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Natural Product ResearchFormerly Natural Product Letters

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Bio-active sesquiterpenoids and norsesquiternoidsfrom the Red Sea octocoral Rhytisma fulvum fulvum

Seif-Eldin Nasr Ayyad, Mohamed Ali Deyab, Tamer Kosbar, Walied MohamedAlarif & Ahmed Hassan Eissa

To cite this article: Seif-Eldin Nasr Ayyad, Mohamed Ali Deyab, Tamer Kosbar, Walied MohamedAlarif & Ahmed Hassan Eissa (2019): Bio-active sesquiterpenoids and norsesquiternoidsfrom the Red Sea octocoral Rhytisma�fulvum�fulvum, Natural Product Research, DOI:10.1080/14786419.2019.1709187

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Bio-active sesquiterpenoids and norsesquiternoids fromthe Red Sea octocoral Rhytisma fulvum fulvum

Seif-Eldin Nasr Ayyada, Mohamed Ali Deyabb, Tamer Kosbara,Walied Mohamed Alarifc and Ahmed Hassan Eissaa

aDepartment of Chemistry, Faculty of Science, Damietta University, Damietta, Egypt; bBotanyDepartment, Faculty of Science, Damietta University, Damietta, Egypt; cDepartment of MarineChemistry, Faculty of Marine Sciences, King Abdulaziz University, Jeddah, Saudi Arabia

ABSTRACTThree previously undescribed nardosinane-type sesquiterpenes(1–3), together with six known compounds (4–9) were isolatedfrom the alcyonacean soft coral Rhytisma fulvum fulvum. 2 and 3are 13-nornardosinane, and 6,7-seco-13-nornardosinane deriva-tives, respectively. 2 could be an artifact due to C-6 epimerizationof the known one 4. Chemical structures were elucidated basedon 1D, 2D NMR and MS spectral data. Compounds 1-8 showedcytotoxic activity against NCI-H1299, HepG2 and MCF-7 withIC50 values between 0.0352–0.0974, 0.0717–0.3745 and0.0341–0.1325mM, respectively. The antibacterial activity of allisolated compounds have examined against a number of Gram-positive (Bacillus cereus, Staphylococcus aureas) and Gram-negative(Escherichia coli, Klebsiella pneumoniae and Pseudomonas sp.) bac-teria. Compounds 6 and 7 showed a high degree of inhibitionagainst B. cereus, S. aureus and Pseudomonas sp. Interestingly,neolemnane (6) showed strong inhibition against two fungi,Aspergillus niger and Fusarium oxysporum; while 8 showed positiveinhibition against Fusarium oxysporum at 150mg/mL.

ARTICLE HISTORYReceived 16 September 2019Accepted 11 December 2019

KEYWORDSRed sea; soft coral;sesquiterpenes; cytotoxic;antimicrobial

CONTACT Seif-Eldin Nasr Ayyad snayyad2@yahoo.com Department of Chemistry, Faculty of Science,Damietta University, Damietta, Egypt.

Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1709187.

� 2019 Informa UK Limited, trading as Taylor & Francis Group

NATURAL PRODUCT RESEARCHhttps://doi.org/10.1080/14786419.2019.1709187

1. Introduction

The Genera Rhytisma, family Alcyoniidae, an Indopacific soft coral exists in twocolour morphs: yellow-brown and grey, was initially identified from the Red Seawaters (Benayahu and Loya 1983; Alderslade 2000). Interestingly, in spite ofits taxonomic classification as an Alcyoniidae member, the secondary metabolitesprofile of Rhytisma revealed its close similarity to those of two Genera Lemnaliaand Paralemnalia belonging to the family Nephtheidae. Nevertheless, The DNA ofRhytisma showed its closeness to the family Xeniidae rather than the Alcyoniidae(Haverkort-Yeh et al. 2013).

To the best of our knowledge, 28 metabolites were reported from the genusRhytisma including: sesquiterpenes, norsesquiterpenes and bis-sesquiterpenes as wellas steroids (Kashman 1979; Bowden et al. 1980; Green et al. 1992; Jurek and Scheuer1993; Wessels et al. 2001; Bishara et al. 2008; Trifman et al. 2016).

