Background and Objective: Invertebrates comprises more than fifty percent of marine animals. Among them, sponges are the most prolific source of bioactive compounds. Suberites aurantiacus (S. aurantiacus) is an abundant sponge in Mexican Pacific, which has been scarcely studied, for this reason authors screened it for its bioactivities. The current study proposed to determine the enzymatic inhibition and scavenger, toxic and antibacterial activities from Suberites aurantiacus. Materials and Methods: S. aurantiacus samples were collected in Magdalena Bay, Mexico. The ethanolic extract and its fractions were assayed for their antioxidant effect using DPPH, ABTS and NO assays, evaluated their toxicity against Artemia salina (A. salina) and their antibacterial activity against Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis), Enterococcus faecalis (E. faecalis), Enterobacter aerogenes (E. aerogenes), Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) as well as their inhibitory effect on α-glucosidase and α-amylase. Results: The results showed median lethal doses (LD50) against A. salina < 1 mg mL1 for two fractions and a moderate antibacterial activity against Gram positive bacteria. Conclusion: In view of these results, S. aurantiacus could be considered a potential source of antibacterial compounds.
PDF Abstract XML References Citation
How to cite this article
Natural products have been explored over the years for their potential applications as food, fragrances, pigments, insecticides and drugs1. Most of the efforts to discover new bioactive metabolites have been focused in plants due to their relative ease of access. However, since the development of appropriate scuba techniques, about 50 years ago, the marine environment has been intensely explored as a source of new bioactive agents2.
This environment covers a wide range of temperatures, pressure and nutrients, housing extensive photic and aphotic zones3, constituting a source of countless products whose properties include antiviral, antibacterial, antiprotozoal, antifungal, cytotoxic and antitumor activities4.
Marine invertebrates comprise approximately 60% of all marine animals, one of the groups with the highest number of bioactive compounds reported, most of them produced as defense mechanism. Due to their lack of swimming speed and physical defense, synthesizing compounds that can be used to discourage predators confers them an adaptive advantage in marine environment5.
Sponges (phylum Porifera) are the oldest multicellular animals and they are present in abundance in tropical reefs, polar latitudes, freshwater lakes and rivers6. More than 5,300 metabolites has been isolated from sponges7, representing 48.8% of all the new marine invertebrates natural products discovered between 1990 and 20118.
Suberites aurantiacus sponge belongs to the Demospongiae class, the Hadromerida order and the Suberitidae family. It inhabits estuarine environments and semi-enclosed bays and is one of the most abundant species in Mexican Pacific estuaries9. In addition, it is known that Suberites sponge produce principally sesterpenes10-14, nevertheless, there is a lack of biological activity studies concerning sponges from the Suberites genus in Mexico. For these reasons, current study proposed to determine the enzymatic inhibition and scavenger, toxic and antibacterial activities from Suberites aurantiacus.
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 mL1) 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 mL1) and 100 μL DPPH (0.12 mg mL1) 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 mL1), 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 mL1) 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 mL1) 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 mL1) 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 mL1) 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.
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 mL1 (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 mL1. The inhibition of α-amylase and α-glucosidase show IC50 higher to 2 mg mL1, while acarbose IC50 were 0.12±0.02 and 0.97±0.08 mg mL1 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.
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 activity10-14. 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 mL1, 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 mL1 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 mL1 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 mL1 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 mL1). 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 mL1. 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.
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.
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.
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).
- Tziveleka, L.A., C. Vagias and V. Roussis, 2003. Natural products with anti-HIV activity from marine organisms. Curr. Top Med. Chem., 3: 1512-1535.
- Cragg, G.M. and D.J. Newman, 2005. Biodiversity: A continuing source of novel drug leads. Pure Applied Chem., 77: 7-24.
- Jha, R.K. and X. Zi-Rong, 2004. Biomedical compounds from marine organisms. Mar. Drugs, 2: 123-146.
- Kazanjian, A. and M. Farinas, 2006. [Biological activity of an aqueons extract of the sponge Aplysina lacunosa (Porifera: Aplysinidae)]. Rev. Biol. Trop., 54: 189-200, (In Spanish).
- Aguila-Ramirez, R.N., C.J. Hernandez-Guerrero and B. Gonzalez-Acosta, 2011. [Biotechnical potential of sponges in new pharmaceutics production: Perspectives and limits]. CICIMAR Oceanides, 26: 31-46, (In Spanish).
- Schmitt, S., P. Tsai, J. Bell, J. Fromont and M. Ilan et al., 2012. Assessing the complex sponge microbiota: Core, variable and species-specific bacterial communities in marine sponges. ISME J., 6: 564-576.
- Kamalakkannan, P., 2015. Marine sponges a good source of bioactive compounds in anticancer agents. Int. J. Pharmaceut. Sci. Rev. Res., 31: 132-135.
- Leal, M.C., M.H. Munro, J.W. Blunt, J. Puga and B. Jesus et al., 2013. Biogeography and biodiscovery hotspots of macroalgal marine natural products. Nat. Prod. Rep., 30: 1380-1390.
- Carballo, J.L., B. Yanez and H. Nava, 2004. Persistence of the sponge Suberites aurantiaca (Duchassaing and Michelotti, 1864) in an estuarine ecosystem (Pacific coast, Mexico). Bull. Mar. Sci., 75: 487-497.
- Shin, J., Y. Seo, J.R. Rho, E. Baek, B.M. Kwon, T.S. Jeong and S.H. Bok, 1995. Suberitenones A and B: Sesterterpenoids of an unprecedented skeletal class from the Antarctic sponge Suberites sp. J. Org. Chem., 60: 7582-7588.
