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Herbicidal activity and bioactive components of Brucea javanica (L.) Merr. residue
⁎Corresponding authors. dulily9@163.com (L.W. Du), wenweitg@163.com (W.W. Tang)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Abstract
There are many nutrients and active substances in the residue of traditional Chinese medicine, which can be processed into organic fertilizer and returned to the field to improve the soil fertility. In this study, we found that Brucea javanica (L.) Merr. residue has herbicidal activity and can be used for weed control in the field, this prompted us to investigate the active constituents in it. The herbicidal activities of extracts of B. javanica residue were evaluated by conducting bioassays against gramineous weed, Eleusine indica (L.) Gaertn. and broad-leaved weed, Bidens pilosa L., respectively. It confirmed that ethyl acetate and n-butanol extracts possessed potent biological activities on seed germination and seedling growth of E. indica at 5 mg/mL. This prompted us to investigate the active constituents in the seeds residue. By the activity-guided fractionation, eight known compounds were isolated, which were bruceines D−F (1–3), pityriacitrin (4), 4-(9H-β-carbolin-1-yl)-4-oxobut-2-enoic acid methyl ester (5), protocatechuic acid (6), vanillic acid (7) and carisphthalate (8). Then, three quassinoids (1–3) were evaluated for their potential herbicidal effects in bioassay. Results showed that bruceines D−F have potential herbicidal activity against the test weeds. In the range of experimental concentration, bruceine D had obvious herbicidal effect on the two tested weeds. At the concentration of 31.25 μg/mL, bruceine D could completely inhibit the root growth of E. indica; at the concentration of 125 μg/mL, bruceine D can inhibit the seed germination and shoot elongation of E. indica and the shoot growth of B. pilosa, with the inhibition reached 100%. In addition, at the same concentration (250 μg/mL), bruceines D−F could completely affected shoot elongation of B. pilosa. These results showed that B. javanica residue is a potential source of botanical herbicide development, which has great agricultural economic value, at the same time, the reuse of B. javanica residue realizes the transformation of waste into treasure, rational utilization of resources and sustainable development.
Keywords
Brucea javanica (L.) Merr.
Residue
Bruceine D
Herbicidal activity
1 Introduction
Weeds do serious harm to farmland, affect crop yield and bring huge economic losses (Araniti et al., 2015; Jabran et al., 2015), so weed control is very necessary. At present, the application of chemical herbicides is the main means of weed control, but the widespread use of chemical herbicides has brought negative effects on the environment and human health (Böcker et al., 2019), and also made weeds resistant (Jabran et al., 2015; Ahmed, 2018). Therefore, effective and low toxic weed control methods are urgently needed.
The residues of traditional Chinese herbal medicine produced by the development and utilization of Chinese herbal medicine have been increasing. Among them, the production of Chinese patent medicine brings the largest amount of waste residue, accounting for about 70% of the total amount of drug residue (Yang et al., 2015). Currently, traditional methods of incineration, landfill and stacking in fixed area are often used to treat the herb residues, causing direct or potential damage to the natural environment (Li et al., 2010). Moreover, the waste residue of traditional Chinese medicine still contains high economic value components (Zhao et al., 2012), which can not be reasonably developed and reused, resulting in waste of resources. Therefore, it is of great significance to protect the ecological environment how to effectively utilize the waste of traditional Chinese medicine and realize the transformation of waste into treasure. Recently, the research on the reuse of traditional Chinese medicine residue value is mainly in the direction of agriculture, planting and breeding. For example, it is used for nursery and cultivation substrates, livestock feed production, cultivation of edible fungi, etc (Yang et al., 2015; Xu et al., 2009).
Brucea javanica (L.) Merr. (Ya-dan-zi in Chinese) is a traditional Chinese herbal medicine in China, and the B. javanica oil extracted from seeds has a variety of pharmaceutical activities (Cui et al., 2010; Kim et al., 2004). Meanwhile, B. javanica oil emulsion is a first-line anti-cancer drug in China (Zhang et al., 2018). However, the waste residue of B. javanica produced after oil extraction is rarely reprocessed, resulting in a huge waste of this resource. In addition, the current research on B. javanica residue has medical activity (anti-tumor and anti-inflammatory) (Yan et al., 2019; Gao et al., 2019) and agricultural insecticidal activity (Mao et al., 2019), with little herbicidal activity. Moreover, B. javanica residue still contains a large number of quassinoid compounds (Wang, 2016), which have phytotoxicity (Heisey and Putnam, 1985). Therefore, the extract of B. javanica residue may be a potential source of botanical herbicides.
The purpose of our study is to explore the herbicidal effect of B. javanica residue and the possibility of its application in field weed control. Hence, the herbicidal activities of B. javanica residue extracts againet two common weeds (Eleusine indica (L.) Gaertn. and Bidens pilosa L.) in upland crop fields of Guangxi in laboratory and greenhouse conditions were determined. Meanwhile, the inhibitory effects of active compounds on weeds were also surveyed.
