Chemical characterization, antioxidant and inhibitory effects of some marine sponges against carbohydrate metabolizing enzymes
© Shaaban et al.; licensee Springer. 2012
Received: 17 January 2012
Accepted: 5 April 2012
Published: 16 August 2012
More than 15,000 marine products have been described up to now; Sponges are champion producers, concerning the diversity of products that have been found. Most bioactive compounds from sponges were classified into anti-inflammatory, antitumor, immuno- or neurosurpressive, antiviral, antimalarial, antibiotic, or antifouling. Evaluation of in vitro inhibitory effects of different extracts from four marine sponges versus some antioxidants indices and carbohydrate hydrolyzing enzymes concerned with diabetes mellitus was studied. The chemical characterizations for the extracts of the predominating sponges; SP1 and SP3 were discussed.
All chemicals served in the biological study were of analytical grade and purchased from Sigma, Merck and Aldrich. All kits were the products of Biosystems (Spain), Sigma Chemical Company (USA), Biodiagnostic (Egypt). Carbohydrate metabolizing enzymes; Î±-amylase, Î±-glucosidase, and Î²-galactosidase (EC188.8.131.52, EC184.108.40.206, and EC220.127.116.11, respectively) were obtained from Sigma Chemical Company (USA).
Four marine sponges; Smenospongia (SP1), Callyspongia (SP2), Niphates (SP3), and Stylissa (SP4), were collected from the Red Sea at Egyptian coasts, and taxonomically characterized. The sponges' extracts exhibited diverse inhibitory effects on oxidative stress indices and carbohydrate hydrolyzing enzymes in linear relationships to some extent with concentration of inhibitors (dose dependant). The extracts of sponges (3, 1, and 2) showed, respectively, potent-reducing power. Purification and Chemical characterization of sponge 1 using NMR and mass spectroscopy, recognized the existence of di-isobutyl phthalate (1), di-n-butyl phthalate (2), linoleic acid (3), β-sitosterol (4), and cholesterol (5). Sponge 3 produced bis-[2-ethyl]-hexyl-phthylester (6) and triglyceride fatty acid ester (7).
Marine sponges are promising sources for delivering of bioactive compounds. Four marine sponges, collected from Red Sea at Egyptian coasts, were identified as Smenospongia (SP1), Callyspongia (SP2), Niphates (SP3), and Stylissa (SP4). The results demonstrated that different sponges extracts exhibited inhibitory effects on oxidative stress indices and carbohydrate hydrolyzing enzymes in linear relationships to some extent with concentration of inhibitors (dose dependant). The extracts of sponges (3, 1, and 2) showed, respectively, potent-reducing power. Chemical characterizations of sponges SP1 and SP3 were discussed. Based on this study, marine sponges are considered as talented sources for production of diverse and multiple biologically active compounds.
Pharmaceutical interest in sponges was aroused in the early 1950s by the discovery of a number of unknown nucleosides: spongothymidine and spongouridine in the marine sponge Cryptotethia crypta [1, 2]. These nucleosides were the basis for the synthesis of Ara-C, the first marine-derived anticancer agent and the antiviral drug Ara-A . Ara-C is currently used in the routine treatment of patients with leukaemia and lymphoma. More than 15,000 marine products have been described up to now [4, 5]; Sponges are champion producers, concerning the diversity of products that have been found . They are responsible for more than 5,300 different products and every year hundreds of new compounds are being discovered . Most bioactive compounds from sponges can be classified as anti-inflammatory, antitumor, immuno- or neurosurpressive, antiviral, antimalarial, antibiotic, or antifouling [5–9].
