Amiaspochalasins I and J, Undescribed Aspochalasins with Antibacterial Activities From Aspergillus micronesiensis

ABSTRACT

Two undescribed aspochalasins named amiaspochalasins I and J (1 and 2), along with a known analogue named aspochalasin U (3) were isolated from the endophytic fungus Aspergillus micronesiensis. Their structures including absolute configurations were elucidated by extensive NMR data, molecular modelling studies, and electronic circular dichroism (ECD) calculations. Usually, there is a double bond at Δ ⁶ in the core structure of aspochalasins, while in compounds 1−3, this double bond has shifted to Δ ⁵. Compound 1 is the first aspochalasin bearing a carbonyl at C‐7, and compound 2 has an additional hydroxyl at C‐17 in comparison of aspochalasin U (3). Compounds 1−3 showed moderate inhibitory effects on Staphylococcus aureus, Escherichia coli, and Salmonella enterica, and the MIC values of 1−3 against Staphylococcus aureus were 32, 64, and 64 µg mL⁻¹, respectively.

Three aspochalasins with an unusual double bond at Δ ⁵ (1−3) were isolated from the solid culture of Aspergillus micronesiensis, among which two were undescribed compounds (1 and 2). Their structures including absolute configurations were elucidated by extensive NMR data, molecular modelling studies, and electronic circular dichroism (ECD) calculations.

1. Introduction

Cytochalasans comprise a diverse group of polyketide–amino acid hybrid metabolites, and can be divided into five classes according to the different types of amino acids involving in the skeleton [1]. Aspochalasin, which involves a leucine in the biosynthetic pathway is one of the largest class of cytochalasans [2]. So far, more than 90 aspochalasins have been reported [3]. In our previous study, eight aspochalasins with a C‐21 ester carbonyl, named amiaspochalasins A−H were discovered from Aspergillus micronesiensis [4], and in the current study, another three aspochalasins (1−3) have been isolated and identified (Figure 1), among which two were new compounds and were named as amiaspochalasins I and J (1 and 2). Notably, a majority of the reported aspochalasins featuring a double bond at Δ ⁶ in their core structures [3], while in compounds 1−3, this double bond has shifted to Δ ⁵. Compound 1 is the first aspochalasin bearing a carbonyl at C‐7, and compound 2 has an additional hydroxyl at C‐17 in comparison of aspochalasin U (3). Although the planar structure of 2 is the same as that of aspochalasin L [5], whose stereochemistry remains unassigned, the different NMR data suggest that they may have different absolute configurations.

FIGURE 1.

Chemical structures of 1−3.

The absolute configurations of 1 and 2 were determined by careful 2D NMR data analyses, molecular modelling studies, ECD calculations, and a comparison of their experimental ECD spectra with that of aspochalasin U (3), an analogue whose absolute configuration had been confirmed by x‐ray single crystal diffraction [6]. The cytotoxic activities of 1−3 against five human cancer cell lines (HL60, SW480, A549, MCF‐7, and Hep3B) were evaluated, however, none of them significantly affected cell viability. The antimicrobial effects were also evaluated using four types of bacteria (Escherichia coli, Staphylococcus aureus, Salmonella enterica, and Pseudomonas aeruginosa) and a fungal strain (Candida albicans), and 1−3 showed moderate inhibitory activities against S. aureus, E. coli and S. enterica, the MIC values against S. aureus were 32, 64, and 64 µg mL⁻¹, respectively.

Herein, we report the isolation, structural elucidation, and bioactivities of the isolated compounds.

