Glycyrrhizic acid-pigeon pea leaf stilbene type self-assembled nanoparticles

Abstract

The invention provides glycyrrhizic acid-pigeonpea leaf stilbene self-assembled nanoparticles which are prepared by the following steps: dissolving pigeonpea leaf stilbene compounds in absolute ethyl alcohol to obtain an organic phase; dissolving glycyrrhizic acid in pure water to obtain a water phase; the preparation method comprises the following steps: respectively injecting an organic phase and a water phase into a micro-channel reactor at the same time through an injection pump, mixing the two phases in the reactor, staying for 10-120 seconds under a water bath condition of 20-100 DEG C, carrying out a molecular self-assembly reaction, collecting effluent, and evaporating to remove an organic solvent, so as to obtain an aqueous solution containing the glycyrrhizic acid-pigeon pea leaf stilbene type self-assembly nanoparticles; the particle size of the prepared nanoparticles is 50-100 nm, and the polydispersity index is less than 0.3. The self-assembly process is accurately controlled through the microfluidic technology, the problems that a traditional kettle type method is low in encapsulation efficiency, uneven in particle size and the like are solved, the encapsulation efficiency of the prepared nanoparticles is 95.3% and is remarkably superior to 79.3% of the traditional kettle type method, the technology is simple, reproducibility is good, continuous production is easy to achieve, and an efficient synthesis strategy is provided for controllable preparation of nano-drugs.

Description

A type of glycyrrhizic acid-pinnea leaf stilbene self-assembled nanoparticles Technical Field

[0001] This invention belongs to the field of nanomedicine formulation and microfluidics technology, specifically relating to a method for preparing nanomedicines, particularly a method for controlling the mixing and reaction of solutions using a microtube reactor and a method for self-assembling stilbene nanoparticles from glycyrrhizic acid-pigeon leaf. Background Technology

[0002] Glycyrrhizic acid is the main active ingredient of licorice, a traditional Chinese medicine, and has various pharmacological activities such as anti-inflammatory, hepatoprotective, and antiviral effects. (LiY, Wang LF, Wang JL, et al. Research on preparation process of andrographolide-glycyrrhizic acid polymeric micelles [J]. China journal of Chinese materia medica. 2018, 43(01): 79-85.) Stilbene compounds are a class of active substances extracted from pigeon pea leaves, which have significant antioxidant, antitumor, and neuroprotective effects. (Chen Mengli. Research progress on the structure, classification and application of stilbene compounds [J]. Polyester Industry, 2024, 37(05): 60-63.) However, the poor water solubility and low bioavailability of stilbene compounds limit their clinical application.

[0003] Nanoparticle drug delivery systems are an effective strategy for improving the bioavailability of poorly soluble drugs in vivo (Zhang Ting'en, Yang Wenzhuo, Zhang Linhui, et al. Preparation and in vitro antitumor evaluation of oleanolic acid and hydroxycamptothecin co-assembled nanoparticles [J]. Chinese Journal of Modern Applied Pharmacy, 2025, 42(11):1862-1870.). Among them, self-assembled nanoparticle technology has attracted attention due to its simple preparation process and the absence of the need for a large amount of synthetic materials. Glycyrrhizic acid, as an amphiphilic triterpenoid saponin, can be used as a natural drug carrier. It can self-assemble with hydrophobic drugs through intermolecular forces such as hydrophobic interactions and π-π stacking to form nanoparticles (Liang Qifan, Cui Jiwei, Zhang Xinru, et al. Preparation and in vitro anti-glioma evaluation of tanshinone IIA-glycyrrhizic acid self-assembled nanomicelles [J]. Journal of Nanjing University of Traditional Chinese Medicine, 2022, 38(06):534-540.).

[0004] Traditional methods for preparing self-assembled nanoparticles, such as solvent injection and thin-film hydration, typically rely on slow mixing processes, resulting in drawbacks such as large batch-to-batch variations, poor particle size uniformity, difficulty in precise process control, and challenges in scale-up production. Microfluidics, however, can precisely control the mixing process of fluids within nanometer-scale channels, forming uniform nanoparticles within milliseconds, providing a novel solution for preparing high-quality, uniform self-assembled nanoparticles.

