Industrial Crops and Products 83 (2016) 509–514

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Industrial Crops and Products

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In vitro photoprotective activity of the Spondias purpurea L. peel crude

extract and its incorporation in a pharmaceutical formulation
Renata V. Silva, Sônia C.C. Costa, Carla R.C. Branco, Alexsandro Branco ∗
Department of Health, Faculty of Pharmacy, State University of Feira de Santana, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: The fruits of Spondias purpurea are found in many parts of the world, where it is an important natural
Received 13 October 2015 source of phenolic compounds such as tannins, phenolic acids and flavonoids. This study targets the
Received in revised form photoprotective capacity of the S. purpurea L. peel crude extract (SPPE) against in vitro UVA and UVB
26 December 2015
rays and its incorporation in a sunscreen formulation as an active principle. The phenolic and flavonoid
Accepted 28 December 2015
contents, scavenger radical activity (DPPH), antimicrobial activity and chemical analysis were also eval-
Available online 25 January 2016
uated. The SPPE was shown to be effective against UVB (sun protection factor 43.78 ± 0.19 in dilution
of 50 mg/mL) and UVA (protection comparable to the rutin and benzophenone-3, used as patterns). The
Keywords:
Spondias purpurea L.
phenolic and flavonoid contents were 28.68 ± 0.046 mg GAE/g and 2.64 ± 0.005 mg EQ/g extract, respec-
Photoprotection tively. The antioxidant activity showed inhibition percentage equal to 74.41, with EC50 27.11 ␮g/mL.
Phenolic compounds The high-performance liquid chromatography coupled with electrospray ionization mass spectrometry
Antioxidant activity (HPLC-ESI-MSn ) analysis allowed the characterization of the compounds dicaffeoyl-glucose, HHDP-
Sunscreen formulation galloyl-glucose, galloyl-bis-HHDP-glucose, rutin and quercetin. The formulation containing 30% of extract
showed excellent activity against UVA rays, with a protection percentage of 46.16 and UVB protection
with SPF 43.01 ± 0.81 for dilution of 50 mg/mL.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction ics, currently extensively sought after by the cosmetics industries

(Badea et al., 2015). Among the established plant extracts used
Ultraviolet A (320–400 nm) and B (290–320 nm) are described as sunscreens that contain antioxidant compounds, we can men-
to cause irreversible damage to skin, such as cancer, and to sig- tion the species Garcinia brasiliensis (Figueiredo et al., 2014) and
nificantly affect both the dermis and the epidermis layers (Saija Malpighia glabra L. (Souza et al., 2013).
et al., 2000; Mishra et al., 2012). The free radicals generated from Spondias purpurea L. is a plant belonging to the Anacardiaceae
sunlight are responsible for the degradation of essential cellular family, which is found in many parts of the world, especially in
components such as DNA and proteins, in addition to the oxida- Brazil, where it is known as seriguela (Augusto et al., 2012). The S.
tive damage from the fatty acids, destabilizing the membrane and purpurea are tropical fruit trees, with fruit in ovoid form of char-
causing irreversible biological effects (Chen et al., 2012). acteristic colors, used by some cultures to treat illnesses such as
The phenolic compounds are natural components of plants that ulcers, diarrhea, and parasitic diseases (Miller and Schaal, 2005;
can be found in large numbers in fruits (peel, pulp and juice), hav- Engels et al., 2012; Gachet et al., 2010). This fruit is an important
ing diverse chemical and biological functions, including the ability natural source of phenolic compounds such as tannins, phenolic
to absorb the solar spectrum and react with free radicals, formed acids and flavonoids (Omena et al., 2012).
from the oxidation metabolic processes, decreasing the harmful This study describes the potential of the crude extract of S. pur-
effects of the sun (Robards and Antolovich, 1997; Agati et al., 2013). purea peels as a sun protection agent through in vitro analytical
Those actions show that fruit-derived phenolic compounds can methods and formulations.
be incorporated in formulations, yielding innovative phytocosmet-
2. Materials and methods

