1 Introduction

The genus Cordyceps, a group of insect-pathogenic fungi, includes many species with important medical applications in Chinese Medicine. The sensu stricto species used in Chinese Medicine are Cordyceps sinensis, Cordyceps militaris, and Cordyceps cicadae [1]. Among the three species, C. sinensis has been used for treating diverse chronic diseases for more than 300 years, while C. militaris is a species which has been widely used as a materia medica, but also used a food supplement. C. cicadae is a parasitic fungus formed by cicada nymphs infected by the fungus C. cicadae (= Isaria cicadae) [2]. Jin Chan Hua, a fungus-insect complex used in Chinese Medicine, is one of the earliest medicines recorded in classics of Traditional Chinese Medicine. Pharmacological experiments results suggested that Jin Chan Hua is effective remedies for convulsions, chronic kidney diseases, measles, asthma, insomnia, and heart palpitations [3]. However, although the medicinal values of C. cicadae have long been recognized, but unlike the C. militaris, the C. cicadae are not cultivatable [4], and thereby, its shortage of supply has hampered the widely application of this species.

Recently, there have been few studies on the chemical composition and pharmacological effects of the C. cicadae strains, which have facilitated the alternative application of the crude fractionated products of this fungus. It is clear that the bioactive substances of C. cicadae include nucleosides, polysaccharides, sterols, cyclic dipeptides, amino acids, and other small organic compounds [5,6,7,8], which have extensive pharmacological effects including immunomodulatory, antitumor [9], neuroprotective [10], antibacterial [11], and anti-diabetic [12] activities. However, in terms of the exact chemical profiles of the natural products derived from this fungus, only a handful of compounds were reported. Cordycicadins A−D, purified from the fermentation broth of the fungus C. cicadae JXCH1, are unprecedented C20 polyketides with unusual exocyclic enol ether bridges [3]. Biological evaluations of these polyketides indicate that they showed significant antifeedant activity against silkworm larvae, implying the potential of these compounds in green agrochemicals development. Therefore, the secondary metabolites of C. cicadae are indeed worth to be further investigated. Herein, we report the purification as well as structural elucidation of three metabolites from the cultures of the fungus C. cicadae JXCH1.

2 Results and discussion

Cordycicadione (1, Fig. 1) was obtained as a pale-yellow oil. It presented an ion peak with highest abundance at m/z 497.28726 [M + Na]+ in the (+)-HRESIMS analysis, which returned a molecular formula of C28H42O6 (calcd for C28H42O6Na, 497.28736), corresponding to eight degrees of unsaturation. The structural elucidation of 1 was accomplished by thorough analysis of the NMR spectra. The 1H NMR spectroscopic data of 1 recorded in methanol-d4 (Table 1) showed resonances for two CH3 singlet protons at δH 1.16 (Me-18), 1.28 (Me-19), and four CH3 doublets at δH 0.74 (d, J = 6.9 Hz, Me-28), 0.76 (d, J = 6.9 Hz, Me-26), 0.93 (d, J = 7.0 Hz, Me-27), 1.08 (d, J = 6.9 Hz, Me-21), four oxygen-attached methine protons at δH 3.30 (overlapped, H-23), 3.76 (s, H-12), 4.19 (br. d, J = 7.6 Hz, H-22), 4.49 (m, H-16). The 13C NMR as well as DEPT spectroscopic data of 1 recorded in methanol-d4 (Table 2) displayed sharp carbon signals that were classified into six CH3, six CH2, nine CH [four oxygenated CH at δC 74.8 (C-23), 80.3 (C-12), 83.5 (C-16) and 85.2 (C-22)], two double bond carbons, three quaternary carbons [sp3 hybridized, one oxygenated, δC 83.7 (C-14)], and two ketone carbons at δC 201.0 (C-11) and 214.1 (C-3). The 1D NMR spectroscopic data of 1 displayed similarity with the data of asperfloroid [13], an ergostane steroid purified from the fungus Aspergillus flocculosus 16D-1 which resides in sponge, except for the substitutions at C-3, C-5, C-6 and C-7. The C-5 and C-6 are double carbons of the reference compound asperfloroid, while C-5 of 1 is a methine, C-6 and C-7 are methylenes. These changes were substantiated by 1H-1H COSY spectrum that showed correlations of H2-4/H-5/H2-6/H2-7 (Fig. 2). The HMBC correlations from H2-1, H2-2, and H2-4 to C-3 at δC 214.1 suggested that C-3 was a ketone carbon in 1, instead of being a hydroxymethine in asperfloroid (Fig. 2). Analysis of the ROESY spectrum recorded in dimethyl sulfoxide-d6 allowed to determine the relative configuration, which enabled the presence of some pivotal exchangeable protons. The ROESY correlations of H-12/Me-18/OH-14 indicated that OH-14 was β orientation, and OH-12 was α orientation. Moreover, the diagnostic ROESY correlations of Me-18/H-20, Me-18/H-15β (δH 1.82), Me-18/H-22, and H-15α (δH 2.20)/H-16/H-17 suggested the S*and R* configurations of C-16 and C-22, respectively (Fig. 2). The relative stereochemistry of both C-23 and C-24 were assigned as R* by comparing the 1H and 13C NMR chemicals shifts with those of asperfloroid. Based on above-mentioned analysis, the ECD calculation method was adopted to assign the absolute configuration of 1. As a result, the calculated ECD of 5 S,10 S,12R,13 S,14 S,16 S,17R,20 S,22R,23R,24R showed compatible curve with that of the experimental CD spectrum (Fig. 3). Therefore, the evidence presents in above texts help to establish the structure of compound 1 (Fig. 1).

