. Int. 2012, 46, 557. and the photoprotective and antioxidant action of phenolic extracts of Spondias purpureaL. fruits peel.44 Silva, R. V.; Costa, S. C. C.; Branco, C. R. C.; Branco, A.; Ind. Crops Prod. 2016, 83, 509. 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 purpureaL. 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 purpureaL. 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 purpureaL. stem bark extract.
Experimental
Chemical
Cetearyl alcohol, lecithin, cetearyl glycoside, octyl methoxycinnamate, and methylparaben were purchased from Mapric (São Paulo, Brazil). The ethylenediaminetetraacetic acid (EDTA) and isopropyl palmitate were purchased from Infinity Pharma (Campinas, Brazil). Propylene glycol and aminomethyl propanol were purchased from Hallstar (Chicago, USA) and Fragon (São Paulo, Brazil), respectively. Ascorbic acid and gallic acid were obtained from Sigma-Aldrich (Saint Louis, USA). Aluminum chloride hexahydrate and sodium acetate were purchased from Vetec (Duque de Caxias, Brazil) and Synth (Diadema, Brazil) respectively. The 2,2-diphenyl-1-picrylhydrazyl (DPPH), 3-(2-pyridyl) 5,6-diphenyl-1,2,4-triazine-p-p’-disulfonic acid (ferrozine), Folin-Ciocalteu reagent, sodium carbonate, ferrous sulfate, and all solvents used were purchased from Sigma-Aldrich (Saint Louis, USA). For all methods, high purity water by Milli-Q system (Bedford, USA) was used.
Plant materials
Stem bark samples of Spondias purpureaL. 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.1616 Singleton, V. L.; Rossi, J. A.; Am. J. Enol. Vitic. 1965, 16, 144. 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 (Na2CO3), 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 (, with coefficient of determination (r2) 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.1717 Lin, J.; Tang, C.; Food Chem. 2007, 101, 140. 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 (AlCl3.6H2O), 0.1 mL of 1 mol L−1 sodium acetate (CH3COONa), 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 (). 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 (IC50) value. The ability of the test sample to scavenge the DPPH• radical was calculated using the following equation 1:
where Ac was the absorbance of the control and As was the absorbance of the sample with DPPH-ethanol solution.
Ferrous ion chelation (FIC) assay
FIC assay was carried out by the method of Chewet al.1818 Chew, Y. L.; Lim, Y. Y.; Omar, M.; Khoo, K. S.; LWT-Food Sci. Technol. 2008, 41, 1067. with some modifications. In this order, 1 mL of 0.1 mmol L−1 ferrous sulfate (FeSO4) 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 IC50.
where Ac was the absorbance of the control and As was the absorbance of the sample with ferrozine-FeSO4 solution.
Analysis of chemical profile using ultra-performance liquid chromatography coupled with an electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MSE)
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 MSE 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).1919 Masslynx v.41 SCN719; Water Corporation, Milford, USA, 2009. 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 Munhozet al.2020 Munhoz, V. M.; Lonni, A. A. S. G.; Mello, J. C. P.; Lopes, G. C.; Rev. Cienc. Farm. Basica Apl. 2012, 33, 225. 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 Mansuret al.2121 Mansur, J. S.; Breder, M. N. R.; Mansur, M. C. A.; Azulay, R. D.; An. Bras. Dermatol. 1986, 61, 121. 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.
Correlation between the erythemogenic effect (EE) and the radiation intensity at each wavelength (I)21
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).
Preparation of a cosmetic formulation and in vitro SPF determination
Six formulations were prepared (Table 2): base formulation (FB), lotion and extract 0.2% (FBE0.2), 2.5% (FBE2.5), 5% (FBE5), 10% (FBE10); base formulation and octyl methoxycinnamate 7.5% (FBOM7.5); and base formulation, extract 10% and octyl methoxycinnamate 7.5% (FBE10OM7.5). The UVB photoprotection of the formulations was measured according to the previous description. A sample solution was obtained (0.2 and 2.0 mg mL−1) and FB was used as blank.
Statistical analysis
All experimental data were represented as mean ± standard deviation (SD). Statistical values were assessed by using the Minitab® software2222 Minitab v.19.2020.1; Minitab LCC, State College, Pennsylvania, USA, 2020. 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.1111 Rasera, G. B.; Hilkner, M. H.; Alencar, S. M.; Castro, R. J. S.; Ind. Crops Prod. 2019, 135, 294. Several studies44 Silva, R. V.; Costa, S. C. C.; Branco, C. R. C.; Branco, A.; Ind. Crops Prod. 2016, 83, 509. ,2323 Remila, S.; Atmani-Kilani, D.; Delemasure, S.; Connat, J. L.; Azib, L.; Richard, T.; Atmani, D.; Eur. J. Integr. Med. 2015, 7, 274.
24 Santos, C. C. S.; Masullo, M.; Cerulli, A.; Mari, A.; Estevam, C. D. S.; Pizza, C.; Piacente, S.; Phytochemistry 2017, 140, 45.
