Nematicidal Action of Microencapsulated Essential Oil of Flesh Fingered Citron

Flesh ﬁngered citron (FFC) essential oil (EO) is susceptible to volatilisation at room temperature. Therefore, its use as a nematicide requires a controlled release. In the present study, we encapsulated FFC EO in β -cyclodextrin by embedding and investigated release from the capsules compared to unembedded EO. We evaluated the structural and thermal properties of the capsules by SEM and TGA. The loading capacity was 32.67%, and the embedding yield was 96.24%, assuming that a core-to-wall quality ratio of 1 :6 is optimal for the carrier. Using Caenorhabditis elegans as a model organism, we explored the toxicity of (1) FFC EO microcapsules (MCs) and (2) four key compounds of the EOs. The MCs enabled sustained release, e.g., 77% mortality after 4 h and 100% within an additional half-hour. The four main compounds in EO can each kill nematodes by reducing antioxidant activity. Since microencapsulation can improve FFC EO stability and prevent product loss due to adverse environments exposed to the air, encapsulating FFC EO in MCs has great potential as a new nematicide.


Introduction
Worldwide, nematodes cost as much as $157 billion (USD) per year in economic damage. Severe nematode diseases are frequent and continue to spread, resulting in a continually decreasing crop yield. Due to nematodes' high species diversity and extremely rapid reproduction, farmers have difficulty selecting an appropriate nematode control method. Synthetic pesticides are expensive, and environmental hazards include contaminated soil and water, high persistence, toxicity to humans and other animals, and residues in food. Farmers need new nematode biocontrol measures that are nonsynthetic, cheaper, and more environmentally friendly [1,2].
In this context, natural products and their derivatives-important sources of novel therapeutics [3]-may be useful. For example, industries including cosmetics, food packaging and processing, and agriculture use plant essential oils (EOs) [4,5]. EOs can exhibit toxic, repellent, and antifeedant effects on insects and nematodes [6][7][8]. EO from flesh fingered citron (FFC; from citrus) are generally extracted mechanically [9]. FFC EO has antimicrobial, antifungal, and anthelmintic properties, and functions through various modes of action: (a) antirespiratory (analogously to fumigants), (b) prevent through exposure or ingestion experiments, (c) antireproductive (including reduced fertility and sterilization), (d) antifeedant, or (e) a combination thereof. However, the biological activity of EOs may be lost by volatilisation or degradation, and repeated applications are necessary to achieve the effective nematicidal activity [10,11].
us, commercial applications of EOs are limited [12]. Microcapsules (MCs) can address these limitations. By encapsulating an active compound, MCs are a potential platform to accomplish nematicide. Microencapsulation to entrap EOs is one of the most effective methods of applying nematicides, due to "packaging" the active ingredients in the wall material, thereby converting the liquid into a more stable powder [13]. e release of the functional agent occurs by diffusion of the capsule wall and/or MC rupture. erefore, MCs provide a durable nematicide repellent and lethal toxin [14].
To test the utility of EOs for biocontrol, Caenorhabditis elegans nematodes are good model organisms in many fields, such as biomedicine, environmental toxicity, aging, metabolism, obesity, and drug discovery. C. elegans are good model organisms because they are easy to grow and inexpensive to maintain in the laboratory, have a relatively short lifecycle, and are small [15].
In this paper, we characterized MCs that consist of β-cyclodextrin (β-CD)-packaged EO of FFC. We evaluated MC properties by measuring the embedding and release rates. We analyzed MC molecular structure and thermal stability. We quantitated MC nematicidal activity. We also verified that each of the four main compounds in the EO acts as a nematicide. Our results will be useful for minimising how frequently EOs must be applied to retain nematicidal activity.

