Method for preparing natural aloe bitter substance film and method for analyzing characteristics thereof

Abstract

The application discloses a preparation method of aloin natural film agent and a characteristic analysis method thereof, and particularly relates to the field of natural film agent, and comprises 2% aloin, 1.5% sodium carboxymethyl cellulose, 0.5% sodium alginate, 0.025% calcium chloride, 2% glycerol and deionized water in balance. The 2% aloin natural film agent is a safe and effective plant protection film, and the effective component aloin is a harmless natural plant product, which has obvious effects on the formation of conidia and the color of conidia of rice blast fungus; the base film sodium carboxymethyl cellulose and sodium alginate are edible film components, the wetting agent glycerol has no obvious harm to rice, organisms and environment, and can also reduce the internal and external turgor pressure of conidia and play a part of blocking invasion effect. Therefore, the 2% aloin natural film agent provides an effective and safe new dosage form for the prevention and control of rice blast in rice production.

Description

Technical Field

[0001] This invention relates to the field of natural film-forming agents, specifically to a method for preparing an aloe vera extract natural film-forming agent and a method for analyzing its properties. Background Technology

[0002] Aloe vera extract is a natural active ingredient extracted from the traditional Chinese medicine aloe vera. It has a strong inhibitory effect on the germination of appressorium spores of rice blast fungus. Its chemical structure is 2-acetone-8-D-glucopyranose-7-hydroxy-5-methyl-p-oxynaphthone, which also has antibacterial effects against a variety of microorganisms (Yang Jiyuan et al., 2001; Lin et al., 2008; Wang Jinxu et al., 2008). Because rice leaves have a waxy layer, the deposition rate of sprayed pesticides on the leaf surface is low. Furthermore, rice plants are affected by factors such as solar radiation, rainfall, and microbial degradation, resulting in short pesticide retention periods and low effective utilization rates (Zhou et al., 2018; Massinon et al., 2017; De et al., 2017). Hydrogels are three-dimensional networks formed by water-soluble molecules through physical cross-linking (hydrogen bonds, host-guest interactions, ionic bonds, etc.) or dynamic chemical bonding (imine bonds, disulfide bonds, etc.) (Feng et al., 2020; Purcell et al., 2014). They possess excellent biomechanical properties and plasticity. Biocompatible hydrogels with dual-membrane structures have great application prospects in the biomedical field and are often used as novel edible films and for constructing medicinal wound dressings. Plant protection films have advantages such as defensiveness, safety, and low cost, and are mainly used in China for the control of pests and diseases.

[0003] Hydrogel protective films for rice blast are currently unavailable. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing a natural aloe vera extract film and a method for analyzing its properties, so as to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] Aloe vera natural film agent, comprising 2% aloin, 1.5% sodium carboxymethyl cellulose, 0.5% sodium alginate, 0.025% calcium chloride, 2% glycerin, and the remainder deionized water.

[0007] The preparation method of aloe vera extract natural film is as follows: First, weigh 1.5g of sodium carboxymethyl cellulose, 0.5g of sodium alginate, and 0.025% calcium chloride, mix them evenly, and then dissolve them in 70ml of deionized water on a magnetic stirrer for about 4 hours. Next, weigh 2g of glycerin and put it into a 50ml beaker, then dissolve it in 10ml of deionized water. Pour the glycerin solution into the mixture of sodium carboxymethyl cellulose, sodium alginate, and calcium chloride, and mix it evenly with a magnetic stirrer. Then, weigh 2g of aloe vera extract, dissolve it in 10ml of deionized water, and pour it into the above mixture. Finally, add water to make up to 100g, let it stand for 8-10 hours until all the bubbles disappear, and you will get a 2% aloe vera extract natural film.

[0008] The analytical methods for the properties of aloe vera extract natural film agents include the following analytical methods:

[0009] 1) The adhesion and film-forming properties of natural film-forming agents were analyzed using a digital camera and scanning electron microscope;

[0010] 2) Fourier transform infrared spectroscopy and X-ray diffraction were used to analyze the natural film-forming agent of aloe vera and the crystalline form of aloe vera.

[0011] 3) Combined thermogravimetric analysis and differential scanning calorimetry (DSC) are used to analyze and determine the possible reaction processes of the material and to identify the nature of the transition peaks;

[0012] 4) Observe the particle size distribution of the aloe vera natural film agent, and measure its viscosity, spreading area, pH value and contact angle, and observe the wetting performance of the aloe vera natural film agent.

[0013] 5) The efficacy of aloe vera extract natural film agent against rice blast and its effect on rice growth and development were tested by inoculating detached rice leaves with abrasives, inhibiting appressorium germination, and measuring the chlorophyll content of rice.

[0014] Compared with the prior art, the beneficial effects of the present invention are:

[0015] 2% Aloe Vera Extract Natural Film is a safe and effective plant protection film. Its active ingredient, aloe vera extract, is a harmless natural plant product that has a significant effect on the formation and color of appressorium of rice blast fungus. The base film, sodium carboxymethyl cellulose and sodium alginate, are both edible film components. The humectant glycerin is also harmless to rice, organisms, and the environment, and it also reduces the internal and external turgor pressure of appressorium, thus playing a partial role in preventing invasion. Therefore, 2% Aloe Vera Extract Natural Film provides an effective and safe new formulation for the control of rice blast in rice production. Attached Figure Description

[0016] Figure 1 a-1e are morphological characteristic diagrams of the initial screening film agent in the examples;

[0017] Figure 2 a-2c are morphological characteristic diagrams of the film containing the wetting agent in the examples;

[0018] Figure 3 The following are morphological characteristics of the film formulations containing aloe-emodin (0%, 2%, and 4%) in the examples;

[0019] Figure 4 This is an experimental diagram illustrating the determination of drug loading in an aloe vera natural film agent, as shown in the example.