25 Tiburski, J. H.; Rosenthal, A.; Deliza, R.; Godoy, R. L. O.; Pacheco, S.; Food Res. Int. 2011, 44, 2326. -2626 Villa-Hernández, J. M.; Mendoza-Cardoso, G.; Mendoza-Espinoza, J. A.; Vela-Hinojosa, C.; León-Sánchez, F. D.; Rivera-Cabrera, F.; Alia-Tejacal, I.; Pérez-Flores, L. J.; J. Food Sci. 2017, 82, 2576. 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 purpureaL. such as fruit pulp and epicarp.44 Silva, R. V.; Costa, S. C. C.; Branco, C. R. C.; Branco, A.; Ind. Crops Prod. 2016, 83, 509. ,2626 Villa-Hernández, J. M.; Mendoza-Cardoso, G.; Mendoza-Espinoza, J. A.; Vela-Hinojosa, C.; León-Sánchez, F. D.; Rivera-Cabrera, F.; Alia-Tejacal, I.; Pérez-Flores, L. J.; J. Food Sci. 2017, 82, 2576. Silvaet 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 purpureaL. fruit. Santoset al.2424 Santos, C. C. S.; Masullo, M.; Cerulli, A.; Mari, A.; Estevam, C. D. S.; Pizza, C.; Piacente, S.; Phytochemistry 2017, 140, 45. 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-Hernándezet al.2626 Villa-Hernández, J. M.; Mendoza-Cardoso, G.; Mendoza-Espinoza, J. A.; Vela-Hinojosa, C.; León-Sánchez, F. D.; Rivera-Cabrera, F.; Alia-Tejacal, I.; Pérez-Flores, L. J.; J. Food Sci. 2017, 82, 2576. 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 purpureaL. 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 IC50. The IC50 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 IC50 of 6.25 ± 0.52 µg mL−1 (Table 3), which is close to that found for ascorbic acid with an IC50 of 9.85 ± 1.65 µg mL−1 (positive control). Also, the value of IC50 for SBE was higher than that found by Silvaet al.44 Silva, R. V.; Costa, S. C. C.; Branco, C. R. C.; Branco, A.; Ind. Crops Prod. 2016, 83, 509. with IC50 of 27.11 µg mL−1 for peels of Spondias purpureaL. 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.2727 Abeywickrama, G.; Debnath, S. C.; Ambigaipalan, P.; Shahidi, F.; J. Agric. Food Chem. 2016, 64, 9342. 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 IC50 of 352.22 ± 15.01 µg mL−1 for SBE of Spondias purpureaL. stem bark. In general, lower metal chelations are observed for phenolic compounds than EDTA.1818 Chew, Y. L.; Lim, Y. Y.; Omar, M.; Khoo, K. S.; LWT-Food Sci. Technol. 2008, 41, 1067.,2727 Abeywickrama, G.; Debnath, S. C.; Ambigaipalan, P.; Shahidi, F.; J. Agric. Food Chem. 2016, 64, 9342. However, phenolic compounds are shown to be potential sources of natural chelating agents of metallic oxidants. Thus, SBE of Spondias purpureaL. may well be used as a source of the secondary antioxidant agent.
Characterization of phytochemical profiles
UPLC-QTOF-MS22 Lacatusu, I.; Arsenie, L. V.; Badea, G.; Popa, O.; Oprea, O.; Badea, N.; Ind. Crops Prod. 2018, 123, 424. analysis was carried out to annotated and compare the major chemical components present in the stem bark extract of Spondias purpureaL.2828 Rodrigues, F. A. M.: Filmes e Revestimentos Ativos à Base de Amido e Bioativos de Spondias purpurea para Aplicação em Manga Minimamente Processada; MSc dissertation, Federal University of Ceará, Fortaleza, Brazil, 2018, available at http://www.repositorio.ufc.br/bitstream/riufc/39134/5/2018_dis_famrodrigues.pdf, accessed in April 2021.
http://www.repositorio.ufc.br/bitstream/...
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 MS22 Lacatusu, I.; Arsenie, L. V.; Badea, G.; Popa, O.; Oprea, O.; Badea, N.; Ind. Crops Prod. 2018, 123, 424. fragment ions (Table 4). The compounds were annotated and characterized by comparing MS and MS22 Lacatusu, I.; Arsenie, L. V.; Badea, G.; Popa, O.; Oprea, O.; Badea, N.; Ind. Crops Prod. 2018, 123, 424. 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.
The chromatogram obtained in the UPLC-QTOF-MS in negative mode (ESI−) of the stem bark extracts from the Spondias purpurea L.
Simple phenolic acids and derivatives
In this study, five simple phenolic acids were annotated. The peak 4 gave ion [M - H]− at m/z 169.0137 and a MS22 Lacatusu, I.; Arsenie, L. V.; Badea, G.; Popa, O.; Oprea, O.; Badea, N.; Ind. Crops Prod. 2018, 123, 424. fragment ion at m/z 125.0252, resulted from the cleavage of −CO2 group. These fragments are typical of gallic acid.1212 Engels, C.; Gräter, D.; Esquivel, P.; Jiménez, V. M.; Gänzle, M. G.; Schieber, A.; Food Res. Int. 2012, 46, 557. Peaks 16 ([M - H]− m/z 183.0293), 26 ([M - H]− m/z321.0249) and 27 ([M - H]− m/z 197.0452) were annotated as methyl gallate (C8H8O5), digallic acid (C14H10O9), and ethyl gallate (C9H10O5), respectively. The common MS22 Lacatusu, I.; Arsenie, L. V.; Badea, G.; Popa, O.; Oprea, O.; Badea, N.; Ind. Crops Prod. 2018, 123, 424. fragment ion at m/z 169.01 in methyl gallate and digallic acid resulting from the fragment of galloyl unit.2929 Abu-Reidah, I. M.; Ali-Shtayeh, M. S.; Jamous, R. M.; Arráez-Román, D.; Segura-Carretero, A.; Food Chem. 2015, 166, 179.
