A preparation method of a hemp leaf essential oil nanostructured lipid carrier

Abstract

A preparation method of a hemp leaf essential oil nanostructured lipid carrier belongs to the field of hemp essential oil preparation. The method is as follows: selecting glycerin monostearate as a solid lipid, selecting caprylic acid / capric acid triglyceride as a liquid lipid, and selecting soybean lecithin and poloxamer 188 as emulsifiers; the solid lipid and the liquid lipid are melted in a 65-75 DEG C water bath kettle at a mass ratio of 0.5-1.5:1, hemp leaf essential oil is added, then the emulsifiers melted at 65-75 DEG C at 2%-4% are added, an initial emulsion is obtained, the initial emulsion is heated and stirred in a magnetic stirrer, then is placed in an ultrasonic pulverizer for ultrasonic treatment, and then is filtered. The preparation process of the melt ultrasonic method adopted in the application is simple in operation, clear in parameters (melt temperature 70 DEG C, stirring speed 1000 rpm, ultrasonic time 14 min, etc.), the raw materials used are common auxiliaries (glycerin monostearate, caprylic acid / capric acid triglyceride, soybean lecithin and poloxamer 188), are widely sourced and controllable in cost, and have a good industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of hemp essential oil preparation, specifically relating to a method for preparing a nanostructured lipid carrier for hemp leaf essential oil. Background Technology

[0002] Hemp essential oil faces severe technical bottlenecks in practical applications: it is mainly composed of highly volatile and heat-sensitive terpenoid compounds, which generally suffer from poor water solubility, low photothermal stability, and easy oxidation and deterioration. During processing, storage, and use, the active ingredients are easily lost or degraded through volatilization, resulting in low bioavailability and severely restricting the development of its high-value products.

[0003] To overcome these shortcomings and improve the stability and bioavailability of essential oils, researchers have attempted to use traditional emulsion or cyclodextrin encapsulation techniques. However, traditional emulsion systems are thermodynamically unstable, prone to Austronesian ripening, stratification, or demulsification during long-term storage, and have relatively limited oil loading capacity. While cyclodextrin encapsulation can improve water solubility, its hydrophobic cavity volume is limited, making it difficult to efficiently encapsulate high-molecular-weight terpene mixtures, and the encapsulation efficiency often fails to meet industrial requirements. Nanostructured lipid carriers (NLCs), as a novel lipid delivery system, have shown irreplaceable advantages. NLCs are constructed by mixing solid and liquid lipids in a certain proportion. The introduction of liquid lipids disrupts the perfect lattice structure of solid lipids, forming more irregular defect spaces. This not only significantly improves the drug loading and encapsulation efficiency of lipophilic active ingredients (such as hemp essential oil), but also effectively prevents drug leakage and precipitation during storage. In addition, NLCs have excellent biocompatibility, controllable sustained-release properties and high physical stability, and have been proven to significantly improve the physicochemical stability of plant essential oils such as tea tree oil and rosemary oil. Summary of the Invention

[0004] In order to overcome the above-mentioned problems in the background art, the present invention provides a method for preparing a nanostructured lipid carrier of hemp leaf essential oil.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A method for preparing a hemp leaf essential oil nanostructured lipid carrier, the method specifically comprising: selecting glyceryl monostearate as a solid lipid, caprylic / capric triglyceride as a liquid lipid, and selecting soybean lecithin and palosham 188 as emulsifiers; melting the solid lipid and liquid lipid in a water bath at 65-75℃ at a mass ratio of 0.5-1.5:1, adding hemp leaf essential oil, and then adding 2%-4% of the emulsifier melted at 65-75℃ to obtain a pre-emulsion; heating and stirring the pre-emulsion in a magnetic stirrer, then ultrasonicating it in an ultrasonic pulverizer, and finally passing it through a membrane to obtain the hemp leaf essential oil nanostructured lipid carrier.

