The current work aims to isolate the bioactive secondary metabolites from the Red Sea green alga T. expeditionis. Its organic extract was partitioned and analyzed using chromatographic and spectroscopic techniques. Four triterpenoids of the cycloartane-carbon skeleton were identified as: 29-norcycloartane-3-en-23,28-diol (1), 29-norcycloartane-5,24-dien-3-ol-23-one (2), 29-norcycloartane-3,24-dien-3-ol-23-one (3), and 29-norcycloartane-5,24-dien-3-ol (4), along with hydroxylated C-18 fatty acid, 3-hydroxyoctadeca-15(Z)-enoic acid (5). The antiproliferative activity of the isolated metabolites was examined against three cancer cell lines, i.e., HeLa, HepG-2, and MCF-7. Compounds 2 and 3 demonstrated a strong antiproliferative effect against all cells with IC50 values ranging from 17.8 ± 1.71 to 23.3 ± 1.66 µM. Compounds 1 and 4 showed a moderate antiproliferative effect against all cell lines with IC50 values ranging from 44.7 ± 2.32 to 65.0 ± 3.66 µM. The antifungal activity of all compounds has been tested against several fungi. Compounds 2–4 revealed strong inhibition against A. flavus and Fusarium oxysporum. Compounds 1–4 showed moderate to weak inhibition activity against A. niger, A. fumigatus, C. albicans, and C. tropicalis. Compound 5 showed weak to non-detected activity against all cell lines and microbes. The results indicated that norcycloartanes exhibit antiproliferative and antifungal activities, especially those with α,β-unsaturated ketones in their side chain.
In the last decade, marine natural product specialists have greatly enhanced the search for new pharmaceutical armamentariums to reserve a distinguished position among today’s drugs. Marine algae continue to act as suppliers of astonishing natural organic substances that possess potential therapeutic effects [1,2]. Based on recently reported surveys, more than 72,000 species of macro-algae worldwide have been identified. The marine macro-algae, including Chlorophyta (green algae), Phaeophyta (brown algae), and Rhodophyta (red alga), constitute the most significant proportion of the total macro-algae on the earth .
As a matter of fact, marine algae are affected by harsh environmental circumstances, including but not limited to temperature, salinity, sunlight, and physiological conditions . Despite various environmental conditions, marine algae can survive and grow through different adaptation strategies. This adaptive nature of marine macro-algae has enabled them to produce exclusive chemical skeletons such as polyketides, polysaccharides, tannins, steroids, terpenoids, shikimates, and alkaloids [3,5]. Chlorophytin seaweeds or marine green algae are common in intertidal and deep waters. Tydemania expeditionis (Udoteaceae) is a feeble calcium carbonate forming green alga, spread among tropical Indian and Pacific waters .
Phytochemical exploration of Tydemania expeditionis collected from Guam, the US territory, yielded three norcycloartene triterpenoids  and a linear diterpenoid . A specimen of the same algal species collected from Micronesia was investigated and was found to contain cycloartanol sulfates . Four cycloartenol sulfates and three β-hydroxyl unsaturated fatty acids were isolated from a sample of T. expeditionis collected in Fiji . A study conducted by Zhang et al.  on a specimen collected in the China Sea revealed the isolation of several steroids, including a new diketosteroid, (E)-stigmasta-24(28)-en-3,6-dione . The isolated new ketosteroid displayed remarkable affinity to the androgen receptor and unexpected weak activity to prostate cancer . Sulfated cycloartenols exhibited antifungal activities  and inhibited the pp60v-src protein tyrosine kinase . Antineoplastic fatty acid derivatives were also identified amongst the constituents of the Fijian green alga T. expeditionis; of these derivatives, polyunsaturated fatty acids (PUFA) of C16 and C18 carbon skeleton along with sphingolipids were isolated . In line with a continued project aiming at the isolation of bioactive agents from the Red Sea macro-algae [13,14,15], a specimen of T. expeditionis collected in the Red Sea of Jeddah coast was phytochemically investigated for the isolation of new chemicals and/or identification of new biological effects of previously isolated compounds.
