THOMAS 2022 Archivage

THÈSE
UNIVERSITE DE PAU ET DES PAYS DE L’ADOUR

École doctorale ED 211
Présentée et soutenue le 11 Juillet 2022

par Martin George Thomas
pour obtenir le grade de docteur
de l’Université de Pau et des Pays de l’Adour
Spécialité : Chimie
Study of the impact of different water matrices and photosensitizers on the

photostability of mycosporines, mycosporine-like amino acid and their
chitosan-based derivatives
MEMBRES DU JURY
RAPPORTEURS
• Cécile PICARD, Professor / Université Le Havre, France
• Eric FOUQUET, Professor / Université de Bordeaux, France
EXAMINATEUR
• Asier MARTINEZ, Dr / Centre de Recherche CIDETEC, Espagne
ENCADRANTE
• Sylvie BLANC, Dr, HDR / Université de Pau et des Pays de l’Adour, France
DIRECTEURS
• Thierry PIGOT, Professor / Université de Pau et des Pays de l’Adour, France
• Susana de MATOS FERNANDES, Dr, HDR / Université de Pau et des Pays de l’Adour, France
Présentée et soutenue le 11 Juillet 2022
par Martin George Thomas
pour obtenir le grade de docteur

de l’Université de Pau et des Pays de l’Adour
Spécialité : Chimie
Study of the impact of different water matrices and photosensitizers on the

photostability of mycosporines, mycosporine-like amino acid and their
chitosan-based derivatives
MEMBRES DU JURY
RAPPORTEURS
• Cécile PICARD, Professor / Université Le Havre, France
• Eric FOUQUET, Professor / Université de Bordeaux, France
EXAMINATEUR
• Asier MARTINEZ, Dr / Centre de Recherche CIDETEC, Espagne
ENCADRANTE
• Sylvie BLANC, Dr, HDR / Université de Pau et des Pays de l’Adour, France
DIRECTEURS
• Thierry PIGOT, Professor / Université de Pau et des Pays de l’Adour, France
• Susana de MATOS FERNANDES, Dr, HDR / Université de Pau et des Pays de l’Adour, France
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Abstract
Due to the increasing amount of UV filters being used for the protection from solar UV radiation
and to the toxic effects of these compounds, nowadays there is an important need to look for
alternatives. This thesis mainly focuses on investigating the photostability of natural UV-
protective sunscreens called mycosporines like gadusol (enolate form, λMax = 298 nm) and
mycosporine-serinol (λMax = 310 nm), and mycosporine-like amino acids (MAAs) like
shinorine, porphyra-334 (λMax = 334 nm), and palythine (λMax = 320 nm), and their chitosan-
based derivatives using shinorine (CS-SH) or porphyra-334 (CS-P334). Interestingly, as all
these compounds and materials have their maximum absorption λMax in the UV-A or UV-B
regions and present high absorption capacity, their photostability have been studied with a solar
simulator in different natural water matrices (river, ocean and estuary waters) and in the
presence of photosensitizers namely riboflavin and porphine. Their photostability was
determined in terms of quantum yield (Φ). This study revealed that these mycosporines and
MAAs were extremely photostable with very low quantum yield of photodecomposition in pure
water and least stable in river water due to the presence of large amount of coloured dissolved
organic matter. For instance, in pure water, among the mycosporines, mycosporine-serinol
(Φ=2.3x10-5) was photostable relative to gadusolate (Φ=10.0x10-5) probably because of steric
factors. Regarding the MAAs, shinorine (Φ=0.14x10-5) and porphyra-334 (Φ=0.18x10-5) were
the most stables and palythine (Φ=6.5x10-5) was the least stable due to its protonated form at
acidic pH. The photodegradation of all these molecules were higher in natural matrices relative
to pure water due to the generation of reactive oxygen species on solar irradiation. Singlet
oxygen was identified using furfuryl alcohol probe in river, estuary and ocean water via high
performance liquid chromatography. Some photoproducts generated after irradiation of the
MAAs were analysed and identified by using Liquid Chromatography coupled with Mass
Spectrometry. The study also pointed out that chitosan conjugates were extremely photostable
relative to the free molecules after successful conjugation, in particular in the presence of
photosensitizers.
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Résumé de la thèse
En raison de la quantité croissante de crèmes solaires utilisées de nos jours pour la protection
contre les rayons UV solaires et les effets potentiellement toxiques de ces composés, il est
urgent de rechercher des alternatives. Cette thèse se concentre principalement sur l'étude de la
photostabilité de mycosporines connues comme étant protectrices contre les UV-(A ou B)
comme le gadusol (forme énolate, λMax = 298 nm) et le mycosporine-serinol (λMax = 310 nm),
et les mycosporines comportant une unité acide aminé (MAA) comme la shinorine, le porphyra-
334 (λMax = 334 nm), palythine (λMax = 320 nm) ainsi que des matériaux chitosane avec
shinorine (CS-SH) et chitosane avec Porphyra-334 (CS-P334). Tous ces composés et matériaux
ont leur absorption maximale λMax dans les régions UV-A ou UV-B et leur photostabilité a été
étudiée grâce à un simulateur solaire dans différentes matrices d'eau (rivières, océaniques et
estuariennes) et en présence des photosensibilisateurs comme la riboflavine ou la porphine.
Leur photostabilité a été comparée grâce à la détermination du rendement quantique de
photodégradation (Φ). L'étude a révélé que ces molécules de mycosporines et de MAA étaient
extrêmement photostables avec un très faible rendement quantique de photodécomposition.
Dans l’eau pure, parmi les mycosporines, le M-serinol (Φ=2.3x10-5) était plus photostable par
rapport au gadusol (Φ=10.0x10-5) probablement à cause de facteurs stériques. Dans les MAA,
la shinorine (Φ=0.14x10-5) et le porphyra-334 (Φ=0.18x10-5) étaient les plus stables et la
palythine (Φ=6.5x10-5) était la moins stable en raison de sa forme protonée à pH acide. La
photodégradation de toutes ces molécules était plus élevée dans les matrices naturelles par
rapport à l'eau pure en raison de la génération d'espèces réactives de l'oxygène lors de
l'irradiation solaire dans ces matrices. L'oxygène singulet a été identifié à l'aide d'une sonde
d'alcool furfurylique dans l'eau des rivières, des estuaires et des océans par chromatographie
liquide à haute performance. Nous avons pu analyser et identifier certains des photoproduits
générés en utilisant la chromatographie liquide couplée à la spectrométrie de masse (LC-MS).
L'étude a également souligné que les conjugués de chitosane gardaient leur photostabilité par
rapport aux molécules libres après une conjugaison réussie, en particulier en présence des
photosensibilisateurs.
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List of Publications
This thesis is written based on the following publications to be submitted in

scientific journals
1. Understanding the effect of natural water matrices and photosensitizers on the

photostability of gadusol and mycosporine-serinol – a kind of natural molecules that
absorb on UV-B (Chapter 2, In preparation)
M.G. Thomas, S. Blanc, M. Le Bechec, J-M. Sotiropoulos, T. Pigot, S.C.M. Fernandes
2. A roadmap to UV-A-absorbing natural sunscreens – photostability in different natural

water matrices and with photosensitizers, and analysis of the ensuing photoproducts
(Chapter 3, In preparation)
M.G. Thomas, M. Parailloux, S. Blanc, M. Le Bechec, J-M. Sotiropoulos, R. Lobinski, T. Pigot,
S.C.M. Fernandes
3. Influence of photosensitisers on the photodegradation of chitosan/mycosporine-like

amino acid derivatives in aqueous solutions - pure and river water (Chapter 4, In
preparation)
M.G. Thomas, F. Samalens, S. Blanc, T. Pigot, S.C.M. Fernandes
Other Publications in scientific journals not included in this thesis
1. Claverie, Marion, Colin McReynolds, Arnaud Petitpas, Martin Thomas, and Susana
Fernandes, Marine-derived polymeric materials and biomimetics: An
overview, Polymers, 5 (2020) 1002
https://doi.org/10.3390/polym12051002
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2. François Samalens, Martin Thomas, Marion Claverie, Natalia Castejon, Yi Zhang, Thierry
Pigot, Sylvie Blanc, Susana C. M. Fernandes, Progresses and future prospects in
biodegradation of marine biopolymers and emerging biopolymer-based materials for
sustainable marine ecosystems, Green Chemistry, 24, (2022) 1762
https://doi.org/10.1039/D1GC04327G
Conferences
Oral presentations in international/national conferences
1. Martin Thomas, Unnimaya Veettil, Sylvie Blanc, Thierry Pigot, Susana C.M. Fernandes,
Study of the photostability of bio-inspired biopolymers-conjugated with bioactive
molecules in different aquatic environments, 7th International Polysaccharide Conference
(EPNOE), October 2021, Nantes, France
Posters in international/national conferences
1. Marion Claverie, Colin Mcreynolds, Martin Thomas, Arnaud Petitpas, Susana C.M.
Fernandes, Development of marine and bio-inspired polymer materials with low environmental
impact, GDR-Polymers & Oceans, June 2019, Créteil, France.
2. Bascans, N. Brugerolle de Fraissinette, Martin Thomas, C. McReynolds, N. Castejon, F.

Samalens, S. Olza, M. Ekoule, U. T. Veettil, A. Petitpas, J-R Dalle, S. C. M. Fernandes, From
the Ocean and for the Ocean: MANTA Chair, an example of bio-inspired research for the
development of a circular Blue Bioeconomy, European Federation of Biotechnology
(EFB2021), 10-14 May 2021, virtual conference
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Acknowledgements
I Martin George Thomas, the author of this PhD Thesis, is grateful to the French government
for funding my predoctoral fellowship. This PhD thesis has been co-funded by Campus France
through ‘’Make My Planet Great Again’’ programme (grant agreement # MOPGA-927921L)
and the Chair E2S UPPA MANTA: Marine Materials for the entire tenure 2018-2022.
I express my sincere gratitude to my supervisors Prof Dr.Thierry Pigot and Dr.Susana CM

Fernandes for their continuous guidance and support thought out the period of my research
work. I am indebted to the director Dr.Ryszard Lobinski and deputy director Dr.Cécile
Courrèges of the Institut des Sciences Analytiques et de Physico-Chimie pour l'Environnement
et les Matériaux at the Université de Pau et des Pays de l'Adour (UPPA) for giving me access
to the research laborataries.
I also express my thanks to Dr.Sylvie Blanc and Dr.Mickael Le Bechec for their mentorship
and providing the required assistance whenever needed. I also thank Manta Marine Materials
research group and my fellow doctoral students Mr.Colin McReynolds, Ms.Sheila Ozla , Mr.
François Samalens, Mr.Arnaud Pettipas, Research Engineer Ms. Nelly Brugerolle and post
doctoral fellows Dr.Marion Clavarie, Dr.Amandine Adrien, Dr.Natalia Castejon, and the
adjunct faculty Dr.Yi Zhang. I also thank Dr. Maroussia Parailloux for the LC-MS
experimentation and analysis. I also thanks the other faculty members, research students and
administrative staff for the cooperation and support that they have extended to me during my
thesis work.
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General Context and Objectives
Lowering the devastating impact of the emerging pollutants in the marine environment
especially sunscreen products is one of the major challenges in the present century. The growing
consciousness of the risks associated with skin exposure to ultraviolet (UV) radiation has led to
an increased use of inorganic and organic sunscreens and, consequently has led to the
introduction of these chemicals into the marine environment leading to a series of
consequences. It is thus, particularly challenging to develop biobased and safe UV-absorbing
materials derived from natural UV protective molecules like mycosporines and mycosporine-
like amino acids (MAAs) that are present in marine organisms like algae, cynobacteria, reef
fish, etc, and have been extracted from algal biomass. These molecules are photostable, do not
generate reactive oxygen species when exposed to UV radiation and have multifunctionality
both in terms of prevention values (antioxidant, sunscreen protection and photoprotection
attributed to the high molar absorptivity) and ecological significance (intracellular nitrogen
storage, etc).
This thesis mainly focuses on investigating the photodegradation of mycosporines,

mycosporine-like amino acids (MAAs) and chitosan-MAAs derivatives, that were
prepared to mimic the functionality of some marine organisms as photoprotective and
environmentally sustainable UV-absorbing materials. This research work mainly emphases
on investigating the photodegradation of mycosporines like gadusol or mycosporine-serinol,
and mycosporine-like amino acids namely shinorine, porphyra-334 and palythine and their
conjugates in pure water and different natural water matrices like river, estuary and ocean and
in the presence of photosensitisers.
To achieve this main objective, the specific objectives of the present work are:
- To irradiate the different mycosporine-based aqueous solutions (pure, river, stuary and
ocean) using a solar radiation simulator at different times;
- To irradiate the different mycosporines or MAAs solutions on pure water in the presence
of the riboflavin and porphine (photosensitisers) with or without azide quencher at
different times;
- To determine the quantum yield of the different compounds;
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- To assess the indirect determination of the singlet oxygen steady state concentration by
quenching experiments;
- To identified and analyse the photoproducts by liquid chromatography mass
spectroscopy (LC-MS);
- To graft MAAs like shinorine and pophyra-334 on chitosan backbone by chemical
coupling via amidation reaction to assess the photostability of these conjugates in pure
water and river water and in the presence of riboflavin and porphine.
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Abbreviations
UV - Ultraviolet
IPD - Immediate pigment darkening
PPD - Persistent pigment darkening
BP-3 - (3-Benzophenone)
MBC-4 - (4-Methylbenzylidene camphor)
BC-3 - 3-benzylidene-camphor
OMC - Octyl-methoxycinnamate
SML - Surface Micro layer
WWTP - Waste water treatment plants
EHMC - 2-ethyl-hexyl-4-trimethoxycinnamate
OCR - Octocrylene
IC50 - Half maximal inhibitory concentration
EC50- Half maximal effective concentration
DPPH - 1,1-diphenyl-2-picrylhydrazyl)
ORAC - Oxygen Radical Absorbance Capacity
ABTS - 2, 2'-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid
ROS - Reactive oxygen species
CTAC - Cetrimonium chloride
SDS - Sodium dodecyl sulfate
ISC - Intersystem Crossing
SPF-Sun Protection Factor
DNA-Deoxyribonucleic Acid
MAA - Mycosporine like Amino Acids
RF - Riboflavin
PPY - Porphine
SH - Shinorine
P334 - Porphyra
PNE - Palythine
Gd - Gadusol
MseOH – Mycosporine Serinol
CDOM - Coloured Dissolved Organic Matter
FFA- Furfuryl Alcohol
HOMO- Highest Occupied Molecular Orbital
LUMO- Lowest Occupied Molecular Orbital
QE (Φ) - Quantum yield of photodegradation
ATR-FTIR - Fourier Transform Infrared Spectroscopy
NMR - Nuclear Magnetic Resonance
DOSY- Diffusion Ordered Nuclear Magnetic Resonance
CS-Chitosan
CS-SH-Chitosan-shinorine conjugates
CS-P334-Chitosan-porphyra-334 conjugates
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Contents
Chapter 1 : Literature Riview
1. Introduction ....................................................................................................................................... 21
1.1 Solar Radiation ........................................................................................................................... 21
1.2 Effects of UV radiation on human skin ...................................................................................... 23
2. Conventional Sunscreens and UV filters ........................................................................................... 25
2.1 Physical/Inorganic UV filters ..................................................................................................... 26
2.2 Chemical/Organic UV filters...................................................................................................... 26
2.3. Common Inorganic UV filters................................................................................................... 27
2.4. Common Organic UV filters ..................................................................................................... 29
2.5 Consequences of conventional sunscreens on humans .............................................................. 30
2.6 Impact of conventional sunscreens in marine ecosystems ......................................................... 31
2.7 Distribution of conventional sunscreens in various marine ecosystems .................................... 32
2.8 Ecotoxicological issues and impact of organic UV filters ......................................................... 34
2.9 Ecotoxicological issues and impact of inorganic UV filters ...................................................... 36
3.Natural UV protective Molecules - Mycosporine and Mycosporine-like Amino Acids (MAAs) ..... 38
3.1 Introduction ................................................................................................................................ 38
3.2 Occurrence and Environmental distribution............................................................................... 42
3.3 Biosynthesis pathway of Mycosporines and MAAs .................................................................. 44
3.4 Evolution of mycosporines/MAAs as UV-absorbing compounds ............................................. 45
3.5 Isolation and Identification of mycosporines and MAAs........................................................... 45
3.6 The different roles of Mycosporines and MAAs........................................................................ 46
3.7 Photoprotection Mechanism of Mycosporines and MAAs ........................................................ 50
3.8 Photostability of Mycosporines: Gadusol .................................................................................. 51
3.9 Photostability of Mycosporines like Amino Acids: Shinorine, Porphyra and Palythine ........... 53
3.10 Mycosporine and MAAs-based products and materials for UV filters .................................... 58
4.Conclusion .......................................................................................................................................... 60
5. References ......................................................................................................................................... 63
Chapter 2 : Photostability of Mycosporines M-serinol and Gadusol in Aquatic Matrices
1.Introduction ........................................................................................................................................ 78
2. Experimental Part .............................................................................................................................. 80
2.1. Materials and Chemicals ........................................................................................................... 80
2.2. Irradiation experiments ............................................................................................................. 80
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2.3. Determination of the photodegradation quantum yield ............................................................. 82
2.4. Indirect determination of singlet oxygen steady state concentration in natural matrices by
quenching experiments ..................................................................................................................... 82
2.5. Antioxidant capacity analysis by DPPH radical-scavenging assay ........................................... 83
3. Results and Discussion ...................................................................................................................... 83
3.1 Photostability in Pure, River, Estuary and Ocean Water............................................................ 85
3.2 Photostability with Photosensitisers Riboflavin and Porphine ................................................... 90
3.3 Quantification of Singlet Oxygen with Furfuryl Alcohol probe ................................................ 97
3.4 Antioxidant Activity: DPPH Assay .......................................................................................... 100
4. Conclusion ....................................................................................................................................... 102
5. References ....................................................................................................................................... 102
Chapter 3 : Comparison of photostability of mycosporines like amino acids in different

natural matrices
1. Introduction ..................................................................................................................................... 112

2. Experimental Part ............................................................................................................................ 114
2.1. Materials and Chemicals ......................................................................................................... 114
2.2. Irradiation experiments ........................................................................................................... 115
2.3. Determination of the photodegradation quantum yield ........................................................... 116
2.4. Indirect determination of singlet oxygen steady state concentration in natural matrices by
quenching experiments ................................................................................................................... 117
2.5. Detection of photoproducts by Liquid Chromatography Mass Spectroscopy (LC-MS) ......... 117
3. Results and Discussion .................................................................................................................... 119
3.1 Photostability in Pure, River, Estuary and Ocean Water.......................................................... 119
3.2 Photostability with Photosensitisers Riboflavin and Porphine ................................................. 129
3.3 Quantification of Singlet Oxygen with Furfuryl Alcohol probe by HPLC .............................. 139
3.4 Photostability of MAA-precursors: Characterization of photoproducts by mass spectrometry142
3.5 Structural identification of MAA-Photoproducts ..................................................................... 144
4. Conclusion ....................................................................................................................................... 148
5. References ....................................................................................................................................... 149
Chapter 4: Photoprotective Polysaccharides Based on Chitosan and Mycosporine Like

Amino Acids
1. Introduction ..................................................................................................................................... 160

2.Materials and Methods ..................................................................................................................... 161
2.1. Materials and Chemicals ......................................................................................................... 161
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2.2. Synthesis and characterization of the chitosan-mycosporines like amino acid derivatives .... 162
2.3 Photostability experiments of the chitosan-MAA derivatives.................................................. 164
3. Results and Discussion .................................................................................................................... 165
3.1 Synthesis of the materials ......................................................................................................... 165
3.2 Characterisation by Fourier Transform Infrared Spectroscopy (FTIR).................................... 167
3.3 Characterisation by Nuclear Magnetic Resonance Spectroscopy (NMR)................................ 168
3.4 Characterisation by UV Spectroscopy ..................................................................................... 170
3.5 Photostability of Chitosan Conjugates vs Pure MAA molecules ............................................. 171
3.6 Photostability of Chitosan Conjugates vs Pure MAA molecules with Photosensitizers
Riboflavin and Porphine................................................................................................................. 173
3.7 Photostability of Chitosan Conjugates in a natural photosensitizer: Acidified River Water vs
Pure MAA molecules ..................................................................................................................... 177
4. Conclusion ....................................................................................................................................... 180
Reference ............................................................................................................................................ 180

Conclusion…………………………………………………………………………………………...187
Future Perspectives ........................................................................................................................... 191
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Chapter 1
Literature Review
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1. Introduction .......................................................................................................................... 21
1.1 Solar Radiation .............................................................................................................. 21
1.2 Effects of UV radiation on human skin ......................................................................... 23
2. Conventional Sunscreens and UV filters.............................................................................. 25
2.1 Physical/Inorganic UV filters ........................................................................................ 26
2.2 Chemical/Organic UV filters ......................................................................................... 26
2.3. Common Inorganic UV filters ...................................................................................... 27
2.3.1 Titanium dioxide (TiO2) ........................................................................................ 27
2.3.2 Zinc oxide (ZnO) ................................................................................................... 28
2.4. Common Organic UV filters ........................................................................................ 29
2.4.1 Benzophenone ........................................................................................................ 29
2.4.2 Methylbenzylidene Camphor ................................................................................. 30
2.5 Consequences of conventional sunscreens on humans.................................................. 30
2.5.1 Organic................................................................................................................... 30
2.5.2 Inorganic ................................................................................................................ 31
2.6 Impact of conventional sunscreens in marine ecosystems ............................................ 31
2.7 Distribution of conventional sunscreens in various marine ecosystems ....................... 32
2.8 Ecotoxicological issues and impact of organic UV filters ............................................ 34
2.8.1 Bioaccumulation .................................................................................................... 34
2.8.2 Ecotoxicological Evaluation .................................................................................. 35
2.9 Ecotoxicological issues and impact of inorganic UV filters ......................................... 36
3.Natural UV protective Molecules - Mycosporine and Mycosporine-like Amino Acids
(MAAs) .................................................................................................................................... 38
3.1 Introduction ................................................................................................................... 38
3.2 Occurrence and Environmental distribution .................................................................. 42
3.3 Biosynthesis pathway of Mycosporines and MAAs...................................................... 44
3.4 Evolution of mycosporines/MAAs as UV-absorbing compounds ................................ 45
3.5 Isolation and Identification of mycosporines and MAAs .............................................. 45
3.6 The different roles of Mycosporines and MAAs ........................................................... 46
3.6.1 As photoprotectants ............................................................................................... 47
3.6.2 As sunscreen compounds ....................................................................................... 47
3.6.4 As intracellular nitrogen storage ............................................................................ 48
3.6.5 In thermal and dessication stress............................................................................ 49
3.6.6 In fungal reproduction ........................................................................................... 49
3.6.7 In wound healing ................................................................................................... 49
3.7 Photoprotection Mechanism of Mycosporines and MAAs ........................................... 50
3.8 Photostability of Mycosporines: Gadusol...................................................................... 51
3.9 Photostability of Mycosporines like Amino Acids: Shinorine, Porphyra and Palythine
............................................................................................................................................. 53
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3.10 Mycosporine and MAAs-based products and materials for UV filters ....................... 58
4.Conclusion ............................................................................................................................. 60
5. References……………………………………………………………………………... 63
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Chapter 1
Literature Review
1. Introduction
1.1 Solar radiation
In the solar spectrum the ultra violet rays belong to 100-400 nm region. The UV rays emanating
from the sun can further be subdivided into three main regions: UV-A (with lower energy and
λ-(320 nm to 400 nm), UV-B (290-320 nm) and UV-C (100-290 nm)1 with higher energies as
shown in Figure 1.
Figure 1: The Electromagnetic Spectra (100 nm - 400 nm) (Reproduced from2).
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The UV-A rays can be further subdivided into two bands i.e., long UV- Al (340-400 nm) and
short UV-A2 (320-340 nm). These three regions have different irradiance (The power of
electromagnetic radiation per unit area, expressed in watts per square meter (Wm -²) and solar
variability (the change in the amount of radiation emitted by the Sun) as shown in Table 1.
Table 1: Sub-regions of the UV spectrum with corresponding parameters

