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Investigating on the Correlation Between Some Biological Activities of Marine Sponge-Associated Bacteria Extracts and Isolated Diketopiperazines Article in Current Microbiology · October 2016 DOI: 10.1007/s00284-016-1144-3

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Curr Microbiol DOI 10.1007/s00284-016-1144-3

Investigating on the Correlation Between Some Biological Activities of Marine Sponge-Associated Bacteria Extracts and Isolated Diketopiperazines Faten K. Abd El-Hady1 • Walid Fayad2 • Carmine Iodice3 • Zeinab A. El-Shahid1 Mohamed S. Abdel-Aziz4 • Egle Crudele3 • Giuseppina Tommonaro3

•

Received: 4 July 2016 / Accepted: 5 October 2016 Ó Springer Science+Business Media New York 2016

Abstract Marine organisms have been considered as the richest sources of novel bioactive metabolites, which can be used for pharmaceutical purposes. In the last years, the interest for marine microorganisms has grown for their enormous biodiversity and for the evidence that many novel compounds isolated from marine invertebrates are really synthesized by their associated bacteria. Nevertheless, the discovery of a chemical communication Quorum sensing (QS) between bacterial cells and between bacteria and host has gained the researchers to expand the aim of their study toward the role of bacteria associated with marine invertebrates, such as marine sponge. In the present paper, we report the evaluation of biological activities of different extracts of bacteria Vibrio sp. and Bacillus sp. associated with marine sponges Dysidea avara and Ircinia variabilis, respectively. Moreover, we evaluated the biological activities of some diketopiperazines (DKPs), previously isolated, and able to activate QS mechanism. The results showed that all extracts, fractions, and DKPs showed low scavenging activity against DPPH and superoxide anion, low cytotoxic and anti-tyrosinase activities, but no antimicrobial and acetylcholinesterase inhibitory activities. One DKP [cyclo-(trans-4-hydroxy-L-prolyl-L& Giuseppina Tommonaro [email protected] 1

Chemistry of Natural Products Department, National Research Center, Giza, Egypt

2

Drug Bioassay-Cell Culture Laboratory, Pharmacognosy Department, National Research Center, Giza, Egypt

3

Institute of Biomolecular Chemistry, National Research Council of Italy, Pozzuoli, NA, Italy

4

Department of Microbial Chemistry, National Research Center, Giza, Egypt

leucine)] has the highest a-glucosidase inhibitory activity even than the standard acarbose.

Introduction The marine environment represents the most promising source of new compounds with application as drugs for many diseases [6]. Secondary metabolites from marine organisms have gained attention for their multiple biological activities such as anticancer, anti-inflammatory, antimicrobial, and others [11, 24]. Among marine organisms, marine invertebrates like sponges represent the richest source providing novel structural compounds with a variety of biological activities [18, 32]. A high microbial diversity occurs within marine sponges, and interesting ‘‘host-symbiont’’ distributions of these bacterial populations have been discovered [27]. To date, the roles bacteria play in sponge biology and ecology have not been elucidated, and few reports describe the contribution of symbiotic microbes to sponge well-being or survival [30]. In the last years, it has been demonstrated that many bacteria employ chemical communication mechanisms known as quorum sensing (QS) to regulate the expression of important virulent phenotypes against hosts [23]. Bacteria synthesize small diffusible compounds (termed ‘‘autoinducer’’) and release them in the surrounding environment where they accumulate. When the concentration of these substances reaches a critical threshold, the bacterial communities ‘‘sense’’ the reached ‘‘quorum’’ and coordinate gene expression and subsequent phenotypic outcomes [10]. It is reported that in Gram-negative bacteria, the signaling system is mainly based on N-acyl homoserine lactones (AHL), while in gram-positive bacteria, QS is mainly regulated by modified peptides, such as

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F. K. Abd El-Hady et al.: Investigating on the Correlation Between Some Biological Activities

diketopiperazines (DKPs) [31]. Several DKPs were isolated from an a-Proteobacterium of the genus Ruegeria associated with the marine sponge Suberites domuncula and from strains of the genera Staphylococcus and Bacillus associated with Ircinia variabilis, and it was suggested that these cyclic dipeptides might play a role in QS mechanisms and could regulate bacterial–sponge interactions [1, 8, 19]. This study will focus on the correlation between the quorum sensing signaling compounds (QSSCs), DKPs, the fractions they were isolated from and other fractions with different secondary metabolites production without QSSCs and some biological activities.

