Original Article Bioactive Secondary Metabolites from the Red Sea soft ...

International Journal of Applied Research in Natural Products Vol. 4 (4), pp. 15-27, Jan 2012 Directory of Open Access Journals ©2012. IJARNP-HS Publication

Original Article Bioactive Secondary Metabolites from the Red Sea soft coral Heteroxenia fuscescens Mohammed R1,*, Seliem MA-E2, Mohammed TAA3, AbedElFatah A4, Abo-Youssef AM5, Thabet MM6 1

Department of Pharmacognosy, School of Pharmacy, Beni Suef University, Egypt. Department of Pharmacognosy, School of pharmacy, Cairo University, Cairo, Egypt. 3 Marine invertebrates, National Institute of Oceanography and Fisheries, Red Sea Branch, Hurghada, Egypt. 4National Center of Research, Cairo, Egypt. 5 Department of Pharmacology, School of Pharmacy, Beni Suef University, Egypt. 6Department of Pharmacognosy, School of Pharmacy, MUST University, Egypt 2

Summary: The Red Sea soft coral Heteroxenia fuscescens has been investigated concerning its secondary metabolites. Analysis of H. fuscescens has led to the isolation of 6-hydroxy -α- muurolene (1), gorgosten-5(E)-3 βol (2), 1-nonadecyloxy-2,3-propanediol (3) and (2S,3R,4E,8E)-N-hexadecanoyl-2-amino 4,8-octadecadiene-1,3diol (4) and sarcoaldosterol A (5). The isolated compounds were reported from several marine organisms and are identified for the first time from the soft coral H. fuscescens collected from the Red Sea. The activity of the alcoholic extract as anti-inflammatory, antipyretic, analgesic, anti oxidant is reported. The activity of the isolated compounds against several pathogenic microbes has been also reported. Industrial Relevance: A huge number of secondary metabolites are produced by soft bodied marine organisms to get over predation and infection. Compounds produced by soft bodied marine organisms are different from those produced by terrestrial organisms, and therefore may yield novel lead for antimicrobial drugs. With the diversity in the secondary metabolites and the new activities and mechanism of action marine animals considered an excellent source for new pharmaceuticals. This work is concerned with the isolation of secondary metabolites isolated form the Heteroxenia fuscescens from the red sea and the evaluation of some biological activities. The alcoholic extract of Heteroxenia fuscescens was found to possess antipyretic and anti-inflammatory activity. 6-hydroxy -αmuurolene was active against Staphylococcus aureus and Escherichia coli with MIC of 19 µg/ml. The alcoholic extract of the organism under study is none toxic so we believe its sterol content could be a good source for safer anti-inflammatory drugs and also the 6-hydroxy -α- muurolene (compound 1) will be a good candidate for more derivatisation studies to optimize its activity and selectivity as antimicrobial. Keywords: Heteroxenia fuscescens; antifungal; 6-hydroxy -α- muurolene; Red Sea.

INTRODUCTION 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 [1-3]. Few studies, however, focused on marine organisms from the Red Sea as a source for drug leads. Soft corals (Octocorallia, Alcyonacea) are a highly diverse group of marine organisms, which are known to contain a rich variety of secondary metabolites. These substances from corals show not only great significance in chemical ecology, but also various biological activities such as antitumor, antibacterial, antiviral and antifungal.[4] The soft corals belonging to genus Xenia and Heteroxenia are rich in sesquiterpenoids, diterpenes and sterols.[5-8] The studied biological activities of most of these compounds were as antitumors, antibacterial and antifungal.[5, 9, 10] The present investigation is depended on H. fuscescens (Ehrenberg, 1834) as a common Xeniidae species at Hurghada, Egypt. ______________________ *Corresponding Author: E-mail: [email protected] Tel.: +0822362211 Available online http://www.ijarnp.org

Bioactive Secondary Metabolites from the Red Sea soft coral Heteroxenia fuscescens