∗ Corresponding author at: Universidade Estadual de Feira de Santana, Departa-

2.1. Materials
mento de Saúde, Laboratório de fitoquímica, CEP 44031 460 Feira de Santana, BA,
Brazil. Fax: +55 75 3224 8010. Methanol p.a, ethanol p.a, 2-diphenyl-1-picrylhydrazyl radi-
E-mail address: [email protected] (A. Branco). cal (DPPH), standard rutin, gallic acid, quercetin, benzophenone-3,

http://dx.doi.org/10.1016/j.indcrop.2015.12.077
0926-6690/© 2015 Elsevier B.V. All rights reserved.
510 R.V. Silva et al. / Industrial Crops and Products 83 (2016) 509–514

2 2.5

1.5 2
Absorbance

Absorbance
1 1.5
y = 0.015x + 0.0164
0.5 R² = 0.9981 1 y = 0.022x
R² = 0.9946
0 0.5
0 20 40 60 80 100 120
0
Concentraon (μg/mL) 0 20 40 60 80 100 120
Concentraon (μg/mL)
Fig. 1. Standard calibration curve of gallic acid.
Fig. 2. Standard calibration curve of quercetin.

resveratrol, Folin Ciocalteu’s reagent, sodium carbonate (Na2 CO3 ), 100.000

aluminum chloride (AlCl3 ), tryptone soy agar (TSA), tryptone soy
80.000
broth (TSB), agar Muller-Hilton, broth Muller Hilton, sulfuric acid

Acvity
(H2 SO4 ), formic acid (CH2 O2 ), barium chloride (BaCl2 ) and dimethyl 60.000
sulfoxide (DMSO) were purchased from Sigma–Aldrich (Steinheim, 40.000 y = 13.66ln(x) + 4.9106
Germany). Amoxicillin 500 mg was purchased from Medley (São R² = 0.9831
20.000
Paulo, Brazil). Lanette N, octyl stearate, imidazolidinyl urea, liq-
uid vaseline, propylene glycol and triethanolamine were purchased 0.000
from Fragon (São Paulo, Brazil). 0 50 100 150 200 250 300
Concentraon (μg/mL)
2.2. Plant material
Fig. 3. Calculation of the logarithmic regression of DPPH radical sequestration by
S. purpurea L. fruits were manually collected in January 2013 methanol extract of S. purpurea peels.
(latitude 12◦ 26 05.9 S and longitude 39◦ 15 34.2 W) in the Santo
Estevão city (Bahia, Brazil). After, the samples were washed and
expressed as mg of quercetin equivalent (QE) to 1.0 g of the dry
prepared for obtaining the crude extract. A voucher specimen was
extract.
deposited in the Herbarium of the State University of Feira de San-
tana with the number HUEFS 211101.
2.4.3. Scavenger radical activity (DPPH)
Solutions of SPPE in ethanol were prepared at concentrations in
2.3. Obtainment of the crude extract the range of 1–250 ␮g/mL. 2.5 mL of each concentration was mixed
with 1.0 mL of 0.3 mM DPPH in ethanol, at room temperature and
The 10 kg of ripe fruits were manually peeled and dried in an in the dark. The samples were kept in the dark for 30 min, and only
oven at 50 ◦ C. After that, the material was powdered and the chem- after that was the absorbance measured at 518 nm in UV–vis spec-
ical components were extracted with methanol (2 L), for 72 h at trophotometer. The blank solution was composed by ethanol. The
room temperature (25 ◦ C). The mixture was vacuum-filtered and negative control solution was prepared by mixing 1.0 mL of 0.3 mM
the solvent evaporated under reduced pressure and controlled tem- DPPH solution with 2.5 mL of absolute ethanol. A curve of free rad-
perature of 60 ◦ C with router evaporator. The yielding of the extract ical inhibition (%) versus the concentration of SPPE was plotted and
was 500 g. used to calculate the concentration of SPPE to inhibit 50% of free
radicals in the solutions (EC50 ), shown in Fig. 3. The experiments
2.4. Crude extract analysis were repeated 3 times to confirm the reproducibility of the data.
The antioxidant activity was expressed as the percentage of DPPH
2.4.1. Total phenolic content (TPC) radical inhibition. The EC50 was calculated by means of logarith-
A stock solution was prepared to 1.0 mg/mL in methanol, where mic regression of the curves obtained by plotting the results of %
1.0 mL of the extract was mixed with 5.0 mL of Folin–Ciocalteu the DPPH inhibition. On these plots, the abscissa represents the
reagent (1:10 in water). After 8 min, the mixture was added with concentration of SPPE and the ordinate represents the antioxidant
4.0 mL of sodium carbonate 7.5% (w/v). After 60 min in the dark activity (Chiari et al., 2012).
and at room temperature (25 ◦ C), its absorbance was read at 765 nm
using UV–vis spectrophotometer against a blank. The concentration 2.4.4. In vitro UVB photoprotection
of the polyphenols in samples was derived from a standard calibra- The evaluation of the in vitro UVB photoprotection was carried
tion curve of gallic acid in the range of 10–50 ␮g/mL (R2 = 0.9981) using the spectrophotometric method described by Mansur et al.
in Fig. 1. The data were expressed as mg of the gallic acid equivalent (1986), according to Eq. (1) (Fig. 4)and Table 1. The stock solution of
(GAE) to 1.0 g of the dry extract (Jarzycka et al., 2013). the crude extract (1 g/mL in ethanol) was provided. From the stock
solution were several other solutions obtained (0.2, 2, 5, 10, 15, 20,
2.4.2. Total flavonoid content (TFC) 30 and 50 mg/mL) to investigate the SPF profile. These solutions
According to Jarzycka et al. (2013), 0.5 mL aluminum trichlo- were read in triplicate in UV–vis spectrophotometer. Ethanol was
ride 2% in methanol was added to 0.5 mL of the extract diluted in used as blank and the readings were made at 290–320 nm.
methanol (1.0 mg/mL). The absorbance value was measured at the
wavelength of 420 nm using UV–vis spectrophotometer against a 2.4.5. In vitro UVA photoprotection
blank, after 60 min in the dark and at room temperature. The final The petri dishes (4.5 cm in diameter) containing 0.1 mg/mL
absorbance of each sample was compared with a standard calibra- resveratrol in ethanol, weighing 0.04 g of extract, were investi-
tion curve made for quercetin (R2 = 0.9946) in Fig. 2. The data were gated. The plates were placed in a closed vessel and subjected
 R.V. Silva et al. / Industrial Crops and Products 83 (2016) 509–514 511

