Quantitative Analysis of Ingenol in Euphorbia species via Validated Isotope Dilution Ultra‐high Performance Liquid Chromatography Tandem Mass Spectrometry

Introduction

Various species of the Euphorbia genus contain diterpene ingenol and ingenol mebutate (ingenol‐3‐angelate), a substance found in the sap of the plant Euphorbia peplus and an inducer of cell death. A gel formulation of the drug has been approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the topical treatment of actinic keratosis.

Objective

To develop a rapid and reliable method for quantification of ingenol in various plant extracts.

Methodology

Methanolic extracts of 38 species of the Euphorbia genus were analysed via ultra‐high performance liquid chromatography with tandem mass spectrometry (UHPLC–MS/MS) after methanolysis and solid‐phase extraction (SPE) purification. The ¹⁸O–labelled ingenol analogue was prepared and used as an internal standard for ingenol content determination and method validation.

Results

The highest ingenol concentration (547 mg/kg of dry weight) was found in the lower leafless stems of E. myrsinites. The screening confirms a substantial amount of ingenol in species studied previously and furthermore, reveals some new promising candidates.

Conclusion

The newly established UHPLC–MS/MS method shows to be an appropriate tool for screening of the Euphorbia genus for ingenol content and allows selection of species suitable for raw material production and/or in vitro culture initiation. Copyright © 2017 John Wiley & Sons, Ltd.     

五种大戟属植物nrDNA的ITS序列分析及其叶的比较解剖学研究

运用PCR直接测序法和石蜡切片法,测定5种安徽产大戟属植物的nrDNA的内转录间隔区(ITS)序列包括5.8SrDNA及其叶片的解剖。结果表明(1)5种大戟属植物的ITS的长度范围为641~662bp,运用Maga软件构件ITS树把5种植物归为两大支大戟、月腺大戟与乳浆大戟聚为一支;地锦和斑地锦聚为另一支。结果表明地锦和斑地锦亲缘关系较近,而大戟、月腺大戟和乳浆大戟亲缘关系较近。(2)5种大戟属植物的叶片结构中,除地锦和斑地锦外,其余3种有明显的栅栏组织和海绵组织之分,但二者在叶肉中的厚度比在种间有一定的差别;而地锦和斑地锦叶的栅栏组织与海绵组织分化不明显,且皆有乳汁管,其他3种植物叶片内未见此结构,叶片结构分析表明大戟、月腺大戟和乳浆大戟叶片结构相近,而地锦草和斑地锦叶片结构也相近。(3)分析表明nrDNA的ITS序列及叶的比较解剖特征具有明显的相关性。以上研究结果为大戟属植物的分类和药用开发提供了实验证据。     

月腺大戟(Euphorbia ebracteolata)根部提取物抑菌作用的测定

以小麦赤霉病病菌（Fusarium graminerum）、油菜菌核病病菌（Sclerotirda scleotiorum）、棉花黄萎病病菌（Verticillium dahliae）、苹果炭疽病病菌（Glomarella cingulata）、甜瓜蔓枯病病菌（Mycosphaerella melonis）为供试病原菌,采用琼胶平板法对月腺大戟根部丙酮提取物进行了离体抑菌活性测定;月腺大戟根部提取物样品供试质量浓度分别设为0．05g／mL,0．1g／mL,0．2g／mL．0．4g／mL四个梯度。测定结果表明：不同浓度月腺大戟根部丙酮提取物对5种供试病原菌均表现为很强的抑菌作用,该研究为月腺大戟根部进一步开发植物性农药提供理论依据。     

A comparative phytochemical study of the diterpenes of some species of the genera Euphorbia and Elaeophorbia (Euphorhiaceae)

Nearly 60 species from the genera Euphorbia L. and Elaeophorbia Stapf were investigated for diterpenes of the classes tiglianes, ingenanes and ortho-esters. The diterpenes were isolated as their acetates by a micro-technique from latex or fresh herb material collected from several countries around the world. Authentication of the diterpenes was by chromatographic means using spectroscopic methods as back-up data. These compounds were absent from only 9% of the species examined. The most commonly occurring diterpene was ingenol, followed closely by 12-Deoxy phorbol. The ingenane derivative 5-Deoxy-ingenol was always detected as a minor companion to ingenol from species of the section Tithymalus. Species of the section Euphorbium contained mainly the diterpene ingenol although both tigliane and ortho-ester diterpenes were also present in some species. Species from the sections Anisophylhtm and Poinsettia did not contain diterpenes of the type in question, whilst species from the genus Elaeophorbia yielded ingenol. These results provide additional chemical evidence concerning recent suggestions on the subgeneric and generic status of Anisophyllum, Poinsettia, Tithymalus and Elaeophorbia.     

