NZ620436B2 - New sunscreen compositions - Google Patents

Abstract

The present description refers to eco-compatible compositions for sunscreening the body, in particular to sunscreens for the protection of human health, compatible with organism life and not harmful to marine ecosystems (in particular for tropical environments with coral reefs). In one embodiment the composition comprise one or more UV filters selected from 2,2’-methylene-bis-(6-(2H-benzotriazole-2-yl)-4-(1,1,3,3-tetramethylbutyl)-phenol, 2-[4-(diethylamine)-2-hydroxybenzoyl]hexyl benzoate, ethylhexyl triazone; and one or more antioxidants selected from tocopheryl acetate, tocopherol, retinol, polyphenolic compounds extracted from green tea. e composition comprise one or more UV filters selected from 2,2’-methylene-bis-(6-(2H-benzotriazole-2-yl)-4-(1,1,3,3-tetramethylbutyl)-phenol, 2-[4-(diethylamine)-2-hydroxybenzoyl]hexyl benzoate, ethylhexyl triazone; and one or more antioxidants selected from tocopheryl acetate, tocopherol, retinol, polyphenolic compounds extracted from green tea.

Description

Trans 12.3 (19.07.2012) NEW SUNSCREEN COMPOSITIONS DESCRIPTION The present description refers to eco-compatible sunscreen compositions, in ular to sunscreens compatible with the marine environment and the life inhabiting it, with specific reference to coral reefs.

STATE OF THE PRIOR ART Coral reefs are among the most biologically productive and e ecosystems in the world, representing hot spots of marine biodiversity, and directly sustaining half a billion people (Moberg and Folke 1999, Wilkinson 2004). Approximately 60% of coral reefs are currently threatened by several natural and anthropogenic impacts s et al 2003; fi et al 2003). Over the last 20 years, coral bleaching (loss of symbiotic zooxanthellae hosted within scleractinian corals) has increased ically, both in frequency and spatial extent -Guldberg 1999; Hughes et al 2003; Knowlton 2001). This phenomenon has been associated with temperature anomalies, excess ultraviolet (UV) radiation or altered availability of photosynthetic radiation, and presence of bacterial pathogens and pollutants (Brown et al 2000; Bruno et al 2007; Douglas 2003; Glynn 1996; Jones 2004).

Production and consumption of personal care and cosmetic sun products are increasing worldwide, with potentially important consequences related to environmental contamination. The release of these products is also linked with the rapid expansion of tourism in marine coastal areas (Wilkinson 2004). Chemical compounds ned in sunscreens and other personal care products can reach detectable levels in seawater ton and Ternes 1999; Giokas et al 2007). These compounds might be potentially harmful to the environment; hence the use of sunscreen products is now banned in a few of the most popular tourist destinations, for example, in marine ks in Mexico (Xcaret 2007; Xel-ha 2007). Because eens are ilic, their UV filters can accumulate in aquatic animals (Giokas et al, 2007) and cause effects similar to those reported for other xenobiotic compounds (Balmer et al 2005; Daughton and Ternes 1999).

Paraben preservatives and some UV absorbers contained in eens have estrogenic activity (Daughton and Ternes 1999; Schlumpf et al 2004). In addition, it has been demonstrated that sunscreen ingredients can o various photodegradations, with the entailed transformation of these agents into toxic by- products (Giokas et al. 2007, and literature therein). It has also been demonstrated that sunscreens have an impact on marine bacteria (Danovaro and Corinaldesi 2003).

Recently, a tight onship among sunscreens and bleaching of hard corals was observed through specific tests worldwide (Danovaro et al. 2008). In particular, in this In one embodiment there is provided an eco-compatible sunscreen composition comprising: - one or more UV filters selected from 2,2’-methylene-bis-(6-(2H-benzotriazoleyl) ,3-tetramethylbutyl)-phenol, 2-[4-(diethylamine)hydroxybenzoyl]hexyl te, ethylhexyl triazone; - one or more antioxidants selected from tocopheryl acetate, tocopherol, retinol, polyphenolic compounds extracted from green tea; - one or more carriers selected from propylene glycol dicaprylate/dicaprate, olive oil, beeswax; - one or more fragrances selected from , lavender, grapefruit, guava and coconut, and free from: - preservatives belonging to the paraben family; - ic acid derivatives; - UV filters selected from bis-ethylhexyloxyphenol methoxyphenyl triazine, ates, benzophenones, camphor derivatives, titanium oxide; AH26(10335840_1):RTK - fragrances selected from melaleuca ial oil, ylang ylang flower extracts, Bambousa Arundinacea extracts, Karité; - carriers selected from animal-derived fats and argan oil.

