Allelopathy and Identification of Five Allelochemicals in the Leaves of the Aromatic Medicinal Tree Aegle marmelos (L.) Correa

Aegle marmelos (L.) Correa is an economically and therapeutically valuable tree. It is cultivated as a fruit plant in southeast Asian countries. In this research, we investigated the allelopathy and possible allelochemicals in the leaves of A. marmelos. Aqueous methanol extracts of A. marmelos exhibited significant inhibitory effects against the growth of Lepidium sativum, Lactuca sativa, Medicago sativa, Echinochloa crusgalli, Lolium multiflorum, and Phleum pratense. Bioassay-directed chromatographic purification of the A. marmelos extracts resulted in identifying five active compounds: umbelliferone (1), trans-ferulic acid (2), (E)-4-hydroxycinnamic acid methyl ester (3), trans-cinnamic acid (4), and methyl (E)-3’-hydroxyl-4’-methoxycinnamate (5). The hypocotyl and root growth of L. sativum were considerably suppressed by these compounds. Methyl (E)-3’-hydroxyl-4’-methoxycinnamate also suppressed the coleoptile and root growth of E. crusgalli. The concentrations of these compounds, causing 50% growth reduction (I50) of L. sativum, were in the range of 74.19–785.4 μM. The findings suggest that these isolated compounds might function in the allelopathy of A. marmelos.


Introduction
A major challenge facing the agricultural sector is controlling weed plant species, which negatively affect crop productivity [1].Herbicides are powerful and effective tools for controlling weeds.However, overuse of agrochemicals for pest control has led to various issues such as environmental pollution, unsafe agricultural products, human health problems [2], and the emergence of resistant weed biotypes [3,4].Because of these negative concerns, investigating alternative methods of weed control is required [5], and allelopathy appears to be one option [6].
Allelopathy is an ecological phenomenon in which a plant releases allelochemicals into the surrounding environment, causing either direct or indirect, positive or negative effects on other plant species (including microbes) [7].Allelopathic plants produce these allelochemicals by leaching, root secretion, or microbial degradation [8].Several secondary metabolites (or allelochemicals), such as phenolic acids, flavonoids, terpenoids, coumarins, alkaloids, and their degraded products, have allelopathic effects and play an important role in the ecosystem [9,10].The primary allelochemicals in plants are thought to be phenolic acids, which include derivatives of cinnamic and benzoic acids [11,12].Phenolic compounds are reported in a wide range of plant groups, including trees, crops, weeds, and medicinal plants [13].Compared with other plants, medicinal plants may contain more bioactive molecules [14].In addition, strong allelopathic effect has been found in Plants 2024, 13, 559 2 of 13 secondary metabolites isolated from medicinal plants [15].Moreover, phenolic compounds derived from medicinal plants are beneficial to human health [16] and can be applied as allelochemicals for weed management [13].Research on the allelopathic properties of medicinal plants offers an effective alternative for diversifying weed management approaches.Therefore, the allelopathy of medicinal plants has attracted attention in recent years.Other studies have also confirmed that medicinal plants possess an allelopathic effect and allelopathy-associated substances [13,17,18].Based on the reported literature, this study assumed that the majority of medicinal plants have allelopathic effects and contain active compounds.
Aegle marmelos (L.) Correa, belonging to the Rutaceae family, is highly reputed for its medicinal properties [19].It is commonly distributed in the dry forests of India, Thailand, Myanmar, Bangladesh, Sri Lanka, and Pakistan [20]. A. marmelos is a slow-growing, smallto medium-sized tree around 25 to 30 feet in height [21] (Figure 1).The tree is aromatic, and all its parts are medicinally important [22].The different plant parts (leaves, fruit, seeds, roots, and bark) are used as folk medicine to treat several ailments and possess anti-inflammatory, antifungal, analgesic, antipyretic, hypoglycemic, antidyslipidemic, immunomodulatory, antiproliferative, anti-fertility, and insecticidal activity [22][23][24].This tree contains more than one hundred phytochemicals, such as tannins, cardiac glycosides, alkaloids, flavonoids, phenol, terpenoids, and steroids [25].Although all parts of the tree have been explored for their medicinal properties [26,27] and plant growth inhibitory potential [28,29], there is still a lack of evidence related to their allelochemicals.Thus, the aim of the current study is to evaluate the allelopathic potential of the leaf extracts of A. marmelos and to investigate the related allelochemicals.
to be phenolic acids, which include derivatives of cinnamic and benzoic acids [11,12].Phenolic compounds are reported in a wide range of plant groups, including trees, crops, weeds, and medicinal plants [13].Compared with other plants, medicinal plants may contain more bioactive molecules [14].In addition, strong allelopathic effect has been found in secondary metabolites isolated from medicinal plants [15].Moreover, phenolic compounds derived from medicinal plants are beneficial to human health [16] and can be applied as allelochemicals for weed management [13].Research on the allelopathic properties of medicinal plants offers an effective alternative for diversifying weed management approaches.Therefore, the allelopathy of medicinal plants has attracted attention in recent years.Other studies have also confirmed that medicinal plants possess an allelopathic effect and allelopathy-associated substances [13,17,18].Based on the reported literature, this study assumed that the majority of medicinal plants have allelopathic effects and contain active compounds.
Aegle marmelos (L.) Correa, belonging to the Rutaceae family, is highly reputed for its medicinal properties [19].It is commonly distributed in the dry forests of India, Thailand, Myanmar, Bangladesh, Sri Lanka, and Pakistan [20]. A. marmelos is a slow-growing, small-to medium-sized tree around 25 to 30 feet in height [21] (Figure 1).The tree is aromatic, and all its parts are medicinally important [22].The different plant parts (leaves, fruit, seeds, roots, and bark) are used as folk medicine to treat several ailments and possess anti-inflammatory, antifungal, analgesic, antipyretic, hypoglycemic, antidyslipidemic, immunomodulatory, antiproliferative, anti-fertility, and insecticidal activity [22][23][24].This tree contains more than one hundred phytochemicals, such as tannins, cardiac glycosides, alkaloids, flavonoids, phenol, terpenoids, and steroids [25].Although all parts of the tree have been explored for their medicinal properties [26,27] and plant growth inhibitory potential [28,29], there is still a lack of evidence related to their allelochemicals.Thus, the aim of the current study is to evaluate the allelopathic potential of the leaf extracts of A. marmelos and to investigate the related allelochemicals.