30 Castro, C. B.; Luz, L. R.; Guedes, J. A. C.; Porto, D. D.; Silva, M. F. S.; Silva, G. S.; Ribeiro, P. R. V.; Canuto, K. M.;Brito,E. S. ; Zampieri, D. S.; Pessoa, C. O.; Zocolo, G. J.; J. Braz. Chem. Soc. 2020, 31, 775.
31 Dorta, E.; González, M.; Lobo, M. G.; Sánchez-Moreno, C.; Ancos, B.; Food Res. Int. 2014, 57, 51. -3232 Erşan, S.; Üstündağ, Ö. G.; Carle, R.; Schweiggert, R. F.; J. Agric. Food Chem . 2016, 64, 5334. Peak 16 (methyl gallate) shown MS22 Lacatusu, I.; Arsenie, L. V.; Badea, G.; Popa, O.; Oprea, O.; Badea, N.; Ind. Crops Prod. 2018, 123, 424. fragment at m/z 124.0053 [M - H - CO2CH3]− and 168.0013 [M - H - CH3]−. In the ethyl gallate (peak 27) the loss of 28 Da to m/z 169.0132 was attributable to the neutral loss of ethylene.3131 Dorta, E.; González, M.; Lobo, M. G.; Sánchez-Moreno, C.; Ancos, B.; Food Res. Int. 2014, 57, 51. ,3333 Shaheen, N.; Lu, Y.; Geng, P.; Shao, Q.; Wei, Y.; J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2017, 1046, 211. Another simple phenolic (peak 8), associated with hexoside, was annotated as dihydroxybenzoic acid hexoside. The spectrum mass showed deprotonated ion m/z 315.0718 and MS(2 fragment 153.0193, related to the loss of monosaccharide unit (−162 Da).1212 Engels, C.; Gräter, D.; Esquivel, P.; Jiménez, V. M.; Gänzle, M. G.; Schieber, A.; Food Res. Int. 2012, 46, 557.
Hydrolysable tannins
Peaks 3, 5, 12, 18 and 25 were annotated as hydrolysable tannins. Peak 3 presented precursor ion [M - H]− at m/z 331.0663 and MS22 Lacatusu, I.; Arsenie, L. V.; Badea, G.; Popa, O.; Oprea, O.; Badea, N.; Ind. Crops Prod. 2018, 123, 424. spectrum observed the fragments ions at m/z 271.0386 [M - H - CO2CH3]−, and 169.0144 and 125.0292 related to the typical loss of galloyl and carboxylic group, respectively (Figure S2, SI section). This compound 3 was annotated as monogalloyl glucose according to previous work.1212 Engels, C.; Gräter, D.; Esquivel, P.; Jiménez, V. M.; Gänzle, M. G.; Schieber, A.; Food Res. Int. 2012, 46, 557. ,2424 Santos, C. C. S.; Masullo, M.; Cerulli, A.; Mari, A.; Estevam, C. D. S.; Pizza, C.; Piacente, S.; Phytochemistry 2017, 140, 45.,3131 Dorta, E.; González, M.; Lobo, M. G.; Sánchez-Moreno, C.; Ancos, B.; Food Res. Int. 2014, 57, 51. The peak 5 showed a deprotonated ion [M - H]− at m/z493.1192 and yielded fragment ions at m/z 331.0661 [M ?H ?162]− (due to the loss of monosaccharide unit), 241.0650 [M - H - 252]− and 271.0373 [M - H - 222]− caused by cross-ring fragmentations of glucose,3434 Muccilli, V.; Cardullo, N.; Spatafora, C.; Cunsolo, V.; Tringali, C.; Food Chem. 2017, 215, 50. and 169.0133 attributable to the loss of a glucosyl moiety. This compound was annotated as monogalloyl di-glucose (C19H26O15), according to the report of Cunhaet al.3535 Cunha, A. G.;Brito, E. S.; Moura, C. F. H.; Ribeiro, P. R. V.; Miranda, M. R. A.; J. Chromatogr. B: Anal. Technol. Biomed. Life Sci . 2017, 1051, 24. and Dortaet al.3131 Dorta, E.; González, M.; Lobo, M. G.; Sánchez-Moreno, C.; Ancos, B.; Food Res. Int. 2014, 57, 51. At peak 12, the precursor ion [M - H]− m/z483.0777 was observed and fragment ions at m/z 331.0714 [M - H - 162]−, 271.0532 [M - H - 212]−, 313.0634 [M - H - 170]− and 169.0132. The compound12 was annotated as digalloyl glucose.2929 Abu-Reidah, I. M.; Ali-Shtayeh, M. S.; Jamous, R. M.; Arráez-Román, D.; Segura-Carretero, A.; Food Chem. 2015, 166, 179.
30 Castro, C. B.; Luz, L. R.; Guedes, J. A. C.; Porto, D. D.; Silva, M. F. S.; Silva, G. S.; Ribeiro, P. R. V.; Canuto, K. M.;Brito,E. S. ; Zampieri, D. S.; Pessoa, C. O.; Zocolo, G. J.; J. Braz. Chem. Soc. 2020, 31, 775.