320 Table 2
FPS = CF . ∑ . EE (λ) . I (λ) . Abs (λ) (Eq. 1) Composition of sunscreen emulsion.
290
Ingredients wt.%

Phase A (oil phase)

CF = correction factor (equal to 10); EE (λ) = erythematous effect of radiation of Lanette N 8
Liquid vaseline 3
wavelength λ; I (λ) = intensity of sunlight at wavelength λ; Abs (λ) = spectrophotometric Octyl stearate 4
Propylparaben 0.05
absorbance reading for preparing the solution at a wavelength (λ). Benzophenone-3 (as used) 5

Fig. 4. Equation Mansur et al. (1986) to calculate of SPF. Phase B (aqueous phase)
Methylparaben 0.15
Glycerin 5
Table 1 Deionized water qsp
Relationship between the erythematogenic effect, and the intensity of the radiation
at each wavelength. Phase C
Imidazolidinyl urea 0.1
 (nm) EE () × I ()
Phase D
290 0.0150 Rutin (as used) 5
295 0.0817 Crude extract 5, 10, 20, 30
300 0.2874
305 0.3278
310 0.1864
315 0.0839 formulations were prepared: lotion lanette (LL), lotion and extract

320 0.0180 5% (LS5), 10% (LS10), 20% (LS20) and 30% (LS30), benzophenone-3
1.0000
lotion containing 5% (LB5) and rutin lotion 5% (LR5). The tests were
performed after 24 h of equilibrium of formulations.
to UV radiation lamp (320–400 nm) exposure at 60.0 W of power.
The photodegradation of resveratrol was evaluated at time 0 (t0) 2.5.1. Photoprotective in vitro UVB and UVA formulation
to 120 min (t120) by means of UV–vis spectrophotometer. The The UVB and UVA photoprotection of the formulations was mea-
absorbance was measured at 306 nm. The photodegradation of sured as item in Sections 2.4.4 and 2.4.5, respectively. LL was used
resveratrol was made in triplicate (Detoni et al., 2012). as blank.

