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Chemical and Biological Technologies in Agriculture
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Prenylated flavonoids isolated from the twigs of Artocarpus champeden as potential activators for tobacco powdery mildew and their mode of actions

Chemical and Biological Technologies in Agriculture volume 10, Article number: 94 (2023) Cite this article

Abstract

Background

Golovinomyces cichoracearum (DC.) is the main pathogen for tobacco powdery mildew fungus disease. Its outbreaks often result in severe harvest losses for the yield and quality of tobacco. Artocarpus champeden is rich in prenylated flavonoids, which are important for the plant’s defensive strategies. With the aim of continuously exploring bioactive natural metabolites for agricultural chemicals, the chemical investigations on the twigs of A. champeden were carried out.

Results

Six new (1–6) and five known (7–11) prenylated flavonoids were isolated. Compound 1 is the first example of flavone whose prenylated side-chain is converted into an unusual 1H-pyrrol-2-yl functional group. Compounds 2 and 3 are rare flavones bearing a 4-methylfuran-2-yl moiety. The frameworks of the above three flavones are reported in natural products for the first time. Interestingly, compound 1 showed high anti-G. cichoracearum activity with an inhibition rate of 88.3% ± 6.2. This rate is higher than that of the positive control (with an inhibition rate of 81.5% ± 6.3) compared to the negative control, compounds 2–11 also showed potential activities with inhibition rates in the range of 50.9%–72.0%. In addition, the mechanistic studies on 1 revealed that it has a potent direct effect on conidiospores of G. cichoracearum and induces systemic acquired resistance for tobacco plants, which may be the reasons for its significant effects against G. cichoracearum.

Conclusions

Powdery mildew is a fungal disease harmful to tobacco. Flavonoids have been identified as the sources of promising antifungal agents. For prenylated flavonoids, the combination of a flavonoid skeleton with prenylated side-chain can give the resultant more potential for biological activities. The successful isolation and structure identification of the above prenylated flavonoids provide new materials for the screening of powdery mildew inhibitors, and also contribute to the improved utilization of A. champeden.

Graphical Abstract

Background

Fungal diseases have harmful effects on the growth and yield of crops. Fungal pathogens attacking can cause serious losses to various crops worldwide. For main crops, their quality and yield will be notably affected once they are infected by pathogenic fungi [1, 2]. Among the most plant pathogenic fungi, Golovinomyces cichoracearum (DC.) is the main pathogen for powdery mildew disease, and can attack a wide range of hosts, such as tobacco, pepper, tomato, eggplant, grape, sunflower, and some melon crops [3,4,5]. Especially for tobacco, the powdery mildew outbreaks often result in the losses by reducing the yield and quality of tobacco leaves [6].

In agriculture, breeding resistant varieties [7, 8], inducing plants resistances [9, 10], improving cultivation [11, 12], biological control [13, 14], chemical pesticides [15, 16], and the like, are the main methods to prevent powdery mildew diseases. As compared with synthetic antifungal chemicals, natural products are highly promising prevention strategies due to their low toxicity with no residual effects [17, 18]. Therefore, they have attracted increasing attention from plant protection scholars, and also led more and more biological companies to commit to developing natural products into new pesticides [19, 20]. Among the numerous of natural products, flavonoids are associated with a group of metabolites with polyphenolic structures which are broadly found in plants. They have undertaken a variety of biological processes [21, 22], and also potentially involved in plant resistances to biotic stresses, such as protections against microbes, insects, and virus [23]. Therefore, flavonoids have been identified as promising antifungal agents [24,25,26].

Artocarpus champeden is a tropical fruit in the Moraceae family, which is native to India, and widely distributed in Southwest Asia. This plant has significant economic values for its fruits, woods, and folk medicine functions [27]. Previous investigations have revealed that A. champeden is rich in prenylated flavonoids [28,29,30,31]. Prenylated flavonoids are the combination of a flavonoid skeleton with a prenylated side-chain, and this combination can give the resultant more potential for biological activities [25]. The vertical zone climates and extensive sunlight environments provide rich plant diversity in Yunnan Province, P.R. China. These also give the good resources for local chemists to search bioactive natural products. Since the prenylated flavonoids have been identified as promising antifungal agents with higher efficiencies [25]. With the aim of continuously exploring bioactive natural products for agricultural chemicals, the chemical investigations on the twigs of A. champeden were carried out. As a result, we discovered six new (1–6), as well as five known (7–11) prenylated flavonoids. Herein, the details of isolation and structure determination for above compounds, and their activities for against tobacco powdery mildew are presented in this manuscript.

Methods

General experimental procedures

UV and IR (KBr) spectra were obtained on an UV-1900 spectrophotometer (Shimadzu, Kyoto, Japan) and a FTS185 spectrophotometer (Bio-Rad, California, USA). NMR experiments were carried out on Bruker DRX-500 NMR spectrometer (Bruker, Karlsruhe, Germany) with TMS as internal standard. ESIMS and HRESI-MS analyses were performed on a 6540 Q-TOF mass spectrometer equipped with Agilent 1290 UPLC (Agilent Technologies, Wilmington, DE, USA). Microscopic observation was examined with a fluorescence biological microscope (Olympus CX33, Tokyo, Japan). 80–100 Mesh or 200–300 mesh Silica gel (Qingdao Marine Chemical, Inc., Qingdao, China) and 75–150 μm MCI CHP20P gel (Mitsubishi Chemical Corporation, Tokyo, Japan) were used for normal column chromatography. The fractions were monitored by thin-layer chromatography (Qingdao Marine Chemical, Inc., Qingdao, China), and the spots were visualized by heating silica gel plates (approximately 120 °C) after sprayed with 5% H2SO4 in ethanol. Semi-preparative HPLC was performed on an Agilent 1260 preparative liquid chromatography (Agilent Technologies, Wilmington, DE, USA) using a Venusil MP C18 column (5 μm, 2.0 cm × 25 cm, Bonna-Agela, Tianjin, China) or a Zorbax PrepHT GF C18 column (5 μm, 2.12 cm × 25 cm, Agilent, Palo Alto, USA).