In The present work the secondary metabolite content of the soft coralRhytisma fulvum fulvum (family Alcyoniidae), collected from the Red Sea Coast atJeddah, Saudi Arabia was investigated. Fractionation and purification of theorganic extract of this animal were achieved by successive column and preparativeThin-Layer chromatography (TLC), led to isolation and identification of ninecompounds (1–9) (Figure 1).

Figure 1. Isolated compounds from Rhytisma fulvum fulvum.

2 S.-E. N. AYYAD ET AL.

2. Results and discussion

2.1. Structure elucidation

Compound 1 was isolated as optical active yellow oil. HRESIMS and 13C NMR assigned themolecular formula as, C17H26O3, and five double-bond equivalents. The 1H- and 13C NMRspectra revealed the presence of one carbonyl (dC 212.9C), an acetyl (dC 170.7C, 20.7 CH3;dH 2.02 s) and a tri-substituted double-bond (dC 137.5C, 123.8 CH; dH 5.56 d) (cf. exp.,Figure S2). The aforementioned functionalities account for three unsaturation degrees, sug-gesting a bicyclic structure. In addition to the methyl of acetate group, 1H NMR spectraindicated the presence two secondary methyls (dH 0.94, d and 0.81, d) and a tertiary one(dH 0.90, s). Further analysis of the 13C and DEPT NMR experiments displayed one sp3 qua-ternary carbon (dC 42.7), three sp3 methines (dC 61.9, 32.8 and 31.3), one oxygenated-methylene (dC 66.1), as well as four upfield-methylenes (40.5, 30.6, 26.6 and 25.5). With theaid of HSQC experiments, all protons were accounted and the compound contains nohydroxyl function. From the 1H-1H COSY spectrum, three separate couplings in the form ofcross-peaks were observed between; the first one being between H-12 and H-11, H-11 andH-13, the second H-2 coupled with H-3, H-3 coupled with H-4, and H-4 coupled with H-14,while the third one noted started from the olefinic proton H-7 resonating at dH 5.56 to H-8, H-8 to H-9 and H-9 to H-10 (As depicted in Figure S1). HMBC correlations establishedthe connections of CH3-14 to C-4 and CH3-15 to C-5, as well as H-11 to C-6, C-13 and C12(Figure S1). The above-mentioned observations and reviewing literatures on metabolitesisolated from the genus Rhytisma reflect the presence of a nardosinane-type sesquiterpene.The locations of the carbonyl and the acetate functions were established from the HMBCspectra as shown in Figure S1. The structure of compound 1, namely, 12-O-acetyl-nardo-sinan-6-en-1-one is shown in Figure 1. The relative stereochemistry of 1 was concluded bystudying the NOESY cross-peaks. NOEs observed between all the methyl protons (Me-13,14 and 15) suggest their co-facial orientation, the b-side; consequently, the a-side is occu-pied by H-4 and H-10. The absolute stereochemistry of 1 was concluded as depicted inFigure 1, on the basis biogenetic considerations and on comparison of the obtained 13CNMR data with those in the literature (Bishara et al. 2008; Daloze et al. 1977).

Compound 2 was isolated as optical active yellow oil. HRESIMS and 13C NMRassigned the molecular formula as, C14H20O3, and five double-bond equivalents.Analysis of its 13C NMR spectrum and DEPT spectra showed the presence of two car-bonyl functions (dC 207.3 and 206.1 ppm), which accounted for two oxygen-atoms (cf.exp., Figure S3). The absence absorption due to hydroxyl functions in the IR spectrum;assigned the third oxygen atom to an etheric function. Based on the absence of reso-nances in the low-field region, compound 2 is a tricyclic skeleton. The presence ofoxirane ring was evidenced from the 1H- and 13C NMR absorptions (dC 65.1 C and 61.8;dH 3.16 d). Extensive studies of HSQC, 1H-1H COSY and HMBC spectral data (Figure S1)indicated a 13-nornardosinane structure (Izac et al. 1982; Jurek and Scheuer 1993,Bishara et al. 2008). On comparing the obtained spectral data of 2 with these of theknown co-occurred 6a-acetyl-4b,5b-dimethyl-1(10)-a-epoxy-7-oxodecalin (4) (Izac et al.1982; Jurek and Scheuer 1993), the orientation of the H-6 proton is being the only dif-ference. The NOESY correlation cross-peaks were observed between the methyl pro-tons of CH3-14 and CH3-15, as well as H-1. No NOEs observed between the latter

