A beta-cyclodextrin-ocimene nanoparticle, a preparation method and application thereof

Abstract

The present application relates to a kind of β-cyclodextrin-linalool nanoparticles, preparation method and its application, the specific preparation method includes mixing reaction, ultrasonic, centrifugal, drying and the like steps, aims at obtaining the particle size suitable, embedding rate high, the β-cyclodextrin-linalool nanoparticles of good stability, and the application of the nanoparticle in the research of plant physiological and biochemical indexes etc., provide new way and method for the application of linalool.

Description

Technical Field

[0001] This invention relates to the field of preparing biological herbicides from nanoparticles, specifically to β-cyclodextrin-ocimene nanoparticles, their preparation methods, and their applications. Background Technology

[0002] Herbicides, as crucial chemical inputs for weed control in modern agriculture, play a vital role in ensuring food security, increasing crop yields, and reducing the cost of manual weeding. They primarily work by inhibiting physiological processes in weeds, such as photosynthesis, amino acid synthesis, and cell division, rapidly reducing weed competition for water, nutrients, and light, thereby enhancing crop growth. As a key pesticide type, herbicides promote large-scale agricultural development by reducing labor costs and precisely controlling weeds. However, the long-term, single-use application of traditional chemical herbicides such as glyphosate and atrazine has led to a series of prominent problems: Firstly, the persistent residues of some long-residual herbicides in the soil can cause "hidden phytotoxicity," affecting not only the normal growth of subsequent sensitive crops but also potentially causing leaf yellowing, hindered root development, and yield reduction. Secondly, long-term, single-use of herbicides accelerates the development of herbicide-resistant weeds, leading to increased weed community succession and control difficulties. Furthermore, herbicide residues can affect the structure and function of soil microbial communities, disrupt the ecological balance of farmland, and enter water bodies through runoff and infiltration, posing a potential threat to ecological and environmental safety. Therefore, there is an urgent need to develop efficient, low-residue, and environmentally friendly green weeding technologies in modern agriculture to reduce the weed damage and ecological risks caused by traditional chemical herbicides.

[0003] Ocimenes are a class of natural monoterpenoid compounds that are widely found in the essential oils of various plants, including those in the genus Ocimum basilicum, the genus Hemerocallis, and citrus. They can be extracted from plant branches, leaves, petals, and other parts by steam distillation and are typical representatives of volatile secondary metabolites of plants.

[0004] Plant-derived herbicidal active substances mainly include terpenes, phenolic acids, alkaloids, and plant essential oils. Their mechanism of action primarily involves disrupting cell membrane structure, inhibiting photosynthesis and respiratory metabolism, interfering with cell division, and inducing the accumulation of reactive oxygen species (ROS), thereby inhibiting weed germination and growth. Compared to traditional herbicides, they have advantages such as natural origin, easy degradation, environmental friendliness, and low likelihood of inducing resistance. However, they also have disadvantages such as high volatility, poor stability, short residual effect, and susceptibility to environmental influences in field application, thus limiting their large-scale application. Traditional formulation technologies cannot solve the problem of volatilization loss; encapsulation technology is currently the main means to overcome this challenge.

[0005] β-Cyclodextrin is a cyclic oligosaccharide composed of seven glucose molecules linked by α-1,4-glycosidic bonds. The molecule has a truncated conical shape, possessing both a hydrophobic interior and a hydrophilic exterior. It can encapsulate hydrophobic guest molecules through van der Waals forces, hydrophobic interactions, and hydrogen bonds to form stable complexes. Although its own water solubility is relatively low (18.5 g / L at 25℃), encapsulation significantly enhances the water solubility and stability of guest molecules. It is currently widely used in drug delivery, the food industry, and catalysis. As a biodegradable carrier, β-cyclodextrin aligns with the trend of green pesticide development. The sustained-release properties of its encapsulated compounds can extend the herbicidal effect to over 14 days, reducing the number of applications and the risk of off-target exposure.

[0006] This study successfully constructed a bio-nano herbicide by encapsulating ocimene in β-cyclodextrin using physical encapsulation technology. This β-cyclodextrin-ocimene system not only effectively improves the shortcomings of ocimene, such as poor water solubility and easy volatility and oxidation, but also reduces the potential risk of soil pollution by relying on a biodegradable carrier. After systematic characterization and process optimization, the obtained nano-formulation exhibited significant herbicidal activity and a sustained-release characteristic lasting more than 96 hours, thereby reducing the frequency of application and the risk of exposure to non-target organisms. This study is the first to achieve the construction of a nano-herbicide by encapsulating ocimene in β-cyclodextrin, which has important theoretical and applied value: theoretically, it can reveal the host-guest encapsulation mechanism, elucidate the regulatory relationship between nanoparticle structure and stability, and enrich the research system of nano-formulations of natural essential oils; practically, it provides core technical support for the industrialization of natural herbicides and the development of "highly efficient, low-toxicity, and environmentally friendly" pesticides. Summary of the Invention

[0007] In view of the above background, the present invention aims to provide β-cyclodextrin-ocimene nanoparticles and their preparation method. By optimizing the preparation process parameters, β-cyclodextrin-ocimene nanoparticles with suitable particle size, high encapsulation rate and good stability are obtained. The application of these nanoparticles in the study of plant physiological and biochemical indicators is also investigated, providing new approaches and methods for the application of ocimene.

[0008] To achieve the above objectives, the following technical solution is adopted: This invention provides β-cyclodextrin-ocimene nanoparticles, which are mainly prepared by mixing β-cyclodextrin and ocimene.

