Marine sponges (phylum Porifera) are among the oldest and simplest animals, which grow in every ocean and have a great capacity to withstand extreme temperatures and pressures . They are filter feeders, and their bodies are full of pores and channels that allows water to circulate through them [2,3]. Moreover, they are well-known for their production of secondary metabolites that constitute an effective defense mechanism against foreign predators [2,3]. So far, about 8000 species of Porifera, inhabiting different marine and freshwater ecosystems, have been described. Since the beginning of the exploration of marine natural products in the 1970s, the investigation of the secondary metabolites of Xestospongia sponges (family Petrosiidae), commonly known as barrel sponges, have been carried out successively in several regions around the world . They have been recognized as rich sources of different chemical classes, such as isoquinoline, macrocyclic quinolizidines, pyridoacridine alkaloids, quinones, sterols, brominated polyacetylenic acids, and esters . Previous investigations on the chemistry and pharmacology of genus Xestospongia have shown that its crude extracts and isolated compounds displayed remarkable bioactivities, such as anti-inflammatory, antioxidant, immunomodulatory, cytotoxicity, antimicrobial, insecticidal, HIV protease inhibition, cardiotonic, vasodilatation, and antiplasmodial activities [5,6,7,8,9]. The currently investigated species, Xestospongia testudinaria, has been the source of indole alkaloids, sterols, sterol esters, and brominated polyunsaturated fatty acids (BPUFAs) [4,10,11].
As part of our continued interest in identifying potential marine-derived bioactive compounds for treatment of contemporary diseases such as cancer and infectious diseases [12,13,14,15], we thoroughly investigated the chemical constituents and cytotoxic activity of the Red Sea sponge Xestospongia testudinaria. In this paper, we report about the purification and identification of thirteen compounds including two new xestersterol esters (2 and 4) with eleven known compounds. In addition, the cytotoxic activities of the compounds against three cancer cell lines will be evaluated.
2. Results and Discussion
Compound 2 (Figure 1) was isolated as a white powder. Its HREIMS displayed [M]+ at 664.6158 consistent with the molecular formula C46H80O2 and seven degrees of unsaturation. The intense IR absorption band at 1735 cm−1 indicated the presence of a carbonyl ester, which was also evident from a quaternary carbon signal, in 13C NMR, at 172.1 ppm (Table 1 and Table 2). The 13C NMR data of 2 (Table 1) possesses the signals for a basic steroid nucleus with 30 carton atoms (xestosterol type) as established by the similarity of its 1H and 13CNMR data with those of xestosterol (1) . The significant differences between Compounds 1 and 2 lie in the downfield shift of H-3/C-3 from δH/C 3.53/71.8 in 1 to δH/C 4.55/73.6 in 2, and an upfield shift of C-4 from δC 42.2 ppm in 1 to δC 38.2 ppm in 2. These differences, together with the presence of a carbonyl ester signal at δC 172.1 ppm in 2, suggested the esterification of 2 at C-3. Based on evidence obtained from HR-EI-MS, 1H, and 13C NMR (Table 1 and Table 2), the ester moiety at C-3 was determined to be a long chain fatty acid (palmitic acid) . The 13C NMR and DEPT experiments revealed the presence of a total of 46 carbons ascribed as 30 carbons for the xestosterol skeleton, while the remaining 16 carbons were assigned to the long chain fatty acid moiety. The 1HNMR spectrum (Table 2) showed a signal at δH 0.79, assigned to the terminal methyl of the long chain fatty acid, beside a long chain methylene at δH 1.29 integrated for 22 protons. The terminal methyl carbon appeared, in 13CNMR, at δC 14.1 ppm, while the methylene carbons of the long chain moiety appeared at δC 34.4, 25.1, 31.9, and 22.7, assigned to C-2’, C-3’, C-14’, and C-15’, respectively, and from 29.1 to 29.8 (11 CH2) for C-4’–C-13’. The complete assignment for all protons and carbons for both the steroid nucleus and the long chain fatty acid moiety was achieved by the aid of 1H–1H COSY and HSQC experiments. A careful interpretation of the HMBC spectra allowed the final structural elucidation of 2, whereas two and three bond correlations were observed from H-4 to C-1’, C-2, C-4, C-6, and C-10; from H-2 to C-1 and C-3; from H-13’ to C-14’ and C-15’; and from H-2‘ to C-1’, C-3’, and C-4’. Thus, Compound 2 was deduced as xestosterol-3 palmitate and is reported here as a new natural product.
Compound 4 (Figure 1) was isolated as a yellowish brown powder. HR-FAB-MS showed [M]+ at 728.4168, consistent with the molecular formula C46H65BrO2 with 14 degrees of unsaturation. IR showed bands at 2240, 1731, and 1469 cm−1, assigned to an acetylenic group, an ester group, and a terminal methylene group, respectively. The 13C NMR (Table 1) and DEPT experiments showed a total of 46 carbon atoms, 30 of which were assigned to a xestosterol nucleus similar to Compounds 1–3. Xestosterol has previously been isolated and partially chemically synthesized from the same sponge . On the other hand, careful analysis of the remaining 16 carbons revealed a close similarity between signals of the brominated polyacetylenic acid 9, except for the free carboxylic acid carbon (C-1, δC 179.8), which was shifted to 173.1 ppm in 4, confirming the presence of an ester moiety. The E configurations at C7’/C8’, C11’/C12’, and C15’/C16’ were secured from the large coupling constants b (15.8–16.2 Hz). Like 2, the downfield shift of H-3, compared to 1, and a 3J cross peak correlation, in the HMBC experiment, from H-3 (δH 4.57) to C-1’ (ester carbonyl at δC 173.1) justified the esterification’s being at position three. Additional cross peak correlations were observed in the HMBC experiment from H-1 to C-3, C-5, and C-19. The above data proved that Compound 4 is a new ester of xestosterol-3-(l6′-bromo-7′E,11′E,l5′E-hexadeca-7′,11′,l5′-triene-5′,13′-diynoic acid) isolated here for the first time from a natural source. It is worth mentioning that xestosterol esters with brominated acetylenic fatty acid moieties have rare occurrences in marine sponges with few published reports [10,11,18].