2.4.6. Antimicrobial activity

2.6. Statistical analyses
The antimicrobial activity was evaluated according to Bauer
et al. (1966) with some modifications. The microorganisms
The TPC, TFC and scavenger radical activity were calculated by
Escherichia coli (1110033 Fiocruz), Pseudomonas aeruginosa
the original values and expressed as mean ± standard deviation
(1010230) and Salmonella dublin (142804) were used in the test.
after analysis in triplicate. The potential sunscreens were analyzed
These microorganisms were enriched in TSB and TSA, sown on
in vitro using the software GraphPad Prism and statistical evalu-
the surface of agar Mueller–Hilton. The preparation of the bac-
ation was determined by using ANOVA followed by Tukey test of
terial suspension in 0.75% saline was made according to the 0.5
multiple variances with P < 0.05.
McFarland scale that corresponds to approximately 106 CFU/mL.
Filter paper disks containing 1 cm of diameter were impregnated
with 20 ␮L of SPPE diluted in DMSO (102.400 ␮g/mL) and placed 3. Results and discussion
on the inoculated agar surface. After incubation for 24 h at 37 ◦ C,
the inhibition halos were observed. The halo measurements were 3.1. Crude extract investigation
performed using a caliper, and the results were given in millime-
ters. The positive and negative controls used were the antibiotic 3.1.1. TPC and TFC
amoxicillin and DMSO, respectively. The TPC shows a value at 28.68 ± 0.046 mg GAE/g. According to
the studies of Tiburski et al. (2011) on Brazilian fruits, the pulp of
2.4.7. HPLC–ESI–MSn analysis Spondias mombin L. showed 260.21 ± 11.89 mg GAE/100 g, lower
The HPLC–ESI–MSn analysis was performed on the Bruker than what has been found in this study. The TFC value was of
Esquire 3000 Plus-Daltonics equipment, capillary 4000 V, nebulizer 2.64 ± 0.005 mg EQ/g extract, which was similar to values found
40 psi, dry gas 9 l/min and temperature 300 ◦ C, reversed phase C18 in the ethanol extracts of the fruit Geophagus brasiliensis at a con-
column. The mobile phase was composed by eluent A (aqueous centration of 3.4 mg EQ/g (Figueiredo et al., 2014). The above results
solution of H2 0/0.1% CH2 O2 ) and eluent B (methanol). The gradient confirm that SPPE contains a high amount of phenolic compounds,
program starting from 0 to 19 min of 75% A and 25% B, 20–24 min 0% wherein 10% corresponds to the flavonoid class.
A and 100% B, ending in 25–30 min with 75% A and 25% B, followed
by washing and reconditioning of the column.
Table 3
Antioxidant activity of SPPE.
2.5. Preparation of sunscreen emulsion
Concentration (␮g/mL) Inhibition (%)
The base oil/water emulsion was prepared by combining the SPPE Rutin
components using four different concentrations of extracts (5, 10,
1 3.29 ± 0.042 30.83 ± 0.037
20 and 30%). The emulsion compositions are described in Table 2. 10 34.23 ± 4.890 50.86 ± 0.071
First, phases A (oil phase) and B (aqueous phase) were heated sep- 25 49.41 ± 0.603 80.19 ± 0.029
arately at 70–80 ◦ C. Then, the aqueous phase was added to the oil 50 62.90 ± 2.562 82.65 ± 0.338
and homogenized at 40 ◦ C, when the phase C was added. The phase 125 71.83 ± 0.488 84.93 ± 0.494
250 74.41 ± 1.255 90.01 ± 1.393
D was added after the emulsion preparation. In all cases, seven
512 R.V. Silva et al. / Industrial Crops and Products 83 (2016) 509–514

Table 4
Sun protection factor of crude extract and formulations.