Growth-Suppressive Activity of A. marmelos
The leaf extracts of A. marmelos had significant growth-suppressive effects against the seedling (hypocotyl/coleoptile and root) growth of L. sativum, L. sativa, M. sativa, E. crusgalli, L. multiflorum, and P. pratense at concentrations of more than 10 mg DW equivalent A. marmelos extract/mL, except the coleoptile growth of E. crusgalli (Figure 2A,B).The concentration of 30 mg DW equivalent A. marmelos extract/mL completely inhibited the growth of the L. sativum hypocotyls and inhibited the hypocotyl/coleoptile growth of L. sativa, M. sativa, E. crusgalli, L. multiflorum, and P. pratense to 28.9, 8.9, 56.4,40.8, and 11.6% of the control, respectively (Figure 2A), whereas the root growth was inhibited to 5.4, 17.2, 11.4, 13.2, 41.2, and 1.5% of the control, respectively (Figure 2B).Moreover, the concentration of 300 mg DW equivalent A. marmelos extract/mL completely suppressed the hypocotyl/coleoptile and root lengths of L. sativum, L. sativa, M. sativa, E.

Growth-Suppressive Activity of A. marmelos
The leaf extracts of A. marmelos had significant growth-suppressive effects against the seedling (hypocotyl/coleoptile and root) growth of L. sativum, L. sativa, M. sativa, E. crusgalli, L. multiflorum, and P. pratense at concentrations of more than 10 mg DW equivalent A. marmelos extract/mL, except the coleoptile growth of E. crusgalli (Figure 2A,B).The concentration of 30 mg DW equivalent A. marmelos extract/mL completely inhibited the growth of the L. sativum hypocotyls and inhibited the hypocotyl/coleoptile growth of L. sativa, M. sativa, E. crusgalli, L. multiflorum, and P. pratense to 28.9, 8.9, 56.4,40.8, and 11.6% of the control, respectively (Figure 2A), whereas the root growth was inhibited to 5.4, 17.2, 11.4, 13.2, 41.2, and 1.5% of the control, respectively (Figure 2B).Moreover, the concentration of 300 mg DW equivalent A. marmelos extract/mL completely suppressed the hypocotyl/coleoptile and root lengths of L. sativum, L. sativa, M. sativa, E. crusgalli, L. multiflorum, and P. pratense, except the coleoptile length of E. crusgalli, compared with control.
Plants 2024, 13, 559 3 of 13 examined plants were significantly greater than for the root growth.Of the six examined plants, the I50 values showed that the P. pratense coleoptiles and roots were more responsive to the A. marmelos extracts, whereas the E. crusgalli coleoptiles and L. multiflorum roots were least sensitive.
The correlation coefficient values between the dose of A. marmelos extract and the seedling growth of the test plants ranged from −0.70 to −0.91 (Table 1).The concentrations of A. marmelos extract needed for 50% growth restriction (I 50 values) of the hypocotyl/coleoptile and root growth of the six examined plants (L.sativum, L. sativa, M. sativa, E. crusgalli, L. multiflorum, and P. pratense) were in the ranges of 3.16-36.14and 1.61-10.06mg DW equivalent A. marmelos extract/mL, respectively (Table 1).The I 50 values for the extract against the hypocotyl/coleoptile growth of the six examined plants were significantly greater than for the root growth.Of the six examined plants, the I 50 values showed that the P. pratense coleoptiles and roots were more responsive to the A. marmelos extracts, whereas the E. crusgalli coleoptiles and L. multiflorum roots were least sensitive.The correlation coefficient values between the dose of A. marmelos extract and the seedling growth of the test plants ranged from −0.70 to −0.91 (Table 1).