Streamlining plant sample preparation: the use of high-throughput robotics to process echinacea samples for biomarker profiling by MALDI-TOF mass spectrometry.

Several species in the genus Echinacea are beneficial herbs popularly used for many ailments. The most popular Echinacea species for cultivation, wild collection, and herbal products include E. purpurea (L.) Moench, E. pallida (Nutt.) Nutt., and E. angustifolia (DC). Product adulteration is a key concern for the natural products industry, where botanical misidentification and introduction of other botanical and nonbotanical contaminants exist throughout the formulation and production process. Therefore, rapid and cost-effective methods that can be used to monitor these materials for complex product purity and consistency are of benefit to consumers and producers. The objective of this continuing research was to develop automated, high-throughput processing methods that, teamed with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, differentiate Echinacea species by their mass profiles. Small molecules, peptide, and proteins from aerial parts (leaf/stem/flowers), seeds, and roots from E. purpurea and E. angustifolia; seeds and roots from E. pallida; and off-the-shelf Echinacea supplements were extracted and analyzed by MS using methods developed on the ProPrep liquid handling system (Genomic Solutions). Analysis of these samples highlighted key MS signal patterns from both small molecules and proteins that characterized the individual Echinacea materials analyzed. Based on analysis of pure Echinacea samples, off-the-shelf products containing Echinacea could then be evaluated in a streamlined process. Corresponding analysis of dietary supplements was used to monitor for product composition, including Echinacea species and plant materials used. These results highlight the potential for streamlined, automated approaches for agricultural species differentiation and botanical product evaluation.     

青藏高原有毒植物瑞香狼毒根抑菌活性初步研究

以人体病原细菌大肠杆菌、金黄色葡萄球菌、深部真菌白色念珠菌、浅部真菌红色毛癣菌、石膏样毛癣菌、石膏样小孢子菌、絮状表皮癣菌为供试菌,采用药敏纸片法对瑞香狼毒活性成分的抑菌活性进行了初步研究。结果表明瑞香狼毒对人体常见病原细菌和真菌有抑制作用,乙酸乙酯萃取物在33．33g／L浓度下对细菌组的抑菌圈直径最高可达12．02mm,对真菌组最高可达9．04mm,并初测瑞香狼毒中的抑菌活性物质主要为中偏强极性成分。     

Development of Diagnostic Microscopic and Chemical Markers of Some Euphorbia Latexes

The latexes of the three Euphorbia species, namely E. antiquorum L., E. nerifolia L., and E. tirucalli L., are highly valued in the Indian system of medicine as purgatives, in addition to their specific and distinct therapeutic activities. In order to distinguish these latexes and develop their diagnostic microscopic and chemical markers, we performed extensive chemical and microscopic studies. The three latexes differ significantly in their microscopic features by exhibiting characteristic starch grain patterns. Although amoebic structures were found to be characteristic of E. antiquorum, dumb-bell and oval structures are characteristic of E. nerifolia and E. tirucalli, respectively. In addition, these latexes showed bone-shaped structures as a common feature, but these differed considerably in their length （10-60, 30-55, and 50-70 μm in length in E. antiquorum, E. nerifolia, and E. tirucalli, respectively）. The chemical markers nerifoliene and euphol were found to be common to both E. antiquorum and E. nerifolia, whereas euphol is the only marker for E. tirucalli. A reverse-phase high-performance thin-layer chromatographic （HPTLC） method was developed to distinguish these three latexes and to generate their standard fingerprinting patterns. Most significantly, the markers nerifoliene and euphol could be resolved by RP-18 F254s precoated aluminium plates and the latexes have been quantitatively estimated with respect to these markers. The developed microscopic, chemical and HPTLC patterns can be used to distinguish the three latexes.     