2. Results and Discussion

Compound 1 was isolated as white amorphous powder, and its molecular formula was determined to be C₂₅H₃₇NO₆ based on ¹³C NMR data and the HRESIMS ion peak at m/z 470.2517 [M + Na]⁺ (calcd for C₂₅H₃₇NO₆Na, 470.2519) (Figure S1), suggesting eight degrees of unsaturation. The ¹H NMR spectrum showed signals for one olefinic proton [δ _H 6.03 (1H, d, J = 10.4 Hz, H‐13)], five methyls [δ _H 1.95 (3H, s), 1.74 (3H, s), 1.41 (3H, s), 0.95 (3H, d, J = 6.6 Hz), and 0.92 (3H, d, J = 6.6 Hz)], and one methoxy group [δ _H 3.43 (3H, s)] (Figure S4). The ¹³C NMR and DEPT spectra displayed a total of 25 carbon resonances assigned to two carbonyls (δ _C 208.2 and 195.9), one amide carbonyl (δ _C 175.0), four olefinic carbons (δ _C 147.9, 140.2, 133.5, and 119.5), one quaternary carbon (δ _C 62.6), seven methines (δ _C 78.0, 77.4, 72.8, 54.9, 52.9, 48.7 and 25.2), four methylenes (δ _C 45.6, 42.9, 37.8 and 29.9), and six methyls (δ _C 57.5, 23.1, 22.0, 19.5, 15.8 and 12.1) (Figures S5−S7). These ¹H and ¹³C NMR data (Table 1) suggested that 1 was possibly an aspochalasin with a 5/6/11 ring system. The HMBC correlations from H₃‐11 to C‐4, C‐5, and C‐6, from H₃‐12 to C‐5, C‐6, and C‐7, from H₃‐25 to C‐13, C‐14, and C‐15, and from H‐8 to C‐1, C‐4, C‐7, C‐9, C‐13, and C‐21 established the gross framework, in which there were two carbon‐carbon double bonds at Δ ^5,13 (Figure S8). The ¹H−¹H COSY cross‐peaks revealed fragments of CH₂(15)−CH₂(16)−OCH(17), OCH(18)−OCH(19)−CH₂(20) (Figure S9), which together with the HMBC correlations from H‐17 to C‐15, from H‐19 to C‐21, from H‐20 to C‐18, C‐19, and C‐21, and from the methoxyl [δ _H 3.43 (3H, s)] to C‐19 (Figure 2), completed the assignment of the 11‐membered macrocycle. Thus, the planar structure of 1 was established.

TABLE 1.

¹H and ¹³C NMR data of compounds 1, 2, and aspochalasin L.

no.	1 a			2 b			aspochalasin L c
	δ H (J, Hz)	δ C		δ H (J, Hz)	δ C		δ H (J, Hz)	δ C
1		175.0	C		174.4	C		174.3	C
3	3.45 m	54.9	CH	3.14 m	54.1	CH	3.15 m	53.5	CH
4	3.26 m	52.9	CH	2.80 s	51.4	CH	2.81 brs	50.8	CH
5		147.9	C		125.7	C		125.4	C
6		133.5	C		134.1	C		133.9	C
7		195.9	C	3.75 brt (10.6)	68.6	OCH	3.75 brm	67.9	OCH
8	3.62 m	48.7	CH	2.44 t (10.6)	47.1	CH	2.45 t (10.5)	46.5	CH
9		62.6	C		61.7	C		61.5	C
10a	1.26 m	45.6	CH2	1.05 m	45.3	CH2	1.07 m	44.9	CH2
10b	1.22 m			1.05 m			1.07 m
11	1.95 s	19.5	CH3	1.64 s	17.3	CH3	1.65 s	16.7	CH3
12	1.74 s	12.1	CH3	1.61 s	14.7	CH3	1.61 s	14.0	CH3
13	6.03 d (10.4)	119.5	CH	5.79 d (10.6)	124.1	CH	5.80 d (10.5)	123.4	CH
14		140.2	C		137.1	C		136.9	C
15a	2.16 m	37.8	CH2	1.95 m	38.4	CH2	1.96 m	37.6	CH2
15b	2.12 m			1.95 m			1.96 m
16a	1.59 m	29.9	CH2	1.47 m	29.5	CH2	1.48 m	28.8	CH2
16b	1.59 m			1.28 m			1.30 m
17	3.74 m	72.8	OCH	3.48 brm	70.6	OCH	3.49 brd (3.6)	69.9	OCH
18	3.74 m	77.4	OCH	3.36 m	77.9	OCH	3.36 brd (9.0)	77.2	OCH
19	3.13 m	78.0	OCH	2.90 m	77.7	OCH	2.92 brd (9.0)	77.0	OCH
20a	3.66 m	42.9	CH2	3.68 dd (18.9, 1.2)	43.3	CH2	3.69 brd (19.0)	42.6	CH2
20b	2.19 m			1.92 m			1.96 m
21		208.2	C		209.5	C		209.3	C
22	1.65 m	25.2	CH	1.56 m	24.4	CH	1.57 m	23.6	CH
23	0.95 d (6.6)	23.1	CH3	0.85 d (6.6)	23.0	CH3	0.86 d (6.6)	22.3	CH3
24	0.92 d (6.6)	22.0	CH3	0.82 d (6.6)	22.0	CH3	0.83 d (6.6)	21.2	CH3
25	1.41 s	15.8	CH3	1.33 s	15.7	CH3	1.33 s	15.0	CH3
1′	3.43 s	57.5	OCH3	3.31 s	57.3	OCH3	3.32 s	56.6	OCH3

^a1H (400 MHz) and ¹³C (100 MHz) in CDCl₃.