Exogenous chemical and endogenous metabolic processes in the human body or in the digestive system might produce highly reactive free radicals, especially oxygen-derived radicals, which are capable of oxidizing biomolecules, resulting in cell death and tissue damage. Almost all organisms are well protected against free radical damage by anti-oxidative enzymes such as superoxide dismutase and catalase (CAT), or by chemicals such as carotenoids, polyphenols, and glutathione . However, when the process of antioxidant protection becomes unbalanced, deterioration of physiological functions may occur resulting in diseases and accelerated aging. There is an increasing evidence, indicating that reactive oxygen species and free radical-mediated reactions are involved in degenerative or pathological events such as aging, cancer, coronary heart ailments, and Alzheimer’s diseases . Moreover, the suppression of the oxidative stress and inflammatory were responded through the inhibition of tumor necrosis factor β-(TNF-β) signaling . Natural triterpenes isolated from different marine sponges inhibited iNOS expression and the activation of NF-β, while polyketides showed antitumoural activity . Most screenings of secondary metabolites of biomedical importance from marine sponge extracts reported an inhibitory effects that turned out to be have strongly cytotoxic effects [14, 15].
In this article, evaluation of in vitro inhibitory effects of different extracts from four marine sponges Smenospongia (SP1), Callyspongia (SP2), Niphates (SP3), and Stylissa (SP4) versus some antioxidants indices and carbohydrate hydrolyzing enzymes concerned with diabetes mellitus. The studied sponges were collected from Red Sea, Hurghada, at Egyptian coasts. Alternatively, the chemical characterizations for two extracts of the predominating sponges; SP1 and SP3 were discussed on the bases of different chromatographic and spectroscopic means. In accordance, di-isobutyl phthalate (1), di-n-butyl phthalate (2), linoleic acid (3), β-sitosterol (4), and cholesterol (5) were obtained from SP1; while SP3 delivered bis-[2-ethyl]-hexyl-phthylester (6) and triglyceride fatty acid ester (7).
Four marine sponges belonging to the genus Smenospongia (SP1), Callyspongia (SP2), Niphates (SP3) and Stylissa (SP4) were collected from Hurghada at El-Gouna and Shaa’b south Giffton island at depth of 5–8 m. Morphologically, the sponges were characterized and specimens of them were deposited at Red Sea Marine parks, P.O. Box 363-Hurgada, Red Sea, Egypt.
The four sponges were individually extracted by DCM–MeOH (2:1), followed by filtration, and the afforded DCM layers were extracted and evaporated in vacuo to dryness. Extracts of sponges were applied to a series of chromatographic purifications including Flash chromatography on silica gel (230–400 mesh), Size exclusion chromatography was done on Sephadex LH-20, and PTLC to isolate their produced bioactive compounds in pure forms. Purity of the yielded compounds were monitored by R f values were measured on Polygram SIL G/UV254 TLC cards. This lead to isolate the following compounds; Linoleic acid; (9Z,12Z)-9, 12-octadecanoic acid (3), β-sitosterol (4), Cholesterol (5), Di-(2-ethylhexyl)phthalate(DEHP)(6), and Triglyceride fatty acids mixture (7) were assigned with the aid of different spectroscopic means as follows; NMR (1 H &13 C NMR) was served using Varian Unity 300 (300.145 MHz; and Varian Inova 600 (150.820 MHz) spectrometers. ESI MS (Thermo Finnigan LCQ with quaternary pump Rheos 4000 (Flux Instrument); Thermo Scientific, USA). EI MS(a Finnigan MAT 95 spectrometer (70 eV); Thermo Scientific, USA. GC-MS was measured on a Trace GC-MS Thermo Finnigan chromatograph, ionization mode EI (70 eV).
The in vitro antioxidant study of the sponges extracts were carried out using Carbohydrate metabolizing enzymes; α-amylase, α-glucosidase, and β-galactosidase. The antioxidant scavenging activity was studied using serial concentrations of different sponge extracts (10:1000 μg/mL) versus DPPH-free radical. The NO-free radical scavenging activity of extracts was determined according to the method of Sreejayan and Rao .
