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BY 4.0 license Open Access Published by De Gruyter Open Access April 27, 2023

Cytotoxic ketosteroids from the Red Sea soft coral Dendronephthya sp.

  • Mohammed A. Ghandourah EMAIL logo
From the journal Open Chemistry

Abstract

A marine specimen of the Red Sea soft coral Dendronephthya sp. was extracted with a mixture of n-hexane, diethyl ether, and methanol. One new cytotoxic steroid dendronestadione (1), five known steroids: dendronesterones A-C (24), dendrotriol (5), and cholesterol (6) along with 4-oxo-pentanoic acid (7) and a polyhydroxy alkane hexitol (8) were isolated from the Dendronephthya sp. extract. The chemical structures of the isolated metabolites were elucidated by the application of several spectroscopic techniques (1D, 2D NMR, IR, and UV) and mass spectrometry. The antiproliferative effect of the isolated compounds was assessed against a panel of human cancer cell lines including HepG2, HT-29, and PC. The obtained results indicated that compounds 14 (dendronesterones A–C) exhibited a higher cytotoxic effect than that of the other co-isolated ones. Among all examined dendronesterones, dendronesterone C showed the highest IC50 values of 19.1 ± 1.81, 32.4 ± 2.84, and 7.8 ± 0.80 µM against the three cancer cells under investigation. Interestingly, all isolated ketosteroids showed potent effects against prostate cancer cells. These findings highlight the role of ketosteroids as an antiproliferative agent against the examined cells in this study.

1 Introduction

The increasing evidence of human diseases like pathogenic bacteria or virus infection and cancer in the current decadal demand attention to discovering new treatment methods [1]. Chemotherapy, radiotherapy, and surgery are common treatment methods, while synthetic-antibiotic drugs are administered to cure microbial infection [2]. However, the administration of synthetic drugs shows side effects [3]. Natural products derived from plants or marine invertebrates have scientifically proven their merit with fewer side effects [4].

Soft corals inhabit competitive and extreme environments that produce secondary metabolites for their defense mechanism [5]. Until 2012, the metabolite from soft coral represented 22% of total new marine metabolites isolated from marine invertebrates [6]. Marine natural products from soft coral possess diverse molecular structures with pharmaceutical properties [7]. Many of them enter the preclinical and clinical studies due to their promising in vivo and in vitro evaluation [8]. The family of Xeniidae, Nephtheidae, Alcyoniidae, or Clavulariidae was the primary source of natural products [9]. Roughly 179 new steroids had been discovered from soft corals worldwide from 2015 to 2020, most of which belong to hydroxysteroids [10]. The noticeable bioactive activity from these newly discovered metabolites was anti-cancer, antibacterial, and anti-inflammatory [11].

Red Sea soft corals are prolific sources of secondary metabolites, including steroids, sesquiterpenoids, diterpenoids, triterpenoids, norterpenoids, ceramides, and several fatty acid derivatives [12,13,14,15]. Alcyonaceans of the genus Dendronephthya (family Nephtheidae) are common in the Indo-Pacific Ocean. Steroids are the most frequently isolated secondary metabolites from the species of Dendronephthya [16,17]. In this article, a specimen of Dendronephthya sp. was collected from the Red Sea waters of Jeddah city. The soft coral extract afforded one new cytotoxic steroid dendronestadione (1) along with seven known metabolites: dendronesterones A–C (24), dendrotriol (5), and cholesterol (6) along with 4-oxo-pentanoic acid (7) and the polyhydroxy alkane hexitol (8) (Figure 1).

Figure 1 
               Molecular structures of compounds 1–8 isolated from Dendronephthya sp.
Figure 1

Molecular structures of compounds 1–8 isolated from Dendronephthya sp.

2 Experimental

2.1 General

The instruments’ specifications and solvents and materials used in this article are detailed elsewhere [17].

2.2 Animal material

A sample of a soft bodied coral Dendronephthya sp. animal was collected in April 2019 off Jeddah city coast, Saudi Arabia. A voucher specimen (DS19-01) was deposited at the Marine biology department, Faculty of Marine Sciences, KAU.

