Gentamicin was used in ribosome engineering to introduce drug-resistance in bacteria. However, it could not be applied to fungi up to now because of its insensitivity. The fungus Penicillium purpurogenum G59 is also insensitive to gentamicin. We assumed that low intracellular concentration of gentamicin is likely to be a possible reason for its insensitiveness to fungi and furthermore the permeability of the fungal membrane to gentamicin may restrict its intracellular concentration. Dimethylsulfoxide (DMSO) is known as a membrane permeabilizer which enhances the penetration of both hydrophilic and hydrophobic molecules into the cells. Thus, DMSO was chosen as first choice accessorial agent to find conditions for introducing gentamicin-resistance into the fungal strain G59 in the present study.

Search of experimental condition for introducing gentamicin-resistance, as well as study of the effects of gentamicin, DMSO and their combinations on the growth of G59 were undertaken at first, after treatment of the G59 spores in the test and control groups in 20% and 50% DMSO (Table 1) at 4 °C for 1–20 days. The treated spores were spread on potato dextrose agar (PDA) plates and incubated at 28 °C to estimate the growth of G59.

The pretreatment of G59 spores at 4 °C in water with gentamicin or in 20% and 50% DMSO without gentamicin did not affect the growth of G59 on PDA plates at 28 °C. Typical examples as shown in Figure 1, of G59 in the control groups grew well throughout on the plate. However, pretreatment of the spores with gentamicin at 4 °C in 20% and 50% DMSO resulted in significant inhibition of G59 growth at 28 °C on PDA plates, allowing the development of resistant mutant colonies. Thus, the conditions and experimental procedure for introducing gentamicin-resistance were decided as follows: First, the G59 spores were treated with the mg/mL amount of gentamicin in 20% or 50% DMSO (Table 1) at 4 °C for 1–20 days. Second, the resistant mutants were selected by single colony isolation using the pretreated G59 spores.

The resistance of the mutants to gentamicin was testified by a resistance test using a mutant 5-1-4 and the parent G59. After pretreatment of the 5-1-4 and G59 spores with 5 mg/mL gentamicin as that for selecting the mutant 5-1-4, both the spores were spread on PDA plates and incubated at 28 °C for 10 days to examine their day-by-day growth. The colonies of mutant 5-1-4 appeared on the second day of incubation and developed to become confluent with the incubation times as shown in Figure 2. In contrast, few colonies of G59 appeared on the fifth day of incubation and developed to give four isolated colonies on the sixth day of incubation (Figure 2), which were kept isolated for the incubation time period examined. These results demonstrated that the treatment of G59 spores with gentamicin in the present study successfully introduced the gentamicin-resistance into the mutant 5-1-4.

As shown in Figure 1, the pretreatment of G59 spores with gentamicin in aqueous DMSO resulted in the development of resistant colonies on PDA plates. Thus, single colony isolation was performed using G59 spores treated with gentamicin in 20% and 50% DMSO at 4 °C for different times by spreading them on PDA plates. The colonies selected are gentamicin-resistant mutants, having survived by acquiring resistance capability to the treatment. A total of 181 mutants were selected with different appearances of colonies on PDA plates, including 136 and 45 mutants from 20% and 50% DMSO test groups, respectively (Table 2).

Both growth and spore formation of 181 of the mutants were not so obviously different from those of the parent strain G59 under the culture conditions at 28 °C on PDA plates. However, they showed obviously different morphology, color of mycelia, or pigment formation, when grown on PDA plates at 28 °C (typical examples are shown in Figure 3). These differences correlated with the different appearances of corresponding colonies observed during mutant selection, which were used as an indicator to select different mutants in single colony isolation. Correspondingly, similar differences in colors of mycelia and culture broths were also observed by fermentation in liquid medium (data not shown).

All EtOAc extracts from the fermentation of G59 and its mutants were subjected to MTT assay at 100 μg/mL. In triplicate MTT tests using samples from three rounds of individual fermentations, nine mutants showed inhibitory effects on the proliferation of K562 cells with mean IR% values over 40% (Table 3). Among them, two were from the 20% DMSO group, which accounts for 1.5% of the 136 mutants from this group; whereas seven were from the 50% DMSO group, accounting for 15.6% of the 45 mutants from this group.

