Lichens – Photophysical studies of potential new sunscreens

Journal of Photochemistry and Photobiology B: Biology 95 (2009) 40–45

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Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol

Lichens – Photophysical studies of potential new sunscreens Fritz Boehm a, Kristy Clarke b, Ruth Edge c,*, Ernesto Fernandez d, Suppiah Navaratnam e,f, Wanda Quilhot d, Fiorenza Rancan a, T. George Truscott b,* a

Department of Dermatology, Humboldt University, 10117 Berlin, Germany Chemistry Section, School of Physical and Geographical Sciences, Keele University, Staffs ST5 5BG, UK c School of Chemistry, The University of Manchester, Oxford Road, Manchester M13 9PL, UK d Chemistry and Pharmacy School, University of Valparaiso, Chile e STFC Daresbury Laboratory, Daresbury WA4 4AD, UK f Bioscience Research Institute, Peel Building, University of Salford, Salford M5 4WT, UK b

a r t i c l e

i n f o

Article history: Received 22 October 2008 Received in revised form 24 November 2008 Accepted 16 December 2008 Available online 24 December 2008 Keywords: Lichens Pulse radiolysis Laser flash photolysis Sunscreens Free radicals Singlet oxygen

a b s t r a c t Fundamental photophysical properties have been obtained for six polyaromatics, calycine, usnic acid, vicanicine, 1-Cl-pannerine and epiphorelic acids I and II, extracted from Antarctic lichens – potential future sunscreens. None of the lichen compounds produced a measurable amount of triplet states and the singlet oxygen quantum yield was also very low ranging from 0.003 to 0.06. However, three exhibited triplet energy levels which may be above that of thymine. The radical cations of calycine and usnic acid were generated via pulse radiolysis and were observed to be quenched by vitamin C, vitamin E and Trolox. Ó 2008 Elsevier B.V. All rights reserved.

1. Introduction Lichens are remarkable symbiotic species (Fig. 1) – they can survive the short-wavelength radiation conditions of outer space [1]. Furthermore, they can survive the extreme conditions of Antarctica, such as ozone deficiency and high altitude with virtually no nutrient supply. Of particular interest is that they can survive in conditions of high intensity short wavelength radiation [2] (UVA (315–400 nm), UVB (280–315 nm) and UVC (200–280 nm)) which would certainly damage man and, of course, under such conditions, in Antarctica, this radiation will lead to the generation of potentially damaging oxy-radicals. Clearly, lichens are able to avoid damage by such radical species. Lichen chromophores absorbing UVA, UVB and UVC might be molecule candidates for a new generation of sunscreens. Certainly, all of the existing commercial sunscreens have drawbacks, such as photo-instability (organic) and inconsistent coatings (inorganic). Furthermore, an ability to scavenge specific free radicals could also make these useful antioxidants as well as natural filters [3]. The aim of this study is to determine if the photophysical properties of such lichen-based

* Corresponding authors. Tel.: +44 0161 2754580 (R. Edge); tel.: +44 01782 583038; fax: +44 01782 610645 (T.G. Truscott). E-mail addresses: [email protected] (R. Edge), [email protected] (T.G. Truscott). 1011-1344/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2008.12.008

molecules, such as singlet oxygen and triplet state yield and radical reactivity, indicate that they may be worthy of further study as potential sunscreens and/or antioxidants. The blue/UV absorbers in lichens are typically polyaromatics containing hydroxy and carbonyl groups and the structures of the compounds studied are presented in Fig. 2. Two types of short-lived product can arise from absorption of UVA and UVB-excited states and radical ions and, both are potentially damaging. The techniques we have used in this study are nano-second pulse radiolysis and laser flash photolysis [4]. The laser-based technique generates excited states, including activated oxygen species (so-called singlet oxygen). Fast electron excitation (pulse radiolysis) generates large amounts of free radicals that, in the presence of oxygen, include a range of oxy-radicals. 2. Materials and methods The UVA/UVB absorbers were extracted from appropriate lichens (10 -chloropannarine from Erioderma leylandii, usnic acid from Xanthoparmelia farinose, calycine from Pseudocyphellaria bereberina, epiphorelic acid I and II from Coelopogon epiphorellus and vicanicine from Teloschistes flavicans as described previously [5,6]. The laser flash photolysis, singlet oxygen time-resolved luminescence and the pulse radiolysis apparatus have all been described previously [7–9].

