The Herbicidal Activity of Nano- and MicroEncapsulated Plant Extracts on the Development of the Indicator Plants Sorghum bicolor and Phaseolus vulgaris and Their Potential for Weed Control

Abstract

Weeds decrease yield in crops through competition for water, nutrients, and light. Due to the circumstances mentioned above and the challenge of the emergence of herbicide-resistant weeds, developing sustainable alternatives becomes imperative. Plant extracts formulated into nano- and micro-encapsulates (NPs) emerge as a viable option for weed management. The objectives of this study were to identify phytochemical compounds within the ethanolic extracts of Carya illinoinensis, Ruta graveolens, and Solanum rostratum; determine their pre-emergence herbicidal activity on the indicator plants Sorghum bicolor and Phaseolus vulgaris; produce and characterize NPs with plant extracts; and assess their phytotoxicity under greenhouse conditions. The extracts were provided by Greencorp Biorganiks de México. Phytochemicals were identified through colorimetric assays and HPLC-MS, while pre-emergence tests were conducted in vitro, assessing concentrations of 12.5, 25, and 50% for each extract. NPs were synthesized using the ionotropic pre-gelation method, with size, zeta potential, and encapsulation efficiency (EE) being characterized. Finally, post-emergence tests were carried out in a greenhouse with seedlings. Compounds belonging to the hydroxycinnamic acid, flavonol, methoxyflavonol, hydroxybenzoic acid, methoxyflavone, tyrosol, stilbene, and lignan families were identified in all extracts. The pre-emergence herbicidal activity was observed for all extracts, with germination percentages ranging from 0 to 41% in both indicator plants. NPs exhibited sizes between 290 and 345 nm, zeta potentials ranging from −30 to −35 mV, and EE up to 94%. Finally, enhanced herbicidal activity was observed with plant extract NPs, with the species S. bicolor being more susceptible. NPs containing plant extracts are a viable option for bioherbicide production; however, continued research is necessary to refine formulations and enhance efficacy.

1. Introduction

Weeds are one of the main problems in agriculture, because they induce yield reduction in all agricultural production systems. Historically, their management has been carried out through chemical synthesis products; nevertheless, these agents stimulate environmental problems and adverse effects on human health. Furthermore, their indiscriminate employment has generated the emergence of herbicide-resistant weeds, decreased the chemical options for weed management [1]. Owing to those reasons mentioned above, plant extracts and their allelopathic activity arise as an ecological alternative within sustainable agricultural production. This option offers advantages such as reduced environmental impact and a diminished likelihood of generating herbicide-resistant weeds. Thus, phytochemical compounds and their biological efficacy can be used as bioherbicides in weed management strategies [2,3].

It has been elucidated that phenolic compounds are the secondary metabolites of plants with higher allelopathic activity. These compounds constitute one of the most significant groups of antioxidant substances and are synthesized by plants as a defense mechanism against many factors [4]. In this context, herbicidal activity toward weed species has been reported for extracts of Canavalia ensiformis, Cirsium setosum, Cynara cardunculus, Juglans nigra, Lantana camara, and Ocimum basilicum. These extracts affected weeds, provoking growth and development reduction, inhibiting germination, inducing oxidative stress, and altering physiological processes in cells. Additionally, these extracts inhibit H+ATPasa activity, causing a reduction in photosynthesis and a decrease in the production of roots, leaves, and cotyledon [5].

Although phytochemical compounds represent a promising solution for managing ecological challenges and herbicide resistance, and several studies document their efficacy, this effectiveness may be reduced by environmental factors such as light exposure, temperature fluctuations, humidity, ultraviolet radiation, and compound leaching [6]. Consequently, these factors can reduce their phytotoxic activity, such that only 0.1% of the applied product reaches its target [6,7,8,9,10]. Therefore, it becomes necessary to develop formulations to enhance the stability and effectiveness of these natural compounds in various applications [11].

Due to the aforementioned factors, the use of nano- and microencapsulated formulations (NPs) based on biopolymers, such as alginate and chitosan, emerges as an option to enhance the efficacy of phytochemical compounds. Thus, utilizing nanoparticles loaded with plant extracts may represent a viable strategy for reducing reliance on chemical herbicides, which inflict environmental harm, pose risks to human health, exhibit extended persistence, and develop herbicide-resistant weeds [12]. This alternative prevents the degradation of compounds and safeguards active agents upon field application. Moreover, due to the properties of biopolymers, the release of active ingredients can be regulated, thereby enabling a sustained level of the active agent over an extended period, lower doses, a reduction in evaporation loss, and the mitigation of leaching effects [12].

In this manner, it can be inferred that the utilization of compounds for producing encapsulated organic pesticides represents a viable option that can be employed for pest management. Studies have demonstrated enhancements in the properties and increased efficacy of various pesticides when encapsulated with different compounds. In this regard, Ge et al. [13] encapsulated the fungicide carbendazim with hydroxypropyl-β-cyclodextrin and observed improvements in solubility and fungicidal activity. Similarly, Fan et al. [14] prepared gallic acid through an inclusion process with hydroxypropyl-cyclodextrin, finding an increase in gallic acid solubility, and the resulting compound exhibited inhibitory activity against bacteria. Lastly, the encapsulation of phytochemical compounds also can prevent their volatilization, making them suitable for use in weed control. Thus, Mejías et al. [15] observed phytotoxic activity of sesquiterpene lactones encapsulated in organic nanotubes against the weeds Phalaris arundinacea, Lolium perenne, and Portulaca oleracea, reducing the compound volatilization.