EO Preparation and GC-MS Analysis.
Mechanical pressing is suitable for extracting easily burnt raw materials such as citrus and lemon because it is easy to maintain the structure and function of EOs. e FFC peels were saturated in 2% (w/v) calcium chloride or calcium hydroxide for 5 h, and then washed with distilled water. e soaked flavedo (50 g) was weighed and turned into fruit juice with 0.25% sodium chloride added to dissolve it. After stratification, the upper oil layer of the mixture was separated and dried over anhydrous sodium sulphate. e resulting EO was kept at 4°C until further analysis. e purification was conducted using molecular distillation (Pope, USA). e extraction rate of the EOs was approximately 0.27%.
A fused silica capillary column (HP-5MS) was used for GC-MS analysis with a Hewlett-Packard 6890 Gas Chromatograph (5% phenyl-methyl siloxane, 60 m × 0.25 mm, film thickness 0.25 μm, Agilent Technologies, USA). We determined the percentage of the oil components in the EO by quantitating the peak area. e relative content of each component was calculated by area normalization [16].

Preparation of Microcapsules.
Microencapsulation was using the embedding method. A certain amount of β-CD was added to water and dissolved at 80°C to obtain a saturated solution. Adding 10% essential oil of ethanol solution under stirring, preparing a solution of EO: β-CD in different proportions 1 : 5, 1 : 6, and 1 : 7, maintaining 50°C and stirring for 2 h, and then naturally cooling to room temperature, at 4°C for 24 hours. en suction was filtered with a Buchner funnel and washed.

Percent Yield and LC Value of Microcapsules.
e embedding yield of the microcapsules is calculated as a percentage of the total amounts of β-CD and essential oils employed during preparation. e percentage yield of the microcapsules was calculated by using the following formula [17]: %yield � actual amount of microcapsules obtained total(amount of oil + amount of β − CD) × 100.
(1) e loading capacity (LC) is defined as the mass percentage of the encapsulated essential oils, which reflects the microcapsule loading ability. Using the thermogravimetric analyzer (TGA) to perform thermal weight test on the blank microcapsules and the essential oil of flesh fingered citrons microcapsules powder respectively, calculates the loading amount of the essential oil in the microcapsules. TGA test conditions are as follows: room temperature ∼600°C; heating rate: 10°C/min; carrier gas: N2; flow 20 ml/min. e load (LC) is calculated according to the following formula: Here, η 1 is weight loss score of essential oil microcapsules in the range of 25∼100°C; η 2 is weight loss score of essential oil microcapsules in the range of 100∼200°C (surface residual and absorbed essential oil); η 3 is fine oil microcapsules in 200∼600°C; η 1' is weight loss score of the blank microcapsules in the 25∼100°C segment; η 2' is the weight loss score of the blank microcapsules in the 100∼200°C segment; η 3' is the weight loss score of the blank microcapsules in the 200∼600°C segment.

Scanning Electron Microscopy
Analysis. SEM analyzed the morphology of microcapsules on a s-3400N scanning electron microscope (Hitachi, Japan), coating microcapsule samples with a thin sputtered gold layer prior to inspection [18].

Comparative Studies of Stability of Essential Oil Free (EO) and Microencapsulated (MCs).
Comparative studies of stability for free and microencapsulated essential oil (EO and MCs, respectively) were conducted during 24 h of monitoring time at 45°C (two replicates), taking the same quality of essential oils and microcapsules, judged by the volatile oil and residual quality of essential oils. For the preliminary studies of stability and biological activity, the microcapsule preparation selected EO: β-CD � 1 : 6.

C. elegans Strains and Handling.
e nematodes used in this study were Bristol wild-type N2. We maintained C. elegans and assayed them on nematode growth medium agar plates carrying a lawn of E. coli OP50 at 20°C. e phytochemicals were dissolved in 0.1% (v/v) DMSO. All chemicals in the nematode growth medium plate and liquid are expressed as final concentrations.

Comparative Nematicide Studies.
Mortality studies were conducted of the activity of (1) free FFC EO compared to (2) encapsulated EO (i.e., in MCs). e EO and MC solutions were prepared using 0.1% DMSO as a solvent. e EO-to-β-CD ratio was 1 : 6, and the loading capacity (LC) was 32.67%. en 3.3 mg of EO and 10 mg of MCs were dissolved in 1 mL of 0.1% DMSO; in the control group, no DMSO was added. One hundred L4 C. elegans were placed in three separate groups, and the mortality was determined for 4 h at 30 min intervals. ree independent biological replicates were performed.