Material Basis of Chinese Herbal Formulas Explored by Combining Pharmacokinetics with Network Pharmacology

The clinical application of Traditional Chinese medicine (TCM), using several herbs in combination (called formulas), has a history of more than one thousand years. However, the bioactive compounds that account for their therapeutic effects remain unclear. We hypothesized that the material basis of a formula are those compounds with a high content in the decoction that are maintained at a certain level in the system circulation. Network pharmacology provides new methodological insights for complicated system studies. In this study, we propose combining pharmacokinetic (PK) analysis with network pharmacology to explore the material basis of TCM formulas as exemplified by the Bushen Zhuanggu formula (BZ) composed of Psoralea corylifolia L., Aconitum carmichaeli Debx., and Cnidium monnieri (L.) Cuss. A sensitive and credible liquid chromatography tandem mass spectrometry (LC-MS/MS) method was established for the simultaneous determination of 15 compounds present in the three herbs. The concentrations of these compounds in the BZ decoction and in rat plasma after oral BZ administration were determined. Up to 12 compounds were detected in the BZ decoction, but only 5 could be analyzed using PK parameters. Combined PK results, network pharmacology analysis revealed that 4 compounds might serve as the material basis for BZ. We concluded that a sensitive, reliable, and suitable LC-MS/MS method for both the composition and pharmacokinetic study of BZ has been established. The combination of PK with network pharmacology might be a potent method for exploring the material basis of TCM formulas.     

Primary structures of hypertrehalosaemic neuropeptides isolated from the corpora cardiaca of the cockroaches Leucophaea maderae, Gromphadorhina portentosa, Blattella germanica and Blatta orientalis and of the stick insect Extatosoma tiaratum assigned by tandem fast atom bombardment mass spectrometry

Hypertrehalosaemic peptides were isolated by reversed-phase high-performance liquid chromatography from corpora cardiaca of four species of cockroaches (Leucophaea maderae, Gromphadorhina portentosa, Blattella germanica, and Blatta orientalis) and one stick insect species (Extatosoma tiaratum), and their primary sequences were assigned by collision-induced decomposition tandem fast atom bombardment mass spectrometry (FABMS/CID/MS). The members of the cockroach families Blaberidae (L. maderae and G. portentosa) and Blattellidae (B. germanica) contained an identical decapeptide (Glu-Val-Asn-Phe-Ser-Pro-Gly-Trp-Gly-ThrNH2), whereas the member of the cockroach family Blattidae (B. orientalis) had two octapeptides (Glu-Val-Asn-Phe-Ser-Pro-Asn-TrpNH2 and Glu-Leu-Thr-Phe-Thr-Pro-Asn-TrpNH2). The structure of the stick insect hypertrehalosaemic compound was assigned as a decapeptide (Glu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-Gly-ThrNH2). The respective synthetic peptides elevated blood carbohydrates in their respective acceptor species. The results are discussed in the light of family-specificity of members of the adipokinetic hormone/red pigment-concentrating hormone family.     

Jolkinolide B from <Emphasis Type="Italic">Euphorbia fischeriana Steud</Emphasis> induces apoptosis in human leukemic U937 cells through PI3K/Akt and XIAP pathways

Jolkinolide B, a bioactive diterpene isolated from the roots of Euphorbia fischeriana Steud, is known to induce apoptosis in cancer cells. However, the molecular mechanism of its anti-cancer activity has not been fully elucidated. In the present study, we found that Jolkinolide B reduced cell viability and induced apoptosis in a dose- and timedependent manner in human leukemic U937. The induction of apoptosis was also accompanied by the downregulation of PI3K/Akt and the inhibitor of apoptosis protein (IAP) family proteins. Moreover, we observed that Jolkinolide B treatment resulted in activation of caspase-3 and -9, which may partly explain the anti-cancer activity of Jolkinolide B. Taken together, our study for the first time suggest that Jolkinolide B is able to enhance apoptosis of U937 cells, at least in part, through downregulation of PI3K/Akt and IAP family proteins. Moreover, triggering of caspase-3 and -9 activation mediated apoptotic induction.     