2 Experimental method
The column chromatography utilized silica gel of 60 mesh size (Fluka). The Thin-layer chromatography (TLC) was silica gel 60 mesh F 254 (Merck). The Preparative thin-layer chromatography (PTLC) was pre-coated glass plates (MACHEREY_NAGEL) G-25 UV254. The visualization employed a spray reagent (50% sulfuric acid in ethanol and p-anisaldehyde) and UV light. The chemicals used were crystal violet stain (1%); dimethyl sulfoxide (DMSO); MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye from Sigma (St. Louis, MO, USA); fetal bovine serum, DMEM, RPMI 1640, HEPES buffer solution, l-glutamine, gentamycin, and 0.25% Trypsin-EDTA from Lonza.
2.2 Extraction of the green alga Tydemania expeditionis
A specimen of T. expeditionis (Figure 1) was collected in June 2021 from the Jeddah coast (20°42′52.36″N; 39°29′20.74″E), Jeddah, Saudi Arabia. The air-dried sample (181.0 g) was macerated with an organic solvent mixture (n-hexane:diethylether:methanol [1:1:1, v/v/v]). A dark green residue (7.7 g) was obtained after complete solvent evaporation of the total extract. The whole algal extract was mixed with a suitable amount of silica gel (60 G) and poured into an open silica gel column. The elution process was started with n-hexane and the polarity was gradually elevated by adding diethyl ether, ethyl acetate, and methanol.
2.3 Isolation of compounds 1–5
Fraction A eluted with n-hexane:diethyl ether (90:10, v/v) was purified using PTLC and solvent system 15% diethyl ether in n-hexane. A dark red band with R f = 0.57 visualized upon spraying with p-anisaldehyde was scraped, and the silica was filtered to yield 1.9 mg of gummy residue (compound 3). Fraction B eluted with n-hexane:diethyl ether (75:25, v/v) was purified using PTLC and solvent system 30% diethyl ether in n-hexane. Three bands were developed in different R f values. The first was a purple band with R f = 0.86 (visualization after spraying p-anisaldehyde reagent to afford a pure oily material, 2.4 mg), yielding compound 5. The second was a crimson-red band with R f = 0.34 (visualization p-anisaldehyde reagent to afford a brown residue, 2.0 mg), yielding compound 4. The third was a crimson-red band with R f = 0.22 (visualization with a UV lamp and p-anisaldehyde reagent to afford a brown residue, 2.0 mg), yielding compound 2. Fraction C eluted with n-hexane:diethyl ether (60:40, v/v) was purified using PTLC and solvent system with 25% ethyl acetate in n-hexane. A dark red band with R f = 0.37 visualized upon spraying with p-anisaldehyde was scraped, and the silica was filtered to yield 1.6 mg of gummy residue (compound 1).
2.4 Characterization of the isolated compounds
2.4.1 29-Norcycloartane-3-en-23,28-diol (1)
Gummy residue; [α]D 22 + 12.3 (CHCl3, 0.02); IR ν max 3,470, 3,398, 2,921, 1,661, and 1,376 cm−1; HR-ESI-MS m/z 429.3740 [M + 1]+ (Calcd for C29H49O2, 429.3733); 1H and 13C NMR (CHCl3) (Table 1).
|C No.||b δ C||δ H (J in Hz) c||C No.||δ C||δ H (J in Hz)|
|1||34.3 (CH2)||1.71, m||16||26.2 (CH2)||1.90, m|
|1.46, m||1.25, m|
|2||23.6 (CH2)||2.11, m||17||52.7 (CH)||1.58, m|
|3||121.8 (CH)||5.37, brs||18||18.3 (CH3)||0.97, s|
|4||138.1 (C)||—||19||22.2 (CH2)||0.48, d (4.4)|
|−0.7, d (4.4)|
|5||40.6 (CH)||2.28, m||20||32.8 (CH)||1.41, m|
|6||25.5 (CH2)||1.50, m||21||18.5 (CH3)||0.91, d (6.8)|
|7||27.8 (CH2)||1.65, m||22||44.9 (CH2)||1.49, m|
|1.35, m||0.94, m|
|8||43.0 (CH)||1.96, m||23||67.1 (CH)||3.77, m|
|9||21.7 (C)||—||24||48.1 (CH2)||1.34, m|
|10||29.5 (C)||—||25||24.8 (CH)||1.30, m|
|11||31.9 (CH2)||1.68, m||26||22.4 (CH3)||0.88, d (6.8)|
|12||34.1 (CH2)||1.30, m||27||23.2 (CH3)||0.88, d (6.8)|
|13||45.5 (C)||—||28||62.2 (CH2)||4.29, dd (11.6, 4.0)|
|4.14, dd (11.6, 6.6)|
|15||32.2 (CH2)||1.49, m|
aAll assignments are based on 1D and 2D measurements (HMBC, HSQC, COSY). bImplied multiplicities were determined by DEPT (C = s, CH = d, CH2 = t). c J in Hz.