AH26(10335840_1):RTK Creams; c) Antioxidants; d) UV s; e) Controls.

Figure 5. Coral nubbins at different incubation times. Panels a and b show control systems at time 0 and after 72 h of incubation. Panels c and d show nubbins of treated corals at time 0 and after 72 h of incubation.

Figure 6. Photostability of UV-tested filters.

DETAILED PTION OF THE INVENTION The present invention refers to sunscreen compositions mpatible with the marine ecosystem of coral reefs.

In the present description, by the definition: “eco-compatible with the marine ecosystem” of the coral reefs it is meant a product causing no harm nor alteration to the marine organisms or to the communities or habitats as a whole. An eco-compatible product is therefore usable without ctions, as it protects the health of environment and biodiversity. In the tests reported herein, the health conditions of marine organisms exposed to the various chemical components were verified in order to select the components absolutely innocuous. Note that a radable product (declaration associated with some marketed products) can be harmful to the nment and ore can be not eco-compatible, as the concept of biodegradation refers only to the decomposition times of the compound.

The compositions of the present invention comprise: - one or more UV filters selected from MBBT, DHHB and EHT; - one or more antioxidants selected from eryl acetate, tocopherol, retinol, polyphenolic compounds extracted from green tea, and which are free from preservatives belonging to the n family.

In the present description, by ens" are meant the esters of 4-hydroxybenzoic acid used as preservatives in the cosmetics industry. The most common parabens are methylparaben, ethylparaben, propylparaben and butylparaben.

An example of polyphenols extracted from green tea is polyphenone—60 (CAS N° 138988-88—2) commercially available from Sigma.

In an embodiment, the compositions of the present invention may comprise one or more fragrances eco-compatible with the marine environment, selected from the group consisting of fragrances of orange, lavender, grapefruit, guava and coconut.

Examples of fragrances suitable for the present invention are the nces commercially available from FAROTTI SRL company, such as guava and coconut, natural lavender, natural grapefruit, preferably in concentrations equal to or lower than 0.3% of the product.

In order to increase the shelf life times, in an ment the compositions comprise as vative sorbic acid, which from the testing reported herein did not prove harmful to coral reefs in conjunction with the other compounds.

The compositions could be prepared in different cosmetic forms, such as, e.g., cream, lotion, ointment, spray, lipstick. The selected UV s, antioxidants, fragrances and preservatives could be mixed with different carriers suitable (according to test results) for the determined cosmetic form that is to be manufactured.

Preferably, carriers having an emollient on will be used, selected in the group consisting of propylene glycol dicaprylate/dicaprate, olive oil, beeswax.

Propylene glycol dicaprylate/dicaprate (CAS number: 685837) is a derivative of propylene glycol, and is a r of propylene glycol and fatty acids, In particular, it is a mixture of ene glycol dicaprylate and propylene glycol dicaprate. This product is commercially available under the name Labrafac.

Beeswax (CAS number:8012—89—3) suitable for the present invention is e.g. commercially available under the name Apifil.

The data reported in the experimental section demonstrate that the s MBBT, DHHB, the antioxidants tocopheryl e and tocopherol and the fragrance of guava and coconut are the compounds having a greater eco-compatibility with corals. ably, the compositions of the present invention will therefore comprise MBBT or DHHB, tocopheryl acetate or Tocopherol and optionally the fragrance of guava and coconut The present invention is based on the selection of compounds suitable to be used in compositions for use as sunscreens compatible with the marine environment and life inhabiting it, with particular reference to coral reefs.