2 Materials and methods
2.1 General experimental information
Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AVANCE III HD 600 Ultra Shield spectrometer (Zurich, Swiss) and calibrated with the residual undeuterated solvent as internal reference. QTOF-MS spectra were acquired on a high-resolution quadrupole time-of-flight (QTOF-MS) system (Xevo G2-S, Waters Corporation, Manchester, UK). Silica gel (100–200, 200–300 mesh) was purchased from Qingdao Marine Chemical Co., Ltd. (Qingdao, China) and Sephadex LH-20 was obtained from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). Rapid medium pressure purification and preparative chromatography CHEETAHMP200 FS-9200S was purchased from Tianjin Agela Technologies Co., Ltd. (Tianjin, China). The other chemicals used in the experiments were of analytical grade. Petroleum ether, ethyl acetate, chloroform, methanol and n-butanol were obtained from Chengdu Kelong Chemical Reagent Factory (Chengdu, China). TLC was performed on silica gel plates GF254 (25 × 75 mm) (Qingdao, China). Spots on TLC were visualized under UV light (254 or 365 nm) or by spraying with 10% sulphuric acid in 90% ethanol followed by heating.
2.2 Plant material and weed seeds
The dried seeds of B. javanica were collected at Tianlin County, Guangxi Province, People’s Republic of China, in September 2019. An authenticated voucher specimen (No. YT2019003) has been deposited at the herbarium of the Agriculture, Guangxi University, Nanning, People’s Republic of China. Seeds of Eleusine indica (L.) Gaerth. and Bidens pilosa L. were collected in Guangxi University farm from September to November 2019. Voucher specimens were deposited in the herbarium of the Agriculture, Guangxi University (GXU201909-EI and GXU201909-BP).
2.3 Extraction procedures
In this study, we mainly discussed the herbicidal activity of B. javanica residue and its possibility in weed control. Therefore, we refer to the methods reported in the previous literature (Kim et al., 2016) and make corresponding improvements in the pretreatment of B. javanica seeds. The dried B. javanica seeds were crushed by a grinder to obtain powder of B. javanica seeds (10 kg), then used to extract oil, and then the residues left after oil extraction were ultrasonically extracted three times with methanol at room temperature. The extract solution was concentrated with a rotary evaporator (54 °C) under reduced pressure to give a dried extract. Then the methanolic extract (M, 2 kg) was dissolved in water and made into suspension (20 L). The suspension was first degreased with petroleum ether. Then, the degreased material was extracted sequentially with ethyl acetate and n-butanol. The extracts were evaporated under vacuum to produce the corresponding fractions of petroleum ether extract (PE, 495 g), ethyl acetate extract (EA, 66 g) and n-butanol extract (n-B, 198 g). The crude extracts were naturally cooled to room temperature, then sealed with preservative film and stored in the refrigerator at −20 °C for subsequent experiments.
2.4 Laboratory herbicidal activity assay
According to Petri dish filter paper method reported by Kaab et al. (2020), it has been improved. Seeds of E. indica as a model of gramineous weed and B. pilosa as a model of broad-leaved weed were sterilised using 10% sodium hypochlorite for 20 min, and then washed three times with sterile deionized water. Crude extracts or compounds were first fully dissolved in methanol (the volume ratio of methanol to the solvent does not exceed 5%), and then diluted to the required concentration with deionized water. Two layers of filter paper [placed on the bottom of the inverted Petri dish (90 × 15 mm) cover] were moistened with 2 mL solution of different concentrations, and then thirty sterilized weed seeds (arranged by 5 × 6) were immediately placed in each Petri dish, and finally 3 mL solution was added. At the same time, the negative control (solvent control) and blank control with the same volume ratio were set up, and each treatment was repeated for three times. After treatment, all Petri dishes were transferred to constant temperature incubator (temperature: 30 ± 2 °C, relative humidity: 75%, light/dark: 14/10 h) for culture. During cultivation, an appropriate amount of deionized water was added to maintain the water required for seed growth. The condition of seed germination was observed every day, and the number of seed germination was recorded for 7 days. After 7 days, the germination number of seeds (root protrusion of 1–2 mm) was counted, the root length and shoot length of germinated seeds were measured, and the inhibition rates were calculated according to equation (1), in which C represents parameters of the negative control group and T represents parameters of the treatment group.
2.5 Greenhouse herbicidal activity assay
The pre-emergent herbicidal activity of extracts against E. indica and B. pilosa was evaluated using pot culture assay (Ma et al., 2018). The specific experimental process has made some changes on the basis of literature reports. Firstly, all the 200 mL disposable plastic cups (3 holes were made at the bottom of the cup with an electric melting gun) were filled with 150 g breeding medium (substrate/sand/clay, v/v/v 1:1:1), then the mixture wetted with 25 mL of water. Ten weed seeds (arranged in 3 × 4 × 3) were placed in each plastic cup, and the seeds were covered with a layer of fine sand. Finally, 25 mL of different concentrations of test solution (prepared by dissolving the extracts in methanol and then diluted with deionized water to final concentrations) were added. At the same time, the solvent control and blank control with the same volume ratio were set up, and each treatment was repeated for three times. After treatment, the self-made breeding pots were placed in greenhouse (temperature: 25 ± 2 °C, relative humidity: 75%, natural light) for cultivation. During the cultivation period, appropriate amount of corresponding test solution should be added to maintain the water needed for seed growth. The emergence of seeds was observed and the emergence number of seeds was recorded every day. The observation lasted for 30 days. After 15 days, the seed emergence inhibition rate was calculated by equation (1).
