Specialized Metabolites of the Lichen Vulpicida pinastri Act as Photoprotective Agents

The extreme resiliency of lichens to UV radiations makes them an interesting model to find new photoprotective agents acting as UV-blockers and antioxidant. In this research, using a new in vitro method designed to overcome the shortage of material associated to many studies dealing with natural products, we show that the three major compounds isolated from the lichen Vulpicida pinastri, vulpinic acid, pinastric acid and usnic acid, were UV blocker agents. Antioxidant assays evidenced superoxide anion scavenging activity. Combination of the most promising compounds against UVB and UVB radiations, usnic acid, vulpinic acid and pinastric acid, increased the photoprotective activity. At the same time, they were found not cytotoxic on keratinocyte cell lines and photostable in the UVA and UVB ranges. Thus, lichens represent an attractive source to find good candidate ingredients as photoprotective agents. Additionally, the uncommon scalemic usnic acid mixture in this Vulpicida species was proven through electronic circular dichroism calculation.


Captions
Spectrum S1 1 H NMR spectrum in CDCl3 + d6-acetone of the extract of Vulpicida pinastri Spectrum S2a 1 H NMR spectrum in CDCl3 of vulpinic acid (1) Spectrum S2b 13 C NMR spectrum in CDCl3 of vulpinic acid (1) Spectrum S3a 1 H NMR spectrum in CDCl3 of pinastric acid (2) Spectrum S3b 13 C NMR spectrum in d6-acetone of pinastric acid (2) Spectrum S4 1 H NMR spectrum in CDCl3 of the extract of (+)-usnic acid (3) Figure S1. Comparison of SPF values of authorized UV filters determined using PMMA plates and from experimental data in solution. (Correlation diagram presented in window).     Figure S7. Isobologram analysis to evaluate the combination effect of vulpinic acid and usnic acid on superoxide scavenging activity (evaluated via NBT assay). Combination Index (CI) values at 0%, 25%, 50%, 75%, 100% of IC50 indicated the efficacy of both compounds at different ratio : CI < 1 means synergy, CI = 1 means additivity, CI > 1 means antagonism. Figure S8. Cytotoxicity before and after irradiation under UVA of the combination effect of pinastric acid and usnic acid on HaCaT cells in the range of concentrations to observe a synergistic effect for antioxidant activity. Table S1. Results of exact mass measurements performed from the mass spectrum of the Figure. 3 related to the PI-DART-MS of a whole piece of Vulpicida pinastri.  Table S4. Photoprotection indexes of organic filters from experimental data obtained in a 96-wells plate (grey line) vs data obtained in a cuvette (white line). Table S5. Superoxide anion scavenging activity (% inhibition) when compound 1 and 3 are combinated Table S6. Superoxide anion scavenging activity (% inhibition) when compound 2 and 3 are combinated Table S7. Percentage of cytotoxicity of compound 1 and 3 before and after UVA Table S1. Percentage of cytotoxicity of compound 2 and 3 before and after UVA irradiation

New method for Photoprotective Indexes' Determination
A new method for photoprotective indexes' determination was developed to face the low availability of products such as secondary metabolites stemming from natural products. The method, first developed with commercial filters, allows the determination of SPF, PF-UVA, λC, SUI and ISP indexes.

Emulsion Preparation
An emulsion O/W was first prepared by dissolving 10 g of sodium lauryl sulfate in 20 g distilled water. Then, under a vigorous mix in a blender, 10 g of liquid paraffin was incorporated to obtain a homogeneous emulsion. The quantity of emulsion required to perform the analysis is much less important than the quantity prepared due to device size. The efficiency of the emulsion was then investigated after freezing of aliquots at −4 °C. The emulsion is stable during 3 months.

In Vitro Sun Protection Indexes Determination
The experimental data were recorded from ethanolic solutions prepared according the expected compound percentage. Absorbances (Aλ) were then transferred for calculations into a specially dedicated Excel spreadsheet. Transmittance (Tλ) was calculated from absorbance according to Eλ is the spectral irradiation of terrestrial sunlight at λ, Iλ is the erythemal action spectrum at λ and Tλ is the spectral transmittance of the sample at λ. SPF values are means of different values calculated from (Eλ × Iλ )relative to Mexico, Melbourne, [1,4,5]. where Aλ is the spectral absorbance at λ and Âλ is the ideal spectral absorbance at λ. Âλ is equal to the mean absorbance between 290 and 385 nm for all wavelengths in this spectral interval. Between 385 and 400 nm, Âλ is given as: where SST is the sum of squared deviation from the mean absorbance over the spectral band 290-400 nm and SSE is the sum of the squared deviations from the ideal absorbance at each wavelength [3]

Method's Development in Cuvette
Twelve commercial UV-filters spanning the 1 to 25 SPF range (Table S3) were used to develop the protocol. Experimental SPF values were compared with published data which were considered as reference values in this work [6][7][8] (Figure S1). For each compound, all indexes were determined from equations described above. The solubility has important bearing on SPF values drawn from assays conducted on anizotriazine and OMC. First, anisotriazine exhibited a SPF value of 22.8 in THF, close to the published value (SPF = 23.9), while the SPF value was 1.8 in DMSO where it was slightly soluble. Compounds were then dissolved either in THF or in DMSO depending on their solubility. Conversely, results were not statistically different for assays carried out on OMC, dissolved either in THF or in DMSO where it was soluble, SPF = 9.6 in THF and SPF = 10.1 in DMSO.
The SPF values were within the span 3.6 to 23.9 and were well correlated with the reference values (r 2 = 0.977). As a general trend, this new method well answered in an extended range of values though it underestimated them, suggesting even higher photoprotection when investigated in formulation. All the results of critical wavelengths and PF-UVA are summarized in Figure S2. All the λc values were below 370 nm and PF-UVA values were under 3 except for anisotriazine ( Figure S2). This was in agreement with the classification of these filters in the UVB and UVA+UVB range (Table S3). The ability of the method to discriminate a UVA filter via these indexes, was illustrated with avobenzone. Its critical wavelength and its PF-UVA value, respectively 377 nm and 3.9, were consistent with the restricted UVA filter profile of this compound.
Recently, new indexes of broad spectrum protection (SUI and ISP) were reported by Diffey [2,3] to focus on protection across the entire ultraviolet spectrum. They calculated the goodness of fit of the spectral profile to the ideal flat profile. The higher the SUI indexes, the better the product meets the ideal spectral requirement of a uniform absorption sunscreen throughout the ultraviolet spectrum. So does a weak ISP value. Anisotriazine, avobenzone, oxybenzone and octocrylene differed clearly  Arising from these results on twelve commercial organic filters, all indexes are in agreement with their well-known photoprotective properties. This new method allows the identification of UVA, UVB or (UVA + UVB) filter candidates using absolute and relative indexes of photoprotection (SPF, PF-UVA, λc, SUI, ISP). .

96-well Plates Indexes vs Cuvette Indexes
In a second time, in order to use the method for high throughput screening, we have adapted it for 96-well plate. 180 µL of solution S4 fill the well, absorbances were recorded and results were obtained as described above. Four commercial UV filters (OMC, anisotriazine, Uvasorb HEB, oxybenzone) were selected to cover a large SPF range. The adequacy of the results was evaluated by comparing the photoprotective indexes of these four filters obtained from absorbances recorded in cuvette and in a 96-well plates. All results collected in Table S5 were self-consistent and have validated the high throughput screening method. Table S5. Photoprotection indexes of organic filters from experimental data obtained in a 96-wells plate (grey line) vs data obtained in a cuvette (white line). CV in % in brackets.