[0020] Figure 5 a-5d are morphological characteristic diagrams of the aloe vera bitter natural film agent in the example;

[0021] Figure 6 Scanning electron microscope images of the film-forming agent and the natural film-forming agent of aloe vera extract, as shown in the examples;

[0022] Figure 7 a-7d are examples of 2% aloe vera natural film, CMC-Na@SA film, and aloe vera FTIR spectrum;

[0023] Figure 8 a-8c are the XRD spectra of CMC-Na@SA film, aloe vera, and 2% aloe vera natural film in the examples;

[0024] Figure 9 a-9c are TGA-DSC curves of CMC-Na@SA film agent, aloe vera extract, and 2% aloe vera extract natural film agent in the examples.

[0025] Figure 10 a-10b are particle size distribution diagrams of the diluted solutions of 2% aloe vera extract natural film and CMC-Na@SA film in Examples;

[0026] Figure 11 The pH and viscosity graphs for the CMC-Na@SA film agent in the example are shown.

[0027] Figure 12 The image shows the contact angle measurements of the CMC-Na@SA membrane and the 2% aloe vera natural film agent in the example.

[0028] Figure 13 The graph shows the wetting properties of aloe vera extract natural film agent.

[0029] Figure 14 Diagram of an in vitro rice leaf inoculation experiment involving scratching;

[0030] Figure 15 a-15b are experimental diagrams illustrating the inhibitory effect of CMC-Na@SA film and 2% aloe-emodin natural film dilution on the germination of appressorium spores of rice blast fungus in Example 1.

[0031] Figure 16The graph shows the effect of 2% aloe vera extract natural film on the chlorophyll content of rice seedlings in Example 1.

[0032] Figure 17 This is a graph showing the effect of 2% aloe vera extract natural film on net photosynthesis in rice seedlings, as described in Example 1. Detailed Implementation Example

[0033] 1. Materials and Methods

[0034] 1.1 Preparation of Experimental Materials

[0035] Aloe vera extract (Xi'an Yunyue Biotechnology Co., Ltd.), chitosan (Zhengzhou Mingrui Chemical Products Co., Ltd.), sodium alginate (chemically pure, Tianjin Guangfu Fine Chemical Research Institute), vegetable oil (Yihai Kerry Arawana Grain & Oil Food Co., Ltd.), sodium carboxymethyl cellulose (Zhengzhou Shengyu Chemical Products Co., Ltd.), glycerol (Tianjin Xinyu Fine Chemical Co., Ltd.), guar gum (Shenzhen 001 Biotechnology Co., Ltd.), pectin (Xuzhou Zhongbo Biotechnology Co., Ltd.), soluble starch (Tianjin Zhiyuan Chemical Reagent Co., Ltd.).

[0036] 1.2 Preparation of Instruments and Equipment

[0037] AL104 electronic balance (Mettler-Toledo Instruments Ltd.), digital display heated magnetic stirrer (Jintan Shenglan Instrument Manufacturing Co., Ltd.), JJ-1 power-boosting electric stirrer (Jintan Fuhua Instrument Co., Ltd.), NDJ-8S digital viscometer (Shanghai Youke Instrument Co., Ltd.), X-ray diffractometer (Brook XD3-Advance), Fourier transform infrared spectrometer (Nicolet iS5), contact angle meter (Dongguan Pusaite Testing Equipment Co., Ltd.), SEM (Q45_XFlash6_30), STA (Netzsch STA 449F3), pH meter (Leici PHSJ-4A), etc.

[0038] 1.3, Preparation of 2% Aloe Vera Extract Natural Film

[0039] 1.3.1 Screening of film-forming substances

[0040] Weigh 2 g of the prepared film-forming material and mix thoroughly with a mixer to prepare a 2% film solution. The hydrogel film-forming material is preliminarily evaluated by comparing its filament strength, elongation, and tensile strength. To improve the physicochemical properties of the film-forming agent, the hydrogel needs to be optimized by adding a wetting agent to enhance its toughness. The ratio of sodium carboxymethyl cellulose (CMC) and sodium alginate hydrogels was screened based on the filament strength, elongation, tensile strength, and film-forming properties of the main film-forming materials. The film-forming effect of the film-forming agent was evaluated to determine the optimal CMC-Na:SA ratio for the base film and the amount of wetting agent to be added.

[0041] 1.3.2. Amount of Aloe Vera Extract Added

[0042] Adding aloin to the film formulation as an antibacterial active ingredient can produce antibacterial activity against rice blast fungus. To understand the effect of aloin on the film formulation, two glycerol addition concentrations (0.5% and 2%, i.e., 0.5g and 2g of glycerol per 100g of film formulation) were selected, and three aloin addition concentrations (0%, 2%, and 4%) were selected. Tensile strength (MPa), elongation (%), and tensile strength (N) were used as evaluation indicators to screen the humectant varieties and determine the optimal addition amount of aloin in the base film.