(Reproduced from 3).
UV Region Irradiance Solar Wavelength

(Wm-2) Variability (nm)
(%)
UV-C 6.4 1- 2 100 - 280
UV-B 21.1 1 280 - 350
UV-A 87.5 1 315 - 400
UV-A and UV-B rays are different in term of spectra of action and are effective in causing a
series of biological reactions. The perilous region of the spectra of solar erythema lies
predominantly in the UV-B band region having a peak around 295 nm and diminishes towards
the UV-A region. UV-B radiation is 1000 times more erythemic (redness of the skin caused by
dilatation, congestion of the capillaries) relative to UV-A radiation. In normal conditions UV-
A radiations does not cause sunburn.
Solar rays emanating from the sun get altered by the ozone layer present in the stratosphere in
the earth’s atmosphere. This layer has a photoprotective function that absorbs the high-energy
radiation that falls in the UV-C band (200-280 nm) and short-wave UV-B band (280-290 nm)1.
The ratio of UV-A to UV-B that reaches the earth’s surface is 20:1. UV-A rays are found to
transmits through window glasses and could be found indoors at very low attenuated doses 4, 5.
Irradiance depends of factors like latitude, aerosols and clouds. The influence of clouds on
irradiance is due to reflection and absorption of the irradiance by cloud particles and depends
strongly on the volume, shape, thickness, and composition of the clouds. UV exposure can take
Exposure due to diffuse irradiation constitute more than 80% on cloudy days. Diffuse and
reflected radiations mainly consists of wavelengths in the UV-A region 5, 2
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1.2 Effects of UV radiation on human skin
The most common acute reaction caused by UV rays is sunburn of white skin also called as
solar erythema. The penetrative effect of UV-A rays on the skin is higher relative to UV-B rays
i.e. about 30% reach the epidermis and 20% reach the dermis and rest 1% reach the
subcutis/hypodermis layer (Figure 2).
Figure 2: Subregions of the UV spectrum penetrating the human skin (Reproduced from6).
The major proportion of UV-B rays i.e., around (70%) is shielded by the topmost layer of the
epidermis i.e., stratum corneum via absorption and scattering 5. As a result, 20% of UV-B rays
reaches the epidermal spinous, 10% in the superficial dermis layer. In the case of UV-A rays
and visible light which is not effectively filtered by the stratum corneum (outermost layer of
the epidermis) about 70% of it is absorbed by melanin, the rest 30% of it reaches the basal cells
of the epidermis and, 20% reach the reticular dermis and 1% of UV-A reaches the
hypodermis 5, 7.
UV rays are scattered by stratum corneum but some amount penetrate up to the reticular dermis.
They are absorbed by endogeneous chromophores like urocanic acids, NADH flavins and
unsaturated lipids that go to an excited state and heat is released by the combining with tissue
oxygen leading to the formation of Reactive Oxygen Species (ROS) leading to DNA damage
which in turn cause degradation of dermal collagen framework leading to photoageing. DNA
damage can cause release of cytokine and inflammatory mediators into the skin causing
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sunburn. DNA damage can also cause tanning response. In small doses immediate pigment
darkening (IPD) is caused where the skin develops greyish colour which appears within 15 min
of exposure and disappears within 2 hours. If exposed to high doses causes brown colour
pigmentation referred to as persistent pigment darkening (PPD) that persist after 2 hours post
exposure. This is due to photo oxidation of melanin in the presence of oxygen. UV-A also
causes immunosuppression by reducing the langerhand cells which are antigen presenting cells
in the skin. This immunosuppression can also lead to carcinogenesis 5. UV-B acts on 7-
dehydrocholesterol present in the skin to form vitamin D-3. It also stimulates mitotic activity
causing light induced callosity thereby protecting underlying living cells. Target molecules like
urocanic acid, melanin and some aromatic amino acids and nucleotides absorb UV-B rays 5, 8.
UV-B absorption by nucleotides lead to the formation of pyrimidine and purine dimers as
photoproducts. UV-B simulates Denovo melanin synthesis from existing melanin monomers
causing delayed tanning. It can have immunosuppressive potential even at sub erythemal doses
causing carcinogenicity in the skin (Figure-3) 4.
Figure 3: Detrimental effects of UV-A and UV-B rays (Reproduced from5).
24
2. Conventional Sunscreens and UV filters
To circumvent the detrimental effects of UV rays protective measures are a prerequisite. To

overcome these consequences topically applied sunscreens have been deployed for more than
5, 9
40 years. The first sunscreen was launched in the year 1930 . These are photochemical
systems that contain organic or inorganic UV filters that absorb or reflect UV light that is used
for the topical application on the skin to protect from detrimental effects of UV. They can be
classified into two types:
a. Chemical/Organic UV-filters
b. Physical/Inorganic UV –filters
The chemical UV-filters can be of three types:
a. UV-A filters (that absorb UV-A-rays),
b. UV-B-filters (that absorb UV-B-rays)
c. UV-A-UV-B filter (wide spectrum that absorbs both UV-A and UV-B simultaneously).
The physical UV filters are broad spectrum filters that absorb, reflect and scatter UV rays 1.
Their corresponding mechanisms of protections are depicted in Figure 4 and Figure 5.
Figure 4: Mechanism of Action of: Organic and Inorganic UV filters (Reproduced from10).
25
Figure 5: Mechanism of Absorption of Organic and Inorganic UV filters
(Reproduced from11).
2.1 Physical/Inorganic UV filters
These materials are able to absorb, scatter and reflect UV rays back to the surroundings. They
mainly act as a physical barrier and block UV rays. The spectrum of absorption of these
compounds are wide as they cover the complete ultraviolet spectrum. These absorb UV rays
resulting in transition of an electron from the valence band to the conduction band (Figures 4
and 5). These filters are also referred to as sun blocks due to fact that they provide a coating
11-13
that blocks sun rays from penetrating the skin . The mode of extinction of inorganic UV
filters is via the combination of absorption and scattering.
2.2 Chemical/Organic UV filters
These are usually aromatic compounds which possess a carbonyl group. On exposure to UV
rays these filters absorb energy and act in 3 main ways:
a. These could undergo conformational/configurational molecular changes;
b. They can emit radiation at higher wavelength (fluorescence or phosphorescence);
c. They can release the incident energy as heat.
26
Post UV exposure these undergo π (HOMO) → π ∗ (LUMO)/n (HOMO) → π ∗
(LUMO) transitions (Figures 4 and 5). The mode of action of these UV filters is normally
reversible so they could be reused 10, 11.
Sunscreen products can be formulated in the form of oils, creams, lotions, sprays, gels etc.
These products are composed of a combination of ingredients in addition to UV filter agents
(sunscreen agent) such as moisturizers, photostabilizer, solvents and antioxidants1, 14
Moisturizers (e.g.: cetyl alcohol, polyethylene glycol) are occlusive agents that create a barrier
that blocks water from escaping from the skin. Photostabilizers (e.g: 2(2′-Hydroxy-5′-
methylphenyl) benzotriazole) are organic compounds that are used to increase photostability,
safety and effectiveness of sunscreens15. Antioxidants (e.g.: butylated hydroxyanisole) are used
in these products to capture the reactive oxygen species generated to prevent skin ageing16. The
extent of protection increases with higher filter concentration in sunscreens, but due to its toxic
nature and the poor cosmetic acceptance the concentration has been limited to a threshold for
in-vivo use in human beings 1. Application of sunscreens regularly on the surface of the skin is
the most effective mode of shielding off the dangerous effects of UV radiation. It is
recommended that sunscreens have to be applied 15 to 30 min before exposure to the sun and
the application must be repeated subsequently every two hours. Substantivity is an essential
condition for sunscreens i.e., the ability to fix itself to the upper epidermis that will confirm
long lasting action 5, 17.
2.3. Common inorganic UV filters
Most commonly used inorganic UV filters are titanium oxide, zinc oxide, kaolin, magnesium
oxide, etc. Some of them are described in detail below.
2.3.1 Titanium dioxide (TiO2)
Titanium dioxide has been used as sunscreen since the 1980s. The particle size distribution is
in the range from 150-300 nm. It appears in the form of white chalky powder due to the
reflection of the incident visible light and perceived by the retina as white. These are commonly
utilized in the form of nanoparticles whose particle size are less than 100 nm. The small particle
27
size aids the nanoparticles to be applied smoothly on the skin. Its transparency is due to very
small amount of reflection of the incident light. TiO2 is more effective in UV-B region (Figure
6). The mode of photoprotection provided by these particles is predominantly via absorption
with small amount of scattering. The ability of particles to protect against UV exposure is
directly related to the particle size. As the particle size decreases the absorption spectrum of
TiO2 shifts predominantly to UV-B region 18.
Figure 6: UV absorption spectra of TiO2 and ZnO filters offering protection against both UV-
A and UV-B (Reproduced from10).
2.3.2 Zinc oxide (ZnO)
ZnO has also been used as sunscreens since the 1980s. The particle size distribution ranges
from 200-400 nm. It is found to be more effective in the UV-A spectrum. To circumvent the
opaque nature in cosmetic applications micro sized ZnO has been replaced by nano sized
ZnO18.The optical refractive index is between 2.3 and 2.0. It is an n-type semiconducting
19
material with a wide bang gap. At a temperature of 77 K the band gap energy is 3.32 eV .
Figure 6 shows the absorption spectra of ZnO in the UV region.
28
2.4. Common organic UV filters
Most commonly used organic UV filters are Benzophenones (BP), 4-Methylbenzylidene

Camphor (MBC), 3-benzylidene-camphor (BC), Octyl-methoxycinnamate (OMC), 2-ethyl-
hexyl-4-trimethoxycinnamate (EHMC), Octocrylene (OCR), Octyl dimethyl PABA (ODP) etc.
Some of them are described in detail below.
2.4.1 Benzophenone
These are a family of compounds that are derivatives of dibenzoylmethane compounds that
belong to the aromatic ketone category. Benzophenones require lower energy and have longer
wavelength for electronic transition. These compounds show resonance phenomenon. The
mode of protection is via absorption mechanism. The main disadvantage of benzophenones as
UV filters is due their toxicity i.e., a mutagen, carcinogen, and endocrine disruptor 20.
Figure 7: Structure of Benzophenone-3 (left) and Benzophenone-4 (right).
When high sun protection factor (minimum erythemal dose on protected skin divided by the
MED on unprotected skin (MED = UV-B - dose when redness of the skin is visible) SPF (20-
30) is required, benzophenone-3 is usually used in combinations with other solubilizers. Most
commonly used benzophenones are BP-3 (C14H12O3-Oxybenzone λMax=290 nm, Figure 7) and
BP-4 (C14H12O6S-sulisobenzone λMax=240 nm, 288 nm)21.
29
2.4.2 Methylbenzylidene camphor
It is an organic sunscreen that provides protection in the UV-B range (290-320 nm) with a λMax
at 301 nm with low photostability (Figure 8). It dissipates energy via cis trans isomerization.
Figure 8: Structure of 4-Methylbenzylidene Camphor.
The quantum yield of E-Z isomerizations found to be in the range 0.13-0.3. It is a highly
lipophilic (logP=3.385) compound which can be absorbed through the skin and was found in
human tissues, including placenta on analysis 22.
2.5 Consequences of conventional sunscreens on humans
Sunscreen can get into the human body by application on the skin which in addition to dermal
exposure also cause gastrointestinal and pulmonary exposure. Swimming in or drinking water
contaminated with sunscreen is yet another route for exposure.
2.5.1 Organic
Organic sunscreens like benzophenone eg: BP-3, BP-4 have poor photo stability. They are
easily degraded into free radicals, accelerate skin ageing, penetrate skin causing damage DNA,
cause allergies and has the potential to cause endocrine disruption and neurotoxic effects in the
human body like peripheral neuropathy, parkinson’s-like effect, encephalopathy etc.22, 23
30
2.5.2 Inorganic
The use of inorganic sunscreens like ZnO and TiO2 has led to many consequences for human
beings. Studies has shown that ZnO nanoparticles penetrated the skin to a limited extent. A
small increase in zinc ions (Zn2+) in the blood and urine was observed in humans who used
sunscreens containing these nanoparticles on healthy skin for five consecutive days.
Investigations have shown that TiO2 nanoparticles are taken up by cells very quickly and in the
absence of irradiation (in the dark), there is reduction in keratinocyte proliferation and the
alteration of calcium homeostasis. In the presence of UV radiation TiO2 nanoparticles induce
cell death and cause DNA damage by releasing reactive oxygen species like singlet oxygen and
hydroxyl radicals which cause cytotoxic and genotoxic effects on human keratinocytes 24, 22 .
2.6 Impact of conventional sunscreens in marine ecosystems
Populations of coastal areas (beaches, river sides etc) and tourism have increased significantly
in the last 20 years. Every year millions of people travel to the beaches where they get exposed
to lot of sunlight. In commensurate there is an upsurge in the use of sunscreens and cosmetics
inoculated with high dosage of UV-filters in their corresponding formulation. The coastal
regions of the world are most susceptible to the detrimental impact of these cosmetics products.
The recent studies show that organic UV-filters and inorganic UV filters as well as many other
components in the cosmetics reach the aquatic environment and cause detrimental effects to the
marine ecosystem. Tourism related to coastal and marine sector is one of the fastest growing
areas of the global tourism industry. Statistics show a tremendous increase in the tourists from
25, 26
463 million in the year 1992 to 763 million in 2004 . A corresponding growth of the
sunscreen market has many implications and aftermath. The sunscreens as a source of chemicals
in the coastal marine system have been reported in a few studies. There are two pathways in
which the UV filters reach into the marine environment: (i) Directly via the recreational water
activities such as swimming, water sports, bathing the aftermath of coastal tourism (ii)
Indirectly via wastewater treatment plants due showering, washing, rubbing off and discharge
after application of sunscreens on skin 27.
31
2.7 Distribution of conventional sunscreens in various marine
ecosystems
The presence of UV filters particularly its organic components have been found in different
aquatic ecosystems like lakes, rivers, sea water and also in waste water treatment plant (WWTP)
influents and effluents, swimming pools, urban groundwater, tap water, soil, sludge and marine
27
organisms via studies conducted . Human activities such as laundering, showering etc can
lead these UV filter compounds to reach WWTP. WWTP together with the natural degradation
that takes place in the environment are not enough for these filters to get decomposed
comprehensively and as a result they end up in effluents, lakes, river water and which finally
culminate in the seas28. The concentration of different UV filters like Benzophenone-3 (BP-3),
4-Methylbenzylidene camphor (MBC-4), 3-benzylidene-camphor (BC-3), Benzophenone-4
(BP-4), Octyl-methoxycinnamate (OMC), Octocrylene (OCR), Octyl dimethyl PABA (ODP)
etc in different water matrices are shown in Table 2.
Table 2: Presence of different organic UV filters in different water matrices (Reproduced26).
UV Filter Structure Water Matrices Concentration

(ngL-1)
River Water up to 114

Sea Water up to 3 300 ± 200
BP-3
Lake Water < 2 - 125
Tap water n.d
Swimming pool up to 3 300
WWTP < 1 - 700
MBC-4 Sea Water up to 798.7
Lake Water up to 1,140 ± 50
Tap water up to 18
Swimming pool up to 330
WWTP up to 6500
32
BC-3 Sea Water 9 - 13
River Water < 3 - 1,980 ± 130

BP- 4
Sea Water <1
Tap water up to 18
WWTP up to 1,947 ± 34
Sea Water up to 389.9

OMC
Lake Water up to 3,009 ± 206
Tap water n.d.
Swimming pool up to 86 ± 7
WWTP up to 505.2
Sea Water up to 2780.7

OCR
Lake Water up to 4381 ± 539
Tap water n.d.
Swimming pool up to 15
WWTP up to 5322 ± 612
Sea Water up to 390 ± 40

ODP
Lake Water up to 34
Tap water up to 2.3
Swimming pool up to 2.1
WWTP up to 224.3
Note: n.d. is not detected
33
2.8 Ecotoxicological issues and impact of organic UV filters
2.8.1 Bioaccumulation
With increased use of UV filters there is increased accumulation of these compounds and their
degradation products in water bodies. Both these have detrimental effects on aquatic life and
are a cause of increasing concern. These compounds are lipophilic (Eg: log Kow BZ-3 and 4-
MBC are 3.79 and 4.95, respectively) and waste water treatment plants cannot (WWTPS) get
rid of them efficiently and they ultimately reach the sea and they get accumulated in the sea
surface microlayer (SML), biota and sediments and get bioaccumulated in the food web. These
UV-filters could be photodegraded to byproducts or photoexcited by sunlight generating
elevated concentrations of reactive oxygen species with toxic effects on phytoplankton 29. The
concentration of these UV filters was found to be highest in the first few centimeters of SML29.
The organic UV-filters due to their lipophilic nature (eg: BP-3, 4-MBC, etc) have been found
to accumulate in the muscle and adipose tissues in natural populations of aquatic organisms
such as mussels, crustacean, fishes, marine mammals and aquatic birds 30. Analysis of tissues
of marine organisms (like clams, oysters, gastropods, fish like D. rerio and crustaceans like D.
pulex) have shown their presence in them 31, 32. A high concentration of octocrylene OCR) i.e.
about 712 ngg-1 lipid was found to be present in the liver of Franciscana dolphin (Pontoporia
blainvillei) that is found in Brazil 33. The mussel Mytilus sp. that is found in France contained
7112 ngg-1 d.w of OCR 34
. The diffusion of 4-MBC, BP-4, ODP and OCR was studied in
mussels (Mytilus galloprovincialis). 4-MBC, BP-4 and OCR were absorbed by mussels
showing high levels on investigation. The levels of BP-3 and ODP were found to be low.
35
Mussels could metabolize or biotransform ODP in the tissues to undetectable levels . The
researchers Danovaro and Corinaldesi showed that sunscreens were toxic to marine ecosystem,
causing viral infection, coral bleaching by promoting lytic viral cycle, killing symbiotic
microalgae and preventing the growth of marine bacterioplankton 36, 37.
34
2.8.2 Ecotoxicological evaluation
The ecotoxicological studies were conducted according to half maximal inhibitory
concentration (IC50), half maximal effective concentration (EC50) in 24, 48 and 72 h culture
experiments on various organism using a series of UV filters such as BP-3, BC-3, MBC-4,
EHMC, and OMC shown in Table 3.
Table 3: IC50 and EC50 for Various UV filters and organisms.
UV Filters IC50 (mgL-1) Organism
BP-3 0.96 Phytoplankton

BC-3 6.99 (Desmodesmus subspicatus)38
MBC-4 7.66×10−6
UV Filters EC50 (mgL-1) Organism
BP-3 7.50 Protozoan

(Tetrahymena thermophile)39
MBC-4 5.1×10−6 24 h culture medium
BP-3 1.67 - 1.9
BP-4 50 Crustacean
BC-3 3.61 (Daphnia magna)38
MBC-4 0.56-0.80 48 h culture medium
OMC 0.29-0.57
Autotrophs
BP-3 13.87
BC-3 74.72×10−6 Microalgae
MBC-4 1714.5×10−3 (Isochryris galbana)40
BP-4 >10 000×10−6 72 h culture medium
EHMC 74.73×10−3
Herbivore
BP-3 3 472×10−6
MBC-4 587.17×10−6 (Mussel)
EHMC 3 118×10−6 Mytilus galloprovincialis40
EHMC 284×10−6
MBC-4 854×10−6 Paracentrotus lividus40
BP-3 3 280×10−6
MBC-4 192.63×10−6
EHMC 199.43×10−6 Siriella armata40
BP-3 710.76×10−6
The above table shows that various conventional organic UV filters are toxic in various
organisms. For phytoplankton D. subspicatus BP-3 was the most toxic according to IC50.
According to EC-50 values the most toxic UV filters were MBC-3 for Protozoan Tetrahymena
35
thermophile, OMC for Crustacean (Daphnia magna), BP-4 for Microalgae (Isochryris
galbana), MBC-4 for Mussel (Mytilus galloprovincialis), EHMC for Paracentrotus lividus,
MBC-4 for Siriella armata.
2.9 Ecotoxicological issues and impact of inorganic UV filters
The global production of nano-ZnO and nano-TiO2 is about 10 000 tons and 5 000 tons per
annum respectively. Out of the total production approximately 60% of nano-TiO2 and 80% of
nano-ZnO are deployed in the cosmetic sector41. These nanoparticles have a high probability to
reach marine ecosystems during various steps in the life cycle such as production, fabrication
and usage stage via air deposition, from the effluents of waste water treatment plants and by
direct release 42, 43. The estimated amount of sunscreens that has been discharged into the coastal
ecosystem is around 250 tons according to Wong et al. 44. If these reach the marine ecosystem
these can interact with aquatic organisms via surface adsorption on microorganisms, cellular
internalization, trapping ingestion by fauna found on the sea bed. Ecotoxicological studies have
26, 45, 46
shown the toxic effects on both fresh water and salt water organisms . The study
according to Miller et al. came to the conclusion that nano ZnO concentrations between 0.5 and
1.0 mgL-1 inhibits the growth of several species of marine phytoplankton diatoms. Toxicity of
nano ZnO in phytoplankton is mainly due to the dissolution, release, and uptake of free zinc
ions. Nano TiO2 particles (0.5 and 1.0 mgL-1) had no effect on the growth of these species 47.
Ma et.al. observed that Daphnia magna (planktonic crustacean) was 100 times more sensitive
to nano-TiO2 in comparison to the Japanese rice fish (Oryzias latipes). Under simulated solar
radiation (SSR) the phototoxicity of nano-TiO2 increases 2 to four 4 folds 48. Jacobasch et al.
studied the exposure of nano-TiO2 on six generations of ladocera D. magna and found that
chronic exposure to 1.78 mgL-1 lead to 100% mortality 49. Chen et al. exposed nano-TiO2 to
zebra fish (D. rerio) for a duration of six months and found that it had bioaccumulated in many
organs like brain, gills, and heart causing morbidity and death 50.
The UV filters that are released into the marine environment are a major source of nutrients like
nitrates (N03-), nitrites (NO2-), phosphates (PO43-), silicates (SiO2) and ammonium (NH4+)
which could simulate algal growth and could be a potential cause of ecological imbalance 51.
The main components of both organic and inorganic UV filters reach the marine ecosystems
such as coastal seawater, lakes, rivers via the recreational activities these has the potential to
36
cause a plethora of ecological consequences on the ecosystem as mentioned earlier. Once it
reaches the water column, the components released from sunscreens accumulate in different
environment such as SML, biota and sediments and get bioaccumulated in the food web
indicated in the schematic diagram (Figure 9).
Figure 9: Conceptual diagram transfer of sunscreen derived-products (Reproduced 26).
Organic and inorganic UV-filters could be photodegraded to byproducts or photoexcited by

sunlight generating elevated concentrations of reactive oxygen species with toxic effects on
phytoplankton 26. In order to get over the aforementioned consequences there is an urgent need
to find natural sunscreens that outperform the conventional sunscreens that are highly efficient,
effective and safe. So, the best strategy is to look into the nature to find and explore natural
sunscreens.
37
3. Natural UV Protective Molecules : Mycosporine
and Mycosporine-like Amino Acids (MAAs)
3.1 Introduction
Mycosporines and mycosporines-like amino acids (MAAs) are secondary metabolites which
can act as natural UV filters showing very high molar extinction coefficients, high UV
52
absorptions, and high photo-stability . These natural photoprotective compounds are
synthesized by marine organisms such as fungi, algae, lichens, and cyanobacteria as a chemical
defense to shield UV rays. They are Schiff bases (enamino ketones) having low molecular
weight (< 400 Da) presenting an aminocyclohexenone (mycoporines) or an
aminocyclohexenimine (MAAs) ring substituted with a nitrogen atom and linked with amino
acid or amino alcohol (Figure 10) 53, 54.
Figure 10: General structure of mycosporines and MAAs.
The ring of the mycosporines contains a glycine subunit and some mycosporines contain sulfate
esters or glycosidic bonds via the imine substituents. The UV absorption in mycosporines vary
according to different side groups and nitrogen substituents attached to them (Table 4) 55.
38
Table 4: Molar absorptivity and λMax of different MAAs and mycosporines 56
Molar Absorbivity
Molecular λMax
Mycosporine/MAA Units
Formula (nm)
(Lmol-1cm-1)
Shinorine
C13H20N2O8 44 668 334
Porphyra-334
C14H22N2O8 42 300 334
Palythine C10H16N2O5 36 200 320
Mycosporine glycine C10H15NO2 28 100 310
Palythinol C13H22N2O6 43 500 332
Palythene
C13H20N2O7 50 000 360
Gadusol C8H12O6 21 800 298
Mycosporine-glycine C10H15NO2 28 100 310
Mycosporine-serinol C11H19NO6 25 516 310
They exhibit maximum absorbance in the ultraviolet spectrum i.e., UV-A (320 nm - 400 nm)
and UV-B range (290-320 nm) and present high molar extinction coefficients ε (28 100-50 000
M−1cm−1)54, 57
. There are mainly two categories of mycosporines: mono-substituted
mycosporines and di-substituted mycosporines. In mono-substituted mycosporines, the carbon
3 spot on the cyclohexenone ring is substituted by an amino compound eg: mycosporine-glycine
(MG λMax= 310 nm) Figure 11-A. The absorption maxima of mono-substituted MAAs is
present in the UV-B region. In di-substituted mycosporines the imine group is substituted with
a protonated nitrogen atom. This substitution leads to the formation of a zwitterion (Zwitter-
ionic form of porphyra-334, Figure 11-B)57
39
Figure 11 A: Mono-substituted mycosporine (M-Gy) and Di-substituted MAA (P334).
Figure 11 B: Zwitterion form of porphyra-334.
There is a delocalization and conjugation of the positive charge on the nitrogen atom on the
ring. This conjugation enhances the UV absorption. The extent of delocalization can affect the
extinction coefficient and absorption maximum of each MAAs. The absorption maxima of di-
substituted MAAs is present in the UV-A region i.e., from 320 to 362 nm. This will vary
depending on the amino acids attached as substituents e.g.: P334 (C1: tyrosine; C3: Glycine
λMax = 334 nm, palythine (C1: NH2; C3: Glycine λMax = 320 nm), and Mycosporine-2 Glycine
(C1: Glycine; C3: Glycine λMax = 331 nm)57, 58
. The MAAs can be stabilized by methylation
with CH2N2 and acetylation with acetic anhydride in pyridine which lead to the formation of
aromatised methyl esters or acetates. Some mycosporines are prone to hydrolysis e.g.:
mycosporine-Gly, resulting in glycine and β-diketone as byproducts 59. The chemical structures
of over 30 different mycosporines have been known till now and some of them are depicted in
Figure 12 59.
40
Figure 12: Chemical structure of different MAAs and mycosporines.
Interestingly, these molecules suppress the UV induced stress in organism by absorbing UV

rays and dissipating it in the form of heat energy without the generation of reactive oxygen
species. MAAs also possess antioxidant, DNA-protection and anti-inflammatory actions. They
enhance the osmotic equilibrium and also take part in cellular interactions57. These compounds
are also highly water soluble due to the formation of zwitterions due to the amino acid
substitutions.
41
3.2 Occurrence and environmental distribution
A UV-B absorbing amino acid was discovered by Wittenburg in 1960 in the epipelagic
Portuguese man of war (Physalia physalis). It was characterized by Price and Forrest in 1969
60
. As mentioned before, till today around 30 structurally different mycosporines/MAAs (Figure
11) were identified in marine and terrestrial organisms 61 that include 152 species (206 strains)62
of bacteria and fungi like (Zygomycetes, Deuteromycetes, Ascomycetes, Basidiomycetes,
63-66 67, 68
Bacillariophyta and Aphyllophorales) , cyanobacteria (blue green algae) ,
69-71 72
phytoplankton (microalgae) macroalgae , marine fishes (in the eyes, skin, tissues)60,
echinoderms, artemia, anaspidea, coral reefs, sea urchins, sea stars, marine molluscs,
72 60,
ascidiacea , scleractinia (in the eggs and mucus), zoantharia, scyphozoan and tridacna
73
.These molecules are quiet prevalent and extensively distributed in the environment especially
in the marine environment (oceans, rivers) 73, 74 The presence of these molecules has been found
73, 75
in several species of corals in the Great Barrier Reef . Some animals can absorb MAAs
from the algae via diet transfer or they can acquire them through symbiosis with algae,
cyanobacteria or association with bacteria and could accumulate MAA or convert to other MAA
forms to function as molecules that have photoprotection ability. Previous studies have also
shown that zebra fishes were able to synthesize the gladusol, the mycosporines precursor 76, 77.
Table 5 shows the distribution of these molecules in various marine organisms.
42
Table 5: Distribution of mycosporines and MAAs in some important marine organisms
(literature) 54.
Type of marine Type of Mycosporine/MAAs

organism
Phytoplankton Porphyra-334, Palythene,

Alexandrium excavatum Shinorine, Usujirene
Prorocenirum micans Mycosporine-glycine, Shinorine.