Dilution (mg/mL)

Formulation 0.2 0.4 2 5 10 15 20 30 50

SPPE 2.70 ± 0.37c 4.30 ± 0.26b 6.53 ± 0.31c 15.37 ± 0.29c 29.31 ± 0.56c 39.92 ± 0.09b 41.88 ± 0.20a 42.59 ± 0.42a 43.78 ± 0.19a
LS5 0d 0e 0f 0g 0g 0g 1.18 ± 0.03g 4.23 ± 0.086f 8.15 ± 0.075e
LS10 0d 0e 0.79 ± 0.18e 1.83 ± 0.12f 4.13 ± 0.10f 6.46 ± 0.055f 6.65 ± 0.13f 13.25 ± 0.23e 21.88 ± 0.13d
LS20 0d 0e 0.70 ± 0.05e 3.18 ± 0.13e 7.82 ± 0.04e 12.53 ± 0.31e 16.33 ± 0.33e 23.14 ± 0.09d 37.33 ± 0.17c
LS30 0d 0.32 ± 0.04d 1.62 ± 0.11d 5.62 ± 0.27d 13.99 ± 0.22d 22.73 ± 0.21d 30.38 ± 0.17d 40.21 ± 0.10b 43.01 ± 0.81a
LB5 4.82 ± 0.09a 7.01 ± 0.12a 38.53 ± 0.32a 38.80 ± 0.14a 38.73 ± 0.12b 39.10 ± 0.08c 38.20 ± 0.06c 39.29 ± 0.16c 39.84 ± 0.23b
LR5 0.36 ± 0.05b 0.67 ± 0.04c 12.27 ± 0.22b 32.67 ± 0.30b 40.02 ± 0.0a 40.58 ± 0.06a 41.08 ± 0.07b 42.37 ± 0.29a 44.27 ± 0.12a

Tukey: equal letters represent statistically similar values for the same dilution.

3.1.2. Scavenger radical activity (DPPH) Table 5

Absorbance after 120 min of UVA irradiation.
The evaluation of the ability of SPPE to sequestrate the DPPH
radical is described in Table 3. The flavonoid rutin used as pos- Formulation Absorbance ()
itive control in the test enables it to sequestrate 90.01% of the SPPE 1.024 ± 0.02a
DPPH radical formed at the concentration of 250 ␮g/mL. The LL 0.423 ± 0.009d
SPPE sequestrated 74.41%. The EC50 found was 27.11 ␮g/mL, LC 0.419 ± 0.005d
values that are greater than those found by Sousa and Vieira DC 1.037 ± 0.01a
LS5 0.480 ± 0.006d
(2011), with EC50 for M. glabra L. (308.07 ± 0.75 ␮g/mL), Psid-
LS10 0.523 ± 0.01c
ium guajava L. (142.89 ± 4.85 ␮g/mL) and Ananas comosus L. LS20 0.566 ± 0.01c
(3293.92 ± 9.89 ␮g/mL). LS30 0.662 ± 0.007b
Several authors have conclusively shown that there is a cor- LB5 1.028 ± 0.04a
LR5 0.981 ± 0.02a
relation between the antioxidant capacity and the phenolic and
flavonoid concentrations in fruits and vegetables (Chiari et al., Tukey: same letters refer to statistically equal values.
2012). However, Melo et al. (2008) points out that the antiox-
idant ability cannot be explained based on its phenolic content
and flavonoids, once that the characterization of the active com-
pound structure is important and necessary since the locations and standard banzophenone-3. However, benzophenone-3 is potent
amounts of hydroxyl groups can influence the effectiveness of the as active principle, reaching maximum absorbency levels at low
antiradical capacity. concentrations.
In comparison with the rutin, the SPPE achieved good results
3.1.3. In vitro UVB and UVA photoprotection in dilutions of 30 and 50 mg/mL. While an ideal sunscreen con-
The method described by Mansur is widely used to measure the taining SPF 15 is adequate for protection in accordance with the
sun protection factor (SPF) in vitro, what proves good correlation international standards (Food and Drug Administration, 2012), the
in substitution for in vivo tests because it relates the absorbance of SPPE can be considered as a promising active ingredient because it
the substance with erythematous effect of radiation and the inten- presents high sun protection factors in small dilutions.
sity of light at wavelengths from 290 to 320 nm (UVB region in the The in vitro UVA photoprotection evaluation was performed
spectrum) (Violante et al., 2009). Table 4 shows the sun protection with the method of trans-resveratrol. The samples were tested for
factor of the SPPE and the formulations containing it as active prin- their potential UVA sunscreen in the range of 0–120 min (Table 5).
ciple. All the SPF test profiles allow the verification that its activity The SPPE showed protection statistically equal to LB5, LR5 and
is proportional to the amount of extract added to the formulation dark control (DC), which consists of wrapped sample location away
(Ribeiro, 2006). from any radiation. The percentage of degradation was 28.95%,
Thus, at the dilution of 0.2 mg/mL, the patterns and the SPPE 28.61%, 31.95% and 27.77%, respectively, indicating that the extract
had values different from zero instead of the formulations. The is a potential protector against UVA radiation. That result can be
SPPE (20 mg/mL) and benzophenone-3 (2 mg/mL) saturation lev- explained because there are photoprotective organic substances
els were 41.88 ± 0.20 and 38.53 ± 0.32, respectively. At dilution of capable of absorbing, scattering and reflecting UV rays (Ribeiro,
50 mg/mL, the SPF has higher ultraviolet B observance with the 2006).