2.4.2 3-Hydroxyoctadeca-15(Z)-enoic acid (5)
Colorless oil; [α]D 22 + 8.1 (CHCl3, 0.01); IR ν max 3,434, 2,961, 1,715, and 1,665 cm−1; HR-ESI-MS m/z 297.2425 [M–H]+ (Calcd for C18H34O3, 297.2430); 1H and 13C NMR (CHCl3) (Table 2).
|C no.||δ C||δ H (J in Hz)|
|18||13.6||0.93, t (6.8)|
2.5 Biological activities
2.5.1 Cytotoxic assay
HeLa (human cervix adenocarcinoma), HepG-2 (human hepatocellular carcinoma), and MCF-7 (human breast carcinoma) cells were obtained from American Type Culture Collection (ATCC) and cultured in RPMI 1640 medium, Gibco, USA. The cytotoxicity assay was performed according to Mosmann .
2.5.2 Antifungal assay
The agar well diffusion method was used to determine the sensitivity of the pathogenic fungi to the isolated compounds (1–5) . Over the surface of Mueller–Hinton agar, 0.5 mL of the bacterial suspension (4 × 106 CFU/mL) was spread, and agar wells (6 mm diameter) were filled with 50 µL of the tested material (10 µg/mL DMSO). On the surface of Sabouraud agar or potato dextrose agar, yeast cells or fungal spore suspensions containing 4 × 104 CFU/mL were spread. After an hour of incubation at room temperature, the inoculated plates were incubated for 24 h at 37°C for bacteria and 25°C for 4 days for fungi. Inhibition zone diameters were measured and the mean diameter of six replicates (mean value ± SD) was calculated. Amphotericin B was used as a positive control and DMSO was used as a negative control.
3 Results and discussion
Sequential chromatographic fractionation of the methylene chloride/methanol extract of the Red Sea green alga T. expeditionis yielded a total of five metabolites (1–5). Four of these metabolites belong to the cycloartane-type sesquiterpenoids, including compound 1: 29-norcycloartane-5,24-dien-3-ol-23-one (fraction C, a dark red band, R f = 0.37); compound 2: 29-norcycloartane-3,24-dien-3-ol-23-one (fraction B, a crimson-red band, R f = 0.22); compound 3: 29-norcycloartane-5-en-3,28-diol (fraction A, a dark red band, R f = 0.57); and compound 4: 29-norcycloartane-5,24-dien-3-ol (fraction B, a crimson-red band, R f = 0.34). Compound 5 belongs to the fatty acids, 3-hydroxyoctadeca-15(Z)-enoic acid (fraction B, a purple band, R f = 0.86). The identity of the isolated metabolites has been elucidated using different spectroscopic analyses in addition to mass spectrometry and comparison of the obtained data with those in the literature (Figure 2; Tables 1 and 2).
