**2. Results and Discussion**

#### *2.1. Chemical Composition of P. lapathifolia EO*

The hydrodistillation of the aerial parts of *P. lapathifolia* via the Clevenger apparatus produced 0.18% (*v*/*w*) of a dark yellow oil accompanied by a slight scent. This yield was lower than those reported from other *Persicaria* species such as Vietnamese *P. odorata* (0.41%) [28], Malaysian *P. odorata* (0.64%) [29], and *P. hydropiper* (0.70%) [15]. Overall, twenty-one volatile components were identified via GC–MS analysis, representing 98.3% of the total mass of oil (Table 1).

Eight classes of the compounds were characterized for the EO, including monoterpenes (hydrocarbons and oxygenated), sesquiterpenes (hydrocarbons and oxygenated), diterpenes (hydrocarbons and oxygenated), carotenoid-derived compounds, and other nonterpenoid compounds (Table 1). Among these components, nonterpenoid constituents represented the predominant type of compounds, with a relative concentration of 58.69%. This result is consistent with the previously described essential oils from Vietnamese [28] and Australian [30] *P. odorata*. In contrast, Dũng, et al. [31] described that sesquiterpene hydrocarbons were the main compounds of the Vietnamese *P. odorata* along with the oxygenated constituents. Herein, 11 nonterpenoids were identified, comprising *n*-dodecanal (22.61%), 2,4-dimethylicosane (8.97%), 2*E*-hexenoic acid (8.04%), and γ-nonalactone (3.51%) as the abundant compounds, while *n*-tricosane (1.18%) was determined as a minor compound. The preponderance of the alkyl aldehyde, *n*-dodecanal, in the EO of *P. lapathifolia* is in harmony with the published data on the EOs of Vietnamese and Australian *P. odorata* [28,30].

Monoterpenes were determined with a relative concentration of 17.23% of the EO mass. They can be divided into monoterpene hydrocarbons (6.80%) and oxygenated monoterpenes (10.43%). Three monoterpene hydrocarbon compounds were assigned, of which limonene (3.09%) was the major and α-pinene was a minor compound. Limonene is not a widely distributed compound in the EOs of *Persicaria* plants, although it has been reported as a major constituent of the EOs of several species such as *Schinus terebinthifolius* [32], *Callistemon viminalis* [33], *Artemisia scoparia* [34], *Heterothalamus psiadioides* [35], and *Carum carvi* [36].

On the other side, 2-methyl butyl isovalerate was the only identified oxygenated monoterpene, with a relative concentration of 10.43%. Isovalerate derivatives have been described in the EOs of several plants such as *Eucalyptus brockwayii* [37], Algerian *Daucus gracilis* [38], and *Chamaemelum fuscatum* [39].


**Table 1.** Essential oil chemical composition from aerial parts of *Persicaria lapathifolia*.

<sup>a</sup> Rt: retention time, <sup>b</sup> average value ± standard deviation, <sup>c</sup> the identification of EO constituents was based on the comparison of mass spectral data and Kovats indices (KI) with those of the NIST Mass Spectral Library (2011) and Wiley Registry of Mass Spectral Data 8th edition and literature, <sup>d</sup> KIpublished: reported Kovats retention indices; <sup>e</sup> KIObserved: experimentally calculated Kovats index relative to C8–C28 *n*-alkanes.

> Sesquiterpenes made up a relative concentration of 15.77% of the total oil mass. They consisted of sesquiterpene hydrocarbons (12.58%) and oxygenated sesquiterpene (3.19%). α-Humulene (11.29%), and *trans*-caryophyllene (1.29%) were the only identified sesquiterpene hydrocarbons. The two compounds are popular in the EOs of the plants belonging to *Persicaria* such as Vietnamese *P. odorata* [28,31] and Australian *P. odorata* [30]. In the EO of *P. lapathifolia*, diterpenes represented 4.86% of the total oil mass, including one diterpene hydrocarbon, phytane (3.78%), and one oxygenated compound, abienol (1.08%). Diterpenes have been recorded in the EOs of *Persicaria* species as traces [28,30,31]. The scarcity of diterpenoids in EOs derived from plants is a common phenomenon with a few exceptions such as *Araucaria heterophylla* [5] and *Calotropis procera* [14].

