A method for preparing nano-embedded materials based on low-temperature ultrafine grinding technology

Abstract

The application discloses a kind of nanometer embedding material preparation methods based on low-temperature ultrafine grinding technology, belong to modified nanometer material technical field.The method includes the following steps: take fresh cow strength rootstock, after washing section program liquid nitrogen freezing, airflow pulverization is obtained nanometer powder;Take anion polysaccharide, neutral polysaccharide nanoparticle, nanometer modified cassava starch and deionized water, after magnetic stirring, ultrasonic treatment, mix 30-45min;Wall material solution is mixed with nanometer powder, ultrasonic treatment forms coating liquid, add calcium chloride and tween 80 and stir;Voltage 40-60kV, flow rate 0.5-1.5mL / h spray into magnesium chloride solution and stir, then with chitosan solution mass ratio 1: (2-4) Mix, adjust pH and stir, freeze-drying is obtained.The application retains cow strength active ingredient, improves embedding efficiency 85-96%, stability, control release, suitable for health products and drug carrier.

Description

Technical Field

[0001] This invention belongs to the field of modified nanomaterials technology, specifically, it relates to a method for preparing nano-embedded materials based on low-temperature ultrafine pulverization technology. Background Technology

[0002] *Achyranthes bidentata*, also known as beautiful cardamom root or *Achyranthes bidentata* rhizome, is a traditional medicinal and edible plant belonging to the Fabaceae family, Papilionoideae subfamily. It is mainly distributed in southern China, such as Guangdong, Guangxi, and Hainan. As an important traditional Chinese medicine, *Achyranthes bidentata* rhizome has been widely used in clinical practice and folk health care since ancient times. Its rhizome is rich in various bioactive components, including flavonoids (such as isoflavones and quercetin derivatives), polysaccharides, alkaloids, saponins, phenolic acids, and other secondary metabolites. These components endow *Achyranthes bidentata* with various pharmacological effects, such as liver protection, anti-fatigue, strengthening muscles and bones, tonifying the lungs, anti-oxidation, anti-inflammation, and immune regulation. According to literature reports, flavonoids in *Achyranthes bidentata*, such as quercetin and kaempferol, have significant antioxidant activity, scavenging free radicals and protecting liver cells from oxidative stress damage; polysaccharides show immune-enhancing effects, stimulating macrophage activity and improving the body's resistance; alkaloids and saponins help combat fatigue and improve muscle function. These medicinal properties make *Niu Dali* a commonly used traditional Chinese medicine for treating chronic fatigue, liver damage, arthritis, and weakened immunity. *Niu Dali* is also used in the development of health foods, such as *Niu Dali* tea, powdered drinks, and functional beverages, which are very popular with consumers.

[0003] Traditional processing methods mainly include water extraction, alcohol extraction, reflux extraction, and conventional pulverization. These methods have historically played a significant role, but they also have many limitations. First, water extraction is the most common traditional extraction process, involving slicing the rhizome of *Smilax glabra* and soaking or boiling it in hot water to extract the active ingredients. However, this method has low extraction efficiency, typically less than 70%, because many fat-soluble components, such as flavonoids and alkaloids, are difficult to completely dissolve. Simultaneously, high-temperature boiling can lead to the degradation of heat-sensitive components; for example, polysaccharides are prone to hydrolysis or oxidation at high temperatures, resulting in a loss of biological activity. Studies show that the loss rate of flavonoids in *Smilax glabra* can reach 20%-30% during traditional water extraction. Second, alcohol extraction uses ethanol or other organic solvents, which can better dissolve fat-soluble components, but solvent residue is a significant problem, potentially introducing toxicity risks, and the extraction process requires large amounts of organic solvents, causing serious environmental pollution. Furthermore, while reflux extraction can improve the extraction rate, it is complex to operate, energy-intensive, and high-temperature reflux can also cause denaturation of active ingredients. Conventional pulverization methods involve drying the rhizome and mechanically pulverizing it into micron-sized powder for use in formulations or direct consumption. However, these powders have relatively large particle sizes (typically >10μm) and small specific surface areas, resulting in poor solubility and low bioavailability (<30%). These powders are also susceptible to oxidative degradation due to humidity, light, and oxygen, leading to poor storage stability. In practical applications, traditionally processed *Niu Da Li* products often have a rough texture and slow absorption, failing to meet the demands of modern pharmaceuticals and health products for high efficiency and stability.

[0004] With the advancement of science and technology, the application of nanotechnology in the modernization of traditional Chinese medicine (TCM) is becoming increasingly prominent. Nanotechnology significantly improves the specific surface area, solubility, and bioavailability of TCM by processing it into nanoscale particles (particle size <100nm). For example, ultrafine grinding technology can reduce the particle size of TCM powder to the nanoscale, enhancing cell permeability and targeting. Literature indicates that nano-sized TCM can improve the intestinal absorption of active ingredients, increasing bioavailability by 2-5 times. However, conventional ultrafine grinding, such as ball milling or air jet milling, is often carried out at room temperature or high temperature, leading to the accumulation of frictional heat and damage to heat-sensitive components. To address this, low-temperature ultrafine grinding technology has emerged. This technology combines liquid nitrogen freezing and air jet milling, operating below -60℃ to avoid high-temperature degradation and preserve the integrity of active ingredients. Studies show that when low-temperature ultrafine grinding is applied to other TCMs such as Astragalus membranaceus and ginseng, the retention rate of polysaccharides and saponins increases by 15%-20%. This technology is particularly suitable for *Smilax glabra*, as its rhizome has a high water content and is easily oxidized by heat during room-temperature grinding. However, the existing low-temperature pulverization process parameters are not optimized enough, such as inaccurate freezing time and pulverization frequency, resulting in uneven particle size distribution (50-500nm) and large fluctuations in specific surface area (<150nm). 2 / g), affecting subsequent applications.

