Evaluation of photoprotective potential and photosafety of a combination of UV-filters and the polyphenols mangiferin and naringenin

KAWAKAMI, C.M. 1; BENEVENUTO, C.G. 1; GASPAR, L.R.1; 1Av. do Café, s/nº, Faculdade de Ciências Farmacêuticas de Ribeirão Preto – Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.

Nowadays it is known that to radiation (UV) is closely related to and the regular application of is one the most effective way to protect the skin from UV-induced damage. is a widely used UV- filter due to its great performance in UVA band; however, it shows photo reactivity and photo degradation under some circumstances and with some combinations. These reactions can lead to the reduction of photoprotective potential and to the formation of reactive intermediates, which can promote phototoxic reactions into skin. Moreover, there is a tendency to combine substances with antioxidant properties to conventional to increase their efficacy and the polyphenols mangiferin and naringenin could be used in these kinds of formulations due to their antioxidant and anti- inflammatory activities. Thus, the aim of this study was to evaluate UV absorption and phototoxicity of a UV-filter combination containing avobenzone and two polyphenols, mangiferin and naringenin, by using 3T3 NRU phototoxicity test. For this purpose a UV-filter combination containing , and avobenzone was supplemented or not (F) with 0.40% of mangiferin (FM), 0.40% of naringenin (FN) or their combination (FMN). The substances, alone or in combination, were subjected to the analysis of the absorption spectra in the UV region. The phototoxicity of avobenzone and UV-filters combination was evaluated by using 3T3 monolayer fibroblast culture, which was submitted to UVA radiation for the determination cell viability in the presence and absence of radiation, according to OECD TG 432. For this purpose, after the evaluation of the fibroblasts sensitivity to the UVA radiation, two 96-well plates were used for each substance, one to determine the cytotoxicity (absence of radiation) and another for the phototoxicity (presence of radiation). For that, after the cell incubation, eight different concentrations (ranging from 6.8 to 100 g/mL) of UV-filter or combination were applied in sextuplicate in the 96-well plates. One plate was irradiated with UVA radiation (5J/cm2), and the other plate was protected from radiation. After the incubation time, neutral red was added to the plates and the relative cell viability was calculated. The data was analyzed by the Phototox Software for the evaluation of phototoxic potential. The results showed that naringenin and mangiferin presented a high absorption on UVB and UVA region, respectively. The phototoxicity study showed that avobenzone presented phototoxic potential. When UV-filters and antioxidants combinations were analyzed, it was observed that the combination of avobenzone and naringenin (CN) presented phototoxic potential. Moreover, it was demonstrated that the addition of mangiferin to this combination (CMN) provided a reduction of phototoxic potential. In conclusion, naringenin is not indicated for sunscreens containing avobenzone, since this combination was considered phototoxic. Moreover, the use of mangiferin can be considered safer than naringenin and, besides that, its high antioxidant activity can improve the photoprotective effects of sunscreens.

Financial support: FAPESP and CNPq

Key Words: UV-filters, phototoxicity, 3T3-NRU

1. Introduction Nowadays, there is a tendency in the use of natural compounds such as polyphenols aiming the improvement of their potential to prevent UV-induced harmful effects, mainly due to some limitation of the organic UV-filters which are characterized by their narrow spectrum of protection and low photostability (JARZYCKA et al., 2013). Polyphenols have several biological properties, like anti-inflammatory, anticarcinogenic and immunomodulatory effects and they can act as a potent antioxidant (NICHOLS; KATIYAR, 2010). In addition, they also absorb UV radiation between 300 and 350 nm, which can suggest that it may have a biologically relevant filter activity as well (GASPAR et al., 2013). Mangiferin is a xanthone glucoside which is abundantly found in fruit, leaves and stem bark of Mangifera indica L (Mango). It exhibits several beneficial pharmacological properties like antioxidant, antiviral, antitumor, anti-inflammatory and hepatoprotective effects (PAL et al., 2013; JAGETIA et al., 2005). Naringenin is a flavanone found especially in many citrus fruits such as orange and grapefruit and it exhibits antioxidant, anti-inflammatory, antiproliferative and antimutagenic properties (COLLINS et al., 2011; MIR et al., 2015). However, some combinations containing UV-filters and antioxidants can present photoreactivity and be subject to degradation reactions upon exposure to UV radiation. The photodecomposition of combinations not only reduces the UV absorptive capacity but can also promote phototoxic or photoallergic contact . Consequently, there is an increasing concern about the phototoxicity and photoallergy of UV filters (GASPAR et al., 2013). In , since the year 2000, the marketing of cosmetics products and their ingredients that have been tested on animals for most of their human health effects is prohibited (VINARDEL, 2015). Consequently, many alternative methods to animal experimentation have been developed in several countries due to changes in legislation and have become a global trend assessment of ingredients and products. The validated 3T3 Neutral Red Uptake Phototoxicity Test (3T3 NRU PT) was accepted in 2000 by the European Community (EC) for regulatory purposes and later by the Organization for Economic Co-operation and Development (OECD) in 2004 as a test Guideline (TG) 432. It compares the cytotoxicity of a chemical when tested in the presence and absence of exposure to a non-cytotoxic dose of UVA/visible light (OECD, 2004; LIEBSCH; SPIELMANN, 2005). Therefore, the aim of this study was to evaluate UV absorption and phototoxicity of a UV-filter combination containing avobenzone and two polyphenols, mangiferin and naringenin, by using 3T3 NRU phototoxicity test.

