1. Introduction
Natural products (NPs) have always been an indispensable source of new drugs [
1]. As an important source of NPs with novel structures and high-value biological activities, plant endophytic fungi always attracted broad attentions from natural product chemists and pharmacologists [
2,
3]. Xylariaceae is one of the largest, most commonly encountered, and highly diverse fungal families of the Ascomycota [
4]. The genus
Xylaria, belonging to the family Xylariaceae, is medicinal fungi commonly found in decaying plant tissues and is widely distributed in temperate, tropical, and subtropical regions[
5,
6]. So far, more than two hundred bioactive compounds (> 100 new ones) were isolated from
Xylaria, including cytochalasins, α-pyrones, cyclopeptides, terpenoids, lactones, and succinic acid derivatives [
7]. Besides, the secondary metabolites produced by species of
Xylaria were found to exert wide range of biological activities, such as anti-inflammatory, antifungal, antibacterial, anti-tumor, and
α-glucosidase inhibitory activities [
7,
8].
As part of our group’s ongoing effort to identify bioactive natural products from medicinal plants and endophytic fungi[
9,
10,
11], the fungus
Xylaria sp. Z184, isolated from the leaves of
Fallopia convolvulus (L.) Á. Löve for the first time, has attracted our attention for its impressive compound abundance in TLC and HPLC analyses (Figure). Our current investigation on this strain led to the isolation of three new pyranone derivatives, called fallopiaxylaresters A–C (
1–
3), a new bisabolane-type sesquiterpenoid fallopiaxylarol A (
4) (
Figure 1), and the first complete set of the spectroscopic data for the previously-disclosed pestalotiopyrone M (
5), as well as a suite of known compounds consisting of six pyranone derivatives (
6–
11), three sesquiterpenoids (
12–
14), three isocoumarin derivatives (
15–
17), and one aromatic allenic ether (
18). Herein, the details of the isolation, structure elucidation of all new compounds and their anti-inflammatory, anti-microbial, and
α-glucosidase inhibitory activities were described.
2. Results and Discussion
Compound
1, named fallopiaxylarester A, was obtained as a white solid. Its molecular formula C
12H
16O
6 was determined by the HRESIMS molecular ion peak at
m/
z 279.0837 [M + Na]
+ (calcd for C
12H
16O
6Na, 279.0839). The IR absorption bands showed the presence of hydroxyl (3426 cm
-1) and carbonyl (1733 cm
-1) functionalities. Detailed comparison of
1H and
13C NMR spectra of
1 and
10 revealed that structure of
1 is almost identical with that of
10, which was supported by the further analysis of 2D NMR spectra (
Table 1). The HMBC correlations of
1 from H-2 (
δH 5.56) to C-1/C-3/C-4, from H
3-12 (
δH 3.87) to C-3, from H-4 (
δH 6.22) to C-3/C-5/C-6, from H-6 (
δH 4.35) to C-5 and C-8, showed the same settlement with compound
10 (
Figure 2). The main difference between
1 and
10 was found at C-10 of the side chain of the pyranone core, replaced by the fragment of methyl valerate group. This deduction was further identified by the HMBC correlations from H
3-11 (
δH 3.66) and H
2-9 (
δH 2.38) to C-10. Thus, the planar structure of
1 was established. Since there was only one chiral center in the molecule, the relative configuration was arbitrarily assigned as 6
R*. The absolute configuration of C-6 was subsequently assigned to be
R by comparing the optical rotational value [
α +56.2 (
c 0.11, MeOH) with [
α +96.0 (
c 0.10, MeOH) of compound
10 [
12]. Besides, the deduction was also confirmed by a time-dependent density functional theory-electronic circular dichroism (TDDFT-ECD) approach. As shown in
Figure 3, the Boltzmann-averaged ECD spectrum of (6
R)-
1 displayed a similar curve compared to the experimental one. Thus, the absolute configuration at C-6 in
1 was unambiguously assigned as 6
R (
Figure 1).
