Dzeha, Thomas

Jaspars, Marcel

Tabudravu, Jioji

Western Indian Ocean Journal of Marine Science

English

Marcel Jaspars

Thomas Dzeha

Jioji Tabudravu

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Clionasterol, a Triterpenoid from the Kenyan Marine Green Macroalga &lt;i&gt;Halimeda macroloba&lt;/i&gt;

Aberdeen University Research

CLIONASTEROL ISOLATED FROM HALIMEDA MACROLOBA 157Corresponding author: MJ.E-mail: m.jaspars@abdn.ac.ukWestern Indian Ocean J. Mar. Sci. Vol. 2, No. 2, pp.157–161, 2003© 2003 WIOMSAClionasterol, a Triterpenoid from the Kenyan Marine GreenMacroalga Halimeda macrolobaThomas Dzeha1, Marcel Jaspars2 and Jioji Tabudravu21Kenya Marine Fisheries Research Institute, P.O. Box 81651 Mombasa, Kenya; 2Dep rtment of Chemistry,University of Aberdeen, AB24 3UE Scotland, UKKey words: clionasterol, macroalga, Halimeda macroloba, spectroscopy, bioprospecting, KenyaAbstract—The triterpenoid clionasterol (1), a 29 carbon structure compound was isolated fromthe less polar extract (20% EtOAc in hexanes) of the green alga H limeda macroloba collectedat Shimoni near Mombasa, Kenya. The structure and relative stereochemistry of this compoundwas elucidated by spectroscopic data, mainly NMR and mass spectrometry. This metabolitewas inactive against DLD-1 cells on the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay. Further experiments on mosquito larvae and brine shrimp lethalityconfirmed this result.INTRODUCTIONIn our continued interest in prospecting for novelpharmaceuticals from Kenyan marine organismsand on the basis of potent antibacterial andantifungal activities of its ‘spore-like’ greenepiphytes, Halimeda macroloba, was selected forinvestigation. In this study, it is established thatthe potent activity exhibited by the epiphytes isnot derived from the host. The epiphytes had a2.2 cm inhibition zone against the fungus A per-gillus niger and 1.9 cm against the gram-negativebacteria Escherichia coli on the 12.5 mm disc.These antimicrobial activities of the epiphytesare in great contrast to the observations made onclionasterol (1), which did not exhibit any activity.Consequently, we report on the isolation andstructure determination of clionasterol, atriterpenoid previously only isolated from theIndian marine red alga Gracilaria edulis (Das etal., 1992) and from an earlier synthesis via a Wittigreaction of its analogue extracted from the spongeVerongia aerophoba (De Luca et al., 1973).MATERIALS AND METHODSGeneral experimental proceduresLRMS ESMS were obtained on a FinniganMasslab Navigator Electrospray MassSpectrometer. For details of NMR instruments andsolvents, see Table 1. Silica gel 60F254 TLC sheetswere purchased from Merck (Germany). Normalphase HPLC utilised a phenomenex sphereclone5m column. The size exclusion sephadex LH-20was from Amersham Pharmacia Biotech AB(Sweden).CollectionHalimeda macroloba algal specimens werecollected from the intertidal zone at Shimoni, 100km south of Mombasa, Kenya in March 2000 andkept in cold conditions in methanol prior toextraction. A voucher specimen can be found atthe University of Aberdeen where extraction andanalysis took place.158 T. DZEHA ET AL.Extraction and isolationA 200 g sample (80 g dry weight) was homogenisedin MeOH. Sequential extraction with MeOH,CH2Cl2 and partitioning with 1:1 CH2Cl2/H2Oresulted into the aqueous and organic phases. Theorganic CH2Cl2 phase was then partitioned withhexane in 90% MeOH/H2O. The hexane extract(98.5 mg) was applied to a sephadex LH-20(Pharmacia) gel filtration column. Elution was with1:1 CH2Cl2/MeOH. The normal (60:40 hexane/EtOAC) TLC chromatography of the Sephadexcolumn collection afforded three sub-samples. Thesecond sub-sample was subjected to normal phaseHPLC (80:20 hexane/EtOAc) on a phenomenexsphereclone 5µ silica column resulting into 11peaks. Clionasterol (1) was collected as a palegreen liquid from the 9th peak at 14:09, 14:32 and14:51 min on a