Table 6
HPLC–MSn results of phenolic compounds in the crude extract of S. purpurea (SPPE).

Peak tr (min) Negative mode (m/z) Positive mode (m/z) Compound

1 2.8 MS : 683.2 [M − H]
1 − – Dicaffeoylglucose
MS2 [683.2]: 341
MS3 [341]: 179.2, 161
2 6.5 MS1 : 655.1 [M + Na − H]− , 633.1 [M − H]− MS1 : 652.5 [M + H2 O + H]+ HHDP-galloyl-glucose
MS2 [633.1]: 463.0, 300.9 MS2 [652.5]: 465.1
MS3 [463.0]: 300.9 MS3 [465.1]: 303.0
3 7.6 MS1 : 953.1 [M + H2 O − H]− , MS1 : 937.3 [M + H]+ , 785 Galloyl-bis-HHPD-
935.4 [M − H]− MS2 [785]: 465.0, 303.0 glucose
MS2 [935.4]: 633.1, 462.9, 300.9
4 10.6 MS1 : 609.2 [M − H]− MS1 : 633.2 [M + Na]+ , 611.2 [M + H]+ Quercetin-3-O-
MS2 [609.2]: 300.9 MS2 [611.2]: 465.0, 303.0 rutinoside
MS3 [465.0]: 303.0 (Rutin)
5 11.6 MS1 : 625.0 [2M + Na − 2H]− MS1 : 648.9 [2M + 2Na]+ , 626.9 [2M + Na]+ Quercetin
MS2 [625.0]: 300.9 [M − H]− MS2 [626.9]: 303.0 [M + H]+
 R.V. Silva et al. / Industrial Crops and Products 83 (2016) 509–514 513

Fig. 5. HPLC-MSn chromatogram of the crude extract of S. purpurea.