Compound
2, a white solid, was determined to possess the molecular formula of C
19H
28O
9 with six degrees of unsaturation by using HRESIMS {
m/z 423.1626 [M + Na]
+, (calcd for C
19H
28O
9Na, 423.1626)}. The spectra of
2 showed the similar absorption bands, indicating the same presence of hydroxyl (3359 cm
-1) and carbonyl (1696 cm
–1) functionalities. The
1H NMR spectra (
Table 2) data showed signals of a typical sugar moiety at
δH 5.12 (d,
J = 3.7 Hz), 3.53 (dd,
J = 10.0, 3.7 Hz), 3.78 (t,
J = 9.3 Hz), 3.45 (t,
J = 9.3 Hz), 3.90 (m), and 3.76 (m). Analysis of the
13C NMR and DEPT spectra of
2 indicated the presence of 19 carbon signals, assignable to two methyl carbons (one methoxyl,
δC 57.0 and 14.4), five methylene carbons (
δC 27.1, 30.0, 32.8, 23.6 and 62.1), eight methine carbons (three olefinic,
δC 89.4, 100.2, 125.0, 102.8, 73.4, 74.4, 71.0, and 75.5), one ester carbonyl carbon (
δC 166.7), and three olefinic quaternary carbons (
δC 174.0, 157.9 and 145.8). These substructures accounted for four out of five degrees of unsaturation, indicating one cyclic system in
2. The
1H–
1H COSY spectrum revealed three spin systems: (a) H-7/H-8/H-9; (b) H-11/H-12 and (c) H-1'/H-2'/H-3'/H-4'/H-5'/H-6' (
Figure 2). And those spin systems were connected by the key HMBC correlations from H-2 (
δH 5.59) to C-1/C-3/C-4, from H
3-13 (
δH 3.87) to C-3, from H-4 (
δH 6.95) to C-3/C-5/C-6, from H-7 (
δH 6.07) to C-5, and from H
2-9 (
δH 1.48) to C-10/C-11.
Subsequently, the long range HMBC correlation from H-1' (
δH 5.12) to the anomeric carbon C-6 suggested the sugar was attached at C-6 of the side chain. Acid hydrolysis of
2 followed by HPLC analysis of the sugar derivative was applied to determine the type of sugar moiety but without success. Then, different deuterated solvent, pyridine-
d5, was used to analyze the proton signals on the sugar. Besides, the isolated anomeric proton signal was probed by a selective 1D-TOCSY experiment. In the 1D-TOCSY experiment, irradiation of the signal at
δH 5.68 (1H, d,
J = 3.7 Hz) enabled the identification of H-2' (
δH 4.27, dd,
J = 10.5, 4.1 Hz), H-3' (
δH 4.71, t,
J = 9.3 Hz), H-4' (
δH 4.35, t,
J = 10.0 Hz), H-5' (
δH 4.66, dt,
J = 10.0, 3.5 Hz), H
2-6' (
δH 4.48, br s) in the same conjugated system (
Figure 4,
Table 2). The
J values of
JH-5'/H-4' (10.0 Hz) and
JH-5'/H-6' (3.5 Hz) in
1H NMR in pyridine-
d5 of
2 combined with the 1D-TOCSY experiment suggested an α-D-glucose. Besides, those chemical shift values of anomeric carbon at
δC 102.8 (C-1'),four tertiary carbons at
δC 73.4 (C-2'), 74.4 (C-3'), 71.0 (C-4'), 75.5 (C-5'), and a methylene oxide carbon at
δC 62.1 (C-6') in methanol-
d4 was highly similar to 6-Buty-AA-2G along with other derivatives of AA-2G in the literature [
13], which also confirmed the inference that the sugar unit was α-D-glucose. Besides, the geometry of the double bond between C-6 and C-7 was inferred by the ROESY spectrum in DMSO-
d6. The ROESY correlations between H
2-8 (
δH 2.33) and H-1' (
δH 4.96)/H-3' (
δH 3.70)/H-5' (
δH 3.54), and between H-4 (
δH 6.98) and H-1'/H-3' demonstrated the
Z geometry of Δ
6,7 (
Figure 5 and
Figure S15). Thus, this undescribed
2 was established as shown in
Figure 1 and named fallopiaxylarester B.