[0005] Currently, studies have been conducted on the preparation of glycyrrhizic acid-encapsulated stilbene formulations using traditional methods. However, this method requires human serum albumin as a carrier and involves complex processes with poor controllability. (Wu Mingfang. Preparation, characterization and activity evaluation of glycyrrhizic acid-conjugated human serum albumin-encapsulated resveratrol liver-targeting formulations [D]. Northeast Forestry University, 2017.)

[0006] Therefore, there is an urgent need in this field to develop a process-controllable, reproducible, and easily scaled-up technique for preparing glycyrrhizic acid-pigeon leaf stilbene-based self-assembled nanoparticles with uniform particle size, good stability, and high encapsulation efficiency. Summary of the Invention

[0007] One of the objectives of this invention is to overcome the shortcomings of the prior art and provide glycyrrhizic acid-pigeon leaf stilbene self-assembled nanoparticles with high encapsulation efficiency and excellent uniformity, and whose preparation process is controllable, reproducible, and easy to scale up.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] This invention provides glycyrrhizic acid-pigeon leaf stilbene compound self-assembled nanoparticles, wherein the nanoparticles are formed by molecular self-assembly of glycyrrhizic acid and pigeon leaf stilbene compound, and the particle size of the nanoparticles is 50-100 nm, with a polydispersity index (PDI) of less than 0.3; the nanoparticles are prepared by the following method:

[0010] A stilbene leaf compound was dissolved in anhydrous ethanol to obtain an organic phase; glycyrrhizic acid was dissolved in pure water to obtain an aqueous phase; the organic phase and the aqueous phase were simultaneously injected into a microchannel reactor using a syringe pump to mix the two phases in the reactor, and the mixture was subjected to a molecular self-assembly reaction under a water bath at 20-100 °C for 10-120 s. The effluent was collected, and the organic solvent was evaporated to remove it, yielding an aqueous solution containing the glycyrrhizic acid-stilbene leaf self-assembled nanoparticles; the mass ratio of the stilbene leaf compound to the glycyrrhizic acid was 1:1-10; the concentration of the stilbene leaf compound in the organic phase was 0.1 mg / mL-5 mg / mL; and the concentration of glycyrrhizic acid in the aqueous phase was 0.1 mg / mL-5 mg / mL.

[0011] Furthermore, the stilbene leaf compounds are extracted by the following method: dried pigeon pea leaf powder is mixed with water at a mass-to-volume ratio of 1:20 to 1:60 (g: mL) (preferably 1:40), and extracted by reflux in a water bath at 50-100℃ (preferably 100℃) for 0.5-2 hours (preferably 1 hour). The resulting extract is cooled and filtered. The resulting residue is dried and mixed with methanol at a mass-to-volume ratio of 1:20 to 1:60 (g: mL) (preferably 1:40). The mixture is ultrasonically vibrated at 20-60℃ (preferably 30℃), cooled, and filtered. The solvent is removed from the filtrate to obtain the pigeon pea leaf compounds.

[0012] Furthermore, the pigeon pea leaf powder is obtained by pulverizing dried pigeon pea leaves and passing them through a 20-100 mesh sieve (preferably 40 mesh).

[0013] Furthermore, the ultrasonic oscillation treatment has an electrical power of 150-500W (preferably 500W) and an ultrasonic time of 10-60min (preferably 20min).

[0014] Furthermore, the molecular self-assembly reaction is carried out at a temperature of 60°C for 30 seconds.

[0015] Furthermore, the mass ratio of the stilbene leaf compound to the glycyrrhizic acid is 1:2.

[0016] Furthermore, the concentration of stilbene compounds in the organic phase is 0.5 mg / mL.

[0017] Furthermore, the concentration of glycyrrhizic acid in the aqueous phase is 1 mg / mL.

[0018] Furthermore, the microchannel reactor is constructed using stainless steel tubing.

[0019] Furthermore, the inner diameter of the microchannel reactor is 0.1-3 mm (preferably 1.56 mm), and the length is 1-10 m (preferably 10 m).

