New Sesquiterpenoids From Plant-Associated Irpex lacteus.

Bacteria produce a large number of virulence factors through the quorum sensing (QS) mechanism. Inhibiting such QS system of the pathogens without disturbing their growth is a potential strategy to control multi-drug-resistant pathogens. To accomplish this, two new tremulane-type sesquiterpenoids, irpexolaceus H (1) and I (2), along with two known furan compounds, irpexlacte B (3) and C (4), were isolated from Orychophragmus violaceus (L.) OE Schulz endophytic fungus Irpex lacteus (Fr.) Fr. Their structures were elucidated by detailed spectroscopic data (NMR, HRESIMS, IR, and UV), single-crystal X-ray diffraction, and electronic circular dichroism (ECD) analysis. Furthermore, those compounds were evaluated for anti-quorum sensing (anti-QS) activity, and compound 3 was found contributing to the potential QS inhibitory activity.

Introduction

Bacterial quorum sensing (QS) is a cell-density-dependent communication process by which cells measure population density and trigger appropriate responses and conduct behavioral regulation, such as luminescence, motility, secretion of virulence factors, and formation of biofilms (Papenfort and Bassler, 2016). QS inhibitor (QSI) inhibits the QS system without affecting bacteria’s growth and reduces its virulence production and biofilm formation; thus, the bacteria are in a low or non-toxicity state, the growth is not inhibited, and therefore it is difficult to cause drug resistance (Jiang and Li, 2013). However, the purpose of traditional antibacterial agents is to kill or inhibit the growth of bacteria, and it is difficult to avoid bacterial resistance (Kalia, 2013). Therefore, finding new QSIs to replace traditional antibacterial agents has become a new strategy in the antibacterial field.

Irpex lacteus (Fr.) Fr. (Phanerochaetaceae) is a basidiomycete that usually colonizes on the deadwood white (i.e., white rot), and it is often used as a traditional Chinese medicine for the treatment of chronic glomerulonephritis. In our previous study on this subject, seven sesquiterpenoids, irpexolaceus A–G, and two new furan derivatives, irpexonjust A–B, were isolated from I. lacteus OV38 (Luo et al., 2022). Furthermore, two new tremulane-type sesquiterpenoids, irpexolaceus H (1) and I (2), were isolated in this study from fungi, as well as two furan compounds, such as irpexlacte B (3) and C (4), were also obtained (Figure 1). These compounds were screened for QS inhibitory activity, and compound 3 exhibited the highest QS inhibitory activity among them. The details of the isolation, structure assignment, and QS inhibitory activities of 1–4 are presented.

FIGURE 1

Chemical structures of 1–4.

Materials and Methods

General Experimental Procedures

Nuclear magnetic resonance (NMR) was obtained on a Bruker AV-400 spectrometer (Bruker Corporation, Karlsruhe, Germany). HRMSESI data were recorded on a Q-Exactive Orbitrap MS system (Thermo Fisher Scientific, Bremen, Germany). UV data were obtained on an Evolution 220 UV-vis spectrophotometer (Thermo Fisher Scientific, Madison, United States). Infrared spectroscopy (IR) spectra were obtained on a Nicolet™ iS10 FTIR spectrometer (Thermo Fisher Scientific, Madison, United States). Optical rotations were recorded on an Autopol VI automatic polarimeter (PerkinElmer, Waltham, MA, United States). The silica gel (100–200 and 200–300 mesh, Qingdao Marine Chemical Factory, Qingdao, China) and Sephadex LH-20 column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) were used for open column chromatography (CC). Fractions were monitored by TLC (HSGF 254, Yantai Jiangyou Silica Gel Development Co., Yantai, China), and spots were visualized by heating silica gel plates after soaking in methanol supplemented with 10% H2SO4. The preparative HPLC was performed with UltiMate 300 HPLC (Thermo Fisher Scientific, Madison, United States) equipped with a YMC-Pack ODS-A column (250 × 10 mmI.D, S-5 μm, 12 nm, and flow speed = 2–3 ml/min, YMC Co., Ltd., Kyoto, Japan).