[0007] Furthermore, the stirring speed was 900~1000 rpm, the colostrum stirring time was 20~24 min, the ultrasonic time was 14 min (5 s intervals of 5 s), and the ultrasonic amplitude was 91%.

[0008] Furthermore, the amount of lipids used is 1% to 1.5%.

[0009] The advantages of this invention over the prior art are as follows:

[0010] (1) A hemp leaf essential oil nanostructured lipid carrier with small particle size, uniform distribution, and stable system is provided: The hemp leaf essential oil nanostructured lipid carrier prepared by this invention has an average particle size of 68.91 nm, exhibits a normal distribution, and has a polydispersity index (PDI) of 0.219, indicating a narrow particle size distribution, uniform nanoparticle size, and that the nanoparticles are not prone to aggregation or agglomeration due to mutual collisions; the zeta potential is -28.2 mV, the surface is negatively charged, the interparticle repulsion is large, and the system has good physical stability. Compared with existing essential oil nano-preparations, this invention has significant advantages in particle size control and system stability.

[0011] (2) Significantly improved the encapsulation rate of hemp leaf essential oil: The present invention uses a nanostructured lipid carrier to encapsulate hemp leaf essential oil, with an encapsulation rate of 75.4%, which can effectively encapsulate the active ingredients of the essential oil and reduce its volatilization and loss during storage and use.

[0012] (3) Overcoming the application defects of plant essential oils such as easy volatility and poor stability: Hemp leaf essential oil, as a plant essential oil, has inherent defects such as easy volatility, poor stability, and weak hydrophilicity, which limits its application in the fields of medicine, cosmetics, and food. This invention encapsulates the essential oil in a nanostructured lipid carrier. Utilizing the unique lipid properties of mixing solid and liquid lipids, it can effectively protect the essential oil from adverse environmental factors, improving its stability, bioavailability, and safety. In addition, the nanostructured lipid carrier has advantages such as good biocompatibility and controlled release, which can prolong the effective action time of the essential oil.

[0013] (4) The process is simple and the parameters are clear, making it suitable for industrial production: The melt ultrasonic method used in this invention is easy to operate and the parameters are clear (melting temperature 70 ℃, stirring speed 1000 rpm, ultrasonic time 14 min, etc.). The raw materials used are all common auxiliary materials (glyceryl monostearate, caprylic / capric triglyceride, soybean lecithin, poloxamer 188), which are widely available and cost controllable, and have good prospects for industrial application. Attached Figure Description

[0014] Figure 1 Figure showing the effect of emulsifier dosage on lipid carrier particle size;

[0015] Figure 2 This is a graph showing the effect of solid-liquid ratio on the particle size of lipid carriers.

[0016] Figure 3 This is a graph showing the effect of lipid dosage on lipid carrier particle size.

[0017] Figure 4 The graph shows the effect of the interaction between emulsifier dosage (%) and solid-liquid ratio (g / g) on ​​particle size.

[0018] Figure 5 This is a particle size distribution diagram of a nanostructured lipid carrier.

[0019] Figure 6 This is a zeta potential distribution diagram of a nanostructured lipid carrier;

[0020] Figure 7 Chromatogram of β-caryophyllene;

[0021] Figure 8 This is a standard curve for β-caryophyllene.

[0022] Figure 9 This is a chromatogram of liposomes.

[0023] Figure 10 The graph shows the effect of the interaction between emulsifier dosage (%) and lipid dosage (%) on particle size.

[0024] Figure 11 The graph shows the effect of the interaction between solid-liquid ratio (g / g) and lipid content (%) on particle size. Detailed Implementation

[0025] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention that do not depart from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention.

[0026] Example 1:

[0027] A method for preparing a hemp leaf essential oil nanostructured lipid carrier, comprising: extracting hemp leaf essential oil; selecting glyceryl monostearate as the solid lipid and caprylic / capric triglyceride as the liquid lipid; and selecting soybean lecithin and palosham 188 as emulsifiers. The solid lipid and liquid lipid are melted in a 70°C water bath at different mass ratios (solid-liquid ratios). Hemp leaf essential oil is added, followed by the addition of emulsifiers melted at 70°C in different proportions to obtain a pre-emulsion. The pre-emulsion is heated and stirred in a magnetic stirrer, then ultrasonically pulverized in an ultrasonic grinder, and finally filtered through a membrane to obtain the hemp leaf essential oil nanostructured lipid carrier.

