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6 New cytotoxic compounds from Heteroxenia ghardaqensis and protective effect of chloroform extract against cadmium toxicity in rats Abdelsamed I. Elshamy a *, Mahmoud I. Nassar a, Abdel-Razik H. Farrag b. a Natural Compounds Chemistry Department, National Research Centre, Dokki, 12311, Cairo, Egypt. b Department of Pathology, National Research Centre, Dokki, 12311, Cairo, Egypt. *Corresponding Author: elshamynrc@yahoo.com. 1

7 Contents Chapter I Introduction Marine natural products Family: Xeniidae Heteroxenia genus Medicinal importance of Heteroxenia genus Chemical constituents of Heteroxenia genus Chapter II Experimental Extraction and isolation Role of chloroform extract of the coral against adrenal glands toxicity induced by cadmium chloride Anticancer activity of pure compounds Antimicrobial activity of chloroform extract of Heteroxenia ghardaqensis and some pure compounds Chapter III Results and discussion Investigation of the chemical constituents of Heteroxenia ghardaqensis chloroform extract Compound 1 Compound 2 Compound 3 Compound 4 Compound 4a Compound 5 Compound 6 Cytotoxic activity of ceramides 1 and 2 Cytotoxic activity of sterols 4, 5 and 6 against human Caco-2 cell lines Role of Heteroxenia ghardaqensis CHCl 3 extract against cadmium-induced adrenal toxicity Antimicrobial activity of chloroform extract and some pure compounds Conclusion References

8 Chapter I 1. Introduction 1.1. Marine natural products The chemistry of marine natural products has grown enormously in the last fifty years. On land, communication between insects is largely by pheromones. Because these must be volatile, their chemical structures are often simple and many are easy to synthesize. In contrast, in an aqueous environment communication between living organisms depends on solubility in water. As a consequence, the chemical compounds used in the communication can have complex structures and large molecular weights as long as there is adequate solubility in water. Since all forms of life are subject to perpetual competition, it is not surprising that the organisms that live in the sea produce an enormous range of biological activity. Besides the compounds that repel predators by their toxicity, there are those which are attractive to make reproduction more probable. (Bhakuni and Rawat, 2005). Given all these factors it is not surprising that marine organisms are a wonderful source of biologically active natural products. It has taken half a century for this to be fully appreciated. In this time the means of collection have been developed so that marine diving, at least in shallow coastal waters, is relatively simple. The result of all this is that there is an avalanche of new and biologically exciting marine natural products (Scheuer, 1978). To date approximately more than 16,000 marine natural products have been isolated from marine organisms and reported in approximately 6,800 publications. Several of the compounds isolated from marine source exhibit biological activity. The ocean is considered to be a source of potential drugs (Scheuer, 1978a; 1979). Marine organisms produce some of the most cytotoxic compounds ever discovered, but the yields of these compounds are invariably so small that natural sources are unlikely to provide enough material for drug development studies (Faulkner, 1987). Marine environment provides different biosynthetic conditions to organisms that live in it. Marine organisms generally live in symbiotic association. The pathway of 3

9 transfer of nutrients between symbiotic partners is of much importance and raises questions about the real origin of metabolites produced by association (Faulkner, 1988). Marine natural products chemistry has passed through several phases of development. Effective methods of isolation provided many potent compounds in pure form. Advancement in instrumentation methods such as nuclear magnetic resonance, mass spectrometric techniques and X-ray diffraction have helped to solve many intricate structural and stereochemical problems (Tziveleka et al., 2003& Berlink et al., 2004). Chemically several metabolites of unusual structure and exhibiting biological activities have been isolated from marine animals. Some of these bioactive metabolites have biomedical potential. The bioactive metabolites that are of interest have been mainly isolated from marine sponges, jelly fish, sea anemones, corals, bryozoans, molluscs, echinoderms, tunicates and crustaceans. The bioactive metabolites isolated from marine animals could be divided into steroids, terpenoids, isoprenoids, nonisoprenoids, quinones, brominated compounds, nitrogen heterocyclics, and nitrogen sulphur heterocyclics (Scheuer, 1980; 1983& Faulkner, 1990) Family Xeniidae The Red Sea represents one of the most promising areas as a source of medicinal and nutritional natural products. Many collections made on the Red Sea reefs led to numerous comprehensive studies on the taxonomy of Red Sea soft corals (Gohar, 1940; Benayahu & Loya, 1987; Benayahu, 1990). Soft corals (Octocorallia, Alcyonacea) are a highly diverse group of marine organisms, which are known to contain a rich variety of secondary metabolites. Soft corals elaborate a large variety of steroids, ceramides, sesquiterpenoids and diterpenoids (Bowden et al., 1986; Anata et al., 2002; Mohamed et al., 2012). 4

10 The Red Sea xeniidae (Alcyonacea) comprises more than 5 genera, the most common, were Anthelia, Cespitularia, Heteroxenia, Sympodium and Xenia species (Benayahu & Loya, 1985& Gohar, 1940) Heteroxenia genus Common names for Heteroxenia sp. are pulse coral, pom-pom coral, and also pom-pom xenia. Heteroxenia sp. are dimorphich, containing both autozooid and siphonozoid polyps. Their stalks terminal in a capitulum from which the polyps emerge. The tentacles on the autozoid polyps extend in a way that resembles a pompom of a cheerleader, hence the common name. These stalks rarely branch. Heteroxenia corals are usually light in color, white or cream. Their polyps pulse faster than other Xeniids. Found commonly on inner reefs in shallow zones, they prefer high light intensity. Due to their prevalence in shallower waters, they may be exposed to air during low tide. It is not necessary to directly feed this coral. They will receive all their nutrients from their zooxanthellae and from absorbing microfauna in the water. These are prime specimens for captive breeding. Their planulae (larva) brood first in their gastrovascular cavity before they move on to pouches in their mesoglea (stiff, gelatinous, connective tissue with lots of air space reminiscent of stale Jell-O). After their planulae are released, they quickly settle on a substrate, metamorphosis into a sessile coral, acquire zooxanthellae, and mature into polyp. For H. fuscescens, this all happens in a month s time. Heteroxenia corals have a high mortality rate in collection and shipping (Alderslade, 2000& Borneman, 2001). Heteroxenia species are very closely related to other xenia species and take an expert eye to identify. Heteroxenia genus include only two species; Heteroxenia fuscescenes (Ehrb.) and Heteroxenia ghardaqensis (sp.n.) (Gohar, 1940& Benayahu& Loya, 1987). 5