2.6 Isolation of compounds
The BuOH extract was subjected to column chromatography (CC) on silica gel (100–200 mesh), eluted with ethyl acetate–methanol (v/v 100:0–0:100) to give 7 fractions (F1-F7). Fraction F1 (14.5 g) was separated by CC on silica gel (0 to 20% methanol in chloroform) furnishing 5 sub-fractions (F1.1-F1.5). Sub-fraction F1.1 (3.8 g) was purified by the rapid medium pressure preparation system in gradient elution of ethyl acetate–methanol and recrystallization in acetone solvent to provide compound 1 (320 mg). Sub-fractions F1.4 (1.7 g) was separated by the rapid medium pressure preparation system (petroleum ether-ethyl acetate and ethyl acetate–methanol) to afford 7 subfractions. The fourth subfraction (F1.4.4, 0.2 g) was purified twice by silica gel CC and then on a Sephadex LH-20 column (CHCl3/MeOH: 1/1), yielding compound 4 (6.6 mg). Sub-fractions F1.5 (2.9 g) was further purified by repeated silica gel CC and the rapid medium pressure preparation system, finally purified with a Sephadex LH-20 column with chloroform/methanol (1/1) to yield pure compound 8 (15 mg). Sub-fractions F1.2 (4.1 g) was subjected to the rapid medium pressure preparation system in gradient elution of petroleum ether/ethyl acetate and ethyl acetate/methanol then led to 8 subfractions. The third subfraction (F1.2.3, 1.1 g) was further isolated by silica gel CC and Sephadex LH-20 (CHCl3/MeOH: 1/1) to provide compound 5 (6.4 mg). The fourth to seventh subfractions (F1.2.4-F1.2.7, 1.9 g) were combined, and then the combined subfraction was separated by three Sephadex LH-20 elution (CHCl3/MeOH: 1/1, 2×; acetone, 1× ) and one silica gel CC purification, respectively, giving compound 6 (11.2 mg). Sub-fractions F1.3 (2.0 g) was separated by the rapid medium pressure preparation system in gradient elution of PE-EtOAc (0–50%) and EtOAc-MeOH (0–20%) into 6 subfractions. The third subfraction (F1.3.3, 500 mg) was separated by repeated Sephadex LH-20 CC (chloroform/methanol: 1/1, 1×; methanol, 1× ) and finally purified by silica gel CC, using a gradient of MeOH in CHCl3 (2–10%) for elution, to afford compound 7 (19.1 mg). One hundred milligram crude product (marked as F2″, 100 mg) was taken out and further purified by silica gel CC (gradient elution of CHCl3-MeOH, 10–30%) to obtain compound 2 (87 mg). Fraction F3 (28.4 g) was fractionated using chromatography over a silica gel column and eluted with CHCl3-MeOH mixtures of increasing polarity (0–50%), getting 6 sub-fractions (F3.1-F3.6). Sub-fraction F3.4 (26.3 g) was separated by the rapid medium pressure preparation system in gradient elution of EtOAc-MeOH (0–50%) and MeOH-H2O (20–50%), respectively, following recrystallization in acetone solvent, yielding compound 3 (280 mg).
2.7 Structural identification
By the activity-guided fractionation, eight compounds (Fig. 1) were isolated from the n-butanol extract of B. javanica residue.
Chemical structures of compounds 1–8.
Compound 1 (bruceine D), colorless needle. 1H NMR (600 MHz, CD3OD): δ 6.06 (1H, dd, J = 3.0, 1.8 Hz, H-3), 5.24 (1H, s, H-15), 5.12 (1H, t, J = 5.4, 3.0 Hz, H-7), 4.61 (1H, d, J = 4.2 Hz, H-11), 4.55 (1H, d, J = 7.8 Hz, H-1), 3.85 (1H, dd, J = 7.8, 1.8 Hz, H-20), 3.77 (1H, d, J = 1.2 Hz, H-12), 2.97 (1H, br. d, J = 12.6 Hz, H-5), 2.42 (1H, dd, J = 4.8, 1.2 Hz, H-9), 2.38 (1H, dt, J = 15.0, 3.0 Hz, H-6α), 1.99 (3H, s, H-17), 1.88 (1H, m, H-6β), 1.44 (3H, s, H-18), 1.19 (3H, s, H-19); 13C NMR (150 MHz, CD3OD): δ 10.1 (C-19), 17.0 (C-18), 21.1(C-17), 27.3 (C-6), 43.0 (C-5), 44.9 (C-9), 48.2 (C-10), 49.4 (C-8), 69.0 (C-20), 69.3 (C-15), 74.1 (C-11), 79.7 (C-7), 80.0 (C-12), 81.0 (C-14), 81.6 (C-1), 83.6 (C-13), 123.8 (C-3), 164.2 (C-4), 174.9 (C-16), 198.5 (C-2). The 1H and 13C NMR data were in accordance with those of bruceine D (NoorShahida et al., 2009).