The objectives of the present study were the characterization of the phytochemical compounds present in ethanolic extracts of the leaf and husk of Carya illinoinensis, Ruta graveolens, and Solanum rostratum; the determination of preemergence herbicidal activity in vitro; and the production, characterization, and evaluation of the phytotoxicity of plant extracts and NPs loaded with plant extracts on Sorghum bicolor and Phaseolus vulgaris as indicator plants under greenhouse conditions. The extract characterizations aim to identify the phytochemical compounds present, thereby elucidating the potential mode of action of the extracts on the test plants. This characterization was conducted using colorimetric methods and HPLC-MS.

2. Materials and Methods

2.1. Obtaining Plant Extracts and Phytochemical Characterization

Ethanol extracts of leaves and shoots from C. illinoinensis, as well as from R. graveolens and S. rostratum, were employed, because ethanolic extracts are more stable and exhibit greater biological efficacy than aqueous extracts, and they are more economically viable than oils. These extracts were provided by the company GreenCorp Biorganiks de México S.A de C.V.

2.1.1. Phytochemical Analysis of Extracts

The identification of phytochemicals was carried out with qualitative techniques, where the presence or absence of phytochemicals was determined through colorimetric methods, observing the color change when a phytochemical was present in the extract. The following test materials were used: alkaloids (Dragendorff and Sonheschain reagents), carbohydrates (Molisch reagent), carotenoids (H₂SO₄ and FeCl₃ reagents), coumarins (Erlich reagent), flavonoids (Shinoda and 1% NaOH reagents), reducing sugars (Fehling and Benedict reagents), cyanogenic glycosides (Grignard reagent), purines (HCl test), quinones (NH₄OH and H₂SO₄ reagents for anthraquinones, and Börntraguer test for benzoquinones), saponins (foam test, Bouchard reagent for steroidal saponins, and Rosenthaler reagent), terpenoids (Ac₂O reagent), and tannins (FeCl₃ and ferrocyanide reagents) [16]. After the reagents for each test were prepared, the extracts at 100% concentration were added to initiate the respective reaction. Subsequently, the extracts changed color, and depending on the color they assumed, the presence or absence of phytochemicals was determined following the established protocols in the methodologies.

2.1.2. Identification of Phytochemical Compounds through High-Performance Liquid Chromatography Coupled with Mass Spectrometry (HPLC-MS)

The detection of phytochemical compounds via HPLC-MS was conducted following the method proposed by Ascacio-Valdés et al. [17]. A Varian HPLC system was employed, comprising an autoinjector (Varian ProStar 410), a ternary pump (Varian ProStar 230I), and a PDA detector (Varian ProStar 330). An ion trap mass spectrometer was also coupled with liquid chromatography, utilizing electrospray ionization as the ion source. Five microliters of the sample were injected into a Denali C18 column, maintained at a constant oven temperature of 30 °C. The eluents employed were formic acid (0.2% v/v, solvent A) and acetonitrile (solvent B). The applied gradient was as follows: initially, 3% B; 0–5 min, linear increase to 9% B; 5–15 min, linear increase to 16% B; 15–45 min, linear increase to 50% B; after which the column was cleaned and reconditioned. The flow rate was set at 0.2 mL/min, and elution was monitored at 245, 280, 320, and 550 nm. The entire effluent was directed into the mass spectrometer source without splitting. All MS experiments were conducted in negative ion mode. Nitrogen was employed as the nebulizing gas, while helium was the collision gas. The ion source parameters were set as follows: spray voltage of 5.0 kV, capillary voltage of 90.0 V, and a temperature of 350 °C. Data analysis was performed using MS Workstation software (V6.9).

2.2. Pre-Emergence in Vitro Herbicidal Activity

Seeds of S. bicolor and P. vulgaris were employed as indicator plants to simulate narrow-leaf and broad-leaf weeds, respectively. The seeds were sterilized with 0.5% sodium hypochlorite for 2 min, and subsequently, the seeds were washed with sterile distilled water for 1 min to remove the hypochlorite. Ethanol extracts from the husk and leaves of C. illinoinensis, R. graveolens, and S. rostratum were utilized. The extracts were diluted with sterile distilled water, considering the crude extract provided by the company as 100%. Thus, the concentrations used were 50, 25, and 12.5%, and the absolute control of distilled water. Subsequently, a filter paper of medium-pore cellulose was placed in a Petri dish of 90 mm, 2 mL of each concentration was added, and ten seeds were distributed in the Petri dish. Three replicates were performed for each extract and concentration. Finally, the Petri dishes were incubated at 25 °C for seven days. The number of germinated seeds was counted, and the length of the radicle and hypocotyl was measured [3]. The percentage of germination and the inhibition rate were calculated using the following formulas:

$$\mathrm{Germination} \mathrm{percentage}=\left(\frac{A}{B}\right)\times 100$$

(1)

where A = number of germinated seeds in the treatment, and B = number of germinated seeds in the control.

$$\mathrm{Inhibition} \mathrm{rate}=\left(\frac{T}{C}\right)\times 100$$

(2)

where T represents the length of roots or hypocotyls of treated seedlings, and C represents the length of roots or hypocotyls of control seedlings.

