MATERIALS AND METHODS

Preparation of Suberites aurantiacus extract and fractionation: Sponges were collected in June, 2009 in Magdalena Bay, Baja California Sur at 1.5 m of depth. Dr. Enrique Ávila Torres collected and identified the specimens, 675 g of animal material were used to obtain an ethanolic extract by maceration. The ethanolic extract was partitioned with water:butanol (1:1), obtaining an aqueous partition (22.79 g) and butanol (4.72 g). The organic phase was mixed and evaporated with silica gel, then fractionated using a stepwise gradient of CH₂Cl₂:EtOH (100:0-0:100). Posteriorly, a Thin Layer Chromatography silica gel (TLC plates) was performed and all the fractions were compared and grouped it into four fractions: E1Bt1 (1.5 g), E1Bt2 (1.2 g), E1Bt3 (1.4 g) and E1Bt4 (0.66 g).

Antibacterial activity: The antibacterial activity was determined by the disk diffusion method using Mueller Hinton agar. Gram-positive (S. aureus, B. subtilis, E. faecalis) and Gram-negative bacteria (E. aerogenes, E. coli, K. pneumoniea) standardized suspension (1.5×10⁸ CFU mL^–1) was applied on the solidified culture medium and sterile paper disks (6 mm) were impregnated with 1.5 mg/disk of the samples. The disk were transferred onto the inoculated agar plates and incubated at 37°C/24 h, then the resulting inhibition zone (clear area without bacterial growth including the disk)¹⁵. Positive and negative controls were gentamicin (15 μg/disk) and dimethyl sulfoxide (DMSO), respectively.

Antioxidant activity: DPPH (2,2-Dipheyl-1-picrylhydrazyl) radical scavenging activity was determined using 100 μL of samples (from 2-0.12 mg mL^–1) and 100 μL DPPH (0.12 mg mL^–1) in microplate. The reaction was completed after 30 min in the dark at room temperature and the absorbance was read at 517 nm, noting a color to turn from purple to yellow¹⁶. Positive and negative controls were trolox and methanol, respectively.

To determine ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging activity, the cation was prepared by mixing 10 mM ABTS stock solution with 2.45 mM potassium persulfate and leaving the mixture for 16 h. The ABTS solution was diluted with ethanol to get an absorbance of 0.7±0.05 at 734 nm. The assay was conducted adding 200 μL of ABTS solution and 20 μL of tested samples (0.25-2 mg mL^–1), measurements were taken at 734 nm after 6 min in dark¹⁶. Trolox (6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was the control.

Nitric oxide (NO) scavenging activity was analyzed using the Griess reaction in microplate. Fifty microliter of sodium nitroprusside (5 mM) was mixed with 50 μL of the samples (2-0.12 mg mL^–1) and it was incubated at room temperature for 2 h. After that, 100 μL of Griess reagent was added and the absorbance was read at 550 nm. Two controls were added: NO production (PBS) and NO inhibition (trolox).

Toxic activity: Toxic activity was determined by Artemia salina bioassay. Brine shrimp eggs were hatched in artificial sea water and after 48 h incubation in a warm room (27°C), nauplii were exposed to three different samples concentration (1000, 100 and 10 μg mL^–1) containing 10 organisms/5 mL and incubated at 27°C for 24 h. Vials were then examined and the number of dead nauplii in each vial was counted. Controls were artificial sea water, DMSO (1%) and potassium dichromate¹⁵.

Enzyme inhibition assays: The α-glucosidase inhibitory activity was evaluated according to the chromogenic method described by Kaskoos¹⁷ with some modifications. After a pre-incubation at 37°C for 15 min, 50 μL of sample and 50 μL of α-glucosidase (0.8 U mL^–1) were incubated in 96 well plates at 37°C for 15 min. After that, 50 μL of 625 mM p-nitrophenyl-α-D-glucopyranoside (PNPG) solution was added to each well and incubated for another 15 min. Then, the reaction was stopped by adding 100 μL of 0.2 M NaCO₃ into each well and absorbance reading was recorded at 405 nm.

