الفهرس 
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Abstract
1.Marine sponge and coral samples (Clathria, Crella, and Xenia) were collected from the Red Sea, Hurghada, and identified by Dr. Mohamed Abdel Latif Ezz El Arab at the National Institute for Marine Sciences and Fisheries, Hurghada. The samples were extracted using methanol and ethyl acetate through intense shaking for 3 hours, then left in darkness at room temperature. The process was repeated for 3 days, with filtration conducted the next day and further solvent added. This extraction sequence was followed by various quantitative assays.
2. Analysis of the marine sponges and corals revealed the presence of eleven groups of biologically active compounds. Using two different solvents (methanol and ethyl acetate), the analysis identified ten chemical groups in the Clathria samples, including alkaloids, tannins, saponins, glycosides, emodin, phenols, flavonoids, terpenoids, steroids, and carotenoids. Saponins were particularly prevalent, indicating high levels in both extracts. Anthocyanins were absent, and there was a discrepancy in the presence of terpenoids between the two extracts.
3. Chemical assays of Crella extracts, using both methanol and ethyl acetate, showed the presence of ten different secondary metabolic products, including alkaloids, tannins, saponins, glycosides, phenols, flavonoids, terpenoids, and steroids, while anthocyanins were missing in the methanol and ethyl acetate extracts.
4. Current results showed that Xenia extracts (methanol and ethyl acetate) contain alkaloids, tannins, saponins, glycoside, emodin, phenols, flavonoids, terpenoids, and others. Anthocyanins were absent in both methanol and ethyl acetate extracts.
Determination of tannins
5. Quantitative estimation of tannins revealed that all three samples contained high concentrations of tannins in methanol extracts compared to ethyl acetate.
Determination of chlorophylls and carotenoids
6. Methanol extracts exhibited chlorophyll a content up to 2.32 mg/g in Crella, while it registered 1.63 mg/g in Clathria and 1.6 mg/g in Xenia. Methanol extractions of chlorophyll b showed a significantly higher content in Clathria and Crella (3.60 and 1.42 mg/g), but ethyl acetate extractions were more productive in Xenia.
7. When estimating the total levels of carotenoids in the three samples under study, Clathria methanol extract showed the highest possible concentration, reaching 297.6 mg/g, followed by Xenia (224.3) and Crella (173.2). The concentrations of carotenoids in the ethyl acetate extract were significantly lower than those in the methanol extract, also showing that methanol is more efficient in extracting carotenoids than ethyl acetate.
Estimation of total phenolic and flavonoids compounds
8. The study addressed the estimation of total phenolic compounds, ranging from 16.17 to 51.08 mg/g in the methanol extract and from 10.47 to 30.97 mg/g in the ethyl acetate extract, with the highest phenolic content observed in Xenia extract and the lowest in Clathria. The results also indicated that the concentrations of phenolic compounds in the methanol extract are consistently higher, suggesting that methanol is more effective and more capable of extracting phenolic compounds than ethyl acetate.
9. The total flavonoid concentrations in the ethyl acetate extract were always higher than those in the methanol extract. The flavonoid concentrations in Xenia ethyl acetate extract showed the highest value at 18.67 mg/g, followed by Clathria extract at 11 mg/g, with the lowest value in Crella extract at about 3.19 mg/g.
Determination of antioxidant activities
10. Antioxidant activity was estimated using the potassium permanganate method, comparing the activities against the standard antioxidant, ascorbic acid, which is used as a natural standard for antioxidants. Activities were measured at various concentrations ranging from 31.25 to 1000 µg/ml compared to potassium permanganate (KMnO4) in methanol. The IC50 value for Clathria was 49.6 µg/ml, indicating moderate antioxidant activity compared to an IC50 value for ascorbic acid of 124.8 µg/ml. While Crella was similar to Clathria, a decrease in antioxidant activity was observed with decreasing concentration. The IC50 was slightly higher at 50.5 µg/ml, indicating similar antioxidant activity to Clathria. Xenia shows a pattern of decreasing activity with dilution, but it appears to have slightly higher antioxidant activity at higher concentrations compared to Clathria and Crella. The IC50 value is 48.4 µg/ml, slightly less than the other two samples, indicating better antioxidant efficiency compared to ascorbic acid at 124.8 µg/ml.