瑞香狼毒茎叶化学成分研究

新鲜瑞香狼毒（Ｓｔｅｌｅｒａｃｈａｍａｅｊａｓｍｅ）茎叶经正己烷提取,脱腊后进行ＧＣＭＳＤＳ联用分析,鉴定出２０个化合物。主要成分为正十八烷;正十九烷;２,６,１０,１４四甲基十六烷;十六烷酸;十六烷酸甲酯;十六烷酸乙酯;９,１２,１５十八碳三烯酸甲酯;９,１２十八碳二烯酸;９,１２,１５十八碳三烯酸和十八烷酸。     

Phenolic acids and depsides from some species of the Erodium genera

Six natural polyphenolic compounds, brevifolin carboxylic acid, brevifolin, ellagic acid, methyl gallate, gallic acid and protocatechuic acid have been isolated from the methanol extract of the whole plant of Erodium cicutarium (L.) L.'Hérit. (Geraniaceae). Structures were determined by conventional methods of analysis and confirmed by MS and NMR spectral analysis. The distribution of these compounds in the other species of the Erodium genera (E. botrys, E. chium, E. ciconium, E. cicutarium, E. glutinosum subsp. dunense, E. gruinum, E. manescavi, E. pelargoniiflorum, E. petraeum) were examined by HPLC with a RP-18 column, and MGD-TLC methods on unmodified silica gel and silica gel chemically modified with polar and nonpolar groups (HPTLC-Si 60 LiChrospher, HPTLC-NH2, HPTLC-DIOL, HPTLC RP-18W).     

Metabolomics Unravel Contrasting Effects of Biodiversity on the Performance of Individual Plant Species

In spite of evidence for positive diversity-productivity relationships increasing plant diversity has highly variable effects on the performance of individual plant species, but the mechanisms behind these differential responses are far from being understood. To gain deeper insights into the physiological responses of individual plant species to increasing plant diversity we performed systematic untargeted metabolite profiling on a number of herbs derived from a grassland biodiversity experiment (Jena Experiment). The Jena Experiment comprises plots of varying species number (1, 2, 4, 8, 16 and 60) and number and composition of functional groups (1 to 4; grasses, legumes, tall herbs, small herbs). In this study the metabolomes of two tall-growing herbs (legume: Medicago x varia; non-legume: Knautia arvensis) and three small-growing herbs (legume: Lotus corniculatus; non-legumes: Bellis perennis, Leontodon autumnalis) in plant communities of increasing diversity were analyzed. For metabolite profiling we combined gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF-MS) and UPLC coupled to FT-ICR-MS (LC-FT-MS) analyses from the same sample. This resulted in several thousands of detected m/z-features. ANOVA and multivariate statistical analysis revealed 139 significantly changed metabolites (30 by GC-TOF-MS and 109 by LC-FT-MS). The small-statured plants L. autumnalis, B. perennis and L. corniculatus showed metabolic response signatures to increasing plant diversity and species richness in contrast to tall-statured plants. Key-metabolites indicated C- and N-limitation for the non-leguminous small-statured species B. perennis and L. autumnalis, while the metabolic signature of the small-statured legume L. corniculatus indicated facilitation by other legumes. Thus, metabolomic analysis provided evidence for negative effects of resource competition on the investigated small-statured herbs that might mechanistically explain their decreasing performance with increasing plant diversity. In contrast, taller species often becoming dominant in mixed plant communities did not show modified metabolite profiles in response to altered resource availability with increasing plant diversity. Taken together, our study demonstrates that metabolite profiling is a strong diagnostic tool to assess individual metabolic phenotypes in response to plant diversity and ecophysiological adjustment.     

NMR spectroscopic analysis of exopolysaccharides produced by Leuconostoc citreum and Weissella confusa

Dextrans are the main exopolysaccharides produced by Leuconostoc species. Other dextran-producing lactic acid bacteria include Streptococci, Lactobacilli, and Weissella species. Commercial production and structural analysis has focused mainly on dextrans from Leuconostoc species, particularly on Leuconostoc mesenteroides strains. In this study, we used NMR spectroscopy techniques to analyze the structures of dextrans produced by Leuconostoc citreum E497 and Weissella confusa E392. The dextrans were compared to that of L. mesenteroides B512F produced under the same conditions. Generally, W. confusa E392 showed better growth and produced more EPS than did L. citreum E497 and L. mesenteroides B512F. Both L. citreum E497 and W. confusa E392 produced a class 1 dextran. Dextran from L. citreum E497 contained about 11% alpha-(1-->2) and about 3.5% alpha-(1-->3)-linked branches whereas dextran from W. confusa E392 was linear with only a few (2.7%) alpha-(1-->3)-linked branches. Dextran from W. confusa E392 was found to be more linear than that of L. mesenteroides B512F, which, according to the present study, contained about 4.1% alpha-(1-->3)-linked branches. Functionality, whether physiological or technological, depends on the structure of the polysaccharide. Dextran from L. citreum E497 may be useful as a source of prebiotic gluco-oligosaccharides with alpha-(1-->2)-linked branches, whereas W. confusa E392 could be a suitable alternative to widely used L. mesenteroides B512F in the production of linear dextran.     