isolated compounds, Vib (A-D) and Bac (A,B), were chemically characterized by NMR analysis (1H, 13C) (Fig. 1). 1H NMR spectra were recorded on a Bruker AMX-400 spectrometer in CD3OD; 13C spectra were recorded on a Bruker AMX-300 spectrometer in CD3OD. DPPH Radical Scavenging Activity The Vib1 and Bac1 extracts, Vib2,3 and Bac2,3 fractions, and the isolated compounds Vib (A–D) and Bac (A,B) were studied for DPPH radical scavenging activity following the modified procedure of Matsushige et al. [17]. The absorbance was measured at 520 nm. All the reactions were performed in triplicate in 96-well microplate.

Materials and Methods Isolation of Strains Samples of Dysidea avara and Ircinia variabilis were collected in the bay of Naples at a depth of 20–25 m. Identification of Strains Isolated strains were identified by PCR analysis. Complete 16S sequences for each bacterial species were obtained as reported by De Rosa et al. [8]. Extraction The cell-free supernatant of Vibrio sp. (from D. avara) and Bacillus sp. (from I. variabilis) were extracted with diethyl ether to give Vib1 and Bac1 extracts, with n-butanol to give Vib2 and Bac2 fractions, and finally with dichloromethane to give Vib3 and Bac3 fractions. Isolation and Chemical Characterization of Compounds In a previous work, the isolation and chemical characterization of compounds belonging to the diketopiperazine class from cell-free supernatant of Vibrio and Bacillus spp. has been described [8]. Briefly, the dichloromethane extracts of cell-free medium of Vibrio sp. (from D. avara) and Bacillus sp. (from I. variabilis) were chromatographed on a Lobar RP-18 column eluted with a gradient of H2O/ CH3OH starting from 9:1 up to pure CH3OH (with steps of 100 mL); fractions of 100 mL were collected. All recovered fractions were assayed for activation of QS by TLCoverlay test using A. tumefaciens NTL4 and CV026 bioreporters [1]. The active fractions were further purified by reversed-phase HPLC (Kromasil RP-18, 5 lm, 4.6 9 250 mm) using 0.05 % TFA water/acetonitrile gradient (flow: 1 mL/min; detection: UV at 215 nm). The

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Superoxide Anion Scavenging Activity Superoxide anion scavenging activity was determined according to a modified method of Matsushige et al. [17]. Reaction mixtures containing 1.4 mL of 50 mM Na2CO3 (pH 10.2), 100 lL of 3 mM xanthine, 100 lL of 3 mM EDTA, 100 lL of BSA (1.5 mg/mL), 100 lL of 75 mM Nitro blue tetrazolium, and 50 lL of each compound (100 lg/mL) were preincubated at 30 °C for 10 min, and 50 lL of xanthine oxidase (0.3 unit/mL) was added. After incubation at 30 °C for 20 min, 200 lL of 6 mM CuCl2 was added to stop the reactions and the absorbance was measured at 560 nm. Evaluation of Cytotoxic Activity MCF-7 human breast carcinoma cell line was obtained from the American type culture collection (ATCC, Minisota, USA). The cell line was cultured in RPMI-1640 medium (Sigma Aldrich Chemical Co., St. Louis. Mo. USA) supplemented with 10 % fetal bovine serum, penicillin (100 U/mL), and streptomycin (2 mg/mL) at 5 % CO2 in a 37 °C incubator. The cells were plated in 96-well plate at a concentration of 5000 cell/200 lL of medium per well. Tested extracts and compounds dissolved in DMSO were added to the wells in triplicates with concentrations of 1.56, 3.13, 6.25, 12.5, 25, and 50 lg/mL for 72 h. 0.5 % DMSO was used as negative control, while 2 lM staurosporine was used as positive control. The cytotoxic activity was determined using MTT [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay as described by Mosmann [20]. In brief, medium was aspirated, 40 lL MTT salt (2.5 lg/mL) was added to each well and incubated for further 4 h. To stop the reaction and dissolve the formed crystals, 150 lL of 10 % sodium dodecyl sulfate in deionized water was added to each well and incubated overnight at 37 °C. The