[0020] Furthermore, the steps for determining the encapsulation efficiency of the glycyrrhizic acid-pigeon leaf stilbene-based self-assembled nanoparticles prepared in this invention are as follows:

[0021] The obtained glycyrrhizic acid-pigeon leaf stilbene nanoparticles were separated by ultrafiltration centrifugation and ultra-high performance liquid chromatography (UPLC), and the encapsulation efficiency of stilbene compounds in the glycyrrhizic acid-stilbene nanoparticles was quantitatively analyzed by internal standard method. 0.5 mL of glycyrrhizic acid-pigeon leaf stilbene nanoparticle solution was added to the upper layer of an ultrafiltration centrifuge tube (3 kDa) and centrifuged at 5000 rpm for 10 min using a Thermo Scientific Biofuge Stratos centrifuge. 100 µL of the lower layer solution was taken and diluted in 100 µL of methanol solution containing a known concentration of resveratrol. The free stilbene compounds (W) were determined by ultra-high performance liquid chromatography. f To determine the content of stilbene compounds, 100 µL of glycyrrhizic acid-stilbene nanoparticle solution was taken and 100 µL of methanol containing resveratrol as an internal standard was added. The content of stilbene compounds in the total drug W1 was determined by UPLC to determine the encapsulation efficiency of stilbene compounds under different conditions.

[0022] The chromatographic conditions were as follows: the column was an ACQUITY UPLC® C18 (2.1 × 100 mm, 1.8 μm), the mobile phase was 0.1% (v / v) formic acid aqueous solution (A) and acetonitrile (B), the flow rate was 0.2 mL / min, the column temperature was 35℃, the detection wavelength was 300 nm (PDA detection range 200-400 nm), and the injection volume was 10 μL. Gradient elution was used, with the following elution program: 0-12 min, 86%-70% B; 12-15 min, 70%-65% B; 15-19 min, 65%-56% B; 19-27 min, 56%-52% B; 27-31 min, 52%-44% B; 31-44 min, 44%-40% B; 44-45 min, 40%-86% B; 45-47 min, 86% B.

[0023] The encapsulation ratio is calculated using the following formula:

[0024] Encapsulation efficiency (%) = (W1 - W) f ) / W1× 100%

[0025] The beneficial effects of this invention are as follows:

[0026] 1) Uniform particle size: By precisely controlling the self-assembly process through microfluidic technology, uniform and rapid nucleation and growth of nanoparticles are achieved. The prepared nanoparticles have a narrow particle size distribution (PDI < 0.3), and the particle size can be precisely controlled within the range of 50-100 nm by adjusting parameters such as reaction time, reaction temperature, mass ratio, and concentration.

[0027] 2) Improved encapsulation efficiency: By precisely controlling the self-assembly conditions and systematically optimizing key process parameters, nanoparticles with high encapsulation efficiency can be obtained. The nanoparticles prepared by the method of this invention have an encapsulation efficiency of up to 95.3%, which is 20.2% higher than that of the traditional batch method (79.3%), effectively improving drug utilization efficiency.

[0028] 3) Good reproducibility and easy to scale up: Microfluidic processes are continuous production processes, avoiding batch-to-batch differences, with stable processes, and the transition from laboratory to industrial production can be easily achieved through the "scale-up" strategy.

[0029] 4) Green and efficient: This method uses low-toxicity organic solvents such as ethanol in small quantities. Through rapid mixing and subsequent removal, it is environmentally friendly and in line with the principles of green chemistry. Attached Figure Description

[0030] Figure 1 is a schematic diagram of the microtube reactor used in this invention;

[0031] Figure 2. Characterization of glycyrrhizic acid-pigeon leaf stilbene nanoparticles: A) Particle size distribution; B) Zeta potential distribution; C) Scanning electron microscopy; D) Tyndall effect; Detailed Implementation

[0032] The present invention will be further described in detail below through specific embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods.

[0033] Example 1: Extraction of stilbene compounds from pigeon pea leaves

[0034] After pulverizing the dried pigeon pea leaves and passing them through a 40-mesh sieve, weigh out 5 g of the powder and add it to 200 mL of purified water. Extraction was performed under reflux in a water bath for 1 hour. After cooling, the extract was filtered, and the residue was dried in an oven at 40°C. The dried residue was mixed with methanol solution at a solid-liquid ratio of 1:40 (g / mL), and ultrasonicated at 30°C and 500W for 20 minutes. After cooling, the mixture was filtered, and the filtrate was evaporated to dryness to obtain the extract of stilbene leaves.