Oxidative Stress Resistance Assays for C. elegans.
Oxidative stress assays were performed essentially as previously described. Wild-type N2 C. elegans was synchronized to the L4 period and pretreated with 10 mg microcapsule and 3 mg EO (LC is approximately 30%), and 0.1% DMSO as the solvent control for 48 h before exposure to K 2 Cr 2 O 7 (8 μL, 10 mM). Culturing was done for 48 h. Worms were observed every 4 h, and survival was determined by touch-provoked movement [19]. At least three independent biological replicates were performed.

Survival Rate.
e steps were similar to those described in Section 2.4.2. Four major compounds (D-limonene, c-terpinene, a-pinene, and β-pinene; 5 mM each) were dissolved in 0.1% DMSO. At least three independent biological replicates were performed using at least 100 C. elegans.

Effect on Oxidative Stress Resistance.
e steps were similar to those described in Section 2.4.3. Four major compounds (D-limonene, c-terpinene, a-pinene, and β-pinene; 5 mM each) were used. At least three independent biological replicates were performed.

Statistical Data Analysis.
For the oxidative stress, we used GraphPad Prism Software and the statistical significance of the difference between the curves was demonstrated by a log-rank test using Kaplan−Meier survival analysis. ANOVA test and subsequent Dunnett test were used to analyze the data (P < 0.05). Statistical analysis was performed using SPSS Statistics software (SPSS). Results are expressed as mean ± standard error of the mean (SEM). Differences were considered significant at * P < 0.05, * * P < 0.01, and * * * P < 0.001 levels [20]. MCs prevent degradation of internal active substances and loss of activity due to external environmental factors [21]. e encapsulated EO as the ratio of β-CD increased from 1 : 5 to 1 : 7, indicating that the embedding yield (EY) at 96.24% (1 : 6) was the maximum percentage yield of the MCs (Table 1). e LC obtained by TGA also changes in accordance with the proportion and is the largest, 32.67%, at 1 : 6 ( Table 1). is result may be due to the higher ratio of leaching from the internal aqueous phase volume of the primary water/ oil emulsion to the external aqueous phase volume of the secondary water/oil/water emulsion [22].

Capsule Morphology.
To characterize the morphology and size distribution of the capsules, a scanning electron microscope (SEM) of the samples was performed [23]. e SEM micrographs of β-CD and microcapsules with EO were depicted in Figure 1. β-CD was irregular in shape, dispersed after dissolution, and was a crystal structure. ere were observed differences in the shape of β-CD and microcapsules and the embedded microcapsules were granular. e diameter of the particles in all the prepared microcapsules ranged from 10 um to 50 um.

Microcapsule Stability.
We conducted comparative studies of the stability for free and microencapsulated EO over the course of 24 h at 45°C [2]. We calculated the percentage retention of EO in microcapsules as follows: EO at elapsed time × 100/EO at zero time . Under these experimental conditions, we detected a very rapid liberation of free EO: less than 15% retention over 24 h (Figure 2). In contrast, MCs exhibited very slow liberation. us, in these experimental conditions, MCs exhibited sustained release of EO and were stable. [24]. e free-EO-induced (3.3 mg/mL) mortality rate was as follows: 2 h, 89%; 4 h, 99%. We also conducted two independent experiments using free EO (3.3 mg/mL), on different days, to study the bioassay's reproducibility and select the appropriate bioassay duration. We observed a very rapid response, a plateau (maximum response) before 2 h of incubation, in both independent experiments for free EO. In contrast, we observed a slower response for MCs (10 mg/mL). We observed considerable mortality differences for free EO and MC treatments at 2 h (97% and 37%, respectively) and 4 h (100%  (Figure 3).

Oxidative Stress Resistance.
We used K 2 Cr 2 O 7 as an oxidative stress inducer because it is a heavy metal oxidizer. It can undergo intracellular redox cycling with superoxide radical (O2•) production in C. elegans and is commonly used to generate intracellular oxidative stress in C. elegans [25]. Pretreatment with free EO and pretreatment with MCs both induced considerable oxidative stress (Figure 4). EO contains a variety of compounds that might exert various biological activities that reduce C. elegans' antioxidant activity. e MCs behaved in a similar manner as free EO.  [26]. e nematode 24 h survival rate was as follows: D-limonene, 30.33%; c-terpinene, 34%; a-pinene, 12%; β-pinene, 10% ( Figure 5). erefore, the compounds that comprise FFC EO had obvious nematicidal effects.