The majority of compounds commonly used in sunscreens proved harmful to these nments, compounds such as, e.g.: - preservatives belonging to the paraben family - ic acid derivatives; - UV filters selected from bis ethylhexyloxyphenol methoxyphenyl triazine (BEMT), cinnamates, benzophenones, camphor derivatives, titanium oxide; - fragrances selected from melaleuca ial oil, ylang ylang flower extracts, Bambousa Arundinacea extracts, Karite; - carriers selected from -derived fats; - Argan oil.

In an embodiment, the sunscreen compositions comprise: - one or more UV filters selected from 2,2’-methylene-bis-(6-(2H-benzotriazole- 2—yl)—4-(1,1,3,3-tetramethylbutyl)—phenol, 2—[4-(diethylamine)—2— hydroxybenzoyl]hexyl benzoate, ethylhexyl triazone; - one or more idants selected from eryl acetate, tocopherol, retinol, polyphenolic compounds extracted from green tea; - one or more rs selected from propylene glycol dicaprylate/dicaprate, olive oil, beeswax and are free from: -preservatives belonging to the paraben family or ic acid derivatives; -UV filters selected from bis ethylhexyloxyphenol methoxyphenyl triazine, cinnamates, benzophenones, camphor derivatives, titanium oxide; -fragrances selected from melaleuca essential oil, ylang ylang flower extracts, Bambousa Arundinacea extracts, Karite; -carriers selected from animal-derived fats and argan oil.

In an embodiment with a very high eco-compatibility, they will further comprise sorbic acid and/or one or more fragrances selected from orange, lavender, ruit, guava and t.

In a further highly eco-compatible embodiment the compositions are ially consisting of: -one or more UV filters selected from 2,2’-methylene-bis-(6-(2H-benzotriazole— 2—yl)—4-(1,1,3,3-tetramethylbutyl)—phenol, 2—[4-(diethylamine)—2— hydroxybenzoyl]hexyl benzoate, ethylhexyl triazone; - one or more antioxidants selected from tocopheryl acetate, tocopherol, retinol, polyphenolic compounds extracted from green tea; - one or more carriers selected from propylene glycol dicaprylate/dicaprate, olive oil, beeswax; - water.

In a further highly eco-compatible embodiment, the compositions are essentially consisting of: -one or more UV filters selected from 2,2’-methylene-bis-(6-(2H-benzotriazole— 4-(1,1,3,3-tetramethylbutyl)—phenol, 2—[4-(diethylamine)—2— hydroxybenzoyl]hexyl benzoate, exyl triazone; - one or more antioxidants selected from tocopheryl acetate, tocopherol, retinol, polyphenolic compounds extracted from green tea; - sorbic acid; - one or more carriers selected from ene glycol dicaprylate/dicaprate, olive oil, beeswax; - water.

In a further ment, the compositions are essentially consisting of: - 2,2’-methylene-bis-(6-(2H-benzotriazole—2—yl)(1,1,3,3-tetramethylbutyl)— phenol and/or 2—[4-(diethylamine)hydroxybenzoyl]hexyl benzoate; - tocopheryl acetate and/or tocopherol; -sorbic acid; -one or more carriers selected from propylene glycol dicaprylate/dicaprate, olive oil, beeswax; -water; - one or more fragrances selected from orange, lavender, grapefruit, guava and coconut In a r embodiment, the compositions are essentially consisting of: - 2,2’-methylene—bis-(6-(2H-benzotriazole—2—yl)(1,1,3,3-tetramethylbutyl)— phenol and/or 2-[4-(diethylamine)hydroxybenzoyl]hexyl benzoate; - eryl acetate and/or tocopherol; -sorbic acid; -one or more carriers selected from propylene glycol ylate/dicaprate, olive oil, beeswax; -water; - one or more fragrances selected from , lavender, ruit, guava and coconut - yethyl cellulose and/or EDTA and/or sodium hydroxide.

In a further embodiment, the itions are essentially consisting of: - 2,2’-methylene—bis-(6-(2H-benzotriazole—2—yl)(1,1,3,3-tetramethylbutyl)— phenol, 2-[4-(diethylamine)—2—hydroxybenzoyl]hexyl benzoate and ethylhexyl triazone; - tocopheryl acetate; -sorbic acid; -propylene glycol dicaprylate/dicaprate, olive oil and beeswax; -water; - hydroxyethyl ose; - EDTA; -sodium hydroxide.

