agriculture

Article
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
Marco Antonio Tucuch-Pérez 1 , Evelyn Isabel Mendo-González 1 , Antonio Ledezma-Pérez 2 , Anna Iliná 1 ,
Francisco Daniel Hernández-Castillo 3 , Cynthia Lizeth Barrera-Martinez 4 , Julia Cecilia Anguiano-Cabello 1 ,
Elan Iñaky Laredo-Alcalá 4, * and Roberto Arredondo-Valdés 1, *

Citation: Tucuch-Pérez, M.A.;
Mendo-González, E.I.;
Ledezma-Pérez, A.; Iliná, A.;
Hernández-Castillo, F.D.;
Barrera-Martinez, C.L.;
Anguiano-Cabello, J.C.;
Laredo-Alcalá, E.I.;
Arredondo-Valdés, R. 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. Agriculture 2023, 13, 2041.
https://doi.org/10.3390/
agriculture13112041
Academic Editor: Luis Antonio de
Avila
Received: 29 August 2023
Revised: 20 September 2023
Accepted: 29 September 2023
Published: 24 October 2023
1 Facultad de Ciencias Químicas, Universidad Autónoma de Coahuila, Unidad Saltillo, Saltillo 25298, Mexico;
martp1216@gmail.com (M.A.T.-P.); mendo.e@uadec.edu.mx (E.I.M.-G.); annailina@uadec.edu.mx (A.I.);
julia.anguiano@ulsasaltillo.edu.mx (J.C.A.-C.)
2 Laboratorio de microbiología, Centro de Investigación en Química Aplicada, Saltillo 25294, Mexico;
antonio.ledezma@ciqa.edu.mx
3 Departamento de Parasitología Agrícola, Universidad Autónoma Agraria Antonio Narro, Buenavista,
Saltillo 25315, Mexico; danielhdzc@gmail.com
4 Centro de Investigación para la Conservación de la Biodiversidad y Ecología de Coahuila,
Universidad Autónoma de Coahuila, Unidad Norte, Cuatro Ciénegas de Carranza 27690, Mexico;
cynthia_barrera@uadec.edu.mx
* Correspondence: elan_laredo@uadec.edu.mx (E.I.L.-A.); r-arredondo@uadec.edu.mx (R.A.-V.)
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.

Keywords: bioherbicides; plant extracts; nanotechnology; nanotechnology; allelopathy

Copyright: © 2023 by the authors.

Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1. Introduction
Weeds are one of the main problems in agriculture, because they induce yield reduc-
tion in all agricultural production systems. Historically, their management has been carried
out through chemical synthesis products; nevertheless, these agents stimulate environ-
mental problems and adverse effects on human health. Furthermore, their indiscriminate

Agriculture 2023, 13, 2041. https://doi.org/10.3390/agriculture13112041 https://www.mdpi.com/journal/agriculture

Agriculture 2023, 13, 2041 2 of 15

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 sustain-
able agricultural production. This option offers advantages such as reduced environmental
impact and a diminished likelihood of generating herbicide-resistant weeds. Thus, phy-
tochemical 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 effi-
cacy, this effectiveness may be reduced by environmental factors such as light exposure,
temperature fluctuations, humidity, ultraviolet radiation, and compound leaching [6]. Con-
sequently, these factors can reduce their phytotoxic activity, such that only 0.1% of the
applied product reaches its target [6–10]. Therefore, it becomes necessary to develop for-
mulations 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 formula-
tions (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 her-
bicides, 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
Agriculture 2023, 13, 2041 3 of 15

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 (H2 SO4 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
(NH4 OH and H2 SO4 reagents for anthraquinones, and Börntraguer test for benzoquinones),
saponins (foam test, Bouchard reagent for steroidal saponins, and Rosenthaler reagent),
terpenoids (Ac2 O 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
Agriculture 2023, 13, 2041 4 of 15

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:
 
A
Germination percentage = × 100 (1)
B

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

seeds in the control.  
T
Inhibition rate = × 100 (2)
C
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 pro-
posed 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:
T0 − S0
 
% EE = × 100 (3)
T0
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.
Agriculture 2023, 13, 2041 5 of 15

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. 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.

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.
Agriculture 2023, 13, 2041 6 of 15

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 di-
verse compounds in all plant extracts (Table 3). 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. grave-
olens 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 hydroxycin-
namic acids, hydroxybenzoic acids, stilbenes, lignans, methoxyflavones, methoxycinnamic
acids, and methoxyflavonols.