[0043] 1.3.3 Drug loading capacity of aloe vera

[0044] Films containing 2% and 4% aloe vera extract were poured into disposable petri dishes and placed in a bellows for 24-48 hours. After the film-forming agent was completely dried, it was observed whether any components were precipitated.

[0045] 1.4 Performance Characterization of 2% Aloe Vera Extract Natural Film

[0046] 1.4.1 Morphology of 2% Aloe Vera Extract Natural Film Formulation

[0047] The surface morphology of the test film was observed and recorded at 200x, 400x, 800x and 1000x magnification using a scanning electron microscope (Q45_XFlash6_30).

[0048] 1.4.2 Fourier Transform Infrared Spectroscopy Analysis

[0049] Approximately 1 mg of the powder sample to be tested was weighed and ground into a fine powder in an agate mortar containing approximately 100 mg of dry potassium bromide. The powder was then pressed into tablets using a mold. The samples were tested using a Fourier transform infrared spectrometer (Nicolet iS5) with a spectral scanning range of 400–4000 cm⁻¹. -1 4 cm resolution -1 .

[0050] 1.4.3 X-ray diffraction spectroscopy analysis

[0051] The X-ray diffraction pattern of the film was analyzed using an X-ray diffractometer (Brook XD3-Advance). After placing the sample, the room temperature conditions were set to 40 kV voltage and 30 mA current, with a scanning angle range of 10-80° and a scanning rate of 8° / min. The XRD spectrum of the sample was recorded and analyzed.

[0052] 1.4.4 Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC)

[0053] The thermal stability of the film was determined using a thermogravimetric analyzer (NETZSCH STA 449F3). Approximately 10 mg of each sample was placed in a standard aluminum pan and heated from 30°C to 600°C at a heating rate of 10°C / min under a nitrogen flow rate of 100 mL / min.

[0054] 1.4.5 Film-forming agent particle size distribution

[0055] Using Malvern laser particle size analysis, the sample was diluted 400 times with water and then directly tested on the instrument.

[0056] 1.4.6. Measurement of pH value of film agent

[0057] The membrane sample was diluted with purified water at 200, 400, 800 and 1000 times, and the pH was directly measured using a pH meter (Leici PHSJ-4A).

[0058] 1.4.7 Measurement of film rotation viscosity

[0059] The film agent samples were diluted with pure water at 200, 400, 800 and 1000 times, and their viscosity values ​​were measured using an NDJ-8S rotational viscometer. Rotor No. 0 was selected, and the test was conducted at room temperature.

[0060] 1.4.8 Contact Angle Measurement

[0061] Under normal indoor temperature conditions, cut (2×2) cm. 2 Fresh, clean rice leaves were horizontally fixed on the sample stage of an automated water droplet angle measuring instrument. 3 μL of film solution was dropped onto the surface of the rice leaf at a fixed height. The droplets on the leaf surface were photographed using a camera on the contact angle measuring instrument. The contact angle of the film solution on the rice leaf surface was calculated using a fitting analysis method.

[0062] 1.4.9 Wettability Test

[0063] Using a video optical contact angle meter, the film agent was diluted 200 times. Under normal indoor temperature conditions, a drop of the diluted solution was pipetted onto the surface of a rice leaf, and its dynamic spreading process was recorded.

[0064] The efficacy and safety of 1.5% and 2% aloe vera extract natural film-forming agents for disease prevention and control.

[0065] 1.5.1. Effect of film-based control

[0066] Rice leaf inoculation was performed using detached rice leaves with puncture wounds. Rice seedlings at 4-6 leaf stage were used. Leaf segments approximately 4-5 cm long from the lower part of the leaf were cut. Several shallow wounds (not penetrating the leaf) were made along the midrib with a dissecting needle. The leaf segments were placed on toothpicks. Then, CMC-Na@SA film and 2% aloe-emodin natural film agent diluted 200, 400, 800, and 1000 times were sprayed onto the rice leaves. After the leaves absorbed the diluted film agent solution, mycelial blocks approximately 0.5 cm × 0.5 cm in size were cut from the edge of colonies cultured on PSA and placed hyphae face down on the wounds on the leaf. The inoculated rice leaves were placed in darkness at 25-28℃ and 85-95% relative humidity for 30-32 hours, followed by light exposure at 25-28℃ and 85-95% relative humidity for 72 hours. The control group was treated with 0.02% (V / V) Tween 20 solution. The disease symptoms on the leaves were observed at any time, and each treatment was repeated 3 times.

[0067] 1.5.2 Microscopic examination

[0068] The bioactivity of CMC-Na@SA membrane and 2% aloe-emodin natural film against rice blast fungus was evaluated using the spore germination inhibition method. The polar growth, spore morphology, and appressorium germination status of conidia of rice blast fungus were observed at dilutions of 200, 400, 800, and 1000 times on CMC-Na@SA membrane and 2% aloe-emodin natural film.

[0069] 1.6% and 2% Aloe Vera Extract Natural Film-Forming Agent on Rice Seedling Growth

[0070] 1.6.1 Chlorophyll content in rice

[0071] To investigate the effects of CMC-Na@SA film and 2% aloe vera extract natural film on the chlorophyll content of rice, the chlorophyll content of rice was measured using a portable chlorophyll meter 7 days after the film was applied.

