Chemical Composition and Biological Activities of Metabolites from the Marine Fungi Penicillium sp. Isolated from Sediments of Co To Island, Vietnam

Marine microorganisms are an invaluable source of novel active secondary metabolites possessing various biological activities. In this study, the extraction and isolation of the marine sediment Penicillium species collected in Vietnam yielded ten secondary metabolites, including sporogen AO-1 (1), 3-indolecarbaldehyde (2), 2-[(5-methyl-1,4-dioxan-2-yl)methoxy]ethanol (3), 2-[(2R-hydroxypropanoyl)amino]benzamide (4), 4-hydroxybenzandehyde (5), chrysogine (6), 3-acetyl-4-hydroxycinnoline (7), acid 1H-indole-3-acetic (8), cyclo (Tyr-Trp) (9), and 2’,3’-dihydrosorbicillin (10). Their structures were identified by the analysis of 1D and 2D NMR data. Among the isolated compounds, 2-[(5-methyl-1,4-dioxan-2-yl)methoxy]ethanol (3) showed a strong inhibitory effect against Enterococcus faecalis with a minimum inhibitory concentration value of 32 µg/mL. Both 2-[(2R-hydroxypropanoyl)amino]benzamide (4) and 4-hydroxybenzandehyde (5) selectively inhibited E. coli with minimum inhibitory concentration values of 16 and 8 µg/mL, respectively. 2’,3’-Dihydrosorbicillin (10) potentially inhibited α-glucosidase activity at a concentration of 2.0 mM (66.31%).


Introduction
Drug-resistant bacteria have been an emerging global problem in the last few decades and are one of the most serious issues affecting public health. In the treatment of infections, there is a sharp increase in the number of pathogens with multiple drug-resistant agents [1]. Among them, biofilm-synthetic small molecules are microbes that survive in hostile environments and raise the resistance by a thousand-fold. Biofilms potential to cause disease often occurs in Gram-negative pathogens such as Streptococcus pneumoniae, Staphylococcus aureus, and Enterococcus faecium, as well as Gram-negative pathogens such as Escherichia coli and Pseudomonas aeruginosa [2]. Of particular note is some bacteria are as Streptococcus pneumoniae, Staphylococcus aureus, and Enterococcus faecium, as well as Gram-negative pathogens such as Escherichia coli and Pseudomonas aeruginosa [2]. Of particular note is some bacteria are naturally resistant to a certain antibiotic, but otherwise, antibiotic resistance is the result of the long-term use of antibiotics to treat infections [3]. Recent research revealed that antimicrobial chemotherapy could prevent the pathogenesis of bacterial infection, and simultaneously may induce drug-resistant mutations in bacteria. As stated above, these contrasting outcomes required the urgent exploitation of specific interactions of drugs and modulation of these interactions, which can raise the susceptibility of the bacteria to therapeutic compounds [3].
Recently, marine microorganisms are important sources of novel active secondary metabolites possessing various biological activities, including antibiotics, anti-cancer, and anti-virus [4][5][6]. In the past few years, a number of biologically active compounds have been isolated from marine microorganisms, which exhibited promising pharmacological applications. Numerous marine natural products are now being tested in the clinical stages [7,8]. The recent progress of advanced technologies in genetic manufacturing and biological activity guided fractionation brought several opportunities to the discovery of marine natural products as lead compounds in the drug development [9,10].
Natural fungi are clarified as one of the huge prolific producers of secondary metabolites. The Penicillium genus, which comprises more than 200 species, is famous since 1920s due to the discovery of its antibiotic metabolite penicillin. This genus was discovered in some uncommon environments, including permafrost soil [11], wastewater from mines [12], and deep ocean sediments [13]. Penicillium species have been found to generate several bioactive compounds that could be used as antibacterial [14,15], anti-fungal [16], immunosuppressants, cholesterol-lowering agents [17], and even mycotoxins [18]. Since the discovery of penicillin, numerous Penicillium isolates had been examined, and new metabolites from these fungi draw a great range of attentions for scientists. These metabolites showed varied chemical structures such as polyketides [19], tetrameric acids [20], phenols and alkaloids [21] that could be potential sources for the treatment of diseases. Although the metabolites from fungi show promising biological benefits, only a limited number of the marine microbes have been investigated as bioactive agents.
Here, we focused on the extract of fermentation broth of the Penicillium strain M30, which was isolated from the sediment that was collected at a depth of 14 m sea at the Co To island, Northern Vietnam. This extract showed significantly antimicrobial, α-glucosidase, and α-amylase inhibitory activities. This study reports the isolation and chemical identification of secondary metabolites from the fermentation broth of the Penicillium strain M30 as well as their biological activity.