^b1H (400 MHz) and ¹³C (100 MHz) in DMSO‐d ₆.

^c1H (500 MHz) and ¹³C (125 MHz) in DMSO‐d ₆.

FIGURE 2.

¹H−¹H COSY and key HMBC correlations of 1 and 2.

The relative configuration of 1 was determined by its ¹H NMR data, ¹H−¹H COSY cross‐peaks, NOESY spectrum and molecular modelling studies. The NOESY correlations of H‐3/H₃‐11, H‐4/H‐8, and H‐4/H₂‐10 were the same as those of aspochalasin U [6], whose configuration was confirmed by the x‐ray diffractions. In other words, the 5/6 ring junction and the macrocyclic ring were cis‐ and trans‐ stereochemistry, respectively, and H‐4 and H‐8 were β‐configurations. The NOESY correlations of H‐8/CH₃‐25, CH₃‐25/H‐15b, and H‐13/H‐15a established the E configuration for the C‐13(14) double bond (Figures S10 and S11). Moreover, the NOESY interaction of H‐19/CH₃‐25 suggested the β‐configuration of H‐19 (Figure 3).

FIGURE 3.

Key NOESY correlations of 1 and 2.

Although it was difficult to directly determine the configurations of H‐17 and H‐18 by the NOESY correlations since their ¹H NMR chromatographic peaks were overlapped, with the help of molecular modelling studies by Chem3D, as well as the ¹H−¹H COSY spectrum of 1, their configurations were elucidated. In the NOESY spectrum, obvious correlations between the protons at δ _H 3.74 (H‐17 and H‐18) with H‐13, H‐15a, H‐20a, and H‐20b were observed (Supporting Information), which indicated that at least one of the two protons (H‐17/H‐18) was α‐configuration. Thus, the configurations of H‐17/H‐18 should be 17α,18α, or 17α,18β, or 17β,18α as shown in Figure 4 (1a, 1b, and 1c). The absence of NOESY correlations of H‐17/H‐19, or H‐17/H3‐25 did not support 17β configuration (1c), and the NOESY correlations of H‐17/H‐20b, or H‐18/H‐20b should not be observed in 1b, in another word, only 1a (17α,18α) satisfied all the NOESY correlations mentioned above. Moreover, when the dihedral angle of two hydrogens is close to 90°, there will no ¹H−¹H COSY correlation between them. In consideration of 1b, the dihedral angle of H‐18 and H‐19 was close to 90°, however, the ¹H−¹H COSY correlation between H‐18 and H‐19 was obviously observed. Therefore, 1b and 1c were not supported, the relative configurations of H‐17 and H‐18 were finally elucidated as 17α,18α.

FIGURE 4.

Three possible stereoisomers of 1 (1a, 1b, and 1c), key NOESY correlations and distance measurement predicted in Chem3D.

Compound 1 showed similar ECD spectrum with that of aspochalasin U (3) (Figure 5), whose absolute configuration was confirmed by x‐ray diffraction analysis [6]. We also calculated ECD spectrum for compound 1 (Tables S1 and S2), as a result, the calculated ECD spectrum matched well with the experimental one (Figure 5), which further supported the absolute configuration of 1. Therefore, the absolute configuration of 1 was assigned as 3S,4R,8R,9R,17R,18R,19R. However, the confirmation of stereo chemistry at C‐17, C‐18, and C‐19 still need more evidence such as the X‐ray diffraction due to the limitation of current experiments.

FIGURE 5.

Experimental and calculated ECD spectra of 1−3.

Notably, in all natural aspochalasins with 5/6/11 ring systems reported so far, the absolute configurations for C‐3 and C‐4 are assigned as 3S,4R because of the diastereofacial selectivity of the c reaction during the biosynthesis [2, 3, 6, 7].