Results and discussion
Extracts of the four marine sponges Smenospongia (SP1), Callyspongia (SP2), Niphates (SP3), and Stylissa (SP4) were applied to a series of chromatographic applications, and hence to identify their bioactive constituents chemically using diverse spectroscopic means. Both sponges, SP1 and SP3, were intensively studied. Five compounds were revealed from SP1; di-isobutyl phthalate (1), di-n-butyl phthalate (2), linoleic acid (3), β-sitosterol (4), and cholesterol (5). The first two esters, di-isobutyl phthalate (1, RT: 13.79, 100%) and di-n-butyl phthalate (2, RT: 14.84, 12%), were established by GC-MS analysis together with further unknown components of RT 19.03 (17%) and 20.01 (78%). Purification of sponge 3 (SP3) afforded bis-[2-ethyl]-hexyl-phthylester (6) and triglyceride fatty acid ester (7). In contrast, working up and purification of the extracts obtained from the remaining two sponges SP2 and SP4 delivered multi-metabolites, however, with insufficient amounts for analysis.
Based on their chromatographic properties, spectroscopic means (NMR and MS), and comparison with authentic samples and literatures, the obtained structures were deduced as (9Z,12Z)-9,12-octadecanoic acid (3) , β-sitosterol (4) [18, 19], cholesterol (5) [20–24], phthalic acid bis-[2-ethyl-hexyl] ester (6)  and triglyceride fatty acid mixture (7) .
DPPH inhibition percent of the four sponges extracts
Extracts of the four sponges
10.85 ± 5.42c
47.7 ± 0.84e
26.31 ± 0.50d
16.39 ± 4.63d
10.77 ± 2.09c
60.53 ± 0.50d
26.00 ± 1.38d
22.38 ± 1.23c
18.09 ± 1.13b
66.45 ± 0.29c
34.04 ± 0.94c
32.54 ± 2.14b
22.81 ± 1.81b
69.52 ± 0.97b
37.39 ± 1.10b
35.94 ± 1.42ab
30.64 ± 0.60a
72.19 ± 0.69a
40.17 ± 0.9a
38.48 ± 0.70a
Inhibition percent of nitric oxide (NO) of the four sponges extracts
Extracts of the four sponges
10.30 ± 4.29c
37.60 ± 1.68e
15.26 ± 6.94d
13.50 ± 5.53d
16.92 ± 3.25c
39.16 ± 9.31d
24.94 ± 2.89c
21.41 ± 4.42c
21.95 ± 2.86b
52.13 ± 6.06c
33.30 ± 2.65bc
27.80 ± 4.32bc
23.59 ± 1.01b
56.58 ± 3.46b
36.29 ± 5.18ab
30.32 ± 2.74b
36.14 ± 2.93a
53.85 ± 5.12a
44.64 ± 4.29a
41.24 ± 3.27a
α -Amylase inhibition percent of four sponges extracts
Extracts of the four sponges
7.41 ± 2.61e
92.00 ± 1.21
18.26 ± 3.97d
15.44 ± 2.68e
18.75 ± 2.04d
94.35 ± 2.69
24.55 ± 4.03c
21.72 ± 3.20d
24.27 ± 3.07c
85.22 ± 4.92
32.56 ± 2.07b
27.38 ± 1.73c
29.05 ± 1.47b
89.38 ± 8.22
37.14 ± 0.89ab
33.75 ± 2.31b
38.13 ± 0.64a
88.40 ± 7.29
37.97 ± 1.86a
44.59 ± 1.55a
α -Glucosidase inhibition percent of the four sponges extracts
Extracts of the four sponges
8.06 ± 3.51c
28.05 ± 1.63c
25.45 ± 4.04c
23.47 ± 4.56c
21.08 ± 4.68b
37.80 ± 2.55b
33.91 ± 3.15b
33.49 ± 1.76b
28.95 ± 1.44a
44.21 ± 2.87a
40.44 ± 4.34a
40.55 ± 3.08a