Identification of Fungus
The fungus M30 was purified successfully as described in the Materials and Methods ( Figure 1). M30 strain's colonies produce white and dry conidiophores.  It is obvious that rRNA is crucial for cell living systems. The rRNA encoding genes are extremely maintained not only in the fungal but also in other kingdoms. Moreover, proteins comprising the ribosomes and rRNA sequences are highly conserved throughout evolution. rRNA analysis is a common method used to investigate microbial diversity and to identify new strains. In this study, the M30 strain, as a member of the Penicillium sp., was characterized using genotyping techniques involving the amplification of the subunit of the 18S rRNA gene. A total of 1173bp of the 18S rRNA gene was sequenced and used for the identification of the isolated fungal strain. Results showed that the gene sequence of M30 strain has 99% sequence matching (Score = 2139 bits, Expect = 0.0) with 18S rRNA gene sequence of Penicillium (GenBank Acc. No.: MH 673731), and thus, this strain belonged to the genus Penicillium.

Biological Activities of the Extract of Penicillium sp.
In the primary experiment, M30 extract was examined for its antibacterial activity against Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Salmonella enterica (ATCC 12228), Enterococcus faecalis (ATCC 13124), Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 13245), and anti-yeast against Candida albicans (ATCC 1023). The results in Table 1 showed that M30 extract inhibited C. albicans with an MIC value of 64 µg/mL. This extract also exhibited moderate inhibitory activity against some Gram (+) bacteria including S. aureus (minimum inhibitory concentration − MIC = 256 µg/mL), B. cereus (MIC = 256 µg/mL) and the Gram (-) bacterium P. aeruginosa (MIC = 128 µg/mL). Screening results of the α-amylase and α-glucosidase inhibitory activity revealed that M30 extract exhibited a positive effect against α-glucosidase with an IC 50 value of 628 µg/mL. In this experiment, acarbose, a positive control, showed an inhibitory effect with an IC 50 value of 423 µg/mL. Considering the potential of the M30 strain, our subsequent study focused on the fermentation of this Penicillium sp. in order to analyze active secondary metabolites contributing to these above biological activities.

Fungus Isolation
The marine sediment samples were collected at 14 m depth with geographic coordinates 2105 11"-107050 57", water at temperature 26 • C in the Co To Island, Vietnam. The samples were collected into 15 mL or 50 mL sterile Falcon tubes and preserved in an ice-box and processed within 24 h. Briefly, 0.5 g of the sample was suspended in 4.5 mL of sterile distilled water, homogenized by vortexing for 1 min, and the suspension was treated using a wet-heat technique (60 • C for 6 min). Next, 0.5 mL of this suspension was transferred to 4.5 mL sterile distilled water, and this step was repeated to set up a ten-fold dilution series to 10 −3 . At the final dilution step, aliquots of 50 µL were spread on medium A1 (soluble starch: 10 g/L; yeast extract: 4 g/L; peptone: 2 g/L; instant ocean: 30 g/L; agar: 15 g/L) supplemented with 50 µg/mL polymycin B to inhibit Gram-negative bacteria. The plates were incubated at 28 • C for seven days. The colony of fungus M30 was transferred onto a new Petri dish of medium A1 for purification.