F. K. Abd El-Hady et al.: Investigating on the Correlation Between Some Biological Activities Fig. 1 Chemical structures of diketopiperazines (Vib A–Vib D) isolated from Vibrio sp. associated with D. avara and (Bac A–Bac B) isolated from Bacillus sp. associated with I. variabilis

absorbance was then measured at 595 nm with a reference wavelength of 690 nm. The equation used for calculation of percentage cytotoxicity was as follows: ½1 ðavðxÞÞ=ðavðNCÞÞ100; where Av is the average, X is the absorbance of test fraction or compound, and NC is the absorbance of negative control. a-Glucosidase Inhibition Assay a-Glucosidase inhibitors which act as competitive inhibitors of intestinal a-glucosidase can delay the digestion and subsequent absorption of elevated postprandial blood glucose levels. The a-glucosidase inhibitory activity was assessed by the standard method with slight modifications [9]. Briefly, a volume of 60 lL of sample solution and 50 lL of 0.1 M phosphate buffer (pH 6.8) containing aglucosidase solution (0.2 U/mL) was incubated in 96-well

plates at 37 °C for 20 min. After pre-incubation, 50 lL of 5 mM p-nitrophenyl-a-D-glucopyranoside (PNPG) solution in 0.1 M phosphate buffer (pH 6.8) was added to each well and incubated at 37 °C for another 20 min. Then the reaction was stopped by adding 160 lL of 0.2 M NaCO3 into each well, and absorbance readings (A) were recorded at 405 nm by microplate reader and compared to a control which had 60 lL of buffer solution in place of the extract. For blank incubation (to allow for absorbance produced by the extract), enzyme solution was replaced by buffer solution and absorbance recorded. Commercially available Acarbose was used as positive control and compared with all extracts. Mushroom Tyrosinase Activity To evaluate the inhibitory action of methanol extract on tyrosinase, tyrosinase isolated from mushrooms was utilized as described previously with a minor modification [14]. In brief, 140 lL 50 mM phosphate buffer (pH 6.8),

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10 lL of extract (1 mg/mL, dissolved in MeOH), 40 lL of 1.5 mM L-tyrosine solution, and 20 lL of Mushroom tyrosinase (1500 U/mL) were added to a 96-well microplate. The assay mixture was incubated at 25 °C for 30 min. Following incubation, the amount of dopachrome produced in the reaction mixture was determined spectrophotometrically at 492 nm in a microplate reader. Vitamin C was used as positive control and compared with all extracts. Antimicrobial Activity Test Disk agar plate method was done to evaluate the antimicrobial activity of bacterial extracts and isolated compounds [26]. Investigated samples were solubilized in methanol. The antimicrobial activities of 0.5-cm-diameter filter paper disk saturated with about 1 mg sample were tested against three different microbial strains, i.e., Staphylococcus aureus (G?ve bacteria), Pseudomonas aeruginosa (G-ve bacteria), and Candida albicans (yeast). Acetylcholinesterase (AChE) Inhibitory Activity The AChE inhibitory activity was performed following the method previously described with slight modification [13]. Electric-eel AChE (Sigma) was utilized; the enzymatic hydrolysis of acetylthiocholine was measured at a wavelength of 412 nm (15 min). All the reactions were performed in triplicate in 96-well microplate.

Results and Discussion Cell-free medium of Vibrio and Bacillus spp. were extracted with diethyl ether to give Vib1 and Bac1 extracts, with n-butanol to give Vib2 and Bac2 fractions, and finally with dichloromethane to give Vib3 and Bac3 fractions. Dichloromethane extracts were purified repeatedly by Lobar RP-18 and reversed-phase HPLC to give pure compounds and six DKPs, Vib (A–D) and Bac (A,B), were isolated (Fig. 1). The isolated compounds were chemically identified by means of NMR analysis (1H, 13C). 1H NMR spectra were recorded on a Bruker AMX-400 spectrometer in CD3OD; 13C spectra were recorded on a Bruker AMX300 spectrometer in CD3OD. Herein, we report the 1HNMR data (d in ppm) for the isolated compounds. Cyclo-(L-prolyl-L-phenylalanine) (Vib A); 1H-NMR (CD3OD): d 4.28 (dd, J = 3 and 9.9 Hz), 5.98 (s), 4.05 (t, J = 7.5 Hz), 1.91, 2.32, 1.88, 3.59, 2.83 (dd, J = 10.0 and 14.5 Hz), 3.54, 7.22, 7.35. Cyclo-(cis-4-hydroxy-D-prolyl-L-leucine) (Vib B); 1 HNMR (CD3OD): d 4.56, 4.46 (dd, J = 8.3 and 8.1 Hz), 4.00, 3.65 (dd, J = 12.3 and 3.5 Hz), 3.49 (dd, J = 12.3