Compound 2 was obtained as white amorphous powder. Its molecular formula was established as C₂₅H₃₉NO₆ based on the HRESIMS ion peak at m/z 472.2688 [M + Na]⁺ (calcd for C₂₅H₃₉NO₆Na, 472.2675) (Figure S12), with two more protons than compound 1. The overall NMR data of 2 were similar to those of 1 (Figures S15−S20), however, the absence of a conjugated carbonyl and the presence of an additional oxygenated methine in the ¹³C NMR spectrum of 2 suggested that the C‐7 carbonyl in 1 had been converted to be an oxygenated methine in 2. This suggestion was further confirmed by the HBMC correlations from H₃‐12 to C‐5, C‐6, and C‐7 (δ _C 77.9), and the ¹H−¹H COSY cross‐peaks of H‐7/H‐8/H‐13 (Figure 2).

The NOESY correlations of H‐3/H₃‐11, H‐4/H‐8, and H‐4/H₂‐10 were the same as those of 1 (Figure S21), and H‐4 and H‐8 were β‐configurations. The big coupling constant between H‐7 and H‐8 (J = 10.6) implied their trans‐configuration, so H‐7 was α‐configuration. The NOESY correlations of H‐8/CH₃‐25 and H‐7/H‐13 established the E configuration for the C‐13(14) double bond (Figure 3). In addition, the NOESY interactions of H‐13/H‐17, H‐13/H‐20a, H‐17/H‐20a, and H‐19/CH₃‐25 suggested the α‐configuration of H‐17 and the β‐configuration of H‐19 (Figure S21). The NOESY correlations of H‐17/H‐18, H‐18/H‐20a, and H‐18/H‐20b indicated that H‐18 was α‐configuration, which was further supported by molecular modelling studies: as shown in Figure 4, if H‐18 in the 11‐membered macro‐ring was β‐configuration, the spatial distance of H‐18 and H‐20a/H‐20b would exceed 3Å, and no NOESY correlation should be observed between H‐18 and H‐20a/H‐20b. Moreover, in the ¹H−¹H COSY spectrum of compound 2, it was clearly that there was no ¹H−¹H COSY correlation between H‐17 and H‐18, which suggested that the dihedral angle of H‐17 and H‐18 was close to 90°, and further supported 18α configuration. Thus, the relative configuration of 2 was elucidated.

The absolute configuration of 2 was suggested to be 3S,4R,7S,8R,9R,17R,18R,19R on the basis of its experimental ECD spectrum, which was identical to that of aspochalasin U (3) (Figure 5). We also calculated ECD spectrum for compound 2 (Tables S3 and S4, Supporting Information), as a result, the calculated ECD spectrum matched well with the experimental one (Figure 5). However, more evidence such as the x‐ray diffraction is needed to confirm the stereo chemistry at C‐17, C‐18, and C‐19.

The planar structure of 2 is identical to that of aspochalasin L [5], whose stereochemistry remains unassigned. In literature, the configuration of H‐7 in aspochalasin L was determined to be β‐orientation since the observed ROESY correlation of H‐7 and H‐8. However, H‐7 and H‐8 are adjacent carbons, it is insufficient to determine their relative configurations based on the ROESY correlation. Moreover, we think that H‐7 should also be α configuration in aspochalasin L because of the large coupling constant between H‐7 and H‐8 (J = 10.5). In conclusion, we think that the configuration of H‐7 in compound 2 and aspochalsin L are the same. However, the different ¹³C NMR data suggest that maybe there is some difference in the configurations of H‐17, H‐18 and H‐19 in compound 2 and aspochalsin L (Table 1).

Compounds 1−3 were initially evaluated for their cytotoxic activities against five human cancer cell lines (HL60, SW480, A549, MCF‐7 and Hep3B), and none of them significantly affected cell viability when the concentration was up to 40 µM. Then, 1−3 were evaluated for their antimicrobial effects against four types of bacteria (Escherichia coli, Staphylococcus aureus, Salmonella enterica and Pseudomonas aeruginosa) and a fungal strain (Candida albicans). As a result, 1−3 showed moderate activity against S. aureus with MIC values of 32, 64, and 64 µg mL⁻¹, respectively. Moreover, compound 2 also showed moderate inhibitory effect on E. coli and S. enterica (Table 2).

TABLE 2.

Antimicrobial activities of 1−3 (MIC, µg mL⁻¹).

