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Home , Fungus, Red algae

Endophytic compounds from marine as photoprotectors

Renata Spagolla Napoleão Tavares1; Olívia Maria C. Maciel1; Daniela Ricardo Engracia Caluz1; Gabriela Thimoteo Amaral1; Carolina Benevenuto1; Hosana Maria Debonsi1; Lorena Rigo Gaspar 1

1University of Sao Paulo, Faculty of Pharmaceutical Sciences of Ribeirao Preto, SP, Brazil

1. Introduction

The deleterious effects generated by UV rays have increased the need to protect the skin. However, conventional compounds with photoprotective activity in the UVA region suffer interactions, instability, and there are few options in the market. In the marine environment, especially in marine algae, environmental adversities, mainly related to sun exposure, increases the natural defenses by the production of secondary metabolites capable of absorbing/ reflecting the solar rays or acting as . Such molecules may be produced by algae, but mainly by the endophytic fungi associated with them (PALLELA et al, 2010). The stygium belongs to the family. This family is distributed around the world and widely studied, and this Annulohypoxylon genre, can produce a variety of secondary metabolites with different biological activities, including cytotoxic activity, antibacterial, antiviral, antiinflammatory and (SURUP et al., 2013; ROBL et al., 2015). The Pterocladiella belongs to Rhodophyta division, , Pterocladiaceae family. The division Rhodophyta is defined by Lee (2008) as a group consisting of eukaryotic algae, whose are surrounded by two envelope membranes. It consists of a single , Rhodophyceae, one of the oldest among the eukaryotic algae, with the record from the (LEE, 2008). Some studies demonstrate that Pterocladiella presents anti-inflammatory agents such as lecithin, found mainly in Pterocladiella capillacea (SILVA et al., 2010). Consequently, the aim of this study was to evaluate the photoprotective potential of fractions of the endophytic fungus A. stygium isolated from a red radicans and the fractions of the red algae Pterocladiella.

2. Methodologies

2.1.Annulohypoxylon stygium

The endophytic fungus A. stygium extracts, were obtained as follows: the was reactivated in solid medium PDA (Potato Dextrose ) and later was cultivated in solid medium Rice /Seawater, in a pilot-scale for 28 days under light. The was grounded and it was extracted three with dichloromethane: methanol (DCM: MeOH / 2: 1). Then, it was separated by vacuum filtration and the filter was concentrated on a rotary evaporator to obtain the organic extract. These extracts were selected between the most promising ones to be cultivated on a large scale, due to their UV absorption, The extracts were subjected to a fractionation using vacuum liquid chromatography (CLV), according to the methodology used in the Scripps Institution of Oceanography - University of California - USA. The obtained fractions were named by the partitioning gradient elution and the three fractions studied were the following: 1. Fraction C 20% Hexane / Ethyl (8/2); 2. Fraction E 60% Hexane / Ethyl acetate (4:6); 3. Fraction F 80% Hexane / Ethyl acetate (2:8).

2.2.Pterocladiella

The specie Pterocladiella sp. was collected on the coast of São Paulo, under the supervision of Dr. S. Nair Yokoya (IBot - Institute - SP) in two dates first December of 2004 and 17th January, 2014. The algal material was washed in seawater to remove contaminants (sand, crustaceans). Then the seaweed was stored in glass bottles with filtered seawater, supplemented with 200 mg /L of chloramphenicol and sterilized by autoclaving. These bottles were packed in coolers and transported to the Organic Chemistry Laboratory of the Marine Environment - FCFRP / USP. The algal material was washed in a freshwater and immediately lyophilized to lately be triturated in liquid nitrogen. The extract was obtained by three times maceration, with DCM: MeOH (2:1) for 12 hours while stirring. The third maceration was carried out for 24 hours while stirring. The extract was subjected to a fractionation by the same method described on item 2.1. The fractions studied were: Fraction D 40% Hexane/ Ethyl acetate (6/4) and Fraction E 60% Hexane/ Ethyl acetate (4/6).