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and 5.6 Hz), 2.53, 2.17, 1.70, 1.54, 0.94 (d, J = 6.1 Hz), 0.91 (d, J = 6.1 Hz). Cyclo-(trans-4-hydroxy-L-prolyl-L-phenylalanine) (Vib C); 1H-NMR (CD3OD): d 7.43, 7.26, 4.66 (t, J = 4.4 Hz), 4.41, 3.80 (dd, J = 12.8 and 5.4 Hz), 3.34 (dd, J = 12.8 and 1.2 Hz), 3.30 (dd, J = 14.1 and 4.5 Hz), 3.18 (dd, J = 14.1 and 4.5 Hz), 2.04 (dd, J = 13.1 and 5.9 Hz), 1.12 (ddd, J = 13.1, 12.8 and 5.0 Hz). Cyclo-(L-prolyl-L-leucine) (Vib D); 1H-NMR (CD3OD): d 3.99 (dd, J = 4.5 and 11.0 Hz), 6.38 (s), 4.11 (t, J = 7.5), 2.36, 2.06, 3.57, 1.86, 1.92, 1.55 (ddd, J = 6.0 and 11.5 Hz), 0.98 (d, J = 8.0), 0.95 (d, J = 8.5). Cyclo-(cis-4-hydroxy-D-prolyl-L-phenylalanine) (Bac A); 1H-NMR (CD3OD): d 7.34, 7.21, 4.41 (t, J = 4.4 Hz), 4.33, 3.58 (dd, J = 12.4 and 3.8 Hz), 3.34 (dd, J = 12.4 and 5.7 Hz), 3.22 (dd, J = 13.6 and 4.2 Hz), 3.06 (dd, J = 13.6 and 5.0 Hz), 2.62 (dd, J = 8.5 and 8.4 Hz), 2.26, 1.86. Cyclo-(trans-4-hydroxy-L-prolyl-L-leucine) (Bac B); 1 HNMR (CD3OD): d 4.44, 4.15, 3.59 (dd, J = 13.1 and 4.2 Hz), 3.32 (d, J = 13.1 Hz), 2.17 (dd, J = 13.4 and 6.3 Hz), 2.00 (ddd, J = 13.4, 13.2 and 4.2 Hz), 1.66, 1.46, 0.76 (d, J = 6.1 Hz). The spectral data were in accordance with those reported in the literature [8]. The crude extracts and isolated DKPs were evaluated for different biological activities. DPPH Radical Scavenging Activity The DPPH radical scavenging activity was evaluated for all fractions and compounds. The QSSCs and their extracts had no DPPH-free radical scavenging activity (Vib3, Bac3, Vib A–D, and Bac A–B, respectively). The ether extract Vib1 showed 39 % scavenging activity (Fig. 2). The obtained data are in agreement with previous studies, as the diketopiperazine cristatumin F showed moderate radical scavenging activity against DPPH radicals [34]. However, previous reports indicated that antioxidant activity of DKPs, and their free radical scavenging properties were not correlated [28]. Superoxide Anion Free Radical Scavenging Activity The fraction Vib2 had the highest free radical scavenging activity against superoxide anion radicals (52 %, Fig. 3), while Vib1 and quorum sensing extracts, Bac3 and Vib3, showed moderate activity (46, 36, and 31 %, respectively). All the other fractions and QSSCs compounds showed low activity. There was a remarkable difference in the scavenging of superoxide free radicals. This finding is suggestive that the isolate in our study could scavenge greater number of superoxide free radicals at the same concentration [28].


