Porphyra-334, a potential natural source for UVA protective sunscreens

Abstract

The mycosporine-like amino acid (MAA), porphyra-334 (λ_max = 334 nm; ε = 42 300 M⁻¹cm⁻¹), was isolated from the aquatic cyanobacterium Aphanizomenon flos-aquae (AFA) and its structure was verified by spectroscopic methods. The UVA absorption properties of the crude methanolic extract were determined against two commercial sun care products in terms of mean critical wavelength, mean UVA/UVB ratios and UVA protection category (Boots the Chemists, Ltd.). The crude methanolic extract from AFA exhibited maximum UVA protection comparable to that determined for Boots SPF 4.

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Introduction

Sunscreens absorb and/or reflect UV radiation. Topical application of sunscreens is widely advocated for prevention of UV-induced sunburn, photoaging, and skin cancer. However, in spite of the increasing use of sunscreens the incidence of malignant melanoma and non-melanoma skin cancer is rising dramatically.¹ An explanation might be found in the fact that sunscreens efficacy is currently evaluated by the sun protection factor (SPF), which is traditionally assessed by its ability to inhibit erythema 24 h after exposure of the skin to UV light. The effectiveness of UV to induce erythema declines rapidly with longer wavelength. Approximately 1000 times more UVA (315–400 nm) is needed to induce erythema compared with UVB (280–315 nm). UVA, on the other hand, is known to induce the formation of singlet oxygen and hydroxyl free radicals which can cause damage to cellular proteins, lipids, and carbohydrates. Like UVB, it can cause structural damage to the DNA, impair the immune system, and possibly lead to cancer. Our present understanding of the immunological and molecular events leading to skin cancer questions the concept that topical sunscreens capable of preventing erythema can also protect against UV induced carcinogenesis. Thus, SPF might be insufficient as an indicator of protection from UV-induced carcinogenesis.^2–8 Moreover, the overwhelming majority of sunscreen products available to consumers provide protection primarily limited to UVB and short-wavelength UVAII (315–340 nm). Only a few compounds such as sulisobenzone, dioxybenzone, avobenzone, titanium dioxide, zinc oxide, Mexoryl SX, Mexoryl XL, Tinosorb M, and Tinosorb S are broad-spectrum UVA filters. However, some of these compounds are problematic in terms of photostability and cross stability with other sunscreen agents. For example, the photoprotective capacity of avobenzone decreased by 50% to 60% after 1 h of exposure to sunlight. Besides, it has been reported to strongly enhance the degradation of octyl methoxycinnamate (OMC).^7,9,10 Since the UVA band constitutes about 5% of the solar spectrum at the Earth's surface, whereas UVB only makes up to 0.5%³ there is an urgent need for new and additional UVA blocking compounds.

A successful UV-screening compound ultimately depends upon simple organic photochemistry. The π-electron system is one of the most effective UV radiation absorbers. π-Electron systems are primarily found in conjugated bond structures that may be represented both in a linear chained molecule with alternating single and double bonds and in many aromatic and cyclic compounds containing electron resonance. π-Electron systems are a common theme in the function and characteristics of natural UV-screening molecules.¹¹ For example, many organisms such as aquatic cyanobacteria, which live in shallow water and normally are exposed to intense radiation, have evolved a variety of photo-adaptive mechanisms and strategies for successful growth. Such strategies include antioxidant activity and production of UV absorbing secondary metabolites such as mycosporine-like amino acids (MAAs).¹¹

MAAs are water-soluble substances characterized by a cyclohexenone or cyclohexenimine chromophore conjugated with the nitrogen substituents of amino acids or their imino alcohols, having absorption maxima ranging from 309 to 360 nm and an average molecular weight of around 300.^8,11–15 Their function as UVR protectors is strongly supported by recent studies on the identification, distribution, and regulation of MAAs in aquatic organisms. Seasonal cycles of MAA in soft corals have been shown to be positively correlated with annual cycles in solar radiation. It was also found that in many algae there is a negative correlation between levels of MAAs and water depth. Their high molar extinction coefficients (ε = 28 100–50 000 M⁻¹ cm⁻¹) support the hypothesis of a photoprotective role.^15–20 Studies on the photo-degradation and photo-physical characteristics of MAAs have shown that MAAs are capable of effectively dissipating absorbed UV radiation.^21,22

MAAs occur in taxonomically diverse organisms. MAAs have been assumed to be synthesized early in the shikimic pathway. Organisms that lack this biochemical pathway can obtain MAAs from algal endosymbionts or from the diet.^11,15,23,24 One such MAA, porphyra-334, was first found in the marine red alga Porphyra tenera by Takano et al.,²⁵ and later found in many marine red algal species.