2. Materials and methods

2.1. Determination of the absorption spectrum The absorption spectrum of substances/combinations under study was determined by analysis of their solutions in a spectrophotometer Hitachi U-2001 in the range 200 to 400 nm.

2.2. In vitro phototoxicity studies 2.2.1. Combinations DMSO stock solutions of combinations containing UV-filters, mangiferin and naringenin in same proportion used in the formulations under study (C: AVO, OMC, OCT; CM: AVO, OMC, OCT, MGF; CN: AVO, OMC, OCT, NRG; CMN: AVO, OMC, OCT, MGF, NRG) were each analyzed. These solutions were diluted to eight different concentrations in a geometric progression (6.8–100 µg mL-1, constant factor = 1.47) in phosphate buffered saline (PBS) containing calcium and magnesium. Stock solutions and working concentrations of the isolated compounds were prepared in the same way as described above for the combinations.

2.2.2. 3T3 Neutral Red Uptake Phototoxicity Test The 3T3 Neutral Red Uptake Phototoxicity Tests were performed according to the INVITTOX Protocol No. 78 (LIEBSCH; SPIELMANN, 2002) and to OECD TG 432 (OECD, 2004) using 3T3 Balb/c fibroblasts. After evaluation of fibroblast sensitivity to UVA radiation, two 96-well plates were used for each substance or combination, one to determine cytotoxicity (in absence of radiation) and another for the phototoxicity test (after radiation). Briefly, 100 µL of a cell suspension of 3T3 fibroblasts in Dulbecco’s Modification of Eagle’s Medium (DMEM) containing Fetal Calf Serum (10% v/v) and antibiotics (1 x 105 cells mL-1, 1 x104 cells/well) was dispensed in two 96-well plates. After a 24 h period of incubation (7.5% CO2, 37 ºC), plates were washed with 150 µL of PBS and different concentrations of the test chemicals or combinations were added in sextuplicate to the 96-well plates. After 1 h incubation, the + UV were irradiated with 5 J cm-2 of UVA radiation from a Philips UVA Actinic BL/10 lamp (Eindhoven, Netherlands). The UVA radiation was calculated with a VLX-3W Vilber Lourmat (Marnela-Vallee, France) Radiant Power Meter equipped with UVA (365 nm) sensor. The -UV plates were kept in a dark box. The test solutions were replaced by culture medium and plates incubated overnight. Neutral Red was added into each well and after an incubation period, cells were washed with PBS and desorbed with an ethanol/acetic mixture. The Neutral Red extracted from viable cells was a homogeneous solution and the +UV and -UV plates were analyzed in a Synergy 2 Multi-Mode Microplate Reader at 540 nm (Bio-Tek Instruments, Inc., Winooski, USA). For concentration–response analysis the Phototox Version 2.0 software (ZEBET, Germany) was employed. The mean photo effect (MPE), which is a statistical comparison of dose–response curves obtained with (+UV) and without (-UV) UV irradiation and test substances, was calculated. The photoirritation factor (PIF), which is factor generated by comparing two equally effective cytotoxic concentrations (IC50) of the test chemical obtained in the absence (-UV) and in the presence (+UV) of a non- cytotoxic irradiation with UVA/vis light (OECD, 2004) was also calculated. According to the OECD Test Guideline 432, a substance is predicted as phototoxic if MPE is higher than 0.15 or PIF is higher than 5. A test substance with an MPE >0.1 and <0.15 (PIF > 1 and < 5) is predicted as ‘‘probably phototoxic’’ (OECD, 2004). Results are the mean of at least two independent experiments +/- SEM. Norfloxacin and L-histidine were used as positive and negative controls, respectively.

