Chemical and Bioactivity Studies of the Red Sea Soft Corals: Sinularia polydactyla (Ehrenberg) and Lobophytum crassum (Marenzellar) (Fam. Alcyoniidae) Thesis submitted by Nabil Mohammed Aboul-Fotouh Selim Assistant Lecturer, Pharmacognosy Department, Faculty of Pharmacy Cairo University For the degree of Ph.D. in Pharmaceutical Sciences (Pharmacognosy) Under the supervision of Prof. Dr. Elsayed Ali Hassan Aboutabl Pharmacognosy Department, Faculty of Pharmacy, Cairo University Prof. Dr. Shadia Mazloum Azzam Pharmacognosy Department, Faculty of Pharmacy, Cairo University Prof. Dr. Camilia George Michel Pharmacognosy Department, Faculty of Pharmacy, Cairo University Prof. Dr. Ahmed Abdel Fattah Hussein Chemistry of Medicinal plants Department, National Research Center, Cairo Pharmacognosy Department Faculty of Pharmacy Cairo University A.R.E 2012 List of Tables List of Tables Table Page No. 1 Diterpenes reported in different Sinularia species…………………….. 2 Sesquiterpenes reported in different Sinularia species ………………... 35 3 Polyhydroxysterols reported in different Sinularia species …………… 41 4 Miscellaneous compounds reported in different Sinularia species……. 48 5 Diterpenes reported in different Lobophytum species…………………. 50 6 Polyhydroxysterols reported in different Lobophytum species………... 66 7 Miscellaneous compounds reported in different Lobophytum species… 71 8 Proximate analysis of the air-dried soft coral; Sinularia polydactyla 104 15 and Lobophytum crassum……………………………………………… 9 Percentages of different elements in the ash of Sinularia polydactyla 105 and Lobophytum crassum……………………………………………… 10 Yield of the EtOAc extracts from Sinularia polydactyla and 107 Lobophytum crassum ………………………………………………….. 11 Fractionation of the EtOAc extract from soft coral Sinularia 108 polydactyla……………………………………………………………... 12 Fractionation results of the fraction XXII……………………………... 110 13 Fractionation results of the fraction XIV5-7……………………………. 111 14 Fractionation of the EtOAc extract from soft coral Lobophytum 114 crassum………………………………………………………………… 15 Fractionation results of fraction IX…………………………………….. 116 16 Fractionation results of fraction XIV…………………………………... 117 17 NMR data for compound S3 (CDCl3, 600 MHz) ……………………... 131 18 NMR data for compound L4 (CDCl3, 600 MHz) …………………….. 156 19 NMR data for compound L5 (CDCl3, 600 MHz) ……………………... 165 iv List of Tables 20 NMR data for compound L6 (CDCl3, 600 MHz) ……………………... 21 Cytotoxic activity and IC50 (µg/mL) of the EtOAc extract of Sinularia 185 171 polydactyla on different human cancer cell lines……………………… 22 IC50 (µg/mL) of compounds (S1-S3) isolated from Sinularia 188 polydactyla and the standards against human cancer cell lines ……….. 23 Cytotoxic activity and IC50 (µg/mL) of the EtOAc extract of 190 Lobophytum crassum on different human cancer cell lines……………. 24 IC50 (µg/mL) of compounds (L1- L6) isolated from Lobophytum 194 crasssum and the standards against human cancer cell lines…………... 25 Antimicrobial screening of the EtOAc extracts of Sinularia 196 polydactyla and Lobophytum crassum…………………………………. 26 Minimum inhibitory concentrations (MIC µg/mL) of the EtOAc 198 extracts of Sinularia polydactyla and Lobophytum crassum................... 27 Antimicrobial screening (inhibition zone in mm) of isolated 200 metabolites (S1- S3) from Sinularia polydactyla ……………………... 28 Antimicrobial screening (inhibition zone in mm) of isolated 201 metabolites (L1- L6) from Lobophytum crassum ……………………... 29 Minimum inhibitory concentrations (MIC µg/mL) of the isolated 202 compounds from Sinularia polydactyla and Lobophytum crassum........ 30 Antiviral activity screening of the EtOAc extracts of Sinularia 204 polydactyla and Lobophytum crassum on HAV-10, HSV-1, HSV-2 and Cox B4 cultured on Vero cells……….............................................. v Introduction 1. Introduction Oceans are considered to be a great source of potential drugs. In contrast to work on terrestrial natural products, marine natural products have attracted the attention of scientists from different disciplines; viz organic chemistry, bioorganic chemistry, pharmacology, biology and ecology, as a result of the increasing demand for new drug discovery. This interest has led to the discovery of over 16,000 marine natural products to-date and