Introduction
Marine organisms account for more than half of the global biodiversity and have proven to be a rich source of pharmacologically active compounds. The discovery of natural products derived from marine sources remains an important issue for the development of drugs (Francesch et al., Reference Francesch, Glaser, Jaspars, Jiménez, Luesch, Mayer, Newman, Pierce and Taglialatela-Scafati2024) and marine bioproducts (Papon et al., Reference Papon, Copp and Courdavault2022).
Turkey is surrounded by the Black Sea, the Aegean Sea, the Mediterranean Sea, and the Marmara. The country has an extensive coastline of over 8300 km and diverse marine ecosystems (Konuklugil and Heydari, Reference Konuklugil, Heydari and Jha2019). These factors have prompted many researchers to focus on finding biological activities, detection of secondary metabolites, and isolation/identification of compounds from marine organisms found in the Turkish waters (Gözcelioğlu and Konuklugil, Reference Gözcelioğlu and Konuklugil2012; Heydari et al., Reference Heydari, Gözcelioğlu and Konuklugil2018, Reference Heydari, Gözcelioğlu and Konuklugil2019; Konuklugil et al., Reference Konuklugil, Heydari, Genç and Őzgen2018; Korpayev et al., Reference Korpayev, Heydari, Koc, Gözcelioğlu and Konuklugil2020; Orhan et al., Reference Orhan, Orhan, Demir and Konuklugil2021, Reference Orhan, Ozcelik, Konuklugil, Putz, Kaban and Proksch2012; Uras et al., Reference Uras, Mutlu, Koç and Konuklugil2022). It is important to highlight that sponges, tunicates, and corals have become a source of interest, since they provide novel natural products with a diverse spectrum of biological activities.
Turkish marine biodiversity is under threat due to the destruction of marine ecosystems and habitats, as well as over-exploitation of marine resources. As a result, many organisms have not been extensively investigated, and much remains unknown about them.
The present work aimed to increase our current knowledge of the biodiversity of the Turkish coasts, providing a phytochemical prospection based on chromatographic profiles of extracts, fractions, and sub-fractions from marine species, and to evaluate their antioxidant and antibacterial activity as well as the inhibition of acetylcholinesterase (AChE).
Material and methods
Sample collection
Sampling was performed between 2013 and 2015 in the Aegean Sea and the Sea of Marmara, Turkey. The samples were collected at different localities by scuba diving at depths ranging from 9 to 40 m and were identified by Dr Bülent Gözcelioğlu. Voucher specimens of this study are deposited at the Hacettepe University Biodiversity Research Center (Biosphere Museum), Ankara, Turkey. The details about the collected samples, such as species, vernacular name, voucher number, family, date, collection site, and biomass, are given in Table 1.
Selected marine species, including family, vernacular name, taxonomic group, voucher, date, collection site, and biomass in Turkey
Sample processing
After collection, the specimens were processed to remove foreign materials using a knife. The material was weighed, cut into small pieces, and stored in a deep freeze to preserve them until required for analysis.
Preparation of crude extracts
The samples were extracted individually with methanol (500 mL), and the extracts were filtered and concentrated under vacuum (Konuklugil et al., Reference Konuklugil, Heydari, Genç and Őzgen2018) to yield the methanolic extracts (MeOH). The latter were stored at 4°C until required for the experiments.
Besides, the MeOH extract of Dictyonella incisa (8.9 g) was partitioned into the n-hexane and water phases. The latter (6.4 g) was partitioned with chloroform (Heydari et al., Reference Heydari, Gözcelioğlu and Konuklugil2018) to afford three fractions (hexanic, chloroformic, and aqueous). The aqueous fraction was chromatographed to obtain three sub-fractions (DIFr 1-3, DIFr 4-18, and DIFr 19-27).