Plant material

The twigs of Artocarpus champeden (Lour.) Stokes, were collected from Xishuangbanna Prefecture, Yunnan Province, on August 2021. The samples were dried at 35–40 °C, and then crushed to 30–60 mesh. The crushed samples were used for extraction and isolation. The species was identified by Prof. Yuan N, and the voucher specimen of the title plant (No. Ynni-21-08-047) had been deposited in the School of Ethnic Medicine, Yunnan Minzu University.

Extraction and isolation

The crushed twigs of A. champeden (approximately 10.0 kg) were extracted with 70% aqueous acetone and filtered, and then the solvent was removed under reduced pressure to yield the crude extract (1.02 kg). The crude extract was partitioned between water and ethyl acetate, and then decolorized with MCI GELCHP20P. The purified extract (482 g) was separated on silica gel (80–100 mesh) column with trichloromethane/methanol gradient system (10:0, 9:1, 8:2, 7:3, 6:4, and 5:5) to afforded six fractions (A–F). Fraction A (9:1, 52.6 g) was separated by silica gel column (200–300 mesh) eluted with trichloromethane/acetone (9:1 to 2:1) to yield sub-fractions B1–B7. Sub-fraction B1 (9:1, 3.85 g) was further subjected to silica gel column (200–300 mesh), and then semi-preparative HPLC (72% methanol/water, 12 mL/min) separation to yield 2 (22.5 mg), 3 (26.4 mg), 4 (26.3 mg), 5 (21.2 mg) and 6 (20.8 mg). Sub-fraction B2 (8:2, 3.21 g) was separated by another silica gel column (200–300 mesh) and subsequently separated by semi-preparative HPLC (65% methanol/water, 12 mL/min) to give 1 (28.4 mg), 7 (18.5 mg), 8 (32.4 mg), 9 (16.4 mg), 10 (13.6 (mg), and 11 (21.2 mg).

4ʹ-Hydroxy-8-methoxy-6-(4-methyl-1H-pyrrol-2-yl)-flavone (1)

C21H17NO4, pale-yellow powder; UV (MeOH) λmax (log ε) 376 (3.72), 275 (3.93), 215 (4.26) nm; IR (KBr): νmax 3418, 3360, 3049, 2987, 2842, 1668, 1616, 1549, 1457, 1362, 1254, 1170, 1062, 870 cm−1; 1H and 13C NMR data (500 and 125 MHz, in CDCl3), see Table 1; ESIMS m/z 370; HRESI-MS m/z 370.1053 [M+Na]+ (calcd C21H17NNaO4 for 370.1050).

Table 1 1H and 13C NMR data for compounds 1–3 (CDCl3, 500 and 125 MHz)
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4ʹ-Hydroxy-8-methoxy-6-(4-methylfuran-2-yl)-flavone (2)

C21H16O5, pale-yellow powder; UV (MeOH) λmax (log ε) 374 (3.75), 278 (3.96), 215 (4.29) nm; IR (KBr): νmax 3412, 3165, 3057, 2982, 2838, 1666, 1615, 1536, 1468, 1354, 1262, 1164, 1069, 848 cm−1; 1H and 13C NMR data (500 and 125 MHz, in CDCl3), see Table 1; ESIMS m/z 371; HRESI-MS m/z 371.0892 [M+Na]+ (calcd C21H16NaO5 for 371.0890).

6,4ʹ-Dimethoxy-7-(4-methylfuran-2-yl)-flavone (3)

C22H18O5, Pale-yellow powder; UV (MeOH) λmax (log ε) 380 (3.74), 282 (3.91), 215 (4.22) nm; IR (KBr): νmax 3152, 3063, 2979, 2832, 1669, 1617, 1542, 1464, 1359, 1270, 1161, 1072, 883 cm−1; 1H and 13C NMR data (500 and 125 MHz, in CDCl3), see Table 1; ESIMS m/z 385; HRESI-MS m/z 385.1049 [M+Na]+ (calcd C22H18NaO5 for 385.1046).

4ʹ-Hydroxy-8-methoxy-6-prenyl-flavone (4)

C21H20O4, Pale-yellow powder; UV (MeOH) λmax (log ε) 362 (3.70), 268 (3.85), 215 (4.16) nm; IR (KBr): νmax 3392, 2928, 2835, 1668, 1644, 1605, 1583, 1435, 1322, 1265, 1143, 1046, 849 cm−1; 1H and 13C NMR data (500 and 125 MHz, in CDCl3), see Table 1; ESIMS m/z 359; HRESI-MS m/z 359.1262 [M+Na]+ (calcd C21H20NaO4 for 359.1254).