11 Taxonomy of Heteroxenia ghardaqensis Kingdom: Animalia Phylum: Cnidaria Class: Anthozoa Order: Alcyonidae Family: Xeniidae Genus: Heteroxenia Species: Heteroxenia ghardaqensis (sp.n.) Figure (1): Photo of Heteroxenia ghardaqensis Medicinal importance of Heteroxenia genus Antifungal activity It was reported that isolated compounds from H. fuscescenes have antifungal activity against Cladosporium cucumerinum (Edrada et al., 2000) Acute toxicity study The alcoholic extract of H. fuscescens has been found to be safe and non toxic (Mohammed et al., 2012) Anti-inflammatory activity The alcoholic extract of H. fuscescens has been reported to induce significant antiinflammatory effect (Mohammed et al., 2012) Analgesic activity The alcoholic extract of H. fuscescens exhibits a significant analgesic effect (Mohammed et al., 2012) Antipyretic activity It was reported that the alcoholic extract of H. fuscescens has significant antipyretic effect (Mohammed et al., 2012). 6

12 Antioxidant activity It was listed that the alcoholic extract of H. fuscescens has mild antioxidant activity (Mohammed et al., 2012) Antimicrobial activity The total alcoholic extract of H. fuscescens has marked effect against bacterial strains and fungus strains (Mohammed et al., 2012) Anticancer activity The alcoholic extract of H. fuscescens has exhibited significant cytotoxic activity against different human tumor cell lines in vitro (Mohammed et al., 2012) Reported chemical constituents of Heteroxenia genus Species of Heteroxenia genus, H. fuscescenes and H. ghardaqensis, are non common, so the chemistry of this genus is very rare. Little reports sheets have been published on the chemical constituents of Heteroxenia species for only H. fuscescenes and there are no reports for H. ghardaqensis. Only two reports for the previously isolated compounds from H. fuscescenes that include sesquiterpenoids (eudosmans), steroids, acyl glycerols and ceramides. These isolated compounds were summarized in Tables

13 Marine Organ Heteroxenia fuscesens Table (1): Sesquiterpenes of Heteroxenia species. Compound References -murrolene (I) Kashman et al., 1978& Mohammed et al., hydroxy- -murrolene (II) Kashman et al., acetoxy- -murrolene (III) 6-hydroxy- -Muurolene (IV) Edrada et al.,

14 HO I -murrolene II 7-hydroxy- -murrolene HO AcO III 7-acetoxy- -murrolene IV 6-hydroxy- - murrolene Chart 1: Structures of sesquiterpenes of Heteroxenia species. 9

15 H. fuscesens Table (2): Steroids, acyl glycerols and ceramides of Heteroxeni species. Gorgosten-5(E)-3 -ol V Mohammed et al., 2012 Sarcoaldosterol A VI Edrada et al., 2000; Mohammed et al., 2012 VII octadecadiene-1,3-diol Mohammed et al., 2012 (2S,3R,4E,8E)-Nhexadecanoyl-2-amino 4,8-1-nonadecyloxy-2,3- propanediol VIII 10

16 HO HO HO OH OH V Gorgosten-5(E)-3 -ol VI Sarcoaldosterol A OCH 2 (CH 2 ) 17 CH 3 OH HO H OH HO HN O VII 1-nonadecyloxy-2,3-propanediol VIII (2S,3R,4E,8E)-N-hexadecanoyl-2- amino 4,8-octadecadiene-1,3-diol Chart 2: Structures of steroids, acyl glycerols and ceramides of Heteroxenia species. 11

17 2. Experimental 2.1. Animal material Chapter II The soft coral Heteroxenia ghardaqensis (4 Kg) (Fig. 1) collected from the Red Sea on May 2010, at a depth of 3-4 m at the front of Hurghada marine station of the National Institute of Oceanography and Fisheries, Hurghada, Egypt. It was kept in sea water and stored at -30 ºC to avoid degradation of the secondary metabolites. The soft coral was identified by both of Assoc. Prof. Hashim Madkour and Assoc. Prof. Tarek Abdel-Aziz, at the Department of Invertebrates, National Institute of Oceanography and Fisheries, Red Sea branch, Hurghada, Egypt Extraction and isolation The frozen marine organism H. ghardaqensis (wet weight 4.0 Kg) was broken down into small pieces at room temperature and extracted with chloroform, several times until no further residue was obtained to ensure complete extraction. The chloroform extract was filtered, and concentrated under reduced pressure at 50 ºC to afford dark brown gum (15 g). The chloroform extract was chromatographed on silica gel column (250 g; 50 cm L x 6 cm D), eluted with a mixture of pet ether (40-60 ºC) / EtOAc according to increasing polarity to give 32 fractions (each fraction 200 ml). They were inspected by TLC eluted by pet ether (40-60 ºC) / EtOAc and using different spraying reagent. The identical fractions were collected to afford nine major fractions (Table 3, Scheme 1). 12

18 Table (3): Major fractions of CHCl 3 extract of H. ghardaqensis Fractions obtained Fraction IV Fraction V Fraction VI Fraction VII Fraction VIII Fraction IX Eluent (Pet ether : EtOAc) 1:1 7:3 4:1 2:3 9.5:0.5 9:1 Weight (g)

19 CHCl3 extract Silica gel CC (Petr ether/ EtOAc) Frac IV Frac V Frac VI Frac VII Frac VIII Frac IX Frac I Frac II Frac III Silica gel CC, (Petr ether:etoac) PTLC 9.5:0.5, (pet ether:etoac) PTLC 3:2, (pet ether:etoac) PTLC 7:3, (pet ether:etoac) PTLC 1:1, (pet ether:etoac) Silica gel CC, (Petr ether:etoac) PTLC 2:3, (pet ether:etoac) Silica gel CC, (Petr ether:etoac) PTLC 4:1, pet ether:etoac 10 SubFra 3 9 SubFra LC ester Sterol Sephadex CC, (MeOH) Sterol Sephadex CC, (MeOH) Sterol LC ester New Sterol Sterol Ceramide Sterol 1 2 Acetylation Ceramide New Ceramide A1 Scheme 1: Fractionation and compounds purification from the soft coral extract 14

20 2.3. Purification of the isolated fractions from chloroform extract Fraction I was applied on glass silica gel PTLC (20 x 20 cm), eluted with pet ether (40-60 C) / EtOAc (8:2) to afford one pure compound gorgosten-5(e)-3 -ol (9, 30 mg). Fraction II was subjected to silica gel column (150 cm L x 4 cm D), with pet ether (40-60 C) / EtOAc according to increasing polarities, each of 10 ml in fractions, to afford one pure compound gorgostan-3,5,6,11 -tetraol (5, 25 mg) from eluent (3 : 7). Fraction III was subjected on PTLC (20 x 20 cm), eluted with pet ether (40-60 C) / EtOAc (2 : 3) to afford one pure compound gorgostan-3,5,6 -triol-11 acetate (6, 10 mg). Fraction IV was chromatographed on PTLC, eluted with pet ether (40-60 C) / EtOAc (3 : 2) to afford one pure compound cholesten-5(e)-3 -ol (7, 10 mg). Fraction V was subjected to PTLC eluted with pet ether (40-60 C) / EtOAc (1:1) and the subfraction (18 mg) was subjected to quickly sephadex column chromatography, eluted with methanol 100% to afford one pure compound 2S,3R- 4E,8E-2-(hexadecanoylamino)-docosa-4,8-diene-1,3-diol (1, 20 mg). Fraction VI was subjected to PTLC eluted with pet ether (40-60 C) / EtOAc (7:3) to afford one pure compound 7 -hydroxy cholesterol (9, 10 mg) along with subfraction (12 mg) that subjected to sephadex column chromatography, eluted with methanol 100% to afford one pure compound 2S,3R-4E,8E-2-(octadecanosylamino)- octadecadiene-1,3-diol (2, 10 mg). Fraction VII was purified on glass PTLC, eluted with pet ether (40-60 C) / EtOAc (3:2) to afford two pure compound cholesterol-3 -acetate (8, 8 mg) and 2S,3R-4E-2-(octadecanosyl amino)-octadec-4-ene-1-ol (9, 10 mg). Fraction VIII was subjected to silica gel column chromatography (150 cm L x 4 cm D), eluted with pet ether (40-60 C) / EtOAc according to increasing polarities, each of 10 ml in fractions, to afford one pure compound cosanyl octadecanoate (10, 12 mg) from eluent (9.5 : 0.5). 15