2.3 Extraction and isolation

The squeeze-dried Dendronephthya sp. sample (900 g) was macerated in acetone at room temperature (three times, 24 h each). The extract was completely dried to provide 37.3 g of solvent-free oil residue. 15.0 g of the residue was well mixed with a suitable amount of normal phase column chromatographic Si gel powder. The homogenized sample-silica gel paste was poured on the top of 1 m silica gel column (3.5 cm diameter). The fractionation process started with a non-polar solvent (n-hexane) and the polarity was increased gradually by adding increased percentages of chloroform in pet. ether and the EtOAc in pet. ether. A total of 678 fractions (25 ml each) were obtained. TLC plates were visualized with the aid of UV lamp (UV254) and p-anisaldehyde-sulfuric acid reagent. PTLC glass plates were used for further purification. Chromatographic fractionation of the animal material was performed as previously described by Alassass et al. [15]. In brief, elution commenced with n-hexane, and then increased amounts of Et2O were added to elevate the polarity and then the polarity increased by EtOAc. The initial fraction eluted with n-hexane was repurified with PTLC (100% n-hexane), to give a pink spot up on spraying with sulfuric acid reagent. This zone was collected (1.1 mg) and assigned as 7. The fraction eluted with Et2O/n-hexane (1:4) was applied to PTLC using the same previous composition, the spot at R f 0.51 (reddish spot with sulfuric acid reagent) was collected (2.0 mg) and assigned as 6. The fraction eluted with EtOAc/n-hexane (1.5:8.5) was applied to PTLC (22% EtOAc in n-hexane), and the spots appeared at R f 0.73, 0.69, and 0.51 were collected and assigned to compounds 2 (1.0 mg), 3 (0.7 mg), and 4 (1.1 mg), respectively. The fraction eluted with EtOAc/n-hexane (1:4) was applied to PTLC (25% EtOAc in n-hexane), and the spot appeared at R f 0.66 was collected to give compound 1 (0.9 mg). The fraction eluted with EtOAc/n-hexane (1:3) was applied to PTLC (35% EtOAc in n-hexane), and the spot appeared at R f 0.49 was collected to give compound 5 (0.7 mg). The fraction eluted with MeOH/EtOAc/n-hexane (5:20:80) was applied to PTLC (15% MeOH in dichloromethane), and the spot appeared at R f 0.66 was collected to give compound 8 (0.9 mg).

2.4 Characterization of the isolated compounds

2.4.1 Dendronestadione (1)

Gummy residue; [α]D 22 + 12.3 (CHCl3, 0.02); UV (MeOH) λ max 238 and 226 nm; IR νmax 3,010, 2,930, 1,686, 1,675, 1,368, and 1,351 cm−1; HR-ESI-MS m/z 396.3109 [M+1]+(Calcd. for C27H41O2, 397.3107); 1H and 13C NMR (CHCl3) (Table 1).

Table 1

1H and 13C NMR (500 and 125 MHz, respectively) spectral data of compound 1 in CDCl3

C No. δ C δ H (J in Hz) C No. δ C δ H (J in Hz)
1 158.6 (CH) 7.16, dd (10.0) 16 27.9 (CH2) 1.68, m,
1.13, m
2 127.4 (CH) 5.85, d (10.0) 17 55.0 (CH) 1.66, m
3 200.3 18 12.1 (CH3) 0.85, s
4 41.1 (CH2) 2.37 dd (18.0, 14.0) 19 13.0 (CH3) 1.01, s
2.21 dd (18.0, 4.5)
5 44.4 (CH) 1.95, m 20 34.6 (CH) 1.95, m
6 28.5 (CH2) 1.73, m 21 18.5 (CH3) 1.02, d (7.0)
1.29, m
7 31.1 (CH2) 1.71, m 22 52.9 (CH2) 2.51, m
0.98, m 2.11, dd (15.0, 3.0)
8 35.6 (CH) 1.48, m 23 204.6 (CH2)
9 50.0 (CH) 1.00, m 24 125.2 (CH) 6.19, br s
10 38.9 (C) 25 156.7 (C)
11 21.1 (CH2) 1.80, m 26 21.0 (CH3) 2.13, s
1.53, m
12 39.8 (CH2) 2.07, m 27 27.5 (CH3) 1.91, d (6.8)
1.18, m
13 43.0 (C)
14 55.8 (CH) 1.19, m
15 24.6 (CH2) 1.57, m
1.14, m

Compounds 2–8 were characterized and identified by comparing their spectral data with the previously reported data in the literature. Those compounds were identified dendronesterones A–C (24), dendrotriol (5), and cholesterol (6) along with, 4-oxo-pentanoic acid (7) and the polyhydroxy alkane hexitol (8) [16,18].

