A new lanostane-type triterpenoid from the marine shellfish symbiotic fungus Ceriporia lacerata CD7-5

Abstract One new lanostane-type triterpenoid, 3β-acetoxy-7,11-dioxolanosta-8,24-dien-21-oic acid (1), together with six known analogues (2–7), were isolated from the cultures of a marine fungus Ceriporia lacerata CD7-5, which was derived from the shellfish Ostrea denselamellosa. Their structures were determined by detailed analysis of spectroscopic data and comparison with the literature reported. The biological activities of these lanostane triterpenoids against marine-derived microalgae, zooplankton, and pathogenic bacteria were also evaluated in this study.


Introduction
Marine organisms such as algae and invertebrates are widely believed to possess chemical defense systems to resist predator predation, pathogen invasion and fouling organism attachment (Fang et al. 2017).In some case, marine microorganisms, especially symbiotic fungi, are thought to be the true producers of some active metabolites found in marine organisms (Yue et al. 2015).So, in recent decades, marine-derived fungi have been recognized as productive sources of unique and active secondary metabolites with significant ecological potential (Ji and Wang 2016), and the symbiosis has proven to be a source of highly diverse natural products (Piel 2006;Spiteller 2015).
Ceriporia lacerata is a common white-rot fungus which has been used for medical purposes (Jang et al. 2012).Recently, some researchers have found that C. lacerata also lives in various soils, plants and even marine environments (Wang et al. 2013;Zhao et al. 2013;Ying et al. 2013aYing et al. , 2013b)).Phytochemical investigations suggested that the tremulane-type sesquitepenoids and lanostane-type triterpenoids are the characteristic metabolites in this fungus (Zhao et al. 2013;Ying et al. 2013aYing et al. , 2013b)), and only two tremulanes (ceriponols F and K) have been reported showing moderate cytotoxicity against some human tumor cell lines (Ying et al. 2013b).According to the literature, so far, there is no report about the biological activity and ecological function of the lanostane triterpenoids derived from C. lacerata strains.
The shellfish Ostrea denselamellosa is a common bivalve invertebrate with high nutritional value and commercial importance, which is mainly distributed the coastal of China, Korean Peninsula, and Japan (Xu and Zhang. 2008).So far, there is no report on the symbiotic fungi, fungal metabolites and ecological relationship relevant to O. denselamellosa.As part of our continuing efforts to explore the bioactive and functional compounds from marine-derived fungal strains (Fang et al. 2021), the secondary metabolites of a symbiotic fungus C. lacerata CD7-5 derived from the shellfish O. denselamellosa was investigated.One new lanostane-type triterpenoid, 3b-acetoxy-7,11dioxolanosta-8,24-dien-21-oic acid (1), together with six known analogues (2-7) (Figure 1) were isolated from the fermentation broth of the strain C. lacerata CD7-5 in this study.Herein, we report the isolation, structure elucidation, and biological activities of these compounds.

Results and discussion
Compound 1 was isolated as white amorphous solid.Its molecular formula was determined as C 32 H 46 O 6 by HREIMS at m/z 526.3303 [M] þ (C 32 H 46 O 6 þ , calcd for 526.3289), indicating 10 degrees of unsaturation.In the 1 H NMR spectrum of 1 (Table S1), the signals of one olefinic proton at d H 5.10 (1H, t, J ¼ 7.1 Hz, H-24), one oxygenated methine proton at d H 4.54 (1H, dd, J ¼ 9.0, 7.4 Hz, H-3), and eight singlet methyl groups at d H  ) exist in 1 than 2, which could be supported by its HMBC experiment (Figure S1).The HMBC correlations of H-3 to C-1 0 , and of H 3 -2 0 to C-3, implied that the acetoxy group was attached to C-3.Similarly, the two additional keto carbonyls were located at C-7 and C-11 deduced from the HMBC correlations of H-5, H 2 -6 to C-7, and of H 2 -12 to C-11.The relative configuration of 1 was determined by NOESY spectrum (Figure S1).The NOESY correlations of H-3 with H-5 and H 3 -28, and of H 3 -30 with H-12a and H-17 suggested that H-3, H-5, H-17, H 3 -28 and H 3 -30 are a-oriented.Furthermore, the NOE correlations from H 3 -19 to H 3 -29, and from H 3 -18 to H-12b and H-20 indicated that 1 has the same configuration as that of 2. Thus, the structure of 1 was established as 3b-acetoxylanosta-8,24-dien-7,11-dione-21-oic acid.
In order to develop the ecological function of above isolates, compounds 1-3, 5 and 7, along with the mixture of 4 and 6 were tested for growth inhibition against three marine harmful microalgae (Prorocentrum donghaiense, Heterosigma akashiwo, and Chattonella marina), one marine zooplankton (Artemia salina), and four marinederived pathogenic bacteria (Vibrio anguillarum, V. harveyi, V. parahemolytics, and V. splendidus) (Miao et al. 2012;Shi et al. 2018).As shown in Table S2, the new compound 1 and the known compound 7 displayed potential inhibition of three microalga species with IC 50 values ranging from 5.3 to 43.0 lg/mL.It is worth noting that compounds 4 and 6 had not been separated successfully in this study, but their mixture (with a 5:2 ratio) exhibited significant inhibitory activities against three microalgae, P. donghaiense, H. akashiwo, and C. marina, with IC 50 values of 4.5, 2.4, and < 1.25 lg/ mL, which was probably due to their synergistic effects.The main metabolite 2 has no activity except for C. marina, but some of its derivatives have shown a certain biological activity.From the perspective of their structures, the acetoxy group at C-3 may be the active group, and the improvement of the oxidation degree of the side chain may also help to improve the activity.Furthermore, compounds 1, 7, and the mixture of 4 and 6 were also showed inhibitory activities against the zooplankton A. salina with 52.9 ± 1.6%, 80.6 ± 2.0%, and 73.9 ± 4.4% inhibition rates at 90 lg/mL, respectively.Therefore, the lanostane-type triterpenoids can be act as chemical defense substances for the host to resist changes in the external environment or the biological threats.However, none of the tested compounds displayed any activities against the four marine Vibrio strains tested at 20 lg/disk, using the disk diffusion method (Miao et al. 2012).