Porphyra-334, Asterina-330
Prorocenirum minimum Shinorine, Palythene
Lingdodinium polyedra Porphyra-334, Mycosporineglycine:

Valine, Palythine
Thalassiosira sp. Polythinol, Palythene, Shinorine,

Porphyra-334
Macroalgae Chondrus Shinorine, Palythine, Palythene,

Crispus Palythinol
Curdlea racovixzae Palythine, Shinorine, Palythinol
Iridaea chordare Palythine, Shinorine; Palythinol,

Palythene
Liihotamnion aniarciicum Shinorine, Porphyra-334
Palnnria decipiens Palythine, Palythene,

Porphyra-334, Palythinol, Shinorine
Phyllophom Antarctica Shinorine, Palythene
Plyllophom appenchculara Shinorine
Porplnra umbilicalis Shinorine, Porphyra-334
Gracilaria cornea Shinorine, Porphyra-334
Gelidum sp. Shinorine
Fucus spiralis Shinorine
43
3.3 Biosynthesis pathway of mycosporines and MAAs
The biosynthesis of mycosporines pathway is adopted by microorganisms such as fungi,

cynobacteria, phytoplankton and macroalgae. Various factors namely UV radiation, photo-
synthetically active radiation (PAR) of various wavelengths, desiccation, nutrients and salt
concentrations play a crucial role in simulating the biosynthetic pathway 54, 55. The precursors
for the biosynthesis of mycosporines are 3-dehydroquinate and 2-epi-5-epi-valiolone (Figure
13). 3-dehydroquinate comes from the shikimate pathway which starts with pyrUV-Ate and 2-
epi-5-epi-valiolone comes from the pentose phosphate pathway which stars with serine these
two precurors combine to form mycosporine glycine. The enzymatic and genetic information
53
of the synthesis is not fully known . Four enzymes coded by a cluster of four genes
dehydroquinate synthase (DHQS), O-methyltransferase (O-MT), ATP-grasp and nonribosomal
peptide synthetase (NRPS) are involved in the biosynthesis. DHQS and OMT convert 3-
dehydroquinate to 4 deoxygadusol. ATP grasp add glycine to 4 deoxygadusol to form
mycosporine glycine. NRPS catalyses the addition of serine to M-Glycine to form shinorine an
MAA.MysD protein add threonine to M-Glycine to form porphyra-334 55, 78.
Figure 13: Proposed pathway of MAAs biosynthesis (Reproduced from55).
44
3.4 Evolution of mycosporines/MAAs as UV-absorbing compounds
Sunlight permeates into the marine bodies with a certain degree of attenuation depending on
the wavelength. In case of clear water, the UV rays would have a prominent biological
consequence up to a depth of 20 m. The wavelengths ranging between 285 and 340 nm in the
UV spectrum would cause destruction of biological cells and tissues and is deleterious to marine
organisms and plants. The UV absorbing molecules in microorganisms play an effective role in
54, 79
circumventing the detrimental effects of UV radiation . In the Proterozoan age, shielding
of the harmful UV rays was obligatory in order to prevent the complex organic molecules from
destruction and this protective role has been taken up by specific organic molecules in the
marine environment. Mycosporines being extensively found in a lot of microorganisms can be
considered as an evidence for its protective role against the negative effects of UV rays. It has
also been understood that these UV absorbing molecules have many other functions such as
80, 81
antioxidant activity, prevention of photo oxidative stress and osmotic regulation . It has
been assumed that these molecules were inherently present to perform these functions and later
82
evolved as UV screening substances . The evolutionary origins of mycosporines is still not
known 54.
3.5 Isolation and identification of mycosporines and MAAs
These mycosporines and MAAs are commonly extracted from red macroalgae Bangia fusco-
purpurea, Gelidium amansii, Gracilaria confervoides, and Gracilaria by techniques like
solvent extraction, ultrasound-assisted extraction, orthogonal experiments by varying a set of
variables like temperature time of extraction, extraction degree and solid liquid ratio. Various
solvents like ethanol (25%), methanol (25%) and distilled water can be used for the extraction
process83, 84
. Chromatographic methods that are used for the isolation and purification of
mycosporines in the form of ethanolic or methanolic extracts are high performance liquid
chromatography (HPLC), reverse phase column chromatography and gel filtration
chromatography. The columns that are used for the chromatography are ion exchange resins,
norite A, carbon and cellulose, and preparative TLC on silica gel 59. Solvents that are used for
the elution are water, ethyl alcohol, hydrochloric acid and many buffers. After elution the
59
mycosporines are obtained in the form of oils, amorphous powders and crystals . The most
frequently used technique for the separation of mycosporine extracts is reverse-phase isocratic
45
high-performance liquid chromatography with simultaneous UV detection. The solvent used
for the mobile phase depends on the polarity of the extracts. For extracts with less polarity a
mixture of water with 0.1% acetic acid is used, methanol being used as the mobile phase 59 for
more polar extracts. Comparison of the UV spectra and the corresponding retention time
obtained in high performance liquid chromatography with values in the literature helps to
identify the corresponding mycosporines. These methods cannot be completely relied on
because some mycosporines show similar UV absorption maxima, same molar extinction
coefficients and retention time in HPLC in two different solvent systems but have different
59, 85
chemical structure eg: glutamic acid-glycine found in Dysidea herbacea (sponge) and
sterina-330 found in Plectropomus leopardus (coral trout) 59, 86 The methods that are deployed
for elucidation of the chemical structure and characterisation of mycosporines are HPLC,
fractionation, alkaline hydrolysis, derivatisation with OPA(o-phthalaldehyde) (to identify the
hydrolysed amino functionality)87, 59
infrared spectroscopy, liquid chromatography-mass
spectrometry (LC/MS), ion trapped liquid chromatography, amino acid analysis and nuclear
magnetic resonance spectroscopy 57, 88.
3.6 The different roles of mycosporines and MAAs
Figure 14 summarize the different roles and applications of mycosporines and MAAs.
Photo-Protection
Fungal Reproduction
Sun Screen
Compounds MAA Intracellular
Nitrogen Storage
Thermal and
Antioxidants Dessiccation Stress
Wound Healing
Figure 14: Applications of Mycosporines and MAAs.
46
3.6.1 As photoprotectants
Exposure to UV rays can cause DNA and cellular damage in organisms due to the formation of
reactive oxygen species (ROS). Mycosporines have the potential to absorb high doses of UV
radiation and dissipate it by converting it into less harmful heat energy and thus prevent
54, 57
oxidative stress damage caused by ROS and hydrogen peroxide . In the case of marine
organisms, the mycosporines they contain absorb the UV wavelengths inside the water. In the
case of cyanobacteria these compounds prevent 30% of the photons from reaching the
cytoplasm89.On the other hand, according to experimental investigations based on irradiation
of A375 human melanoma cells, fibroblast cells IMR-90 90 and HaCaT cells 91 with UV rays in
the presence of mycosporine-glutamine, porphyra-334 and palythine were conducted. And, the
in vivo assay showed that mycosporines and MAAs had a protective effect against UV induced
DNA damage.
3.6.2 As sunscreen compounds
These compounds can be used in cosmetic products to protect skin from damage caused by UV
radiation. Mycosporines can act as sunscreens and they are found to accumulate in the
epidermis of certain organisms that have UV blocking ability eg: dinoflagellate-Phaeocystis
pouchetii, diatom-Guinardia striata, Cyanobacteria-Anabaena sp. Also, it has been found that
UV light induces the biosynthesis of mycosporine in certain organisms to function as sunscreen
compounds 53, 54. Photodegradation, photophysical studies and thermoresponsive studies clearly
indicates that these mycosporines/MAAs are both photostable and thermally stable sun-
sunscreen compounds 58. Two industries in Europe have ventured into sunscreen obtained from
mycosporine shinorine extracted from Porphyra umbilicalis, a type of red algae 92. Researchers
from Oregon State University have extracted mycosporine gadusol (that provide protection
against UV-B) from zebrafish and have started a pharmaceutical industry called as Gadusol
Laboratories to develop and engineer sunscreens based on gladusol and a US patent was also
granted (Patent No: US11072806B2) 93. Paul Long’s research group, in King’s College London
has demonstrated that palythine, a MAA which has been extracted from red algae Chondrus
yendoi that can provide protection against UV-A and UV-B. They filed a patent application
(Patent No: WO2017013441A1) for this molecule and licensed it to a cosmetic company in
92
London . MAAs have only been used in formulations as free molecules. For example,
47
shinorine and porphyra-334 have been used as additives in creams for skin protection leading
to their commercialization as Helioguard 365 sunscreen.
3.6.3 As antioxidants
Most of the mycosporines found in nature have the ability to scavenge the reactive oxygen
species (ROS) such as singlet oxygen superoxide anions, hydroperoxyl radicals, and hydroxyl
radicals that is either due to UV radiation or due to environmental stress, 53, 57 Antioxidant
property of these molecules is attributed to presence of a cetonic group 94. The free radicals are
produced during the oxidation step for energy metabolism in most biological process, these
species activate the cellular mechanisms such as the cell division, inflammation, stress response
and can function as cell signaling molecules. The ROS generation and scavenging are in a state
of equilibrium in these mechanisms. The photodegradation of these molecules in presence of
reactive oxygen species like singlet oxygen indicate its antioxidant activity. Some of the
mycosporines that exhibit good antioxidant potential activity are P334, shinorine, mycosporine-
52
2-glycine and the antioxidant activity could be assessed through DPPH or ABTS assay .
Briefly, the DPPH (1,1-diphenyl-2-picrylhydrazyl) assay is primarily based on the mechanism
by which antioxidants act to inhibit lipid oxidation. The DPPH free radical is very stable and
instantly reacts with compounds that can donate hydrogen atoms and has a UV λMax at 515 nm.
The method is based on the scavenging of DPPH by antioxidants which upon a reduction
reaction decolorizes the DPPH methanol solution95. The ABTS (2,2'-azino-bis(3-
ethylbenzothiazoline-6-sulfonic acid) assay measures the relative ability of antioxidants to
scavenge the ABTS radical generated in aqueous phase when compared to Trolox (water
soluble vitamin E) standard. This radical cation absorbs light at 734 nm in water. When
antioxidants are added to a solution containing ABTS radical cation, the absorption decreases
when the ABTS radical cation scavenged 96.
3.6.4 As intracellular nitrogen storage
MAAs have the ability to act as a nitrogenous reservoir54. They can store two nitrogen atoms
per molecule53. According to the study by Nathalie Korbee Peinado ammonium ions and UV
radiation increase the accumulation of mycosporine in Porphyra columbina algae. The
48
percentage of accumulation was dependent on the specificity of the irradiation. When irradiated
with UV-A and UV-B there was a 29% and 5% increase, respectively. UV-B irradiation was
found to be more efficient in inducing mycosporine synthesis per unit energy in the Porphyra
97
columbina . When intracellular degradation of mycosporines takes place, the nitrogen gets
released 54.
3.6.5 In thermal and desiccation stress
Studies show that there is a high concentration of mycosporine formation in organisms exposed
to high temperature, thermal and desiccation stresses, also the concentration of mycosporine in
soft corals like Lobophytum compactum and Sinularia flexibilis in the Great Barrier Reef
increased due to the presence of stress and high temperature, exposure to UV rays further
increasing the concentration 53, 54. Cynobacterium N. commune, when subjected to external
factors such as desiccation stress, UV radiation and oxidation, showed to accumulate high
content of mycosporines in the extracellular matrix 53.
3.6.6 In fungal reproduction
UV light is one of the prerequisites for the development of reproductive organs in fungi. The
presence of UV light is detected by certain pigments that have absorption at λmax at 210 nm and
310 nm. The compounds that have absorption at 310 nm is referred to as P310. One such
compound extracted from the sporophores of the Basidiomycete, Stereum hirsutum is the MAA
serinol. The P310 usually refers to three substances with different sporogenic activity that
absorb different wavelengths shorter than 310 nm. Lot of mycosporines are ubiquitous among
the fungal classes such as zygomycetes, deuteromycetes, ascomycetes, and basidiomycetes, and
these MAAs aid them to sporulate 53, 54.
3.6.7 In wound healing
MAAs can act as wound healing agents. The molecular mechanism that accelerates and
enhances the wound healing process in cells of the skin is due to the activation of signaling
pathways of the focal adhesion kinases (FAK) and mitogen-activated protein kinases (MAPK).
49
The application of MAAs is found to increase the FAK phosphorylation at Y397 that in turn
facilitates the activation of MAPKs extracellular signal-regulated kinases (ERK).
Characterization of cells treated with MAAs were done by the activation of c-Jun N-terminal
kinases (JNK) i.e. JNK1. Studies led to the conclusion that MAAs could induce and initiate the
skin repair by triggering the activation of both kinases in which the c-Jun N-terminal kinases
(JNK) enzyme is the main driving force for this process 73.
3.7 Photoprotection mechanism of mycosporines and MAAs
The ability of mycosporines/MAAs to protect from UV-A and UV-B rays is well understood
from its absorption spectra and also from its high molar extinction coefficients. Both of these
family of compounds exhibit maximum absorption wavelength (λMax) in the range from 268-
362 nm i.e., encompassing both UV-A and UV-B spectrum depending on there intrinsic nature
and on their molecular structure covering a major proportion of the electromagnetic spectrum
(295-400 nm). The photochemistry of few of these molecules are explored like shinorine (SH),
porphyra-334 (P334), palythine, mycosporine-glycine, gadusol etc.
According to previous studies both mycosporines/MAAs absorb the UV rays and undergo a
transition from the ground state (S0) to the first excited state (S1). The photo-excited states
these molecules can relax mainly via two pathways. From (S1) it can transalate to electronic
ground state (S0) by internal energy conversion via non radiative relaxation culminating in a
controlled release of heat energy with low quantum yield of fluorescence without the generation
of reactive oxygen species (ROS) 98. These molecules also relax by intersystem crossing from
the singlet excited state (S1) to the triplet excited state (T1) (Figure 15). The Photoacoustic
calorimetric studies show that that a major proportion of photon energy (90%) is released to the
external environment as heat reducing the propensity of dangerous photochemical reactions.
Studies have shown that MAA like porphyra-334 and shinorine dissipate 96-98% of absorbed
energy via this mode 99.
50
Figure 15: Jabloski diagrams of UV absorbing molecules (Reproduced from 100).
Various studies have been conducted in order to comprehend the photophysical and
photochemical properties of Mycosporines and Mycosporines like amino acids.
3.8 Photostability of mycosporines: gadusol
Gadusol, the mycosporines precursor, can have two forms depending on its pH i.e. (gadusol) at
2.5 (gadusolate) at 7 with λMax at 268 nm and 296 nm, respectively (Figure 16).
Figure 16: Structure of gadusol; enol and enolate form.
Arbeloa et al. has extensively studied the photostability of gadusol101 in aqueous solutions at
acidic pH 2.5 and neutral pH 7. The photolysis of gadusol solutions were carried out in both air
51
and argon atmospheres (inert), illuminated with a 4 W germicide mercury and 1000 W high-
pressure Xe-Hg lamp with a monochromator (254 nm and 303 nm) for total time period of 6
and 3 min, respectively. There absorption spectra were recorded as a function of time (each 2
min-acidic solution, and 1 min-neutral solution). HPLC analyses of the irradiated samples were
also done at fixed intervals of time during the photolysis.
The results indicate absence of new bands in the (200-900 nm) range of the absorption spectra
of the irradiated samples relative to pure samples, absence of photoproducts in the HPLC
analysis of the irradiated samples (Figure 17).
Figure 17 : Photodegradation of a. gadusol (λmax=268) and b. gadusolate (λmax=296)

(reproduced from101). Insets: Absorbance at λmax vs. irradiation time for the photolysis under
air (●) and argon (O) atmospheres
The QE (Φ) of photodegradation of gadusol and gadusolate in air was found to be (3.65 ±
0.36)×10−2 and (1.42 ± 0.51)×10−4, respectively. The photostability evaluations done in argon
atmosphere (inert) gave a quantum yield of (3.65 ± 0.36)×10−2 for gadusol (3.64 ± 0.49)×10−4
for gadusolate showing that the presence of oxygen did not have much influence on the
photodegradation of both these species.
52
3.9 Photostability of mycosporine like amino Acids: shinorine,
porphyra and palythine
Conde et al. has extensively studied the photostability of MAAs like shinorine, porphyra-334
and palythine. The photostability study of shinorine was studies in aqueous state with
concentration 1.5×10−5 M each102. The solution of shinorine was irradiated at room temperature
with low pressure 4 W UV-B lamp for a total time period of 61 min in both oxygen, nitrogen
and air atmosphere (inert) and evaluation the corresponding absorption spectra at 0, 16.5, 36.0,
and 61 min, respectively (Figure 18).
Figure 18: Photodegradation spectra (Absorption Spectra) of shinorine

The photostability of Porphyra-334 was studied in aqueous state with an initial concentration
of 2×10−5 M 99
. The solution was irradiated at room temperature (21 0C), the total time of
irradiation was 300 min in oxygen, nitrogen and air atmosphere (inert) and analyzing the
corresponding absorption spectra at 0, 130 and 300 min respectively (Figure 19).
53
Figure 19: Photodegradation spectra of porphyra-334
The quantum yield of photodegradation was evaluated using actinometry. The quantum yield
of photodegradation Φ was evaluated by the ratio of the photolysis rate (vi) to the initial
absorbed intensity (Ia). The Steady-state fluorescence spectra of both shinorine and porphyra-
334 with concentrations 6.0×10−6 in its aqueous state were recorded. The quantum yield of
flourescence were also evaluated (Table 6).
Table 6: Quantum yield (Φ), fluorescence and life time of shinorine and porphyra-334
(Literature 99, 102).
Photo-property Shinorine (×10−4) Porphyra-334 (×10−4)
Quantum yield (N2 atmosphere) 3.1 ± 0.7 1.9 ± 0.3

Quantum yield (O2 atmosphere) 4.9 ± 0.8 3.4 ± 0.4
Quantum yield (Air atmosphere) 3.4 ± 0.5 2.4 ± 0.3
Fluorescence quantum yield 1.6 ± 0.2 2.0 ± 0.2
Both shinorine and porphyra-334 degraded upon irradiation by UV-B. The QE of

photodegradation (Φ) of shinorine (3.4 ± 0.5)×10−4 was a slightly higher than porphyra-334 (2.4
± 0.3) ×10−4 . The QE values were both in the order of 10−4 indicating high photostability. The
quantum yields of shinorine and porphyra-334 under different atmospheres indicated that the
MAA were most stable in N2 atmosphere (inert) and lest stable in Air and O2 atmosphere due
to the presence of oxygen that can enhance the probability of photodegradation due to the
formation of reactive oxygen species like singlet oxygen, hydroxyl radicals etc.
54
The photostability studies of palythine was studied with an initial concentration of 1.5×10−5 M
in aqueous state 103. The solution was irradiated with 1000 W Hg–Xe high-pressure lamp with
a high-intensity grating monochromator. The total time of irradiation was 440 min. The
absorption spectra were evaluated at 0, 170, 440 min at λ-Max-320 nm (Figure 20). The
quantum yield of photodegradation was found to be (1.2 ± 0.2)×10−5 .
Figure 20: Photodegradation of palythine (Reproduced from103).
The lower photodecomposition quantum yield of palythine in comparison to shinorine and

porphyra-334 indicate more photostability. In the case of shinorine and porphyra-334 the
substitution of nitrogen atom of the cyclohexenimine unit increases the photochemical
robustness of the molecule. The involvement of competitive relaxation pathways of the excited
states through geometrical isomerization around the C=N bond. In fact, the energy barriers to
C=N rotation and to nitrogen inversion may be substantially influenced by structural effects.
Particularly, nitrogen inversion rates depend markedly on the substituents on nitrogen.
Other authors have also investigated the photodegradation of shinorine, porphyra-334 and
palythine in the presence of photosensitisers and in the presence of different natural matrices104.
The photostability experiments were conducted in a solar simulator (Suntest XLS) with a 1500
W xenon arc lamp with a monochromator to remove all the wavelength below 290 nm. The
intensity of the used was 400 Wm-2. The initial concentration of MAA samples used were
0.1x10-6 M in aqueous medium (distilled water and sea water). Photosensitizers like riboflavin
and rosebengal were also used for this study. The samples were irradiated for a time period
55
between 2 h and 60 h at room temperature (18 0C). The photodegradation of shinorine, porphya-
334 and palythine in presence of photosensitisers like riboflavin, rose bengal a major source of
singlet oxygen in the ratio (1:10) was studied and rate constants were evaluated (Table 7).
Table 7: Photostability of MAA with photosensitisers and natural matrices 104
Mycosporines like Amino Acids Rate Constant

(m2 kJ-1)
Shinorine + Riboflavin 7.39
Porphyra-334 + Riboflavin 4.98
Palythine + Riboflavin 3.17
Palythine + Rose Bengal 0.12
Shinorine + Sea Water 0.018
Porphyra-334 + Sea Water 0.026
Palythine + Sea Water 0.26
Palythine in the presence of rose bengal showed a degradation of 50% post 2 h irradiation (Total
dose=3000 kJm-2). Palythine in combination with riboflavin showed a degradation of 99% post
1 h irradiation (Total dose=1440 kJm-2) (Figure 21).
Figure 21 : Degradation of palythine with riboflavin (o) and rose bengal (Δ) in distilled water
The photodegradation of shinorine and porphyra-334 in the presence of distilled water and with
riboflavin depicted in (Figure 22).
56
Figure 22: Degradation of Porphyra-334 and Shinorine with riboflavin (o) and distilled water (Δ)
The photodegradation of shinorine, porphya-334 and palythine in presence of sea water was
studied by irradiation for 24 h and rate constants were evaluated. Irradiation of palythine in sea
water resulted in 99% degradation for 24 h (Total dose = 38000 kJm-2) with no substantial
change for dark control (Figure 23). The rate constants of all these experiments are showed in
Table 7.
Figure 23: Photodegrdation of palythine in light (o) and dark (Δ) in sea water (Reproduced from104)
57
3.10 Mycosporine and MAAs-based products and materials for UV
filters
To evaluate the photochemical and photophysical behaviour of MAAs shinorine and porphyra-
334 in an environment where they are naturally found, Oralo et al. conducted experiments in
Cetrimonium chloride (CTAS) and Sodium dodecyl sulfate (SDS) a solution of direct ionic
micelles. Their experiments showed that these MAAs had more affinity towards water
(hydrophilic phase) in comparison to the hydrophobic phase. The distribution of MAAs
shinorine and porphyra-334 in an heterogeneous micro environment depends on its acid base
properties. According to electrophoretic studies with shinorine and porphyra-334 at pH values
below 2.0 they are positively charged and at pH 5.7 they are neutral due to its zwitterionic
formation.The photostability of the shinorine and porphyra-334 in micellar solutions were
evaluated by irradiating it in a quartz cell of 1cm path length with a UV lamp and an optical
filter (cut-on 320 nm) for different time periods like 0.1 M SDS (10 h-shinorine, 4.5 h-porphyra)
0.1 M CTAC (12.5 h-shinorine, 5.5 h-porphyra-334) and water (10 h-shinorine, 4.5 h-porphyra-
334 (Figure 24 and Figure 25). The quantum yield of photodegradation was also evaluated
shown in Table 8 105.
Figure 24 : The Plot of absorbance of shinorine at 334 nm in water(-▲-), SDS (-●-) and
CTAC (-■-) (Reproduced from105).
58
Figure 25 : The Plot of absorbance of Porphyra at 334 nm in water
(-▲-), SDS (-●-) and CTAC(-■-) (Reproduced from105).
Table 8: Quantum yield of photodegradation of MAA in different micellar solutions

(Reproduced from105)
MAA CTAC (x 10-4) SDS (x 10-4) Water (x 10-4)

Shinorine 1.2 3.1 3.4
Porphyra-334 1.0 1.9 2.4
The quantum yields of photodegradation are lower in all these media compared to water by
0.04% confirming the high photochemical stability of the molecules in these micellar systems.
The photostability is highest in CTAC followed by SDS. This was due to the cationic nature of
CTAC resulting in attraction of positively charged heads of the detergent with carboxylate
groups of the MAAs contributing to high stability. On the contrary, SDS is an anionic micelle
and it repels the carboxylate group of MAAs forcing the MAAs to migrate to the aqueous phase.
Fernandes et al. have done synthesis on chitosan conjugates with MAA such a shinorine,
porphyra-334 and M-glycine via amidation for UV-absorbing materials based exclusively on
bio-based compounds. Films of these chitosan conjugates were prepared and transmittance of
these films were evaluated for Chitosan-Porphyra-334 (CS-P334), Chitosan-shinorine (CS-SH)
at 334 nm and Chitosan-mycosporine glycine (CS-MGly) at 307 nm (Figure 26). All these
films were found to be UV absorbing. The stability of these conjugate films were evaluated by
irradiating it with a 254 nm UV bench lamp XX-15S UVP of power 0.6 Wcm-2 at room
59
temperature 25 0C for a time period of 12 h and monitoring the absorbance at 307 and 334 nm
every 2 h during the experiments106.
Figure 26: Trasmittance of Chitosan (CS) and chitosan conjugates CS-MGly, CS-SH and CS-
P334 (Reproduced from106 )
The authors showed that the final UV-absorbing materials are photo- and thermoresistant,
exhibit a highly efficient absorption of both UV-A and UV-B radiations and are biocompatible.
Nonetheless, no studies were done regarding the photodegradation of these materials in
different water matrices or in the presence of photosensitisers.
4. Conclusion
The harmful effects of UV radiation on human beings and their mechanism of action have been
clearly brought in the literature survey. The toxic effects of conventional organic and inorganic
sunscreen to flora and fauna have been illustrated. We learn from nature that certain marine
organisms have been endowed with inherent UV protectants mycosporines and MAAs. There
is great scope for development of UV filters from these molecules.
Some studies have shown the photophysical and photochemical characteristics of these
molecules make them good potential candidates to replace the current UV filters in existing
sunscreen formulations or materials. There is great scope in future of exploring the possibility
of making use of these naturally occurring UV filters in human beings without toxic effects.
60
Few research and investigation has been carried out in this arena of photostability of
mycosporines and MAAs in different conditions (different water matrices and in the presence
of photosensitisers). Some of them are:
1a. [Conde, F. R., M. S. Churio, and C. M. Previtali. "The photoprotector mechanism of mycosporine-like amino
acids. Excited-state properties and photostability of porphyra-334 in aqueous solution." Journal of
Photochemistry and Photobiology B: Biology 56, no. 2-3 (2000): 139-144.],
1b. [Conde, F. R., M. S. Churio, and C. M. Previtali. "The photoprotector mechanism of mycosporine-like amino
acids. Excited-state properties and photostability of porphyra-334 in aqueous solution." Journal of
Photochemistry and Photobiology B: Biology 56, no. 2-3 (2000): 139-144.]
Conde et al. have evaluated the photostability in terms of quantum yield in pure water for MAA
like shinorine (QE-3.4×10−4) porphyra-334 (QE-2.4×10−4) and palythine (QE-1.2×10−5) and
came to the conclusion that palythine was more photostable relative to shinorine and porphyra-
334 due to the absence of geometrical isomerism. The photoacoustic calorimetry results point
out that relaxation pathway of these molecules is non radiative.
2. [Whitehead, Kenia, and John I. Hedges. "Photodegradation and photosensitization of mycosporine-like amino
acids." Journal of Photochemistry and Photobiology B: Biology 80, no. 2 (2005): 115-121.] Whitehead et al.
(2005) have evaluated the photostability of shinorine, porphyra-334 and palythine in terms of
rate constant in the presence of photosensitisers like riboflavin, rosebengal and sea water and
concluded that reactive oxygen species accelerates the photodegradation of MAA and the
mechanism of photodegradation is via Type 1.
3. [Arbeloa, Ernesto Maximiliano, Sonia Graciela Bertolotti, and María Sandra Churio. "Photophysics and
reductive quenching reactivity of gadusol in solution." Photochemical & Photobiological Sciences 10, no. 1
(2011): 133-142.]
Arbeloa et al. (2010) have evaluated the phostability of gadusol and gadusolate in aqueous
solutions in terms of quantum yield of photodegrdation and concluded that gadusolate (QE-
1.2×10−5) was more stable relative to gadusol (QE-1.2×10−5).
To the best of our knowledge, the photodegradation of MAA shinorine, Porphyra-334 and
palythine and mycosporines like M-serinol and gadusol have never been investigated in detail
in aquatic matrices like river, estuary, ocean water in terms of QE. The secondary products
61
generated by the irradiation have never been separated and elucidated. The photostability of M-
serinol has never been studied till now. The present thesis expects to go a step further as
described in the different chapters:
In Chapter 2 we have investigated the photostability of mycosporines gadusol and

mycosporine-serinol in pure water and in different aquatic matrices namely river, ocean and
estuary and in pure water in the presence of photosensitisers like riboflavin and porphine.
Analysis and quantification of reactive oxygen species via molecular probes were done. We
have also tried to unravel the photodegradation mechanism and made efforts to analyse the
photodegradation products. The antioxidant potential of these molecules was also evaluated.
In Chapter 3 we have investigated the photostability of MAAs namely shinorine, porphyra-

