A ginseng self-assembly nanobody and its preparation method and application

Abstract

A ginseng self-assembling nanobody and its preparation method and application relate to the field of medical technology, and solve the problems in the prior art that long-term use of drugs for treating atopic dermatitis is prone to adverse reactions and its preparation process is complicated and costly. The present invention crushes and decocts ginseng to obtain a ginseng decoction, adopts a differential combined centrifugation method to obtain nanobodies of different particle sizes, and obtains ginseng self-assembling nanobodies by filtering, purifying, and freeze-drying. The ginseng self-assembling nanobody provided by the present invention improves the solubility and bioavailability of the drug, achieves the accuracy and efficiency of the treatment of atopic dermatitis in mice, reduces the side effects caused by traditional treatments, ensures the safety of long-term use, and at the same time, the preparation process is simple and clear, the cost is low, and it has high economic applicability. The present invention can be applied to the preparation of drugs for treating atopic dermatitis.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a ginseng self-assembly nanobody and a preparation method and application thereof. Background Art

[0002] Atopic dermatitis is a common, chronic, recurring, inflammatory skin disease that manifests clinically as dry skin, eczema-like rashes, and intense itching, severely impacting patients' quality of life. According to statistics, there are approximately 230 million patients worldwide, including over 70 million in my country. The prevalence rate among adults is 10%, and among children it is as high as 25%. The pathogenesis of atopic dermatitis is complex, involving the influence of multiple factors, including genetics, immune abnormalities, skin barrier dysfunction, and environmental factors.

[0003] Currently, pharmacological treatments for atopic dermatitis primarily include topical and systemic medications. Glucocorticoids and calcineurin inhibitors are commonly used topical medications, but long-term use can cause adverse reactions such as skin atrophy, telangiectasia, and hyperpigmentation, with significant toxic side effects. Systemic medications such as antihistamines and immunosuppressants can provide some control, but immunosuppressants like cyclosporine can lead to decreased immune function, severe toxicities such as hypertension and renal impairment, and even an increased risk of infection and tumors. Biologics such as dupilumab, while effective, are expensive, poorly accessible, and carry risks such as ocular inflammation and allergic reactions. Therefore, the development of safe and effective new medications is crucial for the treatment of atopic dermatitis.

[0004] Ginseng, a traditional and precious Chinese medicinal material, has been used in my country for thousands of years and possesses diverse pharmacological activities, including immunomodulatory, anti-inflammatory, and antioxidant properties. The Qianjin Fang and Shengji Zonglu document that ginseng and its prescriptions are effective in treating chapped skin and skin dryness. Modern research indicates that the main active ingredients in ginseng, including ginsenosides, polysaccharides, and proteins, play important roles in regulating immune function and alleviating inflammatory responses. However, the practical application of ginseng's active ingredients has several limitations: Firstly, the poor water solubility of ingredients such as ginsenosides results in low bioavailability, limiting their absorption and distribution in the body. Secondly, due to their complex composition, traditional ginseng extracts may cause unwanted adverse reactions, affecting their clinical effectiveness.

[0005] Advances in nanotechnology have provided new avenues for drug delivery and improving drug efficacy. Nanocarriers, with their small particle size, large surface area, and high modifiability, can improve drug solubility and stability, enhance drug targeting, and reduce drug toxicity and side effects. Formulating ginseng's active ingredients into nanobodies has the potential to overcome its inherent limitations and enhance its efficacy in the treatment of atopic dermatitis. While there have been some reports on ginseng-related nanoformulations, most have focused on other disease areas, with limited research on ginseng self-assembled nanobodies (GSNs) and their applications in atopic dermatitis. Furthermore, existing nanobody preparation methods often suffer from complex processes, high costs, and the use of organic solvents, hindering large-scale production and clinical application. Therefore, developing a simple, efficient, and green method for preparing ginseng self-assembled nanobodies and applying it to the preparation of drugs for the treatment of atopic dermatitis has important theoretical and clinical implications. Summary of the Invention

[0006] To address the problems of long-term adverse reactions and complex and costly preparation processes of existing drugs for treating atopic dermatitis, the present invention proposes a ginseng self-assembling nanobody and its preparation method and application. The technical solution of the present invention is as follows:

[0007] A method for preparing ginseng self-assembled nanobodies comprises the following steps:

[0008] S1: Grind and sieve the ginseng, soak the ginseng powder in distilled water, decoct the soaked material, collect the decoction and filter the ginseng medicinal material;

[0009] S2: adding distilled water to the ginseng material in S1 for decoction, collecting the decoction and filtering it, combining the two decoctions to obtain a ginseng decoction, and cooling it to room temperature;

[0010] S3: The ginseng decoction is centrifuged to remove impurities, the supernatant is collected, and the supernatant is centrifuged again. The above combined centrifugation steps are repeated 3 times, the supernatant is collected, and the purification centrifugation is performed again to obtain the ginseng centrifuge liquid, and the supernatant is collected;

[0011] S4: Redissolve the precipitate in S3 and repeat the steps in S3;

[0012] S5: filtering and purifying the collected supernatant, freezing the filtrate to form, and then freeze-drying to obtain ginseng self-assembled nanobodies;

[0013] Furthermore, the speed of the impurity removal centrifugation in S3 is 800-1200 r / min; the speed of the secondary centrifugation is 1000-12000 r / min, the speed of the purification centrifugation is 10000-14000 r / min, and the centrifugation time is 20-40 min;

[0014] Furthermore, the number of repetitions in S4 is 0 to 4 times;

[0015] Furthermore, the sieving in S1 is performed with a 0-40 mesh screen; the mass ratio of the ginseng powder to distilled water is 1:6-12; the soaking time is 0-3 h; the decoction temperature is 80-120° C.; and the decoction time is 0-3 h.

