Wuchuyuamines A-G, seven new alkaloids isolated from the fruits of Tetradium ruticarpum and their antifungal activities

Abstract

Plant diseases caused by fungal pathogens severely threaten crop yields and food security, driving the need for new, environmentally friendly fungicides. In this study, thirty-six alkaloids, including seven novel ones (1–4, 23, 29, and 30), were isolated from the fruits of Tetradium ruticarpum, and their structures were elucidated by extensive spectroscopic analyses. The antifungal potential of compounds obtained with sufficient quantities was evaluated using the mycelial growth rate method against three phytopathogens (Alternaria solani, Bipolaris sorokiniana, and Fusarium odoratissimum). Compounds 16 and 24 showed notable activity against A. solani, exhibiting approximately 80% and 90% growth inhibition at 100 μg/mL, with IC₅₀ values of 33.39 and 36.02 μg/mL, respectively. To evaluate the antifungal spectrum, the abundant constituents 13, 19, and 25 were further screened against sixteen phytopathogenic fungi. Among them, compound 25 displayed the most potent and broad-spectrum antifungal activity, achieving an IC₅₀ of 13.15 μg/mL against Fusarium graminearum, which was stronger than the positive control hymexazol. Furthermore, SEM and TEM analyses revealed that compound 25 markedly disrupted the surface morphology and cellular ultrastructure, suggesting a potential link to membrane integrity impairment. Additionally, leakage assays showed increased release of nucleic acids and proteins, providing evidence that compound 25 increased membrane permeability and compromised the membrane integrity in F. graminearum. Therefore, compound 25 represents a promising lead structure for the development of novel, eco-friendly fungicides.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1007/s13659-026-00610-6.

Introduction

Plant pathogens cause a wide range of diseases in fruits, vegetables, and other crops, resulting in substantial yield losses and posing a major threat to global agricultural production and food security. Approximately 8000 species of fungi and oomycetes have been reported to be associated with plant diseases [1, 2]. Among fungal pathogens, Magnaporthe oryzae, Botrytis cinerea, Puccinia spp., Fusarium graminearum, and Fusarium oxysporum are the most scientifically and economically important fungal pathogens [3]. Chemical control remains the most widely used approach for managing plant diseases. However, the excessive and long-term application of synthetic fungicides has led to multiple problems, including environmental pollution, potential health risks to humans and animals, and the emergence of fungicide-resistant pathogens [4]. These issues have prompted an urgent demand for safer and more sustainable alternatives.

To meet this demand, the development of plant-derived natural products as sources of fungicidal agents is consistent with future-oriented food and agricultural policies [5]. Among these natural products, alkaloids, a class of nitrogen-containing heterocyclic compounds, have garnered increasing attention due to their relatively low mammalian toxicity, ready biodegradability, and environmental compatibility [6, 7]. Tetradium ruticarpum (A. Juss.) T. G. Hartley (Rutaceae family, Tetradium genus), known as Wu Zhu Yu in traditional Chinese medicine, has been widely used for centuries to treat ailments such as headache, abdominal pain, and vomiting [8]. Phytochemical investigations have revealed that T. ruticarpum contains diverse bioactive constituents, including alkaloids, limonoids, and flavonoids [9–11]. To date, approximately 300 metabolites have been reported in T. ruticarpum, about half of which are alkaloids [12]. Pharmacological studies further indicate that these constituents exhibit diverse biological activities, such as vasodilatory, anti-inflammatory, analgesic, and antibacterial effects [13, 14]. Nevertheless, the antifungal potential of alkaloids derived from T. ruticarpum remains largely unexplored.

In this study, we aimed to systematically isolate and identify the alkaloids from the fruits of T. ruticarpum and evaluate their antifungal activities against phytopathogenic fungi. A total of thirty-six alkaloids were obtained (Fig. 1), including seven new ones (1–4, 23, 29, and 30) and twenty-nine known alkaloids: rutaecarpine (5) [15], 3-hydroxyrutaecarpine (6) [16], evodiamine (7) [15], 3-hydroxyevodiamine (8) [17], N¹⁴-formyldihydrorutaecarpine (9) [18], evodamide A (10) [19], evollionines A (11) [20], goshuyuamide I (12) [21], 1,2,3,4-tetrahydronorharman-1-one (13) [22], goshuyuamide II (14) [21], 3-(2-(1H-indol-3-yl)ethyl)quinazolin-4(3H)-one (15) [23], N-(2-methylaminobenzoyl)tryptamine (16) [24], evodiamide (17) [25], wuchuyuamide II (18) [26], wuchuyuamide I (19) [26], evollionines B (20) [20], atanine (21) [27], edulinine (22) [28], 2-heptyl-1-methyl-4(1H)-quinolinone (24) [29], evocarpine (25) [30], 1-methyl-2-[(Z)-7-tridecenyl]-4(1H)-quinolinone (26) [31], euocarpine A (27) [32], euocarpine B (28) [32], wuchuyuamide III (31) [33], wuchuyuamide IV (32) [34], skimmianine (33) [35], sinometumine I (34) [36], evodiamide A (35) [37], and evodiamide B (36) [37]. Their structures were elucidated based on extensive spectroscopic analyses. Compounds 8, 15, 22, and 34 were reported from T. ruticarpum for the first time. This report described the isolation and structural elucidation of all alkaloids obtained from T. ruticarpum, and the antifungal activities of selected compounds were further assessed.