Identification of the Five Active Compounds
The results (Figure 3) showed that, compared with the aqueous fraction, the ethyl acetate fraction notably restricted the growth of the L. sativum seedlings (hypocotyl and root) with the application of 10 and 30 mg DW equivalent A. marmelos extract/mL.Thus, the ethyl acetate fraction was separated using a sequence of chromatography steps: silica gel, Sephadex LH-20, reverse-phase C 18 cartridges, and HPLC.Consequently, the five substances with growth-suppressive effects were isolated by reverse-phase HPLC and identified using spectroscopy.

Identification of the Five Active Compounds
The results (Figure 3) showed that, compared with the aqueous fraction, the ethyl acetate fraction notably restricted the growth of the L. sativum seedlings (hypocotyl and root) with the application of 10 and 30 mg DW equivalent A. marmelos extract/mL.Thus, the ethyl acetate fraction was separated using a sequence of chromatography steps: silica gel, Sephadex LH-20, reverse-phase C18 cartridges, and HPLC.Consequently, the five substances with growth-suppressive effects were isolated by reverse-phase HPLC and identified using spectroscopy.The chemical formula of compound-1 was determined to be C9H5O3 (isolated as a colorless powder).The 1 H-NMR spectrum of this compound was determined by 500 MHz, acetone-d6.The HRESIMS data, 1 H-NMR spectrum, and optical rotation of this compound are similar to the published literature [30].By comparing its spectral data with other research [31], this compound was identified as umbelliferone (Figure 4a).
Compound-4 was isolated as a colorless powder and the chemical formula C9H8O2 was determined by HRESIMS.The  The chemical formula of compound-1 was determined to be C 9 H 5 O 3 (isolated as a colorless powder).The 1 H-NMR spectrum of this compound was determined by 500 MHz, acetone-d 6 .The HRESIMS data, 1 H-NMR spectrum, and optical rotation of this compound are similar to the published literature [30].By comparing its spectral data with other research [31], this compound was identified as umbelliferone (Figure 4a).
Compound-4 was isolated as a colorless powder and the chemical formula C 9 H 8 O 2 was determined by HRESIMS.The 1 H-NMR spectrum (500 MHz, CDCl 3 ) showed five aromatic proton signals at δ H 7.56 (2H, m) and 7.43-7.39(3H, m) and two olefinic proton signals at δ H 7.78 (1H, d, J = 16.0) and 6.46 (1H, d, J = 16.0).After comparing the spectral data of compound-3 with that in the published literature [34], this compound was identified as trans-cinnamic acid (Figure 4d)., d, J = 15.9) and 6.29 (1H, d, J = 15.9).After comparing these spectral data with information from published documents [35], it was determined that the chemical structure of this compound was methyl (E)-3 ′ -hydroxyl -4 ′ -methoxycinnamate (Figure 4e).The spectroscopic data of the five compounds are provided in the Supplementary File.
The I 50 values for suppressing the growth of the L. sativum hypocotyls and roots were 378.