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
Caffeic acid 4-O-glucoside 5.863 341 Hydroxycinnamic acids
illinoinensis
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
47.812 623.6 Methoxyflavonols
7-O-rhamnoside
Quercetin 3-O-rhamnoside 49.502 477 Flavonols
Husk of Carya
Cinnamoyl glucose 5.243 308.6 Hydroxycinnamic acids
illinoinensis
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
34.22 622.8 Methoxyflavonols
7-O-rhamnosideo
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
33.77 622.8 Methoxyflavones
7-O-rhamnoside
Agriculture 2023, 13, 2041 7 of 15

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 Tables 4 and 5. 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%.

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% 0c 0c
Carya illinoinensis leaf extract at 25% 0c 0c
Carya illinoinensis leaf extract at 50% 0c 0c
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% 0c 0c
Ruta graveolens extract at 12.5% 0c 8.3 b
Ruta graveolens extract at 25% 0c 8.3 b
Ruta graveolens extract at 50% 0c 0c
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% 0c 0c
* Values with the same letter are not significantly different.

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.

Regarding the hypocotyl and radicle inhibition rate, statistical differences were ob-
served 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,
Agriculture 2023, 13, 2041 8 of 15

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. 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.

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

Size Zeta Potential Encapsulation

pH
(nm) (mV) Efficiency (%)
NPs of leaves of C.
313 ± 24 −35.4 ± 1.3 4.62 92
illinoinensis
NPs of husk of C.
343 ± 25 −35.25 ± 1.0 4.62 94
illinoinensis
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 -----

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 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.
 Agriculture 2023, 13, 2041 9 of 15

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

Diameter Dry Weight Height Diameter Dry Weight
Phytotoxicity Height (cm) Phytotoxicity
(mm) (g) (cm) (mm) (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.

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
Agriculture 2023, 13, x FOR PEER REVIEW
compared to the control in plants treated with NPs of leaf extract of C. illinoinensis, with10a of 16
measurement of 1.93 mm (Figure 1).

Figure 1. Cont.
Agriculture 2023, 13, 2041 10 of 15

Figure 1. Phytotoxicity
Figure 1. Phytotoxicityandandeffect
effect on thedevelopment
on the development ofof
thethe indicator
indicator plants
plants Sorghum
Sorghum bicolorbicolor
(a) and(a) and
Phaseolus vulgaris (b) treated with plant extracts and NPs loaded with extracts from Carya
Phaseolus vulgaris (b) treated with plant extracts and NPs loaded with extracts from Carya illinoinensis, illinoinen-
sis,Ruta
Rutagraveolens,
graveolens,and and Solanum
Solanum rostratum.
rostratum. RCi: ethanolic
RCi: ethanolic extractextract
of husk of husk
of C. of C. illinoinensis
illinoinensis 100%; HCi:100%;
HCi:ethanolic extract of leaves of C. illinoinensis 100%; Rg: ethanolic extract of R. graveolens 100%; Sr:100%;
ethanolic extract of leaves of C. illinoinensis 100%; Rg: ethanolic extract of R. graveolens
Sr: ethanolic
ethanolicextract
extract ofrostratum
of S. S. rostratum
100%;100%; NPsNPs
NPs RCi: RCi:
of NPs
husk of husk
of C. of C. illinoinensis;
illinoinensis; NPs HCi: NPs NPsofHCi:
leavesNPs of
leaves
of C. illinoinensis; NPs Rg: NPs of R. graveolens; NPs Sr: NPs of S. rostratum; NpsSe: NPs witouth NPs
of C. illinoinensis; NPs Rg: NPs of R. graveolens; NPs Sr: NPs of S. rostratum; NpsSe:
witouth extract;
extract; TA: Absolute
TA: Absolute control.
control. Values Values
with withletter
the same the same
are notletter are not different.
significantly significantly different.