Fig. 1
figure 1

The chemical structures of compounds 1–3

Full size image
Table 1 1H NMR Spectroscopic data of compounds 1−3 (600 MHz)
Full size table
Table 2 13C NMR spectroscopic data of compounds 1−3 (150 MHz)
Full size table
Fig. 2
figure 2

Key 2D NMR correlations of compounds 1–3

Full size image
Fig. 3
figure 3

Experimental CD and calculated ECD of 1

Full size image

Cordycicadin F (2, Fig. 1), a pale-yellow oil, possesses the molecular formula of C19H26O5 from the ion peak at m/z 335.18518 [M + H]+ (calcd for C19H27O5, 335.18530) in (+)-HRESIMS analysis. The 1H NMR spectroscopic data of 2 (Table 1) showed two doublet methyl signals at δH 1.14 (d, J = 6.2 Hz, Me-19), 1.40 (d, J = 6.2 Hz, Me-1), five oxygen-connected methine protons at δH 3.41 (m, H-18), 3.89 (m, H-15), 3.99 (dd, J = 4.9, 2.1 Hz, H-14), 4.62 (overlapped, H-9), 4.68 (m, H-2). The 13C NMR along with the DEPT spectroscopic data of 2 (Table 2) indicated resonances for 19 carbons attributable to two CH3 carbon at δC 20.6 (C-1) and 22.2 (C-19), seven CH2 carbons at δC 26.1 (C-16), 28.3 (C-17), 31.6 (C-3), 31.8 (C-4), 45.6 (C-11), 45.8 (C-13), 46.3 (C-8), six methines [five oxygenated ones at δC 73.1 (C-18), 75.8 (C-15), 78.0 (C-9), 78.3 (C-14), and 83.0 (C-2)], one pair of double bonds at δC 111.4 (C-6) and 174.1 (C-5), one ketal carbon and one ketone carbon at δC 117.1 (C-12) and δC 206.6 (C-7), respectively. These data of 2 (Tables 1 and 2) resembled those of the known compound opaliferin [14], a polyketide isolated from the entomopathogenic fungus Cordyceps sp. NBRC 106954. The difference between compound 2 and opaliferin is that the C-14 and C-18 of opaliferin are each substituted with a hydroxy group, while the C-14 and C-18 of 2 are attached by an ether bond. This change was reinforced by the HMBC correlations from H-18 to C-14 (Fig. 2). The tetrahydrofuran moiety (C-1–C-5) was established by HMBC correlations from H-2, H2-3, and H2-4 to C-5. The presence of cyclopentanone moiety (C-6–C-10) was confirmed by the HMBC correlations of H-8, H-9 to C-6, C-7, and of H-10, H-11 to C-6. The HMBC correlation from H2-4 to C-6 and chemical shifts of C-5 (δC 174.1), C-6 (δC 111.4) in 13C NMR spectrum suggested the existence of an enol moiety. The HMBC correlations from H2-11 to C-12, H2-13 to C-11, H2-13 to C-12, H-14 to C-12, H-15 and H-9 to C-12, together with the chemical shift of C-12 (δC 117.1) indicated that C-12 was an acetal carbon which connected to C-9 and C-15 by two epoxy bonds. All of these data were highly similar to opaliferin. Furthermore, the configurations of the chiral carbons of 2 were consistent with those of opaliferin, which were assigned as 2R,5E,9S,10R,12R,14S,15S,18R by biosynthetic considerations, comparison with the NMR data, and the ROESY correlations of H-14/H-15/H-18, and H-9/H-10 (Fig. 2), and confirmed by ECD calculation (Fig. 4). Therefore, compound 2 was established and was trivially named cordycicadin F.

