Penigrisacids A–D, Four New Sesquiterpenes from the Deep-Sea-Derived Penicillium griseofulvum

Four new (penigrisacids A–D, 1–4) and one known (5) carotane sesquiterpenoids were isolated from the deep-sea-derived fungus Penicillium griseofulvum, along with four known compounds (6–9). The planar structures and relative configurations of the new compounds were determined by extensive analysis of the NMR and HRESIMS data. The absolute configurations were established by comparison of the experimental and calculated ECD (electronic circular dichroism) spectra or OR (optical rotation) value. Compound 9 exhibited potent anti-food allergic activity with IC50 value of 28.7 μM, while 4 showed weak cytotoxicity against ECA-109 tumor cells (IC50 = 28.7 μM).


Introduction
Carotanes (also called daucanes) are bicyclic sesquiterpenes mainly isolated from the plants of the Umbelliferae family and the fungus Aspergillus terreus [1][2][3][4]. Some of these compounds showed significant biological activities, for example, aspterric acid was found to inhibit Arabidopsis pollen development at meiosis [5]. More recently, studies showed aspterric acid could be used as a herbicide [6]. In our current investigations on the bioactive secondary metabolites from the deep-sea-derived microorganisms [7][8][9][10], the crude extract of Penicillium griseofulvum, a fungus isolated from the Indian Ocean, showed potent in vitro anti-food allergic activity. Accordingly, a systematic isolation was conducted, which led to the isolation of five carotanes (1-5, Figure 1) and four other compounds (5)(6)(7)(8)(9). Herein, we report the isolation, structure elucidation, as well as anti-allergic and anti-tumor bioactivities of these compounds.

Introduction
Carotanes (also called daucanes) are bicyclic sesquiterpenes mainly isolated from the plants of the Umbelliferae family and the fungus Aspergillus terreus [1][2][3][4]. Some of these compounds showed significant biological activities, for example, aspterric acid was found to inhibit Arabidopsis pollen development at meiosis [5]. More recently, studies showed aspterric acid could be used as a herbicide [6]. In our current investigations on the bioactive secondary metabolites from the deep-sea-derived microorganisms [7][8][9][10], the crude extract of Penicillium griseofulvum, a fungus isolated from the Indian Ocean, showed potent in vitro anti-food allergic activity. Accordingly, a systematic isolation was conducted, which led to the isolation of five carotanes (1-5, Figure 1) and four other compounds (5)(6)(7)(8)(9). Herein, we report the isolation, structure elucidation, as well as anti-allergic and anti-tumor bioactivities of these compounds.

Results and Discussion
Compound 1 was obtained as a colorless oil. Its molecular formula was established as C15H22O4 on the basis of the sodium adduct ion peak at m/z 289.1411 [M + Na] + in its HRESIMS spectrum,

t).
Since H-13a (δ H 5.01 s) was correlated to H-6 (δ H 1.72 m) in the NOESY spectrum, the hydroxy group at the C-10 position was deduced to be β-orientation. Therefore, compound 4 was established as 10,11-dihydro-10β-hydroxy-11,13-dehydroaspterric acid, and was named penigrisacid D. All detailed data of theoretical calculations, NMR, and HRESIMS spectra of 1-4 can be found in the Supplementary Materials Figures S1-S30 Tables S1 and S2.
All the nine isolates were tested for anti-food allergic activity. Compound 9 exhibited potent effect with an IC 50 value of 28.7 µM, compared to 91.6 µM of the positive control, loratadine. While compound 6 showed moderate effect with an IC 50 value of 97.3 µM. Therefore, 9 could be a promising lead compound for anti-food allergic agent. Moreover, 1-9 were also subjected to cytotoxicity assay against five different cancer cells, i.e., BIU-87, Bel-7402, ECA-109, Hela-S3, and PANC-1. However, only 4 showed weak effect on ECA-109 tumor cells with IC 50 value of 28.7 µM.

Fermentation and Extraction
The fungus was isolated from the deep-sea-sediment (−1420 m) of the Indian Ocean in 2005 by Prof. De-Zan Ye of the Third Institute of Oceanography. It was purchased from the Marine Culture Collection of China with the accession number of MCCC 3A00225. The fungus was cultured on a PDA plate at 25 • C for 3 days. The fresh mycelia and spores were inoculated to 10 × 250 mL Erlenmeyer flasks containing 120 mL of the ISP medium 2 (1.0 L contains 4.0 g yeast extract, 10.0 g malt extract, 4.0 g dextrose, and 20.0 g agar), which were incubated in a 180 rpm rotary shaker at 28 • C for 5 days. Then the spore cultures were used to inoculate 100 × 1 L Erlenmeyer flasks containing corn medium (100 g corn and 120 mL tap water for each flask) to perform the large-scale fermentation. After 62 days, the fermentation broth was extracted with EtOAc three times to provide a crude extract (55.4 g).

Theoretical Calculations
As reported previously [7], the preliminary conformational analyses were carried out using RDKit Toolkit [16] by Genetic algorithm at MMFF94 force field. Subsequently, the dominating conformers were re-optimized using density functional theory (DFT) at the B3LYP/6-31+G(d) level. Further calculation in the same level with PCM were conducted for ECD in ACN and OR in MeOH. The ECD spectra of different conformers were simulated by the overlapping Gaussian function [17]. The final spectrum was averaged according to the Boltzmann distribution theory and their relative Gibbs free energy (∆G).

Anti-Allergic Experiment
As reported previously [18], anti-allergic bioassay was conducted on RBL-2H3 cells. In brief, RBL-2H3 cells were seeded into 96-well cell culture plates to incubate with dinitrophenol specific IgE overnight. IgE-sensitized RBL-2H3 cells were pre-treated with tested compounds for 1 h and stimulated with dinitrophenyl-bovine serum albumin. Phosphate-buffered saline (PBS) buffer and loratadine were used as negative and positive controls, respectively. The bioactivity was quantified by measuring the fluorescence intensity of the hydrolyzed substrate in a fluorometer.

Cytotoxicity Assay
The in vitro antiproliferative assay was performed using MTT method according to the previously reported protocol [19]. Five different cancer cell lines (BIU-87, Bel-7402, ECA-109, Hela-S3, and PANC-1) were seeded into 96-well cell culture plates. After 24 h, different concentrations of tested compounds were added, and the incubation was continued for another 48 h. Then 20 µL MTT solution were added and cell viability was evaluated by measuring the absorbance at 570 nm.

Conflicts of Interest:
The authors declare no conflict of interest.