27.24 ± 3.45a
41.19 ± 1.59ab
37.42 ± 1.14ab
36.01 ± 1.78ab
27.65 ± 2.37a
42.5 ± 2.24a
39.32 ± 2.60ab
38.11 ± 2.70ab
β -galactosidase inhibition percent of four sponges extracts
Extracts of the four sponges
11.45 ± 11.54b
51.35 ± 5.24b
23.09 ± 8.33b
17.24 ± 6.39c
31.58 ± 22.03ab
39.62 ± 8.53bc
34.78 ± 11.03b
35.35 ± 9.72b
27.37 ± 13.18ab
34.49 ± 11.82c
36.49 ± 8.35b
33.53 ± 11.38b
46.58 ± 8.22a
67.82 ± 3.94a
62.63 ± 1.89a
54.13 ± 2.44a
42.21 ± 5.28a
66.86 ± 3.79a
62.12 ± 4.37a
55.08 ± 5.11a
The NMR spectra were measured on Varian Unity 300 (300.145 MHz) and Varian Inova 600 (150.820 MHz) spectrometers. ESI MS was recorded on a Thermo Finnigan LCQ with quaternary pump Rheos 4000 (Flux Instrument); Thermo Scientific, USA). EI mass spectra were recorded on a Finnigan MAT 95 spectrometer (70 eV); Thermo Scientific, USA. GC-MS was measured on a Trace GC-MS Thermo Finnigan chromatograph, ionization mode EI (70 eV), instrument equipped with a capillary column CP-Sil 8 CB for amines (length: 30 m; inside diameter: 0.25 mm; outside diameter: 0.35 mm; film thickness: 0.25 μm); Thermo Scientific., USA. The analysis was carried out at a programmed temperature: initial temperature 40°C (kept for 1 min), then increasing at a rate of 10°C/min and final temperature 280°C (kept for 10 min), injector temperature was 250°C and detector (mode of ionization: EI) temperature at 250°C, He was used as carrier gas at a flow rate of 1 mL/min, total run time 27 min, injection volume 0.2 μL. Flash chromatography was carried out on silica gel (230–400 mesh). R f values were measured on Polygram SIL G/UV254 TLC cards (Macherey-Nagel GmbH & Co. Germany). Size exclusion chromatography was done on Sephadex LH-20 (Lipophilic Sephadex, Amersham Biosciences Ltd. (purchased from Sigma-Aldrich Chemie, Steinheim, Germany). All chemicals served in the biological study were of analytical grade, which were purchased from Sigma, Merck and Aldrich. All kits were the products of Biosystems (Spain), Sigma Chemical Company (USA), Biodiagnostic (Egypt).
Sponge materials, collection, and taxonomy
Extraction and isolation
The four sponges, Smenospongia (SP1), Callyspongia (SP2), Niphates (SP3) and Stylissa (SP4), were individually cut into small pieces and homogenized mechanically (Figure 1), treated with DCM–MeOH (2:1) and kept at approximately 5°C for 8 days. After filtration, the DCM layers were extracted and evaporated in vacuo to dryness affording 1.59, 0.57, 0.39, and 0.64 g from sponges SP1, SP2, SP3, and SP4, respectively. In contrast with sponges SP1 and SP3, both sponges SP2 and SP4 were applied to a series of chromatographic purifications using silica gel, Sephadex, and PTLC affording no desired and inadequate compounds amounts.