21 Fraction IX was subjected to silica gel column, eluted with pet ether (40-60 C) / EtOAc according to increasing polarities to afford one pure compound hexadecanyl octadecanoate (11, 17 mg) from eluent (9.5 : 0.5) Acetylation of compound 4 Gorgosten-5(E)-3 -ol (4, 15 mg) was acetylated by addition of an excess of acetic anhydride (3 ml) in presence of one drop of pyridine. This mixture was kept at room temperature for 48 hr (Scheme 2). The reaction mixture was poured onto icecold water (Shen et al., 2001& Tanaka et al., 2002). A white precipitate was formed R f = 0.80 (hexane/etoac 4:1), filtered off and quickly purified by silica gel column chromatography eluted with hexane / EtOAc to afford one product gorgosten- 5(E)-3 -acetate (4a, 8 mg, mp C; {[ ] 25 D (c 0.01, CHCl 3 )})., gave dark violet color under UV light, gave dark orang color with both Ce(SO 4 ) 2 and MeOH/H 2 SO 4 (3:1) spraying reagents. HO 4 Acetic anhydride excess Pyridine (one drop) 24 hrs, Room temp AcO Scheme 2: Acetylation reaction of compound 4. 4a 2.4. Methanolysis (acid hydrolysis) of ceramides and long chains esters The methanolysis of long chain ester and ceramide compounds was carried out by treated with a certain amount of 1 N HCl in an excess of methanol at 90 C under reflux for 18 h with magnetic stirring. The fatty acid methyl ester so obtained was extracted with n-hexane and analyzed by GLC and GC/MS. Then the reaction mixture was dissolved in distilled water and the sphingosine (long chain base) was extracted be EtOAc ( Mukhtar et al., 2002; Abdel-Razik et al., 2014). 16

22 2.5. GC-MS conditions of methanolysis products of ceramides GC-MS analysis of methanolysis fatty acid methyl ester was performed on a Varian gas chromatograph interface to SSQ 3400 coupled to mass selective detector, the columns used was a DB5, 30M, X0.25 mm, 0.5Mm film thickness. Injector and ion source temperature was 220 ºC, the ionization energy was set at 70 ev, and the volume injected was 0.88 l at 270ºC. The oven temperature was programmed from 50 ºC for 32 minutes, isothermal, then heating by 10 ºC /min to 150 ºC,, isothermal, then heating by 5 ºC /min to 270 ºC, and isothermally for 3min. at 270 ºC. 17

23 2.6. Role of H ghardaqensis chloroform extract against adrenal glands toxicity induced by cadmium chloride Cadmium (Cd 2+ ) is a ubiquitous industrial and environmental pollutant that accumulates in humans. The tissues that accumulate Cd 2+ include the kidneys, lung, reproductive organs and nervous system. Cd 2+ causes genotoxicity and cancer in some tissues and nonmalignant chronic toxicity in others. Cadmium accumulates in the adrenal gland after entering the body. The order of important organs that contain cadmium is adrenal gland > liver > kidney > hypothalamus > cerebral cortex, with the adrenal gland ranked as No. 1 (Min et al., 2008; Leal et al., 2007). The present investigation deals with the histopathological effects of cadmium chloride on adrenal gland of rats. Besides, the study aims at detecting the protective role of chloroform extract of Heteoxenia ghardaqensis on cadmium chloride toxicity Chemicals (CdCl 2 ) Cadmium Chloride was purchased from E. Merck Ag Darmstadt as colorless, odorless crystals. The empirical formula is CdCl 2.H 2 O and the molecular weight is It is freely soluble in water and acetone, slightly soluble in methanol and ethanol, and partially soluble in ether. The oral LD 50 of cadmium chloride to rats is 75 mg/kg.b.w (Weil, 1952). Cadmium solutions were prepared by dissolving cadmium chloride in distilled water. Doses were expressed as milligram of cadmium chloride per kilogram body weight The experimental Animals Male albino rats (Laboratory Animal Colonies, National Research Centre, Cairo, Egypt) weighting g were used in this study. The animals were housed in groups of 5 rats in stainless steel community cages at 22 ± 2 o C for 12 hr. light/dark cycle and allowed to acclimatize for a period of 15 days prior to experimental use. Throughout the experiment, the rats were allowed free access feed (rats dietary pellets prepared by Cairo Company of Oil & Soap, Egypt) and water. All procedures concerning animal treatment and experimentation were in accordance with the 18

24 guiding principles in the care and use of animals, approved and adopted by the local Ethical Commission in the National Research Centre Experimental Design Thirty male rats were used and classified into 5 groups (6 rats / group). Group I: The control group; Group II: Rats given one dose of cadmium chloride daily equivalent to 1/10 LD 50 (8.8 mg/kg.b.w) (Koriem, et al., 2009) for 10 days; Group III: Rats given one dose of cadmium chloride daily equivalent to 1/10 LD 50 (8.8 mg/kg.b.w) and chloroform extract (20 mg/kg.b.w) for 10 days; Group IV: Rats given one dose of cadmium chloride daily equivalent to 1/10 LD 50 (8.8 mg/kg.b.w) and chloroform extract (40 mg/kg.b.w) for 10 days; Group V: Rats given one dose of cadmium chloride daily equivalent to 1/10 LD 50 (8.8 mg/kg.b.w) and chloroform extract (60 mg/kg.b.w) for 10 days. In this work the following investigations were carried out: Histopathological study of adrenal glands Adrenal glands were dissected out and fixed instantaneously in 10% formal saline for 24 hours. The specimens were washed in tap water, dehydrated in ascending grades of ethanol, cleared in xylene, embedded in paraffin wax (m. p o C). Paraffin sections of 6 m thicknesses were prepared and stained with haematoxylin and eosin (Drury and Wallington, 1980). In this method the paraffin sections were stained in hematoxylin for 5 minutes. Sections were washed in running water for bluing and then stained in 1% watery Eosin for 2 minutes, washed in water, dehydrated, cleared and mounted in Canada balsam. The cytoplasm stained shades of pink and red and the nuclei gave blue color. 19