Fig. 1.

The structures of compounds 1–36

Results and discussion

Structure elucidation and identification

Wuchuyuamine A (1) was acquired as a yellow amorphous powder and its molecular formula C₁₉H₂₁N₃O₂ was determined by HRESIMS at m/z 346.1533 [M + Na]⁺ (calcd. for C₁₉H₂₁N₃O₂Na, 346.1526) with 11 degrees of unsaturation. The ¹H NMR data (CD₃OD, 500 MHz; Table 1) of compound 1 revealed two 1,2-disubstituted aromatic rings [δ_H 7.49 (1H, d, J = 7.8 Hz, H-9), δ_H 6.98 (1H, ddd, J = 8.0, 7.0, 1.1 Hz, H-10), δ_H 7.06 (1H, ddd, J = 8.1, 7.1, 1.2 Hz, H-11), δ_H 7.29 (1H, m, H-12); δ_H 6.68 (1H, d, J = 8.4 Hz, H-16), δ_H 7.29 (1H, m, H-17), δ_H 6.56 (1H, dd, J = 8.0, 7.0 Hz, H-18), δ_H 7.46 (1H, dd, J = 7.9, 1.6 Hz, H-19)], three aliphatic methylene signals [δ_H 4.66 (2H, s, H-3), δ_H 3.78 (2H, t, J = 6.8 Hz, H-5), δ_H 3.03 (2H, t, J = 6.8 Hz, H-6)], and an N-CH₃ signal [δ_H 2.83 (3H, s, N-CH₃)]. In addition, the ¹³C NMR, DEPT, and HSQC data showed seven quaternary carbon signals, including six aromatic carbon signals [δ_C 133.9 (C-2), 110.4 (C-7), 129.4 (C-8), 137.4 (C-13), 151.6 (C-15), 116.9 (C-20)] and an amide carbon signal [δ_C 172.2 (C-21)]. These data indicated that compound 1 was an indolequinazoline alkaloid. The structure could be determined through further 2D NMR analysis. In the ¹H-¹H COSY spectrum, the cross peak of H₂-5/H₂-6 confirmed the presence of a CH₂CH₂ alkyl fragment. The HMBC correlations from H₂-5 to C-7 and from H₂-6 to C-2/C-7/C-8 indicated that C-7 on the indole ring was connected to the CH₂CH₂ alkyl fragment. The HMBC correlations from H₂-3 to C-2/C-7/C-21 demonstrated that the quinazoline unit was connected to the indole ring through C-3 (Fig. 2). Along with the molecular formula of compound 1 and the chemical shift of C-5 (δ_C 63.4) suggested that C-5 was connected to a hydroxyl group. Comparing with the structure of goshuyuamide I (12) [21], the notable difference was that 1 was a C-seco indolequinazoline alkaloid with a 5-hydroxyl group. Therefore, the structure of compound 1 was established as depicted in Fig. 1.

Table 1.

¹H NMR and ¹³C NMR spectroscopic data for 1–4 (δ in ppm, J in Hz)