Fig. 4
figure 4

Experimental CD and calculated ECD of 2

Full size image

The molecular formula of the colorless crystals 7-hydroxybassiatin (3, Fig. 1) was determined as C15H19NO4 by the protonated ion peak at m/z 278.13851 [M + H]+ (calcd for C15H20NO4, 278.13868) in the HRESIMS analysis, requiring seven degrees of double bond equivalence. The proton NMR data of 3 (Table 1) showed three singlet methyls [two methyl protons at δH 1.02 (Me-9) and 1.07 (Me-8), one N-methyl protons at δH 3.05 (Me-17)], two methine protons at δH 3.10 (s, H-6) and 4.67 (t, J = 4.8 Hz, H-3), one pair of methylene protons at δH 3.28 (dd, J = 14.2, 4.8 Hz, H-10), 3.37 (dd, J = 14.2, 4.8 Hz, H-10), one exchangeable proton at δH 4.80 (s, OH-7), and five aromatic protons. The 13C NMR and DEPT spectroscopic data of 2 (Table 2) presented carbon signals for three methyl carbons at δC 24.0 (C-8), 27.0 (C-9) and 32.3 (C-17), one methylene carbon at δC 37.4 (C-10), seven methines [one oxygenated at δC 80.4 (C-6), the others at δC 63.1 (C-3), 128.7 (C-14), 129.8 (C-13), 129.8 (C-15), 130.7 (C-12), 130.7 (C-16)], two quaternary carbons at δC 72.2 (C-7) and 136.1 (C-11), and two ketone carbons at δC 166.9 (C-2) and 167.5 (C-5). The aforementioned data exhibited high similarity to bassiatin, which was isolated from the cultured broth of the fungus Beauveria bassiana K-717 [15]. The 2D NMR spectroscopic analysis revealed that the major structural discrepancy between 3 and bassiatin was that the substituted pattern of C-7. The C-7 of bassiatin is a methine while C-7 of 3 is attached by a hydroxy group. The difference was fully corroborated by the HMBC correlations from OH-7 to C-6, C-7 and C-9. Therefore, the absolute configuration of 3 was assigned as 3S,6R by single-crystal X-ray diffraction analysis with the Flack parameter of 0.01(4) (Fig. 5). Hence, the structure of compound 3 was established (Fig. 1), and trivially named 7-hydroxybassiatin.

Fig. 5
figure 5

X-ray ORTEP drawing of compound 3

Full size image

The compounds isolated were tested for their cytotoxicity toward A549 cell line (human lung cancer), and the anti-inflammatory activity in murine RAW264.7 macrophages by evaluating the production of nitric oxide (NO). However, the results suggested that they were devoid of any activity.

