Three New Heptelidic Acid Derivatives from the Culture of Mushroom Lentinellus ursinus

Abstract Three new heptelidic acid derivatives (1–3) including two new dimeric esters and two known heptelidic acid analogues (4 and 5) were isolated from the solid culture of mushroom Lentinellus ursinus. The structures of new compounds were confirmed by the analysis of NMR and HRESIMS spectroscopic data. The biosynthetic origin of compounds 1–5 was postulated. Compounds 1–5 exhibited no antibacterial activity against Staphylococcus aureus and Escherichia coli at the dose of 100 μM. Graphical Abstract Electronic supplementary material The online version of this article (10.1007/s13659-018-0168-8) contains supplementary material, which is available to authorized users.


Introduction
The mushrooms in the genus of Lentinellus are white rot, wood decay, and characterized with rough-walled and amyloid spores. Eighteen species and varieties of Lentinellus have been described all over the world. There have been eleven species reported in China [1,2]. Mushroom-derived natural products draw much attention of chemists and biologists due to their diverse structural skeletons and interesting biological activities [3]. Mushrooms have been known as a prolific source of structurally diverse sesquiterpenes [4][5][6][7]. Heptelidic acid and its analogues are a group of significant secondary metabolites with interesting biological activities, such as cytotoxic, antimicrobial, antimalarial activities [8][9][10]. So far, heptelidic acid and its derivatives have been reported from the genus of fungi Lentinellus, Gliocladium, Chaetomium, Trichoderma, Xylaria, Phyllosticta, and Acremonium [8,9,[11][12][13]. In our ongoing search for new sesquiterpenes from mushrooms, the EtOAc extract from the solid culture of Lentinellus ursinus was investigated. To date, only one sesquiterpene lentinellic acid was reported from the liquid culture of L. ursinus [14]. In this study, five heptelidic acid derivatives including a new acetylated compound and two new dimeric sesquiterpenoid esters were isolated from the fungus L. ursinus. Herein, we report the isolation, structure elucidation, and antibacterial activity evaluation of new compounds 1-3 (Fig. 1).

Results and Discussion
The fungus L. ursinus was fermented on rice medium. The EtOAc extract of its rice culture was subjected to silica gel, ODS, Sephadex LH-20, and HPLC chromatography to afford three new compounds 3-O-acetylheptelidic acid A (1) lentisinic acid A (2) and B (3), and two known compounds hydroheptelidic acid (4) [12] and xylaric acid D (5) [11].
The sesquiterpene lactone of compounds 1-5 are structurally related to each other and might be originated from heptelidic acid. A proposed biogenetic pathway for these compounds is shown in Fig. 5. Heptelidic acid derived from the 1,10-and 1,6-cyclization of FPP [15] was converted into 6 by hydrolysis and oxidation cleavage. 4 was formed from 6 by dehydration, and further transformed into 1 and 5 by acetylation or dehydration, respectively. The intermidate 7 derived from heptelidic acid was further reacted with 4 and 5 to give new dimeric compounds 2 and 3, respectively.
In conclusion, three previously undescribed cadinanetype sesquiterpenes including one acetylated heptelidic acid derivative (1), two dimeric esters (2-3), and two known heptelidic acid analogues (4)(5), were isolated from the solid culture of L. ursinus. The current study enriches the secondary metabolites from this mushroom.

General Experimental Procedures
HPLC separation was conducted on Agilent 1200 HPLC system equipped with Agilent G1315D DAD detector, using a YMC-Pack ODS-A column (5 lm; 9.4 9 250 mm). NMR spectra were measured on a Bruker Avance-500 spectrometer using solvent signals (CD 3  Mass spectra were obtained on an Agilent Accurate-Mass-Q-TOF LC/MS 6520 spectrometer. Optical rotations were recorded on a polarimeter with sodium light (589 nm) by using a Perkin-Elmer 241 polarimeter. UV and IR spectra were recorded on a Thermo Genesys-10S UV-Vis spectrophotometer and a Nicolet IS5FT-IR spectrophotometer, respectively.

Fungal Material
The strain of L. ursinus was isolated from the fruiting bodies of mushroom L. ursinus collected in Meilixueshan (Yunnan, China) by Junjie Han, and identified on the basis of the morphological characteristics and ITS sequences.  The strain was cultured on PDA plates at 25°C for 14 days. Agar plugs were inoculated into a 250 mL Erlenmeyer flask containing 100 mL PDB medium cultured at 25°C on a rotary shaker at 180 rpm for 14 days. Large-scale cultivation was carried out in 20 9 500 mL Fernbach culture flasks each containing 80 g of rice and 100 mL of distilled water. Each flask was inoculated with 5 mL of culture medium and incubated at 25°C for 40 days in dark.

Extraction and Isolation
The cultivated rice substrate was extracted repeatedly with EtOAc (3 9 10 L) at room temperature and 20.

Antimicrobial Assay
The antimicrobial assay was conducted as our previous described method [16]. The bacterial strains S. aureus (ATCC 6538) and E. coli (ATCC 25922) were grown in Lysogeny Broth (LB) medium. The inhibition rate was calculated and plotted versus test concentrations to afford the MIC. MIC values were defined as the minimum concentration of compound that inhibited visible microbial growth.