Isolation, antimicrobial and antitumor activities of a new polyhydroxysteroid and a new diterpenoid from the soft coral Xenia umbellata

1 Introduction

In recent years, the search for and use of dietary supplements and drugs derived from marine organisms have accelerated. Marine animals, including soft corals, are known to provide natural compounds with great significance in medicinal applications [1], [2]. Soft corals of the genus Xenia (Alcyoniidae) are a proliferative source of bioactive diterpenoids having a nine-membered ring structure [3], [4], [5], [6]. Five groups of this class of diterpenoids were differentiated: xenicins, xeniolides, xeniaphyllanes, xeniaethers and azamilides [7]. These terpenoid compounds showed appreciable antitumor, antibacterial, and antifungal activities [5], [6], [7], [8].

In the course of our research program aimed at the discovery of bioactive and/or new metabolites from marine organisms, the constituents of the shallow water Red Sea soft coral Xenia umbellata Lamarck (Family Alcyoniidae, Order Alcyonacea, subclass Octocorallia, class Anthozoa, phylum Coelenterata) were investigated.

2 Experimental

3 Results and discussion

The CHCl₃-soluble fraction of the CH₂Cl₂/MeOH extract of the soft coral X. umbellate was fractionated on a silica gel column. Similar fractions were combined and purified using PTLC.

Two new metabolites from this marine organism were identified: the polyhydroxysteroid 3β-,5α-,6β-,11α-, 20β-pentahydroxygorgosterol (1) and the diterpenoid xeniumbellal (2), which were isolated along with three known sesquiterpenes (3–5) (Figure 1).

Figure 1:

Compounds isolated from the soft coral Xenia umbellate.

Compound 1 was isolated as a white powder and was assigned the molecular composition C₃₀H₅₂O₅ indicated by mass measurement (HREIMS m/z 492.3804, calculated 492.3815), implying five unsaturation sites. The consecutive appearance of fragment ions separated by 18 mass units from the parent peak indicates the existence of hydroxyl functions. The IR absorption spectrum showed a broad band at 3392 cm⁻¹, supporting the presence of the hydroxyl function. The ¹³C NMR spectra of 1indicated resonances for 30 carbons, categorized by a DEPT NMR experiment into 7 CH₃, 9 CH₂, 10 CH and four non-protonated carbons (Table 1).

No signals were observed in the downfield region of the ¹³C NMR spectrum, which favored the presence of a pentacyclic carbon skeleton. The ¹H and ¹³C NMR spectra showed the presence of seven methyl groups; three doublets resonating at δ_H/δ_C 0.95/22.4, δ_H/δ_C 0.95/15.9, and δ_H/δ_C 0.85/21.0, and four singlets resonating at δ_H/δ_C 1.25/17.3, δ_H/δ_C 1.02/21.5, δ_H/δ_C 0.91/14.5, and δ_H/δ_C 0.68/13.4. Such a methylation pattern can be attributed to a steroidal nucleus [18]. The ¹H NMR spectrum exhibited signals in its upfield region (δ_H=−0.1 to 0.43 ppm), which were unambiguously ascribed to a cyclopropane-bearing gorgosterol-type side chain resonating at δ_H 0.43 (dd, 9.0, 5.4 Hz), δ_H 0.19 (dd, 11.4, 9.6) and δ_H −0.10 (dd, 9.0, 4.8). This information, together with the methylation pattern and several other features appearing in the 1D and 2D NMR spectra (Table 1), suggested a gorgosterol-type sterol. The presence and nature of five hydroxyl functionalities were revealed from ¹³C NMR, DEPT and HSQC spectra, where signals due to five oxygenated carbons [δ_H 79.0 (s), 76.6 (s), 76.3 (d), 68.6 (d), and 67.3 (d)] were noticed. The methine proton resonating at δ_H 3.97 (dddd, 10.8, 10.8, 6.6, and 4.8 Hz) should be located at C-3, since this is the only available position flanked by two methylene groups. H-H COSY and HSQC spectra established the positions of the other hydroxyl groups; H-3 is correlated with the quaternary carbon at δ_C 79.0, and this carbon is also correlated with the proton resonating at δ_H 3.48 (brs), which implies that positions 5 and 6 are both hydroxylated (Figure 2). The position of the fourth hydroxyl group was concluded by observing the proton resonating at δ_H 3.81, which appeared as doublet of doublet of doublets with J values of 10.8, 10.8, and 10.8 Hz. This proton could be located at several positions within the carbon skeleton, but owing to the HMBC correlations (Figure 2) between this proton and the two quaternary carbons assigned to C-10 and C-13, we were able to locate the OH group at C-11.

Figure 2:

Selected ¹H-¹H COSY and HMBC correlations of 1 and 2.

The position of the fifth OH group was deduced to be at C-20 from the HMBC correlations between the upfield protons assigned to the cyclopropanyl moiety and the signal at δ_C 76.6 together with the disappearance of the signal due to Me-21 frequently encountered in steroids. These conclusions were further supported by comparison of our data with those in the literature [19]. The stereochemistry of the hydroxyl groups was deduced from the values of the coupling constant and also from the NOE correlations with the well-established β−orientation of the two methyls Me-18 and Me-19, to be 3β-,5α-,6β-,11α-, 20β- (Figure 3). Hence, compound 1 can be assigned as 3β-,5α-,6β-,11α-,20β-pentahydroxygorgosterol.

