e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 928–935
Available online at www.sciencedirect.com
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Cytotoxic effects of three new metabolites from Red Sea marine sponge, Petrosia sp. Ahmed Abdel-Lateff a,f , Walied M. Alarif b,∗ , Hany Z. Asfour c , Seif-Eldin N. Ayyad d,g , Alaa Khedr c , Farid. A. Badria h , Sultan S. Al-lihaibi b a
Department of Natural Products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University, PO Box 80260, Jeddah 21589, Saudi Arabia b Department of Marine Chemistry, Faculty of Marine Sciences, King Abdulaziz University, PO. Box 80207, Jeddah 21589, Saudi Arabia c Department of Pharmaceutical Chemistry and Phytochemistry, Faculty of Pharmacy, King Abdulaziz University, PO Box 80260, Jeddah 21589, Saudi Arabia d Department of Chemistry, Faculty of Science, King Abdulaziz University, PO. Box 80203, Jeddah 21589, Saudi Arabia f Department of Pharmacognosy, Faculty of Pharmacy, Minia University, Minia 61519, Egypt g Department of Chemistry, Faculty of Science, Dammietta University, New Dammietta, Egypt h Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
a r t i c l e
i n f o
a b s t r a c t
Article history:
Marine sponges represent an affluent source of biogenetically unprecedented array of bio-
Received 28 October 2013
logically active compounds. This study revealed the isolation of ten compounds from marine
Received in revised form
sponge of Petrosia sp. Their chemical structures were determined by using 1D and 2D
2 March 2014
NMR, UV, IR and MS measurements. A polyoxygenated steroid (3,7,9␣-trihydroxycholest-
Accepted 5 March 2014
5-en (1), a purine-derivative (3,7-dimethyl-2-(methylamino)-3H-purin-6(7H)-one (2) and a
Available online 15 March 2014
sphingolipid (N-((3S,E)-1,3-dihydroxytetracos-4-en-2-yl)stearamide (3) proved to be new compounds. Meanwhile, seven known compounds; (4–10) were also identified. The cyto-
Keywords:
toxicity of the total extract and the isolated compounds were subjected to cytotoxicity
Sponges
evaluation employing two cancer cell lines; HepG2 and MCF-7. All tested compounds exhib-
Sphingolipids
ited cytotoxic effect on both cancer cell lines with IC50 in range of 20-500 M. The proposed
Sterols
mechanism of cytotoxic activities was examined through its molecular affinity to the DNA.
Cytotoxicity
Compound 5 showed the highest affinity to the DNA with IC50 30 g/mL.
HepG2 MFC-7
∗
Corresponding author. Tel.: +966 0560352034. E-mail address: [email protected] (W.M. Alarif).
http://dx.doi.org/10.1016/j.etap.2014.03.005 1382-6689/© 2014 Published by Elsevier B.V.
© 2014 Published by Elsevier B.V.
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 928–935
1.
Introduction
Genus Petrosia (Demospongiae, Petrosiidae) is a tropical marine sponge, was recognized for its veritable cornucopia metabolites; steroids, alkaloids, polyketides, terpenoids and polyacetylenes (Kim et al., 1998; Lim et al., 2001; Shen et al., 2004; Hoshino et al., 2003; Nukoolkarn et al., 2008; Giner et al., 1999; Cimino et al., 1989). These compounds possessed interesting pharmacological effects as anti-fungal, immunosuppressant, anti-inflammatory, anti-neoplastic, and cytotoxic compounds (Chelossi et al., 2004). For instance; polyacetylenetriol was isolated from mediterranean sponge Petrosia sp. and found to be a potent inhibitor of DNA polymerases (Loya et al., 2002). Moreover, 2-bromoamphimedine and petrosamine were isolated from the same genus and showed strong acetylcholinesterase inhibitory activities approximately six times higher than that of the reference galanthamine (Nukoolkarn et al., 2008). The main objective of the current study is the isolation of bioactive metabolites from marine sponge, particularly, Petrosia sp. Extensive fractionation on NP-Silica led to the isolation of ten metabolites (1–10), (Fig. 1).
fractions were collected. The fractions were monitored by TLC pattern using UV254 lamp and 50%-sulfuric acid in methanol as spraying reagent. The fractions were combined into 6 pools (P001–P006). P001 (n-hexane:ether, 9:1) was further purified by fractionation on NP-silica gel (˚ = 30, L = 100 cm, 10 mL each) employing flash column with fixed solvent system n-hexane: diethyl ether (9.5:0.5), 50 fractions, 20 mL each, were collected. The fractions (29 and 30) were promising according to the TLC pattern, were purified by employing PTLC to yield 5 (6 mg). P002 was fractionated on NP-silica using n-hexane:diethylether (7:3), was re-purified by PTLC, employing the same solvent system to give 8 (2 mg), 9 (2 mg) and 7 (7 mg) as polarity increased. P003 (n-hexane:ether 5:5) was re-fractionated on VLC-NP-silica using n-hexane:diethyl ether (5:5), 30 fractions were collected, 5 ml each, yielding 2, 10 and 4 which were purified by PTLC employing the same solvent system giving 5, 8 and 1 mg, respectively. P005 (n-hexane:ethylacetate, 7:3) was fractionated on PTLC-NP silica using CHCl3 :MeOH:acetic acid (9:1:0.5), yielding 6 (1 mg), 3 (4 mg)and 1 (3 mg). Much effort had been paid to purify P004 and P006 but unfortunately, no isolates were obtained. Finally, all compounds were purified by employing Sephadex LH 20, using methanol as eluent.
2.3.1.
2.
Experimental
2.1.
General
Optical rotations were measured on ATAGO POLAX-L 2 polarimeter. GC-MS analyses were carried out using RTX-1 column (30 m, 0.25 mm). 1D and 2D NMR spectra were recorded on Bruker AVANCE III WM 600 MHz spectrometers and 13 C NMR at 150 MHz. Chemical shifts are given in ıc (ppm) relative to TMS as internal standard. Thin layer chromatography was performed on silica gel (Kieselgel 60, F254 ) of 0.25 mm layer thickness. Gel filtration was carried out using Sephadex LH20. Spots were detected by using 50% ethanol/sulfuric acid as spray reagent.
2.2.
Animal material
Marine sponge Petrosia sp. (Demospongiae, Petrosiidae) was collected from the North of Jeddah Saudi Arabia Red Sea coast (21◦ 29 31 N 39◦ 11 24 E), at a depth of 1–2 m, in June 2013. After collection of the fresh material of the sponge, it was squeezed by hand, then weighed and extracted with organic solvent. A voucher specimen (IP-2010-1) was deposited in the faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia. Petrosia sp. was red–purple to brown in color. It has a compact, hard texture, with spherical oscula irregularly spread over the surface. It is similar to some extent to Petrosia ficiformis.
2.3.
Extraction and isolation
The fresh Petrosia sp. (4.0 kg) was extracted with a mixture of CHCl3 :MeOH (1:1) (22 ◦ C, 3 × 10 L) and yielded viscous brown extracts (6 g). The total extract was fractionated on NP-Silica (˚ = 50, L = 100 cm, 100 mL each) employing gradient elution from n-hexane to EtOAc, and washed with methanol, 60
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3ˇ,7ˇ,9˛-Trihydroxycholest-5-en(1)
Amorphous powder, mp 221–223 ◦ C (3 mg); Rf = 0.15, Benzene:ethyl acetate:MeOH (5:5:0.5), yellowish brown spot developed upon spraying with 50%-sulfuric acid in ethanol); IR max (film) cm−1 : 3421, 1665, 2971, 2876; ESIMS m/z (rel. int.): 418 [M]+ (C27 H46 O3 ), 400 [M–H2 O]+ , 367 [M–2H2 O–CH3 ]+ , 349 [M–3H2 O–CH3 ]+ , 287 [M–H2 O–C8 H17 ]+ , 269[M–2H2 O–C8 H17 ]+ , 251 [M–3H2 O–C8 H17 ]+ , 113 [C8 H17 ]. HRESI-MS m/z: 418.3435 [M]+ (calculated for C27 H46 O3 418.3447); 1 H NMR and 13 C NMR (CDCl3 ) (Table 1).
2.3.2. (2)
3,7-dimethyl-2-(methylamino)-3H-purin-6(7H)-one
Amorphous powder, (5 mg); IR max (film) cm−1 : 3120, 2876; ESIMS m/z (rel. int.): 194 [(M + H]+ , 163 [M–CH4 N]+ , 137 [M–C2 H4 N2 ]+ , 108, 93. HRESIMS (pos.) m/z: 180.0880 [(M + H)–CH2 ](calculated for C7 H10 N5 O 180.0885); 1 H NMR (CDCl3 , 600 MHz), ı (ppm): 3.41 (s, CH3 -2), 3.59 (s, CH3 -3), 3.99 (s, CH3-9), 7.51 (s, 1H-8); 13 C NMR (CDCl3 , 150 MHz), ı (ppm) 107.7 (C-5), 148.7(C-4), 151.8 (C-2), 141.4(C-8), 155.5 (C-6).
2.3.3. N-((3S,E)-1,3-dihydroxytetracos-4-en-2yl)stearamide(3) Yellowish brown viscous material (4 mg); IR max (film) cm−1 : 3340, 3320 and 1640 cm−1 ; HRESI-MS m/z: 649.6368 [M]+ (Calculated for C42 H83 NO3 649.6373); 1 H NMR and 13 C NMR (CDCl3 ), (Table 1).
2.3.4.
Dehydroepiandrosterone(4)
NMR (CDCl3 ), ı (ppm): 1.04 (s, 3H, CH3 -19), 0.98 (s, 3H, CH3 18), 5.39 (brs, H-6), 3.55 (dddd, 10.8, 10.8, 6.6 and 4.8 Hz, 1H, H␣ -3); 13 C NMR (CDCl3 ), ␦ (ppm): 37.2 (C-1), 31.4 (C-2), 71.6 (C3), 42.2 (C-4), 141.0 (C-5), 120.9 (C-6), 30.9 (C-7), 31.5 (C-8), 50.2 (C-9), 36.6 (C-10), 20.3 (C-11), 31.6 (C-12), 47.6 (C-13), 51.8 (C-14), 21.9 (C-15), 35.9 (C-16), 221.3 (C-17), 13.0 (C-18), 19.4 (C-19). 1H
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e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 928–935
Figure 1 – Compounds isolated from Petrosia sp. (2).
2.3.5.
