RF-3192C and other polyketides from the marine endophytic Aspergillus niger ASSB4: structure assignment and bioactivity investigation

Abstract

Chemical investigation of the methanolic extract of endophytic Aspergillus niger SB4, isolated from the marine alga Laurencia obtuse, afforded the pentacyclic polyketide, RF-3192C (1), the dimeric coumarin orlandin (2), fonsecin B (3), TMC-256A1 (4), cyclo-(Leu-Ala) (5), and cerebroside A (6).The chemical structure of RF-3192C (1) is assigned herein for the first time using 1D/2D NMR and HRESI-MS. Additionally, the revision of the NMR assignments of orlandin (2) was reported herein as well. Investigation of the antimicrobial activities of isolated compounds revealed the high activity of RF-3192C (1) against Pseudomonas aeruginosa and Bacillus subtilis, and moderate activity against yeast. Moreover, an in vitro cytotoxic activity against liver (HEPG2), cervical (HELA), lung (A549), prostate (PC3), and breast (MCF7) cancer cell lines of the isolated compounds was evaluated. The isolation and taxonomical characterization of the producing fungus was reported as well.

Graphical Abstract

Introduction

Polyketides (PKSs), varied structural scaffolds with diverse biological activities, are widely distributed in nature including plants, insects, marine organisms, and microorganisms [1–5]. PKSs span a large range of medicinally important compounds, such as antibiotics [6], antiparasitic, anticancer agents [7], immunosuppressants [8, 9], and cholesterol-lowering agents [10] and they are a validated source of drugs [1, 11]. Fungi (Penicillium, Fusarium, and Alternaria species) and bacteria (Actinomycetes) serve as the primary sources for PKSs [4, 12–14].

In the course of our screening for fungal metabolites having promising bioactivities, the methanolic extract of Aspergillus niger SB4 was chemically investigated affording the patented pentacyclic polyketide RF-3192C (1) [15], the dimeric coumarin orlandin (2) [16] (Fig. 1) fonsecin B (3) [17], TMC-256A1 (4) [18], cyclo(Leu-Ala) (5) [19], and cerebroside A (6; also known as cordycerebroside A) [20]. Structures of the isolated compounds were assigned by 1D and 2D NMR (Fig. 4, Tables 1, 2; Supplementary Information, Fig. S1 and Tables S1–3) and mass spectrometric measurements. Detailed NMR structural assignment of RF-3192C (1) is reported herein for the first time, and the revision of the NMR assignments of orlandin (2) is presented as well using 1D/2D NMR. Biological activity studies of the fungal metabolites including antimicrobial and in vitro cytotoxicity, along with the isolation and detailed taxonomical characterization of the producing fungus, are also presented.

Fig. 1.

Chemical structures of compounds 1–6, produced by the endophytic Aspergillus niger SB4

Fig. 4.

H,H COSY () and selected HMBC () correlations of RF-3192C (1) and orlandin (2)

Table 1.

¹³C (125 MHz) and ¹H (500 MHz) NMR data of RF-3192C (1) in acetone-d₆ (δ in ppm)

Position	δ C	δH (m, J [Hz])	Position	δ C	δH [ppm] (m, J [Hz])
1	190.2		1'	198.5
2	99.4	5.81 (s)	2'	49.1	3.37 (s)
3	171.6		3'	111.8
4	122.6		4'	145.1
4a	135.1		4a'	130.8
5	105.1	7.68 (d, 2.3)	5'	109.1	7.28 (d, 2.3)
6	162.1		6'	165.5
7	104.8	6.41 (d, 2.2)	7'	105.2	6.52 (d, 2.3)
8	164.8		8'	164.5
8a	108.3		8a'	109.7
8 -OH		13.83 (s)	8' -OH		12.68 (s)

Table 2.

¹³C (125 MHz) and ¹H (500 MHz) NMR data of orlandin (2) in DMSO-d₆ in comparison with literature data (δ in ppm)

Position	Orlandin (2) [experimental]		2 [Literature data [16]
	δ C	δH (m, J [Hz])	δC [ppm]	δH [ppm] (m, J [Hz])
2	162.2		169.4a
3	86.9	5.58 (s)	86.3	5.53
4	170.2		153.7a
5	137.6		136.8
6	116.1	6.73 (s)	115.3	6.68
7	154.4		158.3
8	106.2		105.9
9	159.0		161.4
10	106.6		105.5
11	57.0	3.95 (s)	56.4	3.93
12	23.7	2.60 (s)	23.1	2.62
7-OH		10.25 (br s)