31 Dorta, E.; González, M.; Lobo, M. G.; Sánchez-Moreno, C.; Ancos, B.; Food Res. Int. 2014, 57, 51. -3232 Erşan, S.; Üstündağ, Ö. G.; Carle, R.; Schweiggert, R. F.; J. Agric. Food Chem . 2016, 64, 5334. ,3535 Cunha, A. G.;Brito, E. S.; Moura, C. F. H.; Ribeiro, P. R. V.; Miranda, M. R. A.; J. Chromatogr. B: Anal. Technol. Biomed. Life Sci . 2017, 1051, 24.
Peak 18 showed ion [M - H]− m/z 635.0892 and MS22 Lacatusu, I.; Arsenie, L. V.; Badea, G.; Popa, O.; Oprea, O.; Badea, N.; Ind. Crops Prod. 2018, 123, 424. fragments ions at m/z 483.0891 [M - H - 152]− (loss of a galloyl unit from a gallate ester), 465.0908 [M - H - 170]−, loss of a gallic acid) and 169.0121 (Figure 2). This fragmentation pattern for trigalloyl glucose was previously reported in the literature.2929 Abu-Reidah, I. M.; Ali-Shtayeh, M. S.; Jamous, R. M.; Arráez-Román, D.; Segura-Carretero, A.; Food Chem. 2015, 166, 179.,3434 Muccilli, V.; Cardullo, N.; Spatafora, C.; Cunsolo, V.; Tringali, C.; Food Chem. 2017, 215, 50. Another compound (peak25) was annotated as tetra-O-galloyl hexoside (C34H28O22) based in research of Abu-Reidahet al.2929 Abu-Reidah, I. M.; Ali-Shtayeh, M. S.; Jamous, R. M.; Arráez-Román, D.; Segura-Carretero, A.; Food Chem. 2015, 166, 179. and Muccilliet al.3434 Muccilli, V.; Cardullo, N.; Spatafora, C.; Cunsolo, V.; Tringali, C.; Food Chem. 2017, 215, 50. This peak 25 showed ion [M - H]−, m/z 787.0999 and MS22 Lacatusu, I.; Arsenie, L. V.; Badea, G.; Popa, O.; Oprea, O.; Badea, N.; Ind. Crops Prod. 2018, 123, 424. fragments in m/z 635.1156 [M - H - 152]−, deriving from the loss of a galloyl unit, and m/z 169.0118.3434 Muccilli, V.; Cardullo, N.; Spatafora, C.; Cunsolo, V.; Tringali, C.; Food Chem. 2017, 215, 50.
Chemical structures and representative fragmentation in the gas phase of some peak annotation compounds in the stem bark extract of Spondias purpurea L. (adapted from references 31, 34 and 37).
Flavonoid derivatives
Flavonoids and their glycosides are one of the major classes of compounds annotated in Spondias purpureaL.1212 Engels, C.; Gräter, D.; Esquivel, P.; Jiménez, V. M.; Gänzle, M. G.; Schieber, A.; Food Res. Int. 2012, 46, 557. A total of 11 flavonoid derivatives were detected in SBE sample.
Peak 6 ([M - H]− m/z 609.1454) was annotated as rutin (C27H30O16), showing a MS(2 fragments at m/z 301.0458 (quercetin unit) after the loss of two monosaccharide units.2929 Abu-Reidah, I. M.; Ali-Shtayeh, M. S.; Jamous, R. M.; Arráez-Román, D.; Segura-Carretero, A.; Food Chem. 2015, 166, 179. This compound has already been quantified in Spondias tuberosa leaves.3636 Silva, A. R. A.; Morais, S. M.; Marques, M. M. M.; Oliveira, D. F.; Barros, C. C.; Almeida, R. R.; Vieira, I. G. P.; Guedes, M. I. F.; Pharm. Biol. 2012, 50, 740. The hexoside derivative of myricetin was annotated at peak 15 ([M - H]− m/z479.0828) from the MS22 Lacatusu, I.; Arsenie, L. V.; Badea, G.; Popa, O.; Oprea, O.; Badea, N.; Ind. Crops Prod. 2018, 123, 424. fragmentation pattern reported in the literature.3535 Cunha, A. G.;Brito, E. S.; Moura, C. F. H.; Ribeiro, P. R. V.; Miranda, M. R. A.; J. Chromatogr. B: Anal. Technol. Biomed. Life Sci . 2017, 1051, 24.,3737 Li, Z.; Guo, H.; Xu, W.; Ge, J.; Li, X.; Alimu, M.; He, D.; J. Chromatogr. Sci. 2016, 54, 805. Compound 23 showed a MS22 Lacatusu, I.; Arsenie, L. V.; Badea, G.; Popa, O.; Oprea, O.; Badea, N.; Ind. Crops Prod. 2018, 123, 424. fragment ion detected at m/z 301.0613 [M - H - 308]− concerning to aglycone ion for quercetin, and m/z 271.0435 [M - H - 162]− which indicates the loss of the unit of monosaccharide.3838 Brito, A.; Ramirez, J. E.; Areche, C.; Sepúlveda, B.; Simirgiotis, M. J.; Molecules 2014, 19, 17400. Therefore, this compound with the molecular formula was annotated proposed to be quercetin pentoside.1212 Engels, C.; Gräter, D.; Esquivel, P.; Jiménez, V. M.; Gänzle, M. G.; Schieber, A.; Food Res. Int. 2012, 46, 557. ,3535 Cunha, A. G.;Brito, E. S.; Moura, C. F. H.; Ribeiro, P. R. V.; Miranda, M. R. A.; J. Chromatogr. B: Anal. Technol. Biomed. Life Sci . 2017, 1051, 24.