Fungal Material and Fermentation Conditions

The strain Irpex lacteus was isolated from the healthy flowers and stems of Orychophragmus violaceus (L.) OE Schulz collected from Nanjing University of Science and Technology (NJUST) (Luo et al., 2021). The fungus I. lacteus was fermented in 250-ml Erlenmeyer flasks containing 100 ml Fungus No. 2 medium (2% sorbitol, 2% maltose, 1% glutamine, 1% glucose, 0.3% yeast extract, 0.05% tryptophan, 0.05% KH2PO4, and 0.03% MgSO4, pH 6.4) at 28°C and 140 rpm for 15 d (Zhou et al., 2017).

Extraction and Purification of Secondary Metabolites

The supernatant (total 100 L) of I. lacteus fermentation was extracted three times by equal ethyl acetate (EtOAc) and concentrated under reduced pressure to give a crude extract (99.49 g), which was subjected to column chromatography (CC) over a silica gel (100–200 mesh) eluted with a gradient of dichloromethane/methyl alcohol (CH2Cl2/MeOH, 100:0-0:100) to obtain a fraction G (Fr. G) and other 13 fractions (Fr. A–F and H–M). Fr. A (19.61 g) was purified by CC over a silica gel (200–300 mesh) eluted with the gradient systems of petroleum ether/EtOAc (20:1–1:1) and EtOAc/MeOH (100:1–10:1) to yield six sub-fractions Fr. A1–A6. Fr. A6 (4.79 g) was subjected to CC over a silica gel eluted with a gradient system of CH2Cl2/MeOH (80:1–0:100) to yield five sub-fractions, that is, Fr. A6.1–A6.5. Fr. A6.1 (97.4 mg) was further purified by HPLC to give 3 (40.1 mg, 45% MeOH, flow speed = 2.5 ml/min, and t = 31.0 min) and 4 (5.0 mg, 45% MeOH, flow speed = 2.5 ml/min, and t = min). Fr. G (37.30 g) was continually separated by CC over a silica gel (200–300 mesh) eluted with a gradient system of CH2Cl2/MeOH (100:0–0:100) to give nine sub-fractions Fr. G1–G9. Fr. G2 (7.76 g) was repeatedly separated using CC over a silica gel, Sephadex LH-20 (100% MeOH), and finally purified by HPLC to give 1 (30.3 mg, 50% MeOH, flow speed = 3.0 ml/min, and t R = 14.1 min) and 2 (4.90 mg, 50% MeOH, flow speed = 3.0 ml/min, and t R = 25.6 min).

Irpexolaceus H (1). white amorphous powder; [α]18 D + 17.2 (c 0.19, MeOH); UV (MeOH) λmax (log ε) 203 (3.99) nm and 290 (2.65) nm; IR (KBr): υmax 3380.60, 2932.72, 2872.93, 1752.01, and 1027.87 cm−1; 1H NMR (400 MHz in MeOD) and 13C NMR (100 MHz in MeOD) data are shown in Table 1; and HRESIMS m/z 311.1502 [M + COOH]- (calcd for C16H23O6, 311.1500).

TABLE 1

1H (400 MHz) and 13C (100 MHz) NMR spectroscopic data of 1 and 2 in CD3OD.