Effects of Four
e MCs loaded with individual EOs killed nematodes somewhat slower than the corresponding free EO compounds, possibly due to slow sustained release.

Oxidative Stress
Resistance. Different components like monoterpenoids are responsible for insecticidal properties and physiological discrepancies of insects [27,28]. As shown in Figure 6, D-limonene, c-terpinene, a-pinene, and β-pinene all affect the oxidative stress response of C. elegans, which shortens lifespan.

Conclusions
In recent years, researchers have used plant EOs and extracts to hinder nematode survival [29]. Given that EOs are potential pest control agents, environmental impact assessments of EOs have received much attention [30]. Most plant extracts used in pest control inhibit-rather than kill-insects and nematodes, e.g., inhibit feeding. We used MCs to encapsulate EO extracted from FFC as a plant-derived nematicide to inhibit C. elegans. We found that FFC EO is a complex mixture of terpenes (monoterpenes, sesquiterpene, and derivatives) that degrade rapidly, are selective, exhibit low mammalian toxicity, and have a minimal environmental impact [31]. We used β-CD to embed the EO of FFC and found that an LC of 1 : 6 is optimal. MCs exhibit good sustained release.
After 4 h, the nematode mortality rate induced by EO and MCs was 100% and 77%, respectively. Due to the strong volatility of EOs, the residual activity is short, and repeated application is required. However, MCs exhibit controlled release and stabilise EOs prior to release. Furthermore, oxidative stress experiments showed that the EO and MCs shorten nematode survival times by reducing antioxidant activity. Four isolated individual terpene compounds considerably increased mortality, indicating that they are the source of nematicidal activity. e MCs also play a nematicidal role by slowly releasing monoterpenes. In conclusion, our microencapsulation procedure will be useful for preparing plant-derived pesticides and controlled release of active ingredients. In this manner, EO is protected from the external environment during product application and is slowly released, providing a more persistent nematicidal effect compared to no encapsulation [2].

Data Availability
e numerical data used to support the findings of this study are included within the article. Results include at least three independent biological replicates. Statistical significance of the difference between the curves was demonstrated by a log-rank test using Kaplan−Meier survival analysis. Error bars represent the standard error, and differences compared to the solvent (0 mg/ml, 0.1% DMSO) were considered significant at * P < 0.05, * * P < 0.01, and * * * P < 0.001 by one-way ANOVA and the LSD post hoc test. Results include at least three independent biological replicates. Error bars represent the standard error, and differences compared to the control (for each column of 0 mg/ml, 0.1% DMSO) were considered significant at * P < 0.05, * * P < 0.01, and * * * P < 0.001 by one-way ANOVA and the LSD post hoc test. Solvent D-Limonene * * * γ-Terpinene * * * α-Pinene * * * β-Pinene * Figure 6: Four components in the flesh fingered citrons essential oil affect the oxidative stress resistance of wild-type C. elegans. At least three independent biological replications were performed, and the statistical significance of the differences between curves was demonstrated by a log-rank test using Kaplan−Meier survival analysis. Error bars represent the standard error, and differences compared to the solvent (0 mg/ml, 0.1% DMSO) were considered significant at * P < 0.05, * * P < 0.01, and * * * P < 0.001 by one-way ANOVA and the LSD post hoc test.

Additional Points
Highlights. (1) A plant-derived nematicide is proposed. (2) e embedding conditions of flesh fingered citron (FFC) essential oil (EO) microcapsules (MCs) and the MC's slowrelease effect were explored. (3) e authors investigated the nematicidal mechanism of the four main components of FFC EO. (4) Our microencapsulated EO can replace synthetic nematicides, in that MCs can store EO for long periods of time and are environmentally friendly.

Conflicts of Interest
e authors declare that they have no conflicts of interest.