Antioxidant cytotoxic antitumor and protective DNAFX抗氧化细胞毒等.docx

1、Antioxidant cytotoxic antitumor and protective DNAFX抗氧化细胞毒等窗体顶端窗体底端Pharmacognosy Res. 2011 Jul-Sep; 3(3): 160165. doi: 10.4103/0974-8490.85000PMCID: PMC3193615Copyright : Pharmacognosy ResearchAntioxidant, cytotoxic, antitumor, and protective DNA damage metabolites from the red sea brown alga Sargas

2、sum spSeif-Eldin N. Ayyad, Saleh T. Ezmirly, Salim A. Basaif, Walied M. Alarif,1 Adel F. Badria,2 and Farid A. Badria3Department of Chemistry, Faculty of Science, King Abdul Aziz University, P. O. 80203, Jeddah 21589, KSA1Department of Marine Chemsitry, Faculty of Marine Sciences, King Abdulaziz Uni

3、versity, Jeddah 21589, P. O. 80207, KSA2Tissue Engineering Lab., Faculty of Dentistry, Alexandria University, Alexandria, Egypt3Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura 35516, EgyptAddress for correspondence: Dr. Seif-Eldin N Ayyad, Faculty of Sciences, King Ab

4、dulaziz University, Jeddah, Saudi Arabia. E-mail: snayyad2 Received February 1, 2011; Revised April 6, 2011; Accepted September 16, 2011.This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported, which permits unrestricted u

5、se, distribution, and reproduction in any medium, provided the original work is properly cited.AbstractBackground:Macroalgae can be viewed as a potential antioxidant and anti-inflammatory sources owing to their capability of producing compounds for its protection from environmental factors such as h

6、eat, pollution, stress, oxygen concentration, and UV radiations.Objective:To isolate major compounds which are mainly responsible for the pharmacological activity of brown alga under investigation, Sargassum sp.Materials and Methods:Algal material was air dried, extracted with a mixture of organic s

7、olvents, and fractionated with different adsorbents. The structures of obtained pure compounds were elucidated with different spectroscopic techniques, and two pure materials were tested for protection of DNA from damage, antioxidant, antitumor, and cytotoxicity.Results:Four pure compounds were obta

8、ined, of which fucosterol (1) and fucoxanthin (4) were tested; it was found that fucoxanthin has strong antioxidant and cytotoxicity against breast cancer (MCF-7) with IC50 = 11.5 g/ml.Conclusion:The naturally highly conjugated safe compound fucoxanthin could be used as antioxidant and as an antitum

9、or compound.Keywords: Antioxidant, cytotoxicity, fucoxanthin, Sargassum spOther Sections oAbstract oINTRODUCTION oMATERIALS AND METHODS oRESULTS AND DISCUSSION oREFERENCESINTRODUCTIONThe genus Sargassum belongs to brown algae of class Phaeophyceae in the order Fucales. Numerous species are distribut

10、ed throughout the tropical and subtropical oceans of the world, where they generally inhabit shallow water and coral reefs. However, the genus may be well known for its planktonic (free-floating) species.1 It was clear from the literatures that more than 62 species of the genus Sargassum has been in

11、vestigated,2 and indicated its productivity with impressive diversity of natural compounds. For instances, the secondary metabolites contain different structural classes such as plastoquinones,35 chromanols,6 chromenes,7,8 steroids,9 and glycerides.10 The publications showed that, Sargassum metaboli

12、tes have wide range of biological activities, which include antibiotic, anti-HIV, anticoagulant, anticonvulsant, anti-inflammatory, antineoplastic, and antitumor.1114In continuation of our search program, which interested in the isolation of secondary metabolites from marine sources, especially macr

13、oalgae collected from Red Sea,1517 a marine macroalgae, identified as Sargassum sp., collected from El- Shuaiba lagoon 80 km south of Jeddah, was investigated. The total extract (Pet. ether: Chloroform: Methanol 1 : 1 : 1) had been fractionated using different chromatographic techniques and afforded