[0072] 1.6.2 Net photosynthetic rate of rice

[0073] The net photosynthetic rate of rice seedlings directly affects rice yield. Seven days after spraying CMC-Na@SA film and 2% aloe vera natural film agent, the net photosynthetic rate of rice seedlings was measured using a 3051D photosynthesis meter.

[0074] 1.7 Statistical Analysis

[0075] The experimental results were analyzed using DPS software. p < 0.05 indicated a significant difference, and p < 0.01 indicated a highly significant difference.

[0076] 2. Results and Analysis

[0077] 2.1. Development process of 2% Aloe Vera Extract Natural Film Agent

[0078] 2.1.1 Initial screening of film-forming substances

[0079] Natural substances with excellent hydrogel film-forming properties were screened based on four indicators: tensile strength (MPa), elongation (%), tensile strength (N), and film-forming properties. Six substances were selected for screening: sodium carboxymethyl cellulose (CMC-Na), sodium alginate (SA), guar gum, pectin, chitosan, and starch, all possessing suitable properties for hydrogel film formation and good water solubility. The results are shown in Table 1 and... Figure 1 As shown in Table 1, the film-forming effect evaluation table for film-forming substances is as follows: Figure 1 (a) Membrane solution, (b) Primary screening membrane, (c) 2% CMC-Na membrane, (d) 2% SA membrane, (e) 1% CMC-Na + 1% SA membrane. Most of the membrane-forming materials used to create hydrogels did not produce ideal results. Only sodium alginate, sodium carboxymethyl cellulose, and 1.0% sodium carboxymethyl cellulose + 1.0% sodium alginate exhibited strong toughness. Furthermore, the tensile strength, elongation, and tensile strength increased with increasing sodium carboxymethyl cellulose content, resulting in membranes with strong toughness. Figure 1 Sodium carboxymethyl cellulose (CMC) and sodium alginate both dissolve in water to form transparent hydrogels. CMC possesses certain thickening, emulsifying, and stability properties. Sodium alginate exhibits good film-forming, thickening, and water-retention properties and is biodegradable, making it suitable as an excipient for pharmaceutical preparations, requiring stability, solubility, viscosity, and safety. Because the film formed by the combination of these two materials exhibits strong toughness, CMC and sodium alginate can be used as the primary film-forming materials for subsequent hydrogel film preparation.

[0080] Table 1

[0081] After determining sodium carboxymethyl cellulose (CMC) and sodium alginate as the main film-forming substances, and considering that sodium alginate (SA) and calcium chloride can coagulate to form hollow, colorless sodium alginate particles, adding an appropriate amount of calcium chloride (CaCl2) can significantly increase the viscosity of sodium alginate and induce a hydrogel state. Calcium chloride was added at 5% of the amount of sodium alginate. The film-forming performance of the hydrogel CMC-Na:SA ratio was screened using four indicators: tensile strength (MPa), elongation (%), tensile strength (N), and film-forming properties. The main film-forming substances CMC-Na:SA ratios were 1:5, 1:3, 1:1, 3:1, and 5:1. Tensile strength (MPa), elongation (%), and tensile strength (N) were used as evaluation indicators to screen the CMC-Na:SA ratio. As shown in Table 2, which evaluates the film-forming effect of sodium carboxymethyl cellulose (CMC) and sodium alginate (SA) in different ratios, the film-forming effect is optimal when the CMC-Na:SA ratio is 3:1. Therefore, the optimal CMC-Na:SA ratio for the base film is determined to be 3:1.

[0082] Table 2

[0083] 2.1.2, Amount of lubricant added

[0084] Through screening of film-forming substances, CMC-Na:SA (3:1) was ultimately selected as the base film. To further improve the film-forming effect, the humectants were screened by measuring tensile strength and elongation. Common humectants such as polyethylene glycol 4000 (PEG 4000), vegetable oil, and glycerol were used for initial screening. Tensile strength (MPa), elongation (%), and tensile strength (N) were used as evaluation indicators to screen the humectants. The results are shown in Table 3. Table 3 is an evaluation table of the film-forming effect of humectants on sodium carboxymethyl cellulose-sodium alginate film. The humectant polyethylene glycol showed good tensile strength and tensile strength, but its elongation was very low. The humectant glycerol (glycerin) achieved an elongation of 276.94±20.49% for the film, but its tensile strength and tensile strength were very low (Table 3).

[0085] Table 3

[0086] The humectants polyethylene glycol (PEG) and glycerin were screened again, with tensile strength (MPa), elongation (%), and tensile strength (N) used as evaluation indicators to screen the film-forming effect of the humectants in order to obtain the optimal type and ratio of humectants. As shown in Table 4, which is the evaluation table of the film-forming effect of polyethylene glycol on sodium carboxymethyl cellulose-sodium alginate film, the PEG content was screened at proportions of 0.5%, 1.0%, 3.0%, 5.0%, and 10.0% of the sodium carboxymethyl cellulose-sodium alginate film (2g). With the increase of polyethylene glycol, there was no significant change in tensile strength (MPa), elongation (%), and tensile strength (N), indicating that the humectant effect was not obvious.