The α-amylase inhibitory activity was evaluated according to Sudha et al.¹⁸ with some modifications. After a pre-incubation at 37°C for 30 min, 50 μL of sample and 50 μL of α-amylase (1 U mL^–1) was incubated in 96 well plates at 37°C for 15 min. After that, 50 μL of 0.5% starch solution in phosphate buffer was added to each well and the reaction was incubated at 37°C for 20 min. The reaction was stopped with 20 μL of 1 M HCl, followed by the addition of 50 μL of iodine reagent and absorbance was read at 750 nm.

Statistical analysis: All the experiments were performed in triplicate and the data were expressed as mean±standard deviation (SD). The median lethal dose (LD₅₀), half maximum effective concentration (EC₅₀) and half maximum inhibitory concentration (IC₅₀) values were determined using a probit regression with the SPSS version 17.0 software.

RESULTS

The present results demonstrated the scavenging effect, the carbohydrate hydrolyzing enzymes inhibition and the toxic and bactericide effect of the ethanolic extract and fractions from S. aurantiacus. The fraction obtained with CH₂Cl₂:EtOH (2:8) from butanolic phase showed bactericide effect only against Gram-positive bacteria, the best activity was against S. aureus and B. subtilis with 9 mm of inhibition zone. (Table 1), this sample also showed toxicity in the Artemia salina assay with median lethal dose of 637.9±120 μg mL^–1 (Table 2). Nevertheless, all the samples obtained from S. aurantiacus possess a poorly effectiveness as DPPH, ABTS and NO radical scavengers, since the results show EC₅₀ higher to 2 mg mL^–1. The inhibition of α-amylase and α-glucosidase show IC₅₀ higher to 2 mg mL^–1, while acarbose IC₅₀ were 0.12±0.02 and 0.97±0.08 mg mL^–1 for α-glucosidase and α-amylase, respectively. The observed biological effect for the fraction E1Bt3 is acceptable, considering that the active compound could be diluted. It is possible that isolating the active compound or compounds will provide better effect.

DISCUSSION

Antibiotic use has contributed significantly to the increased resistance of many bacterial strains and as a consequence of an increasing demand in screening for new therapeutic drugs from natural products. There is a greater interest towards marine organisms¹⁹, only from marine sponges more than 800 antibiotic compounds have been isolated²⁰.

Table 1:	Bactericidal activities from Suberites aurantiacus
Values are in inhibition zone (mm), n = 3±SD, Fractionation obtained with gradients CH2Cl2:EtOH: E1Bt1(100:0), E1Bt2 (1:1), E1Bt3 (2:8) and E1Bt4 (0:100)

Table 2:	Median lethal doses (LD50) from S. aurantiacus against A. salina
Medial lethal dose (LD50), n=3±SD. Fractionation obtained with gradients CH2Cl2:EtOH: E1Bt1(100:0), E1Bt2 (1:1), E1Bt3 (2:8) and E1Bt4 (0:100)

For these reasons, authors decided to studied the antibacterial activity of Suberites aurantiacus extract and its fractions using the disk diffusion method, which showed a moderate bactericide effect against Gram positive bacteria for fractions E1Bt2 and E1Bt3, this could be due to a minority compound, the sesterpenes have been the more common isolated natural compounds from sponges with bactericidal activity^10-14. Antibacterial studies from sponges has been reported since marine natural products started, Burkholder and Rutzler²¹ assayed antibacterial activity of 31 sponges and found a major inhibition of Gram-positive bacteria, due to the permeability provided by the cell wall or to the membrane accumulation tactics of Gram negative bacteria²², this was according to current results and may be due to the many sterols present in the S. aurantiacus extract²³.

Turk et al.²⁴ reported the antibacterial activity of ethanolic extracts from Antarctic marine sponges, where only 8 out from the 33 sponges were active with Minimum Inhibitory Concentration (MIC) values between 4 and 400 μg mL^–1, in contrast to their present study where they studied the aqueous and organic extracts from tropical marine sponges against B. subtilis and E. coli and most of the organic extracts showed MIC values between 0.1 and 36 μg mL^–1 for B. subtilis²⁵.