3.1 Structure elucidation
Compound 1 was isolated as a brown residue. Mass spectrometry showed a molecular ion [M + 1]+ at m/z 429.3733, assigned to a molecular formula of C29H48O2, with six sites of unsaturation. The IR spectrum (cm−1) showed bands due to hydroxyl function (3,470 and 3,398), carbon-carbon double bond (1,661), and gem-dimethyl (1,376). 13C NMR spectrum confirmed the presence of 29 carbon signals, 2 of them are present in the olefinic region (δ C 138.1 and 121.8 ppm), accounting for 1 degree of unsaturation. The presence of a pentacyclic carbo-structure in compound 1 was gleaned from the 13C NMR spectrum that showed 27 carbon signals appear below δ C 70.0 ppm. Distortionless enhancement by polarization transfer (DEPT) experiment characterized the carbon atoms in compound 1 into 5 methyls, 12 methylenes, 7 methines, and 5 non-protonated carbons. 1H NMR spectrum exhibited three secondary methyl groups resonating at δ H 0.91, 0.88, and 0.88 ppm and 2 tertiary methyls resonating at δ H 0.97 and 0.87 ppm. 1H, 13C NMR, and heteronuclear single quantum coherence (HSQC) experiments clarified the presence of an oxygenated methine (δ H/δ C: 3.77/67.1), an oxygenated methylene group (4.29 and 4.14/62.1), and a cyclopropyl methylene group resonating in the up-field region (0.48 and −0.07/22.2). Literature surveys on the constituents of this green alga species, Tydemania expeditionis, revealed cycloartane-type triterpenoids [7,9], diterpenoids , and fatty acid derivatives as frequently encountered constituents . All the previous information favors the presence of a pentacyclic nor-triterpenoid carbon skeleton. 1H–1H correlation spectroscopy (COSY) confirmed the presence of an isopropyl function (proton signal resonating at δ H 1.30 ppm is correlated with signal due to 2 methyls resonating at δ H 0.88 ppm. The same methine proton (δ H 1.30 ppm) is connected with the methylene protons resonating at δ H 1.34 and 1.12 ppm which are, in turn, correlated with the oxygenated methine proton resonating at δ H 3.77 ppm. The former methine proton is linked with other methylene protons resonating at δ H 1.48 and 0.91 ppm, which are correlated with the methine proton resonating at δ H 1.70 ppm. This methine proton is correlated with a methyl proton resonating at δ H 0.91 ppm. From the previous discussion, along with the heteronuclear multiple bond correlation (HMBC) spectrum’s investigation and the published data in the literature [7,9], the side chain was concluded to be (CH3)2CHCH2CHOHCH2CHCH3– (Figure 1). The location of the side chain was deduced from the COSY correlation between proton resonating at δ H 1.70 ppm and that at δ H 1.58 ppm. The HMBC correlations show a linkage between the latter proton and the 2 quaternary carbons resonating at δ C 45.5 and 49.5 ppm and the methyl carbons at δ C 16.5, 18.3, and 18.5 ppm. The HMBC correlations observed between the low-frequency protons resonating at δ H 0.48 and −0.07 ppm and the quaternary carbon signals (δ C 29.5 and 21.7 ppm), the methine carbon signals (δ C 43.0 and 40.6 ppm), and the methylene carbon signals (δ C 34.3 and 31.9 ppm) unambiguously the location of the cyclopropyl group between C-9–C-10–C-19. The location of the second hydroxy function and the trisubstituted double bond were deduced from the HMBC correlation observed between the oxygenated methylene protons resonating at δ H 4.29 and 4.13 ppm and the carbon signals at δ C 138.1, 121.1, and 40.6 ppm. The previous discussion indicated the chemical structure of compound 1 as 29-norcycloartane-3-en-23,28-diol.
Compound 5 was isolated as colorless oily material. The molecular formula was established as C18H34O3 from the quasi-molecular ion [M–H] peak at m/z 297.2425 in the HRESIMS analysis. The obtained molecular formula suggested 2 sites of unsaturation. The IR spectrum revealed the presence of hydroxyl function (3,434 cm−1), CH (2,961 cm−1), acid carbonyl (1,715 cm−1), and a carbon-carbon double bond (1,665 cm−1). 13C NMR spectrum confirmed the presence of 18 carbon signals, 2 of them are present in the olefinic region (δ C 133.5 and 124.3 ppm), and a carboxyl group (δ C 178.3) accounted for the 2 degrees of unsaturation. The presence of a long-chain fatty acid 5 was gleaned from the 13C NMR spectrum that showed 18 carbon signals. Hence, the DEPT experiment characterized the carbon atoms in compound 5 into 1 methyl, 14 methylene carbons, 3 methine carbons including an oxygenated methine carbon resonating at δ C 70.6 ppm and a disubstituted carbon-carbon double bond, and a non-protonated carbon. 1H NMR spectrum exhibited 1 primary methyl group resonating at δ C 0.93 ppm and 2 olefinic protons resonating at δ C 5.34 and 5.10 ppm. 1H, 13C NMR, and HSQC experiments clarified the presence of an oxygenated methine (δ H/δ C: 4.10/70.6), a disubstituted carbon-carbon double bond, and a primary methyl (δ H/δ C: 0.93/13.6). From the previous discussion, compound 5 is a long-chain fatty acid of 18 carbons decorated with 1 carbon–carbon double bond and a secondary hydroxyl function. The location of these function groups was assigned from both 1H–1H COSY and HMBC spectra, where the highest field proton signal δ H 5.34 ppm is correlated with the methylene protons resonating at δ H 2.10 ppm, which are in turn correlated with the methyl function. These correlations and that in the HMBC between H (δ H 5.34 ppm) and carbon signals resonating at δ C 20.7 and 13.6 ppm confirmed the location of the double bond at C15═C16. A similar combination between 1H–1H COSY and HMBC spectra located the hydroxyl function in the b-position to the carboxyl function. From the previous position, the chemical structure of compound 5 can be concluded and identified as 3-hydroxyoctadeca-15(Z)-enoic acid (Figure 2).