2.3. Production and Characterization of NPs Using the Ionotropic Gelation Method

The production of NPs was carried out through the ionotropic gelation method proposed by Sarmento et al. [18]. A total of 3.75 mL of a CaCl₂ solution was added to 59 mL of a sodium alginate solution (0.063%, pH 4.9), using a peristaltic pump under continuous and vigorous agitation. Subsequently, 12.5 mL of a chitosan solution (0.07%, pH 4.6) was added to the CaCl2 and sodium alginate solution mixture, maintaining constant agitation for 90 minutes. This procedure was in the presence and absence of the plant extracts.

2.3.1. Characterization of NPs

The size of NPs was determined through the dynamic light scattering (DLS) technique. The samples were diluted, and subsequently, triplicate analyses were conducted at 25 °C with light scattered at an angle of 90°, using the NanoSight NS, Malvern. The zeta potential (mV) was determined for triplicated with the samples at 25 °C, with the Colloid Metrix ZETA-Check system. To evaluate formulation component degradation, pH levels were assessed using an Ion Meter450, Corning potentiometer developed by Corning.

2.3.2. Encapsulation Efficiency

The encapsulation efficiency was determined using a spectrophotometer with the methodology proposed by Taban et al. [19]. An absorbance measurement of plant extracts was performed, and subsequently, the NPs were centrifuged, followed by an absorbance measurement of the supernatant. Encapsulation efficiency (EE) was calculated using the formula:

$$\% \mathrm{EE}=\left(\frac{T0-S0}{T0}\right)\times 100$$

(3)

where T0 is the absorbance of the plant extract, and S0 represents the absorbance of the supernatant from NPs loaded with plant extracts.

2.4. Post-Emergence Herbicidal Activity under Greenhouse Conditions

The post-emergence herbicidal activity was determined with the indicator plants S. bicolor and P. vulgaris to simulate narrow-leaf and broad-leaf weeds, respectively. Polypropylene pots with a capacity of 200 mL were utilized. The pots were filled with a substrate composed of peatmoss and soil (1:1). Subsequently, three seeds were sown in the pots. After seven days, two seedlings were removed from each pot, leaving one seedling. Treatments were applied through direct leaf spraying using a manual atomizer. The treatments employed are presented in

Table 1. Treatments used in post-emergence bioassay on indicator plants Sorghum bicolor and Phaseolus vulgaris.

Treatment
T1	100% ethanolic extract of C. illinoinensis husk (RCi)
T2	100% ethanolic extract of C. illinoinensis leaf (HCi)
T3	100% ethanolic extract of R. graveolens (Rg)
T4	100% ethanolic extract of S. rostratum (Sr)
T5	C. illinoinensis husk NPs (NPsRCi)
T6	C. illinoinensis leaf NPs (NPsHCi)
T7	R. graveolens NPs (NPsRg)
T8	S. rostratum NPs (NPsSr)
T9	NPs without extract (NpsSe)
T10	Absolute control (TA)

.

The experimental design was a completely randomized design with three replicates per treatment. The phytotoxic effect on plants was evaluated with the scale proposed by the European Weed Research Society (EWRS) (1: complete death, 2: very good control, 3: good control, 4: practically sufficient control, 5: moderate control, 6: regular control, 7: poor control, 8: very poor control, and 9: no effect) [20]; additionally, plant height, stem diameter, and dry weight were assessed.

2.5. Statistical Analysis

The data were analyzed using the Statistical Analysis System software (SAS), version 9.0. All data were analyzed using ANOVA, and multiple mean comparisons were made using Duncan’s multiple range test (at p < 0.05).

3. Results

3.1. Phytochemical Analysis of Extracts

The phytochemical compounds identified from ethanolic plant extracts from the species C. illinoinensis, R. graveolens, and S. rostratum, are described in

Table 2. Phytochemical compounds identified in ethanolic extracts of leaves and husks of Carya illinoinensis, Ruta graveolens, and Solanum rostratum have been documented.

Extract	A	C	F				GC	AZ	S		T			Q			Cu	P	Ca
			F1	F2	F3	F4			S1	S2	T1	T2	T3	Q1	Q2	Q3
Leaf of C. illinoinensis	+	-	-	-	+	-	-	+	+	+	+	-	+	+	+	-	-	+	+
Husk of C. illinoinensis	+	+	+	+	+	-	+	+	+	+	+	-	+	+	-	-	-	+	+
R. graveolens	+	+	+	-	-	-	-	+	-	-	-	+	+	+	-	-	+	-	+
S. rostratum	+	+	+	-	-	-	-	+	-	-	-	+	+	+	-	-	-	-	-

+:Phytochemical present; -: phytochemical absent; A: alkaloids; C: carbohydrates; F: flavonoids; GC: cyanogenic glycosides; AZ: reducing sugars; S: saponins; T: tannins; Q: quinones; Cu: coumarins; P: purines; Ca: carotenoids; F₁: anthocyanins; F₂: flavones; F₃: flavonones; F₄: chalcones; S₁: triterpenoids; S₂: steroidal; T₁: gallic acid derivatives; T₂: catechol derivatives; T₃: phenols; Q₁: anthraquinones; Q₂: benzoquinones; Q₃: anthrones.

. The presence of alkaloids, flavonoids, reducing sugars, tannins, and quinones was detected in all extracts. Moreover, the C. illinoinensis leaf extract presented purines and carotenoids, in contrast to the husk extract, which encompassed carbohydrates within its phytochemical composition. Meanwhile, in the extract from R. graveolens, carbohydrates, coumarins, and carotenoids were detected, while the S. rostratum extract only contained carbohydrates. Plants, in their metabolisms, have the intrinsic capacity to synthesize a huge amount of phytochemical compounds with biological activity, which affect the metabolic process and the cellular structure of organisms that come into contact with them.