[0016] Furthermore, the mass ratio of the ginseng medicinal material to distilled water in S2 is 1:4-8; the decoction temperature is 80-120° C.; and the decoction time is 0-3 h.

[0017] Furthermore, the filtration in S5 uses filter membranes with pore sizes of 0.4 μm and 0.22 μm respectively; the freezing molding temperature is -20°C, and the freezing molding time is 6 to 18 hours;

[0018] Furthermore, the freeze-drying pressure in S5 is 0.5 MPa, the freeze-drying temperature is -80°C, and the freeze-drying time is 80-120 h.

[0019] A ginseng self-assembling nanobody prepared by the above preparation method, wherein the ginseng self-assembling nanobody uses ginseng glycoprotein as a carrier and synergistically completes self-assembly with an active ginsenoside composition;

[0020] Furthermore, based on dry weight, the mass fraction of the ginseng glycoprotein is 72-92%, and the ginseng glycoprotein includes polysaccharides and proteins, and the mass ratio of the polysaccharides to proteins is 14-22:1; the mass fraction of the active ginsenoside composition is 2-7%; the types of the ginsenoside composition include ginsenoside Re (Ginsenoside Re), ginsenoside Rg1 (Ginsenoside Rg1), ginsenoside Rf (Ginsenoside Rf), ginsenoside Rb1 (Ginsenoside Rb1), ginsenoside mRb1 (Ginsenoside mRb1), ginsenoside Rc (Ginsenoside Rc), ginsenoside Rg2 (Ginsenoside Rg2), ginsenoside Ro (Ginsenoside Ro), ginsenoside Rb2 (Ginsenoside Rb2), ginsenoside Rh1 (Ginsenoside Rh1), ginsenoside Rd (Ginsenoside Rd), ginsenoside Rg6, ginsenoside Rg4, ginsenoside Rk3, ginsenoside Rh4, ginsenoside Rg3, 20-gluco-ginsenoside Rf and notoginsenoside R1, and the mass proportions of various ginsenosides in the ginsenoside composition are as follows, based on dry weight: Ginsenoside Re 0-9 parts; Ginsenoside Rg1 0-19 parts; Ginsenoside Rf 0-5 parts; Ginsenoside Rb1 6-20 parts; Ginsenoside mRb1 1-8 parts; Ginsenoside Rc 5-11 parts; Ginsenoside Rg2 2-5 parts; Ginsenoside Ro 3~10 parts; GinsenosideRb2 2~6 parts; Ginsenoside Rh1 1~5 parts; Ginsenoside Rd 4~7 parts; Ginsenoside Rg6 0~1 part; Ginsenoside Rg4 0~2 parts; Ginsenoside Rk3 0~2 parts; Ginsenoside Rh4 0~3 parts; GinsenosideRg3 0~4 parts; 20-gluco-ginsenoside Rf 0~2 parts; Notoginsenoside R1 0~3 parts.

[0021] An application of the ginseng self-assembled nanobody in preparing a drug for treating atopic dermatitis.

[0022] Compared with the existing technology, the present invention solves the problem that long-term use of drugs for treating atopic dermatitis is prone to adverse reactions and their preparation process is complex and costly. The specific beneficial effects are:

[0023] 1. The first invention provides ginseng self-assembled nanobodies for the preparation of drugs for the treatment of atopic dermatitis: This invention utilizes the non-covalent interactions of the active ingredients in ginseng (such as ginsenosides) to prepare ginseng self-assembled nanobodies for the preparation of drugs for the treatment of atopic dermatitis. The invention specifies that the ginseng self-assembled nanobodies are composed of 72-92% ginseng glycoproteins and 2-7% active ginsenoside compositions (both by dry weight), and the content of each active ginsenoside composition is determined. Ginseng glycoproteins can encapsulate ginsenosides, forming a stable nanostructure. This improves the stability and bioavailability of the ginseng self-assembled nanobody drugs in the body, significantly enhances their solubility, and strengthens their ability to penetrate the skin barrier, allowing them to more effectively target sites of inflammation, reduce irritation to healthy tissue, and thus minimize adverse reactions.