[0005] Nanoencapsulation technology is another key advancement, achieving protection, controlled release, and targeted delivery of active ingredients by embedding them into nanocarriers. Traditional Chinese medicine extracts are complex and multi-component, easily affected by gastric acid, enzymatic hydrolysis, and oxidation, resulting in low bioavailability. Nanoencapsulation uses wall materials such as polysaccharides, starch, and chitosan to form nanoemulsions or microcapsules to encapsulate the core substance. Anionic polysaccharides (such as sodium alginate and pectin) provide negative charge stability, neutral polysaccharide nanoparticles (such as β-cyclodextrin) enhance inclusion, and nano-modified cassava starch improves hydrophobicity. After modification with octenyl succinic anhydride, the particle size of cassava starch is reduced to 30-60 nm, achieving a hydrophilic-lipophilic balance and improving encapsulation efficiency. Existing research shows that nano-encapsulation can control the release rate of active ingredients in traditional Chinese medicine (TCM) within 30%-50% (within 24 hours), but challenges remain: uneven wall material composites lead to agglomeration, resulting in a zeta potential >-20mV and poor stability; incomplete cross-linking and curing cause serious leakage of active ingredients; improper pH control during self-assembly affects chitosan encapsulation. Furthermore, multi-component TCMs, such as *Achyranthes bidentata* extract, are prone to phase separation in nanosystems, making quality control difficult. Patent literature on similar technologies mostly targets single components, such as flavonoid nanocapsules, but lacks comprehensive methods specifically for *Achyranthes bidentata* rhizomes.

[0006] Specific problems with existing technologies include: (1) Low retention rate of active ingredients. In traditional extraction, the loss rate of flavonoids and polysaccharides is high; although nano-pulverization improves this, there is no low-temperature protection, and the retention rate is <80%. (2) Poor bioavailability and stability. Micron-sized powders are slowly absorbed, and although nano-encapsulation improves this, the wall material is singular, and the particle size change during storage is >10%. (3) Weak release control. Traditional products release rapidly (>60% within 24 hours), and the nano-system needs to optimize cross-linking and self-assembly. (4) Industrialization barriers. Existing process parameters are broad, resulting in large batch-to-batch differences and high costs. (5) Safety hazards. Organic solvent residues and nano-toxicity need to be assessed. Summary of the Invention

[0007] Traditional processing methods for *Smilax glabra* rhizome mainly rely on water extraction, alcohol extraction, or conventional pulverization. While these methods are simple and easy to implement, they have significant drawbacks: low extraction efficiency, significant loss of active ingredients, large particle size, poor bioavailability, and weak stability. Although nanotechnology has been introduced, existing low-temperature pulverization parameters are suboptimal, wall material composites are uneven, and cross-linking is incomplete, resulting in low encapsulation efficiency, unstable zeta potential, and excessively rapid release. To solve the above problems, this invention adopts the following technical solution.

[0008] To solve the above problems, the present invention adopts the following technical solution.

[0009] A preparation method of nano-encapsulation material based on low-temperature ultrafine grinding technology, comprising the following steps: (1) Low-temperature ultrafine grinding: Take fresh Millettia speciosa Champ. rhizomes, wash and cut them into slices with a thickness of 2-5 mm, perform programmed liquid nitrogen freezing treatment, place the frozen Millettia speciosa Champ. rhizomes in an ultrafine grinder for grinding treatment to obtain nano-powder; (2) Preparation of composite wall material solution: By mass fraction, take 15-25 parts of anionic polysaccharide, 4-8 parts of neutral polysaccharide nanoparticles, 5-10 parts of nano-modified cassava starch and 80-100 parts of deionized water, and under the condition of magnetic stirring at 600-800 rpm, then perform ultrasonic treatment, and then mix at 4-8 °C for 30-45 min to form a uniform composite wall material solution; (3) Formation of nano-emulsion: Mix the composite wall material solution in step (2) with the nano-powder in step (1) according to a mass ratio of (30-50):1, perform ultrasonic treatment to form a coating solution, then add calcium chloride with a mass of 0.1-0.2 times that of the coating solution and Tween 80 with a mass of 0.5-0.8 times that of the coating solution, and stir at 30-40 °C for 60-90 min to obtain a nano-emulsion; (4) Crosslinking and curing treatment: Perform crosslinking and curing treatment on the nano-emulsion obtained in step (3) to obtain an embedding solution; (5) Self-assembly embedding: Mix the embedding solution obtained in step (4) with a chitosan solution according to a mass ratio of 1:(2-4), adjust the pH to 4.0-5.8, and stir at 800-1000 rpm for 80-120 min to obtain an embedding wall material; (6) Low-temperature drying treatment: Place the embedding wall material obtained in step (5) in a freeze dryer at -60 °C for 24-60 h to obtain the finished nano-encapsulation material product.

[0010] Among them, the pH of each step is adjusted and maintained by dropping under the monitoring of a pH meter by selecting dilute hydrochloric acid (0.1-1.0 mol / L) and sodium hydroxide (0.1-1.0 mol / L); all pH values are measured based on 25±2 °C.

[0011] For the Millettia speciosa Champ. rhizomes used as the source of Millettia speciosa Champ., the mass fraction of water in the rhizomes is 58-61%.

[0012] Preferably, the method of programmed liquid nitrogen freezing in step (1) is as follows: First pre-cool at -20 °C for 30-60 min, and then transfer to a liquid nitrogen environment at -80 °C for quick freezing for 2-4 h; the parameters of the grinding treatment in step (1) are as follows: airflow grinding at a temperature of -60 °C, with a frequency of 15000-25000 Hz and a time of 30-60 min.

[0013] Preferably, the parameters of the nano-powder in step (1) are as follows: particle size 50-200 nm, specific surface area 150-200 m 2 / g.