Cholest-5-en-3ˇ-yl formate (5)
(CDCl3 ), ı (ppm): 0.86 (d, J = 6.6 Hz, 3H, CH3 -27), 0.85 (d, J = 6.6 Hz, 3H, CH3 -26), 0.91 (d, J = 6.6 Hz, 3H, CH3 -21), 0.68 (s, 3H, CH3 -18), 1.03 (s, 3H, CH3-19), 4.74 (m, 1H, H -3), 5.36 (brs, H-6), 2.00 (m, 1H, Ha-7), 1.51 (m, 1H, Hb-7), 8.04 (s, 1H, H-COO); 13 C-NMR (CDCl3), ␦ (ppm): 160.7 (C-formyl group), 36.9 (C-1), 27.7 (C-2), 74.0 (C-3), 38.0 (C-4), 139.3 (C-5), 123.0 (C-6), 31.8 (C7), 31.7 (C-8), 50.0 (C-9), 36.5 (C-10), 21.0 (C-11), 39.6 (C-12), 42.3 (C-13), 56.7 (C-14), 24.3 (C-15), 28.2 (C-16), 56.1 (C-17), 11.8 (C18), 19.3 (C-19), 35.7 (C-20), 18.7 (C-21), 36.1 (C-22), 23.8(C-23), 39.5 (C-24), 29.0 (C-25), 22.4 (C-26), 22.8 (C-27). 1 H-NMR
2.3.6.
5˛,6˛-epoxycholest-8(14)-ene-3ˇ,7˛-diol(6)
(C-19), 32.1 (C-20), 19.0 (C-21), 35.8 (C-22), 23.6 (C-23), 39.5 (C24), 25.0 (C-25), 22.6 (C-26), 22.8 (C-27).
2.3.7.
5˛, 8˛-epidioxycholesta-6-en-3ˇ-ol (7)
1 H NMR (CDCl ):3.97 (1H, m, H-3), 6.25 (1H, d, J = 9 Hz, H-6), 6.52 3
(1H, d, J = 9 Hz, H-7), 0.80 (3H, d, 3.6, H3 -18), 0.89 (3H, s, H3 -19), 0.92 (3H, d, J = 6.6 Hz, H3 -21), 0.85 (3H, d, J = 6.6 Hz, H3 -26), 0.87 (3H, d, J= 6.6 Hz, H3 -27), 13 C NMR (CDCl3 ), 30.2 (C-1), 34.8 (C-2), 66.5 (C-3); 39.4 (C-4), 82.2 (C-5), 135.4 (C-6), 130.8 (C-7), 79.4 (C8), 51.3 (C-9), 37.2 (C-10), 21.0 (C-11), 37.1 (C-12), 44.7 (C-13), 51.6 (C-14), 23.7 (C-15), 28.7 (C-16), 56.4 (C-17), 12.6 (C-18), 18.6 (C19), 36.9 (C-20), 14.2 (C-21), 36.8 (C-22), 22.7 (C-23), 39.2 (C-24), 27.9 (C-25), 22.6 (C-26), 22.7 (C-27).
1H
NMR (CDCl3 ):3.92 (1H, m, H-3), 3.16 (1H, d, J = 3.6 Hz, H-6), 4.43 (1H, bd, J = 3.0 Hz, H-7), 0.86 (3H, s, H3 -18), 1.25 (3H, s, H3 19), 0.92 (3H, d, J= 6.6 Hz, H3 -21), 0.86 (3H, d, J = 6.6 Hz, H3 -26), 0.87 (3H, d, J = 6.6 Hz, H3 -27), 13 C NMR (CDCl3 ), 34.5 (C-1), 29.6 (C-2), 68.6 (C-3); 31.0 (C-4), 65.1 (C-5), 61.3 (C-6), 67.8 (C-7), 125.0 (C-8), 38.6 (C-9), 35.9 (C-10), 22.5 (C-11), 36.6 (C-12), 43.1 (C-13), 152.7 (C-14), 24.9 (C-15), 26.5 (C-16), 56.7 (C-17), 16.5 (C-18), 18.9
2.3.8.
Cholest-5,7-en-3ˇ-ol (9)
NMR (CDCl3 ); ı (ppm): 0.88 (d, J = 6.6 Hz, 3H, CH3 -27), 0.87 (d, J = 6.6 Hz, 3H, CH3 -26), 1.02 (d, J = 7.2 Hz, 3H, CH3 -21), 0.62 (s, 3H, CH3 -18), 0.95 (s, 3H, CH3 -19), 3.63 (dddd, J = 11.2, 11.2, 4.4, 4.0 Hz, 1H, H␣ -3), 5.58 (dd, J = 5.6, 2.4 Hz, H-6), 5.38 (tt, J = 2.8, 2.4 Hz, 1H, H-7); 13 C NMR (CDCl3 ), ı (ppm): 38.4 (C-1), 31.9 (C-2), 1H
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Table 1 – NMR spectral data of compounds 1 and 3 in CDCl3 (ı, ppm, J, Hz).a Carbon No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 24 25 26 27 a b
1 ı 1 H (m, J, Hz) 1.23 (m) 1.90 (m) 1.45 (m) 1.87 (m) 4.08 (dddd, 10.8, 10.8, 6.6, 4.8) 2.15 (m) 1.89 (m) – 5.70 (brs) 3.63 (d, 10.2) 1.95 (m) 1.66 (m) – 1.55 (m) 2.10 (m) 1.60 (m) – 1.90 (m) 1.65 (m) 1.16 (m) 1.91 (m) 1.51 (m) 1.25 (m) 0.59 (s) 1.08 (s) 1.46 (m) 0.93 (d, 6.6) 1.00 (m) 1.32 (m) 1.63 (m) 1.31 (m) 1.15 (m) 1.35 (m) 0.87 (d, 6.6) 0.88 (d, 6.6)
3
Carbon No. ı13 Cb
ı 1 H (m, J, Hz)
ı13 C
33.8
1a
3.70 (dd, 10.8, 3.6)
62.5
30.8 – 67.7 39.4
1b 2 3 4 5 6 7 8–22 23 24 1 2 3 4 -12 13 14 NH
3.95 (dd, 11.4, 3.6) 3.91 (m) 4.32 (m) 5.54 (dd, 11.2, 6.6) 5.78 (dd, 11.2, 6.6) 2.15 (q, 6.6) 2.08 (q, 6.6) 1.25 (m) 1.30 (m) 0.88 (t, 7.2) – 2.23 (t, 7.2) 1.64 (m) 1.25 (m) 1.30 (m) 0.87 (t, 7.6) 6.27 (d, 7.2)
54.8 74.7 128.8 134.4 32.4 32.2 22.7–36.9 25.8 14.2 174.0 36.8 32.3 22.7-36.9 22.7 14.2 –
144.0 117.5 73.6 43.4 76.0 37.1 22.0 39.5 43.9 54.7 22.9 27.8 56.2 12.1 19.0 36.1 12.1 36.0 23.9 39.3 28.0 22.6 22.7
All assignments are based on 1D and 2D measurements (HMBC, HSQC, COSY). Implied multiplicities were determined by DEPT (C = s, CH = d, CH2 = t).
70.5 (C-3), 40.7 (C-4), 139.8 (C-5), 116.2 (C-6), 119.6 (C-7), 141.4 (C-8), 46.2 (C-9), 36.0 (C-10), 21.3 (C-11), 39.2 (C-12), 42.92 (C13), 54.6 (C-14), 23.1 (C-15), 28.4 (C-16), 55.8 (C-17), 11.9 (C-18), 16.3 (C-19), 40.5 (C-20), 21.3 (C-21), 37.1 (C-22), 23.9 (C-23), 39.1 (C-24), 27.2 (C-25), 22.6 (C-26), 22.8 (C-27).
2.3.9.
Indole-3-carboxaldehyde (10)
NMR (CDCl3 ), ı (ppm): 7.86 (1H, H-2), 8.32 (1H, H-4), 7.32 (1H, H-5), 7.34 (1H, H-6), 7.45 (1H, H-7) 8.78 (NH), 10.08 (CHO); 13 C NMR (CDCl ), ı (ppm): 135.3 (C-2), 119.7 (C-3), 122.0 (C-4), 3 123.1 (C-5), 124.5 (C-6), 111.5 (C-7), 124.4 (C-8), 136.6 (C-9), 185.2 (CHO). 1H
2.4.
Biological evaluation
2.4.1. Cytotoxicity bioassays(Alarif et al., 2013; Krizkova et al., 2009; Grem, 1990; El-Ansari et al., 1991; Paula et al., 2002) The cytotoxic activity of the isolated compounds was tested against human hepatocellular liver carcinoma (HepG2), and human breast adeno-carcinoma (MCF-7). The IC50 of tested compounds is presented in Table 2.
The stock samples were diluted with RPMI-1640 medium to desired concentrations ranging from 10 to 1000 g/mL(0–250 M). The final concentration of dimethylsulphoxide (DMSO) in each sample did not exceed 1% v/v. The cancer cells were batch cultured for 10 days, then seeded in 96 well plates of 10 × 103 cells/well in fresh complete growth medium in 96-well Microtiter plastic plates at 37 ◦ C for 24 h under 5% CO2 using a water jacketed carbon dioxide incubator (Shedon.TC2323.Cornelius, OR, USA). The medium (without serum) was added and cells were incubated either alone (negative control) or with different concentrations of sample to give a final concentrations of (1000, 500, 200, 100, 50, 20, 10 g/mL). Cells were suspended in RPMI-1640 medium, 1% antibiotic–antimycotic mixture (104 g/mL potassium penicillin, 104 g/mL streptomycin sulfate and 25 g/mL amphotericin (B) and 1% l-glutamin in 96-well flat bottom micro-plates at 37 ◦ C under 5% CO2 . After 96 h of incubation, the medium was again aspirated, trays were inverted onto a pad of paper towels, the remaining cells rinsed carefully with medium, and fixed with 3.7% (v/v) formaldehyde in saline for at least 20 min. The fixed cells were rinsed with water, and examined. The cytotoxic activity was identified as confluent,
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Table 2 – In-vitro cytotoxic activities of the isolated compounds towards cancer cell lines. Compound No.
IC50 MCF-7b
Total extract 1 2 3 5 7 10 5-FUd a b
c
d
a
Compounds HepG2
19.8 ± 0.0197 153.0 ± 0.012c 130.0 ± 0.017 23.0 ± 0.017 86.1 ± 0.014 60.0 ± 0.017 500 ± 0.170 16.0 ± 0.005
a
18.1 ± 0.015 230.0 ± 0.031 145.0 ± 0.120 20.0 ± 0.120 887 ± 0.032 67.0 ± 0.120 480.0 ± 0.102 63.0 ± 0.001
TheIC50 of the total extract was calculated by g/mL. Human Hepatocellular Liver Carcinoma (HepG2) and Human Caucasian Breast Adenocarcinoma (MCF-7). IC50 of the compounds was calculated as M, deviation means that values given in mean ± SD are significant. 5-Fluorouracil was used as positive control.
relatively unaltered mono-layers of stained cells treated with compounds. The IC50 was calculated based on the 50% loss of monolayer and 5-fluorouracil was used as a positive control. To calculate IC50 , a series of dose-response data (e.g., drug concentrations x1, x2, . . ., xn and growth inhibition y1, y2, . . ., yn), were plotted and the values of y are in the range of 0–1. The simplest estimate of IC50 is to plot X–Y and fit the data with a straight line.