7,4ʹ-Dihydroxy-8-methoxy-6-prenyl-flavone (5)

C21H20O5, Pale-yellow powder; UV (MeOH) λmax (log ε) 359 (3.73), 265 (3.82), 215 (4.13) nm; IR (KBr): νmax 3402, 2933, 2840, 1665, 1648, 1607, 1580, 1432, 1326, 1269, 1148, 1042, 864 cm−1; 1H and 13C NMR data (500 and 125 MHz, in CDCl3), see Table 1; ESIMS m/z 375; HRESI-MS m/z 375.1205 [M+Na]+ (calcd C21H20NaO5 for 375.1203).

6-(2,2-Dimethyl-2H,6H-pyrano[3,2-g])-4ʹ-hydroxy-8-methoxy-flavone (6)

C21H18O5, pale-yellow powder; UV (MeOH) λmax (log ε) 370 (3.76), 284 (3.85), 215 (4.19) nm; IR (KBr): νmax 3397, 2946, 2835, 1668, 1639, 1610, 1571, 1446, 1370, 1263, 1156, 1049, 880 cm−1; 1H and 13C NMR data (500 and 125 MHz, in CDCl3), see Table 1; ESIMS m/z 373; HRESI-MS m/z 373.1049 [M+Na]+ (calcd C21H18NaO5 for 373.1046).

Microscopic observation

The conidiospores of G. cichoracearum were peeled from the leaves surface using transparent tapes and then installed them on a microscope slides for observation. The slides were examined and photographed with a fluorescence biological microscope (Olympus CX33, Tokyo, Japan) at 400 (10 × 40) and 1600 (16 × 100) magnifications, respectively.

Antifungal activity assays

For antifungal activity assays, the inhibition rates for compounds were tested according to the previous literatures [10, 32]. For compounds with significant activities in inhibition rates assay, their protective and the curative effects on G. cichoracearum were also evaluated. The detailed procedures are listed in Additional file 1: Figure S2.

Analysis of defense enzymes activities

The activities of phenylalanine ammonia lyase (PAL), peroxidase (POD), polyphenol oxidase (PPO), superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) were determined using enzyme assay reagent kits according to the manufacturer’s instructions (Jiancheng Bioengineering Research Institute, Nanjing) and literature [33]. The N. tabacum cv. HD plants were used as hosts. After 24 h of infection with G. cichoracearum, the tobacco leaves were sprayed with 250 μg/mL of the compounds. Then, the leaves were harvested on 1, 3, 5, and 7 days, and used for the determination of the activities of above enzymes.

Analysis of SA, JA, MDA, and CHL accumulation

The amount of salicylic acid (SA), jasmonic acid (JA), malondialdehyde (MDA), and chlorophyll (CHL) were determined by SA, JA, MDA, and CHL assay reagent kits in accordance with the manufacturer's instructions (Comin Bioengineering Institute, Suzhou, P. R. China). The N. tabacum cv. HD plants were infected with G. cichoracearum. After 24 h of infection, the plants were sprayed with 250 μg/mL of the compounds. Then the leaves were harvested on 1, 3, 5, and 7 days, and used for the determination of SA, JA, MDA, and CHL contents. The CHL contents were expressed as the plus of chlorophyll-a and chlorophyll-b.

Quantitative real-time PCR analysis of defense-related genes

The quantitative real-time PCR analysis of defense-related genes (PR-1, PR-5, PAL and Chit-1) was performed according to previous literatures [34, 35]. The materials used, detailed procedures, and the primer pairs are listed in Additional file 1: Figure S3.

Molecular docking

The molecular docking calculations were executed using AutoDock Vina software with Tubulin (G. cichoracearum) proteins as target. The protein sequence was got from the NCBI database (GenBank: RKF84170.1, https://www.ncbi.nlm.nih.gov/protein/RKF84170.1) [36]. The 3D protein structures were built by homology model using Modeller10.1, and the ligands’ structures were generated by chem3D. For molecular docking calculations, the pdbqt files for the proteins and ligands were prepared according to the AutoDock protocol. All docking parameters were conserved to their default values, except the maximum number of energy evaluation (eval) and the number of genetic algorithms (GA) runs. The docking grids were made to binding sites for the receptor with a grid size of 40 Å * 40 Å * 40 Å. The grid spacing values were adjusted to 0.375 Å. Gasteiger atomic partial charges were assigned for all investigated ligands.

Results and discussion

Structure characterization of compounds 1–11

The extract obtained from the twigs of A. champeden was repeatedly separated by various column chromatography and preparative HPLC to afford six new (1–6), along with five known prenylated flavonoids (7–11). The structures of compounds 1–11 are shown in Fig. 1, and the 1H and 13C NMR data of 1–6 are listed in Tables 1 and 2. The known compounds were identified as artochamin C (7) [37], 2″,2″-dimethylpyran-(5″,6″:6,7)-5,4′-dihydroxy-4′-methoxy-flavonol (8) [38], corylifol C (9) [39], 6-prenyl-5,7,4′-trihydroxy-flavonol (10) [38], and artoindonesianin A-2 (11) [40], respectively, by the comparison of their NMR data with those of reported in the literatures.

Fig. 1
figure 1

Isoprenylated flavones from the twigs of A. champeden

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Table 2 1H and 13C NMR data for compounds 4–6 (CDCl3, 500 and 125 MHz)
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To best of our knowledge, compound 1 is the first example of flavone which prenylated side-chain converted into an unusual 1H-pyrrol-2-yl functional group, and 2 and 3 are rare flavones bearing an unusual 4-methyl furan-2-yl moiety. The frameworks of above three new flavones are reported in natural products for the first time.