According to literature, sponges produce high levels of cytotoxic chemicals by the emission of mucus containing toxins as defense of predation and against fouling organisms²⁶, for this reason the toxic effect of S. aurantiacus using the brine shrimp lethality assay was determined, which is one of the most useful tools for the preliminary assessment of toxicity/cytotoxic of organic extracts, considered good candidates for the search of antineoplastics when it shows a LD < 1 mg mL^{–1 15,27}. Current results showed a toxic effect for fractions E1Bt1 and E1Bt3, this could suggest also two different bioactive compounds and being that fraction E1Bt2 were not active. In general, sponge extracts have shown a wide variation of lethal doses, for example, Sonia et al.²⁸, demonstrated the toxic effect from many sponges against A. salina nauplii and lethal doses (LD₅₀) fluctuating between 0.5 and 9 mg mL^–1 was observed.

Natural products have been reported as carbohydrate-hydrolyzing enzymes, associating the observed effects with the phenolic content and antioxidant activity of the extracts²⁹. This could explain current results, which did not show carbohydrate-hydrolyzing enzymes inhibition neither antioxidant activity. Orhan et al.³⁰ evaluated biological activity from seven marine sponges from Mediterranean sea and showed a low radical scavenging activity (>2 mg mL^–1). Shaaban et al.³¹ evaluated the antioxidant activity by DPPH and NO methods and the α-glucosidase and α-amylase inhibitory effect of different extracts from four marine sponges and only one extract showed EC₅₀ and IC₅₀ values <1 mg mL^–1. Berne et al.³² assayed 24 Antarctic marine sponges for a range of bioactivities, where four showed inhibitory effect on α-amylase, additionally, the extracts were tested on a variety of pathogenic and multiresistant Gram-negative and Gram-positive bacterial strains whereby all the tested sponge extracts showed weak to moderate inhibitory activities against at least one strain of the bacteria tested, nevertheless, the assay was carry out with 20 mg/disk, obtaining inhibitory zone diameter of 7-13 mm.

Although, sponges have been an interesting source of bioactive compounds, present study results and studies cited formerly, showed moderate Bioactivities from different genus of sponges. Mehbub et al.³³ reviewed the marine sponge derived natural products between 2001 and 2010 and reported a total of 1,615 new compounds from different orders of marine sponges with different Bioactivities, which showed some orders with over 200 bioactive compounds, meanwhile there are orders with less than 10 bioactive molecules. From Hadromerida order, the review showed 74 bioactive molecules, 2 and 25 out of them with antibacterial and cytotoxic activities, respectively.

These studies were according with current study, in which ethanolic extract and fractions of S. aurantiacus is not as promising as antiradical and α-glucosidase and α-amylase inhibitor. Nevertheless, it is moderately active against Gram positive bacteria and A. salina nauplii.

CONCLUSION

Marine invertebrates have been an important source of bioactive metabolites with a wide spectrum of interesting activities, in relation to the above, the marine sponge S. aurantiacus possesses compounds with bactericidal effect against Gram negative bacteria and toxic/cytotoxic activity. The best effect was showed by the fraction E1Bt3 proposing the isolation of bactericidal and/or cytotoxic compounds from these fraction for futures studies.

SIGNIFICANCE STATEMENT

The present study reports the bactericidal and toxic activities of the fractions from Suberites aurantiacus extract, which although it is an abundant specie in pacific coast of Mexico, it had not been study before for these bioactivities. It aims to expand upon the knowledge concerning the bioactive profile of sponges from Baja California Sur, what opens the possibility for the technological use of regional biodiversity.

ACKNOWLEDGMENT

Dr. Enrique Ávila Torres for the donation and identification of the sponge. Sara García-Davis received a fellowship for Ph.D. studies from CONACYT (No. 290666).