[0035] Example 2: Preparation of glycyrrhizic acid-pigeon leaf stilbene-based self-assembled nanoparticles

[0036] Weigh 5 mg of the extract obtained in Example 1, dissolve it in 10 mL of anhydrous ethanol, and sonicate for 3 minutes to prepare an organic phase of stilbene compounds with a concentration of 0.5 mg / mL. Weigh 10 mg of glycyrrhizic acid, dissolve it in 10 mL of ultrapure water, and magnetically stir at 60°C and 500 rpm for 10 minutes to prepare an aqueous phase of glycyrrhizic acid with a concentration of 1 mg / mL.

[0037] The two phase solutions were each loaded into a 10 mL syringe and fixed to a microfluidic injection pump. The flow rates of the two phases were set so that the volumetric flow rate ratio of the organic phase to the aqueous phase was 1:1 (total flow rate of 2 mL / min). Both phases were simultaneously pumped into a stainless steel microchannel reactor with an inner diameter of 1.56 mm and a length of 10 m, and the reactor was placed in a 60°C water bath for constant temperature. The residence time of the two phases in the reactor was approximately 30 seconds, and the effluent was collected in a sample vial. The basic device diagram of the microtube reactor is shown in Figure 1.

[0038] The collected effluent was rotary evaporated at 50°C (vacuum degree -0.09 MPa) for 20 minutes to remove anhydrous ethanol, thus obtaining an aqueous solution of glycyrrhizic acid-pigeon leaf stilbene self-assembled nanoparticles.

[0039] The nanoparticle solution was characterized by dynamic light scattering (DLS), showing a particle size of 73.79 nm, a polydispersity index (PDI) of 0.293, and a zeta potential of -30.4 mV. The encapsulation efficiency was determined to be 95.3% by ultrafiltration-centrifugation combined with ultraperformance liquid chromatography (UPLC).

[0040] Example 3:

[0041] The mass ratio of glycyrrhizic acid to stilbene compounds was changed to 1:1, while other conditions and operating procedures were the same as in Example 2. The particle size of the glycyrrhizic acid-pigeon leaf stilbene compound self-assembled nanoparticles was measured to be 103.0 nm, PDI was 0.263, zeta value was -25.7 mV, and encapsulation efficiency was 84.1%.

[0042] Example 4:

[0043] The mass ratio of glycyrrhizic acid to stilbene compounds was changed to 5:1, while other conditions and operating procedures were the same as in Example 2. The particle size of the glycyrrhizic acid-pigeon leaf stilbene compound self-assembled nanoparticles was measured to be 82.30 nm, PDI was 0.289, zeta value was -21.4 mV, and encapsulation efficiency was 92.1%.

[0044] Example 5:

[0045] The mass ratio of glycyrrhizic acid to stilbene compounds was changed to 7:1, while other conditions and operating procedures were the same as in Example 2. The particle size of the glycyrrhizic acid-pigeon leaf stilbene compound self-assembled nanoparticles was measured to be 80.46 nm, PDI was 0.333, zeta value was -16.1 mV, and encapsulation efficiency was 89.6%.

[0046] Example 6:

[0047] The mass ratio of glycyrrhizic acid to stilbene compounds was changed to 10:1, while other conditions and operating procedures remained the same as in Example 2. The measured particle size of the glycyrrhizic acid-pigeon leaf stilbene compound self-assembled nanoparticles was 81.02 nm, the PDI was 0.367, the zeta value was -15.9 mV, and the encapsulation efficiency was 88.1%.

[0048] Comparing Examples 2-6, it can be seen that when the mass ratio of glycyrrhizic acid to stilbene compounds is 2:1, the encapsulation rate of stilbene compounds in the obtained glycyrrhizic acid-pigeon leaf stilbene self-assembled nanoparticles is the highest.

[0049] Example 7:

[0050] The reaction temperature was changed to 20℃, while other conditions and operating procedures were the same as in Example 2. The particle size of the glycyrrhizic acid-pigeon leaf stilbene self-assembled nanoparticles was measured to be 195.1 nm, PDI was 0.434, zeta value was -34.5 mV, and encapsulation efficiency was 60.6%.

[0051] Example 8:

[0052] The reaction temperature was changed to 40℃, while other conditions and operating procedures were the same as in Example 2. The particle size of the glycyrrhizic acid-pigeon leaf stilbene self-assembled nanoparticles was measured to be 193.4 nm, PDI was 0.370, zeta value was -37.0 mV, and encapsulation efficiency was 83.3%.