Compound
3, named fallopiaxylarester C, was isolated as a white solid. Its molecular formula of C
19H
30O
9 with five degrees of unsaturation was based on HRESIMS analysis {
m/z 425.1780 [M + Na]
+, (calcd for C
19H
30O
9Na, 425.1782)}. Like
2, the presence of
α,
β-unsaturated
γ-lactone and hydroxyl groups in
3 was obvious by its IR absorption bands at
vmax 3380, and 1700 cm
–1. Besides, with the analysis of 1D NMR spectra, a sugar moiety in
3 was also quickly recognized. Besides, the HMBC correlation from H-6 (
δH 4.52) to C-1' suggested the same set with
2. The main difference between these two compounds was the hydrogenation of the trisubstituted olefinic group at C-6/C-7 in
2, according to 2 mass unit difference between
2 and
3. Subsequently, an acid hydrolysis of
3 afforded the products including a pyrone aglycone
3a and a sugar moiety
3b. The absolute configuration of C-6 in
3a was assigned to be
R form by comparing its optical value [
α +52.5 (
c 0.11, MeOH) with +67.6 (
c 0.25, MeOH) of nodulisporipyrones A [
14]. According to the detailed analysis of
1H NMR and 1D-TOCSY experiment of
3, H-1' (
δH 5.42, d,
J = 3.8Hz), H-2' (
δH 4.22, dd,
J = 9.6, 3.8 Hz), H-3' (
δH 4.67, t,
J = 9.6 Hz), H-4' (
δH 4.24, t,
J = 9.6 Hz), H-5' (
δH 4.44, t,
J = 9.6 Hz), H
2-6' (
δH 4.57, dd,
J = 9.6, 5.6 Hz;
δH 4.43, m), the sugar moiety was indicated as α-D-glucose (
Figure 4,
Table 3). Moreover, large similarities were observed by comparison of NMR data in DMSO-
d6 of
3 with 5-(
α-D-glucopyranosyloxymethyl)-2-furancarboxylic acid and other analogs in the literature [
15]. Thus, the structure of
3 was established as shown (
Figure 1).
Compound
4 was isolated as a colorless oil. Its molecular formula of C
16H
28O
5 with three degrees of unsaturation was also based on HRESIMS analysis {
m/
z 323.1829 [M + Na]
+, (calcd for C
16H
28O
5Na, 323.1829). The IR spectrum of
4 demonstrated characteristic absorption bands for hydroxyl (3425 cm
−1) and carbonyl (1687 cm
−1) groups. The 1D NMR and HSQC spectra of
4 revealed 16 carbon signals, including four methyl groups, five sp
3 methylene groups, one sp
2 methine, two sp
3 methine groups and four quaternary carbons (three oxygenated carbons). The above information accounted for two degrees of unsaturation, indicating one cyclic system in compound
4. The
1H–
1H COSY spectrum revealed two spin systems: (a) H-5/H-6/H-1/H-2/H-3 and (b) H-8/H-9/H-10 (
Figure 2). Furthermore, the HMBC correlations from H
3-15 (
δH 1.04) to C-3/C-5; from H
2-2 (
δH 1.59, 1.53)/H-3 (
δH 3.36) to C-4; from H
2-2 (
δH 1.59, 1.53)/H
2-6 (
δH 1.29) to C-7; from H
3-14 (
δH 0.96) to C-1/C-8; from H-10 (
δH 6.71) to C-8/C-12/C-13; from H
3-13 (
δH 1.77) to C-11/C-12 and from H
3-16 (
δH 3.64) to C-12 made those two spin systems connected.
Thus, the planar structure of 4 was established as shown (
Figure 1) and named fallopiaxylarol A
.