[0024] 2. Simple and clear preparation process: This invention utilizes a differential combined centrifugation method. Initially, low-speed centrifugation (1000 r / min) removes larger impurity particles and unassembled components. Then, fractionated centrifugation is performed at different speeds (1000, 3000, 5000, 7000, and 12000 r / min), leveraging the differences in sedimentation coefficients of different particles to gradually remove smaller impurity particles and enrich nanobodies of varying sizes. Finally, high-speed centrifugation (12000 r / min) precipitates and purifies the target nanobodies, achieving efficient fractionation and purification of ginseng self-assembled nanobodies. This simple and clear preparation process requires no complex equipment, is quick and easy to operate, and is cost-effective, making it highly economical. While ensuring the safety and efficacy of traditional Chinese medicine, this method can further enhance the medicinal value of ginseng, broaden its application prospects in pharmaceuticals and health foods, and provide new technical support for the development of the ginseng industry.

[0025] 3. Verifying the Formation of Ginseng Self-Assembled Nanobodies Using Visual Detection: Because ginseng self-assembled nanobodies contain numerous nanoparticles ranging in size from 1 to 100 nm, the centrifuge fluid of ginseng that has successfully self-assembled undergoes the Tyndall effect when exposed to light. This is a rapid, intuitive, and low-cost method that can provide a preliminary assessment of the formation of ginseng self-assembled nanobodies. Scanning electron microscopy and dynamic light scattering further verified the distribution and size of the ginseng self-assembled nanobodies, providing strong evidence for the successful preparation of ginseng self-assembled nanobodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a photograph of the Tyndall effect of the ginseng centrifuge prepared in Example 1-5;

[0027] Figure 2 is the scanning electron microscope (SEM) image of GSNs-3k;

[0028] Figure 3 is the dynamic light scattering (DLS) particle size distribution diagram of GSNs-3k;

[0029] Figure 4 is the standard curve of ginsenoside Re;

[0030] Figure 5 is the standard curve of total polysaccharides;

[0031] Figure 6 is the bovine serum albumin standard curve;

[0032] Figure 7 This is the total ion chromatogram of ginsenosides of GSNs-1k (negative ion mode);

[0033] Figure 8 This is the identification result of the compound in the negative ion mode of GSNs-1k;

[0034] Figure 9 This is the total ion chromatogram of ginsenosides of GSNs-3k (negative ion mode);

[0035] Figure 10 This is the identification result of the compound in the negative ion mode of GSNs-3k;

[0036] Figure 11 This is the total ion chromatogram of ginsenosides of GSNs-5k (negative ion mode);

[0037] Figure 12 This is the identification result of the compound in the negative ion mode of GSNs-5k;

[0038] Figure 13 This is the total ion chromatogram of ginsenosides of GSNs-7k (negative ion mode);

[0039] Figure 14 This is the identification result of the compound in the negative ion mode of GSNs-7k;

[0040] Figure 15 This is the total ion chromatogram of ginsenosides of GSNs-12k (negative ion mode);

[0041] Figure 16 This is the identification result of the compound in the negative ion mode of GSNs-12k;

[0042] Figure 17The effect of GD and GSNs treatment on the proliferation rate of Hacat cells;

[0043] Figure 18 The effects of GD and GSNs on the regulation of inflammatory cytokine expression levels;

[0044] Figure 19 The effect of GSNs-3k on the gene expression levels of inflammatory factors IL-6 and IL-10;

[0045] Figure 20 The effects of GD and GSNs-3k on the skin of atopic dermatitis mice;

[0046] Figure 21 Effects of GD and GSNs-3k on HE staining of skin tissues in mice with atopic dermatitis;

[0047] Figure 22 This is the effect of GD and GBSNs-3k on the gene expression levels of inflammatory factors in the tissues of atopic dermatitis model mice. DETAILED DESCRIPTION

[0048] In order to make the technical solution of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the specification of the present invention. It should be noted that the following embodiments are only used to better understand the technical solution of the present invention and should not be understood as limiting the present invention.

[0049] Example 1.

[0050] S1: Ginseng was crushed at room temperature and sieved using a 20-mesh sieve. The sieved ginseng powder was added to distilled water and soaked for 1 hour (the mass ratio of ginseng powder to distilled water was 1:10). The soaked ginseng liquid was decocted at 100°C for 2 hours, and the decoction was collected and filtered.

[0051] S2: Add distilled water to the ginseng material in S1 and boil at 100°C for 1.5 h (the mass ratio of ginseng material to distilled water is 1:6). Collect the decoction and filter it. Combine the two filtered decoctions to obtain GD, which is then cooled to room temperature for later use.

[0052] S3: Centrifuge the GD prepared in S2 at room temperature at 1000 r / min for 20 min to obtain the supernatant, continue centrifuging at 1000 r / min for 20 min, repeat the above steps three times, obtain the supernatant and centrifuge again at 12000 r / min for 20 min to obtain ginseng centrifuge solution -1k. Collect the supernatant in a transparent, clean glass container and use a parallel beam of light as a light source to observe the Tyndall effect.

[0053] S4: The collected supernatant was filtered through filter membranes with pore sizes of 0.45 μm and 0.22 μm, respectively. The filtered liquid was frozen at -18°C for 12 h, and then freeze-dried at 0.5 MPa (5 bar) and -80°C for 96 h to obtain ginseng self-assembled nanobodies-1k (GSNs-1k).