Ampelopsin and its glycosylated derivatives were annotated at peaks 30 ([M - H]− m/z 319.0533) and 28 ([M - H]− m/z 481.0990), respectively. Peak 30 (C15H12O8) showed MS22 Lacatusu, I.; Arsenie, L. V.; Badea, G.; Popa, O.; Oprea, O.; Badea, N.; Ind. Crops Prod. 2018, 123, 424. fragments at m/z 125.0441, 153.1123, 179.0817, and 193.0112 (Figure S2, SI section) related to aglycone fragmentation of ampelopsin.2929 Abu-Reidah, I. M.; Ali-Shtayeh, M. S.; Jamous, R. M.; Arráez-Román, D.; Segura-Carretero, A.; Food Chem. 2015, 166, 179. Compound 28 () has been annotated assigned as ampelopsin glucoside.2929 Abu-Reidah, I. M.; Ali-Shtayeh, M. S.; Jamous, R. M.; Arráez-Román, D.; Segura-Carretero, A.; Food Chem. 2015, 166, 179. MS22 Lacatusu, I.; Arsenie, L. V.; Badea, G.; Popa, O.; Oprea, O.; Badea, N.; Ind. Crops Prod. 2018, 123, 424. 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.3939 Woo, H.; Kanga, H.; Nguyena, T. T. H.; Kim, G.; Kim, Y.; Park, J.; Kime, D.; Cha, J.; Moo, Y.; Mam, S.; Xia, Y.; Kimura, A.; Kim, D.; Enzyme Microb. Technol. 2012, 51, 311.
A total of six flavan-3-ols (flavonoid subgroup) and derivatives were annotated at peaks 9, 11, 19, 20, 24, and 29. Peak 11 with precursor ion [M - H]− m/z 593.1296 was annotated as epicatechin-3,5-O-digallate (C29H22O14). This compound showed MS22 Lacatusu, I.; Arsenie, L. V.; Badea, G.; Popa, O.; Oprea, O.; Badea, N.; Ind. Crops Prod. 2018, 123, 424. fragmentation ions m/z 441.0632 [M - H - 152]−, and 289.0535 [M - H - 152]− resulted after the successive loss of galloyl unit (152 Da). This fragmentation pattern was previously described by Shokoet al.4040 Shoko, T.; Maharaj, V. J.; Naidoo, D.; Tselanyane, M.; Nthambeleni, R.; Khorombi, E.; Apostolides, Z.; BMC Complementary Altern. Med. 2018, 18, 54. for Sclerocarya birrea (Anacardiaceae family). Peak 29 with the molecular formula C29H22O15 and having the precursor ion at m/z 609.0882 was being annotated as epigallocatechin-3,5-O-digallate.4141 Wang, D.; Lu, J.; Miao, A.; Xie, Z.; Yang, D.; J. Food Compos. Anal. 2008, 21, 361. In the MS22 Lacatusu, I.; Arsenie, L. V.; Badea, G.; Popa, O.; Oprea, O.; Badea, N.; Ind. Crops Prod. 2018, 123, 424. spectrum, this compound produced ions fragment at m/z 125.0312, 169.0101, and 457.1022 [M - H - 152]− (Figure 2).
Compounds 9 (tR = 2.27 min) and 19 (tR = 3.54 min) had deprotonated ions [M - H]− at m/z 305.0654 and 305.0665, respectively. Based on QTOF-MS data, retention time, and the previous literature,3030 Castro, C. B.; Luz, L. R.; Guedes, J. A. C.; Porto, D. D.; Silva, M. F. S.; Silva, G. S.; Ribeiro, P. R. V.; Canuto, K. M.;Brito,E. S. ; Zampieri, D. S.; Pessoa, C. O.; Zocolo, G. J.; J. Braz. Chem. Soc. 2020, 31, 775. ,4040 Shoko, T.; Maharaj, V. J.; Naidoo, D.; Tselanyane, M.; Nthambeleni, R.; Khorombi, E.; Apostolides, Z.; BMC Complementary Altern. Med. 2018, 18, 54.,4141 Wang, D.; Lu, J.; Miao, A.; Xie, Z.; Yang, D.; J. Food Compos. Anal. 2008, 21, 361. these compounds have been annotated as gallocatechin isomers (C15H14O7). These compounds were suggested as gallocatechin (trans-isomer) and epigallocatechin (cis-isomer). Other isomers were found at peaks 20 ([M - H]− m/z 457.0778) and 24 ([M - H]− m/z 457.0768). These isomers were annotated as epigallocatechin-3-O-gallate and gallocatechin-3-O-gallate based on the calculated molecular formula (), MS(2 fragments, and retention times position (tR = 3.61 and 3.92 min) reported in the literature.4141 Wang, D.; Lu, J.; Miao, A.; Xie, Z.; Yang, D.; J. Food Compos. Anal. 2008, 21, 361.