[0014] Preferably, in step (2), the anionic polysaccharide is sodium alginate (CAS No.: 9005-38-3), pectin (CAS No.: 9000-69-5), or sodium carboxymethyl cellulose (CAS No.: 9004-32-4); in step (2), the neutral polysaccharide nanoparticles are raw starch (CAS No.: 9005-25-8) or β-cyclodextrin (CAS No.: 7585-39-9), with a particle size of 200-800 nm.

[0015] Preferably, the ultrasonic power in step (2) is 300-500W and the ultrasonic frequency is 20kHz-40kHz.

[0016] Preferably, the preparation method of nano-modified cassava starch in step (2) is as follows: dry cassava starch (CAS No.: 9005-25-8) is mixed with deionized water at a mass ratio of 1:(10-20), and the pH value is adjusted to 6-6.5. Then, 0.1-0.3 times the mass of cassava starch is added with ascorbic acid (CAS No.: 50-81-7) or gallic acid (CAS No.: 149-91-7), and the mixture is magnetically stirred at 300-500 rpm and 40-50℃ to form a uniform slurry. Then, the pH value of the slurry is adjusted to 4-5.5 to obtain a pre-hydrolyzed slurry. The slurry is ultrasonicated for 10-30 min at a power of 400-500W and a frequency of 20-30kHz, and then subjected to high pressure of 80-120MPa. Homogenize 3-5 times to obtain starch nanoparticles of 30-60 nm. Add 5-10 times the mass of deionized water to obtain a resuspension. Add octenyl succinic anhydride (CAS No.: 26680-54-6) and sodium tripolyphosphate (CAS No.: 68915-31-1) to the resuspension at 40-60℃ and 300-400 rpm. The mass of octenyl succinic anhydride is 0.2-0.4 times the mass of the resuspension, and the mass of sodium tripolyphosphate is 0.1-0.3 times the mass of the resuspension. Maintain the pH at 8-9 and react for 4-8 hours to obtain a modified suspension. Treat the modified suspension overnight using an 8-14 kDa dialysis bag. Finally, freeze-dry under vacuum at -50℃ for 24-36 hours to obtain the product.

[0017] Preferably, the ultrasonic treatment power in step (3) is 200-300W, the ultrasonic treatment frequency is 10-20kHz, and the ultrasonic treatment time is 10-20min; the stirring speed in step (3) is 600-800rpm.

[0018] Preferably, the parameters for the crosslinking curing treatment in step (4) are as follows: the crosslinking agent is sprayed into the emulsion at a voltage of 40-60kV and a flow rate of 0.5-1.5mL / h, wherein the crosslinking agent is a magnesium chloride solution with a mass percentage of 20-30%, and the mixture is stirred at 500-800rpm for 30-60min.

[0019] Preferably, the mass percentage of the chitosan solution in step (5) is 2-6%.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: The invention provides a method for preparing nano-embedded materials based on low-temperature ultrafine pulverization technology. Through the optimized integration of steps such as programmed liquid nitrogen freezing combined with airflow pulverization, composite wall material solution preparation, nanoemulsion formation, cross-linking curing, self-assembly embedding, and low-temperature drying, the limitations of the processing of *Smilax glabra* root and rhizome in the prior art are significantly overcome.

[0021] Significantly Improves the Retention Rate of Active Ingredients: Traditional water extraction, alcohol extraction, or conventional pulverization methods result in a 20%-30% loss rate of active ingredients such as flavonoids and polysaccharides in the rhizome of *Smilax glabra*, with a retention rate of less than 80%. This invention employs programmed liquid nitrogen freezing (pre-cooling at -20℃ for 30-60 min followed by rapid freezing at -80℃ for 2-4 h) and airflow pulverization at -60℃ (frequency 15000-25000 Hz, time 30-60 min), effectively preventing the degradation of heat-sensitive components. The nanoparticle size is controlled at 50-200 nm, and the specific surface area is 150-200 m². 2 / g. Test results show that the retention rate of active ingredients reaches 89.5%-96.1% (93.2% average in the examples), which is 15%-20% higher than the existing technology, ensuring the integrity of bioactive substances such as flavonoids, alkaloids, and polysaccharides in the rhizome of *Smilax glabra*.

[0022] Improving Encapsulation Efficiency and Material Stability: Existing nano-encapsulation technologies suffer from uneven wall material composites and Zeta potentials >-20mV, leading to aggregation and active material leakage, resulting in encapsulation efficiency <80%. This invention uses a composite wall material (15-25 parts anionic polysaccharide, 4-8 parts neutral polysaccharide nanoparticles, and 5-10 parts nano-modified cassava starch), combined with stirring with calcium chloride (0.1-0.2 times) and Tween 80 (0.5-0.8 times), and cross-linking and curing by spraying a 20-30% magnesium chloride solution at a voltage of 40-60kV and a flow rate of 0.5-1.5mL / h. The self-assembly encapsulation is then mixed with a 2-6% chitosan solution at pH 4.0-5.8. The results showed that the encapsulation efficiency was 85.1%-96.1% (average 90.4% in the examples), the Zeta potential was -27.8mV to -37.4mV (average -32.6mV), and the storage stability (particle size change rate at 4℃ for 30 days) was 2.4%-4.3% (average 3.4%). Compared with the existing technology, the encapsulation efficiency was improved by 10%-20%, the stability was improved by 2-3 times, and the loss due to oxidation and enzymatic hydrolysis was significantly reduced.

[0023] Achieving controlled release and improved bioavailability: Traditional products release too quickly (>60%), with bioavailability <30%. The nano-encapsulated material of this invention achieves a release rate of 37.2%-48.4% in a simulated intestinal environment over 24 hours (average 42.8% in the examples), achieving slow, controlled release and improving bioavailability by 2-5 times. Benefiting from the nano-sized particles (average particle size 48.9-203.6 nm) and the hydrophobic-hydrophilic balance of the modified wall material (e.g., nano-modified cassava starch modified with 0.2-0.4 times octenyl succinic anhydride and 0.1-0.3 times sodium tripolyphosphate), it enhances cell permeability and targeting, making it suitable for health products (such as Niu Dali tea and functional beverages) and drug carriers (such as liver-protecting and anti-fatigue preparations).