[0087] Table 4

[0088] Table 5 shows the evaluation of the film-forming effect of glycerol on sodium carboxymethyl cellulose-sodium alginate film. Screening was conducted using glycerol contents of 0.5%, 1.0%, 2.0%, 3.0%, and 4.0% per 100g of water in the sodium carboxymethyl cellulose-sodium alginate film. The results showed that with increasing glycerol content, tensile strength (MPa) and tensile force (N) decreased, while elongation (%) increased sharply. Furthermore, the film's toughness and viscosity increased, transforming from a thin, transparent dry film into a more viscous, soft gel, making it unsuitable for film preparation. Figure 2 c) Figure 2 The formulations include (a) a polyethylene glycol film-forming agent, (b) a glycerin film-forming agent, and (c) a film-forming agent containing 3% glycerin. Therefore, the type of wetting agent in the film-forming agent formulation is determined to be glycerin, and the addition amount is 2%.

[0089]

[0090] Table 5

[0091] 2.1.3. Amount of Aloe Vera Extract Added

[0092] Adding aloin to film-forming agents as an antibacterial active ingredient can produce antibacterial activity against rice blast fungus. To understand the effect of aloin on film-forming agents, this design selected two glycerol addition concentrations (0.5% and 2%, i.e., 0.5g and 2g of glycerol per 100g of film-forming agent), and three aloin addition concentrations (0%, 2%, and 4%). Tensile strength (MPa), elongation (%), and tensile strength (N) were used as evaluation indicators to screen the humectant varieties. The results are shown in Table 6. Table 6 is an evaluation table of the film-forming effect of aloin on sodium carboxymethyl cellulose-sodium alginate film-forming agents. In the film-forming agent containing 0.5% glycerol, the addition of aloin resulted in a decrease in film toughness, leading to a fragile film that could not be peeled off for measurement. In the film-forming agent containing 2% glycerol, although the toughness of the film decreased slightly with the increase of aloin, the elongation (%) remained high and the toughness was strong, and the film was in a soft gel-like state (2c). Based on the principle of reducing the cost of pharmaceuticals within a similar elongation range, the addition ratio of aloe vera extract in the film formulation was determined to be 2%.

[0093]

[0094] Table 6

[0095] 2.1.4 Drug loading capacity of aloe-emodin

[0096] Drug loading assay test, such as Figure 4 As shown, when the aloin content in the film poured into the plate was 0%, 2%, and 4%, the aloin was completely dissolved, and no components were precipitated within 48 hours. This indicates that the film can achieve 100% loading capacity for aloin content of 4% or less.

[0097] 2.1.5 Preparation process of 2% Aloe Vera Extract Natural Film

[0098] Through screening of film-forming substances, optimization of film formulation, and addition of the natural antibacterial product aloe vera extract, the hydrogel formulation of the aloe vera extract natural film was determined to be: 1.5% sodium carboxymethyl cellulose, 0.5% sodium alginate, 0.025% calcium chloride, 2% glycerin, 2% aloe vera extract, and deionized water to bring the total to 100%. The specific process is as follows: First, weigh 1.5 g of sodium carboxymethyl cellulose, 0.5 g of sodium alginate, and 0.025% calcium chloride, mix them thoroughly, and then dissolve them completely in 70 ml of deionized water using a magnetic stirrer (approximately 4 hours). Next, weigh 2 g of glycerin and place it in a 50 ml beaker, then dissolve it completely in 10 ml of deionized water. Finally, pour the glycerin solution into the mixture of sodium carboxymethyl cellulose, sodium alginate, and calcium chloride, and mix thoroughly using a magnetic stirrer. Next, weigh 2 grams of aloe vera extract and dissolve it fully in 10 ml of deionized water. Then pour it into the above mixture and add water to make up to 100 grams. Let it stand for 8-10 hours until all the bubbles disappear. This will give you a 2% aloe vera extract natural film (Table 7). Table 7 is the formula for the 2% aloe vera extract natural film.

[0099]

[0100] Table 7

[0101] 2.2 Physicochemical Properties of 2% Aloe Vera Extract Natural Film-Forming Agent

[0102] 2.2.1 Morphology of 2% Aloe Vera Extract Natural Film Formulation

[0103] The morphological characteristics and microstructure of the drug-free CMC-Na@SA film and the natural film containing 2% aloe vera extract were photographed and recorded using a digital camera and a scanning electron microscope, respectively. Figure 5 As shown, Figure 5 (a) CMC-Na@SA film solution, (b) 2% aloe vera natural film solution, (c) CMC-Na@SA film agent, (d) 2% aloe vera natural film agent; the 2% aloe vera natural film agent and the CMC-Na@SA film agent were both transparent hydrogels and exhibited good adhesion to the walls of small beakers, indicating that both film agents had good adhesion and film-forming properties. Furthermore, the 2% aloe vera natural film agent had better adhesion and a slightly brownish-yellow hue. Figure 5 a, b). Both film-forming agents have smooth and transparent surfaces, strong toughness, are not easily broken, and have excellent film-forming properties. Figure 5 c). By Figure 6 visible, Figure 6Electron microscopy results showed that the CMC-Na@SA film solution (200x, 500x, 1000x, 2000x, and 4000x) and the aloe vera natural film solution (200x, 500x, 1000x, 2000x, and 4000x) had a smooth and flat surface, demonstrating good adhesion and film-forming properties. Aloe vera was uniformly dispersed in the film solution in a wetted state.