No	1a		2b		3c		4a
	δH	δC	δH	δC	δH	δC	δH	δC
2		133.9		179.8	7.08 (1H, s)	124.5		132.4
3	4.66 (2H, s)	36.0			4.21 (2H, s)	68.1	5.98 (1H, s)	67.9
5	3.78 (2H, t, 6.8)	63.4	3.58 (2H, m)	37.8	3.80 (2H, t, 7.0)	48.5	3.80 (2H, m)	63.3
6	3.03 (2H, t, 6.8)	28.5	2.18 (2H, m)	31.3	3.08 (2H, t, 7.0)	25.0	3.06 (2H, m)	28.4
7		110.4	3.53 (1H, d, 6.0)	44.8		113.0		113.9
8		129.4		130.7		129.1		128.8
9	7.49 (1H, d, 7.8)	119.2	7.40 (1H, d, 7.4)	125.1	7.09 (1H, d, 2.4)	101.2	7.58 (1H, d, 8.0)	119.8
10	6.98 (1H, ddd, 8.0, 7.0, 1.1)	119.7	7.00 (1H, dd, 7.6, 7.5)	122.5		155.0	7.04 (1H, ddd, 8.0, 6.9, 1.0)	120.1
11	7.06 (1H, ddd, 8.1, 7.1, 1.2)	122.4	7.20 (1H, dd, 7.8, 7.6)	128.6	6.74 (1H, dd, 8.7, 2.4)	112.9	7.14 (1H, ddd, 8.2, 7.0, 1.1)	123.7
12	7.29 (1H, m)	112.0	6.92 (1H, d, 7.7)	110.2	7.22 (1H, d, 8.7)	112.9	7.36 (1H, d, 8.2)	112.6
13		137.4		143.5		133.3		138.1
15		151.6		151.7		151.2		151.0
16	6.68 (1H, d, 8.4)	112.0	6.64 (1H, d, 8.3)	111.4	6.72 (1H, d, 8.4)	113.3	6.91 (1H, m)	114.0
17	7.29 (1H, m)	133.8	7.27 (1H, dd, 8.6, 7.1)	133.2	7.40 (1H, dd, 8.3, 7.2)	134.9	7.48 (1H, dd, 7.3, 1.7)	135.8
18	6.56 (1H, dd, 8.0, 7.0)	115.9	6.52 (1H, dd, 7.9, 7.1)	114.8	6.86 (1H, dd, 7.5, 7.5)	119.6	6.91 (1H, m)	119.5
19	7.46 (1H, dd, 7.9, 1.6)	129.3	7.54 (1H, d, 7.9)	128.6	7.83 (1H, d, 7.7)	129.3	7.88 (1H, dd, 7.9, 1.7)	129.2
20		116.9		116.1		118.6		117.2
21		172.2		170.4		165.6		166.4
N-CH3	2.83 (3H, s)	29.9	2.82 (3H, s)	30.0	2.63 (3H, s)	35.6	2.65 (3H, s)	34.2
OCH3					3.72 (3H, s)	56.2

^a500 MHz in CD₃OD; 125 MHz in CD₃OD

^b600 MHz in (CD₃)₂CO; 150 MHz in (CD₃)₂CO

^c600 MHz in CD₃OD; 150 MHz in CD₃OD

Fig. 2.

Key ¹H–¹H COSY and HMBC correlations of compounds 1–4, 23, 29, and 30

Wuchuyuamine B (2) was obtained as a white amorphous powder. Its molecular formula was determined as C₁₈H₁₉N₃O₂ based on HRESIMS ion peak at m/z 310.1547 [M + H]⁺ (calcd. for C₁₈H₂₀N₃O₂, 310.1550). The ¹H NMR (CD₃OD, 600 MHz; Figure S12.1) signals of compound 2 were significantly broadened, rendering the spin–spin coupling constants unmeasurable and thereby complicating the structural analysis. Therefore, derivatization of compound 2 was attempted by using chemical approach with hydrogen chloride-methanol solution. After converting 2 into the wuchuyuamine B chloride, the signal broadening disappeared, and coupling constants could be easily measured. The 1D NMR data (¹H NMR, 600 MHz; ¹³C NMR, 150 MHz), recorded in (CD₃)₂CO (see Table 1), indicated that compound 2 had a high degree of structural similarity to N-(2-methylaminobenzoyl)tryptamine (16) [24]. However, compound 2 had an indolinone unit rather than an indoline unit due to the signals of the indole ring showed significant changes compared with those in N-(2-methylaminobenzoyl)tryptamine (16), particularly the downfield shift of C-2 (δ_C 179.8, Δδ + 56.3 ppm) and the upfield shift of C-7 (δ_C 44.8, Δδ—68.7 ppm). Furthermore, the HMBC correlations from H-7 to C-2/C-5/C-13 were observed, indicating that the double bond placed at C-2 and C-7 in N-(2-methylaminobenzoyl)tryptamine (16) was replaced by a carbonyl group in compound 2 (Fig. 2). Although compounds N-(2-methylaminobenzoyl)tryptamine (16) and 2 had similar structures, the ¹H NMR spectrum of N-(2-methylaminobenzoyl)tryptamine (16) did not exhibit broadening signals. The broadening signals of the ¹H NMR spectrum of compound 2 were attributed to the existence of an amide moiety, and the 4-NH as an active hydrogen capable could form the intermolecular hydrogen bond with the carbonyl group at C-2. In addition, the specific rotation value of compound 2 was close to zero. Combined with ECD experiments showing no obvious Cotton effect (Figure S17) and the presence of only one chiral center at C-7, compound 2 was determined to be a racemic mixture, and named wuchuyuamine B.

Wuchuyuamine C (3) was obtained as a white amorphous powder. Based on the HRESIMS ion peak at m/z 358.1529 [M + Na]⁺ (calcd. for C₂₀H₂₁N₃O₂Na, 358.1526), its molecular formula was determined as C₂₀H₂₁N₃O₂. Analysis of the ¹H and ¹³C NMR data (600 and 150 MHz, respectively, in CD₃OD; Table 1) for compound 3 revealed a similar structure to 10-methoxygoshuyuamide-II [19]. The molecular weight of compound 3 was 14 less than that of 10-methoxygoshuyuamide-II, and this difference was due to the presence of a carbonyl substitution at C-3 in 10-methoxygoshuyuamide-II. The HMBC correlations from H₂-3 to C-5/C-15/C-21/N–CH₃, and from H₂-5 to C-3/C-7/C-21 confirmed the above speculation (Fig. 2), and therefore the structure of compound 3 was determined as wuchuyuamine C.