3 Conclusion

Three new compounds isolated from the cultures of the entomopathogenic fungus C. cicadae JXCH1. The isolates in this study were subjected to screening their inhibition against the proliferation of the A549, a human lung cancer cell line, and the production of nitric oxide in murine macrophages RAW264.7. Although they were devoid of any activity in the biological activity screening model used in this study, but these compounds may play important or untapped roles during the process of fungal invasion into the insect hosts. Besides, this study extends the diversity of secondary metabolites in the fungus C. cicadae, which also promotes the understanding of chemical basis of traditional Chinese medicine. In subsequent experiments, we also hope to find molecules with novel skeleton or better activity from C. cicadae to provide more lead compounds for drug research and development.

4 Experimental section

4.1 General experimental procedures

The laboratory instruments used in this study are as same as those reported in the literature [16,17,18].

4.2 Fungal material

A flower-like cicadae larva-fungi complex was collected in Dajueshan Natural Reserve, Fuzhou, Jiangxi Province, China, in June 2018. The “flower” part of the complex was wash by disinfectant distilled water for three times. The inner part of the “flower” which contained powder-like spores was transferred onto the potato-dextrose-agar (PDA) plate supplemented with ampicillin and streptomycin. The fungus was purified by several times of efforts, and it was identified by sequencing the internal transcribed spacer region, which was amplified from the genomic DNA of this fungus with the primers ITS1 and ITS4. As a result, the sequencing data (GenBank Accession No. OP591391) suggest that the fungus was C. cicadae. A voucher specimen of the cicadae larva-fungi complex and the fungus (JXCH1) was kept by the Mushroom Bioactive Natural Products Research Group of South-Central Minzu University.

The fresh strain of C. cicadae JXCH1 was grew on PDA medium for seven days (25 ℃). Then the plate agar was cut into small cubes to inoculate 200 Erlenmeyer flasks (500 mL), each containing 250 mL of culture medium which consists of 5 g glucose, 1.5 g pork peptone, 5 g yeast extract, 0.5 g KH2PO4, 0.5 g MgSO4 per liter. The flasks were cultured on dark shakers at 25 °C and 160 rpm for 25 days.

4.3 Extraction and isolation

Based on previous experience on handling the cultures of this fungus [3], 50 L of methanol was added into the cultures and soaked for more than 3 days in room temperature, followed by centrifugation to break apart the mycelia and broth. The liquid phase was evaporated in vacuo to 3 L, and it was further partitioned with ethyl acetate (EtOAc) (3 × 3 L). The EtOAc layer was gathered and concentrated to give the extract A (20.2 g). The mycelia part was soaked in 5 L MeOH (3 × 3 days). The MeOH extract was combined and removed in reduced pressure, then dissolved in water and partitioned against EtOAc to give the extract B (33.3 g). The total crude extract (A + B, 53.5 g) was fractionated by MPLC with a stepwise gradient of MeOH-H2O (20:80–100:0) to afford fifteen fractions (A–O).

Fraction I was subjected to a size-exclusion CC (Sephadex LH-20) eluting with acetone and yielded ten subfractions (I1–I10). Subfraction I2 further separated by prep-HPLC (H2O-MeCN: 75:25–50:50, 25 min, 4 mL min−1) to gain six subfractions (I2a–I2f). Subfraction I2b was separated into three subfractions (I2b1–I2b3) by prep-HPLC (H2O-MeOH: 60:40–40:60, 30 min, 3 mL min−1). Subfraction I2b1 was purified on prep-HPLC (H2O-MeCN: 69:31–57:43, 15 min, 4 mL min−1) to yield compounds 3 (tR = 14.5 min, 3.9 mg).

Fractions K-M were combined and subjected to a size-exclusion CC (Sephadex LH-20) eluting with acetone and yielded fifteen subfractions (K1–K15). Compounds 1 (tR = 23.26 min, 2.4 mg) and 2 (tR = 19.55 min, 3.0 mg) were isolated from subfraction K6 by prep-HPLC (H2O-MeCN: 70:30–46:54, 30 min, 4 mL min−1).