[0024] Optimizing process parameters reduces costs and improves industrial applicability: Existing technologies have broad parameters, large batch-to-batch variations, and high energy consumption. This invention precisely controls parameters at each step (e.g., ultrasonic power 200-500W, frequency 10-40kHz), avoids organic solvent residue, and ensures high safety (biodegradable wall materials reduce nano-toxicity). Comparison of examples and comparative examples shows that this invention outperforms cases where key components are missing or parameters exceed limits (e.g., comparative examples show encapsulation efficiency of 67.7%-81.2% and release rate of 56.8%-66.0%), exhibits good batch-to-batch consistency, reduces costs by 15%-25%, and facilitates large-scale production.

[0025] Environmental friendliness and broad application: This method involves no high temperatures and no organic solvents, reducing environmental pollution; it is applicable to the nano-sizing of other Chinese medicinal materials, promoting the modernization of traditional Chinese medicine. Test results verify the advanced nature of this invention: the average indicators of the examples are 20%-50% better than those of the comparative examples, and meet national standards (such as GB / T19627-2005).

[0026] In summary, this invention retains >90% of the active ingredients of *Achyranthes bidentata*, has an encapsulation efficiency of 85%-96%, a zeta potential of <-27mV, a release rate of 37%-48%, and a particle size change of <5%, comprehensively surpassing existing technologies. It is suitable for health products and drug carriers, and has significant economic and social benefits. Attached Figure Description

[0027] Figure 1 This is a transmission electron microscope image of the composite wall material solution prepared in Example 1.

[0028] Figure 2 This is a transmission electron microscope image of the starch nanoparticles prepared in Example 1.

[0029] Figure 3 This is a photograph of the nanoemulsion prepared in Example 1. Detailed Implementation

[0030] The present invention will now be described in detail through specific embodiments. However, these illustrative embodiments are for the purpose of illustrating the invention only and do not constitute any limitation on the actual scope of protection of the invention, nor are they intended to limit the scope of protection of the invention to these embodiments. For parameter ranges not mentioned, intermediate values ​​are selected. Furthermore, for mass percentages or weight percentages not explicitly stated or mentioned, they generally refer to the final concentration after addition.

[0031] Example 1

[0032] Low-temperature ultrafine pulverization: Take 100g of fresh *Smilax glabra* root and rhizome, wash and cut into 3.5mm thick slices, and process using programmed liquid nitrogen freezing (pre-cool at -20℃ for 45min, then quick-freeze in a -80℃ liquid nitrogen environment for 3h). Place the frozen *Smilax glabra* root and rhizome in an ultrafine pulverizer and pulverize using airflow at -60℃, a frequency of 20000Hz, and a time of 45min to obtain nanoparticles (particle size 125nm, specific surface area 175m²). 2 / g). Preparation of composite wall material solution: By mass, take 20g of sodium alginate, 6g of neutral polysaccharide nanoparticles (raw starch, particle size 500nm), 7.5g of nano-modified cassava starch, and 90g of deionized water, and stir magnetically at 700rpm, followed by ultrasonic treatment (power 400W, frequency 30kHz). Then mix at 6℃ for 37.5min to form a homogeneous composite wall material solution, such as... Figure 1 As shown. The preparation method of nano-modified cassava starch is as follows: 30g of dried cassava starch and 450g of deionized water are mixed at a mass ratio of 1:15, and the pH value is adjusted to 6.25. Then, ascorbic acid (6g) with a mass ratio of 0.2 times that of the cassava starch is added, and the mixture is magnetically stirred at 45℃ and 400rpm to form a uniform slurry. The pH value of the slurry is then adjusted to 4.75 to obtain a pre-hydrolyzed slurry. This slurry is then sonicated for 20min at a power of 450W and a frequency of 25kHz, followed by homogenization four times under a high pressure of 100MPa to obtain 45nm starch nanoparticles (e.g., ...). Figure 2As shown in the figure, 337.5 g of deionized water (7.5 times the mass of starch nanoparticles) was added to obtain a resuspension slurry. Octenyl succinic anhydride and sodium tripolyphosphate were added to the resuspension slurry at 50°C and 350 rpm. The mass of octenyl succinic anhydride was 0.3 times the mass of the resuspension slurry (202.5 g), and the mass of sodium tripolyphosphate was 0.2 times the mass of the resuspension slurry (135 g). The pH was maintained at 8.5, and the reaction was carried out for 6 h to obtain a modified slurry. The modified slurry was treated overnight using an 11 kDa dialysis bag and finally freeze-dried under vacuum at -50°C for 30 h to obtain the product. Nanoemulsion formation: 123.5g of the above composite wall material solution and 3.5g of the above nanopowder were mixed at a mass ratio of 35:1, and ultrasonically treated (power 250W, frequency 15kHz, time 15min) to form a coating solution. Then, 0.15 times the mass of the coating solution of calcium chloride (i.e., 19.275g) and 0.65 times the mass of the coating solution of Tween 80 (i.e., 83.525g) were added, and the mixture was stirred at 35°C (speed 700rpm) for 75min to obtain the nanoemulsion. Figure 3 As shown. Crosslinking and curing treatment: The nanoemulsion obtained above was subjected to crosslinking and curing treatment. A crosslinking agent, which was a 25% (w / w) magnesium chloride solution, was sprayed into the emulsion at a voltage of 50kV and a flow rate of 1.0mL / h. The mixture was stirred at 650rpm for 45min to obtain the embedding solution. Self-assembly embedding: The embedding solution obtained above was mixed with a chitosan solution (4% (w / w)) at a mass ratio of 1:3. The pH was adjusted to 4.9, and the mixture was stirred at 900rpm for 100min to obtain the embedded wall material. Low-temperature drying treatment: The embedded wall material obtained above was freeze-dried at -60℃ for 42h to obtain the finished nanoemulsion material.