[0053] Example 9:

[0054] The reaction temperature was changed to 80℃, while other conditions and operating procedures were the same as in Example 2. The particle size of the glycyrrhizic acid-pigeon leaf stilbene self-assembled nanoparticles was measured to be 184.8 nm, PDI was 0.412, zeta value was -34.5 mV, and encapsulation efficiency was 93.4%.

[0055] Example 10:

[0056] The reaction temperature was changed to 100℃, while other conditions and operating procedures were the same as in Example 2. The particle size of the glycyrrhizic acid-pigeon leaf stilbene self-assembled nanoparticles was measured to be 124.1 nm, the PDI was 0.350, the zeta value was -31.3 mV, and the encapsulation efficiency was 93.1%.

[0057] Comparing Examples 2 and 7-10, it can be seen that when the reaction temperature is 60℃, the encapsulation rate of stilbene compounds in the obtained glycyrrhizic acid-pigeon leaf stilbene self-assembled nanoparticles is the highest.

[0058] Example 11:

[0059] The residence time was changed to 10 s, and other conditions and operating procedures were the same as in Example 2. The particle size of the glycyrrhizic acid-pigeon leaf stilbene self-assembled nanoparticles was measured to be 337.6 nm, the PDI was 0.266, the zeta value was -27.0 mV, and the encapsulation efficiency was 88.5%.

[0060] Example 12:

[0061] The residence time was changed to 60 s, while other conditions and operating procedures were the same as in Example 2. The particle size of the glycyrrhizic acid-pigeon leaf stilbene self-assembled nanoparticles was measured to be 61.26 nm, PDI was 0.315, zeta value was 22.9 mV, and encapsulation efficiency was 92.3%.

[0062] Example 13:

[0063] The residence time was changed to 90 s, and other conditions and operating procedures were the same as in Example 2. The particle size of the glycyrrhizic acid-pigeon leaf stilbene self-assembled nanoparticles was measured to be 331.7 nm, PDI was 0.073, zeta value was -33.8 mV, and encapsulation efficiency was 91.9%.

[0064] Example 14:

[0065] The residence time was changed to 120 s, and other conditions and operating procedures were the same as in Example 2. The particle size of the glycyrrhizic acid-pigeon leaf stilbene self-assembled nanoparticles was measured to be 342.5 nm, PDI was 0.200, zeta value was -29.6 mV, and encapsulation efficiency was 90.6%.

[0066] Comparing Examples 2 and 11-14, it can be seen that when the residence time is 30 s, the encapsulation rate of stilbene compounds in the obtained glycyrrhizic acid-pigeon leaf stilbene self-assembled nanoparticles is the highest.

[0067] Example 15:

[0068] The concentration of the stilbene leaf compound was changed to 0.1 mg / mL, while other conditions and procedures were the same as in Example 2. The particle size of the glycyrrhizic acid-stilbene leaf self-assembled nanoparticles was measured to be 107.6 nm, the PDI was 0.413, the zeta value was -13.8 mV, and the encapsulation efficiency was 89.3%.

[0069] Example 16:

[0070] The concentration of the stilbene leaf compound was changed to 1 mg / mL, while other conditions and procedures were the same as in Example 2. The particle size of the glycyrrhizic acid-stilbene leaf self-assembled nanoparticles was measured to be 82.98 nm, the PDI was 0.283, the zeta value was -6.99 mV, and the encapsulation efficiency was 90.6%.

[0071] Example 17:

[0072] The concentration of the stilbene leaf compound was changed to 2.5 mg / mL, while other conditions and procedures were the same as in Example 2. The particle size of the glycyrrhizic acid-stilbene leaf self-assembled nanoparticles was measured to be 370.5 nm, the PDI was 0.156, the zeta value was -7.48 mV, and the encapsulation efficiency was 90.2%.

[0073] Example 18:

[0074] The concentration of the stilbene leaf compound was changed to 5 mg / mL, while other conditions and procedures were the same as in Example 2. The particle size of the glycyrrhizic acid-stilbene leaf self-assembled nanoparticles was measured to be 244.8 nm, the PDI was 0.408, the zeta value was -20.3 mV, and the encapsulation efficiency was 89.9%.