2.5.
DNA affinity (Abdelhafez et al., 2011)
2.5.1.
DNA binding assay (DNA/compound using RP-TLC)
TLC plates (RP-18 F254 ; 0.25 mm; Merck) were redeveloped with MeOH–H2 O (8:2 v/v). Tested compounds were then applied (1 mg/ml in MeOH) at the origin, followed by addition of DNA (1 mg/ml in H2 O and MeOH mixture) at the same position at the origin. The plates were then developed with the sane solvent system and the position of DNA was determined by spraying with anisaldehyde reagent which yield a blue color on reaction with DNA, the intensity of the color is proportional to the quantity of DNA added to the plate. Ethidium bromide was used as positive control.
2.5.2.
Table 3 – DNA binding activities employing DNA-methyl green displacement assay.
Colorimetric assay for compounds that bind to DNA
DNA methyl green (20 mg/Sigma, St., Louis, MO, USA) was suspended in 100 ml of 0.05 M Tris-HCl buffer, PH 7.5, containing 7.5 mM MgH2 SO4 and stirred at 37 ◦ C with a magnetic stirrer for 24 h. Unless otherwise indicated, samples to be tested were dissolved in EtOH in Eppendorff tubes. Solvent was removed under vacuum and 200 L of DNA methyl green solution was added to each tube. The absorption maxima for DNA/methyl green complex is 642.5–645 nm. Samples were incubated in the dark at ambient temperature. After 24 h, the final absorbance of samples was determined. Readings were corrected for initial absorbance and normalized as a percentage of the untreated DNA/methyl green complex absorbance value. IC50 values were determined for each compound as shown in Table 3.
IC50 , g/mla 78 ± 1.1 68 ± 1.2 40 ± 1.3 30 ± 1.3 72 ± 1.4 70 ± 1.1 78 ± 1.1 45 ± 1.4
1 2 3 5 6 7 8 10 a
Values represent the concentration (mean ± SD, n = 3–5) required for 50% decrease in the initial absorbance of DNA-methyl green solution.
2.5.3.
Bleomycin-dependent DNA damage assay
To the reaction mixtures in a final volume of 1.0 ml, the following reagents at the final concentrations stated were added: DNA (0.2 mg/ml), bleomycin (0.05 mg/ml), FeCl3 (0.025 mM), magnesium chloride (5 mM), KH2 PO4 –KOH buffer pH 7.0 (30 mM), and ascorbic acid (0.24 mM) or the test fractions diluted in MeOH to give a concentration of (0.1 mg/ml). The reaction mixtures were incubated in a water-bath at 37 ◦ C for 1 h. At the end of the incubation period, 0.1 ml of ethylenediaminetetraacetic acid (EDTA) (0.1 M) was added to stop the reaction (the iron–EDTA complex is unreactive in the bleomycin assay). DNA damage was assessed by adding 1 ml 1% (w/v) thiobarbituric acid (TBA) and 1 ml of 25% (v/v) hydrochloric acid (HCl) followed by heating in a water-bath maintained at 80 ◦ C for 15 min. The chromogen formed was measured at 532 nm.
3.
Results and discussion
3.1.
Chemistry
Compound 1 was isolated as amorphous powder. It gave a molecular ion peak in the HRESIMS at 418.3435 corresponding to a molecular formula of C27 H46 O3 requiring five unsaturation sites. The ESIMS contained five significant mass peaks at m/z 400 [M–H2 O]+ , 367 [M–2H2 O-CH3 ]+ , 349 [M–3H2 O–CH3 ]+ , 287[M–H2 O–C8 H17 ]+ , 269[M–2H2 O–C8 H17 ]+ and 251 [M–3H2 O–C8 H17 ]+ , that exhibited the presence of three hydroxyl groups and an aliphatic chain of C8 H17 . The existence of hydroxyl function was supported by IR absorption (max 3421 cm−1 ). The 13 C NMR spectrum and DEPT (1 H decoupled) experiments of 1 indicated 27 resolved resonances, which are attributable to five methyls, ten methylenes, eight methines and four quaternary carbons. The 1 H NMR spectral data (Table 1) indicated the presence of two singlets resonating at ıH 0.59 (H3 -18) and 1.08 (H3 -19) and three doublets at ı 0.93 (J = 6.6 Hz, H3 -21), 0.87 (J = 6.6 Hz, H3 -26) and 0.88 (J = 6.6 Hz, H3 -27), proved the presence of a cholestan skeleton and confirmed by methylation pattern, which is frequently encountered (John Goad & Akihisa 1977). Since four of the five double-bond equivalents are accounted for by the steroidal skeleton, the remaining one
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 928–935
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Figure 2 – Selected 1 H–1 H COSY and HMBC correlations of 1 (9).
Figure 3 – Fragmentation pattern of 3 (10).
must be due to trisubstituted double bond. The proton spectrum had a proton signal at ı 5.35 (br d) ascribed to an olefinic proton, on the other hand the 13 C NMR spectrum showed resonances due to C=CH (trisubstituted double bond) at ıC 144.0 and 117.5 ppm (John Goad & Akihisa 1977). IR spectral data showed absorption band of C=C bond at max 1665 cm−1 . Thus, compound 1 has a cholestane skeleton with C=C bond. The oxygen atoms appeared in the molecular formula is accounted as three hydroxyl functions (absence of oxymethyl signal in 13 C NMR). The interpretation of 1 H and 13 C NMR spectra indicated the presence of two oxymethine groups (CH) resonating at ıH /ıC 4.08/67.7 and 3.63/73.6 ppm, an oxygenated quaternary carbon at ıC 76.0 ppm and two of the triad resonances (ıH /ıC 4.08/67.7 and 3.63/73.6 ppm) were due to two secondary hydroxyl carbons, while the third signal ıC 76.0 ppm, was attributed to tertiary alcohol carbon. Thus, 1 has a trihydroxycholestene skeleton. The position and orientation of the functional groups can be deduced. The shape of the seven-line multiplet and the chemical shift value at ıH 4.08 in the 1 H NMR spectrum of 1 is suggested to be in the normal 3␣-proton (3-hydroxy) of the normal 3-hydroxy5 steroids. Evidence that strengthens this deduction came from the 1 H–1 H COESY spectrum of 1 (Fig. 2), where correlation peak due to the four couplings of 3␣-proton with the adjacent protons of H2 -2 and H2 -4. The latter protons also H2 -4 showed no couplings but the mutual correlation between both of them and with H-3 indicated that C-5 was a quaternary carbon. The final confirmation was deduced from the HMBC spectrum; H2 -4 is correlated with C-3, C-5 and C-6 which locate the double bond at C-5. The second hydroxyl group was positioned at C-7 based on the proton resonating at ıH 3.63 (H-7), which was correlated to the olefinic proton (ıH 5.36 ppm, H6). The third hydroxyl group was located at C-9 as the HMBC spectrum showed correlations between H-8, H2 -11 and H3 -19 with the oxygenated quaternary carbon resonating at ␦C 76.0. Stereochemistry was based on coupling constant values. H-7 proton appeared as doublet at ıH 3.63 (J = 10.2 Hz), then indicating its axial nature which implies -orientation of the hydroxyl group at C-7. The third OH-group at C-9 should be oriented as ␣-owing to its natural occurring (John Goad & Akihisa 1977). From the above discussion 1 can be assigned as 3,7,9␣-trihydroxycholest-5-en. Compound 2 was isolated as amorphous solid, its molecular formula was deduced to be C8 H11 N5 O by HRESIMS (positive mode) m/z 180.0880 [(M + H)–CH2 ] indicating the presence of six unsaturation sites (positive Dragendorff’s reagent supported the presence of N-containing compound). The 1 H NMR
spectral data of 2 (CDCl3 , 600 MHz), has three deshielded methyl groups resonating at ı 3.41 (s), 3.59 (s) and 3.99(s), a methine proton at ı 7.51 (s), suggesting the purine nature of 2. This was further substantiated by 13 C NMR data, which showed signals for four quaternary carbons resonating at ı 107.7, 148.7, 151.8 and 155.5, one methine carbon at ı 141.4, three N-methyl carbons at ı 33.6, 29.8 and 28.0. HMBC correlations observed from the N-Me at ı 3.99 with carbons at ı 107.7 and 141.4 and from the N-methyl group at ı 3.60 with carbon at ı 151.8 and 148.7 and N-methyl group at ı 3.41 with carbon at ı 151.8 laced them on two adjacent atoms. This information suggested the presence of a guanine base which substituted with three N-methyls. This was confirmed by the fragment ion at m/z 120, observed in the ESIMS, corresponding to the loss of the CH NCO moiety. A computer survey including Science finder indicated that 2 is new purine derivative (3,7-dimethyl2-(methylamino)-3H-purin-6(7H)-one) Compound 3 has a molecular formula of C42 H83 NO3 which was determined from HRESIMS (m/z 649.6368 [M]+ ). The MS fragmentation pattern is illustrated in Fig. 3. The IR spectra showed absorption bands attributable to hydroxyl, amide (NH) and CONH groups at 3340, 3320 and 1640 cm−1 , respectively. The 1 H NMR spectrum (Table 1) exhibited the presence of two primary methyls at ı 0.87 (6H, t, J = 7.6 Hz), two hetero bearing-methines at ı 3.91 and 4.32 and oxygenated methylene protons at ı 3.70 and 3.95, two olefinic protons at ı 5.54 and 5.78, an NH proton at ı 6.27 and a huge methylene envelope at ı 1.25. The 13 C and DEPT NMR spectral data of 3 supported the above analysis, showing a carbonyl group at ıC 174.0, a double bond at ıc 134.4 and 128.8, three oxygenated or other hetero atomized carbons at ıC 74.7, 62.5 and 54.8, aliphatic methylenes at ıc 22.7–36.9 and two methyls at ıc 14.1. The downfield doublet at ı 6.27 (NH) was deuterium-exchangeable, and there was no correlation between this signal and any carbon in the HMQC spectrum. On the other hand, a correlation from ı 6.27 (NH) to ı 3.91 (m), and the correlations from ı 6.27 (NH) to ıc 174.0(C-1 ), 36.9 (C-2 ), 62.5 (C-1), 54.8 (C-2) and 74.7 (C-3) were observed in the 1 H–1 H COSY and HMBC spectra, respectively. All the above data suggested 3 is a ceramide (sphingolipid) (Loukaci et al., 2000; Gaver and Sweeley, 1965). In order to determine the lengths of sphingosine and fatty acid chains, the positions of double bonds and the absolute configuration of 3, the acid methanolysis method according to Gaver and Sweeley (Tao et al., 2010), which yield a FAME methyl octadecanoate m/z 268 detected by GCMS, the presence of C-18 fatty acid moiety confirmed by the characteristic
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 928–935
3.2.