All embodiments listed above are exclusively consisting of compounds tested individually and as a whole to have a full eco-compatibility with the coral reef and (with no need of addition of other compounds potentially harmful for the marine ecosystem) have a high photostability and prolonged storage in a suitable cosmetic formulation.

In the compositions of the present invention, the MBBT, DHHB and EHT could be in a percentage comprised between 3 and 6% by weight of the composition.

EXAMPLES The following examples are merely illustrative and do not limit the invention (e.g., the weighings and concentrations are merely indicative, as the SPFs and the B absorbance ratios have to be specifically formulated to conform to the nominal SPFs of the product). Amounts of individual compounds are sed as weight percentages.

Example 1: In a ic embodiment, the invention has the following formulation in the form of cream: APIFIL LABRAFAC 15.5% Oliveon Tocopheryl acetate Hydroxyethyl cellulose EDTA 0.05% Sodium hydroxide 0.4% Sorbic acid Guava and coconut 0.3% H20 remaining % Examgle 2: In a specific embodiment, the invention has the following formulation in the form of cream: Ingredients wlw MBBT DHHB APIFIL LABRAFAC Olive oil Tocopheryl acetate yethyl ose EDTA 0.05% Sodium hydroxide 0.4% Sorbic acid Lavender Example 3: In a specific embodiment, the invention has the following formulation in the form of cream: APIFIL LABRAFAC 13.5% Olive oil Tocopheryl acetate Hydroxyethyl cellulose EDTA 0.05% Sodium hydroxide 0.4% Sorbic acid Orange Example 4: preparing the compositions Compositions are ed by the process described hereinafter: Phases 1 and 3, indicated in Table 1 are dissolved together at 65°C using a stirrer.

Phase 2 is dissolved separately at the same temperature, always under continuous stirring. Upon reaching the same temperature, phase 2 is added to phases 1+3 and the whole is mixed for some minutes at 65°C. To obtain a product having homogeneous consistency, it is mixed at room temperature and only at the end, then phase 4 is added. The three compositions thus prepared were tested as described in the experimental section of the present description.

