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

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.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.^12,13Spondias 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.^14,15 Some studies have reported the chemical profile of fruits¹² and the photoprotective and antioxidant action of phenolic extracts of Spondias purpureaL. 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 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.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 (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 ($\left(\mathit{y}=0.0156\mathit{x}+0.0073\right.$, 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.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 (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 ($\left(\mathit{y}=0.001\mathit{x}+0.007,{\mathit{r}}^2=0.9988\right)$). 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:

(1) ${\mathrm{DPPH}}^{•}\mathrm{scavenging}\mathrm{rat}\left(\%\right)=\frac{\left(A_c-A_s\right)}{A_c}\times 100$

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 Chewet al.¹⁸ 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.

(2) $\mathrm{FIC}\left(\%\right)=\frac{\left(A_c-A_s\right)}{A_c}\times 100$

where A_c was the absorbance of the control and A_s 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/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 Munhozet 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 Mansuret 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) $\mathrm{SPF}=\mathrm{CF}\times {{\sum }_{290}^{320}\mathrm{EE}}_{\left(\lambda \right)}\times I_{\left(\lambda \right)}\times {\mathrm{abs}}_{\left(\lambda \right)}$

Table 1
Correlation between the erythemogenic effect (EE) and the radiation intensity at each wavelength (I)²¹

λ / nm	EE(λ ) × I(λ )
290	0.0150
295	0.0817
300	0.2874
305	0.3278
310	0.1864
315	0.0839
320	0.0180

• EE_{(λ )}: erythematous effect of radiation of wavelengths (λ ); I_{(λ )}: intensity of sunlight at wavelength (λ ).

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% (FBE_0.2), 2.5% (FBE_2.5), 5% (FBE₅), 10% (FBE₁₀); base formulation and octyl methoxycinnamate 7.5% (FBOM_7.5); and base formulation, extract 10% and octyl methoxycinnamate 7.5% (FBE₁₀OM_7.5). 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.

Table 2
Composition of formulations prepared for the photoprotective assay

Component	FB	FBE0.2	FBE2.5	FBE5	FBE10	FBOM7.5	FBE10OM7.5
Cetearyl alcohol / wt.%	0.5	0.5	0.5	0.5	0.5	0.5	0.5
Lecithin and cetearyl glycoside / wt.%	1.0	1.0	1.0	1.0	1.0	1.0	1.0
Isopropyl palmitate / wt.%	3.0	3.0	3.0	3.0	3.0	3.0	3.0
Propylene glycol / wt.%	2.0	2.0	2.0	2.02	2.0	2.0	2.0
Methylparaben / wt.%	0.05	0.05	0.05	0.05	0.05	0.05	0.05
Propylparaben / wt.%	0.015	0.015	0.015	0.015	0.015	0.015	0.015
Octyl methoxycinnamate / wt.%	-	-	-	-	-	7.5	7.5
SBEof S. purpurea / wt.%	-	0.2	2.5	5.0	10	-	10
EDTA / wt.%	0.05	0.05	0.05	0.05	0.05	0.05	0.05
Aminomethyl propanol / wt.%	q.s.	q.s.	q.s.	q.s.	q.s.	q.s.	q.s.
Water / wt.%	q.s.	q.s.	q.s.	q.s.	q.s.	q.s.	q.s.

• FB: base formulation; FBE: base formulation with extract; OM: octyl methoxycinnamate; SBE: stem bark extract; EDTA: ethylenediaminetetraacetic acid; q.s.: quantum sufficit.

Statistical analysis

All experimental data were represented as mean ± standard deviation (SD). Statistical values were assessed by using the Minitab^® software²² 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 purpureaL. such as fruit pulp and epicarp.^4,26 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.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.