NATURAL PRODUCT RESEARCH 3

protons (b-side) and H-6, which assigns a-configuration to H-6. Analysis of coupling con-stants revealed the absence of w-coupling between H-6 and Heq-8, confirmed the C-6b-substituent. Compound 2 was assigned as 6b-acetyl-1(10)-a-13-nornardosin-7-one.

The new compound 2 is an isomer to 4 (which previously isolated from Paralemnaliathyrsoide) (Izac et al. 1982). Compound 4, also isolated in this article from R. fulvum ful-vum and the 1H and 13C NMR spectral data of 4 was in good agreement with thosereported in the literature (Izac et al. 1982). However, adsorption of 4 on Si gel for hoursresulted in epimerization at C-6 and provided a mixture of both compounds 2 and 4.Therefore, generation of 2 from 4 during isolation cannot be excluded.

Compound 3 was isolated as faint yellow oil. EIMS, HRESIMS and 13C NMR assignedthe molecular formula of 3 as, C15H24O4, with four unsaturation equivalents. Two sites ofunsaturation were accounted as two carbonyl groups; a ketone and an ester functions(dC 208.6 and 173.7; respectively), implies a bicyclic skeleton. DEPT experiments showedthe presence of 4 methyl, 5 methylene, 2 methine and 4 quaternary carbons. 1H- and13C NMR spectra, as well as HSQC experiments (Figure S4), evidenced the presence of atri-substituted epoxide ring (dC 62.9 C, 55.6 CH; dH 2.89 t), an oxygenated methyl (dC51.4 CH3; dH 3.64 s), one secondary methyl (dC 15.8 CH3; dH 0.84 d) and two tertiarymethyls (dC/dH 18.0 CH3/1.00 s and 32.4 CH3/2.19 s). After accounting for all oxygen-atoms, compound 3 is a monocarbocyclic structure fused with an oxirane ring (cf. exp.).The 1H-1H COSY spectrum indicated the presence of isolated methylene protons, H-6protons and two separate couplings in the form of cross-peaks between; the first onebeing between proton signals from H-1 to H-14 and the second between H-8 and H-9protons (Figure S1). HMBC correlations established the position of the: oxirane ring (H-1to C-2 and C-10), Me-14 and Me-15 (H-15 to C-10, C-4 and C-6), and the two carbonyls(H-6 to C-5 and C-11; H-8 to C-7 and C-10, respectively). After careful examination thespectral data of 3 and on comparing with those reported in the literature (Jurek andScheuer 1993, Bishara et al. 2008; Daloze et al. 2010), compound 3 is a 6,7-seco-13-nornardosinane. The relative stereochemistry of 3 was concluded by studying the NOESYcross-peaks. NOEs observed are closely similar to those of compound 2, indicating theb-orientation of H-14 and H-15, along with the a-position of the epoxide ring.

In addition to three new compounds (1–3), we have isolated several knowncompounds (Figure 1) from R. fulvum fulvum: 6a-acetyl-1(10)-a-13-nornardosin-7-one(4) (which previously isolated from Paralemnalia thyrsoide) (Izac et al. 1982),12-Acetoxy-l(10)-aristolene (5) and 4-Acetoxy-2,8-neolemnadien-5-one (6) (which previ-ously isolated from Lemnalia africana) (Jurek and Scheuer 1993), besides, a cembra-noid-diterpene Nephthenol (7) (which previously isolated from Nephthea sp.) (Schmitzet al. 1974) and two steroids 24-Methylcholesterol (8) and 23,24-methylenecholesterol(9) (which previously isolated from diverse natural sources, in particular from softcorals such as Sinularia sp., Sinularia dura, Lobophytum sp. and others) (Subrahmanyamet al. 1992; Radwan et al. 2008; Putra et al. 2012).

