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Article

Allelopathic Activity of a Novel Compound and Two Known Sesquiterpene from Croton oblongifolius Roxb.

by
Seinn Moh Moh
1,2,
Shunya Tojo
3,
Toshiaki Teruya
4 and
Hisashi Kato-Noguchi
1,2,*
1
Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki 761-0795, Japan
2
The United Graduate School of Agricultural Sciences, Ehime University, Matsuyama 790-8566, Japan
3
Graduate School of Engineering and Science, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan
4
Faculty of Education, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan
*
Author to whom correspondence should be addressed.
Agronomy 2024, 14(4), 695; https://doi.org/10.3390/agronomy14040695
Submission received: 29 February 2024 / Revised: 18 March 2024 / Accepted: 25 March 2024 / Published: 28 March 2024
(This article belongs to the Special Issue Extraction and Analysis of Bioactive Compounds in Crops—Series II)
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Versions Notes

Abstract

:
Plant extracts with allelopathic activity and their related compounds have been investigated for a long time as an eco-friendly approach to sustainable weed management. Croton oblongifolius (Roxb.) is a traditional medicinal plant valued for its diverse source of bioactive compounds that have been used to treat various diseases. C. oblongifolius leaf extract was previously described to involve a number of allelochemicals. Therefore, we conducted this research to explore more of the allelochemicals in the leaves of C. oblongifolius. The leaf extracts showed significant inhibitory activity against two test plants, Lolium multiflorum (monocot) and Medicago sativa (dicot). The bioassay-directed chromatographic purification of the leaf extracts yielded three compounds, including one novel compound, identified using spectral data, as follows: (1) alpinolide peroxide, (2) 6-hydroxy alpinolide, and (3) 3-hydroxy-5-isopropyl-3-methyl-2,3-dihydro-1H-inden-1-one (a novel sesquiterpene). These compounds considerably limited the growth of L. sativum. The compound concentrations affecting a 50% growth limitation (IC50) of L. sativum varied from 0.16 to 0.34 mM. Therefore, these characterized compounds may be allelopathic agents that cause the allelopathy of C. oblongifolius.