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F. Boehm et al. / Journal of Photochemistry and Photobiology B: Biology 95 (2009) 40–45

Briefly, the pulse radiolysis experiments were carried out at the Free Radical Research Facility at STFC Daresbury Laboratory, UK, with a 12 MeV Radiation Dynamics Ltd. (UK) electron linear accelerator. We used single pulses of duration 0.22 to 2 ls and with a peak current of about 30 mA. The accelerator is normally operated at 10 Hz but the single pulse mode is achieved by modifying the pulses to the electron gun [9]. The detection system consists of a Xe arc lamp with a pulsing unit, high radiance Kratos monochromator and quartz optics. The sample cell, constructed by Spectrosil quartz, had an optical path length of 25 mm [10]. Optical transmissions at various wavelengths selected with the monochromator were observed as a function of time before and

after the pulse using photoelectric detection. The output of the photomultiplier (EMI9558Q) was displayed on a Tektronix TDS 380 digitising oscilloscope then transferred to a PC and fitted using in house software. Absorbed doses were determined from the transient ðSCNÞ 2 formation in air-saturated 10 mM KSCN [11,12]. Saturation of such solutions with N2O results in a doubling of the ðSCNÞ 2 yield. In water, three different primary radicals are produced by irradiation: solvated electrons (eaq) and hydrogen atoms (H), which are reducing species and hydroxyl radicals (HO), which are oxidising species. To generate almost exclusively HO, aqueous solutions were saturated with N2O which converts eaq into hydroxyl radicals by dissociative electron attachment:

Fig. 1. (a) Pseudocyphellaria bereberina, a lichen containing Calycine. (b) Xanthoparmelia farinosa a lichen containing usnic acid – see Fig. 2 for lichen structures.

O

O O

HO

OH

O O

HO OH

O

Usnic Acid

O

O

Cl

O

Cl

Cl

HO

O O Cl

O 1-Cl-Pannerine

Vicanicine O

O

O O

O O O

O O OH

HO

OH

O

OH

O

O HO

O O Calycine

O

Epiphorelic Acid I

Epiphorelic Acid II

Fig. 2. Structures of UV/Visible absorbing compounds extracted from lichens.

OH

O

OH

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F. Boehm et al. / Journal of Photochemistry and Photobiology B: Biology 95 (2009) 40–45

N2 O þ eaq þ H2 O ! N2 þ HO þ HO In some cases, when oxidising conditions are required, milder oxidants may be needed, because the hydroxyl radical can react with the solute via addition as well as electron transfer. Hydroxyl radicals can be converted into milder one-electron oxidants via the addition of bromide or azide ions:

HO þ Br ! Br þ HO Br þ Br ! Br 2 HO þ N3 ! N3 þ HO Pulse radiolysis was also used to generate the triplet states of usnic acid, 1-Cl-pannerine and calycine via energy transfer from triplet biphenyl in toluene. Using a range of oxygen concentrations allowed us to measure the rate constants for the reaction of these triplets with oxygen. In flash photolysis experiments, solutions were excited at 355 nm or 266 nm using a Spectron Q-switched Nd:YAG laser [7]. The detection system, similar to that used in the pulse radiolysis experiments, was supplied by Applied Photophysics Ltd. and consists of a Xe arc lamp and a pulsing unit, high radiance monochromator and quartz optics. The singlet oxygen yields were obtained in chloroform using time-resolved luminescence detection at 1270 nm with a Ge photodiode. The zero time luminescence intensity (S0), immediately after the laser pulse, is compared to a reference compound with a well established and high singlet oxygen quantum yield (UD). Phenalenone (UD = 0.98 ± 0.15) [13] was used and comparisons made under the same conditions for several laser energies. The organic solvents used were super purity from Romil Ltd., UK, the water was purified ‘in house’ via chromatography and the detergent, Triton X 100 was purchased from Fisher Ltd., UK. Normal standard buffers were use and samples were bubbled with argon, nitrous oxide, oxygen and air as required and described in the results. 3. Results and discussion