25 2.7. Anticancer activity of some pure compounds Cell Culture Human colorectal adenocarcinoma cell line (Caco-2) and Human hepatocarcinoma cell line (Hep-G2), which were purchased from ATCC, USA, was used to evaluate the cytotoxic effect of the tested compounds. Cells were routinely cultured in Eagle's Minimum Essential Medium which was supplemented with 10% fetal bovine serum (FBS), 2 mm L-glutamine, containing 100 units/ml penicillin G sodium, 100 units/ml streptomycin sulphate, and 250 μg/ml amphotericin B. Cells were maintained at sub-confluence at 37ºC in humidified air containing 5% CO 2. For sub-culturing, monolayer cells were harvested after trypsin/edta treatment at 37 C. Cells were used when confluence had reached 75%. Tested compounds were weighed, dissolved in dimethyl sulphoxide (DMSO), and diluted thousand times in the assay to begin with the intended concentration. All cell culture material was obtained from Cambrex BioScience (Copenhagen, Denmark). All chemicals were purchased from Sigma/Aldrich, USA, except mentioned. All experiments were repeated three times, unless mentioned Reagents preparation MTT solution: 5 mg/ml of MTT in 0.9% NaCl. Acidified isopropanol: 0.04 N HCl in absolute isopropanol Calculation Percentage of relative viability (V) was calculated using the following equation: A treated V% = X 100 A cont Where: V%: Percentage of relative viability; A treated : Absorbance of treated cells; and A cont :Absorbance of control cells The half maximal inhibitory concentration (IC 50 ) was calculated from the equation of the dose response curve. 20

26 Anti-tumor activity Cytotoxicity of the isolated ceramides 1 and 2 and against Hep-G2 and also the isolated gorggostane steroids 4, 5 and 6 against colon cell lines were measured using the MTT cell viability assay. MTT (3-[4,5-dimethylthiazole-2-yl]-2,5- diphenyltetrazolium bromide) assay is based on the ability of active mitochondrial dehydrogenase enzyme of living cells to cleave the tetrazolium rings of the yellow MTT and form a dark blue insoluble formazan crystals which is largely impermeable to cell membranes, resulting in its accumulation within healthy cells. Solubilization of the cells results in the liberation of crystals, which are then solubilized. The number of viable cells is directly proportional to the level of soluble formazan dark blue color. The extent of the reduction of MTT was quantified by measuring the absorbance at 570 nm (Hansen et al., 1989). Cells (0.5X105 cells/ well), in serum-free media, were plated in a flat bottom 96-well microplate, and treated with 20μl of serial dilutions of the tested compounds for 48 h at 37ºC, in a humidified 5% CO 2 atmosphere. After incubation, media were removed and 40 μl MTT solution / well were added and incubated for an additional 4 h. MTT crystals were solubilized by adding 180 μl of acidified isopropanol / well and plate was shaked at room temperature, followed by photometric determination of the absorbance at 570 nm using microplate ELISA reader. Triplicate repeats were performed for each concentration and the average was calculated. Data were expressed as the percentage of relative viability compared with the untreated cells compared with the vehicle control for the solid sample only, with cytotoxicity indicated by <100% relative viability. 21

27 2.8. Antimicrobial activity of chloroform extract of Heteroxenia ghardaqensis and some pure compounds Antimicrobial activity of the extract and some isolated compounds (2; 4 and 11) in comparison with that of the control drug thiophenicol (Sanofi Aventis, Cairo, Egypt) was evaluated. The used microbes in this test were gram positive bacteria (Bacillus subtilis NRRL-B-4219, Staphylococcus aureus ATCC and Micrococcus luteus B-287), gram-negative bacteria (Escherichia coli ATCC 25922, Klebsiella pneumonia and Alcaligenes faecalis B-170) and fungi (Candida albicans ATCC and Aspergillus niger NRRL-363) by the agar diffusion technique (Domig et al., 2007). All microorganisms used were obtained from the culture collection of the Department of Chemistry of Natural and Microbial Products, National Research Centre, Cairo, Egypt. The microorganisms were passaged at least twice to ensure purity and viability. The bacteria were maintained on nutrient agar medium while fungi were maintained on potato dextrose agar medium. DMSO showed no inhibition zone. The compounds were mounted on a concentration of 500 g/disc. Agar media were incubated with different microorganism cultures tested. After 24 h of incubation at 37 C for bacteria and 30 C for fungi, the diameter of inhibition zone in mm was measured. Thiaphenicol was used as a positive control for antimicrobial activity in a concentration of 100 g/disc Preparation of the discs Compounds (2, 4 and 11) and extracts together with the positive control thiaophenicol were mounted on a paper disc prepared from blotting paper (5 mm D) with the help of a micropipette on a concentration of 500 g/10 L DMSO/disc. The discs were applied on the microorganism-grown Agar plates Preparation of agar plates Minimal agar was used for the growth of specific microbial species. The preparation of agar plates for Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Micrococcus luteus, Alcaligenes faecalis and Klebsiella pneumonia (bacteria) utilized nutrient agar (2.3 g; obtained from Panreac Quimica SA, Spain) suspended in freshly distilled water (100 ml), and potato dextrose agar medium (3.9 g/100 ml; 22

28 obtained from Merck) for Candida albicans and Aspergillus niger. This was allowed to soak for 15 min and boiled on a water bath until the agar was completely dissolved. The mixture was autoclaved for 15 min at 120 ºC and then poured into previously sterilized Petri dishes and stored at 30 ºC for inoculation Procedure of inoculation Spore suspension was prepared with the help of a platinum wire loop to reach a microbial concentration equivalent to 0.5 Mac-Farland Application of the discs Sterilized forceps was used for the application of the paper disc on previously inoculated agar plates. When the discs were applied, they were incubated at 37 ºC for 24 h for bacteria and at 30 C for fungi. The zone of inhibition around the disc was measured in millimeters Minimum inhibitory concentration (MIC) The isolated compounds were tested for their antimicrobial activities against gram positive bacteria, gram-negative bacteria, and fungi by paper disc diffusion method (Cooper, 1972). The discs were mounted with samples at concentrations 1000, 500, 250, 125, 64, 32 and 16 μg/l, respectively and applied on previously inoculated agar plates. The lowest concentration of the sample that visibly inhibited growth after overnight incubation was considered to be the MIC. The MICs were read in μg/l. 23