^aIncorrectly assigned in literature

Results and discussion

Isolation and taxonomical identification of the producing strain

The endophytic fungus Aspergillus sp. ASSB4 was isolated from marine red algae Laurencia obtuse (Fig. 2A) collected from Red Sea at the area of Nabq Bay, Egypt. The strain ASSB4 was cultivated on potato-dextrose agar (PDA). Phenotypically, the cultures of ASSB4are rapidly growing and appear black in color. Conidia heads are globose to ellipsoidal and compact (Fig. 2B). Conidiophores are small, septate and hyaline. Phylogenetic analyses of the aligned segment of 18S rRNA gene revealed close similarity (98%) of ASSB4 to Aspergillus niger, (97%) to A. awamori and A. costaricaensis (Fig. 3). The sequence has been recorded in GenBank database (accession no. MN593358) [21].

Fig. 2.

A Laurencia obtuse. B Light microscopy of Aspergillus niger

Fig. 3.

Neighbor-joining phylogenetic tree of strain ASSB4 based on 18S rRNA gene sequences, showing its close relationship to Aspergillus spp

Structure elucidation

The physicochemical properties of compounds 1–6 are summarized in Supplementary Information File. Compound 1, a relatively polar reddish solid, was characterized on TLC displaying strong UV-absorbance and gray staining with anisaldehyde/sulfuric acid and heating. The molecular weight of 1 was deduced as 380 Dalton by both positive and negative modes HR-ESI-MS with corresponding molecular formula C₂₀H₁₂O₈ and fifteen degrees of unsaturation (DBE).The ¹H NMR data (Table 1) exhibited eight resonances classified into: two downfield peri-hydroxy protons (δ_H 13.83 and 12.68 ppm), five sp² methines, four of them being for two m-coupled aromatic residues (J 2.3 Hz) were shown between 7.68 and 6.41 (H-5/H-7, H-5′/H-7′), one singlet at δ_H 5.81 (H-2), one singlet sp³ methylene (δ_H 3.37, H₂-2′).The ¹³C/HSQC NMR spectra displayed 20 carbon signals (Table 1), classified into two ketonic carbonyls, eleven sp² carbons (among them five phenolics and five sp² methines), an oxygenated sp³ quaternary, and sp³ methylene, with summation of C₂₀H₇O₈. The less 5H atoms are being for OH–groups. In agreement with the 15 DBE revealed by the molecular formula, compound 1 possesses a pentacyclic skeleton. A further full assignment of 1 was supported by long range coupling of 2D NMR (H,H-COSY and HMBC) experiments (Fig. 4, Table 1 and Supplementary Data). Hence, to first time, structure RF-3192C (1) was herein assigned based on 1D and 2D NMR and MS spectral data, filling in the gap in literature [15].

Compound 2 was isolated as a colorless solid, having a strong UV-absorbance on TLC (254 nm), however, it showed no color staining with anisaldehyde/sulfuric acid and heating. The molecular weight of 2 was deduced from HR-ESI-MS as 410 Daltons with a corresponding molecular formula C₂₂H₁₈O₈ and 14 DBE.

The ¹H NMR data (Table 2) exhibited five singlets of different categories representing an acidic broad singlet (δ_H 10.25 ppm, 7–OH), two sp² methines (δ_H 6.73 [H-6], 5.58 [H-3]), one methoxy (δ_H 3.95 [H-11]) and an sp²-attached methyl (δ_H 2.60). The ¹³C/HSQC NMR spectra displayed 11 carbon signals being for one lactone carbonyl (162.2), three phenolic sp² quaternary, three non-oxygenated sp² quaternary, two sp² methines, one methoxy, and an sp²-bounded CH₃, which are equivalent to the emperical formula C₁₁H₉O₄; representing the half of the main molecular formula (C₂₂H₁₈O₈). Hence, this greatly supported the dimeric nature of compound 2. Thus, an intensive HMBC study of structure 2 was performed confirming the structure as depicted in Fig. 4. A survey in the literature revealed a great matching of the NMR data for compound 2 with those reported for orlandin [16], however, with some imprecisions in assignment as the previous study was mainly based on one dimensional NMR data (Table 2). It is worthy to refer herein that, the difference between the NMR equipment reported in our data and those in literature, have not greatly affect the chemical shifts but only on the signals resolutions (frequencies). Chemical shifts for the same molecule might be changed if measured in different solvents [22] or different temperatures [23]. In Table 2, only values for C-2 and C-4 are labeled with asterisk as they are mutually assigned to each other. Based on 1D NMR and for first instance, the carbonyl (C-2) will be given the higher value but according to HMBC connectivities, C-4, highly de-shielded atom, has the higher value being attached to highly electronegative atom (oxygen) in the β-position of a carbonyl group and has no hydrogen atoms for compensation.