Calycine Usnic Acid Vicanicine 1-Cl-Pannerine Epiphorelic Acid I Epiphorelic Acid II

60000

-1

Molar Absorbtion Cofficient/M cm

-1

The UV absorption spectra of the lichen compounds studies are presented in Fig. 3 and the molar absorption coefficients given in Table 1 and are all rather high and comparable to most commercial sunscreens [14]. As can be seen from Fig. 3, calycine in particular and usnic acid, have the largest wavelength absorption bands and have more conjugation than the other lichen compounds studied–indeed calycine forms a yellow solution.

50000 40000 30000 20000 10000 0 250

300

350

400

450

500

Wavelength/nm Fig. 3. UV-visible absorption spectra in chloroform.

550

Table 1 Molar absorption coefficients at maximum absorptions in the UVA, UVB and UVC regions in chloroform. Lichen compound

k (nm)

e (dm3 mol1 cm1)

e (nm)

e/ (dm3 mol1 cm1)

Calycine Usnic acid Vicanicine 1-Cl-Pannerine Epiphorelic acid I Epiphorelic acid I

– 283 258 242 260 260

– 45,000 9250 22,100 15,388 15,065

388 340 342 342 312 312

22,800 12,300 570 3340 5800 5520

Calycine shows the strongest UVA absorption, though usnic acid, vicanicine and 1-Cl-pannerine also have some absorption in UVA. Usnic acid and the epiphorelic acids are UVB absorbers, with vicanicine and 1-Cl-pannerine absorbing mainly in the UVC region. In addition, calycine, with a yellow/orange colour has strong absorption in the visible region, extending to 470 nm. This may make calycine unacceptable as a sunscreen, on the other hand, carotenoids, are frequently studied as potential sunscreens and are much more intensly coloured than the lichen compounds [15]. As can be seen, this range of structures gives UVA only absorption, UVB only absorption and both UVA and UVB absorption. Such flexibility may be useful in the wide range of properties needed for future sunscreens. 3.1. Excited states and singlet oxygen As well as efficient UVA and UVB absorption, low yields of triplet states, low yields of singlet oxygen and triplet energy levels below that of the DNA bases such as thymine (sensitisers with triplet energies higher may sensitise cyclo-butane derivative formation possibly leading to mutations) are preferable for sunscreens. Ideally, if small amounts of triplet states [3L*] are formed the lifetime of these excited states should be as short as possible to minimise reactions with molecular oxygen [O2] to generate singlet oxygen ½1 O2 , i.e. to minimise the following reaction:

L þ O2 ! L þ 1 O2

3 

Laser excitation produced no detectable triplet states (indeed no transients were observed whatsoever) suggesting the yields from UVA/UVB absorption are either below about 0.05, the triplet lifetimes are below 10 ns or the molar absorption coefficients are extremely small. However, it is convenient to use pulse radiolysis to generate and study triplet states, because they are formed in high yield via a different mechanism (ion recombination) than in laser flash photolysis [4]. These pulse radiolysis experiments to generate triplet states can only be performed in non-polar solvents. The lichens are rather insoluble in non-polar solvents but sufficiently soluble (1  105 mol dm3) to generate the lichen triplets via energy transfer from biphenyl in toluene (this route allows generation of predominantly triplet states and the observation of oxygen quenching of these transients further confirms they are triplet states). This allowed us to determine the triplet lifetimes and the triplet–singlet difference absorption spectra for calycine, usnic acid and 1-Cl-pannerine – these are given in Fig. 4 and Table 2 below: in addition, the rate constants for oxygen quenching of the triplets were found to be in the region of 2.5 ± 0.6  109 dm3 mol1 s1 (see Table 2), which is near the 1/9th diffusion-controlled limit for such reactions [16]. In other solvents, especially chloroform, the solubility is much higher and is typically about 102 mol dm3. Thus chloroform was a suitable solvent to prepare the detergent (Triton X 100) micellar systems for the radical studies of lichen molecules via pulse radiolysis (water-based solvents are preferred for such radical studies), see below.