Compound 2 (bruceine E), colorless needles. 1H NMR (600 MHz, CD3OD): δ 5.41 (1H, d, J = 1.8 Hz, H-3), 5.15 (1H, s, H-15), 5.08 (1H, t, J = 5.4, 2.4 Hz, H-7), 4.64 (1H, d, J = 7.2 Hz, H-11), 4.01 (1H, m, H-1), 3.84 (1H, dd, J = 7.2, 1.2 Hz, H-20), 3.77 (1H, d, J = 0.6 Hz, H-12), 2.43 (1H, d, J = 12.6 Hz, H-5), 2.18 (1H, dt, J = 15.0, 3.0 Hz, H-6), 2.09 (1H, d, J = 3.6 Hz, H-9), 1.74 (1H, dd, J = 13.2, 1.2 Hz, H-6), 1.67 (3H, d, J = 12.6 Hz, H-17), 1.43 (3H, s, H-18), 1.24 (3H, s, H-19); 13C NMR (150 MHz, CD3OD): δ 10.8 (C-19), 17.0 (C-18), 19.7 (C-17), 27.2 (C-6), 42.4 (C-5), 44.0 (C-10), 45.3 (C-9), 49.6 (C-8), 69.2 (C-20), 69.4 (C-15), 72.8 (C-2), 74.4 (C-11), 79.8 (C-7), 80.6 (C-12), 81.0 (C-14), 81.4 (C-1), 83.4 (C-13), 123.8 (C-3), 135.2 (C-4), 175.1 (C-16). The 1H and 13C NMR data were in accordance with those of bruceine E (NoorShahida et al., 2009).
Compound 3 (bruceine F), colorless needles. 1H NMR (600 MHz, DMSO‑d6): δ 5.31 (1H, s, H-3), 4.88 (2H, m, H-1, H-7), 4.46 (1H, d, J = 7.2 Hz, H-20), 4.38 (1H, t, J = 10.8, 5.4 Hz, H-12), 3.95 (1H, m, H-18), 3.81 (1H, s, H-15), 3.70 (3H, m, H-2, H-18, H-20), 3.39 (1H, m, H-11), 2.27 (1H, d, J = 12.6 Hz, H-5), 1.96 (1H, m, H-6), 1.93 (1H, m, H-9), 1.57(3H, s, H-17), 1.53 (1H, m, H-6), 1.08 (3H, s, H-19); 13C NMR (150 MHz, DMSO‑d6): δ 12.1 (C-19), 21.2 (C-17), 27.6 (C-6), 42.3 (C-5), 44.2 (C-10), 44.9 (C-9), 49.7 (C-8), 62.8 (C-18), 69.3 (C-7), 69.8 (C-20), 72.4 (C-15), 74.4 (C-12), 75.9 (C-2), 79.3 (C-1), 81.6 (C-11), 82.5 (C-14), 83.5 (C-13), 125.7 (C-3), 134.1 (C-4), 174.2 (C-16). The 1H and 13C NMR data were in accordance with those of bruceine F (Li and Zuo, 1980).
Compound 4 (pityriacitrin), yellow feathery microcrystalline. QTOF-ESI-MS m/z 312.1235 [M + H]+ (calcd for C20H13N3O). 1H NMR (600 MHz, CDCl3): δ 9.48 (1H, d, J = 3.0 Hz, H-2′), 8.60 (1H, d, J = 4.8 Hz, H-3), 8.21 (1H, br. dd, J = 7.8, 0.6 Hz, H-5), 8.18 (1H, d, J = 4.8 Hz, H-4), 7.64 (1H, m, H-7), 7.64 (1H, m, H-8), 7.52 (1H, d, J = 7.8 Hz, H-7′), 7.43 (1H, m, H-5′), 7.38 (1H, m, H-6), 7.34 (1H, m, H-6′); 13C NMR (150 MHz, CDCl3): δ 111.3 (C-7′), 112.0 (C-8), 115.9 (C-3′), 117.8 (C-4), 120.4 (C-6), 120.9 (C-5a), 121.8 (C-5), 122.7 (C-4′), 122.9 (C-5′), 123.7 (C-6′), 127.5 (C-3a′), 129.0 (C-7), 131.5 (C-4a), 135.5 (C-7a′), 136.6 (C-9a), 136.8 (C-3), 137.5 (C-2′), 138.3 (C-1), 141.1 (C-8a), 189.0 (C-10). The 1H and 13C NMR data were in accordance with those of pityriacitrin (Mayser et al., 2002) isolated from the yeast Malassezia furfur. This compound has been reported as a secondary metabolite of marine bacteria and synthetic compound (Zhang et al., 2011) in previous studies.