Wuchuyuamine D (4) was isolated as a white amorphous powder. The molecular formula C₁₉H₁₉N₃O₂ was determined by HRESIMS ion peak at m/z 322.1554 [M + H]⁺ (calcd. for C₁₉H₂₀N₃O₂, 322.1550). The ¹H NMR (500 MHz, CD₃OD) and ¹³C NMR (125 MHz, CD₃OD) data (Table 1) displayed signals corresponding to indole and quinazoline rings, indicating that it was an indolequinazoline alkaloid with a similar skeleton to evodiamine (7) [15]. Comparison with the ¹³C NMR data of evodiamine (7) suggested that C-5 (δ_C 63.3) and C-6 (δ_C 28.4) in compound 4 were shifted downfield and the chemical shifts were close to C-5 (δ_C 63.4) and C-6 (δ_C 28.5) in compound 1. The HMBC spectrum of compound 4 also lacked the correlations from H-5 to C-3/C-21 (Fig. 2). Similar to compound 1, the C ring of compound 4 was broken with the bond disruption of the C-5 and N-4, and a hydroxyl group was linked to C-5. The specific rotation was close to zero, and the experimental ECD spectra showed no obvious Cotton effect (Figure S34). Therefore, compound 4 was determined to be a racemic mixture, the structure was depicted as shown in Fig. 1.

Wuchuyuamine E (23) was obtained as a yellow oil. Its molecular formula was determined to be C₂₅H₃₁NO₃ based on the HRESIMS ion peak at m/z 394.2380 [M + H]⁺ (calcd. for C₂₅H₃₂NO₃, 394.2377). The UV spectrum showed absorption maxima at 215, 239, 322, and 335 nm, which were characteristic of the N-methyl-4(1H)-quinolone skeleton, indicating that compound 23 was a quinolone alkaloid. The ¹H and ¹³C NMR data (500 and 125 MHz, respectively, in CD₃OD; Table 2) also displayed characteristic signals of the N-methyl-4(1H)-quinolone skeleton [δ_H 6.31 (1H, s, H-3), 8.32 (1H, dd, J = 8.1, 1.5 Hz, H-5), 7.47 (1H, ddd, J = 7.9, 6.8, 1.1 Hz, H-6), 7.80 (1H, ddd, J = 8.7, 6.8, 1.6 Hz, H-7), 7.86 (1H, dd, J = 8.8, 1.3 Hz, H-8), 3.89 (3H, s, N-CH₃); δ_C 158.8 (C-2), 111.1 (C-3), 179.5 (C-4), 126.9 (C-4a), 126.6 (C-5), 125.1 (C-6), 133.9 (C-7), 117.9 (C-8), 143.5 (C-8a), 35.5 (N-CH₃)]. Combined with the ¹H-¹H COSY and HSQC, the signals of the aliphatic side chain were assigned as [δ_C 35.6, 29.5, 30.2, 29.9, 28.2, 133.1, 126.0, 36.0,72.5, 148.0, 128.9, 145.3, 131.1, 201.4, 27.1 (C1′-C15′)]. In the ¹H-¹H COSY spectrum, the correlations of H-7′/H-8′/H-9′/H-10′, combined with HMBC correlations from H-10′ to C-8′/C-12′, from H-11′ to C-9′/C-13′ (Fig. 2), along with their chemical shifts, indicated the hydroxyl group located at the position of C-9′. Additionally, HMBC correlations from H-3 to C-1′ and from H-2′ to C-2 confirmed that the aliphatic carbon chain was connected to C-2. Based on the coupling constants (³J_{H-6′,H-7′} = 18.1 Hz, ³J_{H-10′,H-11′} = 15.3 Hz, ³J_{H-12′,H-13′} = 15.6 Hz), the configurations of the couple of olefinic carbons between C-6′ and C-7′, between C-10′ and C-11′, between C-12′ and C-13′ in compound 23 were elucidated as E-form. Compound 23 was further confirmed to be a racemate because it showed no Cotton effect and its optical rotation value was close to zero (Figure S43). From above mentioned results, the structure of 23 was elucidated as wuchuyuamine E.

Table 2.