This work focused on isolation, purification and identification of the secondary metabolites from the Red Sea soft coral Heteroxenia fuscescens and evaluation of its biological activities such as: anti cancer, antimicrobials, analgesic, antipyretic and anti-inflammatory. The structures of the isolated compounds were elucidated on the basis of spectral analysis e.g. (MS, 1H NMR, 13C NMR, and 2D NMR). MATERIALS AND METHODS The soft coral (wet weight 1 Kg), (Figure 1) collected on March 2008, at a depth of 2 - 3 m at the front of Hurghada marine station of the national institute of oceanography and fisheries. It was kept in seawater and stored at -20 ºC to avoid degradation of the secondary metabolites. The soft coral was identified at the department of invertebrates, National Institute of Oceanography and Fisheries, Red Sea Branch, Hurghada, Egypt. A voucher sample was kept in the department of pharmacognosy Beni Suef University under the code RT1-2008.

Figure 1. Red Sea soft Coral: Heteroxenia fuscescens

Materials used for chromatography study include Pre-coated silica TLC plates 60 F 254 (20x20 cm) (E. Merck), Silica gel for column chromatography (CC) (E. Merck) and Sephadex LH-20 (Pharmacia Fine Chemicals AB) for Column Chromatography. Vanillin sulphuric acid for sterols and triterpenes was used as spray reagent [26] The solvents used in this work, viz. petroleum ether, n-hexane, chloroform, ethyle acetate, acetone, and methanol were purified adopting the procedures described by Vogel (1966).[27] Absolute ethanol, ethanol (95%) and n-butanol used were analytical pure grade. Water used was double-distilled and deionized. Materials used for pharmacological screening include alcoholic extracts of organism under investigation, Experimental animals (Albino mice, 25-30 g body weight, Adult male Albino rats, 130-150 g body weight, Adult female Albino rats, 125-150 g body weight). Equipments: Jeol NMR spectrometer, 200 MHz, Jeol mass spectrophotometer, 70 ev, Shimadzu-IR435 Infrared spectrophotometer, Shimadzu-265 spectrophotometer used for determination of ultraviolet absorption spectra. All animals were kept in a temperature-controlled room. The allocation of animals to different groups was randomized and the experiments were carried out under blind conditions. Animals were housed in colony cages (6 per cage), maintained on a standard diet with water and left for two days for acclimatization before the experimental sessions. The food was withheld the day before the experiment, but animals were allowed to have free access to water. Drugs and chemicals: Indomethacin: Nile Pharmaceutical Company, Egypt. Carrageenan: Sigma Co. – Aldrich. Glucose kit: Biolab. SA, France. Brewer Yeast: Fluka. DPPH (2,2-diphenyl-1picrylhydrazyl) used for assessment of the antioxidant activity. Pharmacological screening: Acute anti-inflammatory activity. Carrrgeenan-induced paw inflammation was produced according to the method described by Winter et al. (1962).[18] Twenty four male albino rats, weighing 130-150 g were divided into four groups, each of six animals. The first group received 1 ml saline and kept as control. The second and third groups received oral dose of 400 mg/kg and 800 mg/ kg of alcoholic extract of Heteroxenia fuscescens respectively. The forth group

