Chemical characterization, antioxidant and inhibitory effects of some marine sponges against carbohydrate metabolizing enzymes

Background 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. Methods 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 (EC3.2.1.1, EC3.2.1.20, and EC3.2.1.23, respectively) were obtained from Sigma Chemical Company (USA). Results 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). Conclusion 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.


Background
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 [3]. 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 [6]. They are responsible for more than 5,300 different products and every year hundreds of new compounds are being discovered [4]. Most bioactive compounds from sponges can be classified as anti-inflammatory, antitumor, immuno-or neurosurpressive, antiviral, antimalarial, antibiotic, or antifouling [5][6][7][8][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 [10]. 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 [11]. Moreover, the suppression of the oxidative stress and inflammatory were responded through the inhibition of tumor necrosis factor β-(TNF-β) signaling [12]. Natural triterpenes isolated from different marine sponges inhibited iNOS expression and the activation of NF-β, while polyketides showed antitumoural activity [13]. 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].
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 [16].

Biological study
The present results demonstrate the inhibitory effect of different extracts of marine sponges, on antioxidant indices and carbohydrate hydrolyzing enzymes in vitro. The 2,2-diphenyl-1-picrylhydrazyl (DPPH)-free radical scavenging effects of different extracts from marine sponge were shown in Table 1 and Figure 1. All the tested extracts showed appreciable free radical scavenging activities. Extract of SP2 has the strongest radical scavenging activity at different concentrations compared to other extracts followed by SP3 and SP4. However, SP1 showed the lowest radical scavenging activity. A dose-response relationship was found in the DPPH radical scavenging activity, at where the activity increased as the increase of extracts concentrations. SP2 extract was able to reduce the stable radical DPPH to the yellow color to give significant inhibitory percent 47.7 ± 0.84, 60.53 ± 0.50,  Contrarily, SP1 exhibited the lowest reducing activity compared with aforementioned sponges extracts. The demonstrated inhibitory activity of the DPPH by the sponges extracts might be mainly attributed to their containing of some terpenoidal analogs [26]. Nitric oxide synthase (NOS) is catalyzing the production of nitric oxide (NO). Inducible NOS (iNOS) is expressed by vascular endothelial cells and smooth muscle cells in response to cytokines, unlike the two other types of NOS, which are constitutive. NO produced by iNOS is implicated in inflammatory diseases [27]. NO-free radicals scavenging capacity of the different marine sponges extracts were illustrated in Table 2 and Figure 2. The most reducing capacity was as well considered for SP2, which showed significant ability to reduce the activity of NO by 37.60 ± 1.68, 39.16 ± 9.13, 52.13 ± 6.06, 56.58 ± 3.46, and 53.85 ± 5.12% at concentrations of 10-1000 μg/mL. Alternatively, extract of SP3 exhibited lower significant reducing activity of NO (15.26 ± 6.94, 24.94 ± 2.89, 33.30 ± 2.65, 36.29 ± 5.18, and 44.64 ± 4.29%) with lower extent than those of SP2. Based on the percentage scavenging values, it was remarked that SP1 and SP4 exhibited moderate scavenging effects with linear relationships in a dosedependent manner. Consequently, extracts of SP1 and SP4 recorded potent reducing capability of 41.24 ± 3.27 and 38.17 ± 3.01%, respectively, at a concentration of 1000 μg/mL. Tasi et al. [28] reported that food and phytochemicals exerts NO-suppressing activity via three different pathways: the blocking of iNOS expression, inactivation of iNOS catalytic function, and the scavenging NO. While NO suppressing effect was primarily NO is expressed as%; data are mean ± SD of 3 replicates; statistical analysis is carried out using one way analysis of variance (ANOVA) using CoStat: computer program; unshared superscript letters between treatments are significance values at P < 0.001. through regulation of cellular iNOS expression. The extracts' effects on the suppressing activity of NO production might be attributed to their containing of polyphenolic compounds or the triterpenes [29].
In alternative manner, extracts of the four sponges were tested against α-amylase carbohydrate hydrolyzing enzyme activity (Table 3, Figure 3). The four sponges showed potent α-amylase inhibitory activity, which may be potentially useful in control of obesity and diabetes. The inhibition of α-amylase by SP3 and SP4 was remarked to be as dose dependent, exhibiting the highest significant reducing activity 44.59 ± 1.55 and 43.64 ± 1.79%, respectively, at a concentration of 1000 μg/mL. Furthermore, extract of SP2 exhibited the most dramatic inhibiting effect at 10 and 50 μg/mL, displaying insignificant reducing activity 92.00 ± 1.21 and 94.35 ± 2.69%, respectively. In addition, the inhibitory activity of SP2 recorded 88.40 ± 7.29% at 1000 μg/mL. Consequently, SP3 recorded a significant inhibitory percent of 18.26 ± 3.97, 24.55 ± 4.03, 32.56 ± 2.07, 37.14 ± 0.89, and 37.97 ± 1.86% in a dose-dependent manner at 10, 50, 500, and 1000 μg/mL, respectively. The anti-amylase inhibitory activity may be due to the ability of phenolic compounds to interact with and/or inhibit proteins enzymes [30].
One therapeutic approach for treating diabetes is to decrease the post-prandial hyperglycemia. This is done by retarding the absorption of glucose through the inhibition of the carbohydrate hydrolyzing enzymes αamylase, α-glucosidase, and β-galactosidase in the digestive tract. Inhibition of these enzymes delay carbohydrate digestion and prolong overall carbohydrate digestion time, causing a reduction in the rate of glucose absorption and consequently blunting the post-prandial plasma glucose rise [31]. Many natural resources have been α-amylase is expressed as %; data are mean ± SD of 3 replicates; statistical analysis is carried out using one way analysis of variance (ANOVA) using CoStat: computer program; unshared superscript letters between treatments are significance values at P < 0.001. investigated with respect to the antidiabetic and suppression of glucose. The inhibitory effects of the four sponges extracts against α-glucosidase carbohydrate hydrolyzing enzyme activity were further studied as listed in Table 4 and Figure 4. were deduced at concentrations of 100 and 1000 μg/mL, respectively. Hence, the inhibition percent was significantly correlated with the increase in concentration of inhibitors. The fact that α-glucosidase and α-amylase showed different inhibition kinetics seemed to be due to structural differences related to the origin of the enzymes [32]. Manosroi et al. [33] attributed the anti-diabetic, antiinflammatory, anti-tumor, and anti-proliferative effect of many species, to their constituents of mono, sesquiterpenes, phenolic compounds, and flavonoids such as cinnamic acid, caffeic acid, and rosmarinic acid. β-Galactosidase inhibitory activity was finally studied versus the sponges extracts as summarized in Table 5 and Figure 5. Accordingly, SP2 and SP3 provided additional support for the previous finding by having the strongest reducing activity at various concentrations. Hence, SP2 at 500 and 1000 μg/mL displayed significantly the highest inhibitory percent amounted 67.82 ± 3.94 and 66.86 ± 3.79%, respectively, followed by SP3 (62.63 ± 1.89 and 62.12 ± 4.37%, respectively) and SP4 (54.13 ± 2.44 and 55.08 ± 5.11%, respectively). In contrast, SP1 displayed a comparable insignificant inhibitory activity of 45.89 ± 4.91, 43.77 ± 4.5% at 500 and 1000 μg/ mL, respectively. From the manipulated results, it was deduced significant increase in reducing activity with the increase in concentrations of the individual extract (linear relationship).