[0009] This invention also provides a method for preparing β-cyclodextrin-ocimene nanoparticles, which includes the following steps: (1) Weigh a certain amount of β-cyclodextrin, add it to distilled water, and treat it in a water bath at 30°C to completely dissolve it, so as to obtain a β-cyclodextrin solution; Weigh a certain amount of ocimene and dissolve it in anhydrous ethanol to obtain an ocimene solution; (2) While stirring, slowly add the ocimene solution dropwise into the β-cyclodextrin solution. After the addition is complete, continue stirring for 15-20 minutes to obtain mixture A. (3) The above mixture A is subjected to ultrasonic treatment to obtain a nanoemulsion; (4) Centrifuge the above nanoemulsion at a speed of 3500-4500 rpm, a temperature of 25 ℃, and a centrifugation time of 10-15 min. (5) Wash the precipitate after centrifugation with anhydrous ethanol to remove the unencapsulated ocimene. Centrifuge the washed emulsion again. Place the precipitate in a -20℃ refrigerator and let it stand for 12-15 h until it freezes into a solid. Then freeze-dry it in a freeze dryer for 48-72 h to obtain nanoparticles. Centrifuge again at a speed of 3500-4500 rpm, a temperature of 25℃, and a centrifugation time of 10-15 min.

[0010] Preferably, in the preparation method of the β-cyclodextrin-ocimene nanoparticles, the mass ratio of β-cyclodextrin to distilled water in step 1 is 14:110-130; the mass ratio of ocimene dissolved in anhydrous ethanol is 1:0.8-1.5; and the mass ratio of β-cyclodextrin to ocimene is 8:0.8-1.2.

[0011] Preferably, the method for preparing β-cyclodextrin-ocimene nanoparticles is characterized in that: in step 1, the mass ratio of β-cyclodextrin to distilled water is 14:125; the mass ratio of ocimene dissolved in anhydrous ethanol is 1:1; and the mass ratio of β-cyclodextrin to ocimene is 8:1.

[0012] Preferably, in the preparation method of the β-cyclodextrin-ocimene nanoparticles, step 2 involves a stirring speed of 300-600 rpm, a stirring time of 30-120 min, and a stirring temperature of 20-60 ℃.

[0013] Preferably, in the preparation method of the β-cyclodextrin-ocimene nanoparticles, the ultrasonic treatment conditions in step 3 are: ultrasonic power of 120 W and treatment time of 40 min.

[0014] This invention also investigates the application of β-cyclodextrin-ocimene nanoparticles for the preparation of bio-nano herbicides.

[0015] Compared with the prior art, the present invention can achieve the following technical effects: 1. This invention encapsulates ocimene within β-cyclodextrin using physical encapsulation technology, successfully constructing β-cyclodextrin-ocimene nanoparticles, which can be used as bio-nano herbicides. This β-cyclodextrin-ocimene system not only effectively improves the shortcomings of ocimene, such as poor water solubility and easy volatility and oxidation, but also reduces the potential risk of soil pollution by relying on a biodegradable carrier.

[0016] 2. After optimization of the preparation method of the present invention, the resulting nano-formulation exhibits significant herbicidal activity and has a sustained-release characteristic of more than 96 hours, thereby reducing the frequency of application and the risk of exposure to non-target organisms.

[0017] 3. This invention is the first to achieve the construction of nano-herbicides by encapsulating ocimene with β-cyclodextrin, which has important theoretical and applied value: theoretically, it can reveal the host-guest encapsulation mechanism, clarify the regulatory relationship between nanoparticle structure and stability, and enrich the research system of nano-formulation of natural essential oils; practically, it provides core technical support for the industrialization of natural herbicides and the development of "highly efficient, low-toxicity, and environmentally friendly" pesticides.

[0018] Of course, any product implementing this invention does not necessarily need to achieve all of the technical effects described above at the same time.

[0019] The following will describe the implementation of the present invention in detail with reference to the embodiments, so that the process of how the present invention uses technical means to solve technical problems and achieve technical effects can be fully understood and implemented accordingly.

[0020] Through analysis and testing commissioned by the Zhengzhou University Analysis and Testing Center, we have for the first time achieved the construction of nano-herbicides by encapsulating ocimene with β-cyclodextrin, which has both important theoretical and practical value: theoretically, it can reveal the host-guest encapsulation mechanism, clarify the regulatory relationship between nanoparticle structure and stability, and enrich the research system of nano-formulation of natural essential oils; practically, it provides core technical support for the industrialization of natural herbicides and the research and development of "highly efficient, low-toxicity, and environmentally friendly" pesticides. Attached Figure Description

[0021] Figure 1 Line graph showing the orthogonal experiment and weighted scoring of process parameters for the preparation of β-cyclodextrin-ocimene nanoparticles; Figure 2 FTIR image of β-cyclodextrin-ocimene nanoparticles; Figure 3 Scanning electron microscope image of β-cyclodextrin-ocimene nanoparticles; Figure 4 The cumulative release rate of β-cyclodextrin-ocimene nanoparticles within 96 hours; Figure 5 Pre-germination results of dandelion and foxtail grass seeds under different treatments after 7 days; Figure 6 This is a standard curve diagram for maltose. Figure 7 Figures showing different enzyme activities after 7 days of treatment under different conditions: Content of α-amylase activity (A), superoxide dismutase activity (C), catalase activity (E), peroxidase activity (G) and dehydrogenase activity (H) in dandelion; content of α-amylase activity (B), superoxide dismutase activity (D), catalase activity (F) and dehydrogenase activity (I) in foxtail grass leaves; Figure 8 The inhibition of β-cyclodextrin-ocimene nano herbicide on dandelion (A, C) and foxtail (B, D) 7 days after application, and the comparison of the rhizomes of dandelion (C) and foxtail (D); Figure 9 A graph showing the relative conductivity measurements of root length (A), stem length (C), and fresh weight (E) of dandelion under different treatments after 7 days, and root length (B), stem length (D), and fresh weight (F) of foxtail grass. Figure 10 A comparative graph showing the relative conductivity (A), malondialdehyde (C), chlorophyll a (E), chlorophyll b (G), total chlorophyll (I), and O2- content (K) of dandelion leaves after 7 days of treatment under different conditions; and the relative conductivity (B), malondialdehyde (D), chlorophyll a (F), chlorophyll b (H), total chlorophyll (J), and O2- content (L) of foxtail grass leaves. Detailed Implementation Example

[0022] This invention provides β-cyclodextrin-ocimene nanoparticles, which are mainly prepared by mixing β-cyclodextrin and ocimene.