3.2. Identification of Phytochemical Compounds through High-Performance Liquid Chromatography Coupled with Electrospray Mass Spectrometry (HPLC-MS)

The high-performance reverse-phase liquid chromatography analysis detected diverse compounds in all plant extracts (

Table 3. Phytochemical compounds detected in the leaf and husk extract of Carya illinoinensis, Ruta graveolens, and Solanum rostratum using high-performance liquid chromatography in reverse-phase mode (HPLC-MS).

Extract	Compound	Retention time (Min)	Mass	Family
Leaves of Carya illinoinensis	Caffeic acid 4-O-glucoside	5.863	341	Hydroxycinnamic acids
	1-Caffeoylquinic acid	22.258	353	Hydroxycinnamic acids
	3-p-Coumaroylquinic acid	26.359	337	Hydroxycinnamic acids
	4-p-Coumaroylquinic acid	34.554	336.9	Hydroxycinnamic acids
	Myricetin 3-O-glucoside	38.492	479	Flavonols
	Quercetin 3-O-glucoside	41.58	463	Flavonols
	Quercetin	43.0	300.0	Flavonols
	Isorhamnetin 3-O-glucoside 7-O-rhamnoside	47.812	623.6	Methoxyflavonols
	Quercetin 3-O-rhamnoside	49.502	477	Flavonols
Husk of Carya illinoinensis	Cinnamoyl glucose	5.243	308.6	Hydroxycinnamic acids
	Caffeic acid 4-O-glucoside	6.139	340.9	Hydroxycinnamic acids
	Protocatechuic acid 4-O-glucoside	18.887	314.9	Hydroxycinnamic acids
	3,7-dimethylquercetin	48.53	329	Methoxyflavones
Ruta graveolens	Caffeic acid 4-O-glucoside	6.76	340.8	Hydroxycinnamic acids
	3,4 DHPEA-EA	6.76	340.8	Tyrosols
	Protocatechuic acid 4-O-glucoside	18.97	314.9	Hydroxybenzoic acids
	1-Caffeoylquinic acid	20.39	352.8	Hydroxycinnamic acids
	3-p-Coumaroylquinic acid	21.907	336.9	Hydroxycinnamic acids
	3-Feruloylquinic acid	24.49	366.9	Methoxycinnamic acids
	4-Feruloylquinic acid	27.26	366.8	Methoxycinnamic acids
	Quercetin 3-O-xylosyl-glucuronide	31.78	608.8	Flavonoles
	Isorhamnetin 3-O-glucoside 7-O-rhamnosideo	34.22	622.8	Methoxyflavonols
	1,2-Apigenin diglucoside	35.29	752.7	Methoxycinnamic acids
	Quercetin 3-O-rhamnosyl-galactoside	44.08	608.7	Flavonols
	p-Coumaric acid 4-O-glucoside	48.67	324.9	Hydroxycinnamic acids
Solanum rostratum	Caffeic acid 4-O-glucoside	5.7	340.9	Hydroxycinnamic acids
	Protocatechuic acid 4-O-glucoside	17.417	314.7	Hydroxybenzoic acids
	Resveratrol 3-O-glucoside	17.96	389.9	Stilbenes
	Protocatechuic acid 4-O-glucoside	16.842	314.8	Hydroxybenzoic acids
	Secoisolariciresinol	20.338	364.7	Lignans
	Sinensetin	28.397	370.8	Methoxyflavones
	Ferulic acid 4-O-glucoside	31.085	354.7	Methoxycinnamic acids
	Tetramethylscutellarin	34.40	340.8	Methoxyflavones
	Isorhamnetin 3-O-glucoside 7-O-rhamnoside	33.77	622.8	Methoxyflavones

). The leaves and husks of C. illinoinensis extracts exhibited compounds from the families of hydroxycinnamic acids, flavonols, methoxyflavonols, hydroxybenzoic acids, and methoxyflavones. Meanwhile, the R. graveolens extract presented components from the families of hydroxycinnamic acids, tyrosols, hydroxybenzoic acids, methoxycinnamic acids, flavonols, and methoxyflavonols. Finally, in the S. rostratum extract, the phytochemicals detected were from families of hydroxycinnamic acids, hydroxybenzoic acids, stilbenes, lignans, methoxyflavones, methoxycinnamic acids, and methoxyflavonols.

3.3. Pre-Emergence Herbicidal Activity of Ethanolic Extracts in Vitro

The pre-emergence herbicidal activity of ethanolic extracts from the leaves and husks of C. illinoinensis, R. graveolens, and S. rostratum is in

Table 4. Germination percentage of Sorghum bicolor and Phaseolus vulgaris seeds treated with ethanolic extracts at different concentrations.