[0104] 2.2.2 Fourier Transform Infrared Spectroscopy (FT-IR Spectroscopy)

[0105] The infrared absorption peaks of aloe vera natural film, aloe vera, and a blank gel film were determined using the potassium bromide pellet method. The formation of the mixture was confirmed by observing changes in the absorption peaks. The IR values ​​of the aloe vera natural film are shown below. Figure 7 a. The IR of CMC-Na@SA film is shown in [reference needed]. Figure 7 IR of b and aloe-emodin can be found in [link / reference]. Figure 7 c. A comparison of the three reveals that the infrared spectrum of aloe vera extract from pharmaceutical products and the infrared spectrum of aloe vera extract from natural film preparations show characteristic peaks of aloe vera extract at 915.02 cm⁻¹, 1035.80 cm⁻¹, 1089.33 cm⁻¹, and 1651.83 cm⁻¹ in the pharmaceutical film preparation. Figure 7 Figure (a) shows the FTIR spectrum of 2% aloe vera natural film, (b) shows the FTIR spectrum of CMC@SACMC-Na@SA film, (c) shows the FTIR spectrum of aloe vera, and (d) shows the comparison between the three FTIR spectra.

[0106] 2.2.3 X-ray diffraction (XRD) analysis of 2% aloe vera extract natural film-forming agent

[0107] Aloe verain is a white crystalline substance. For example... Figure 8 As shown, Figure 8 XRD patterns of CMC@SACMC-Na@SA film, (b) XRD pattern of aloe vera, (c) XRD pattern of 2% aloe vera natural film; after aloe vera is made into a film with sodium carboxymethyl cellulose and sodium alginate, its state changes from crystalline to amorphous. Figure 8 a) Aloe-emodin ( Figure 8 b) and 2% aloe vera extract natural film ( Figure 8The XRD characterization of c) is shown in the figure. A characteristic peak of aloin appears at 20.15. The characteristic peaks of the 2% aloin natural film and aloin in the XRD match, a phenomenon consistent with Fourier transform infrared spectroscopy. Furthermore, a large number of characteristic crystalline peaks of aloin can still be observed in the XRD spectrum of the 2% aloin natural film. The absence or weakening of many characteristic peaks of aloin indicates that the state of aloin in the physical mixture remains unchanged. This further confirms that compared to the crystalline form of aloin, the amorphous form of aloin in the natural film has higher solubility, dissolution rate, molecular energy, and mobility, thus being more conducive to improving the bioavailability of aloin.

[0108] 2.2.4 Thermogravimetric (TGA) and Differential Scanning Calorimetry (DSC) Analysis of 2% Aloe Vera Extract Natural Film

[0109] Thermogravimetric analysis (TGA) is commonly used to assess the decomposition rate and thermal stability of compounds. TGA reflects the mass change of a sample during heating. Figure 9 It can be seen that, Figure 9 The TGA-DSC curves for CMC@SA, CMC-Na@SA film, (b) Aloe vera, and (c) 2% Aloe vera natural film are shown in the figures. The solid line represents mass, and the dashed line represents the derivative of the solid line, i.e., the rate of mass change. As can be seen from the figures, CMC-Na@SA film, aloe vera, and 2% Aloe vera natural film all exhibit a period of rapid weight loss at temperatures of 208.49℃, 225.97℃, and 243.90℃ (where the downward peak of the curve is largest). The mass retention rates are 19.90%, 41.03%, and 30.19%, respectively. This indicates that the weight loss temperature of the pharmaceutical film is higher than that of the CMC-Na@SA film and aloe vera, and the combination of the two significantly improves the thermal stability of the film.

[0110] 2.2.5 Particle size distribution of 2% aloe vera extract natural film agent

[0111] To observe the particle morphology of CMC-Na@SA membrane and 2% aloe vera extract natural film agent after a 400-fold dilution. Particle size distribution results showed ( Figure 10The particle sizes (Dv(50), Dv(75), Dv(90), and Dv(95)) of the 2% aloe-emodin natural film were 144.50, 288.33, 436.38, and 537.38 μm, respectively; while those of the CMC-Na@SA film solution were 136.94, 226.41, 307.9, and 358.8 μm, respectively. This indicates that the CMC-Na@SA film has smaller particle size and more uniform dispersion. After the addition of the drug, the difference between the two films was not significant. Both films easily adhere to rice leaves and around the rice blast fungus, increasing their biological activity against the fungus.

[0112] 2.2.6 Physicochemical properties of 2% aloe vera extract natural film-forming agent

[0113] To clarify the physicochemical properties of CMC-Na@SA film and aloe vera extract natural film, their viscosity, spreadable area, pH value, and contact angle were measured. The results are as follows: Figure 11 Regarding pH, the CMC-Na@SA membrane and the 2% aloe vera natural film both have a pH between 6.2 and 7.4. The CMC-Na@SA membrane is weakly alkaline, while the 2% aloe vera natural film is weakly acidic. This means that the addition of the weakly acidic aloe vera makes the film slightly acidic. The optimal pH range for microbial growth is 6.5-7.5, and since the 2% aloe vera natural film is weakly acidic, it exhibits a certain antibacterial effect. Regarding viscosity, the viscosity of both CMC-Na@SA film and 2% aloe vera natural film gradually decreased with increasing dilution ratio. Furthermore, the viscosity of aloe vera natural film at the same dilution ratio was lower than that of CMC-Na@SA film. Additionally, the viscosity of CMC-Na@SA film gradually decreased with increasing dilution ratio, while the viscosity of 2% aloe vera natural film was higher at the same dilution ratio. The 2% aloe vera natural film formed after adding aloe vera dissolved immediately upon contact with water, indicating a relatively low viscosity.