[0054] Example 2.

[0055] The precipitate after centrifugation in Example 1 S3 was dissolved in an appropriate amount of purified water. After vortex mixing, the mixture was centrifuged at 1000 r / min for 20 min. The supernatant was collected and centrifuged at 3000 r / min for 20 min. The above steps were repeated three times. The supernatant was collected and centrifuged at 12000 r / min for 20 min to obtain ginseng centrifuge solution-3k. The supernatant was collected in a transparent, clean glass container and a beam of parallel light was used as a light source to observe the occurrence of the Tyndall effect.

[0056] The collected supernatant was filtered through filter membranes with pore sizes of 0.45 μm and 0.22 μm in sequence. The filtered liquid was frozen at -18°C for 12 h and then freeze-dried at 0.5 MPa (5 bar) and -80°C for 96 h to obtain ginseng self-assembled nanobodies-3k (GSNs-3k).

[0057] Example 3.

[0058] The precipitate after centrifugation in Example 2 was dissolved in an appropriate amount of purified water. After vortex mixing, the mixture was centrifuged at 1000 r / min for 20 min. The supernatant was collected and centrifuged at 5000 r / min for 20 min. The above steps were repeated three times. The supernatant was collected and centrifuged at 12000 r / min for 20 min to obtain ginseng centrifuge solution -5k. The supernatant was collected in a transparent, clean glass container and a beam of parallel light was used as a light source to observe the occurrence of the Tyndall effect.

[0059] The collected supernatant was filtered through filter membranes with pore sizes of 0.45 μm and 0.22 μm in sequence. The filtered liquid was frozen at -18°C for 12 h and then freeze-dried at 0.5 MPa (5 bar) and -80°C for 96 h to obtain ginseng self-assembled nanosomes-5k (GSNs-5k).

[0060] Example 4.

[0061] The precipitate after centrifugation in Example 3 was dissolved in an appropriate amount of purified water. After vortex mixing, the mixture was centrifuged at 1000 r / min for 20 min. The supernatant was collected and centrifuged at 7000 r / min for 20 min. The above steps were repeated three times. The supernatant was collected and centrifuged at 12000 r / min for 20 min to obtain ginseng centrifuge solution -7k. The supernatant was collected in a transparent, clean glass container and a parallel beam of light was used as a light source to observe the occurrence of the Tyndall effect.

[0062] The collected supernatant was filtered through filter membranes with pore sizes of 0.45 μm and 0.22 μm in sequence. The filtered liquid was frozen at -18°C for 12 h and then freeze-dried at 0.5 MPa (5 bar) and -80°C for 96 h to obtain ginseng self-assembled nanosomes-7k (GSNs-7k).

[0063] Example 5.

[0064] The precipitate after centrifugation in Example 4 was dissolved in an appropriate amount of purified water. After vortex mixing, the mixture was centrifuged at 1000 r / min for 20 min. The supernatant was collected and centrifuged at 12000 r / min for 20 min. The above steps were repeated three times. The supernatant was collected and centrifuged at 12000 r / min for 20 min to obtain ginseng centrifuge solution-12k. The supernatant was collected in a transparent, clean glass container and a parallel beam of light was used as a light source to observe the Tyndall effect.

[0065] The collected supernatant was filtered through filter membranes with pore sizes of 0.45 μm and 0.22 μm in sequence. The filtered liquid was frozen at -18°C for 12 h and then freeze-dried at 0.5 MPa (5 bar) and -80°C for 96 h to obtain ginseng self-assembled nanobodies-12k (GSNs-12k).

[0066] like Figure 1 Photos of the Tyndall effect of ginseng centrifuges prepared in Examples 1-5 show that all ginseng centrifuges exhibited the Tyndall effect, demonstrating the successful preparation of ginseng micro- and nanoparticles. This is due to the naturally amphiphilic molecular structure of the active ingredients in ginseng. For example, the molecular structure of ginsenosides contains a hydrophobic steroidal core and a hydrophilic glycoside group. This structure enables these two groups to spontaneously aggregate in solution through non-covalent interactions (such as hydrogen bonds and hydrophobic interactions) to form nanostructures. Ginseng centrifuge 3k exhibited the most pronounced Tyndall effect, suggesting its potential advantages in terms of particle size, stability, and degree of self-assembly.

[0067] like Figure 2This is a scanning electron microscope (SEM) image of GSNs-3k. As can be seen from the image, GSNs-3k presents a layered petal-like microstructure and fish-scale lamellar channels on the surface. This unique structure indicates that the nanobody has a high specific surface area, uniformly distributed layered channels, can stack more substances per unit volume, and has stronger permeability.

[0068] like Figure 3 The dynamic light scattering (DLS) particle size distribution of GSNs-3k shows a relatively concentrated particle size distribution, primarily around 100 nm, exhibiting a typical nanostructure. GSNs-3k exhibits good size uniformity and is evenly dispersed in solution. Furthermore, the zeta potential of GSNs-3k is -25 mV, demonstrating strong electrostatic repulsion between nanoparticles and good system stability, effectively preventing particle aggregation and maintaining the integrity of the nanostructure.