[0023] The preparation method of the β-cyclodextrin-ocimene nanoparticles includes the following steps: (1) Weigh a certain amount of β-cyclodextrin, add it to distilled water, and treat it in a water bath at 30°C until it is completely dissolved to obtain a β-cyclodextrin solution; weigh a certain amount of ocimene and dissolve it in anhydrous ethanol to obtain an ocimene solution; the mass ratio of β-cyclodextrin to distilled water in step 1 is 14:110-130; the mass ratio of ocimene to anhydrous ethanol is 1:0.8-1.5; the mass ratio of β-cyclodextrin to ocimene is 8:0.8-1.2; (2) While stirring, slowly add the ocimene solution to the β-cyclodextrin solution. After the addition is complete, continue stirring for 15-20 minutes to obtain mixture A. In step 2, the stirring speed is 300-600 rpm, the stirring time is 30-120 minutes, and the stirring temperature is 20-60℃. (3) The above mixture A is subjected to ultrasonic treatment to obtain nanoemulsion; wherein the ultrasonic treatment conditions are: ultrasonic power of 120 W; treatment time of 40 min.

[0024] (4) Centrifuge the above nanoemulsion at a speed of 3500-4500 rpm, a temperature of 25 ℃, and a centrifugation time of 10-15 min. (5) Wash the precipitate after centrifugation with anhydrous ethanol to remove the unencapsulated ocimene. Centrifuge the washed emulsion again. Place the precipitate in a -20℃ refrigerator and let it stand for 12-15 h until it freezes into a solid. Then freeze-dry it in a freeze dryer for 48-72 h to obtain nanoparticles. Centrifuge again at a speed of 3500-4500 rpm, a temperature of 25℃, and a centrifugation time of 10-15 min. Example

[0025] This invention provides a method for preparing β-cyclodextrin-ocimene nanoparticles, which includes the following steps: (1) Weigh a certain amount of β-cyclodextrin, add it to distilled water, and treat it in a water bath at 30°C until it is completely dissolved to obtain a β-cyclodextrin solution; weigh a certain amount of ocimene and dissolve it in anhydrous ethanol to obtain an ocimene solution; the mass ratio of β-cyclodextrin to distilled water is 14:125; the mass ratio of ocimene to anhydrous ethanol is 1:1; the mass ratio of β-cyclodextrin to ocimene is 8:1; (2) Under stirring, the ocimene solution was slowly added dropwise to the β-cyclodextrin solution. After the addition was completed, the mixture was stirred for 20 minutes to obtain mixture A. In step 2, the stirring speed was 600 rpm, the stirring time was 40 minutes, and the stirring temperature was 25 ℃. (3) The above mixture A is subjected to ultrasonic treatment to obtain nanoemulsion; wherein the ultrasonic treatment conditions are: ultrasonic power of 120 W; treatment time of 40 min.

[0026] (4) The above nanoemulsion was centrifuged and precipitated at a speed of 4000 rpm, a temperature of 25 ℃, and a centrifugation time of 15 min. (5) The precipitate after centrifugation was washed with anhydrous ethanol to remove the unencapsulated ocimene. The washed emulsion was centrifuged again. The resulting precipitate was placed in a -20℃ refrigerator and allowed to stand for 12-15 h. After it was frozen into a solid, it was placed in a freeze dryer and freeze-dried for 48-72 h to obtain nanoparticles. The centrifugation speed was 4000 rpm, the temperature was 25℃, and the centrifugation time was 15 min.

[0027] This invention also provides an application of β-cyclodextrin-ocimene nanoparticles for the preparation of bio-nano herbicides.

[0028] This invention determines the gradient range of single factors in orthogonal experiments through single-factor experiments: Five groups of single-factor experiments (ratio of β-cyclodextrin to ocimene, ultrasonic time, stirring time, stirring speed, and stirring temperature) were conducted to determine the effects of each factor on the nanoparticle size and encapsulation efficiency. The effects of the β-cyclodextrin to ocimene wall-to-core ratio (6:1, 7:1, 8:1, 9:1, 10:1), ultrasonic time (20, 40, 60, 80, 100 min), stirring time (30, 60, 90, 120 min), stirring speed (300, 400, 500, 600 rpm / min), and stirring temperature (20, 30, 40, 50, 60 ℃) were set at five levels. The results are as follows: Figure 1 As shown.

[0029] See Figure 1 The optimal conditions for preparing β-cyclodextrin-ocimene nano-herbicides were determined through orthogonal experiments and weighted scoring. Based on the results of single-factor experiments, a wall-to-core ratio of 8:1 and an ultrasonic time of 40 min were determined as the optimal conditions. Orthogonal experiments were conducted at three levels: stirring speed of 400, 500, and 600 rpm / min; stirring temperature of 30, 40, and 50 ℃; and stirring time of 60, 90, and 120 min, to comprehensively evaluate the best scheme for preparing ocimene nanoparticles.

[0030] Table 1. Orthogonal experimental table for the preparation of β-cyclodextrin-encapsulated ocimene nanoparticles

[0031] The experiment used particle size and encapsulation efficiency as evaluation indicators for the preparation of nanoparticles. Considering that the prepared nanoparticles would be applied to weed control experiments in subsequent experiments, the encapsulation efficiency was considered more important than particle size. A weighted scoring method was used for analysis. Specifically, the encapsulation efficiency score had a weight of 60%, and the particle size score had a weight of 40%. Analysis of the orthogonal experimental results showed that the range of stirring speed (R=0.01), stirring time (R=4.47), and stirring temperature (R=4.46) had the following order of influence: stirring time > stirring temperature > stirring speed. Based on the K value, the optimal combination for preparing nanoparticles was determined to be: a wall-to-core ratio of 8:1; ultrasonic time of 40 min; stirring speed of 600 rpm; stirring time of 40 min; and stirring temperature of 30 ℃. Specific results are shown in Table 1. Nanopesticides were prepared using the optimal combination, and the experiment was repeated three times. The average embedding rate was 90.42% and the average particle size was 225.5 nm, indicating that the preparation scheme was stable and had good repeatability, which was consistent with the results of the optimal preparation scheme in the orthogonal table. The results are shown in Table 2.