334 and palythine in pure water and in different aquatic matrices like river, ocean, estuary and
photosensitisers like riboflavin, porphine. Analysis and quantification of reactive oxygen
species via molecular probes were done. We have also looked into the mechanism of
photodegradation and the photodegradation products generated were identified and analysed
via LC-MS.
In Chapter 4, chitosan-MAA derivatives such as shinorine and porphyra via amidation reaction
were produced and characterised using ATR-FTIR, NMR and UV spectroscopy. The
photostability of these conjugates were studied using a solar simulator to irradiate the solutions
prepared in pure and river water. The photodegradation of these conjugates in the presence of
photosensitisers like riboflavin and porphine were also studied.
62
5. References
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Kockler, J., Oelgemöller, M., Robertson, S., & Glass, B. D. (2012). Photostability of
sunscreens. Journal of Photochemistry and Photobiology C: Photochemistry Reviews, 13(1),
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2
Svobodova, Alena, Daniela Walterova, and Jitka Vostalova. "Ultraviolet light induced
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3
Stamnes, K. (2003). Ultraviolet radiation
4
Kullavanijaya, Prisana, and Henry W. Lim. "Photoprotection." Journal of the American
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5
Bens, Guido. "Sunscreens." Sunlight, vitamin D and skin cancer (2014): 429-463
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Pérez-Sánchez, A., Barrajón-Catalán, E., Herranz-López, M., & Micol, V. (2018).
Nutraceuticals for skin care: A comprehensive review of human clinical
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Dermatology in General Medicine: Textbook and Atlas, 3rd ed.; Fitzpatrick, T. B., Ed.;
McGraw-Hill: New York, 1987.
8
Young, Antony R. "Chromophores in human skin." Physics in Medicine & Biology 42.5
(1997): 789.
9
Urbach, Frederick. "The historical aspects of sunscreens." Journal of photochemistry and
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70
71
72
Chapter 2
Photostability of Mycosporines : M-serinol and

Gadusol in Aquatic Matrices
73
74
1.Introduction ........................................................................................................................... 78
2. Experimental Part ................................................................................................................. 80
2.1. Materials and Chemicals .............................................................................................. 80
2.2. Irradiation experiments ................................................................................................. 80
2.2.1. In pure and natural water matrices (river, estuary and ocean) .............................. 80
2.2.2. In presence of photosensitisers - riboflavin and porphine .................................... 81
2.2.3 Photostability in Riboflavin with sodium azide quencher ..................................... 81
2.3. Determination of the photodegradation quantum yield ................................................ 82
2.4. Indirect determination of singlet oxygen steady state concentration in natural matrices
by quenching experiments ................................................................................................... 82
2.5. Antioxidant capacity analysis by DPPH radical-scavenging assay .............................. 83
3. Results and Discussion ......................................................................................................... 83
3.1 Photostability in Pure, River, Estuary and Ocean Water ............................................... 85
3.2 Photostability with Photosensitisers Riboflavin and Porphine ...................................... 90
3.3 Quantification of Singlet Oxygen with Furfuryl Alcohol probe ................................... 97
3.4 Antioxidant Activity: DPPH Assay ............................................................................. 100
4.Conclusion ........................................................................................................................... 102
5. References .......................................................................................................................... 102
75
76
Chapter 2: Photostability of Mycosporines : M-serinol and
Gadusol in Aquatic Matrices
Adapted from Understanding the effect of natural water matrices and photosensitizers on
the photostability of gadusol and mycosporine-serinol – a kind of natural molecules that
absorb on UV-B, M.G. Thomas, S. Blanc, M. Le Bechec, J-M. Sotiropoulos, T. Pigot,
S.C.M. Fernandes, manuscript in preparation
Abstract
Mycosporine serinol and gadusol (enolate form)/gadusolate were exposed to UV radiation via
a solar simulator and the photostability was assessed in pure water and different natural matrices
like river, estuary and ocean water. The photostability of M-serinol was found to be higher than
that of gadusolate for all water matrices. The study also revealed that the photodegradation of
these mycosporines are higher in these natural matrices than in pure water due to the generation
of singlet oxygen on UV irradiation. The effect of photosensitisers riboflavin and porphine on
the photostability of these mycosporines in pure water was also evaluated. The DPPH assay
studies revealed the antioxidant potential of these molecules.
77
1. Introduction
Among the natural molecules acting as ultra-violet (UV)-sunscreens, mycosporines and

mycosporine-like amino acids (MAAs) are between the most prominent exemplars1-3. They
present strong UV-absorption capacity in both UV-B (mycosporines) and UV-A (MAAs) with
maximum wavelengths from 310 to 365 nm, very high molar extinction coefficients (Ɛ, from
28 100 to 60 000 M-1cm-1), ability to absorb UV-radiation by dissipating it as heat energy
1-4
without the generation of oxidative photoproducts, and high photo- and thermostability .
These two families of secondary metabolites are biosynthesized by algae, fungi and
cyanobacteria and ingested or accumulated by marine animals like fish, molluscs and
arthropods to protect themselves from the harmful effects of UV-radiation 1, 2, 4. Mycosporines
and MAAs are low-molecular weight (< 400 Da) and water-soluble molecules; and their basic
chemical structure contains a cyclohexenone or a cyclohexenimine unit, respectively. Their
precursors, gadusol and 6-deoxygadusol, consist of cyclohexenones compounds absorbing in
the UV-B spectrum 3. Because of some controversy related to side effects provoked by the
conventional synthetic sunscreens on both human and environment health, and their limited
photostability and cross-stability with other sunscreen agents; these natural sunscreens have
attracted the interest of the academic community, scientists and industrials5, 6. For instance, due
to wide spread use of synthetic sunscreens along with the recreational activities these molecules
have been found in aquatic ecosystems like ocean, rivers and estuaries and also in marine
organisms such as fishes, coral reefs, etc7 causing neurotoxicity, mortality in fish, reduce coral
reproduction and coral reef bleaching 8. Thus, these UV-absorbing natural molecules are
suitable potential candidates as an alternative to conventional synthetic UV-filters in sunscreen
4, 9
formulations and design of UV-absorbing and protective biomaterials10 Indeed, the
assessment and understanding of their photostability in different water matrices and on the
presence of photosensitizers seems to be crucial. Herein, a particular focus is done to the natural
molecules absorbing in UV-B (Figure 1). The penetration of UV-B rays into the earth’s
atmosphere can have a series of consequences in humans such as sunburn, immunosuppression,
generation of reactive oxygen species (ROS) leading to DNA damage and carcinogenesis
(melanoma and non-melanoma skin cancer)11.
78
Figure 1: Chemical structure of mycosporines; M-serinol (right) and gadusol (left).
Only two studies have investigated the photostability of gadusol and its derivatives. Arbeloa et
al. 12 studied the photostability and photophysics of gadusol in aqueous solution at neutral pH
7 (gadusolate-enolate form with λMax =298 nm) and acidic pH 2.5 (gadusol-enol form with λMax
=269 nm) under air atmosphere. Under physiological conditions, they demonstrated the high
photostability of gadusol/gadusolate even if gadusolate present a lower quantum yields of
photodecomposition (13×10-05) than gadusol (3600×10-5). With gadusolate, the authors
concluded that the reductive quenching reactivity (electron transfer from gadusolate to excited
triplet state sensitizer) could be considered as one of the fundamental mechanisms that support
the antioxidant capacity of gadusol in biological environments. Interestingly, the same authors
demonstrated also that gadusolate react very efficiently with by singlet oxygen arising from
energy transfer from an excited state sensitizer (Type 2 mechanism), which also contributes to
the antioxidant properties of gadusolate 13.
In this work, the evaluation of the photostability of gadusolate and M-serinol in different natural
water matrices (ocean, river and estuary) is reported. These natural matrices are known to
generate Reactive Oxygen Species that can react with these molecules and thus contribute to
their degradation in natural environments. Complementary experiments have been also
conducted with photosensitisers (riboflavin and porphine). On the other hand, to the best of our
knowledge, the photostability of mycosporine-serinol has never been reported before.
To sum up, this study aims to investigate the photostability of these mycosporines under
realistic conditions (solar irradiation, natural waters) and to better understand the role of ROS
in the abiotic degradation process.
79
2. Experimental Part
2.1. Materials and chemicals
Gadusol (C8H12N2O6, Ɛ 268 nm (molar absorptivity) = 12 400 14, 15

or Ɛ264nm = 12 90016 L.mol-
1
cm-1, gadusolate Ɛ296 nm=21 800 14
or 22 200 15
L.mol-1.cm-1, Figure 1) produced by yeasts,
was kindly offered by Gadusol Laboratories, Oregon, USA. The sample was received in 2019
as a lyophilized pure extract and stored at -20 °C until use. Mycosporine-serinol (M-serinol,
C11H19NO6, Ɛ310 nm = 25516 or Ɛ310 nm = 27270 14
L.mol-1.cm-1, Figure 1) extracted from the
marine lichen Lichina pygmaea in the Andalusian coast in 2020, was purchased from the
Laboratory of Photobiology of the Central Research Services of the University of Málaga
(Spain). M-serinol was received as lyophilized pure extract and stored at -20 °C until use. The
extraction, purification and characterization of the of M-serinol were done as described
previously17. Ocean water was collected from Bay of Biscay (Anglet, France) in March 2021.
Estuary water was collected in Adour estuary (Bayonne, France) in March 2021. River water
was collected from Gave de Pau (Pau, France) in 2021. Pure water was obtained from a
Millipore Milli-Qapparatus, Riboflavin (7,8-Dimethyl-10-((2R,3R,4S)-2,3,4,5-
tetrahydroxypentyl)benzo-[g]-pteridine-2,4-(3H,10H)-dione) (98% purity), porphine
((4,4′,4′′,4′′′-(porphine-5,10,15,20 tetrayl) tetrakis (benzenesulfonic acid) tetrasodium salt
hydrate) (98% purity), sodium azide ( > 99.5%) were purchased from Merck, Germany.
Furfuryl alcohol was freshly distilled before used and stored in the dark.
2.2. Irradiation experiments
2.2.1. In pure and natural water matrices (river, estuary and ocean)
In order to make the irradiation experiments, M-serinol and gadusol were dissolved in pure
water and in the above natural water matrices in order to have a solution with an absorbance of
around 1 with an optical path of 0.2 cm. The volume of each mycosporine solution used for
irradiation was around 0.6 mL in rectangular quartz cuvettes (3 cm × 1 cm with a path length
of 0.2 cm). These solutions were then irradiated in a sun test solar simulator (Sun Test XLS
80
from Atlas Material Testing Solutions, USA) with a closed chamber of1 170 cm2, a Xenon Arc
lamp (1700 W) and a Light day filter. The irradiation was performed with an irradiance of 590
W.m-2 and dosage per hr of 2000 KJ.m-2 in the wavelength (λ) range of 200 to 700 nm, the
temperature was kept at 30 °C during the irradiation. The above experimental conditions were
chosen to fit the conditions of natural solar radiation18. The photon flux, the lamp was measured
by a spectroradiometer (microprocessor controlled Seimyuing Vectron UV). The total time of
irradiation (T) was chosen as 6 h (total dosage of 12 000 KJ.m-2). The absorption spectra of the
different solutions were recorded using a Perkin Elmer UV/VIS/NIR Lambda-750
Spectrophotometer (USA) at subsequent time interval (t) every 2 h during the irradiation
experiment to monitor the kinetics of photodegradation. These experiments were done in
triplicate.
2.2.2. In presence of photosensitisers - riboflavin and porphine
The photostability of M-serinol and gadusolate in the presence of riboflavin (RF) and porphine
(PPY) was done only on pure water. The molar ratio of Mycosporine:Photosensitisers were
Mserinol:RF-260.8:1, Gd:RF-332:1, Mserinol:PPY-1904:1, Gd:PPY-2433:1 and these
solutions were irradiated with total dosage of 2 000 KJ.m-2 per hr at 30 0C. Gadusolate and M-
serinol solutions were irradiated in the presence of riboflavin for a total period of 20 and 60 s,
respectively. Their corresponding spectra were analysed in a time interval of 5 and 10 s,
respectively during the irradiation experiments. Similarly, M-serinol and gadusolate were
irradiated in the presence of porphine for a total time period of 5 min each and the
corresponding spectra were analysed in a time interval every min during the irradiation
experiments.
2.2.3 Photostability in riboflavin with sodium azide quencher
The photodecomposition study of the mycosporines were also conducted with 6.6×10-11 M
riboflavin in combination with 5 ×10-3 M sodium azide in pure water as solvent and with the
irradiated with a dosage of 2 000 KJ.m-2 per h at 30 0C. Gadusolate and M-serinol were
irradiated in the presence of riboflavin and sodium azide quencher for a total time period (T) of
81
20 s. Their corresponding spectra were analysed in a time interval (t) every 5 s during the
irradiation experiments.
2.3. Determination of the photodegradation quantum yield
The photostability of these compounds were compared using quantum yield of

photodecomposition (Φ) defined as in equation 1a:
number of molecules photodecomposed

Φ= Eq. 1a
number of photons absorbed
Number of molecules photodecomposed = roV t NA where ro is the rate of photo decomposition,

V the volume of solution (L) NA is Avogadro number, t is the time in s.
Number of photons absorbed = I0 (1- 10-(A)), I0 is the number of photons emitted within the
absortion band of mycosporine, A is the absorbance of the mycosporine solution.
2.4. Indirect determination of singlet oxygen steady state concentration

in natural matrices by quenching experiments
Natural matrices and photosensitizers porphine = 4×10-7 M and riboflavin = 1.34×10-5 M were
irradiated in presence of furfuryl alcohol (FFA, 10-4 M). The natural water matrices were
irradiated for time periods of 0.5 and 1 h for river, 2 and 4 h for ocean water and 1, 2 and 4 h
for estuary water. The photosensitizers solutions were irradiated for time periods of 1 min for
riboflavin and 3 min for porphine. The major product of singlet oxygen reaction with FFA is 6-
hydroxy-(2H)-pyran-3-one (6-HP-one). It was quantified as a function of irradiation time using
HPLC. HPLC analysis was carried out with Agilent 1290 equipped with a Supelco Lichrosphere
RP18-5 (25 mm × 4.6 mm, 5µm) column, 20 µL injection, eluent 80% water with 0.1 % H3PO4,
20% acetonitrile, rate 2 mL.min-1, UV detection at 205 nm and 218 nm. The retention times of
FFA and 6-HP-one were 2.9 min and 1.8 min, respectively. The steady state concentration of
singlet oxygen ([1O2]ss) was determined using the method of Haag and Hoigne 19, 20
.
82
2.5. Antioxidant capacity analysis by DPPH radical-scavenging assay
The antioxidant activity was assessed by the determination of the radical scavenging activity
using 2,2- diphenyl-1-picrylhydrazyl (DPPH) as described by Wu et al.21 with minor
modifications. An aliquot of 0.5 mL of 2.2×10-5 M gadusolate or M-serinol solution was mixed
with 1 mL of 100 μM DPPH solution which was freshly prepared in 95% ethanol. After
incubation in the dark for 30 min at 25 °C, the absorbance was measured at 517 nm. DPPH
radical scavenging activity (%) was calculated using the Eq 1b:
Scavenging effect (%) = [1 - (Asample - Asample blank)/Acontrol)] ×100 Eq. 1b
Where: Asample is the absorbance of the test sample (DPPH solution plus test sample), Asample
blank is the absorbance of the sample only (sample without DPPH solution), and Acontrol is the
absorbance of the control (DPPH solution without sample).
3. Results and Discussion
The photostability of gadusolate and M-serinol were assessed in pure water and natural matrices
by UV spectrometry following irradiation and the absorbance and concentration of was
measured periodically and the quantum yield was measured. The mechanism of degradation
(Type 1 and Type 2) of these molecules in the presence of photosensitizes riboflavin and
porphine was also investigated. Detection and quantification of singlet oxygen in natural
matrices were carried out with a furfuryl alcohol probe via high performance liquid
chromatography.
Before the experimental tests, absorption spectra of these UV absorbing mycosporines and the
spectral irradiance of the solar lamp used in the solar simulator were recorded in the UV region.
As expected, gadusolate and M-serinol are found to be UV absorbing as displayed in Figure 2.
This feature could be attributed to its high molar extinction coefficient (Ɛ > 20 000 M-1cm-1) as
in accordance with literature 3. This property of these molecules is due to the highly polar
structure consisting of cyclohexenone chromophoric ring in the case gadusolate and
cyclohexenone chromophoric ring along with a conjugation of a nitrogen substituent to its
imino alcohol with various hydroxyl groups attached in the case of M-serinol 2. M-serinol and
83
gadusolate both absorbs in the UV-B region and show an absorption maximum (λmax) at 310
nm and 298 nm, respectively 2, 22
.
Figure 2: Spectra of M-serinol (MseoH) and gadusolate (Ɛ vs Wavelength), and spectral

irradiance of the solar lamp used in the solar simulator (Irradiance vs Wavelength overlayed).
As described before, studies by Arbeloa et al. showed that absorption spectra of gadusol
behaved differently in acidic (enol form-gadusol) at pH 2.5, and in neutral media (enolate form-
gadusolate) at pH 7 and λmax were found to be 269 nm and 298 nm, respectively 12. In this study,
the pH of the 2 studied mycosporine post dissolution in pure water (pH around 6) were mildly
acidic. Their individual pH are 5.3 for M-serinol and 5.4 for gadusolate (enolate form). Thus,
in the present study, gadusol is in its enalote form (Figure 3). The pH of all solutions was found
to be unchanged after 6 h of irradiation.
Figure 3: Structure of Gadusol - enol and enolate form (in equilibrium).
84
3.1 Photostability in pure, river, estuary and ocean water
M-serinol and gadusolate were found to photodegrade differently upon UV exposure for a time
period of 6 h in pure water as shown in Figure 4A & 4B, and Figure 5.
Figure 4 : Time dependent absorption spectra and kinetics of photodegradation (inset) for: A.
M-serinol (λmax = 310 nm), and B. Gadusolate (λmax =298 nm) in pure water (T0 , T2 , T4 ,
T6 are the time periods of irradiation after 0, 1, 2, 4, 6 h, respectively).
85
Figure 5: Comparison of the kinetics of photodegradation of M-serinol and gadusolate in
pure water after 0, 1, 2, 4, 6 h of irradiation respectively).
A relative comparison among rate of photostability of these according to the quantum yield of
photodegradation revealed that M-serinol presents a QE of (2.3±0.46)×10−5 showing higher
photostability compared to gadusolate that presented a QE of (10±3)×10−5 (Table 1). As a
result, only 10% of M-serinol is decomposed after 6 h of irradiation whereas almost 50% of
gadusolate is decomposed in the same conditions.
These molecules on UV absorption gets excited to higher energy levels (singlet and/or triplet
states) (π-π* transitions) from the ground state and some of these molecules take part in
photoreactions and others subsequently returns to the ground state via nonradiative relaxation
as heat energy culminating in its degradation.
Table 1: Quantum yield of photodecomposition of M-serinol and Gadusolate.
Quantum Yield of Photodegradation (Φx10−5)

Mycosporines
Pure Water Ocean Water Estuary Water River Water
M-serinol 2.3 ± 0.46 5.4 ± 1 9.5 ± 2 20 ± 5
Gadusolate 10 ± 3 20 ± 4 28 ± 7 40 ± 10
86
The higher stability of M-serinol over gadusolate could be due to steric factor of the bulky side
chain (NH-CH-(CH2OH)2 attached to the cyclohexanone ring that makes the molecule more
robust which is absent in gadusolate. The photostability investigations by Arbeloa et al. of
gadusol and gadusolate gave a QE of 3640×10−5 and 13.8×10−5, respectively12. The QE of our
observed in this work are in accordance with this study.
Photodegradation of these mycosporines were also evaluated in natural matrices like, river, and
estuary and ocean water as detailed in Table 1. These matrices were found to be slightly basic
in nature with pH 8.1 for ocean, 8.4 for river and 8.5 for estuary water and supposed to be a
rich source of reactive oxygen species (ROS) on UV exposure23. These natural matrices contain
a mix of abiotic components such as organic, and inorganic along with the biotic components.
Both of these factors could simultaneously play a significant role in the generation of ROS. The
prime reasons for the formation of ROS in these natural waters is due to the photochemical
reactions involving coloured dissolved organic matter (CDOM)24. CDOM are peculiar
chromophoric organic molecules formed by leaching from decaying detritus of plant and animal
organic matter. The prime peculiarities of these molecules are their light absorbing nature in
broad range of visible and UV region, yellowish in colour due to the absorption of blue light
and the capability of exhibiting blue fluorescence together with high photo reactivity. The
spectrum of absorption of CDOM ranges from 280 nm to 700 nm. The abiotic photoreactions
involving CDOM generates ROS such as predominantly singlet oxygen together with hydroxyl
radical, superoxide radicals etc 24, 25
. CDOM acts like a natural photosensitiser in the presence
24
of sunlight. The main components of CDOM are humic acid, fulvic acid etc . The
concentration of CDOM is higher in river and estuary water and least in ocean water due to its
high salinity26. The higher absorption in the 280-700 nm region in river and estuary water can
substantiate the high CDOM content. A high concentration of nitrates is also present in these
matrices in the 200-250 nm band 27 (Figure 6).
87
Figure 6: UV Absorption spectra of pure, ocean, river and estuary water with the solar
spectrum overlayed.
The rate of photodegradation of M-serinol and gadusolate were evaluated in terms of QE by

irradiating it in these matrices for 6 h (Table 1, Figure 7 & Figure 8).
Figure 7: Kinetics of photodegradation of M-serinol (λ-310 nm) in river, ocean and estuary
water at 0, 2, 4, and 6 h
88
Figure 8 : Kinetics of photodegradation of gladusolate (λ-298 nm) in river, ocean and estuary
water at 0, 2, 4, 6 h.
As listed in Table 1, the quantum yield of photodegradation of both M-serinol and gadusolate
in these matrices are higher relative to pure water indicating low photostabilty and high
photodegradation. On comparison the photodegradation in terms of QE for both of these
molecules were found to be highest in river water and the least in ocean water and estuary water
in the middle. The photosensitizing effect of singlet oxygen generated from CDOM (Type 1
and Type 2) and hydroxyl radicals generated from nitrates (Type 1) respectively on irradiation
of these matrices seems to be responsible for this increase in photodecomposition of both these
mycosporines by oxidation (Eq-4 and Eq-5) 28, 29.
Mechanism of by photodegradation by CDOM (by Type 2)

hv
1CDOM → 1CDOM ∗ (𝐸𝑞 2)
ISC
1CDOM ∗ → 3CDOM ∗ (𝐸𝑞 3)
3O2
3CDOM ∗ → 1CDOM + 1O2 (𝐸𝑞 4)
1O2 + Gd− /MseoH → Oxidised Products (𝐸𝑞 5)
The biotic degradation was also evaluated keeping all the other conditions constant at 30 0C in
the dark and it was found to be negligible (Figure 9A & B).
89
Figure 9: Kinetics of biotic degradation of A. M-serinol B. Gadusolate in river, ocean and
estuary water at 0, 2, 4, 6 h.
In the next part, it will be proved that photosensiting reactions play a major role in the
photodecomposition of mycosporines.
3.2 Photostability with photosensitisers riboflavin and porphine
Photostability studies of these molecules in the presence of photosensitizers such as riboflavin