¹H NMR (500 MHz) and ¹³C NMR (125 MHz) spectroscopic data for 23 in CD₃OD (δ in ppm, J in Hz)

No	δH	δC	No	δH	δC
N-CH3	3.89 (3H, s)	35.5	4′	1.48 (2H, m)	29.9
2		158.8	5′	2.10 (2H, m)	28.2
3	6.31 (1H, s)	111.1	6′	5.51 (1H, dt, 18.2, 7.3)	133.1
4		179.5	7′	5.43 (1H, dt, 18.1, 7.2)	126.0
4a		126.9	8′	2.32 (2H, m)	36.0
5	8.32 (1H, dd, 8.1, 1.5)	126.6	9′	4.21 (1H, dt, 6.6, 5.3)	72.5
6	7.47 (1H, ddd, 7.9, 6.8, 1.1)	125.1	10′	6.27 (1H, dd, 15.3, 5.7)	148.0
7	7.80 (1H, ddd, 8.7, 6.8, 1.6)	133.9	11′	6.41 (1H, dd, 15.4, 11.0)	128.9
8	7.86 (1H, dd, 8.8, 1.3)	117.9	12′	7.25 (1H, dd, 15.7, 10.8)	145.3
8a		143.5	13′	6.14 (1H, d, 15.6)	131.1
1′	2.89 (2H, t, 7.8)	35.6	14′		201.4
2′	1.73 (2H, m)	29.5	15′	2.25 (3H, s)	27.1
3′	1.48 (2H, m)	30.2

Wuchuyuamine F (29) was a white amorphous powder. The HRESIMS ion peak at m/z 320.1362 [M + Na]⁺ (calcd. for C₁₇H₁₉N₃O₂Na, 320.1369) and 1D NMR data (¹H NMR, 600 MHz; ¹³C NMR, 150 MHz), recorded in CD₃OD (Table 3), led to the determination of the molecular formula as C₁₇H₁₉N₃O₂. ¹³C NMR data of 29 indicated a similar structure to wuchuyuamide III (31), except for the absence of a carbonyl signal. In the HMBC spectrum (Fig. 2), compound 29 lacked the key correlations from H-9′ to C-2 and from N–CH₃ to C-2. Therefore, the absent carbonyl group was located at the position of C-2 and the structure of compound 29 was constructed (Fig. 1).

Table 3.

¹H NMR and ¹³C NMR spectroscopic data for 29 and 30 (δ in ppm, J in Hz)

No	29a		30b
	δH	δC	δH	δC
4		172.3		170.0
5	7.39 (1H, dd, 7.8, 1.6)	129.1	7.30 (1H, m)	127.3
6	6.58 (1H, m)	115.9	6.58 (1H, dd, 7.6, 7.6)	114.6
7	7.29 (1H, ddd, 8.6, 7.1, 1.6)	133.6	7.30 (1H, m)	133.2
8	6.68 (1H, d, 8.4)	111.9	6.66 (1H, d, 8.3)	111.3
9		151.4		150.8
10		117.4		114.8
N-CH3	2.83 (3H, s)	29.9	2.85 (3H, d, 5.0)	29.8
1′		118.6		121.6
2′		152.7		140.1
3′	6.74 (1H, d, 8.4)	118.3	8.76 (1H, d, 8.5)	121.9
4′	7.23 (1H, ddd, 8.5, 6.9, 1.5)	135.5	7.58 (1H, dd, 8.0, 7.9)	135.7
5′	6.58 (1H, m)	116.2	7.17 (1H, dd, 7.8, 7.7)	123.4
6′	7.80 (1H, dd, 8.3, 1.5)	132.4	7.94 (1H, d, 8.0)	131.1
7′		202.5		204.0
8′	3.27 (2H, t, 6.8)	39.5	3.39 (2H, t, 5.7)	39.8
9′	3.69 (2H, t, 6.8)	36.8	3.82 (2H, dt, 5.9, 5.8)	34.7
CHO			8.52 (1H, s)	160.0
NH-CHO			11.5 (1H, s)

^a600 MHz in CD₃OD; 150 MHz in CD₃OD

^b600 MHz in CDCl₃; 150 MHz in CDCl₃

Wuchuyuamine G (30) was obtained as a white amorphous powder. Its molecular formula, C₁₈H₁₉N₃O₃, was deduced from the HRESIMS ion peak at m/z 326.1499 [M + H]⁺ (calcd. for C₁₈H₂₀N₃O₃, 326.1499). Since 30 exhibited similar UV, IR, and NMR spectral characteristics to 29, it was supposed that compound 30 had the same skeleton as 29. Analysis of the ¹H and ¹³C NMR spectra of 30, recorded in CDCl₃ at 600 and 150 MHz (Table 3), showed an additional aldehyde signal (δ_H 8.52; δ_C 160.0) compared with 29. Combined with the HMBC correlation from aldehyde proton (δ_H 8.52) to C-2′ (Fig. 2), it could be concluded that the aldehyde group and C-2′ were connected through the amino group. The structure of compound 30 was determined as depicted in Fig. 1.

