Biological Secondary Metabolites from the Lumnitzera littorea-Derived Fungus Penicillium oxalicum HLLG-13

Five new compounds, including two cyclopiane diterpenes conidiogenones J and K (1–2), a steroid andrastin H (5), an alkaloid (Z)-4-(5-acetoxy-N-hydroxy-3-methylpent-2-enamido) butanoate (6), and an aliphatic acid (Z)-5-acetoxy-3-methylpent-2-enoic acid (7), together with ten known compounds (3–4 and 8–15) were isolated from the EtOAc extract of the fermentation broth of the Lumnitzera littorea-derived fungus Penicillium oxalicum HLLG-13. Their structures were elucidated by 1D, 2D NMR, and HR-ESI-MS spectral analyses. The absolute configurations of 1, 2, 5, and 8 were determined by quantum chemical electronic circular dichroism (ECD) calculations, and the absolute configuration of 8 was determined for the first time. Compound 15 was a new natural product, and its NMR data were reported for the first time. Compounds 5 and 9–14 exhibited antibacterial activities against Staphylococcus epidermidis and Candida albicans, with MIC values ranging from 6.25 to 25 μg/ mL. Compounds 1–6 and 9–14 showed significant growth inhibition activities against newly hatched Helicoverpa armigera Hubner larvae, with IC50 values ranging from 50 to 200 μg/mL.


Introduction
Lumnitzera littorea (Jack) Voigt is a mangrove tree that has been included on the list of national key protected wild plants (the first batch) (Level II) approved by the State Council of China on 4 August 1999. According to the literature reports, different types of active compounds from Lumnitzera have been isolated, such as hepatoprotective flavonoids and phenolic glycosides [1], antileishmanial macrolides [2], cytotoxic polyketones [3], and anti-angiogenic and anti-inflammatory neolignans [4]. Due to the shortage of L. littorea, the study of bioactive secondary metabolites from the L. littorea-derived endophytic fungus is necessary. Only four articles about the secondary metabolites from the endophytic fungi of Lumnitzera have been reported [5][6][7][8], including an article about antibacterial terpenoids [5], one about cytotoxic polyketones [6], another about cytotoxic oxygenated meroterpenoids [7], and one about steroids with α-glucosidase inhibitory activity [8].
During our exploration of the structurally diverse and bioactive compounds from mangrove-derived fungi, some new bioactive compounds have been found [9][10][11][12]. In the previous study, cytotoxic oxygenated meroterpenoids and steroids with α-glucosidase inhibitory activity had been isolated from the secondary metabolites of two endophytic fungi: Penicillium sp. HLLG-122 and Penicillium sclerotiorum HLL113, which were both isolated from the roots of the L. littorea. [7,8]. In our continuing research, the endophytic fungus Penicillium oxalicum HLLG-13, obtained from the roots of L. littorea and collected from the Tielugang Mangrove Reserve in Sanya, was selected for further research because its EtOAc extract showed antibacterial activity and growth inhibition activity against newly hatched H. armigera Hubner larvae. Five new compounds (1-2 and 5-7) and ten known compounds (3-4 and 8-15) ( Figure 1) were isolated from the EtOAc extract of the fermentation broth from P. oxalicum HLLG-13. In this study, we report the isolation, structure elucidation, antibacterial activity, and growth inhibition activity of these compounds against newly hatched H. armigera Hubner larvae.
The relative configuration of compound 5 was determined by the NOESY correlations ( The absolute configuration was assigned by the experimental and calculated ECD spectra. The ECD spectrum of (3R, 5S, 8S, 9S, 10S, 13R, 14R)-5 (5a) and its enantiomer (5b) were calculated using TDDFT in MeOH. As shown in Figure 4, the calculated spectrum of 5a agreed well with the experimental spectrum. Therefore, the absolute configuration of 5 was assigned as 5a and named as andrastin H.
Compound 8 was isolated as a yellow amorphous powder. Its molecular formula was established by HR-ESI-MS (m/z 379.1529 [M + Na] + , calcd. for 379.1521) to be C 21 H 24 O 5 with ten degrees of unsaturation. Compared with that of the literature [15], the 1D NMR data (Tables 1 and 2) of 8 closely resembled those of stocksiloate, which was isolated from Vincetoxicum stocksii, and the absolute configuration remained to be determined due to certain limitations. In order to determine the absolute configuration of 8, the theoretical ECD spectra of two possible stereoisomers of 2R and 2S were calculated using TDDFT calculation, and the calculated ECD curve of isomer 2R revealed a good agreement with the experimental one ( Figure 4). Therefore, the absolute configuration of 8 was assigned as 2R-form and named as methyl 2R-stocksiloate.
The growth inhibition activities against newly hatched H. armigera Hubner larvae were tested using the assay described by Guo [32]. Compound 5 exhibited obvious insecticidal activity against newly hatched H. armigera Hubner larvae, with an IC 50 value of 50 µg/mL, which was equivalent to that of the positive control (azadirachtin); and compounds 1-4, 6, and 9-14 also showed growth inhibition activities against newly hatched H. armigera Hubner larvae, with IC 50 values ranging from 100 to 200 µg/ mL (Table 4).