Discussion
The A. marmelos extracts exhibited a growth-suppressive effect against the se (hypocotyl/coleoptile and root) growth of the dicot (L.sativum, L. sativa, and M. and monocot plants (E.crusgalli, L. multiflorum, and P. pratense) (Figure 2).Moreov correlation coefficients (r) revealed a significant negative relationship between th pocotyl/coleoptile and root lengths of the bioassayed plant species and the extrac centrations (Table 1).These results suggested that the A. marmelos extract concentr have the growth inhibitory activity of bioassays plants.Additionally, the differ values of the A. marmelos extracts for the hypocotyl/coleoptile and root growth of tivum, L. sativa, M. sativa, E. crusgalli, L. multiflorum, and P. pretense indicate that t

Discussion
The A. marmelos extracts exhibited a growth-suppressive effect against the seedling (hypocotyl/coleoptile and root) growth of the dicot (L.sativum, L. sativa, and M. sativa) and monocot plants (E.crusgalli, L. multiflorum, and P. pratense) (Figure 2).Moreover, the correlation coefficients (r) revealed a significant negative relationship between the hypocotyl/coleoptile and root lengths of the bioassayed plant species and the extract concentrations (Table 1).These results suggested that the A. marmelos extract concentrations have the growth inhibitory activity of bioassays plants.Additionally, the different I 50 values of the A. marmelos extracts for the hypocotyl/coleoptile and root growth of L. sativum, L. sativa, M. sativa, E. crusgalli, L. multiflorum, and P. pretense indicate that the inhibitory effect of the extracts depends on the six examined plant species (Table 1).Similar results of dose-and species-dependent allelopathic properties have been documented by other researchers [36][37][38].The growth inhibitory activity of the A. marmelos extracts against the six plant species investigated in this study indicates that these extracts may involve active compounds that have growth-suppressive properties.
Trans-ferulic acid (or (E)-3-hydroxy-4-methoxycinnamic acid) is a stereoisomer of ferulic acid and is widely present in cereal crops (rice and wheat), fruits (bananas and pineapples), and vegetables (peanuts, eggplants, and tomatoes) [44].Based on the reports of other studies, this compound has many pharmaceutical properties such as antioxidant [45], and antimicrobial and antifungal [44] properties.Additionally, ferulic acid inhibits the root growth of Vigna radiata [46], and the net rate of photosynthesis and stomatal conductance in Rhododendron delavayi [47].
Trans-cinnamic acid (or (2E)-3-phenylprop-2-enoic acid) is a natural aromatic carboxylic acid and is widely present in plants such as Cinnamomum cassia and Panax ginseng, fruits, whole grains, vegetables, and honey [48].The antimicrobial, antioxidant, and antifungal properties of trans-cinnamic acid have led to its widespread use in food, cosmetics, and pharmaceuticals [49,50].Moreover, trans-cinnamic acid exhibits allelopathic effects on the seed sprouting and seedling growth of Glycine max [51], Lactuca sativa [52], and Chrysanthemum coronarium [53].Trans-cinnamic acid is liberated into the soil through plant tissue decomposition, leaf leachate, and root secretion, e.g., Elytrigia repens [51], Cucumis sativus [52], and Medicago sativa [53].Anh et al. (2021) [54] reported that trans-cinnamic acid inhibits Glycine max seedlings through the disorder and interference of the uptake and transport of ions in the plant and, consequently, causes morphological distortion of the roots.