1. Introduction

In agricultural systems worldwide, weeds are more detrimental to the economy than other pests (insects, bacteria, and fungi) [1] and a main factor limiting crop productivity [2]. Weed infestations decrease the quantity and quality of agricultural products, which results in financial distress for farmers [3]. Nowadays, farmers most frequently employ the chemical control of weeds, which involves using synthetic herbicides [4]. However, the overuse of herbicides, fungicides, and fertilizers in modern agricultural practices has negatively affected soil quality and its chemical composition, leading to soil and water contamination [5], as well as the development of weeds that are tolerant to herbicides. Presently, there are about 272 weed species (including 117 monocots and 155 dicots) that have developed increased resistance to 168 herbicides [6]. Additionally, several countries have recently banned various herbicides because of the health and environmental risks associated with their widespread use [7]. To decrease the use of herbicides, it is possible to search for and use an eco-friendly approach that includes allelopathy to restrict weeds through cultivating specific crops or using fields sprayed with extracts from allelopathic plants [8].
Allelopathy is the result of plants or microbes naturally competing with one another by obstructing their competitors’ growth through the production and release of plant bioactive substances known as allelochemicals [9]. Using plant extracts with allelopathic properties can effectively control insect pests and weeds [9]. Extracts that are acquired from different plants have been utilized for therapeutic or nutritional purposes and could be used to create eco-friendly herbicides for weed management [10]. Bioherbicides, which are composed of phytotoxins generated by plant extracts, insects, or microbes, serve as a crucial tool for natural weed control [11]. Additionally, plant-extract-based bioherbicides have shown efficacy against various weeds. Certain plant extract compounds have very specific weed-growth-suppressive potential without negatively affecting crop plants [12]. Moreover, numerous compounds that are allelopathic have been identified in different plant species [13,14,15]. The chemical structure of the allelochemicals results in them being more environmentally friendly than synthetic herbicides [16]. Unlike synthetic herbicides, these naturally occurring compounds function by obstructing the biochemical pathways of weeds. As a result, they could be regarded as a sustainable substitute for artificial herbicides because they are environmentally safe [17,18]. Among the phytotoxic compounds, terpenoids are one of the most representative classes of major compounds [19]. Some terpenoids are extremely valuable, because they can be used in industry, agriculture, and medicine [20]. The majority of terpenoids, particularly mono- and sesquiterpenes, are found in the essential oils of medicinal plants [21]. Recently, medicinal plants have gained attention because of their comparatively high allelopathic activity [22] and for the secondary metabolites or active components that they contain [23]. Many researchers have recently reported that the growth of test plant species, including weeds, was inhibited by the phytotoxic substances found in medicinal plants [24,25,26]. Kato-Noguchi (2023) [27] reported that many medicinal plants are highly allelopathic and have yielded over 100 allelochemicals, including novel compounds that exhibit different levels of biological activities and functions. These findings showed that medicinal plants contain different kinds of phytotoxic substances that could exert allelopathic activity, and these plants are probable candidates that could be used to isolate and identify potent allelopathic substances that can be used as bioherbicides for sustainable weed management.
Croton oblongifolius (Roxb.) (family Euphorbiaceae), known locally as “Thetyin-gyi” in Myanmar, is well known for its therapeutic properties. It is normally widespread in Myanmar and other countries in Asia [28,29] (Figure 1). The whole plant is used to treat liver diseases, increased blood pressure, dysmenorrhea, dyspepsia and dysentery [30], and gastric ulcers and gastric cancers [31], and as a purgative [32]. Methanolic leaf extracts of C. oblongifolius have been found to exhibit anticancer, antibacterial, antihepatotoxic, and hepatoprotective activity [31,33,34]. The reported phytochemicals of this plant are as follows: monoterpenes, sesquiterpenes (patchoulenone) [35], diterpenoids (nasimalun A), crovatin, evatin, (−)-hardwickiic acid) [36], croblongifolin [37], and crotocembraneic acid [38]. Based on its pharmacological and bioactive molecules, C. oblongifolius may have active substances with a lot of allelopathic activity. Nonetheless, there is little information on the allelopathic potential and allelochemicals of C. oblongifolius. In our earlier research, C. oblongifolius extracts significantly restricted the growth of four plants (Lactuca sativa, Lepidium sativum, Phleum pratense, and Echinochloa crusgalli), and we also identified one C13 nor-isopenoid and three sesquiterpenes in the leaf extracts [39]. Moreover, we previously observed that one other fraction of C. oblongifolius leaf extracts was strongly allelopathic, suggesting that other constituents of interest remain to be identified. Therefore, this research was conducted to further investigate the allelopathic potential and possible allelochemicals in the leaves of C. oblongifolius. This present and previous research focuses on the allelopathy of C. oblongifolius and reports on the isolation of allelochemicals (including novel compounds) and the evaluation of the allelopathic activity of these compounds under laboratory conditions. However, further research is necessary in laboratory or soil conditions in order to study the molecular targets of the active compounds in the plant cell and their modes of action for the development of bioherbicides.

2. Materials and Methods

2.1. Plant Materials

The immature and mature leaves of C. oblongifolius were brought from Khin-U, Sagaing region, Myanmar (95°48′12″ E; 22°49′4″ N) (Figure 1), in May 2020. The leaves were pulverized to obtain a fine leaf powder after drying in the shade. Two plant species, L. multiflorum (monocot) and M. sativa (dicot), were selected as representative bioassay plants.

2.2. Extraction

The dry leaf powder (300 g) was extracted using 70% (v/v) aqueous methanol (1.5 L) for two days (48 h). The extract was passed through a sheet of filter paper (No. 2, 125 mm; Toyo Ltd., Tokyo, Japan) to obtain the first filtrate. Then, the residues were extracted once again by soaking with an equal volume of methanol (1.5 L) for one day (24 h) to obtain the second filtrate. To produce an aqueous solution of crude extracts, the two filtrates were condensed in a rotavapor at 40 °C.