Biological evaluation
The total extract and isolated metabolites were tested toward two cancer cell lines HepG2 and MCF-7and showed moderate cytotoxic activities against both of them (Table 2). The mechanism of action was investigated by employing DNA affinity assays (cf exp.). Compound 5 showed significant affinity to DNA (Table 3). These results directed us to investigate the protection ability of the current isolated compounds. The results obtained are significantly important that this class of compounds showed protection activities. DNA is an important target of anticancer drugs; it was proved that the molecules which showed high affinity to bind with DNA showed significant inhibitory effect as alkylating agents. Different strategies are currently developed to potentiate the use of classical alkylating agents. (Pourquier P. Agents
100
% of DNA Protection
100
93
93
89
93
92
50
10
7
d un po om C
om C
po om C
po
un
un
d
d
5
4 d un po om C
C
om
po
un
m in
d
1
C
0 ita
ion at m/z 211 [CH3 (CH2 )12 CO]+ . So the molecular formulas of FAME and sphingosine are C18 H35 O and C24 H48 NO2 , respectively. The double bond and hydroxyl groups should be in sphingosine moiety and their positions could be determined by inspection of 1 H–1 H COSY spectrum, two methylene (C-1) protons at ı 3.70 and 3.95 correlated with the methine proton (C-2) at ı 3.91 which is correlated with the methine (C-3) proton at ı 4.32, the methine (C-3) proton at ı 4.32 correlated with the olefinic (C-4) proton at ␦ 5.54 (dt, J = 15.0, 6.6 Hz) which is in turn correlated with another olefinic (C-5) proton at ␦ 5.78 (dt, J = 15.0, 6.6 Hz), the olefinic proton at ı 5.78 correlated with two methylene (C-6) at ␦ 2.23 (q, J= 6.6 Hz) that correlated with another two methylene (C-7) protons at ␦ 2.06 (q, J = 6.6 Hz). The above discussion implies that the two OH groups are at C-1 and C-3, and a double bond at C-4/C-5 is trans oriented owing to the values of chemical shifts of allylic methylene ıc > 30 and the J values) (Su and Takaishi, 1999). Consideration of biogenesis and steric hindrance of such class of metabolites, generally were acknowledged to determine the absolute stereochemistry of the phytosphingosine moiety. On the basis of the 13 C NMR spectral data, the relative stereochemistries at C–3 (ı 74.7) were deduced to be 3S (Yaoita et al., 2000). Thus, the structure of 3 was established as N-((3S,E)-1,3-dihydroxytetracos-4-en2-yl)stearamide. Compound 4 was isolated as solid crystals. The 1 H, 13 C and DEPT NMR spectral data of 4 showed the presence of 19 carbon atoms; two methyls, eight methylenes, one sp2 methine (ıC 120.9 ppm) and five quaternary carbons (ıC 36.6, 47.6, 141.0 and 221.3). The chemical shift value and shape of the signals resonating at ıH 3.55 (dddd, J = 10.8, 10.8, 6.6 and 4.8 Hz, 1H, H␣ -3), 0.98 (s, 3H, CH3 -18) and 1.04 (s, 3H, CH3 -19) suggested a steroidal nucleus with no side chain attached at C-17. The absence of any methyl doublet and the down field value of CH3 -18 favored locating carbonyl group at C-17, this deduction was supported by ıC 221.0 and IR absorption. Compound 4 was identified by comparison of its 1 H and 13 C NMR data (cf. exp.) with those in the literature (Yang et al., 2008, Burgess et al., 2006) to be dehydroepiandrosterone. Compounds 5–10 were identified by comparing their spectral data with literature (Li et al., 2005; Migliuolo et al., 1993; Kobayashi and Kanada, 1991; John Goad & Akihisa, 1977; Pouchert, 1983).
V
934
Figure 4 – Bleomycin-dependent DNA damage assay of the tested compounds (13).
alkylants. Bull Cancer; 98: 1237- 1251). A brief description of the current method is that fixed amount of ligand is spotted on the RP18 TLC plates followed by addition of known quantity of DNA. The TLC was developed and the position of the DNA was assigned by spraying the plates with anisaldehyde reagent. It is easy to establish if the response of the test system is dependent on the dose of compounds. In case of increasing the quantities of DNA intercalators of the DNA lead to form a complex, and consequently, the free DNA can be detected as a blue spot (MeO:HeH O, 8:2) on RP-18 TLC after spraying with anisaldehyde reagent. The compounds of high binding to DNA were retained on the base line. However, when the DNA was mixed with compounds with which it is known to interact, the complex was retained at the origin when MeOH:H2 O (8:2) was used for elution. The inactive compounds did not cause the DNA to be retained at the origin. Moreover, methyl green reversibly binds polymerized DNA forming a stable complex when pH (7). The max for the DNA–methyl green complex is 642–645 nm. This colorimetric assay was employed to measure the displacement of methyl green from DNA by metabolites with DNA. The degree of displacement was determined spectrophotometrically by measuring the change in the initial absorbance of the DNA-methyl green solution in the presence of reference compound. Results from DNA binding assay (Table 3) revealed that compounds 5, 7 and 10 showed the highest affinity to DNA was measured IC50 (concentration required for 50% decrease in the initial absorbance of the DNA–methyl green solution). The remaining compounds showed weak activity. Bleomycin-dependent DNA damage has been evaluated as a specific method estimate the pro-oxidant drugs. Degradation of DNA could have been occurred when the assayed compounds reduced the bleomycin-Fe3+ to bleomycin-Fe2+ resulting in the production of MDA which combined with TBA to give a pink color. All compounds which decreased the absorbance and bleomycin-Fe3+ is not converted into bleomycin-Fe2+ , thus, preventing the DNA degradation. These results (Fig. 4) showed clearly that compounds (5 and 10) have the ability to protect DNA from the induced damage by bleomycin.
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 928–935
4.
Conclusion
Three new metabolites; polyoxygenatedcholestane-, purineand ceramide-derivatives (1–3), along with seven known metabolites (4–10) were obtained from Saudi marine sponge Petrosia sp. Their chemical structures were determined by interpretation of the spectral data of 1D and 2D NMR, UV, IR and MS measurements. The cytotoxicity of the total extract and the isolated pure compounds was evaluated by employing two cancer cell lines; HepG2 and MCF-7. All tested compounds showed anticancer activity with IC50 in range 20-500 M. The mechanism of anticancer activities was determined through its molecular affinity to the DNA. Compound 5 showed significant affinity to the DNA.
Conflict of Interest The authors declare no conflict of interests.
Acknowledgment This project was funded by the Saudi Basic Industries Corporation (SABIC) and the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under grant no. MS/14/318/1433. The authors, therefore, acknowledge with thanks SABIC and DSR technical and financial support. We also thank Dr. Yahia Folos, Marine Biology Department, Faculty of Marine Sciences, King Abdulaziz University, for collection and identification of the sponge sample.
references
Abdelhafez, O.M., Abdelatif, N.A., Badria, F.A., 2011. DNA binding, antiviral activities and cytotoxicity of new fluorochromone and benzene derivatives. Arch. Pharm. Res. 34, 1623–1632. Alarif, W.M., Abdel-Lateff, A., Al-Abd, A.M., Basaif, S.A., Badria, F.A., Shams, M., Ayyad, S.-E., 2013. Selective cytotoxic effects on human breast carcinoma of new methoxylated flavonoids from Euryopsarabicus grown in Saudi Arabia. Eur. J. Med. Chem. 66, 204–210. Burgess, J.P., Wintermute, J.S., Thomas, B.F., Kapetanovic, I.M., 2006. Spectral assignments and reference data. Mag. Res. Chem. 44, 1051–1053. Chelossi, E., Milanese, M., Milano, A., Pronzato, R., Riccardi, G., 2004. Characterisation and antimicrobial activity of epibiotic bacteria from Petrosiaficiformis (Porifera demospongiae). J. Exp. Mar. Biol. Ecol. 309, 21–33. Cimino, G., De Giulio, A., De Rosa, S., Di Marzo, V., 1989. High molecular weight polyacetylenes from Petrosiaficiformis: further structural analysis and biological activity. Tetrahedron Lett. 30, 3563–3566. El-Ansari, M.A., Barron, D., Abdalla, M.F., Saleh, N.A.M., Le Quere, J.L., 1991. Flavonoid constituents of Stachysaegyptiaca. Phytochemicals 30, 1169–1173. Gaver, R.C., Sweeley, C.C., 1965. Methods for methanolysis of sphingolipids and direct determination of long-chain bases by gas chromatography. J. Am. Oil Chem. Soc. 42, 294–298.