General Experimental Procedures
Optical rotations were measured on an Anton paar MCP 5100 modular circular polarimeter (JASCO, Tokyo, Japan). ECD spectra were recorded on a Boilogic Mos-500 spectrometer (JASCO, Tokyo, Japan). IR spectra were recorded on a Nicolet 6700 spectrophotometer (Thermo Scientific, Madison, WI, USA). UV spectra were recorded on a Beckman DU 640 spectrophotometer (JASCO, Tokyo, Japan). The 1D and 2D NMR spectra were obtained with a Bruker AV spectrometer (400 MH Z for 1 H and 100 MH Z for 13 C, (Bruker Corporation, Basel, Switzerland) or a JNM-ECZS spectrometer (600 HM Z for 1 H and 125 MH Z for 13 C, (JEOL, Tokyo, Japan), using Methanol-d 4 or DMSO-d 6 as a solvent. TMS was used as an internal standard. HR-ESI-MS spectra were measured on a Bruker APEX II spectrometer (Billerica, MA, USA). Silica gel (Qing Dao Hai Yang Chemical Group Co., Qingdao, China; 200-300 mesh) and octadecylsilyl silica gel (YMC; 12 µm-50 µm) were used for column chromatography (CC). Precoated silica gel plates (Yan Tai Zi Fu Chemical Group Co., Yan Tai, China; G60, F-254) were used for thin layer chromatography (TLC). Semi-preparative HPLC was performed on an Agilent 1260 LC series with a DAD detector using an Agilent Eclipse XDB-C 18 column (250 × 9.4 mm, 5 µm, Agilent Corporation, Santa Clara, CA, USA).

Fungal Materials
The fungus HLLG-13 was isolated from the roots of the mangrove L. littorea (Jack) Voigt. The L. littorea was collected in Tielugang, Sanya city, Hainan province, in November 2018 and identified by Yukai Chen, associate professor of the College of Life Sciences, Hainan Normal University. This strain was deposited in the Key Laboratory of Tropical Medicinal Resource Chemistry of Ministry of Education, College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou, Hainan, China. The fungus was identified according to its morphological traits and a molecular protocol by amplification and sequencing of the DNA of the ITS region of the rRNA gene. Its base pair ITS sequence had 99% sequence identity to that of P. oxalicum. Therefore, the fungal strain was identified as P. oxalicum. The sequence data have been submitted to GenBank, with an accession number of OK560165.

Fermentation, Extraction, and Isolation
The seed culture was prepared in a potato liquid medium (30 g sea salt in 1 L of potato infusion in 1 L × 4 Erlenmeyer flasks, each containing 300 mL seed medium), and incubated on a rotary shaker (160 rpm) for 3 days at 28 • C. In total, 50 mL seed culture was then transferred into 1 L Erlenmeyer flasks with a solid rice medium, for a total of 200 bottles of fermentation (each flask contained 80 g rice, 3.0 g sea salt, and 100 mL water) at 28 • C for 28 days. The medium was extracted repeatedly with EtOAc to obtain the corresponding extracts.

Antibacterial Activity
The antibacterial activities of all compounds against eight pathogenic bacteria (S. aureus (ATCC 25923), E. coli (ATCC 25922), C. albicans (ATCC 14053), S. epidermidis (ATCC 49134), P. aeruginosa (ATCC 17749), V. harveyi (ATCC 25919), V. alginolyticus (ATCC 33787), and V. parahaemolyticus (ATCC 27969)) were determined by the microplate assay method. The activated pathogenic bacteria were inoculated into the nutrient broth medium. The concentration of the test group and positive control was 1 mg/mL. The antibacterial effect was evaluated by full wavelength multifunctional microplate reader measurement at 630 nm; the broth medium containing pathogenic bacteria was used as the blank group and DMSO as the negative control; and ciprofloxacin was used as the positive control.

Growth Inhibition Activities against Newly Hatched H. armigera Hubner Larvae
In the test, there were three groups, each containing three neonate larvae of H. armigera Hubner, and the tested compounds were dissolved in DMSO at a concentration of 1 mg/mL. The insecticidal activity was investigated by adding the serial dilution of the isolated compounds and the positive control azadirachtin at 200, 100, and 50 µL/well, with 3 replicates per treatment to the artificial diet for the newly hatched larvae, and the bioassay diet was placed into six-well plates. Newly hatched larvae were incubated at 25 • C and a relative humidity of 80%. DMSO was used as the negative control, azadirachtin was used as the positive control, and an artificial diet was used as the blank control. The number of dead larvae was recorded on the second, fourth, sixth, and eighth day after treatment.

Conclusions
Five new compounds, including two cyclopiane diterpenes conidiogenones J and K (1-2), a steroid andrastin H (5), an alkaloid (Z)-4-(5-acetoxy-N-hydroxy-3-methylpent-2-enamido) butanoate (6), and an aliphatic acid (Z)-5-acetoxy-3-methylpent-2-enoic acid (7), together with ten known compounds (3-4 and 8-15) were isolated from the EtOAc extracts of the fermentation broth of the L. littorea-derived fungus P. oxalicum HLLG-13. Compounds 5 and 9-14 exhibited strong antibacterial activities against S. epidermidis and C. albicans, with MIC values ranging from 6.25 to 25 µg/ mL. Compounds 1-6 and 9-14 exhibited significant growth inhibition activities against newly hatched H. armigera Hubner larvae, with IC 50 values ranging from 50 to 100 µg/ mL. Author Contributions: Y.W. and Z.X. performed the experiments for the isolation and structure elucidation and prepared the manuscript; Q.B. contributed to the antibacterial activity and growth inhibition activity against newly hatched H. armigera Hubner larvae; X.Z. and M.B. contributed to part of the structure determination; W.C., C.Z. and G.C. supervised the research work and revised the manuscript. All authors have read and agreed to the published version of the manuscript.