29 Chapter III 3. Results and Discussion 3.1. Investigation of the chemical constituents of Heteroxenia ghardaqensis chloroform extract The frozen marine organism, H. ghardaqensis (4 Kg) was broken down into small pieces at room temperature and extracted with sufficient amount of chloroform, several times until no further residue was obtained to ensure complete extraction. The chloroform extract was filtered, and concentrated under reduced pressure at 50ºC to afford dark brown gum (15 g). The residue (15 g) was subjected to glass silica gel (250 g) column (50 cm L x 6 cm D), eluted with petroleum ether (40-60)/ ethyl acetate step gradient in order of increasing polarity (each fraction 200 ml). Final purification of the compounds occurred by using different chromatographic methods as described in experimental section afforded eleven compounds A new ceramide, 2S,3R-4E,8E-2-(hexadecanoylamino)-docosa-4,8-diene-1,3- diol (1), along with two known ceramides, namely, 2S,3R-4E,8E-2- (octadecanoylamino)- octadeca-4,8-diene-1,3-diol (2) (Inagaki et al., 2004), 2S,3R- 4E-2- (octadecanoylamino)-octadec-4-ene-1-ol (3) (figure 2) (Tian et al., 2001) were isolated from the chloroform extract of H. ghardaqensis (Fig 1A). This is the first report of the spectroscopic data of ceramide (3). Also a new steroid was isolated and identified as gorgostan-3,5,6 -triol-11 -acetate (4) in addition to a new semisynthetic gorgostane derivative (4a). 24

30 O 3` HN 1` 2` n HO m OH 1 n = 11, m = 12 ; n = 13, m = 8 HO HN O RO RO HO OH OH 4 R = H, 4A R = COCH 3 5 R = H; 6 R = COCH 3 R R 1 7 R = OH; R 1 = H; 8 R = OCOCH 3, R 1 = H 9 R = R 1 = OH Figure (2A): Structures of isolated compounds. O n 14 O 10 n = n = 12 NH 2 O HO 12 O OH 1a 1b Figure (2B): Structures of methanolysis products of a new ceramide

31 3.2. Identification of the isolated compounds Compound 1 Compounds identification occurred by chemical methods and modern spectroscopic measurements, including FT-IR, 1D-NMR, 2D-NMR, MS and different chemical elucidation methods. Ceramide 1 was obtained as white crystals (15 mg, mp C [á] 25 D - 7.6º (c 0.01, CHCl 3 )}), HRESI-MS and ESI-MS of compound (1) showed a pseudomolecular ion peak ESI-MS: [M+H] + at m/z: 593, corresponding to the molecular formula C 38 H 74 NO 3. IR spectrum (figure 11) indicated the presence of secondary amide at max 1642 cm -1, and hydroxyl groups at max 3316 cm -1. The 1 H-NMR and 13 C-NMR spectral data (table 4) of (1) indicated that it had a ceramide like structure. The 1 H-NMR spectrum (figure 5) showed signals at 4.29 (t, H-3), 3.90 (m, H-2), 3.88 (dd, H-1b), 3.68 (dd, H-1a) which agree well with those reported for N-acyl-Derythro-sphingosine (Huang et al., 2010; Liu et al., 2010). The spectrum showed proton signals for four olefinic protons at 5.36 (dd, H-9), 5.40 (dd, H-8), (dd, H-4) and 5.76 (dd, H-5). The E geometry was deduced from the trans coupling constants for H-4/H-5, H-8/H-9 and comparing these values with those reported before (Wang et al., 2000; Phillips et al., 2009). 13 C-NMR spectrum (figure 6) showed 18 + n CH 2 carbon resonances. The spectrum revealed the presence of carbonyl carbon at 174.1, as well as the four olefinic carbon signals at (C- 4), (C-8), (C-9) and (C-5). The signals of two methyl groups at 0.88 (6H, t, J=7 Hz) and the very strong signal of polymethylene (CH 2 )n at 1.28 (br s) in the 1 H-NMR spectrum but lack of upfield methine and tertiary carbon signals in the 13 C NMR spectrum revealed that 1 must contain two long branchless carbon chains. It also showed signal for carbon adjacent to an amide group at DEPT spectrum (figure 7) showed two methyl signals, 9 + n CH 2 methylene signals and 7 methine groups. The protonated carbons were assigned by HMQC (figure 8) experiment. Location of double bonds was established by 1 H, 1 H-COSY and HMBC (Fig 3; 9 and 10). In 1 H- 1 H COSY, there were correlations for H-4/H-3, H-5/H-6, and H-7/H- 26

32 8. In HMBC experiment, there were correlations for H-3/C-5 (J 3 ) and H-4/C-3 (J 2 ) which confirmed the location of double bond at C-4. Furthermore, the location of other double bond at C-8 was deduced by correlations of H-8/C-7 (J 2 ), H-9/C-10 (J 2 ) and H-9/C-7 (J 3 ). The relative stereochemistry of ceramide (1) was assigned by comparing the coupling constants values with those previously reported (Huang et al., 2010; Yue et al., 2001; Liu et al., 2010; Han et al., 2005; Abdel-Razik et al., 2014). O HN 10 HO OH 11 : 1 H- 1 H COSY correlations : HMBC correlations Figure (3): 1 H- 1 H COSY and key HMBC correlations of ceramide 1. The length of the long chain base (LCB) and the fatty acid moiety (FA) were determined by ESI-MS (figure 4 and 12) and methanolysis. Two diagnostic fragment aliphatic ions at m/z 394 (calcd for C 24 H 44 NO 3 : 393) and at m/z 198 (calcd for C 14 H 29 : 197) were observed due to -cleavage (McLafferty rearrangement). This indicated the presence of palmityl group as FA in compound (1). Furthermore, the secondary amide position was assigned on basis of typical fragment ions at m/z 256 and m/z 338 which were formed by -cleavage and aliphatic chain fragment. Moreover, the 4,5 and 8,9 double bonds were confirmed on allylic cleavage fragment ions at m/z 368 [C 22 H 42 NO 3 ] + and 224 [C 16 H 31 ] +, which were formed by elimination of dodecene. 27

33 C 22 H 41 O HN O. C 24 H 44 NO C 16 H 32 NO 254. HO C 18 H 36 NO OH.. C 20 H 37 O 293 C 16 H C 12 H Figure (4): ESI-MS fragmentation of ceramide 1. Methanolysis of compound (1) yielded sphingosine (1a) and fatty acid methyl ester (1b) (Fig 1B). The fatty acid methyl ester (1b) was analyzed using GC/MS (figure 13) and FT-IR (figure 14). The GC/MS showed that the molecular ion peak was at m/z 270 (C 16 H 32 OCH 3, Rt 39.83) methyl hexadecanoate). Thus, the lengths of the sphingosine and the fatty chain moieties of compound (1) were unambiguously determined as C22 and C16 for amino alcohol (sphingosin) and fatty acid moities, respectively. According to the above mentioned data, ceramide (1) was characterized as 2S,3R-4E,8E-2-(hexadecanoylamino)-docosa-4,8-diene-1,3-diol which is reported here for the first time in nature. 28

34 Table (4): 1 H- and 13 C-NMR data of 1 No H (J value Hz) 1a 3.91 dd (11.7, 3.8) 1b 3.68 dd (11.7, 3.5) m m dd (15.8, 6.18 ) dt (15.8, 6.18 ) m m dt (15.2, 6.54) dt (15.2, 6.5) m 1` ` 2.22 t (7.5) 3` 1.62 m 16`, brs 17`, brs 18`, t (6.8) (CH 2 )n 1.28 brs NH 6.27 d (7.5) OH 2.93 C