Compound 1 was obtained as a pale-yellow powder. Its molecular formula C21H17NO4 was obtained from the quasimolecular ion peak at m/z 370.1053 [M+Na]+ in HRESI-MS (calcd 370.1050) with 14 degrees of unsaturations. The 1H, 13C NMR, and DEPT spectral data of 1 (Table 1) displayed 21 carbon and 17 hydrogen atoms, respectively. These signals can be classified as a 1,2,3,5-tetrasubstituted benzene ring (C-5–C-10, H-5 and H-7), a 1,4-disubstitued benzene ring (C-1ʹ–C-6ʹ, H2-2ʹ,6ʹ and H2-3ʹ,5ʹ), an α,β-unsaturated carbonyl (C-2–C-4, H-3), a 4-methyl-1H-pyrrol-2-yl moiety (C-2″–C-6″, H-3″, H-5″, H3-6″, and –NH) [41, 42], a methoxy group (δC 56.2 and δH 3.83), and a phenolic hydroxy group (δH 10.22). In addition to eight degrees of unsaturations for two benzene rings, two degrees of unsaturations for α,β-unsaturated carbonyl, three degrees of unsaturations for pyrrole ring, the still on ring needed to support 14 degrees of unsaturations in its molecule. By further analysis of its NMR data, the existence of two oxidized aromatic quaternary carbons (C-9 and C-2) suggested that C-9 and C-2 should be linked by an oxygen atom to form a pyran ring, and 1 should be the flavone skeleton [43]. This deduction also supported by the HMBC correlations (Fig. 2) from H-3 to C-4/C-10/C-1′, from H-5 to C-4/C-9/C-10, and from H-2′ to C-2. Furthermore, the existence of 3-methylpyrrol-2-yl moiety was also supported by the HMBC correlations from H-3″ to C-2″/C-4″/C-5″/C-6″, from H-5″ to C-2″/C-3″/C-4″, from H-6″ to C-3″/C-4″/C-5″, and from –NH to C-2″/C-3″/C-4″/C-5″.

Fig. 2
figure 2

The key HMBC correlations of compounds 1–6

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Since the flavone skeleton and the main substituents were determined, the positions of substituents can also be determined by further analyzed of its HMBC correlations (Fig. 2). The HMBC correlations from methoxy proton signal (δH 3.83) to C-8 indicated that the methoxy group located at C-8. The 4-methyl-pyrrol-2-yl moiety located at C-6 was supported by the HMBC correlations from H-3″ to C-6, from H-5 and H-7 to C-2″, and from –NH to C-6. Finally, the phenolic hydroxy group located at C-4ʹ was supported by the HMBC correlations of phenolic hydroxy proton (δH 10.22) with C-4ʹ/C-3ʹ,5ʹ. Thus, the structure of 1 was elucidated, and given the systematic name of 4ʹ-hydroxy-8-methoxy-6-(4-methyl-1H- pyrrol-2-yl)-flavone.

Compound 2 was obtained as a pale-yellow powder. It has the molecular formula C21H16O5 from HRESI-MS (m/z: 371.0892 [M+Na]+, calcd 371.0890). The 1H and 13C NMR spectral data of 2 were highly similar to these of 1 in C-2–C-10 and C-1ʹ–C-6ʹ. The obvious differences were attributed to the disappearance of 4-methyl-1H-pyrrol-2-yl moiety, and appearance of a 4-methylfuran-2-yl moiety (C-2″–C-6″, H-3″, H-5″, and H3-6″) [44] in 2. The existence of 4-methylfuran-2-yl moiety was also supported by the HMBC correlations from H-3″ to C-2″/C-4″/C-5″/C-6″, from H-5″ to C-2″/C-3″/C-4″, from H-6″ to C-3″/C-4″/C-5″. In addition, the 4-methylfuran-2-yl moiety located at C-6, the methoxy group located C-8, and the phenolic hydroxy located at C-4ʹ can also be confirmed by further analysis of its HMBC correlations (Fig. 2). Therefore, the structure of 4ʹ-hydroxy-8-methoxy-6-(4-methylfuran-2-yl)-flavone (2) was assigned as shown.

6,4ʹ-Dimethoxy-7-(4-methylfuran-2-yl)-flavone (3) is also a pale-yellow powder. It molecular formula C22H18O5 was confirmed by HRESI-MS (m/z 385.1049 [M+Na]+, calcd 385.1046). The 1H and 13C NMR spectral data of 3 were also highly similar to these of 2 in B and C rings. The major differences were due to the replacement of a pair of doublets [δH 7.84 (d) 1.6 and 7.38 (d) 1.6] to a pair singlets (δH 7.44 s and 7.41 s), and the hydroxy group to a methoxy group at C-4ʹ. Moreover, the HMBC correlations (Fig. 2) from H-3″ to C-7, from H-8 to C-2″ suggested that the 4-methylfuran-2-yl moiety was located at C-7. The HMBC correlations from two methoxy protons (δH 3.77 and 3.80) to C-6 and C-4ʹ indicated that two methoxy groups were attached to C-6 and C-4ʹ, respectively. The structure of 3 was therefore defined.

6-Prenyl-8-methoxy-4ʹ-hydroxy-flavone (4) was obtained as a pale-yellow powder, and which had the molecular formula of C21H20O4 based on the HRESI-MS data. Detailed NMR spectroscopic analyses indicated that the structural differences between 2 and 4 were resulted from the replacement of a 4-methylfuran-2-yl moiety to a prenyl group [–CH2CH=(CH3)2, C-2″–C-6″, H2-2″, H-3″, H3-5″, and H3-6″] [45]. Therefore, compound 4 was elucidated as 4ʹ-hydroxy-8-methoxy-6-prenyl-flavone.