Table 3
Polyphenols, flavonoids and antioxidant activity in Spondias purpurea stem bark extracts

Sample	TPC / (mg GAE g-1)	TFC / (mg QE 100 g-1 SBE)	DPPH•assaya			FICassaya
			IC50 / (µg mL-1)	RSA / %		IC50 / (µg mL-1)	CA / %
SBE	523.26 ± 18.11	40.2 ± 4.21	6.25 ± 0.52	96.57 ± 0.9		352.22 ± 15.01	43.57 ± 0.95

• ^a
  The test concentrations ranged from 5 to 250 µg mL^-1; data are described as mean ± standard deviation (SD), n = 3. TPC: total phenolics content; TFC: total flavonoids content; GAE: gallic acid equivalent; QE: quercetin equivalent; SBE: stem bark extract; IC₅₀: half maximal inhibitory concentration; DPPH: 2,2-diphenyl-1-picrylhydrazyl; FIC: ferrous ion chelation; RSA: radical scavenging activity; CA: chelation ability.

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.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 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.⁴ 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.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 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.^18,27 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-MS² analysis was carried out to annotated and compare the major chemical components present in the stem bark extract of Spondias purpureaL.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 MS² fragment ions (Table 4). The compounds were annotated and characterized by comparing MS and MS² 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.

Table 4
Compounds annotated in SBE using UPLC-QTOF-MS^E in negative mode

Peak No.	tR / min	[M - H]- m/z	Product ions (MS2)	Molecular formula	Error / ppm	Proposed compound	Reference
1	0.89	207.0141	189.0022; 127.0039	$C_6{H}_8{O}_8$	0.0	hydroxycitric acid	46
2	1.27	191.0193	85.0295; 111.0097	$C_6{H}_8{O}_7$	0.5	citric acid	31,47,48
3	1.53	331.0663	271.0451; 169.0137; 125.0224	$C_{13}{H}_{16}{O}_{10}$	-0.6	monogalloyl glucose	12,31
4	1.76	169.0137	125.0252	$C_7{H}_6{O}_5$	0.0	gallic acid	12,30
5	1.84	493.1192	169.0133; 241.0650; 271.0373; 331.0661	$C_{19}{H}_{26}{O}_{15}$	-0.2	monogalloyl di-glucose	31,35
6	2.05	609.1454	151.0441; 301.0458	$C_{27}{H}_{30}{O}_{16}$	-0.3	rutin	29
7	2.07	235.0453	-	$C_8{H}_{12}{O}_8$	-0.4	n.i.	-
8	2.23	315.0718	153.0193	$C_{13}{H}_{16}{O}_9$	0.0	dihydroxybenzoic acid hexoside	12