Compound 5 [4-(9H-β-carbolin-1-yl)-4-oxobut-2-enoic acid methyl ester], yellow green solid. QTOF-ESI-MS m/z 281.0940 [M + H]+ (calcd for C16H12N2O3). 1H NMR (600 MHz, CDCl3): δ 8.85 (1H, d, J = 16.2 Hz, H-11), 8.64 (1H, d, J = 4.8 Hz, H-3), 8.23 (1H, br. dd, J = 4.8, 0.6 Hz, H-4), 8.21 (1H, br. dd, J = 7.8, 0.6 Hz, H-5), 7.67 (1H, m, H-8), 7.62 (1H, m, H-7), 7.40 (1H, m, H-6), 7.13 (1H, d, J = 15.6 Hz, H-12), 3.91 (3H, s, H-14); 13C NMR (150 MHz, CDCl3): δ 52.3 (C-14), 112.1 (C-8), 119.8 (C-4), 120.7 (C-5a), 121.1 (C-6), 122.0 (C-6), 129.5 (C-7), 131.4 (C-12), 131.8 (C-4a), 135.5 (C-1), 135.9 (C-9a), 136.5 (C-11), 138.7 (C-3), 141.1 (C-8a), 166.2 (C-13), 191.2 (C-10). The 1H and 13C NMR data were in accordance with those of 4-(9H-β-carbolin-1-yl)-4-oxobut-2-enoic acid methyl ester, previously isolated from terrestrial Streptomycetes (Fotso et al., 2008) and the rhizomes of Polygonatum sibiricum Red. (Zhao et al., 2018).
Compound 6 (protocatechuic acid), colorless needle. 1H NMR (600 MHz, CD3OD): δ 7.46 (1H, m, H-2), 7.44 (1H, m, H-6), 6.83 (1H, d, J = 7.8 Hz, H-5); 13C NMR (150 MHz, CD3OD): δ 114.4 (C-5), 116.3 (C-2), 121.7 (C-6), 122.5 (C-1), 144.7 (C-3), 150.1 (C-4), 168.9 (HOOC-1′). The 1H and 13C NMR data were in accordance with those of protocatechuic acid (Colombo et al., 2010).
Compound 7 (vanillic acid), colorless solid. 1H NMR (600 MHz, CD3OD): δ 7.58 (1H, m, H-2), 7.57 (1H, m, H-6), 6.86 (1H, d, J = 9.0 Hz, H-5), 3.91 (3H, s, H3CO-3); 13C NMR (150 MHz, CD3OD): δ 55.0 (H3CO-3), 112.4 (C-2), 114.4 (C-5), 123.9 (C-1), 147.3 (C-3), 151.3 (C-4), 168.1 (HOOC-1′). The 1H and 13C NMR data were in accordance with those of vanillic acid (Colombo et al., 2010).
Compound 8 (carisphthalate), white crystalline solid. QTOF-ESI-MS m/z 577.1328 [M + H]+ (calcd for C30H24O12). 1H NMR (600 MHz, CDCl3): δ 8.13 (12H, s, H-2, 3, 5, 6, 2′′, 3′′, 5′′, 6′′, 2′′′′, 3′′′′, 5′′′′, 6′′′′), 4.69 (12H, s, H-1′, 2′, 1′′′, 2′′′, 1′′′′′, 2′′′′′); 13C NMR (150 MHz, CDCl3): δ 62.8 (C-1′, 2′, 1′′′, 2′′′, 1′′′′′, 2′′′′′), 129.8 (C-2, 3, 5, 6, 2′′, 3′′, 5′′, 6′′, 2′′′′, 3′′′′, 5′′′′, 6′′′′), 133.8 (C-1, 4, 1′′, 4′′, 1′′′′, 4′′′′), 165.3 (C-7, 8, 7′′, 8′′, 7′′′′, 8′′′′). The above data were basically consistent with the spectral data of carisphthalate isolated from the methanol extract of Carissa opaca (Parveen et al., 2017) or the marine-derived actinomycetes Streptomyces sp. G278 (Cao et al., 2018). In addition, the compound was previously reported as a synthetic derivative (Kint et al., 2003).
2.8 Statistical analyses
The results were presented as mean and standard deviation of all replicates. An one-way analysis of variance (ANOVA) was used to determine whether there were significant (p < 0.05) treatment effects between groups, and the means were compared using Duncan’s multiple range test (DMRT).
3 Results
3.1 Herbicidal activity of B. javanica extracts in laboratory
The herbicidal activities of organic extracts of B. javanica residue on seed germination and seedling growth (root and shoot elongation) of E. indica and B. pilosa in laboratory were investigated (Table 1). The inhibitory effects on E. indica and B. pilosa at 10 mg/mL (methanol extract) and 5 mg/mL (petroleum ether, ethyl acetate and n-butanol extract) on germination and seedling growth varied significantly. Both ethyl acetate and n-butanol extracts of B. javanica residue completely suppressed germination of E. indica seeds. In addition, ethyl acetate extract also showed excellent inhibitory effect against seed germination of B. pilosa, while the methanolic extract showed a good inhibitory activity on the germination of E. indica and B. pilosa, with inhabition of 84.08% and 60.65%, respectively.