	E. coli	S. aureus	S. enterica	P. aeruginosa	C. albicans
1	128	32	>128	>128	>128
2	64	64	64	>128	>128
3	>128	64	>128	>128	>128

3. Conclusions

Even though more than 90 aspochalasins have been reported so far, only seven members featuring a characteristic double bond at Δ ⁵, namely aspochalasin L [5], aspochalasin U [6], aspochalasin W [8], trichalasin C [9], trichalasin E [10], trichoderone B [11], and periconiasin I [12], respectively. In the current study, another two new aspochalasins with an unusual double bond at Δ ⁵ (1 and 2) were isolated from the solid culture of A. micronesiensis, and their structures including absolute configurations were elucidated by NMR data, molecular modelling studies, and ECD spectra. Compound 1 is the first aspochalasin bearing a carbonyl at C‐7. Although the planar structure of 2 is the same as that of aspochalasin L, whose stereochemistry remains unassigned, the different NMR data suggest that they have different configurations, and the configuration 2 was elucidated with the help of Chem3D. We tested the antimicrobial activities of these aspochalasins for the first time, as a result, compounds 1−3 showed moderate inhibitory effects on S. aureus, E. coli and S. enterica, and the MIC values of 1−3 against S. aureus were 32, 64, and 64 µg mL⁻¹, respectively. Our findings enriched the chemical diversity and pharmacological activity of aspochalasin‐type cytochalasans.

4. Experimental Section

4.1. General Experimental Procedures

Optical rotations were determined in MeOH on a Perkin‐Elmer 341 polarimeter. UV spectra were obtained with a Varian Cary 50 spectrometer. ECD spectra were obtained with a JASCO J‐810 spectrometer. IR spectra were acquired on a Bruker Vertex 70 FT‐IR instrument (Bruker, Karlsruhe, Germany). NMR spectra were obtained on a Bruker AM‐400 spectrometer, and chemical shifts were referenced to the residual peaks for CDCl₃ (δ _H 7.26 and δ _C 77.16) and DMSO‐d ₆ (δ _H 2.50 and δ _C 39.52). High‐resolution electrospray ionization mass spectroscopy (HRESIMS) was conducted on a Thermo Fisher LC‐LTQ‐Orbitrap XL spectrometer. Semipreparative high‐performance liquid chromatography (HPLC) was performed using a Dionex Ultimate 3000 HPLC (Dionex, Sunnyvale, CA, USA) with UV detector and an Ultimate XB‐C₁₈ (10 × 250 mm, 5 µm) column. Silica gel (100−200 mesh and 200−300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), ODS (50 µm, YMC, Japan), and Sephadex LH‐20 (Pharmacia Biotech AB, Uppsala, Sweden) were used for column chromatography (CC). Thin‐layer chromatography (TLC) was performed with RP‐C₁₈ F₂₅₄ plates (Merck, Germany) and silica gel 60 F₂₅₄ (Yantai Chemical Industry Research Institute).

4.2. Fungal Material

The strain in our work was derived from the root of the traditional Chinese medicinal plant Phyllanthus glaucus, which was collected from LuShan Mountain, Jiangxi Province, China, and was identified by Prof. Jianping Wang of Huazhong University of Science and Technology. The sequence data of the strain have been submitted to the DDBJ/EMBL/GenBank database with the accession no.MH938722. A voucher sample (ZYH20150717) has been preserved in the culture collection center of Tongji Medical College, Huazhong University of Science and Technology.

4.3. Fermentation and Isolation

The fungal strain was cultured on potato dextrose agar (PDA) for 5 days to prepare the seed culture. Agar plugs were inoculated into 96 Erlenmeyer flasks (1 L), previously sterilized by autoclaving, each containing 200 g of rice and 200 mL of water. All flasks were incubated at 28°C for 21 days. The fermented rice substrate was extracted seven times in 95% aqueous EtOH at room temperature, and the solvent was evaporated under vacuum to afford a residue. The residue was suspended in H₂O and successively partitioned with EtOAc.