[0114] 2.2.7. Determination of the contact angle of 2% aloe vera extract.

[0115] Comparing CMC-Na@SA film and 2% aloe vera natural film at the same 200-fold dilution, the results showed that the average contact angle of the CMC-Na@SA film was 32.51±4.03°, with a left contact angle of 31.22±2.89° and a right contact angle of 33.80±5.18°. The average contact angle of the 2% aloe vera natural film was 14.59±1.08°, with a left contact angle of 14.30±2.73° and a right contact angle of 14.87±0.56°. At the same 200-fold dilution, the 2% aloe vera natural film had a smaller contact angle than the CMC-Na@SA film. The contact angle of the 2% aloe vera natural film was below 30°. Figure 12 The results indicate that 2% aloe vera extract natural film agent can spread rapidly after being dripped onto the surface of rice leaves.

[0116] 2.2.8. Wetting properties of 2% aloe vera extract natural film-forming agent

[0117] To observe the dynamic wetting and spreading effect of a 2% aloe vera natural film-forming agent on rice leaves, the agent was diluted 200 times and dripped onto the surface of rice leaves, and its dynamic spreading process was recorded. Figure 13 As shown, after a drop of 2% aloe vera natural film-forming agent is applied to the surface of rice leaves, it takes 25 seconds to fully spread. It is worth noting that when the 2% aloe vera natural film-forming agent is diluted 400 times, it is instantly absorbed upon application to the rice leaves, making measurement and recording impossible. This is mainly because the 2% aloe vera natural film-forming agent has excellent water solubility, meaning it also has excellent diffusion and wetting properties. This indicates that it has good wetting properties, suggesting minimal droplet loss after field spraying and a low risk of off-target application. This result is consistent with the low contact angle of the aloe vera natural film-forming agent on the rice leaf surface.

[0118] 2.3. The efficacy and safety of 2% aloe vera extract natural film-forming agent for disease prevention and control.

[0119] Given the unclear efficacy and safety of CMC-Na@SA film and 2% aloe-emodin natural film against rice blast, this study investigated their inhibitory effects on spore germination. CMC-Na@SA film and 2% aloe-emodin natural film were sprayed on rice seedlings to assess their safety. The study primarily used measurements of chlorophyll content and photosynthesis to elucidate the efficacy of CMC-Na@SA film and 2% aloe-emodin natural film against rice blast and their impact on rice growth and development, aiming to provide a safe new formulation for rice blast control in rice production.

[0120] 2.3.1 Inoculation by scrambling detached rice leaves

[0121] Rice leaf inoculation was performed using detached rice leaf scuffing inoculation (Zhang Haiwang et al., 2014), such as... Figure 14 As shown, a 2% aloe vera natural film agent diluted 800× resulted in mild disease development, while a 1000× dilution showed more pronounced symptoms. Furthermore, spraying rice leaves with CMC-Na@SA film also demonstrated some inhibitory and disease-preventing effects.

[0122] 2.3.2 Appendices Germination Inhibition Method

[0123] The bioactivity of CMC-Na@SA film and 2% aloe-emodin natural film formulation against rice blast fungus was evaluated using the appressorium germination inhibition method. Results are as follows: Figure 15 As shown, Figure 15 (a) CMC-Na@SA film and (b) 2% aloe-emodin natural film; CMC-Na@SA film and 2% aloe-emodin natural film were used to treat conidia of *Bacillus oryzae* at the same dilution ratio for the same duration (6 h), which affected the germination and appressorium formation of *Bacillus oryzae* spores. *Bacillus oryzae* spores treated with CMC-Na@SA film and 2% aloe-emodin natural film resulted in a prolonged appressorium formation time. The smaller the dilution ratio, the more likely appressorium formation occurred at 800 and 1000 times dilution, but the appressorium color was lighter than the control (CK), and the number was less. The conidial germ tubes of *Bacillus oryzae* treated with 2% aloe-emodin natural film were longer. Therefore, CMC-Na@SA film can delay the formation of *Bacillus oryzae* appressorium, and 2% aloe-emodin natural film has a good inhibitory effect on the germination of conidia and the formation of appressorium.

[0124] 2.3.3 Effects of 2% Aloe Vera Extract Natural Film on Rice Seedling Growth

[0125] Rice chlorophyll content

[0126] To investigate the effects of CMC-Na@SA film and 2% aloe vera extract natural film on the chlorophyll content of rice, the chlorophyll content of rice was measured using a portable chlorophyll meter 7 days after the film was applied. The results are as follows: Figure 16 As shown, the chlorophyll content of rice treated with CMC-Na@SA membranes (200, 400, 800, and 1000 times dilution) was 24.8, 25.7, 25.3, and 24.5 SPAD, respectively, which was not significantly different from the chlorophyll content of the control group (25.6 SPAD). The chlorophyll content of rice treated with 2% aloe vera extract natural film (200, 400, 800, and 1000 times dilution) was 29.8, 29.7, 29.3, and 30.0 SPAD, respectively, which was also not significantly different from the control group. Therefore, CMC-Na@SA membranes and 2% aloe vera extract natural film have little effect on chlorophyll synthesis in rice at dilution ratios of 200 to 1000 times.