14、 four metabolites; fucosterol (1), saringosterone (2), saringosterol (3), and fucoxanthin (4). 1 and 4 had been tested toward the Bleomycin-dependent DNA damage, cytotoxicity against HepG2 (human hepatocellular liver carcinoma cell line), WI 38 (Skin carcinoma cell line), Vero (cell line was initiat

15、ed from the kidney of a normal adult African green monkey), MCF-7 (breast cancer cell lines), antitumor, and antioxidant using 2,2-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS).Other Sections oAbstract oINTRODUCTION oMATERIALS AND METHODS oRESULTS AND DISCUSSION oREFERENCESMATERIALS AND MET

16、HODSApparatus and materialChromatographic material: Aluminum oxide type 60-120 mesh was used for column chromatography. Thin-layer chromatography (TLC) silica gel GF 254 was used for TLC. Preparative thin-layer chromatography (PTLC) was performed on aluminum oxide plates (20 20 cm) of 250-mm thickne

17、ss. Electron impact mass spectra were determined at 70 eV on a Kratos MS-25 instrument. 1D and 2D NMR (Nuclear Magnetic Resonance) spectra were recorded on Bruker AVANCE III WM 600 MHz spectrometers and 13C NMR at 150 MHz Chemical shifts are given in (ppm) relative to TMS (Tetramethyl silane) as int

18、ernal standard. The Infra Red (IR) spectra were recorded on a Perkin Elmer spectrometer model 100. The spray reagent used is 50%-sulfuric acid in methanol as spraying reagent. The chromatoplate was heated after spraying at 100 to 105C until the spots attained maximum color intensity. The alga was de

19、scribed as Sargassum (Family Sargassaceae) and was collected by hand from El- Shuaiba lagoon 80 km south of Jeddah, Saudi Arabia, in the Red Sea, in June 2010.Extraction of Sargassum spThe air-dried algal material (350 g) was extracted by equal volume of mixture of Pet. ether, chloroform, and methan

20、ol (2 6 l, 24 hours for each batch) at room temperature. The extract was concentrated under reduced pressure to obtain 10 g residue. This material was chromatographed on a column of Silica gel.Column chromatographySilica gel column (500 g, 80 2.5 cm) was used for resolution. The residue (10 g) was h

21、omogenized with small amount of silica gel (50 g) and poured on to the top of the column which was packed in Pet. ether (40: 60). The eluent were used successively (Pet. ether- Ether, Pet. ether- Ethyl acetate). Fractions were collected (50 ml); Pet. ether- Ether mixture of increasing polarity by di

22、ethyl ether and then ethyl acetate and followed by TLC using silica-gel chromatoplates, appropriate solvent system and 50% sulfuric acid in methanol as spraying reagent. If the material was not pure, preparative TLC was applied using the appropriate solvent system and the adsorbent aluminum oxide fo

23、r purification.Isolated compoundsThe fraction A eluted by Pet. ether-Ether (6 : 4) was collected and rechromatographed over PTLC of silica gel using Pet. Ether-ethyl acetate (8 : 2) to give three compounds: 1 , 2, and 3. The fraction B eluted by Pet. ether-Ethyl acetate (6 : 4) was collected and rec

24、hromatographed on Sephadex LH-20 using a mixture of MeOH-CHCl3 (9 : 1) and then finally purified by preparative TLC of silica gel using chloroform methanol (9.5 : 0.5) to afford a pure compound 4.Fucosterol (1), (24E)-Stigmasta-5, 24(28)-diene-3-ol: white solid (30 mg, 0.008% dry wt) m.p. 133-135C.