(RF) and porphine (PPY) were evaluated. This study was carried out to substantiate that reactive
oxygen species primarily singlet oxygen (1O2) was the responsible factor that played a crucial
role in degrading both gadusolate and M-serinol in natural water matrices. Both riboflavin and
porphine are water soluble and have a series of absorption bands in the UV region and the
visible region riboflavin λRb (Max)=222, 266, 373 and 447 nm respectively and porphine
λPPY(Max)=414, 517, 555, 580 and 635 nm respectively (Figure 10 A). 414 nm is the most intense
band referred to as soret band and the other four of low intensity corresponds to the Q band
(Figure 12 A). Both of these sensitizers possess a high molar absorptivity (ƐRb=13 222 M-1cm-
1
(447 nm), ƐPPY =21 9000 M-1cm-1 (414 nm)) and capable of generating ROS such as singlet
oxygen (1O2) on UV irradiation 30, 31. Photosensitization reaction can take place via Type 1 or
Type 2 mechanism. The photosensitisation process of riboflavin take place via the combination
90
of both Type 1 and Type 2. However, in the case of porphine, it takes place predominantly via
Type 2 mechanism 30, 32.
Type 1 mechanism of photosensitisation of Riboflavin

hv
1RF → 1RF ∗ (𝐸𝑞 6)
ISC
1RF ∗ → 3RF ∗ (𝐸𝑞 7)
3RF ∗ + Gd− /MseoH → 3RF .− + MseoH .+ /Gd. (𝐸𝑞 8)
302
3RF .− → 3RF + O2 −. (𝐸𝑞 9)
Gd. /MseoH + + O2 −. → GdOO. /MseoHOO. (𝐸𝑞 10)
The Type 1 mechanism of riboflavin (same for porphine) is depicted in Eqn 6- Eqn 10. In Type
1 mechanism riboflavin upon irradiation by UV rays undergoes excitation from the ground state
(1RF) to the excited singlet state (1RF*) as in Eq-6. The lifetime of 1RF* is transient (10-10-10-9
s) so prevents photosensitisation reactions from taking place at this stage. 1RF* can move to its
triplet state 3RF* via intersystem crossing (ISC) of same vibrational energy since intersystem
crossing is an expected pathway for most photosensitisers as in Eq-7. In its triplet state 3RF*
reactions are favourable due to its high life time (10-6-10-3) s 33
. The triplet state favours the
reaction of the activated riboflavin with gadusolate (Gd-) and M-serinol (MseoH) resulting in
electron transfer reaction from the mycosporines leading to the formation of two radicals, the
riboflavin anion (3RF-) and gadusolate/M-serinol cation (Gd.- and MseoH+) or vice versa
depending on the redox potential of the pair of these individual molecules (Eq-8). The presence
of ground state dioxygen from water can lead to the transfer of newly acquired electron to oxygen
by the riboflavin radical 3RF- resulting in superoxide radical (O2-) radical formation and the
riboflavin returns to the ground state (Eq-9). Gadusolate and M-serinol cation (Gd.- and MseoH+)
radicals can react with superoxide radical (O2-) to generate oxygenated peroxy radicals like
GdOO., MSeoHOO. (Eq-10). Arbeloa et al. has estimated the redox potential of gadusolate 0.60
V 34. The redox potential of triplet state of riboflavin and porphine was found to be to be 1.7 V
and 1.54 V respectively 35, 36. The E0 (O2/O2-) reduction potential of molecular oxygen (3O2) was
found to be -0.33 V 37. These reduction potential values make Eq-8 and Eq-9 thermodynamically
favourable.
91
hv
1RF → 1RF ∗ (𝐸𝑞 11)
ISC
1RF ∗ → 3RF ∗ (𝐸𝑞 12)
302
3RF ∗ → 1RF + 1O2 (𝐸𝑞 13)
1O2 + Gd− /MseoH → GdOO. /MseoHOO. (𝐸𝑞 14)
The Type 1 mechanism of riboflavin (same for porphine) is depicted in Eqn 11- Eqn 14. In
Type 2 mechanism riboflavin 3RF* in the exited state (triplet state) transfers its excess
excitation energy to ground state molecular dioxygen (3O2) resulting in the formation of reactive
oxygen species (eg: singlet oxygen) and thereby regenerating ground state riboflavin (1RF).
Energy dissipation of 1O2 is via heat dissipation either by physical quenching with water or
reacting with Gd-/MseoH to produce oxidised products (peroxy radicals GdOO. and
MseoHOO.) (Eq-14). Photosensitization of porphine takes place mainly via this mechanism.
M-serinol and gadusolate show substantial photodegradation in the presence of riboflavin and
porphine (Figures 10 A & B, 11 A & B, 12 A & B). No photobleaching was observed for
riboflavin for any of its absorption bands. In the case of porphine there is broadening and an
increase followed by a decrease of the intensity of soret absorption band corresponding to λ(Max)-
414 nm on irradiation (Figure 12 A). The increases could be due to the non-luminescent
aggregation of porphine dye molecules with the water molecules acting as a connecting bridge
between porphine through hydrogen bonding. This aggregation is due to the structure of the
chromophoric dyes mainly due to the large dipole moments, alternation of opposite charges and
the flat structure of the chromophore which results in high energy of intermolecular
interaction38. The intensity decrease is due to photobleaching. There is a subtle interplay
between photobleaching and aggregation. The QE of both M-serinol (17000±4000)×10−5 and
gadusolate (18000±5000)×10−5 are higher relative to the unsensitised ones indicating low
photostability. The QE values of both of these molecules are almost similar in the case of
riboflavin. But in the case of porphine gadusolate has a higher QE (2000±500)×10−5 compared
to M-serinol (970±150)×10−5 indicating low photostability.
92
Figure 10: Spectra of photodegradation of: A. M-serinol (λ-310 nm) with Riboflavin; B.
(magnified region-250-400 nm) and kinetics (inset) (where T0 sec, T5 sec, T10 sec , T15 sec,
T20 sec are the time periods of irradiation after 0, 5, 10, 15, 20 s, respectively)
93
Figure 11 A: Spectra of photodegradation A.M-serinol (λ-310 nm) with Porphine; B.
(magnified region-250-350nm) and kinetics (inset) where T0 , T1 , T2 , T3 , T4 , T5 are the
time periods of irradiation after 0, 1, 2, 3, 4, 5 s, respectively).
94
Figure 12: Spectra of photodegradation of Gadusolate (λ-298 nm) with A. Riboflavin; B.
Porphine where T0 , T5 , T10 , T15 , T20 and T1 , T2 , T3 , T4 , T5 are the time periods of
irradiation after 0, 5, 10, 15, 20 s and 0, 1, 2, 3, 4, 5 min, respectively)
To evaluate the respective fraction of degradation of mycosporine by riboflavin by electron

transfer (Type 1) and energy transfer (Type 2) sodium azide was used as a physical quencher
to scavenge singlet oxygen as in Eq-15 with rate constant (k) 5.0 × 108 M-1 s-1 39 (Figure 13 A
& B).
Physical quenching by sodium azide
NaN3 + 1O2 → NaN3 + 3O2 (𝐸𝑞 15) 39
95
Figure 13: A: Time dependent absorption spectra and kinetics of photodegradation (inset) M-
serinol in the presence of riboflavin with sodium azide (where T0 sec, T5 sec, T10 sec, T15
sec, T20 sec are the time periods of irradiation after 0, 5, 10, 15, 20 s, respectively); B : Time
dependent absorption spectra and kinetics of photodegradation (inset) -Gadusolate in presence
of riboflavin with sodium azide (where T0 , T5, T10 , T15, T20 are the time periods of
irradiation after 0, 5, 10, 15, 20 s, respectively).
Upon evaluating the respective proportion of degradation of these two mycosporines by energy
transfer i.e., via Type 1 individually, the extent of degradation was 55 % for M-serinol and 7%
for gadusolate (Table 2). The mode of degradation via singlet oxygen is higher in case of
gadusolate (93%) relative to M-serinol (45%). This indicates that degradation of M-serinol
mainly takes place via Type 1 mechanism this points out the reason behind the low QE (970 ±
150)×10−05 of it in presence of porphine compared to gadusolate.
96
Table 2 : Quantum yield of photodecomposition of mycosporines in combination with
riboflavin (Type 1 + Type 2) and porphine (Type 2).
Quantum Yield of Photodegradation (Φ)x10−5
Riboflavin Porphine
Mycosporine
TYPE 1 + TYPE 2 TYPE 1 TYPE 2
M-serinol 17000 ± 4000 9300 ± 2000 970 ± 150
Gadusolate 18000 ± 5000 1400 ± 200 2000 ± 500
3.3 Quantification of singlet oxygen with furfuryl alcohol probe
The molecular probe furfuryl alcohol (FFA) was used to detect and quantify singlet oxygen
generated from the natural matrices (river, estuary, ocean water) and also from photosensitisers
(riboflavin, porphine). This probe is used due to its high selectivity and a high second order rate
constant (k) with singlet oxygen (k=1.2×108 M-1s-1). The furfural alcohol gets oxidised in the
presence of singlet oxygen to a 6-hydroxy-(2H)-pyran-3-one (Figure 14)40.
Figure 14: Formation of 6-hydroxy-(2H)-pyran-3-one by reaction of FFA with singlet

oxygen.
The concentrations of singlet oxygen [1O2]ss generated by porphine and riboflavin is estimated
2500×10−14 M and 7500×10−14 M, respectively which were evaluated from slope (kapp) of
97
Ln[FFA]0/[FFA] vs time shown in Table 3 by Eqn : [1O2]ss = kapp /k and Figure 15 D & E.
Similarly, the concentration of singlet oxygen was evaluated in river, ocean and estuary water
(Figure 15 A, B & C). Irradiation of river water for 1 h leads to a stationary concentration of
16×10−14 M singlet oxygen on the contrary ocean and estuary water up on irradiation for 4 h
generated 5.8×10−14 M and 8.3×10−14 M singlet oxygen, respectively shown in Table 3. The
concentration of [1O2]ss was found to be highest in river and least in ocean and midway between
the two in estuary water (river > estuary > ocean). This was in accordance with the
photostability of gadusolate and M-serinol in these matrices (river > estuary > ocean). These
results of highest and lowest concentration of singlet oxygen in river and ocean water fortifies
the data for the corresponding photostabilities of M-serinol and gadusolate in these matrices.
98
Figure 15: Ln[FFA]0/[FFA] vs Time of natural matrices and photosensitisers A. River Water
B. Estuary Water C. Ocean Water D. Riboflavin E. Porphine
Table 3 : Concentration of singlet oxygen generated by natural matrices
Matrices kapp (S-1) x10−3 [1O2]ss = kapp/k (M) x10−14 Total Irradiation Time (hrs)
Ocean Water 0.007 5.8 4
River Water 0.02 16 1
Estuary Water 0.01 8.3 4
Riboflavin 9 7500 0.01
Porphine 3 2500 0.05
99
3.4 Antioxidant activity: DPPH assay
Antioxidant activity have been evaluated according DPPH assay. This test is based on the
difference of absorption spectrum between DPPH and its reduced form. It must be recalled that
DPPH (α,α-difenil-β-picrylhydrazyl) is a stable free radical. The presence of delocalized
electrons with seven conjugated double bonds gives it a deep purple colour with absorption
band in methanol solution at 517 nm. M-serinol and gadusolate were found to degrade in the
presence of photosensitisers; riboflavin and porphine via Type 1 (electron transfer) and Type 2
(singlet oxygen) mechanisms. Orallo et al. 2020 has stated that photooxidative quenching of
gadusolate supports a Type 2 mechanism initiated by the addition to its C-C double bond hinting
antioxidation potential 13. This has prompted us to evaluate the antioxidant potential of these
molecules via DPPH assay. DPPH radical strongly reacts with reacts with M-serinol and
gadusolate to produce new bonds, thus changing the colour of the solution from purple (DPPH-
Oxidised) to yellow (DPPH2 reduced) (Figure 16 & Figure 17). The reduction of its intensity
of its purple colour is mainly due to the decrease of the chromophore in DPPH. The relative
comparison of the DPPH values in Table 4 by Eq 1b indicate that gadusolate is a strong
antioxidant and M-serinol is a weak antioxidant. The rate of photodegradation of gadusolate
with porphine (Type 2 photosensitiser) in terms of quantum yield (2000 ± 500)×10−5 was found
to be higher than M-serinol (970±150)×10−05 indicating that the antioxidant activity is indirectly
linked to Type 2 mechanism showing that higher the Type 2 mechanism higher the antioxidant
potential. The results are in accordance with as stated by Orallo et al13.
100
Figure 16: DPPH radical reduction by mycosporine serinol (MseoH), gadusolate and control
as an antioxidant compound.
Table 4 : DPPH radical scavenging activity of Gadusolate and M-serinol.
Mycosporines Scavenging activity (%)
M-serinol 11
Gadusolate 58
Figure 17: DPPH radical reduction reaction from purple to yellow (517 nm) by
mycosporines.
101
4. Conclusion
Mycosporines like M-serinol and Gadusolate could be potential candidates to be used as UV-B
filters in sunscreen formulations. In comparison to the current existing conventional sunscreens
these molecules are of biobased origin and have high photostability. This study infers that M-
serinol was more photostable relative to gadusolate due to its bulkier and robust structure. The
photodegradation of both M-serinol and gadusolate are higher in natural matrices like river,
estuary and ocean water compared to pure water due to the presence of coloured dissolved
organic matter. The photodegradation was found to be highest in river water and least in ocean
water. Gadusolate was found to be a better antioxidant compared to M-serinol.
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105
106
Chapter 3
Comparison of Photostability of Mycosporines like

Amino acids in Different Natural Matrices
107
108
1. Introduction ........................................................................................................................ 112
2. Experimental Part ............................................................................................................... 114
2.1. Materials and Chemicals ............................................................................................ 114
2.2. Irradiation experiments ............................................................................................... 115
2.2.1. In pure and natural water matrices (river, estuary and ocean) ............................ 115
2.2.2. In presence of riboflavin and porphine (photosensitisers) .................................. 116
2.2.3 Photostability in riboflavin with sodium azide quencher .................................... 116
2.3. Determination of the photodegradation quantum yield .............................................. 116
2.4. Indirect determination of singlet oxygen steady state concentration in natural matrices
by quenching experiments ................................................................................................. 117
2.5. Detection of photoproducts by Liquid Chromatography Mass Spectroscopy (LC-MS)
........................................................................................................................................... 117
2.5.1 Chromatographic separation ................................................................................ 117
2.5.2 Evaluation of the photostability of MAA-precursors .......................................... 118
2.5.3 Untargeted Screening of MAA-photoproducts .................................................... 118
3. Results and Discussion ....................................................................................................... 119
3.1 Photostability in Pure, River, Estuary and Ocean Water ............................................. 119
3.2 Photostability with Photosensitisers Riboflavin and Porphine .................................... 129
3.3 Quantification of Singlet Oxygen with Furfuryl Alcohol probe by HPLC ................. 139
3.4 Photostability of MAA-precursors: Characterization of photoproducts by mass
spectrometry ...................................................................................................................... 142
3.5 Structural identification of MAA-Photoproducts ........................................................ 144
4. Conclusion .......................................................................................................................... 148
5. References .......................................................................................................................... 149
109
110
Chapter 3: Comparison of Photostability of Mycosporine
like Amino acids in Different Natural Matrices
Adapted from A roadmap to UV-A-absorbing natural sunscreens – photostability in different

natural water matrices and with photosensitizers, and analysis of the ensuing photoproducts,
M.G. Thomas, M. Parailloux, S. Blanc, M. Le Bechec, J-M. Sotiropoulos, R. Lobinski, T. Pigot,
S.C.M. Fernandes, manuscript in preparation
Abstract
This study deals with the investigation of photostability of mycosporine like amino acids
(MAAs) namely shinorine, porphyra-334 and palythine in different natural matrices like pure,
river, estuary and ocean water. Shinorine and porphyra-334 were found to be most stable and
palythine was the least stable due to its protonated form at acid pH. The photostability of these
amino acids was lower in natural matrices due to the generation of Reactive Oxigen Species
(ROS) like singlet oxygen on UV irradiation. The effect of photosensitisers riboflavin and
porphine on the photostability of these amino acids were also studied. The photodegradation
products on UV irradiation were isolated and analysed by LC-MS.
111
1. Introduction
Most living organisms are exposed to the effects of ultraviolet radiation (UVR) from the sun i.e
both UV-B: 280 nm-315 nm and UV-A: 315 nm-400 nm. Sunlight presents numerous benefits
like the stimulation of vitamin D production, the increase of energy and mood, the plants growth
etc, but also have a plethora of adverse effects namely skin and eyes damages, immune system
injury, etc 1-7.
In particular, UV-A, that comprises 90-98% of UVR in terrestrial sunlight 8, 9, can pass through
the glass and penetrates the dermal layer of human skin and reach both the epidermis and
dermis7. In general, UV-A: (i) provokes the formation of reactive oxygen species (ROS) that
damage the cell membrane by reacting with its lipids and amino acids; (ii) induces photo
carcinogenesis and DNA mutations as UV-B; (iii) results in immunosuppression, affecting both
the induction and the elicitation of immune responses; and (iv) has an important role in
6, 7, 10-12
photoaging . To avoid such detrimental effects of UV-A, photoprotective compounds
namely avobenzone, zinc oxide (ZnO), titanium oxide (TiO2), drometriazole trisiloxane among
others have been used for several decades. Nevertheless, these UV-filters/sunscreens have been
found to negatively impact both human health (e.g. systemic absorption of UV-filters in the
skin, production of ROS, easy degradation into free radicals, etc)13 and aquatic ecosystems
equilibria (e.g. high concentration of organic UV-filters in ocean and river waters and in marine
organisms such as fish, coral reef bleaching, etc) 14-16.
Thanks to the discovery of natural sunscreens such as gadusols and its derivatives namely
mycosporines and mycosporines-like amino acids (herein mentioned to as MAAs)17, new
directions on the design and development of highly efficient photoprotective and
environmental-friendly UV-protective compounds can be exploited and contribute to the
medical and cosmetic fields 18-21. Such natural sunscreens are ubiquitous in nature, commonly
found in macroalgae, cyanobacteria, fungi, phytoplankton and accumulated through ingestion
17, 22, 23
in high order animals to protect themselves from UVR . In this family of natural
sunscreens, the MAAs exhibit the highest UV-absorbing capacity in UV-A spectrum. They are
water-soluble low molecular weight molecules with the ability to absorb high energy UV
radiation and dissipate it as weak heat energy avoiding undesirable photochemical reactions as
the formation of photoproducts 24-26. Chemically, these secondary metabolites share a common
cyclohexenimine core, and present additional functional groups determining their specific
112
absorption maximum (λmax) (Figure1). Among the MAAs, shinorine (λmax-334 nm), porphyra-
334 (λmax-334 nm) and palythine (λmax-320 nm)are widely studied and used for commercial
usages 17, 18, 20, 21, 27-31.
Figure 1: Chemical Structure of mycosporines-like amino acids (MAAs): shinorine,

porphyra-334 and palythine.
Different studies have been conducted on the photostability analysis of these three MAAs. In
the early 2000s, Conde et al. investigated the photostability by the determination of the quantum
yields of shinorine, porphyra-334 and palythine in aqueous solutions (shinorine and porphyra-
32-34
334 and palythine in pure water) . They demonstrated that shinorine and porphyra-334
exhibited low quantum yield of photolysis 30×10-05 and 19×10-05 for shinorine and porphyra-
334, respectively) and fluorescence 1.6×10-04 and 2.0×10-04 for shinorine and porphyra-334,
32, 33
respectively) indicating high stability and low emissivity . In the case of palythine, the
quantum yield was found to be 1.2×10-05, indicating a high stability 34. The results obtained in
these studies, showed that around 96 to 98% of the absorbed photon energy by shinorine or
porphyra-334 is promptly delivered as heat to the surroundings, and the remaining energy is
stored by the triplet state of the MAAs. Consequently, the on setting of indirect or direct
photochemistry in the exposed organisms which may render harmful to living tissues is avoid.
Also, a comparative study done by the same co-authors on the photodegradation quantum yields
and photophysical properties of shinorine, porphyra-334 and palythine, suggested that
geometrical isomerization around the C=N bond of the cyclohexenimine unit may contribute to
34 35
the rapid deactivation of these MAAs . Whitehead and Hedges , chosen palythine (in
distilled or sea water, and with photosensitizers like riboflavin or rose bengal) and other MAAs
113
(in distilled water, and with riboflavin) to investigate their photodegradation rate constants and
production of MAA-derived photoproducts. In this work, the co-authors demonstrated that
palythine dissolved in natural sea water showed slow photodegradation rate constants, and that
75% of the initial porphyra-334 and shinorine remaining in sea water after 4 h of irradiation. In
the same study, they also observed indirect photodegradation of MAAs occurred with all
photosensitizers tested, and when riboflavin was used as opposed to a singlet oxygen source
(rose bengal), the photodegradation was rapid indicating that Type 1 photooxidation may be
the most successful mechanism for MAAs photodegradation. It seems that the stability of the
MAAs is dependent of the photoreactivity of the surrounding dissolved chromophoric
constituents and their potential to form reactive radicals. This study revealed that shinorine and
porphyra-334 were the most and palythine the least stable. Finally, the results from Whitehead
and Hedges work highlighted the inherent ability of MAAs to resist photodegradation, and once
again to support their assigned role as potent and effective sunscreen compounds 35.
Although many articles exist analyzing the photodegradation and photosensitization of MAAs,
a complete and comparative study under realistic conditions such as solar irradiation and using
natural water matrices such as river, estuary and ocean, is, to the best of our knowledge, lacking.
Natural waters are known to present naturally occurring photosensitizers that can react with the
MAAs contributing to their degradation in natural environments. Thus, this information can
provide greater insight into better understand the role of ROS in the abiotic degradation process.
Experiments in the presence of riboflavin and porphine (photosensitisers) were also conducted.
Finally, the potential production of MAA-photoproducts was monitored.
2. Experimental Part
Shinorine (SH, C13H20N2O8, Ɛ (molar absorbivity) = 44668 Lmol-1cm-1), porphyra-334 (P-334,

C14H22N2O8, Ɛ = 42300 L.mol-1cm-1) and palythine (PNE, C10H16N2O5, Ɛ = 36200 L.mol-1cm-
1
) were extracted from the macroalgae Gymnogongrus devoniensis, Porphyrarosengurrtii and
Asparragopsisarmata, respectively, collected in the Andalusian coast between 2019 and 2020.
The three mycosporines-like amino acids (MAAs) used in this study, were purchased from the
Laboratory of Photobiology of the Central Research Services of the University of Málaga,
114
Spain. The samples were received as lyophilized pure extract and stored at -20 °C until use.
The extraction, purification and characterization of the of the three MAAs were done as
described previously 36, 37. All the natural water matrices were collected in March 2021: river
water was gathered from Gave de Pau (Pau, France); estuary water was collected from Adour
estuary (Bayonne, France); and ocean water was gathered from Bay of Biscay (Anglet, France).
Pure water was obtained from a Millipore Milli-Q apparatus (France). Porphine [4,4′,4′′,4′′′-
(porphine-5,10,15,20 tetrayl) tetrakis (benzenesulfonic acid) tetrasodium salt hydrate] with
98% purity, riboflavin 7,8-Dimethyl-10-((2 R,3 R,4 S )-2,3,4,5-
tetrahydroxypentyl)benzo[g]pteridine-2,4-(3H,10H)-dione) with 99% purity, sodium azide
with > 99.5% purity and furfuryl alcohol with 98% purity were purchased from Merck,
Germany. Furfuryl alcohol was freshly distilled before used and stored in the dark. Methanol
and acetonitrile used for extraction and analytical experiments were LC-MS grade and
purchased from Honeywell (Morris Plains, NJ). Ammonium acetate was LC-MS grade and
purchased from Sigma Aldrich, France.
2.2. Irradiation experiments
2.2.1. In pure and natural water matrices (river, estuary and ocean)
Before the irradiation tests, shinorine (SH), porphyra-334 (P-334) and palythine (PNE) were
dissolved in pure and in the natural water matrices. The solutions were prepared in order to
have an absorbance of around 1 with an optical path of 0.2 cm. 0.6 mL of each MAAs solution
were used for irradiation in rectangular quartz cuvettes (3 cm× 1 cm with a path length of 0.2
cm). The solutions were irradiated in a sun test solar simulator (Sun Test XLS from Atlas
Material Testing Solutions, USA), equipped with a Xenon Arc lamp (1700 W), a light day filter
and a closed chamber of 1170 cm2. The experimental conditions were chosen to fit the
conditions of natural solar radiation as follows: the irradiation was performed in the wavelength
range from 700 to 200 nm with an irradiance of 590 W.m-2 and a dosage per hr of 2000 KJm-2,
the temperature was kept at 30 °Call along the irradiation tests. The photon flux was measured
with a spectroradiometer microprocessor controlled Seimyuing Vactron UV (Korea). The total
time of irradiation was chosen as 6 hours (total dosage of 12 000 KJm-2), and the absorption
spectra of the different solutions were recorded every 2 hours (T0 h, T2 h, T4 h and T6 h) using
115
a Perkin Elmer UV/VIS/NIR Lambda-750 Spectrophotometer (USA) to monitor the kinetics of
photodegradation. These experiments were done in triplicate. The biotic degradation was also
evaluated keeping all variables same in the dark.
2.2.2. In presence of riboflavin and porphine (photosensitisers)

The photostability of the three MAAs was also assessed in the presence of two photosensitisers:
riboflavin (RF) and porphine (PPY) in pure water. The respective molar ratios of
MAA:Photosensitisers were P334:RF-197:1, SH:RF-205:1 and P334:PPY-1460:1, SH:PPY-
1412:1. Then, these MAA-solutions were irradiated with total dosage of 2000 KJ m-2 per hr at
30°C in the presence of riboflavin for a total period 20 sec each. Their spectra were analyzed
every 5 seconds all along the irradiation experiments. Similarly, the MAAs were also irradiated
in the presence of porphine for a total time period of 60 minutes each and the corresponding
spectra were analysed every 15 minutes during the irradiation experiments.
2.2.3 Photostability in riboflavin with sodium azide quencher

The photodecomposition study of the MAA were also conducted with 6.6×10-11 M riboflavin
in combination with 5×10-3 M sodium azide in pure water as solvent. The solutions were
irradiated with a dosage of 2000 KJm-2 per h at 30°C for a total time period of 20 seconds for
SH and P-334, and 60 seconds for PNE. During the irradiation experiments, their corresponding
spectra were investigated every 5 seconds for SH and P-334, and 10 seconds for PNE.
2.3. Determination of the photodegradation quantum yield
The photostability of the MAAs were assessed and compared using the photodegradation
quantum yield (Φ)as described in Eq-1:
Number of molecules photodecomposed

Φ= Eq. 1
Number of photons absorbed
Number of molecules photodecomposed = roV t NA where ro is the rate of photo decomposition,

V the volume of solution (L) NA is Avogadro number, t is the time in sec
116
Number of photons absorbed = I0(1- 10-(A)), I0 is the number of photons emitted within the
absorption band of mycosporine, A is the absorbance of the mycosporine solution.
2.4. Indirect determination of singlet oxygen steady state concentration

in natural matrices by quenching experiments
The natural water matrices, 4.0×10-7 M of porphine and 1.3×10-5 M of riboflavin were irradiated
in presence of furfuryl alcohol (FFA,1.0×10-4 M). The natural water matrices were irradiated
for time periods of 0.5 and 1 h for river, 1, 2 and 4 h for estuary water and 2 and 4 h for
oceanwater. The photosensitizers solutions were irradiated for 1 and 3 minutes for riboflavin
and porphine, respectively. The major product of singlet oxygen reaction with FFA is 6-
hydroxy-(2H)-pyran-3-one(6-HP-one), and it was quantified as a function of irradiation time
using High Performance Liquid Chromatography (HPLC). HPLC analyses were carried out
with Agilent 1290 (USA) equipped with a Supelco Lichrosphere RP18-5 (25 mm×4.6 mm,
5µm) column, 20 µL injection, eluent 80% water with 0.1% H3PO4, 20% acetonitrile, rate 2
mLmin-1, and UV detection at 205 nm and 218 nm. The retention times of FFA and 6-HP-
onewere 2.9 and 1.8 minutes, respectively. The steady state concentration of singlet oxygen
([1O2]ss) was determined using the method of Haag and Hoigne et al 38.
2.5. Detection of photoproducts by liquid Chromatography mass

spectroscopy (LC-MS)
The MAA-solutions analyses were carried out using an Ultimate 3000 RSLC system
(ThermoFisher Scientific, Germany) coupled with an Orbitrap Fusion Lumos Tribrid mass
spectrometer (ThermoFisher Scientific, Waltham, MA, USA) operated in positive mode.
2.5.1 Chromatographic separation

Photoirradiated MAAs were diluted 100-fold with mobile phases (90% A: acetonitrile; 10% B:
ammonium acetate 5 × 10-3 M) for HPLC-ESI MS and MS2/MS3 analysis. Mycosporine-like
amino-acid precursors and photoproducts were separated on a HILIC Osaka Soda Capcell Core
PC column (2.1 × 150 mm, 2.7 μm, 90Å) from BGB Analytics (Saint-Jean de Gonville, France).
117
The mobile phases were: 5x10-6 M ammonium acetate in water at pH 6.5 (A) and acetonitrile
(B). The flow rate was fixed at 0.35 mLmin-1. The HPLC separation was carried out with the
following gradient elution profile: 0-2min, 10% B; 2-12 min, 10 to 60% B; 12-14min, 60%; 14-
15.5min, 60 to 80%; 15.5-17.5 min 80%; 17.5-19.5 min, 80 to 10% B; 19.5-23 min, 10% B. A
15 µL aliquot of diluted extract was injected.
2.5.2 Evaluation of the photostability of MAA-precursors

To study the photostability of the MAA-precursors, a SIM analysis (Selected Ion Monitoring)
was performed in order to monitor the variation of the signal intensity of the MAA precursors
at T0 h, T3 h, and T6 h For LC-MS experiments, the ESI conditions were: sheath gas 50 (arb),
auxiliary gas 10 (arb), sweep gas 1 (arb), ion transfer tube and vaporizer temperature 350 °C,
rf lens 50% and positive ionization voltage 3500 V. SIM analysis settings: isolation width 1
min, resolution 60000.
2.5.3 Untargeted screening of MAA-photoproducts

