Development of broad spectrum mycosporine loaded sunscreen formulation from Ulva fasciata delile

Ba.ckground: Sunscreen formulations primarily offer protection against UV induced damages however nowadays it also maintains skin natural physiological conditions. Current global market is flooded with numerous sunscreen products which offer protection to skin against several UV induced damages. However most of these sunscreen formulations offers narrow spectrum protection against UV and also suffer from stability as well as toxicity related issues. Methods: Present work aims to isolate mycosporine amino acid (Mgy) from green alga namely Ulva fasciata (U. fasciata) and study its sunscreen potential against widely used domestic marketed formulation. Stability evaluations were also performed for almost 90 days. Results: Results demonstrated that the isolated compound, mycosporine glycine (Mgy) preserved physicochemical properties of the product and offered good stability for all formulations throughout the experimental period. Furthermore, Mgy loaded carbopol gel showed better sunscreen protection against marketed formulation in a concentration dependent manner. (7.709). Conclusion: (6.806) Novel Myg loaded gel was proved to demonstrate several quality characteristics that may unlock new prospects for the production of more efficient, safe, and economic skin-care products.


Introduction
Ultraviolet (UV) irradiation and its interaction with living system is a critical issue from several years. UV irradiation represents UV region (280 to 400 nm) which is divided in to three components: UV-B irradiation (280 to 315 nm), UV-A (315-400 nm) and UV-C (200 to 280 nm) [1][2][3][4][5]. UV-C is not of biological relevance as it's totally absorbed by the atmosphere. Due to global warming (ozone depletion) there is a marked increase in the UV-B (7-8 W mK 2 ) irradiation. Marked increase in UV-A (45-50 W mK 2 ) also observed in some of the regions [6][7][8][9]. This is considered as one of the major global environmental threat which can increase injurious effects of UV radiation over health. Thus utilization of sunscreen agents is always encouraged globally to prevent chances of skin injuries caused by UV radiations. Most of the marketed formulations are specifically prepared to block UV-B absorption. These formulations not only prevent the absorption but also reflect UV-B. One of the most common approaches to evaluate potential of the sunscreen is Sun Protection Factor (SPF). SPF of any formulation can be calculated by evaluating its potential in inhibiting erythema (redness due to inflammation) and edema (swelling or inflammation). These skin reactions can be noticed 24 h after exposing skin against UV radiations [10][11][12][13][14][15][16][17]. UVA (315-400 nm) is having longer wavelength, thus it's not efficient in inducing erythema or any other skin reactions whereas UVB (280-315 nm) is required to induce erythema within less period of time. Present sunscreen formulations which are already lites) such as mycosporine-like amino acids (MAAs) [33][34][35][36][37][38][39]. In our previous study we have reviewed and investigated the major characteristics of these water-soluble compounds namely MAAs (309 to 360 nm) and explore in-vitro sun protection of Porphyra-334 from Porphyra vietnamensis. Ulva contains high amount of carbohydrate, protein, lipid, moisture, dietary fiber, vitamin C and ash content. Moreover this green alga is also enriched with minerals and amino acids. A unique class of amino acid mainly mycosporine glycine (Mgy) which is rarely available in green alga has been identified in this study. Current study is to identify, isolate and evaluate stability of sun protective compounds derived from U. fasciata. Furthermore isolated compound was further assessed for its sun screening potential against "Lotus gel" [40,41]. The sufficient amount of fresh Ulva fasciata sample (100 g) was preserved by glutaraldehyde (3%), and sample was entirely washed before its usage. Ethanolic fraction was isolated from available in the market offer protection mainly against UVB and only few formulations are broad-spectrum UVA filters. Additionally these sunprotective compounds are more sensitive against other excipients present in the formulations which causes several stability problems. So the current Indian market requires the most compatible, stable and broad spectrum compounds or formulations which can provide a protective screening shield to the human skin.
These compounds once identified, isolated and toxicological verified against designed pharmacological models can be considered as good candidate for formulation and development. Recent reports suggested the presence of unique class of sun protective compounds present in algae called as mycosoorine amino acids (MAAs). These MAAs are comparatively more present in seaweeds which can be considered as a vital source for sunprotective compounds. Ulva fasciata, a green marine alga belonging to family Ulvaceae, is widely available in east and west coast of India. The first report of Ulva (U. lactuca L.) was from the India which was evidenced by Boergesen (1934) [31]. This alga is commonly found in the intertidal region along the coastline typically lithophytic or epiphytic. So far 14 species have been evidenced from Indian origin [32]. Ulva is not reported so far for its sun protective action but still carries photo-adaptive mechanisms to protect against intense sun radiations. This type of mechanism includes radical scavenging potential and triggering the synthesis of sun protective compounds (secondary metabo-