Herein, we report the isolation and identification of porphyra-334 (Fig. 1) from Aphanizomenon flos-aquae (AFA), as well as its UVA absorption properties versus two commercial sunscreens.

Fig. 1 Chemical structure of porphyra-334. Single end arrows represent Heteronuclear Multiple Bond Correlations (HMBC), and double end arrows refer to Nuclear Overhauser Effect SpectroscopY (NOESY) correlations.

Experimental

Biological material

The absorbing compound was isolated from Vegicaps soft capsules (Solgar Laboratories, Leonia, NJ, USA), containing AFA as lyophilized powder. The capsules were opened and 10 g of powder was obtained.

Extraction and isolation procedure

Extraction of the powder was carried out with a mixture of methanol–water (80 : 20, v/v), followed by CH₂Cl₂.^26–28 The aqueous phase was lyophilized till dryness, to yield 4.3 g of a pale yellow powder. An aliquot of the powder was redissolved in 80% MeOH and checked for its UV absorbance. A sharp peak with an extinction coefficient of 42 300 M⁻¹cm⁻¹ was obtained at 334 nm (Fig. 2). The resulting powder was chromatographed on a silica gel column, using a gradient condition ranging from 10% MeOH and 90% EtOH to 90% MeOH and 10% EtOH, to yield 320 mg of a pale yellow powder. Purity of the isolated compound was confirmed by HPLC analysis, which showed a single sharp peak with a retention time of 2.7 min. The analysis was carried out on a reversed phase semi-preparative column (Symmetry Prep C₁₈7 µm, 7.8 × 300 mm) protected with a guard column using a mixture of 98% DDW–2% acetonitrile at a flow rate of 2 mL s⁻¹ and revealed a new, highly polar water soluble compound that absorbs strongly in the UVA region. The collected fractions were evaporated and then lyophilized till dryness to yield 48 mg of a colorless powder. Its structure was determined by using different techniques, including 1D/2D NMR and Q-TOF-micro-LC-MS.

Fig. 2 UV spectrum of the crude methanolic extract of Aphanizomenon flos-aquae.

Instrumentation

UV-spectra were measured on a UV IKON _XS BIO-TEK instrument. HPLC analysis was carried out on a Waters 600 instrument, using a reversed phase semi-preparative column (Symmetry Prep C₁₈7 µm, 7.8 × 300 mm) connected to a symmetry guard column, outfitted with a Waters 996 photodiode array detector. IR spectra were recorded on a Perkin-Elmer 2000 Fourier transformed infrared instrument. Spectrometry analysis was made on a Q-TOF-micro-LC mass spectrometer (MicroMass, Manchester, UK) in Bar-Ilan University. NMR spectra were recorded on a Bruker 400 MHz (Bruker BioSpin Corp., MA, USA). The ¹H NMR chemical shifts (referenced to D₂O observed at 4.8 ppm) were assigned using a combination of data from correlation spectroscopy (COSY) and heteronuclear multiple quantum correlation (HMQC) experiments. Optical activity was measured using CD-ORD Jobin Yvon spectroscopy.

UVA transmittance spectrometry

The UVA/UVB critical wavelength ratio was determined for the methanolic extract of AFA (see: Extraction and isolation procedure) against control products. The test method was based on an in vitro technique previously described by Diffey et al.⁵ Radiation from a UV source was directed onto the surface of a smooth quartz glass plate and the quantity of radiation transmitted through the plate was measured. The test product was then applied to a roughened quartz glass plate. Ten minutes later, radiation from a UV source (290–400 nm) was directed onto the surface of the plate and the quantity of radiation transmitted through the plate was measured. This procedure was repeated 3 times. Two internal control sun care products (Nivea SPF 20 and Boots SPF 4) were tested before and after the test product for validation purposes. The mean critical wavelength, the mean UVA/UVB ratios, and the UVA product category (Boots the Chemists Ltd.) were then calculated.

UV irradiation

The UV source was a dual diode array spectrometer with a Xenon flash lamp, optimized for UV emission, and with an integrating sphere providing instantaneous spectral acquisition (Labsphere UV-1000S). Roughened quartz glass plates were chosen because they are UV transparent, non-fluorescent, photostable, non-reactive, and compatible with all vehicle ingredients and distribute topically applied products in a manner similar to human skin. The diffuse illumination geometry of the sphere measures the transmittance from all path lengths through the sample and utilizes the total energy from the xenon flash lamp for optimal signal-to-noise performance. The smooth quartz glass plate was mounted in a sample holder. UV transmission through the plate was then measured at 5 nm increments throughout the UVB and UVA regions (290–400 nm) at single discrete regions of the plate to determine 100% transmission. The test product dissolved in water at a concentration of 10 mg ml⁻¹ was then applied in a series of small dots to a roughened quartz glass plate using a micro-pipette and then spread evenly using a gloved finger to achieve a typical uniform application rate of 0.75 mg cm⁻² of the sample. Care was taken to ensure a uniform film. UV transmission was then measured at 5 nm increments from 290 nm to 400 nm at 6 discrete regions of the plate ca. 10 min after product application. Subsequently, transmission measurements obtained before and after product application at discrete wavelength intervals from 290 nm to 400 nm were used to give an indication of the protection potential of the product throughout the UVA and UVB regions.