Treatments	Sorghum bicolor	Phaseolus vulgaris
Untreated	100 a*	100 a
Carya illinoinensis leaf extract at 12.5%	0 c	0 c
Carya illinoinensis leaf extract at 25%	0 c	0 c
Carya illinoinensis leaf extract at 50%	0 c	0 c
Carya illinoinensis husk extract at 12.5%	16.6 bc	8.3 b
Carya illinoinensis husk extract at 25%	25 bc	8.3 b
Carya illinoinensis husk extract at 50%	0 c	0 c
Ruta graveolens extract at 12.5%	0 c	8.3 b
Ruta graveolens extract at 25%	0 c	8.3 b
Ruta graveolens extract at 50%	0 c	0 c
Solanum rostratum extract at 12.5%	41.6 b	8.3 b
Solanum rostratum extract at 25%	41.6 b	8.3 b
Solanum rostratum extract at 50%	0 c	0 c

* Values with the same letter are not significantly different.

and

Table 5. Hypocotyl and radicle inhibition rate of Sorghum bicolor and Phaeolus vulgaris seeds treated with ethanolic extracts at different concentrations.

	Sorghum bicolor		Phaseolus vulgaris
Treatments	Hipocotyl	Radicle	Hipocotyl	Radicle
Untreated	0.0 d*	0.0 c	0.0 b	0.0 b
Carya illinoinensis leaf extract at 12.5%	100.0 a	100.0 a	100.0 a	100.0 a
Carya illinoinensis leaf extract at 25%	100.0 a	100.0 a	100.0 a	100.0 a
Carya illinoinensis leaf extract at 50%	100.0 a	100.0 a	100.0 a	100.0 a
Carya illinoinensis husk extract at 12.5%	95.4 ab	86.5 b	91.6 a	86.1 a
Carya illinoinensis husk extract at 25%	90.9 abc	88.0 b	91.6 a	88.5 a
Carya illinoinensis husk extract at 50%	100.0 a	100.0 a	100.0 a	100.0 a
Ruta graveolens extract at 12.5%	100.0 a	100.0 a	100.0 a	97.6 a
Ruta graveolens extract at 25%	100.0 a	100.0 a	100.0 a	98.1 a
Ruta graveolens extract at 50%	100.0 a	100.0 a	100.0 a	100.0 a
Solanum rostratum extract at 12.5%	86.0 bc	100.0 a	100.0 a	97.6 a
Solanum rostratum extract at 25%	84.2 c	100.0 a	100.0 a	98.1 a
Solanum rostratum extract at 50%	100.0 a	100.0 a	100.0 a	100.0 a

* Values with the same letter are not significantly different.

. The allelopathic effect of phytochemical compounds present in the extracts was evident in the germination process and subsequent development of seeds from both plant species. In the case of S. bicolor, highly significant differences were observed compared to the untreated control. It is noteworthy that the majority of treatments caused complete inhibition, reaching 100% in seed germination. Only the treatments corresponding to doses of 12.5% and 25% husk extracts of C. illinoinensis and S. rostratum did not achieve 100% suppression of germination, showing germination percentages ranging from 16% to 41%. Similarly, for P. vulgaris seeds, similar results were observed, with statistical differences among the treatments compared to the control. Likewise, similar to S. bicolor seeds, most treatments led to complete 100% suppression in germination. However, treatments with the husk extracts of C. illinoinensis, R. graveolens, and S. rostratum at 12.5% and 25% exhibited germination percentages of 8.3%.

Regarding the hypocotyl and radicle inhibition rate, statistical differences were observed between the treatments and the untreated control in both species. In S. bicolor-treated seeds, the inhibition of hypocotyl and radicle development was 100% in all treatments, except for seeds treated with the husk extracts of C. illinoinensis and R. graveolens at 12.5% and 25%, which showed hypocotyl inhibition at a rate ranging from 84% to 95%, while radicle inhibition fluctuated between 86% and 100%. Similarly, the seeds of P. vulgaris were entirely inhibited by all treatments except for treatments with the husk extracts of C. illinoinensis, R. graveolens, and S. rostratum at doses of 12.5 and 25%.

3.4. Characterization of NPs

The size of the NPs with plant extracts ranged from 290 to 345 nm (Table 3). The variation in size suggests that adding phytochemical compounds increased the size of the NPs. The concentration of chitosan and alginate is a factor that can determine the size of the NPs. Zeta potential values were −35.4, −35.25, −30.2, and −30.45 mV for the NPs with plant extracts, and −28 mV for the NPs without plant extracts (Table 3). The zeta potential is an important factor because it represents an electrostatic potential that exists between the layers surrounding a particle, being a factor that prevents their agglomeration. Concerning the chemical stability of the evaluated polymers in this study, the final pH of the NPs with plant extracts was 4.62, 4.62, 4.59, and 4.63 (Table 3), and 4.45 for the NPs without plant extracts.

3.5. Encapsulation Efficiency

The encapsulation efficiency for plant extracts is in

Table 6. Values derived from the assessment of distinct variables in NPs formulations incorporating plant extracts and in those devoid of plant extracts.

	Size (nm)	Zeta Potential (mV)	pH	Encapsulation Efficiency (%)
NPs of leaves of C. illinoinensis	313 ± 24	−35.4 ± 1.3	4.62	92
NPs of husk of C. illinoinensis	343 ± 25	−35.25 ± 1.0	4.62	94
NPs of R. graveoelens	291 ± 26	−30.2 ± 2	4.59	91
NPs of S. rostratum	340 ± 23	−30.45 ± 1.5	4.63	90
NPs without extract	150 ± 23	−30 ± 2	4.45	-----

. In this context, the formulation with the highest encapsulation efficiency was the NPs of C. illinoinensis husk at 94%, followed by NPs of the leaf of C. illinoinensis at 92%. Finally, the formulations with the lowest encapsulation efficiencies were the NPs of S. rostratum and NPs of R. graveolens at 91% and 90%, respectively. Encapsulation efficiencies exceeding 90% show a strong interaction of phytochemical compounds with the biopolymers used in the plant extract formulation.