No	1		2
	δ C, type	δ H (J in Hz)	δ C, type	δ H (J in Hz)
1	126.8, C	–	148.0, C	–
2	139.9, C	–	134.1, C	–
3	38.7, CH	3.62, br d (14.4)	42.1, CH	3.31, m
4	30.9, CH2	1.99, m and 1.84, m	40.2, CH2	2.27, dd (14.0 and 7.3) and 2.11, br d (15.6)
5	73.3, CH	3.96, t (5.0)	181.5, C	–
6	39.7, CH	1.87, m	143.6, CH	5.81, m
7	40.8, CH	3.39, m	46.3, CH	3.43, m
8	40.4, CH2	1.72, m; 1.40, dd (12.9 and 11.0)	43.8, CH2	1.93, ddd (13.2, 8.7, and 1.4) and 1.31, dd (13.2 and 7.4)
9	44.9, C	–	44.0, C	–
10	42.1, CH2	2.17, d (16.2) and 1.81, m	41.6, CH2	2.57, br d (15.6) and 2.35, dd (14.0 and 7.3)
11	70.8, CH2	4.82, d (13.0) and 4.73, d (13.0)	60.2, CH2	4.10, d (11.6) and 4.00, d (11.6)
12	182.3, C	–	66.4, CH2	3.60, dd (10.8 and 6.6); 3.50, m
13	11.8, CH3	0.90, d (7.0)	114.0, CH2	5.05, dt (17.1 and 1.48) and 4.94, br d (10.2)
14	23.9, CH3	1.11, s	24.5, CH3	1.07, s
15	68.6, CH2	3.21, dd (12.2 and 11.4)	69.5, CH2	3.26, d (11.0) and 3.21, d (11.0)

Irpexolaceus I (2). Yellow oil; [α]18 D + 21.6 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 205 (3.57) nm; CD (MeOH) 195 (Δε—4.72) and 215 (Δε + 8.61) nm; IR (KBr): υmax 3359.87, 1564.95, 1397.66, and 1020.64 cm−1; 1H NMR (400 MHz in MeOD) and 13C NMR (100 MHz in MeOD) data are shown in Table 1; and HRESIMS m/z, 283.1551 [M-H]- (calcd for C15H23O5 and 283.1551).

Crystallographic Data of Compound 1

Crystal data for Cu _0m. C15H22O4, M = 266.33, a = 6.6684 (4) Å, b = 12.1018 (5) Å, c = 8.4219 (4) Å, α = 90°, β = 93.164 (4)°, γ = 90°, V = 678.61 (6) Å3, Z = 2, T = 170.0 K, space group P212121, μ(Cu Kα) = 0.760 mm−1, Dcalc = 1.303 g/cm3, 10,065 reflections measured (10.520° ≤ 2θ ≤ 127.318°), and 2210 unique (R int = 0.0504, R sigma = 0.0357) which were used in all calculations. The final R 1 was 0.0321 [I > 2σ(I)]. The final wR 2 was 0.0752 [I > 2σ(I)]. The final R 1 was 0.0346 (all data). The final wR 2 was 0.0772 (all data). The goodness of fit on F 2 was 1.091. Flack parameter = −0.02 (12), which was determined using 1101 quotients [(I+)−(I−)]/[(I+)+(I−)]. CCDC: 2133065 (www.ccdc.cam.ac.uk).

Electronic Circular Dichroism Calculation of Compound 2

The experiment was performed as previously described (Luo et al., 2022). The conformers with Boltzmann population (over 1%) were initially optimized at B3LYP/6–31+G. The ECD spectra of all conformers were conducted by time-dependent density functional theory (TD-DFT) methodology at ωB97X/def2-TZVP, CAM-B3LYP/TZVP, and M062X/def2-TZVP using IEFPCM in MeOH (Neuhaus and Loesgen, 2020). ECD spectra were generated using the program SpecDis 1.71 from dipole-length rotational strength by applying Gaussian band shapes with sigma = 0.2 eV (Bruhn et al., 2013).

Anti-Quorum Sensing Activity

The efficacy of purified compounds on inhibiting QS mechanisms was screened using indicator organisms Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136. Overnight grown C. violaceum CV026 culture (1 ml) (OD600 ≈ 0.1) was added into 100 ml LB agar medium supplemented with kanamycin (20 μg/ml) and C6-HSL (5 μM), mixed, and poured into the plates (Kumar et al., 2021). After the medium was solidified, 2 μl of samples (1–4) (50 mg/ml) were dropped onto the plates and then incubated at 28°C for 24 h, recording the violacein changes in color. The absence of violacein production in CV026 represents compounds that inhibit the QS system. Furthermore, QS inhibitory activity-screened A. tumefaciens A136 was determined as described earlier (Zhu et al., 2018). X-gal (50 μg/ml) and C10-HSL (5 μM) were added into the LB agar medium supplemented with A. tumefaciens A136. Each experiment was repeated three times.