Prior to MTT treatment of K562 cells in the MTT assay, the morphology of K562 cells treated with 100 μg/mL of EtOAc extracts was observed under a reversed phase microscope. Corresponding to the MTT assay results (Table 3), morphological observations (full data not shown) revealed that the K562 cells treated with a G59 sample showed no morphological changes in comparison with the control (Figure 4). However, a portion of the cells treated with a mutant 5-1-2 sample showed heteromorphosis with club-shaped cells, revealing an effect of the sample on cytoskeleton of inhibiting cell division (Figure 4). Furthermore, a major portion of the cells treated with the other eight mutant samples showed necrotic cell morphology, as shown for mutant 5-1-4 in Figure 4, indicating cytotoxic effects of the eight mutant samples on K562 cells.

The EtOAc extracts from G59 and nine mutants in Table 3, prepared for the third test of MTT assay, were subjected to TLC and HPLC analysis. By comparison with the strain G59, several major components detected in the G59 sample by HPLC disappeared in all nine mutant samples almost completely, and instead, the secondary metabolites newly produced by the mutants were detected both by TLC (Supplementary Data S1) and HPLC (Figure 5 and Supplementary Data S1). HPLC profiles detected at different wave lengths and TLC chromatograms in Supplementary Data S1 provided more detailed information including differences between one another. These results revealed that the introduction of gentamicin-resistance resulted in the alteration of secondary metabolisms to activate the production of some dormant secondary metabolites in G59.

During the course of mutant 5-1-4 fermentation, relative percentage of the mycelial volume and inhibitory effect of the EtOAc extract at 100 μg/mL reached their maxima at 11 and 12 days of the fermentation, respectively, while pH of the supernatants (pH 6.0) remained unchanged throughout fermentation period tested. Thus, the producing fermentation of mutant 5-1-4 was carried out for 12 days to obtain total 49 L of liquid cultures for the isolation of antitumor metabolites.

Total liquid cultures of mutant 5-1-4 were extracted with EtOAc to obtain an active EtOAc extract. The EtOAc extract was then separated by chromatographic means, tracing active components absent in the metabolites of G59 by direct comparison with the sample from G59, under guidance of bioassay and chemical examination. This has resulted in the isolation of four antitumor metabolites 1–4 (Figure 6) newly produced by the mutant 5-1-4 (see Supplementary Data S2).

Compound 1 giving pseudo-molecular ion peak at m/z 285 [M + H]⁺ in ESI-MS and compound 2, [α]²⁰_D −175.4 (c 0.24, CHCl₃), showing pseudo-molecular ion peak at m/z 444 [M + H]⁺ in ESI-MS were identified as janthinone [51] (1, Figure 6) and fructigenine A [52] (2, Figure 6) on the basis of their NMR data, respectively.

Compound 3 was obtained as a colorless oil with [α]²⁰_D −59 (c 1.0, MeOH) and its molecular formula could be determined as C₁₆H₂₄O₄ by HR-ESI-MS measurement. The ¹H and ¹³C NMR spectra of 3 in CD₃OD closely resembled those of aspterric acid [53], except for additional signals ascribable to a methoxy group (δ_H3.72 and δ_C52.4), and detailed analysis of its DEPT, ¹H–¹H COSY, HMQC and HMBC spectra led to the assignment of 3 as aspterric acid methyl ester.

Compound 4, [α]²⁰_D −17.3 (c 1.0, EtOH), giving pseudo-molecular ion peak at m/z 251 [M + H]⁺ in ESI-MS, could be identified as citrinin [54,55] based on its ¹H and ¹³C NMR data.

The antitumor activity of 1–4 was tested preliminarily by the MTT assay using K562 cells, coupled with the morphological examination of K562 cells under a reversed phase microscope. In MTT assay, all of 1–4 showed to a certain extent an inhibitory effect on K562 cells in the inhibition intensity order of 4 > 2 > 1 and 3 (Table 4), and the IC₅₀ values for 4 and 2 could be determined as 52.6 and 58.4 μg/mL, respectively. Morphological examination of the K562 cells treated with 1–4 at 100 μg/mL showed that 1–4 could inhibit K562 cells mainly by their cytotoxic effect except for an additional, weak but significant effect of 4 on cytoskeleton to inhibit cell division, revealed by a small portion of club-shaped K562 cells similar to those shown for mutant 5-1-2 in Figure 4.