> Carotenoid-derived components were represented by only the common compound, hexahydrofarnesyl acetone (1.75%), which has been characterized in the EOs of many plants such as *Launaea mucronata*, *Launaea nudicaulis* [40], and *Heliotropium curassavicum* [13].

#### *2.2. Allelopathic Activity of P. lapathifolia EO*

The extracted EO of *P. lapathifolia* exhibited substantial allelopathic activity against the germination, seedling root, and shoot growth of the weed *E. colona* in a dose-dependent manner (Figure 2). At the highest concentration (100 mg L−1), the germination, seedling

root, and shoot growth were inhibited by 64.25, 82.48, and 95.25%, respectively. Additionally, the EO showed IC50 values of 77.27, 60.84, and 33.80 mg L−1, respectively (Figure 2). It is clear that the seedling growth was more inhibited than germination. However, the root growth of *E. colona* was more sensitive to the EO than the shoots, and this observation was in harmony with other studies [13,41]. This could be attributed to the permeability of the root cells and the direct contact with the medium that contained the EO [14,41,42].

**Figure 2.** Allelopathic activity of the *Persicaria lapathifolia* essential oil. (**a**) Inhibitory effect on the germination, seedling root, and shoot growth of the weed *Echinochloa colona*; (**b**) IC50 values. Different letters mean a significant difference in values after Tukey's HSD test (*p* < 0.05).

To our knowledge, the allelopathic activity of *P. lapathifolia* EO has not been described yet. Herein, the EO of *P. lapathifolia* was found to exhibit allelopathic effects on *E. colona*. Many published data have revealed the principal and direct relationship between the allelopathic properties of EOs derived from plants and their chemical compositions [4,14,40–42]. Therefore, the observed allelopathic activity of *P. lapathifolia* EO might be ascribed to its chemical constituents, especially the main compounds *n*-dodecanal, α-humulene, 2,4 dimethylicosane, and 2*E*-hexenoic acid, γ-nonalactone, and limonene. These compounds could act either singularly or in a synergistic manner as allelochemicals that inhibit germination and seedling growth [41]. The allelopathic effect of allelochemicals may occur by inhibition of cell division, reduction of respiration, affecting photosynthesis, inhibition of enzymatic systems, affecting nucleic acid, or induction of reactive oxygen species in plant cells [43–45]. Humulene has been reported as a major compound in the EO of *Symphyotrichum squamatum*, which exhibited significant allelopathic activity on the weed *Bidens pilosa* [12]. Also, limonene has been reported as a major compound in the EOs of various plants that exhibited considerable allelopathic activity such as *Heterothalamus psiadioides* [35], *Agastache rugosa* [46], *Schinus terebinthifolius* [32], *Callistemon viminalis* [33], *Artemisia scoparia* [34], *Pinus pinea* [47], and *Carum carvi* [36].

It is worth mentioning here that *E. colona* has been reported to have allelopathic activity against various crops and weeds such as rice, soybean [48], and *Avena fatua* [49]. Also, the allelopathic activity of various plant extracts, such as those of *Sorghum bicolor*, *Helianthus annuus*, *Brassica campestris* [50], *Mikania micrantha*, *Clidemia hirta*, *Dicranopteris linearis*, and *Ageratum conyzoides* [51], has been studied against *E. colona*. However, no study has revealed the activity of the essential oil of *P. lapathifolia* against this weed. In this line, the present study showed that the EO of the aerial parts of *P. lapathifolia* could be used in the management of weeds as a green, ecofriendly herbicide, particularly against species of *Echinochloa*, including *E. colona*, which has been reported to be resistant against herbicides [52].