many of the compounds have shown very promising biological activities. The first significant study of marine natural products started just 60 years ago with the pioneering work of Bergman on nucleosides from the sponges (Bergman and Feeney, 1951). Despite the fact that sea offers a rich source of biodiversity from which a series of potential drugs, particularly in the area of cancer chemotherapy, have already been discovered; yet research into the use of marine natural products as medicinal agents is still in its infancy. This may be due to the lack of ethnomedical history and the difficulties involved in the collection of marine organisms. But, with the implementation of scuba diving tools, development of new diving techniques and remote-operated machines, collection of marine organisms has been possible. In addition, the development of sophisticated techniques as well as the establishment of many oceanographic institutes all over the coastal territories of the world facilitates collection, isolation and elucidation of structures from marine natural products. Marine organisms have been found to be storehouses of a variety of secondary metabolites (Faulkner, 2002). In particular, soft corals are a rich source of terpenoids, mainly cembranoid diterpenes, polyhydroxylated steroids and other compounds with amazing diverse structures (Coll, 1992; 1 Introduction Venkateswarlu et al., 2001). Soft corals of the genera Sinularia and Lobophytum (Alcyoniidae) are widely distributed over most habitat types with colonies of various forms having grey, yellow, brown, or green colours (Fabricius and Alderslade, 2001). The ease of their collection facilitated extensive chemical investigation of their diverse and unique metabolites (Tursch et al., 1978 a). Aim of the Work: This study aims to investigate the bioactive metabolites of the Red Sea soft corals Sinularia polydactyla (Ehrenberg, 1834) and Lobophytum crassum (Marenzellar, 1886), Family Alcyoniidae. The main goals are: isolation, purification and identification of active constituents and evaluation of the biological activities including; antimicrobial, anticancer, antiviral and anti-inflammatory activities. The present work includes:1) Review of Literature. 2) Taxonomy of the corals. 3) Collection of Sinularia polydactyla and Lobophytum crassum from a specific location on the Egyptian Red Sea coast. 4) Confirmation of the identity of the soft corals collected from the Red Sea. 5) Preliminary chemical screening and proximate analysis of the samples under investigation. 6) Extraction with suitable organic solvents. 7) Bioactivity-guided fractionation of extracts and isolation of active constituents from biologically 2 active fractions by different Introduction chromatographic techniques: viz, TLC, vacuum liquid chromatography, Gel chromatography, CC on Silica gel, Sephadex LH20, etc. 8) Elucidation of structure of isolated pure bioactive compounds by spectral tools (UV, MS, 1H-NMR, 13C-NMR, 2D-NMR). 9) Evaluation of certain bioactivities (anti-inflammatory, cytotoxicity and antimicrobial) of the pure compounds isolated from the soft coral under investigation. 3 Review of Literature 2. Review of Literature 2.1. Drug Development from Marine Natural Products: Drug development from natural products is dominated by medicinal chemists. The development phase, where there is the pressing need for supply of these compounds, will need strong leadership from natural products chemists, as not all marine-based drugs can be synthesized, or obtained by fermentation technology. There will be a need for new and innovative approaches to the aquaculture of many species from phyla that traditionally have not been subjected to aquaculture. For those compounds that do become marketable drugs, there will be the challenge to grow the producing organisms commercially by aquaculture. The chemists will continue to search for new leads. Marine compounds have different biological and pharmacological effects e.g. anthelmentic, antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial, antiplatelet, antiprotozoal, antituberculosis and antiviral activities. Other marine compounds have significant effects on the cardiovascular, immune and nervous systems and some isolated marine compounds act on specific molecular targets