The original strain Penicillium purpurogenum G59 used in the present study is a marine-derived wild-type fungal strain G59 isolated from a soil sample collected at the tideland of Bohai Bay around Lüjühe in Tianjin, China, in September, 2004 [50], which was identified later by L. Guo, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China. The strain was deposited at the China General Microbiological Culture Collection Center under the accession number CGMCC No. 3560. The EtOAc extract of G59 fermentation culture did not show any effect on K562 cells and its inhibition rate at 1000 or 100 µg/mL was always lower than 6.9% in repeated tests using different samples from individually fermented cultures.

Human chronic myelogenous leukemia cell line K562 was provided by S. Li (Beijing Institute of Pharmacology and Toxicology). The cells were routinely maintained at 37 °C in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum in the presence of 100 µg/mL penicillin and streptomycin under a humidified atmosphere of 5% CO₂ and 95% air.

Fresh spores formed by cultivation of strain G59 on PDA (potato dextrose agar) plates at 28 °C for 3–4 days were harvested, suspended in the appropriate amount of sterilized, distilled water with several glass beads in 50 mL cone-shaped flasks, and shaken well to prepare a crude spore suspension. A 100 μL portion of this crude spore suspension was added into a well of 96-well plates, diluted with water with its OD at 600 nm measured using a VERSAmax-BN03152 plate reader, and the dilution ratio was recorded when the OD value reached 0.35. Then, the whole crude spore suspension remaining was diluted with sterilized, distilled water in the same proportion to obtain a G59 spore suspension. This G59 spore suspension was used in the following experiments to keep the same spore density in all experiments concerned. The spore suspension for mutant 5-1-4, a gentamicin-resistant one, obtained by treatment of G59 with 5 mg/mL gentamicin in 50% (v/v) DMSO at 4 °C for 1 d (Table 3), was prepared in the same way as mentioned above for G59.

To nine of the sterilized 2 mL Eppendorf tubes was added 0, 1, 2, 4, 10, 20, 40, 100 and 200 mg of gentamicin, respectively, and then 1600 μL of G59 spore suspension was added to dissolve gentamicin by shaking. Then, 400 μL DMSO was added to each tube to obtain a series of spore suspensions in 20% (v/v) DMSO. The one without gentamicin was used as a control and the other eight with 0.5, 1, 2, 5, 10, 20, 50 and 100 mg/mL gentamicin were used as the test group. To each of nine sterilized 2 mL Eppendorf tubes was added 0, 1, 2, 4, 10, 20, 40, 100 and 200 mg of gentamicin, respectively, as controls without DMSO, and then 1600 μL of G59 spore suspension was added and diluted with 400 μL of sterilized, distilled water to obtain 4:1 diluted G59 spore suspensions with the same spore density as in 20% (v/v) DMSO, which contained 0, 0.5, 1, 2, 5, 10, 20, 50 and 100 mg/mL gentamicin, respectively.

Similarly, to each of the six sterilized 2 mL Eppendorf tubes was added 0, 2, 4, 10, 20 and 40 mg of gentamicin, respectively, and 1000 μL of G59 spore suspension was added to dissolve gentamicin. Then, 1000 μL DMSO was added to each tube to obtain a series of spore suspensions in 50% (v/v) DMSO. The one without gentamicin was used as a control and the other five with 1, 2, 5, 10 and 20 mg/mL gentamicin were used as the test group in 50% (v/v) DMSO. Similarly, the 1:1 water diluted G59 spore suspensions containing 0, 1, 2, 5, 10 and 20 mg/mL gentamicin were used as the controls without DMSO, having the same spore density as in 50% (v/v) DMSO.

G59 spores in the above 20% (v/v) and 50% (v/v) DMSO tubes were treated at 4 °C for 1–20 days along with the controls in water. Each 90 μL of the treated spore suspensions was spread on PDA plates with a 24 h interval and incubated at 28 °C for 3–4 days. The mutant strains from the gentamicin-treatment test groups in 20% (v/v) and 50% (v/v) DMSO were obtained by single colony isolation, selecting colonies with different appearances during the incubation period.