Benzophenones
In this study, two metabolites were annotated from the benzophenones class (peaks 17 and 21). Peak 17 gave ion [M - H]− m/z 575.1016 and molecular formula C26H24O15. In the MS22 Lacatusu, I.; Arsenie, L. V.; Badea, G.; Popa, O.; Oprea, O.; Badea, N.; Ind. Crops Prod. 2018, 123, 424. spectrum was observed the ions fragment values of m/z 423.0760 [M - H - 152]−, 303.0494 [M - H - 272]− (referring to cross-ring cleavages of the glucose moiety), 285.0258 [M - H - 272 ? H2O]−, and neutral loss for the gallic acid unit at m/z 169.0069 (Figure 2). This compound was annotated as maclurin-3-C-(2-O-galloyl)-β-D-glucoside from the fragmentation pattern (Figure S2, SI section) reported in the literature.3131 Dorta, E.; González, M.; Lobo, M. G.; Sánchez-Moreno, C.; Ancos, B.; Food Res. Int. 2014, 57, 51. ,4242 Galvão, W. R. A.; Braz Filho, R.; Canuto, K. M.; Ribeiro, P. R. V.; Campos, A. R.; Moreira, A. C. O. M.; Silva, S. O.; Mesquita Filho, F. A.; Santos S. A. A. R.; Melo Jr., J. M. A.; Gonçalves, N. G. G.; Fonseca, S. G. C.; Bandeira, M. A. M.; J. Ethnopharmacol. 2018, 222, 177. The other compound was found at peak 21 that showed an ion [M - H]− at m/z 727.1132 and the molecular formula C33H28O19. The fragment ion of MS22 Lacatusu, I.; Arsenie, L. V.; Badea, G.; Popa, O.; Oprea, O.; Badea, N.; Ind. Crops Prod. 2018, 123, 424. spectrum was detected at m/z 575.1180 [M - H - 152]− caused by loss of galloyl unit. The resulting fragment m/z 575 produced product ions at m/z 407.0898 [M - H - 152 - 168]− indicating the loss of further gallic acid moiety.3131 Dorta, E.; González, M.; Lobo, M. G.; Sánchez-Moreno, C.; Ancos, B.; Food Res. Int. 2014, 57, 51. 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.3131 Dorta, E.; González, M.; Lobo, M. G.; Sánchez-Moreno, C.; Ancos, B.; Food Res. Int. 2014, 57, 51. ,4343 Barreto, J. C.; Trevisan, M. T. S.; Hull, W. E.; Erben, G.;Brito,E. S.; Pfundstein, B.; Owen, R. W.; J. Agric. Food Chem . 2008, 56, 5599.
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.3131 Dorta, E.; González, M.; Lobo, M. G.; Sánchez-Moreno, C.; Ancos, B.; Food Res. Int. 2014, 57, 51. ,4444 Marto, J.; Gouveia, L. F.; Chiari, B. G.; Paiva, A.; Isaac, V.; Pinto, P.; Ribeiro, H. M.; Ind. Crops Prod. 2016, 80, 93. However, glycosylated benzophenones derivatives were annotated in leaves extracts,4545 Pan, J.; Yi, X.; Zhang, S.; Cheng, J.; Wang, Y.; Liu, C.; He, X.; Ind. Crops Prod. 2018, 111, 400. peel and seed3131 Dorta, E.; González, M.; Lobo, M. G.; Sánchez-Moreno, C.; Ancos, B.; Food Res. Int. 2014, 57, 51. of Mangifera indicaL. (Anacardiaceae family). This is the first study to report these compounds in the specie Spondias purpureaL.
Other metabolites
Peak 1 had a precursor ion [M - H]− at m/z 207.0141 and showed MS22 Lacatusu, I.; Arsenie, L. V.; Badea, G.; Popa, O.; Oprea, O.; Badea, N.; Ind. Crops Prod. 2018, 123, 424. fragments at m/z 189.0022 [M - H - H2O]− and 127.0039 [M - H - 2H2O - CO2]−. The compound was annotated as hydroxycitric acid.4646 Pandey, R.; Chandra, P.; Kumar, B.; Srivastva, M.; Aravind, A. P. A.; Shameer, P. S.; Rameshkumar, K. B.; Ind. Crops Prod. 2015, 77, 861. Citric acid (C6H8O7) is proposed for compound 2 with ion [M - H]− m/z 191.0193, and its MS22 Lacatusu, I.; Arsenie, L. V.; Badea, G.; Popa, O.; Oprea, O.; Badea, N.; Ind. Crops Prod. 2018, 123, 424. spectrum gave a fragments ion at m/z 111.0097 [M - H - 2H2O - CO2]− and 85.0295 [M - H - 106]−.3030 Castro, C. B.; Luz, L. R.; Guedes, J. A. C.; Porto, D. D.; Silva, M. F. S.; Silva, G. S.; Ribeiro, P. R. V.; Canuto, K. M.;Brito,E. S. ; Zampieri, D. S.; Pessoa, C. O.; Zocolo, G. J.; J. Braz. Chem. Soc. 2020, 31, 775. ,3131 Dorta, E.; González, M.; Lobo, M. G.; Sánchez-Moreno, C.; Ancos, B.; Food Res. Int. 2014, 57, 51. ,4747 Carvalho-Silva, L. B.; Dionísio, A. P.; Pereira, A. C. S.; Wurlitzer, N. J.; Brito, E. S.; Bataglion, G. A.; Brasil, I. M.; Eberlin, M. N.; Liu, R. H.; LWT-Food Sci. Technol. 2014, 59, 1319. ,4848 Luz, L. R.; Porto, D. D.; Castro, C. B.; Silva, M. F. S.; Alves Filho, E. G.; Canuto, K. M.;Brito, E. S.; Becker, H.; Pessoa, C. O.; Zocolo, G. J.; J. Chromatogr. B: Anal. Technol. Biomed. Life Sci . 2018, 1099, 97.