[0033] Example 2-18

[0034] Examples 2-18 refer to the process flow of Example 1, with some parameters adjusted. The specific formula and process parameters are shown in the table.

[0035] Comparative Examples 1-16 were designed to verify the necessity of key components and technical conditions. They demonstrated the disadvantages of using key components by omitting them (such as nano-modified cassava starch or anionic polysaccharides) or exceeding the parameter range, or by replacing them with other commonly used similar components (e.g., replacing sodium alginate with agar or calcium chloride with sodium chloride). Specific formulations and process parameters are shown in the table, with the parameters arranged in the order of the process flow. Comparative Example 1: Unlike Example 1, nano-modified cassava starch was not added; the remaining steps were the same as in Example 1. Comparative Example 2: Unlike Example 1, anionic polysaccharides were not added; an equal amount of deionized water was used instead. The remaining steps were the same as in Example 1. Comparative Example 3: Unlike Example 1, neutral polysaccharide nanoparticles were not added; an equal amount of deionized water was used instead. The remaining steps were the same as in Example 1. Comparative Example 4: Unlike Example 5, the nanoparticle size was 250 nm (out of the range of 50-200 nm); the remaining steps were the same as in Example 5. Comparative Example 5: Unlike Example 5, the mass ratio of composite wall material solution to nanopowder is 55:1 (out of range 30-50:1), and the remaining steps are the same as in Example 5. Comparative Example 6: Unlike Example 5, the ultrasonic treatment power is 350W (out of range 200-300W), and the remaining steps are the same as in Example 5. Comparative Example 7: Unlike Example 8, octenyl succinic anhydride is not added in the preparation of nano-modified cassava starch; an equal amount of water is used instead. The remaining steps are the same as in Example 8. Comparative Example 8: Unlike Example 8, the mass of octenyl succinic anhydride in the preparation of nano-modified cassava starch is 0.5 times the mass of the resuspension slurry (out of range 0.2-0.4 times), and the remaining steps are the same as in Example 8. Comparative Example 9: Unlike Example 8, the ultrasonic treatment power in the preparation of the composite wall material solution is 550W (out of range 300-500W), and the remaining steps are the same as in Example 8. Comparative Example 10: Unlike Example 10, magnesium chloride solution was not added during the crosslinking and curing process; instead, an equal amount of water was used. The remaining steps were the same as in Example 10. Comparative Example 11: Unlike Example 10, the voltage during the crosslinking and curing process was 70 kV (out of range 40-60 kV). The remaining steps were the same as in Example 10. Comparative Example 12: Unlike Example 10, the chitosan solution mass percentage during self-assembly embedding was 7% (out of range 2-6%). The remaining steps were the same as in Example 10. Comparative Example 13: Unlike Example 12, the pre-cooling time during low-temperature ultrafine grinding was 70 min (out of range 30-60 min). The remaining steps were the same as in Example 12. Comparative Example 14: Unlike Example 12, the anionic polysaccharide (sodium alginate) was replaced with agar. The remaining steps were the same as in Example 12. Comparative Example 15: Unlike Example 12, calcium chloride was replaced with sodium chloride. The remaining steps were the same as in Example 12. Comparative Example 16: Unlike Example 15, the freeze-drying time was 70 hours (out of the range of 24-60 hours), and the remaining steps were the same as in Example 15.

[0036] Table 1: Formulations and Process Parameters for Examples 1-6

[0037]

[0038] As shown in Table 1, Examples 1-6, based on the same amount of *Smilax glabra* root (100g), optimized the low-temperature ultrafine grinding process parameters by adjusting the slice thickness (2-5mm), pre-cooling time (30-60min), quick-freezing time (2-4h), grinding frequency (15000-25000Hz), and grinding time (30-60min) to obtain particle sizes of 50-200nm and specific surface areas of 150-200m². 2 / g of nanoparticles.

[0039] Table 2: Formulations and Process Parameters for Examples 1-6

[0040]

[0041] As shown in Table 2, Examples 1-6 optimized the nanoparticle characteristics (particle size 50-200 nm, specific surface area 150-200 m²). 2 The process involved several steps: using a mixture of 2.5-4.5 g of chitosan and 2.5-4.5 g of sodium chloride, along with a composite wall material formulation (15-25 g of anionic polysaccharide, 4-8 g of neutral polysaccharide, 5-10 g of nano-modified cassava starch, and 80-100 g of deionized water), ultrasonic treatment (power 200-500 W, frequency 10-40 kHz), stirring conditions (600-800 rpm, 30-40℃, 60-90 min), cross-linking curing (voltage 40-60 kV, flow rate 0.5-1.5 mL / h, magnesium chloride 20-30%), self-assembly embedding (chitosan concentration 2-6%, pH 4.0-5.8), and freeze drying (-60℃, 24-60 h). This process enabled the efficient preparation of nano-embedded materials.

[0042] Table 3: Formulations and Process Parameters for Examples 7-12

[0043]

[0044] As shown in Table 3, Examples 7-12 achieved their intended results by precisely controlling the slice thickness (2-5 mm) of the *Achyranthes bidentata* rhizome, the liquid nitrogen freezing conditions (pre-cooling for 30-60 min, quick-freezing for 2-4 h), the low-temperature ultrafine grinding parameters (-60℃, 15000-25000 Hz, 30-60 min), and the nanoparticle characteristics (particle size 50-200 nm, specific surface area 150-200 m²). 2The preparation process of the nano-embedded material was optimized by adjusting the formula of the composite wall material (15-25g of anionic polysaccharide, 4-8g of neutral polysaccharide, 5-10g of nano-modified cassava starch, and 80-100g of deionized water), ultrasonic and stirring conditions, and cross-linking curing parameters. This laid the foundation for subsequent self-assembly embedding and freeze-drying, ensuring efficient embedding and stability.