9	2.27	305.0654	219.0548; 137.0332; 125.0261	$C_{15}{H}_{14}{O}_7$	-0.3	gallocatechin	30,40,41
10	2.31	593.1508	241.0158; 305.0642	$C_{27}{H}_{30}{O}_{15}$	0.3	n.i.	-
11	2.35	593.1296	441.0632; 289.0535; 169.0150; 125.0174	$C_{29}{H}_{22}{O}_{14}$	0.2	epicatechin-3,5-O-digallate	40
12	2.43	483.0777	169.0132; 271.0532; 313.0634; 331.0714	$C_{20}{H}_{20}{O}_{14}$	0.4	digalloyl glucose	29
13	2.81	761.1355	-	$C_{37}{H}_{30}{O}_{18}$	0.1	n.i.	-
14	3.10	453.1033	169.0136; 179.0011; 313.0612	$C_{20}{H}_{22}{O}_{12}$	0.0	hydroxymethoxyphenyl-O-(O-galloyl)-hexose	29
15	3.22	479.0828	271.0215; 287.0614; 317.0499	$C_{21}{H}_{20}{O}_{13}$	0.4	myricetin 3-O-hexoside	32,35
16	3.23	183.0293	124.0053; 168.0013	$C_8{H}_8{O}_5$	0.0	methyl gallate	29,31
17	3.29	575.1016	169.0069; 285.0258; 303.0494; 423.0760	$C_{26}{H}_{24}{O}_{15}$	1.4	maclurin-3-C-(2-O-galloyl)-b-D-glucoside	31,42
18	3.44	635.0892	483.0891; 465. 0908; 169.0121	$C_{27}{H}_{24}{O}_{18}$	1.3	trigalloyl hexoside	29,34
19	3.54	305.0665	219.0776; 137.0319; 125.0247	$C_{15}{H}_{14}{O}_7$	1.3	epigallocatechin	30,40,41
20	3.61	457.0778	125.0260; 169.0125; 305.0726	$C_{22}{H}_{18}{O}_{11}$	-0.7	epigallocatechin-3-O-gallate	41
21	3.71	727.1132	169.0152; 303.0419; 407.0898; 575.1180	$C_{33}{H}_{28}{O}_{19}$	-2.1	maclurin-3-C-(2.3-di-O-galloyl)-b-D-glucoside	31,43
22	3.76	479.1194	169.0130; 271.0358; 331.1218	$C_{22}{H}_{24}{O}_{12}$	0.8	4.9-dihydroxypropiophenone-9-O-(6'-O-galloyl)-b-D-glucopyranoside	24
23	3.81	433.0762	151.0077; 271.0435; 301.0613	$C_{20}{H}_{17}{O}_{11}$	-2.1	quercetin pentoside	12,35
24	3.92	457.0768	125.0381; 169.0128; 305.0873	$C_{22}{H}_{18}{O}_{11}$	-0.7	gallocatechin-3-O-gallate	41
25	3.98	787.0999	169.0118; 635.1156	$C_{34}{H}_{28}{O}_{22}$	0.6	tetra-O-galloyl hexoside	29,34
26	4.15	321.0249	125.0275; 169.0159	$C_{14}{H}_{10}{O}_9$	0.6	digallic acid	29,32
27	4.24	197.0452	169.0132	$C_9{H}_{10}{O}_5$	1.0	ethyl gallate	31,33
28	4.44	481.0990	301. 0513; 319.0241	$C_{21}{H}_{22}{O}_{13}$	1.7	ampelopsin glucoside	29
29	4.52	609.0882	125.0312; 169.0101; 305.0735; 457.1022	$C_{29}{H}_{22}{O}_{15}$	-1.6	epigallocatechin-3,5-O-digallate	41
30	4.97	319.0533	125.0441; 153.1123; 179.0817; 193.0112	$C_{15}{H}_{12}{O}_8$	-0.3	ampelopsin	29