2.2. Biological activities

All isolated compounds showed cytotoxic activity against different human cancer celllines HepG2 (hepatocellular liver carcinoma), NCI-H1299 (lung carcinoma) and MCF-7

4 S.-E. N. AYYAD ET AL.

(breast carcinoma). Compounds 1, 5, 6 and 7 displayed strong significant cytotoxicityagainst cell line NCI-H1299 and moderate cytotoxicity against HepG2 cell line.Compounds 2-4 showed a moderate cytotoxicity against NCI-H1299 and weakresponse for HepG2. Only compounds 7 and 8 have been evaluated against MCF-7cell, where they showed strong and weak responses, respectively (Table S1).

Compounds 1�8 were tested for their antimicrobial activity, against two Gram-positive bacteria (Bacillus cereus and staphylococcus aureus), three bacteria of Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae and Pseudomonas sp.) andthree fungi (Aspergillus niger, Fusarium oxysporum and Candida albicans). Compounds 6and 7 showed a high degree of inhibition against B. cereus, staphylococcus aureus andPseudomonas sp. while showed a mild inhibition against E. coli and K. pneumoniae incomparison with penicillin G as a standard drug (Table S2). For compounds 3 and 8, noinhibition activities were observed, while compounds 1, 2, 4 and 5 showed appreciatedactivities in some bacteria (Table S2). Compound 6 showed good inhibitions againsttwo fungi (Aspergillus niger, Fusarium oxysporum), while compound 8 showed reason-able inhibition against only the fungus, Fusarium oxysporum at 150 mg/mL. For all othercompounds, no activity was observed in the other three tested fungi (Table S3).

3. Experimental

3.1. General

1D and 2D NMR spectra were recorded in CDCl3 on Joel ECA- II 500MHz NMR spec-trometer and Bruker Avance III 400MHz NMR spectrometer, at NMR Unit Center,Faculty of Science and Faculty of Pharmacy, Mansoura University. EIMS analyses werecarried out on a Thermo, USA Spectrometer with detector MS (Ultra DSQ II) at Centrallab, National Institute of Oceanography and Fisheries, Alexandria, Egypt. Thin-layerchromatography (TLC) was performed on silica gel G60 F254 (Merck, Darmstadt,Germany) of 0.2mm layer thickness and Silica gel 60 for column chromatography(60–120 mesh LR). P-anisaldehyde was the spray reagent.

3.2. Animal material

The soft coral R. fulvum fulvum was collected from the Red Sea Coast at Jeddah, SaudiArabia, in April 2017, and was identified by Prof. Mohsen El-Sherbiny, Marine biologydepartment, Faculty of Marine Sciences, King Abdulaziz University. A voucherspecimen (RFF18-77) was deposited at Marine biology department, Faculty of MarineSciences, King Abdulaziz University.

3.3. Extraction and isolation

The partially dried animals (200.0 g) were extracted with a mixture of equal volume ofCHCl3/MeOH (3� 1 L) at room temperature. Evaporation to dryness yielded 15.0 g of anoily residue. The residue was homogenised with an amount of silica gel 60 A�, and wasloaded in column chromatography (60� 2.5 cm) using silica gel 60 A� (400.0 g) andeluted using petroleum ether with increasing proportions of CH2Cl2 and EtOAc to yield

NATURAL PRODUCT RESEARCH 5

fractions, each of 50ml. The fraction eluted with petroleum ether-CH2Cl2 (9:1) affordedcompound 5, with petroleum ether-CH2Cl2 (6:4) afforded compound 6 and 7, with pet-roleum ether-CH2Cl2 (5:5) to give compound 1, with petroleum ether-CH2Cl2 (4:6)afforded compounds 8 and 9, and the fraction eluted with petroleum ether- EtOAc (9:1)gave compounds 2–4. All fractions were purified by preparative TLC plate (20� 20 cm,0.25mm thickness) with appropriate solvent systems to give nine compounds (1–9).