Treatments
Conc. (mg/mL)
E. indica
B. pilosa
Seed germination
Inhibition rate (%)
Seed germination
Inhibition rate (%)
Blankb
–
25.00 ± 0.65
–
28.00 ± 0.35
–
CKc
–
27.75 ± 0.31
–
29.00 ± 0.29
–
M
10
4.50 ± 0.75
84.08 ± 2.59
11.50 ± 1.01
60.65 ± 3.19
PE
5
23.75 ± 1.11a
14.74 ± 3.25b
22.50 ± 0.32a
22.44 ± 0.55c
EA
5
0.00 ± 0.00b
100.00 ± 0.00a
3.50 ± 0.43c
88.04 ± 1.44a
n-B
5
0.00 ± 0.00b
100.00 ± 0.00a
17.25 ± 1.03b
40.77 ± 3.13b
However, root elongation was essentially remarkably restrained for extracts of B. javanica (Fig. 2). At 10 mg/mL, methanol crude extract showed significant inhibitory activity against root growth of E. indica and B. pilosa. At 5 mg/mL, ethyl acetate and n-butanol extracts completely inhibited the seedling growth of E. indica. Similarly, ethyl acetate extract and n-butanol extract also had significant effect on the root length of B. pilosa.
Effect of organic extracts of B. javanica on E. indica (a) and B. pilosa (b). Effect of B. javanica extracts on the root (c) and shoot (d) length of E. indica and B. pilosa. The data in the figure were given as mean ± SD for three repetitions. Different letters indicated significantly different (p < 0.05) among the columns with same color, based on DMRT. (Blank: blank control; CK: negative control; M: methanol extract, 10 mg/mL; PE: petroleum ether extract, EA: ethyl acetate extract, n-B: n-butanol extract, 5 mg/mL).
On the other hand, the shoot growth of E. indica and B. pilosa were also severely inhibited by the extracts (Fig. 2). At the same concentration (5 mg/mL), the shoot elongation of E. indica was completely affected by ethyl acetate and n-butanol extracts. Moreover, methanol extract, ethyl acetate extract and n-butanol extract could completely prevent the shoot growth of B. pilosa under the corresponding concentrations.
3.2 Herbicidal activity of B. javanica extracts under greenhouse conditions
The inhibitory effect of the tested extracts on emergence of E. indica and B. pilosa 15 d after treatment (Table 2 and Fig. 3). The methanol, ethyl acetate and n-butanol extracts strongly inhibited the emergence of these two weeds at the corresponding tested concentrations. Remarkably, n-butanol extract completely inhibited on emergence of E. indica and B. pilosa. However, methanol and ethyl acetate extracts only inhibited the emergence of E. indica completely.
Treatments
Conc. (mg/mL)
E. indica
B. pilosa
Seedling emergence
Inhibition rate (%)
Seedling emergence
Inhibition rate (%)
M
10
0.00 ± 0.00
100.00 ± 0.00
1.00 ± 0.20
86.61 ± 2.56
PE
5
1.75 ± 0.13a
63.75 ± 1.88b
4.25 ± 0.24a
33.48 ± 2.04b
EA
5
0.00 ± 0.00b
100.00 ± 0.00a
0.25 ± 0.13b
96.88 ± 1.56a
n-B
5
0.00 ± 0.00b
100.00 ± 0.00a
0.00 ± 0.00b
100.00 ± 0.00a
Effect of organic extracts of B. javanica on emergence of E. indica (a) and B. pilosa (b). Effect of B. javanica extracts on the growth of B. pilosa (c). (Blank: blank control; CK: negative control; M: methanol extract, 10 mg/mL; PE: petroleum ether extract, EA: ethyl acetate extract, n-B: n-butanol extract, 5 mg/mL).
After 30 days of pot experiment, the effect of different extracts on the growth of seedlings of B. pilosa changed significantly (Fig. 3). Compared with the results of 15 days investigation, the n-butanol extract of B. javanica not only completely inhibited the emergence of B. pilosa, but also inhibited its growth. However, methanol extract and ethyl acetate extract had some inhibition on the emergence of B. pilosa, but the inhibition on the growth of seedlings is weak, and they were weaker than n-butanol extract.
3.3 Structural elucidation
The bioassay-guided separation of the components in the n-butanol extract of B. javanica residue revealed eight chemical constituents. All the compounds were purified by column chromatography, and their structures were characterized by 1H NMR, 13C NMR and ESI-MS. Additionally, their structures were confirmed by direct comparisons with previously reported spectroscopic data (NoorShahida et al., 2009; Li and Zuo, 1980; Mayser et al., 2002; Fotso et al., 2008; Colombo et al., 2010; Parveen et al., 2017). On the basis of the spectroscopic data, the constituents were identified as the known compounds, bruceine D (1), bruceine E (2), bruceine F (3), pityriacitrin (4), 4-(9H-β-carbolin-1-yl)-4-oxobut-2-enoic acid methyl ester (5), protocatechuic acid (6), vanillic acid (7) and carisphthalate (8). Among them, pityriacitrin, 4-(9H-β-carbolin-1-yl)-4-oxobut-2-enoic acid methyl ester and carisphthalate were isolated from B. javanica residue for the first time.
3.4 Herbicidal activity of three quassinoid compounds
The laboratory herbicidal activities of compounds bruceines D–F (1–3) were evaluated using E. indica and B. pilosa at 250 μg/mL. In the herbicidal activity assay, all of the three quassinoid compounds exhibited potential inhibition against the seed germination of E. indica with inhibition of 100.00%, 43.29% and 49.96%, respectively (Table 3). At this concentration, bruceine D showed the greatest germination inhibition in E. indica, which was far stronger than atrazine (−4.82%), a commercial herbicide co-assayed as the positive control. Conversely, bruceine D had weaker inhibitory activity against the seed germination of B. pilosa, but the inhibitory rate (46.67%) was much greater than those of atrazine (10.00%). However, the inhibition of bruceine E and F on seed germination of B. pilosa was very weak or not at all.