3.6. Post-Emergence Herbicidal Activity under Greenhouse Conditions

Phytotoxicity arises from reactions in which plant cells lose their integrity, leading to damage, developmental alterations, physiological changes, and morphological disruptions. In this context,

Table 7. Phytotoxicity and effect on the development of the indicator plants Sorghum bicolor and Phaseolus vulgaris treated with plant extracts and NPs loaded with extracts from Carya illinoinensis, Ruta graveolens, and Solanum rostratum.

Treatment	S. bicolor				P. vulgaris
	Phytotoxicity	Height (cm)	Diameter (mm)	Dry Weight (g)	Phytotoxicity	Height (cm)	Diameter (mm)	Dry Weight (g)
RCi	7.3 ± 0.6 bc*	11.3 ± 2.6 ab	0.79 ± 0.2 b	0.07 ± 0.01 a	8.0 ± 0.1 b	9.5 ± 0.8 bc	2.50 ± 0.1 a	0.37 ± 0.07 a
HCi	7.3 ± 0.6 bc	11.5 ± 1.7 ab	1.11 ± 0.2 ab	0.08 ± 0.02 a	8.0 ± 0.1 b	11.1 ± 0.4 ab	2.60 ± 0.1 a	0.32 ± 0.06 a
Rg	7.7 ± 0.6 bc	11.6 ± 2.9 ab	1.09 ± 0.2 ab	0.10 ± 0.01 a	8.3 ± 0.1 ab	10.4 ± 1.0 b	2.50 ± 0.1 a	0.35 ± 0.09 a
Sr	7.0 ± 1 bc	11.1 ± 3.6 ab	1.07 ± 0.2 ab	0.09 ± 0.04 a	8.7 ± 0.6 ab	9.0 ± 0.1 bc	2.70 ± 0.3 a	0.39 ± 0.14 a
NPs RCi	6.3 ± 0.6 cd	8.7 ± 1.7 b	1.00 ± 0.1 b	0.07 ± 0.02 a	9.0 ± 0.6 a	7.7 ± 1.2 c	2.53 ± 0.1 a	0.31 ± 0.06 a
NPs HCi	6.0 ± 0.1 d	10.1 ± 2.0 ab	1.26 ± 0.1 a	0.05 ± 0.02 a	8.7 ± 0.1 ab	11.2 ± 2.3 ab	1.93 ± 0.3 b	0.37 ± 0.05 a
NPs Rg	5.0 ± 0.1 e	12.8 ± 0.6 a	1.27 ± 0.2 a	0.07 ± 0.01 a	8.3 ± 0.6 ab	9.6 ± 1.7 bc	2.70 ± 0.3 a	0.31 ± 0.14 a
NPs Sr	7.7 ± 0.1 bc	7.5 ± 1.7 b	1.26 ± 0.1 a	0.14 ± 0.09 a	8.7 ± 0.6 ab	8.7 ± 0.6 bc	2.53 ± 0.3 a	0.33 ± 0.06 a
NPsSe	9.0 ± 0.6 a	12.9 ± 2.7 ab	1.29 ± 0.0 a	0.07 ± 0.01 a	8.7 ± 0.6 ab	13.3 ± 1.5 a	2.90 ± 0.2 a	0.40 ± 0.8 a
TA	9.0 ± 0.6 a	10.3 ± 1.4 ab	1.33 ± 0.1 a	0.27 ± 0.43 a	9.0 ± 0.1 a	12.9 ± 1.6 a	2.83 ± 0.1 a	0.47 ± 0.06 a

RCi: ethanolic extract of husk of C. illinoinensis 100%; HCi: ethanolic extract of leaves of C. illinoinensis 100%; Rg: ethanolic extract of R. graveolens 100%; Sr: ethanolic extract of S. rostratum 100%; NPs RCi: NPs of husk of C. illinoinensis; NPs HCi: NPs of leaves of C. illinoinensis; NPs Rg: NPs of R. graveolens; NPs Sr: NPs of S. rostratum; NpsSe: NPs witouth extract; TA: Absolute control. * Values with the same letter are not significantly different.

show the phytotoxic effect and the height, diameter, and dry weight of indicator plants subjected to different treatments. The phytotoxicity was highest in the S. bicolor species, showing statistical differences among treatments compared to the absolute control. The most affected plants were those treated with NPs of R. graveolens extract and NPs of husk and leaf extracts from C. illinoinensis, with 5.0, 6.0, and 6.3, respectively. However, in the P. vulgaris species, only plants treated with husk and leaf extracts from C. illinoinensis showed a statistical difference compared to the control.

On the other hand, concerning morphometric characteristics in S. bicolor species, a statistical difference in height was observed between NPs loaded with an extract of S. rostratum and the husk of C. illinoinensis compared to the control, with measurements of 7.5 and 8.7 cm, respectively. Similarly, differences in stem diameter were statistically significant between the absolute control and an extract of the husk of C. illinoinensis, as well as between the control and NPs loaded with an extract of the husk of C. illinoinensis, with values of 0.79 and 1.00 mm. Regarding the P. vulgaris species, the plants with reduced height were those sprayed with NPs of an extract of the husk of C. illinoinensis (7.7 mm) and those treated with R. graveolens extract (10.4 mm), showing statistical differences compared to the control. Moreover, the plant diameter only decreased and exhibited statistical differences compared to the control in plants treated with NPs of leaf extract of C. illinoinensis, with a measurement of 1.93 mm (Figure 1).