Initially, the ROESY correlation between H
3-13 and H
2-9 and the lack of correlation of H
3-13/H-10 assigned the
E-geometry of Δ
10, 11, which was also supported by the
J value of H-10 (7.5) (
Figure 5). Besides, the ROESY correlations of H-1/3-OH/H
3-15/H-5α/H-6α suggested the
cis orientation of the H-1, 3-OH, and H
3-15. Furthermore, the literature survey revealed that the NMR data of the six-membered ring and the optical rotation values of
4 were almost identical to those of (1
S,3
R,4
R,7
S)-3,4-dihydroxy-
α-bisabolol [
16]. Thus, the absolute configuration of
4 was tentatively determined as shown in
Figure 1.
Table 4.
1H NMR (δH, 600 MHz) and 13C NMR (δC, 150 MHz) data for 4.
Table 4.
1H NMR (δH, 600 MHz) and 13C NMR (δC, 150 MHz) data for 4.
No. |
δH, mult (J Hz)a
|
δC, Typea
|
δH, mult (J Hz)b
|
δC, Typeb
|
1 |
1.72, m |
39.1, CH |
1.59, m, overlap |
38.8, CH |
2 |
1.80, m |
29.3, CH2
|
1.59, m, overlap |
29.2, CH2
|
1.53. m |
3 |
3.66, br s |
74.0, CH |
3.36, m, overlap |
72.6, CH |
4 |
|
70.9, C |
|
69.5, C |
5 |
1.74, m |
33.6, CH2
|
1.49, m |
33.5, CH2
|
1.55, m |
1.29, m, overlap |
6 |
1.49, m |
22.1, CH2
|
1.29, m, overlap |
21.7, CH2
|
1.40, m |
7 |
|
74.0, C |
|
72.0, C |
8 |
1.60, m |
38.8, CH2
|
1.41, m |
38.1, CH2
|
9 |
2.26, m |
22.9, CH2
|
2.18, q (8.0) |
22.7, CH2
|
10 |
6.77, td (7.5, 1.2) |
142.6, CH |
6.71, td (7.5, 0.9) |
143.5, CH |
11 |
|
127.6, C |
|
126.4, C |
12 |
|
168.8, C |
0.90, t (6.8) |
167.7, C |
13 |
1.84, s |
12.4, CH3
|
1.77, s |
12.2, CH3
|
14 |
1.14, s |
23.7, CH3
|
0.96, s |
23.8, CH3
|
15 |
1.26, s |
27.6, CH3
|
1.04, s |
27.9, CH3
|
16 |
3.73, s |
51.8, CH3
|
3.64, s |
51.6, CH3
|
3-OH |
|
|
4.36, d (4.0) |
|
4-OH |
|
|
3.96, s |
|
7-OH |
|
|
3.88, s |
|
After literature survey, as for the secondary metabolites produced by the genus
Xylaria, the main structural differences between the co-isolated new pyranone derivatives in this case and the other analogues of the genus are the variation of substituents in the side chain attached pyranone core [
7,
12,
16]. Although compound
5 has previously been reported as a natural product from fermentation extracts of endophytic fungi [
17], this is the first report of its existence to be accompanied by a full suite of supporting spectroscopic data. The fourteen known compounds, pestalotiopyrone M (
5), 4-methoxy-6-nonyl-2-pyrone (
6) [
18], xylariaopyrone A (
7) [
19], xylariaopyrone H (
8) [
12], xylariaopyrone I (
9) [
12], xylapyrone D (
10) [
20], scirpyrone H (
11) [
21], 1
α,10
α-epoxy-3
α,13-dihydroxyeremophil-7(11)-en-12,8
β-olide (
12) [
4], 3
α-hydroxymairetolide A (
13) [
4], mairetolide A (
14) [
22], (−)-5-methylmellein (
15) [
23], diaporthin (
16) [
24], mucorisocoumarin B (
17) [
25], eucalyptene (
18) [
26], were also isolated from
Xylaria sp. Z184. The structures of these compounds (
5–
18) were identified by comparing the spectral data to those reported in the respective references.