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received oral dose of reference drug indomethacin (20 mg/ kg). One hour later all the animals had a sub planter injection of 0.1 ml of 1% carrageenan solution in saline in right hind paw and 0.1 ml saline in the left hind paw. Four hours after drug administration, the rats were sacrificed. Both hind paws were excised and weighed separately where their difference represents the weight of oedema. Analgesic activity. Analgesic effect was determined using hot- plate test as described by Eddy and Leimback, (1953).[19] The rats used for this study were divided into four groups each of six rats, two groups received the extracts (400 and 800mg/kg), while the remaining two groups received saline (control) and indomethacin (20 mg/kg). A 600-ml glass beaker was placed on a hot-plate (with adjustable temperature). The temperature of the hot-plate was then adjusted to 55 ± 0.5 ◦C. Each rat was placed in the glass beaker (on the hot-plate) in order to obtain the animal’s response to heat induced pain (licking of the forepaws and eventually jumping out of the glass beaker). Jumping out of the beaker was taken as an indicator of the animal’s response to heat-induced pain. The time taken for each rat to jump out of the beaker (i.e. reaction time) was noted and recorded as the response latency (in second). The mean of the latency for each group was determined. Hypoglycemic activity. Serum glucose level was determined according to the method described by Trinder, (1969) [21] using glucose kit (BIOLABO SA, FRANCE) for the enzymatic determination of glucose using glucose oxidase method. Principle of the assay depends on that, in the presence of glucose oxidase, glucose is oxidized to gluconic acid and hydrogen peroxide. Hydrogen peroxide reacts, in the presence of oxidase, with phenol and 4-aminophenazone to form a colored compound. The color is measured at a wavelength of 500 nm. The intensity of the pink color formed is proportional to the glucose concentration. Antioxidant activity. Cuvette assay of DPPH radical scavenging activity according to the method of Sreejayan and Rao (1996). [22] Where 50 µl sample was added to 2.95 ml DPPH solution (4.5 mg DPPH in 100 ml methanol) in a disposable cuvette. The absorbance at 517 nm was then monitored at 15 seconds interval from 0 to 5 min. The extract at concentration of (1, 2, 4, 6, 8 and 10 mg/ml) was tested; methanol was used as the blank solution and ascorbic acid (0.1M) as a positive control. Antimicrobial screening: Bacterial and fungal strains used include Bacillus subtilis, Staphylococcu aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Syncephalastrum racemosum, Aspergillus fumigatus and Penicillium italicum. Chloramphenicol impregnated discs served as antibacterial standard and Terbinafin impregnated discs as antifungal standard. Media used include MacCONKEY Agar used as media for Gm +ve bacterial growth and Nutrient Agar used as media for Gm -ve bacterial growth. Cytotoxic screening: Human tumor cell lines: Human liver tumor cell lines (HEPG-2), Human cervix tumor cell lines (HELA), Human colon tumor cell lines (HCT-116), Human Breast carcinoma cell lines (MCF-7), Human normal melanocytes cell lines (HFB-4) and Human larynx carcinoma cell lines (HEP-2) were used in the antitumor study. Human liver tumor cell lines (HEPG-2), Human cervix tumor cell lines (HELA), Human colon tumor cell lines (HCT-116), Human Breast carcinoma cell lines (MCF-7), Human normal melanocytes cell lines (HFB-4) and Human larynx carcinoma cell lines (HEP-2) were used in this study. They were obtained frozen in liquid nitrogen (-180 0C) from the American Type Culture Collection. The tumor cell lines were maintained in the National Cancer Institute, Cairo, Egypt, by serial sub-culturing. Sulphorhodamine-B (SRB) assay of cytotoxic activity was carried out. Preparation of the alcoholic extracts: The sample of the soft coral was extracted with sufficient amount 70% Ethanol, until no further residue was obtained to ensure complete extraction. The combined extracts were concentrated under reduced pressure. The residue was washed by methanol into small vial and labeled. Extract was stored at -20ºC to be used in pharmacological study. Testing solution for in-vitro cytotoxic effect: Two mg of the alcoholic extract of the soft coral under investigation was dissolved in 2 ml dimethyl sulfoxide (DMSO) to give a concentration of 100 µg/0.1 ml. Solution for in-vitro antimicrobial screening. The alcoholic extract of the soft coral under investigation was dissolved in dimethyl sulfoxide (DMSO) as 10 mg of sample in 1 ml of DMSO and then 10µl were aseptically transferred into sterile paper discs (Schleicher & Schuell, Spain) with a diameter of 8 mm. Extraction, fractionation and isolation of pure metabolites: Extraction. The frozen sample of the marine organism (wet weight one Kg) was left to defrost. The sample was broken down into small pieces and extracted at room temperature with sufficient amount (6 X 3L) of 70% Ethanol, until no further residue was obtained to ensure complete extraction. The combined extracts were filtered through Whatman no. 1 filter paper and then concentrated under reduced pressure at 50ºC. The residue was kept in a small vial and labeled. All extracts were stored at -20ºC to avoid degradation of the secondary metabolites.