Biological activity

Antimicrobial activity assay

Based on agar diffusion method on paper disc, the fungal extract (ASSB4) gave low to moderate antibacterial and antifungal activities against S. aureus, C. albicans, B. subtilis, and S. cerevisiae and no activity against P. aeruginosa. In contrast, RF-3192C (1) exhibited notable activity against B. subtilis and P. aeruginosa but low activity against S. aureus and S. cerevisiae. Orlandin (2) exhibited moderate activity against B. subtilis while all other metabolites (3–6) exhibited no antimicrobial activity (Table 3).

Table 3.

Antimicrobial activity of the fungal extract (ASSB4) and compounds RF-3192C (1) and Orlandin (2)

Extract/compound	Pseudomonas aeruginosa	Staphylococcus aureus	Bacillus subtilis	Candida albicans	Saccharomyces cerevisiae
ASSB4 extract	-	10	12	10	9
RF-3192C (1)	15	7	14	-	9
Orlandin (2)	-	-	12	-	-
Gentamycin	18	20	16	-	-

(-) = no activity investigated

Cytotoxicity assay

The in vitro cytotoxic efficiency of compounds 1–4 and 6 compared with the original extract, were evaluated against the liver cancer HEPG2-cell line (Fig. 5, Table 4, and Supplementary Information Table S4), and compounds 2, 3, and 6 against HELA at different concentrations (0–100 μg/mL) (Table 4, and Supplementary Information Table S4 and Fig. 5B) utilizing doxorubicin as reference. Based on this study, cerebroside A (6) exhibited the most potency (0.6 SF/12.5 μg mL⁻¹) against the desired cell line of HEPG2 (Table 4, and Supplementary Information, Fig. 5A). Previous reports indicate gluco-cerebrosides such as 6 to be more cytotoxic than their galacto-cerebroside counterparts [24]. Naphthopyrones fonsecin B (3) and TMC-256A1 (4) each displayed moderate cytotoxicity (EC₅₀ ~30 μg mL⁻¹) (Table 4 and Supplementary Information, Fig. 6B). Meanwhile, orlandin (2) showed the lowest EC₅₀ (45 μg mL⁻¹) against HEPG2-cell line (Table 4 and Supplementary Information, Fig. 6B). Meanwhile testing RF-3192C (1) against MCF7 (breast cancer cell line) and A549 (non-small cell lung cancer) showed a moderate to low activity (Table 4, and Supplementary Information, Table S4 and Fig. 6B). However, orlandin (2) astonishingly gave a remarkable activity when separately tested against PC3 (prostate cancer) at 80 μM concentration with about 25% viability resazurine viability assay and showed inferior activity against A549 cancer cell line (with about 60% viability) at the same concentration (Table 4 and Supplementary Information, Fig. 6A).

Fig. 5.

A Dose–response of compounds of compounds 1–4, 6 and the fungal extract against liver HEPG2-cell line versus Log concentration in μg mL⁻¹; B In vitro cytotoxic activity of compounds 2, 3, and 6 and the fungal extract against liver HELA-cell line versus the concentration in μg mL⁻¹. For EC₅₀ values, see Table 4 and Supplementary Information, Table S4

Table 4.

EC₅₀ (μg/mL) of antitumor activity of compounds 1–4, 6 and the fungal extract against five cell lines

Compound/extract	EC50 μg/mL
	HEPG2	HELA	MCF7	A549	PC3
RF-3192C (1)	NTa	NT	47	48.7	NT
Orlandin (2)	45	-	NT	>8.2b	>8.2b
Fonscein B (3)	29.5	-	NT	NT	NT
TMC-256A1 (4)	30	-	NT	NT	NT
Cerebroside A (6)	20.8	-	NT	>58b	>58b
Crude extract	9.34	-	NT	NT	NT
Doxorubicin	4.28	1.45	17.44	8.64	NT

(-) = no activity investigated

^aNT = not tested

^bCompounds 2 and 6 were tested against A549 and PC3 cell lines in triplicates using a single dose experiment at 80 μM concentration (Supplementary Information, Fig. S3A). For more details, see Supplementary Information, Table S4 and Figs S2–3

Fig. 6.