[0045] Table 4: Formulations and Process Parameters for Examples 7-12 (Part 2)

[0046]

[0047] As shown in Table 4, Examples 7-12 optimized the preparation process of the nano-embedded materials by adjusting the parameters of self-assembly embedding (mass ratio of embedding solution to chitosan solution 1:2 to 1:4, chitosan concentration 2-6%, pH 4.0-5.8, stirring speed 800-1000 rpm, stirring time 80-120 min) and freeze-drying conditions (-60℃, 24-60 h).

[0048] Table 5: Formulations and Process Parameters for Examples 13-18

[0049]

[0050] Table 5 shows the key formulations and process parameters for Examples 13-18 in the preparation method of nano-embedded materials based on low-temperature ultrafine grinding technology (mainly covering the first half of step 1, low-temperature ultrafine grinding, and step 2, preparation of composite wall material solution). In all examples, the amount of *Smilax glabra* rootstock used was 100g, the pre-cooling temperature was -20℃, the quick-freezing temperature was -80℃, and the grinding temperature was -60℃. The parameters exhibit a regular variation: the parameter patterns of Examples 13-15 are highly similar to those of Examples 16-18 (13 corresponds to 16, 14 to 17, and 15 to 18), reflecting the combined optimization of minimum, intermediate, and maximum values.

[0051] Table 6: Formulations and Process Parameters for Examples 13-18 (Part 2)

[0052]

[0053] As shown in Tables 5 and 6, these parameters ensure the high encapsulation efficiency, stability, and controlled release performance of the nano-encapsulation materials, making them suitable for health supplements and drug carriers.

[0054] Table 7: Formulations and Process Parameters of Comparative Examples 1-8

[0055]

[0056] As shown in Table 7, Comparative Examples 1-8 maintained the same amount of *Smilax glabra* rhizome (100g) but adjusted the slice thickness (3.5-5mm), pre-cooling time (45-60min), quick-freezing time (3-4h), grinding frequency (20000-25000Hz), and grinding time (45-60min). However, the nanoparticle size (250nm) of Comparative Example 4 exceeded the optimized range, potentially leading to a decrease in subsequent encapsulation efficiency and stability. While other comparative examples partially met the requirements, the subsequent processes lacked key components (such as nano-modified cassava starch and anionic polysaccharides) or exceeded parameter limits (such as the mass ratio of composite wall material solution to nanoparticles and ultrasonic power in Comparative Examples 5-8), resulting in performance inferior to the examples.

[0057] Table 8: Formulations and Process Parameters of Comparative Examples 1-8

[0058]

[0059] As shown in Table 8, the performance of these comparative examples (encapsulation efficiency 67.7%-81.2%, Zeta potential -14.9 to -20.1 mV, storage stability 10.4%-14.8%, release rate 56.8%-66.0%) was significantly lower than that of the examples (encapsulation efficiency 85.1%-96.1%, Zeta potential -27.8 to -37.4 mV, storage stability 2.4%-4.3%, release rate 37.2%-48.4%), verifying the necessity of optimizing key components and process parameters.

[0060] Table 9: Formulations and Process Parameters of Comparative Examples 1-8

[0061]

[0062] As shown in Table 9, the parameters of Comparative Examples 1-8 in the crosslinking curing treatment, self-assembly embedding, and freeze-drying stages were similar to those of the Examples. However, due to the absence of key components or the exceeding of parameters in the preceding steps (steps 2 and 3), the overall performance was significantly lower than that of the Examples. The test results showed that the embedding efficiency of Comparative Examples 1-8 was 67.7%-81.2%, the Zeta potential was -14.9 to -20.1 mV, the storage stability (particle size change rate) was 10.4%-14.8%, and the release rate was 56.8%-66.0%, all of which were far inferior to those of the Examples (embedding efficiency 85.1%-96.1%, Zeta potential -27.8 to -37.4 mV, storage stability 2.4%-4.3%, release rate 37.2%-48.4%).

[0063] Table 10: Formulations and Process Parameters of Comparative Examples 9-16

[0064]

[0065] Table 10 shows that Comparative Examples 9-16, based on the same amount of *Achyranthes bidentata* rhizome (100g), adjusted the slice thickness (3.5-5mm), pre-cooling time (45-70min), quick-freezing time (3-4h), pulverizing frequency (20000-25000Hz), and pulverizing time (45-60min). However, the pre-cooling time (70min) of Comparative Example 13 exceeded the 30-60min range required by the patent, which may affect the freezing effect and powder uniformity. In addition, deviations in other parameters or components in subsequent processes (such as ultrasonic power of 550W in Comparative Example 9, magnesium chloride-free solution in Comparative Example 10, voltage of 70kV in Comparative Example 11, chitosan concentration of 7% in Comparative Example 12, agar as a substitute for anionic polysaccharide in Comparative Example 14, sodium chloride as a substitute for calcium chloride in Comparative Example 15, and freeze-drying time of 70h in Comparative Example 16) resulted in significantly lower performance than the examples. Test results showed that the encapsulation efficiency of comparative examples 9-16 was 67.7%-81.2%, the zeta potential was -14.9 to -20.1 mV, the storage stability (particle size change rate) was 10.4%-14.8%, and the release rate was 56.8%-66.0%, which was far inferior to the excellent performance of the examples, verifying the necessity of optimizing the process and key components.

[0066] Table 11: Formulation and Process Parameters of Comparative Example 9-16 II

[0067]

[0068] Table 11 lists the formulations and process parameters for the preparation of composite wall material solutions (step 2) and nanoemulsion formation (step 3) in the preparation methods of nano-embedded materials based on low-temperature ultrafine grinding technology in Comparative Examples 9-16.

[0069] Table 12: Formulation and Process Parameters of Comparative Examples 9-16

[0070]

[0071] Table 12 lists the formulations and process parameters for comparative examples 9-16 in the preparation method of nano-embedded materials based on low-temperature ultrafine grinding technology, from cross-linking curing treatment (step 4) to freeze drying (step 6).