Experimental
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 β-galactosidase is expressed as %; data are mean ± SD of 3 replicates; statistical analysis is carried out using one way analysis of variance (ANOVA) using CoStat computer program; unshared superscript letters between treatments are significance values at P < 0.001.  β-galactosidase is expressed as%; data are mean ± SD of 3 replicates; statistical analysis is carried out using one way analysis of variance (ANOVA) using CoStat computer program; unshared superscript letters between treatments are significance values at P < 0.001. Taxonomically, the first species (SP1) was belonging to the genus Smenospongia, family Thorectidae. Morphologically, the SP1 is Massive with oscular mounds, displaying a bright to dark green coloration. The sponge exuded abundant mucus when handled, exhibiting blunt ends of primary protrude fibers on the surface [34,35]. The second sponge was belonging to the genus Callyspongia and family Callyspongiidae [36,37]. Morphologically, it showed bluish to pinkish tubes and sticky massive. Moreover, tissues of the sponge were clear away easily, leaving the clean skeleton. They inhabit as well in the coral reef habitat attached to corals or rocks. The third sponge was belonging to the genus Niphates and family Niphatidae. Morphologically, it is massive or encrusting, showing a bluish to purplish grayish cushions and repent branches [36,37]. Finally, the fourth sponge was belonging to the genus Stylissa and family Dictyonellidae [36,37]. Morphologically, it is bushy with orange color and tough consistency (Figures 7 and 8).

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 cm 2 ) 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.

The antioxidant scavenging activity
The activity of serial concentrations of different sponge extracts (10:1000 μg/mL) on DPPH-free radical will performed according to the method of McCue et al. [36] and Katsube et al. [37]. The decrease in optical density of DPPHis calculated compared with a control substance as follows: Determination of NO-free radical scavenging activity NO-scavenging activity of extracts was determined according to the method of Sreejayan and Rao [16].
Determination of α-amylase α-Amylase was determined according to the method of Bernfeld [38].
Determination of β-galactosidase activity β-galactosidase was measured by the method of Sánchez and Hardisson [39].
Estimation of α-glucosidase activity α-glucosidase activity was determined according to the method of Kapustka et al. [40] and Kim et al. [32].