Given the secondary metabolites generated by stains of
Xylaria usually show obvious anti-inflammatory and antifungal activities [
7,
8]. In this case, compounds
2–
10 and
15–
18 and the crude extract were selected to evaluate the antimicrobial, anti-inflammatory and α-glucosidase inhibition activities due to the limitation of samples. In antimicrobial assay, compounds
5,
7, and
8 displayed weak activity against
Staphylococcus areus subsp.
aureus with inhibition ratios of 25.9%, 31.5% and 25.3% at a concentration of 100
μM. Unfortunately, in anti-inflammatory and α-glucosidase assay, only the crude extract potently inhibited LPS-induced NO production in RAW264.7 mouse macrophages, with an inhibition rate of 77.28 ± 0.82% at a concentration of 50
μg/mL, although it was cytotoxic at this concentration, reducing the concentration to 6.25
μ/mL abrogated the cytotoxicity (
Table 5).
Table 5.
Inhibitory activities of compounds selected and crude extract on LPS-stimulated NO production.
Table 5.
Inhibitory activities of compounds selected and crude extract on LPS-stimulated NO production.
Compounds |
Concentration |
NO production inhibition (%)a
|
2 |
50 μM |
4.51 ± 0.35 |
3 |
50 μM |
–3.93 ± 2.43 |
4 |
50 μM |
1.85 ± 3.18 |
5 |
50 μM |
–1.61 ± 0.53 |
12 |
50 μM |
0.92 ± 2.97 |
13 |
50 μM |
4.67 ± 2.36 |
14 |
50 μM |
3.54 ± 1.26 |
18 |
50 μM |
–0.92 ± 2.21 |
Crude extract |
50 μg/mL |
77.28 ± 0.82 |
|
6.25 μg/mL |
7.78 ± 3.29 |
L-NMMAb |
50 μM |
53.75 ± 1.28 |
3. Materials and Methods
3.1. General Experimental Procedures
Optical rotations were determined with a PerkinElmer 341 polarimeter (PerkinElmer, Waltham, MA, USA). UV absorptions were obtained by using a Waters UV-2401A spectrophotometer equipped with a DAD and a 1 cm path length cell. Methanolic samples were scanned from 190 to 400 nm in 1 nm steps. Measurements of IR spectra were performed using a Bruker Vertex 70 FT-IR spectrometer (Bruker, Karlsruhe, Germany). NMR spectra were recorded on Bruker AM-400 and AM-600 NMR spectrometers (Bruker, Karlsruhe, Germany) with TMS as internal standard, and NMR data were referenced to selected chemical shifts of methanol-d4 (1H: 3.31 ppm, 13C: 49.0 ppm), chloroform-d (1H: 7.26 ppm, 13C: 77.0 ppm) and dimethyl sulfoxide-d6 (1H: 2.50 ppm, 13C: 39.5 ppm), respectively. HRESIMS data were acquired on a Thermo Fisher LTQ XL LC/MS (Thermo Fisher, Palo Alto, CA, USA). Semi-preparative HPLC was performed on an Agilent 1220 instrument equipped with a UV detector with a semi-preparative column (RP-C18, 5 μm, 250 × 10 mm, Welch Materials, Inc.). Column chromatography was performed using SephadexTM LH-20 gel (40–70 μm; Merck KGaA, Darmstadt, Germany), and precoated silica gel plates (GF254, Qingdao Marine Chemical Co. Ltd., Qingdao) were used for TLC analyses. Spots were visualized by heating silica gel plates sprayed with 10% H2SO4 in EtOH. All HPLC solvents were purchased from Guangdong Guanghua Sci-Tech Co. Ltd (Guangzhou, China). All solvents were of analytical grade (Guangzhou Chemical Regents Company, Ltd., Guangzhou, China).