[0032] Table 2 Validation of Optimal Preparation Conditions

[0033] The β-cyclodextrin-ocimene nano-herbicide prepared under the optimal conditions obtained in the examples was characterized by Fourier transform infrared spectroscopy. Figure 2 .

[0034] An appropriate amount of ocimene nanoparticles, β-cyclodextrin, and ocimene solution prepared under optimal conditions were co-ground with KBr for pretreatment. Using air as the scanning background, β-CD, OC, and β-CD@OC were characterized by Fourier transform infrared spectroscopy (FTIR). The results showed that the O–H stretching vibration peak of β-CD at 3410 cm⁻¹ broadened significantly after embedding (to 3411 cm⁻¹), indicating a change in its hydrogen bonding environment. The C=C characteristic peak of OC at 1641 cm⁻¹ shifted slightly to 1636 cm⁻¹ in the embedding system, and its intensity decreased. Simultaneously, the olefin characteristic peaks at 984 cm⁻¹ and 895 cm⁻¹ were significantly weakened, indicating restricted guest molecule vibrations. Furthermore, the C–O–C characteristic peak of β-CD at 1031 cm⁻¹ still existed, but its peak shape changed. The above results demonstrate that OC successfully entered the hydrophobic cavity of β-CD and formed an inclusion complex through non-covalent interactions (mainly hydrophobic interactions and hydrogen bonding), proving the successful encapsulation of OC by β-CD; specific results are as follows. Figure 2 As shown, Figure 2 Among them, (a) β-CD@OC, (b) OC, (c) β-CD.

[0035] Morphological observation of β-cyclodextrin-ocimene nanoparticles An appropriate amount of ocimene nanoparticles prepared under optimal conditions were diluted 100 times with deionized water and sonicated for 10 min to ensure uniform dispersion. Then, 6 μL of the treated nanoparticle solution was dropped onto a silicon wafer. After drying with nitrogen, the silicon wafer containing the sample was fixed to the sample stage with conductive adhesive, and the morphology of the nanoparticle sample was observed using a scanning electron microscope (SEM). SEM observation revealed that the β-CD@OC nanoparticles exhibited an irregular rhomboid morphology, with a significantly rougher surface than pure β-CD, and accompanied by a certain degree of agglomeration. Figure 3 Meanwhile, the nanoparticle size is generally concentrated around 200-300 nm, indicating that β-cyclodextrin can effectively embed and assemble ocimene into well-structured nanoparticles. We speculate that the introduction of OC changes the crystal structure or surface properties of β-CD.

[0036] Evaluation of in vitro sustained-release effect Weigh 10 mg of ocimene nanoparticles and place them in a dialysis bag. Add 5 mL of PBS solution (pH=7.4) to completely dissolve them. Then place the dialysis bag in a beaker containing PBS solution and shake it on a shaker at a constant temperature (37 ℃, 100 r / min). At different time points, take samples of the solution from the beaker, measure the absorbance, and calculate the release amount.

[0037]

[0038] In the formula: Cn is the mass concentration of the sample taken at each time interval (μg / mL), V1 is the sampling volume (mL), V2 is the volume of the dissolution medium (mL), and W is the mass of ocimene (μg).

[0039] Specific results are as follows Figure 4 As shown, free ocimene releases extremely rapidly, with a cumulative release of 95% within 96 hours, indicating its difficulty in retention within the system. In contrast, the release of ocimene nanoparticles encapsulated with β-cyclodextrin is significantly delayed, with a cumulative release of only 58% after 96 hours, a stark contrast to free ocimene. This fully demonstrates that β-cyclodextrin encapsulation can effectively delay ocimene release, increase its retention time in the system, and achieve a good sustained-release effect. It also confirms that nanoparticles can effectively delay drug release and prolong their action time in the environment.

[0040] The herbicidal effect of the β-cyclodextrin-ocimene nano-herbicide prepared under the optimal conditions obtained in the examples was evaluated.

[0041] Pre-germination rate was significantly inhibited One hundred seeds each of foxtail grass and dandelion were taken and placed separately in petri dishes. The plants were treated with deionized water, β-cyclodextrin solution, ocimene solution, and ocimene nanoparticle solution, and the germination rate was measured on the 7th day of cultivation. The results are as follows: Figure 5 As shown, the germination rates of dandelion and foxtail grass in the deionized water treatment group were 46% and 33%, respectively; the germination rates of the two weeds in the β-cyclodextrin solution treatment group decreased to 36% and 24%, respectively; while the germination rates of foxtail grass and dandelion seeds in the ocimene solution and ocimene nanoparticle solution treatment groups were both 0%. This confirms that ocimene nanoparticle herbicides can effectively inhibit weed seed germination, demonstrating excellent pre-emergence weed control potential.

