Biochemistry & Analytical Biochemistry

Herbicidal Quassinoids Isolated from Ailanthus altissima Leaves

Young Sook Kim¹, Surk-Sik Moon^2* and Jung Sup Choi^{1* 1}Eco-friendly and New Materials Research Center, Korea Research Institute of Chemical Technology, Daejeon, South Korea
²Department of Chemistry, Kongju National University, Gongju 314-701, Republic of Korea
^*Correspondence: Surk-Sik Moon, Department of Chemistry, Kongju National University, Gongju 314-701, Republic of Korea, Email: Jung Sup Choi, Eco-friendly and New Materials Research Center, Korea Research Institute of Chemical Technology, Daejeon, South Korea, Email:

Received: 16-Nov-2023, Manuscript No. BABCR-23-23962; Editor assigned: 20-Nov-2023, Pre QC No. BABCR-23-23962 (PQ); Reviewed: 04-Dec-2023, QC No. BABCR-23-23962; Revised: 11-Dec-2023, Manuscript No. BABCR-23-23962 (R); Published: 18-Dec-2023, DOI: 10.35248/2161- 1009.23.12.517

Abbreviations

COSY: Correlation Spectroscop Y; HMBC: Heteronuclear Multiple Bond Correlation; HPLC: High-Performance Liquid Chromatography; HRTOFMS: High-Resolution Time-Of-Flight Mass Spectrometry; NMR: Nuclear Magnetic Resonance; NOE: Nuclear Overhauser Effect; HSQC: Heteronuclear Single Quantum Correlation; ROESY: Rotating-Frame Nuclear Overhauser Effect Spectroscopy

Introduction

Quassinoids are a group of degraded triterpenes found in the family Simaroubaceae that undergo extensive oxidative biodegradation, leaving their carbon skeleton highly oxygenated [1-4]. Quassinoids are classified into five groups according to their basic structure, C-18, C-19, C-20, C-22, and C-25. The C-20 quassinoids have been extensively investigated due to their antileukemic activity discovered in the early 1970s [5]. Since 1960, hundreds of quassinoids have been isolated and identified from plants, but they seem harder to synthesize due to the presence of their highly oxygenated carbon framework. Many quassinoids display a wide range of biological activities in vitro or in vivo, including antitumor [6], antimalarial [7], antiviral [8], anti-inflammatory [9], antifeedant [10], insecticidal [11], antiulcer [12], and herbicidal activities [13]. In our ongoing investigations of structurally unique bioactive agents, such as herbicides derived from traditional plants, systematic phytochemical studies of Ailanthus altissima resulted in the isolation of one new quassinoid (3) and three known quassinoids (1, 2, and 4). In this paper, we describe the isolation, structural elucidation, and evaluation of the herbicidal activities of all isolates obtained.

Materials and Methods

Experimental details

Plant material: Seeds of 10 weed species (S. bicolor, E. crus-galli, A. smithii, D. sanguiinalis, P. dichotomiflorum, S. nigrum, A. indica, A. avicennae, X. strumarium, C. japonica) were germinated in flats in a commercial greenhouse substrate and watered with tap water. The weeds were grown in a greenhouse at 30 ± 3/20 ± 3°C day/night temperature with a 14-h photoperiod.

Biological activity: The activity assay was performed 12 days after sowing for the foliar application of samples at each concentration with a laboratory spray gun. The herbicidal activity of the foliar application was evaluated by visual injury 14 days after treatment (0, no damage; 100, complete control).

Extraction and isolation: A. altissima leaves (5 kg) collected from Nonsan, Korea were dipped in MeOH at room temperature and filtered after three days. After concentrating the methanol, the extract was defatted with hexane, then partitioned between EtOAc and H₂O. A portion of the active EtOAc fraction (6 g) was purified by gradient elution on a flash silica gel column using CHCl₃:MeOH (50:1, 20:1, 10:1, 5:1, and 100% MeOH). Fraction II [1.03 g, CHCl₃-MeOH (20:1)] and fraction III [1.32 g, CHCl₃: MeOH (10:1)], which were active in the bioassay, were further purified by ODS Sep-pak cartridge (Alltech, Deerfield, IL, USA) chromatography eluted with an increasing methanol concentration gradient (0%–100%) in water. The active fraction was finally purified by reversed-phase HPLC. Preparative HPLC (C18, 5 μ, 20 × 250 mm; COSMOSIL) was performed using 30%–50% aqueous MeOH, UV detection at 254 nm, a flow rate of 12 mL/min, and gradient elution for 50 min.