Results and Discussion

Structure Elucidation

Irpexolaceus H (1), a white amorphous powder, was assigned the molecular formula of C15H24O4 with five degrees of unsaturation based on HRESIMS data at m/z 311.1502 [M + COOH]- (calcd for C16H23O6, 311.1500). The 1H and 13C NMR spectra of 1 (Table 1) revealed 15 carbon resonances, including two methyls at δ C 11.8 and 23.9, three methylene carbons at δ C 30.9, 40.4, and 42.1, two oxygenated methylene carbons at δ C 70.8 and 68.6, three methine carbons at δ C 38.7, 39.7, and 40.8, one oxygenated methine carbon at δ C 73.3, three sp2 quaternary carbons at δ C 126.8, 139.9, and 182.3, and one sp3 quaternary carbon at δ C 44.9. These data demonstrated high similarity to irpexolaceus F (Luo et al., 2022), with the main difference being that the low field at δ C 139.9 and 126.8 (1) shifted downfield to δ C 165.9 and 128.5 in irpexolaceus F, indicating differences in the position of the carbon–carbon double bonds, which was confirmed by the HMBC correlations from H2-8 (δ H 1.72, 1.40) and H2-10 (δ H 2.17, 1.81) to C-1 (δ C 126.8) and from H2-4 (δ H 1.99, 1.84), H2-10 (δ H 2.17, 1.81), and H2-11 (δ H 4.82, 4.73) to C-2 (δ C 139.9), in combination with the 1H–1H COSY correlations of H-3 (δ H 3.62)/H2-4 (δ H 1.99, 1.84)/H-5 (δ H 3.96)/H-6 (δ H 1.87)/H-7 (δ H 3.39)/H2-8 (δ H 1.72, 1.40) (Figure 2). The mentioned data combined with previously reported data (Luo et al., 2022) determined the planar structure of 1. In addition, the relative configuration of 1 was determined by the NOESY correlations of H-3 (δ H 3.62)/H-6 (δ H 1.87)/H-7 (δ H 3.39) and Me-13 (δ H 0.90)/H-5 (δ H 3.96)/Me-14 (δ H 1.11) (Figure 2). Combined with the single-crystal X-ray diffraction analysis (Figure 3), the absolute configuration of 1 was confirmed as 3S,5S,6S,7R,9S.

FIGURE 2

Key 1H–1H COSY (), HMBC (), and NOESY () correlations of 1 and 2.

FIGURE 3

Perspective ORTEP drawing for 1.

Irpexolaceus I (2), a yellow oil, was assigned the molecular formula of C15H24O5 with four degrees of unsaturation based on HRESIMS data at m/z 283.1551 [M-H]- (calcd for C15H23O5, 283.1551). The 1H and 13C NMR spectra of 2 (Table 1) revealed 15 carbon resonances, including one methyl at δ C 24.5, four methylene carbons at δ C 40.2, 43.8, 41.6, and 114.0, three oxygenated methylene carbons at δ C 60.2, 66.4, and 69.5, three methine carbons at δ C 42.1, 143.6, and 46.3, three sp2 quaternary carbons at δ C 148.0, 134.1, and 181.5, and one sp3 quaternary carbon at δ C 44.0. There were two C=C and one carbonyl group, and the remaining one unsaturation was a ring, which was confirmed by the 1H-1H COSY correlations of H2-8 (δ H 1.93, 1.31)/H-7 (δ H 3.43)/H-6 (δ H 5.81)/H2-13 (δ H 5.05, 4.94), and the HMBC correlations from H-7 (δ H 3.43) to C-1 (δ C 148.0) and C-10 (δ C 41.6), from H2-8 (δ H 1.93, 1.31) to C-1 (δ C 148.0), C-7 (δ C 46.3), C-9 (δ C 44.0), and C-10 (δ C 41.6), and from H2-10 (δ H 2.57, 2.35) to C-1 (δ C 148.0), C-7 (δ C 46.3), C-8 (δ C 43.8), and C-9 (δ C 44.0). Moreover, a high-field quaternary carbon, C-9 (δ C 44.0), was linked to C-14 (δ C 24.5) and C-15 (δ C 69.5). Combined with another set of 1H–1H COSY correlations of H-3 (δ H 3.31)/H2-4 (δ H 2.27, 2.11)/H2-12 (δ H 3.60, 3.50), the planar structure of 2 was determined as shown in Figure 1, which was highly similar to ceriponol P (Ying et al., 2014), with a difference of a hydroxyl group binding to δ C 69.5. In addition, the relative configuration of 2 was confirmed by the NOESY correlations of H-6 (δ H 5.81)/H-3 (δ H 3.31)/Me-14 (δ H 1.07) (Figure 2). The calculated and experimental ECD spectra of 2 showed excellent fit (Figure 4), indicating that the absolute configuration of 2 was 2S,7S,9R.