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Giner, J.-L., Gunasekera, S.P., Pomponi, S.A., 1999. Sterols of the marine sponge Petrosiaweinbergi: implication for the absolute configurations of the antiviralorthoesterols and weinbersterols. Steroids 64, 820–824. Grem, J.L., 1990. Fluorinated pyrimidines. In: Chabner, B.A., Collins, J.M. (Eds.), Cancer Chemotherapy, Principles and Practice. JB Lippincott Co, Philadelphia, pp. 197–200. Hoshino, A., Mitome, H., Miyaoaka, H., Shintani, A., Yamada, Y., van Soest, R.W.M., 2003. New strongylophorines from the Okinawan Marine sponge Petrosia (strongylophora) cortcata. J. Nat. Prod. 66, 1600–1605. John Goad, L., Akihisa, T., 1977. Analysis of Sterols, 1st ed. Blackie Academic & Professional. Kim, J.S., Im, K.S., Jung, J.H., Kim, Y.-L., Kim, J., Shim, C.J., Lee, C.-O., 1998. New bioactive polyacetylenes from the marine sponge Petrosia sp. Tetrahedron 54, 3151–3158. Kobayashi, M., Kanada, F., 1991. Marine sterols 18. Isolation and structure of four oxygenated sterols from a gorgonian coral Melithaeaocracea. J. Chem. Soc. Perkin Trans. 1, 1177–1179. Krizkova, J., Burdova, K., Stiborova, M., Kren, V., Hodek, P., 2009. The effects of selected flavonoids on cytochromes P450 in rat liver and small intestine. Interdisc. Toxicol. 2, 201–204. Li, G., Deng, Z., Guan, H., van Ofwegene, L., Proksch, P., Lin, W., 2005. Steroids from the soft coral Dendronephthya sp. Steroids 70, 13–18. Lim, Y.J., Park, H.S., Im, K.S., Lee, C.-O., Hong, J., Lee, M.-Y., Kim, D.-K., Jung, J.H., 2001. Additional cytotoxic polyacetylenes from the marine sponge Petrosia species. J. Nat. Prod. 64, 46–53. Loukaci, A., Bultel-ponce, V., Longeon, A., Guyot, M., 2000. New lipids from the tunicate cystodytes cf. dellechiajei, as PLA2 inhibitors. J. Nat. Prod. 63, 799–802. Loya, S., Rudi, A., Kashman, Y., Hizi, A., 2002. Mode of inhibition of HIV-1 reverse transcriptase by polyacetylenetriol, a novel inhibitor of RNA- and DNA-directed DNA polymerases. Biochem. J. 362, 685–692. Migliuolo, A., Piccialli, V., Sica, D., Giordano, F., 1993. New 8 - and 8(14) - 5␣,6␣-epoxysterols from the marine sponge Spongiaofficinalis. Steroids 58, 134–140. Nukoolkarn, V.S., Sean-oon, S., Rungrotmongkol, T., Hannongbua, S., Ingkaninan, K., Suwanborirux, K., 2008. Petrosaminee, a potent anticholinesterase pyridoacridine alkaloid from a Thai marine sponge Petrosian sp. Bioorg. Med. Chem. 16, 6560–6567. Paula, V.F., Barbosa, L.C.A., Errington, W., Howarth, O.W., Cruz, M.P., 2002. Chemical constituents from Bombacopsisglabra (Pasq.) A. Robyns: Complete 1 H and 13 C NMR Assignments and X Ray Structure of 5-Hydroxy-3,6,7,8,4 -pentamethoxyflavone. J. Braz. Chem. Soc. 13, 276–280. Pouchert, J., The Aldrich library of NMR spectra, Ed. II, 1983, 2(2), 533A. Shen, C.S., SaiParakash, C.V., Guh, J.-H., 2004. New pentacyclicpolyketidedimeric peroxide from a Taiwanese marine sponge Petrosiaelastic. Tetrahedron Lett. 45, 2463–2466. Su, B.-N., Takaishi, Y., Morinins, H.-K., 1999. Four novel phenylpropanol ester lipid metabolites from morinachonesis. J. Nat. Prod. 62, 1325–1327. Tao, W.W., Yang, N.-Y., Liu, L., Duan, J.A., Wu, D.-K., Qian, D.-W., Tang, Y.-P., 2010. Two new cerebrosides from the pollen of Typhaangustifolia. Fitoterapia 81, 196–199. Yang, J.-H., Ho, Y., Tzou, D-L.M., 2008. A 13 C solid-state NMR analysis of steroid compounds. Magn. Reson. Chem. 46, 718–725. Yaoita, Y., Ishizuka, T., Kakuda, R., Machida, K., Kikuchi, M., 2000. Ceramide constituents from five mushrooms. Chem. Pharm. Bull. 48, 1356–1358.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/etap
Cytotoxic effects of three new metabolites from Red Sea marine sponge, Petrosia sp. Ahmed Abdel-Lateff a,f , Walied M. Alarif b,∗ , Hany Z. Asfour c , Seif-Eldin N. Ayyad d,g , Alaa Khedr c , Farid. A. Badria h , Sultan S. Al-lihaibi b a
Department of Natural Products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University, PO Box 80260, Jeddah 21589, Saudi Arabia b Department of Marine Chemistry, Faculty of Marine Sciences, King Abdulaziz University, PO. Box 80207, Jeddah 21589, Saudi Arabia c Department of Pharmaceutical Chemistry and Phytochemistry, Faculty of Pharmacy, King Abdulaziz University, PO Box 80260, Jeddah 21589, Saudi Arabia d Department of Chemistry, Faculty of Science, King Abdulaziz University, PO. Box 80203, Jeddah 21589, Saudi Arabia f Department of Pharmacognosy, Faculty of Pharmacy, Minia University, Minia 61519, Egypt g Department of Chemistry, Faculty of Science, Dammietta University, New Dammietta, Egypt h Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
a r t i c l e
i n f o
a b s t r a c t
Article history:
Marine sponges represent an affluent source of biogenetically unprecedented array of bio-
Received 28 October 2013
logically active compounds. This study revealed the isolation of ten compounds from marine
Received in revised form
sponge of Petrosia sp. Their chemical structures were determined by using 1D and 2D
2 March 2014
NMR, UV, IR and MS measurements. A polyoxygenated steroid (3,7,9␣-trihydroxycholest-
Accepted 5 March 2014
5-en (1), a purine-derivative (3,7-dimethyl-2-(methylamino)-3H-purin-6(7H)-one (2) and a
Available online 15 March 2014
sphingolipid (N-((3S,E)-1,3-dihydroxytetracos-4-en-2-yl)stearamide (3) proved to be new compounds. Meanwhile, seven known compounds; (4–10) were also identified. The cyto-
Keywords:
toxicity of the total extract and the isolated compounds were subjected to cytotoxicity
Sponges
evaluation employing two cancer cell lines; HepG2 and MCF-7. All tested compounds exhib-
Sphingolipids
ited cytotoxic effect on both cancer cell lines with IC50 in range of 20-500 M. The proposed
Sterols
mechanism of cytotoxic activities was examined through its molecular affinity to the DNA.
Cytotoxicity
Compound 5 showed the highest affinity to the DNA with IC50 30 g/mL.
HepG2 MFC-7
∗
Corresponding author. Tel.: +966 0560352034. E-mail address: [email protected] (W.M. Alarif).
http://dx.doi.org/10.1016/j.etap.2014.03.005 1382-6689/© 2014 Published by Elsevier B.V.
© 2014 Published by Elsevier B.V.
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 928–935
1.
Introduction
Genus Petrosia (Demospongiae, Petrosiidae) is a tropical marine sponge, was recognized for its veritable cornucopia metabolites; steroids, alkaloids, polyketides, terpenoids and polyacetylenes (Kim et al., 1998; Lim et al., 2001; Shen et al., 2004; Hoshino et al., 2003; Nukoolkarn et al., 2008; Giner et al., 1999; Cimino et al., 1989). These compounds possessed interesting pharmacological effects as anti-fungal, immunosuppressant, anti-inflammatory, anti-neoplastic, and cytotoxic compounds (Chelossi et al., 2004). For instance; polyacetylenetriol was isolated from mediterranean sponge Petrosia sp. and found to be a potent inhibitor of DNA polymerases (Loya et al., 2002). Moreover, 2-bromoamphimedine and petrosamine were isolated from the same genus and showed strong acetylcholinesterase inhibitory activities approximately six times higher than that of the reference galanthamine (Nukoolkarn et al., 2008). The main objective of the current study is the isolation of bioactive metabolites from marine sponge, particularly, Petrosia sp. Extensive fractionation on NP-Silica led to the isolation of ten metabolites (1–10), (Fig. 1).
fractions were collected. The fractions were monitored by TLC pattern using UV254 lamp and 50%-sulfuric acid in methanol as spraying reagent. The fractions were combined into 6 pools (P001–P006). P001 (n-hexane:ether, 9:1) was further purified by fractionation on NP-silica gel (˚ = 30, L = 100 cm, 10 mL each) employing flash column with fixed solvent system n-hexane: diethyl ether (9.5:0.5), 50 fractions, 20 mL each, were collected. The fractions (29 and 30) were promising according to the TLC pattern, were purified by employing PTLC to yield 5 (6 mg). P002 was fractionated on NP-silica using n-hexane:diethylether (7:3), was re-purified by PTLC, employing the same solvent system to give 8 (2 mg), 9 (2 mg) and 7 (7 mg) as polarity increased. P003 (n-hexane:ether 5:5) was re-fractionated on VLC-NP-silica using n-hexane:diethyl ether (5:5), 30 fractions were collected, 5 ml each, yielding 2, 10 and 4 which were purified by PTLC employing the same solvent system giving 5, 8 and 1 mg, respectively. P005 (n-hexane:ethylacetate, 7:3) was fractionated on PTLC-NP silica using CHCl3 :MeOH:acetic acid (9:1:0.5), yielding 6 (1 mg), 3 (4 mg)and 1 (3 mg). Much effort had been paid to purify P004 and P006 but unfortunately, no isolates were obtained. Finally, all compounds were purified by employing Sephadex LH 20, using methanol as eluent.
2.3.1.
2.
Experimental
2.1.
General
Optical rotations were measured on ATAGO POLAX-L 2 polarimeter. GC-MS analyses were carried out using RTX-1 column (30 m, 0.25 mm). 1D and 2D NMR spectra were recorded on Bruker AVANCE III WM 600 MHz spectrometers and 13 C NMR at 150 MHz. Chemical shifts are given in ıc (ppm) relative to TMS as internal standard. Thin layer chromatography was performed on silica gel (Kieselgel 60, F254 ) of 0.25 mm layer thickness. Gel filtration was carried out using Sephadex LH20. Spots were detected by using 50% ethanol/sulfuric acid as spray reagent.
2.2.
Animal material
Marine sponge Petrosia sp. (Demospongiae, Petrosiidae) was collected from the North of Jeddah Saudi Arabia Red Sea coast (21◦ 29 31 N 39◦ 11 24 E), at a depth of 1–2 m, in June 2013. After collection of the fresh material of the sponge, it was squeezed by hand, then weighed and extracted with organic solvent. A voucher specimen (IP-2010-1) was deposited in the faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia. Petrosia sp. was red–purple to brown in color. It has a compact, hard texture, with spherical oscula irregularly spread over the surface. It is similar to some extent to Petrosia ficiformis.
2.3.
Extraction and isolation
The fresh Petrosia sp. (4.0 kg) was extracted with a mixture of CHCl3 :MeOH (1:1) (22 ◦ C, 3 × 10 L) and yielded viscous brown extracts (6 g). The total extract was fractionated on NP-Silica (˚ = 50, L = 100 cm, 100 mL each) employing gradient elution from n-hexane to EtOAc, and washed with methanol, 60
929
3ˇ,7ˇ,9˛-Trihydroxycholest-5-en(1)
Amorphous powder, mp 221–223 ◦ C (3 mg); Rf = 0.15, Benzene:ethyl acetate:MeOH (5:5:0.5), yellowish brown spot developed upon spraying with 50%-sulfuric acid in ethanol); IR max (film) cm−1 : 3421, 1665, 2971, 2876; ESIMS m/z (rel. int.): 418 [M]+ (C27 H46 O3 ), 400 [M–H2 O]+ , 367 [M–2H2 O–CH3 ]+ , 349 [M–3H2 O–CH3 ]+ , 287 [M–H2 O–C8 H17 ]+ , 269[M–2H2 O–C8 H17 ]+ , 251 [M–3H2 O–C8 H17 ]+ , 113 [C8 H17 ]. HRESI-MS m/z: 418.3435 [M]+ (calculated for C27 H46 O3 418.3447); 1 H NMR and 13 C NMR (CDCl3 ) (Table 1).