Extraction and isolation procedure
Twenty gram of dried algal material was powdered and treated with a mixture of ethanol and water (75: 25, v/v) followed by treatment with chloroform. Non polar fraction was removed whereas polar fraction was lyophilized ultimately to yield two gram of a pale yellow powder. Weighed quantity of powder was re-treated with 80% ethanol.

Isolation of bioactive compound
To isolate bioactive compound, powder derived from the initial procedure was eluted through a silica gel column (60-120 mesh) loaded with 20% methanol +0.2% acetic acid. TLC analysis was done to ensure the homogeneity of loaded fractions. Out of 2 gram of loaded sample 377 mg of pale yellow powder was obtained. Percentage yield [(Wt of bioactive compound: 377 mg) / Wt of raw material: 2000 mg) ×100] was calculated. Resulting fraction was evaporated and lyophilized till dryness to ultimately yield 270 mg of a colorless powder.

HPLC analysis
To ensure purity of the sample isolated fraction was further analyzed by HPLC with PDA detector (SPD-M30A, 85 mm optical path length, 0.4 × 10 -5 AU noise level) with ODS (RP-18 column) and a guard (ODS, 5 μm, 4.0 × 10 mm). 20 % methanol +0.2% acetic acid (mobile phase) was run at the rate of one millilitre per minute. By using auto-injector fifty micro litre sample was injected into the HPLC column. 20% methanol +0.2% acetic acid was loaded, which showed retention time of 2.8 min. To record absorption spectra of sample, detector was scanned from 200 to 450 nm. HPLC peak was recorded at wavelength of MAA was 320 nm. Pure sample (MAA) was collected by using fraction collector which was again lyophilized. The whole investigation was performed on highly polar water soluble compound (mycosporine glycine) that absorbs strongly in the 10 nm to 400 nm (UVA region) [42].

Development of MAA loaded carbopol gel
MAA loaded Gel was prepared by using 1% of carbopol (carbopol 934), 1% of MAA, EDTA (0.03%), propylene glycol (3%) as major ingredients. Whole mixture was dissolved in water and heated upto 5 min at 50°C. Prepared mixture was kept for 48 h in dark place. To achieve the uniform consistency of gel and to stabilize its pH, a crosslinker (triethanolamine) was added in a drop wise fashion.

Physiochemical properties of MAA loaded gel
Morphological characterization of gel: Physical appearance and color was monitored by visual observations over period of time from 1-80 days.
pH: pH of drug loaded gel was determined by means of pH meter (calibrated). This was done at various at different twenty days time interval (1, 20, 40,   cro System, Surrey, UK). Other parameters such as GT, MT, MW were determined by methods introduced by Craigie and Leigh [44] and Rochas and Lahaye [45]. SA was determined by measuring spread diameter of one gram of gel. This was performed by keeping one gram of gel between two plates of dimensions 20 cm each.
Skin erythema and edema evaluation: Male Swiss mice of weight range 25 g was used for this study. Optimal conditions were maintained at a controlled temperature (22 ± 2 °C) for 12hours light/dark cycle. To prevent food interference with reference/test drugs absorption, eight hours before each experiment animals received only water. The experimental studies were conducted with the consent of the procedure by the local Institutional Ethics Committee-IAEC/ABMRCP/2016-2017/11. Prepared gel and gel base as control were applied after removing hairs of the selected animal. Visual observations were made after an interval of seven days of gel application [46].