Antifungal activity in vitro

The antifungal activities of compounds 2, 4, 12–14, 16, 18, 19, 24–26, 31, and 33, available in sufficient amounts, were evaluated against three phytopathogenic fungi. The results are summarized in Table S1. Among the tested compounds, 16 and 24 exhibited pronounced inhibitory activity against Alternaria solani, with inhibition rates approaching 80% and 90% at 100 μg/mL, respectively. To further assess their antifungal potency, the IC₅₀ values of 16 and 24 against A. solani were determined (Table 4). Both compounds 16 and 24 demonstrated dose-dependent inhibition, although their activities were slightly lower than that of the commercial fungicide hymexazol. These results suggest that the structural frameworks of 16 and 24 may confer selective antifungal activity toward A. solani.

Table 4.

Antifungal activities with IC₅₀ of compounds 13, 16, 24, 25

Test fungi	Compounds	IC50 (μg/mL)	95% CI	R2
A. solani	16	33.39	29.58–38.26	0.9964
	24	36.02	32.26–40.79	0.9977
	Hymexazola	14.90	11.23–19.92	0.9872
F. graminearum	13	> 100	–	–
	25	13.15	8.51–21.36	0.9710
	Hymexazola	30.86	23.61–43.67	0.9829

^aHymexazol was used as the positive control

To further investigate the broad-spectrum antifungal potential of the major alkaloids in T. ruticarpum, compounds 13, 19 and 25, identified as the high-content constituents in the fruit extract, were selected for testing against sixteen phytopathogenic fungi, including Botrytis sp., Ceratocystis fimbriata, Colletotrichum capsici f. nicotianae, Colletotrichum sp., Curvularia lunata, Exserohilum turcicum, Fusarium graminearum, Fusarium moniliforme, Fusarium oxysporum, Gerlachia nivalis, Neofusicoccum sp., Pestalotiopsis maculans, Pestalotiopsis sp., Phytophthora capsici, Rhizoctonia solani, Verticillium dahliae, with hymexazol serving as the positive control (Table S2). Among the three alkaloids, compound 25 presented the most pronounced antifungal activity, with inhibition rates varying between 7.2% and 83.5%. Notably, compound 25 showed stronger inhibitory activity against F. graminearum than hymexazol, with an IC₅₀ value of 13.15 μg/mL (Table 4), highlighting its potential as a promising lead compound for the development of plant-derived fungicides. Additionally, compound 13 showed moderate inhibition activity, with a maximum inhibition rate of 72.9% at 100 μg/mL, depending on the pathogen tested. Unfortunately, compound 19 exhibited negligible inhibitory activity across all tested fungi, indicating limited antifungal potential.

Observation of morphology and ultrastructure in F. graminearum

Scanning Electron Microscopy (SEM) was employed to observe the morphological effects of 25 on F. graminearum hyphae, in which the mycelia of F. graminearum treated by DMSO were used as blank controls. As shown in Fig. 3A, the hyphae on the PSA medium containing DMSO were slender, uniform, and smooth. In contrast, 25 (15 μg/mL) caused disordered growth and distortion, with a roughened surface, localized collapse and hyphal breakage. These results indicated that 25 caused severe damage to the hyphae of F. graminearum.

Fig. 3.

A SEM images [200 × (a, d), 500 × (b, e) and 1000 × (c, f) magnification] of F. graminearum hyphae treated with DMSO (a-c) and 25 at 15 µg/mL (d-f). B TEM images [22,000 × (a, d-f), 17,500 × (b) and 28,000 × (c) magnification] of F. graminearum hyphae treated with DMSO (a-c) and 25 at 15 µg/mL (d-f). Scale bars are provided in each image in (A) and (B). CW, cell wall; PM, plasma membrane; S, septum; V, vacuole; MI, mitochondrion. C Leakage of nucleic acids (OD_{260 nm}) and proteins (OD_{280 nm})

Transmission Electron Microscopy (TEM) observations further confirmed ultrastructural disruption (Fig. 3B). Control hyphae displayed intact cell walls with well-defined plasma membranes, preserved mitochondria, and normal vacuoles. After treatment with 25 (15 μg/mL), the cell wall contour became deformed and the plasma membrane was indistinct, accompanied by pronounced cytoplasmic vacuolization and markedly wrinkled vacuolar membranes. In addition, some mitochondria showed disorganization and compromised membrane integrity. These observations suggest that 25 disrupted the cellular ultrastructure of F. graminearum.

Effect of 25 on the cytoplasmic content leakage of F. graminearum

Membrane damage was evaluated by measuring leakage of intracellular components. To assess the effect of 25, nucleic acid (OD_{260 nm}) and protein (OD_{280 nm}) leakage were quantified. As shown in Fig. 3C, OD_{260 nm} and OD_{280 nm} values in the DMSO control remained relatively stable over time. In contrast, treatment with 25 (15 µg/mL) led to a time-dependent increase in OD_{260 nm} and OD_{280 nm} within 4 h. This demonstrates that 25 enhanced cell membrane permeability, resulting in membrane rupture and subsequent leakage of intracellular contents.