[0072] To comprehensively evaluate the performance of nanomaterials embedded using low-temperature ultrafine grinding technology, the following test methods were designed, covering particle size distribution, embedding efficiency, surface potential, retention rate of active ingredients, storage stability, and release characteristics. The test methods referenced relevant national standards (such as GB / T19627-2005, "Methods for Determination of Particle Size Distribution of Nanomaterials") to ensure the scientific validity and reasonableness of the results.

[0073] Average particle size (nm) testing method

[0074] The particle size distribution of the nano-embedded material was determined using dynamic light scattering (DLS) with a particle size analyzer. The sample was dispersed in deionized water (concentration 0.1 mg / mL) and tested at 25°C. Test conditions: laser wavelength 532 nm, scattering angle 173°, three repeated measurements were taken, and the average value was recorded.

[0075] Significance: To evaluate the nanoscale size uniformity of materials, with a target value of 50-200 nm.

[0076] Embedding efficiency (%) test method

[0077] The difference in the content of active ingredients (polysaccharides) of *Smilax glabra* before and after encapsulation was determined by high performance liquid chromatography (HPLC). Encapsulation efficiency = (content of active ingredient after encapsulation / content of initial active ingredient) × 100%. Test conditions: mobile phase: methanol-water (50:50), flow rate: 1.0 mL / min, detection wavelength: 280 nm.

[0078] Significance: Reflects the encapsulation effect of the encapsulation process on the active ingredient; target value >85%.

[0079] Zeta potential (mV) test method

[0080] The surface potential of the sample in pH 7.0 phosphate buffer was determined using a Zeta potential analyzer. Test conditions: temperature 25℃, conductivity 0.1 mS / cm, measurements repeated 5 times.

[0081] Significance: To assess the stability of materials, with a target value of <-25mV (negative values ​​indicate good stability).

[0082] Test method for retention rate (%) of active ingredients

[0083] After storing the sample at -40℃ for 7 days, the loss of active ingredients was determined by HPLC. Retention rate = (content after storage / initial content) × 100%. Test conditions: relative humidity of storage environment 50%, detection same as encapsulation efficiency.

[0084] Significance: To assess the integrity of active ingredients under low-temperature conditions, with a target value >90%.

[0085] Storage stability (particle size change rate, %) test method

[0086] The particle size change of the sample was measured using DLS before and after storage at 4℃ for 30 days. Change rate = (particle size after storage - initial particle size) / initial particle size × 100%. Test conditions: Stored in a sealed container, away from light.

[0087] Significance: Reflects the long-term stability of the material; target value <5%.

[0088] Release rate (%) test method

[0089] The intestinal environment was simulated using dialysis (pH 6.8 phosphate buffer, 37℃), and the release of the active ingredient within 24 hours was determined using a UV spectrophotometer. Release rate = (release amount / total encapsulation amount) × 100%. Test conditions: stirring speed 100 rpm, sampling interval 2 hours.

[0090] Significance: To evaluate controlled release performance, with a target value of 30-50% (slow release).

[0091] Table 13: Test results of particle size and encapsulation efficiency in the examples and comparative examples

[0092]

[0093] Analysis of the results in Table 13 shows that in Examples 1-18, the average particle size ranged from 48.9 to 203.6 nm, which meets the requirements of nanoscale; the encapsulation efficiency was 85.1%-96.1%, and the Zeta potential ranged from -27.8 to -37.4 mV, indicating good stability and efficient encapsulation; the retention rate of active ingredients was 89.5%-96.1%, the storage stability (particle size change rate) was 2.4%-4.3%, and the release rate was 37.2%-48.4%, demonstrating excellent low-temperature protection and controlled release performance. In Comparative Examples 1-16, the absence of key components (e.g., Comparative Example 1 lacked nano-modified cassava starch, Comparative Example 2 lacked anionic polysaccharides, and Comparative Example 3 lacked neutral polysaccharide nanoparticles) led to a significant decrease in performance, with average particle size ranging from 149.6 to 262.3 nm (increased agglomeration), encapsulation efficiency from 67.7% to 81.2%, and Zeta potentials from -14.9 to -20.1 mV (poor stability). Parameters exceeding the range (e.g., Comparative Example 4 had particle size exceeding the limit, Comparative Example 5 had mass ratio exceeding the limit, and Comparative Example 6 had ultrasonic power exceeding the limit) or being replaced (e.g., Comparative Example 14 used agar, and Comparative Example 15 used sodium chloride) further worsened the results, with active ingredient retention rates of 73.8% to 81.2%, storage stability of 10.4% to 14.8%, and release rates of 56.8% to 66.0% (excessively rapid release). The examples were comprehensively superior to the comparative examples, validating the necessity of key components (e.g., nano-modified cassava starch and sodium alginate) and process parameters. The test results are highly consistent with the target parameters of 50-200 nm particle size and activity integrity in the claims, demonstrating the advanced nature and practicality of the technology.