FIGURE 4

Experimental and calculated ECD spectra of 2.

In addition to the aforementioned compounds, two furans (3 and 4) were obtained and identified as irpexlacte B and C according to the previously reported data (Luo et al., 2022).

Proposed Biotransformation Pathway

Tremulane (i) and (ii) were biosynthesized based on the relative structures of 1 and 2 by the cyclization and rearrangement of farnesyl pyrophosphate (FPP) (Ayer and Browne, 1981; Ayer and Cruz, 1993). After a multi-step reaction such as esterification, cyclization, and dehydration in microbes, tremulane (i) was possibly converted to irpexlaceus H (Figure 5). Moreover, lactarane skeletons ii-1 and ii-2 were produced by a series of methyl migrations of tremulane (ii)) (Ayer and Cruz, 1993), which differed from tremulane (i)) in the configuration of C-1 and C-7. Lactarane skeleton ii-2 was transformed to 5,6-secotremulane under the 5,6-cleavage (He et al., 2020) and then possibly converted to irpexlaceus I by a series of oxidation and dehydration (Figure 5).

FIGURE 5

Proposed biosynthetic pathway for 1 and 2.

Biological Activity

Compounds 1–4 were evaluated for their QS inhibitory activities at 50 mg/ml against biomarker strains C. violaceum CV026 (Figure 6A) and A. tumefaciens A136 (Figure 6B). We assessed the inhibitory effect of the compound by assaying the inhibition of C6-HSL-induced violacein production by C. violaceum CV026, and C10-HSL-induced β-galactosidase expression (blue pigment) by A. tumefaciens A136. Compounds 1–2 showed no inhibitory activities against both QS systems of biomarker strains. However, compound 3, in which the binding position of hydroxyl was closer to the furan ring than 4, exhibited stronger inhibition activity against the production of violacein in C. violaceum CV026 than that of the latter but weaker inhibitory activity against the production of blue pigment in A. tumefaciens A136. The results demonstrated that the binding position of hydroxyl was vital for QS inhibitory activity.

FIGURE 6

Screening of QS inhibitory activity by C. violaceum CV026 (A) and A. tumefaciens A136 (B). Compounds 1 (A), 2 (B), 3 (C), and 4 (D).

Conclusion

Two new tremulane-type sesquiterpenoids, irpexolaceus H (1) and I (2), were isolated from the liquid fermentation of I. lacteus. Their structures were established based on NMR, HRESIMS, IR, single-crystal X-ray diffraction, and ECD analysis. These compounds (1–4) were evaluated for QS inhibitory activities against C. violaceum CV026 and A. tumefaciens A136 at 50 mg/ml. The results found that compound 3 exhibited a significant QS inhibitory activity against C. violaceum CV026, and compound 4 showed a weaker activity. In addition, compound 3 also showed a weak QS inhibitory activity against A. tumefaciens A136. But interestingly, the hydroxyl binding to α-C in the furan ring showed a stronger QS inhibitory activity than that of 4 (hydroxyl binding to β-C in the furan ring), which suggested that the position of hydroxyl in the furan ring was possibly vital for QS inhibitory activity against C. violaceum CV026.