[0069] Determination of the content of ginseng centrifuge:

[0070] (1) Determination of ginsenoside content: Accurately weigh 10 mg of the test sample, ginseng self-assembled nanobodies, add 10 mL of methanol to dissolve it, and pipette 100 μL of this solution into a 10 mL EP tube. Place the EP tube in an 85°C water bath to completely evaporate the methanol. Accurately pipette 10 μL, 20 μL, 30 μL, 40 μL, 60 μL, 80 μL, and 100 μL of ginsenoside Re reference solution into 10 mL EP tubes, respectively, and place them in a water bath not exceeding 90°C to evaporate the methanol. Add 0.5 mL of 8% vanillin anhydrous ethanol test solution and 5 mL of 72% sulfuric acid test solution to the test and reference solutions, respectively. Shake thoroughly to mix, then heat in a 60°C constant temperature water bath for 10 min. Immediately cool with ice water for 10 min after heating, and shake well. Using a blank solution containing only the reagent as a control, the absorbance of each tube of solution was measured at a wavelength of 530.6 nm using spectrophotometry. The concentration of the reference solution was used as the horizontal axis and the absorbance as the vertical axis. Figure 4The standard curve for ginsenoside Re in GSNs-3k is shown. Linear regression was performed according to the method specified in Appendix XI of the Pharmacopoeia of the People's Republic of China (2020 Edition, Part II) to produce a regression equation. The measured absorbance of the test sample was substituted into the regression equation to calculate the concentration of ginsenosides in the test solution, and then the content of total ginsenosides in the test sample was calculated. Determined using this method, the total saponin content in GSNs-3k was 5.5%. The hydrophilic-hydrophobic properties of ginsenosides help regulate the self-assembly behavior, drug loading capacity, and stability of nanobodies, optimize the drug release characteristics of nanocarriers, and improve bioavailability. Furthermore, ginsenosides can synergize with nanomaterials to enhance their targeting and transmembrane absorption capabilities, promote long-term drug circulation in the body, and tissue penetration, thereby improving therapeutic efficacy.

[0071] (2) Determination of polysaccharide content: The polysaccharide content was determined using the phenol-sulfuric acid method, with glucose as the reference. 3 mg of ginseng self-assembled nanobodies were accurately weighed and diluted to a 25 mL volumetric flask with purified water. 1 mL of the test solution was placed in a 2 mL graduated test tube with a stopper, and purified water was added to 2 mL. 1.0 mL of 5% phenol solution was added and shaken well. 5 mL of sulfuric acid solution was quickly added and shaken for 5 min. After standing for 10 min, the solution was placed in a boiling water bath and heated for 20 min. The solution was taken out and cooled to room temperature. The absorbance was measured at a wavelength of 488.2 nm using the reagent blank as a reference. At the same time, accurately pipette 0 mL, 0.1 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL, and 1.2 mL of glucose standard solution into 25 mL stoppered graduated test tubes, add water to 2.0 mL, add 1.0 mL of 5% phenol solution to each tube and shake well, quickly add 5 mL of concentrated H2SO4 solution, shake for 5 minutes, let it stand for 10 minutes, heat it in a boiling water bath for 20 minutes, take it out and cool it to room temperature, and measure the absorbance at a wavelength of 490 nm with the reagent blank as a reference. With the concentration of the glucose standard solution as the horizontal axis and the corresponding absorbance as the vertical axis, plot the following: Figure 5 The figure shows a standard curve for total polysaccharide in GSNs-3k, showing a calculated polysaccharide content of 85.3%. This indicates that the polysaccharide, as the primary backbone component of the nanobody, can enhance its physical and chemical properties, such as increasing the viscosity of the system to improve rheological properties and enhance system stability. Furthermore, the polysaccharide can enhance the dispersion stability and targeting of the nanobody by regulating its surface charge, thereby increasing its application value in drug delivery and biomedicine.

[0072] (3) Determination of protein content: The protein content was determined by Bradford method, with bovine serum albumin (BSA) as the reference substance. 0 mL, 0.1 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of BSA standard solution and 1 mg of ginseng self-assembled nanobodies were added to the test tubes, respectively. Distilled water was added to the test tubes to 1.0 mL, and then 5 mL of Coomassie brilliant blue solution was added. The mixture was vortexed quickly and reacted at room temperature for 15 min. The absorbance at 595 nm was measured in a UV-visible spectrophotometer. The concentration of the BSA standard solution was used as the horizontal axis, and the corresponding absorbance value was used as the vertical axis. Figure 6 The bovine serum albumin standard curve for GSNs-3k is shown. The protein content in GSNs-3k was calculated to be 6.02% based on the bovine serum albumin standard curve. During the preparation of ginseng centrifuge, proteins can participate in the surface functionalization of nanobodies through self-assembly, electrostatic adsorption, or covalent bonding. Furthermore, the emulsification, film-forming, and biorecognition capabilities of proteins can enhance the targeting and biocompatibility of ginseng centrifuge, improving its in vivo stability and delivery efficiency, offering potential advantages for drug delivery and biomedical applications.