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Fractionation of ethanolic extract. The concentrated ethanolic extract (20 g) was dissolved in the least amount of distilled water and (100 mL) was partitioned with successive portions of n-hexane (2 L), CHCL3 (2 L) and EtOAc (2 L) respectively. The combined hexane, CHCl3 and EtOAc extracts were concentrated separately under reduced pressure and labeled fraction A, B and C. The EtOAc fraction (fraction C) was the richest fraction with major spots on TLC and was furthered investigated. The ethyl acetate fraction (2 g) was chromatographed on Silica gel using gradient elution technique from nhexane to 100% ethyl acetate. The effluent was collected in fractions (50 ml each). Similar fractions were combined together according to their TLC pattern. All combined fractions were concentrated, transferred into small vials and labeled. All fractions were stored at -20ºC for further analysis. Purification of compounds. Fraction C-1A (100 mg) was chromatographed on Silica gel using gradient elution technique using n-hexane and ethyl acetate. The collected fractions were examined by TLC using the solvent system n-hexane: EtOAC (9:1) and similar fractions were combined. Subfraction C-1A-2 (16-25) was found to contain major spot, so it was concentrated to yield yellow residue (50 mg). This residue was dissolved in the least amount of methanol and purified on sephadex LH20 using methanol as eluent. 15 fractions were collected, 2 ml each. These fractions were monitored by TLC and similar fractions were combined together. Fractions 7-10 showed similar TLC pattern and evaporated to yield compound 1 as yellow oil (10 mg).Fraction C-5A was concentrated to yield yellow residue. The residue was dissolved in the least amount of chloroform: methanol (3:2) and purified on sephadex LH20 (30x1 cm) using chloroform: methanol (3:2) as eluent. The collected fractions were monitored by TLC using the same solvent system (n-Hexane: EtOAC (8:2)) and similar fractions were combined together. Sub-fraction C-5A-2 (5-10) recrystallized from methanol to give compound 2 (7 mg) as white powder. Fraction C-9A concentrated to yield yellow residue and purified on sephadex LH20 (30 x 1 cm) using methanol as eluent. The collected fractions were monitored by TLC using the same solvent system (n-Hexane: EtOAC (6:4)) and similar fractions were combined together. Sub-fraction C-9A-2 was evaporated to dryness to yield compound 3 as white powder (5 mg). Fraction C-15A concentrated to yield yellow residue and purified on sephadex LH20 (30x1 cm) using methanol as eluent. 14 fractions were collected, 2 ml each and were monitored by TLC using the same solvent system (n-Hexane: EtOAC (6:4)) and similar fractions were combined together. Sub-fraction C-9A-2 (4-10) was evaporated to dryness to yield compound 4 as white powder (4 mg).Fraction C-27A concentrated to yield yellowish white residue (30 mg). It was chromatographed on silica gel using Gradient elution technique starting by n-hexane and increasing polarity by using ethyl acetate. The collected fractions were examined by TLC using the solvent system n-hexane: EtOAC (4:6) and similar fractions were combined. Sub-fraction C-27A-4 was concentrated to yield white residue. The residue was dissolved in the least amount of methanol and purified on sephadex LH20 (30x1 cm) using methanol as eluent. 10 fractions were collected, 2 ml each. The fractions were monitored by TLC and similar fractions were combined together and evaporated to yield compound 5 as white powder (3 mg). RESULTS AND DISCUSSION Isolation and structure elucidation of secondary metabolites from H. fuscescens: Compound 1 (Figure 2) was obtained as a yellow viscous oil (10 mg), and was deduced to have the molecular formula C15H24O by positive mode HRESI-MS; [M+H-H2O]+ m/z 203.1830 (calcd. 203.1800). This fragmentation pattern was previously observed in mass spectra of bovine insulin.[11] The 1H NMR (CDCl3, 200 MHz) data of compound 1 showed two characteristic signals for isopropyl-methyl groups at δH 0.86 (d, J = 7 Hz) and δ 0.95 (d, J = 7 Hz), and isopropyl proton signal appeared at δ 2.09 as (br m). It also showed one broad intense peak at δ 1.67 for two vinyl methyl groups and characteristic peak at δ 5.47 for two vinylic protons. The aforementioned data indicated the basic skeleton of cadinene type of sesquiterpenes. The 13C NMR and DEPT-135 (Table 1) confirmed this data and showed four methyls at (δC 17.7, 21.87, 21.99, 23.45), three methylene at (δC 30.29, 26.543, 21.398), five methines at (δC 126.96, 121.5, 49.68, 43.57, 25.9) and three quaternary carbons at (δC 135.5, 133.99, 72.96). The 13 C NMR spectrum indicated the presence of one quaternary carbon bearing hydroxyl group at δC 72.96. It also showed four characteristic signals for two quaternary carbons at (δC 135.5, 133.99) and two methine carbons at (δC 126.96, 121.5) which confirmed the presence of two double bonds in two vinyl groups. From the above spectral data and discussion, the structure of compound 1 could be assigned as 6-hydroxy -α- muurolene (1). The NOESY spectrum showed correlation between the proton resonating at δH 1.79 and the proton resonating at δH 1.57, this information helped in the assigning the relative stereochemistry of compound 1 at C1 and C7 to be of the S configuration. The 1H NMR and 13C NMR were compared with the published data.[8] This compound is first reported from this species collected in Hurghada reefs.