Treatments
Conc. (μg/mL)
E. indica
B. pilosa
Seed germination
Inhibition rate (%)
Seed germination
Inhibition rate (%)
Blankb
–
26.75 ± 0.55
–
29.25 ± 0.13
–
CKc
–
26.75 ± 0.38
–
30.00 ± 0.00
–
Atrazined
250
28.00 ± 0.20a
−4.82 ± 0.95c
27.00 ± 0.35a
10.00 ± 1.18b
Bruceine D
250
0.00 ± 0.00c
100.00 ± 0.00a
16.00 ± 0.54b
46.67 ± 1.80a
Bruceine E
250
15.25 ± 0.75b
43.29 ± 2.08b
27.25 ± 0.38a
9.17 ± 1.25b
Bruceine F
250
13.50 ± 1.01b
49.96 ± 3.16b
25.75 ± 0.63a
14.17 ± 2.08b
Furthermore, bruceine D, E and F could significantly affected seedling growth of these two weeds. The 7-day investigation results (Fig. 4) showed that bruceine D represented the strongest inhibitory effect on seedling growth of E. indica and the root elongation of B. pilosa at 250 μg/mL compared with atrazine and the negative control. Further, the shoot elongation of B. pilosa was also completely suppressed at this concentration for bruceine E and F.
Effect of bruceines D–F from B. javanica on E. indica (a) and B. pilosa (b). Effect of compounds D–F on the root (c) and shoot (d) length of E. indica and B. pilosa. The data in the figure were given as mean ± SD for three repetitions. Different letters indicated significantly different (p < 0.05) among the columns with same color, based on DMRT. (Blank: blank control; CK: negative control; Atrazine: positive control, 1: bruceine D, 2: bruceine E, 3: bruceine F, 250 μg /mL).
The herbicidal effect of bruceine D at different concentrations (31.25, 62.5, 125, 250, 500 μg/mL) was further determined (Fig. 5). Bruceine D showed significant inhibitory effect on seed germination and growth of E. indica in the range of experimental concentrations, and which was far stronger than that of positive control. In particular, bruceine D completely inhibit root growth of E. indica at concentrations as low as 31.25 μg/mL, while it caused complete inhibition of seed germination and shoot elongation of E. indica when the concentration was at least 125 μg/mL. Furthermore, bruceine D also completely suppressed the shoot growth of B. pilosa at a concentration of 125 μg/mL. However, the inhibitory effect of bruceine D on the germination of B. pilosa seeds was the greatest when the concentration reached the maximum.
Effects of different concentrations (31.25, 62.5, 125, 250 and 500 μg/mL) of bruceine D on E. indica (a) and B. pilosa (e). Effects of different concentrations of bruceine D on the seed germination (b), root (c) and shoot (d) length of E. indica. Effects of different concentrations of bruceine D on the seed germination (f), root (g) and shoot (h) length of B. pilosa. The data in the figure were given as mean ± SD for three repetitions. Different letters indicated significantly different (p < 0.05) at each same concentration, based on DMRT. (CK: negative control; Atrazine: positive control).
4 Discussion
In this study, the methanol, ethyl acetate and n-butanol extracts of B. javanica residue had certain inhibitory effects on the gramineous weed (E. indica) and the broad-leaved weed (B. pilosa). In particular, ethyl acetate and n-butanol extracts showed the most significant inhibition on seed germination and growth of E. indica. These results indicated that B. javanica residue has potential herbicidal activity, which are consistent with the previous reports (Wang, 2016; Heisey and Putnam, 1985). It has been previously reported that there are still a large number of chemical components with high activity in the residues produced after the extraction of B. javanica oil, among which the content of quassinoids is relatively high (Wang, 2016). In addition, quassinoid compounds have phytotoxicity (Heisey and Putnam, 1985).
The total amount of plant residues, especially waste residue of medicinal plants (i.e. herbal residues) is huge. If it is not handled properly, it will cause waste of resources and environmental pollution (Li et al., 2010). At present, there are many ways to utilize traditional herbal residues as resources (Yang et al., 2015; Xu et al., 2009), which effectively alleviates the situation of difficult comprehensive utilization of herbal residues. Herbal residue is a kind of high quality light substrate raw material and organic fertilizer, which can be used to raise seedlings and cultivate substrates and improve soil fertility. In addition, it can make the seedlings grow and strengthen the disease resistance (Yang et al., 2015). Therefore, the residues of B. javanica seed can be processed into organic fertilizer to improve the field fertility, improve the utilization efficiency of the waste resource, turn the waste into treasure, and realize the sustainable development of environment and agriculture.