[0042] Pre-emergence seed α-amylase activity was significantly reduced See Figure 6The determination of α-amylase activity during seed germination reflects the ability of stored starch in seeds to convert into soluble sugars. α-amylase hydrolyzes starch in the endosperm or cotyledons, and its activity changes are closely related to seed germination rate and seedling growth. Generally, increased α-amylase activity indicates accelerated starch degradation and sufficient energy supply, which is beneficial to seed germination; while decreased activity means that starch conversion is hindered, which may lead to delayed or inhibited germination. In this experiment, the 3,5-dinitrosalicylic acid (DNS) colorimetric method was used to determine the α-amylase activity of seeds. Dandelion and foxtail grass seeds were accurately weighed at 0.50 g each, and 5.0 mL of pre-cooled 0.05 mol·L⁻¹ phosphate buffer (pH 6.8) was added. The mixture was thoroughly homogenized under ice bath conditions. The homogenate was centrifuged at 4 ℃ and 10,000 r·min⁻¹ for 15 min, and the supernatant was used as the crude enzyme extract. Mix 1.0 mL of 1% (w / v) soluble starch solution with 0.5 mL of phosphate buffer, preheat to 37 °C, then add 0.5 mL of enzyme extraction solution, mix well, and react in a 37 °C water bath for 10 min. Immediately after the reaction, add 1.0 mL of DNS reagent to terminate the reaction, and heat in a boiling water bath for 5 min for color development. After cooling to room temperature, bring the volume to 10 mL with distilled water and mix well. Zero the volume with a reagent blank and measure the absorbance at 540 nm. Each treatment was repeated in triplicate. An enzyme blank was also set up (the enzyme was inactivated by adding DNS reagent before adding the substrate) to correct for absorbance caused by non-enzymatic reactions. Prepare solutions of different concentration gradients using maltose as a standard, develop the color using the above method, plot a standard curve, and calculate the amount of reducing sugar generated based on the sample absorbance.

[0043] α-Amylase activity is expressed as the amount of maltose produced per unit time, and is calculated using the following formula:

[0044] In the formula: C represents the maltose mass (μg) obtained from the standard curve. V is the total volume of the reaction system (mL); t is the reaction time (min); m is the sample mass (g) like Figure 7As shown in A and B, after 7 days of water and β-cyclodextrin treatment, there was no significant difference in α-amylase activity in dandelion and foxtail millet seeds. However, in dandelion, the α-amylase activity decreased to approximately 150 U / g in the ocimene-treated group and approximately 100 U / g in the nano-preparation-treated group. In foxtail millet, the α-amylase activity decreased to approximately 100 U / g in the ocimene-treated group and approximately 65 U / g in the nano-preparation-treated group, significantly lower than that in the ocimene group. β-CD@OC treatment significantly reduced α-amylase activity during seed germination, indicating that it inhibited the conversion of starch to soluble sugars, thereby limiting the energy supply required for germination.

[0045] Pre-emergence seed superoxide dismutase activity was significantly inhibited During seed germination, superoxide dismutase (SOD) is one of the key antioxidant enzymes for scavenging reactive oxygen species (ROS). It catalyzes the dismutation of superoxide anion radicals (O2⁻) to generate hydrogen peroxide and oxygen, thereby mitigating the oxidative damage of ROS to cell structures. Measuring SOD activity reflects the ability of the seed's antioxidant defense system during germination and its response to oxidative stress. Generally, moderately elevated SOD activity helps maintain intracellular redox balance and promotes normal seed germination; while decreased or imbalanced SOD activity may lead to the accumulation of ROS, causing membrane lipid peroxidation and cell damage, thereby inhibiting seed germination and seedling growth. Therefore, SOD activity is also of great significance to this study. In this experiment, the NBT method was used to determine SOD activity. 0.50 g of dandelion or foxtail grass seeds were added to 5.0 mL of pre-cooled 0.05 mol·L⁻¹ phosphate buffer (pH 7.8) and homogenized under ice bath conditions. Centrifuge at 4 ℃ and 10000 r·min⁻¹ for 15 min, and collect the supernatant as the crude enzyme extract. Add the following to a test tube sequentially: 1.5 mL of 0.05 mol·L⁻¹ phosphate buffer (pH 7.8), 0.3 mL of 130 mmol·L⁻¹ methionine (Met), 0.3 mL of 750 μmol·L⁻¹ NBT, 0.3 mL of 100 μmol·L⁻¹ EDTA, 0.3 mL of 20 μmol·L⁻¹ riboflavin, and 0.1 mL of enzyme solution. Mix well and react under light conditions (approximately 4000 lx) for 10–20 min. A light control (without enzyme) and a dark control (no light) are also provided. Immediately after the reaction, measure the absorbance at 560 nm.

[0046]

[0047] In the formula: A0 represents the absorbance of the control tube (without enzyme solution). A1 represents the absorbance of the sample tube (after adding enzyme solution). 0.5 is defined as the amount of enzyme that inhibits the NBT photoreduction reaction by 50%, and is defined as 1 enzyme activity unit (U). Vt (mL) is the total volume of the enzyme extract. Vs (mL) is the volume of enzyme solution added to the reaction system. FW(g) is the fresh weight of the sample. In this study, the SOD activity trend was consistent with that of α-amylase. In dandelion, the enzyme activity in the ocimene-treated group decreased to 246 U / g FW, while that in the nano-preparation-treated group was around 150 U / g FW. In foxtail grass, the enzyme activity in the ocimene-treated group decreased to 264 U / g FW, while that in the nano-preparation-treated group was around 116 U / g FW, which was significantly lower than that in the ocimene group.

[0048] Pre-emergence seed CAT activity was significantly reduced During seed germination, catalase (CAT) is one of the key enzymes for scavenging reactive oxygen species (ROS), primarily by catalyzing the decomposition of hydrogen peroxide (H2O2) into water and oxygen. Generally, increased CAT activity helps reduce ROS toxicity and maintain cell structural stability, thereby promoting seed germination and seedling growth; conversely, decreased CAT activity may lead to H2O2 accumulation, causing membrane lipid peroxidation and cell damage, thus inhibiting seed germination. Furthermore, CAT activity is highly sensitive to exogenous treatments (such as allelochemicals, nanomaterials, and environmental stress), and is therefore often used as an important physiological indicator for evaluating seed vigor and stress response. CAT activity was determined using a modified H2O2 ultraviolet spectrophotometric method. 0.50 g of dandelion or foxtail millet seeds were added to 5.0 mL of pre-cooled 0.05 mol·L⁻¹ phosphate buffer (pH 7.0) and homogenized under ice bath conditions. Centrifuge at 4 ℃, 10,000 r·min⁻¹ for 15 min, and collect the supernatant as crude enzyme extract. Add 2.9 mL of phosphate buffer (pH 7.0) and approximately 30 mmol·L⁻¹ of H₂O₂ solution to a test tube. After mixing the reaction system, quickly add 0.1 mL of enzyme solution and immediately measure the absorbance at 240 nm. Record the absorbance change every 15–30 s for 1–2 min, and calculate the rate of absorbance decrease (ΔA240 / min). CAT activity is calculated using the following formula:

[0049] in: ΔA240: Absorbance change per minute V: Total reaction volume (mL) ε: Molar absorptivity of H2O2 (39.4 L·mmol⁻¹·cm⁻¹) l: Optical path length (1 cm) t: Reaction time (min) m: Sample mass (g) In this study, the CAT activity showed a consistent trend with α-amylase activity and SOD activity. In the ocimene-treated group, the dandelion enzyme activity decreased to 18 U / g FW, and the foxtail enzyme activity decreased to 11 U / g FW. In the nano-preparation-treated group, the dandelion enzyme activity was 14 U / g FW, and the foxtail enzyme activity decreased to 6 U / g FW, which were significantly lower than those in the ocimene group.

[0050] Pre-emergence seed POD activity assay During seed germination, peroxidase (POD) is an important component of the plant antioxidant enzyme system. It catalyzes the oxidation of various substrates using hydrogen peroxide (H2O2) as an electron acceptor, thereby participating in H2O2 scavenging and mitigating oxidative damage to cells caused by reactive oxygen species (ROS). Measuring POD activity can reflect the ROS scavenging capacity and antioxidant defense level during seed germination. In this study, the guaiacol method was used to determine POD activity. 0.50 g of dandelion or foxtail millet seeds were added to 5.0 mL of pre-cooled 0.05 mol·L⁻¹ phosphate buffer (pH 6.0–7.0) and thoroughly homogenized under ice bath conditions. The homogenate was centrifuged at 4 ℃ and 10,000 r·min⁻¹ for 15 min, and the supernatant was used as the crude enzyme extract. The reaction system was prepared as follows: 30 mL phosphate buffer + 10 mL 50 mM guaiacol solution + 10 mL 2% hydrogen peroxide solution. After mixing the reaction system, add 0.1 mL of enzyme solution to start the reaction. Immediately measure the absorbance at 470 nm. Record the absorbance every 15–30 s and continue measuring for 1–3 min. Calculate the rate of change of absorbance (ΔA470 / min) using the following formula:

[0051] ε is the molar absorptivity, with a value of 26.6 mM⁻¹·cm⁻¹. l represents the optical path length of the cuvette, with a value of 1 cm. ΔA 470 The change per unit time V represents the total enzyme volume (mL). m = sample amount (g) Depend on Figure 8 As shown in Figure C, the POD activity of the dandelion water treatment group and the β-CD treatment group was not significantly different, while the dandelion enzyme activity in the ocimene treatment group decreased significantly to 511 U / g FW, which was significantly lower than that in the ocimene group; the dandelion enzyme activity in the nano-preparation treatment group was 228 U / g FW.

[0052] Pre-emergence seed dehydrogenase activity assay During seed germination, dehydrogenases are important redox enzymes involved in cellular respiratory metabolism, widely present in the glycolysis pathway and the tricarboxylic acid cycle (TCA cycle). Generally, increased dehydrogenase activity indicates enhanced seed respiratory metabolism and improved cell activity, which is beneficial for seed germination and seedling growth; conversely, decreased activity signifies suppressed respiratory metabolism and insufficient energy supply, potentially leading to decreased seed vigor or inhibited germination. The triphenyltetrazolium chloride (TTC) reduction method is commonly used to determine dehydrogenase activity. The principle is that dehydrogenases can reduce colorless TTC to red formazan, thus enabling quantitative evaluation of seed vigor. Take approximately 0.50 g of dandelion or foxtail grass seeds, wash them with distilled water, pat dry, and place them in a petri dish. Add the following solutions: 0.4% TTC solution: 2.0 mL; phosphate buffer (pH 7.0): 2.0 mL. The reaction mixture was placed at 30–37 ℃ in the dark for 2–4 h. After rinsing with distilled water, 5–10 mL of 95% ethanol was added, and the mixture was extracted in a water bath at 60 ℃ for 30 min (or until completely decolorized). The absorbance of the extract was measured at 490 nm with the extract as a blank control. The activity was directly expressed by the OD value. In this study, the dehydrogenase activity and catalase activity showed the same trend. In the ocimene-treated group, the dandelion enzyme activity decreased to 0.051 U / g FW, and the foxtail grass enzyme activity decreased to 0.056 U / g FW. In the nano-preparation-treated group, the dandelion enzyme activity was 0.01 U / g FW, and the foxtail grass enzyme activity decreased to 0.048 U / g FW, which were significantly lower than those in the ocimene group.

[0053] The scavenging effect of β-cyclodextrin-ocimene nano-herbicide on weed seedlings Thirty uniformly sized, robust, and well-developed foxtail and dandelion plants were transplanted and treated with deionized water, β-cyclodextrin solution, ocimene solution, and ocimene nanoparticle solution, respectively. Growth was observed on days 1, 2, and 3, and the fresh weight, root length, and plant height of the foxtail and dandelion plants were measured on day 3. Specific results are shown below. Figure 8 , 9 As shown, observations revealed that the growth of foxtail grass and dandelion seedlings treated with ocimene nanoparticle solution significantly declined after 7 days. Quantitative data further indicated that the treatment significantly inhibited the fresh weight, root length, and plant height of the two weed seedlings, further confirming the application value of ocimene nanoparticles in post-emergence weed control.