The HRESI-MS of compound 5 showed an [M+Na]+ ion peak at m/z 375.1205, and correlated with a molecular formula of C21H20O5. The UV, IR and NMR spectral data of 5 were highly similar to these of 4, except that 5 contained an additional phenolic hydroxy proton (δH 9.79 s) and disappeared an aromatic proton on ring-A. This indicated that a hydroxy group should be substituted on the A-ring of 5. The HMBC correlations (Fig. 2) from hydroxy proton (δH 9.79 s) to C-6/C-7/C-8 revealed that the hydroxy group located C-7. Thus, the structure of 5 was elucidated as a new 7-hydroxy analogue of 4.

Compound 6 was obtained as a pale-yellow powder. Its molecular formula was deduced as C21H18O5 by HRESI-MS [M+Na]+ 373.1049 (calcd C21H18NaO5 for 373.1046). Its spectral data were comparable to those of 5, except that the prenyl group was converted to a gem-dimethylchromene moiety (–CH=CH–C(CH3)2–O–; C-2″–C-6″; H-2″, H-3″, and H6-5″,6″) [46]. This can be deducted that the prenyl should be connected to ring-A by an oxygen atom to form a gem-dimethylchromene ring. Moreover, two mass units less than that of 5 in MS data were also supported this deduction. Long-range correlations (Fig. 2) from H-2″ to C-5/C-6/C-7, from H-3″ to C-6, and from H-5 to C-2″ were observed. These supported that the gem-dimethylchromene moiety was fused at C-6 and C-7, and C-2″ was linked to ring-A. Hence, compound 6 was determined and systematically named as 6-(2,2-dimethyl-2H,6H-pyrano[3,2-g])-4ʹ-hydroxy-8-methoxy-flavone.

The possible biogenetic pathway of compounds 1–6

The possible biogenetic pathway of compounds 1–6 was proposed as shown in Fig. 3. In plants, flavones are synthesized by the flavonoid pathway, which is part of phenylpropanoid metabolism [47]. For the biosynthesis of flavonoids, the phenylalanine was converted into coumarin-CoA through the phenylpropane pathway, then the coumarin-CoA enters the flavonoids synthesis pathway and combines with 3 molecules of malonyl-CoA to form chalcones. After this, the dihydroflavones were generated by an intramolecular cyclization reaction, and the dihydroflavones are the main precursor of other flavonoids [48]. The dihydroflavones and isoprenyl-CoA could be converted to the isoprenylflavones by isopentenylation reactions [49]. Then, compounds 1–6 should be derived from the isoprenylflavones by a series of oxidation, amino substitution, and epoxy reactions on pentenyl side chains, also along with the hydroxylation and methoxylation reactions on flavone nucleus.

Fig. 3
figure 3

The possible biogenetic pathway of compounds 1–6. PAL, phenylalanine ammonia lyase; C4H, cinnamic acid 4-hydroxylase; 4CL, 4-coumarate; malonyl-Co A, malonyl-coenzyme A; CHS, chalcone synthase; CHI, chalcone isomerase; FNS, plant flavonoids synthase; PT, prenyltransferases; DH, dehydroxylase; OMTs, O-methyltransferases; FH, flavone hydroxylase

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Antifungal (G. cichoracearum) activity assays

Since certain of the flavonoids exhibit potential antifungal activities [50, 51], and the fungus G. cichoracearum (DC.) is the main pathogen of tobacco powdery mildew disease [52], Compounds 1–11 were tested for their anti-G. cichoracearum (DC.) activities.

The antifungal activity was tested according to previous literature [10, 32], and carbendazim was used as a positive control. The results (Table 3, Additional file 1: Figure S31) revealed that 1 showed high anti-G. cichoracearum (DC.) activity with an inhibition rate of 88.3% ± 6.2. This rate is higher than that of the positive control (with an inhibition rate of 81.5% ± 6.3). By compared to the negative control, compounds 2–11 also showed notable anti-G. cichoracearum activities with inhibition rates in the range of 50.9%–72.0%.

Table 3 The inhibition G. cichoracearum effects of compounds 1–11 on tobacco leaf
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The IC50 values of compounds 1–11 were also tested. The results (Table 3) revealed that 1 exhibited IC50 value of 51.5 µg/mL. The efficiency was higher than that of carbendazim (with IC50 value of 70.3 µg/mL). Compounds 2–11 also exhibited IC50 values in the range of 106–189 µg/mL. By treated N. tabacum cv. HD with different concentrations of 1, the results in Additional file 1: Figure S32 revealed that the inhibition rates were increased with the increase of the concentrations of 1, and showed a good dose–effect relationships for the infected G. cichoracearum on tobacco leaves.

Since the inhibition rates of 1, 2, 8 and 9 are higher than 70%, the protective effects of 1, 2, 8 and 9 on G. cichoracearum were also evaluated. In the protective assay, the tobacco plants were treated with the solutions of compounds (250 µg/mL). After 24 h of treatment, the G. cichoracearum was inoculated, and the incidences were count at day 7. The results (Additional file 1: Figure S33) revealed that 1 showed higher protective effect on the host plants with inhibition rate of 90.2% ± 6.4, and this rate is higher than that of positive control (84.2% ± 6.2). By comparing to the negative control, compounds 2, 8 and 9 also showed good protective effects for N. tabacum cv. HD which infected with G. cichoracearum. These results indicated that the pretreatment of host plants with compounds 1, 2, 8 and 9 were markedly increased their resistances to G. cichoracearum infection.