The 5-1-4 and G59 spore suspensions with 5 mg/mL gentamicin in 50% DMSO at the same spore density were prepared according to the procedure described in Sections 4.2 and Sections 4.3. The suspensions were stored at the same 4 °C condition for 1 day to treat the spores with gentamicin in the manner as that for selecting the mutant 5-1-4. Then, each 100 μL of the treated spore suspensions was spread on PDA plates, incubated at 28 °C, and examined day-by-day for growth up to 10 days of incubation.

G59 and its mutants were inoculated into a 500 mL cone-shaped flask containing 200 mL of liquid medium (glucose 2%, maltose 1%, mannitol 2%, glutamic acid 1%, peptone 0.5% and yeast extract 0.3% in distilled water, adjusted to pH 6.0 prior to sterilization) and fermented at 28 °C for 13 days on a rotary shaker at 180 rpm. After fermentation, 200 mL broth was extracted with 400 mL acetone by ultrasonication for 1.5 h. The aqueous acetone solution obtained by filtration was concentrated under reduced pressure to remove acetone. The remaining water layer was then extracted three times with equal volumes of EtOAc to obtain the crude extract for MTT assay and chemical analysis (TLC, HPLC).

The EtOAc extracts and pure compounds were dissolved in MeOH to prepare 10.0 mg/mL stock solutions, and serial dilutions were made for MTT assay. Exponentially growing K562 cells were suspended in fresh RPMI-1640 medium at the density of 1 × 10⁵ cells/mL, seeded into 96-well plates, and incubated at 37 °C for 1 h. Then 2 μL of MeOH for control or sample solutions was added to each well and the cells were cultured at 37 °C for 24 h. MTT (20 μL; 5 mg/mL in PBS) was added into each well, incubated at 37 °C for 4 h, and centrifuged at 2000 rpm for 20 min. After removal of the supernatant by aspirating, 150 μL DMSO was added into each well, and shaken for 5 min to dissolve formazan crystals. The OD value at 570 nm was read for each well using the VERSAmax-BN03152 plate reader. Each three wells were set for control and test groups, respectively, and the inhibition rate (IR%) was calculated using the formula, IR% = (OD_control − OD_sample)/OD_control × 100%. The IC₅₀ value for a sample was obtained from the IR% values of the sample at different concentrations.

TLC analysis was carried out on precoated silica gel GF254 plates using CHCl₃-MeOH (20:1, 15:1, 9:1 or 5:1), CH₂Cl₂-MeOH (15:1, 12:1, 9:1 or 5:1) or bp 60–90 °C Pet.Ether-Acetone (10:1, 5:1 or 1:1) as developing solvents. TLC spots were detected under UV light (254 and 365 nm) or by using iodine, Vaughan’s reagent [24 g of ammonium molybdate tetrahydrate (NH₄)₆Mo₇O₂₄·4H₂O) and 1 g of ceric sulfate Ce(SO₄)₂ dissolved in 500 mL of 10% H₂SO₄] or 10% sulfuric acid reagent. HPLC analysis was performed using an analytical Venusil MP C18 column (5 μm, 100 Å, 4.6 mm × 250 mm; Agela Technologies) on a Waters HPLC system equipped with Waters 600 controller, Waters 600 pump, Waters 2414 refractive index detector, Waters 2996 photodiode array (PDA) detector and Waters Empower™ software. Each 10 μL of sample solutions (8.3 mg/mL) in MeOH was injected into the column and eluted with MeOH–H₂O in linear gradient (20% MeOH at initial time 0 min→100% MeOH at 60 min→100% MeOH at 90 min) as mobile phase (flow rate, 1 mL/min). The acquired photodiode array (PDA) data were processed by Empower PDA software to obtain targeted HPLC data.

Spore suspension of mutant 5-1-4 (2 mL) was inoculated into a 500 mL cone-shaped flask with 200 mL of liquid medium (glucose 2%, maltose 1%, mannitol 2%, glutamic acid 1%, peptone 0.5% and yeast extract 0.3% in distilled water, adjusted to pH 6.0 prior to sterilization) and fermented at 28 °C for 14 days on a rotary shaker at 180 rpm. Sampling was performed for a 15 mL portion with a 24 h interval from the third day of fermentation. After centrifugation of the sampled broth at 3000 rpm for 10 min, followed by measurement of the pH value of supernatant and the relative percentage (%) of mycelial volume, the whole broth (15 mL) was extracted with 30 mL acetone by ultrasonication for 1.5 h. The obtained aqueous acetone solution was concentrated under reduced pressure to remove acetone and the remaining water layer was extracted three times with equal volumes of EtOAc to obtain the extracts for assay.