4. Discussion

In nature, plants are exposed to an extensive array of biotic and abiotic factors that lead to the differential expression of genes and the activation of various metabolic pathways for the production of phytochemical compounds [21]. Diverse researchers have documented a huge amount of phytochemical compounds in the species C. illinoinensis, such as tannins, flavonoids, phenolic compounds, saponins, carotenoids, and quinones [22,23,24]. On the other hand, R. graveolens is a perennial plant that has emerged as a source of extracts in treatments for various conditions because it has bioactive compounds such as alkaloids, flavonoids, saponins, and tannins [25,26,27,28]. Finally, S. rostratum is considered an invasive plant species, hard to eradicate in various regions worldwide. Consequently, it has recently been used for the extraction of plant extracts due to its composition of phytochemical compounds, like alkaloids, flavonoids, and tannins [29,30]. All these compounds act as allelochemicals in the metabolism of the treated plants, affecting physiological functions such as membrane integrity, photosynthesis, respiration, hormonal activity, and ion uptake [3].

Phenolic compounds such as caffeic acid and ferulic acid are considered the group with the highest allelopathic activity, and their presence has been reported in the species C. illinoinensis, R. graveolens, and S. rostratum [26,29,31]. It has been elucidated that phenolic compounds induce the production of reactive oxygen species (ROS) and inhibit the generation of detoxifying enzymes and growth hormones. Also, they affect the photochemistry of photosystem II, thereby disrupting electron transport and the production of ATP and NADPH [32,33].

On the other hand, it has been postulated that flavonoids such as quercetin, myricetin, and isorhamnetin have the capacity to inhibit auxin transport, and under specific conditions, they exhibit prooxidant properties, augmenting the generation of ROS and resulting in the alteration of membrane permeability, stomatal closure, the induction of water stress, the disruption of photosynthesis and protein synthesis, as well as the stimulation of overproduction of phenoxy radicals associated with lipid peroxidation and ROS accumulation within the cell, resulting in damage to biological molecules. [3,34]. The flavonoids mentioned above have been identified in extracts of C. illinoinensis, R. graveolens, and S. rostratum [35,36].

The allelopathic activity of phytochemical compounds influences plants across multiple stages of their developmental continuum. Effects can manifest from germination and the emergence of the radicle and hypocotyl, extending to instances where plants have progressed to true leaf formation and other associated structures. Consequently, an imperative arises to systematically investigate allelopathic activity across diverse phases of plant maturation in pursuit of cultivating sustainable alternatives to chemically synthesized herbicides. The inhibition of seed germination in many plant species during pre-emergence when treated with phytochemical compounds has been extensively documented [3]. Several studies have elucidated that phenolic compounds are one of the main groups with herbicidal activity, inhibiting germination through the disruption of the photosynthetic process and cell division [33]. Consequently, the reduction in germination might be because allelochemical compounds inhibit amylase enzymes and gibberellins, altering the mobilization process of essential reserves for embryo development [2]. Concerning radicle development, the literature mentions that flavonoids can present allelopathic activity regarding root development, affecting seedling growth [37].

Our results revealed the presence of phytochemical compounds such as caffeic acid, ferulic acid, quercetin, myricetin, isorhamnetin, and other phenolic compounds and flavonoids. In this context, Kaab et al. [3] documented inhibitory effects on germination and radicle and hypocotyl development in multiple weed species following exposure to Cynara cardunculus extract. Similarly, Anwar et al. [2] evaluated the effects of extracts from Ricinus communis, Artemisia santolinifolia, and Triticum aestivum on the germination of weeds Sinapis arvensis and Lolium multiflorum, observing inhibition in seed germination. Regarding root development, Fernández-Aparicio et al. [34] reported the inhibitory activity of the flavonoid quercetin present in Fagopyrum esculentum extracts on the root development of Phelipanche ramosa seedlings. Likewise, Javid et al. [38] produced extracts from Mangifera indica leaves and tested them on Parthenium hysterophorus seeds, observing significant inhibition in germination, hypocotyl length, and root length.

The production of biopolymer-based encapsulates is an alternative for enhancing the efficacy of plant extracts, given that the physicochemical properties inherent to bioactive compounds and biopolymers have facilitated the formulation of constructs, which, upon in vivo model testing, have evidenced an augmentation in biological effectiveness. This could be attributed to the interaction between biopolymers and phytochemical compounds in the plant extracts [39]. In this regard, several authors have described different sizes of NPs with plant extracts, attributed to the use of varying concentrations of biopolymers; Mohammadi et al. [40] produced NPs from Zataria multiflora extract with a particle size of 125–175 nm; meanwhile, Santo-Pereira et al. [10] reported particles made from alginate and chitosan with a size of 450 nm.