Conclusion

In summary, thirty-six alkaloids, including seven novel compounds, wuchuyuamines A-G (1–4, 23, 29, and 30), were isolated and identified from the fruits of T. ruticarpum. The antifungal activities of representative alkaloids were systematically evaluated against a range of phytopathogenic fungi. Compounds 16 and 24 exhibited moderate inhibitory activity against A. solani, with IC₅₀ values of 33.39 and 36.02 μg/mL, respectively. Notably, compound 25 demonstrated marked broad-spectrum antifungal efficacy, particularly against F. graminearum, where its inhibitory potency (IC₅₀ = 13.15 μg/mL) exceeded that of the commercial fungicide hymexazol. The SEM and TEM analyses indicated that 25 disrupted the surface morphology and cellular ultrastructure of F. graminearum hyphae. Compound 25 induced leakage of intracellular contents, resulting in metabolic dysregulation and ultimately leading to cell death. These findings not only expand the chemical diversity of alkaloids from T. ruticarpum but also highlight the antifungal potential of quinolone alkaloids as promising lead structures for the development of novel, efficient, and environmentally friendly fungicides.

Experimental

General experimental procedures

HRESIMS data were acquired on an Agilent 1260 UPLC/6540 Q-TOF MS spectrometer (Agilent, USA). Optical rotations were measured in MeOH using an Autopol VI spectropolarimeter (Rudolph Research Analytical). ECD spectra were recorded on a Chirascan instrument (Applied Photophysics Ltd., Leatherhead, UK). 1D and 2D NMR spectra were obtained on Bruker 500 or 600 MHz spectrometer with chemical shifts (δ) given in ppm with reference to the solvent and coupling constants (J) in Hz (Bruker, Germany). UV spectra were obtained on a Shimadzu UV-2401PC spectrophotometer (Shimadzu, Tokyo, Japan). IR spectra were recorded on a Bruker Vertex 70 IR spectrometer (Bruker, Germany). Column chromatography was performed using Sephadex LH-20, RP-18, and silica gel. An X-Bridge C₁₈ column (Waters, 10 mm × 250 mm, 5 μm) and a YMC-Triart C₁₈ column (YMC, 10 mm × 250 mm, 5 μm) were used for HPLC analysis.

Plant material

The fruits of T. ruticarpum were purchased from Chongqing Zhongmiao Pharmaceutical Co., Ltd. (Chongqing, China) in September 2023 (Batch No. 230201). The plant material was identified by Dr. Chun-Lei Xiang, Kunming Institute of Botany, Chinese Academy of Sciences. A voucher specimen (NO. KIBH20230915) was deposited at the State Key Laboratory of Phytochemistry and Natural Medicines, Kunming Institute of Botany, Chinese Academy of Sciences.

Fungal materials

Plant pathogenic fungi A. solani, Bipolaris sorokiniana and Fusarium odoratissimum were obtained from the Institute of Microbiology, Chinese Academy of Sciences. The remaining fungal strains were supplied by the State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, China.

Extraction and isolation

The separation and isolation procedures for compounds 1–36 and spectroscopic data of 1–4, 23, 29, and 30 are provided in the Supporting Information (SI).

Spectroscopic data

(1): yellow amorphous powder; UV (MeOH) λ_max (log ε) 207 (4.07), 219 (4.13), 257 (3.58), 278 (3.35), 343 (3.20) nm; IR (KBr) ν_max 3433, 1632, 1520, 1384, 1275, 747 cm⁻¹; ¹H and ¹³C NMR data (CD₃OD, 500 and 125 MHz), see Table 1; HRESIMS m/z 346.1533 [M + Na]⁺ (calcd. for C₁₉H₂₁N₃O₂Na, 346.1526).

(2): white amorphous powder; UV (MeOH) λ_max (log ε) 204 (4.53), 241 (4.05), 307 (3.48) nm; IR (KBr) ν_max 3390, 2920, 1617, 1582, 1412, 1062, 750 cm⁻¹; ¹H and ¹³C NMR data ((CD₃)₂CO, 600 and 150 MHz), see Table 1; HRESIMS m/z 310.1547 [M + H]⁺ (calcd. for C₁₈H₂₀N₃O₂, 310.1550).

(3): white amorphous powder; UV (MeOH) λ_max (log ε) 207 (4.08), 221 (4.15), 297 (3.20), 340 (2.97), 349 (2.94) nm; IR (KBr) ν_max 3434, 2924, 1638, 1384, 1215, 759 cm⁻¹; ¹H and ¹³C NMR data (CD₃OD, 600 and 150 MHz), see Table 1; HRESIMS m/z 358.1529 [M + Na]⁺ (calcd. for C₂₀H₂₁N₃O₂Na, 358.1526).