Fermentation and Extraction
Strain M30 (Penicillium sp.) was cultured in high-nutrient medium (30 g of instant ocean, 10 g of starch, 4 g of yeast, 2 g of peptone, 1 g of calcium carbonate, 40 mg of iron sulfate, and 100 mg of potassium bromate) for seven days at 25 • C while stirring at 200 rpm in a bioreactor (Yuin, Seoul, Korea). The culture solution (30 L) was directly extracted with EtOAc (15 L × 3 times). The EtOAc extract solution was concentrated to dryness by a rotary evaporator (Eyela, Tokyo, Japan).

Antimicrobial Assay
The antimicrobial assay was carried out using E. coli (ATCC25922), P. aeruginosa (ATCC27853), S. enterica (ATCC12228), E. faecalis (ATCC13124), S. aureus (ATCC25923), B. cereus (ATCC13245), and C. albicans (ATCC1023). Stock solutions of samples were prepared in DMSO, and the antimicrobial assays were carried out in 96-well microtiter plates against the microbial strains (5 × 10 5 CFU/mL) using a modification of the published method. After incubation for 24 h at 37 • C, the absorbance at 650 nm was measured using a microplate reader. Streptomycin and nystatin were used as reference compounds.

α-Glucosidase Inhibition Assay
The inhibition of α-glucosidase activity was determined using the modified, published assay [31]. Briefly, the reaction mixture consisting of 50 µL of the sample at the indicated concentrations was incubated with 100 µL of 0.1 M potassium phosphate buffer (pH 6.8) containing α-glucosidase solution (0.5 U/mL) in 96-well plates at 37 • C for 10 min. After pre-incubation, 50 µL of 4-Nitrophenyl β-D-glucopyranoside (pNPG) was added to each well to start the reaction. After incubating at 37 • C Molecules 2019, 24, 3830 7 of 11 for 10 min, absorbance readings were recorded at 405 nm in a microplate reader (Biotek, Winooski, VT, Abbr of State, USA). The control sample was added to 50 µL of buffer solution in place of the sample. Acarbose was used as a positive control of α-glucosidase inhibitor. The α-glucosidase inhibitory activity was calculated according to the equation below: where A control is the absorbance of the control and A sample is the absorbance of the tested samples.

α-Amylase Inhibition Assay
The assay was carried out following the standard protocol with slight modifications [32]. Starch azure was suspended in 0.05 M Tris-HCl buffer (pH 6.9) containing 0.01 M CaCl 2 . The tubes containing the substrate solution were boiled for 5 min and then pre-incubated at 37 • C for 5 min. A total of 100 µL of each sample and 100 µL of substrate solution and 50 µL of porcine pancreatic amylase in Tris-HCl buffer (2 units/mL) were incubated at 37 • C for 10 min. Then, 250 µL of acetic acid 50% was added in each tube to stop the reaction. After, the reaction tubes were centrifuged at 3000 rpm for 5 min at 4 • C, the absorbance of the resulting supernatant was measured at 595 nm using a microplate reader (Biotek, USA).
The α-amylase inhibitory activity was expressed as the percentage of inhibition and was calculated as follows: where A control is the absorbance of the control and A sample is the absorbance of the tested samples.

Statistical Analysis
Data were expressed as the mean ± standard deviations (SD). Statistical significance was assessed by the two-tailed unpaired Student's t-test, and P values less than 0.05 were considered statistically significant.

Conclusions
In recent years, ordinary natural-derived Penicillium sp. is among the most attentive fungi in the science of natural products. In our experiments, we identified the Penicillium strain M30 from sediment collecting at a depth of 14 m at the Co To island, the North of Vietnam's sea. After fermentation, we found that the extract of fermentation broth of this sp. showed significant antimicrobial and α-glucosidase/α-amylase inhibitory activities. Extraction and isolation results yielded a new natural product, 3-acetyl-4-hydroxycinnoline (7), and nine known metabolites as sporogen AO-1 (1), 3-indolecarbadehyde (2) (10) potentially inhibited activity at a concentration of 2.0 mM (66.31% α-glucosidase). This study highlights the value of Penicillium sp. derivation from sediment as a source of antimicrobial, antifungal, and anti-α-glucosidase compounds with the potential to combat afflicting pathogens and diabetic diseases.