The zeta potential of NPs loaded with plant extracts is suitable since it aims to ensure the dispersion of the NPs, enhancing their mobility when transported through the plant’s xylem or phloem. The pH is closely related to the encapsulation capacity and the final size of the particles; in this context, Santo-Pereira et al. [10] reported nanoencapsulates based on chitosan and alginate with a pH value of 4.5. The interactions between phytochemical compounds and biopolymers involve various functional groups present in the structure of the bioactives, which participate in hydrogen bonding and electrostatic interactions, respectively, with groups present in the biopolymers [10]. In this context, Taban et al. [19] reported encapsulation efficiency for the essential oil of Dracocephalum kotschyi in the 76% to 91% range. On the other hand, Singh et al. [41] mentioned that the encapsulation of Echinochloa crus-galli oil within an Arabic gum biopolymeric matrix exhibited an encapsulation efficiency of 70%.

Hence, just as it is imperative to delve into the allelopathic impact of phytochemical compounds on germination and seedling development, a parallel need exists to scrutinize their herbicidal activity during post-emergence stages. This is vital to gauge the extent to which a phytochemical product perturbs the vegetal tissues of maturing plants. The phytotoxic activity exhibited by the plant extracts in the present study can be attributed to the presence of phenolic compounds, alkaloids, and flavonoids, which influence plant metabolic processes [3]. In this regard, the extracts in this research contained phytochemicals belonging to hydroxycinnamic acids, methoxycinnamic acids, flavonols, and methoxyflavonols, to which the phytotoxic activity can be attributed. Among the documented phytotoxic phytochemicals belonging to the aforementioned groups, it has been established that caffeic acid increases ROS levels, triggering oxidative damage through suppressing antioxidant enzymes such as peroxidase, superoxide dismutase, and catalase, which structurally damage plant cells [32].

On the other hand, another phenolic compound with phytotoxic properties, ferulic acid, disrupts CO₂ assimilation, resulting in stomatal closure and provoking the production of toxic ROS. Additionally, it induces lipid and protein oxidation, reduces the efficiency of photosystem II, and affects the thylakoid membrane structure, disrupting electron transport. Thus, the reduction in the photochemistry of photosystem II and in photosynthetic electron transport impacts the photosynthetic machinery’s function, causing the absorbed photon energy to be dissipated as heat rather than being utilized, consequently affecting plant tissues [33].

Phenolic compounds such as myricetin and quercetin identified in our plant extracts are involved in several physiological effects accompanying phytotoxicity expression in plants, such as membrane permeability alteration, the induction of water stress, detrimental impacts on photosynthesis, and protein synthesis. Furthermore, they trigger the overproduction of phenoxy radicals, directly linked to lipid peroxidation and intracellular ROS, which can damage DNA and affect cell division [3]. Nevertheless, despite the herbicidal capacity of phytochemical compounds in the extracts, their volatility and rapid degradation may limit their biological activity and applicability. Thus, the formulation of plant extracts into NPs emerges as an alternative to enhance their efficacy; in this regard, biopolymers like alginate and chitosan provide a viable option for NPs preparation, as they enhance the solubility, stability, and cellular uptake of compounds and are non-toxic and biodegradable [10].

The NPs with plant extracts produced and applied in this study exhibited characteristics that indicate they are stable for agricultural applications, potentially leading to an enhancement in efficacy compared to non-encapsulated plant extracts. This enhancement could be attributed to increased reactivity, improved stability, and mobility. Moreover, encapsulation efficiencies higher than 90% suggest that the NPs with extracts increased their efficacy through encapsulating a high quantity of the plant extracts. Several studies have been conducted on encapsulating phytochemical compounds using biopolymers to increase their phytotoxic effects on diverse plant species. In this context, Taban et al. [42] developed encapsulated essential oil from Satureja hortensis with biopolymers to assess its herbicidal activity, achieving up to 100% control over Amaranthus retroflexus. In another investigation, Alipour et al. [43] encapsulated essential oil from Rosmarinus officinalis, using starch as a biopolymeric matrix to test its phytotoxic capability against A. retroflexus and Rhaphanus sativus, and they reported an inhibitory effect on germination, along with reductions in morphometric characteristics such as leaf area, dry and fresh weight, and stem and root length.

5. Conclusions

Weed control worldwide will persist as a challenge owing to climate shifts and the emergence of weed biotypes resistant to synthetic herbicides. Consequently, the imperative for sustainable alternatives arises, ensuring food production while safeguarding the prospects and well-being of forthcoming generations. The production and utilization of plant extracts for weed management have gained prominence in recent years. Nonetheless, their efficacy can be augmented through formulating them within biopolymeric matrices, such as alginate and chitosan.

The ethanolic extracts of C. illinoinensis, R. graveolens, and S. rostratum encompass a substantial array of phytochemical compounds that confer the capacity to impede plant growth through allelopathic activity. They exhibit pre-emergence herbicidal activity by means of thwarting germination and the development of primary plant structures through diverse mechanisms of action. Moreover, they manifest post-emergence herbicidal activity, as evidenced by their phytotoxic impact on the indicator plants S. bicolor and P. vulgaris. Nonetheless, their efficacy can be enhanced and elevated through formulation within alginate and chitosan biopolymeric matrices, amplifying phytotoxicity and detrimental effects on test plants. Among these, the narrow-leaved weed species S. bicolor displays the most pronounced susceptibility to nanoparticles (NPs) and extracts.

While various studies have presently explored plant extracts as a bioherbicide development alternative, alongside the advancement of formulations grounded in biopolymers to heighten the biological efficacy of diverse phytochemical bioactives, with documented satisfactory outcomes, the pursuit of research into plant extracts and biopolymers remains essential. Such endeavors aim to refine efficacy, ultimately leading to the eventual development of a bioherbicide applicable under field conditions within the purview of sustainable agriculture.