4.4 Characterization data

4.4.1 Cordycicadione (1)

Pale-yellow oil; \( {{\left[\alpha \right]}^{23.0}_{\text{D}}}\)  + 46.9 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 245.0 (3.50) nm; 1H NMR (600 MHz, CD3OD and DMSO-d6) data, see Table 1, 13C NMR (150 MHz, CD3OD and DMSO-d6) data, see Table 2; HRESIMS m/z 497.28726 [M + Na]+ (calcd for C28H42O6Na, 497.28736).

4.4.2 Cordycicadin F (2)

Pale-yellow oil; \( {{\left[\alpha \right]}^{25.0}_{\text{D}}}\) + 17.3 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 280.0 (3.97) nm; 1H NMR (600 MHz, CD3OD) data, see Table 1, 13C NMR (150 MHz, CD3OD) data, see Table 2; HRESIMS m/z 335.18518 [M + H]+ (calcd for C19H27O5, 335.18530).

4.4.3 7-Hydroxybassiatin (3)

Colorless crystals; \( {{\left[\alpha \right]}^{25.0}_{\text{D}}}\) + 14.2 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 210.0 (4.03) nm; 1H NMR (600 MHz, CD3COCD3) data, see Table 1, 13C NMR (150 MHz, CD3COCD3) data, see Table 2; HRESIMS m/z 278.13851 [M + H]+ (calcd for C15H20NO4, 278.13868).

4.5 Computational details

The Gaussian 16 package was used as the software for all the calculation jobs [19]. Firstly, conformational analyses of the candidate molecules were searched at MMFF94s force field. The returned conformers which occupy more than 1% proportion were optimized by the density functional theory (DFT) method at the B3LYP/6-31G(d) level. The optimized conformers were subjected to electronic CD calculations. The method used in ECD calculation was time-dependent DFT (TD-DFT) at B3LYP/6-31G(d,p) level, the solvent model was IEFPCM model in MeOH (Additional file 1). The ECD calculation results were processed by the SpecDis software (v1.71) [20], the calculated ECD and experimental CD curves were plotted in the Microsoft Office Excel program.

4.6 Single-crystal X-ray diffraction data of 3

A colorless block-like sample with the molecular formula of C15H19NO4, M = 277.31, approximate dimensions 0.228 mm × 0.247 mm × 0.322 mm, was used for the X-ray crystallographic analysis on the BRUKER D8 QUEST diffractometer. The integration of the data using an orthorhombic unit cell yielded a total of 20,646 reflections to a maximum θ angle of 79.51° (0.78 Å resolution), of which 3218 were independent (average redundancy 6.416, completeness = 99.4%, Rint = 3.45%, Rsig = 2.93%) and 3160 (98.20%) were greater than 2σ(F2). The final cell constants of a = 6.7753(4) Å, b = 12.8266(8) Å, c = 17.1279(11) Å, α = 90.00°, β = 90.00°, γ = 90.00°, volume = 1488.48(16) Å3, T = 273(2) K. Data were corrected for absorption effects using the Multi-Scan method (SADABS). The structure was solved and refined using the Bruker SHELXTL Software Package, using the space group P212121, with Z = 4, µ(CuKα) = 1.54178. The final anisotropic full-matrix least-squares refinement on F2 with 189 variables converged at R1 = 2.87%, for the observed data and wR2 = 7.66% for all data. The goodness-of-fit was 1.053. The absolute configuration was determined by the Flack parameter = 0.01(4), which was determined using 1303 quotients [(I+)-(I−)]/[(I+)+(I−)] [21]. The crystallographic data have been deposited to the Cambridge Crystallographic Data Centre (CCDC 2250285).

4.7 Biological activity assays

4.7.1 Anti-proliferative assay

The anti-proliferative of the isolated compounds in this study against the five cancer cell lines were conducted by the same protocol in Ref. [22].

4.7.2 Anti-inflammatory activity assay

The anti-inflammatory of the isolated compounds against the murine RAW264.7 macrophages were conducted by the same protocol in Ref. [22].