Results and discussion

The absorption spectrum of the methanolic extract of AFA indicated that the cyanobacterium presented a metabolite with absorption in the UVA region (315–400 nm). The absorption spectrum was characterized by a sharp absorption maximum at 334 nm. Porphyra-334 (Fig. 1) had a molecular formula of C₁₄H₂₂N₂O₈ which was determined by MS in conjunction with ¹H and ¹³C NMR data (Tables 1 and 2). The ¹³C-NMR data revealed a good correlation with previously published chemical shift data for porphyra-334²⁵ and structurally similar MAAs.^29–30

Table 1 NMR data for porphyra-334 in D₂O^a

Position	δ ^13C	δ ^1H	HMBC	NOESY
a Heteronuclear Multiple Bond Correlations (HMBC), Nuclear Overhauser Effect SpectroscopY (NOESY), and (/) no correlation was observed.
1	161.6	/	/	1
2	126	/	8	/
3	163.2	/	9	/
4	32.5	2.75	5	9
5	71.3	/	4, 6	/
6	33	2.77	5	/
7	67.1	3.61(s)	/	/
8	59	3.73(s)	2	/
9	47	4.07(s)	3, 10	4
10	177.6	/	9	/
11	64	4.12(d, J = 5.0 Hz)	14	14
12	178	/	/	/
13	68	4.33(m, J = 5.0, J = 6.4 Hz)	14	14
14	19	1.26(d, J = 6.4)	11, 12	/

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Table 2 Physical and chemical data for porphyra-334

mp	162–164 °C (dec)
[Θ]	−2.13 × 10^4 deg cm^2 dmol^−1(H2O)
UV-VIS (MeOH)	334 nm (42 300 M^−1cm^−1)
IR (KBr pellets)	3300, 1600, 1540, 1380, 1080 cm^−1
MS (positive ions)	347 [MH^+], 369 [MNa^+], 391 [(M-1 + Na) Na^+]
MS (negative ions)	345 [MH^−], 301 [MH^−–COO^−], 257 [MH^−–2COO^−]

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In order to investigate the efficacy of porphyra-334 as a potential sunscreen, mean critical wavelength and the UVA/UVB ratio were measured. Mean critical wavelength is defined as the wavelength below which 90% of sunscreen's UV absorbance occurs. In this method, the breadth or the width of UV protection is determined, whereas the SPF is a reflection of the amplitude or the depth of protection.⁵ The UVA/UVB ratio gives an indication of the UVA absorbance properties of a test product, relative to UVB, rather than as a ratio of protection factors. The UVA/UVB ratios obtained for the test product and for the two commercial internal control sun care products (measurements from both before and after the study products), and the corresponding UVA protection categories are presented in Table 3. The UVA protection categories were then determined according to the UVA symbol system operated by Boots the Chemists, Ltd.³¹ The data presented in this study suggest that porphyra-334 can serve as a UVA protecting sunscreen by providing wide protection against UV radiation. Furthermore, this MAA dissipates UV energy without creating any reactive species which might cause a phototoxic effect on living organisms.^22,29,32

Table 3 Sun protection factor (SPF), mean critical wavelength, UVA/UVB ratio and the corresponding UVA protection categories determined for the test and the control products

Sample	SPF	Mean critical wavelength/nm	Mean UVA/UVB ratio	UVA protection category descriptor
Nivea (Nivea Moisturising Sun Lotion, batch 10932751	20	377	0.64	Superior
Boots (Boots Soltan Extra Moisturising Sun Lotion, batch 1Z)	4	380	0.88	Maximum
Methanolic extract of AFA	4	388	0.95	Maximum

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The unusual tolerance of cyanobacteria to withstand long-term exposure to the potentially damaging effects of UV radiation and the high efficacy of their UV-absorbing compounds (MAAs),^26–28 suggest potential commercial application in the cosmetic industry. However, to fulfill the sunscreen demands, the amino acid or the amino alcohol functions in the MAAs must be replaced by alkyl amino groups to reduce their hydrophilic properties.^11,15

Acknowledgements

This work was financially supported by the Horowitz Foundation administered by Hadassit. Avital Torres thanks the School of Pharmacy for a scholarship. Morris Srebnik is affiliated with the David R. Bloom Center of Pharmacy.

References

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