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F. Boehm et al. / Journal of Photochemistry and Photobiology B: Biology 95 (2009) 40–45

Table 3 The singlet oxygen quantum yield of six lichen compounds in chloroform and, for comparison, those of several commercial sunscreens with the solvent given in parentheses.

0.02

0.00 Absorbance Change

Absorbance Change

0.04

-0.02

-0.04

0.006 0.004 0.002 0.000 -0.002

0

20 Time/µs

40

-0.06 300 350 400 450 500 550 600 650 700 750 Wavelength/ nm Fig. 4. Triplet minus singlet difference absorption spectra for three lichen absorbers in toluene (calycine–black squares; usnic acid–red circles; 1-Cl-pannerine–green triangles). The inset shows typical transient decays (calycine–black, 520 nm; usnic acid–red 480 nm; 1-Cl-pannerine–green 470 nm). (For interpretation of the references in colour in this figure legend, the reader is referred to the web version of this article.)

For calycine, usnic acid and 1-Cl-pannerine the triplet energy levels (ET) are shown to be below 142.6 kJ mol1 (the ET of biphenyl) and they would be unable to damage DNA via energy transfer to thymine (ET = 163.3 kJ mol1). However, it is important to note that no energy transfer was apparent for vicanicine or the epiphorelic acids (I and II), indicating that they have triplet energy levels greater than biphenyl and, as such, they may be able to generate thymine triplets and cause DNA damage. This result may suggest that these three phenolic lichen molecules need further investigation before they can be considered as suitable sunscreens for man. For 1-Cl-pannerine there is no ground state absorption above 380 nm, therefore, this difference spectrum is equivalent to the triplet absorption spectrum above this wavelength. However, for usnic acid and calycine ground state depletion will contribute to these difference spectra. Nevertheless, as can be seen from Fig. 4 and Table 2, the triplet states of all three potential sunscreens are sufficiently long-lived to, for example, generate singlet oxygen. Thus, for these molecules to be useful it is necessary that the triplet and singlet oxygen yield are very small. Pulse radiolysis also allows us to obtain the triplet molar absorption coefficients (in wavelength regions where there is no ground state absorption – Table 2) using DADonor/DeDonor = DAAcceptor/ DeAcceptor (DeDonor = 27000 dm3 mol1 cm1 at 360 nm) where donor refers to the biphenyl triplet and acceptor to the lichen triplet, with appropriate corrections for the decay of triplet donor by routes other than energy transfer. These results show that the laser excitation is not generating significant quantities of triplet states of the lichen molecules because their molar absorption coefficients and lifetimes are sufficiently high for them to be readily detected. Thus, the pulse radiolysis experiments show that both the lifetime and absorption properties of the potential sunscreen are not a factor

Lichen compound or sunscreen

UD

Calycine Usnic acid Vicanicine 1-Cl-Pannerine Epiphorelic acid I Epiphorelic acid II 4-t-Butyl-40 -methoxy dibenzoylmethane [15] 2-Ethylhexyl-p-methoxy cinnamate [15] 3-(40 -Methylbenzylidine camphor) [15] p-Amino benzoic acid (PABA) [15] Octyl dimethyl PABA [15] Terephthalylidine dicamphor sulphonic acid [17]