[0028] I. Single-factor experiment: When conducting the single-factor experiment to investigate the formulation, the mass of hemp leaf essential oil was 0.1g, the melting temperature was 70℃, the stirring speed was 1000rpm, the stirring time of the initial emulsion was 22min, the ultrasonic time was 14min (5s intervals of 5s), and the ultrasonic amplitude was 91%.

[0029] (1) Selection of emulsifier dosage: The emulsifier ratio was 1:1, the lipid dosage was 2%, and the solid-liquid ratio was 1:1. The effects of five different emulsifier dosages (2%, 3%, 4%, 5%, 6%) on the particle size of the hemp leaf essential oil nanostructure lipid carrier were investigated.

[0030] (2) Selection of solid-liquid ratio: The emulsifier ratio was 1:1, the emulsifier dosage was 3%, and the lipid dosage was 2%. The effects of five different solid-liquid ratios (glyceryl monostearate: caprylic / capric triglyceride) (2.5:1, 2:1, 1.5:1, 1:1, 0.5:1) on the particle size of the hemp leaf essential oil nanostructure lipid carrier were investigated.

[0031] (3) Selection of lipid dosage: The emulsifier ratio was 1:1, the emulsifier dosage was 3%, and the solid-liquid ratio was 1:1. The effects of five different lipid dosages (0.5%, 1%, 1.5%, 2%, 2.5%) on the particle size of the hemp leaf essential oil nanostructure lipid carrier were investigated.

[0032] II. Response Surface Optimization Experiment: Based on the single-factor experiment, the particle size (Y) of the hemp leaf essential oil nanostructure lipid carrier was used as the evaluation index, and the three factors of emulsifier dosage (X1), solid-liquid ratio (X2), and lipid dosage (X3) were used as independent variables. The experimental factors and level design are shown in Table 1.

[0033] Table 1. Box-Behnken Design Factors and Levels

[0034]

[0035] Particle size, polydispersity index (PDI), and zeta potential analysis: Particle size analysis is one of the main characterization aspects of nanoparticle drug delivery systems. It involves accurately measuring the particle size of nanoparticles and analyzing their distribution using specific detection methods. The polydispersity index (PDI) is a measure of particle size distribution; a smaller PDI value indicates a narrower particle size distribution and more uniform nanoparticle size. A larger PDI value indicates a wider particle size distribution range, but also suggests the possibility of nanoparticles agglomerating due to collisions, resulting in poor physical stability. Zeta potential refers to the shear surface potential of a particle. A larger absolute value indicates more positive or negative charges carried by the particle, stronger repulsive forces between particles, and a relatively stable system; conversely, a smaller value indicates poorer stability. Hemp essential oil nanostructured lipid carriers were diluted 100-fold, and their particle size distribution and zeta potential were measured using a Malvern nanoparticle size and zeta potential analyzer. The measurements were performed in triplicate, and the average value was calculated.

[0036] Standard curve plotting and encapsulation ratio calculation

[0037] (1) Gas chromatography conditions: The chromatographic column was an ON-5 capillary column (30m×0.32mm×0.25um); the carrier gas was nitrogen, with a flow rate of 2.0mL / min; the split ratio was 9:1, and the injection volume was 1μL; the temperature program was as follows: the initial temperature was 60℃, held for 2min, then increased to 120℃ at a rate of 20℃ / min, then increased to 146℃ at a rate of 2℃ / min, held for 1min, and then increased to 280℃ at a rate of 20℃ / min, held for 10min; the detector was FID, with a temperature of 250℃; the injection port temperature was 250℃.