2.3. Growth Bioassay

A total of 30 g (dry weight) of concentrated crude extracts was dissolved in 20 mL of methanol. Then, the crude extracts with various doses (1 (0.4 μL), 3 (1.2 μL), 10 (4 μL), 30 (12 μL), 100 (40 μL), and 300 (120 μL) mg dry weight (DW) equivalent C. oblongifolius extract/mL) were placed in filter papers in 2.8-cm Petri dishes. The solvent in the Petri dishes was dried in a laminar flow cabinet. Following drying, 0.6 mL of an aqueous solution of Tween 20 (0.05%, Nacalai Tesque, Inc., Kyoto, Japan) was applied to the filter papers. The seeds of L. multiflorum were germinated for 36 to 48 h at 25 °C to obtain sprouted seeds and to break down the seed dormancy. Accordingly, ten seeds of M. sativa and ten sprouted seeds of L. multiflorum were added into each Petri dish and germinated for 48 h at 25 °C. The seeds or sprouted seeds that had been moistened with an aqueous Tween 20 solution (without the concentrated crude extracts) were used as controls. The lengths of the hypocotyl/coleoptile and roots of the seedlings were measured to evaluate the growth-inhibitory activity. A completely randomized design (CRD), carried out two times with three replications (10 seedlings per replication), was used to conduct the growth assay experiment (n = 60).

2.4. Separation of the Active Substances

The dry leaf powder (3200 g) was extracted using the methodology outlined in the sections on extraction and bioassay. The biological activities of each of the isolated fractions are usually determined by measuring the hypocotyl and root growth of L. sativum. The residue was adjusted to a pH of 7.0 with 1 M NaOH, which was then separated six times using the same volume (150 mL each time) of EtOAc. The EtOAc was then placed into a silica gel column (70–230 mesh; Merck, Rahway, NJ, USA), after being concentrated until it was dry and eluted, with seven fractions of an ethyl acetate and n-hexane mixture 20:80 (F1), 30:70 (F2), 40:60 (F3), 50:50 (F4), 60:40 (F5), 70:30 (F6), and 80:20 (F7); v/v; 150 mL each step, EtOAc (150 mL) (F8), and MeOH (300 mL) (F9). Among the nine fractions, the third most active fraction (F4) and the second most active fraction (F6) were selected for further isolation steps.
The third most active fraction of the silica gel column (F4) was dried, and its residue was separated by chromatography using a Sephadex LH-20. The active peak fraction (F3), which was eluted with 60% aqueous methanol, exhibited the highest inhibitory activity in the Sephadex LH-20. The most active fraction (F3) was then further purified using a reverse-phase C18 cartridge. It was discovered that the active fraction, which was eluted with 40% aqueous methanol (F3), had some activity. The F3 residue that remained after it evaporated was purified through the use of reverse-phase HPLC (flow rate of 0.5 mL/min) with 42% aqueous MeOH. Two active peak fractions (fractions 1 and 2) were observed at the retention periods of 88–93 and 100–113 min, respectively. Consequently, active compounds 1 and 2 (derived from the third most active fraction of the silica gel column) were measured using spectral analysis.
After that, the second most active fraction of the silica gel column (F6) was evaporated, and the residue was separated by chromatography using the Sephadex LH-20. Fraction F3, which was loaded with 60% aqueous methanol, exhibited the highest inhibitory activity. Active fraction F3 was further separated using a reverse-phase ODS cartridge. The active fraction was loaded with 40% aqueous MeOH (F3). The F3 residue that remained after it evaporated was purified through the use of reverse-phase HPLC (flow rate of 0.5 mL/min) with 45% aqueous MeOH. Active peak fraction 3 was observed at the retention period of 132–135 min. Finally, active compound 3 (derived from the second most active fraction of the silica gel column) was measured using spectral data.

2.5. Measurement of NMR Spectral Data and Chemical Shifts of Identified Compounds

The molecular structures of the three compounds were identified using 1H and 13C-NMR spectra (500 MHz, CDCl3) and specific rotation. The specific rotations were measured with a JASCO P-1010 polarimeter (Jasco Co., Tokyo, Japan). All NMR spectral data were documented on a Bruker AVANCE Ⅲ NMR spectrometer (500 MHz) (Bruker Switzerland AG, Fallanden, Switzerland). The molecular (chemical) shifts of novel compounds were stated relative to the signal of the remaining solvent ((CDCl3: δH 7.26, δC 77.16) and (acetone-d6: δH 2.05)). HR-ESI-MS was achieved on a Thermo Scientific Orbitrap Exploris 240 Mass Spectrometer (Thermo-Fisher K.K., Tokyo, Japan).

2.6. Bioassays of the Isolated Compounds

The isolated compounds (1, 2, and 3) were diluted in 2 mL of methanol, and six treatment doses of 0.01, 0.03, 0.1, 0.3, 1, and 3 mM were prepared. The activities of the three compounds were evaluated using an L. sativum bioassay, as described in Section 2.2. The percentage of seedling growth was determined by measuring the lengths of the hypocotyl and root. The bioassay studies were set with a CRD design with three replications, with 10 seedlings of L. sativum per replication (n = 30).