[0127] Rice net photosynthetic rate

[0128] The net photosynthetic rate of rice seedlings directly affects rice yield. Seven days after spraying with CMC-Na@SA film and 2% aloe vera extract natural film agent, the net photosynthetic rate of rice seedlings was measured. Results are as follows: Figure 17 The net photosynthetic rates of rice treated with CMC-Na@SA membranes (200, 400, 800, and 1000 times) were 11.3, 14.5, 10.3, and 10.3 μmol / m, respectively. 2 •s, compared to a net photosynthetic rate of 13.8 μmol / m in the control group. 2 The differences were not significant. The net photosynthetic rates of rice treated with 2% aloe vera extract natural film agent (200, 400, 800, and 1000 times dilution) were 12.3, 13.5, 13.3, and 12.8 μmol / m², respectively. 2 The results showed no significant difference compared to the control group. Therefore, CMC-Na@SA membrane and 2% aloe vera extract natural film agent had no significant effect on the net photosynthetic rate of rice seedlings.

[0129] 3. Conclusions and Discussion

[0130] The results of a study on the control efficacy and safety of 2% aloe-emodin natural film agent showed that a 2% aloe-emodin natural film agent diluted 800× resulted in mild disease, while a 1000× dilution showed more pronounced symptoms. Furthermore, spraying rice leaves with CMC-Na@SA film also had a certain inhibitory and disease-preventing effect. This may be because the film agent contains the wetting agent glycerol, which reduces the turgor pressure difference in appressoria, resulting in insufficient turgor pressure for appressoria to invade rice leaves. Treatment of rice blast fungus appressoria formation with CMC-Na@SA film and 2% aloe-emodin natural film agent at the same dilution ratio and for the same duration (6h) significantly affected the formation of appressoria. Rice blast fungus (CK) could form round, black appressoria within 6h. Rice blast fungus spores treated with CMC-Na@SA film and 2% aloe-emodin natural film agent resulted in a prolonged appressoria formation time, and the formed appressoria were lighter in color, fewer in number, and had longer conidial germ tubes than CK. Therefore, 2% aloe vera extract natural film and CMC-Na@SA film can delay the formation of appressorium of rice blast fungus, and have a good inhibitory effect on the germination of conidia and the formation of appressorium. Chlorophyll plays an important role in light absorption during photosynthesis and is the main pigment for photosynthesis in rice and other plants. CMC-Na@SA film and 2% aloe vera extract natural film, when diluted 200 to 1000 times, have little effect on chlorophyll synthesis in rice. The net photosynthetic rate of rice seedlings directly affects rice yield. CMC-Na@SA film and 2% aloe vera extract natural film have no significant effect on the net photosynthetic rate of rice seedlings. Therefore, 2% aloe vera extract natural film is harmless to rice plants and has high safety for rice.

[0131] 2% Aloe Vera Extract Natural Film is a safe and effective plant protection film. Its active ingredient, aloe vera extract, is a harmless natural plant product that significantly affects the formation and color of appressorium spores of rice blast fungus. The base film, composed of sodium carboxymethyl cellulose and sodium alginate, is an edible film component. The wetting agent, glycerin, is also harmless to rice, organisms, and the environment, and even reduces the internal and external turgor pressure of appressorium spores, thus playing a partial role in preventing invasion. Therefore, 2% Aloe Vera Extract Natural Film provides an effective and safe new formulation for the control of rice blast in rice production.

Claims

1. Aloe vera bitter element natural film agent, characterized in that, Includes 2% aloe vera extract, 1.5% sodium carboxymethyl cellulose, 0.5% sodium alginate, 0.025% calcium chloride, 2% glycerin, and the remainder deionized water.

2. A method for preparing a natural aloe vera film agent, characterized in that, First, weigh out 1.5g of sodium carboxymethyl cellulose, 0.5g of sodium alginate, and 0.025% calcium chloride, mix them thoroughly, and then dissolve them completely in 70ml of deionized water using a magnetic stirrer. Next, weigh out 2g of glycerin and dissolve it completely in 10ml of deionized water in a 50ml beaker. Then, pour the glycerin solution into the mixture of sodium carboxymethyl cellulose, sodium alginate, and calcium chloride, and mix thoroughly using a magnetic stirrer. Then, weigh out 2g of aloe vera extract, dissolve it completely in 10ml of deionized water, and pour it into the above mixture. Finally, add water to make up to 100g, let it stand for 8-10 hours until all the bubbles disappear, and you will get a 2% aloe vera extract natural film.

3. The method for analyzing the characteristics of the aloe vera bitter substance natural film agent as described in claim 1, characterized in that, The physicochemical properties analysis of aloe vera bittering natural film agent included the following analytical methods: 1) The adhesion and film-forming properties of natural film-forming agents were analyzed using a digital camera and scanning electron microscope; 2) Fourier transform infrared spectroscopy and X-ray diffraction were used to analyze the natural film-forming agent of aloe vera and the crystalline form of aloe vera. 3) Combined thermogravimetric analysis and differential scanning calorimetry (DSC) are used to analyze and determine the possible reaction processes of the material and to identify the nature of the transition peaks; 4) Observe the particle size distribution of the aloe vera natural film agent, and measure its viscosity, spreading area, pH value and contact angle to observe the wetting performance of the aloe vera natural film agent. 5) The efficacy of aloe vera extract natural film agent against rice blast and its effect on rice growth and development were tested by inoculating detached rice leaves with abrasives, inhibiting appressorium germination, and measuring the chlorophyll content of rice.

Images