0.003 0.065 0.041 0.012 0.021 0.051 0.0015 (C6D6) 0.0012 (C6D6) 0.0023 (C6D6) 0.0029 (C6D6) 0.018 (C6D6) 0.09 (acetonitrile)

in the very low triplet yields indicated by our laser experiments. This, in turn, suggests these molecules may be useful sunscreens. The singlet oxygen yields were obtained in chloroform using time-resolved luminescence detection as described above and Table 3 gives the values obtained. As can be seen, UD is low for all compounds with calycine having a value close to that of many commercial sunscreens [14,17]. For comparison the reported values for such sunscreens are quoted in Table 3. Possibly, some of the lichen compounds have too high a singlet oxygen quantum yield to be of use as a commercial sunscreen. On the other hand, it must be realised that such values, in simple solvents, are only an indicator - the value in the ‘real-life’ situation could be much lower. Indeed, that must be the case for the UVA sunscreen terephthalylidine dicamphor sulphonic acid, where the reported value, in acetonitrile (0.09) is higher than any of the lichen compounds studied in this work [17]. Overall, these values are controlled by three factors. The triplet quantum yield (UT) the fraction of triplets quenched by oxygen (SQ) and the fraction of encounter complexes which yield singlet oxygen (SD).

Ud ¼ UT SQ SD Since the triplet lifetimes are sufficiently long to allow energy transfer to oxygen, and, see Table 2, such energy transfer is indeed very efficient, the value of SQ must be close to 1. Thus, the low UD must be a consequence of low UD or SD values. The carotenoids are well known ‘natural sunscreens’ [15] occurring in all green plants and these molecules not only quench any chlorophyll triplets produced but also any singlet oxygen produced [18] and it is of interest to see whether the lichen compounds behave in the same manner. We used methylene blue as a sensitiser to produce singlet oxygen (this allowed us to use 532 nm laser excitation where the lichen compounds do not absorb). Also, by using chloroform as solvent, we could use high concentration of the lichen compounds. However, even at concentrations of 1  103 mol dm3 we could observe no quenching of the singlet oxygen. This allows a limit to be calculated of the singlet oxygen quenching rate constant (kq). 3

1

K q  7  105 dm mol Table 2 The wavelengths of maximum absorption, molar absorption coefficients, lifetimes and rate constants for reaction of oxygen of the triplets of three lichen compounds in toluene. 3

1

1

3

Lichen compound

kmax (nm)

eT (dm mol cm )

sT (ls)

k (dm mol

Calycine Usnic acid 1-Cl-Pannerine

510 460 390

7800 4700 5800

35.7 8.5 16.2

1.9  109 3.1  109 2.1  109

1

s

1

)

s1

Thus, the lichen compounds generate virtually no singlet oxygen nor react with it. 3.2. Radicals Radical studies were undertaken on calycine, usnic acid and 1Cl-pannerine in 2% Triton X 100 aqueous micellar solutions. Sev   eral oxidising radicals (NO2 ; Br 2 ; N3 ; OH andðSOÞ4 ) were studied with 1-Cl-pannerine and we found no reaction, i.e. the radical cat-