(E)-4-Hydroxycinnamic acid methyl ester and methyl (E)-3 ′ -hydroxyl -4 ′ -methoxycinna mate are part of the group of hydroxycinnamic acids (HCAs) that are derivatives of phenolic compounds [55,56].Trans-cinnamic acid derivatives that include HCAs are reported to have anti-bacterial, anti-inflammatory, and antifungal activities [55,57].Whole grains, coffee, tea leaves, fruits, and vegetables are rich sources of HCAs [58][59][60].DellaGreca et al. ( 2007) [61] reported that cinnamic ester derivatives obtained from Oxalis pes-caprae affect the seedling growth of L. sativum.Other studies have found that some phenolic acids affect ion transport and metabolism [62], interfere with cell division and deform cellular structures [63], and reduce photosynthetic rates [64].Although the phytochemical properties of the characterized compounds from all parts of A. marmelos have been described for a long time, this study is the first account of the growth-suppressive activity of umbelliferone, trans-ferulic acid, (E)-4-hydroxycinnamic acid methyl ester, cinnamic acid, and methyl (E)-3 ′ -hydroxyl-4 ′ -methoxycinnamate from A. marmelos leaves.
In this study, umbelliferone, trans-ferulic acid, (E)-4-hydroxycinnamic acid methyl ester, trans-cinnamic acid, and methyl (E)-3 ′ -hydroxyl-4 ′ -methoxycinnamate significantly reduced the growth of L. sativum in a dose-dependent manner (Figure 5A-F).Allelochemicals such as phenols, coumarin, and terpenoids cause lipid peroxidation [65].In addition, phenolic acids, including trans-cinnamic acid, disturb membrane permeability and thus affect plant growth processes [65][66][67].Comparing I 50 values, trans-cinnamic acid exhibited the most allelopathic effects against L. sativum (Table 2).The strong growth-suppressive effect of this compound may be linked to its hydrophobicity and the -OH and -OCH 3 groups in the molecules.Pinho et al. (2017) [68] stated that phenolic compounds appear to be less allelopathic to plants when the number of -OH and -OCH 3 groups are increased.The I 50 values also showed that the sensitivity to the five compounds was greater against the roots than the hypocotyls.These results indicate that the roots are the first parts to be exposed to the phenolic allelochemicals [69,70].
According to the results of this study, A. marmelos leaves possess allelopathic agents (umbelliferone, trans-ferulic acid, (E)-4-hydroxycinnamic acid methyl ester, trans-cinnamic acid, and methyl (E)-3 ′ -hydroxyl-4 ′ -methoxycinnamate), which account for the growthsuppressive activity of this tree.Additionally, the fresh and dry aromatic leaves of A. marmelos and their extract can be used for living or dead mulch and foliar spray as weed-suppressive resources for controlling weeds.Therefore, the allelopathy of A. marmelos and its allelochemicals may be helpful in developing environmentally safer herbicides Plants 2024, 13, 559 9 of 13 for weed management, as well as minimizing or eliminating the pollution problems of agrochemicals in the environment.

Plant Materials and Test Plant Species
Aegle marmelos leaves were brought from Khin U Region, Sagaing Division, Myanmar (22 • 49 ′ 4 ′′ N and 95 • 48 ′ 12 ′′ E) during July-August 2020.The leaves were shade dried and ground to obtain fine powder.Before starting the experiment, the powder was retained in plastic bags at 2 • C. To assess the allelopathic potential of A. marmelos, three dicot (L.sativum, L. sativa, and M. sativa) and three monocot (E.crusgalli, L. multiflorum, and P. pratense) plant species were selected for bioassays.