Figure 3:

Selected NOESY correlations of 1 and 2.

Compound 2 was isolated as an optically active colorless oil [a]_D²¹+51 (c 0.05, CHCl₃). It was assigned the molecular composition C₂₀H₂₈O₅ indicated by mass measurement (HREIMS m/z 348.1927, calculated 348.1937), implying seven unsaturation sites. The IR spectrum showed absorption bands due to hydroxyl (3440 cm⁻¹), conjugated aldehyde (1699, 1635 cm⁻¹) and epoxide (1264 cm⁻¹) functions, respectively. The absorption band at λ_max 236 nm in the UV spectrum referred to a conjugated carbonyl group. Signals due to 20 carbons appearing in the ¹³C NMR spectra of 2 were categorized, by applying DEPT technique, into 2 CH₃, 6 CH₂, 8 CH, and four quaternary carbons (Table 1). ¹H and ¹³C NMR spectra, together with a HSQC experiment, revealed the presence of the following characters: an aldehyde group [δ_H/δ_C 9.43/195.0], six oxygenated carbons [δ_C: 55.7 (s), 58.4 (d), 59.2 (d), 63.2 (t), and 71.5 (s)] an exocyclic double bond [δ_H/δ_C 5.34, 5.33/120.0, and 143.7 (s)], an α, β, γ, δ-unsaturated aldehyde [δ_H/δ_C: 9.43/195.0, 141.8 (s), 6.95 (d, 11.4)/152.7, 7.10 (dd, 15, 11.4)/122.4, and 6.39 (d, 15)/122.5], a disubstituted epoxy [δ_H/δ_C: 2.95/58.4 and 3.20/58.4], another disubstituted epoxy [δ_H/δ_C; 3.48, 3.17/54.4 and 55.7 (s)], and a carbinol group (CH₂OH) [δ_H/δ_C; 3.23, 3.07/63.2], respectively. All this information suggested the presence of a nine-membered ring structure that is frequently found in this genus of soft corals.

H-H COSY and HMBC spectra (Figure 2) established the connections: C-3/C-4/C-12/C-13/C-14/C-15/C-16(C-17), C-1/C-4a/C-4/C-5/C-6, and C-8/C-9/C-10. The structure of 2 was well supported by comparison of its spectral data with those in literature [3], [4], [5], [6], and it was given the trivial name xeniumbellal (Figure 1) as a new natural product. After establishing the gross structure of compound 2, the relative stereochemistry of 2 was determined by the analysis of the coupling constant (J) values, by comparison of its spectroscopic data to those of Xenia diterpenes and from the NOESY cross-peaks (Figure 3). A cis-configuration was assigned to the epoxy moiety 8(9) due to the small measured (4.2 Hz) coupling constant between them. A trans-configuration for H-4a and H-11a was concluded due to the large J (11.4 Hz) observed between them. The E-geometry was assigned to the ∆^4,12 double bond on the basis of the NOE cross-peaks observed between H-3 and H-12 and between H-13 and H-4a. The E-geometry of the ∆¹³ double bond was established by the large coupling constant (J=15 Hz) observed between H-13 and H-14. The suggested structure of the epimeric 7(18) epoxide was deduced based on NOESY cross-peaks (Figure 3) and by comparison of its spectral data with those of the similar diepoxy compound havannahine [20].

Compounds 3–5 were identified as aromadendrene, palustrol and viridiflorol by comparison of their spectral data with those in the literature [9], [10], [11].

After having established the chemical structures of compounds 1–5, a literature search suggested that polyhydroxysteroids, Xenia diterpenoids, and aromadendrane-type sesquiterpenoids may possess various antimicrobial and antitumor activities [3], [4], [5], [6], [7], [8], [21], [22], [23].

The crude extract and compounds 1–5 were subjected to a preliminary screening against a panel of Gram-negative and Gram-positive bacteria. The crude extract showed the highest antibacterial activity (diameter of inhibition zone ranging from 18 to 34 mm) compared with the individual five isolated compounds (Table 2).

The more potent inhibition by the crude extract could be due to the synergism between all components of the extract, or to the presence of an un-isolated factor. 1 and 2 showed antibacterial activities against all tested bacteria, and the highest activity was found against A. baumannii with MICs of 0.22 and 0.28 mM, respectively, and the lowest activities were against S. epidermidis (Table 3). Resistance of A. baumannii to different antibiotics in hospitals has been well documented, due to its ability to resist desiccation and to survive on artificial surfaces. This bacterium has been found to be responsible for 19% of ventilator-associated pneumonia cases [24]. Furthermore, fractions 1 and 2 showed weak antifungal activities against Candida albicans and Aspergillus niger (MIC, 0.41 and 0.56–0.7 mM, respectively). No antimicrobial activities were recorded for compounds 3–5.

None of the compounds caused lethality in the brine shrimp Artemia salina (LD₅₀ higher than 1.14 mM for 2, and even higher for the other compounds). Except for 2(LD₅₀, 200 µg/ml=0.57 mM), no cytotoxicity of the compounds was observed against the lymphoma cell line, while compounds 1–5 showed no activity against the Ehrlich sarcoma cell line (Table 4).

4 Conclusion

These results are promising, and product 2 may be used as a good antibacterial agent against multidrug resistant bacteria as well as a chemotherapeutic agent against tumor cells.