2.3.2. (2)
3,7-dimethyl-2-(methylamino)-3H-purin-6(7H)-one
Amorphous powder, (5 mg); IR max (film) cm−1 : 3120, 2876; ESIMS m/z (rel. int.): 194 [(M + H]+ , 163 [M–CH4 N]+ , 137 [M–C2 H4 N2 ]+ , 108, 93. HRESIMS (pos.) m/z: 180.0880 [(M + H)–CH2 ](calculated for C7 H10 N5 O 180.0885); 1 H NMR (CDCl3 , 600 MHz), ı (ppm): 3.41 (s, CH3 -2), 3.59 (s, CH3 -3), 3.99 (s, CH3-9), 7.51 (s, 1H-8); 13 C NMR (CDCl3 , 150 MHz), ı (ppm) 107.7 (C-5), 148.7(C-4), 151.8 (C-2), 141.4(C-8), 155.5 (C-6).
2.3.3. N-((3S,E)-1,3-dihydroxytetracos-4-en-2yl)stearamide(3) Yellowish brown viscous material (4 mg); IR max (film) cm−1 : 3340, 3320 and 1640 cm−1 ; HRESI-MS m/z: 649.6368 [M]+ (Calculated for C42 H83 NO3 649.6373); 1 H NMR and 13 C NMR (CDCl3 ), (Table 1).
2.3.4.
Dehydroepiandrosterone(4)
NMR (CDCl3 ), ı (ppm): 1.04 (s, 3H, CH3 -19), 0.98 (s, 3H, CH3 18), 5.39 (brs, H-6), 3.55 (dddd, 10.8, 10.8, 6.6 and 4.8 Hz, 1H, H␣ -3); 13 C NMR (CDCl3 ), ␦ (ppm): 37.2 (C-1), 31.4 (C-2), 71.6 (C3), 42.2 (C-4), 141.0 (C-5), 120.9 (C-6), 30.9 (C-7), 31.5 (C-8), 50.2 (C-9), 36.6 (C-10), 20.3 (C-11), 31.6 (C-12), 47.6 (C-13), 51.8 (C-14), 21.9 (C-15), 35.9 (C-16), 221.3 (C-17), 13.0 (C-18), 19.4 (C-19). 1H
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e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 928–935
Figure 1 – Compounds isolated from Petrosia sp. (2).
2.3.5.
Cholest-5-en-3ˇ-yl formate (5)
(CDCl3 ), ı (ppm): 0.86 (d, J = 6.6 Hz, 3H, CH3 -27), 0.85 (d, J = 6.6 Hz, 3H, CH3 -26), 0.91 (d, J = 6.6 Hz, 3H, CH3 -21), 0.68 (s, 3H, CH3 -18), 1.03 (s, 3H, CH3-19), 4.74 (m, 1H, H -3), 5.36 (brs, H-6), 2.00 (m, 1H, Ha-7), 1.51 (m, 1H, Hb-7), 8.04 (s, 1H, H-COO); 13 C-NMR (CDCl3), ␦ (ppm): 160.7 (C-formyl group), 36.9 (C-1), 27.7 (C-2), 74.0 (C-3), 38.0 (C-4), 139.3 (C-5), 123.0 (C-6), 31.8 (C7), 31.7 (C-8), 50.0 (C-9), 36.5 (C-10), 21.0 (C-11), 39.6 (C-12), 42.3 (C-13), 56.7 (C-14), 24.3 (C-15), 28.2 (C-16), 56.1 (C-17), 11.8 (C18), 19.3 (C-19), 35.7 (C-20), 18.7 (C-21), 36.1 (C-22), 23.8(C-23), 39.5 (C-24), 29.0 (C-25), 22.4 (C-26), 22.8 (C-27). 1 H-NMR
2.3.6.
5˛,6˛-epoxycholest-8(14)-ene-3ˇ,7˛-diol(6)
(C-19), 32.1 (C-20), 19.0 (C-21), 35.8 (C-22), 23.6 (C-23), 39.5 (C24), 25.0 (C-25), 22.6 (C-26), 22.8 (C-27).
2.3.7.
5˛, 8˛-epidioxycholesta-6-en-3ˇ-ol (7)
1 H NMR (CDCl ):3.97 (1H, m, H-3), 6.25 (1H, d, J = 9 Hz, H-6), 6.52 3
(1H, d, J = 9 Hz, H-7), 0.80 (3H, d, 3.6, H3 -18), 0.89 (3H, s, H3 -19), 0.92 (3H, d, J = 6.6 Hz, H3 -21), 0.85 (3H, d, J = 6.6 Hz, H3 -26), 0.87 (3H, d, J= 6.6 Hz, H3 -27), 13 C NMR (CDCl3 ), 30.2 (C-1), 34.8 (C-2), 66.5 (C-3); 39.4 (C-4), 82.2 (C-5), 135.4 (C-6), 130.8 (C-7), 79.4 (C8), 51.3 (C-9), 37.2 (C-10), 21.0 (C-11), 37.1 (C-12), 44.7 (C-13), 51.6 (C-14), 23.7 (C-15), 28.7 (C-16), 56.4 (C-17), 12.6 (C-18), 18.6 (C19), 36.9 (C-20), 14.2 (C-21), 36.8 (C-22), 22.7 (C-23), 39.2 (C-24), 27.9 (C-25), 22.6 (C-26), 22.7 (C-27).
1H
NMR (CDCl3 ):3.92 (1H, m, H-3), 3.16 (1H, d, J = 3.6 Hz, H-6), 4.43 (1H, bd, J = 3.0 Hz, H-7), 0.86 (3H, s, H3 -18), 1.25 (3H, s, H3 19), 0.92 (3H, d, J= 6.6 Hz, H3 -21), 0.86 (3H, d, J = 6.6 Hz, H3 -26), 0.87 (3H, d, J = 6.6 Hz, H3 -27), 13 C NMR (CDCl3 ), 34.5 (C-1), 29.6 (C-2), 68.6 (C-3); 31.0 (C-4), 65.1 (C-5), 61.3 (C-6), 67.8 (C-7), 125.0 (C-8), 38.6 (C-9), 35.9 (C-10), 22.5 (C-11), 36.6 (C-12), 43.1 (C-13), 152.7 (C-14), 24.9 (C-15), 26.5 (C-16), 56.7 (C-17), 16.5 (C-18), 18.9
2.3.8.
Cholest-5,7-en-3ˇ-ol (9)
NMR (CDCl3 ); ı (ppm): 0.88 (d, J = 6.6 Hz, 3H, CH3 -27), 0.87 (d, J = 6.6 Hz, 3H, CH3 -26), 1.02 (d, J = 7.2 Hz, 3H, CH3 -21), 0.62 (s, 3H, CH3 -18), 0.95 (s, 3H, CH3 -19), 3.63 (dddd, J = 11.2, 11.2, 4.4, 4.0 Hz, 1H, H␣ -3), 5.58 (dd, J = 5.6, 2.4 Hz, H-6), 5.38 (tt, J = 2.8, 2.4 Hz, 1H, H-7); 13 C NMR (CDCl3 ), ı (ppm): 38.4 (C-1), 31.9 (C-2), 1H
931
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 928–935
Table 1 – NMR spectral data of compounds 1 and 3 in CDCl3 (ı, ppm, J, Hz).a Carbon No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 24 25 26 27 a b
1 ı 1 H (m, J, Hz) 1.23 (m) 1.90 (m) 1.45 (m) 1.87 (m) 4.08 (dddd, 10.8, 10.8, 6.6, 4.8) 2.15 (m) 1.89 (m) – 5.70 (brs) 3.63 (d, 10.2) 1.95 (m) 1.66 (m) – 1.55 (m) 2.10 (m) 1.60 (m) – 1.90 (m) 1.65 (m) 1.16 (m) 1.91 (m) 1.51 (m) 1.25 (m) 0.59 (s) 1.08 (s) 1.46 (m) 0.93 (d, 6.6) 1.00 (m) 1.32 (m) 1.63 (m) 1.31 (m) 1.15 (m) 1.35 (m) 0.87 (d, 6.6) 0.88 (d, 6.6)
3
Carbon No. ı13 Cb
ı 1 H (m, J, Hz)
ı13 C
33.8
1a
3.70 (dd, 10.8, 3.6)
62.5
30.8 – 67.7 39.4
1b 2 3 4 5 6 7 8–22 23 24 1 2 3 4 -12 13 14 NH
3.95 (dd, 11.4, 3.6) 3.91 (m) 4.32 (m) 5.54 (dd, 11.2, 6.6) 5.78 (dd, 11.2, 6.6) 2.15 (q, 6.6) 2.08 (q, 6.6) 1.25 (m) 1.30 (m) 0.88 (t, 7.2) – 2.23 (t, 7.2) 1.64 (m) 1.25 (m) 1.30 (m) 0.87 (t, 7.6) 6.27 (d, 7.2)
54.8 74.7 128.8 134.4 32.4 32.2 22.7–36.9 25.8 14.2 174.0 36.8 32.3 22.7-36.9 22.7 14.2 –
144.0 117.5 73.6 43.4 76.0 37.1 22.0 39.5 43.9 54.7 22.9 27.8 56.2 12.1 19.0 36.1 12.1 36.0 23.9 39.3 28.0 22.6 22.7
All assignments are based on 1D and 2D measurements (HMBC, HSQC, COSY). Implied multiplicities were determined by DEPT (C = s, CH = d, CH2 = t).
70.5 (C-3), 40.7 (C-4), 139.8 (C-5), 116.2 (C-6), 119.6 (C-7), 141.4 (C-8), 46.2 (C-9), 36.0 (C-10), 21.3 (C-11), 39.2 (C-12), 42.92 (C13), 54.6 (C-14), 23.1 (C-15), 28.4 (C-16), 55.8 (C-17), 11.9 (C-18), 16.3 (C-19), 40.5 (C-20), 21.3 (C-21), 37.1 (C-22), 23.9 (C-23), 39.1 (C-24), 27.2 (C-25), 22.6 (C-26), 22.8 (C-27).
2.3.9.