11. Current results clarify the antioxidant activity of ethyl acetate extracts from marine sponges (Clathria, Crella, Xenia) and ascorbic acid with KMnO4. The results showed that Clathria has an IC50 of 94.5 µg/ml, indicating that it is the strongest among the marine sponge extracts. Compared with ascorbic acid at about 124.8 µg/ml. While Crella has an IC50 of 70 µg/ml and Xenia has an IC50 of 67.7 µg/ml. IC50 values in all extracts are higher in ethyl acetate extracts compared to methanol extracts, suggesting that methanol extracts may have better solubility or compatibility with test conditions or higher inherent antioxidant activities.
12. Antioxidant activity of methanol extracts was estimated compared to ascorbic acid, and IC50 values were calculated, which indicate the concentration required to prevent 50% of DPPH radical activity. The results showed that the IC50 value for Clathria was 19.5 µg/ml, the best value compared to other types. While the IC50 value for Crella was 20.2 µg/ml, for Xenia, it has an IC50 of 19.9 µg/ml. All methanol extracts tested (Clathria, Crella, and Xenia) have stronger antioxidant activities than the standard ascorbic acid, as they all contain lower IC50 values.
13. The antioxidant activity of ethyl acetate extracts was estimated. The results showed that the Xenia sample had the lowest IC50 value at 19.43 µg/ml compared to other types and ascorbic acid as a standard measure. While Clathria has an IC50 value of 21.2 µg/ml and Crella has an IC50 value of 23.57 µg/ml.
14. The study addressed measuring and comparing the antioxidant activity using methylene blue dye in methanol extract. The results showed that all methanol extracts (Clathria, Crella, and Xenia) have IC50 values lower than ascorbic acid, indicating that they have stronger antioxidant activities in the methylene blue test. Specifically, Xenia has the lowest IC50 value at about 53.8 µg/ml, indicating that it is the strongest antioxidant among the extracts tested in this particular setting.
15. Also, the antioxidant activity of ascorbic acid against methylene blue in ethyl acetate extract from marine sponges and corals was compared. The data showed that all ethyl acetate extracts showed stronger antioxidant activity than ascorbic acid at about 122.7 µg/ml, as evidenced by the low IC50 values, with relatively close values for Clathria, Crella, and Xenia around (27.8, 27.39, and 30.3 µg/ml). This indicates that they have similar antioxidant efficacy in this context.
16. All methanol extracts showed stronger antioxidant activity than ascorbic acid, as evidenced by the low IC50 values, which are about 18.7 µg/ml. Crella showed the strongest antioxidant activity among the extracts tested, followed by Clathria at about 19.68 µg/ml, with antioxidant activity in Xenia slightly less than the other two extracts but still more than ascorbic acid.
17. In the current study, the results of antioxidant tests using DCPIP with ethyl acetate extracts from marine sponges and corals were presented. As can be seen, the IC50 values, which represent the concentration required to inhibit 50% of DCPIP, are as follows: Clathria IC50 of 30.1 µg/ml, Crella IC50 of 29.7 µg/ml, and Xenia IC50 of 28.9 µg/ml. These results indicate that all three ethyl acetate extracts have stronger antioxidant activities compared to ascorbic acid, as they contain lower IC50 values. Among the extracts, Xenia shows the strongest activity, closely followed by Crella and Clathria.
18. The FRAP test is a method used to measure antioxidant strength, where a higher Trolox equivalent indicates greater antioxidant capacity. Antioxidant activity in extracts from marine sponges and corals was compared using two different solvents, methanol and ethyl acetate. The results showed that Clathria recorded about 497.5 ± 1.9 micrometer TE / mg, Crella about 40.27 ± 0.2 micrometer TE / mg, and Xenia 105.39 ± 3.58 micrometer TE / mg, respectively, in ethyl acetate extract while the results in the methanol extract showed the highest value compared to the receding ethyl acetate in Xenia about 705.7 ± 6.44 micrometer TE / mg, this high value of the methanol extract by Clathria is 497.5 ± 1.9 micrometer TE / mg and Crella is 222.3 ± 7.3 micrometer TE / mg, respectively.