Chemical profile by thin-layer chromatography
Fourteen MeOH extracts and three sub-fractions of D. incisa (Table 2) were subjected to phytochemical screening on thin layer chromatography (TLC) using methods described by Wagner and Bladt (Reference Wagner and Bladt2001). The MeOH extract of D. incisa was not evaluated since there was no available material. The samples and standards were prepared at a 1.0-mg/mL concentration, and the analyses were performed on silica gel 60 F254 aluminium plates (Merck, Darmstadt, Germany). An aliquot (10 µL) of each solution was applied, in duplicate. In this study, two independent experiments were carried out. Briefly, to characterize the presence of terpenes/steroids (TS), hexane:ethyl acetate (7:3) was used as an eluent and the plates were individually sprayed with the anisaldehyde-sulphuric reagent followed by heating at 100°C; to detect the alkaloids (ALKs), the plates were elueted with ethyl acetate:methanol:water (100:13:10) and sprayed with Dragendorff reagent; to characterize the flavonoids, the eluent was ethyl acetate:formic acid:acetic acid:water (50:5:5:2) and NP/PEG reagent to spray for flavonoids, as well as ethyl acetate:formic acid:acetic acid:water (50:5:5:2); and a mixture of sodium carbonate solution 20% and Folin-Ciocalteu reagent (1:1) was used for detecting the polyphenols.
Phytochemical prospection, extraction yields, antioxidant and anticholinesterase activity of MeOH extracts, fractions, and sub-fractions of marine species
ALK, alkaloids; DIFr, sub-fraction; %I, percentage of inhibition; IC50, inhibitory concentration 50%; NA, not active; NT, not tested; PCA, phenol carboxylic acids; POS, positive; SD, standard deviation; TS, terpenes/steroids.
Differences between the mean inhibition of the extracts were analysed by ANOVA, followed by Tukey’s test (p < 0.05). Different letters within the columns indicate significant differences.
* Data represented above are means of three replicates (biological replicates) with three technical replicates.
Evaluation of antioxidant activity
β-carotene/linoleic acid co-oxidation assay by microplate
The antioxidant activity using the β-carotene/linoleic acid co-oxidation assay was performed according to the methodology of Duarte-Almeida et al. (Reference Duarte-Almeida, Santos, Genovese and Lajolo2006) with modifications as described by Rocha et al. (Reference Rocha, Campana, Scoaris, Almeida, Lopes, Silva, Pieters and Silva2018). Quercetin (20 µg/mL, Sigma, St. Louis, USA) and MeOH were used as positive and negative controls, respectively. The absorbance was recorded at 470 nm on a microplate reader (EL808IU-Biotek model, Biotek, Winooski, VT, USA). The antioxidant activity of the samples was expressed as %I of lipid peroxidation inhibition, and determined by Equation (1):
\begin{align}\% I &= \text{Ac} \left( \text{{initials\ abs - final\ abs}} \right) \nonumber\\
&\quad - \text{Aam} \left( \text{{final\ abs - initial\ abs}} \right) / \text{Ac} \times \text{100}\end{align}The experiment was carried out in triplicate (biological replicates) each time two technical replicates were used. The IC50 values were expressed as mean ± standard deviation (SD) of three independent experiments and were determined by non-linear regression analysis using GraphPad Prism (version 6.0, San Diego, CA, USA) software.
In this assay, samples were considered active when they exhibited IC50 values less than 150 µg/mL.
TLC-DPPH assay
For a rapid screening of antioxidant compounds in the samples, the TLC-2,2-diphenyl-1-picrylhydrazyl (DPPH) was carried out based on the bioautography method (Glavind and Holmer, Reference Glavind and Holmer1967). All the samples were solubilized in MeOH (HPLC, Merck) at 10 mg/mL. An aliquot (10 µL) of each solution was applied, in duplicate, as bands onto conventional TLC pre-coated plates (silica gel 60 F254, Merck, Darmstadt, Germany), which was eluted with Hex:AcOEt (7:3) and sprayed with 0.02% DPPH solution (Sigma, St. Louis, MO, USA). Quercetin (1.0 mg/mL, Sigma, St. Louis, USA) was used as a positive control. The samples with yellow zones after spraying with DPPH solution were taken as positive results. In this study, two independent experiments were carried out.