The EtOAc partition fraction (80.0 g) was subjected to normal phase silica gel CC (eluting with CH₂Cl₂−MeOH, 100:1−0:1) to obtain three fractions (Fr. A−C). Fraction B (14.0 g) was chromatographed on C₁₈ reversed phase (RP‐18) silica gel CC (gradient elution with MeOH−H₂O, 50:50−100:0) to give four subfractions (B1−B4). Fr. B2 (eluted with MeOH−H₂O, 70:30) was chromatographed on Sephadex LH‐20 (CH₂Cl₂−MeOH, 1:1) to give three subfractions. Fr. C4.2 was subjected to normal phase silica gel CC (CH₂Cl_2–MeOH, from 2:0 to 3:1), and then purified by semipreparative HPLC (MeCN–H₂O, 60:40, v = 2.0 mL/min, λ = 210 nm) to give compounds 1−3 (t _R = 16.1, 20.4 and 25.5 min, respectively).

Amiaspochalasin I (1): white amorphous powder; [α]²⁰ _D –56.0 (c 0.7, MeOH); ECD (MeOH) λ (Δε) 230 (+17.0), 294 (–7.4) nm; UV (MeOH) λ _max (log ε) 203 (4.30) nm, 245 (4.06) nm; IR v _max 3433, 2927, 1696, 1633, 1441, 1384, 1312, 1279, 1027 cm–¹; ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 470.2553 [M + Na]⁺ (calcd for C₂₅H₃₇NO₆Na, 470.2519).

Amiaspochalasin J (2): white amorphous powder; [α]²⁰ _D –16.0 (c 0.6, MeOH); ECD (MeOH) λ (Δε) 210 (+8.0), 294 (–1.5) nm; UV (MeOH) λ _max (log ε) 203 (4.13) nm. IR v _max 3428, 2959, 2929, 1689, 1632, 1446, 1384, 1273, 1130, 1102, 1068 cm–¹; ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 472.2688 [M + Na]⁺ (calcd for C₂₅H₃₉NO₆Na, 472.2675).

4.4. Cytotoxic Assay

The Human promyelocytic leukemia cell line HL60 (ATCC CCL‐240; RRID:CVCL_0002), human lung adenocarcinoma cell line A549 (ATCC CCL‐185; RRID:CVCL_0023), human hepatocellular carcinoma cell line Hep3B (ATCC HB‐8064; RRID:CVCL_0326), human breast adenocarcinoma cell line MCF‐7 (ATCC HTB‐22; RRID:CVCL_0031), and human colon adenocarcinoma cell line SW480 (ATCC HTB‐22; RRID:CVCL_0546) were purchased from American Type Culture Collection (VA, USA). HL60 was cultured in RPMI‐1640 (HyClone, UT, USA), and the other cell lines were cultured in DMEM (HyClone, UT, USA). Cell viability was performed using a CCK‐8 kit according to the manufacturer's instructions.

4.5. Antimicrobial Assay

Four types of bacteria: E. coli (ATCC25922), S. aureus (ATCC29213), S. enterica (ATCC14028), and Pseudomonas aeruginosa (ATCC27853) were purchased from China General Microbiological Culture Collection Center (CGMCC), and the fungus Candida albicans (ATCC10231) was purchased from MicroBioLogics (USA). The antimicrobial assay was performed using a modified broth micro‐dilution method as previously described [13]. Briefly, the phytopathogenic fungi were incubated on potato dextrose agar (PDA) at 28°C for a week. Inocula of fungi were prepared by grinding hyphae (fresh weight 0.6 mg) into 10 mL of potato dextrose (PD) medium, and then diluting with PD (1:500); the tested compounds were dissolved in the mixture of 4 µL DMSO and 96 µL PD to make a top dose of 512 µg mL⁻¹. Afterwards, four 2‐fold serial dilutions afforded five concentrations (256, 128, 64, 32, and 16 µg mL⁻¹) in a row. Finally, 100 µL fungal inocula were added to each well and the plates were incubated at 28°C for 48 h. Penicillin G, ceftazidime, and amphotericin B were used as positive controls. The solution of equal concentration of DMSO was used as negative control. 200 µL PD without compounds and fungi was used as blank control. Minimum inhibitory concentration (MIC) values were determined according to the Clinical and Laboratory Standards Institute (CLSI, 2021) guidelines.

Author Contribution

Y. Z., X. Z., and H. Z. conceived and designed the experiment. Z. W. was responsible for the isolation, structural elucidation, and initial drafting of the manuscript. W. S. and H. Z. conducted the biological activity tests. C. C. and Q. L. performed 2D NMR analyses, and Q. L. made significant contributions to the revision of the manuscript. All authors have given approval to the final version of the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File 1: cbdv71594‐sup‐0001‐SuppMat.docx

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.