Fig. 2 Free radical scavenging activity of Vibrio (a) and Bacillus spp. (b) and their isolated compounds in the DPPH radical assay. Values are expressed as mean ± SD, n = 3 at a concentration of 100 lg/mL for extracts, and 10 lM/mL for all tested compounds

Fig. 3 Free radical scavenging activity of Vibrio (a) and Bacillus spp. (b) and their isolated compounds in the xanthine-XOD assay. Values are expressed as mean ± SD, n = 3 at a concentration of 100 lg/mL for extracts, and 10 lM/mL for all tested compounds

According to growing evidence that ROS (reactive oxygen species) are involved in damage of biomolecules and contribute to the etiology of many human diseases, the increase in antioxidants in the body content may help in protecting the cell from damage and the development of chronic diseases [12]. Cytotoxic Activity Oxidative stress resulting from excessive reactive oxygen/ nitrogen/electrophilic species (ROS/RNS/RES) can lead to diseases such as cancer [21]. The cytotoxic activity against human breast carcinoma (MCF-7) cell line was evaluated for all fractions and compounds. At concentration 50 lg/ mL, the fractions Vib2 and Bac2 showed low cytotoxicity (37 and 26 %, respectively), while quorum sensing fractions (Bac3 and Vib3) showed very low cytotoxicity (25 and 9 % respectively). At concentration 50 lM, compounds Vib C and Bac A had low and moderate cytotoxicity (30 and 42 % respectively), i.e., the cis-isomer of cyclo-(4-hydroxy-L-prolyl-L-phenylalanine) is more active than its trans-isomer (Figs. 1, 3). Lower tested concentrations of either fractions or compounds showed lower

activities, and thus only results of 50 lg/mL for fractions and 50 lM for compounds were shown. All the other compounds had very low activity (Fig. 4). Staurosporine at 2 lM concentration caused 69 % cytotoxicity. Diketopiperazines exert an array of biological activities; the esters of rodriguesic acid and of the rodriguesic acid hydroxamate were evaluated in cytotoxicity assays against MCF-7, but they did not exhibit any significant activity [22].

a-Glucosidase Inhibitory Activity Vibrio and Bacillus spp. cell-free medium extracts, fractions, and the QSSCs were investigated for their in vitro aglucosidase inhibitory activity. The results showed that the compound BacB had the highest significant inhibitory activity (36 %) and the compound VibA had the lowest activity (14 %) (Figs. 1, 5). Compound BacB appeared to be more potent in a-glucosidase inhibitory potential if compared to the positive control Acarbose (31 %), the standard used for the evaluation of a-glucosidase inhibitory action [3].

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Fig. 4 Average % cytotoxicity of Vibrio (a) and Bacillus (b) spp. and their isolated compound on MCF7 breast carcinoma cell line. Values are expressed as mean ± SD, n = 3 at a concentration of 50 lg/mL for all tested extracts (fractions), 50 lM for all tested compounds

Fig. 5 The a-Glucosidase inhibitory activity of Vibrio (a) and Bacillus (b) spp. and their isolated compounds. Values are expressed as mean ± SD, n = 3 at a concentration of 200 lg/mL for all tested extracts and acarbose. 10 lM/mL for all tested compounds

A comparative study for a-glucosidase inhibitory activity of the diketopiperazines (VibA–VibD) isolated from Vibrio sp. associated with D. avara. and (BacA– BacB) isolated from Bacillus sp. associated with I. variabilis revealed that L-leucine derivatives BacB [cyclo(trans-4-hydroxy-L-prolyl-L-leucine)] has the highest inhibitory activity even than the standard Acarbose, while its cis- isomer compound VibB [cyclo-(cis-4-hydroxy-D-prolyl-L-leucine)] has no a-glucosidase inhibitory activity (Figs. 1, 5). The L-phenylalanine derivative Vib A [cyclo(L-prolyl-L-phenylalanine)] showed a moderate activity. The potential to inhibit a- and b-glucosidases of a series of chiral piperazine-2,5-dione derivatives was investigated: they exhibited very low inhibitory activity toward b-glucosidase, whereas some were more active against a-glucosidase [5]. It was reported that diketopiperazine alkaloid amauromine B exhibited more potent a-glucosidase inhibitory activities than the positive control acarbose [25]. The diketopiperazine cyclo(dehydroala-L-Leu) inhibited alpha-lucosidase [16]. a-Glucosidase is a key enzyme of carbohydrate digestion and it is one of the glucosidases located in the brush-