AChE inhibitory activity assay
The AChE inhibitory activity was evaluated following Ellman et al. (Reference Ellman, Courtney, Junior and Featherstone1961) with modifications. The test sample (400 μg/mL) was prepared in Tris/HCl buffer (50 mM, pH 8.0). AChE (2,000 U/mL, Sigma-Aldrich, USA) was diluted in 0.1% BSA-Tris/HCl buffer and stored at −20°C. The assay was performed in a 96-well microplate with 25 μL of the test sample, 50 μL of 0.1% BSA-Tris/HCl buffer, 125 μL of DTNB (3 mM), and 25 μL of acetylthiocholine iodide (ATCI) (14 mM). A blank was prepared by replacing the sample with its diluent.
The reaction was initiated by adding 25 μL of AChE (0.2 U/mL), followed by incubation at 37°C. Absorbance was measured at 405 nm every 5 min for 25 min using a Multiskan Go (Thermo-Fisher, USA) microplate reader. Galantamine (Sigma‐Aldrich, USA) was used as a positive control.
The assay was performed in triplicate with three technical replicates. Enzyme inhibition (%I) was calculated according to Equation (2):
\begin{equation}\text{Percentage\ inhibition} = \left( {a - b/a} \right) \times 100\end{equation}where a is the ∆A/min of control; b is the ∆A/min of test sample; and ∆A is the change in absorbance.
The IC₅₀ values were determined for samples with %I above 50%. Data were expressed as mean ± SD and analysed using Microsoft Excel.
Antibacterial bioassay
The antibacterial activity of an aqueous fraction of D. incisa and the sub-fraction DIFr 4-18 was investigated against the microorganisms Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 11775. The samples (5.0 mg) were solubilized in 100 μL of dimethylsulfoxide (DMSO), and each solution (50 mg/mL) was diluted in Mueller Hinton Broth with cation adjustment. The assay was performed according to Rocha et al. (Reference Rocha, Campana, Scoaris, Almeida, Lopes, Silva, Pieters and Silva2018). Chloramphenicol (Sigma-Aldrich, St. Louis, MO, USA) was used as a positive control, and DMSO (Sigma-Aldrich, St. Louis, MO, USA) as the negative control. The reading was performed on a microplate reader (Multiskan Go, Thermo Scientific, Waltham, MA, USA) at λ = 492 nm. The result was expressed as the percentage of inhibition (%I) of the samples compared with the microbial growth control (Fukuda et al., Reference Fukuda, Ohkoshi, Makino and Fujimoto2006). The minimal inhibitory concentration of 60% of bacterial growth (MIC60) was determined for samples with %I higher than 60% as well as to calculate the IC50 (inhibitory concentration of 50% of bacterial growth). The samples were evaluated in three independent experiments with three technical replicates, using concentrations of 1000, 500, 250, 125, and 62.5 μg/mL in the wells. There was no available material for the sub-fractions DIFr 1-3 and DIFr 19-27, and both were not evaluated for antimicrobial activity. The %I and IC50 values were expressed as mean ± SD and were calculated using an Excel spreadsheet in Microsoft Office.
Statistical analysis
Inhibition of AChE by extracts and microbial was compared using analysis of variance (ANOVA), followed by Tukey’s test for post hoc multiple comparisons, with a significance level set at p < 0.05. Statistical analysis was performed using GraphPad Prism software, version 6.0 (GraphPad Software Inc.). The results were expressed as mean ± SD, and differences were considered statistically significant when p < 0.05.
Results and discussion
Sample material and extracts preparation
In total, 14 different marine species (Figure 1) were collected in situ from five sampling locations (Ayvalik, Keşan, Gölcük, Izmir, and Seferihisar) in the Turkish Coasts of the Aegean and Marmara Seas (Table 1). The collected samples at different depths were used for preparing voucher specimens, and to prepare extracts aiming to obtain their chemical profile through TLC and to evaluate their bioactivity. For each marine species, the amount of fresh biomass collected is presented in Table 1. The MeOH extracts and the aqueous fraction of D. incisa sponge (DI), as well as the three sub-fractions DIFr 1-3, DIFr 4-18, and DIFr 19-27 obtained, were stored (4°C). Values of extraction yields are presented in Table 2.