Extraction and Growth Bioassay
Dry powdered leaf (250 g) was extracted by soaking in 70% (v/v) aqueous methanol (1500 mL) for 48 h.The extract was filtered through a single layer of filter paper (No. 2, 125 mm; Toyo Ltd., Tokyo, Japan).After filtration, the extract residue was re-extracted by soaking in methanol (1500 mL) for 24 h and filtered.The two extracts obtained after filtration were then mixed and concentrated in a rotavapor to obtain an aqueous solution at 40 • C. The crude extracts (100 g dry weight) were dissolved in 30 mL of methanol, and various bioassay doses (1 (0.18 µL), 3 (0.54 µL), 10 (1.8 µL), 30 (5.4 µL), 100 (18.2 µL), and 300 (54.5 µL) mg dry weight (DW) equivalent A. marmelos extract/mL) were prepared in Petri dishes (2.8 cm).Concentrated extract (600 µL) and control (only methanol) were added to sheets of filter paper (No. 2; Toyo Ltd.) in Petri dishes.The methanol in the Petri dishes was then evaporated in a draft chamber.After drying, 600 µL of a 0.05% aqueous Tween 20 solution (Nacalai Tesque, Inc., Kyoto, Japan) was added to the filter papers.Ten seeds of L. sativum, L. sativa, and M. sativa and ten sprouted seeds (to avoid dormancy of the seed and incubated at 25 • C for 36-48 h) of E. crusgalli, L. multiflorum, and P. pratense were placed in the Petri dishes and incubated at 25 • C for 48 h.The lengths of the hypocotyls and roots of the test plant species were then measured with a ruler and the growth-suppressive potential was determined.The growth assay experiment was carried out using a completely randomized design (CRD) with six replications (10 seedlings/replicate, n = 60).
The NMR spectral data were documented on a Bruker AVANCE III 500 MHz NMR spectrometer.Chemical shifts were described to the residual signal of solvent (acetone-d 6 , CD 3 OD and CDCl 3 ).HRESIMS was measured on a Thermo Scientific Orbitrap Exploris 240 Mass Spectrometer.

Bioassays of the Identified Compounds
The identified compounds (1, 2, 3, 4, and 5) were dissolved in 1000 µL of methanol.Assay doses of each compound (10, 30, 100, 300, 1000, 3000, and 6000/10,000 µM) were then prepared and added to filter paper (No. 2) in Petri dishes (2.8 cm).The Petri dishes were placed inside a draft chamber.After the methanol evaporated, the filter papers were moistened with 600 µL Tween 20.Ten uniform seeds of L. sativum and 10 sprouted seeds of E. crusgalli (to avoid dormancy of the seed and incubated at 25 • C for 48 h) were placed on the filter papers and incubated at 25 • C for 48 h.The bioassays experiment for each test plant included three replicates, each with 10 seedlings (n = 30).The hypocotyl/coleoptile and root lengths were then measured by ruler to calculate the percentage of seedling growth as mentioned in Section 2.2 above.

Statistical Analysis
The growth assay experiment was carried out using a CRD with six replications.The results are shown as mean ± SE.ANOVA of all the data was analyzed using SPSS (version 16.0) (SPSS Inc., Chicago, IL, USA).Tukey's test was used to evaluate the significant differences between the control and treated plant groups at a p-value of 0.05.The doses of the A. marmelos extracts and each compound needed for 50% growth restriction (I 50 ) of each examined plant species were calculated using GraphPad Prism 6.0 (Software of GraphPad, Inc., La Jolla, CA, USA).