Weeds reduce the yield and quality of crops (Jabran et al., 2015), so weed control is imminent. Currently, the application of chemical herbicides is still the main means of weed control (Böcker et al., 2019), but its limitations urge us to find new efficient and low toxic weed control methods. In recent years, the plant crude extract with herbicidal activity has gradually become a research hotspot. The reasons are as follows: on the one hand, it is the basis of separating and identifying high herbicidal activity compounds and developing botanical herbicides; on the other hand, many plant crude extracts can be directly applied to weed control in the field. The crude extracts of many plants can severely inhibit seed germination and growth of weeds. Theoretically, this preparation can be used as a natural herbicide (Duke et al., 2002; Yang, 2008). Therefore, B. javanica waste residue can be used to control of weeds in the field, especially for the control of grass weeds, because of its pre-emergent sealing effect on grass weeds, the herbicidal activity is significant. In this study, bruceine D, an active herbicidal compound, was obtained from B. javanica residue by activity tracing. In addition, our results indicated that bruceine D can inhibit the seed germination and growth of grass weeds and broad-leaved weeds at a certain concentration, which is expected to be used as a leading compound to develop botanical herbicides.
In previous reports, bruceine D performanced significant anti-cancer activity, which usually inhibited the proliferation of cancer cells or caused apoptosis by regulating some biological processes in cells (Fan et al., 2020; Shen et al., 2020; Tian et al., 2020). In addition, bruceine D also had hypoglycaemic (Choo et al., 2012), anthelmintic (Wang et al., 2011), antibabesial (Subeki et al., 2007) and antitobacco mosaic virus (TMV) activities (Yan et al., 2010). Similarly, bruceine E, which is similar to bruceine D in structure, also has cytotoxic (Liu et al., 2011; Liu et al., 2012) and hypoglycemic activities (Choo et al., 2012). In contrast, there are almost no studies on the activity of bruceine F. Therefore, in the present work, bruceine D, E and F, which were obtained from B. javanica residue, exhibited a certain inhibitory effect on E. indica and B. pilosa. Among them, bruceine D can completely inhibit the root elongation of E. indica at 31.25 μg/mL, and can completely inhibit the seed germination and growth of E. indica and the shoot elongation of B. pilosa at 125 μg/mL. This is similar to the result that bruceine D aqueous solution reported in the invention patent can completely inhibit the seed germination of monocotyledonous weed (Avena fatua L.) and dicotyledonous weed (Brassica napus L.) at the concentration of 200 and 400 μg/mL, respectively (Li et al., 2016). Therefore, bruceine D can significantly inhibit the germination of weed seeds at a lower concentration. In addition, bruceine E and F can completely inhibit the shoot growth of B. pilosa at 250 μg/mL. These results are supported by the phytotoxicity of the previously reported quassinoids (Heisey and Putnam, 1985).
Furthermore, our results showed that the inhibitory effects of bruceines D–F (1–3) at the same concentration on E. indica and B. pilosa were slightly different. At the concentration of 250 μg/mL, bruceine D had the strongest inhibitory effect on the seed germination of E. indica and the general inhibitory effect on the germination of B. pilosa seeds. However, bruceine E and F had a general inhibitory effect on the germination of E. indica seeds and very weak inhibitory effect on the seed germination of B. pilosa. By analyzing their structure–activity relationship (SAR), it can be concluded that the hydroxylation of ketone group at C-2 position is the main factor affecting their herbicidal activity, while the alkyl chain at C-13 position has no obvious effect on their herbicidal activity. The above results are similar to those of SAR of bruceines D−F in vitro anticomplement activity (Zhan et al., 2019). Therefore, the results of this study may provide a basis for further structure optimization of herbicide lead compounds and herbicides development with high efficiency and low toxicity.
At present, the research of B. javanica residue mainly focuses on its medical activities. For example, antitumor (Yan et al., 2019) and anti-inflammatory (Gao et al., 2019). However, there are still many active ingredients in the waste residue of B. javanica (Wang, 2016), which is necessary for its research and development. In this study, the herbicidal activity of B. javanica residue was systematically determined, and bruceine D, one of the active components from B. javanica residue, was determined. It not only makes full use of the waste resources of B. javanica, but also provides a foundation for its further application in agriculture.
5 Conclusion
The crude methanol extract, ethyl acetate extract and n-butanol extract of B. javanica residue have significant inhibitory effects on both E. indica and B. pilosa. Among them, n-butanol extract has the most significant inhibitory effect on the seed germination and growth of E. indica. Therefore, B. javanica residue can be used to control gramineous weeds in the field. At the same time, as a member of industrial Chinese medicine waste residue, B. javanica residue is used as organic fertilizer for weeding, realizing the real transformation of waste into treasure. In addition, bruceine D, E and F, isolated from the n-butanol extraction layer of B. javanica residue, have certain inhibitory activities against both E. indica and B. pilosa. In particular, bruceine D can significantly inhibit the germination of weed seeds at low concentrations. It has the potential to be used as a leading structure of herbicides for subsequent research and development, and has broad prospects for the control of gramineous weeds.
Acknowledgments
This work was supported by Guangxi innovation-driven development special project (Guike AA18118015), Key project of Guangxi Natural Science Foundation [No. 2017GXNSFDA198049], and the National Natural Science Foundation of China [No. 31660529].
Declaration of Competing Interest
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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