The other isolated compounds were identified as 29-norcycloartane-5,24-dien-3-ol-23-one (2), 29-norcycloartane-3,24-dien-3-ol-23-one (3), and 29-norcycloartane-5,24-dien-3-ol (4), after careful comparison of their spectral data with those in the literature [7,9].
3.2 Biological activities
The antiproliferative activity of the isolated metabolites was examined against three cancer cell lines: HeLa, HepG-2, and MCF-7. Compounds 2 and 3 demonstrated a robust antiproliferative effect against all cell lines with IC50 values ranging from 17.8 ± 1.71 to 23.3 ± 1.66 µM. Compounds 1 and 4 showed a moderate antiproliferative effect against all cell lines with IC50 values ranging from 25.6 ± 2.45 to 42.7 ± 3.32 µM. Compound 5 showed a weak cytotoxic effect against all cell lines (Table 3).
|Compound no.||IC50 (µM)|
|1||30.9 ± 2.85||42.7 ± 3.32||30.7 ± 2.67|
|2||17.8 ± 1.71||19.5 ± 1.44||23.1 ± 1.66|
|3||20.4 ± 1.78||23.1 ± 2.00||18.0 ± 1.74|
|4||31.9 ± 2.23||42.0 ± 3.66||25.6 ± 2.45|
|5||>50.0||44.2 ± 4.04||>50.0|
|Doxorubicin||1.9 ± 0.04||1.8 ± 0.01||3.1 ± 0.15|
Human cervix adenocarcinoma (HeLa), hepatocellular carcinoma (HepG-2), and breast adenocarcinoma (MCF-7).
Data are presented as mean value ± SD; n = 6.
The antifungal activity of all isolated compounds has been tested against several fungi (Aspergillus niger, A. fumigatus, A. flavus, Candida albicans, C. tropicalis, and Fusarium oxysporum). Compounds 2–4 revealed potent inhibition against A. flavus and Fusarium oxysporum. Compounds 1–4 showed moderate to weak inhibition activity against A. niger, A. fumigatus, Candida albicans, and C. tropicalis. Compound 5 showed weak to non-detected activity against all microbes (Table 4).
|Aspergillus flavus||A. fumigatus||A. niger||Fusarium oxysporum||Candida albicans||C. tropicalis|
|1||11 ± 0.91||9 ± 1.00||8 ± 0.59||10 ± 0.96||8 ± 0.80||9 ± 0.76|
|2||14 ± 1.11||12 ± 1.13||11 ± 1.06||16 ± 1.43||11 ± 1.01||10 ± 0.97|
|3||11 ± 0.98||9 ± 0.66||7 ± 0.54||12 ± 1.08||10 ± 0.86||9 ± 0.78|
|4||15 ± 1.17||13 ± 1.00||13 ± 1.18||17 ± 1.56||12 ± 1.05||7 ± 0.68|
|5||7 ± 0.67||ND||ND||6 ± 0.51||ND||ND|
|Amphotricin B||29 ± 1.93||32 ± 2.38||34 ± 2.71||26 ± 2.28||28 ± 2.56||29 ± 2.64|
Amphotricin B: is a standard drug.
ND: not detected.