- Diaz-Marrero, A.R., I. Brito, E. Dorta, M. Cueto, A. San-Martin and J. Darias, 2003. Caminatal, an aldehyde sesterterpene with a novel carbon skeleton from the Antarctic sponge Suberites caminatus. Tetrahedron Lett., 44: 5939-5942.
- Diaz-Marrero, A.R., E. Dorta, M. Cueto, A. San-Martin and J. Darias, 2004. Conformational analysis and absolute stereochemistry of 'spongian'-related metabolites. Tetrahedron, 60: 1073-1078.
- Diaz-Marrero, A.R., I. Brito, M. Cueto, A. San-Martin and J. Darias, 2004. Suberitane network, a taxonomical marker for Antarctic sponges of the genus Suberites? Novel sesterterpenes from Suberites caminatus. Tetrahedron Lett., 45: 4707-4710.
- Lee, H.S., J.W. Ahn, Y.H. Lee, J.R. Rho and J. Shin, 2004. New sesterterpenes from the antarctic sponge Suberites sp. J. Nat. Prod., 67: 672-674.
- Viveros-Valdez, E., O.A. Oranday-Cardenas, C. Rivas-Morales, M.J. Verde-Star and P. Carranza-Rosales, 2015. Biological activities of Morus celtidifolia leaf extracts. Pak. J. Pharmaceut. Sci., 28: 1177-1180.
- Viveros-Valdez, E., R. Rivas-Morales, P. Carranza-Rosales, S. Mendoza and G. Schmeda-Hirschmann, 2008. Free radical scavengers from the Mexican herbal tea poleo (Hedeoma drummondii) Zeitschrift Naturforschung C, 63: 341-346.
- Kaskoos, R.A., 2013. In-vitro α-glucosidase inhibition and antioxidant activity of methanolic extract of Centaurea calcitrapa from Iraq. Am. J. Essent. Oils Nat. Prod., 1: 122-125.
- Sudha, P., S.S. Zinjarde, S.Y. Bhargava and A.R. Kumar, 2011. Potent α-amylase inhibitory activity of Indian ayurvedic medicinal plants. BMC Complement. Altern. Med., Vol. 11.
- Shanmughapriya, S., A. Manilal, S. Sujith, J. Selvin, G.S. Kiran and K. Natarajaseenivasan, 2008. Antimicrobial activity of seaweeds extracts against multiresistant pathogens. Ann. Microbiol., 58: 535-541.
- Torres, Y.R., R.G.S. Berlinck, G.G.F. Nascimento, S.C. Fortier, C. Pessoa and M.O. de Moraes, 2002. Antibacterial activity against resistant bacteria and cytotoxicity of four alkaloid toxins isolated from the marine sponge Arenosclera brasiliensis. Toxicon, 40: 885-891.
- Burkholder, P.R. and K., Rutzler, 1969. Antimicrobial activity of some marine sponges. Nature, 222: 983-984.
- Adwan, K. and N. Abu-Hasan, 1998. Gentamicin resistance in clinical strains of Enterobacteriaceae associated with reduced gentamicin uptake. Folia Microbiol., 43: 438-440.
- Ponomarenko, L.P., O.A. Vanteeva, S.A. Rod’kina, V.B. Krasokhin and S.S. Afiyatullov, 2010. Metabolites of the marine sponge Suberites cf. aurantiacus. Chem. Nat. Compd., 46: 335-337.
- Turk, T., J.A. Avgustin, U. Batista, G. Strugar and R. Kosmina et al., 2013. Biological activities of ethanolic extracts from deep-sea Antarctic marine sponges. Mar. Drugs, 11: 1126-1139.
- Sepcic, K., S. Kauferstein, D. Mebs and T. Turk, 2010. Biological activities of aqueous and organic extracts from tropical marine sponges. Mar. Drugs, 8: 1550-1566.
- Perdicaris, S., T. Vlachogianni and A. Valavanidis, 2013. Bioactive natural substances from marine sponges: New developments and prospects for future pharmaceuticals. Nat. Prod. Chem. Res., Vol. 1.
- Dhinakaran, D.I., V. Manohari, B. Atchya, K. Tamilselvi and A.P. Lipton, 2012. Antifungal and cytotoxic activities of some marine sponges collected from the South East Coast of India. J. Applied Pharmaceut. Sci., 2: 52-55.
- Sonia, G.A.S., A.P. Lipton and R.P. Raj, 2009. Lethal concentration of methanol extract of sponges to the brine shrimp, Artemia salina. J. Mar. Biol. Assoc. India, 51: 122-125.
- Lordan, S., T.J. Smyth, A. Soler-Vila, C. Stanton and R.P. Ross, 2013. The α-amylase and α-glucosidase inhibitory effects of Irish seaweed extracts. Food Chem., 141: 2170-2176.
- Orhan, I.E., B. Ozcelik, B. Konuklugil, A. Putz, U.G. Kaban and P. Proksch, 2012. Bioactivity screening of the selected turkish marine sponges and three compounds from Agelas oroides. Rec. Nat. Prod., 6: 356-367.
- Shaaban, M., H.I. Abd-Alla, A.Z. Hassan, H.F. Aly and M.A. Ghani, 2012. Chemical characterization, antioxidant and inhibitory effects of some marine sponges against carbohydrate metabolizing enzymes. Org. Med. Chem. Lett., Vol. 2.
- Berne, S., M. Kalauz, M. Lapat, L. Savin and D. Janussen et al., 2016. Screening of the Antarctic marine sponges (Porifera) as a source of bioactive compounds. Polar Biol., 39: 947-959.
- Mehbub, M.F., J. Lei, C. Franco and W. Zhang, 2014. Marine sponge derived natural products between 2001 and 2010: Trends and opportunities for discovery of bioactives. Mar. Drugs, 12: 4539-4577.