The curative effects for 1 was also tested on N. tabacum cv. HD. In this experiment, compound 1 (250 µg/mL) was sprayed onto the tobacco seedlings which had been infected with powdery mildew disease, and the infected seedlings without sprayed the compound was used as a negative control. The results (Fig. 4) revealed that the powdery mildew was markedly alleviated over time after spraying with 1. Compared to the negative control (Fig. 4a), after 24 h of spraying (Fig. 4b), the spot had obviously atrophied. After day 3 of spraying (Fig. 4c), the obvious disappearance of disease spots had been observed, and no obvious spots observed on newly grown leaves. After day 7 of spraying (Fig. 4d), the disease spots had almost completely disappeared. As compared to the negative control at the same stage (Fig. 4h), the growth of tobacco seedlings was normal and vigorous. For the tobacco seedlings without spraying with 1 (Fig. 4e–h), no obvious changes were observed in powdery mildew, and the disease spots were obviously shown in the newly grown leaves. In addition, the growth of infected seedlings was also slower than that of the 1 treated group. These results indicated that 1 has a good therapeutic effect for powdery mildew disease.

Fig. 4
figure 4

The curative effects for compound 1 on infected N. tabacum cv. HD. a Tobacco seedling infected with G. cichoracearum; b–d the growth of infected tobacco seedling at day 1, day 3, day 5, and day 7, after treated with 250 µg/mL of compound 1 in 0.1% Tween-20 solution; e control (tobacco seedling infected with G. cichoracearum), f–h the growth of control at day 1, day 3, day 5, and day 7, after treated with 0.1% Tween-20 solution

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The mechanism studies

Based on the above studies, 1 has the most significant activity, and the activity of 1 may involve the protective and curative effects, as well as the induction of plant resistances. Thus, the direct action of 1 on G. cichoracearum were observed through microscope. The results showed that when treated with 1, the conidiospores were seriously shrunken (Fig. 5). It was clearly seen that after 24 h of spraying with 1, most of the conidiospores were shrunken and some of the spore walls broke. In addition, the internal structures of the spores were also significantly deformed. After sprayed at day 3 (Fig. 5c), the conidiospores were further shrunken and deformed, and the normal spores were almost invisible. The above information indicted that a potent direct effect on conidiospores might be the reason for 1 against G. cichoracearum with significant effects.

Fig. 5
figure 5

The effects of compound 1 on the conidiospores of G. cichoracearum under light microscope. a–c 10 × 40 magnification; d–f 16 × 100 magnification. a, d Untreated with compound 1 (control); b, e after treated with 250 µg/mL of compound 1 at day 3; c, f after treated with 250 µg/mL of compound 1 at day 5

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In previous studies, the binding modes between small molecule metabolites and tubulin (G. cichoracearum) proteins were commonly used to evaluate the fungicides against powdery mildew activities [53, 54]. To further understanding the binding modes of 1–11 with tubulin proteins, the docking analysis was performed between 1–11 and the proteins. The docking result of 1 is shown in Fig. 6, and results of 2–11 are shown in Additional file 1: Figures S34 and S35.

Fig. 6
figure 6

The binding modes (a and b) of 1 with tubulin (G. cichoracearum) protein. Key residues are represented as stick models; hydrogen bonds are depicted as dotted yellow lines and pi–pi stacking interaction is depicted as dotted blue lines

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By the docking poses analyses, 1–11 showed the similar docking scores to the original ligands in the crystal structure, and can strongly interact with the catalytic pocket on tubulin. This is the fundamental for anti-G. cichoracearum activity. For structure–activity relationships, the –C=O on nucleus of 1 can forms a hydrogen bond with Ser243, and form a π–π stacking with Phe257. The –NH on pyrrole ring can form a hydrogen bond with Ile240, while 2–4 lose the hydrogen bond with Ile240, and resulting in a decrease of activities. Moreover, the –OMe group on 5 has a clash with Leu261, resulting in a further decrease of activity. Compounds 6–10 also lost the hydrogen bond of Ile240, and this may cause the weaker activities when compared with 1. The spatial clash with Phe257 may result in a further decrease in activity for 11. These docking results were consistent with the above in vitro antifungal experiment.

The docking results reveal that flavonoids nucleus can interacted with Tubulin proteins, and this maybe the fundamental for direct effect on G. cichoracearum and play the disease resistances. In addition, the 4-methyl-1H-pyrrol-2-yl moiety substituted on flavone can notably increases the activity. This structure–activity relationship is proposed by our work in natural product for the first time, and it is helpful to find new antifungal activities inhibitors.