3.2. Fungal Material
The fungus Xylaria sp. Z184 was isolated from the leaves of Fallopia convolvulus (L.) Á. Löve collected in Zhuyang Town, Henan province, P. R. China (N 34°14′12″ W 110°47′09″) in June 2022. Leaves of F. convolvulus (L.) Á. Löve were processed within 24 hours and rinsed with sterile water. On a sterile workbench, after 30 minutes of ultraviolet light exposure, the leaves underwent sequential treatment with a 5% sodium hypochlorite solution, sterile water, and 75% ethanol, either soaked or rinsed, followed by drying with sterile filter paper. Leaves were trimmed into small squares with sterile scissors and placed into previously prepared PDA monoclonal agar plates, inoculating three petri plates in parallel. These plates were incubated at 30 °C for 3–7 days, until mycelial growth was observed extending from the inside of the tissue block to its surroundings. Distinct morphological colonies were subsequently transferred to new media for continued cultivation. This procedure was repeated until the fungal strains showed uniform growth, leading to the isolation of purified strains.
To identify the strains, the standardized operating procedure was performed, which included genomic DNA extraction, 16
S/18
S amplification, PCR product detection and purification, and comparison of sequencing results with the NCBI-BLAST database (
https://www.ncbi.nlm.nih.gov/), using ITS1 and ITS4 primers for both amplification and sequencing. The sequence data for this strain was submitted to the GenBank under accession No. KU645984. The fungal strain was deposited on 20% aqueous glycerol stock in a −80 °C freezer at the School of Pharmaceutical Sciences (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, China.
Figure 6.
Photo of the fungus Xylaria sp. Z184.
Figure 6.
Photo of the fungus Xylaria sp. Z184.
3.3. Fermentation, Extraction and Isolation
Xylaria sp. Z184 was cultured on potato dextrose agar for 5 days at 28 °C to prepare the seed culture. The cultured agar plates were cut into small pieces, which were then inoculated into 30 previously-autoclaved Erlenmeyer flasks (350 mL), each containing 50 g of rice and 45 mL of distilled water. All flasks were incubated at 28 °C for 40 days. Cultural media was extracted with methanol four times, and the solvent was evaporated under reduced pressure at 45 °C. Then the extract was suspended in water and extracted four times with ethyl acetate. The combined ethyl acetate layers were concentrated under reduced pressure to yield a brown extract (7.5 g).
The crude extract was chromatographed on Sephadex LH-20 (MeOH) to give eight fractions (Fr.1–Fr.8). Fr. 3 (2.8 g) was separated with silica gel column chromatography (CC) with petroleum ether (PE)/EtOAc (20:1–0:1, v/v) to give seven subfractions (Fr. 3.1–Fr. 3.7). Fr. 3.3 (306.2 mg) was purified with silica gel CC using PE/EtOAc (15:1–1:1, v/v) to yield six further subfractions (Fr. 3.3.1–Fr. 3.3.6). Fr. 3.3.4 (25.8 mg) was purified by semi-preparative HPLC (MeOH/H2O, 48:52, v/v, 3.0 mL/min) to yield compound 7 (4.3 mg, tR 