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Mohammed et al

15

21

H

22 18

H

1 9

19

4 14

16

23

28

24

29

26 27

9 5

3

OH

13

11

1

5

H

20

3

30

7

HO H

12

13

Compound 1

1

Compound 2

19`

1`

OH

OCH2(CH2)17CH3

1

HO

HO

2

3

4

8 7

H HN

3

1`

2`

OH

12

O

Compound 3

Compound 4 21 18

HO

1 3

10

20 17

14

9

28

23

12

19

30

22

15

26

29 27

8

5

HO HO HO

Compound 5 Figure 2. Bioactive compounds isolated from Heteroxenia fuscescens

19

16`

18


Table 1. NMR data for compound 1(CDCl3, 200 MHz) Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

δC 49.67 26.54 30.29 135.54 126.96 72.96 43.57 21.39 121.50 133.99 25.99 17.73 21.98 23.45 21.87

DEPT-135

δH [mult., J (HZ)]

CH CH2 CH2 C CH C CH CH2 CH C CH CH3 CH3 CH3 CH3

1.79 (bd, 13.2) 1.34 (m), 1.93 (bm) 1.93 (bm), 1.99 (bm) 5.47 (bs) 1.57 (m) 1.93 (bm), 1.99 (bm) 5.47 (bs) 2.09 (m) 0.86 (d, 7) 0.95 (d, 7) 1.67 (bs) 1.67 (bs)