[0038] (2) Preparation of reference solution

[0039] Take 0.1 ml of β-caryophyllene reference standard (concentration of 0.905 g / ml) and add anhydrous ethanol to prepare a reference standard stock solution with a mass concentration of 9.05 mg / mL. Store it in a refrigerator at 4℃. When using, dilute it again to prepare a working solution of the reference standard at the required concentration level.

[0040] (3) Preparation of the test solution

[0041] Accurately measure 0.5 mL of the nanostructured lipid carrier and place it in a 10 mL centrifuge tube. Add 2 mL of methanol and sonicate in a water bath at room temperature for 10 min. Then add 2 mL of n-hexane and sonicate in a water bath at room temperature for 5 min. Let it stand, take the n-hexane layer, and remove the n-hexane using a nitrogen blower. Add 1 mL of anhydrous ethanol and filter through a 0.45 μm filter membrane to obtain the final product.

[0042] (4) Plotting the standard curve

[0043] Accurately measure the reference standard stock solution and prepare a series of β-caryophyllene ethanol solutions of different concentrations. Measure the peak area at each concentration. Plot a standard curve with the concentration of β-caryophyllene in the reference standard solution as the ordinate and the peak area integral value as the abscissa.

[0044] (5) Encapsulation ratio calculation

[0045] Accurately measure 500 μL of lipid carrier and place it in the inner tube of a 100 KD ultrafiltration tube. Centrifuge at 7830 rpm for 60 min to collect nanoparticles. Prepare the test solution according to method (3). Measure under the chromatographic conditions of (1), record the peak area of ​​β-caryophyllene, and substitute it into the standard curve drawn in (4) to determine the mass concentration. Calculate the encapsulation rate. The formula is: Encapsulation rate = Encapsulated drug amount / Total drug amount × 100%.

[0046] Data Processing: Based on the Box-Behnken central composite design principle and combined with the results of single-factor experiments, three factors—emulsifier dosage (X1), solid-liquid ratio (X2), and lipid dosage (X3)—were selected as independent variables. The particle size (Y) of the hemp leaf essential oil nanostructure lipid carrier was used as the response value. Design-Expert 8.0.6 software was used to perform regression analysis on the experimental data to obtain the optimal formulation. SPSS 17.0 was used for Duncan multiple comparison analysis of the data, and graphs were generated using GraphPad Prism 5. Each experiment was repeated three times, and the average value was taken.

[0047] III. Results and Analysis

[0048] Single-factor experiment results

[0049] (1) Effect of emulsifier dosage on particle size of hemp leaf essential oil nanostructured lipid carrier

[0050] The effect of emulsifier dosage on the particle size of hemp leaf essential oil nanostructured lipid carriers is shown in [reference needed]. Figure 1 As shown in the figure, the particle size is smallest when the emulsifier dosage is 3%. When the emulsifier dosage is greater than 3%, the particle size begins to increase. Therefore, an emulsifier dosage of 3% is preferable.

[0051] (2) Effect of solid-liquid ratio on particle size of hemp leaf essential oil nanostructured lipid carrier

[0052] The effect of solid-liquid ratio on the particle size of hemp leaf essential oil nanostructured lipid carriers is shown in [reference needed]. Figure 2 As shown in the figure, the particle size decreases with increasing solid-liquid ratio. The particle size is smallest when the solid-liquid ratio is 1:1, and subsequently, the particle size increases with further increases in the solid-liquid ratio. Therefore, a solid-liquid ratio of 1:1 is preferable.

[0053] (3) Effect of lipid dosage on the particle size of hemp leaf essential oil nanostructured lipid carrier

[0054] The effect of lipid dosage on the particle size of hemp leaf essential oil nanostructured lipid carriers is shown in the figure. Figure 3 As shown in the figure, the particle size is smallest when the lipid content is 1.5%. As the lipid content increases, the particle size begins to increase. Therefore, a lipid content of 1.5% is appropriate.