2.7. Statistical Analysis

All assay experiments were carried out two times for the growth assays and one time for the compound assays, which were performed using a CRD design with three replications and 10 seedlings per replication. The mean ± standard error (SE) represents the obtained results. SPSS (version 16.0) software was used to calculate the analysis of variance (ANOVA) data (SPSS Inc., Chicago, IL, USA). The significant differences between the treated and control plant groups were assessed using Tukey’s test (p < 0.05). The IC50 of each bio-assayed plant species was evaluated using GraphPad Prism software ®Ver. 6.0, La Jolla, CA, USA.

3. Results

3.1. Growth-Suppressive Activity of C. oblongifolius

The leaf extracts restricted the seedling growth of M. sativa (dicot) and L. multiflorum (monocot) at concentrations higher than 10 mg DW equivalent C. oblongifolius extract/mL (Figure 2A,B). The dose of 30 mg DW equivalent C. oblongifolius extract/mL inhibited the hypocotyl/coleoptile growth of M. sativa and L. multiflorum to 24.18 and 39.85% of the control, respectively, and inhibited the root growth to 18.00 and 34.55% of the control, respectively. Additionally, the dose of 300 mg DW equivalent C. oblongifolius extract/mL extract concentration totally suppressed the root and hypocotyl/coleoptile growth of M. sativa and L. multiflorum.
The doses of the C. oblongifolius leaf extracts necessary for 50% growth restriction (IC50) of the root and hypocotyl/coleoptile of M. sativa and L. multiflorum ranged from 3.23 to 13.85 mg DW equivalent C. oblongifolius extract/mL (Table 1). The IC50 values for the root growth of M. sativa (3.23) and L. multiflorum (9.87) were significantly lower than that of their hypocotyl/coleoptile growth (5.44 and 13.85, respectively).

3.2. Characterization of the Allelopathic Compounds

The ethyl acetate (EtOAc) fraction exhibited more inhibitory activity than the aqueous fraction in partition [39]. Therefore, EtOAc was chosen for a subsequent isolation step and loaded onto a silica gel column, resulting nine fractions (Figure 3). According to the growth assay result, the second most active fraction (F6), which was eluted with the 70:30 ethyl acetate and n-hexane mixture, and the third most active fraction (F4), which was eluted with the 50:50 ethyl acetate and n-hexane mixture, both exhibited allelopathic activity. At a concentration of 0.1 g DW equivalent C. oblongifolius extract/mL, F4 inhibited the hypocotyl and root growth of L. sativum to 40.99 and 42.58%, and F6 inhibited the growth to 18.63 and 13.77% of the control growth, respectively (Figure 3). These active fractions (F4 and F6) were selected for further chromatographic purification steps, as follows: Sephadex LH-20, reverse-phase C18 cartridge, and HPLC. Finally, three active compounds with growth-suppressive activity were identified via spectral data analysis.
The chemical formula of compound 1 was C15H22O5 (found as a colorless oil), as revealed by (HR) ESI-MS at m/z 305.1360 [M + Na]+ (calcd for C15H22O5Na 305.1359). The 1H NMR (500 MHz, CDCl3) spectrum of this compound showed the following: δH 5.96 (1H, d, J = 1.2), 1.41 (3H, s), 1.27 (3H, d, J = 6.8), 1.20 (3H, d, J = 6.9), and 0.83 (3H, d, J = 7.3). These obtained 1H-NMR data were in accordance with the previously published research [40], and the compound was determined to be alpinolide peroxide (Figure 4A).
The chemical formula of compound 2 was C15H22O4 (found as a colorless oil), as determined by (HR) ESI-MS at m/z 289.1411 [M + Na]+ (calcd for C15H22O4Na 289.1410). The 1H NMR (500 MHz, CDCl3) spectrum of this compound showed the following: δH 5.81 (1H, s, J = 0.9), 3.36 (1H, m), 2.32 (3H, s), 1.29 (3H, d, J = 6.8), 1.24 (3H, d, J = 6.8), and 0.92 (6H, d, J = 7.0). The data obtained from 1H-NMR were similar to those found in the literature [40], and this compound was identified as 6-hydroxy alpinolide (Figure 4B). The detail chemical composition of compounds 1 and 2 are provided in the Supplementary File.
Compound 3 was found as a colorless oil, [α]D27 = +7.3 (c 0.06, CHCl3), and its chemical formula, C13H17O2Na, was determined by (HR) ESI-MS m/z 205.1223 [M + H]+ (calcd for C13H17O2Na, 205.1223) (Figure 4C). The NMR data of this compound are shown in Table 2.