2.5 Biological activities

2.5.1 Cytotoxic assay

HepG-2, HT-29, and PC-3 (human hepatocellular carcinoma, colorectal carcinoma, and prostate carcinoma) cells were obtained from American Type Culture Collection (ATCC) and cultured in RPMI1640 medium (Gibco, USA). The cytotoxicity test was carried out according to Mosdam [19].

3 Results and discussion

Sequential chromatographic fractionation and purification of the organic extract of the Red Sea soft coral resulted in the identification of eight secondary metabolites; six of them belong to steroids with C-27 and C-28 carbocyclic skeletons and the remaining compounds belong open chain alkanes.

Compound 1 was isolated as an optically active gummy substance. Its thin layer chromatography profile exhibited a positive response for steroids up on spraying with p-anisaldehyde-sulfuric acid reagent (blue turned brown color). Compound 1 showed absorption bands at 238 and 226 nm in the UV spectrum, indicating the presence of two a,b-unsaturated carbonyl functions. The molecular formula was found to be C27H40O2 from HRESIMS (requiring eight unsaturation sites) supported by twenty-seven signals appeared in the 13C NMR spectrum. No absorption due to hydroxyl function was found in the IR absorption spectrum; however, absorption bands due a,b-unsaturated carbonyls (1,686 and 1,675 cm−1) and gem-dimethyl (1,368 cm−1) were observed. The 1H NMR spectrum indicated the presence of one signal of secondary methyl protons resonating at δ H 1.02 ppm, four tertiary methyl protons resonating at δ H 0.85, 1.01, 1.91, and 2.13 ppm, and three olefinic protons resonating at δ H 7.16, 6.19, and 5.85 ppm (Table 1). The methylation pattern, the carbon number signals, the unsaturation degrees, and previous publications from the same genus call to the mind the probability of a steroid with a cholestane skeleton. The 13C NMR spectrum of 1 showed the presence of signals due to carbonyl functions resonating at δ C 204.6 and 200.3 ppm, four olefinic carbon signals resonating at 158.6, 156.7, 127.4, and 125.2 ppm. DEPT NMR experiments showed the presence of five unprotonated, nine methine, eight methylene, and five methyl carbons. HSQC NMR enabled the direct connection between all carbons and protons. Therefore, compound 1 contains no protonated hetero atoms. The presence of two carbonyls and two carbon–carbon double bonds accounted for three sites of unsaturation, which pointed out to the presence of a tetracyclic carbo-structure. The 13C NMR signals at δ C (204.6, 200.3), along with absorption bands in the IR and UV spectra suggested the presence of separate a,b-unsaturated carbonyls. 1H–1H COSY spectrum showed a distinct proton sequence through the correlation observed between H-1 (δ H 7.17) and H-2 (5.85). Other proton sequences were observed and clarified in Figure 2. The HMBC correlations observed from H-1 to the carbonyl carbon at 200.3 (C-3), 38.9 (C-10), and 13.0 (C-19) established the location of an a,b-unsaturated carbonyl function (C-1–C-3). The correlations observed from H-24 to the carbonyl carbon at 204.6 (C-23), and the gem-dimethyl function signals at 156.7, 27.5, and 21.0 established the presence of the second a,b-unsaturated carbonyl moiety at the forked tail (side chain). With the aid of 1H–1H COSY and HMBC NMR, the gross structure of 1 was concluded to be a cholestane skeleton with two carbonyl functions at C-3 and C-23 and two carbon–carbon double bonds at C-1 and C-24. A literature survey of chemical structures isolated from the genus Dendronephthya revealed that compound 1 was not previously isolated metabolite and was identified as dendronestadione (1).

Figure 2 
               Selected 1H–1H COSY and HMBC correlations of compounds 1 and 2.
Figure 2

Selected 1H–1H COSY and HMBC correlations of compounds 1 and 2.