[0055] Response surface analysis

[0056] (1) Box-Behnken experimental design and results

[0057] Based on the results of the single-factor experiments, a response surface methodology optimization experiment was conducted on the formulation of hemp leaf essential oil nanostructured lipid carrier. The results are shown in Table 2.

[0058] Table 2. Experimental Design and Results of Response Surface Analysis

[0059]

[0060] (2) Establishing a mathematical model and performing analysis of variance

[0061] The results in Table 2 were analyzed using Design-Expert 8.0.6 software, and the regression equation for the actual variables (emulsifier dosage (X1), solid-liquid ratio (X2), and lipid dosage (X3)) was obtained as follows: Y = 106.7325 - 11.2318X1 - 11.004X2 - 25.8635X3 - 8.05X1X2 + 0.455X1X3 - 3.52X2X3 + 2.92925X1 2 +23.767X2 2 +10.257X3 2 The results of the analysis of variance (Table 3) show that the model is significant overall (F=23.41, p=0.0002), and the model determination coefficient R0 is [value missing]. 2 =0.9678, indicating that the model can explain 96.78% of the response value variation; adjusted coefficient of determination R 2 adj =0.9265 and the predictive coefficient of determination R 2 pred =0.7122, a relatively small difference, and a model precision (Adeq Precision) of 16.035 (much greater than 4), further verifying the model's reliability and predictive ability. Meanwhile, the lack-of-fit term was not significant (p=0.3661), eliminating significant deviations between the model and pure error, proving a good model fit. The coefficient of variation (CV%) was 1.91%, less than 10%, demonstrating high experimental precision and good repeatability. Significance analysis of various factors and interactions showed that: the single-factor emulsifier dosage (X1) had no significant effect on particle size, the solid-liquid ratio (X2) had an extremely significant effect (p=0.0002), and the lipid dosage (X3) had a significant effect (p=0.0289); the interaction X1X2 was significant (p=0.0008), while X1X3 and X2X3 were not significant (p>0.05); the quadratic term X1... 2 X3 2 All were highly significant (p<0.01), X2 2 The results were extremely significant (p<0.0001), indicating a clear surface effect between the factors and particle size.

[0062] Table 3 Response Surface ANOVA

[0063]

[0064] Note: p<0.05 indicates a significant difference; p<0.01 indicates that the difference is extremely significant; p<0.001 indicates that the difference is extremely significant.

[0065] (3) Response surface analysis and condition optimization

[0066] Using Design Expert software, response surface plots were drawn based on the regression equation, and the interaction between each factor and the response value was visually analyzed. Figure 4 , Figure 10 , Figure 11 The figure represents the effect of the other two variables on particle size when any two of the variables—emulsifier dosage (X1), solid-liquid ratio (X2), and lipid dosage (X3)—are taken at zero levels. A steeper response surface indicates a stronger interaction between the two factors. The figure shows that the interaction between emulsifier dosage (X1) and solid-liquid ratio (X2) is highly significant, while the interaction between emulsifier dosage (X1) and lipid dosage (X3) is not significant, nor is the interaction between solid-liquid ratio (X2) and lipid dosage (X3). This result is consistent with the analysis of variance results in Table 3.

[0067] (4) Optimal formulation conditions and verification

[0068] The optimal formulation, obtained through response surface methodology, was 2.956% emulsifier, 0.831:1 solid-liquid ratio, and 1.338% lipid. Under these conditions, the predicted liposome size was 68.264 nm, with a desirability of 0.966, indicating that this combination can efficiently obtain liposomes of the target size. To verify the model's reliability, three parallel validation experiments were conducted using the adjusted optimal formulation (3% emulsifier, 0.8:1 solid-liquid ratio, and 1.3% lipid). The actual average particle size was measured to be 68.91 nm, with a relative error of less than 3% compared to the predicted value, indicating that the model is accurate, reliable, and has practical value.