3.3. Bioactivity of Compounds 1, 2, and 3

Alpinolide peroxide (compound 1), 6-hydroxy alpinolide (compound 2), and 3-hydroxy-5-isopropyl-3-methyl-2,3-dihydro-1H-inden-1-one (compound 3) significantly restricted the seedling growth of L. sativum. The response of the hypocotyls and roots depended on the dose of the compound. The dose of compound that was greater than 0.1 mM notably suppressed the growth of the L. sativum hypocotyls and roots (Figure 5A–C).
The doses of each compound needed for 50% growth limitation (IC50) of the hypocotyls and roots of L. sativum were 0.21 and 0.17 mM for alpinolide peroxide, 0.26 and 0.23 mM for 6-hydroxy alpinolide, and 0.34 and 0.16 mM for 3-hydroxy-5-isopropyl-3-methyl-2,3-dihydro-1H-inden-1-one, respectively (Table 3).

4. Discussion

Many compounds of terpenoids, or terpenes, are a structurally diverse and abundant class of secondary substances (or metabolites) in plants and are involved in interactions between plants and insects, pathogens, and other plants [41]. These compounds serve as defense compounds against plants, microbes, and herbivores [42,43]. Terpene allelopathic substances can lead to autotoxicity when they are released into the environment, causing plants to compete for available space and struggle to maintain population density and obtain growth advantages [19,44]. These terpenes mainly exhibit their allelopathic mechanism by inhibiting plant growth, disrupting cell membrane function, and impeding physiological metabolism [42]. The findings of the present study show that the leaf extracts of C. oblongifolius significantly suppressed the seedling growth of a dicot (M. sativa) and a monocot (L. multiflorum), with dose-dependent inhibitory activity (Figure 2A,B). These growth-inhibitory effects are in agreement with the findings from our earlier investigation. In our earlier study, we found that C. oblongifolius extracts inhibited the growth of four plants (two dicots, L. sativum and L. sativa, and two monocots, E. crusgalli and P. pratense) [39]. Additionally, the IC50 values of the extracts used against the hypocotyls/coleoptiles and roots of M. sativa and L. multiflorum (in this present research) showed species-dependent inhibitory activity (Table 3). Other researchers have documented similar results regarding the species- and dose-dependent allelopathic properties for the extracts of Anredera cordifolia, Nicotiana glauca, Callistemon viminalis, and Aegle marmelos [45,46,47,48].
Based on the IC50 results, the root growth of the two plant species in the current study and the four plant species in the prior study [39] showed greater susceptibility to the C. oblongifolius leaf extracts than the hypocotyl/coleoptile growth. Roots are more susceptible to plant extracts than hypocotyls or coleoptiles, because they come into direct contact with the allelochemicals during germination [49,50]. In addition, root growth depends on cell expansion, which is affected by allelochemicals [51]. Thus, the growth-inhibitory effects of C. oblongifolius against the seedling growth of all of the bio-assayed plants suggest that leaf extracts may contain allelochemicals. In our previous research, we identified four allelochemicals, 2-hydroxy alpinolide, (3R,6R,7E)-3-hydroxy-4,7-megastigmadien-9-one, epialpinolide, and alpinolide, from C. oblongifolius [39]. In the current research, another three allelochemicals, alpinolide peroxide, 6-hydroxy alpinolide, and 3-hydroxy-5-isopropyl-3-methyl-2,3-dihydro-1H-inden-1-one, were isolated and identified from extracts of C. oblongifolius (Figure 4A–C).
Compound 1 (alpinolide peroxide) is a sesquiterpene of the secoguaiane type, connected with a cyclic peroxide bond. The infrared (IR) spectrum of this compound shows an α- and β-unsaturated butenolide, but it lacks either a methyl ketone or a lactonic methine proton. The 1H-NMR and IR spectra showed carbon atoms, a hydroxyl group, and a peroxide linkage between C6 and C10 [40].
Compound 2 (6-hydroxy alpinolide) is also a secoguaiane type of sesquiterpene. The IR spectrum of this compound has an α- and β-unsaturated butenolide and a methyl ketone group. The spectroscopic data indicate that 6-hydroxy alpinolide has a hydroxyl group at the C-6 position when compared to that of alpinolide [40].