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border surface membrane of intestinal cells. In the small intestine, a-glucosidase inhibitors block the actions of aglucosidase enzymes, thus limiting the conversion of oligosaccharides and disaccharides to monosaccharides, necessary for gastrointestinal absorption. Postprandial glucose peaks may be attenuated by delayed glucose absorption. The main benefits attributable to a-glucosidase inhibitors are reductions in both postprandial glycemic levels and in the total range of postprandial glucose levels [2]. a-Glucosidase inhibitors became a new class of antidiabetic drug, they slow down the process of digestion and absorption of carbohydrates by competitively blocking the activity of glucosidase. Consequently, the peak concentration of postprandial blood glucose is reduced and the blood sugar level comes under control [33].

Tyrosinase Inhibitory Activity Vibrio and Bacillus spp. cell-free medium extracts, fractions, and the QSSCs were investigated for their in vitro


Fig. 6 Inhibition of the tyrosinase activity of Vibrio (a) and Bacillus (b) spp. and their isolated compounds. Values are expressed as mean ± SD, n = 3 (200 lg/mL for extracts and 10 lM/mL for all tested compounds)

tyrosinase inhibitory activity. The results showed that compounds (VibB–D) had low inhibitory activity (15, 26, and 19.6 %, respectively) and the compound BacA had low activity (17.4 %) (Figs. 1, 6). Bacillus thuringiensis contain active tyrosinases and they become heavily pigmented in the presence of L-tyrosine [4]. Thermophilic Bacillus sp. is known as potent inhibitor of tyrosinase activity [15]. The increase in production and accumulation of melanins are the cause of increased skin-acquired hyper-pigmentation. Tyrosinase inhibitor [melanogenesis inhibitors] action mainly resides in some interference in melanin formation, regardless of any direct inhibitor/enzyme interaction. Many putative inhibitors are examined in the presence of tyrosine or dopa as the enzyme substrate, and activity is assessed in terms of dopachrome formation. Thus, experimental observation of the inhibition of tyrosinase activity can be accomplished by some mechanisms: one of them was by reducing agents which cause chemical reduction of dopaquinone, such as ascorbic acid [it was used as positive control here] which is used as a melanogenesis inhibitor because of its capacity to reduce Odopaquinone back to dopa, thus avoiding the formation of dopachrome and melanin [7]. Kojic acid is one of the popular chemicals used in whitening cosmetics, but its chronic, cytotoxic, and mutagenic effects have been proved. To avoid this problem, more attention has been paid to the use of natural products instead of chemical agents as anti-tyrosinase substances [29].

Acetylcholinesterase (AChE) Inhibitory Activity Vibrio and Bacillus spp. cell-free medium extracts, fractions, and the QSSCs were investigated for their in vitro acetylcholinesterase inhibitory activity. The results showed no activity.

Conclusions The isolation of diketopiperazine derivatives from cell-free medium of Vibrio and Bacillus spp. revealed that there is no correlation between the QSSCs DKPs, the fractions they were isolated from and other fractions with different secondary metabolite production without QSSCs and some biological activities. One DKP, BacB [cyclo-(trans-4-hydroxy-L-prolyl-L-leucine)], has been found to have the highest a-glucosidase inhibitory activity even than the standard Acarbose. Acknowledgments This work was financially supported by the bilateral projects within the Executive Programmes for Scientific and Technological Cooperation between Arab Republic of Egypt and Italian Republic for the years 2013–2016, Project no. [A2-12-15]. This work was supported by Ministero degli Affari Esteri e della Cooperazione Internazionale, Direzione Generale per la Promozione del Sistema Paese, Italia-Progetto di Grande Rilevanza ‘‘Applicazioni biotecnologiche di microorganismi associati alle spugne marine e valutazione biologica di composti bioattivi’’ Italia-Egitto 2013–2016. Compliance with Ethical Standards Conflict of Interest No conflict of interest declared.

Antimicrobial Activity Test

References There is no activity for all samples tested against three different microbial strains, i.e., Staphylococcus aureus (G?vebacteria), Pseudomonas aeruginosa (G-ve bacteria), and Candida albicans (yeast).

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