22.0 min). Fr. 4 (1.7 g) was separated by silica gel CC with CH2Cl2/MeOH (80:1–0:1, v/v) to obtain seven subfractions (Fr. 4.1–Fr. 4.7). Fr. 4.3 (277.5 mg) was purified by semi-preparative HPLC (MeCN/H2O, 25:75, 0–14 min, then MeCN/H2O, 50:50, 14.01–33 min, v/v, 3.0 mL/min) to yield compounds 13 (4.6 mg, tR 12.1 min), 14 (1.6 mg, tR 27.6 min), and 6 (4.1 mg, tR 31.5 min). Fr. 4.5 (239.9 mg) was purified by semi-preparative HPLC (MeCN/H2O, 20:80, 0–10 min, then MeCN/H2O, 40:60, 10.01–21 min, v/v, 3.0 mL/min) to yield compounds 10 (8.7 mg, tR 13.2 min) and 4 (6.0 mg, tR 19.5 min). Fr. 4.6 (225.9 mg) was purified by semi-preparative HPLC (MeOH/H2O, 40:60, v/v, 3.0 mL/min) to yield compounds 2 (3.9 mg, tR 30.1 min) and 3 (12.1 mg, tR 35.5 min). Fr. 5 (1.3 g) was separated with silica gel CC with CH2Cl2/MeOH (25:1–0:1, v/v) to give six subfractions (Fr. 5.1 – Fr. 5.6). Fr. 5.1 (44.0 mg) was further purified by semi-preparative HPLC (MeOH/H2O, 55:45, 0–23 min, then MeOH/H2O, 65:35, 23.01–47 min, v/v, 3.0 mL/min) to yield compounds 15 (3.1 mg, tR 21.8 min) and 18 (1.4 mg, tR 45.5 min). Fr. 5.3 (399.7 mg) was purified by semi-preparative HPLC (MeOH/H2O, 35:65, 0–19 min, then MeOH/H2O, 54:46, 19.01–40 min, v/v, 3.0 mL/min) to yield compounds 1 (1.2 mg, tR 6.0 min), 17 (8.3 mg, tR 18.1 min), 16 (2.7 mg, tR 31.9 min), and 11 (2.4 mg, tR 38.2 min). Similarly, Fr. 5.4 (355.8mg) was purified by semi-preparative HPLC (MeCN/H2O, 10:90, v/v, 3.0 mL/min) to yield compounds 5 (3.5 mg, tR 6.5 min), 8 (1.3 mg, tR 8.1 min), 9 (8.3 mg, tR 10.9 min), and 12 (2.1 mg, tR 15.5 min).
3.4. Spectral and Physical Data of Compounds 1–5
Fallopiaxylarester A (
1): White solid; [
α +56.2 (
c 0.11, MeOH); UV (MeOH)
λmax (log
ε): 279 (0.19), 204 (0.72) nm; IR (KBr)
vmax: 3426, 2922, 1733, 1648, 1569, 1457, 1412, 1384, 1247, 1032, 832 cm
–1; ECD (MeOH)
λmax (Δε): 279 (+3.0), 206 (–4.0) nm.
1H and
13C NMR data, see
Table 1; HRESIMS (
m/
z): 279.0837 [M + Na]
+ (calcd for C
12H
16O
6Na, 279.0839).
Fallopiaxylarester B (
2): White solid; [
α +102.9 (
c 0.38, MeOH); UV (MeOH)
λmax (log
ε): 310 (0.16), 260 (0.06), 219 (0.41) nm; IR (KBr)
vmax: 3359, 2928, 2858, 1696, 1623, 1560, 1456, 1409, 1260, 1230, 1080, 1018, 817, 539 cm
–1; ECD (MeOH)
λmax (Δε): 310 (+3.4), 231 (–4.1), 205 (–3.3) nm.
1H and
13C NMR data, see
Table 2; HRESIMS (
m/
z): 423.1626 [M + Na]
+ (calcd for C
19H
28O
9Na, 423.1626).
Fallopiaxylarester C (
3): White solid; [
α +124.2 (
c 0.39, MeOH); UV (MeOH)
λmax (log
ε): 281 (0.11), 204 (0.45) nm; IR (KBr)
vmax: 3380, 2927, 2857, 1700, 1649, 1569, 1458, 1414, 1384, 1250, 1025, 836, 700 cm
–1; ECD (MeOH)
λmax (Δε): 280 (+4.5), 232 (+0.9), 205 (+3.0) nm;
1H and
13C NMR data, see
Table 3; HRESIMS (
m/
z): 425.1780 [M + Na]
+ (calcd for C
19H
30O
9Na, 425.1782).
Fallopiaxylarol A (
4): Colorless oil; [
α –31.8 (
c 0.10, MeOH); UV (MeOH)
λmax (log
ε): 205 (0.36), 218 (0.48) nm; IR (KBr)
vmax: 3546, 3426, 2945, 2930, 1688, 1287, 1150, 1036 cm
–1;
1H and
13C NMR data, see
Table 4; HRESIMS (
m/
z): 323.1829 [M + Na]
+ (calcd for C
16H
28O
5Na, 323.1829).