(4): white amorphous powder; UV (MeOH) λ_max (log ε) 227 (4.83), 268 (4.15), 347 (3.48) nm; IR (KBr) ν_max 3390, 3281, 1657, 1488, 1041, 749 cm⁻¹; ¹H and ¹³C NMR data (CD₃OD, 500 and 125 MHz), see Table 1; HRESIMS m/z 322.1554 [M + H]⁺ (calcd. for C₁₉H₂₀N₃O₂, 322.1550).

(23): yellow oil; UV (MeOH) λ_max (log ε) 215 (3.27), 239 (3.28), 274 (2.88), 322 (2.91), 335 (2.91) nm; IR (KBr) ν_max 3433, 2922, 1620, 1598, 1557, 1384, 1268, 1179, 763 cm⁻¹; ¹H and ¹³C NMR data (CD₃OD, 500 and 125 MHz), see Table 2; HRESIMS m/z 394.2380 [M + H]⁺ (calcd. for C₂₅H₃₂NO₃, 394.2377).

(29): white amorphous powder; UV (MeOH) λ_max (log ε) 208 (3.82), 219 (3.85), 257 (3.45), 355 (3.20) nm; IR (KBr) ν_max 3448, 2920, 1637, 1520, 1277, 1210, 1166, 750 cm⁻¹; ¹H and ¹³C NMR data (CD₃OD, 600 and 150 MHz), see Table 3; HRESIMS m/z 320.1362 [M + Na]⁺ (calcd. for C₁₇H₁₉N₃O₂Na, 320.1369).

(30): white amorphous powder; UV (MeOH) λ_max (log ε) 207 (4.07), 219 (4.14), 225 (4.14), 257 (3.87), 329 (3.48) nm; IR (KBr) ν_max 3405, 2922, 1641, 1520, 1277, 1200, 1172, 752 cm⁻¹; ¹H and ¹³C NMR data (CDCl₃, 600 and 150 MHz), see Table 3; HRESIMS m/z 326.1499 [M + H]⁺ (calcd. for C₁₈H₂₀N₃O₃, 326.1499).

Antifungal activity assay in vitro

Compounds 2, 4, 12–14, 16, 18, 19, 24–26, 31 and 33 were evaluated for their antifungal activities against A. solani, B. sorokiniana and F. odoratissimum at 100 μg/mL following the mycelium growth rate method [38]. For compounds exhibiting appreciable inhibition, dose–response assays were conducted at serial concentrations, and IC₅₀ values were calculated from the fitted inhibition curves using the same method. Hymexazol was used as a positive control. All results were expressed as the mean ± SD. Broad-spectrum antifungal activities of compounds 13, 19 and 25 were further evaluated against sixteen phytopathogenic fungi using the same mycelium growth rate method.

SEM and TEM observations

Fusarium graminearum was treated with compound 25 at a concentration of 15 μg/mL, while the solvent served as the control. Subsequently, SEM and TEM were used to observe alterations in mycelial morphology and intracellular ultrastructure, respectively [39].

Effect of 25 on the cytoplasmic content leakage of F. graminearum

The mycelium (400 mg) was suspended in 40 mL of sterile water containing 25 (15 µg/mL) and incubated at 26 ℃ for 0, 1, 2, or 4 h, with DMSO as the solvent control. After incubation, the supernatant absorbance at 260 and 280 nm was measured using a SpectraMax iD3 microplate reader (Molecular Devices, USA) to assess nucleic acid and protein leakage, respectively [40].

Statistical analysis

All experiments were conducted with three independent biological replicates, with consistent results among replicates. The half-maximal inhibitory concentration (IC₅₀) values were calculated by nonlinear regression (log[inhibitor] vs. normalized response, Variable slope) using GraphPad Prism (GraphPad Software, USA). Absorbance at 260 and 280 nm (OD_260 nm and OD_280 nm) was analyzed and plotted in GraphPad Prism.

Author contributions

Meng-Ting Chen: writing—original draft, investigation, formal analysis, data curation. Jian Zhang: formal analysis, data curation. Qian Zhao: formal analysis, data curation. Su-Min Zhang: investigation, data curation. Yin-Ling Wei: investigation. Ying Zhang: data curation. Xiao-Jiang Hao: writing—review and editing, supervision, project administration, funding acquisition, conceptualization. All authors read and approved the final manuscript.

Funding

The present study was supported by the grants from the National Natural Science Foundation of China (82293683), CAMS Innovation Fund for Medical Sciences (CIFMS, 2021-I2M-5-004), Key Research and Development Project of Yunnan Province (202203AC100009), the Project of Yunnan Characteristic Plant Screening and R&D Service CXO Platform (2022YKZY001), and the Open Research Fund of Yunnan Characteristic Plant Extraction Laboratory (YKKF2024005).

Data availability

The data underlying this study are available in the published article and its Supporting Information.

Declarations

Competing interests

The authors declare that there is no competing financial interest in this paper.