3.1.4. Antimicrobial activity when applied topically, has the ability to protect the skin against
The SPPE demonstrated activity against the Gram negative P. UVB-induced erythema in vitro and in vivo (Kullavanijaya and Lim,
aeruginosa (170 mm inhibition zone) and S. dublin (310 mm); also, 2005). Prasad et al. (2009) irradiated lymphocytes together with
it was sensitive to strains of E. coli with the disc diffusion method. caffeic acid and proved that this substance reduces both the levels
DMSO, negative control, did not possess any activity to strain, of markers of lipid peroxidation and the cytotoxic effects induced
confirming that the activity was given by the samples being evalu- by UVB rays, confirming their photoprotective activity. Tannins are
ated. The positive control, amoxicillin, was effective against strains potent antioxidants that can protect the skin cells against free rad-
of E. coli (450 mm) and S. dublin (490 mm), proving no activity icals caused by exposure to ultraviolet rays, reducing the risk of
against Pseudomonas. Islam et al. (2013) observed similar results cancer (Svobodová et al., 2003). Romani et al. (2012) showed that
when assessing the antimicrobial power of Spondias dulcis. Its fruit the epicarp of Punica granatum (Pomegranate) contains galloyl phe-
showed activity against Gram-negative strains such as P. aeruginosa nolic compounds such as glucose, ellagic acid and several other
and E. coli, and also against Gram-positive Staphylococcus aureus. types of flavonols glycosylated, as well as those found in the S.
Although the inhibition zone test is a screening tool for the activ- purpurea peels with potent antioxidants. According to Saewan and
ity against microorganisms, it is suggested that the antimicrobial Jimtaisong (2013), quercetin is one of the most powerful antioxi-
activity of SPPE can occur by the existence of phenolic compounds dants because of the number of flavonol hydroxyl groups replaced
(Rios and Recio, 2005). in its molecule. This compound protects mice against UVA radiation
and eliminates species of free radicals. Kreft et al. (2003) found out
that flavonoids such as rutin and quercetin protect cells against UVB
3.1.5. HPLC–ESI–MSn analysis
rays, preventing radiation from modifying vital molecules such as
The chromatogram (negative mode) and the spectral data are
DNA and RNA. Solovchenko and Schmitz-Eiberger (2003) demon-
shown in Fig. 5 and Table 6, respectively (Flamini, 2013). Compound
strated the increased amount of glycosylated quercetin, the main
1 (Rt = 2.8 min), with a pseudomolecular ion [M − H]− at m/z 683,
flavonoid featured in apple epicarp exposed to sunlight. They also
originated MS2 fragmention at m/z 341. This fact shows that the
demonstrated that this compound contributes positively to the UVB
ion at m/z 683 is a dimer formatted for two units of ion at m/z
protection of these fruits because they absorb radiant energy in the
341. Fragmentation of ion at m/z 341 furnished fragment at m/z
spectrum range between 290 and 320 nm.
179 (attributed to caffeic acid unit) due to the loss of a hexose unit
(162 Da). The compound 1 was characterized as a dimer of caffeic
acid O-hexoside (Gouveia and Castilho, 2011; Barreira et al., 2014). 3.2. In vitro UVB and UVA photoprotection of the formulations
Compound 2 (Rt = 6.5 min) showed signal deprotonated
molecule [M − H]− at m/z 633, corresponding to a hexahydrox- The UVB photoprotection of the formulation at 5, 10 and 20%
ydiphenoyl (HHDP) galloyl glucose, classified as an ellagitannin. did not reach saturation levels that are observed in SPPE, where
MS2 fragmentation signals generated m/z 463 [M − H − 170]− small amounts of active ingredient absorb much of the radiated UVB
related to the loss of gallic acid and m/z 301 [M − H − 170 − 162]− . energy. It is suggested that the lotion 30% at a dilution of 50 mg/mL
On the other hand, the structure of galloyl bis-HHDP-glucose is is close to saturation levels as compared with the crude extract, due
suggested for the compound 3 (Rt = 7.6 min) due to the [M − H]− to the fact that their results are statistically equal.
at m/z 935. The fragmentation (MS3 ) conducted the ions at m/z At a dilution of 0.4 mg/mL, 5–20% formulations obtained SPF
633 [M − 302 − 2H]− which is the result of the loss of HHDP. The equal to zero. Only the lotion containing 30% of this result is dif-
fragment at m/z 463 [M − 170 − H]− is the result of the loss of a ferent from zero (0.32 ± 0.04). However, for photoprotection, this
gallic acid unit and m/z 301 [M − 162 − H]− is due to the loss of a value is considered irrelevant. From the dilution of 2 mg/mL, all
hexose (Romani et al., 2012; Bora et al., 2014). formulations contained SPPE, except for LS5, where SPF values dif-
Compound 4 showed pseudomolecular ion at m/z 609 [M − H]− . ferent from zero were obtained, demonstrating UVB absorption.
The MS2 furnished the fragment at m/z 301, which confirmed the Analyzing the data in the 30% lotion, it was observed that its SPF
aglycone quercetin after the loss of sugar rutinose (Tiberti et al., is very close to the benzophenone-3 in 30 mg/mL dilutions. This
2007). Compound 5 (Rt = 11.6) showed signal at m/z 625 due to the phenomenon is also observed comparing the lotion raw extract
formation of an adduct molecule [2M + Na − 2H]− , whose fragmen- and rutin, where the profiles intersect at a dilution of 50 mg/mL
tation (MS2 ) furnished ion at m/z 301 [M − H]− . Thus, the compound equivalent to SPF.
4 and 5 can be characterized as rutin and quercetin, respectively. Adil et al. (2010) presented a study with Emblica officinalis fruit,
Out of the compounds described above, only the flavonols widely used in folk medicine in India as antiulcerogenic, against
quercetin and rutin have been described in S. purpurea (Engels et al., inflammatory and metabolic diseases, suggesting that the extract
2012). Along with this fact, the other components found in this effectively inhibits the aging fibroblasts induced by UVB. In the
study contributed to the photoprotective effect. The caffeic acid, future, it may be used in cosmetics applications. UVA photopro-
514 R.V. Silva et al. / Industrial Crops and Products 83 (2016) 509–514