Structural analysis: Melting points were measured on a Fisher melting point apparatus and uncorrected. Optical rotations were measured on a Perkin Elmer 341-LC polarimeter. NMR spectra were recorded on a Bruker 900 and 700 spectrometer with standard pulse sequences, operated at 900 and 700 MHz for ¹H NMR and 226 and 176 MHz for ¹³C NMR. Chemical shifts, measured in ppm, were referenced to solvent peaks (δ_H 2.50 and δ_C 39.5 for DMSO-d₆). HR-TOF-MS spectra were recorded in the positive ESI mode on a Waters Synapt G2 at the Korea Basic Science Institute.

Compound 1 (ailanthone):

¹H NMR (DMSO-d₆, 900 MHz) δ: 8.23 (¹H, s, 11-OH), 7.08 (¹H, d, J = 2.8 Hz, 1-OH), 5.99

(¹H, d, J = 0.9 Hz, H-3), 5.40 (¹H, d, J = 4.5 Hz, 12-OH), 5.05 (¹H, d, J = 0.9 Hz, Ha-21), 5.02

(¹H, d, J = 0.9 Hz, H_b-21), 4.55 (¹H, t, J = 2.8 Hz, H-7), 4.28 (¹H, s, H-1), 3.81 (¹H, d, J = 9.0

Hz, Ha-20), 3.27 (¹H, d, J = 9.0 Hz, H_b-20), 3.67 (¹H, d, J = 3.6 Hz, H-12), 2.88 (¹H, d, J = 10.8

Hz, H-5), 2.98 (¹H, dd, J = 18.0, 14.4 Hz, Ha-15), 2.81 (¹H, s, H-9), 2.77 (¹H, dd, J = 13.5, 5.4,

H-14), 2.44 (¹H, dd, J = 18.0, 5.4 Hz, H_b-15), 2.03 (2H, m, H-6), 1.93 (3H, s, H-18), 1.06 (3H, s,

H-19) ppm; ¹³C NMR (225 MHz, DMSO-d₆): 197.1 (C2), 169.1 (C16), 162.4 (C4), 146.6 (C13),

125.0 (C3), 117.6 (C21), 108.8 (C11), 82.4 (C1), 79.0 (C12), 77.5 (C7), 71.1 (C₂₀), 46.1 (C14),

44.5 (C8 and C10), 43.3 (C9), 41.2 (C5), 34.3 (C15), 25.1 (C6), 22.4 (C18), and 9.5 (C19) ppm;

HRTOFMS (positive ESI mode) m/z 399.1415 [M + Na]⁺ (calcd for C₂₀H₂₄O₇+Na, 399.1420).

Compound 2 (13, 18-dehydroglaucarubinone):

¹H NMR (DMSO-d₆, 700 MHz) δ: 8.33 (¹H, s, 11-OH), 7.16 (¹H, d, J = 2.1 Hz, 1-OH), 5.99

(¹H, q, J = 0.7 Hz, H-3), 5.64 (¹H, br s, H-15), 5.35 (¹H, br s, 12-OH), 5.11 (¹H, s, 2’-OH), 5.09

(¹H, d, J = 1.4 Hz, H_a-21), 4.99 (¹H, br s, H_b-21), 4.70 (¹H, t, J = 2.8 Hz, H-7), 4.42 (¹H, d, J =

2.1 Hz, H-1), 3.81 (¹H, d, J = 8.4 Hz, H_a-20), 3.67 (¹H, d, J = 4.9 Hz, H-12), 3.32 (¹H, dd, J =

8.4 Hz, H_b-20), 3.08 (¹H, d, J = 11.9 Hz, H-5), 3.04 (¹H, d, J = 11.4 Hz, H-14), 2.97 (¹H, s, H-9),

2.06 (2H, m, H-6), 1.93 (3H, s, H-18), 1.70 (¹H, dq, J = 140.0, 7.0 Hz, H_a-3’), 1.53 (¹H, dq, J =

140.0, 7.0 Hz, H_b-3’), 1.30 (3H, s, H-5’), 1.06 (3H, s, H-19), 0.81 (3H, t, J = 7.0 Hz, H-4’) ppm;