or have miscellaneous mechanisms of action in the body (Mayer and Hamann, 2002:2005). In an early review covering the marine literature up to early 1986, (Munro et al., 1987), 185 bioactive compounds were reported. Statistical data from the US National Cancer Institute (NCI) screening program clearly indicated that marine invertebrates are a great source of bioactive compounds with pharmaceutical potential, due to the much higher molecular suitability of significant cytotoxic activity (Garson, 1994). At present, due to the increasing number of victims annually, require an outstanding potential to be directed for searching of new drugs or lead compounds. Almost 60% of drugs approved for cancer treatment are of natural origin. Vincristine, irinotecan, etoposide and taxanes are all examples of plant origin. 4 Review of Literature Dactinomicine, anthracyclines, mitomycin and bleomycin are anticancer agents derived from microbial sources. There is still great need to discover new chemotherapeutic agents, especially for solid human cancers such as lung, colon, breast, ovarian, prostate, pancreas and brain (Cassady, 1990; Mayer and Gustafson, 2004). Since the conditions in marine environment are so different from those on land, most classes of marine organisms show molecules with unique structural features. Marine organisms, such as sponges and soft corals, are sessile with soft fragile tissues and without physical defense capability against their potential predators. However, these animals can survive in the competitive environments, because of their chemical defense strategy. They have evolved the ability to synthesize toxic compounds or to obtain them from marine microorganisms (symbiotic relationships). These compounds are released into water to defend the animal against its natural predators (Changyun et al., 2008). 2.2. Ecological Roles of Secondary Metabolites in Soft Corals:The secondary metabolites in marine organisms play a number of ecological roles including predator defense, interspecific competition for space, anti-fouling, and reproduction (Bakus et al., 1986). 2.2.1. Predator Defense Soft corals are marine benthic sessile invertebrates that usually attach to the reefs and lack hard exoskeleton for defense. When encountering their predators, they cannot escape freely and quickly. However, they can survive in the competitive coral reef environment against numerous predators, depending on their toxic and antifeeding properties of secondary metabolites (Changyun et al., 2008). For example, the crude extracts of the tropical soft 5 Review of Literature corals Sinularia polydactyla and another Sinularia sp. exhibited antifeeding activities on fish predators (Alstyne et al., 1994). Studies on many soft corals showed that terpenes and sterols are the most common antifeeding secondary metabolites. Bioassay-guided fractionation of the extract of Lobophytum schoedei led to the isolation of a new cembrane-type diterpenoid, lobophynin C, with ichthyotoxicity against mosquito fish O. latipes and the lethal activity towards brine shrimps. Sarcotol and 13membered carbocyclic cembranoids, were isolated from the unidentified soft coral Sarcophyton species, and these compounds showed strong ichthyotoxic activity against Japanese killifish (mosquito fish) Oryzia latipes (Iwagawa et al., 1995). The toxicity effect ranges from respiratory stress to mortality. This test is used as an indicator of the presence of cytotoxic compounds. 2.2.2. Competition for Space Many studies have been carried out on the allelopathic effects observed in the interactions between alcyonacean (soft) and scleractinian (hard) corals. These effects include retardation of growth and tissue necrosis or mortality of scleractinian via direct tissue to tissue contact. Mortality may also occur as a result of passage of allelochemicals through the water column in the absence of contact (Coll et al., 1982). In this way, soft corals are able to maintain and expand their living space (Sammarco et al., 1983(. The effectiveness of the allelopathic compounds responsible for these harmful responses varies greatly between species. The susceptibility of the competing species to the released toxins is also highly species-specific (La Barre et al., 1986; Sammarco et al., 1985). Terpenoids