Marine species collected in situ at depths ranging from 9 to 40 m in the coastal area of Turkey: (A) Paramuricea clavata; (B) Halocynthia papillosa; (C) Cladocora caespitosa; (D) Eunicella cavolini; (E) Ascidiella aspersa; (F) Axinyssa digitada; (G) Limaria hians; (H) Clavelina lepadiformis; and (I) Anemonia viridis. The samples were used to prepare voucher specimens, to obtain chromatographic profiles, and to evaluate their bioactivity.
Thin-layer chromatography profile of marine extracts, fractions, and sub-fractions
The MeOH extracts revealed spots with a colouration violet in the visible light characteristic of terpenes/steroids (TS) (Table 2). It was observed a common spot with an intense violet colour in the samples except in D. incisa (DI) and DIFr 1-3. Moreover, the sub-fractions DIFr 4-18, DIFr 19-27, and DIFr 1-3 also showed a blue spot (Rf = 0.83) (Supplementary Figure S1). After spraying the plates with Dragendorff reagent, H. papillosa (HP-29), E. cavolini (EC-33), M. polymorphus (MP-42), L. hians (LH-45), A. aspersa (AA-53), D. incisa (DI), and both sub-fractions DIFr 4-18 and DIFr 19-27 showed orange-brown spots (Supplementary Figure S1) indicating ALKs, underscoring their prevalence in these samples (Table 2). The possible presence of ALKs in D. incisa corroborates with the literature, since sponges synthesize these special metabolites as an evolutionary adaptation in response to environmental stressors (Mehbub et al., Reference Mehbub, Yang, Cheng, Franco and Zhang2024). Flavonoids were not detected in any samples; however, the latter showed blue fluorescence under UV after NP/PEG spraying (Supplementary Figure S1), which may indicate the presence of phenol carboxylic acids (Table 2). With regard to polyphenols, the plates were sprayed with Folin-Ciocalteu, and blue-greyish spots were observed under visible light in most of the samples with the exception of the sub-fraction DIFr 1-3. Moreover, dark blue-grey spots were detected in the samples DI, PC-27, EC-33, CI-41, MP-42, CH-45, AV-52, and AA-53 indicating a high content of polyphenols. Despite the therapeutic potential of marine polyphenols (Pereira and Cotas, Reference Pereira and Cotas2023), they have been less investigated in marine species than in their terrestrial counterparts (Matulja et al., Reference Matulja, Vranješević, Kolympadi Markovic, Pavelić and Marković2022).
These preliminary assessments of the special metabolites in the marine extracts suggest that some species should be further investigated. Concerning their chemical composition, only few phytochemical studies have been reported so far.
In vitro antioxidant activity
The antioxidant activity was evaluated by two complementary test systems with different mechanisms, and the results are given in Table 2. The β-carotene/linoleic acid system determines the activity of a sample to protect a lipid substrate from oxidation, whereas the DPPH radical inhibition is based on the transfer of electrons from an antioxidant compound to an oxidant (Duarte-Almeida et al., Reference Duarte-Almeida, Santos, Genovese and Lajolo2006).
The MeOH extract from P. clavata (sample PC-27) inhibited oxidative stress related to lipid peroxidation according to our results in the β-carotene/linoleic acid co-oxidation assay (Table 2). This extract was active, with an IC50 value of 124.96 ± 9.38 µg/mL, while the reference compound quercetin showed an IC50 value of 0.3 ± 0.1 µg/mL. Moreover, the sample PC-27 had the potential to reduce DPPH radical in the bioautographic assay, which is in concordance with the findings of other radical-scavenging methods as reported previously (Korpayev et al., Reference Korpayev, Heydari, Koc, Gözcelioğlu and Konuklugil2020). According to these authors, the crude extract of this coral showed moderate antioxidant activity, with an IC50 values of 231.23 ± 0.4 µg/mL, 228.9 ± 0.32 µg/mL, and 243.8 ± 0.46 µg/mL in DPPH, superoxide (SO), and nitric oxide (NO), respectively. The content of phenolic compounds, phenolic acids, and polyphenols in P. clavata may be responsible for the moderate antioxidant activity observed. It is interesting to note that P. clavata showed antioxidant activity in both assays, and thus could be a candidate for further studies.