Estimation of Cytotoxic effects of extracts:
(1) Estimation of cytotoxicity of marine sponge and methanol coral extract on Huh-7 liver cancer cell line.
19. The cytotoxic effects of methanol extract of Clathria, Crella, and Xenia against the Huh-7 cell line were studied. Cell viability was evaluated using the MTT assay. The results showed that Xenia methanol extract had the strongest cytotoxic effect with an IC50 of 16.78 ± 0.09 µg/ml. At a concentration of 1000 µg/ml, the extract left only 2.87% of the cells viable, indicating high toxicity. While Crella methanol extract showed moderate efficacy with an IC50 of 159.9 ± 0.87 µg/ml. At the highest concentration tested, the viability was 27.97%. While Clathria methanol extract showed moderate efficacy with an IC50 of 78.78 ± 0.89 µg/ml. Cell viability at 1000 µg/ml was very low (2.68%), similar to Xenia extract.
(2) Estimation of cytotoxicity of marine sponge and ethyl acetate coral extract on Huh-7 liver cancer cell line.
20. The cytotoxic effects of the ethyl acetate extract of Clathria, Crella, and Xenia against the Huh-7 cell line were studied. Cell viability was evaluated using the MTT assay. Xenia’s extract showed that the highest concentration tested (1000 µg/ml) resulted in the lowest cell growth viability (2.58%), indicating high toxicity. Cell viability increases with decreasing concentration. The IC50 value is 19.11 ± 0.1 µg/ml, which is relatively low and indicates that the extract has a significantly cytotoxic activity. While Crella ethyl acetate extract also showed high toxicity at the highest concentration (1000 µg/ml) with a cell survival rate of 96.97%, a gradual increase in cell viability was observed with decreasing concentrations. The IC50 value is 71.63 ± 0.67 µg/ml, which is higher than Xenia extract, indicating that it is less effective. Clathria showed high toxicity at the highest concentration (1000 µg/ml) with a viability rate of 3.55%. Cell viability improves with decreasing concentration. The IC50 value is 124.34 ± 2.23 µg/ml, indicating that this extract is the least effective among the three in terms of cytotoxic activity.
(3) Estimation of cytotoxicity of marine sponge and methanol coral extract on MDA- MB231 breast cancer cell line.
21.The cytotoxic effects of the methanol extract of Clathria, Crella, and Xenia against the MDA MB231-cell line were studied. Cell viability was evaluated using the MTT assay. The data showed that Xenia methanol extract showed that the IC50 value is 17.6 ± 0.13 µg/ml, indicating strong cytotoxicity. The extract showed high toxicity at 1000 µg/ml with a viability of only 2.43%. While Crella methanol extract has an IC50 value of 83.67 ± 0.23 µg/ml, indicating moderate cytotoxicity compared to Xenia. At the highest concentration tested, the viability was 3.86%. While Clathria methanol extract showed an IC50 of 82.2 ± 1.46 µg/ml, which is similar to Crella, indicating moderate cytotoxicity. The viability rate at 1000 µg/ml is 4.41%, which is slightly higher compared to Crella and Xenia at the same concentration. The results indicate that the methanol extracts of these marine organisms have a cytotoxic effect on the MDA MB231 breast cancer cell line, with Xenia extract being the strongest, followed directly by Clathria and Crella.
(4) Estimation of cytotoxicity of marine sponge and ethyl acetate coral extract on MDA-MB231 breast cancer cell line.
22. The cytotoxic effects of the ethyl acetate extract of Clathria, Crella, and Xenia against the MDA MB231 cell line were studied. Cell viability was evaluated using the MTT assay. The data showed that Xenia ethyl acetate extract showed high toxicity at the highest concentration (1000 µg/ml) with a viability of 2.43%. And an IC50 relatively low at 18.6 ± 0.11 µg/ml, indicating a cytotoxic effect against the MDA MB231 cell line. While Crella ethyl acetate extract also showed high toxicity at 1000 µg/ml with a cell survival rate of 3.26%. The IC50 is higher than Xenia at 20.32 ± 0.15 µg/ml, indicating slightly less effectiveness but still notable cytotoxic activity. While Clathria ethyl acetate extract shows a viability of 3.48% at the highest concentration tested, the IC50 value is 79.39±0.8 µg/ml, which is much higher than the other two extracts, indicating that it is the least effective among the three. The general pattern observed is that all three extracts show a dose-dependent cytotoxic effect on the MDA MB231breast cancer cell line, with Xenia and Crella showing greater effectiveness compared to Clathria, as evidenced by their low IC50 values.