[0073] Table 1 below summarizes the component contents of the ginseng self-assembled nanobodies prepared in Examples 1-5. The ginseng self-assembled nanobodies primarily comprise polysaccharides (68%-85%), total saponins (2%-7%), and a small amount of protein (3%-6%) (all calculated by dry weight). The polysaccharides, proteins, and active saponins act synergistically: the polysaccharides provide stability and bioactivity, the proteins act as a backbone and transporter, and impart targeting, and the active saponins exert key pharmacological effects and regulate signaling pathways, collectively enhancing the therapeutic efficacy and bioavailability of the nanobodies.

[0074] Table 1

[0075]

[0076] Detection of ginsenoside compounds:

[0077] The ginseng self-assembled nanobody samples were analyzed by ultra-performance liquid chromatography-quadrupole electrostatic field orbitrap tandem mass spectrometry (UPLC-Q-Exactive-MS / MS) technology. The detection was carried out by electrospray ionization (ESI) in positive and negative ion dual mode, and the scanning range was set from 100 to 1500 m / z. Figure 7 The total ion chromatogram of ginsenosides of GSNs-1k (negative ion mode) shows that the retention time (RT) and mass-to-charge ratio (m / z) of different ginsenosides are significantly different. Figure 8This is the identification result of the compounds in the negative ion mode of GSNs-1k. As can be seen from the figure, the present invention successfully detected 12 ginsenosides in GSNs-1k, namely 20-gluco-ginsenoside Rf, Notoginsenoside R1, GinsenosideRg1, Ginsenoside Rf, Ginsenoside Rb1, Ginsenoside mRb1, Ginsenoside Rc, Ginsenoside Rg2, Ginsenoside Ro, Ginsenoside Rb2, Ginsenoside Rh1, and GinsenosideRd. Based on dry weight, the mass ratios of the above ginsenosides are 1:2:13:3:8:3:5:2:4:1:1:3, respectively.

[0078] like Figure 9 This is the total ion chromatogram of ginsenosides of GSNs-3k (negative ion mode). The results show that the retention time (RT) and mass-to-charge ratio (m / z) of different saponins are significantly different, and the secondary mass spectrometry fragmentation characteristics are clear, which helps to distinguish isomers (such as Rg1 and Rf, Rb1 and mRb1). Among them, ginsenosides Rb1 (m / z 1131.5959) and Rg3 (m / z 829.4858) elute later in the chromatogram, indicating that their polarity is lower, while Rg1 and Re have a higher degree of glycosylation and stronger polarity, and elute earlier. Figure 10 This is the identification result of the compound in the negative ion mode of GSNs-3k. It can be seen from the figure that the present invention successfully detected 16 ginsenosides in GSNs-3k, namely Ginsenoside Re, Ginsenoside Rg1, Ginsenoside Rf, Ginsenoside Rb1, Ginsenoside mRb1, Ginsenoside Rc, Ginsenoside Rg2, GinsenosideRo, Ginsenoside Rb2, Ginsenoside Rh1, Ginsenoside Rd, Ginsenoside Rg6, GinsenosideRg4, Ginsenoside Rk3, Ginsenoside Rh4, and Ginsenoside Rg3, as shown in Table 2 below. The ratios are 5:10:4:5:1:4:3:3:3:2:4:1:2:2:3:3, respectively.

[0079] like Figure 11 and Figure 12The figures are the total ion chromatogram of ginsenosides in GSNs-5k (negative ion mode) and the identification results of compounds in negative ion mode. It can be seen from the figure that the present invention successfully detected 11 ginsenosides in GSNs-5k, namely Ginsenoside Re, Ginsenoside Rg1, Ginsenoside Rf, GinsenosideRb1, Ginsenoside mRb1, Ginsenoside Rc, Ginsenoside Rg2, Ginsenoside Ro, Ginsenoside Rb2, Ginsenoside Rh1, and Ginsenoside Rd, as shown in Table 2 below, and their ratios are 1:17:4:11:5:7:2:6:6:2:5, respectively.

[0080] like Figure 13 and Figure 14 The figures are the total ion chromatogram of ginsenosides in GSNs-7k (negative ion mode) and the identification results of compounds in negative ion mode. It can be seen from the figures that the present invention successfully detected 11 ginsenosides in GSNs-7k, namely Ginsenoside Re, Ginsenoside Rg1, Ginsenoside Rf, GinsenosideRb1, Ginsenoside mRb1, Ginsenoside Rc, Ginsenoside Rg2, Ginsenoside Ro, Ginsenoside Rb2, Ginsenoside Rh1, and Ginsenoside Rd, as shown in Table 2 below, and their ratios are 7:11:2:8:3:5:2:4:4:1:3, respectively.

[0081] like Figure 15 and Figure 16 The figures are the total ion chromatogram of ginsenosides in GSNs-12k (negative ion mode) and the identification results of compounds in negative ion mode. It can be seen from the figures that the present invention successfully detected 8 ginsenosides in GSNs-12k, namely Ginsenoside Rb1, Ginsenoside mRb1, Ginsenoside Rc, Ginsenoside Ro, Ginsenoside Rg2, Ginsenoside Rb2, Ginsenoside Rh1, and GinsenosideRd, as shown in Table 2 below, and their ratios are 7:3:4:3:2:1:2:2, respectively.