[0069] Particle size, PDI, and Zeta potential analysis: The particle size distribution, PDI, and Zeta potential of the hemp essential oil nanostructured lipid carrier were determined using a Malvern nanoparticle size and Zeta potential analyzer. The analysis was performed in triplicate, and the average value was calculated. Figure 5 It can be seen that the hemp essential oil liposomes exhibit a normal distribution in particle size, with an average particle size of 68.91 nm and a PDI of 0.219, indicating that the liposomes have a relatively suitable particle size and a relatively uniform particle size distribution. Figure 6 It can be seen that the liposome has a Zeta potential of -28.2mV, exhibits negative surface charge, and is relatively stable.

[0070] Calculation of liposome encapsulation efficiency: Accurately measure the reference standard stock solution and prepare a series of β-caryophyllene ethanol solutions with concentrations of 0.1810, 0.2715, 0.3620, 0.5430, 0.6335, and 0.7240 mg / mL. Measure the peak area at each concentration. The gas chromatogram of the β-caryophyllene standard is shown below. Figure 7 A standard curve was plotted with the concentration of β-caryophyllene in the reference solution as the ordinate and the peak area integral as the abscissa, yielding the curve y = 4 × 10⁻⁶. -7 x-0.1165, R 2 =0.9979, see Figure 8 Within this range, the linear relationship is good. The encapsulation efficiency of the hemp leaf essential oil nanostructured lipid carrier prepared under the optimal process formulation, calculated using the standard curve, is 75.4%. The gas chromatogram of the liposomes is shown below. Figure 9 .

[0071] Based on single-factor experiments, the formulation of hemp leaf essential oil nanostructured lipid carrier was optimized using response surface methodology. Analysis of variance showed that the single-factor effect of emulsifier dosage (X1) on particle size was not significant, the effect of solid-liquid ratio (X2) was extremely significant, and the effect of lipid dosage (X3) was significant. The interaction X1X2 was significant (p=0.0008), while X1X3 and X2X3 were not significant (p>0.05). The quadratic term X1... 2 X3 2 All were highly significant (p<0.01), X2 2 The results were highly significant (p<0.0001), indicating a clear surface effect between the factors and particle size. Considering practical feasibility, the optimal formulation parameters were adjusted to: emulsifier dosage 3%, solid-liquid ratio 0.8:1, and lipid dosage 1.3%. Validation experiments were conducted based on these formulation parameters, and the actual average particle size was measured to be 68.91 nm, with a relative error of less than 3% compared to the predicted value of 68.264 nm, indicating that the model is accurate, reliable, and has practical value. The hemp essential oil liposomes exhibited a normal particle size distribution with a PDI of 0.219, indicating that the liposomes have a suitable particle size and a relatively uniform particle size distribution. The liposomes had a Zeta potential of -28.2 mV, exhibited negative surface charge, and showed good system stability. The encapsulation efficiency of the lipid carrier was 75.4%.

Claims

1. A method for preparing a nanostructured lipid carrier of hemp leaf essential oil, characterized in that: The method specifically involves: selecting glyceryl monostearate as a solid lipid, caprylic / capric triglyceride as a liquid lipid, and selecting soybean lecithin and palosham 188 as emulsifiers; melting the solid lipid and liquid lipid in a water bath at a mass ratio of 0.5~1.5:1 at 65~75℃, adding hemp leaf essential oil, and then adding 2%~4% of the emulsifier melted at 65~75℃ to obtain a pre-emulsion; heating and stirring the pre-emulsion in a magnetic stirrer, then ultrasonicating it in an ultrasonic pulverizer, and finally passing it through a membrane to obtain a hemp leaf essential oil nanostructured lipid carrier.

2. The method for preparing a hemp leaf essential oil nanostructured lipid carrier according to claim 1, characterized in that: The stirring speed was 900~1000 rpm, the colostrum stirring time was 20~24 min, the ultrasonic time was 14 min (5 s intervals of 5 s), and the ultrasonic amplitude was 91%.

3. The method for preparing a hemp leaf essential oil nanostructured lipid carrier according to claim 1, characterized in that: The lipid dosage is 1%~1.5%.

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