Based on the result of the time course experiment mentioned, spore suspension of mutant 5-1-4 (2 mL) was inoculated into each of 245 cone-shaped 500 mL flasks with 200 mL of liquid medium (glucose 2%, maltose 1%, mannitol 2%, glutamic acid 1%, peptone 0.5% and yeast extract 0.3% in distilled water, adjusted to pH 6.0 prior to sterilization) and fermented at 28 °C for 12 days on a rotary shaker at 180 rpm. The whole broth (49 L) was filtrated to separate into a filtrate and a mycelial cake. The filtrate was extracted three times with equal volumes of EtOAc to give an extract (14.3 g). The mycelial cake was extracted with 3 L of acetone-water (2:1) by ultrasonication for 1.5 h and the aqueous acetone solution obtained by filtration was evaporated under reduced pressure to remove acetone. The water layer remained was extracted three times with equal volumes of EtOAc to give another extract (16.3 g). The combined EtOAc extracts (30.6 g) showed an inhibitory effect on K562 cells, with an IR% value of 42.8% at the 100 µg/mL, from which the antitumor secondary metabolites 1–4 were isolated.

G59 spore suspension (2 mL) was inoculated into each of two 500 mL cone-shaped flasks containing 200 mL of liquid medium with the same composition and fermented at 28 °C for 12 days under the same conditions. The fermented broth (400 mL) was extracted as described above for mutant 5-1-4 to obtain an EtOAc extract (200 mg), which did not show any inhibitory effect on K562 cells (an IR% value of 6.0% at 100 µg/mL). This extract was used as negative control in the experiments for tracing antitumor secondary metabolites newly produced by the mutant 5-1-4.

The EtOAc extract (30.6 g) of mutant 5-1-4 was separated into 15 fractions (Fr-1 to Fr-15) by vacuum liquid chromatography (VLC) using a silica gel column (bed 6.2 × 10 cm) dry-packed with 150 g of silica gel H (100–200 meshes) through stepwise elution with bp 60–90 °C petroleum ether (P)-ethyl acetate (E) (100:0→0:100) and then dichloromethane (D)-methanol (M) (90:10→0:100). Six fractions, Fr-4 (1.6 g) eluted by PE (9:1→8:2), Fr-5 (3.7 g) eluted by PE (8:2), Fr-6 (2.0 g) eluted by PE (8:2→7:3), Fr-7 (4.6 g) eluted by PE (7:3→4:6), Fr-8 (2.5 g) eluted by PE (2:8) and Fr-12 (5.1 g) eluted by DM (9:1→8:2), showed inhibitory effects against K562 cells, with IR% values of 34.4, 31.4 36.5, 52.8, 43.0, and 49.4%, respectively, at 100 μg/mL.

Fr-4 (1.6 g) was subsequently subjected to Sephadex LH-20 column (bed 1.5 × 135 cm in DM 1:1) chromatography using DM (1:1) as eluting solvent to obtain seven fractions, Fr-4-1 to Fr-4-7, in the order of elution. Fr-4-3 (1.45 g) was further separated by VLC using a silica gel column (bed 2.5 × 6 cm) dry-packed with 12 g of silica gel H (200–300 meshes) by a stepwise elution with PE (100:0→0:100) and EM (100:0→0:100) to give seven fractions, Fr-4-3-1 to Fr-4-3-7. Among them, Fr-4-3-2 (420 mg) eluted by PE (99:1→97:3) gave yellow crude crystals at room temperature, which were filtered and then repeatedly recrystallized from dichloromethane–methanol (1:1) to obtain 1 (28 mg) as yellowish needles.

Fr-5 (3.7 g) was further separated into 12 fractions, Fr-5-1 to Fr-5-12, by VLC on a silica gel column (bed 2 × 12 cm) dry-packed with 25 g of silica gel H (100–200 meshes) through stepwise elution with PE (100:0→0:100) and DM (90:10→0:100). Fr-5-2 (160 mg) eluted by PE (99:1→96:4) was separated by Sephadex LH-20 column (bed 1.5 × 135 cm in DM 1:1) chromatography eluted with DM (1:1) to obtain five fractions, Fr-5-2-1 to Fr-5-2-5, in the order of elution. Fr-5-2-1 (70 mg) was separated by semi-preparative HPLC on a reversed-phase Senshu Pak C18 column (8 × 250 mm) using 65% aqueous MeOH as eluting solvent (flow rate 1.5 mL/min, detector wave length 246 nm) to obtain a fraction corresponding to the peak with a retention time (t_R) 38 min. This fraction was recrystallized in dichloromethane to give 2 (30 mg) as colorless needles.