4. Discussion
4. Discussion
In In nature,plants
nature, plants are
areexposed
exposed to to
an an
extensive arrayarray
extensive of biotic
of and abiotic
biotic andfactors
abioticthat lead that
factors
to the differential expression of genes and the activation of various metabolic pathways for
lead to the differential expression of genes and the activation of various metabolic path-
the production of phytochemical compounds [21]. Diverse researchers have documented a
ways for the production of phytochemical compounds [21]. Diverse researchers have doc-
huge amount of phytochemical compounds in the species C. illinoinensis, such as tannins,
umented
flavonoids, a huge amount
phenolic of phytochemical
compounds, compounds
saponins, carotenoids, andinquinones
the species C. illinoinensis,
[22–24]. On the other such
as tannins, flavonoids, phenolic compounds, saponins, carotenoids, and
hand, R. graveolens is a perennial plant that has emerged as a source of extracts in treatments quinones [22–24].
Onfor thevarious
other conditions
hand, R. graveolens is a bioactive
because it has perennial plant thatsuch
compounds has as emerged asflavonoids,
alkaloids, a source of ex-
saponins,
tracts and tannins
in treatments for[25–28].
variousFinally, S. rostratum
conditions is considered
because an invasive
it has bioactive plant species,
compounds such as
hard to eradicate in various regions worldwide. Consequently, it has recently
alkaloids, flavonoids, saponins, and tannins [25–28]. Finally, S. rostratum is considered an been used for
the extraction
invasive of plant hard
plant species, extracts
to due to its composition
eradicate in various of phytochemical
regions worldwide.compounds, like
Consequently, it
alkaloids, flavonoids, and tannins [29,30]. All these compounds act as allelochemicals in
has recently been used for the extraction of plant extracts due to its composition of phy-
the metabolism of the treated plants, affecting physiological functions such as membrane
tochemical compounds, like alkaloids, flavonoids, and tannins [29,30]. All these com-
integrity, photosynthesis, respiration, hormonal activity, and ion uptake [3].
poundsPhenolic
act as allelochemicals
compounds such in as
thecaffeic
metabolism
acid andofferulic
the treated
acid are plants, affecting
considered physiolog-
the group
icalwith
functions suchallelopathic
the highest as membrane integrity,
activity, photosynthesis,
and their presence has been respiration,
reported inhormonal
the speciesactivity,
C.
andillinoinensis,
ion uptakeR.[3]. graveolens, and S. rostratum [26,29,31]. It has been elucidated that phenolic
compounds
Phenolic induce
compoundsthe production
such asofcaffeic
reactive oxygen
acid and species
ferulic(ROS) and considered
acid are inhibit the genera-
the group
tion
with theof highest
detoxifying enzymes and
allelopathic growth
activity, andhormones. Also, they
their presence hasaffect
been the photochemistry
reported in the species
C. of photosystem
illinoinensis, R. II, thereby disrupting
graveolens, electron transport
and S. rostratum [26,29,31]. and
It the
hasproduction of ATP that
been elucidated and phe-
NADPH [32,33].
nolic compounds induce the production of reactive oxygen species (ROS) and inhibit the
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 condi-
tions, 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 accumu-
lation 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 mul-
tiple stages of their developmental continuum. Effects can manifest from germination
Agriculture 2023, 13, 2041 11 of 15

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 im-
perative 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]. Sev-
eral 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 be-
cause allelochemical compounds inhibit amylase enzymes and gibberellins, altering the
mobilization process of essential reserves for embryo development [2]. Concerning radi-
cle 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, fer-
ulic 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 devel-
opment, 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 phy-
tochemical 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 phyto-
Agriculture 2023, 13, 2041 12 of 15

chemicals belonging to hydroxycinnamic acids, methoxycinnamic acids, flavonols, and

methoxyflavonols, to which the phytotoxic activity can be attributed. Among the docu-
mented 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, detri-
mental impacts on photosynthesis, and protein synthesis. Furthermore, they trigger the
overproduction of phenoxy radicals, directly linked to lipid peroxidation and intracellu-
lar 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 character-
istics 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 reduc-
tions 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 impera-
tive 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
Agriculture 2023, 13, 2041 13 of 15

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 devel-
opment 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 re-
mains 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.

Author Contributions: M.A.T.-P.: conceptualization, methodology, validation, investigation, data

curation, writing—original draft preparation, and writing—review and editing. E.I.M.-G.: method-
ology, validation, and investigation. A.L.-P.: methodology, software, resources, and supervision.
A.I.: methodology and resources. F.D.H.-C.: methodology, resources, and validation. C.L.B.-M.:
methodology and validation. J.C.A.-C.: validation, formal analysis, and methodology. E.I.L.-A.:
conceptualization, methodology, supervision, and funding acquisition. R.A.-V.: conceptualization,
methodology, data curation, supervision, writing—review and editing, project administration, and
funding acquisition. All authors have read and agreed to the published version of the manuscript.
Funding: This study was supported by the National Council for Science and Technology of Mexico
through the project of frontier science “Nano and microencapsulated bioherbicides loaded with
plant extracts from the Chihuahuan semi-desert for the control of plant development” with reference
number 320692.
Institutional Review Board Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: The authors acknowledge the support of the National Council for Science
and Technology of Mexico, for the assistance provided through the scholarship number 708037,
corresponding to the “Postdoctoral Stays in Mexico 2022” program.
Conflicts of Interest: The authors declare no conflict of interest.

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