Indole-3-carboxaldehyde (10)
NMR (CDCl3 ), ı (ppm): 7.86 (1H, H-2), 8.32 (1H, H-4), 7.32 (1H, H-5), 7.34 (1H, H-6), 7.45 (1H, H-7) 8.78 (NH), 10.08 (CHO); 13 C NMR (CDCl ), ı (ppm): 135.3 (C-2), 119.7 (C-3), 122.0 (C-4), 3 123.1 (C-5), 124.5 (C-6), 111.5 (C-7), 124.4 (C-8), 136.6 (C-9), 185.2 (CHO). 1H
2.4.
Biological evaluation
2.4.1. Cytotoxicity bioassays(Alarif et al., 2013; Krizkova et al., 2009; Grem, 1990; El-Ansari et al., 1991; Paula et al., 2002) The cytotoxic activity of the isolated compounds was tested against human hepatocellular liver carcinoma (HepG2), and human breast adeno-carcinoma (MCF-7). The IC50 of tested compounds is presented in Table 2.
The stock samples were diluted with RPMI-1640 medium to desired concentrations ranging from 10 to 1000 g/mL(0–250 M). The final concentration of dimethylsulphoxide (DMSO) in each sample did not exceed 1% v/v. The cancer cells were batch cultured for 10 days, then seeded in 96 well plates of 10 × 103 cells/well in fresh complete growth medium in 96-well Microtiter plastic plates at 37 ◦ C for 24 h under 5% CO2 using a water jacketed carbon dioxide incubator (Shedon.TC2323.Cornelius, OR, USA). The medium (without serum) was added and cells were incubated either alone (negative control) or with different concentrations of sample to give a final concentrations of (1000, 500, 200, 100, 50, 20, 10 g/mL). Cells were suspended in RPMI-1640 medium, 1% antibiotic–antimycotic mixture (104 g/mL potassium penicillin, 104 g/mL streptomycin sulfate and 25 g/mL amphotericin (B) and 1% l-glutamin in 96-well flat bottom micro-plates at 37 ◦ C under 5% CO2 . After 96 h of incubation, the medium was again aspirated, trays were inverted onto a pad of paper towels, the remaining cells rinsed carefully with medium, and fixed with 3.7% (v/v) formaldehyde in saline for at least 20 min. The fixed cells were rinsed with water, and examined. The cytotoxic activity was identified as confluent,
932
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 928–935
Table 2 – In-vitro cytotoxic activities of the isolated compounds towards cancer cell lines. Compound No.
IC50 MCF-7b
Total extract 1 2 3 5 7 10 5-FUd a b
c
d
a
Compounds HepG2
19.8 ± 0.0197 153.0 ± 0.012c 130.0 ± 0.017 23.0 ± 0.017 86.1 ± 0.014 60.0 ± 0.017 500 ± 0.170 16.0 ± 0.005
a
18.1 ± 0.015 230.0 ± 0.031 145.0 ± 0.120 20.0 ± 0.120 887 ± 0.032 67.0 ± 0.120 480.0 ± 0.102 63.0 ± 0.001
TheIC50 of the total extract was calculated by g/mL. Human Hepatocellular Liver Carcinoma (HepG2) and Human Caucasian Breast Adenocarcinoma (MCF-7). IC50 of the compounds was calculated as M, deviation means that values given in mean ± SD are significant. 5-Fluorouracil was used as positive control.
relatively unaltered mono-layers of stained cells treated with compounds. The IC50 was calculated based on the 50% loss of monolayer and 5-fluorouracil was used as a positive control. To calculate IC50 , a series of dose-response data (e.g., drug concentrations x1, x2, . . ., xn and growth inhibition y1, y2, . . ., yn), were plotted and the values of y are in the range of 0–1. The simplest estimate of IC50 is to plot X–Y and fit the data with a straight line.
2.5.
DNA affinity (Abdelhafez et al., 2011)
2.5.1.
DNA binding assay (DNA/compound using RP-TLC)
TLC plates (RP-18 F254 ; 0.25 mm; Merck) were redeveloped with MeOH–H2 O (8:2 v/v). Tested compounds were then applied (1 mg/ml in MeOH) at the origin, followed by addition of DNA (1 mg/ml in H2 O and MeOH mixture) at the same position at the origin. The plates were then developed with the sane solvent system and the position of DNA was determined by spraying with anisaldehyde reagent which yield a blue color on reaction with DNA, the intensity of the color is proportional to the quantity of DNA added to the plate. Ethidium bromide was used as positive control.
2.5.2.
Table 3 – DNA binding activities employing DNA-methyl green displacement assay.
Colorimetric assay for compounds that bind to DNA
DNA methyl green (20 mg/Sigma, St., Louis, MO, USA) was suspended in 100 ml of 0.05 M Tris-HCl buffer, PH 7.5, containing 7.5 mM MgH2 SO4 and stirred at 37 ◦ C with a magnetic stirrer for 24 h. Unless otherwise indicated, samples to be tested were dissolved in EtOH in Eppendorff tubes. Solvent was removed under vacuum and 200 L of DNA methyl green solution was added to each tube. The absorption maxima for DNA/methyl green complex is 642.5–645 nm. Samples were incubated in the dark at ambient temperature. After 24 h, the final absorbance of samples was determined. Readings were corrected for initial absorbance and normalized as a percentage of the untreated DNA/methyl green complex absorbance value. IC50 values were determined for each compound as shown in Table 3.
IC50 , g/mla 78 ± 1.1 68 ± 1.2 40 ± 1.3 30 ± 1.3 72 ± 1.4 70 ± 1.1 78 ± 1.1 45 ± 1.4
1 2 3 5 6 7 8 10 a
Values represent the concentration (mean ± SD, n = 3–5) required for 50% decrease in the initial absorbance of DNA-methyl green solution.
2.5.3.
Bleomycin-dependent DNA damage assay
To the reaction mixtures in a final volume of 1.0 ml, the following reagents at the final concentrations stated were added: DNA (0.2 mg/ml), bleomycin (0.05 mg/ml), FeCl3 (0.025 mM), magnesium chloride (5 mM), KH2 PO4 –KOH buffer pH 7.0 (30 mM), and ascorbic acid (0.24 mM) or the test fractions diluted in MeOH to give a concentration of (0.1 mg/ml). The reaction mixtures were incubated in a water-bath at 37 ◦ C for 1 h. At the end of the incubation period, 0.1 ml of ethylenediaminetetraacetic acid (EDTA) (0.1 M) was added to stop the reaction (the iron–EDTA complex is unreactive in the bleomycin assay). DNA damage was assessed by adding 1 ml 1% (w/v) thiobarbituric acid (TBA) and 1 ml of 25% (v/v) hydrochloric acid (HCl) followed by heating in a water-bath maintained at 80 ◦ C for 15 min. The chromogen formed was measured at 532 nm.
3.
Results and discussion
3.1.
Chemistry
Compound 1 was isolated as amorphous powder. It gave a molecular ion peak in the HRESIMS at 418.3435 corresponding to a molecular formula of C27 H46 O3 requiring five unsaturation sites. The ESIMS contained five significant mass peaks at m/z 400 [M–H2 O]+ , 367 [M–2H2 O-CH3 ]+ , 349 [M–3H2 O–CH3 ]+ , 287[M–H2 O–C8 H17 ]+ , 269[M–2H2 O–C8 H17 ]+ and 251 [M–3H2 O–C8 H17 ]+ , that exhibited the presence of three hydroxyl groups and an aliphatic chain of C8 H17 . The existence of hydroxyl function was supported by IR absorption (max 3421 cm−1 ). The 13 C NMR spectrum and DEPT (1 H decoupled) experiments of 1 indicated 27 resolved resonances, which are attributable to five methyls, ten methylenes, eight methines and four quaternary carbons. The 1 H NMR spectral data (Table 1) indicated the presence of two singlets resonating at ıH 0.59 (H3 -18) and 1.08 (H3 -19) and three doublets at ı 0.93 (J = 6.6 Hz, H3 -21), 0.87 (J = 6.6 Hz, H3 -26) and 0.88 (J = 6.6 Hz, H3 -27), proved the presence of a cholestan skeleton and confirmed by methylation pattern, which is frequently encountered (John Goad & Akihisa 1977). Since four of the five double-bond equivalents are accounted for by the steroidal skeleton, the remaining one
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 928–935
933
Figure 2 – Selected 1 H–1 H COSY and HMBC correlations of 1 (9).
Figure 3 – Fragmentation pattern of 3 (10).