Peak 14 presented the deprotonated ion [M - H]− m/z453.1033 with molecular formula C20H22O12 with MS22 Lacatusu, I.; Arsenie, L. V.; Badea, G.; Popa, O.; Oprea, O.; Badea, N.; Ind. Crops Prod. 2018, 123, 424. fragment at m/z 313.0612 [M - H - 140]− referring to a C-glycoside pattern fragmentation was annotated as hydroxymethoxyphenyl-O-(O-galloyl)-hexose. This compound was previously annotated by Abu-Reidahet al.2929 Abu-Reidah, I. M.; Ali-Shtayeh, M. S.; Jamous, R. M.; Arráez-Román, D.; Segura-Carretero, A.; Food Chem. 2015, 166, 179.
The compound 4,9-dihydroxypropiophenone-9-O-(6’-O-galloyl)-β-D-glucopyranoside was annotated in peak 22 with ion [M - H]− m/z 479.1194 and MS(2 fragment ions at m/z 331.1218 [M - H - 148]−, 271.0358 [M - H - 208]− and 169.0130 according to Santoset al.2424 Santos, C. C. S.; Masullo, M.; Cerulli, A.; Mari, A.; Estevam, C. D. S.; Pizza, C.; Piacente, S.; Phytochemistry 2017, 140, 45. Peaks 7 (C8H12O8), 10 (C27H30O15), and 13 (C37H43O22) with precursor ions [M - H]− at m/z 235.0453, 593.1508, 761.1355, respectively, are not annotated compounds. These compounds showed similar fragment ions in m/z 125 and 169, indicating the characteristic fragments of galloyl and gallic acid units. Thus, these compounds are possibly derived from gallic acids.
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.4949 Almeida, W. A. S.; Antunes, A. S.; Penido, R. G.; Correa, H. S. G.; Nascimento, A. M.; Andrade, Â. L.; Santos, V. R.; Cazati, T.; Amparo, T. R.; Souza, G. H. B.; Freitas, K. M.; Santos, O. D. H.; Sousa, L. R. D.; Santos, V. M. R.; Rev. Bras. Farmacogn. 2019, 29, 373.,5050 Kanlayavattanakul, M.; Kasikawatana, N.; Lourith, N.; J. Cosmet. Sci. 2016, 67, 167. 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 Wróblewskaet al.1010 Wróblewska, K. B.; Baby, A. R.; Guaratini, M. T. G.; Moreno, P. R. H.; Ind. Crops Prod. 2019, 130, 208. extracts containing polyphenols such as flavonoids generally show peaks of absorbing ultraviolet between 240-280 nm. Silvaet al.44 Silva, R. V.; Costa, S. C. C.; Branco, C. R. C.; Branco, A.; Ind. Crops Prod. 2016, 83, 509. correlated the photoprotection action in peels extracts from Spondias purpureaL. 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 Mansuret al.2121 Mansur, J. S.; Breder, M. N. R.; Mansur, M. C. A.; Azulay, R. D.; An. Bras. Dermatol. 1986, 61, 121. 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.5151 Violante, I. M. P.; Souza, I. M.; Venturini, C. L.; Ramalho, A. F. S.; Santos, R. A. N.; Ferrari, M.; Rev. Bras. Farmacogn. 2009, 19, 452. According to Yanget al.5252 Yang, S. I.; Liu, S.; Brooks, G. J.; Lanctot, Y.; Gruber, J. V.; J. Cosmet. Dermatol. 2018, 17, 518. 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 30/20125353 Agência Nacional de Vigilância Sanitária (ANVISA): Resolução RDC No. 30, de 1 de junho de 2012, Aprova o Regulamento Técnico Mercosul sobre Protetores Solares em Cosméticos e dá outras Providências, available at http://bvsms.saude.gov.br/bvs/saudelegis/anvisa/2012/rdc0030_01_06_2012.html, accessed in May 2021.
http://bvsms.saude.gov.br/bvs/saudelegis...
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): Medinaet al.5454 Medina, C. O.; Louchard, B. O.; Gonçalves, T.; Rev. Cienc. Farm. Basica Apl. 2015, 36, 391. obtained a SPF of 1.44 for Byrsonima crassifolia leaf extract; de Carvalhoet al.5555 de Carvalho, W. L.; Moreira, L. C.; Valadares, M. C.; Diniz, D. G.; Bara, M. T.; Pharmacogn. Mag. 2019, 15, 176. showed SPF equal to 8 ± 0.31 for hydroethanolic extract of Pterodon emarginatus, fruit; and Lima-Saraivaet al.5656 Lima-Saraiva, S. R. G.; Oliveira, F. G. S.; Oliveira Jr., R. G.; Araújo, C. S.; Oliveira, A. P.; Pacheco, A. G. M.; Rolim, L. A.; Amorim, E. L. C.; César, F. C. S.; Almeida, J. R. G. S.; Hindawi 2017, 1713921. 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.