In this study, the other evaluated MeOH extracts showed no antioxidant activity (IC50 value ˃ 200 µg/mL) in the β-carotene bleaching test (Table 2). On the other hand, the majority of the extracts showed antioxidant activity revealed by the yellow spots on the TLC plate after spraying with DPPH solution (Table 2).
The aqueous fraction of D. incisa (sample DI) and the sub-fraction DIFr 4-18 did not show antioxidant activity in the β-carotene/linoleic acid system (Table 2). It was not possible to evaluate the other two sub-fractions (DIFr 1-3 and DIFr 19-27) because there was not enough material. In the TLC-DPPH assay, the three sub-fractions were active, although the sample DIFr 19-27 showed more yellow spots. Previously, Heydari et al. (Reference Heydari, Gözcelioğlu and Konuklugil2018) showed that MeOH extract of D. incisa has the ability to scavenge SO anion in a dose-dependent way (24.62% inhibition in 800 μg/mL). This species still requires further study, as there are limited published reports about its chemistry.
In order to survive in the marine environment, sponges have developed a highly effective chemical defence mechanism to protect them against environmental stress factors (Li et al., Reference Li, Lu, Zhang, Zhang, Liu, Wang and Liu2023). Moreover, they have been at the centre of attention due to their wide spectrum of biologically active special metabolites (Lee et al., Reference Lee, Cho and Tran2021). Sponges exhibit the highest concentration of natural antioxidants when compared to other marine species (Souiba et al., Reference Souiba, Santin, Yettefti, Abdessadek and Amraoui2023), albeit there is a lack of knowledge about their biological effects (Martignago et al., Reference Martignago, Soares-Silva, Parisi, Silva, Granito, Ribeiro, Renno, Souza and Aguiar2023).
Anticholinesterase activity
Alzheimer’s disease (AD), the major cause of adult-onset dementia, might reach 131 million people in 2050 (Arvanitakis et al., Reference Arvanitakis, Shah and Bennett2019; Cummings et al., Reference Cummings, Lee, Ritter, Sabbagh and Zhong2020). Its treatment still requires much research, as there is no cure (Korczyn and Grinberg, Reference Korczyn and Grinberg2024). The inhibitors to cholinesterase enzyme (AChEI) remain the drugs mostly used to treat patients with mild to moderate dementia (Zuliani et al., Reference Zuliani, Zuin, Romagnoli, Polastri, Cervellati and Brombo2024). To address this challenge, marine organisms have been investigated as a source of bioactive compounds and their potential as AChE inhibitors, as presented in a review by Moodie et al. (Reference Moodie, Sepčić, Turk, Frangež and Svenson2019).
In our study, the inhibition of AChE and the IC50 value were investigated in vitro using a modified microplate quantitative assay. All the MeOH extracts revealed to be inactive. On the other hand, the aqueous fraction of D. incisa exhibited the highest inhibition among the samples (%I = 92.98 ± 1.06) with an IC₅₀ value of 23.98 ± 5.42 µg/mL, showing no significant difference (p < 0.05) compared to galantamine (%I = 85.84 ± 2.50). The sub-fractions showed less AChE inhibition, with DIFr 4-18 (%I = 59.43 ± 3.66; IC₅₀ 76 ± 8.00 µg/mL), DIFr 1-3 (%I 45.35 ± 8.26), and DIFr 19-27 (%I 45.38 ± 4.18). In addition, no statistical differences were observed among the sub-fractions (p < 0.05).
The results from the phytochemical screening indicated the presence of ALKs in D. incisa, and this requires further research into their chemical characterization. One interesting approach would be to investigate the link between ALKs in D. incisa and neuroprotective effects, taking into account that inhibition of AChE is a therapeutic strategy in the treatment of AD.