Compound 2 (Figure 2) was obtained as a white powder (7 mg), and was deduced to have the molecular formula C30H50O by positive mode HRESI-MS; [M+H-H2O]+ m/z 409.3857 (calcd. 409.3834). The IR spectrum, showed a broad band at 3421-3444 cm-1 which was attributable to hydroxyl group. The 1H NMR (CDCl3, 200 MHz) data of compound 2 showed characteristic signals at δH -0.13 (1H, H-30), δ 0.192 (2H, m, H-22, H-24), and at δ 0.44 (1H, H-30), thus requiring the presence of a cyclopropyl ring. It also showed signals for seven methyls at δ 0.66 (3H) and δ 0.899 – 1.01 (18H). The spectra also showed signal at δ 3.55 (1H, bs, H-3) indicate the presence of methine proton attached to hydroxyl group. The spectra also showed signal for olefinic proton at δ 5.36 (1H, b s, H-6). The characteristic signals due to a cyclopropyl moiety were identical with those of gorgosterol with the signals for seven methyls were also consistent with a gorgosterol skeleton. [12, 13] The aforementioned data indicated the presence of gorgosterol type sterol with a hydroxyl group at C-3 and double bond between C-5 and C-6. The 13C NMR and DEPT-135 confirmed the proposed structure and showed seven methyls signals, ten methine groups, and four quaternary carbons. The 13C NMR spectrum indicated the presence of one methine carbon bearing hydroxyl group at δ 71.8 and two olefinic carbons at δ 121.7 and at δ 140.7 From the above spectral data and discussion, the structure of compound 2 could be assigned as gorgosten-5(E)-3 β-ol (Fig. 41). It was confirmed by comparison of its spectral data with published values. [12, 13] This compound was previously reported from soft coral Sarcophyton trocheliophorum [14] and from gorgonian Isis hippuris [13] but to the best of our knowledge, this compound has not been isolated from Heteroxenia species. Compound 3 (Figure 2) was obtained as a white powder (5 mg), and was deduced to have the molecular formula C22H46O3 by positive mode HRESI-MS; [M+K]+ m/z 397.4189 (calcd. 397.3084). The IR spectrum showed a broad band at 3417 cm-1 which was attributable to hydroxyl group. The 1H NMR (CDCl3, 200 MHz) data of compound 3 showed signals for two pairs of oxymethylene protons at δH 3.453 (br s), one pair of methylene protons bearing a hydroxyl at δ 3.611 (br s) and one oxymethine proton bearing hydroxyl group at δ 3.809 (br s). It also showed strong signal at δ 1.22 (br s) for aliphatic long chain methylene groups and characteristic signal at δ 0.88 (br s) for terminal methyl group. The aforementioned data indicated that characteristic signals for the glycerol ether derivative with characteristic proton signals at δH 3.809, 3.611 and 3.453. The 13C NMR confirmed this suggestion and showed four characteristic signals for glycerol ether moiety (three oxymethylene carbons at δC 72.2 (C-1), δ 71.8 (C-1'), and δ 63.9 (C-3), and an oxymethine at δ 70.5 (C-2), it also showed strong signal at δC 29.6-29.3 for 14 methylenes and signal at δ 14.03 for terminal methyle. From the above spectral data and discussion, the structure of compound 3 could be assigned as 1nonadecyloxy-2,3-propanediol. It was confirmed by comparison of its 1H and 13C-NMR data with the published data.[15] This compound was previously reported from Dendronephthya gigantean soft coral.[15] To the best of our knowledge, it has not been isolated from Heteroxenia species. Compound 4 (Figure 2) was obtained as a white powder (4 mg), and was deduced to have the molecular formula C22H46O3 by positive mode HRESI-MS; [M + H]+ m/z 536.5086 (calcd. 536.5043). The IR spectrum showed a characteristic band at 1639 cm-1 for carbonyl group, a band at 3298 cm-1 indicated the presence of NH group and broad band at 3356 cm-1 for hydroxyl groups. The 1H NMR (CDCl3, 200 MHz) data of compound 4 showed characteristic proton signal for an amide group