Cloning of casbene and neocembrene synthases from Euphorbiaceae plants and expression in Saccharomycescerevisiae

A large number of diterpenes have been isolated from Euphorbiaceae plants, many of which are of interest due to toxicity or potential therapeutic activity. Specific Euphorbiaceae diterpenes of medical interest include the latent HIV-1 activator prostratin (and related 12-deoxyphorbol esters), the analgesic resiniferatoxin, and the anticancer drug candidate ingenol 3-angelate. In spite of the large number of diterpenes isolated from these plants and the similarity of their core structures, there is little known about their biosynthetic pathways. Other than the enzymes involved in gibberellin biosynthesis, the only diterpene synthase isolated to date from the Euphorbiaceae has been casbene synthase, responsible for biosynthesis of a macrocyclic diterpene in the castor bean (Ricinus communis). Here, we have selected five Euphorbiaceae species in which to investigate terpene biosynthesis and report on the distribution of diterpene synthases within this family. We have discovered genes encoding putative casbene synthases in all of our selected Euphorbiaceae species and have demonstrated high-level casbene production through expression of four of these genes in a metabolically engineered strain of Saccharomyces cerevisiae. The only other diterpene synthase found among the five plants was a neocembrene synthase from R. communis (this being the first report of a neocembrene synthase gene). Based on the prevalence of casbene synthases, the lack of other candidates, and the structure of the casbene skeleton, we consider it likely that casbene is the precursor to a large number of Euphorbiaceae diterpenes. Casbene production levels of 31 mg/L were achieved in S. cerevisiae and we discuss strategies to further increase production by maximizing flux through the mevalonate pathway.     

Establishing the occurrence of major and minor glucosinolates in Brassicaceae by LC-ESI-hybrid linear ion-trap and Fourier-transform ion cyclotron resonance mass spectrometry

Glucosinolates (GLSs) are sulfur-rich plant secondary metabolites which occur in a variety of cruciferous vegetables and among various classes of them, genus Brassica exhibits a rich family of these phytochemicals at high, medium and low abundances. Liquid chromatography (LC) with electrospray ionization in negative ion mode (ESI-) coupled to a hybrid quadrupole linear ion trap (LTQ) and Fourier transform ion cyclotron resonance mass spectrometer (FTICRMS) was employed for the selective and sensitive determination of intact GLSs in crude sample extracts of broccoli (Brassica oleracea L. Var. italica), cauliflower (B. oleracea L. Var. Botrytis) and rocket salad (Eruca sativa L.) with a wide range of contents. When LTQ and FTICR mass analyzers are compared, the magnitude of the limit of detection was ca. 5/6-fold lower with the FTICR MS. In addition, the separation and detection by LC-ESI-FTICR MS provides a highly selective assay platform for unambiguous identification of GLSs, which can be extended to lower abundance (minor) GLSs without significant interferences of other compounds in the sample extracts. The analysis of Brassicaceae species emphasized the presence of eight minor GLSs, viz. 1-methylpropyl-GLS, 2-methylpropyl-GLS, 2-methylbutyl-GLS, 3-methylbutyl-GLS, n-pentyl-GLS, 3-methylpentyl-GLS, 4-methylpentyl-GLS and n-hexyl-GLS. The occurrence of these GLSs belonging to the saturated aliphatic side chain families C(4), C(5) and C(6), presumably formed by chain elongation of leucine, homoleucine and dihomoleucine as primary amino acid precursors, is described. Based on their retention behavior and tandem MS spectra, all these minor compounds occurring in plant extracts of B. oleracea L. Var. italica, B. oleracea L. Var. Botrytis and E. sativa L. were tentatively identified.     