A Viability of A549 (non-small cell lung cancer) and PC3 (prostate cancer) human cell lines at 80 μM concentration of compounds 2 (32.8 μg mL⁻¹) and 6 (58 μg mL⁻¹). B Summary chart of EC₅₀ ([μg mL⁻¹] of in vitro cytotoxic activity of compounds 1–4 and 6 against HEPG2, HELA, MCF7, A549, and PC3 cell line. For EC₅₀ values, see Table 4 and Supplementary Information, Table S4

Conclusion

PKSs are widely distributed diverse compounds in nature including plants, insects, marine organisms, and microorganisms, announcing a broad medicinal potency as anti-biotics, anti-parasites, anti-cancers, immunosuppressants, and cholesterol-lowering agents. Fungi inhabit in diverse environments represent one of the most prolific sources of PKSs. In this study, the endophytic Aspergillus niger SB4, isolated from the marine alga Laurencia obtuse, afforded the pentacyclic polyketide, RF-3192C (1) and the dimeric coumarin orlandin (2) along with further four divers PKS. The chemical structure of 1 was fully assigned for the first time using 1D/2D NMR and HRESI-MS, and the assignment revision of orlandin (2) was reported as well. RF-3192C (1) revealed high activity against Pseudomonas aeruginosa and Bacillus subtilis. The obtained compounds were in vitro investigated against liver (HEPG2), cervical (HELA), lung (A549), prostate (PC3), and breast (MCF7) cancer cell lines.

Experimental section

General experimental procedure

Column chromatography was carried out on silica gel (0.06–0.2 mm, Merck, Darmstadt, Germany). Gel filtration was carried out on Sephadex LH-20 (GE Healthcare, Uppsala, Sweden). Preparative TLC (0.5 mm thick) and analytical TLC was performed on Merck pre-coated silica gel 60 PF_254+366 plates (Merck, Darmstadt, Germany). R_f values of the bioactive compounds and visualization of their chromatograms was carried out under UV light (254 and 366 nm) and further by spraying with anisaldehyde/sulfuric acid followed by heating. High Resolution ESI-MS was done on a Micromass AC-TOF micromass spectrometer (Micromass, Agilent Technologies 1200 series, Waldbronn, Germany). 1D NMR and 2D (COSY, HMQC, HMBC) NMR spectra were recorded on an Avance 500 MHz spectrometer (Bruker, Rheinstetten, Germany) at 500 MHz (¹H) and 125 MHz (¹³C) at 298 K using the residual solvent peaks as a reference.

Isolation and taxonomic characterization of the producing fungus

The alga sample of Laurencia obtuse was aseptically rinsed twice with sterile seawater followed by sterilization with 70% EtOH for 30 s then washed with sterile seawater again. Subsequently, the marine organism was aseptically cut with sterile scalpel to reach the inner tissue surface then transferred to conical flask (100 ml) containing 50 ml sterile seawater and agitated using reciprocal water bath (30 °C) for 30 min. The prepared suspension was subjected to serial dilutions (10⁻¹–10⁻⁶) and aliquots (0.1 mL) of serially diluted samples were used to inoculate petri dishes containing PDA (Potato-Dextrose Agar) medium (Agar 15 g/L, dextrose 20 g/L, potato extract, 4 g/L). The plates were then incubated for 6 weeks at 30 °C. The colonies with distinct morphological characteristics were selected and transferred onto freshly prepared solid media (slants) and stored in refrigerator at 4 °C until use.

DNA isolation and 18S rDNA gene sequencing

The fungal strain was inoculated in 100 mL Erlenmeyer flasks each containing 50 mL of ISP2 (International Streptomyces Project; details are shown in the reference list) [25] medium composition (gL⁻¹): malt extract, 10; yeast extract, 4; glucose 4 (pH 7.2) at 30 °C for 3 days. 3 mL of culture were centrifuged at 5000 × g. The cell pellet was used for isolation of DNA. Genomic DNA of the strain was isolated, purified and sequenced according to our described previous work [26]. The produced gene sequences were compared to the available database at GenBank by using BLAST software (blastn) on the National Center for Biotechnology Information (NCBI) [21]. The phylogenetic tree was constructed using neighbor-joining tree method via MEGA X software.

Phenotypic characterization of fungal strain ASSB4

The phenotypic characterization of ASSB4 was examined after its growth on PDA media. The micromorphology was studied on a culture grown at 35 °C for 10 days on PDA medium. The spore’s morphology was examined under light microscope (Olympus CH-2) at magnification of (×1200).