Compound 2 was isolated as an optically active substance. It gave a positive response for steroids up on spraying with p-anisaldehyde-sulfuric acid reagent (blue turned brown color) on thin layer chromatography. Compound 2 showed an absorption band at 226 nm in the UV spectrum, indicating the presence of a,b-unsaturated carbonyl function. The molecular formula was found to be C27H42O, from HREIMS (required seven unsaturation sites) supported by 27 signals appeared in the 13C NMR spectrum. No absorption due to hydroxyl function was found in the IR absorption spectrum; however, absorption bands due a,b-unsaturated carbonyl (1,688 cm−1), carbon–carbon double bond (1,655 cm−1), and gem-dimethyl (1,360 cm−1) were observed. The 1H NMR spectrum indicated the presence of three secondary methyl protons resonating at δ H 1.00, 0.87, and 0.85 ppm, two tertiary methyl protons resonating at δ H 1.01 and 1.00 ppm, and four olefinic protons resonating at δ H 7.16, 5.85, 5.22, and 5.28 ppm. The 13C NMR spectrum of 2 suggested the presence of a,b-unsaturated ketone (δ C 200.6, 159.0, and 127.4) and carbon–carbon double bond (δ H 138.0 and 126.5). DEPT NMR experiments showed the presence of three unprotonated, eleven methine, eight methylene, and five methyl carbons. HSQC NMR enabled the direct connection between carbons and protons. The presence of one carbonyl and two carbon–carbon double bonds accounted for three sites of unsaturation which pointed to the presence of a tetracyclic carbo-structure. With the aid of 1H–1H COSY and HMBC NMR, the gross structure of 2 was concluded to be a cholestane skeleton with a carbonyl function at C-3 and two carbon–carbon double bonds at C-1 and C-22. The geometry of the C-22–C-23 was proved to be trans-geometry due to the large coupling constant (J = 15.3 Hz). A literature survey of chemical structures isolated from the genus Dendronephthya revealed that compound 2 was previously isolated from Dendronephthya gigantea and was identified as dendronesterone A [16]. Compounds 3, 4, 7, and 8 were identified by the comparison of their spectral data with those reported in the literature [18]. Compound 5 was identified by the comparison of its spectral data with that reported in the literature [18].

In this work, eight compounds were isolated from the Res Sea animal, Dendronephthya sp. The antiproliferative action of these metabolites was investigated against a panel of three cancer cell lines, HepG2, HT-29, and PC. Compound 1 showed the highest cytotoxic effects, where it had IC50 values of 19.1 ± 1.81, 32.4 ± 2.84, and 7.8 ± 0.80 µM in the treatment of HepG2, HT-29, and PC-3 cells respectively. Compound 2 showed good cytotoxic activity against PC-3 cells with an IC50 value of 43.7 ± 3.12 µM. By contrast to compounds 2 and 3, compound 4 showed reasonable activity against all examined cells (Table 2). The data in Table 2 strictly clarified the following points: all examined steroids are more active toward PC-3 cells; decoration of the side chain with α,β-unsaturated ketone might affect the bioactivity exerted by the examined steroids; ketosteroids are more cytotoxic than the co-isolated fatty acid derivatives and the 3-hydroxy steroids.

Table 2

Cytotoxicity of compounds 18 isolated from Dendronephthya sp.

Compound No. IC50 (µM)*
HepG2 HT-29 PC-3
1 19.1 ± 1.81 32.4 ± 2.84 7.8 ± 0.80
2 89.0 ± 4.22 >100 43.7 ± 3.12
3 >100 >100 91.0 ± 6.44
4 81.0 ± 7.91 94.1 ± 9.00 66.7 ± 6.75
5 >100 >100 >100
6 >100 >100 >100
7 >100 >100 >100
8 >100 >100 >100
Doxorubicin 1.9 ± 0.03 1.8 ± 0.01 3.1 ± 0.15

*Four replicates were used for each treatment. Doxorubicin = positive control.

4 Conclusion

The Red Sea soft coral animal Dendronephthya sp. is a rich source of the ketosteroids of C-27 and C-28 carbocyclic skeletons in addition to keto-fatty acids. All ketosteroids showed antiproliferative effects with varying degrees of activity. Compound 1, which was characterized by the presence of two α,β-unsaturated carbonyls, showed the highest potency among all compounds. It is worth noting that the maximum antiproliferative effect of all ketosteroids was observed against prostate adenocarcinoma (PC-3) cells.

Acknowledgments

The authors gratefully acknowledge technical and financial support from the Ministry of Education and King Abdulaziz University, Jeddah, Saudi Arabia. The author is extremely grateful to Dr. Magda M. Ali (Department of Biology, Faculty of Science, King Abdulaziz University) for the support during conducting the biological work.

  1. Funding information: This research work was funded by Institutional Fund Projects under grant no IFPRC-046-150-2020.

  2. Author contributions: The author confirms sole responsibility for the following: study conception and design, data collection, analysis and interpretation of results, and article preparation.

  3. Conflict of interest: This article does not have any conflict of interest. The corresponding author approves the above statement as well.

  4. Ethical approval: The conducted research is not related to either human or animal use.

  5. Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Received: 2023-03-16
Revised: 2023-04-05
Accepted: 2023-04-12
Published Online: 2023-04-27

© 2023 the author(s), published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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