function as allelopathic agents in interspecific competition for space in certain alcyonaceans (La Barre et al., 1986; Sammarco et al., 1983). These chemically-mediated competitive abilities in the Alcyonacea may be enhanced by various behavioral and physiological adaptations. Such 6 Review of Literature adaptation may include: (1) alteration of internal hydrostatic pressure to cause bending of the organism away from its competitor, (2) secretion of a polysaccharides cuticle to protect itself against its competitor’s offensive or defensive mechanisms; (3) redirected growth away from and sometimes over its competitor and (4) sweeper tentacles in some Caribbean and Indo-Pacific gorgonaceans (La Barre and Coll 1982; La Barre et al., 1986; Sebens and Miles, 1988). The competitive balance between certain species of corals can also be controlled by environmental factors (i.e., temperature, wave action, light, predation, and food, etc. (Alino et al., 1992). 2.2.3. Antifouling A range of cembranoid diterpenes isolated from Lobophytum pauciflorum soft coral inhibited the growth of the common filamentous alga Ceramium codii (Coll et al., 1987). Recent investigations of associations between soft corals and the red alga Plocamium hamatum have revealed that the chlorinated monoterpene, chloromertensine present in this alga can kill the soft coral Sinularia cruciata (De Nys et al., 1991). Chloromertensine exhibited antifeedant and antifouling properties (Hay et al., 1989). 2.2.4. Reproduction Many ranges of secondary metabolites also appear to play an important role in reproduction in the alcyonaceans. The diterpenes, epi-thunbergol and pukalide, are present in the eggs of alcyonaceans Lobophytum compactum and Sinularia abrupt, respectively (Bowden et al., 1985; Coll et al., 1989 a). These egg-specific terpenes do not appear to be effective antifeedants against reef fish, as fish consume large amounts of the eggs. These also don’t appear to be effective antibiotics against marine bacteria. However, recent evidence suggests that they play a role in spawning. Both epi-thunbergol and pukalide 7 Review of Literature stimulate polyp contraction and thus egg release (Sammarco and Coll, 1992; Pass et al., 1989). Soft corals are found in reefs all around the world. It is estimated that they cover about 37% of the reef area in the Great Barrier Reef, but they can be found wherever there are reefs, even in Antarctica. They are more conspicuous, and therefore more often studied, in the Red Sea and in the Indo-Pacific as compared to, for example, the Caribbean or Hawaii. Because they are sessile filter feeders, they fill a niche similar to that of hard corals and sponges. Ideal conditions for colonies to develop depend on the species. Generally, soft corals prefer water with a relatively high pH, from 8.2-8.4 (Fenner 1998). Symbiotic microorganisms (Zooxanthellate) species need large amounts of light, and are therefore likely to be found fairly close to the surface of soft corals. All soft corals rely on ocean currents to bring them food, nutrients, and oxygen, so they do not often grow in highly sheltered areas. Ideal substrates for soft corals include rocks, hard corals, and crustose coralline algae (Alderslade, 2001). It is also beneficial to the corals to settle in a small crack or under an overhang on the substrate, as this helps protect the founder polyp from being eaten by fish and sea urchins. Early, Savigny (1817), Ehrenberg (1834), Klunzinger (1877), Kükenthal (1902, 1904, 1913), Thomson and McQueen (1907), Gohar (1940), and others established numerous new taxa of soft corals based on scientific expeditions and investigations in the region (Gohar 1940: Benayahu, 1990). These early investigations into the soft corals formed the cornerstone for later biodiversity and taxonomic studies on this group, not only in the Red Sea, but also throughout the Indian and Pacific Oceans. Later collections from the Red Sea reefs led to numerous comprehensive studies on the taxonomy of their soft corals (Benayahu, 1990; Reinicke, 1997; 8 Review of Literature Verseveldt, 1982; Verseveldt and Benayahu, 1983; and 1978). These publications stimulated an awareness of the high diversity of soft corals in the Red Sea, where they constitute the second most important benthic component on the reefs (Fishelson, 1970; Benayahu and Loya, 1977; and 1987; Sheppard et al., 1992). 