• t_R: retention time; MS: mass spectrometry; n.i.: not identified.

Figure 1
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 MS² fragment ion at m/z 125.0252, resulted from the cleavage of −CO₂ group. These fragments are typical of gallic acid.¹² 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 MS² fragment ion at m/z 169.01 in methyl gallate and digallic acid resulting from the fragment of galloyl unit.^29-32 Peak 16 (methyl gallate) shown MS² 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.^31,33 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).¹²

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 MS² 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.^12,24,31 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,³⁴ 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.³⁵ and Dortaet al.³¹ 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.^29-32,35

Peak 18 showed ion [M - H]⁻ m/z 635.0892 and MS² 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.^29,34 Another compound (peak25) was annotated as tetra-O-galloyl hexoside (C₃₄H₂₈O₂₂) based in research of Abu-Reidahet al.²⁹ and Muccilliet al.³⁴ This peak 25 showed ion [M - H]⁻, m/z 787.0999 and MS² fragments in m/z 635.1156 [M - H - 152]⁻, deriving from the loss of a galloyl unit, and m/z 169.0118.³⁴

Figure 2
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.¹² 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.²⁹ This compound has already been quantified in Spondias tuberosa leaves.³⁶ The hexoside derivative of myricetin was annotated at peak 15 ([M - H]⁻ m/z479.0828) from the MS² fragmentation pattern reported in the literature.^35,37 Compound 23 showed a MS² 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.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 annotated at peaks 30 ([M - H]⁻ m/z 319.0533) and 28 ([M - H]⁻ m/z 481.0990), respectively. Peak 30 (C₁₅H₁₂O₈) showed MS² fragments at m/z 125.0441, 153.1123, 179.0817, and 193.0112 (Figure S2, SI section) related to aglycone fragmentation of ampelopsin.²⁹ Compound 28 ($C_{29}{H}_{22}{O}_{15}$) has been annotated assigned as ampelopsin glucoside.²⁹ MS² 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, 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 MS² 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.⁴⁰ 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.⁴¹ In the MS² spectrum, this compound produced ions fragment at m/z 125.0312, 169.0101, and 457.1022 [M - H - 152]⁻ (Figure 2).

Compounds 9 (t_R = 2.27 min) and 19 (t_R = 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,^30,40,41 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 ($\left(C_{20}{H}_{18}{O}_{11}\right)$), MS⁽² fragments, and retention times position (t_R = 3.61 and 3.92 min) reported in the literature.⁴¹

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 MS² 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.^31,42 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 MS² 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.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 seed³¹ 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 MS² fragments at m/z 189.0022 [M - H - H₂O]⁻ and 127.0039 [M - H - 2H₂O - CO₂]⁻. The compound was annotated as hydroxycitric acid.⁴⁶ Citric acid (C₆H₈O₇) is proposed for compound 2 with ion [M - H]⁻ m/z 191.0193, and its MS² spectrum gave a fragments ion at m/z 111.0097 [M - H - 2H₂O - CO₂]⁻ and 85.0295 [M - H - 106]⁻.^30,31,47,48

Peak 14 presented the deprotonated ion [M - H]⁻ m/z453.1033 with molecular formula C₂₀H₂₂O₁₂ with MS² 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.²⁹

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.²⁴ 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.^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 Wróblewskaet al.¹⁰ extracts containing polyphenols such as flavonoids generally show peaks of absorbing ultraviolet between 240-280 nm. Silvaet al.⁴ 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.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 Yanget 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 30/2012⁵³ 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.⁵⁴ obtained a SPF of 1.44 for Byrsonima crassifolia leaf extract; de Carvalhoet al.⁵⁵ showed SPF equal to 8 ± 0.31 for hydroethanolic extract of Pterodon emarginatus, fruit; and Lima-Saraivaet 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.

Table 5
Sun protection factor of SBE, octyl methoxycinnamate (OM) and formulations containing SBE and OM

Sample/formulations	SPF (0.2 mg mL-1)	SPF (2.0 mg mL-1)
SBE	14.37 ± 0.009a	26.16 ± 0.013a
OM	14.19 ± 0.010b	21.84 ± 0.038b
FBE0.2	0.495 ± 0.001h	2.29 ± 0.098h
FBE2.5	0.702 ± 0.002g	6.89 ± 0.006g
FBE5	1.39 ± 0.008f	10.15 ± 0.005f
FBE10	1.58 ± 0.006e	10.84 ± 0.012e
FBOM7.5	2.15 ± 0.012d	17.16 ± 0.005d
FBE10OM7.5	2.27 ± 0.004c	15.87 ± 0.010c

• Values expressed are means ± standard deviation; n = 3. Different letters in the same column indicate differences in measurements (p < 0.05). SPF: sun protection factor; FB: base formulation; FBE: base formulation with extract; SBE: stem bark extract; OM: octyl methoxycinnamate.

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.^8,57-59 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 Hwanget 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, 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.⁴ 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 (FBOM_7.5) and the analysis of the synergistic effect (FBE₁₀OM_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 purpureaL., showed high potential as antioxidant and photoprotective agents, corroborating with the elevated value of phenolic content and chemical profile obtained by UPLC-QTOF-MS². 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