Working up and purification of smenospongia (SP1)
The afforded greenish-brown crude extract of sponge 1(1.59 g) was subjected to silica gel (column 3 × 60 cm2) and eluted with a cyclohexane-hexane/DCM/MeOH gradient. Based on the TLC monitoring, visualized by UV and spraying with anisaldehyde/sulfuric acid, five fractions were obtained: FI (0.1 g), FII (0.2 g), FIII (0.3 g), FIV (0.3 g), and FV (0.4). The fast oil fraction I was applied to GC-MS analysis, displaying a base signal (RT: 25.04 nm, 100%) of unknown component. Fraction II was likely subjected to GC-MS analysis showing four signals representing four components (RT: 13.79, 100%), (14.84, 12%), (19.03, 17%), and (20.01, 78%); the first two of them were of unknown structures, while the last two were assigned as di-isobutyl phthalate (1) and di-n-butyl phthalate (2). TLC monitoring of the remaining fractions (III, IV, and V) recognized their similarity, and they were combined therefore (1.0 g). Consequently, the combined fractions were then chromatographed on silica gel using cyclohexane-DCM–MeOH gradient and after monitoring by TLC, six sub-fractions; PIa (80 mg), PIb (35 mg), PIc (70 mg), PId (95 mg), PIe (38 mg), and PIf (27 mg). An application of the sub-fractions to further purification using Sephadex LH-20 (DCM/MeOH, 60:40) was carried out. In accordance, sub-fractions PIa, PIc afforded a colorless semisolid of linoleic acid (3, 55 mg), while purification of sub-fraction PIb afforded a colorless solid of β-sitosterol (4, 27 mg). Purification of sub-fraction PId afforded a colorless oil of an olefinic fatty acid (23 mg). Similarly, purification sub-fraction PIe yielded a colorless oil of an additional olefinic acid. Finally, an application of the sub-fraction PIf to Sephadex LH-20 (DCM/MeOH, 60:40) resulted in β-sitosterol (4, 3 mg) and cholesterol (5, 3 mg).
Linoleic acid; (9Z,12Z)-9, 12-octadecanoic acid (3)
Colorless oil (55 mg) was detected as non-polar UV absorbing band at 254 nm and stained to blue when sprayed by anisaldehyde/sulfuric acid and heated. –C 18 H 32 O 2 (280). – R f = 0.90 (CHCl3/MeOH, 10%). - 1 H NMR (CDCl3, 300 MHz): δ = 8.98 (s, br, 1 H, COOH), 5.43–5.28 (m, 4 H, 9,10,12,13-CH), 2.78 (t, 3 J = 6.0 Hz, 2 H, 11-CH2), 2.38 (t, 3 J = 7.2 Hz, 2 H, 2-CH2), 2.08 (m, 4 H, 8,14-CH2), 1.63 (m, 2 H, 3-CH2), 1.42–1.23 (m, 14 H, 4,5,6,7,16,17-CH2), 0.85 (m, 3 H, 18-CH3). – 13 C/APT NMR (CDCl3, 50 MHz): δ = 180.1 (CO, Cq), 130.1 (CH-13), 129.9 (CH-9), 128.0 (CH-10), 127.8 (CH-12), 31.5 (CH2-2), 29.6 (CH2-16), 29.6 (CH2-11), 29.5 (CH2-14), 29.3 (CH2-8), 29.1 (CH2-7), 29.0 (CH2-6), 29.0 (CH2-5), 27.1 (CH2-4), 25.6 (CH2-3), 24.7 (CH2-15), 22.5 (CH2-17) 14.0 (CH2-18). –EI MS (70 eV): m/z (%) = 280 (80), 264 (28), 137 (10), 124 (15), 110 (28), 95 (60), 81 (84), 67 (100), 55 (92), 41 (92).
Colorless solid, UV non-absorbing, turned blue on spraying with anisaldehyde/sulfuric. –C 29 H 50 O (414). –R f = 0.51(CH2Cl2/CH3OH 9: 0.5). – 1 H NMR (CDCl3, 300 MHz): δ = 5.36 (d, J = 4.7 Hz, 1 H, H-6), 3.53 (m, 1 H, H-3), 2.36–2.21 (m, 4 H, H2-1, H2-4), 2.01–1.93 (m, 2 H, H2-6), 1.85–1.75 (m, 4 H), 1.58–1.43 (m, 5 H), 1.21–1.03 (m, 14 H), 0.99 (s, 3 H, CH3-19), 0.94 (d, 3 H, J = 6.1 Hz, CH3-21), 0.86 (d, 3 H, J = 6.2 Hz, CH3-26), 0.84 (d, 3 H, J = 6.2 Hz, CH3-27), 0.79 (t, 3 H, J = 6.7, CH3-29), 0.67 (s, 3 H, CH3-18). –EI-MS (70 eV): m/z (%) = 414 ([M]+, 100), 396 ([M-H2O]+, 37), 381 (21), 329 (34), 303 (41), 283 (16), 259 (10), 241 (22), 227 (9), 206 (11), 189 (18), 173 (22), 151 (13), 135 (25), 123 (21), 109 (18), 83 (13), 43 (15).