3. Photostability of evaluation by UV spectrometry The fractions of the fungus A. stygium and the seaweed Pterocladiella were subjected to the analysis of the absorption spectra in the UV region as well as to photodegradation evaluation. This is determined by absorption spectrum analysis of their solutions at 100μg / ml in Agilent 8453 spectrophotometer at 200 to 400 nm range, which was submitted or not to a UV dose of 9.5 mW / cm2 emitted by a lamp Philips UVA Actinic BL / 10 (Eindhoven Netherlands) for 48 minutes, providing a total dose of 27.6 kJ / cm2 (CAMBON et al, 2001; WHITEHEAD; HEDGES, 2005; GASPAR; MAIA CAMPOS, 2006). For the determination of the photostability, the ratio of the mean UVA (320–400 nm) to the mean UVB (280–320 nm) absorbances was calculated as (DIFFEY, 1994):

4. Phototoxicity test in culture (3T3 NRU)

The fractions were submitted to phototoxicity test by the neutral red uptake in 3T3 murine fibroblasts. In this test 3T3 cells Balb/c cultured on two 96 well microtiter plates were pre-incubated with eight different concentrations of the chemical being analyzed. A plate was then exposed to UVA radiation and the other was kept sheltered from the light, for determining cell viability in the presence and absence of radiation for later determination of cyto- and phototoxicity of the substance (LIEBSCH et al., 2005; GASPAR et al., 2013). For concentration–response analysis Phototox Version 2.0 software (obtained from ZEBET, ) was employed. A test substance is predicted as having a potential phototoxic if the photoirritation factor (PIF), calculated as the ratio of toxicity for each substance with and without UV light, is higher than 5 (SPIELMANN et al., 1998). The mean photoeffect (MPE) was also calculated. The MPE is a statistical comparison of the dose–response curves obtained with and without UV and a test substance is predicted as phototoxic if MPE is higher than 0.1. According to the Organisation for Economic Cooperation and Development (OECD) Test Guideline 432, a test substance with a PIF >2 and <5 or an MPE >0.1 and <0.15 is predicted as ‘‘probably phototoxic’’ (OECD, 2004).

5. Results and Discussion The three fractions of A. stygium showed a significant absorbance in the UVB region. The fraction “C” Hexane/ Ethyl acetate (8:2), “E’ Hexane/ Ethyl acetate (4:6) and “F” Hexane/ Ethyl acetate (2/8) were tested under 9.5 mW/cm2 UVA dose. Two of them, “E” and “F”, were considered photostable. Only the “C” fraction was not considered photostable, Figure 1, due the large decrease in the ratio over 95%.

1,4

1,2 fr C ni fr C i 1,0 fr E ni fr E i 0,8 fr F ni fr F i

0,6 Absorbance

0,4

0,2

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

Figure 1. Photodegradation of three different fractions C, E and F of Annulohypoxylon stygium under irradiation (i), or not (ni). Two fractions of Pterocladiella (“D” and “E”) showed significant absorption in the UVA region, but were not considered photostable, Figure 2, with a decrease in the ratio over 59%.

1,1

1,0

0,9 Fr D 0,8 Fr D ni Fr E i 0,7 Fr E ni 0,6

0,5 Absorbance 0,4

0,3

0,2

0,1

250 300 350 400 450 Wavelenght (nm)

Figure 2. Photodegradation of two different fractions D and E of Pterocladiella under irradiation (i), or not (ni).

For the fungi A.stygium, the fractions “C” and “E” were considered phototoxic (MPE: 0.542 and 0.468, respectively) and the fraction “F” was considered probably phototoxic (MPE: 0.103, 0.108) (Table 1). Studying the isolated compounds of the fraction “E” and ‘F”, two of them were not considered phototoxic, and two others were considered phototoxic, showing that not all of the complex mixture have the same results when the compounds are analyzed separated. The fractions of the seaweed Pterocladiella presented phototoxicity and cytotoxicity potential (Table 1).

Table 1. Phototox - Phototoxicity Assay Test chemical Run IC 50 -UV IC50 +UV MPE PIF Result

A stygium Fr C 1 -* 16,971 0.542 5.2 Phototoxic

A stygium Fr E 1 - 20,719 0.468 4.833 Phototoxic

A stygium Fr F 1 - 79,335 0.103 1.264 Probably phototoxic

A stygium Fr F 2 - 72,271 0.108 1.385 Probably phototoxic

Pterocladiella 1 32.458 2.764 0.75 12.266 Phototoxic / Cytotoxic Fr D Pterocladiella 2 32.458 1.441 0.79 112.38 Phototoxic / Cytotoxic Fr D Pterocladiella 1 12.73 1.682 0.571 48.27 Phototoxic Fr E Pterocladiella 2 9.428 2.781 0.179 4.042 Phototoxic Fr E *No result was obtained (-).

Conclusion

 The results with de endophytic fungi Annulohypoxylon stygium showed a great potential source of chromophores to be used as UVB-filters in sunscreens;  Pterocladiella has a high potential to be employed as a biological UVA- filter due to its high UVA absorption, however subsequent studies with their isolated compounds should be investigated to evaluate which molecules are responsible for the UV absorption and if the phototoxic/cytotoxic potential could be reduced with purification.

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