Preliminary Data Regarding Pharmaceutical Forms Type Gels Based on Marine Algae Extracts with Antioxidant Activity

Particularly interest on herbs and plant extracts is due to their content of active principles with remarkable pharmacological properties. Different vegetal species contain natural antioxidants (polyphenols) used for their dermatological anti-inflammatory and healing activity, justified by their action on free radicals. The aim of this paper was to obtain selected phytopharmaceuticals formulations type gels with an increased antioxidant potential based on a mixture of fluid extracts from seaweeds species in the phylum Chlorophyta, Cladophora vagabunda (L.) C. Hoek, in the family Cladophoraceae, Ulva lactuca (L.) syn. Ulva rigida (L.), in the family Ulvaceae (sea lettuce) and species in the phylum Rhodophyta, Ceramium rubrum C. Agardh, in the famiy Ceramiaceae, frequently presents along the Romanian Black Sea coast. Fluid extracts concentration 10% were obtained using cold maceration extraction method, in solvents ethylic alcohol 40% and 70%. The obtained vegetal extracts were mixed in different ratio and analysed for physico-chemical properties, for the content of total phenols, chlorophylls, cartenoids, flavonoids and antioxidative capacity by photochemiluminescence method (ACL, Analytik Jena AG procedure). The selected mixed fluid extracts with greatest content of polyphenols and antioxidant activity were used for obtaining pharmaceutical forms type gels ISSN 2601-8705 (Print) ISSN 2601-8691 (Online) European Journal of Natural Sciences and Medicine July December 2021 Volume 4, Issue 2 27 which were analyzed for the physical-chemical properties appearance, pH, spredability and total antioxidative capacity. Preliminary results emphasized that proposed pharmaceutical forms type gels had an appreciable antioxidant activity correlated with a good stability, an increased polyphenols content and would represent a possible new dermatological anti-inflammatory and healing preparation.


Abstract
Particularly interest on herbs and plant extracts is due to their content of active principles with remarkable pharmacological properties. Different vegetal species contain natural antioxidants (polyphenols) used for their dermatological anti-inflammatory and healing activity, justified by their action on free radicals. The aim of this paper was to obtain selected phytopharmaceuticals formulations type gels with an increased antioxidant potential based on a mixture of fluid extracts from seaweeds species in the phylum Chlorophyta, Cladophora vagabunda (L.) C. Hoek, in the family Cladophoraceae, Ulva lactuca (L.) syn. Ulva rigida (L.), in the family Ulvaceae (sea lettuce) and species in the phylum Rhodophyta, Ceramium rubrum C. Agardh, in the famiy Ceramiaceae, frequently presents along the Romanian Black Sea coast. Fluid extracts concentration 10% were obtained using cold maceration extraction method, in solvents ethylic alcohol 40% and 70%. The obtained vegetal extracts were mixed in different ratio and analysed for physico-chemical properties, for the content of total phenols, chlorophylls, cartenoids, flavonoids and antioxidative capacity by photochemiluminescence method (ACL, Analytik Jena AG procedure). The selected mixed fluid extracts with greatest content of polyphenols and antioxidant activity were used for obtaining pharmaceutical forms type gels