Waters pump attached to a PerkinElmer differential refractometer 40.The hexane fraction was examined by 1H NMRspectroscopy. This extract was fractionated onSephadex LH-20. Final purification was achievedby normal-phase HPLC as outlined in theexperimental section.Brine shrimp toxicity assayAbout 20 newly hatched brine shrimp (Artemiasalina) in ca. 0.5 ml seawater were added to eachwell containing different concentrations of samplein 50 ml EtOH and 4.5 ml  brine shrimp media(BSM). The experiment was run in triplicate. After24 h at 25 oC, the brine shrimp were observed andcounted under a dissecting light microscope. Thepercentage of live shrimp against total number ofshrimp was used to determine LD50 values.DLD-1 MTT assayA solution of clionasterol was made in MeOH asit proved insoluble in DMSO. The cytotoxicity ofclionasterol against DLD-1 cells was comparedwith that of a vehicle drug control and a controlblank at concentrations ranging between 0 and 100mM using a modification of the MTT-microtitreplate tetrazolium cytotoxicity assay as originallydescribed by Mossman and cited elsewhere(Swaffar et al., 1994).Mosquito larvae toxicityA twelve-well system was used to determine thelethal dose of clionasterol against 5 mosquitolarvae in ca. 0.5 ml saline media at differentconcentrations of the sample in MeOH. The LD50was arrived at through a percentage of live againsttotal larvae.Antibacterial and antifungal activityDisc diffusion assays used conventional methods(Chand et al., 1994). Briefly, a lawn ofmicroorganisms was prepared by pipetting andspreading evenly overnight cultures of E. coli (IFO3545) and A. niger [from the Hebrew UniversitySchool of Pharmacy] (conc. 106–107 CFU/ml) ontoagar and media in Petri dishes. The media for theE. coli was made up of neopeptone, agar, glucoseand maltose whereas that for the fungi utilisedsabaraud prepared in a similar fashion. A 12.5 mmsterile filter disc was used to which 50 ml of thetest compound dissolved in solvent were added.The plates were inverted, stored in the refrigeratorfor 3 hours prior to overnight incubation at 35 oCafter which the diameters of the zone of inhibitionaround the discs were measured. Controlexperiments were performed with equivalentvolumes of solvents without the test compound.RESULTS AND DISCUSSIONThe molecular formula of clionasterol was deducedas C29H50O from a combination of MS with 13CNMR spectroscopy. Initially the compound wasthought to be C28H49OH but closer examination ofthe APT revealed an overlapped carbon on C4 atδ43.3. Negative electron impact mass spectrometry(EIMS) gave the [M-H]+ at 413.3, [M+ Na] + at437.4 and there was an evident [M+ Na + MeOH]+peak displayed at 469.5.ClionasterolPale green liquid; C29H50O; EIMS m/z obs [M -H]+ 413.3, [M + Na]+ 437.4, [M + Na + MeOH]+469.5 (calc’d for C29H50O, 414); elution time 14:09,14:32, 14:51 min; for 1H and 13C NMR data seeTable 1.CLIONASTEROL ISOLATED FROM HALIMEDA MACROLOBA 159Table 1. 1H and 13C NMR assignments for clionasterol (1)C# δC DEPT (mult) δH COSY HMBC1 38.3 CH2 1.80 m, 1.03 m C3,C192 32.7 CH2 1.78 m, 1.44 m C3,C4/4'3 72.8 CH 3.47 dddd; 3.52 m C1/1', C2/2', C4/4'4/4' 43.3 CH2 + C 2.24 m, 2.16 m C4-C6,C2,C2'//C4'-C17,C12,C185 141.8 C – C1/1',C4/4',C7/7', C196 122.8 CH 5.31 d; 5.35 m C4/4',C1/C1',C7/7'7 33.0 CH2 1.92 m, 1.46 m C68 33.0 CH 1.46 m C79 51.2 CH 0.89 m C1',C2',C7',C8,C11,C12,C1910 37.5 C – C2,C4/4',C611 22.1 CH2 1.46 m C1212 40.8 CH2 1.96 m, 1.11 m C17,C1814 57.8 CH 0.96 m C2,C7',C8,C11,C12,C17,C1815 25.3 CH2 1.55 m C1,C2,C14,C1616 29.3 CH2 1.82 m, 1.22 m C17,C2017 57.1 CH 1.06 m C16,C2118 12.9 CH3 0.64 s; 0.65 s C1219 20.6 CH3 0.97 s; 1.00 s C220 37.3 CH 1.32 m C2121 19.9 CH3 0.88 d; 0.90 d22 35.0 CH2 1.30 m, 0.93 m C2123 27.4 CH2 1.32 m C2824 47.1 CH 0.88 m C28/28',C26,C2725 30.0 CH 1.64 m C26,C2726 20.0 CH3 0.77 d; 0.82 d27 20.4 CH3 0.79 d; 0.81 d28 24.0 CH2 1.28 m, 1.11 m C2929 13.4 CH3 0.81 t; 0.85 tAll spectra in CDCl3; 1H