[0082] The following Table 2 shows the detection results of ginsenoside types in the ginsenoside self-assembled nanobodies prepared in Examples 1-5. The proportion of ginsenoside content was obtained by normalization calculation. Based on dry weight, the mass fraction of each type of ginsenoside in the ginsenoside composition is as follows: Ginsenoside Re 0-9 parts; Ginsenoside Rg1 0-19 parts; Ginsenoside Rf 0-5 parts; Ginsenoside Rb1 6-20 parts; Ginsenoside mRb1 1-8 parts; Ginsenoside Rc 5-11 parts; Ginsenoside Rg2 2-5 parts; Ginsenoside Ro 3-10 parts; Ginsenoside Rb2 2-6 parts; GinsenosideRh1 1-5 parts; Ginsenoside Rd 4-7 parts; Ginsenoside Rg6 0-1 part; Ginsenoside Rg4 0-2 parts; Ginsenoside Rk3 0-2 parts; Ginsenoside Rh4 0-3 parts; Ginsenoside Rg3 0-4 parts; 20-gluco-ginsenoside Rf 0-2 parts; Notoginsenoside R1 0-3 parts. This demonstrates that the present invention breaks through the existing gap in the research on the ratio of different types of ginsenosides. It also further verifies that GSNs-3k contains the richest variety of ginsenosides, which greatly enhances the synergistic effect between ginsenosides and glycoproteins. The hydrophobic groups of ginsenosides can bind to the hydrophobic regions of glycoproteins to form stable self-assembled nanobodies. Their active hydroxyl groups can also form weak interactions such as hydrogen bonds with the carbohydrate portion of glycoproteins, changing the glycoprotein conformation, activating related signaling pathways, and enhancing the overall activity effect.

[0083] Table 2

[0084]

[0085] Therapeutic effect of ginseng self-assembled nanobodies on atopic dermatitis:

[0086] (1) Investigating the therapeutic effect of ginseng self-assembled nanobodies on atopic dermatitis based on human immortalized keratinocytes (HaCaT cells):

[0087] GD and multiple groups of GSNs were used to treat TNF-α-induced human immortalized keratinocyte (HaCaT cell) inflammation model. Figure 17Figure 2 shows the effect of GD and GSNs treatment on the proliferation rate of HaCaT cells. In the figure, #### indicates P<0.0001 compared with the control group; **** indicates P<0.0001 compared with the model group; ** indicates P<0.01 compared with the model group. As can be seen from the figure, the proliferation rate of HaCaT cells increased after treatment with GD and GSNs, proving that both GD and GSNs have a positive promoting effect on the proliferation of HaCaT cells. Among them, the proliferation rate of HaCaT cells treated with GSNs-3k was the most significant. In addition, the levels of pro-inflammatory factors IL-1β, IL-6 and TNF-α were detected by enzyme-linked immunosorbent assay (ELISA). Figure 18 Figure 2 shows the effects of GD and GSNs on the regulation of inflammatory cytokine expression. In the figure, #### indicates P < 0.0001 compared with the control group; **** indicates P < 0.0001 compared with the model group; *** indicates P < 0.001 compared with the model group; ** indicates P < 0.01 compared with the model group; and * indicates P < 0.05 compared with the model group. As can be seen from the figure, the levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were significantly decreased, while the levels of anti-inflammatory cytokines IL-10, IL-4, and TGF-β were significantly increased, indicating that GD, GSNs-1k, GSNs-3k, GSNs-5k, GSNs-7k, and GSNs-12k all possess certain anti-inflammatory activities.

[0088] The present invention further used quantitative real-time polymerase chain reaction (qRT-PCR) technology to evaluate the effect of GSNs-3k on the mRNA expression of the inflammatory factor IL-6 and the anti-inflammatory factor IL-10 in HaCaT cells. HaCaT cells were treated with different concentrations of GSNs-3k (25, 50, and 100 μg / mL). Total RNA was extracted from HaCaT cells, and the A values ​​at 260 and 280 nm were measured. Figure 19 Figure 2 shows the effect of GSNs-3k on the gene expression levels of the inflammatory factors IL-6 and IL-10. In the figure, #### indicates P < 0.0001 compared with the control group; *** indicates P < 0.001 compared with the model group; and ** indicates P < 0.01 compared with the model group. As can be seen from the figure, GSNs-3k at different concentrations exhibited an inhibitory effect on IL-6, with 100 μg / mL of GSNs-3k having a particularly significant inhibitory effect on IL-6 (P < 0.01), demonstrating its strong anti-inflammatory capacity. GSNs-3k at different concentrations also exhibited an enhancing effect on IL-10, with 100 μg / mL of GSNs-3k having the most significant effect on IL-10 (P < 0.01), indicating that it can effectively enhance the expression of anti-inflammatory factors and improve the anti-inflammatory capacity of cells.