Table 1. Composition of the 3 preparations of examples 1 ,2 and 3 having a ent SPF. -—mmm Tocopheryl acetate Tocopheryl acetate Tocopheryl acetate Tocopheryl acetate 0.5% 0.5% 0.5% 0.5% DHHB 4% DHHB 6% DHHB 8% EHT 1% EHT 2% EHT 3% MBBT 4% MBBT 6% MBBT 8% iWater 57.22 Water 53.05 % Water 51.05% Water 49.05% yethyl Hydroxyethyl Hydroxyethyl Hydroxyethyl ose 0.2% cellulose 0.2% cellulose 0.2% ose 0.2% EDTA 0.05% EDTA 0.05% EDTA 0.05% EDTA 0.05% Sorbic acid 1% Sorbic acid 1% Sorbic acid 1% Sorbic acid 1% Labrafac 1% Labrafac 1% Labrafac 1% Labrafac 1% Sodium ide Sodium hydroxide Sodium hydroxide Sodium hydroxide 0.4% 0.4% 0.4% 0.4% Fragrance 0.3% Fragrance 0.3% Fragrance 0.3% Fragrance 0.3% OLOGY AND EXPERIMENTAL DATA 3.1. Study area Field activities were conducted at the Marine y Laboratory of Onong Resort, Siladen Island, Indonesia. The study area is located in Bunaken National Park, considered one of the most important Marine Protected Areas in the world. Bunaken National Park is located in the centre of a reef-building coral ersity triangle, which roughly asses the Philippines, Indonesia and the Great Barrier Reef. In total, about 605 zooxanthellate coral species were recorded in the Coral Triangle (Veron et al., 2009), of which 66% are common to all ecoregions of the area. This diversity s to 76% of the world’s total species. About 450 coral species have been described so far in the Indonesian archipelago. Bunaken National Park and nt waters support diverse hermatypic (reef-building) coral communities, composed of more than 390 species, 63 genera and 15 families of hard corals, including also some species yet undescribed. The corals form different community types, broadly distributed in relation to depth, slope angle and other environmental factors. The area supports various regionally important populations of a wide array of coral species, including some 20 species considered to be ly rare. Bunaken National Park exhibits exceptional levels of within-location diversity. Richest locations host more than - 30 % of the reef-building coral species complement of the entire Indo-Pacific region. In particular, in the study area, recent surveys reported that the reef-building corals accounted for about 90% of total coral species, with highest coral ity occurring in shallow , likely related to the vely higher habitat geneity produced by changes in slope angle, re and illumination among other factors, from the w reef slope to the crest to the reef flat. In the study area, the occurrence of many endemic species of the genus Acropora were recently reported ce et al., 2001). Acropora represent one of the larger taxonomic group in the reefs surrounding Siladen Island. Acropora is the most diverse hermatypic coral genus, with 114 species recognised ide and 91 species being identified for the Indonesian archipelago (Wallace, 1999). “Structural species” such as Acropora are a vital tem component, and variations in their abundance are critical to the dynamics of entire reef communities (Connell et al., 1997). Acropora spp. plays a dominant role in the species composition and abundance of many modern day Indonesian reefs (Suharsono, 1992). 3.2. Experimental design In situ tests were performed on nubbins (i.e., small branches of about 3-6 cm) of the stony coral Acropora sp. The nubbins were incubated in microcosms in presence of known concentrations of various ingredients potentially to be included in the composition of the eco-compatible sunscreen cream. In particular, coral nubbins (including more than 3000 polyps each) were collected by scuba divers from the same colony, d at a depth of about 3 m on the edge of the reef slope. Sampling of nubbins was performed by means of forceps. The nubbins were washed with seawater filtered onto 0.02-um filters to ate viruses and prokaryotes. Each microcosm was prepared with a replicate set of s (n=3) immersed in e polyethylene bags (Whirl-pack, Nasco, Fort Atkinson, WI, USA) filled with 2 L filtered seawater. A total of 27 microcosms were prepared, in order to test 20 ingredients with the respective positive and negative controls. The list of treatments, the concentrations of compounds used and the control systems are reported in Table 2. The osms were incubated at in situ temperature and light conditions for 72 hours.

Table 2. List of microcosms, treatment types, concentrations of compounds used and incubation times. code compound D-01 Natural grapefruit fragrance D-02 Tea Tree oil (Mela/euca) fragrance D-03 YIang-ylang fragrance I D-04 Natural orange fragrance D-05 Natural lavender nce Essencesl D-06 Argan oil Fragrances D-07 Guava & Coconut 22661 D-08 Bamboo 21842 D-09 Karite 20471 D-10 Mare d’inverno 4/381 MC D-11 Joyful 60029MC Type-1 base cream Type-2 base cream Base cream Type-3 base cream Alpha-tocopherol (SIGMA code T3251- idant DL-tocopherol-acetate (SIGMA code T3376-5G) 2-phospho-ascorbic acid ium salt (SIGMA code 49752-10G) D-19 retinyl palmitate (SIGMA code R3375- 3% D-20 Retinol (SIGMA code R7632—250MG) 3% Polyphenol 60 from green tea (SIGMA D-21 3% code P1204-25G) MBBT (filter) BEMT (filter) EHT (filter) DHHB (filter) Mitomycin C (agent inducing the lytic cycle in viral infections).

- Control Both negative and positive control systems (controls) were used. The negative ones are s in which a substance has been used, the antibiotic mitomycin C, which triggers viral infections, as it s with use of all creams currently marketed.

Therefore, it serves to indicate the levels of undesired response of a compound. The positive ones, denoted by BLK PG and BLK, are simply microcosms without addition of any compound.