The NMR spectroscopic data of compound 3, as measured in CDCl3, presented with three aromatic protons (δH 7.65, 7.52, and 7.36), three methyl groups (δH 1.72 and 1.31), one methine proton (δH 3.04), and two methylene protons (δH 2.89 and 172). In the 13C-NMR spectrum, thirteen carbon signals were found, comprising one carbonyl carbon (δC 202.6), six aromatic carbons (δC 159.1, 157.8, 133.6, 128.3, 123.3, and 121.1), three methyl carbons (δC 29.3 and 23.8), one quaternary carbon (δC 74.5), one methine carbon (δC 34.9), and one methylene carbon (δC 54.2). A specific analysis of this compound’s COSY spectrum showed two partial structures, C6-C7 and C10-C9-C11 (Figure 6). The connectivity of these fragments was clarified using the HMBC spectrum, as follows: H2 (δH 2.89) to C1 (δC 202.6); H4 (δH 7.52) to C3 (δC 74.5), C6 (δC 128.3), and C7a (δC 133.6); H6 (δH 7.36) to C4 (δC 121.1) and C7a (δC 133.6); H7 (δH 7.65) to C1 (δC 202.6), C3a (δC 159.1), and C5 (δC 157.8); H8 (δH 1.72) to C2 (δC 54.2), C3 (δC 74.5), and C3a (δC 159.1); and H9 (δH 3.04) to C5 (δC 157.8). The 2D NMR correlations of this compound were determined, as shown in Figure 6. According to the NMR spectral data, compound 3 is a novel compound, and is defined as 3-hydroxy-5-isopropyl-3-methyl-2,3-dihydro-1H-inden-1-one.
The characterized compounds alpinolide peroxide and 6-hydroxy alpinolide are secoguaiane-type sesquiterpenes, and 3-hydroxy-5-isopropyl-3-methyl-2,3-dihydro-1H-inden-1-one is a guaiane-type sesquiterpene. These compounds are commonly found in A. japonica (Thunb.) Miq and many other Zingiberaceae plant species [52,53]. Several researchers have also described the pharmaceutical and pest protection abilities of monoterpenoids and sesquiterpenoids from medicinal plants [21,54]. Many sesquiterpene compounds have been documented to have a variety of bioactivities, including cytotoxic [55], antioxidant [56], anti-aging, anti-inflammation, anti-anaphylaxis, antimicrobial, and antifungal activities [52,53,57,58]. Liu et al. (2022) [59] stated that four sesquiterpenes isolated from Ambrosia artemisiifolia had inhibitory effects on Chenopodium album, Digitaria sanguinalis, Setaria viridis, and Arabidopsis thaliana. Four drimane sesquiterpenes from the roots of D. brasiliensis were found to be phytotoxic to the coleoptiles of Triticum aestivum at a high concentration [60]. The sesquiterpene secreted by Juncus effuses has shown autotoxicity, and it restricts the growth of its own seedlings [42]. The volatile monoterpenoids α-pinene and camphor inhibit the germination, synthesis of DNA, and the proliferation of meristem cells in Brassica campestris seedlings [61]. Some terpenoids, such as limonoids and pyrethrin, are used for insecticide production [21,62]. Macias et al. (2000) [63] stated that one of the sesquiterpene lactones, dehydrozaluzanin C, identified from the Compositae family, showed bioherbicidal potential activity against dicotyledonous plants. Nevertheless, this study is the first to isolate the sesquiterpene allelopathic substances (alpinolide peroxide, 6-hydroxy alpinolide, and 3-hydroxy-5-isopropyl-3-methyl-2,3-dihydro-1H-inden-1-one) with growth-suppressive activity from C. oblongifolius.
In this current research, alpinolide peroxide, 6-hydroxy alpinolide, and 3-hydroxy-5-isopropyl-3-methyl-2,3-dihydro-1H-inden-1-one significantly restricted the seedling growth of L. sativum (Figure 5A–C). Based on the IC50 values of the three compounds, alpinolide peroxide has a higher growth-suppressive potential than 6-hydroxy alpinolide and 3-hydroxy-5-isopropyl-3-methyl-2,3-dihydro-1H-inden-1-one. The inhibitory properties exhibited by these compounds could be attributed to the structural differences in the phytotoxic agents [64]. In our investigations, we have found that C. oblongifolius (mature and immature) leaves have growth-suppressive activity, and their characterized compounds, 2-hydroxy alpinolide, (3R,6R,7E)-3-hydroxy-4,7-megastigmadien-9-one, epialpinolide, alpinolide (previous research) [39], alpinolide peroxide, 6-hydroxy alpinolide, and 3-hydroxy-5-isopropyl-3-methyl-2,3-dihydro-1H-inden-1-one (current research), may contribute to their allelopathy. Additionally, C. oblongifolius leaves are allelopathic to the monocot and dicot test plant species, which are affected by the different biological activities of the identified compounds. Thus, the extracts of the mature and immature leaves of this plant can be used as foliar sprays, and fallen leaf residues can be applied as surface mulches and residue incorporation for sustainable weed management.