tection of the LS5 formulation failed in protecting deterioration of Figueiredo, S.A., Vilela, F.M., da Silva, C.A., Cunha, T.M., dos Santos, M.H., Fonseca,
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LL, which was used as a blank. The percentages of degradation of and wine polyphenols. ISRN Spectrosc., 1–46.
Gachet, M.S., Lecaro, J.S., Kaiser, M., Brun, R., Navarrete, H., Munõs, R.A., Bauer, R.,
the three samples were, respectively, 66.59%, 70.83% and 70.62%. Schühly, W., 2010. Assessment of anti-protozoal activity of plants traditionally
The LS10 and LS20 also protected against deterioration of resvera- used in Ecuador in the treatment of leishmaniasis. J. Ethnopharmacol. 128,
trol, since their data were statistically equal. It was concluded that 184–197.
Gouveia, S., Castilho, P.C., 2011. Characterization of phenolic acid derivatives and
there was no advantage in increasing extract concentration from
flavonoids from different morphological parts of Helichrysum obconicum by a
10 to 20%. Its degradation percentages were 64.44% and 60.69%. RP–HPLC–DAD-(−)-ESI–MSn method. Food Chem. 129, 333–344.
The lotion that mostly protected against UVA was the LS30, with a Islam, S.M.A., Ahmed, K.T., Manik, M.K., Wahid, M.A., Kamal, C.S.I., 2013. A
comparative study of the antioxidant, antimicrobial, cytotoxic and
percentage of 53.81%.
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The fruits of the S. purpurea contain natural compounds capable Trop. Biomed. 3, 682–691.
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The S. purpurea peel crude extract (SPPE) is shown to have pho- Melo, E.A., Maciel, M.I.S., Lima, V.L.A.G., Nascimento, R.J., 2008. Capacidade
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identified the phenolic compounds that are considered in the liter- Miller, A., Schaal, B., 2005. Domestication of Mesoamerican cultivated fruit tree,
Spondias purpurea. PNAS 102, 12801–12806.
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first description of the potential sunscreen containing the peel of protection factor of Calendula officinalis L. (Asteraceae) essential oil
S. purpurea as active principle. In addition, this extract has also the formulation. J. Young Pharm. 4, 17–21.
Omena, C.M.B., Valentim, I.B., Guedes, G.S., Rabelo, L.A., Mano, C.M., Bechara, E.J.H.,
potential to combat the harmful effects against ROS, along with its
Sawaya, A.C.H.F., Trevisan, M.T.S., Costa, J.G., Ferreira, R.C.S., Sant’Ana, A.E.G.,
ability to absorb UV rays. Thus, the S. purpurea L. peel is an active Goulart, M.O.F., 2012. Antioxidant, anti-acetylcholinesterase and cytotoxic of
ingredient for innovative phytocosmetics formulations. ethanol extracts of peel, pulp and seeds of exotic Brazilian fruits. Food Res. Int.
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Prasad, N.R., Jeyanthimala, K., Ramachandran, S., 2009. Caffeic acid modulates
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Ribeiro, C., 2006. Cosmetologia aplicada a dermoestética, 2a ed. Pharmabooks, São
We would like to thank CNPQ, FAPESB, the State University of
Paulo (in Portuguese).
Feira de Santana and the Graduate Program in Biotechnology. Rios, J.L., Recio, M.C., 2005. Medicinal plants and antimicrobial activity. J.
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