Sun protection factor of SBE, octyl methoxycinnamate (OM) and formulations containing SBE and OM
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.88 Santhanam, R. K.; Akhtar, M. T.; Ahmad, S.; Abas, F.; Ismail, I. S.; Rukayadi, Y.; Shaari, K.; Ind. Crops Prod. 2017, 96, 165. ,5757 Baloglu, M. C.; Llorent-Martínez, E. J.; Aumeeruddy, M. Z.; Mahomoodally, M. F.; Altunoglu, Y. C.; Ustaoglu, B.; Ocal, M.; Bene, S. G. K.; Sinan, K. I.; Zengin, G.; Ind. Crops Prod. 2019, 134, 33.
58 Guitard, R.; Paul, J. F.; Nardello-Rataj, V.; Aubry, J. M.; Food Chem. 2016, 213, 284.-5959 Lesjak, M.; Beara, I.; Simin, N.; Pintać, D.; Majkić, T.; Bekvalac, T.; Orčić, D.; Mimica-Dukić, N.; J. Funct. Foods 2018, 40, 68. These substances have conjugated bonds and chemical groups that absorb UV radiation at different wavelengths.6060 Mota, M. D.; Boa Morte, A. N.; Silva, L. C. R. C.; Chinalia, F. A.; J. Photochem. Photobiol. , B 2020, 205, 111837. Adding to this, we still have benzophenones, which are classes of metabolites that have powerful antioxidant and photoprotective action.3131 Dorta, E.; González, M.; Lobo, M. G.; Sánchez-Moreno, C.; Ancos, B.; Food Res. Int. 2014, 57, 51. Hwanget al.6161 Hwang, E.; Park, S.; Lee, H. J.; Lee, T. Y.; Sun, Z.; Yi, T. H.; Phytother. Res. 2014, 28, 1778. 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.6060 Mota, M. D.; Boa Morte, A. N.; Silva, L. C. R. C.; Chinalia, F. A.; J. Photochem. Photobiol. , B 2020, 205, 111837.
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.6262 Chiari-Andréo, B. G.; Almeida, F. B.; Yamasaki, P. R.; Santos, J. L.; Corrêa, M. A.; Chin, C. M.; Isaac, V. L. B.; Rodriguésia 2020, 71, e03072018. 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 FBE2.5, FBE5, and FBE10 were found to possess SPF values of 6.89 ± 0.006, 10.15 ± 0.005, 10.84 ± 0.012, respectively. Silvaet al.44 Silva, R. V.; Costa, S. C. C.; Branco, C. R. C.; Branco, A.; Ind. Crops Prod. 2016, 83, 509. in a study with Spondias purpureaL. 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 (FBOM7.5) and the analysis of the synergistic effect (FBE10OM7.5). 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.6363 Mansur, M. C. P. P. R.; Leitão, S. G.; Cerqueira-Coutinho, C.; Vermelho, A. B.; Silva, R. S.; Presgrave, O. A. F.; Leitão, A. A. L.; Leitão, G. G.; Ricci-Júnior, E.; Santos, E. P.; Rev. Bras. Farmacogn. 2016, 26, 251. 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.6464 Dutra, E. A.; Oliveira, D. A. G. C.; Kedor-Hackmann, E. R. M.; Santoro, M. I. R. M.; Braz. J. Pharm. Sci. 2004, 40, 381. 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.1010 Wróblewska, K. B.; Baby, A. R.; Guaratini, M. T. G.; Moreno, P. R. H.; Ind. Crops Prod. 2019, 130, 208. ,5050 Kanlayavattanakul, M.; Kasikawatana, N.; Lourith, N.; J. Cosmet. Sci. 2016, 67, 167.,6262 Chiari-Andréo, B. G.; Almeida, F. B.; Yamasaki, P. R.; Santos, J. L.; Corrêa, M. A.; Chin, C. M.; Isaac, V. L. B.; Rodriguésia 2020, 71, e03072018.
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.6060 Mota, M. D.; Boa Morte, A. N.; Silva, L. C. R. C.; Chinalia, F. A.; J. Photochem. Photobiol. , B 2020, 205, 111837.
Conclusions
In this study, the stem bark extract of Spondias purpureaL., showed high potential as antioxidant and photoprotective agents, corroborating with the elevated value of phenolic content and chemical profile obtained by UPLC-QTOF-MS22 Lacatusu, I.; Arsenie, L. V.; Badea, G.; Popa, O.; Oprea, O.; Badea, N.; Ind. Crops Prod. 2018, 123, 424. . 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 purpureaL. 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.
Acknowledgments
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) - Finance Code 001 (PROEX 23038.000509/2020-82). The authors thank Embrapa Tropical Agroindustry. The corresponding author thanks CNPq for the research grant (N.M.P.S.R-No. 307837/2017-3) and Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP) for the support.
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Publication Dates
-
Publication in this collection
01 Oct 2021 -
Date of issue
Oct 2021
History
-
Received
23 Oct 2020 -
Accepted
07 June 2021