In vitro sun protection determination method
Selected formulation was evaluated for sun protective efficiency by utilizing simple and rapid in vitro sun protection determination method [25,26]. Marketed Lotus gel formulation was selected as reference formulation. Three MAA loaded carbapol gel with different dilutions from 50-150 µ/ml were prepared in methanol. UV spectrophotometer was used to measure absorbance of all dilutions [46]. This was recorded at 5 nm intervals from 250-400 nm. Each determination was made in triplicate (Mean ± SD). The observed absorbance values were calculated according to the equation given below.
The aliquots prepared were scanned between 290 and 320 nm, and the obtained absorbance values were multiplied with the respective EE (l) values. Then, their summation was taken and multiplied with the correction factor (10) [10].

Characterization of isolated MAA
From taxonomic features (Fig. 1) it was confirmed that harvested alga was U. fasciata. Average percentage yield of MAA is 13.5%. Isolated bioactive compound was characterized initially by UV spectrophotometer and HPLC-PDA detector. UV analysis revealed appearance of sharp peak with an extinction coefficient of 31,177 M −1 cm −1 which was obtained at 320 nm (Fig.  2). From UV/Vis, LC-MS and HPLC analysis it was almost clear that isolated compound was mycosporine-glycine (Mgy) with λmax 320 nm, retention time (RT) 2.8 and mass charge ratio (m/ z) 247.9 as depicted in Fig. 2 HPLC-PDA study of the fraction of bioactive compound (MAA) has shown a prominent peak at 2.8 min (Fig. 3) with a UV absorption maximum (UVλmax) at 320 nm (Fig. 2). LCMS analysis of HPLC-PDA purified MAA showed a sharp ion peak of a protonated molecule ([M + H]+) at m/z 247.9 (Fig. 4).

Physiochemical properties of MAA loaded gel
Physiochemical properties of formulated gel in comparison with marketed formulation (Lotus gel) are mentioned in

In vitro sunscreen potential of MAA loaded gel
In present work a green alga harvested from Indian coast namely Ulva. Ethanolic extract U. fasciata was considered as a source for sun protective compound which was assessed for their UV absorbance potential against marketed formulation namely Lotus gel. Based on the results, it was concluded that UV screening potential of MAA loaded formulations was increased in a concentration dependent manner (50-150 µl) as mentioned in Table 2. Absorbance values of bioactive compound and marketed formulation at different concentrations (50-150 µl) were considered to calculate the in-vitro sun protection factor. This calculation was done by using the erythemogenic effect values (EE values). These values are relevant only for the UV-B (290-320 nm). As represented in Fig. 5 and table 2 MAA loaded Ulva gel was more active in screening UV radiations in comparison to standard Lotus gel. It was proven that MAA loaded Ulva gel broadly prevents UV radiations, signifying more potential against Lotus gel.

Conclusion
Several reports have confirmed the presence of MAA among different marine sources and their fundamental way to protect marine organism against UV induced stress. Marine organisms are more vulnerable against UV induced injuries because seawater absorbs light much more strongly than air does and effects of ultraviolet radiation are greater in the coastal zone than in inland areas, due to reflection of sunlight from sand and the sea surface. Thus these marine organisms have various photo-adaptive mechanisms to protect against UV induced stress conditions. MAA is a family of amino acids which are diversely present among different marine organism especially seaweeds to protect them against photo-oxidative stress induced by UV irradiations. Thus in present work we have successfully isolated and formulated Mgy to test its sunscreen potential against marketed formulation. Results   obtained from this work permit the conclusion that this sunprotective formulation constitutes the most prominent and potent amino acid which can in future become a promising molecule in UV-related dermatological ailments.