Since the activities of defense enzymes (PAL, POD, SOD, PPO, CAT, and PAX) are significantly related to plant resistances [55, 56], the activities of six enzymes in 250 µg/mL of 1 treated tobacco leaves were also analyzed. As shown in Fig. 7, the activities of six enzymes for the T+G, C+G, and 1+G groups were higher than those of in mock group. Notably, the PAL, CAT, and PAX activities had the most obvious change. In Fig. 7b, the PAL activity of 1+G treated group in Day 5 had the highest activity; it was 2.78-fold higher than that of the mock group, 2.09-fold higher than that of the T+G group, and 2.29-fold higher than that of the C+G group. PAL is involved in the conversions of phenylpropanoids to cinnamic acid and can produce SA for defense against pathogens. The significantly induced PLA activity may be a cause of increased resistances. In Fig. 7e, the CAT activity of 1+G treated group in day 3 had the highest activity. After fungal infection, the activity of CAT was significant increased. However, compared with the T+G group, a marked decrease in CAT activity was observed. As an important redox marker, CAT is activated to scavenge any resulting reactive oxygen species (ROS), and reduces hydrogen peroxide to oxygen and water. The results suggested that upon powdery mildew infection, the marked decreasing of CAT activity is critical for tobacco to maintain a balance ROS concentration, and therefore enhance resistance. In Fig. 7f, the APX activity of 1+G treated group had the highest activity in Day 5. It was 1.74-fold higher than that of the T+G group. APX plays a very important role in physiological processes, such as plant growth and development and stress response. Especially when plants are subjected to stress, APX can quickly remove excess H2O2 produced in cells, and protect the plant cells from the damage caused by reactive oxygen species.

Fig. 7
figure 7

The activities of SOD (a), PAL (b), POD (c), PPO (d), CTA (e) and PAX (f) in tobacco which treated with 250 μg/mL of compound 1. Mock: healthy tobacco (negative control); T+G: G. cichoracearum in 0.1% of Tween-20 solution; C+G: 250 μg/mL of carbendazim in 0.1% of Tween-20 solution. 1+G: 250 μg/mL of compound 1 in 0.1% of Tween-20 solution. All results are expressed as the average value of three determinations for all groups

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Salicylic acid (SA) and jasmonic acid (JA) are also natural plant defense hormones against pathogens [57, 58]. Since PAL can induce SA and JA, the SA and JA contents were also determined in tobacco plants. The results (Fig. 8a, b) indicated that after treated with 1, the contents of SA and JA in tobacco plants were increased. The changes of SA content were more significant than JA. In 1+G group, the SA contents reached a peak at day 3, and the decreased gradually from day 3 to day 7. The variation trends of 1+G treated group were surpassed than those of in C+G and T+G treated groups. Hence, pretreated with 1 might be notably increase the SA contents and enhance plant resistance to diseases.

Fig. 8
figure 8

The effect of compound 1 (250 μg/mL) on SA (a), JA (b), MDA (c), and CHL (d) accumulations in tobacco leaves. Mock: healthy tobacco (negative control); T+G: G. cichoracearum in 0.1% of Tween-20 solution; C+G: 250 μg/mL of carbendazim in 0.1% of Tween-20 solution. 1+G: 250 μg/mL of compound 1 in 0.1% of Tween-20 solution. All results are expressed as the average value of three determinations for all groups

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As a physiological indicator of cell membrane damage and an indicator of lipid peroxidation [59], the malondialdehyde (MDA) content can reflect the degrees of cell membrane lipid peroxidation and the strength of plants’ response to stress conditions. When the disease-resistant substances are used to treat the infected plants, they can inhibit the increase of MDA content in the plants, thus producing a protective effect. Therefore, the MDA contents were analyzed. As can be seen from Fig. 8c, the changes of MDA content in mock group is not obvious and remains at a low level. In T+G group, the contents of MDA were significantly increased. By contrast with T+G group, the content of MDA was significantly inhibited after treated with 1, which was also better than that of C+G group. This result showed that 1 can inhibit the increase of MDA content in tobacco leaves, thereby reducing the extent of plant damage caused by fungal infections.

In addition, chlorophylls are the major components of chloroplasts, they play an important role in photosynthesis and can provide energy for plant growth [60]. As depicted in Fig. 8d, the chlorophyll contents of tobacco leaves were decreased gradually from day 1 to day 7 after inoculated with G. cichoracearum. In contrast, after treated with 1, the chlorophyll contents were increased from 2.10 to 2.82 mg/g from day 1 to day 7. This indicated that treated with 1 can enhanced the photosynthetic ability of the leaves, thus improved the resistances of tobacco.

The plant resistance to fungus is closely related to the expression levels of plant defense genes. PAL gene is closely related to the formation of antibacterial functional products in the phenylpropanoid metabolism pathway, and strength of resistance to powdery mildew [61]. Chit1 gene can express chitinase, and effectively degrade the chitin components in the cell walls of higher fungi, thereby can inhibit or kill various plant pathogens [62]. PR-1 and PR-5 (the marker genes for plant disease resistances) play the functions of attack pathogens, degrade cell wall macromolecules, and degrade pathogenic toxins [63]. Therefore, their expressions of above four genes in 1+G group were examined. The results are shown in Fig. 9. For the CK− and mock groups, the changes of expression levels were not so obvious. However, in 1+G and C+G groups, the notable up-regulation of AL, Chit1, PR-1 and PR-5 were obtained. In 1+G group, the strongest expression levels at day 3 for PAL, Chit1, PR-1 (Fig. 9a, c, d), and at day 5 (Fig. 9b) for PR-5 were observed. These results revealed that when treated with 1, the notable up-regulation of defense-related genes also might be the causes of enhance disease resistance.

Fig. 9
figure 9

The changes of the transcriptional levels for PR-1 (a) and PR-5 (b), PAL (c), and Chit-1 (d) gene in tobacco leaves treated with 250 μg/mL of compounds. Mock: healthy tobacco (negative control); T+G: G. cichoracearum in 0.1% of Tween-20 solution; C+G: 250 μg/mL of carbendazim in 0.1% of Tween-20 solution. 1+G: 250 μg/mL of compound 1 in 0.1% of Tween-20 solution. All results are expressed as the average value of three determinations for all groups

Full size image

Based on the above mechanistic studies, the mode of actions of compound 1 against G. cichoracearum may be involved in the potent direct effects on the conidiospores of G. cichoracearum; and also trigger several plant defense responses to induce the systemic acquired resistance (SAR) for the tobacco. Thus, lead to pathogen suppression and resistance to powdery mildew.