Several literature articles clarified the role of metabolites isolated from the orders Caulerpales and Bryopsidales abundant in tropical and subtropical waters [18,19]. The tropical and subtropical waters are suitable marine environments for the growth of T. expeditionis and seaweed predators such as sea urchins and fishes. It is believed that the defense adoption of this green alga basically depends on the excretion of chemicals. Sulfated cycloartane-type triterpenes showed good inhibitory activity against VZV and CMV protease enzymes , anti-inflammatory , cytotoxicity, and antifungal activity [6,10]. Interestingly, desulfated, 29-nor, saponin-types cycloartane triterpenes, in some instances, showed even higher biological effects than the corresponding sulfated compounds . Stigmastane-type steroids also contribute to the activity of T. expeditionis, (E)-stigmasta-24(28)-en-3,6-dione showed potent affinity to the androgen receptor with an IC50 value of 7.19 ± 0.45 μM. In the current work, all isolated metabolites showed cytotoxic effects against the examined cell lines, the most potent cytotoxic compounds are 2 and 3. The main apparent difference between these two compounds and the others is the presence of α,β-unsaturated carbonyl function.
Terpenoids are one of the largest class of secondary metabolites with great potency for anticancer candidates, some of which are ready to enter the clinical trial, like mipsagargin (G-202) and thapsigargin . A complex mechanism is involved in the anticancer properties of cycloartane-type terpenoids. The variety of anticancer activity of terpenoids is affected by the diversity of attached functional groups in the side chain. For instance, the sulfation pattern in the glycon unit and the presence of sugar moiety of triterpenoid from various sea cucumbers determine the differences in cytotoxic properties against a wide range of cancer cells . Meanwhile, the replacement of different functional group at C-26 of cycloartane-type triterpenoids from Tetragonula sapiens Cockerell significantly affects their α-inhibitory properties . The antiproliferation of cycloartane-type triterpenes, like 27-hydroxymangiferolic acid, 23-hydroxymangiferolic acid, and mangiferolic acid from Trigona minor, against human pancreatic cancer cells PANC-1 is significantly affected by the presence of the –OH group at C-23 and C-27 . Apart from cytotoxicity and antiproliferation, the cycloartane-type triterpenoid like cycloartan-24-ene-1α,2α,3β-triol from Commiphora myrrha is proven for its apoptosis activity against prostatic cancer PC-3 cells. The anticancer activity involves the upregulation of caspase-3, p53, Bcl-2-associated X protein (Bax), and B-cell lymphoma 2 (Bcl-2) in the cancer cell .
The antifungal activity of all isolated compounds has been tested against several fungi. Compounds 2–4 revealed strong inhibition against A. flavus and Fusarium oxysporum. Compounds 1–4 showed moderate to weak inhibition activity against A. niger, A. fumigatus, C. albicans, and C. tropicalis. Compound 5 showed weak to non-detected activity against all cell lines and microbes. The results indicated that norcycloartanes exhibit antifungal–antiproliferative activities, especially those with a,b-unsaturated ketones in their side chain. Like the famous antifungal drugs carvacrol, other terpenoids may alter Ca2+ transients, vacuolar and cytosolic pH, and metabolic activity of the fungi .
The Red Sea green alga Tydemania expeditionis is a rich source of the norcycloartane-type triterpenoids and omega-3 fatty acids. The compounds isolated from this alga exhibited antiproliferative-antifungal activity. Compounds 2 and 3 showed a strong antiproliferative activity against HeLa, HepG-2, and MCF-7 cell lines with IC50 values ranging from 17.8 ± 1.71 to 23.3 ± 1.66 µM. Compounds 2–4 demonstrated a strong inhibition against A. flavus and Fusarium oxysporum. It is expected that the whole alga might possess nutritional value due to the presence of ω-3 fatty acids.
The author is extremely grateful to Dr Serag Eldin I. Elbehairi (Department of Biology, Faculty of Science, King Khalid University) for the support while conducting the biological work.
Funding information: This research received no external funding.
Author contributions: All the work described in the present article was performed in its entirety by HA, as the sole author.
Conflict of interest: The author states no competing interest.
Ethical approval: The conducted research is not related to either human or animal use.
Data availability statement: All data generated or analyzed during this study are included in this published article (and its supplementary information files).
Sample availability: Samples of the compounds are available from the author.
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