Colorless solid, UV non-absorbing, turned blue on spraying with anisaldehyde/sulfuric. –C 27 H 46 O (386). –R f = 0.47(CH2Cl2/CH3OH 9:0.5). – 1 H NMR (CDCl3, 300 MHz): δ = 5.37 (d, J = 4.7 Hz, 1 H, H-6), 3.51 (m, 1 H, H-3), 2.35–2.19 (m, 4 H, H2-1, H2-4), 2.02–1.94 (m, 2 H, H2-6), 1.85–1.75 (m, 4 H), 1.58–1.43 (m, 6 H), 1.18–1.02 (m, 12 H), 0.99 (s, 3 H, CH3-19), 0.94 (d, 3 H, J = 6.1 Hz, CH3-21), 0.86 (d, 3 H, J = 6.2 Hz, CH3-26), 0.84 (d, 3 H, J = 6.2 Hz, CH3-27), 0.67 (s, 3 H, CH3-18). – 13 C NMR (CDCl3, 75 MHz): δ = 42.7 (Cq, C-13), 36.7 (CH, C-1), 140.6 (Cq, C-5), 56.4 (CH, C-14), 50.2 (CH, C-9), 31.8 (CH, C-8), 56.3 (CH, C-17) 121.8 (CH, C-6), 40.0 (CH2, C-24), 32.0 (CH2, C-16), 21.0 (CH2, C-11), 24.3 (CH2, C-15), 37.3 (Cq, C-10), 28.2 (CH, C-25), 42.3 (Cq, C-4), 35.9 (CH2, C-12), 12.1 (CH3, C-18), 31.7 (CH2, C-7), 31.2 (CH2, C-2), 71.5 (CH, C-3), 19.4 (CH3, C-19), 24.0 (CH2, C-23), 36.3 (CH2, C-22), 18.8 (CH3, C-21), 35.8 (CH, C-20), 22.6 (CH3, 26), 22.6 (CH3, 27). –EI-MS (70 eV): m/z (%) = 386 ([M]+, 100), 368 ([M-H2O]+, 36), 353 (20), 301 (32), 275 (40), 255 (18), 231 (12), 213 (21), 199 (8), 178 (12), 161 (16), 145 (21), 133 (12), 107 (24), 95 (20), 81 (17), 55 (14), 43 (16).
Working up and purification of callyspongia (SP3)
The afforded reddish-brown crude extract of sponge 3 (0.39 g) was subjected to silica gel column (2 × 50 cm) and eluted with a cyclohexane-hexane/DCM/MeOH gradient. According to TLC monitoring, visualized by UV and spraying with anisaldehyde/sulfuric acid, three fractions were obtained: FIa (140 mg), FIb (70 mg), and FIc (60 mg). Purification of FIa using Sephadex LH-20 (DCM/MeOH, 60:40) afforded a colorless oil of phthylester (6, 80 mg). Purification of FIb by Sephadex LH-20 (DCM/MeOH, 60:40) resulted in a colorless oil of triglyceride fatty acid ester mixture (7, 35 mg). An application of fraction FIc to purification with Sephadex LH-20 (DCM/MeOH, 60:40) afforded a colorless solid of cholesterol (5, 28 mg).