Introduction
Seaweeds -are aquatic photosynthetic organisms used largely due to the variety of bioactive compounds in their composition that have begun to attract the attention of the food, pharmaceutical and cosmetic industries. Functional components extracted from algae biomass are widely used as food and health supplements with a variety of applications in food science and technology. The applications of algae in dermocosmetic products are increasing due to the fact that they have a high potential in various treatments for dermal diseases.
There is a growing concern for valorization marine phytobenthic bioresources in cosmetic therapy and treatments, by using new biotechnologies to obtain innovative pharmaceutical and dermo-cosmetic preparations, for biocompatible external use, based on soft fluid extracts or dried purified green algae containing natural bioactive compounds, with antibacterial, antifungal, antiproliferative, antioxidant activity, with maximum efficiency and minimal side effects, non-allergenic, regenerative and with skin protection effect against UV radiation (www.seaweed.ie/; www.irishseaweeds.com/sea-lettuce-ulva-lactuca; www.hawaii.edu/reefalgae/invasive_algae/chloro/cladophora_vagabunda.htm; www.algaebase.org/search/species/; Lindberg M., 2010; Mshigeni K.E., 1991; Drum R.; Leandro A., 2020; Noel Vinay, 2013; Kennedy J., 2020; Pooja S., 2014; Ajit Kandale, 2011) Marine green algae species along Black Sea coast, in the phylum Chlorophyta, Cladophora vagabunda (L.) C. Hoek, in the family Cladophoraceae, Ulva lactuca (L.) syn. Ulva rigida (L.), in the family Ulvaceae (sea lettuce), respectively red algae species in the phylum Rhodophyta, Ceramium rubrum C. Agardh, in the famiy Ceramiaceae, are used in traditional medicine most due to their content in polyphenolic acids with increased antioxidative capacity mainly useful in anti-inflammatory diseases The aim of this paper is the evaluation of the total phenols compounds, chlorophylls, cartenoids, flavonoids content in hydroalcoholic extracts of Cladophora vagabunda (L.) C. Hoek, Ulva lactuca (L.) syn. Ulva rigida (L.), and Ceramium rubrum C. Agardh species along Romanian Black Sea coast, knowing that these three vegetal products present an increased antioxidant activity with a real phytotherapeutic potential. The selected fluid extracts with increased content of polyphenols were used for hydrogels obtaining (Negreanu-Pirjol T., 2015; Sirbu R., 2010; Negreanu-Pirjol T., 2014; Negreanu-Pirjol T., 2014). The pharmaceutical formulations type gels had a good stability in time and the most increased total antioxidant capacity, better than the fluid vegetal extracts used as raw materials.   The immediately pretreatment process after harvesting involves washing the biomass thoroughly several times with distillated water, to remove adhering sand particles and impurities and dried at room temperature, for 24 hours, in the dark. Fresh marine algae biomass was weighed to determine the loss of water through drying process. The dried algae material from three species, Cladophora vagabunda, Ulva lactuca and Ceramium rubrum was macerated to a fine powder (homogeneous, as well as a higher surface-to-volume ratio) and investigated for chlorophyll pigment, carotenoids, total flavonoids and total phenolic content and total antioxidant capacity, with triplicate samples of each determination.

Material and Methods
For fluid extract obtaining -maceration method at cold of vegetal product was used: ethylic alcohol 40% and 70% 1:10 ratio, stir and allow soaking in the dark for 10 days in a constant temperature and stirred periodically. After 10 days, the extract is filtered and brought to a concentration of 10% with a 100 mL volumetric flask. After the 10 days obtained extract was filtered and stored at 4 °C (Negreanu-Pirjol B.S., 2015, Negreanu-Pirjol T., 2019). For the fluid extracts, pH, relative density were determined.
pH was achieved using a Consort electronic pH-meter with temperature sensor For relative density, the pycnometer method was used For chlorophyll and carotenoid content, 0.1 g ground dry algal material was extracted in 10 mL 80% acetone (triplicate samples for each species), 1% final, through UV-Vis spectrophotometric analysis (using a S106 WPA spectrophotometer) at 470, 647 and 663 nm wavelenghts. The concentrations of chlorophyll a, chlorophyll b (only in chlorophytes) and total xanthophyll and carotin were calculated by using the trichromatic equations (Lichtenthaler & Buschmann, 2001, Miazek K., 2011 For total flavonoid determinations, 1 g ground vegetal tissue was extracted in 5 mL methanol and filtered (triplicate samples). 0.5 mL of extract was diluted in 4 mL water and 8 mL methanol mixture, and the spectrophotometric absorbance was read against a methanol blank at 340 nm wavelength (Szabo I., 2012).
For determining the concentration of total phenolic compounds, a spectrophotometric version of the Folin-Ciocâlteu method was used. 0.1 g fruit tissue was extracted in 10 mL methanol and filtered. 1 mL of extract was reacted with 5 mL Folin-Ciocâlteu reagent (10%) and 4 mL sodium bicarbonate solution (7.5%) for 30 minutes. Spectrophotometric absorbance was read against a blank at 765 nm. A calibration curve was prepared, by using different gallic acid concentrations [10][11][12]. The resulting concentration was expressed as mg/kg dry weight and mg/kg DW gallic acid equivalent (GAE), (    Carbopol 940 1% gel based on C-U-C fluid extracts obtaining: disperse carbopol 940 in the preservative solution, shake vigorously (with a high speed electric stirrer) to avoid the formation of agglomerates The dispersion was neutralized gradually with triethanolamine solution so as not to include air. Fill with preservative solution to 100 g add and mixed the fluid extracts.
Stock solution for semisolid pharmaceutical forms type gels, were prepared as folloys: 5 g of C-U-C gels were diluted with 5 mL ethyl alcohol 95%. From the stock solutions, were taken 10 µL working volume, according with Antioxidative Capacity in Lipidsoluble substances (ACL) (procedure, Analytik Jena AG, 2004).
The total antioxidant capacity of the samples were quantified by comparison with the standard Trolox (constructing a calibration curve with Trolox®) (Fig. 2)  From stock solutions were prepared the samples, as follow (Table 3):