The mining of MAA-photoproducts was performed using the untargeted electrospray orbitrap
MS²/MS3 method developed by Parailloux et al. (2020) 39
. For LC-MS experiments, the ESI
conditions were: sheath gas 50 (arb), auxiliary gas 10 (arb), sweep gas 1 (arb), ion transfer tube
and vaporizer temperature 350 °C, rf lens 50 % and positive ionization voltage 3500 V. Full
MS Orbitrap (OT) settings were: resolution 120000, mass range m/z 150 - 500, dynamic
exclusion 5 sec and intensity threshold 2×104. The ddMS² OT settings were: resolution 60000
for HCD70 MS² scans and 30000 for HCD50 and CID30 MS² scans, isolation width 2 Da. The
ddMS3 ion-trap (IT) settings were: scan rate 33333 Da.s-1, peak width ≤ 0.5 FWHM, isolation
width 2 Da. A set of eight characteristic fragment ions and a dozen of neutral losses appearing
in the course of the radical demethylation pathways of MAAs were selected as relevant
fragmentation patterns to screen MAA-photoproducts in a data-dependent MS²/MS3 method.
Fragmentation conditions at HCD70 and CID30 MS² scans were used as the best conditions to
generate MAA-specific fragmentation patterns. Data-treatment of the acquired raw data was
carried out on Compound Discoverer 2.1TM software (ThermoFisher Scientific, Waltham, MA,
USA). An untargeted workflow was designed to sort out the MAA-photoproducts detected for
which the signal intensity was above 1×104 and the number of characteristic fragment ions was
greater than or equal to 5 in their HCD70 MS² scan.
118
The phostability of shinorine, porphyra-334 and palythine were assessed in pure water and
natural matrices following UV irradiation and the absorbance and concentration of these were
measured periodically and the quantum yield was measured via UV spectrometry. The
mechanism of degradation (Type 1 and Type 2) of these molecules in the presence of
photosensitizes riboflavin and porphine were also looked into. Detection and quantification of
singlet oxygen in natural matrices were carried out with a furfuryl alcohol probe by HPLC. The
potential photodegradation products post UV exposure were separated and analysed via LC-
MS. All these are discussed below.
3.1 Photostability in pure, river, estuary and ocean Water
As confirmed by Figure 2, shinorine and porphyra-334 were UV absorbing in the UV-A region
and found to possess λMax at 334 nm, palythine in UV-B with λMax at 320 nm. The spectra of the
lamp is also shown in Figure 2.
Figure 2: Spectra of MAA (Molar Extinction Coefficient Ɛ vs Wavelength Spectral irradiance

of the Xenon lamp used in the solar simulator (Irradiance vs Wavelength overlayed).
119
The pH of these molecules post dissolution in pure distilled water were mostly acidic. The
individual pH of these solutions was 5.2 for shinorine, 5.2 for porphyra-334 and 2.5 for
palythine. The pH of shinorine and porphyra-334 were found to be unchanged after irradiation.
Figures 3 A, B & C and Figure 4 display the potential photodegradation of these MAAs after
UV irradiation for a time duration of 6 h. These molecules on UV absorption gets excited to
higher energy levels from the ground state (π-π* transitions) and some of these molecules take
part in photoreactions and others subsequently return to the ground state predominantly via non
radiative relaxation as heat energy culminating in its degradation. The quantum yield of
fluorescence of these molecules according to studies by Conde et al. is very low, i.e., 1.6×10-04
for shinorine and 2×10-04 for porphyra-334 33
. A relative comparison among rates of
photodegradation of these MAAs according to the quantum yield of photodegradation were
estimated by the determination of the ratio between the number of photodegraded molecules
and the number of photons absorbed measured by radiometry.
120
A
Figure 3: Time dependent absorption spectra and normalised kinetics of photodegradation (in
inset) ofA. Shinorine (334 nm), B. Porphyra-334 (334 nm) and C. Palythine (320 nm) in pure
water (Blue shift in Palythine after 4 h from 320 to 315 nm) T0, T2, T4, T6 are the time
periods of irradiation after 0, 2, 4, 6 h, respectively).
121
Figure 4: Normalised kinetics of photodegradation of Shinorine (334 nm), Porphyra-334 (334
nm), Palythine (320 nm) in pure water at 0, 7200 14400, 21600 seconds (0, 2, 4, 6) h
The photostability data according to QE (Φ) revealed that shinorine (0.14±0.02)×10-05 and
porphyra-334 (0.18±0.040)×10-05 were found to be extremely stable and palythine
(6.54±1.4)×10-05 the least stable (Table 1). Both shinorine and porphyra-334 exhibited
zwitterionic behaviour (neutral form) at pH-5.2. The lower stability of palythine relative to
shinorine and porphyra-334 could be due to the formation of its protonated form at pH < 2.5
(Figure 5). In this form the corresponding absorption spectra of palythine after 4 h of irradiation
(λ Max) exhibited a hypochromic shift/blue shift from 320 nm to 315 nm (Figure 3C). On
irradiation of palythine solutions in acetate buffer at pH of 5 (pH ≥ 2.5) by keeping all other
variables unchanged revealed that it was extremely stable with low QE (0.77±0.19)×10-05 and
devoid of hypochromic shift due to its zwitter-ionic form at the corresponding pH (Figure 6).
Thus, pH of these solutions could be a major factor that decides the stability of these molecules.
This could be confirmed by studying the photostability of MAAs in different buffers solutions.
122
Figure 5: Zwitter-ionic (pH = 5) and protonated form (pH = 2.5) of palythine.
Table 1: Quantum yield of photodecomposition of mycosporine like amino acids (MAA) in

Pure water and in Natural matrices (Ocean, estuary and river water).
Quantum Yield of Photodegradation (Φ) x 10-05
Mycosporines
Like Amino
Acids (MAA) Pure Water Ocean Water Estuary Water River Water
Shinorine 0.14 ± 0.02 0.18 ± 0.036 0.2 ± 0.04 0.35 ± 0.08
Porphyra-P334 0.18 ± 0.040 0.2 ± 0.04 0.25 ± 0.05 0.3 ± 0.06
Palythine 6.54 ± 1.4 7 ± 1.4 8.2 ± 1.6 11.5 ± 2.4
123
Figure 6: Palythine in acetate buffer (320 nm) (T0, T2, T4, T6 are the time periods of
irradiation after 0, 2, 4, 6 h, respectively).
The QE and order of stability that we obtained for shinorine, porphyra-334 and palythine are in
contrary to the results in the literature by Conde et al.. Using a monochromatic source ensuring
that λ~(325 nm and 320 nm) reached the source and fixing the initial pH to about 6.8 and 6
respectively they obtained QE of: 34×10-05 for shinorine, 24×10-05 for porphyra-334 and
1.2×10-05 for palythine, and found an order of stability of PNE > SH ~ P334) 33, 34
. The high
photostability of palythine as explained in these studies relative to shinorine and porphyra
molecules was due to the absence of substitution in the nitrogen atom of the cyclohexenimine
ring that reduces the photochemical robustness of the molecule. This influences the competitive
relaxation pathways of the excited states through geometrical isomerization around the C=N
bond. This geometrical isomerism causes deactivation of shinorine and porphyra-334 and this
is absent in palythine. The energy barriers to C=N rotation and to nitrogen inversion are
influenced by structural effects that are dependent on the substituents on nitrogen 34.
The photostability of shinorine and porphyra-334 according to our study were lower in
comparison to the previous studies. This contradiction arose due to difference in band width of
the irradiation source 200-700 nm (xenon arc lamp) leading to an increased number of photons
absorbed and also due to the lower initial rate photodecomposition (no of photons emitted)
124
resulting in low QE. This difference in order of photostability between our study (SH ~ P334 >
PNE) and that of Conde et al. arises due the experimentation of palythine at acidic pH 2.5 and
additionally the subsequent lowering of pH during irradiation could be responsible for the
hypochromic shift. At acidic pH the protonation of the unbounded lone pair of electrons of the
nitrogen atom would prevent the resonance delocalization of the molecule. This prevention
would increase the energy requirements of the electronic transition in the ultraviolet spectrum
and causes a hypochromic shift as reported by Ogawa et al. for porphyra-334 when pH
decreases from > 3 to 140. This degree of resonance delocalization can also affect the molar
extinction coefficient. The subsequent irradiation of palythine after fixing the pH at 5 with
acetate buffer for palythine gives it a reasonable stability relative to shinorine and porphyra-
334 confirming the results of Conde.et al. from these experiments, we can conclude that the
photostability of palythine decreases in acidic medium.
The natural matrices that were used for the study are river, estuary and ocean waters. These
41
matrices are a rich source of ROS on UV irradiation . The presence of ROS in marine
ecosystems were first detected by Baalen and Marler in 1966 who confirmed the presence of
hydrogen peroxide (H2O2) on the surface sea water via a sensitive scopoletin-peroxidase based
42
fluorescence assay . ROS can accelerate the degradation of both organic substances and
macromolecular materials to low molecular weight compounds enhancing the propensity for
microbial metabolism thus having a good impact on the ecosystem. These species indirectly aid
cycling of carbon, oxygen, sulphur, nitrogen and trace metals. These natural matrices are a mix
of organic and inorganic constituents at very low concentration together with the biotic
components. Many variables such as the pH, temperature and proportion of organic and
inorganic constituents fluctuate according to the time and geographical location. Due to this
complexity, it is difficult to anticipate the reactions that lead to the formation and destruction
of ROS43. Both the biotic and abiotic factors play a significant role in the generation and
dynamics of ROS in these matrices. These ROS are transient intermediates with varying
lifetime like 3×10-06 s for singlet dioxygen (1O2,) 44, 1×10-06 s for hydroxyl radicals OH, 1 s for
42
organic peroxyl radicals (RO2) in water . This has made the detection of ROS quite
challenging. However, it is possible to study them either via direct detection by coupling Laser
flash Photolysis and time resolved spectroscopy or via molecular probes able to selectively
react with ROS and generate a stable compound more easily analysed.
The prime reasons for the formation of ROS in the natural waters is due to the photochemical
43
reactivity of coloured dissolved organic matter (CDOM) . The spectrum of absorption of
125
CDOM ranges from 280 nm to 700 nm. These molecules can act like a photosensitizer in the
presence UV light generating ROS such as singlet oxygen, hydroxyl radical, superoxide radicals
etc (Refer Chapter 2) 43, 45 , 46. The concentration of CDOM decreases with salinity, and were
found to be highest in river water and estuaries compared to oceans water. The main chemical
constituents of CDOM are humic- like, fulvic acid, etc 43 (Refer Chapter 2).
The photostability of MAAs were the investigated in river, estuary and ocean water. These
waters were found to be slightly basic in nature with pH 8.4 for river, 8.5 for estuary and 8.1
for ocean water. The absorption spectra of these matrices are depicted in Figure 7 with the
spectrum of sun simulator used for the irradiation experiments.
Figure 7: UV Absorption spectra of pure, river, estuary and ocean water juxtaposed with the
solar spectra
These matrices show higher absorption relative to pure water both in the visible (400-700 nm)
and the UV region (280-400 nm) and this can be attributed to the presence of CDOM) and their
presence in river water and estuary water is quite evident (higher absorption in this region) in
the spectra. Concentration of CDOM is found to be lowest in ocean water. The higher
47
absorption band in 200 to 250 nm is probably mainly due to the presence of nitrate ions .
48
Irradiation of nitrate ions could also lead to the generation of hydroxyl radicals . The time
period of irradiation was fixed to 6 h and the spectra were analysed after every 2 h to monitor
the photodegradation. The order of photostability shinorine ~ porphyra-334 > palythine is the
same as that which has been observed in pure water (Table 1) and was in accordance with the
35
literature studies by Hedges et al. . Hedges et al. has investigated the photostability of
126
shinorine, porphyra-334 and palythine in sea water and found the rate constant of degradation
(k) 0.018 m2 kJ-1 for shinorine, 0.026 m2 kJ-1 for porphyra-334 and 0.26 m2 kJ-1 for palythine
35
with order of stability SH ~ P334 > PNE . The degradation of shinorine, porphyra-334 and
palythine in all the three matrices were found to be higher than in pure water. On a relative
comparison among the three matrices the degradation was highest in river water and the least
in ocean water and estuary water in the intermediate by taking in account the initial rate between
0 and 2 h (Figure 8, Figure 9A & B and Table 1). This is due to the presence of mixture of
CDOM (organic) and nitrate (inorganic) in these matrices depicted in the spectra in Figure 7.
Its concentration follows the order River > Estuary > Ocean. The photosensitizing action of
CDOM and nitrite leading to the generation of singlet oxygen and hydroxyl radicals on
irradiation are responsible for the oxidation of these MAAs as represented in Eq-4 and
Eq-5 49.The biotic degradation was also evaluated keeping all the other conditions constant in
the dark and it was found to be negligible (Figure 10 A, B & C).
Figure 8: Kinetics of photodegradation of Shinorine in pure, ocean, estuary and river water
respectively at (0, 2, 4, 6 h)
127
Figure 9 A: Kinetics of photodegradation of Porphyra-334 in pure, ocean, estuary and river
water respectively at (0, 2, 4, 6 h)
Figure 9 B: Kinetics of photodegradation of Paythine in pure, ocean, estuary and river water
respectively at (0, 2, 4, 6 h).
128
Figure 10: Kinetics of biodegradation of A. Shinorine B. Porphyra-334 C. Paythine in Ocean,
Estuary and River water respectively at (0, 2, 4, 6 h).
Mechanism of photosensitisation by CDOM

hv
1CDOM → 1CDOM ∗ (𝐸𝑞 2)
ISC
1CDOM ∗ → 3CDOM ∗ (𝐸𝑞 3)
302
3CDOM ∗ → 1CDOM + 1O2 (𝐸𝑞 4)
1O2 + SH/P334/PNE → Oxidised Products (𝐸𝑞 5)
3.2 Photostability with photosensitisers riboflavin and porphine
A study of the photostability of these molecules in the presence of photosensitizers such a

riboflavin and porphine was conducted to fortify what we observed with the natural water
matrices. The fundamental hypothesis is that reactive oxygen species primarily singlet oxygen
are able to degrade MAAs. Both riboflavin and porphine are water soluble photosensitizers.
129
Both have a series of absorption bands both in the UV region and visible region. The bands for
riboflavin are 222, 266, 373 and 447 nm (λRb (Max))50 (Figure 11 A).
Figure 11: Absorption spectra of A. Riboflavin and B. Porphine.
In the case of porphine an intense absorption band at 414 nm which is known as B or Soret
band is seen. This band is narrower when its purity increases, for unprotonated forms and when
no aggregations take place. The Soret band is due to the π-π * electronic transition from ground
state to a higher excited state. There are other four bands at wavelengths 517, 555, 580, 635 nm
that are often referred to as Q bands. The intensity of these bands are 10 to 20 times lower than
the Soret band (Figure 11 B). These absorptions result from a weak π-π* electronic transition
leading the compound from its ground state to the first excited state (S0-S1). Both of these
sensitizers have a high molar extinction coefficient: Riboflavin-(ƐRb-13222 M-1cm-1 (447 nm),
10300M-1cm-1 (373 nm), 31600 (266 nm), 28500 (222 nm)) Porphine-(ƐPPY -219000 M-1cm-1
(414 nm), 1-20000 M-1cm-1 (517, 555, 580, 635 nm)51. These photosensitizers upon UV
irradiation are capable of ROS such as singlet oxygen (1O2), radicals or ions such as superoxide
(O2-), hydrogen peroxide (H2O2), hydroxyl radicals (OH-), peroxy radicals (HO2) 52, 53.
130
The photosensitization reaction can take place via Type 1 (Electron transfer-(O2-), (H2O2),
(OH-), (HO2)) or Type 2 mechanism (Energy transfer-1O2) or both (Figure 12). The
photosensitisation process of riboflavin takes place via the combination of both Type 1 and
54
Type 2 . However, in the case of porphine, it takes place predominantly via
Type 2 mechanism 52, 53 , 55, 56.
Figure 12: Mechanism of photosensitisation by Type 1 and Type 2 (Reproduced from57).
The Type 1 mechanism of photosensitisation of riboflavin is depicted in Eq-6 - Eq-9. In Type

1 mechanism riboflavin upon irradiation by UV rays undergoes excitation from the singlet
ground state (1RF) to the higher singlet excited states (1RF*) of lower energy as in Eq-6. The
lifetime in these singlet excited states is very short about 3 to 5 ns 58. This in fact prevents the
photosensitisation reactions taking place at this stage. A monomolecular deactivation of the
excited electronic states of the photosensitiser (1RF*) takes place by spontaneous emission via
radiative fluorescence to the ground state or by non-radiative processes such as internal
conversion or intersystem crossing leading to triplet state (3RF*) of similar vibrational energy
since Intersystem crossing is an expected pathway for most photosensitisers in Eq-7 59. In The
triplet state lifetime is higher around 120 μs for riboflavin 60
. This long lifetime in the triplet
state favours the photosensitisation reaction of the activated riboflavin with shinorine/porphyra-
334/palythine resulting in a one electron transfer (abstraction of electron) or proton transfer
reaction from the SH, P334 and PNE leading to the formation of two radicals i.e. riboflavin
anion (3RF-) and MAA cation (SH+, P334+ and PNE+) or vice versa depending on the redox
potential of the pair of these individual molecules. The redox potential of triplet riboflavin and
131
porphine was found to be 1.7 V and 1.54V respectively 61, 62 (The redox potential of shinorine,
porphyra-334 and palythine are not known so its quite difficult to predict the thermodynamic
favourability of Eq-8. The presence of ground state dioxygen from solvent (water) can lead to
the transfer of newly acquired electron to oxygen by riboflavin radical 3RF- resulting in
superoxide radical (O2-) formation and subsequently riboflavin returns to the ground state. This
superoxide radical anion can react directly with SH+, P334+ and PNE+ resulting in oxidised
products (SHOO., P334OO., PNEOO. Radicals etc (Eq-10))63 or act as the precursor of other
reactive oxygen species like hydrogen peroxide (H2O2) and hydroxyl radicals (OH.). The
electronically excited triplet state of riboflavin 3RF* and the ground state MAAs (SH-H, P334-
H and PNE-H) can act as hydrogen donor (Eqn-11, Eq-12). The radical species of MAAs
resulting from these Type 1 phenomenon can actively participate in different reactions (Eq-13,
Eq-14). In the presence of oxygen, oxidized forms of MAA substrate can react with O2 to yield
peroxyl radicals initiating a radical chain auto-oxidation (Eq-14) 64.
hv
1RF → 1RF ∗ (Eq 6)
ISC
1RF ∗ → 3RF ∗ (Eq 7)
3RF ∗ + SH/P334/PNE → 3RF .− + SH .+ /P334.+ /PNE.+ (𝐸𝑞 8)
3O2
3RF .− → 3RF + O2 −. (𝐸𝑞 9)
SH + /P334+ /PNE+ + O2 −. → SHOO. /P334OO. /PNEOO. (𝐸𝑞 10)
3RFH ∗ + SH/P334/PNE → 3RF . + SH . /P334. /PNE . (𝐸𝑞 11)
3RF ∗ + SHH/P334H/PNEH → 3RFH . + SH . /P334. /PNE . (𝐸𝑞 12)
SH . /P334. /PNE . + O2 → SHOO. /P334OO. /PNEOO.(𝐸𝑞 13)
SHOO. /P334OO. /PNEOO. + SHH/P334H/PNEH → SH . /P334. /PNE. + SHOOH/P334OOH/
PNEOOH (𝐸𝑞 14)
The Type 2 mechanism of photosensitisation of riboflavin (same for porphine) is depicted in Eq-
15 - Eq-17. In Type 2 mechanism the photosensitiser riboflavin 3RF* in the exited state transfers
its excess excitation energy to ground state dioxygen (3O2) resulting in the formation of active
oxygen intermediates such as singlet oxygen (1O2), and thereby regenerating ground state
132
photosensitiser (RF). Singlet oxygen dissipates energy in the form of heat via physical quenching
or reacts with SH, P334 and PNE resulting in the formation of oxidised products (Eq-18).
hv
1RF → 1RF ∗ (𝐸𝑞 15)
ISC
1RF ∗ → 3RF ∗ (𝐸𝑞 16)
3O2
3RF ∗ → 1RF + 1O2 (𝐸𝑞 17)
1O2 + SH/P334/PNE → SHOO. /P334OO. /PNEOO. (𝐸𝑞 18)
Photosensitization of porphine occurs mainly via this phenomenon. MAAs show substantial
degradation in the presence of riboflavin and porphine (Figure 13 A & B, 14 A & B, 15 A & B,
16 A & B). These sensitizers can photodegrade these molecules via Type 1 and Type 2
mechanisms at a fast pace. No photobleaching was observed for riboflavin for any of its
absorption bands (Figure 13 A). In the presence of riboflavin where both Type 1 and Type 2
phenomenon can occur concomitantly the quantum yield of all these MAAs are higher than the
unsensitized ones indicating low photostability. Both shinorine and porphyra-334 are unstable
upon irradiation in presence of riboflavin and have high quantum yield of photodegradation
(2700±700)×10-05 and (3000±600)×10-05 respectively (relative to unsensitised ones in pure
water) when compared to palythine with low QE (700±150)×10-05 (Table 2). This makes
palythine stable in the presence of riboflavin relative to the other two. This is in contrary to its
behaviour observed with pure water on irradiation. In the presence of porphine (PPY) where
Type 2 mechanism is more predominant photobleaching of the Soret band of porphine at λ (Max)-
414 nm is observed on irradiation (Figure 15 A). Shinorine, porphyra-334 and palythine showed
almost similar stability in terms of QE (Table 2).
133
Figure 13: A. Spectra of photodegradation of Shinorine (λ-334 nm) in Riboflavin + Pure
water B. Spectra of photodegradation of Shinorine (λ-334 nm) (250-400nm magnified region)
(where T0, T5, T10, T15, T20 are the time periods of irradiation after 0, 5, 10, 15, 20 s,
respectively)
134
Figure 14: Spectra of photodegradation of A. Porphyra-334 (λ-334 nm) with Riboflavin B. Spectra of
photodegradation of Palythine with Riboflavin (λ-320 nm) (where T0, T5, T10, T15, T20 are the time periods of
irradiation after 0, 5, 10, 15, 20 s, respectively).
Figure 15: A. Spectra of photodegradation of Shinorine (λ-334 nm) in Porphine + Pure water B. Spectra of
photodegradation of Shinorine (λ-334 nm) (270-400nm-magnified Region) (where T0, T15, T30, T45, T60 are
the time periods of irradiation after 0, 15, 30, 45, 60 min, respectively)
135
Figure 16 : Spectra of photodegradation of A. Porphyra-334 (λ-334 nm) with Porphine B. Spectra of
photodegradation of Palythine with Porphine (λ-334 nm) (where T0 , T15, T30, T45, T60 are the time periods of
irradiation after 0, 15, 30, 45, 60 min, respectively)
To evaluate the respective fraction of degradation of MAAs by riboflavin by electron transfer

(Type 1) sodium azide was used as a physical quencher to trap and scavenge singlet oxygen as
in (Eq-19) with rate constant (k) 5.0 × 108 M-1s-1 65, 66
. This quencher can enter vibrational or
electronic excited states and deactivate the excited state of singlet oxygen. Physical quenching
by sodium azide takes place by catalysis of singlet oxygen to the ground state oxygen via spin
orbit coupling or triplet state energy transfer.
Physical quenching by sodium azide
NaN3 + 1O2 → NaN3 + 3O2 (𝐸𝑞 19)65
Upon evaluating the respective proportion of degradation via Type 1 individually, the extent of
degradation is as follows: shinorine (15%), porphyra-334 (7%), palythine (29%) (Table 2,
Figure 17 A & B, Figure 18). Separate evaluation of the quantum yield of photodegradation
136
in the presence of riboflavin via Type 1 and Type 2 mechanism concludes that MAAs degrade
mostly by energy transfer i.e. via singlet oxygen. The approximate percentage of degradation
via Type 2 mechanism can be estimated to: shinorine-85%, porphyra-93% and palythine-71%.
The triplet energy of palythine was about 330 KJ/mol in contrast to ground state shinorine and
porphyra-334 with 250 KJ/mol each 67. The triplet energy of 3RF* was about 200 KJ/mol 68.
This triplet energy difference between 3RF* and palythine could prevent energy transfer to a
considerable extent and may lead to quenching of the triplet state of riboflavin by palythine.
This could be one of reasons for the diminution of Type 2 phenomenon according to our
hypothesis.
Figure 17: Time dependant absorption spectra and kinetics of photodegradation (inset) of A.
Shinorine B. Porphyra-334 presence of riboflavin with sodium azide (where T0, T5, T10,
T15, T20 are the time periods of irradiation after 0, 5, 10, 15, 20 s, respectively)
137
Figure 18 : Time dependant absorption spectra and kinetics of photodegradation (inset)
Palythine presence of riboflavin with sodium azide (where T0, T5, T10, T15, T20, T30, T60
are the time periods of irradiation after 0 , 5, 10, 15, 20, 30, 60 s, respectively)
Table 2: Quantum yield of photodecomposition of MAA in combination with riboflavin

(Type 1 + Type 2) and porphine (Type 2).
Quantum Yield of Photodegradation (Φ)x10-05
Mycosporine Like Riboflavin Porphine