Table 3. Concentrations of tested filters FILTERS Max Low SPF concentration MBBT ——-.- DHHB ——-.- 3.3. ination of viral and rokar otic abundances Subsamples (15 mL) of seawater nding coral nubbins were collected at the start of the ment (T0) and after 3 and 6 hours of incubation. After subsampling, sea water was transferred into sterile test tubes without use of fixatives and stored at -20°C until laboratory analysis for quantification of viral and prokaryotic abundance. Once in laboratory, the water samples were immediately sed. Briefly, 200 uL sea water were diluted 1:10 with prefiltered MilliQ water, through 0.02-um pore filters (Anodisc filter; 25-mm diameter, Alzos; Whatman) and stained with 20 uL SYBR Green (stock solution diluted 1:20; lnvitrogen, Carlsbad, CA, USA). Filters were incubated in the dark for 15 min and mounted on glass slides with a drop of antifade consisting of 50% phosphate buffer (6.7 mM, pH 7.8) and 50% glycerol containing 0.25% ascorbic acid (Shibata et al. 2006; Helton et al. 2006; Wen et al. 2004). Slides were stored at -20°C until microscopy is. Virus and prokaryote counts were obtained by epifluorescence microscopy fication, X1000; Zeiss Axioplan) by examining at least 10 optical fields, that is, at least 200 cells or viruses per ate. Viral (and prokaryotic) enrichment was calculated as ratio between virus (and prokaryote) abundances and virus (and prokaryote) abundance at the end of the experiment. 3.4 Zooxanthellae counts Subsamples (50 mL) of seawater surrounding coral nubbins were collected at the start of the ment (T0) and after 6, 12, 24, 36, 48 and 72 hours of incubation. The subsamples were fixed in 3% glutaraldehyde and then about 6.0 ml of each replicate was filtered through 2.0-um polycarbonate filters. Filters were mounted on glass slides and zooxanthellae counted under epifluorescence microscopy (Zeiss Axioplan, Carl Zeiss lnc., Jena, Germany; X 400 and X1000) using standard and UV light. Based on color, autofluorescence and gross cell structure, zooxanthellae released or ted from nubbins were classified as a) healthy (H, bright yellow color, intact/in good conditions zooxanthellae; b) pale (P, pale yellow color, vacuolated, partially degraded zooxanthellae; transparent (T, lacking pigmentations, empty zooxanthellae; Mise and Hidaka 2003). 3.5 Quantification of coral bleaching In order to quantify the levels of coral ing (Siebeck et al. 2006) a colorimetric analysis was performed on digital photographs of corals, taken at the beginning of the experiment and after 6, 12, 24, 36, 48 and 72 hours of incubation. Photographs were taken under cal illumination with a Canon PowerShot A620 digital camera (Canon Inc, Tokyo, Japan) with a scale meter on the background. The photos were successively analyzed with a photo-editing software (Adobe Photoshop CS2) for color composition [cyan, a, yellow, black ]. Levels of bleaching were measured as the difference n the corals' color at the beginning of the experiment and after ents. Variations in the percentage of the ent color components (CMYK) were analyzed with one-way analysis of variance (ANOVA). To rank the bleaching effect due to the compounds tested, a Bray—Curtis similarity matrix was prepared and a multidimensional scaling analysis of the shifts in CMYK color ition of treated corals was performed using Primer 5.0 software (Primer-E Ltd., Plymouth, UK). Bleaching rates were measured as the dissimilarity percentage in CMYK color composition between treated and control corals using the SIMPER tool of Primer 5.0 software (Primer-E Ltd). 4. Results 4.1. Viral and prokaryotic abundance Viral abundances significantly increased of about 1 order of magnitude during the incubation experiments. Viral ment ranged from 1.9 to 37.4, in the systems BLK PG and D08 (Figure 1). Viral abundances at the end of the experiment ranged from 0.44 to 31.2 x 105 viruses ml'1 in systems BLK PG and D15, respectively. yotic abundances increased of 1-2 orders of magnitude during the incubation experiment.

Prokaryiotes enrichment factors fluctuated within a wide range (Figure 2). The lowest enrichment factor (1.88) was ed in the BLK PG , whereas in system D11 the enrichment factor was 78.16. A slight reduction of ratios between viral and prokaryotic abundance (VPR) after incubations was observed in all systems, with the exception of systems D07 and BLK, where VPR remained stable over time. VPR sed up to 10 times in system D22. 4.2. Zooxanthellae abundance At the start of the incubations, in the microcosms no ed zooxanthellae were found. The number of zooxanthellae increased over time in all microcosms considered.