There are no reports in the literature of the evaluation of the anticholinesterase activity of D. incisa, but other sponges have already demonstrated in vitro AChE inhibitory activity. Earlier studies have shown that sponges from the genera Axinella, Ircinia, and Dysidea exhibited a moderate inhibition of AChE with a %I above 37% (Orhan et al., Reference Orhan, Ozcelik, Konuklugil, Putz, Kaban and Proksch2012). In another study, extracts from 45 Mauritian sponges were evaluated for AChE by both quantitative and qualitative methods (microplate and TLC). Based on the microplate assay, the AcOEt extracts from the species Pericharax heteroraphis and Amphimedon navalis presented a potent AChE inhibitory activity (%I higher than 90% at 0.1 mg/mL). With regard to the TLC results, both AcOEt extracts presented AChE inhibition that could be attributed to terpenoids (Beedessee et al., Reference Beedessee, Ramanjooloo, Surnam-Boodhun, van Soest and Marie2013).
Evaluation of antibacterial activity
Previous studies have shown the antimicrobial potential of marine sponges from Turkey assessed against bacteria and their clinically isolated strains, and fungi (Orhan et al., Reference Orhan, Ozcelik, Konuklugil, Putz, Kaban and Proksch2012). The MeOH extracts were the most active against S. aureus (ATCC 25923) with MIC values from 0.5 to 1.0 μg/mL. In this work, the reference compounds were ofloxacin and ampicillin, which showed MIC values of 0.25 and less than 0.12 μg/mL, respectively. The authors also reported that variations in the local of collection lead to changes in the biological activities of sponges.
Furthermore, the sponges Spongia officinalis, S. agaricina, and Aplysina aerophoba, collected from the Gulf of Saros (Turkey), were evaluated against bacterial and fungal strains of microorganisms (Canakay and Yapici, Reference Canakay and Yapici2016) and had different levels of antimicrobial activity. Similar investigations by Konuklugil et al. (Reference Konuklugil, Heydari, Genç and Őzgen2018) showed that extracts of coral, tunicates, and anemone from Turkish coasts have the best antibacterial results against Gram-positive bacteria and yeast strains.
Despite the great diversity of sponges in Turkey, there is a shortage of studies on Turkish species (Heydari et al., Reference Heydari, Gözcelioğlu and Konuklugil2019). Therefore, the search for antibacterial and/or antifungal activities of extracts from these organisms can increase their potential therapeutic approaches.
In the present study, the two samples of D. incisa evaluated (aqueous fraction and sub-fraction DIFr 4-18) have shown activity against both Gram-negative and Gram-positive bacteria, although they displayed a variable degree of bacterial growth. In this assay, chloramphenicol (positive control) showed %I of 100 at 50 μg/mL.
The aqueous fraction possessed antimicrobial activity, showed greater inhibition (p < 0.05) against Gram-negative bacteria E. coli, exhibiting %I of 85.62 ± 16.80 at 1 mg/mL, and yielded an MIC value of 500 μg/mL with an IC50 value of 193.34 ± 10.97 μg/mL. The sub-fraction DIFr 4-18 showed %I of 57.95 ± 8.20 as shown in Table 3.
Antibacterial activity of an aqueous fraction and sub-fraction from D. incisa
D. incisa, aqueous fraction; DIFr, sub-fraction; %I, percentage of inhibition; IC50, 50% inhibitory concentration; NA, not active; NT, not tested; MIC60, minimal inhibitory concentration of 60% of bacterial growth; SD, standard deviation.
Differences between the mean inhibition of the extracts were analysed by ANOVA, followed by Tukey’s test (p < 0.05). Different letters within the columns indicate significant differences.
* Data represented above are means of three replicates with three technical replicates.