¹³C NMR (176 MHz, DMSO-d₆): 197.0 (C2), 174.2 (C1’), 166.7 (C16), 162.4 (C4), 142.4 (C13),

124.8 (C3), 119.8 (C21), 108.6 (C11), 82.1 (C1), 78.7 (C12), 77.8 (C7), 73.9 (C2’), 70.7 (C₂₀),

68.2 (C15), 50.2 (C14), 46.4 (C8), 44.5 (C10), 44.0 (C9), 40.7 (C5), 32.8 (C3’), 25.8(C5’), 24.6 (C6), 22.2 (C18), 9.5 (C19), and 8.0 (C4’) ppm; HRTOFMS (positive ESI mode) m/z 499.194

[M + Na]⁺ (calcd for C₂₅H₃₂O₉+Na, 499.1944) and m/z 975.3983 [2M + N]⁺ [calcd for (C₂₅H₃₂O₉)₂+Na, 975.3990].

Compound 3 (6-α-tigloyloxyailanthone):

¹H NMR (DMSO-d₆, 700 MHz) δ: 8.15 (¹H, s, 11-OH), 7.28 (¹H, d, J = 2.8 Hz, 1-OH), 6.91 (dq,

J = 7.7, 2.1 Hz, H3’), 6.02 (¹H, s, H-3), 5.55 (¹H, d, J = 4.2 Hz, 12- OH), 5.49 (¹H, dd, J = 11.9,

2.8 Hz, H-6), 5.02 (¹H, d, J = 1.4 Hz, H_a-21), 5.01 (¹H, s, H_b-21), 4.63 (¹H, d, J = 2.8 Hz, H-7),

4.39 (¹H, s, H-1), 3.91 (¹H, d, J = 8.4 Hz, H_a-20), 3.69 (¹H, t, J = 4.9 Hz, H-12), 3.46 (¹H, d, J =

11.9 Hz, H-5), 3.39 (¹H, dd, J = 8.4 Hz, H_b-20), 3.01 (¹H, dd, J = 18.4, 13.3 Hz, H_a-15), 2.85

(¹H, dd, J = 13.3, 5.6 Hz, H-14), 2.83 (¹H, s, H-9), 2.47 (¹H, dd, J = 18.4, 5.6 Hz, H_b-15), 1.94

(3H, s, H-18), 1.829 (3H, s, H5’), 1.823 (3H, d, J = 4.2 Hz, H4’), 1.21 (3H, s, H-19) ppm; ¹³C

NMR (176 MHz, DMSO-d₆): 196.6 (C2), 168.2 (C16), 165.9 (C1’), 161.2 (C4), 146.1 (C13),

139.2 (C3’), 127.9 (C2’), 127.6 (C3), 117.5 (C21), 109.0 (C11), 82.4 (C1), 79.1 (C12), 77.5 (C7),

70.2 (C₂₀), 67.3 (C6), 47.2 (C10), 46.1 (C14), 45.2 (C8), 44.1 (C5), 42.0 (C9), 34.0 (C15), 24.7 (C18), 14.4 (C4’), 11.9 (C5’), and 10.9 (C19) ppm; HRTOFMS (positive ESI mode) m/z 497.1785 [M + Na]⁺ (calcd for C₂₅H₃₀O₉+Na, 497.1788) and m/z 971.3670 [2M + N]⁺ [calcd for (C₂₅H₃₀O₉)₂+Na, 971.3678].

Compound 4 (6-a-tigloyloxychaparrinone):

¹H NMR (DMSO-d₆, 700 MHz) δ: 8.0 (¹H, s, 11-OH), 7.19 (¹H, d, J = 2.8 Hz, 1-OH), 6.91 (dq,

J = 7.7, 2.1 Hz, H3’), 6.0 (¹H, s, H-3), 5.49 (¹H, dd, J = 11.9, 2.8 Hz, H-6), 5.14 (¹H, d, J = 4.9

Hz, 12-OH), 4.56 (¹H, d, J = 2.8 Hz, H-7), 4.30 (¹H, d, J = 2.8 Hz, H-1), 3.93 (¹H, d, J = 8.4 Hz,

H_a-20), 3.64 (¹H, dd, J = 8.4 Hz, H_b-20), 3.39 (¹H, d, J = 11.9 Hz, H-5), 3.21 (¹H, t, J = 4.9 Hz,