2.3. Chemical Diversity Coral reefs represent an extraordinary diverse biota in tropical environments, and soft corals often constitute a dominant part of the reef biomass. Soft corals attracted considerable attention, because of the wide range of bioactive secondary metabolites, generated by these marine invertebrates. The interest began with the isolation of the prostaglandin [(15R)-PGA2] from the gorgonian Plexaura homomalla Esper, 1792 (Weinheimer and Spraggins, 1969). This was followed, in the early 1970s, with the examination of the Alcyonacean octocorals. Chemists came to the conclusion that terpenoid chemistry predominates across the Octocorallia and Tursch et al. (1978 a) surveyed the known distribution of terpenoids across the Octocorallia. Coll (1992) reviewed the chemistry and chemical ecology of Octocorals and made several contributions to the ecological reasons for the structural chemical diversity. 2.3.1. Bioactive Constituents of the Genus Sinularia: The genus Sinularia is one of the most widely distributed soft corals. It constitutes a dominant portion of the biomass in the tropical reef environment. Sinularia elaborates a rich harvest of secondary metabolites including sesquiterpenes, diterpenes, polyhydroxylated steroids and polyamine compounds. These metabolites were recently shown to possess a range of biological activities, such as: antimicrobial, anti-inflammatory and cytotoxic activities (Venkateswarlu et al., 2001). During the past decade, 9 Review of Literature Sinularia has yielded many new structures with novel skeletons. Several of the previously published secondary metabolites have been reexamined for their pharmacological properties, and the results strongly support further investigations. Terpenoids of the soft coral genus Sinularia and their pharmacological significance were reviewed by Kamel and Slattery, (2005). A number of questions were raised in the past regarding, the origin of terpenes in symbiotic associations between marine invertebrates and algae (Zooxanthellae). Although, Kobbe et al., (1984) came to the conclusion that the zooxanthellae do not make terpenes and that the coral polyp was the active organelle in terpenoid biosynthesis, the subject is still in question. Since 1995, the number of research papers investigating the chemical constituents of the soft coral genus Sinularia has exceeded 100; the majority reporting new and novel terpenoids. 2.3.1.1. Terpenes: Cembranoid diterpenes and their cyclized derivatives are the most abundant metabolites of soft corals (Faulkner 2002; Krebs, 1986; Tursch et al., 1978 a). Cembranoids are a class of diterpenes possessing a 14membered ring skeleton. They are produced as a defense against predators, such as other corals and fishes and against settlement of microorganisms, such as fungi and bacteria (Coll et al., 1987; Coll et al., 1989 b). Previous bioassay results have shown that cembrane analogues possess significant biological activities, including ichthyotoxic (Kusumi et al., 1988), cytotoxic, anti-inflammatory, and antiarthritic (Norton and Kazlauskas, 1980), Caantagonistic (Kobayashi et al., 1983 a), HIV-inhibitory activities (Coll et al., 1985; Anjaneyulu et al., 1999; Su et al., 2000; Rashid et al., 2000) and antimicrobial properties. Overall, the antitumor effects have been the most important activity of cembranoids reported so far. The genus Sinularia is a rich source of cembranoid diterpenes that have been extensively studied 10 Review of Literature (Duh and Hou, 1996; Su et al., 2000). Other classes of terpenoids were also reported as Sesquiterpenes, Norcembranoid and Non-cembranoid diterpenes. Polymaxenolide is a novel terpenoid metabolite isolated from the hybrid soft coral Sinularia maxima x S. polydactyla. To date, no bioactivity has been reported for this compound (Kamel et al., 2004). The different classes of terpenes isolated and the biological activities ascribed to them are compiled in table 1 and 2, respectively. 