must be due to trisubstituted double bond. The proton spectrum had a proton signal at ı 5.35 (br d) ascribed to an olefinic proton, on the other hand the 13 C NMR spectrum showed resonances due to C=CH (trisubstituted double bond) at ıC 144.0 and 117.5 ppm (John Goad & Akihisa 1977). IR spectral data showed absorption band of C=C bond at max 1665 cm−1 . Thus, compound 1 has a cholestane skeleton with C=C bond. The oxygen atoms appeared in the molecular formula is accounted as three hydroxyl functions (absence of oxymethyl signal in 13 C NMR). The interpretation of 1 H and 13 C NMR spectra indicated the presence of two oxymethine groups (CH) resonating at ıH /ıC 4.08/67.7 and 3.63/73.6 ppm, an oxygenated quaternary carbon at ıC 76.0 ppm and two of the triad resonances (ıH /ıC 4.08/67.7 and 3.63/73.6 ppm) were due to two secondary hydroxyl carbons, while the third signal ıC 76.0 ppm, was attributed to tertiary alcohol carbon. Thus, 1 has a trihydroxycholestene skeleton. The position and orientation of the functional groups can be deduced. The shape of the seven-line multiplet and the chemical shift value at ıH 4.08 in the 1 H NMR spectrum of 1 is suggested to be in the normal 3␣-proton (3-hydroxy) of the normal 3-hydroxy5 steroids. Evidence that strengthens this deduction came from the 1 H–1 H COESY spectrum of 1 (Fig. 2), where correlation peak due to the four couplings of 3␣-proton with the adjacent protons of H2 -2 and H2 -4. The latter protons also H2 -4 showed no couplings but the mutual correlation between both of them and with H-3 indicated that C-5 was a quaternary carbon. The final confirmation was deduced from the HMBC spectrum; H2 -4 is correlated with C-3, C-5 and C-6 which locate the double bond at C-5. The second hydroxyl group was positioned at C-7 based on the proton resonating at ıH 3.63 (H-7), which was correlated to the olefinic proton (ıH 5.36 ppm, H6). The third hydroxyl group was located at C-9 as the HMBC spectrum showed correlations between H-8, H2 -11 and H3 -19 with the oxygenated quaternary carbon resonating at ␦C 76.0. Stereochemistry was based on coupling constant values. H-7 proton appeared as doublet at ıH 3.63 (J = 10.2 Hz), then indicating its axial nature which implies -orientation of the hydroxyl group at C-7. The third OH-group at C-9 should be oriented as ␣-owing to its natural occurring (John Goad & Akihisa 1977). From the above discussion 1 can be assigned as 3,7,9␣-trihydroxycholest-5-en. Compound 2 was isolated as amorphous solid, its molecular formula was deduced to be C8 H11 N5 O by HRESIMS (positive mode) m/z 180.0880 [(M + H)–CH2 ] indicating the presence of six unsaturation sites (positive Dragendorff’s reagent supported the presence of N-containing compound). The 1 H NMR
spectral data of 2 (CDCl3 , 600 MHz), has three deshielded methyl groups resonating at ı 3.41 (s), 3.59 (s) and 3.99(s), a methine proton at ı 7.51 (s), suggesting the purine nature of 2. This was further substantiated by 13 C NMR data, which showed signals for four quaternary carbons resonating at ı 107.7, 148.7, 151.8 and 155.5, one methine carbon at ı 141.4, three N-methyl carbons at ı 33.6, 29.8 and 28.0. HMBC correlations observed from the N-Me at ı 3.99 with carbons at ı 107.7 and 141.4 and from the N-methyl group at ı 3.60 with carbon at ı 151.8 and 148.7 and N-methyl group at ı 3.41 with carbon at ı 151.8 laced them on two adjacent atoms. This information suggested the presence of a guanine base which substituted with three N-methyls. This was confirmed by the fragment ion at m/z 120, observed in the ESIMS, corresponding to the loss of the CH NCO moiety. A computer survey including Science finder indicated that 2 is new purine derivative (3,7-dimethyl2-(methylamino)-3H-purin-6(7H)-one) Compound 3 has a molecular formula of C42 H83 NO3 which was determined from HRESIMS (m/z 649.6368 [M]+ ). The MS fragmentation pattern is illustrated in Fig. 3. The IR spectra showed absorption bands attributable to hydroxyl, amide (NH) and CONH groups at 3340, 3320 and 1640 cm−1 , respectively. The 1 H NMR spectrum (Table 1) exhibited the presence of two primary methyls at ı 0.87 (6H, t, J = 7.6 Hz), two hetero bearing-methines at ı 3.91 and 4.32 and oxygenated methylene protons at ı 3.70 and 3.95, two olefinic protons at ı 5.54 and 5.78, an NH proton at ı 6.27 and a huge methylene envelope at ı 1.25. The 13 C and DEPT NMR spectral data of 3 supported the above analysis, showing a carbonyl group at ıC 174.0, a double bond at ıc 134.4 and 128.8, three oxygenated or other hetero atomized carbons at ıC 74.7, 62.5 and 54.8, aliphatic methylenes at ıc 22.7–36.9 and two methyls at ıc 14.1. The downfield doublet at ı 6.27 (NH) was deuterium-exchangeable, and there was no correlation between this signal and any carbon in the HMQC spectrum. On the other hand, a correlation from ı 6.27 (NH) to ı 3.91 (m), and the correlations from ı 6.27 (NH) to ıc 174.0(C-1 ), 36.9 (C-2 ), 62.5 (C-1), 54.8 (C-2) and 74.7 (C-3) were observed in the 1 H–1 H COSY and HMBC spectra, respectively. All the above data suggested 3 is a ceramide (sphingolipid) (Loukaci et al., 2000; Gaver and Sweeley, 1965). In order to determine the lengths of sphingosine and fatty acid chains, the positions of double bonds and the absolute configuration of 3, the acid methanolysis method according to Gaver and Sweeley (Tao et al., 2010), which yield a FAME methyl octadecanoate m/z 268 detected by GCMS, the presence of C-18 fatty acid moiety confirmed by the characteristic
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 928–935
3.2.
Biological evaluation
The total extract and isolated metabolites were tested toward two cancer cell lines HepG2 and MCF-7and showed moderate cytotoxic activities against both of them (Table 2). The mechanism of action was investigated by employing DNA affinity assays (cf exp.). Compound 5 showed significant affinity to DNA (Table 3). These results directed us to investigate the protection ability of the current isolated compounds. The results obtained are significantly important that this class of compounds showed protection activities. DNA is an important target of anticancer drugs; it was proved that the molecules which showed high affinity to bind with DNA showed significant inhibitory effect as alkylating agents. Different strategies are currently developed to potentiate the use of classical alkylating agents. (Pourquier P. Agents
100
% of DNA Protection
100
93
93
89
93
92
50
10
7
d un po om C
om C
po om C
po
un
un
d
d
5
4 d un po om C
C
om
po
un
m in
d
1
C
0 ita
ion at m/z 211 [CH3 (CH2 )12 CO]+ . So the molecular formulas of FAME and sphingosine are C18 H35 O and C24 H48 NO2 , respectively. The double bond and hydroxyl groups should be in sphingosine moiety and their positions could be determined by inspection of 1 H–1 H COSY spectrum, two methylene (C-1) protons at ı 3.70 and 3.95 correlated with the methine proton (C-2) at ı 3.91 which is correlated with the methine (C-3) proton at ı 4.32, the methine (C-3) proton at ı 4.32 correlated with the olefinic (C-4) proton at ␦ 5.54 (dt, J = 15.0, 6.6 Hz) which is in turn correlated with another olefinic (C-5) proton at ␦ 5.78 (dt, J = 15.0, 6.6 Hz), the olefinic proton at ı 5.78 correlated with two methylene (C-6) at ␦ 2.23 (q, J= 6.6 Hz) that correlated with another two methylene (C-7) protons at ␦ 2.06 (q, J = 6.6 Hz). The above discussion implies that the two OH groups are at C-1 and C-3, and a double bond at C-4/C-5 is trans oriented owing to the values of chemical shifts of allylic methylene ıc > 30 and the J values) (Su and Takaishi, 1999). Consideration of biogenesis and steric hindrance of such class of metabolites, generally were acknowledged to determine the absolute stereochemistry of the phytosphingosine moiety. On the basis of the 13 C NMR spectral data, the relative stereochemistries at C–3 (ı 74.7) were deduced to be 3S (Yaoita et al., 2000). Thus, the structure of 3 was established as N-((3S,E)-1,3-dihydroxytetracos-4-en2-yl)stearamide. Compound 4 was isolated as solid crystals. The 1 H, 13 C and DEPT NMR spectral data of 4 showed the presence of 19 carbon atoms; two methyls, eight methylenes, one sp2 methine (ıC 120.9 ppm) and five quaternary carbons (ıC 36.6, 47.6, 141.0 and 221.3). The chemical shift value and shape of the signals resonating at ıH 3.55 (dddd, J = 10.8, 10.8, 6.6 and 4.8 Hz, 1H, H␣ -3), 0.98 (s, 3H, CH3 -18) and 1.04 (s, 3H, CH3 -19) suggested a steroidal nucleus with no side chain attached at C-17. The absence of any methyl doublet and the down field value of CH3 -18 favored locating carbonyl group at C-17, this deduction was supported by ıC 221.0 and IR absorption. Compound 4 was identified by comparison of its 1 H and 13 C NMR data (cf. exp.) with those in the literature (Yang et al., 2008, Burgess et al., 2006) to be dehydroepiandrosterone. Compounds 5–10 were identified by comparing their spectral data with literature (Li et al., 2005; Migliuolo et al., 1993; Kobayashi and Kanada, 1991; John Goad & Akihisa, 1977; Pouchert, 1983).
V
934
Figure 4 – Bleomycin-dependent DNA damage assay of the tested compounds (13).
alkylants. Bull Cancer; 98: 1237- 1251). A brief description of the current method is that fixed amount of ligand is spotted on the RP18 TLC plates followed by addition of known quantity of DNA. The TLC was developed and the position of the DNA was assigned by spraying the plates with anisaldehyde reagent. It is easy to establish if the response of the test system is dependent on the dose of compounds. In case of increasing the quantities of DNA intercalators of the DNA lead to form a complex, and consequently, the free DNA can be detected as a blue spot (MeO:HeH O, 8:2) on RP-18 TLC after spraying with anisaldehyde reagent. The compounds of high binding to DNA were retained on the base line. However, when the DNA was mixed with compounds with which it is known to interact, the complex was retained at the origin when MeOH:H2 O (8:2) was used for elution. The inactive compounds did not cause the DNA to be retained at the origin. Moreover, methyl green reversibly binds polymerized DNA forming a stable complex when pH (7). The max for the DNA–methyl green complex is 642–645 nm. This colorimetric assay was employed to measure the displacement of methyl green from DNA by metabolites with DNA. The degree of displacement was determined spectrophotometrically by measuring the change in the initial absorbance of the DNA-methyl green solution in the presence of reference compound. Results from DNA binding assay (Table 3) revealed that compounds 5, 7 and 10 showed the highest affinity to DNA was measured IC50 (concentration required for 50% decrease in the initial absorbance of the DNA–methyl green solution). The remaining compounds showed weak activity. Bleomycin-dependent DNA damage has been evaluated as a specific method estimate the pro-oxidant drugs. Degradation of DNA could have been occurred when the assayed compounds reduced the bleomycin-Fe3+ to bleomycin-Fe2+ resulting in the production of MDA which combined with TBA to give a pink color. All compounds which decreased the absorbance and bleomycin-Fe3+ is not converted into bleomycin-Fe2+ , thus, preventing the DNA degradation. These results (Fig. 4) showed clearly that compounds (5 and 10) have the ability to protect DNA from the induced damage by bleomycin.
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 928–935
4.
Conclusion
Three new metabolites; polyoxygenatedcholestane-, purineand ceramide-derivatives (1–3), along with seven known metabolites (4–10) were obtained from Saudi marine sponge Petrosia sp. Their chemical structures were determined by interpretation of the spectral data of 1D and 2D NMR, UV, IR and MS measurements. The cytotoxicity of the total extract and the isolated pure compounds was evaluated by employing two cancer cell lines; HepG2 and MCF-7. All tested compounds showed anticancer activity with IC50 in range 20-500 M. The mechanism of anticancer activities was determined through its molecular affinity to the DNA. Compound 5 showed significant affinity to the DNA.
Conflict of Interest The authors declare no conflict of interests.
Acknowledgment This project was funded by the Saudi Basic Industries Corporation (SABIC) and the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under grant no. MS/14/318/1433. The authors, therefore, acknowledge with thanks SABIC and DSR technical and financial support. We also thank Dr. Yahia Folos, Marine Biology Department, Faculty of Marine Sciences, King Abdulaziz University, for collection and identification of the sponge sample.
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