Conclusion

In this study, six new (1–6), along with five known (7–11) prenyl flavones were isolated from the twigs of A. champeden. Compound 1 is the first example of flavone bearing a 4-methyl-1H-pyrrol-2-yl functional group, and 2 and 3 are rare flavones bearing an unusual 4-methylfuran-2-yl moiety. The frameworks of above three flavones are reported in natural products by our group for the first time. Interestingly, 1 showed high activity with inhibition rate of 88.3% ± 6.2. This rate is higher than that of positive control (with inhibition rate of 81.5% ± 6.3). Compared to the negative control, 2–11 also showed potential activities with inhibition rates in the range of 50.9%–72.0%. Flavonoids are potentially involved in plant resistances to biotic stresses, and they also had been identified as promising antifungal agents. However, our studies firstly reported that the prenylated flavonoids had significant effects against tobacco powdery mildew. The successful isolation and structure identification of the above prenylated flavonoids provide a new source of antifungal agents for the control of tobacco powdery mildew.

The mechanism studies also revealed that the mode of action of 1 on G. cichoracearum involved in the spoiling the conidiospores, and accompanied by inducing the actives of defense enzymes (PAL, CAT, and PAX), adjusting the plant hormone (SA, JA, MDA, and CHL), up-regulating the expression of defense-related genes in tobacco plant. This is also helpful for the further discovery of antifungal pesticides.

In addition, prenylated flavonoids are characterized by the presence of a prenylated side-chain, and the bioactivities of routine flavonoids can be increased by prenylation. Our study confirmed that the twigs of A. champeden are a rich source of prenylated flavonoids. A. champeden has the characteristics of rapid growth and high biological yield, which can provide the cheaper raw material sources for the extraction and utilization of prenylated flavonoids. Thus, this study also provides the beneficial proof for expanding the utilization of A. champeden resources.

Availability of data and materials

The additional materials used for this study are available in Additional file 1.

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Acknowledgements

We appreciate the Kunming Institute of Botany, Chinese Academic Sciences, for use of the Schrodinger Suite software.

Funding

This work was supported financially by the National Natural Science Foundation of China (32260111), the Foundation of China Tobacco Monopoly Bureau (110202101011(XJ-03)), the Foundation of Yunnan Basic Research Youth Program (2019FD119), the Candidates of the Young and Middle Aged Academic Leaders of Yunnan Province (2019HB016), and the Foundation for Key Laboratory of Yunnan Provincial Department of Education (2022HD021).

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Authors and Affiliations

  1. Key Laboratory of Natural Products Synthetic Biology of Ethnic Medicinal Resources, State Ethnic Affairs Commission, Yunnan Minzu University, Kunming, 650504, People’s Republic of China

    Qiu-Fen Hu, Sui Yao, Yue-Yu Ma, Rui-Feng Xiong, Miao Dong, Wei-Guang Wang, Min Zhou & Yin-Ke Li

  2. Yunnan Academy of Tobacco Agricultural Sciences, Kunming, Yunnan, 650021, People’s Republic of China

    Guang-Hui Kong, Yu-Ping Wu & Gao-Kun Zhao

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  1. Qiu-Fen Hu
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Contributions

QH, MZ, YL, and WW designed the experiment. MD and YL prepared the samples. SY, YM, GK, RX, YW, QH, MZ, MZ, YL and GZ performed the experiments, analyzed data and wrote the paper. QH, YL, MZ, and WW reviewed and checked all the details. All authors read and approved the final manuscript.

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Correspondence to Min Zhou or Yin-Ke Li.

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Supplementary Information

Additional file 1: Figures S1

–S5. 13C, DEPT, 1H, HSQC, HMBC NMR, and HRMS spectra of 1. Figures S5–S10. 13C, DEPT, 1H, HSQC, HMBC NMR, and HRMS spectra of 2. Figures S11–S15. 13C, DEPT, 1H, HSQC, HMBC NMR, and HRMS spectra of 3. Figures S16–S20. 13C, DEPT, 1H, HSQC, HMBC NMR, and HRMS spectra of 4. Figures S21–S25. 13C, DEPT, 1H, HSQC, HMBC NMR, and HRMS spectra of 5. Figures S26–S30. 13C, DEPT, 1H, HSQC, HMBC NMR, and HRMS spectra of 6. Figure S31. The inhibitory effects of 1–11 on G. cichoracearum (DC.). Figure S32. The dose–effect relationships of compound 1 for G. cichoracearum. Figure S33. The protective effects of compounds 1, 2, 8 and 9 on tobacco plant. Figure S34. The binding modes of compounds 2–7 and Tubulin (G. cichoracearum) protein. Figure S35. The binding modes of compounds 8–11 and Tubulin (G. cichoracearum) protein. S2. Anti-fungi activity assays. S3. Quantitative real-time PCR analysis of defense-related genes. Table S1. The primer pairs.

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Hu, QF., Yao, S., Ma, YY. et al. Prenylated flavonoids isolated from the twigs of Artocarpus champeden as potential activators for tobacco powdery mildew and their mode of actions. Chem. Biol. Technol. Agric. 10, 94 (2023). https://doi.org/10.1186/s40538-023-00457-w

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