2.3.1.2. Polyhydroxylated Sterols: Marine organisms produce sterols with a remarkable variety of side chains, unconventional nuclear structures, and assorted hydroxylation patterns. Sterol patterns in marine invertebrates reflect the complexity of sterols arising through the food chain. The capability for biochemical modification of dietary sterols makes the sterol composition even more complex. The symbiotic relationships between organisms also complicate the sterol composition (Goad, 1978). Worldwide chemical investigation of the steroidal content of soft corals of genus Sinularia have afforded various polyhydroxylated steroids as derivatives of 24-methyl- and 24- methylenecholestan-3β-ol and their glycosides (Su et al., 1989; Li and Long, 1992; Anjaneyulu et al., 1995; Sheu et al., 2000; Ahmed et al., 2003 b; Jagodzinska et al., 1985; Jin et al., 2005; Tellekeratne and Liyanage, 1989; Rao et al., 1993). The different classes of polyhydroxylated sterols isolated and the biological activities ascribed to them are compiled in table 3. 2.3.1.3. Miscellaneous Compounds: Different classes of compounds with unusual nuclei are reported in table 4. 11 Review of Literature 2.3.1.4. Quinolone Derivatives: 7-hydroxy-8-methoxy-4(1H)-quinolone, isolated from the Chinese soft coral Sinularia polydactyla, increased the blood flow in the brain and heart of mice, protected against hypoxia and pituitrin-induced acute ischemia in myocardium, and mitigated aconitine-induced arrhythmia. No mice died within 3 days after an i.p. dose at 150 mg/kg (Kanghou et al., 1984). OMe H HO N O 2.3.2. Bioactive Constituents of the Genus Lobophytum: Alcyonaceans (soft corals, Phylum: Coelenterata) of the genus Lobophytum, consists almost of 50 species, are rich storehouse of terpenoids and polyhydroxysteroids, of which some have been reported to possess HIVinhibitory activity (Rashid et al., 2000), cytotoxic (Duh et al., 2000; Wang et al., 2007 and 1992; Morris et al., 1998) and anti-inflammatory activities (Radhika et al., 2005). The terpenoid and steroid content of Alcyonaceans, particularly, Lobophytum species, vary considerably, based on the geographical location and seasons of collection (Parameswaran et al., 1989 and 1991; Anjaneyulu et al., 1994). 2.3.2.1. Terpenes: The genus Lobophytum elaborates a variety of sesqui- and diterpenes of the eudesmane, lobane and cembranoid type (Krebs, 1986). The different classes of terpenes isolated and the biological activities ascribed to them are compiled in table 5. 12 Review of Literature 2.3.2.2. Polyhydroxysterols: Lobophytum species studied so far contain more or less complex mixtures of monohydroxysterols such as cholesterol, 24 methylcholesterol, 24-methylenecholesterol, brassicasterol and other common marine sterols. The different classes of sterols and lipoidal compounds isolated, as well as the biological activities ascribed to them are compiled in table 6. 2.3.2.3. Miscellaneous Compounds: Different classes of lipids, fatty acids and sphingolipids, as well as Lobocalone; a novel secondary metabolite isolated from the soft coral Lobophytum caledonense (Su et al., 1993) are reported in table 7. 2.3.2.4. Lobozoanthamine Alkaloids: Lobozoanthamine, a new zoanthamine-type alkaloid, was isolated from the Indonesian soft coral Lobophytum sp. It is the recently reported isolation of a complex heptacyclic alkaloid belonging to the zoanthamine class, which is the first alkaloid from the soft coral belonging to the genus Lobophytum (Fattorusso et al., 2008). H HO O H H O O N O 2.4. Composition of Sclerites in Sinularia polydactyla and Lobophytum crassum: Soft corals contain small spicules of calcium carbonate called "sclerites", which are biomineralized structures composed of an organic matrix and a mineral fraction. Since information concerning the structure of 13 Review of Literature sclerites, their crystalization properties and mineral composition are scarce; these parameters were studied in the alcyonarian, Sinularia polydactyla. In the first step, the structure and shape of the sclerites were studied. The polymorphs of calcium carbonate were studied both by x-ray diffraction and Raman microprobe analysis. A mineral phase in the sclerites was identified as calcite. This analysis indicates that the sclerite has both Ca2+ and Mg2+ bearing calcite, in which Mg2+ showed a relatively high concentration (5-10 mol.