Fr-6 (2.0 g) was also further separated by Sephadex LH-20 column (bed 1.5 × 135 cm in DM 1:1) chromatography using DM (1:1) as eluents to give seven fractions, Fr-6-1 to Fr-6-7, in the order of elution. Fr-6-4 (490 mg) was separated by preparative TLC on silica gel GF254 plates (0.5 mm thickness, 20 × 20 mm) using cyclohexane-acetone-ammonia water (30:10:1) as the developing solvents. The silica gel on the band with the R_f value of 0.6 under 254 nm UV light was collected to afford 3 (8 mg) as colorless oil from MeOH solution.

Fr-8 (2.5 g) gave yellow crude needles at room temperature in MeOH. The crystals were filtered and repeatedly recrystallized from MeOH to obtain 4 (40 mg) as lemon-yellow needles.

Janthinone (1): yellowish needles (CH₂Cl₂-MeOH), mp 197–198 °C. Positive ESI-MS m/z: 285 [M + H]⁺, 307 [M + Na]⁺. ¹H NMR (400 MHz, CDCl₃) δ: 12.06 (1H, s, 8-OH), 7.65 (1H, dd, J = 8.5, 7.3 Hz, H-3), 7.43 (1H, dd, J = 8.5, 1.0 Hz, H-4), 7.22 (1H, dd, J = 7.3, 1.0 Hz, H-2), 6.65 (1H, br s, H-5), 6.53 (1H, br s, H-7), 3.95 (3H, s, H₃-11), 2.34 (3H, s, H₃-12). ¹³C NMR (100 MHz, CDCl₃) δ: 180.4 (C-9), 169.6 (C-10), 161.4 (C-8), 155.9 (C-4a), 155.6 (C-4b), 149.3 (C-6), 134.7 (C-3), 133.5 (C-1), 122.4 (C-2), 119.4 (C-4), 117.5 (C-9a), 111.6 (C-7), 107.3 (C-5), 106.9 (C-8a), 53.1 (C-11), 22.6 (C-12). The above data for 1 are identical with those reported for janthinone [51].

Fructigenine A (2): colorless needles (CH₂Cl₂), mp 102–105 °C, [α]²⁰_D −175.4 (c 0.24, CHCl₃). Positive ESI-MS m/z: 444 [M + H]⁺; negative ESI-MS m/z: 442 [M − H]⁻. ¹H NMR (400 MHz, CDCl₃) δ: 7.94 (1H, br s, H-7), 7.30–7.11 (7H, m, H-8,10,14,15,16,17,18), 7.06 (1H, td, J = 7.6, 1.1 Hz, H-9), 5.98 (1H, br s, H-2), 5.70 (1H, dd, J = 17.6, 10.8 Hz, H-20), 5.57(1H, br s, H-5a), 5.06 (1H, d, J = 10.8 Hz, Hcis-21), 5.04 (1H, d, J = 17.6 Hz, Htrans-21), 4.16 (1H, dd, J = 10.4, 2.8 Hz, H-3), 3.73 (1H, dd, J = 11.7, 5.5 Hz, H-11a), 3.48 (1H, dd, J = 14.4, 2.8 Hz, Ha-12), 2.74 (1H, dd, J = 14.4, 10.4 Hz, Hb-12), 2.59 (3H, s, H₃-23), 2.50 (1H, dd, J = 11.7, 5.5 Hz, Ha-11), 2.20 (1H, t, J = 11.7 Hz, Hb-11), 1.06 (3H, s, H₃-19b), 0.91 (3H, s, H₃-19a). ¹³C NMR (100 MHz, CDCl₃) δ: 170.1 (C-22), 168.1 (C-4), 164.8 (C-1), 143.3 (C-7a), 143.0 (C-20) , 135.3 (C-13), 131.7 (C-10a), 129.4 (2C, C-15,17), 129.1 (3C, C-8,14,18), 127.7 (C-16), 124.6 (2C, C-7,10), 119.1 (C-9), 114.6 (C-21), 79.5 (C-5a), 60.9 (C-10b), 59.1 (C-11a), 56.0 (C-3), 40.4 (C-19), 37.0 (C-11), 36.0 (C-12), 23.6 (C-19b), 23.2 (C-19a), 22.4 (C-23). The above data for 2 are identical with those of fructigenine A [52].