35 Figure (5): 1 H-NMR of compound 1 30

36 Figure (6): 13 C-NMR of compound 1 31

37 Figure (7): DEPT of compound 1 32

38 Figure (8): HMQC of compound 1 33

39 Figure (9): 1 H, 1 H, COSY of compound 1 34

40 Figure (10): HMBC of compound 1 35

41 Figure (11): FT-IR of compound 1 Figure (12): ESI-MS of compound 1 36

42 Figure (13): GC/MS of long chain fatty acid 1a 37

43 Figure (14): FT-IR of compound 1b 38

44 Compound 2 It was obtained as a yellow powder (10 mg, mp C). R f = 0.55 (pet ether (40-60 ºC)/EtOAc), colorless under both long and short UV light, gave brown color with both Ce(SO 4 ) 2 and brightness white with MeOH/H 2 SO 4 (3:1) spraying reagents. IR max (KBr) cm 1 ; 3315, 2920, 2850, 1638, 721. ESI-MS (M+H) + ; at m/z 564 (M +, 100%), 521(40%), 451 (35%), 395 (45%), 339 (35%), 327 (60%), 283 (80%), 282 (75%), 238 (60%), 225 (53%), 168 (55%), 113 (40%). 1 H and 13 C-NMR (CDCl 3, 600 MHz) spectral data [Table (5)]. The IR spectrum (Fig 21) data indicated the presence of secondary amide at 1638 cm -1, hydroxyl groups at 3315 cm -1, and the long alphatic chains at 721 cm -1. ESI-MS of compound 2 (Fig 22) showed a molecular ion peak at [M+H] + m/z: 564 corresponding to the molecular formula C 36 H 69 NO 3. 1 H-NMR and 13 C-NMR spectra of compound 2 indicated that this compound has a ceramide like structure and very closed to ceramide 1. 1 H-NMR spectrum (Fig 15) revealed signals at 4.02 (t, H-3), 3.82 (m, H-2) 3.66 (dd, H-1b) and 3.86 (dd, H-1a) which agreed well with those reported for unusual N-acyl-D-erythro sphingosine. In addition, the spectrum showed also an intense broad singlet at 1.28 (brs) for poly methylene (CH 2 ) n of fatty moieties. The spectrum showed the four proton signals for four olefinic protons at 5.44 (d, H-4), 5.68 (d, H-5), (d, H-8) and (d, H-9). In addition, the spectra displayed signal for one pair of methylene protons attached to carbonyl group at 2.17 (t, H-2'). The E geometry was deduced from the vicinal coupling constants between H-4/H-5, H-8/H-9 and comprising with structurally related compounds (Phillips et al., 2009). 13 C-NMR spectrum (Fig 16) showed 18 + n CH 2 carbon resonances. It revealed the presence of carbonyl carbon at C 174.8, as well as the four olefinic carbon signals at (C- 4), (C-8), (C-9) and (C-5). It also showed signal for carbon adjacent to an amide group at DEPT 135º experiments (Fig 17) showed two methyl signals, 9 + n CH 2 methylene signals and 6 methine groups. The protonated carbons were assigned by HMQC experiment (Fig 18). Location of double bonds at C-4, and C-8 was established by 1 H, 1 H, COSY and HMBC. In 1 H, 1 H, COSY (Fig 39

45 19), there was a correlations for H-4/H-5, H-4/H-3, H-5/H-6, H-7/H-8 and H-8/H-9. In HMBC, there was a correlation for H-3/C-5 and H-4/C-3, (J 2 ). Also HMBC (Fig 20) showed a correlation for H-5/C-6 (J 2 ); C-7 (J 3 ). Furthermore, the location of other double bond at C-8 was deduced by correlation of H-8/C-7 (J 2 ) H-9/C-10 (J 2 ), and H- 9/C-7 (J 3 ). The relative stereochemistry of ceramide 2 was assigned by comparing the coupling constants values with reported data (Wang et al., 2000; Patra et al., 2003; Huang et al., 2010; Han et al., 2005; Mohamed et al., 2012). The length of the long chain base (LCB) and the fatty acid (FA) were determined by ESI-MS. Two diagnostic fragment ions at m/z 327 (calcd for C 20 H 40 NO 2 : 326) and at m/z 238 (calcd for C 16 H 29 O: 237) were observed due to - cleavage (Mclaeverty rearrangement). This is indicated the presence of stearyl group in FA and octadiene in compound 2 (Scheme 4). Furthermore, the secondry amide position was assigned on basis of typical fragment ions at m/z 282 and m/z 327 which were formed by -cleavage and aliphatic chain fragment at m/z 283. Moreover, the 4,5 and 8,9 double bonds was confirmed on the fragment ion at m/z 238 [C 18 H 34 ] + and 170 [C 12 H 26 ] +, which were formed by elimination of dodecene via Mclaeverty rearrangement (scheme 2). According to the aforementioned data, ceramide 2 was characterized as 2S,3R- 4E,8E-2-(octadecanoylamino)-octadeca-4,8-diene-1,3-diol (Inagaki et al., 2004) which has not reported previously from this species especially H. ghardaqensis. 40

46 Table (5): 1 H- and 13 C-NMR data of 2 No H (J value Hz) 1a 3.86 dd (11.5, 3.6) 1b 3.66 d dd (11.5, 3.6) m t (7.5) d (7.5 and 15.7) d (8.8 and 15.5) m m d (7.5 and 15.7) d (8.8 and 15.5) m 1` ` 2.17 t (7.5) 3` 1.57 m 16`, brs 17`, brs 17`, t (7.5) (CH 2 )n 1.28 brs NH 5.39 d (7.0) C

47 Fig (15): 1 H-NMR of compound 2 Fig (16): 13 C-NMR of compound 2 42

48 Fig (17): DEPT of compound 2 Fig (18): HMQC of compound 2 43

49 Fig (19): 1 H, 1 H, COSY of compound 2 Fig (20): HMBC of compound 2 44

50 Fig (21): FT-IR of compound 2 Fig (22): ESI-MS of compound 2 45

51 C 18 H 34 O 2 m/z: 283 C 20 H 37 NO 3 m/z:340 O C 16 H 32 m/z: 225 C 18 H 37 NO m/z: 284 HN 13 HO 6 OH C 24 H 46 NO 3 m/z:397 C 12 H 24 m/z:169 C 28 H 53 NO 3 m/z: 452 C 16 H 29 O m/z: 238 C 8 H 16 m/z:113 Scheme 4: Suggested mass fragmentation of compound 2 46