The final abundance of zooxanthellae found in water surrounding the coral nubbins ranged from 0.85 to 4.45 x 104 cell ml'1, respectively in systems D11 (inoculated with Joyful 60029MC) and D18 (inoculated with 2-phospho-L-ascorbic acid ium salt), (Figure 3 a, b, c, d, e).

In some cases, abundance of released zooxanthellae was lower in systems treated than in controls. Statistical analyses revealed that only in the case of systems D15, D16, D17 and D18 the abundance of released cells was significantly higher than in the BLK system (P<0.01). For all other compounds no significant ences were found between control and d systems, suggesting that the presence of the compounds tested in the experimental systems (with the exception of D15, D16, D17 and D18) causes no increase in the rate of zooxanthellae e from corals.

During microscopic analyses, observed zooxanthellae were split into three main groups (healthy, pale and transparent) as described in the Materials and methods section. At the end of the incubations, each group exhibited different abundances in the water surrounding the coral nubbins (Figure 4). Pale and transparent zooxanthellae were, in all experimental systems, the dominant , on average representing 44% and 50% of total released zooxanthellae. Healthy thellae always represented the lesser fraction, ranging from 0 to 21%.

The low abundance of healthy cells suggests that the incubation time of the experiments causes a high level of damage to zooxanthellae in all s considered, negative controls (BLK) included. 4.3. Coral bleaching In all replicates of every microcosm (control systems included), the release of coral mucous (comprised of zooxanthellae and coral tissue) was ed within the first 48 h of incubation. This observation is in accordance with the results of the release of zooxanthellae reported in section 4.2. Mucous release by coral fragments could be related to the mechanical stress occurred during sampling and preparation of microcosms. In all microcosms (BLK systems included) after 72 h of incubation a slight change in color was observed in coral nubbins, potentially occurred as a consequence of the stress d by incubations. The most evident changes were observed in systems D18 and MITO, where all 4 color channels were significantly d at the end of the tions. ln systems D05, D08, and BLK PG, 3 out of 4 color channels showed alterations. In all other systems, 2 or 1 color channels showed significant changes. The systems D03, D09, D10, D11, D21, D21 showed changes in 2 color channels, whereas systems D01, D04, D05, D13, D14, D19, D20 and BLK showed changes only in 1 color channel. In all other experimental systems, no color changes were detected in tested s. 4.4 tability of the sunscreen cream exposed to UVA rays (~ 275 kJ/m2) 2 mg cm'2 of cream were spread on glass slides (5 x 5 cm) and exposed to UVA rays.

. UVA absorbance, 320-400 nm (peak corresponding to the DHHB filter) is too high with respect to the UVB one, 290-320 nm (peak corresponding to the EHT filter). UVA absorbance should be about 1/3 of the UVB one. o The spectrum of the wide-spectrum filter, MBBT, is not visible since it has an extinction coefficient lower than DHHB and EHT, therefore is covered by the absorbance of the latter. MBBT should appear as a wide spectrum without m and minimum peaks.

. At the moment, obtained absorbance falls within the range of a sunscreen cream with a high and low SPF.

. According to the results, it can be ded that the filters are highly photostable. 4.5 Summary of criteria used to test eco-compatibility of tested products with coral reefs: In order to select the most eco-compatible products for each group of compounds (i.e., base cream, nce, antioxidants and filters) the following criteria were selected: 1. coral bleaching level; 2. degree of release of symbiotic zooxanthellae; 3. zooxanthellae health conditions; 4. viral enrichment in the incubation medium with respect to controls.