Regarding S. aureus, both samples showed promising results at the same concentration, although the sub-fraction DIFR4-18 was more potent (p < 0.05). The latter showed the highest inhibition results (%I of 94.33 ± 5.20), and an IC50 value of 215.90 ± 29.99 μg/mL at 1.0 mg/mL. The aqueous fraction presented %I of 74.14 ± 16.14 at 1 mg/mL and an IC50 value of 359.55 ± 24.08 μg/mL. The MIC’s value of the sub-fraction was 500 μg/mL, whereas the value for the aqueous fraction was 1000 μg/mL as shown in Table 3.
The aqueous fraction had a bactericidal effect on E. coli at 1000 μg/mL, while for S. aureus, this fraction and the sub-fraction DIFr 4-18 had a bacteriostatic effect at all concentrations tested.
According to an earlier work, the crude extract of D. incisa was evaluated against Gram-positive and Gram-negative bacteria (Heydari et al., Reference Heydari, Gözcelioğlu and Konuklugil2018). The extract was active against S. aureus (ATCC 43300) and S. epidermidis (ATCC 12228), having an MIC value of 62.5 μg/mL for both strains, while no activity was observed against E. coli (ATCC 25922), contrasting with our results (Table 3). It is noteworthy to mention that by using E. coli (ATCC 11775) in our experiments, we observed different results compared to other strains. Strain-specific variations have a critical role in determining the outcomes of antibacterial activity. In addition, Dogan et al. (Reference Dogan, Demir, Sertdemir, Saracli and Konuklugil2018) reported that this extract showed antimicrobial activity against Acinetobacter haemolyticus (ATCC 19002), A. septicum (GATA-NRB 239), Cryptococcus neoformans (NIH 68), and C. gattii (NIH 112) with an MIC value of 32 μg/mL for all of them. The extract was also active against S. aureus (JCSC 4744), S. epidermidis (ATCC 35984), and K. pneumoniae (CDC 529) with an MIC value of 64.0 μg/mL.
Finally, it is important to consider that the geographic location in which sponges grow has an essential role in their chemical composition and, to some extent, affects their bioactivity (Bayona et al., Reference Bayona, van Leeuwen, Erol, Swierts, van der Ent, Voogd and Choi2020).
Conclusions
The outcome of this study may contribute to an increase in the knowledge of the organisms evaluated from the marine biodiversity of Turkey. The chromatographic profiles and data on bioactivities were given with a focus on low-cost and rapid assays widely available. Moreover, this work demonstrates that combined chemical and biochemical assays are useful approaches for screening complex matrices such as species marine-based extracts.
Our findings suggest that the MeOH extract from P. clavata exhibits antioxidant activity, being a good scavenger of radicals as well as being able to prevent the β-carotene bleaching, which may be further studied.
The phytochemical profile of D. incisa still needs to be investigated. It might be interesting to extend the research carried out with an aqueous fraction of the sponge D. incisa and the sub-fraction DIFr 4-18, and further investigate the content of special metabolites, mainly ALKs, that might be present in both. These samples could be a possible source of an ALK scaffold with promising biological activity, and how this could be useful in the search for new drugs.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0025315425100325.
Data availability statement
The data that supports the findings of this study are available within the article.
Acknowledgements
The authors are grateful to ‘Fundação Ezequiel Dias’, Brazil, where the in vitro assays were carried out. We also acknowledge J. R. de Vasconcelos for her assistance during the preparation of the manuscript and A. W. A. Linghorn for English revision.
Author contributions
Conceptualization: B.K. and C.G.S.; B.G. collected and identified the marine organisms; data curation: B.K., B.G., V.L.d.A., and C.G.S.; funding acquisition: B.K.; formal analysis: K.L.L., M.P.R., and V.L.d.A.; resources: B.K., B.G., and V.L.d.A.; project administration: V.L.d.A. and C.G.S.; supervision: C.G.S.; and writing – original draft: B.K., K.L.L., and C.G.S.; writing – review and editing: V.L.d.A. and C.G.S. All the authors contributed to the final version of the manuscript.
Funding
This study was partially supported by the Scientific and Technological Research Council of Turkey.
Competing interests
The authors declare none.
Ethical standards
Ethical approval was waived for this study because no live animals were used for the experiments.