[0075] Comparing Examples 2 and 15-18, it can be seen that when the concentration of stilbene leaf compound is 0.5 mg / mL, the encapsulation rate of stilbene compound in the obtained glycyrrhizic acid-stilbene leaf self-assembled nanoparticles is the highest.

[0076] Example 19: Optimization and Validation of Response Surface Methodology

[0077] Based on the above single-factor experimental results, three key factors were selected: reaction temperature (A), residence time (B), and mass ratio (C). With encapsulation rate as the response value, a three-factor, three-level response surface analysis was conducted using a Box-Behnken design.

[0078] Through regression model fitting and optimization, the optimal process parameters were predicted as follows: reaction temperature 66.1℃, residence time 34.2 s, mass ratio (stilbene:glycyrrhizic acid) 2.2:1, and predicted encapsulation efficiency 95.0%. Considering the convenience of actual operation, the parameters were adjusted to: reaction temperature 66℃, residence time 34 s, and mass ratio 2.2:1.

[0079] Three verification experiments were conducted using this optimized process, achieving an average encapsulation efficiency of 94.8% (RSD < 2%). The average particle size of the nanoparticles prepared in the three repeated experiments was 68.5 nm, and the average PDI was 0.115. These results confirm the stability and reproducibility of the optimized process, and the obtained product fully meets the excellent performance standards described in this invention (particle size 50-100 nm, PDI < 0.3).

[0080] Example 20:

[0081] The microchannel reactor was replaced with a PTFE tube instead of a stainless steel tube, while other conditions remained the same as in Example 2. The results showed that the effluent was clear and transparent, and no nanoparticle signal was detected by dynamic light scattering, indicating that self-assembled nanoparticles could not be formed under these conditions. This comparative example demonstrates that the stainless steel material is crucial for the successful implementation of this invention.

[0082] Example 21:

[0083] The concentrations of glycyrrhizic acid and stilbene compounds were increased to 20 mg / mL (mass ratio 2:1), with other conditions remaining the same as in Example 2. The resulting mixture was a gel with poor flowability, making normal nanoparticle characterization and encapsulation efficiency determination impossible.

[0084] Example 22:

[0085] The mass ratio of glycyrrhizic acid to stilbene compounds was changed to 1:2, while other conditions and operating procedures remained the same as in Example 2. After self-assembly, a large amount of stilbene compounds precipitated in the system, resulting in extremely low encapsulation efficiency. The obtained glycyrrhizic acid-pigeon leaf stilbene compound self-assembly exhibited the phenomenon of unencapsulated stilbene compounds precipitating.

[0086] Example 23: Comprehensive Characterization of Nanoparticles

[0087] Glycyrrhizic acid-pigeon leaf stilbene-based self-assembled nanoparticles were prepared according to the optimized process of Example 19 and were fully characterized.

[0088] Particle size, PDI and Zeta potential: 1.5 mL of nanoparticle solution was precisely measured and measured by dynamic light scattering method. The results showed that the nanoparticle size was 71.07 nm, the PDI was 0.126 and the Zeta potential was -37.2 mV (Figure 2A, 2B).

[0089] Scanning electron microscopy (SEM) morphology observation: An appropriate amount of lyophilized glycyrrhizic acid-based stilbene nanoparticles were evenly distributed on a conductive adhesive. After coating with a platinum layer using a gold sputtering machine, the sample was placed in the SEM sample chamber. The particle morphology of the sample powder was observed under an accelerating voltage of 15.0 kV (Figure 2C). The nanoparticles were found to be regular spherical or near-spherical in shape and uniform in size.

[0090] Tyndall effect: The nanoparticle solution exhibits a significant Tyndall effect under laser pen irradiation (Figure 2D), further confirming its colloidal nanoparticle characteristics.

[0091] Example 24: Preparation of Nanoparticles by Traditional Stir-Fry Method

[0092] Weigh out the same amount of stilbene and glycyrrhizic acid as in Example 2, and prepare them by the conventional solvent injection method: slowly inject the ethanol solution of stilbene (organic phase) into the glycyrrhizic acid aqueous solution (aqueous phase, 60°C) under vigorous stirring (500 rpm) within 1 minute, and then perform the same rotary evaporation steps as in Example 2.