Using a combination of 1D, 2D NMR spectra (Table 1 and Table 2) and HREIMS determinations and by comparing their spectral data with those in the literature, the known compounds were identified as xestosterol (24-methylene,26,27-dimethylcholest-5-en-3β-ol) (1) , xestosterol ester of 18′-bromo-9′E,17′E-octadecadiene-7′,15′-diynoic acid (3) , (5E,11E,15E,19E)-20-bromoeicosa-5,11,15,19-tetraene-9,17-diynoic acid (5) , 18,18-dibromo-9E-octadeca-9,17-diene-5,7-diynoic acid (6) , 18-bromooctadeca-9E,17E-diene-7,15-diynoic acid (7) , 18-bromooctadeca-9E,13E,17E-triene-7,15-diynoic acid (8) , l6-bromo-7E,11E,l5E-hexadeca-7,11,l5-triene-5,13-diynoic acid (9) , 2-methyl maleimide-5-oxime (10) , maleimide-5-oxime (11) , tetillapyrone (12) , and nortetillapyrone (13) (Figure 1) .
The isolated compounds were evaluated for their antitumor activity against three cancer cell lines—human hepatocellular carcinoma (HepG-2), human medulloblastoma (Daoy), and human cervical cancer (HeLa) cells—using an MTT assay, as described previously , using dasatinib as a positive reference drug. Five concentrations (0–50 µg/mL) of each compound were prepared and incubated with each cell line, and the survival fraction curves were obtained to calculate the concentration that produced 50% cell growth inhibition (IC50).
As shown in Table 3, the total ethanolic extract of X. testudinaria, n-hexane fraction, and Compound 6 exhibited broad spectrum inhibition of all tested cancer cell lines. In this context, the potency can be arranged in the following descending order: Compound 6 > n-hexane fraction > the total ethanolic extract on human cervical cancer (HeLa) and Daoy cells, while on HepG-2 cells, 6 > the total ethanolic extract > n-hexane fraction. Compounds 7 and 9 showed moderate selectivity against HeLa and Daoy cells. Compound 7 was more potent than 9, since the IC50 of 7 was 30.38 and 23.1 µg/mL on HeLa and Daoy cells, respectively, compared to 9, which exhibited IC50 values of 44.41 and 24.57 µg/mL on HeLa and Daoy cells, respectively. With regard to the sensitivity of cancer cells to the compounds, the Daoy cell line appeared to be the most sensitive towards the tested compounds, followed by HepG-2 and HeLa cell lines. Structure-activity relationships are proposed by comparing the IC50 values of the isolated brominated polyacetylenic fatty acids. The results suggest that the cytotoxic activity was dramatically enhanced by the presence of an extra bromine atom as in Compound 6.
With regard to the brominated compounds (5, 6, 7 and 9), the dibrominated compound (6) showed stronger cytotoxic activity than the monobrominated ones. On the other hand, the presence of a conjugated triple bond in Compound 6 may have contributed to the improved activity.
To write a narrative essay, you’ll need to tell a story (usually about something that happened to you) in such a way that he audience learns a lesson or gains insight.
To write a descriptive essay, you’ll need to describe a person, object, or event so vividly that the reader feels like he/she could reach out and touch it.
Tips for writing effective narrative and descriptive essays:
- Tell a story about a moment or event that means a lot to you--it will make it easier for you to tell the story in an interesting way!
- Get right to the action! Avoid long introductions and lengthy descriptions--especially at the beginning of your narrative.
- Make sure your story has a point! Describe what you learned from this experience.
- Use all five of your senses to describe the setting, characters, and the plot of your story. Don't be afraid to tell the story in your own voice. Nobody wants to read a story that sounds like a textbook!
How to Write Vivid Descriptions
Having trouble describing a person, object, or event for your narrative or descriptive essay? Try filling out this chart:
What do you smell?
What do you taste?
What do you see?
What do you hear?
What might you touch or feel?
Remember: Avoid simply telling us what something looks like--tell us how it tastes, smells, sounds, or feels!
- Virginia rain smells different from a California drizzle.
- A mountain breeze feels different from a sea breeze.
- We hear different things in one spot, depending on the time of day.
- You can “taste” things you’ve never eaten: how would sunscreen taste?
Using Concrete Details for Narratives
Effective narrative essays allow readers to visualize everything that's happening, in their minds. One way to make sure that this occurs is to use concrete, rather than abstract, details.
…makes the story or image seem clearer and more real to us.
...makes the story or image difficult to visualize.
…gives us information that we can easily grasp and perhaps empathize with.
…leaves your reader feeling empty, disconnected, and possibly confused.
The word “abstract” might remind you of modern art. An abstract painting, for example, does not normally contain recognizable objects. In other words, we can't look at the painting and immediately say "that's a house" or "that's a bowl of fruit." To the untrained eye, abstract art looks a bit like a child's finger-painting--just brightly colored splotches on a canvas.
Avoid abstract language—it won’t help the reader understand what you're trying to say!
Abstract: It was a nice day.
Concrete: The sun was shining and a slight breeze blew across my face.
Abstract: I liked writing poems, not essays.
Concrete: I liked writing short, rhythmic poems and hated rambling on about my thoughts in those four-page essays.
Abstract: Mr. Smith was a great teacher.
Concrete: Mr. Smith really knew how to help us turn our thoughts into good stories and essays.