3.4. Spectral data

3.4.1. 12-O-acetyl-nardosinan-6-en-1-one (1)Yellowish oil (4.2mg, 0.0021%); [a] D

22 - 128.0 (c 0.01, CH2Cl2); IR Vmax (film) cm�1:2964 and 2927 (C-H), 1738 (Ac), 1704 (C¼O), 1460, 1368, 1234, 1181, 1034; 1H NMR(CDCl3, 500MHz): 2.41 (1 H, ddd, 15.0, 3.5, 2.0 Hz, H-2a), 2.30 (1 H, ddd, 15.0, 6.5, 4.0 Hz,H-2b), 1.46 (1 H, m, H-3a), 1.42 (1 H, m, H-3b), 2.14 (1 H, m, H-4), 5.56 (1 H,d, 5.5 Hz, H-7), 2.63 (1 H, m, H-8a), 2.45 (1 H, m, H-8b), 2.12 (1 H, m, H-9a), 1.97 (1 H, m, H-9b), 2.33(1 H, dd, 3.5, 1.5 Hz, H-10), 2.15 (1H, m, H-11), 4.51 (1 H, dd, 10.5, 3.5 Hz, H-12a), 3.68(1 H, dd, 10.5, 10.5 Hz, H-12b), 0.94 (3 H, d, 7.0 Hz, H-13), 0.81 (3 H, d, 7.0 Hz, H-14), 0.90(3 H, s, H-15), 2.02 (3 H, s, CH3C¼O), 13C NMR (CDCl3, 125MHz): 212.9 (C, C-1), 40.5(CH2, C-2), 26.6 (CH2, C-3), 32.8 (CH, C-4), 42.7 (C, C-5), 137.5 (C, C-6), 123.8 (CH, C-7),30.6 (CH2, C-8), 25.5 (CH2, C-9), 61.9 (CH, C-10), 31.3 (CH, C-11), 66.1 (CH2, C-12), 17.6(CH3, C-13), 15.1 (CH3, C-14), 21.6 (CH3, C-15), 170.7 (C¼O, Ac), 20.7 (CH3, Ac); EIMS m/z 278 (6) [M, C17H26O3]

þ, 263 (8) [M – CH3], 203 (44) [M–C3H7O2]þ, 135 (89)

[M–C8H15O2]þ, 107 (100) [M–C10H19O2]

þ; HRESIMS (positive mode) m/z¼ 279.1952[MþH]þ (Calculated m/z¼ 279.1960 for C17H27O3).

3.4.2. 6-b-1(10)-a-13-nornardosin-7-one (2)Yellowish oil, isolated as mixture with 4 (4.0mg, 0.0020%); IR Vmax (film) cm�1: 2964and 2935 (C-H), 1728 and 1701 (C¼O), 1464, 1357, 1242, 1165, 1038; 1H NMR (CDCl3,500MHz): 3.16 (1 H, brd, 2.0 Hz, H-1), 1.91-1.97 (2 H, m, H-2), 1.25 (1 H, m, H-3a), 1.15(1 H, m, H-3b), 1.92 (1 H, m, H-4), 3.74 (1 H, s, H-6), 2.70 (1 H, ddd, 15.0, 11.0, 6.0 Hz, H-8a), 2.45 (1 H, ddd, 15.0, 8.5, 5.5 Hz, H-8b), 1.33-1.37 (2 H, m, H-9), 2.20 (3 H, s, H-12),0.65 (3 H, d, 6.5 Hz, H-14), 1.21 (3 H, s, H-15), 13C NMR (CDCl3, 125MHz): 61.8 (CH, C-1),22.2 (CH2, C-2), 25.4 (CH2, C-3), 33.9 (CH, C-4), 45.0 (C, C-5), 69.1 (CH, C-6), 206.1 (CH,C-7), 38.4 (CH2, C-8), 29.9 (CH2, C-9), 65.1 (C, C-10), 207.3 (CH, C-11), 34.1 (CH2, C-12),18.0 (CH3, C-14), 14.1 (CH3, C-15); EIMS m/z 236 (46) [M, C14H20O3]

þ, 221 (12) [M–CH3],163 (62) [M–C4H9O]

þ, 109 (100) [M–C7H11O2]þ; HRESIMS (positive mode) m/

z¼ 237.1483 [MþH]þ (Calculated m/z¼ 237.1491 for C14H21O3).