[0093] The obtained nanoparticles were measured to have an average particle size of 423.2 nm, a PDI of 0.290, a Zeta potential of -14.7 mV, and an encapsulation efficiency of 79.3%.

[0094] Compared with the results of Examples 2 and 19 of the present invention, the conventional method is significantly inferior to the microfluidic method of the present invention in terms of particle size control, distribution uniformity (PDI) and encapsulation efficiency, which fully demonstrates the inventiveness and technological progress of the present invention.

[0095] The above experimental results show that the microfluidic preparation method and the product obtained by the present invention significantly surpass traditional technologies in key performance indicators.

[0096] The above embodiments are only used to illustrate specific implementations of the present invention and are not intended to limit the scope of protection of the present invention.

Claims

1. A glycyrrhizic acid-pigeon leaf stilbene-based self-assembled nanoparticle, characterized in that, The nanoparticles are prepared by the following method: a stilbene leaf compound is dissolved in anhydrous ethanol to obtain an organic phase; glycyrrhizic acid is dissolved in pure water to obtain an aqueous phase; the organic phase and the aqueous phase are simultaneously injected into a microchannel reactor using a syringe pump, allowing the two phases to mix within the reactor. The mixture is then subjected to a molecular self-assembly reaction under a water bath at 20–100 °C for 10–120 s. The effluent is collected, and the organic solvent is evaporated to remove it, yielding an aqueous solution containing the glycyrrhizic acid-stilbene leaf self-assembled nanoparticles. The mass ratio of the stilbene leaf compound to the glycyrrhizic acid is 1:1–10; the concentration of the stilbene leaf compound in the organic phase is 0.1 mg / mL–5 mg / mL; and the concentration of glycyrrhizic acid in the aqueous phase is 0.1 mg / mL–5 mg / mL.

2. The glycyrrhizic acid-pigeon leaf stilbene-based self-assembled nanoparticles as described in claim 1, characterized in that, The stilbene leaf compounds are extracted by the following method: dried pigeon pea leaf powder is mixed with water at a mass-to-volume ratio of 1:20 to 1:60 (g:mL), and extracted by reflux in a water bath at 50 to 100°C for 0.5 to 2 hours. The resulting extract is cooled and filtered. The resulting residue is dried and mixed with methanol at a mass-to-volume ratio of 1:20 to 1:60 (g:mL). The mixture is ultrasonically vibrated at 20 to 60°C, cooled, and filtered. The solvent is removed from the filtrate to obtain the stilbene leaf compounds.

3. The glycyrrhizic acid-pigeon leaf strychnine self-assembled nanoparticles as described in claim 2, characterized in that, The aforementioned pigeon pea leaf powder is obtained by pulverizing dried pigeon pea leaves and passing them through a 20-100 mesh sieve.

4. The glycyrrhizic acid-pigeon leaf stilbene-based self-assembled nanoparticles as described in claim 2, characterized in that, The ultrasonic oscillation treatment described herein uses an electrical power of 150-500W and an ultrasonic time of 10-60min.

5. The glycyrrhizic acid-pigeon leaf strychnine self-assembled nanoparticles as described in claim 1, characterized in that, The molecular self-assembly reaction was carried out at a temperature of 60°C for 30 seconds.

6. The glycyrrhizic acid-pigeon leaf stilbene-based self-assembled nanoparticles as described in claim 1, characterized in that, The mass ratio of the stilbene leaf compound to the glycyrrhizic acid is 1:

2.

7. The glycyrrhizic acid-pigeon leaf stilbene self-assembled nanoparticles as described in claim 1, characterized in that, The concentration of stilbene compounds in the organic phase is 0.5 mg / mL.

8. The glycyrrhizic acid-pigeon leaf stilbene-based self-assembled nanoparticles as described in claim 1, characterized in that, The concentration of glycyrrhizic acid in the aqueous phase is 1 mg / mL.

9. The glycyrrhizic acid-pigeon leaf stilbene-based self-assembled nanoparticles as described in claim 1, characterized in that, The microchannel reactor is constructed using stainless steel tubing.

10. The glycyrrhizic acid-pigeon leaf stilbene-based self-assembled nanoparticles as described in claim 1, characterized in that, The microchannel reactor has an inner diameter of 0.1-3 mm and a length of 1-10 m.

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