General experimental procedures
EI and HR-EI mass spectra were determined on an Autospec Premier P776 mass spectrometer (Waters Corp., Milford, MA, USA).IR spectra were obtained on a JASCO FT/IR-4100 Fourier Transform InfraRed spectrometer (JASCO, Tokyo, Japan).Optical rotations were measured on an SGW-3 polarimeter (INESA, Shanghai, China).UV and ECD spectra were recorded on a Chirascan CD spectrometer (Applied Photophysics Ltd., Surrey, UK). 1 D and 2 D NMR were recorded on a Bruker Avance III 500 instrument (Bruker Corp., Billerica, MA, USA) with TMS as an internal standard.HPLC separation was operated on an Agilent HPLC system (1260 infinity quaternary pump, 1260 infinity diodearray detector) using an Eclipse SB-C18 (5 lm, 9.4 Â 250 mm) column (Agilent Technologies Inc., Santa Clara, CA, USA).Column chromatography (CC) was performed with silica gel (200-300 mesh, Yantai Xinde Chemical Co., Ltd., Yantai, China), RP-18 reversed-phase silica gel (AAG12S50, YMC Co. Ltd., Kyoto, Japan), and Sephadex LH-20 (GE, Uppsala, Sweden).Thin-layer chromatography (TLC) was carried out with precoated silica gel plates Yantai Xinde Chemical Co.,Ltd.,Yantai,China).All solvents were of analytical grade except for the spectral-grade MeCN for HPLC.The zooplankton, algae and bacteria for this study were purchased or isolated from the special marine environments in our group.

Fungal material and fermentation
The fungal strain C. lacerata CD7-5 was isolated from the inner fresh tissue of the shellfish Ostrea denselamellosa, which collected from the intertidal zone of Changdao Island (latitude 37 55 0 14.17 00 N, longitude 120 43 0 57.64 00 E), Yantai, Shandong Province of China in July 2016.The fungus was identified by analysis of the morphology and ITS regions of its rDNA.The sequence data have been deposited at GenBank with the accession number ON139022, and the strain has been preserved at the Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences.The fungal strain was grown on potato dextrose agar (PDA) plates at 28 C for several days, which was then cultured statically at room temperature for 30 days in 100 Â 1 L Erlenmeyer flasks.Each flask contained 300 mL of the modified potato dextrose broth (PDB) medium (50% natural seawater collected from the coast of Yantai), which was prepared by adding 20.0 g glucose, 6.6 g sucrose, 6.6 g mannitol, 3.0 g sodium glutamate, 0.3 g CaBr 2 , 5.0 g peptone, and 5.0 g yeast extract powder into 1 L potato (200 g) broth.

Conclusion
Seven lanostane-type triterpenoids (1-7), including a new one named 3b-acetoxy-7,11dioxolanosta-8,24-dien-21-oic acid (1), were isolated from the cultures of a marine fungus C. lacerata CD7-5, derived from the shellfish O. denselamellosa.Their structures were mainly determined by detailed analysis of spectroscopic data.All the isolates were tested for inhibitory activities against marine harmful microalgae, marine zooplankton A. salina, and marine-derived pathogenic Vibrio strains.Among these compounds, the new compound 1 showed potential inhibitory activities of three microalga species with IC 50 values ranging from 5.7 to 26.3 lg/mL and the zooplankton A. salina with 52.9 ± 1.6% inhibition rate at 90 lg/mL.Interestingly, the mixture of compounds 4 and 6 (with a 5:2 ratio) exhibited significant inhibitory activities against three microalgae, P. donghaiense, H. akashiwo, and C. marina, with IC 50 values of 4.5, 2.4, and < 1.25 lg/mL, respectively.But, none of the isolated compounds displayed any activities against the four marine Vibrio strains when tested at 20 lg/disk.

Disclosure statement
No potential conflict of interest was reported by the authors.