5. Conclusions

The extracts of mature and immature leaves of C. oblongifolius have revealed significant growth-inhibitory potential on M. sativa and L. multiflorum. Three compounds were identified through bio-guided chromatographic purification steps, and were identified through spectroscopy as alpinolide peroxide, 6-hydroxy alpinolide, and 3-hydroxy-5-isopropyl-3-methyl-2,3-dihydro-1H-inden-1-one (a novel sesquiterpene). These compounds have a significant growth-inhibitory effect on L. sativum hypocotyls and roots. The allelopathy of C. oblongifolius leaves may be responsible for the growth-inhibitory activities of the isolated compounds. Therefore, based on the outcomes of this study, we suggest plant extracts and fallen leaf residues of C. oblongifolius might be useful for foliar sprays, surface mulches, and residue incorporation to suppress weed growth in crop fields. Additionally, its allelochemicals may be used to develop plant-based bioherbicides. However, more research is needed in soil conditions to confirm the allelopathy of C. oblongifolius and to evaluate the modes of action of its active compounds.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14040695/s1, The detail chemical composition of compounds 1 and 2.

Author Contributions

Investigation, H.K.-N.; methodology, S.M.M., T.T., S.T. and H.K.-N.; conceptualization, S.M.M. and H.K.-N.; validation, T.T., S.T. and H.K.-N.; software, S.M.M.; formal analysis, S.M.M.; data curation and visualization, S.M.M.; writing (original draft preparation), S.M.M.; writing (review and editing), H.K.-N.; supervision, H.K.-N. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financially supported by the government of Japan through a MEXT scholarship (Grant Number MEXT 202139).

Data Availability Statement

There are no supporting data in this study.