52 Compound 3: It was obtained as a white amorphous solid (10 mg, mp C). R f = 0.70 (pet ether (40-60) / EtOAc 9:1), colorless under both long and short UV light, gave brownish red color with MeOH/H 2 SO 4 (3:1) and grey color with both Ce(SO 4 ) 2. IR max (KBr) cm 1 ; 3445, 2922, 2853, 1642, 723. ESI-MS (M+H) + ; at m/z 550 (100%, M +, C 36 H 71 NO 2 ), 507 (45%, C 33 H 65 NO 2 ), 481 (35%, C 31 H 61 NO 2 ), 381 (25%, C 24 H 46 NO 2 ), 325 (55%, C 20 H 40 NO 2 ), 283 (80%, C 18 H 36 NO), 269 (75%, C 18 H 35 O), 225 (60%, C 16 H 23 ), 168 (30%, C 12 H 23 ). 1 H, 13 C-NMR (CDCl 3, 600 MHz) spectral data [Table (6)]. The IR (Fig 29) spectral data of compound 3 showed that it also has ceramide structure. It revealed a peak for secondary amide at 1642 cm -1, hydroxyl group at 3445 cm -1, and the long aliphatic chains at 723 cm -1. ESI-MS of compound 3 (Fig 30) showed a molecular ion peak at [M+H] at m/z 550 corresponding to the molecular formula C 36 H 71 NO 2. 1 H-NMR and 13 C-NMR spectra of compound 3 was similar to those for ceramides 1 and 2, except the absence of the signals characteristic to the hydroxyl group at C-3 and the double bond C-8. 1 H NMR spectrum (Fig 23) showed signals at 4.02 (H-1a), 3.64 (H-1b) and 3.85 (H-2) typical for N-acyl-D-erythro sphingosine structures. In addition, only two olefinic protons signals at 5.36 (H-4) and 5.38 (H-5). The E geometry was deduced from the vicinal coupling constants between H-4/H-5 and comprising with structurally related compounds (Phillips et al., 2009). 13 C-NMR spectrum (Fig 24) showed 12 + n CH 2 carbon resonances. It showed a signal at corresponding to the polymethylene carbons of fatty moieties. In addition, the spectrum showed two signals for two olefinic carbons at and corresponding to C-4 and C-5 respectively. DEPT 135º (Fig 25) experiment showed one signal for two methyls, 7 + n CH 2 methylene signals and three methine groups. The protonated carbons were assigned by HMQC experiment (Fig 26). 1 H, 1 H, COSY (Fig 27) showed correlation between H-2/H-3 and H-3/H-4 confirming the position of double bond. The location of the double bond at C-4 was confirmed by HMBC (Fig 28) in which a correlation between H-2 and C-4 (J 3 ). The relative 47

53 stereochemistry of ceramide 3 was assigned by comparing the coupling constants values with reported data (Wang et al., 2000; Huang et al., 2010; Han et al., 2005; Mohamed et al., 2012). The length of the long chain base (LCB) and the fatty acid (FA) were determined by ESI-MS through the presence of two important fragment ions at m/z 283 (calcd for C 18 H 36 NO: 282) and at m/z 268 (calcd for C 18 H 35 O: 267) were observed due to -cleavage (Mclaeverty rearrangement) (Scheme 5). In addition, the location of the double bond at C-4 was confirmed by the fragment ion at m/z 381 (C 24 H 46 NO) and 169 (C 12 H 24 ) disconnections due to Mclaeverty rearrangement. Based upon these data, compound G9 was determined as 2S,3R-4E-2- (octadecanoylamino)-octadec-4-ene-1-ol (Yano et al., 1998) which has been reported from Heteroxenia species especially H. ghardaqensis. Also this is the first report for the spectroscopic data for this compound. 48

54 Table (6): 1 H- and 13 C-NMR data of 3 No 1a 1b ` 2` 3` 16`, 16 17`, 17 18`, 18 (CH 2 )n NH H (J value Hz) 4.02 (dd, 11.5, 3.8) 3.64 (dd, 11.5, 3.8) 3.85 m 2.25 m 5.30 m 5.35 m 1.98 m t (7.5) 1.58 m 1.26 brs 1.26 brs 0.86 t (7.5) 1.26 brs 5.32 br d (7.0) C

55 Fig (23): 1 H-NMR of compound 3 Fig (24): 13 C-NMR of compound 3 50

56 Fig (25): DEPT of compound 3 Fig (26): HMQC of compound 3 51

57 Fig (27): 1 H, 1 H, COSY of compound 3 Fig (28): HMBC of compound 3 52

58 Fig (29): FT-IR of compound 3 Fig (30): ESI-MS of compound 3 53

59 C 18 H 34 O m/z: 264 O C 20 H 39 NO 2 m/z:326 C 16 H 32 m/z: 225 C 18 H 37 NO m/z: 284 HO HN C 16 H 32 m/z: 238 Scheme 5: Suggested mass fragmentation of compound 3 C 24 H 46 NO 2 m/z:381 C 12 H 24 m/z:169 54

60 Compound 4: It was obtained as a white powder (30 mg, mp 227 C). R f = 0.50 (hexane / EtOAc 4:1), gave brown color under UV light, gave dark brown color with vanillin/h 2 SO 4 also grey color with both Ce(SO 4 ) 2 and MeOH/H 2 SO 4 (3:1) spraying reagents. EI-MS (M+H) + ; m/z 426 (10%), 425 (20%), 423 (70%), 409 (100%), 400 (35%), 383 (35%), 311 (15%), 255 (8%). 1 H, 13 C-NMR (CDCl 3, 600 MHz) spectral data as reported in Table (7). Compound 4 was deduced to have the molecular formula C 30 H 50 O by positive mode EI-MS (Fig 36); at m/z H NMR spectrum of compound 4 (Fig 31) showed characteristic signals at H (1H, H-30), 0.15 (2H, m, H-22, H-24), and at 0.44 (1H, H-30), thus requiring the presence of a cyclopropyl ring. It also showed signals for seven methyls. The spectra also showed signal at 3.51 (1H, m, H-3) indicate the presence of methane proton attached to hydroxyl group. Also showed signal for olefinic proton at 5.34 (1H, b s, H-6). The characteristic signals due to a cyclopropyl moiety were identical with the signals for seven methyls were consistent with a gorgostane skeleton. The 13 C NMR (Fig 31) showed 30 carbon resonances The 13 C NMR spectrum showed one methine carbon bearing hydroxyl group at 71.8 in addition of two olefinic carbons at and at DEPT 135º (Fig 32) confirmed the proposed structure and showed seven methyls signals, ten methine groups, and four quaternary carbons. The protonated carbons were assigned by HMQC (Fig 33) experiment. The 1 H, 1 H, COSY (Fig 34) showed correlations between both H-2/H-3 and H-3/H-4 that induced that the hydroxyl group located in C-3. Also the correlation between H-6/H-7 indicated the presence of double bond between C-5 and C-6. HMBC (Fig 35) experiment that showed cross peaks correlations between H-2/C-3, H-3/C-4, H-4/C-5 and H-7/C-6 that supported the location of hydroxylated C-3 in addition to C-5, C-6 double bond. From the above data, the structure of compound 1 could be assigned as gorgosten-5(e)-3- -ol. It was confirmed by comparison of its spectral data with published data (Wagner et al., 1970; Musmar et al., 1983). This compound was previously reported from soft coral Sarcophyton trocheliophorum and from gorgonian Isis hippuris (Tanaka et al., 1982; 55