at 400 MHz, 13C at 100 MHz; assignments by 1H–1H COSY, 1H–13CCOSY, and HMBC experiments. Acquired on a Varian-Unity INOVA spectrometer. Note: C4' issynonymous with C13 in the text. The assignments in bold refer to the original assignments ofclionasterol (Das et al., 1992).moiety at δ3.47. Overall, APT and MF datadescribes the compound as being composed of 3quarternary carbons, 9 methines, 11 methylenesand 6 methyl groups with a typical 4-ring steroidstructure (Fig. 1). Interpretation of the 1H and 13CNMR (Table 1), 1H–1H COSY and 1H–13C COSYdata along with HMBC information confirmed thesubstructures shown  in Fig. 2.The partial sterol parent substructure wasdetermined effectively by HMBC couplingsassigning two of the six methyl groups to C18 andC19 respectively. The two methyl carbons of thesefunctionalities resonate at δ12.9 and 20.6. 1H-1HCOSY correlated the olefinic hydrogen H6 atδ5.31 d with H27 at δ1.92 m and 1.46 m. It alsocorrelated with H24/4' at δ2.24 m and 2.16 m.Thecorrelation between H21 and H6, though evident,Fig. 1. Structure of clionasterolThis molecular composition requiredclionasterol to have 5 double bond equivalentswith 1 olefinic bond at δ141.8 on the 13C NMRspectra. The 1H spectrum revealed the previouslydescribed olefinic bond at δ5.31 and a hydroxyOH160 T. DZEHA ET AL.was weak at 4 bonds away. Other 1H-1H COSYcorrelations suggesting the parent sterol structureare those between H22/2' (δ1.78 m/1.44 m) andH3 at δ3.47 dddd and to H24/4' (δ2.24 m/ 2.16 m).The nOe between H22 and H3 and H24 require thatthe hydroxyl group on C3 should be down,suggesting that the protons at C2, C3, C4 and C7are axial and oriented on the same side of the sterolring structure.The long range heteronuclear 1H-13C coupling(HMBC) between C5 at δ141.8 and the methylhydrogens H319 at 0.97 s; that between C14 atδ57.8 and the H318 at δ0.64 and the ‘solenoid’-like correlations between C3 at δ72.8 and H22 atδ1.78 m, 1.44 m; C5 at δ141.8 and H319 at 0.97 s;C14 at δ57.8 and H318 at δ0.64 confirms the parentsterol substructure (Fig. 2a). Other correlationsconcluding substructure 2a are shown in Table 2.Substructure 2a was connected to substructure 2bby the coupling between C17 and H321 at δ57.1and δ0.88d respectively and to the couplingbetween C16 at δ29.3 to H20 at δ1.32 m on theHMBC. There were 1H-13C correlations betweenC22 m at δ35.0 and the H321 doublet protons atδ0.88; between C23 m at δ27.4 and the methyleneprotons H228 at δ1.28 m and δ1.11 m. The strongcorrelations between C24 at δ47.1 and the H3 ofC26 and C27 at δ0.77 d and δ0.79 d respectivelyas well as those of C24 and the H228 protonscompletely describes the structure of the side chainleaving an unassigned methylene group welldefined by a tertiary C29 methyl signal at δ13.4.Most HMBC were within 2 bonds (2JCH) awayor 3 bonds (3JCH) away at most. Although thestructure elucidation of clionasterol isolated fromthe Kenyan algae was largely afforded by acombination of mass spectrometry and 1D and 2DNMR experiments, most of the assignments of theclionasterol isolated from the Indian marine algaeG. edulis were through the interpretation of theobserved mass spectra (Das et al., 1992).Antibacterial and antifungal activitiesThe epiphytes of H. macroloba, which had a deepgreen color, had potent antibacterial and antifungalactivities based on the inhibition zones of the crudemethanolic extracts (Table 3). The experimentswere done on a 12.5 mm diffusion disc. Theinhibition zone was 2.2 cm against the fungus A.niger strain and 1.9 cm against the gram-negativebacterium E. coli. The mass of these epiphytes (8 g),however, was inadequate for isolation of the activemetabolites. This led to investigating the potencyof the host (H. macroloba), which resulted in theisolation of compound 1.Table 2. Important 2D NMR HMBC correlations forsubstructure 2aC1  ~  H3 3JCHC2  ~  H3 2JCHC3  ~  H1/1', H2/2', H4/4' 3JCH, 2JCH, 2JCHC4  ~  H6 3JCHC4' ~  