Acknowledgments

We are grateful to Dennis Murphy from UGAS, Ehime University, Japan, for the manuscript’s English editing.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Agronomy 14 00695 g001
Figure 1. (A) Immature leaf, (B) mature leaf, and (C) plant of C. oblongifolius.
Figure 1. (A) Immature leaf, (B) mature leaf, and (C) plant of C. oblongifolius.
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Agronomy 14 00695 g002
Figure 2. The growth of M. sativa hypocotyl and root (A) and L. multiflorum coleoptile and root (B) was affected by different doses of C. oblongifolius extract. The various letters show significant differences at p < 0.05 (Tukey’s test).
Figure 2. The growth of M. sativa hypocotyl and root (A) and L. multiflorum coleoptile and root (B) was affected by different doses of C. oblongifolius extract. The various letters show significant differences at p < 0.05 (Tukey’s test).
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Agronomy 14 00695 g003
Figure 3. The biological activity of the nine fractions obtained from the silica gel column at a dose of 0.1 g DW equivalent C. oblongifolius extract/mL on the growth of L. sativum hypocotyls and roots. The different letters indicate significant differences between the controls and leaf-extract-treated groups (Tukey’s test; p < 0.05).
Figure 3. The biological activity of the nine fractions obtained from the silica gel column at a dose of 0.1 g DW equivalent C. oblongifolius extract/mL on the growth of L. sativum hypocotyls and roots. The different letters indicate significant differences between the controls and leaf-extract-treated groups (Tukey’s test; p < 0.05).
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Agronomy 14 00695 g004
Figure 4. Molecular structures of alpinolide peroxide (A), 6-hydroxy alpinolide (B), and 3-hydroxy-5-isopropyl-3-methyl-2,3-dihydro-1H-inden-1-one (C).
Figure 4. Molecular structures of alpinolide peroxide (A), 6-hydroxy alpinolide (B), and 3-hydroxy-5-isopropyl-3-methyl-2,3-dihydro-1H-inden-1-one (C).
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Agronomy 14 00695 g005
Figure 5. Effects of alpinolide peroxide (A), 6-hydroxy alpinolide (B), and 3-hydroxy-5-isopropyl-3-methyl-2,3-dihydro-1H-inden-1-one (C) against the growth of L. sativum hypocotyls and roots. The different letters show significant differences among the treatments at less than the 0.05 probability level (Tukey’s test).
Figure 5. Effects of alpinolide peroxide (A), 6-hydroxy alpinolide (B), and 3-hydroxy-5-isopropyl-3-methyl-2,3-dihydro-1H-inden-1-one (C) against the growth of L. sativum hypocotyls and roots. The different letters show significant differences among the treatments at less than the 0.05 probability level (Tukey’s test).
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Agronomy 14 00695 g006
Figure 6. Planner structure of 3-hydroxy-5-isopropyl-3-methyl-2,3-dihydro-1H-inden-1-one based on 2D NMR (COSY-bold and HMBC-arrow) correlations.
Figure 6. Planner structure of 3-hydroxy-5-isopropyl-3-methyl-2,3-dihydro-1H-inden-1-one based on 2D NMR (COSY-bold and HMBC-arrow) correlations.
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Table 1. The dose of C. oblongifolius extracts that limited 50% of the hypocotyl/coleoptile and root growth (IC50) of M. sativa (dicot) and L. multiflorum (monocot).
Table 1. The dose of C. oblongifolius extracts that limited 50% of the hypocotyl/coleoptile and root growth (IC50) of M. sativa (dicot) and L. multiflorum (monocot).
Plant SpeciesIC50 (mg DW Equivalent C. oblongifolius Extract/mL)
Hypocotyl/ColeoptileRoot
DicotM. sativa5.443.23
MonocotL. multiflorum13.859.87
Table 2. NMR spectral data for compound 3 in CDCl3.
Table 2. NMR spectral data for compound 3 in CDCl3.
PositionδH Mult (J in Hz) aδC b
1 202.6
22.89, s54.2
3 74.5
3a 159.1
47.52, d (1.4)121.1
5 157.8
67.36, dd (8.0, 1.4)128.3
77.65, d (8.0)123.3
7a 133.6
81.72, s29.3
93.04, m34.9
10/111.31, d (6.9)23.8
a Noted at 500 MHz. b Assignments for protonated and quaternary carbons were acquired based on HMBC and HMQC spectra.
Table 3. The dose of the three compounds required for 50% growth limitation (IC50) of the hypocotyls and roots of L. sativum.
Table 3. The dose of the three compounds required for 50% growth limitation (IC50) of the hypocotyls and roots of L. sativum.
Identified CompoundIC50 (mM)
L. sativum
HypocotylRoot
alpinolide peroxide0.210.17
6-hydroxy alpinolide0.260.23
3-hydroxy-5-isopropyl-3-methyl-2,3-dihydro-1H-inden-1-one0.340.16
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Moh, S.M.; Tojo, S.; Teruya, T.; Kato-Noguchi, H. Allelopathic Activity of a Novel Compound and Two Known Sesquiterpene from Croton oblongifolius Roxb. Agronomy 2024, 14, 695. https://doi.org/10.3390/agronomy14040695

AMA Style

Moh SM, Tojo S, Teruya T, Kato-Noguchi H. Allelopathic Activity of a Novel Compound and Two Known Sesquiterpene from Croton oblongifolius Roxb. Agronomy. 2024; 14(4):695. https://doi.org/10.3390/agronomy14040695

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Moh, Seinn Moh, Shunya Tojo, Toshiaki Teruya, and Hisashi Kato-Noguchi. 2024. "Allelopathic Activity of a Novel Compound and Two Known Sesquiterpene from Croton oblongifolius Roxb." Agronomy 14, no. 4: 695. https://doi.org/10.3390/agronomy14040695

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