Aspterric acid methyl ester (3): colorless oil (MeOH), [α]²⁰_D −59 (c 1.0, MeOH), molecular formula C₁₆H₂₄O₄. Positive ESI-MS m/z: 281 [M + H]⁺, 303 [M + Na]⁺. Positive HR-ESI-MS m/z: measured 281.1757 [M + H]⁺, calculated for C₁₆H₂₅O₄ [M + H] 281.1753. ¹H NMR (400 MHz, CD₃OD) δ: 4.31 (1H, d, J = 8.4 Hz, H-2), 3.71 (3H, s, H₃-16), 3.67 (1H, br d, J = 8.2 Hz, Ha-13, gave correlation peak with H-6 at δ2.31 in ¹H–¹H COSY), 3.34 (1H, dd, J = 8.2, 1.2 Hz, Hb-13, gave correlation peak with H-6 at δ2.31 in ¹H–¹H COSY), 2.37–2.45 (2H, m, Ha-4/Ha-5), 2.32 (1H, br d, J = 9.4 Hz, H-6, gave correlation peaks with Ha-13 at δ3.67 and Hb-13 at δ3.34 in ¹H-¹H COSY) 2.25–2.35 (1H, m, Ha-9), 2.16 (1H, dd, J = 13.2, 8.4 Hz, Ha-1), 2.01 (1H, d, J = 13.2 Hz, Hb-1), 1.96–2.07 (1H, m, Hb-9), 1.73 (3H, s, H₃-14), 1.71 (1H, td, J = 12.0, 3.9 Hz, Ha-8), 1.62–1.67 (1H, m, Hb-5), 1.60 (3H, s, H₃-15), 1.49 (1H, dd, J = 12.0, 7.5 Hz, Hb-8), 1.42 (1H, br t, J = 12.5 Hz, Hb-4). ¹³C-NMR (100 MHz, CD₃OD) δ: 176.2 (C-12), 136.6 (C-10), 125.2 (C-11), 84.9 (C-2), 79.8 (C-3), 76.5 (C-13), 56.7 (C-6), 54.3 (C-7), 52.4 (C-16), 36.6 (C-1), 35.2 (C-4), 35.0 (C-8), 33.1 (C-9), 24.7 (C-5), 23.4 (C-15), 21.0 (C-14). The above NMR data for 3 assigned by the analysis of DEPT, ¹H-¹H COSY, HMQC and HMBC spectra closely resembled those of aspterric acid [53], except for additional signals due to a methoxy group (δ_H3.72 and δ_C52.4, H₃-16 and C-16), leading to assignment of 3 as aspterric acid methyl ester.

Citrinin (4): lemon yellow needles (MeOH), mp 178–179 °C, [α]²⁰_D −17.3 (c 1.0, EtOH). Positive ESI-MS m/z: 251 [M + H]⁺, 273 [M + Na]⁺, 289 [M + K]⁺. ¹H NMR (400 MHz, CDCl₃) δ: 15.91 (1H, s, 12-OH), 15.13 (1H, s, 8-OH), 8.25 (1H, s, H-1), 4.79 (1H, q, J = 6.8 Hz, H-3), 3.00 (1H, q, J = 7.0 Hz, H-4), 2.02 (3H, s, H₃-11), 1.35 (3H, d, J = 6.8 Hz, H₃-9), 1.23 (3H, d, J = 7.0 Hz, H₃-10). ¹³C NMR (100 MHz, CDCl₃) δ: 183.7 (C-6), 177.1 (C-8), 174.5 (C-12), 162.9 (C-1), 139.1 (C-4a), 123.0 (C-5), 107.3 (C-8a), 100.2 (C-7), 81.6 (C-3), 34.5 (C-4), 18.5 (C-11), 18.2 (C-9), 9.4 (C-10). The above data are identical with those reported for citrinin [54,55].