H12, H17, H3182JCH, 2JCH, 2JCHC5  ~  H1/1', H4/4', H7/7',H3193JCH, 2JCH, 3JCH, 3JCHC6  ~  H4/4', H7/7' 3JCH, 2JCHC7  ~  H6 2JCHC9  ~  H1, H12, H3193JCH, 3JCH, 3JCHC10 ~ H6 3JCHC14 ~ H12, H17, H3183JCH, 3JCH, 3JCHC17 ~ H21 3JCHH3 H4' H6H7'H7H4H1'H1OHH2H2'119181417 16172122 242927262816(b)(a)Fig. 2. Substructures for clionasterol showing the sterol moiety (a) and its side  chain (b) with selected nOesCLIONASTEROL ISOLATED FROM HALIMEDA MACROLOBA 161Mosquito larvae, brine shrimp andDLD-1 MTT toxicityClionasterol had no activity on mosquito larvaeeven at elevated concentrations; neither did it haveany toxicity against brine shrimp and DLD-1cancer cell line (LC-50 >100 mM/Ml) (Table 3).In conclusion, although C29 compounds havebeen reported in marine organisms (Faulkner,1996; Schmitz, 1978), this is the first time thatclionasterol has been isolated from a Kenyanmarine green macroalgae.Acknowledgement—We are grateful to ShadrackTunje of the Kenya Marine Fisheries ResearchInstitute (KMFRI) for his assistance in thecollection of the algal material and to JacquelineUku of the same institute for identifying the taxa.From Aberdeen University, we acknowledge DrsBerg [Botany department] for confirming thetaxonomic identification, Heather Wallace[Department of Medicine and Therapeutics] for theDLD-1 MTT assay and Peter Billingsley[Department of Zoology] for the mosquito larvaelethality experiments. We thank Dr Linda Lawton[Robert Gordon University], in whose laboratorythe brine shrimp toxicity tests were performed. Wecannot forget the invaluable contribution ofemeritus Professor Shmuel Halevy of the Schoolof Pharmacy, Hebrew University, Jerusalem, whoalso provided the microbial strains used in theantimicrobial bioassays. This work was supportedby a grant ICB/34896/00 8D from the Organisationfor the Prohibition of Chemical Weapons (OPCW)of the Hague, the Netherlands and the Faculty ofScience and Engineering of the University ofAberdeen in Scotland.REFERENCESChand, S., Lusunzi, I., Veal, D.A, Williams, L.R &Karuso, P. (1994) Rapid screening of theantimicrobial activity of extracts and naturalproducts. J. Antibiot. 47: 1295–1304.Das, B., Venkateswarlu, Y., Srinivas, K.V.N.S & RamaRao, A.V. (1992) 5-poriferast-9 (11)-en-3-ol fromthe marine red alga, Gracilaria edulis. Phytochem.31: 1054–1055.De Luca, P., De Rosa, M., Minale, L., Puliti, R.,Sodano, G., Giordano, F. & Mazzarella, L. (1973)Synthesis of 24,28-didehydroaplysterol and X-raycrystal structure of aplysterol: unusual marinesterols. J. Chem. Soc. Chem. Comm. 1: 825–826.Faulkner, D.J. (1996) Marine natural products. J. RoyalSoc. Chem. Natural Products Reports 13: 75–125.Schmitz, F.J. (1978) Uncommon marine steroids. In:Scheur, P. J. (ed.) Marine natural products:Chemical and biological perspectives Vol.1.Academic Press, Inc., 111 5th Av. N.Y 10003.Swaffar, D.S., Ireland, C.M. & Barrows, L.R. (1994)A rapid mechanism-based screen to detectpotential anti-cancer agents. Anti-cancer Drugs,5: 15–23.Table 3. Activity of clionasterol and on the epiphytes of Halimeda macrolobaHalimeda  macrolobaepiphytes Clionasterol1. Antimicrobial activitya. Aspergillus niger 2.2 cm Nilb. Escherichia coli 1.9 cm Nil2. DLD-1 cancer cell line N/d >100 µm/ml3. Brine shrimp toxicity N/d Nil4. Mosquito larvae N/d NilAntimicrobial activity was measured as zones of inhibition on a 12.5 mm sterilefilter disc (Chand et al., 1994). DLD-1 cancer cell line was measured as theLC50 (Swaffar et al., 1994)  whereas the brine shrimp toxicity and the mosquitolarvae bioassays were both determined as the LD50. N/d implies that theexperiments were not done. Nil represents lack of activity.

Clionasterol, a triterpenoid from the Kenyan marine green macroalga halimeda macroloba

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Clionasterol, a triterpenoid from the Kenyan marine green macroalga halimeda macroloba

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