H-12), 2.72 (¹H, dd, J = 18.2, 13.3 Hz, H_a-15), 2.61 (¹H, s, H-9), 2.40 (¹H, dd, J = 18.2, 5.6 Hz,

H_b-15), 2.09 (¹H, m, H-13), 2.05 (¹H, m, H-14), 1.94 (3H, s, H-18), 1.82 (3H, br s, H5’), 1.81

(3H, d, J = 7.7 Hz, H4’), 1.22 (3H, s, H-19), 0.85 (3H, d, J = 7.0 Hz, H-21) ppm; ¹³C NMR (176

MHz, DMSO-d₆): 196.5 (C2), 168.9 (C16), 165.8 (C1’), 161.2 (C4), 139.0 (C3’), 127.8 (C2’),

127.5 (C3), 109.1 (C11), 82.5 (C1), 78.1 (C12), 77.6 (C7), 69.3 (C₂₀), 67.3 (C-6), 47.1 (C10),

45.7 (C8), 44.1 (C5), 41.8 (C9), 40.6 (C14), 30.2 (C13), 29.3 (C15), 24.5 (C18), 14.3 (C4’), 12.5 (C21), 11.8 (C5’), and 10.8 (C19) ppm; HRTOFMS (positive ESI mode) m/z 499.1944 [M + Na]⁺ (calcd for C₂₅H₃₂O₉+Na, 499.1944) and m/z 975.3983 [2M + N]⁺ [calcd for (C₂₅H₃₂O₉)₂+Na, 975.3990].

Results and Discussion

The MeOH extract of Ailanthus altissima leaves at 10 mg/mL showed 100% control of Sorghum bicolor, Digitaria sanguinalis, Aeschynomene indica, and Abutilon avicennae, and >90% control of Echinochlia crus-galli, Panicum dichotomiflorum, Xanthium strumarium, and Calystegia japonica. Herbicidal activity against Agropyron smithii was slightly weaker (Table 1). The main characteristic was its quick action upon foliar application. External symptoms began to appear within 24 h of exposure, and the weeds were completely controlled five days after treatment. MeOH extract showing herbicidal activity was purified by ethyl acetate fractionation and a series of chromatographic techniques, including silica gel column, C18 Sep- pak cartridge, Sephadex LH20, and preparative High-Performance Liquid Chromatography (HPLC). Four quassinoids, 1-4 (Figure 1), were isolated from the MeOH extract of Ailanthus altissima leaves and showed herbicidal activity of 98, 95, 85, and 75%, respectively, when applied to D. sanguiinalis at a concentration of 10 μg/mL (Table 2 and Figure 2).

Table 1: Herbicidal activity of foliar application of methanol extracts from Ailanthus altissima to several weeds in a greenhouse condition.

Table 2: Herbicidal activity of isolated compounds against Digitaria sanguinalis.

Figure 1: Structures of quassinoids compounds 1-4.

Figure 2: Herbicidal activity against Digitaria sanguinalis at same concentration of 10 μg/mL.