[0094] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. A method for preparing a nano-embedded material based on low-temperature ultrafine grinding technology, characterized by: The method comprises the following steps: (1) low-temperature ultrafine grinding: fresh aconite root is washed and cut into thick slices, and then subjected to programmed liquid nitrogen freezing treatment, wherein the programmed liquid nitrogen freezing treatment method is as follows: pre-cooling at-20 DEG C for 30-60 min, and then rapidly freezing in a-80 DEG C liquid nitrogen environment for 2-4 h; the frozen aconite root is subjected to grinding treatment in an ultrafine grinder to obtain nanometer powder, wherein the particle size of the nanometer powder ranges from 50 nm to 200 nm; (2) preparation of composite wall material solution: 15-25 parts of anionic polysaccharide, 4-8 parts of neutral polysaccharide nanoparticles, 5-10 parts of nanometer modified cassava starch and 80-100 parts of deionized water are taken according to the mass fraction, and then subjected to magnetic stirring at 600-800 rpm, followed by ultrasonic treatment at a power of 300-500 W, and then mixed to form a uniform composite wall material solution; wherein the anionic polysaccharide is sodium alginate, pectin or carboxymethyl cellulose, the neutral polysaccharide nanoparticles are raw starch or beta-cyclodextrin, and the nanometer modified cassava starch is prepared as follows: dry cassava starch is mixed with deionized water and the pH value is adjusted, then ascorbic acid or gallic acid is added and subjected to magnetic stirring to form a uniform slurry, the pH value of the slurry is adjusted to obtain a pre-hydrolyzed slurry, and then the slurry is subjected to ultrasonic treatment and then homogenization to obtain starch nanoparticles, deionized water is added to obtain a resuspended slurry, octenyl succinic anhydride and sodium tripolyphosphate are added to the resuspended slurry, wherein the mass of the octenyl succinic anhydride is 0.2-0.4 times the mass of the resuspended slurry, and the mass of the sodium tripolyphosphate is 0.1-0.3 times the mass of the resuspended slurry, and the reaction is maintained at the pH value to obtain a modified slurry, which is treated in a dialysis bag overnight, and finally vacuum freeze-dried to obtain the product; (3) formation of nanometer emulsion: the composite wall material solution of step (2) and the nanometer powder of step (1) are mixed according to a mass ratio of (30-50):1, and subjected to ultrasonic treatment to form a coating liquid, wherein the power of the ultrasonic treatment is 200-300 W, then calcium chloride in an amount of 0.1-0.2 times the mass of the coating liquid and Tween 80 in an amount of 0.5-0.8 times the mass of the coating liquid are added and subjected to stirring treatment to obtain a nanometer emulsion; (4) cross-linking and solidification treatment: the nanometer emulsion obtained in step (3) is subjected to cross-linking and solidification treatment, wherein the parameters of the cross-linking and solidification treatment in step (4) are as follows: the voltage is 40-60 kV, and the cross-linking agent (magnesium chloride solution) is sprayed into the emulsion at a flow rate of 0.5-1.5 mL / h to obtain an embedding liquid; (5) self-assembly embedding: the embedding liquid obtained in step (4) and a chitosan solution are mixed according to a mass ratio of 1:(2-4), the mass percentage of the chitosan solution is 2-6%, the pH value is adjusted, and stirring is performed to obtain an embedding wall material; (6) low-temperature drying treatment: the embedding wall material obtained in step (5) is subjected to freeze-drying at-60 DEG C for 24-60 h to obtain a nanometer embedding material finished product.

2. The method for preparing a nano-embedded material based on low-temperature ultrafine grinding technology according to claim 1, characterized in that: The size of the thick slice in step (1) ranges from 2 to 5 mm; the parameters of the crushing treatment in step (1) are as follows: air flow crushing at a temperature of -60℃, a frequency of 15000-25000 Hz, and a time of 30-60 min.

3. The method for preparing a nano-embedded material based on low-temperature ultrafine grinding technology according to claim 1, characterized in that: The parameters of the nanometer powder in step (1) are as follows: specific surface area 150-200 m 2 / g.

4. The method for preparing a nano-embedded material based on low-temperature ultrafine grinding technology according to claim 2, characterized in that: The particle size of the neutral polysaccharide nanoparticles in step (2) is 200-800 nm.

5. The method for preparing a nano-embedded material based on low-temperature ultrafine grinding technology according to claim 1, characterized in that: The frequency of the ultrasonic treatment in step (2) is 20-40 kHz; the mixing method in step (2) is as follows: mixing at 4-8℃ for 30-45 min.

6. The method for preparing a nano-embedded material based on low-temperature ultrafine grinding technology according to claim 1, characterized in that: The preparation method of the nanomodified cassava starch in step (2) is as follows: mixing the dried cassava starch with deionized water at a mass ratio of 1: (10-20), adjusting the pH value to 6-6.5, then adding ascorbic acid or gallic acid at 0.1-0.3 times the mass of the cassava starch, performing magnetic stirring at 300-500 rpm and 40-50℃ to form a uniform slurry, then adjusting the pH value of the slurry to 4-5.5 to obtain a pre-hydrolyzed slurry, performing ultrasonic treatment at a power of 400-500 W and a frequency of 20-30 kHz for 10-30 min, then performing homogenization at a high pressure of 80-120 MPa for 3-5 times to obtain starch nanoparticles with a size of 30-60 nm, adding deionized water at 5-10 times the mass of the starch nanoparticles to obtain a resuspended slurry, adding octenyl succinic anhydride and sodium tripolyphosphate to the resuspended slurry at 40-60℃ and 300-400 rpm, wherein the mass of the octenyl succinic anhydride is 0.2-0.4 times the mass of the resuspended slurry, and the mass of the sodium tripolyphosphate is 0.1-0.3 times the mass of the resuspended slurry, maintaining the pH value at 8-9, and reacting for 4-8 h to obtain a modified slurry, treating the modified slurry with a dialysis bag with a molecular weight cutoff of 8-14 kDa overnight, and finally performing vacuum freeze-drying at a temperature of -50℃ for 24-36 h to obtain the product.

7. The method of claim 1, wherein the method is characterized by: The stirring speed in step (3) is 600-800 rpm; the frequency of the ultrasonic treatment in step (3) is 10-20 kHz, and the ultrasonic treatment time is 10-20 min; the parameters of the stirring treatment in step (3) are as follows: stirring treatment at 30-40℃ for 60-90 min.

8. The method for preparing a nano-embedded material based on low-temperature ultrafine grinding technology according to claim 1, characterized in that: The parameters of the crosslinking and solidification treatment in step (4) are as follows: the crosslinking agent is a magnesium chloride solution with a mass percentage of 20-30%; meanwhile, stirring is performed at 500-800 rpm for 30-60 min; in step (5), the pH value is adjusted to 4.0-5.8, and stirring is performed at 800-1000 rpm for 80-120 min.

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