UV-absorbing (254 nm) turned intensive violet on spraying with anisaldehyde/sulfuric acid after heating, and changed latter as blue. –C 24 H 38 O 4 (390). – R f = 0.90; CHCl3. – 1 H NMR (CDCl3, 300 MHz): δ = 7.70 (m, 2 H), 7.50 (m, 2 H), 4.25 (d, 3 J = 5 Hz, 2 H), 1.80–1.20 (br m, 18 H), 1.00–0.75 (br m, 12 H). – 13 C NMR (CDCl3, 75 MHz): δ = 167.7 (Cq-1′,1″), 132.4 (Cq-1,2), 130.9 (CH-3,6), 128.8 (CH-4,5), 68.1 (CH2-2′, 2″), 38.7 (CH-3′,3″), 30.3 (CH2-4′,4″), 28.9 (CH2-5′,5″), 22.9 (CH2-6′,6″), 14.0 (CH3-7′,7″), 23.7 (CH2-8′,8″), 10.9 (CH3-9′,9″). –EI-MS (70 eV): m/z: 390 (3), 279 (20), 149 (64). –CI-MS (NH3): 798 ([2 M + NH4]+, 62 %), 408 ([M + NH4]+, 100), 391 ([M + H]+, 65).
Triglyceride fatty acids mixture (7)
Colorless oil, turned violet by anisaldehyde/sulfuric acid; –R f = 0.78 (CH2Cl2). – 1 H NMR (300 MHz, CDCl3): δ = 5.37 (m, 2 H), 5.34 (m 2 H), 5.27 (m, 1 H), 4.30 (dd, 2 H, J = 11.9, 4.3 Hz), 4.14 (dd, 2 H, J = 11.9, 6.0 Hz), 2.81 (m, 2 H), 2.31 (m, 2 H), 2.02 (m, 4 H), 1.61 (m, 2 H), 1.40–1.20 (m, 22 H), and 0.88 (t, 9 H, J = 6.9 Hz). – 13 C NMR (75 MHz, CDCl3): δ = 172.9 (2Cq, CO), 172.5 (Cq, CO), 129.5 (2CH), 129.1 (2CH), 132–127 (further mCH), 68.8 (CH), 62.5 (2CH2), 34.0 (CH2), 31.9 (CH2), 29.8 (CH2), 29.7 (CH2) 29.6 (CH2) 29.5 (CH2) 29.4 (CH2) 29.3 (CH2) 29.2 (CH2) 29.0 (CH2) 29.1 (CH2), 29.0 (CH2), 27.2 (CH2), 25.6 (CH2), 24.9 (CH2), 22.7 (CH2) and 14.1 (CH3).
In vitro antioxidant study
Carbohydrate metabolizing enzymes; α-amylase, α-glucosidase, and β-galactosidase (EC18.104.22.168, EC22.214.171.124, and EC126.96.36.199, respectively) were obtained from Sigma Chemical Company (USA).
The antioxidant scavenging activity
Determination of NO-free radical scavenging activity
NO-scavenging activity of extracts was determined according to the method of Sreejayan and Rao .
Determination of α-amylase
α-Amylase was determined according to the method of Bernfeld .
Determination of β-galactosidase activity
β-galactosidase was measured by the method of Sánchez and Hardisson .
Estimation of α-glucosidase activity
In conclusion, this study was performed to investigate the effects of the extracts of four marine sponges on some biochemical parameters including antioxidant and three different carbohydrate hydrolyzing enzymes (α-amylase, β-galactosidase, and α-glucosidase). These sponges were collected from Red Sea at Egyptian coasts, which were taxonomically belonged to the genus of Smenospongia (SP1), Callyspongia (SP2), Niphates (SP3), and Stylissa (SP4). The results demonstrated that different extracts exhibited inhibitory effects on oxidative stress indices and carbohydrate hydrolyzing enzymes in linear relationships to some extent with concentration of inhibitors (dose dependant). The extracts of sponges (3, 1, and 2) showed, respectively, potent-reducing power. Chemical characterizations of sponges SP1 and SP3 were discussed, at where di-isobutyl phthalate (1), di-n-butyl phthalate (2), linoleic acid (3), β-sitosterol (4), and cholesterol (5) were obtained from sponge SP1; while sponge SP3 produced bis-[2-ethyl]-hexyl-phthylester (6) and triglyceride fatty acid ester (7).
The authors are deeply thankful to Prof. H. Laatsch for his Lab facilities and unlimited support. This research work has been financed during German Egyptian Scientific Projects (GESP) No. 7.
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