[0054] Measurement of relative conductivity Leaves from different treatment groups before and after treatment were placed in test tubes, submerged in distilled water for 2 hours, then the liquid was poured out, and the initial conductivity C1 was measured. The liquid was then poured back into the original test tubes and boiled in a water bath for 15 minutes. Afterward, the liquid was removed, cooled to room temperature, and shaken well, and the final conductivity C2 was measured. The control group test tube used distilled water, and the conductivity C0 was measured. The conductivity was calculated using the formula:

[0055] Relative conductivity is a classic physiological indicator characterizing the permeability and integrity of plant cell membranes, and it has important application value in assessing herbicide damage to plant cells. When cell membranes are subjected to abiotic stress, their selective permeability is disrupted, leading to a large outflow of intracellular electrolytes such as potassium ions and organic acids. This results in an increase in the conductivity of cell extracts, and the magnitude of the conductivity change is significantly positively correlated with the degree of membrane damage. In this experiment, *Setaria viridis* and *Taraxacum officinale* plants were treated with deionized water, β-cyclodextrin solution, ocimene solution, and ocimene nanoparticle solution for 7 days, and the relative conductivity of the leaves was measured. Specific results are as follows: Figure 9 As shown in Figures A and B, the REC values ​​of dandelion leaves after 7 days were 56.76%, 56.77%, 75.83%, and 83.34%, respectively, while those of foxtail grass leaves were 42.52%, 40.94%, 71.73%, and 85.14%, respectively. Compared with the control and β-cyclodextrin groups, the REC of the ocimene nanoparticle group was significantly increased (P<0.05). Malondialdehyde content increased significantly Following Heath's method with slight modifications, leaves from different treatment groups were ground evenly in 5 mL of 10% (w / v) TCA solution, centrifuged, and the supernatant was added to an equal volume of thiobarbituric acid (TBA). After mixing, the mixture was centrifuged again, and the absorbance of the supernatant was measured to calculate the MDA content. CMDA (mol / L) = 6.45×10-6×A532 - 0.56×10-6×A450 Malondialdehyde (MDA) is a hallmark product of cell membrane lipid peroxidation, and its content directly reflects the degree of damage to plant cell membranes. It is a key indicator for assessing abiotic stress in weeds and elucidating the mechanism of action of plant-derived herbicides. When weeds are under stress, unsaturated fatty acids in the cell membrane undergo peroxidation to produce MDA, and its accumulation is significantly positively correlated with the degree of membrane damage. This experiment used the thiobarbituric acid (TBA) colorimetric method, combined with optimized pretreatment steps based on weed leaf characteristics, to determine the MDA content in leaves. *Setaria viridis* and *Taraxacum officinale* were treated with deionized water, β-cyclodextrin solution, ocimene solution, and ocimene nanoparticle solution for 7 days. Specific results are as follows: Figure 9As shown in C and D, the data showed that after 7 days, the MDA content of dandelion leaves was 5.35, 5.12, 11.49, and 14.86 μmol / g, respectively, while the MDA content of foxtail grass leaves was 4.45, 4.48, 8.78, and 12.49 μmol / g, respectively. Compared with the control, there was no significant difference in the β-cyclodextrin and ocimene solution groups (P>0.05), while the ocimene nanoparticle group showed a significant increase (P<0.05), and the trends of the two weeds were consistent.

[0056] The chlorophyll content was significantly reduced. Following Barnes' method with slight modifications, leaves from different treatment groups were soaked in 20 mL of 95% ethanol and extracted overnight in the dark. Using 95% ethanol as a blank control, OD values ​​at 663 nm and 645 nm were measured. Chlorophyll content (mg / g) was calculated using the following formula: Ca = 12.72A663 - 6.88A645 Cb = 22.88A645 - 4.67A663 Ah, CT = Ca + Cb

[0057] In the formula: Ca is the concentration of chlorophyll a, Cb is the concentration of chlorophyll b, CT is the total concentration of chlorophyll, V is the volume of the extract (mL), and W is the fresh weight of the sample (g).

[0058] Chlorophyll, as the core pigment of plant photosynthesis, is directly related to photosynthetic efficiency and is a key physiological indicator characterizing plant growth status. In herbicide mechanism studies, changes in chlorophyll content are often used to assess the degree of damage to the plant's photosynthetic system. When weeds are under herbicide stress, chlorophyll synthesis is inhibited or degradation is accelerated, leading to a decrease in photosynthetic rate and ultimately inhibiting plant growth. This characteristic makes chlorophyll content measurement an important tool for elucidating weed control mechanisms, especially suitable for assessing the physiological interference effects of plant-derived compounds on weeds. In this experiment, foxtail grass and dandelion were treated with deionized water, β-cyclodextrin solution, ocimene solution, and ocimene nanoparticle solution for 7 days. Specific results are as follows: Figure 10 As shown in EJ, the data indicates that the chlorophyll a content in dandelion leaves was 4.52, 4.46, 3.30, and 2.15 mg / g, and the chlorophyll b content was 1.86, 1.81, 0.75, and 0.36 mg / g, respectively. In contrast, the chlorophyll a content in foxtail grass leaves was 2.74, 2.52, 1.70, and 0.84 mg / g, and the chlorophyll b content was 0.73, 0.69, 0.40, and 0.036 mg / g, respectively. It is evident that treatment with ocimene nanoparticles significantly reduced the chlorophyll a and b content in both weeds, indicating disruption of their photosynthetic pigment systems.

[0059] O2- content increased significantly Following Upadhyaya's method with slight modifications, leaves from different treatment groups were taken, thoroughly ground with phosphate buffer, and centrifuged. Then, 1 mL of the supernatant was taken, and 0.75 mL of phosphate buffer and 0.25 mL of hydroxylamine hydrochloride were added. The mixture was reacted in a water bath at 25°C for 30 min. Then, 2 mL of α-naphthylamine and 2 mL of p-aminobenzenesulfonic acid were added, and the mixture was reacted in a water bath at 30°C for 30 min. The 530 nm OD value was then measured.