小花假泽兰精油超临界CO_2提取工艺及其成分和抑菌活性研究

采用正交试验法研究超临界CO_2萃取法提取小花假泽兰精油的提取条件,采用GC-MS法分析测定精油的化学成分,并初步测定精油的抑菌效果.结果表明:超临界CO_2萃取小花假泽兰精油的较佳工艺条件为:采用先静态后动态的萃取方式,静态萃取时间20 min,动态萃取条件为萃取温度55℃,萃取压力35 Mpa,CO_2体积为40 mL/g,CO_2 流速为0.5～1.0 mL/min;从超临界CO_2萃取的小花假泽兰精油中鉴定了64种成分,占精油总量的79.80%,主要有萜类、醇类、脂肪酸、酯类、甾体类等;生物测定结果表明,超临界CO_2萃取物对小麦赤霉病菌和小麦纹枯病菌菌丝生长的EC_(50)分别为119.55 mg/L和78.27 mg/L. 果.结果表明:超临界CO_2萃取小花假泽兰精油的较佳工艺条件为:采用先静态后动态的萃取方式,静态萃取时间20 min,动态萃取条件为萃取温度55℃,萃取压力35 MPa,CO_2体积为40 mL/g,CO_2 流速为0.5～1.0 mL/min;从超临界CO2萃取的小花假泽兰精油中鉴定了64种成分,占精油总量的79.80%,主要有萜类、醇类、脂肪酸、酯类、甾体类等;生物测定结果表 ,超临界CO_2萃取物对     

Radical scavenging activity of spring mountain herbs in the Shikoku mountain area and identification of antiradical constituents by simple HPLC detection and LC-MS methods

The functionality of spring mountain herbs, which were collected in the Kajigamori mountain area of Shikoku area in Japan, was investigated in the course of our studies for utilizing local plant resources. The radical scavenging activity of the extracts from seventeen herbs was measured. Among these herbs, two extracts from Polystichym ovato-paleaceum (Japanese name: Tsuyanashiinode) and Sambucus racemosa subsp. sieboldiana (Japanese name: Niwatoko) showed potent DPPH radical scavenging activity. The material evidence for the potent activity of the extracts was studied by a combination of our developed method for detecting antiradical compounds, LC-MS/MS, and enzymatic hydrolysis.     

Chemical Characterization of Euphorbia heterophylla L. Essential Oils and Their Antioxidant Activity and Allelopathic Potential on Cenchrus echinatus L.

The genus Euphorbia attracted the attention of many researchers worldwide from natural products, bioactivity, and ecological perspective. The essential oils (EOs) of Euphorbia heterophylla are poorly studied. Therefore, the present study aimed to provide a detailed profile of the E. heterophylla EOs as well as to determine their antioxidant and allelopathic activities. The EOs from aerial parts of E. heterophylla were extracted using hydrodistillation and analyzed via GC/MS. The antioxidant activity was determined based on scavenging of the free radical, 1,1‐diphenyl‐2‐picrylhydrazyl and H₂O₂. Various concentrations of the EOs were tested against the noxious weed, Cenchrus echinatus. Thirty‐five compounds were identified representing 100 % of the total mass. Four classes of components were characterized, among which terpenoids were the main components (88.70 %). Monoterpenes represented the main class (69.48 %), followed by sesquiterpenes (18.63 %), and only one diterpenoid, kaur‐16‐ene, was identified. 1,8‐Cineole (32.03 %), camphor (16.54 %), β‐elemene (5.92 %), endo‐borneol (4.94 %), limonene (4.27 %), pentatriacontane (3.91 %), and α‐pinene (3.89 %) were the major compounds. The EOs composition of Egyptian E. heterophylla ecospecies was comparable to that of other reported Euphorbia species, although it showed no correlation with Nigerian E. heterophylla ecospecies. The EOs from E. heterophylla aerial parts exhibited significant antioxidant activity. Moreover, a concentration of 100 μL L^?1 of the EOs reduced the germination, root, and shoot growth of C. echinatus by about 93.95 %, 84.6 %, and 57.8 %, respectively. Therefore, the EOs from E. heterophylla could be integrated into the control of this weed, as eco‐friendly biocontrol method. Further study is needed to characterize their allelopathic activity under field conditions as well as to evaluate their durability and biosafety.