Among these, 3 was a new compound, and the characterization and spectroscopic analysis of this compound were performed by data comparison with literature values [14]. The ¹H and ¹³C NMR (nuclear magnetic resonance) data for these compounds are shown in the experimental section. The ¹H (Table 3) and ¹³C NMR (Table 4) signals assignments were aided by HSQC (Heteronuclear Single Quantum Correlation), HMBC (Heteronuclear Multiple Bond Correlation), and ¹H-¹H COSY (Correlation Spectroscop Y) experiments. The other compounds were identified as ailanthone (1) [15], 13, 18-dehydroglaucarubinone (2) [16], and 6-α-tigloyloxychaparrinone (4) [17,18]. Compound 3 was obtained as a white solid. The molecular formula was established as C₂₅H₃₂O₉ based on a quasi-molecular ion at m/z 497.1785 [M + Na]⁺ (calcd 497.1788) in its HRTOFMS. The ¹H NMR spectrum of 3 displayed signals for two olefinic protons [δ_H 6.91 (¹H, dq, J = 7.7, 2.1, H-3’), 6.02 (¹H, s, H-3)], an exo-methylene [δ_H 5.02 (¹H, d, J = 2.1, H_a-21), 5.01 (¹H, s, H_b-21)], four oxygenated methines [δ_H 5.49 (¹H, dd, J = 11.9, 2.8, H-6), 4.63 (¹H, d, J = 2.8, H-7), 4.39 (¹H, s, H-1), 3.69 (¹H, d, J = 3.5, H-12)], one oxygenated methylene [δ_H 3.91 and 3.39 (each ¹H, d, J = 8.4, H-20)], one methylene proton [δ_H 3.01 (¹H, dd, J = 18.4, 13.3, H_a-15) and 2.40 (¹H, dd, J = 18.4, 5.6, H_b-15)] and four methyl groups [δ_H 1.94 (3H, s, H-18), 1.829 (3H, br s, H-5’), 1.823 (3H, d, J = 7.7, H-4’), and 1.21 (3H, s, H-19)]. The ¹³C NMR spectrum exhibited 25 carbon signals, including three carbonyl signals, six olefinic carbon signals, one hemiketal carbon signal, four methyl carbon signals, two methylene carbon signals, seven methine carbon signals, and two quaternary carbon signals. Comparison of the NMR data of 3 with those of ailanthone (1) revealed that the two compounds possessed the same quassinoid moiety, except that the C6 position in ailanthone (1) was substituted with a tigloyloxy group in 3. The ¹H-¹H COSY between H-3’ (δ_H 6.91) and H-4’ (δ_H 1.823) and the HMBC correlation between H-3’ (δ_H 6.91), H-5’ (δ_H 1.829), and C1’ (δ_C 165.8) and H-4’ (δ_H 1.81) and C-2’ (δ_C 127.8) confirmed the existence of a tigloyloxy group and that it was connected to the C6 position of 3, established by a long-range correlation between δ_H 5.49 (H-6 of quassinoid moiety) and carboxylic carbon (δ_C 165.9, C1’ of tigloyloxy group) in the HMBC spectrum of 3. The correlations between H-9 and H-1/H-5 and between H-7 and H-14 in the ROESY spectrum suggested that the configuration of 3 was the same as that of ailanthone (1). And the ROESY correlation between H-3’ and H-5’ indicated that the geometry of the double-bond of the tigloyloxy group was Z-form. Thus, the structure of compound 3 was determined to be 6-α- tigloyloxyailanthone. The relative stereochemistry of 3 was confirmed from its 2D-ROESY NMR spectrum. The NOE correlations are shown by arrows in Figure 3 (2D-ROESY).

Table 3: ¹H-NMR Data (DMSO-d₆) for compounds 1-4.

Table 4: ¹³C-NMR Data (DMSO-d₆) for compounds 1-4.

Figure 3: 2D ROESY (NOE) correlation of 3.

Conclusion

Herbicidal activity of methanol extract of A. altissima leaves with foliar application on D. sanguiinalis at 2, 5, and 10 mg mL-1 was 90, 100 and 100%, respectively, the main herbicidal symptoms were chlorosis or burn-down and followed by necrosis and eventual death. On 10 weed species, the methanol extract showed strong herbicidal activity and it showed excellent herbicidal activity particularly on A. indica and A. avicennae even at the lowest concentration of 1 mg mL-1. Three known quassinoid compounds (1, 2, 4) and one unknown compound (3) isolated from methanol extract showed herbicidal activity of 98, 95, 75, and 85%, respectively, against D. sanguinalis at 10 μ mL-1. Compound 3, a white solid, was identified as 6-α- tigloyloxyailanthone with a molecular formula of C₂₅H₃₂O₉ by the analyses of HR-TOF-MS and ¹H and ¹³C NMR and 2D NMR spectral data.

This study showed that A. altissima extracts have potential as bioherbicide and one unknown compounds may be used as lead compounds for development of new herbicides.

Supporting Information

Structures and spectra of compound 1, 2, 3 and 4 (Figures S1-S16).

Author Contributions

The authors confirm contribution to the paper as follows: study conception and design: Y. S. Kim, S. S. Moon; data collection: J. S. Choi, S. S. Moon; analysis and interpretation of results: Y. S. Kim, S. S. Moon, J. S. Choi; draft manuscript preparation: Y. S. Kim, J. S. Choi. All authors reviewed the results and approved the final version of the manuscript.

Acknowledgments

This work was carried out with the support of the Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ0161282023), Rural Development Administration, Republic of Korea.

Competing Interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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