Amino Acids (MAA)
Shinorine 2700 ± 700 400 ± 100 20 ± 4
Porphyra-334 3000 ± 600 200 ± 40 15 ± 3
Palythine 700 ± 150 200 ± 50 20 ± 3
138
3.3 Quantification of singlet Oxygen with furfuryl alcohol probe by
HPLC
Furfuryl alcohol probe (FFA) was used to quench the singlet oxygen generated from the
photosensitisers (riboflavin, porphine) as well as from the CDOM from natural matrices river,
estuary and ocean water post UV exposure. FFA is used because of its high selectivity and a
high second order rate constant with singlet oxygen (k =1.2×1008 M-1s-1). The furfural alcohol
69
gets oxidised in the presence of singlet oxygen to 6-hydroxy-(2H)-pyran-3-one . Its
chromatogram is depicted in (Figure 19 A & B) and the structure of 6-hydroxy-(2H)-pyran-3-
one is depicted in Figure 20
Figure 19: A. HPLC Chromatograms of river, estuary and ocean water post irradiation 1 hr,
4h and 4 h respectively with furfuryl alcohol B. Magnified region of the chromatogram
corresponding to λ-209 nm (6-hydroxy-(2H)-pyran-3-one).
139
Figure 20: Formation of 6-hydroxy-(2H)-pyran-3-one by reaction of FFA and singlet oxygen.
F
3CDOM ∗/3RF/PPY ∗ + 3O2 → CDOM/RF/PPY + 1O2 (𝐸𝑞 20)
Singlet oxygen is produced photochemically at a constant rate ‘ F ‘ in Eq-20, the bimolecular

reaction of singlet oxygen with the probe (FFA) with a second order rate constant k to generate
the oxidised product 6-hydroxy-(2H)-pyran-3-one 38, 70. The concentration of furfuryl alcohol
(FFA) in combination with sensitizers and natural matrices upon post irradiation shows a
decrease in comparison to the control (λmax FFA-219 nm)71. This was due to the oxidation of
FFA by singlet oxygen [1O]ss generated on exposure to UV. The oxidised product i.e., 6-
hydroxy-(2H)-pyran-3-one was detected by HPLC with a UV detector at λmax 205 nm 71 (Figure
19 A & B). The concentrations of singlet oxygen [1O2]ss generated by porphine and riboflavin
were 2500×10-14 M and 7500×10-14 M respectively shown in (Table 3) which were evaluated
from the slope (kapp) of Ln[FFA]0/[FFA] vs Time (Figure 21 D &E). Similarly, the
concentration of singlet oxygen was evaluated in river, estuary and ocean water post irradiation
(Figure 21 A, B & C). Irradiation of river water for 1 h leads to generation of 16×10-14 M
singlet oxygen and on the contrary ocean and estuary water upon irradiation for 4 h generated
5.8×10-14 M and 8.3×10-14 M singlet oxygen respectively as shown in Table 3. The
chromatograms of these show the formation of 6-hydroxy-(2H)-pyran-3-one corresponding to
the retention time of 2.30-2.50 min and the retention time of 2.7-3.1 min corresponds to furfuryl
alcohol. The concentration of [1O2]ss was found to be highest in river and least in ocean water
and estuary water in middle (river > estuary > ocean). This was in accordance with the
photodegradation results obtained by UV (river >estuary > ocean). The highest and lowest
concentration of singlet oxygen in river and ocean water supports the results of the lowest and
highest photostability of MAAs in river and ocean water. This indirectly suggests that the
presence CDOM was found to be highest in river water followed by estuary and least in ocean
140
water. The absorption spectra of these matrices (Figure 6) are also substantial evidences for
this.
Figure 21: Ln[FFA]0/[FFA] vs Time for natural matrices and photosensitisers A. River Water
B. Estuary Water C. Ocean Water D. Riboflavin E. Porphine.
141
Table 3: Concentration of singlet oxygen generated by natural matrices and photosensitisers
Matrices kapp (S-1) x10−3 [1O2]ss = kapp/k (M) x10−14 Total Irradiation Time (hrs)
Ocean Water 0.007 5.8 4
River Water 0.02 16 1
Estuary Water 0.01 8.3 4
Riboflavin 9 7500 0.01
Porphine 3 2500 0.05
3.4 Photostability of MAA-precursors: characterization of

photoproducts by mass spectrometry
The photostability of the MAA-precursors was evaluated by the monitoring of their relative
signal intensity over 6 h after SIM analysis. The results illustrated in Figure 22 A, B & C shows
that porphyra-334 and shinorine displayed the greatest stability in all water matrices tested in
comparison to palythine. Indeed, the relative intensity of palythine decreased drastically in river
and ocean water after 3 h and disappeared completely after 6 h.
142
Figure 22: Relative Signal intensities of A. Shinorine B. Porphyra and C. Palythine in pure,
river, and ocean water by Mass Spectrometry.
Interestingly, the relative intensity of porphyra-334 and shinorine decreased linearly over 6 h
but did not reduce below 80 and 60 %, respectively. Moreover, both demonstrated high stability
in ocean water with a relative intensity above 90 %. Contrary to palythine, shinorine was highly
stable in pure and river water with a decrease of solely 15 % of the relative intensity after 6 h.
These observations suggested that porphyra-334 was the least impacted by the UV-exposure
over 6 h and displayed similar photostability in ocean water. Besides, the nature of the water
matrix exhibited a significant effect on the photostability of MAAs, owing to their composition
in mineral and organic materials. The nature of the matrix produces an impact on their retention
times (Table 4). The chromatographic separation of the MAA-precursors showed that all the
three presented the same retention times in river, ocean waters and different in the case of pure
water.
143
Table 4: Retention times of MAA-precursors observed in in pure water, river and ocean
water.
MAA-precursors Pure Water (min) River Water (min) Ocean Water (min)
Shinorine 7.33 7.23 7.22
Porphyra-334 7.13 7.07 7.07
Palythine 7.68 7.75 7.79
3.5 Structural identification of MAA-photoproducts
The untargeted MS²/MS3 method allowed the detection of photoproducts appearing in the
course of the photodecomposition of the MAA precursors, porphyra-334 and shinorine. Indeed,
five MAA-derivatives were detected with at least five characteristic fragment ions in their
HCD70 MS² scans in each of the water matrix as is shown in Table 5. In pure and ocean water,
two identified photoproducts were found with a number of fragment ions below the intensity
threshold. Nonetheless, their targeted fragmentation in HCD70 fragmentation conditions
confirmed their appearance in these water matrices. In porphyra-334 extract, the photoproducts
mycosporine-methyl amine threonine and, especially, aplysiapalythine A were the most
abundant in the different water matrices. In shinorine extract, the three photoproducts were
mainly observed in pure water. The photoproduct palythine produced in porphyra-334 and
shinorine extract was predominant in river water. These observations assume that the ionic and
organic composition of the water matrix also had an impact on the photodecomposition
pathways of the MAA-precursors. In each MAA-extract, the photodegradation products were
detected and elucidated using the fragmentation dataset collected in their CID30 and HCD50
MS² scans. The structural relationships between the MAA-precursors and the photoproducts
are given in Figure 23.
144
Table 5. Photoproducts identified in porphyra-334 and shinorine in the different water matrices tested. The MAA-
.
photoproducts were listed after applying filtering criteria fixing an intensity threshold at 1x10 4 and a number of characteristic
fragment ions (CFI) greater than or equal to five in every water matrix. Structural elucidation of MAA-photoproducts was
carried out on the basis of the set of fragment ions collected in CID30 and HCD50 MS² scans
Number of CFI (/8) found in

HCD70 MS² scans
Precursor Photoproducts Formula [M+H]+ Fragment ions Pure River Ocean
(m/z) CID30/HCD50 MS² Water Water Water
288.1312; 285.1440;
Aplysiapalythine A C13H22O6N2 303.1551 244.1413; 230.0900; 8 8 8
226.1310; 200.1153
Mycosporine-methyl- C13H22O6N2 303.1551 288.1315; 259.1284;
amine threonine 244.1415; 227.1026 5 ND 6
145
C10H16O5N2 245.1132 230.0894; 227.1034;
Porphyra-334 209.0927; 197.0927;
Palythine 186.1000; ND 6 ND
274.11557; 230.1259;
Asterina-330 C12H20O6N2 289.1394 212.1150; 186.1000 5 ND <5
274.1160; 256.1058;
Mycosporine-methyl- C12H20O6N2 289.1394 230.1256; 212.1142 5 ND ND
amine serine
Shinorine 230.0894; 227.1034;
Palythine C10H16O5N2 245.1132 209.0927; 197.0927; <5 7 <5
186.1000;
Figure 23: Structural elucidation of the isomeric couples mycosporine-methyl amine
threonine/aplysiapalythine A and mycosporine-methyl amine serine/asterina-330 on the basis
of their fragmentation data acquired in CID30 MS² scan.
146
The structural elucidation of two isomeric couples in porphyra-334 and shinorine extracts was
based on the comparison of mass spectra and revealed distinct fragment ions, allowing the
confirmation of the presence of mycosporine-methyl amine threonine/aplysiapalythine A (m/z
303.1551) and mycosporine-methyl amine serine/asterina-330 (m/z 289.1394).
Aplysiapalythine was identified by the appearance of the fragment ion m/z 285.1445 after its
dehydration. Moreover, the dealkylation C2H4O (44.026 Da) confirmed the specific structure
of its functional group. Finally, the dealkylation C3H6O (58.042 Da) starting from the fragment
ion m/z 288.1312 [M+H]+ - CH3.) in CID30 MS² spectrum of aplysiapalythine A proved that
there was a decarboxylated functional group on the C1 carbon of the skeleton ring independent
of the glycine group on the C3 carbon. In parallel, the photoproduct asterina-330 produced from
shinorine showed three fragment ions already reported in literature whereas mycosporine-
methyl amine serine revealed an intense fragment ion m/z 256.1058 suggesting its dehydration
to the radical methyl fragment ions m/z 274.1160 72, 73.
Both porphyra-334 and shinorine produced mycosporine-methyl amino-acid derivatives,

namely mycosporine-methyl-amine serine and threonine. The nature of these photoproducts
assumed that decarboxylation mechanisms of amino-groups occurred during their
photodegradation. Similarly, decarboxylation can explain the production of the isomeric
photoproducts asterina-330 and aplysiapalythine A. Interestingly, porphyra-334 and shinorine
displayed a common MAA-photoproduct for which the exact mass and the fragmentation
matched with the palythine (m/z 245.1132).
Structural comparisons between porphyra-334 and its photoproducts permits to demonstrate

that its photodecomposition generated a MAA threonine-derivative by decarboxylation of
amino-groups located on C1 or C3 of the skeleton ring. Likewise, the photoirradiation of
shinorine produced either MAA serine- or glycine-derivative according to the location on which
the decarboxylation occurred. The generation of palythine as photoproduct in both extracts
suggests a dealkylation of the amino-groups, serine on shinorine and threonine on porphyra-
334, positioned on C1. These observations confirmed that photodegradation of MAAs involved
minor structural modifications of the cyclohexenimine ring. This structural decomposition
observed in the experimental dataset were in accordance with previous studies assuming that
the photodegradation induced amino- and imino acid loss similar to what had been observed in
mass spectral fragmentation of MAA-precursor 35, 74.
147
The developed untargeted MS²/MS3 Orbitrap method was efficient to provide the full coverage
of MAA-derivatives appearing in the course of the photoirradiation of porphyra-334 and
shinorine. The nature of the water matrix impacts drastically the photodegradation pathways of
MAAs, related to their ionic and organic composition. The decomposition of the structurally
related shinorine and porphyra-334 induced mainly palythine and mycosporine-like threonine
and serine photoproducts. MAA-photoproducts of shinorine and porphyra-334 were the result
of progressive losses of their functional groups located on C1 and C3, by decarboxylation or
dealkylation. The study of the structural photodecomposition porphyra-334, shinorine,
palythine and mycosporine-serinol brought a novel insight into their resistance to UV-lights
and their applicability as valuable UV-protective sunscreens. The MAA-extracts containing
high levels of di-acidic MAAs may be more resistant to photodegradation, and consequently
more effective. Both shinorine and porphyra-334 have a cyclohexenimine ring with functional
groups both on C1 and C3 carbon. Presumably, these substituents may delay the overall
degradation and protect the skeleton core.
Assuming that the MAA-photoirradiation involved mainly fragmentation mechanisms affecting

75
amino- and imino-groups on C1 and C3 of the skeleton , the hypothesis that MAAs do not
produce putative oxidative MAA-photoproducts by addition of ROS and ring opening under
UV-exposure has not been verified yet. Hence, the development of an untargeted mass
spectrometry approach to flag and annotate oxidative forms and MAA-compounds may
contribute to a deeper exploration of the photodecomposition process in further analysis.
4. Conclusion
MAAs could be a potential replacement for the conventional existing organic and inorganic
synthetic UV blocking agents like oxybenzone, dioxybenzone, ZnO, MgO etc in sunscreen
products due to the biobased origin, non toxicity and high photostability. MAA degradation
occurs via non-radiative relaxation as heat energy not via ROS generation on UV irradiation.
The observations according to this study was that shinorine and porphyra-334 have almost
similar photostability. The neutral/zwitter-ionic form of palythine is highly photostable relative
to its protonated form. All these MAAs could be photodecomposed in natural matrices like
river, estuary and ocean water at a faster pace relative to pure water due to the generation of
148
singlet oxygen from the coloured dissolved organic matter. The concentration of singlet oxygen
on irradiation of these matrices were found to be highest in river water and least in ocean water.
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154
Chapter 4
Photoprotective Polysaccharides Based on Chitosan

and Mycosporine Like Amino Acids
155
156
1. Introduction ........................................................................................................................ 160
2.Materials and Methods ........................................................................................................ 161
2.1. Materials and Chemicals ............................................................................................ 161
2.2. Synthesis and characterization of the chitosan-mycosporines like amino acid
derivatives .......................................................................................................................... 162
2.2.1. Synthesis of chitosan-shinorine (CS-SH) and chitosan-porphyra-334 (CS-P334)
...................................................................................................................................... 162
2.2.2. Characterization of CS-SH and CS-P334 derivatives......................................... 163
2.3. Photostability experiments of the chitosan-MAA derivatives .................................... 164
2.3.1. Irradiation tests ................................................................................................... 164
2.3.2. Quantum Yield determination ............................................................................ 165
3. Results and Discussion ....................................................................................................... 165
3.1 Synthesis of the materials ............................................................................................ 165
3.2 Characterisation by Fourier Transform Infrared Spectroscopy (FTIR) ....................... 167
3.3 Characterisation by Nuclear Magnetic Resonance Spectroscopy (NMR) ................... 168
3.4 Characterisation by UV Spectroscopy ......................................................................... 170
3.5 Photostability of Chitosan Conjugates vs Pure MAA molecules ................................ 171
3.6 Photostability of Chitosan Conjugates vs Pure MAA molecules with Photosensitizers
Riboflavin and Porphine .................................................................................................... 173
3.7 Photostability of Chitosan Conjugates in a natural photosensitizer: Acidified River
Water vs Pure MAA molecules ......................................................................................... 177
4. Conclusion .......................................................................................................................... 180
5. References .......................................................................................................................... 180
157
158
Chapter 4: Photoprotective Polysaccharides Based on
Chitosan and Mycosporine Like Amino Acids
Adapted from Influence of photosensitisers on the photodegradation of chitosan/mycosporine-

like amino acid derivatives in aqueous solutions - pure and river water, M.G. Thomas, F.
Samalens, S. Blanc, T. Pigot, S.C.M. Fernandes, manuscript in preparation
Abstract
Conjugates of chitosan with UV protective mycosporine like amino acids shinorine and
porphyra-334 were synthesized by chemical grafting by amidation. The photophysical and
photochemical properties of these conjugates were characterized by UV, FTIR and NMR
spectroscopy. The photostability of these conjugates were assessed in the presence and absence
of photosensitisers riboflavin and porphine, and also in acidified river water. These conjugates
had extremely low quantum yield of photodegradation due to their high photostability while in
the presence of the photosensitisers and river water , they were unstable do to the generation of
ROS like singlet oxygen.
159
1. Introduction
The discovery, extraction, chemical identification and characterization of natural sunscreens

known as mycosporine-like amino acids (MAAs), may contribute to the rational design of novel
products or materials 1-10.
These highly efficient sunscreens-based materials might be an alternative to conventional UV-

filter products existing today overcoming several drawbacks of the current products such as
side effects on both human (e.g., penetration in the skin and low photostability of such
compounds) and marine environment (e.g., accumulation of such compounds in ocean and
marine organisms) 11-14 that will outperform the currently used UV-protective products.
Mycosporine-like amino acids are secondary metabolites that have been detected in several
marine organisms namely micro and macroalgae, cyanobacteria, zebra fish and marine fishes
2-4, 15-17
(by accumulation via dietary or symbiont origin) . MAAs such as porphyra-334,
shinorine, asterina or palythine, are low molecular weight (188-1050 Da) and water-soluble
molecules consisting of a cyclohexenimine core with different functional groups exhibiting
high absorbing capacity with very high molar extinction coefficients (28 000-50 000 M-1 cm-1)
2, 18, 19,
in the UV-A range (320-360 nm) . Interestingly, these molecules absorb high energy
20-23
UV radiation and dissipate it as less harmful heat energy . They present antioxidant
7, 24
properties and are considered as free radical scavengers that can take up the Reactive
6, 25
Oxygen Species (ROS) reducing cell damage formed during oxidative stress . Because of
these unique properties, MAAs have been used as UV-absorbing/protective compounds in
9, 10, 24, 26
different uses like additives for skin protection photostabilizers to enhance the
durability of plastics, coatings1, 27 and to improve both anionic and cationic micellar systems,
28 29
antioxidants or radical scavengers in food and active molecules for the development of
UV-absorbing polymeric materials 8.
We have been interested on the extraction and identification of MAAs 3, 4 on the study of their
photodegradation in different natural and artificial water matrices and in the presence of
photosensitizers (work of the present PhD thesis), and on the design and development of UV-
absorbing polymeric materials 8. Chemical conjugation and/or immobilization of MAAs on
natural polymers, in particular chitosan (CS), either via covalent or hydrogen bonding can
30-42
facilitate the engineering and preparation of multifunctional materials as we have
demonstrated in our previous work about the preparation of chitosan/mycosporine-like amino
160
acid derivatives (CS-MAAs) conjugates 8. Briefly, in this former study, solutions and
transparent films based on chitosan as a matrix on which MAAs were linked through amide
bond formation were made inspired by the reef fish mucus. It was demonstrated that the ensuing
materials were able to absorb both UV-A and UV-B radiations, were thermoresistant, non-
cytotoxic and compatible with both cell proliferation, and adhesion, and maintain their UV
absorption capacity after UV irradiation.
In the present study, we go one step further by investigating the photodegradation of these
chitosan/mycosporine-like amino acid derivatives under realistic conditions, i.e., solar
irradiation and in pure or natural water matrices (river). The experiments were conducted in
natural water matrices to measure the photodegradation with naturally occurring
photosensitizers like singlet oxygen and nitrates present in river water. These materials were
also photosensitized by the addition of riboflavin and porphine in pure water. A deeper
knowledge about the influence of naturally occurring photosensitizers or riboflavin, porphine
the photodegradation of this kind of chitosan derivatives may contribute to the field of medical,
cosmetic, textile and outdoor materials design.
2. Materials and Methods
Chitosan (CS) with medical-grade (molecular weight (Mw) of 350000 gmol-1 and deacetylation
43
degree of 98% calculated in house was purchased from Mahtani Chitosan PVT Ltd, India.
The MAAs porphyra-334 (P-334, with absorption maximum (λmax)-334 nm and molar
absorptivity (Ɛ)-42300 M-1cm-1) 18
and shinorine (SH, with λmax -334 nm and
Ɛ-44670 M-1cm-1)44 were purchased from the Laboratory of Photobiology of the Central
Research Services of the University of Málaga, Spain. P-334 was extracted from
Porphyrarosengurrtii and SH from Gymnogongrusdevoniensis macroalgae both collected in
the Andalusian coast between 2019 and 2020. The extraction, purification and characterization
of the of the three MAAs were done as described previously 24. P-334 and SH were received as
lyophilized pure extracts and stored at -20 °C until use.
River water was collected from Gave de Pau (Pau, France) in Mars 2021. The sample was ultra
filtered with PVDF syringe filter (0.45 µm pore size, 33 mm diameter, Millex-HV Durapore)
161
in order to remove solid and dissolved organic compounds greater than 30 kDa; and, afterwards,
stored for 4 days (time necessary for the incubations) at 5 °C in glass vials with acid washed
plastic tops. This filtered water was used to record the spectra and unfiltered water was used to
study the photodegradation. River water was selected because of the presence of inorganic and
organic compounds known to function as natural photosensitizers; in particular, nitrates and
dissolved organic carbon that provide a photochemical source for reactive oxygen species
(ROS) via direct photochemical reactions 45. Pure water was obtained from a Millipore Milli-
Q apparatus (France). Riboflavin (7,8-Dimethyl-10-((2R,3R,4S)-2,3,4,5-tetrahydroxypentyl)
benzo[g]pteridine-2,4-(3H,10H)-dione) with 99% purity, porphine [4,4′,4′′,4′′′-(porphine-
5,10,15,20 tetrayl) tetrakis (benzenesulfonic acid) tetrasodium salt hydrate] with 98% purity,
sodium azide with > 99.5% purity and furfuryl alcohol with 98% purity were purchased from
Merck, Germany. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) with commercial
grade was purchased from Sigma Aldrich (Saint Louis, MO).
2.2. Synthesis and characterization of the chitosan-mycosporine like

amino acid derivatives
2.2.1. Synthesis of chitosan-shinorine (CS-SH) and chitosan-porphyra-334 (CS-

P334)
The chitosan-mycosporines-like amino acid (CS-MAAs) derivatives were synthesized via

carbodiimide-based grafting MAAs onto CS backbone by forming amide bonds between the
carboxylic acid groups of MAAs and the primary amino groups of CS, as described in our
previous study 8. Succinctly, 1% (w/v) of CS solution was prepared by dissolving 1g of CS in
100 mL 0.08 M HCl. The molar concentration of carbodiimide added was the same as the
chitosan solution. The molar ratios of unit of CS: MAAs were 1:0.03 for CS:P334 and 1:0.06
for CS:SH. The synthesis was performed in the dark under constant stirring for 24 h at pH 5
and 25 °C. The ensuing derivatives were further dialysed in deionised water for 72 h to remove
the isourea and unreacted MAAs with membranes of molecular weight cut-off of 12000-14000
Da. The samples were then lyophilised to obtain the CS-MAAs derivatives in solid form and
stored at 5 °C before used.
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2.2.2. Characterization of CS-SH and CS-P334 derivatives
Fourier-Transform Infrared Spectroscopy (FTIR) in Attenuated Total Reflectance (ATR) mode

with a Nicolet IS50 FTIR spectrophotometer (Thermo Fisher, USA) equipped with a diamond
crystal and a DLa TGS detector was used to verify the chemical structure of the samples. The
spectra were recorded between 400 and 4000 cm-1 in absorbance mode with a resolution of 4
cm-1 and 128 scans for signal averaging the spectra to ensure a high signal-to-noise ratio. The
acquisitions were fulfilled with the OMNIC Spectra software. Proton (1H) Nuclear Magnetic
Resonance (NMR) spectroscopy and Diffusion Ordered Spectroscopy (DOSY) were done to
verify the functionalization of CS and determine the degree of substitution (DS). NMR spectra
were recorded at 85 °C in D2O/DCl at 5 g L-1 on a Bruker AVANCE DPX-400 spectrometer
(USA). Data acquisition was fulfilled using the Bruker Topspin software 4.1.0 with 5 mm
BBFO probe and a gradient amplifier, which provides a z-direction gradient strength of up to
47.5 G.cm−1. The temperature was maintained constant within 85±0.1 °C by means of the BCU
05 unit. All NMR DOSY experiments were performed using the bipolar longitudinal eddy
current delay pulse sequence (BPLED). The spoil gradients were also applied at the diffusion
period and the eddy current delay. Typically, a value of 4.5 ms was used for the gradient
duration (δ) 200 ms for the diffusion time (Δ), and the gradient strength (g) was varied from
1.67 G cm−1 to 31.88 G cm−1 in 32 steps.
The DS of CS/P334 derivative was determined according to Eq 1a:
𝐼𝐻8 (𝑃334) /3
𝐷𝑆 = Eq.1a
𝐼𝐻1 (𝐶𝑆)
Where IH8 (P334) is the integral of the 3 protons (peak position 8-in Figure 3C) of P334 between
1.14 and 1.2 ppm and IH1 (CS) is the integral of the proton (peak position 1-in Figure 3) of
chitosan between 4.4 and 4.9 ppm. Absorption spectra were recorded using a Perkin Elmer
UV/VIS/NIR Lambda-750 spectrophotometer (USA) in the UV-visible in the range of 200-700
nm at room temperature. Before the measure, each sample was dissolved in acidified pure water
(pH = 5.5) with a concentration of 0.40 mg mL-1 for CS-SH and of 0.18 mg mL-1 for CS-P334.
The DS of SH and P334 were evaluated by Beer Lamberts law by assuming that the molar
163
extinction coefficients remain unchanged (CS-SH, Ɛ = 44 668 Lmol-1cm-1 and CS-P334, Ɛ = 42
300 Lmol-1cm-1).
2.3 Photostability experiments of the chitosan-MAA derivatives
2.3.1. Irradiation tests
A Suntest XLS Atlas Material Testing Solutions (USA) equipped with a 1700-W xenon arc
lamp and a lightday filter was used for all irradiation experiments. The experimental conditions
were chosen to mimic the natural solar radiation conditions 46 i.e., irradiance of 590 W m-2 with
a dosage of 2 000 kJ m-2 per hr in the wavelength range from 200 to 700 nm at 30 °C. In order
to evaluate the photodegradation of CH-SH or CH-P334 derivatives, the conjugates were
dissolved at a concentration of around 2 mg mL-1 in either pure and river water slightly acidified
(pH = 5.5 adjusted with 0.1 M HCl) for complete dissolution of the derivatives. The total
inrradiation time was up to 6 hrs and the absorbance spectra of the derivatives were analyzed
every 2 hrs (T0 hrs, T2 hrs , T4 hrs and T6 hrs) to monitor the photodegradation kinetics in
both waters. Solutions of riboflavin (RF) at 1.32×10-05 M and porphine (PPY) at 4.54×10-06 M
were prepared in pure water in order to use them as photosensitizers of the chitosan-MAAs
derivatives. These photosensitizers were added at a molar ratio of CS-MAA:RF 1:3x10-06 and
CS-MAA:PPY 1:1.9x10-08. In the presence of riboflavin, the derivatives were irradiated up to
20 seconds, and the absorbance spectra were recorded every 5 seconds (T0 sec, T5 sec, T10
sec, T15 sec and T20 sec); in the presence of porphine the samples were irradiated up to 60
min, and the absorbance spectra were recorded every 15 minutes (T0 min, T15 min, T30 min,
T45 min and T60 min). 5x10-3 M sodium azide quencher in combination with 6x10-11 M
riboflavin in pure water was also irradiated for 20 seconds, and the absorbance spectra were
recorded every 5 seconds (T0 sec, T5 sec, T10 sec, T15 sec and T20 sec). The irradiation time
was up to 20 seconds and the absorbance spectra were analyzed every 5 s (T0, T5, T10, T15
and T20) for CS-SH and CS-P334- riboflavin:azide. All incubation tests were carried out in
small quartz cuvettes (3 cm × 1 cm) with 2 mm path length using a volume of 0.6 mL solution,
and done in triplicate.
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2.3.2. Quantum yield determination
The photodegradation quantum yield (Φ) (Eq-1b) was used to evaluate the photostability of the
CS-MAAs derivatives in the different conditions.
Number of molecules photodecomposed

Φ= Eq. 1b
Number of photons absorbed
Number of molecules photodecomposed = roV t NA, Number of photons absorbed= I0(1- 10-(A))
A = maximum absorbance, ro= rate of photo decomposition, V = volume of solution (L),
NA = Avogadro number, t = time (sec), I0 = number of photons emitted by the lamp in the
absortion band wavelength interval.
3.1 Synthesis of the materials
The synthesis of chitosan conjugates with MAA shinorine and porphyra-334 by chemical
grafting with the reaction mechanism is depicted in Figure 1.
165
Figure 1: Schematic representation of reaction and mechanism of Chitosan-Shinorine (CS-
SH) and Chitosan-Porphyra-334 (CS-P334) conjugates.
166
3.2 Characterisation by Fourier Transform Infrared Spectroscopy
(FTIR)
The infrared spectra of chitosan, chitosan grafted shinorine (CS-SH) and porphyra-P334 (CS-
P334) were analysed (Figure 2). The absorption peaks at 3365 cm-1 corresponds to the hydroxyl
groups (OH), 1152 cm-1 corresponds to anti-symmetric stretching of the C-O-C bridge in alpha
glyosidic bond, 1650 cm-1 corresponds carbonyl (C=O) stretching vibrations coupled to the N-
H bending vibrations, 1590 cm-1 corresponds to NH2 bending of amino groups, 1084 cm-1 and
1040 cm-1 correspond to skeletal vibrations involving the C-O stretching in the chitosan
macromolecule.
Figure 2: ATR-FTIR spectra of chitosan and Chitosan-Shinorine (CS-SH), Chitosan-Porphyra-334