3.4.3. 6,7-seco-13-nornardosinane (3)Pale yellow oil, (7.0mg, 0.0035%); [a] D

22 þ 181.0 (c 0.01, CH2Cl2); IR Vmax (film) cm�1:2954 and 2931 (C-H), 1738 (C¼O), 1704 (C¼O), 1438, 1358, 1261, 1169, 1039; 1HNMR (CDCl3, 500MHz): 2.89 (1 H, t, 2.5 Hz, H-1), 1.81-1.85 (2 H, m, H-2), 1.22-1.29 (1 H,m, H-3a), 1.12-1.20 (1 H, m, H-3b), 1.83 (1 H, m, H-4), 2.57 (2 H, dd, 13.5, 13.5 Hz, H-6),2.23 (1 H, m, H-8a), 2.14 (1 H, m, H-8b), 2.25 (1 H, m, H-9a), 2.14 (1H, m, H-9b), 2.19(3 H, s, H-12), 0.84 (3 H, d, 7.0 Hz, H-14), 1.00 (3 H, s, H-15), 3.64 (3 H, s, OCH3),

13C NMR

6 S.-E. N. AYYAD ET AL.

(CDCl3, 125MHz): 55.6 (CH, C-1), 21.6 (CH2, C-2), 24.0 (CH2, C-3), 31.1 (CH, C-4), 40.8 (C,C-5), 48.2 (CH, C-6), 173.7 (C, C-7), 28.0 (CH2, C-8), 25.0 (CH2, C-9), 62.9 (C, C-10), 208.6(CH, C-11), 32.4 (CH3, C-12), 15.8 (CH3, C-14), 18.0 (CH3, C-15), 51.4 (CH3, OCH3); EIMSm/z 268 (4) [M, C15H24O4]

þ, 195 (54) [M–C3H5O2]þ, 135 (40) [M–C6H13O3]

þ, 109 (100)[M–C8H15O3]

þ; HRESIMS (positive mode) m/z¼ 269.1753 [MþH]þ (Calculated m/z¼ 269.1746 for C15H25O4).

3.5. Biological evaluation

3.5.1. Cytotoxicity assayHuman tumour carcinoma cell lines HepG2 (hepatocellular carcinoma) NCI-H1299(lung carcinoma), MCF-7 (human breast carcinoma) used in this study were obtainedfrom American Type Culture Collection (ATCC, Minnesota, USA). The tumour cell lineswere maintained at the National Cancer Institute, Cairo, Egypt, by serial sub-culturing.The cytotoxicity potential was carried out using sulforhodamine B (SRB) colorimetricassay (Vichai and Kirtikara 2006).

3.5.2. Antimicrobial activity assayAgar well diffusion method was applied to investigate the antimicrobial activity of thecompounds at concentrations of 50, 100 and 150lg/mL in DMSO, which was alsoapplied alone as a control. The antibacterial activities against (Bacillus cereus,Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumo-niae) were tested using nutrient agar medium. The antifungal activities were alsotested against three local fungal species (Aspergillus niger, Fusarium oxysporum andCandida sp.) using DOX agar medium. Wells (10mm) were made by a sterile corkborer in agar medium that was inoculated with the tested microorganisms. In eachwell, we applied 200 lL of the tested compound. We kept the agar plates at 4 �C forat least 30minutes, until the complete diffusion of the tested derivative; then plateswere incubated at 37 �C or 30 �C for bacteria and fungi, respectively. Penicillin G andmiconazole were used as standard antibacterial and antifungal, respectively. The anti-microbial activities were assayed in terms of inhibition zone diameters after 24 hoursand 7days for both bacteria and fungi, respectively.

4. Conclusion

Three new nardosinane-type sesquiterpenes along with six known compounds wereisolated for the first time from Rhytisma fulvum fulvum. The diversity of metabolitesrefers to the richness of the genus Rhytisma in secondary metabolites and the highlyeffect of the environment on the production of such metabolites. Generally, the iso-lated compounds displayed antiproliferation and antimicrobial activities, amongstthese; the antifungal activity of the lemnane- derivative (6) is of considerable interest.

Acknowledgments

The authors wish to thank Mr. Kamal Al-dahodi, Faculty of Maritime studies, King AbdulazizUniversity, for sample collection.

NATURAL PRODUCT RESEARCH 7

Disclosure statement

No potential conflict of interest was reported by the authors.

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