Pestalotiopyrone M (5): White solid; UV (MeOH) λmax nm (log ε) 206 (0.45), 293 (0.16); IR (KBr) νmax 3311, 2961, 2928, 1711, 1565, 1365, 1014, 989 cm–1; 1H NMR (methanol-d4, 600 MHz) δH: 4.54 (2H, s, H-7), 4.41 (2H, s, H-8), 4.18 (3H, s, H-10), 2.35 (3H, s, H-9); 13C NMR (methanol-d4, 150 MHz) δC: 171.6 (C, C-4), 167.4 (C, C-2), 163.0 (C, C-6), 115.2 (C, C-5), 110.1 (C, C-3), 63.1 (CH3, C-10), 55.5 (CH2, C-8), 55.1 (CH2, C-7), 17.3 (CH3, C-9); HRESIMS (m/z): 223.0578 [M + Na]+ (calcd for C9H12O5Na, 223.0577).
3.5. Computational Details (TDDFT-ECD) of 1
The conformational search of (6
R)-
1 was performed by using the torsional sampling (MCMM) conformational searches with OPLS_2005 force field within an energy window of 21 kJ/mol. Conformers above 1% Boltzmann populations were re-optimized at the B3LYP/6-31G(d) level with the IEFPCM solvent model for methanol. The following TDDFT calculations of the re-optimized geometries were all performed at the B3LYP/6-311G(d,p) level with the IEFPCM solvent model for methanol. Frequency analysis was performed as well to confirm that the re-optimized geometries were at the energy minima. Finally, the SpecDis 1.62 [
27] software was used to obtain the Boltzmann-averaged ECD spectra of
1 and visualize the result.
3.6. Biological Assays
3.6.1. Antimicrobial Activity
Compounds
2–
10 and
15–
18, and the crude extract were evaluated for antimicrobial activities against
Staphylococcus aureus subsp.
aureus, and fluconazole-resistant
Candida albicans. The antimicrobial assay was conducted according to a previously-described method [
28]. Samples were added into a 96-well culture plate with a maximum test compound concentration of 100
μM. Bacterial liquid was added to each well until the final concentration was 5 × 10
5 CFU/mL. The plate was then incubated at 37 °C for 24 h, and the OD values at 595 nm were measured using a microplate reader. Blank bacterial medium served as control.
3.6.2. Anti-inflammatory Activity
The RAW 264.7 cells (2 × 10
5 cells/well) were incubated in 96-well culture plates with or without 1 µg/mL lipopolysaccharide (LPS, Sigma Chemical Co., USA) for 24 h in the presence or absence of the test compounds. Supernatant aliquots (50 µL) were then treated with 100 µL Griess reagent (Sigma Chemical Co., USA). The absorbance was measured at 570 nm by using a Synergy TMHT microplate reader (BioTek Instruments Inc., USA). In this study, N
G-methyl-L-arginine acetate (L-NMMA, Sigma Chemical Co., USA) was used as a positive control. In the remaining medium, an MTT assay was carried out to determine whether the suppressive effect was related to cell viability. The inhibitory rate of nitric oxide (NO) production = (NO level of blank control – NO level of test samples)/NO level of blank control. The percentage of NO production was evaluated by measuring the amount of nitrite concentration in the supernatants with Griess reagent, as described previously [
29].
3.6.3. Alpha-Glucosidase Inhibition Activity
The α-glucosidase inhibition was assessed according to the slightly modified method of Ma
et al [
30]. All samples were dissolved in DMSO at a concentration of 50
μM. The α-glucosidase (Sigma Chemical Co., USA) and substrate (4–Nitrophenyl α-D-glucopyranoside, PNPG, Sigma Chemical Co., USA) were dissolved in potassium phosphate buffer (0.1 M, pH 6.7). The samples were preincubated with α-glucosidase at 37 °C for 10 min. Then, PNPG was quickly added to the 96-well enzyme label plate to start the reaction, and the plate was incubated at 37 °C for 50 min. At the same time, a blank control without samples and a positive control of quercetin (10 mM) were set up. All samples were thoroughly mixed and analyzed in triplicate. The OD value was measured at 405 nm using a microplate reader. The inhibition percentage (%) was calculated by the following equation: Inhibition (%) = (1 – OD
sample)/OD
control blank × 100.