F. Boehm et al. / Journal of Photochemistry and Photobiology B: Biology 95 (2009) 40–45

ion was not formed even from the extremely oxidising sulphate radical (E = 2.43 V). Furthermore, 1-Cl-pannerine and the other lichen compounds studied did not react with the superoxide radical. For calycine and usnic acid we generated the radical cations as described below. Also, all three lichen compounds were readily reduced to the corresponding radical anions – see below. 3.3. Radical cations For calycine and usnic acid the radical cation (one-electron oxidised) spectra are shown in Fig. 5: As can be seen the kmax is 530 nm for calycine and 520 nm for usnic acid radicals. However, the transient is much weaker for usnic acid suggesting a much lower molar absorption coefficient for the radical cation of this absorber. (The corresponding data for the radical anions, see below, also exhibits a weaker transient for usnic acid.) Vitamin E quenches these radical cations with rate constants of 2.2  107 dm3 mol1 s1 and 6.8  107 dm3 mol1 s1 for calycine and usnic acid respectively. For Trolox (a water soluble vitamin E analogue) the corresponding values are somewhat higher 2.0  108 dm3 mol1 s1 and 1.9  108 dm3 mol1 s1, respectively. For Ascorbic acid the radical cations of calycine is quenched with a kq value of 3  107 dm3 mol1 s1. For usnic acid we could not obtain a totally consistent value but it is similar to or somewhat faster than that for calycine. Overall, these results suggest there could be benefits in using vitamins E and C with these potential sunscreens, since this would remove any radical cations produced. Interestingly, the values for Trolox (in the aqueous phase) are more efficient than for vitamin E (in the lipid phase). Also these results suggest a possible use for the polyaromatics extracted from lichens as antioxidants, particularly in conjuction with vitamins E and C. 3.4. Radical anions Calycine, usnic acid and 1-Cl-pannerine all reacted with the aqueous electron and CO 2 (generated via hydroxyl radical reaction with formate) to produce the one-electron reduced products (radical anions). Typical spectra are shown in Fig. 6, with kmax = 450 nm for calycine, 360 nm for usnic acid and 480 nm for 1-Cl-pannerine (very weak transient).

Absorbance Change

0.14 0.12 0.10 0.08 0.06 0.04 0.02

0.06

Absorbance Change

44

0.05 0.04 0.03 0.02 0.01 0.00 -0.01 300 350 400 450 500 550 600 650 700 750

Wavelength/nm Fig. 6. Calycine (black squares), usnic acid (red circles) and 1-Cl-pannerine (green triangles) radical anion spectra in 2% Triton X 100 aqueous micellar solution at pH 6. (For interpretation of the references in colour in this figure legend, the reader is referred to the web version of this article.)

4. Conclusions The photophysical properties of six polyaromatic, potential sunscreens, extracted from lichens, have been measured and suggest that some of these compounds may have suitable photo-properties for further research into their value as sunscreens. The UV/Vis spectra are reported in Fig. 1 and all six were shown to produce no transients following laser excitation. Three of the molecules quench biphenyl triplets and we have reported the corresponding triplet spectra and lifetimes. The other three (vicanicine and epiphorelic acid I and II) did not quench biphenyl triplets, possibly suggesting their triplet energy levels are too high for them to be useful sunscreens, since they may be capable of generating thymine triplets leading to DNA damage. The singlet oxygen quantum yields are reported for all six lichen-based compounds, with values ranging over a factor of 20, indicating possibly only calycine and 1Cl-pannerine are suitable for consideration as potential sunscreens. On the other hand, as noted above, all values are below that of the commercially used terephthalylidine dicamphor sulphonic acid and all may be worthy of future study as sunscreens. In summary, none of the lichen compounds produce significant yields of singlet oxygen, however, neither do they act as sufficient singlet oxygen quenchers if this damaging species should arise via some other energy transfer route. The radical cations of calycine and usnic acid were efficiently scavenged by both vitamin C and vitamin E suggesting combinations with these small molecule antioxidants may be useful in sunscreen and/or antioxidant formulations. The radical anions of calycine, usnic acid and 1-Cl-pannerine are generated via the formate radical suggesting radical anions may arise if reducing species are present. Finally, our photophysical studies, together with the studies of Rancan et al. [6] suggest that calycine is the most worthy of further study.

0.00 Acknowledgments

-0.02 -0.04 300 350 400 450 500 550 600 650 700 750

Wavelength/nm Fig. 5. Calycine (black squares), usnic acid (red circles) radical cation spectra in 2% Triton X 100 aqueous micellar solution at pH 6.4. (For interpretation of the references in colour in this figure legend, the reader is referred to the web version of this article.)

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