23.These qPCR results provide valuable transcriptional changes induced by the methanolic and ethyl acetate extract of Clathria ,crella and xenia in huh-7 and MDA cell line . By analyzing the relative quantification (RQ) values, we obtained the expression levels of these key genes (p53, BCL2, HIF1α) comparing to the control housekeeping gene. These results revealed a high expression of p53, with RQ value of 8.6778,2.4838,5.3688 and 2.5313 respectively comparing with control and low expression of Bcl2(0.3107,0.8273,0.2368 and 0.5771), HIF1α (1.881,1.5562,1.3889 and 1.9291 )with RQ value of respectively.
Activity of extracts as antibacterial agents
24.This study examined eight distinct strains of marine pathogenic bacteria against G+ve bacteria including Staphylococcus aureus, Enterococcus faecalis, Bacillus cereus, as well as G-ve strains including E. coli, Vibrio Fluvialis, Pseudomonas aeruginosa, and Salmonella typhimurium. The extracts showed an antibacterial effect against all the strains tested, resulting in average inhibition zones ranging from 18.0 ± 0.1 to 20.0 ± 0.3 mm. The extract from Clathria had a very positive effect on the bacteria examined, showing the highest effect on both Staphylococcus aureus (NIOF-B16), Escherichia coli (NIOF-B17), Enterococcus faecalis (NIOF-B21), and Vibrio fluvialis (NIOF-B24) giving an inhibition zone diameter of 18 mm (18 mm). However, this extract had a moderate effect on both Pseudomonas aeruginosa (NIOF-B23), Vibrio damsel (NIOF-B29) and Salmonella typhimurium (NIOF-B35) with inhibition zones of 16.0 ± 0.2, 14.0 ± 0.1 and 14.0 ± 0.2 mm diameter respectively. Finally, there was an acceptable effect on Bacillus cereus (NIOF-B33) with inhibition zones measuring (12.0 ± 0.4 mm). Xenia extract had a strong effect on Staphylococcus aureus (NIOF-B16), as evidenced by inhibition zones measuring 20.0 ± 0.3 mm. Furthermore, the extract showed an effect on other pathogenic bacteria in an effective manner, with inhibition zones ranging from 16.0 ± 0.1 mm to 10.0 ± 0.1 mm. The antibacterial effect of Crella coral extract was excellent and acceptable on all types of bacteria.
25. To evaluate the sensitivity of the tested strains to all extracts, the Minimum Inhibitory Concentration (MIC) was assessed. Clathria extract showed the lowest MIC against Escherichia coli (NIOF-B17) (8.0 ± 0.5 mg/ml). MIC values for Staphylococcus aureus (NIOF-B16), Enterococcus faecalis (NIOF-B21), Pseudomonas aeruginosa (NIOF-B23), Vibrio Fluvialis (NIOF-B24), Vibrio damsel (NIOF-B29), Bacillus cereus (NIOF-B33), and Salmonella typhimurium (NIOF-B35) were 8.5 ± 0.4, 10.0 ± 0.5, 9.5 ± 0.1, 10.0 ± 0.2, 12.5 ± 0.1, 15.0 ± 0.2, and 11.5 ± 0.2 mg / ml, respectively. On the other hand, Xenia extract showed the lowest MIC values represented against Staphylococcus aureus (NIOF-B16) and Escherichia coli (NIOF-B17) where they reached 4.5 ± 0.5, and 7.5 ± 0.1 mg/ml, respectively, while the highest MIC concentration. Against Bacillus cereus (NIOF-B33) (15.0 ± 0.03 mg/ml). In addition, the values of MIC concentrations for the compound crella ranged from 14.0 ± 0.1 to 5.5±0.4 mg/ml.