Conclusions
The extracts of A. marmelos exhibited significant allelopathic potential against the seedlings of the six examined plants (L.sativum, L. sativa, M. sativa, E. crusgalli, L. multiflorum, and P. pretense).Five phenolic compounds with growth-suppressive effects were separated from the A. marmelos leaves and characterized as umbelliferone, trans-ferulic acid, (E)-4hydroxycinnamic acid methyl ester, trans-cinnamic acid, and methyl (E)-3 ′ -hydroxyl-4 ′methoxycinnamate.These allelochemicals greatly inhibited the hypocotyl and root lengths of L. sativum.Trans-cinnamic acid showed the most allelopathic effect against L. sativum.The growth-suppressive properties of these characterized compounds might be related to A. marmelos leaf allelopathy.Therefore, the fresh and dry aromatic leaves of A. marmelos might be useful for living and dead mulch as weed-suppressive or soil-additive resources, and its allelochemicals could be useful in developing an allelochemical-based natural herbicide in sustainable agriculture.However, further experiments with the allelopathy of A. marmelos and the mode of action of its allelochemicals are necessary to examine in both laboratory and field settings for the development of natural herbicides.
Author Contributions: S.M.M.: conceptualization, formal analysis, methodology, software, visualization, and writing-original draft.S.T.: methodology, resources, and validation.T.T.: resources, methodology, and validation.H.K.-N.: conceptualization, data curation, methodology, investigation, resources, supervision, validation, and writing-review and editing.All authors have read and agreed to the published version of the manuscript.

Figure 2 .
Figure 2. The hypocotyl/coleoptile growth (A) and root growth (B) of the six examined plants at the different doses of A. marmelos leaf extract.The vertical bars express mean ± standard error (SE) (six replicates × 10 seedlings).Different letters specify significant differences between the A. marmelos treatments and the control at the 5% probability level (Tukey's test).

Figure 2 .
Figure 2. The hypocotyl/coleoptile growth (A) and root growth (B) of the six examined plants at the different doses of A. marmelos leaf extract.The vertical bars express mean ± standard error (SE) (six replicates × 10 seedlings).Different letters specify significant differences between the A. marmelos treatments and the control at the 5% probability level (Tukey's test).

Figure 3 .
Figure 3. L. sativum growth response to the two fractions of A. marmelos (ethyl acetate and aqueous) extracts.The different letters specify significant difference at the 5% probability level (Tukey's test).

Figure 3 .
Figure 3. L. sativum growth response to the two fractions of A. marmelos (ethyl acetate and aqueous) extracts.The different letters specify significant difference at the 5% probability level (Tukey's test).

13 compound 5
2 and 153.1 µM for compound-1, 785.4 and 552.6 µM for compound-2, 179.3 and 83.8 µM for compound-3, 119.6 and 74.1 µM for compound-4, and 260.5 and 181.8 µM for compound-5, respectively (Table 2).The I 50 values of all the compounds for suppressing L. sativum hypocotyl growth were 2.4, 1.4, 2.01, 1.6, and 1.4 times larger than for the roots, respectively.The I 50 values of compound-4 showed more growth allelopathic potential than the remaining four compounds.The I 50 values of the five compounds for suppressing the hypocotyl and root lengths of L. sativum ranged from 74.1 to 785.4 µM.The I 50 values of Plants 2024, 13, 559 6 of for suppressing the coleoptile and root growth of E. crusgalli were 885.1 and 453.1 µM, respectively.

Table 1 .
The doses of A. marmelos extract that suppressed 50% of the hypocotyl/coleoptile and root growth (I50) and correlation coefficients of the six examined plants by treatment with the A. marmelos extracts.

Table 1 .
The doses of A. marmelos extract that suppressed 50% of the hypocotyl/coleoptile and root growth (I 50 ) and correlation coefficients of the six examined plants by treatment with the A. marmelos extracts.
Asterisks indicate statistical significance at the 0.05% probability level (Tukey's test).

Table 2 .
The doses of the five identified compounds that suppressed 50% of the hypocotyl and root growth (I 50 ) of L. sativum.The I 50 values of compound-5 against the coleoptile and root length of E. crusgalli.

Table 2 .
The doses of the five identified compounds that suppressed 50% of the hypocotyl an growth (I50) of L. sativum.The I50 values of compound-5 against the coleoptile and root lengt crusgalli.