%) (Rahman and Oomori, 2008). Analysis of proteinaceous components of calcified sclerites revealed the significance of protein component to calcification. Acidic proteins are generally thought to control mineral formation and growth in biocalcification. Analysis of proteinaceous components in the soluble and insoluble matrix fractions of sclerites in Sinularia polydactyla indicated that aspartic acid composes about 60% of the insoluble and 29% of the soluble matrix fractions, while analysis of aspartic acids in the matrix fractions of sclerites soft coral, Lobophytum crassum, indicated that (insoluble = 17 mol%; soluble = 38 mol%) which showed comparatively lower aspartic acidrich proteins than S. polydactyla. Thus, characterization of highly acidic proteins in the organic matrix of this species is an important first step toward linking function to individual proteins in soft corals. It was shown that aspartic-acid rich proteins can control the CaCO3 polymorph in vitro. The CaCO3 precipitates in vitro in the presence of aspartic acid-rich proteins and 50 mM Mg2+ was verified by Raman microprobe analysis. These results strongly suggested that the aspartic acid-rich proteins within the organic matrix of soft corals play a key role in biomineralization regulation (Rahman and Oomori, 2009). 14 Review of Literature Table 1: Diterpenes reported in different Sinularia species:Compounds isolated (+)-Polydactylide (I) 7α,8β-dihydroxydeepoxy-sarcophine(II) OH H O O O O HO HO OH OH Coral species and locality Sinularia polydactyla collected from the Gulf of Suez, Egypt. Remarks The molecular formula of polydactylide Grote et al., C20H30O5 was determined by (HR-ESI-MS) 2006. which showed a peak at m/z 373.1951. Compound II; ESIMS, m/z 357 [M+Na]+. II I The hybrid Sinularia polydactyla x Sinularia maxima. Biocatalytic transformation studies of 5episinuleptolide using Streptomyces lavendulea resulted in the isolation and O O characterization of two new metabolites. The H OH new metabolites were less active in O cytotoxicity assay, but were not toxic to Vero O cells. Australian soft Pukalide showed low antiproliferative - Pukalide Sinularia activity against L-929, K-562 cell lines and - 13α-acetoxy-11β,12β epoxypukalide (I) coral polydactyla. low cytotoxic effect against HeLa, while - 13α-acetoxypukalide (II) O CO Me OMe 13α-acetoxypukalide exhibited good O activities against L-929 and K-562. O 5-Episinuleptolide O H O 2 OMe O O O O O O O References O OAc O I O OAc O II 15 Kamel et al., 2007. Bowden et al., 1989. Review of Literature Taiwanese soft Three new cembrane diterpenes designated Lo et al., 2010. coral Sinularia sinuladiterpenes G, H and I. flexibilis. Sinuladiterpenes G and H O OAc OH OH AcO O O G O H Sinuladiterpenes I O O HO MeO Gyrosanolide A, B and C R OH O O OH HO O I O O O O II R= S-OH III R= R-OMe O Gyrosanolide D and E O OH O O O O IV O O O O V Sinularia gyrosa Gyrosanin A displayed cytotoxicity against Cheng et al., collected at the P-388 cell line. However, the other tested 2010 a. Dongsha Atoll, compounds were non-cytotoxic to P-388, ATaiwan. 549, and HT-29 cells (>20.0 µg/mL). In addition, the antibacterial activity assays revealed that none of the compounds exhibited any antibacterial activity. Gyrosanolide A, B and C at a concentration of 10 µM did not inhibit the COX-2 protein expression, but significantly reduced the levels of the iNOS protein by LPS stimulation exhibiting anti-inflammatory 16 Review of Literature activity. Gyrosanolide F and gyrosanin A O O O O O O O VI O OH OMe HO O OH O VII Formosan Soft Sinularia flexibilis. Thioflexibilolide A HO O O S O O OH Hainan soft coral Sinularia parva collected at Lingshui Bay, Hainan Province, China, Sinularia flexibilis collected at Green island, Taiwan. Sinulaparvalides A and B O O O O H O O O OH OH O OH A B Sinuladiterpenes A, B and C O O OOH R A: R = β-OH O O B: R = α-OH AcO A structurally unique symmetric sulphur- Chen biscembranoid, possessed 2010. coral containing significant anti-inflammatory and neuroprotective activities. HO O C O et al., The in vitro cytotoxic activities of these Li et al., 2009. compounds were tested on HCT-116, HL60, and A-549 tumor cell lines, but all of them were inactive. Compounds A, B and C possessed Lo et al., 2009. cytotoxicity against Hela, KB, Daoy, and WiDr human tumor cells with IC50 values of 0.09, 0.12, 0.07 and 0.07 μg/mL, respectively. 17