25、IR (cm-1): 3480 (OH), 1945 (C = C-H), 1640 (C = C), 1370 (gem. Di-Me); EI-MS m/z: 412 M, C29H48O + , 397 M-CH3+, 379 M-CH3-H2O+, 314 (100) M-C6H10O+. 1H NMR (CDCl3) (ppm) 0.86- 0.88 (d, J = 6.6 Hz, 6H, Me-26 and Me-27), 0.91 (d, J = 6.6 Hz, 3H, Me-21), 0.68 (s, 3H, Me-18), 1.00 (s, 3H, Me-19), 1.59

26、(d, J = 6.6 Hz, 3H, Me-29), 3.53 (m, H, H-3), 5.35 (m, H, H-6), 5.11 (q, H, H-28), 2.22 (sep, J = 6.6 Hz, H, H-25). 13C NMR (CDCl3) (ppm) (39.75, C-1), (35.20, C-2), (71.80, C-3), (42.33, C-4), (140.74, C-5), (121.70, C-6), (36.14, C-7), (35.78, C-8), (50.10, C-9), (39.72, C-10), (28.23, C-11), (42.

27、28, C-12), (42.30, C-13), (56.74, C-14), (31.64, C-15), (34.78, C-16), (56.72, C-17), (11.85, C-18), (19.40, C-19), (39.50, C-20), (24.32, C-21), (37.23, C-22), (31.90, C-23), (146.00, C-24), (33.90, C-25), (22.50, C-26), (22.23, C-27), (115.94, C-28), (18.90, C-29).Saringosterone (2), 24-vinyl chol

28、est -4-ene -3-one: a colorless oil (10 mg, 0.003% dry wt). IR (cm-1): 3420 (OH), 1675 (C = O), 1630 (C = C); EIMS m/z (rel. int.): 426 (12) M, C29H46O2+, 383 (21) M+-C3H7, 313 (30), 271(35), 269 (100). HREIMS: m/z 426.3413 (calcd. 426.3349) C29H46O2; 1H-NMR (CDCl3) 5.76 (dd, J = 17, 12 Hz, H, H-28),

29、 5.73 (br s, H, H-4), 5.27 (d, J = 12 Hz, H, H-29), 5.15 (d, J = 17 Hz, H, H-29), 1.16 (s, 3H, Me-19), 0.95 (d, J = 6.5 Hz, 3H, Me-21), 0.86 (d, J = 7.0 Hz, 3H, Me-26), 0.84 (d, J = 7.0 Hz, 3H, Me-27), 0.72 (s, 3H, Me-18). 13C-NMR (36.54, C-1), (34.65, C-2), (200.35, C-3), (124.43, C-4), (172.35, C-

30、5), (33.64, C-6), (32.65, C-7), (36.32, C-8), (54.41, C-9), (39.25, C-10), (21.65, C-11), (40.22, C-12), (43.08, C-13), (56.26, C-14), (24.85, C-15), (28.86, C-16), (56.55, C-17), (12.63, C-18), (17.34, C-19), (36.84, C-20), (18.35, C-21), (32.54, C-22), (28.95, C-23), (89.76, C-24), (29.05, C-25),

31、(19.46, C-26), (18.07, C-27), (137.78, C-28), (117.12, C-29).Saringosterol (3), 24-vinyl cholest-5-ene-3, 24-diol, saringosterol: colorless oil (5 mg, 0.001% dry wt.). IR(cm-1): 3445 (OH), 1644 (C=C); EIMS m/z (rel. int.): 428 (12) M, C29H48O2+, 410 (6) M+-H 2O, 314 (40), 273(20), 271 (100), 255 (28

32、), 228 (22), 213 (40), 145 (64). 1H NMR (CDCl3) d 5.73 (dd, J=17, 12 Hz, H, H-28), 5.34 (br s, H, H-6), 5.28 (d, J = 12 Hz, H, H-29), 5.17 (d, J = 17 Hz, H, H-29), 3.53 (m, H, H-3), 1.02 (s, 3H, Me-19), 0.97 (d, J = 6.6 Hz, 3H, Me-21), 0.88 (d, J = 6.6 Hz, 3H, Me-26, 0.86 (d, J = 6.6 Hz, 3H, Me-27), 0.67 (s, 3H, Me-18, 13C-NMR d (37.17,C-1), (32.54, C-2), (72.43, C-3), (37.94, C-4), (141.43, C-5), (122.31, C-6), (32.35, C-7), (36.84, C-8), (50.74, C-9), (37.93, C-10), (21.75, C-11