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appeared at δH 6.29 (br d, J = 7.4), the spectra also showed four proton signals for two olefinic protons at δ 5.76 (br m, H-5) , δ 5.57 (br d, J = 5.8, H-4), δ 5.49 ( br d, J = 6, H-9) and δ 5.4 (m, H-8), it also showed proton signal for one methine bearing hydroxyl group at δ 4.31 (br s, H-3), two proton signals for one methylene bearing hydroxyl group at δ 3.93 (br m, H-1) and δ 3.69 (br d, J = 8.4, H-1) , one proton signal for one methine attached to amide group at δ 3.93 (br m, H-2) and broad signal for two protons of two hydroxyl groups at δ 2.9 (br s, OH). In addition, the spectra displayed signal for on pair of methylene protons attached to carbonyl group at δ 2.23 (br t, H-2'), a strong signal for nineteen methylene at δ 1.25 (br s, CH2 x 19) indicated the fatty acid moiety and a signal at δ 0.88, revealing terminal methyl groups (m, H-18, H-16'). The 13C NMR confirmed this data and showed signal for carbonyl carbon at δC 173.98, four signals for four olefinic carbons, two signals for two carbons bearing hydroxyl group at δ 74.59 and δ 62.4 and signal for carbon adjacent to an amide group at δ 54.4. In addition it displayed an intense signal at δ 29.68-29.271 for 15 carbons which confirmed the presence of fatty acid moiety. From the above spectral data and discussion, the structure of compound 4 could be assigned as (2S,3R,4E,8E)-N-hexadecanoyl-2-amino 4,8-octadecadiene-1,3-diol. It was confirmed by comparison of its 1H and 13C-NMR data with the published data.[15] This compound was previously reported from Dendronephthya gigantean soft coral.[15] To the best of our knowledge, this compound has not been isolated from Heteroxenia species. Compound 5 (Figure 2) was obtained as a white powder (3 mg), and was deduced to have the molecular formula C30H52O4 by positive mode HRESI-MS; [M + K]+ m/z 515.3536 (calcd. 515.3503). The IR spectrum showed a broad band at 3417 cm-1 which was attributable to hydroxyl groups. The 1 H NMR (CDCl3, 200 MHz) data of compound 5 showed characteristic signals at δH 3.39 (1 H, br s, H6) and δ 3.83 (2 H, m, H-3, H-11) for three hydroxyl-methine protons. It also showed signals characteristic to a cyclopropane-bearing gorgosterol- type side chain at [δH -0.19 (1H, dd, J = 4.5, 5.4, H-30), δ 0.1(2H, m, H-22, H-24), δ 0.4 (1H, dd, J = 4, 4.4)]. It also contained seven methyl singals at δ 0.6 (3H, s, Me-18), δ 0.79 (3H, d, J = 6.6, Me-26), δ 0.83 (3H, s, Me-29), δ 0.86 (3H, d, J = 3.4, Me28), δ 0.89 (3H, d, J = 3.2, Me-27), δ 0.95 (3H, s, Me-21), δ 1.2 (3H, s, Me-19) which also consistent with the gorgosterol skeleton.[15] The 13C NMR and DEPT-135 showed seven methyl signals, eight methylene signals, eleven methine groups, and four quaternary carbons. The 13C NMR spectrum indicated the presence of four oxygenated carbons (three methines and one quaternary carbon). From the above spectral data and discussion, the structure of compound 5 could be assigned as sarcoaldosterol A. It was confirmed by comparison of its spectral data with published values.[16] This compound was previously reported from soft coral Sarcophyton species collected from Okinawa Island. [16] To the best of our knowledge, this compound has not been isolated from Heteroxenia fuscescens from the Red Sea. Pharmacological screening of alcoholic extract of Heteroxenia fuscescens Acute toxicity study (LD50). The (LD50) of the alcoholic extracts of Heteroxenia fuscescens was determined according to the method of Kerber (1941),[17] using 48 male albino mice (25-30 g). The LD50 of the alcoholic extract of Heteroxenia fuscescens is 6.4 g/kg b. Wt. which indicates that the alcoholic extract of Heteroxenia fuscescens is safe and non toxic. Acute anti-inflammatory activity. Carrrgeenan-induced paw inflammation was produced according to the method described by Winter et al. (1962).[18] The percentage of oedema and the percentage of oedema inhibition (% of change) were calculated according to the following equations: % oedema = wt. of right paw – wt. of left paw x 100 / wt. of left paw. % oedema inhibition (% of change) = (Mc – Mt) x 100 / Mc. Where Mc is the mean oedema in control rats and Mt is the mean oedema in drug-treated animals. Results of anti-inflammatory activity of alcoholic extract are shown in Table (2). Results in Table (2) indicated that, both tested doses of alcoholic extract of Heteroxenia fuscescens induce significant antiinflammatory effect compared with the indomethacin and the control group. Also the alcoholic extract of Heteroxenia fuscescens at a dose 400 mg/kg b. wt. reduce the oedema of the control by 48.7% , while at a dose 800 mg/kg b. wt. reduced it by 59%.

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Table 2. Acute anti-inflammatory activity of Indomethacin and alcoholic extract of Heteroxenia fuscescens in male albino rats (n=6) using carrageenan -induced rat hind paw oedema. Treatments

Dose mg/kg b.wt.

Weight of rat’s hind paw ± S.E.

% inhibition of inflammation

Right

Left

Difference

1.43 ± 0.16

0.65 ± 0.04

0.78 ± 0.04

--

1ml saline Control Indomethacin

20

1.16 ± 0.03

0.91 ± 0.07

0.25 ± 0.02*

68.0%

Alcoholic extract of H. fuscescens

400

1.35 ± 0.06

0.95 ± 0.05

0.40 ± 0.03*

48.7%

Alcoholic extract of H. fuscescens 800 1.15 ± 0.06 0.83 ± 0.04 0.32 ± 0.03* Data was expressed as mean± s.e.m. : Significantly different from the normal control group at P