3. Results and discussion 3.1. Determination of the absorption spectrum The mangiferin absorption spectrum showed that this substance has high absorption in the UVA range, presenting absorption peaks at 319 and 368 nm. The absorption spectrum of naringenin showed that this substance has high absorption in the UVB range, with peak absorption at 292 nm (Figure 1a). The absorption spectrum of combinations under study showed that all combinations presented high absorption in the UVB and UVA region (Figure 1b). a) b)

Mangiferin C 2,0 Naringenin CM 1,8 CN 1,8 CMN 1,6 1,6

1,4 1,4

1,2 1,2

1,0 1,0

0,8 0,8

Absorbance 0,6

Absorbance 0,6

0,4 0,4

0,2 0,2 0,0 0,0 -0,2 280 300 320 340 360 380 400 -0,2 280 300 320 340 360 380 400 Wavelength (nm) Wavelength (nm)

Figure 1. Absorption spectrum of a) mangiferin (100µg/mL) and naringenin (25µg/mL) b) combinations under study. C: Avobenzone, octyl methoxycinnamate, octocrylene; CM: avobenzone, octyl methoxycinnamate, octocrylene and mangiferin; CN: avobenzone, octyl methoxycinnamate, octocrylene, naringenin; CMN: avobenzone, octyl methoxycinnamate, octocrylene, mangiferin and naringenin.

3.2. Phototoxicity test in cell culture (3T3 NRU) According to the acceptance criteria determined in the study method validation (OECD, 2004), the controls showed acceptable values: norfloxacin positive control MPE: 0.476 and 0.500 (OECD: 0.34 to 0.90), and the negative control L- histidine MPE: 0.064 (OECD: 0.05-0.10) (OECD, 2004). The results obtained in the 3T3 NRU PT showed that only avobenzone was considered phototoxic, since it presented MPE values of 0.220 and 0.340 (Figure 2). The findings in this study are in agreement with the results of another in vitro experiment using cultured 3T3 murine fibroblasts (GASPAR, et al., 2013). However, the poor penetration of avobenzone into human skin means that its concentration in the viable epidermis after topical application is probably several times lower than that required to induce acute phototoxicity (FREITAS et al., 2015).

(A) MGF (non phototoxic) (B) NRG (non phototoxic)

MPE= 0,005; -0,203 MPE= 0,020; -0,040

(C) OCT (non phototoxic) (C) MTX (non phototoxic)

MPE= 0.011; -0.342 MPE= -0.143; -0.065

(E) AVO (phototoxic)

MPE= 0.340; 0.220

Figure 2. Dose response curves of the substances under study obtained by Software Phototox 2.0. The blue and yellow dots refer, respectively, to non-irradiated substances (-UV) and irradiated ones (+UV). MGF: mangiferin; NRG: naringenin; OCT: octocrylene; OMC: octyl methoxycinnamate; AVO: avobenzone.

When UV-filters and antioxidant combinations were analyzed, it was observed that combination containing avobenzone (C) had no phototoxic potential (Figure 3) showing MPEs bellow the cut-off values (OECD, 2004). The combination containing avobenzone and mangiferin (CM) did not present any phototoxic event as well, since it presented MPE values of 0.004 and 0.000. However, when naringenin was added to this combination (CN) there was an increase of MPE values (MPE: 0.213 and 0.162), which classifies this combination as phototoxic. On the other hand, the combination containing OMC, OCT, AVO, mangiferin and naringenin (CMN) showed no phototoxic potential (MPE: -0.004; 0.036), which suggests that mangiferin provided a reduction of CN phototoxic potential (Figure 3). It is suggested that photoprotective effect of mangiferin it is based on its high antioxidant capacity, since it works through scavenging ability of different ROS, like and superoxide anion, formed after of UV-filters (JAGETIA et al., 2005; MASIBO et al., 2008).

(A) C (non phototoxic) (B) CM (non phototoxic)

MPE= 0.006; 0.042 MPE= 0.004; 0.000

(C) CN (phototoxic) (D) CMN (non phototoxic)

MPE= 0.213; 0.162 MPE= -0.004; 0.036

Figure 3. Dose response curves of the substances under study obtained by Software Phototox 2.0. The blue and yellow dots refer, respectively, to non-irradiated substances (-UV) and irradiated ones (+UV). MFG: mangiferin; NRG: naringenin; OCT: octocrylene; OMC: octyl methoxycinnamate; AVO: avobenzone. 4. Conclusion The phototoxicity study showed that avobenzone presented phototoxic potential. When UV-filters and antioxidants combinations were analyzed, it was observed that the combination of avobenzone and naringenin (CN) presented phototoxic potential. Moreover, it was demonstrated that the addition of mangiferin to this combination (CMN) provided a reduction of phototoxic potential. In conclusion, naringenin is not indicated for sunscreens containing avobenzone, since this combination was considered phototoxic. Moreover, the use of mangiferin can be considered safer than naringenin and, besides that, its high antioxidant activity can improve the photoprotective effects of sunscreens.

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