[0060] O2⁻, as a core component of reactive oxygen species (ROS) in plant cell oxidative stress responses, is a key indicator for assessing the degree of cellular abiotic stress. When plants encounter abiotic stresses such as herbicides, the ROS generation and scavenging mechanisms become unbalanced, leading to a large accumulation of O2⁻. This causes lipid peroxidation, protein oxidation, and DNA damage, ultimately resulting in cell dysfunction and even death. Therefore, measuring O2⁻ content is a crucial method for elucidating the oxidative stress pathway of herbicides and has significant reference value for understanding the herbicidal mechanisms of plant-derived compounds. In this experiment, foxtail grass and dandelion were treated with deionized water, β-cyclodextrin solution, ocimene solution, and ocimene nanoparticle solution for 7 days. Specific results are as follows: Figure 10 As shown in K and L, the data indicates that after 7 days, the O2⁻ content in dandelion leaves was 12.32, 12.18, 15.78, and 20.85 μM / g, respectively, while that in foxtail grass leaves was 8.60, 8.37, 12.37, and 18.64 μM / g. This demonstrates that treatment with ocimene nanoparticles significantly increased the O2⁻ accumulation in the leaves of both weeds.

[0061] Allelopathic effects refer to the ecological phenomenon where plants release specific chemical substances to inhibit the growth of surrounding plants. Based on this principle, our laboratory's previous research found that ocimene, as a plant-derived allelochemical, possesses significant herbicidal activity, low residue, and environmental compatibility, demonstrating its potential for developing green herbicides. This study, for the first time, utilizes this plant-derived allelochemical, with ocimene as the core active ingredient and β-cyclodextrin as the carrier. To the extent that no related research has been reported in the current literature, a β-cyclodextrin-ocimene complex was prepared using ultrasound-assisted encapsulation technology. This strategy breaks through the traditional "carrier + active ingredient" construction model and provides a new approach to solving key issues in sustainable herbicide formulation design.

[0062] After system optimization, the optimal preparation conditions were determined to be: a wall-to-core ratio of 8:1; ultrasonication time of 40 min; stirring speed of 600 rpm; stirring time of 40 min; and stirring temperature of 30 ℃. Under these conditions, the encapsulation efficiency was 90.42%, the particle size was 225.5 nm, and scanning electron microscopy results showed that the particles had a regular rhomboid morphology. Fourier transform infrared spectroscopy confirmed that ocimene was successfully encapsulated in the β-cyclodextrin cavity, indicating that the formulation is stable and feasible. β-cyclodextrin encapsulation achieved effective controlled release: within 96 hours, the cumulative release rate of ocimene from the β-cyclodextrin-ocimene nanoparticles was only 58%, while the release rate of free ocimene exceeded 95%. This encapsulation strategy significantly reduced volatility, prolonged herbicidal activity, and effectively solved the problem of limited scalability of ocimene in practical applications.

[0063] Bioactivity assays showed that the bio-nano herbicide β-cyclodextrin-ocimene inhibited the germination and growth of foxtail grass and dandelion. Its herbicidal effect is achieved through multiple pathways—including disrupting cell membrane integrity, inhibiting photosynthesis, and inducing oxidative stress—thereby reducing weed resistance, which aligns with the principles of green agriculture. Encapsulating ocimene with β-cyclodextrin provides a feasible strategy for developing highly efficient, low-toxicity, and environmentally friendly green herbicides, contributing to the promotion of sustainable agricultural development.

[0064] This invention can be summarized in other specific forms that do not depart from the spirit or essential features of the invention. Therefore, in all respects, the above embodiments of the invention should be considered illustrative only and not limiting, while the claims define the scope of the invention. The foregoing description does not define the scope of the invention; therefore, any variations within the meaning and scope equivalent to the claims should be considered to be included within the scope of the claims.

Claims

1. A β-cyclodextrin-ocimene nanoparticle, characterized in that: It is mainly prepared by mixing β-cyclodextrin and ocimene.

2. The method for preparing β-cyclodextrin-ocimene nanoparticles according to claim 1, characterized in that... It includes the following steps: (1) Weigh a certain amount of β-cyclodextrin, add it to distilled water, and treat it in a water bath at 30°C to completely dissolve it, so as to obtain a β-cyclodextrin solution; Weigh a certain amount of ocimene and dissolve it in anhydrous ethanol to obtain an ocimene solution; (2) While stirring, slowly add the ocimene solution dropwise into the β-cyclodextrin solution. After the addition is complete, continue stirring for 15-20 minutes to obtain mixture A. (3) The above mixture A is subjected to ultrasonic treatment to obtain a nanoemulsion; (4) Centrifuge the above nanoemulsion at a speed of 3500-4500 rpm, a temperature of 25 ℃, and a centrifugation time of 10-15 min. (5) Wash the precipitate after centrifugation with anhydrous ethanol to remove the unencapsulated ocimene. Centrifuge the washed emulsion again. Place the precipitate in a -20℃ refrigerator and let it stand for 12-15 h until it freezes into a solid. Then freeze-dry it in a freeze dryer for 48-72 h to obtain nanoparticles. Centrifuge again at a speed of 3500-4500 rpm, a temperature of 25℃, and a centrifugation time of 10-15 min.

3. The method for preparing β-cyclodextrin-ocimene nanoparticles according to claim 2, characterized in that: In step 1, the mass ratio of β-cyclodextrin to distilled water is 14:110-130; the mass ratio of ocimene dissolved in anhydrous ethanol is 1:0.8-1.5; and the mass ratio of β-cyclodextrin to ocimene is 8:0.8-1.

2.

4. The method for preparing β-cyclodextrin-ocimene nanoparticles according to claim 2, characterized in that: In step 1, the mass ratio of β-cyclodextrin to distilled water is 14:125; the mass ratio of ocimene dissolved in anhydrous ethanol is 1:1; and the mass ratio of β-cyclodextrin to ocimene is 8:

1.

5. The method for preparing β-cyclodextrin-ocimene nanoparticles according to claim 2, characterized in that: In step 2, the stirring speed is 300-600 rpm, the stirring time is 30-120 min, and the stirring temperature is 20-60 ℃.

6. The method for preparing β-cyclodextrin-ocimene nanoparticles according to claim 2, characterized in that: The conditions for ultrasonic treatment in step 3 are: ultrasonic power 120 W; treatment time 40 min.

7. An application of β-cyclodextrin-ocimene nanoparticles, characterized in that: It is used to prepare bio-nano herbicides.

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