Results and Discussions
The values of the pH and relative density parameters did not significantly modified, proving a good compatibility between the selected bases and C-U-C seaweeds fluid extracts (Table. 3). -Macroalgae fluid extracts chlorophylls pigments content determination -Chlorophylls pigments contents of green algae fluid extracts at 10% concentration of algal extracts, emphasize an increased values for both species, as follows: Chlorophyll a concentration was the highest in Cladophora (488,427 mg/kg), while maximum chlorophyll b content was found in Ulva lactuca (321,700 mg/kg), Fig. 3.     (Table 4).

Table 4. Physical-chemical parameters for semisolid pharmaceutical preparations type gels based on C-U-C seaweeds fluid extracts
Parameter Gels based on UCT fluid extracts (G1 -G4) appearance translucent mass, yellow -greenish color, sweet taste, characteristic smell homogeneity homogenous aspect without air bubbles, drops or particle clusters pH 5.6 -6.5 spreadability (cm) 10. 56 -32.448 Total antioxidant capacity (TEAC) for seaweeds C-U-C fluid extracts, respectivelly for gels G1 -G4 based on seaweeds C-U-C fluids extracts in presented in Table 5. The results obtained for total antioxidat capacity (TEAC) determinations, emphasize the follows:

Table 5. Total antioxidant capacity (TEAC) of seaweeds C-U-C fluids extracts and gels based on seaweeds C-U-C fluid extracts
at the working solution volume (10 μL) according to the procedure, it was observed a valuable TEAC for all the analyzed semisolid pharmaceuticals preparations type gels (G1 -G4), between 1.127 -2.991 nmol equiv. Trolox / volume sample, but more decreased compare with the raw material -seaweeds C-U-C fluid extracts. The most increased total antioxidant capacity value for sample Gel 4 (2.991 nmol equiv. Trolox / volume sample), was registered.
at the working solution volume (10 μL) according to the procedure, it was observed an increased TEAC for seaweeds C-U-C fluid extracts samples, with values between 1.112 -3.702 nmol equiv. Trolox / volume, more increased compare with the registered values of gels (G1 -G4) obtained from the same seaweeds C-U-C fluids extracts. The most increased total antioxidant capacity value for sample C-U-C fluid extract (1:2:2) ratio in ethyl alcohol 70% (3.702 nmol equiv. Trolox / volume sample), was registered.

Conclusions
Gels based on C-U-C seaweeds fluid extracts did not have modifications in quality parameters change during 30 days of observation, Total antioxidant capacity was highest for semisolid pharmaceuticals preparations Gel 4 and seaweeds C-U-C fluids extracts used for obtaining G4 were extracted under maceration with alcohol 70%, mixed in 1:2:2 ratio.
Method of extraction, nature of used vegetable products, fluid extracts ratio determined good values for total antioxidant capacity, in this case, nature of the pharmaceutical base used for gels obtaining, did not interfere with total antioxidant capacity, which was increased for semisolid pharmaceuticals preparations Gel 4.
The obtained semisolid preparations G4 could be further submitted to other tests for its antioxidant properties valorification. Pharmaceutical formulations type gels based on marine algae, had an increased total antioxidant capacity correlated with a good stability and increased polyphenols content and would represent a possible new dermatological anti-inflammatory and healing preparations with antioxidant potential against free radicals action.