61 Mohamed et al., 2012). But, this is the first time for isolation of this compound from Egyptian Red Sea H. ghardaqensis. Table (7): 1 H- and 13 C-NMR data of 4 No H (J value Hz) 2.33 m, 1.43 m 1.84 m, 1.46 m 3.5 m 2.22 m, 1.23 m m 1.88 m, 1.44 m 1.88 m 1.04 m m 1.57 m, 1.42 m m 1.53 m, 1.07 m 1.66 m, 1.17 m 1.24 m 0.66 s 1.46 s 1.04 m 0.99 s 0.15 ddd (4, 6, 9) dq (7, 8) 1.54 m 0.94 d (6) 0.83 d (6) 0.92 d (6) 0.88 s dd (4, 4); 0.44 dd (4, 9) C

62 Fig (31): 1 H-NMR of compound 4 57

63 Fig (3): 13 C-NMR of compound 4 Fig (32): DEPT of compound 4 58

64 Fig (33): HMQC of compound 4 Fig (34): 1 H, 1 H, COSY of 4 59

65 Fig (35): HMBC of compound 4 Fig (36): EI-MS of compound 4 60

66 Compound 4a: It was obtained as a white powder (8 mg, mp C; {[ ] 25 D (c 0.01, CHCl 3 )}). R f = 0.80 (hexane / EtOAc 4:1), gave dark violet color under UV light, gave dark orang color with both Ce(SO 4 ) 2 and MeOH/H 2 SO 4 (3:1) spraying reagents. IR max (KBr) cm 1 ; 2956, 1748, 1446, 1382, 1372, 1261, 1022, 858; EI- MS(M+H) + : 469 (15%), 468 (5%), 423 (20%), 410 (100%), 391 (15%), 329 (10%), 282 (10%), 266 (30%), 252 (40%), 237 (20%), 179 (10%), 147 (10%), 83 (15). 1 H- NMR (CDCl 3, 500 MHz) spectral data as reported in Table (8). Compound 4a was deduced to have the molecular formula C 32 H 52 O 2 by positive mode EI-MS (Fig 40); m/z 469. The acetylation of compound 4 to 4a (Shen et al., 2001) was deduced by IR (Fig 39). The absence of the broad beak of OH in average and the appearance of peak at 1748 cm -1 indicating presence of carbonyl group. The 1 H NMR spectrum (Fig. 37) showed the downfield shift of proton H-3 at 4.71, and methyl signal at 2.02 (s, 3H). 13 C-NMR spectrum (Fig. 38) showed the downfield shieft of C-3 at 74.1 with appearance of methyl carbon signal at 21.5 that indicate presence of acetoxy group at C-3. The structure of compound 4a was deduced to be gorgosten-5(e)-3- -acetate. This is the first report for this compound, naturally or synthetically. 61

67 Table (8): 1 H- and 13 C-NMR data of 4a No H (J value Hz) 2.12 m, 1.42 m 1.84 m, 1.48 m 4.71 m 2.31 m, 1.26 m m 1.88 m, 1.44 m 1.88 m 1.09 m m 1.56 m, 1.48 m m 1.54 m, 1.09 m 1.58 m, 1.18 m 1.24 m 0.65 s 1.26 s 1.15 m 1.00 s 0.16 ddd (4, 6, 9) dq (7, 8) 1.54 m 0.94 d (6) 0.83 d (6) 0.92 d (6) 0.88 s dd (4, 4); 0.45 dd (4, 9) (s, 3H) C

68 Fig (37): 1 H-NMR of compound 4a 63

69 Fig (38): 13 C-NMR of compound 4a Fig (39): FT-IR of compound 4a 64

70 Fig (40): EI-MS of compound 4a 65

71 Compound 5: It was obtained as a white powder (25.0 mg, mp 283 C). R f = 0.40 (EtOAc 100%) and 0.65 (EtOAc/MeOH 9:1), gave dark brown color under UV light, gave brownish yellow color with both Ce(SO 4 ) 2 and MeOH/H 2 SO 4 (3:1) spraying reagents. ESI-MS (M+H) + : at m/z 476 (15%), 475 (35%), 443 (5%), 393 (35%), 368 (100%), 340 (5%), 315 (10%), 123 (10%). 1 H-NMR, 13 C-NMR (CDCl 3, 600 MHz) spectral data see Table (9). Compound 5 was deduced to have the molecular formula C 30 H 52 O 4 by positive mode ESI-MS; [M + H] + at m/z 476 (Fig. 47). 1 H-NMR (Fig. 41) data of compound 5 showed characteristic signals at H 3.40 (1 H, br s, H-6), 3.79 (1 H, m, H-11) and 3.96 (1 H, m, H-3) for three hydroxyl-methine protons. It also showed signals characteristic to a cyclopropane-bearing gorgosterol- type side chain at (1H, dd, J = 4, 4, H-30), 0.45 (1H, dd, J = 4, 9, H-30) and 0.17 (1H, ddd, J = 4, 6, 9, H- 22). It also contained seven methyl singals at 0.68 (3H, s, Me-18), 1.23 (3H, s, CH 3-19), 1.01 (3H, s, CH 3-21), 0.94 d (3H, d, J = 6, CH 3-26), 0.90 (3H, s, CH 3-29), 0.85 (3H, d, J = 6, CH 3-27), and 0.95 (3H, d, J = 6, CH 3-28) which also consistent with the gorgosterol skeleton (Tanaka et al., 1982). The 13 C NMR showed 30 carbon resonances. The 13 C-NMR (Fig. 42) spectrum showed four hydroxylated carbons 66.1, 67.3, 74.7 and 75.5 of C-3, C-11, C-6 and C-5 respectively. DEPT 135º (Fig. 43) showed seven methyl signals, eight methylene signals, eleven methine groups, and four quaternary carbons. The protonated carbons were assigned by HMQC (Fig. 44) experiment. 1 H, 1 H, COSY (Fig. 45) showed correlations between H- 2/H-3, H-3/H-4, H-6/H-7, H-9/H-11 and H-11/H-12 that indicated the presence of four hydroxyl groups at C-3, C-5, C-6, and C-11. The location of hydroxyl groups was supported by HMBC (Fig. 46) experiment that showed cross peaks correlations between H-2/C-3, H-3/C-4, H-4/C-5, H-6/C-7, H-9/C-11 and H-12/C-11. The above spectral data and discussion, the structure of compound 5 was assigned as gorgostan- 3,5,6,11 -tetraol (sarcoaldosterol A). It was confirmed by comparison of its spectral data with published values (Umeyama et al., 1996). This compound was previously reported from soft coral Sarcophyton species collected from Okinawa 66