Large scale fermentation, working up, and isolation

The spore suspension of the fungal strain was inoculated into 100 mL of ISP2 medium composition: malt extract, 10 g L⁻¹; yeast extract, 4 g L⁻¹ and glucose, 4 g L⁻¹ at 35 °C for 3 days as seed culture. 5 mL of seed culture were used to inoculate 1 L Erlenmeyer flasks (6 flasks) containing rice medium composition: 100 g commercial rice; 100 mL of 50% sea water. The flasks were incubated for 14 days at 37 °C. After filtration, the water/methanol fraction was evaporated to remove methanol using rotary evaporator (Heidolph) [27]. After evaporation of methanol, the water residue was re-extracted using ethyl acetate. The obtained ethyl acetate extracts were finally evaporated in vacuo to dryness and then applied to working up stages.

The fungal crude extract (6.0 g) was subjected to a silica gel column chromatography (60 × 3 cm). A stepwise elution of the column with cyclohexane–DCM–MeOH gradient [0.5 L cyclohexane: DCM (1:1), 1 L DCM, 1.5 L DCM: MeOH (99:1), 0.5 L DCM: MeOH (97:3), 0.5 L DCM: MeOH (95:5), 0.5 L DCM: MeOH (90:10)] monitored by TLC afforded five fractions FI–FV. Fractions I and II were highly non-polar, mostly fats, and were neglected. Fraction III (0.783 g) was applied to silica gel column and eluted with DCM–MeOH gradient to produce two subfractions FIIIa (0.063 g) and FIIIb (0.389 g). Purification of FIIIa on silica gel column and elution with DCM–MeOH gradient afforded orlandin (2, 5 mg) as colorless solid while FIIIb was applied to Sephadex LH-20 (MeOH) followed by PTLC (DCM/5% MeOH) to afford yellow solids of fonsecin B (3, 4 mg) and TMC-256A1 (4, 3 mg). Subfractions FIVa (0.117 g) and FIVb (0.032 g) were obtained by application of fraction FIV (0.223 g) to sephadex LH-20 (DCM/40% MeOH) followed by silica gel column using DCM–MeOH gradient. An application of FIVa to Sephadex LH-20 (DCM/40% MeOH) afforded cyclo(Leu-Ala) (5, 3 mg) as colorless solid, meanwhile RF-3192C (1, 4 mg) was obtained from sub-fraction FIVb as reddish solid after its application to PTLC (DCM/5%MeOH) followed by sephadex LH-20 (MeOH). A final purification of the last fraction FIV (0.103 g) using sephadex LH-20 (DCM/40% MeOH) led to isolation of cerebroside A (6, 8 mg) as colorless solid.The physicochemical properties of isolated compounds 1–6 are summarized in Supporting Information file.

Biological activity

Antimicrobial activity assay

Antimicrobial activity testing of the fungal crude extract and the isolated compounds were carried out against a set of microorganisms using paper-disk diffusion assay [28] with some modifications according to our previous work [29]. In accordance, antimicrobial activity assay of the crude extract together with the pure compounds against a set of microorganisms using the agar diffusion technique. Paper-disk diffusion assay with some modifications has been followed to measure the antimicrobial activity. Twenty milliliters of medium seeded with test organism was poured into 9 cm sterile Petri dishes. After solidification, the paper disks (6 mm diameter) were placed on inoculated agar plates and allowed to diffuse the loaded substances at 4 °C (in refrigerator) for 2 h. Then, the plates were incubated for 24 h at 35 °C. Both bacteria and yeasts were grown on a nutrient agar medium: 3 gL⁻¹ beef extract, 10 gL⁻¹ peptone, and 20 gL⁻¹ agar. The pH was adjusted to 7.2. Fungal strain was grown on a potato-dextrose agar medium (gL⁻¹): potato extract, 4; dextrose, 20; agar No. 1, 15 (pH 6). After incubation, the diameters of inhibition zones were measured with a wide panel of test microorganisms comprising Gram-positive bacteria (B. subtilis ATCC6633 and S. aureus ATCC6538-P), Gram-negative bacteria (Pseudomonas aeruginosa ATCC 27853), yeasts (C. albicans ATCC 10231, S. cerevisiae ATCC 9080), and the fungus (A. niger NRRL A-326).

Cytotoxicity assays

Cytotoxic assaying of the fungal extract and obtained compounds against the human cervix carcinoma cells KB-3–1 was carried out according to our previous work [29, 30]. Cytotoxic assaying against Liver (HEPG2), cervical (HELA), breast (MCF7), and lung (A549) cancer cell lines was carried out according to [31] methodology. Cytotoxicity against human lung (A549) and prostate (PC3) cancer cell lines were determined utilizing a rezasurine viability assay by treating cells with 80 μM of compounds in triplicate following previously reported protocols [32–35]. Vehicle (DMSO) was used as the negative control and actinomycin D was used as positive control (details of the cytotoxic methodology are shown in Supplementary File).