Table 4. Summary of eco-compatible compounds grouped by category. nd group Ingredients mpatibility Base cream Type 1 Highly compatible Type 2 Compatible Fragrances Antioxidants UV filters Table 5. Viral abundance and enrichment during the incubations. Incubation times (Incub.), total viral abundances (TVN) and the viral enrichment factor (VEF) are also reported. Standard deviation values (STD) and variation cients are reported for all data. -----_- ------- -----_- ------- -----_- ------- ------- -----_- ------- mn_----- ------- ------- -----_- ------- M_----- ------- -----_- ------- -----_- ------- -----_- ------- -----_- ------- -----_- -----_- ------- 447 _---46 -42h ---- ms _----s-oo -42h ---- 42h ---- _---48 42h ---- _---22 22h ---- _---77 22h ---- _---40 22h ---- _---97 22h ---- _---79 22h ---- MITo _---09 22h ---- BLK _---42 -22h ---- um0-44 -42h ---- Table 6. Bacterial nce (total bacterial number, TBN) during incubations, and related standard deviations and coefficient of variation. ----_ ----- ----_ ----- ----_ ----- ----_ ----- ----_ ----- m_--- ----- ----_ ----- ----_ ----- M_--- ----- ----_ ----- ----_ ----- ----_ ----- ----_ ----- ----_ ----- ----_ -____ _____ 1.42E+06 3.82E+05 2. 70E+01 7E+01 _-99E+01 _-67E+01 _--memo _-76E+01 _-3oe+01 ---_new Table 7. Number of healthy, pale and arent (i.e., dead) zooxanthellae (in cells ml'1) released in each microcosm.

Table 8. Percentage of healthy, pale and transparent zooxanthellae ed in the microcosms.

Systems Total Healthy Pale Trasparent m m B 5_8 Q Q m 2 E E Q m E Q E M m a E Q Q m 2 E 7_3 E m 2 fl 5_0 M m 2 fl 5_2 M m Z % Q Q m m E 2 i m 3 Q E J m 2 E E i m é fl 5_9 i m E Q 5_8 m m é fl 5_8 i m 1_ fl fl i m 2 fl 5_4 m m E 5_2 Q i m 2 Q E l m é fl fl A M Q fl fl % m 2 fl 5_9 % m é fl 5_8 % m 1 fl 5_4 % m 2 5_4 M m m 9 a 7_6 % m 2 5_4 fl BLK PG 100 g Q 5_8 Table 9. Output of one-way ANOVA is for the different color components of corals. Probability level reported (**=P<0.01; *=P<0.05; NS = not significant). plecode Cyan (C) genta (M) Yellow (Y) Black (K) REFERENCE Balmer M.E., Buser H.R., Muller M.D., Poiger T. (2005). Occurrence of some organic UV filters in wastewater, in surface waters, and in fish from Swiss lakes. Environmental Science Technology, 39: 953—962.

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Claims (12)

1. CLAIM : olive oil, beeswax: - one or more fragrances selected from orange, er, grapefruit, guava and coconut, and free from: - preservatives belonging to the paraben family; - ascorbic acid derivatives; - UV filters selected from bis-ethylhexyloxyphenol yphenyl triazine, cinnamates, benzophenones, camphor derivatives, titanium oxide; - nces selected from melaleuca essential oil, ylang ylang flower extracts, Bambousa Arundinacea extracts, Karité; - carriers selected from animal-derived fats and argan oil.
2. 2. The composition according to claim 1, further sing sorbic acid.
3. 3. The composition according to claim 1, consisting of:
4. 4. The composition according to claim 1, consisting of: AH26(10335840_1):RTK
5. 5. The ition according to claim 1, consisting of:
6. 6. The composition according to claim 1, consisting of:
7. 7. The composition according to claim 1, consisting of: AH26(10335840_1):RTK
8. 8. The composition according to claim 1, consisting of: - 2,2’-methylene-bis-(6-(2H-benzotriazoleyl)(1,1,3,3-tetramethylbutyl)-phenol, 2-[4-(diethylamine)hydroxybenzoyl]hexyl benzoate and ethylhexyl triazone; - tocopheryl acetate; - sorbic acid; - propylene glycol ylate/dicaprate, olive oil and beeswax; - water; - hydroxyethyl cellulose; - EDTA; - sodium ide; - fragrance selected from lavender or orange or guava and coconut.
9. 9. The composition according to any one of claims 1 to 8, wherein said UV filters are present in a concentration comprised between 3 and 6% by weight.
10. 10. The composition according to any one of claims 1 to 9, in the form of cream, lotion, ointment, spray, or ck.
11. 11. The composition according to any one of claims 1 to 10, characterized in that it is not harmful to the marine environment.
12. 12. The composition of claim 11 which is not harmful to coral reefs. Universita Politecnica delle Marche By the Attorneys for the Applicant SPRUSON & FERGUSON Per: AH26(10335840_1):RTK