(CS-P334) conjugates.
The two chitosan conjugates CS-SH and CS-P334 exhibited prominent absorption peaks at
1530 cm-1 and 1630 cm-1 respectively that is the indication of the presence mycosporine like
amino acid (MAA) molecules in the conjugate. The band 1530 cm-1 is attributed to N-H bending
and 1630 cm-1 is attributed to N-C=O stretching corresponding to the amide II bond showing
that the grafting has successfully taken place via covalent bond formation between COOH of
MAAs and NH2 of chitosan. The absorption band at 2920 cm-1 is attributed to the C-H stretching
of the methylene groups (CH2) and methyl groups (CH3) groups of the MAA which is detected
167
only in CS-SH and CS-P334. The absorption signal at 1380 cm-1 of high intensity in CS-SH
and CS-P334 relative to chitosan is attributable to the symmetrical angular deformation of the
methyl group.
3.3 Characterisation by Nuclear Magnetic Resonance Spectroscopy

(NMR)
1D 1H NMR spectra of CS (Figure 3 A) shows: three protons of N-acetyl glucosamine as a

singlet at 2.00 ppm; a peak position at δ=3.15 ppm (peak no 2) corresponding to the H2 proton
of the D-glucosamine residue; peaks ranging from δ=3.6 to 4.0 ppm corresponding to the
protons (peak no 3, 4, 5, 6) of chitosan; and a peak at δ=4.78 ppm (peak no 1) attributed to H1
proton. The peak at δ=4.15 ppm corresponds to the protons of the solvent. The 1H NMR
spectrum of the CS-P334 derivate (Figure 3C) shows peaks corresponding to the protons of
CS as well as three protons (peak no 8) of P334 as a doublet at 1.18 ppm with a coupling
constant of J=6.4 Hz.
Figure 3: NMR Spectra of A. Chitosan, B. Chitosan-Shinorine (CS-SH), C. Chitosan-Porphyra-334

(CS-P334).
168
The multiplet between 2.6 and 2.8 ppm corresponds to the signal of the protons (peak no 10)
and (peak no 11) of P334. Finally, the singlet at 3.5 ppm correspond to the three protons (peak
no 7) of P334. Regarding the 1H NMR spectrum of the CS-SH derivate (Figure 3B), it displays
peaks corresponding to the protons of CS as well as two protons (peak no 8) of SH as a singlet
at 3.85 ppm. The degree of substitution (DS)/Percentage of grafting found for the CS-P334
derivate is 0.01 (± 0.002) which means that 1/3 of the P334 has been grafted to CS. CS-P334
contains 2% (± 0.2%) of P334. Concerning the CS-SH derivative, the 1H NMR analysis is not
sufficient to conclude on a DS value because the peaks corresponding to the protons of CH
coincide with the peaks corresponding to the protons of CS. Also, the CS-SH derivate was
partially insoluble in D2O/DCl, which could explain the low intensity of the peaks of SH.
2D DOSY NMR (Figure 4) was also relied on to verify the CS functionalization.
Figure 4: Diffusion ordered spectroscopy (DOSY) of A. Chitosan-Porphyra (CS-P334) B. Chitosan-

Shinorine (CS-SH).
The diffusion coefficients pertaining to all the signals of CS-P334 and CS-SH conjugates were
between 1 and 3 µms-1. This provides evidence of grafting via covalent bonds, eliminating the
presence of free MAA molecules or molecular associations via hydrogen bonding.
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3.4 Characterisation by UV spectroscopy
The UV absorption spectra of chitosan, chitosan-shinorine (CS-SH) and chitosan-porphyra

conjugates (CS-P334) were analysed in acidified pure distilled water (pH 5.5) at concentrations
of 0.4mg mL-1 for CS-SH and 0.18 mg mL-1 for CS-P334 (Figures 5).
Figure 5 : UV spectroscopy of chitosan, chitosan-Shinorine (CS-SH) and chitosan-Porphyra-

334 (CS-P334).
The absorption bands of these conjugates fall in the UV-A region (320-400 nm) with maxima
(λMax) at 334 nm similar to free shinorine and porphyra-334 molecules. On the contrary, pure
chitosan does not exhibit absorption in UV-A region. The absorption λMax at 334 nm of CS-SH
and CS-P334 are 0.28 and 0.44, respectively.
The percentage of grafting of shinorine and porphyra were evaluated by Beer Lamberts law by
assuming that the molar extinction coefficients remain unchanged (CS-SH)-44668 Lcm-1M-1,
(CS-P334)-42300 Lcm-1M-1. CS-P334 contains 1.99 % (w/w) of Porphyra-334.
The concentration of Porphyra-334 in CS-P334 calculated by UV-vis Spectroscopy (1.99%) is

consistent with the one calculated by NMR 1H (2%) corresponding to a DS of 0.01. However,
considering CS-SH, a conclusion on the DS cannot be given. As mentioned before, the derivate
170
is partially insoluble in acidified water. Although the UV-visible spectroscopy shows an
absorbance at 334 nm corresponding to a concentration of 0.22 % (w/w) of Shinorine, the real
concentration of Shinorine and the degree of substitution cannot be calculated.
3.5 Photostability of chitosan conjugates vs pure MAA molecules
The spectra of the solar lamp, and CS-SH and CS-P334 are shown in Figure 6.
Figure 6: Overlayed Spectra of chitosan-shinorine (CS-SH), chitosan-porphyra-334 (CS-P334) (Molar

Extinction Coefficient Ɛ vs Wavelength) and Spectral irradiance of the solar simulator (Irradiance vs
Wavelength).
The chitosan conjugates were found to slightly degrade on exposure to UV irradiation for a
time period of 6 h monitoring the absorbance every 2 h (Figure 7 A & B). For CS-SH we can
observe a linear decreasing of absorbance with time indicating an apparent zero order kinetic.
For CS-P334 the photodecomposition seems to begin only after 2 hours of irradiation.
171
Figure 7: Time dependent UV spectra and kinetics of photodegradation of A. Chitosan-
Shinorine (CS-SH), B. Chitosan-Porphyra-334 (CS-P334).
Table 1: Quantum yield of photodecomposition of conjugates and free MAA molecules.
Conjugates and MAAs Quantum Yield of

Photodecomposition (Φ) x10-5
Chitosan-Shinorine 0.263 ± 0.065
Shinorine 0.14 ± 0.02
Chitosan-Porphyra-334 0.130 ± 0.030
Porphyra-334 0.18 ± 0.04
The pH of chitosan-shinorine and chitosan-porphyra-334 conjugates post dissolution in

acidified pure water was found to be 5.5. The conjugates upon UV absorption get excited to the
higher energy levels from the ground state and take part in photoreactions and there after
172
reinstates to the ground state via non radiative relaxation as heat energy culminating in its
degradation47. The quantum yield of photodecomposition of the CS-SH and CS-P334
conjugates were (0.263 ± 0.065)x10-05 and (0.13 ± 0.030)x10-05 respectively indicating that the
conjugates were photostable on irradiation. A comparison can be made regarding the
photodegradation of the chitosan conjugates CS-SH and CS-P334 with the pure MAA
molecules shinorine and Porphyra-334 in terms of QE (Refer Chapter 3) by taking into account
that except the pH (5.5 for conjugates and 7 for pure MAA molecules) all the conditions were
uniform (Table 1). The results indicate that these chitosan conjugates after successful grafting
via chemical bonds have almost similar photostability to the free molecules in the order of
0.1x10-05 and were found to be more stable than work reported in the literature by Conde et al.
34×10-05 for SH and 24×10-05 for P334 20, 21. In other words, photochemical stability of SH and
P334 seems to be not affected by grafting on chitosane.
3.6 Photostability of Chitosan Conjugates vs pure MAA molecules

with photosensitizers riboflavin and porphine
Photodegradation studies of these CS-SH and CS-P334 conjugates were assessed in presence
of photosensitizers like riboflavin and porphine. This was done to study the influence of reactive
oxygen species primarily singlet oxygen on chitosan grafted conjugates. Both riboflavin and
porphine are water soluble and have a series of absorption bands in the UV and visible region:
The λRB(Max) of riboflavin 222, 266, 373 and 447 nm 48 and the λPPY(Max) of porphine- 414, 517,
555, 580 and 635 nm 49 (Figure 8). Photosensitization mediated degradation reactions can take
place mainly by Type 1 (Energy Transfer-Singlet Oxygen) or Type 2 mechanisms (Electron
Transfer)50. The photosensitisation process of riboflavin occurs by combination of Type 1 and
Type 251 and in the case of porphine, it occurs mainly by Type 2 52. The rate of photodegradation
of the conjugates were assessed in term of QE (Table 2).
173
Figure 8: Absorption spectra of chitosan-Porphyra-334 conjugates (CS-P334) in the presence
of A. riboflavin and B. porphine before irradiation.
Figure 9: Time dependant absorption spectra and kinetics of photodegradation of chitosan-

porphyra-334 conjugates (CS-P334) (Magnified spectra: 280-400 nm) in the presence of A.
riboflavin after T0, T5, T10, T15, T20 s and B. porphine after T0, T15, T30, T45, T60 min.
174
Figure 10: Time dependant absorption spectra and kinetics of photodegradation of Chitosan-
Shinorine conjugates (CS-SH) in the presence of A. riboflavin after T0, T5, T10, T15, T20 s
and B. porphine after T0, T15, T30, T45, T60 min.
CS-SH and CS-P334 conjugates photodegrade in the presence of photosensitisers like riboflavin
by irradiating it for a total time of 20 sec with 5 sec time interval and porphine by irradiating it
for a total time of 5 min with 1 min time interval (Figure 9 A & B and Figure 10 A & B). The
QE of photodegradation of both CS-SH and CS-P334 in the presence of both photosensitisers are
higher relative to the unsensitised ones (Table 2 and Table 1) indicating ROS are able to
photodegrade these conjugates at a fast pace by Type 1 and Type 2. The quantum yield values of
both conjugates in the presence of riboflavin is higher compared to porphine. The concentration
of singlet oxygen [1O]ss in riboflavin and porphine was estimated to be around 7500×10-14 and
2500×10-14
A comparison can be made regarding the QE of photodegradation of the chitosan conjugates CS-
SH and CS-P334 relative to pure shinorine and porphyra-334 in terms of QE (Refer Chapter 3)
with the photosensitisers by taking into account that except the pH (5.5 for conjugates and 7 for
175
pure MAA molecules) all the conditions were uniform. The results point out (Table 2) that the
conjugates are more stable relative to the free molecules in presence of ROS generated from
riboflavin and porphine.
Table 2: Quantum yield of photodecomposition of conjugates and free MAA molecules with
riboflavin and porphine.
Quantum Yield of Photodegradation (Φ) x10-5
Riboflavin Porphine
Conjugates
Chitosan-Shinorine 830 ± 200 200 ± 50 6 ± 1.5
Shinorine 2700 ± 700 400 ± 100 20 ± 4
Chitosan-Porphyra-334 1190 ± 300 320 ± 80 7 ± 1.7
Porphyra-334 3000 ± 600 200 ± 40 15 ± 3
To evaluate the respective fraction of degradation of CS-SH and CS-P334 conjugates by

riboflavin by electron transfer (Type 1) and energy transfer (Type 2) sodium azide was used as a
physical quencher to scavenge singlet oxygen 53.
Up on evaluating the respective proportion of degradation of both conjugates by energy transfer

i.e. via Type 1 individually, the extent of degradation is as follows (Table 2): CS-SH (24 %) and
CS-P334 (29 %). (Figure 11 A and B) This concludes the fact that the mode of degradation of
these conjugates is predominantly by Type 2 mechanism. This gives an insight that these
materials can act as good potential candidate for antioxidant formulations.
176
Figure 11 : Time resolved absorption spectra and kinetics of photodegradation of A.
Chitosan-Porphyra-334 (CS-P334) B. Chitosan-Shinorine (CS-SH) conjugates in the presence
of riboflavin + sodium azide after T0, T5, T10, T15, T20 s.
3.7 Photostability of Chitosan Conjugates in a natural photosensitizer:

acidified river water vs pure MAA molecules
Since the chitosan conjugates degrade in the presence of reactive oxygen species generated
from photosensitizers the photodegradation study of these conjugates has been extended to
natural matrices from which ROS can be generated on irradiation as seen in Chapter 3. The
natural matrices that we used for the analysis was river water with a pH of 8.4 which prevented
the dissolution of the conjugates. For the dissolution of CS-SH and CS-P334 conjugates in river
water the pH was reduced to 5.5 with 0.1M HCl. The conjugate solutions were irradiated with
UV for 6 h and the rate of photodegradation was obtained in terms on quantum yield (Figure
12 A & B). Evaluation of the photostability in terms of quantum yield (QE) revealed that these
conjugates possess higher QE of photodegradation in river water (0.71±0.17)×10-05 for CS-SH
177
(0.66±1.65)×10-05 for CS-P334 relative to acidified pure water, (0.263 ± 0.065)×10-05 for CS-
SH, (0.130 ± 0.030)×10-05 for CS-P334 (Table 3) indicating the fast pace of degradation in river
water in presence of UV. This higher rate can be associated to the presence of reactive oxygen
species (ROS) like singlet oxygen in river water on UV irradiation54, the concentration of
singlet oxygen [1O]ss in river water being estimated to be around 16×10-14 as shown in (Refer
Chapter 3).
Table 3 : Quantum yield of photodecomposition of conjugates and pure molecules in river

water
Conjugates Natural Quantum Yield of

Photosensitiser Photodecomposition (Φ) x10-05
Chitosan-Shinorine 0.710 ± 0.17

Acidified
River Water
Chitosan-Porphyra-334 pH (5.5) 0.66 ± 0.16
Shinorine 0.35 ± 0.08

River Water
pH (8.4)
Porphyra-334 0.3 ± 0.06
178
Figure 12 : Time dependant absorption spectra and kinetics of photodegradation of chitosan
conjugates A. Chitosan-Porphyra-334 (CS-P334) and B. Chitosan-Shinorine (CS-SH) in the
presence of acidified river water after T0, T2, T4, T6 h (where T0, T2, T4, T6 are time after
0, 2, 4, 6 h, respectively).
The ROS could accelerate the degradation of the UV absorbing conjugates CS-SH and CS-
P334 to low molecular weight compounds making them readily available for microbial
metabolism thus having a good impact on the marine ecosystems. The prime reason for the
formation of ROS in these natural waters is due to the photochemical reaction involving
55
predominately CDOM (coloured dissolved organic matter) which are distinct organic
molecules formed from decaying detritus of plant and animal organic matter by leaching and
are capable of generation of ROS like singlet oxygen on UV irradiation 56. On comparison of
the conjugates with the pure shinorine and porphyra-334 in river water QE by taking into
account that except the pH (5.5 for conjugates and 8.4 for pure MAA molecules) all other
conditions were uniform. The results indicate that these chitosan conjugates have almost
comparable stability relative to the pure (Table 3).
179
4. Conclusion
Marine organisms such as red algae, cynobacteria, reef fish, zebra fish, red algae and
cyanobacteria have an inherent biological mechanism to circumvent the harmful UV radiation
from the sun. To emulate such natural biotic systems the strategy was to develop
photoprotective materials that combine individual components normally encountered in natural
biological systems. These materials were based on chitosan as the matrix functionalised with
MAA through a covalent amide linkage formed between the amino groups of chitosan and
carboxyl groups of MAA using 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide as coupling
agent. These conjugates are able to provide protection in the UV-A region. In pure water, the
quantum yield of photodegradation of these conjugates were (0.263 ± 0.065)×10-05 for CS-SH
and (0.130 ± 0.030)×10-05 for CS-P334 indicating almost similar photostability according to
our study (0.14 ± 0.02)×10-05 for shinorine (SH) and (0.18 ± 0.040)×10-05 for porphyra-334.
These conjugates were high photostable relative to these individual free molecules according
to the work reported in the literature by Conde et al. 34×10-05 for shinorine and 24 ×10-05 for
20, 21
porphyra-334 . These conjugates were biodegradable since their individual components
were obtained from biosources. These conjugates photodegrade more readily in the presence of
reactive oxygen species generated from CDOM from natural aquatic ecosystems while still
remaining photostable relatively which should make it possible to use these materials as solar
screens.
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185
186
Conclusion
In a time when the world is moving towards greener and more eco-friendly products and
materials, from linear economy with a culture of take, make, use and discard to circular
economy with a culture of little wastage, preserving all natural resources and recycling to the
maximum possible, we introduce here natural UV filters - mycosporines and mycosporines-like
amino acids (MAAs) - which are biodegradable and non eco- or cito-toxic and sustainable, and
therefore very pertinent to the core concept of circular economy. These biomolecules can be
extracted from algae being potential natural UV-absorbing molecules for the development of
novel UV-protective products and materials as alternative to the synthetic ones. Thus, a better
understanding of the photostability of these natural molecules under realistic conditions
(solar irradiation, natural waters) and to better understand the role of ROS in their
abiotic degradation process is important and timely. This was the main objective of this
thesis.
In this study, the photostability of mycosporines namely M-serinol and gadusol (enolate form)
and MAAs like shinorine, porphyra-334 and palythine were studied in pure water and other
natural matrices as river, estuary and ocean waters. These natural matrices are known to
generate ROS that can react with these molecules and thus contribute to their degradation in
natural environments. Complementary experiments have been also conducted with
photosensitisers (riboflavin and porphine). Also, as in nature, mycosporines and MAAs are not
only found in the free form but also linked to oligosaccharides and proteins (e.g., in the mucus
and lenses of reef fishes), herein, MAAs were grafted to chitosan backbone, to mimic this
condition found in nature and the possibility to do UV-protective materials, their photostability
was assessed under the same conditions as the free molecules.
The main conclusions of this work are:
(i) In general, mycosporines and MAA were most stable in pure water and least stable in river
water. This was due to the presence of large amount of coloured dissolved organic matter
187
(CDOM) in river water and least in ocean water. UV irradiation generates singlet oxygen from
CDOM. This singlet oxygen degrades mycosporines and MAAs to oxidised photoproducts.
(ii) M-serinol was found to be more photostable than gadusol (enolate form) whereas among
the MAAs studied shinorine and porphyra-334 were more stable than palythine in pure water
and all natural matrices.
(iii) The antioxidant potential of M-serinol and gadusol (enolate form) were investigated and it
was found that gadusol was a good antioxidant and M-serinol a weak one because of the
increased Type 2 mechanism of photodegradation.
(iv) Photoprotective conjugates of chitosan with shinorine and porphyra-334 were synthesised
via amidation giving shielding in the UV-A region and these conjugates were found to be almost
as photostable as the free molecules.
(v) The photostability of the chitosan-MAAs conjugates was studied in pure and river water
after acidifying the solutions to pH 5.5 at which they were soluble. CDOM in river water was
found to act like a natural photosensitiser in the photodegradation of these conjugates. These
conjugates were found to degrade by Type 1 (electron transfer) and Type 2 (singlet oxygen)
mechanisms by photosensitisation studies with riboflavin and porphine.
(vi) Very interestingly, as is possible to see in the summary table (below), as already mentioned
chemical grafting of the MAAs onto chitosan macromolecules did not alter the photostability
of the MAAs under UV radiation in the different water matrices. Nonetheless, the conjugates
showed to be most more photostable in the presence of photosensitisers when compared with
the free molecules.
188
Summary Table of the Quantum Yields of Photodegradation
Mycosporines/MAA/
Pure Water Ocean Water Estuary Water River Water
Conjugates
(10 ± 3) x 10−05
Porphine x 10-05 Riboflavin x 10-05

Gadusolate (20 ± 4) x 10-05 (28 ± 7) x 10-05 (40 ± 10) x 10-05
Type 2 Type 1 + Type 2 Type 1
2000 ± 500 18000 ± 5000 1400 ± 200
(2.3 ± 0.46) x 10−05

M-Serinol (5.4 ± 1) x10-05 (9.5 ± 2) x 10-05 (20 ± 5) x 10-05
970 ± 150 17000 ± 4000 9300 ± 2000
(0.14 ± 0.02) x 10−05

Shinorine (0.18± 0.036) x10-05 (0.2 ± 0.04) x 10-05 (0.35 ± 0.08)x10-05
20 ± 4 2700 ± 700 400 ± 100
(0.18 ± 0.040) x 10−05

Porphyra-334 (0.2 ± 0.04) x10-05 (0.25 ± 0.05) x 10-05 (0.3 ± 0.06)x10-05
15 ± 3 3000 ± 600 200 ± 40
(0.263 ± 0.065) x 10−05

CS-Shinorine NA NA 0.71 ± 0.17 x10-05
6 ± 1.5 830 ± 200 200 ± 50
(0.130 ± 0.030) x 10−05

CS-Porphyra-334 NA NA 0.66 ± 0.16 x 10-05
7 ± 1.7 1190 ± 0.30 320 ± 80
189
190
Future Perspectives
Taking in account the obtained results, there are still much more to do regarding these
biomolecules that are not fully explored in this work. More research investigations are
suggested:
(i) Characterizing more deeply the antioxidant properties of the prepared materials could give
important informations on their application fields valuating of SPF properties of the conjugates
could be an important information on their potential use of these materials for sunscreens.
(ii) Synthesising new materials with these biomolecules for engineering applications could be
a strategy to move to sustainability in the near future:
✓ Chitosan conjugates grafted with M-serinol and gadusol is worth looking into for
providing protection in the UV-B region. Conjugates with dual protection both in
UV-A and UV-B spectra could be obtained by grafting shinorine or porphyra-334
and M-serinol and gadusol in the same chitosan biomacromolecule;
✓ Exploring other biopolymers such as collagen, bacterial cellulose for skin protection
or reparation and hyaluronic acid for ophthalmic applications for eyes protection,
for the synthesis of novel conjugates with these biomolecules.
(iii) Understanding why the conjugates are more photostable in the presence of photosensitisers
than free molecules.
(iv) To assess the potential impact of the MAAs-based materials (conjugates) on marine
organisms like small invertebrates and corals via ecotoxicological tests. The toxicity could be
measured in vitro according to the half maximal effective concentration and the hormonal
effects could be assessed using cells of the mentioned organisms.
(v) As coral bleaching has negative impacts on biodiversity and functioning of reef ecosystems,
the effect of the MAAs-based materials (conjugates) could be also assessed in laboratory.
(vi) From a fundamental aspect, a better understanding of the reactivity and the photoreactivity
of MAAs and mycosporines could be undertaken both from a theoretical and experimental point
of view:
191
✓ DFT calculations on mycosporines and MAAs would give a detailed knowledge of
the electronic structure of these molecules and would allow explaining their high
reactivity towards ROS.
✓ Time-resolved spectroscopic techniques could help characterize the excited states
of these compounds and better understand their (photo)reactivity.
192
193
ECOLE DOCTORALE : ED 211, L’ÉCOLE DOCTORALE DES SCIENCES EXACTES ET
LEURS APPLICATIONS, UNIVERSITE DE PAU ET DES PAYS DE L'ADOUR
LABORATOIRE : INSTITUT DES SCIENCES ANALYTIQUES ET DE PHYSICO-CHIMIE
POUR L'ENVIRONNEMENT ET LES MATERIAUX
CONTACT
Martin George Thomas
martin.thomas@univ-pau.fr

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THÈSE

UNIVERSITE DE PAU ET DES PAYS DE L’ADOUR

Présentée et soutenue le 11 Juillet 2022

Study of the impact of different water matrices and photosensitizers on the

pour obtenir le grade de docteur

Study of the impact of different water matrices and photosensitizers on the

This thesis is written based on the following publications to be submitted in

1. Understanding the effect of natural water matrices and photosensitizers on the

2. A roadmap to UV-A-absorbing natural sunscreens – photostability in different natural

3. Influence of photosensitisers on the photodegradation of chitosan/mycosporine-like

Other Publications in scientific journals not included in this thesis

Oral presentations in international/national conferences

Posters in international/national conferences

2. Bascans, N. Brugerolle de Fraissinette, Martin Thomas, C. McReynolds, N. Castejon, F.

I express my sincere gratitude to my supervisors Prof Dr.Thierry Pigot and Dr.Susana CM

This thesis mainly focuses on investigating the photodegradation of mycosporines,

Chapter 1 : Literature Riview

Chapter 2 : Photostability of Mycosporines M-serinol and Gadusol in Aquatic Matrices

Chapter 3 : Comparison of photostability of mycosporines like amino acids in different

1. Introduction ..................................................................................................................................... 112

Chapter 4: Photoprotective Polysaccharides Based on Chitosan and Mycosporine Like

1. Introduction ..................................................................................................................................... 160

Reference ............................................................................................................................................ 180

1.1 Solar radiation

Figure 1: The Electromagnetic Spectra (100 nm - 400 nm) (Reproduced from2).

Table 1: Sub-regions of the UV spectrum with corresponding parameters

UV Region Irradiance Solar Wavelength

Figure 3: Detrimental effects of UV-A and UV-B rays (Reproduced from5).

To circumvent the detrimental effects of UV rays protective measures are a prerequisite. To

The chemical UV-filters can be of three types:

a. UV-A filters (that absorb UV-A-rays),

b. UV-B-filters (that absorb UV-B-rays)

2.1 Physical/Inorganic UV filters

2.2 Chemical/Organic UV filters

a. These could undergo conformational/configurational molecular changes;

b. They can emit radiation at higher wavelength (fluorescence or phosphorescence);

c. They can release the incident energy as heat.

2.3. Common inorganic UV filters

2.3.1 Titanium dioxide (TiO2)

2.3.2 Zinc oxide (ZnO)

Most commonly used organic UV filters are Benzophenones (BP), 4-Methylbenzylidene

Figure 7: Structure of Benzophenone-3 (left) and Benzophenone-4 (right).

Figure 8: Structure of 4-Methylbenzylidene Camphor.

2.5 Consequences of conventional sunscreens on humans

2.6 Impact of conventional sunscreens in marine ecosystems

Table 2: Presence of different organic UV filters in different water matrices (Reproduced26).

UV Filter Structure Water Matrices Concentration

River Water up to 114

MBC-4 Sea Water up to 798.7

Lake Water up to 1,140 ± 50

River Water < 3 - 1,980 ± 130

River Water up to 153

Sea Water up to 389.9

River Water up to 283

Sea Water up to 2780.7

Tap water n.d.

WWTP up to 5322 ± 612

Sea Water up to 390 ± 40

Note: n.d. is not detected

Table 3: IC50 and EC50 for Various UV filters and organisms.

UV Filters IC50 (mgL-1) Organism

BP-3 0.96 Phytoplankton

UV Filters EC50 (mgL-1) Organism