[0089] (2) Investigating the therapeutic effect of ginseng self-assembled nanobodies on atopic dermatitis in mice:

[0090] After one week of adaptive feeding, 36 KM mice were induced with 2,4-dinitrochlorobenzene (DNCB) to establish an atopic dermatitis model. Mice were anesthetized with an intraperitoneal injection of 50 mg / kg sodium pentobarbital, and abdominal hair was removed using a depilatory cream combined with shaving. The mice were randomly divided into six groups (n=6): blank control, model, high-dose GD, low-dose GD, high-dose GBSNs-3k, and low-dose GBSNs-3k. Except for the blank control group, 100 μL of a 5% DNCB solution was applied to the abdomen of mice every three days for 14 days to induce atopic dermatitis-like skin lesions. After modeling, mice were gavaged with drugs. The blank control group received no treatment, the model group only received modeling, the GD high-dose group received 0.18 g / kg, the GD low-dose group received 0.09 g / kg, the GSNs-3k high-dose group received 0.6 g / kg, and the GSNs-3k low-dose group received 0.3 g / kg. The drugs were administered once a day for 10 days. Figure 20 Figure 2 shows the effects of GD and GSNs-3k on the skin of mice with atopic dermatitis. As shown in the figure, after DNCB treatment, the abdominal skin of all six groups of mice developed typical pathological features of atopic dermatitis, including erythema, papules, exudation, crusting, scaling, and lichenification. Following treatment, the erythema area in the GD group decreased, and the crusts partially fell off, but mild scaling persisted. The GSNs-3k group (particularly the high-dose group) showed significant improvement in skin lesions, with only minor dryness and scaling observed. New hair appeared normal in appearance, and erosions were completely healed with no exudative lesions.

[0091] like Figure 21 Figure 2 shows the effects of GD and GSNs-3k on HE staining of skin tissue in mice with atopic dermatitis. The figure shows that in the model group, the squamous epithelium in the epidermis proliferated with shedding and the stratum corneum thickened. The collagen fibers in the dermis were disordered, with a large number of inflammatory cells (lymphocytes, plasma cells, and neutrophils) infiltrating, and the hair follicle structure was destroyed. In the high-dose GBSNs-3k group, the epidermis thickness was close to normal, with no significant proliferation. The degree of inflammatory cell infiltration in the dermis was significantly reduced (P<0.05), with a small number of scattered lymphocytes and intact hair follicle structure. Figure 22Figure 2 shows the effects of GD and GBSNs-3k on the gene expression levels of inflammatory factors in the tissues of mice with atopic dermatitis. As shown in the figure, inflammatory factor levels in the skin tissues of mice treated with a high-dose GBSNs-3k treatment group decreased compared to the model group, with IL-1β decreasing by 20.15% and TNF-α decreasing by 25.41% (P < 0.05). This suggests that high-dose GBSNs-3k treatment significantly alleviates DNCB-induced atopic dermatitis, promoting the resolution of acute inflammatory reactions such as erythema and exudation, and repairing the skin barrier. Furthermore, inflammatory cell infiltration in the dermis decreased, and collagen alignment returned to normal. Furthermore, GBSNs-3k inhibited the inflammatory response by downregulating IL-1β and TNF-α expression (P < 0.05), demonstrating its therapeutic effect in atopic dermatitis by ameliorating skin inflammation.

[0092] In summary, the present invention utilizes non-covalent interactions (such as hydrogen bonds, van der Waals forces, and hydrophobic interactions) among the active ingredients in ginseng (such as ginsenosides) to naturally aggregate and form nanostructures. This allows the preparation of ginseng self-assembled nanobodies for use in the preparation of drugs for the treatment of atopic dermatitis. The present invention also demonstrates that the ginseng self-assembled nanobodies are composed of 72-92% ginseng glycoproteins and 2-7% active ginsenoside compositions (all by dry weight), and the content of each active ginsenoside composition is determined. This improves the solubility and bioavailability of the drug. Furthermore, the ginseng self-assembled nanobodies achieve precise and effective treatment of atopic dermatitis in mice, reduces the side effects of traditional treatments, and ensures safety for long-term use. Furthermore, the present invention's preparation process is simple and clear, and the formation of the ginseng self-assembled nanobodies can be visually verified by observing the Tyndall effect. Combining centrifugation with observation of the Tyndall effect allows precise control of the particle size and purity of the nanobodies, thereby enabling the preparation of efficient and stable nanomedicines.

[0093] The above embodiments are only intended to help understand the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by those skilled in the art, and such improvements and modifications also fall within the scope of protection of the claims of the present invention.

[0094] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing ginseng self-assembled nanobodies, characterized in that: The following steps are involved: S1: Grind and sieve the ginseng, soak the ginseng powder in distilled water, decoct the soaked material, collect the decoction and filter the ginseng medicinal material; S2: adding distilled water to the ginseng material in S1 for decoction, collecting the decoction and filtering it, combining the two decoctions to obtain a ginseng decoction, and cooling it to room temperature; S3: The ginseng decoction was centrifuged at 1000 r / min for 20 min to remove impurities, and the supernatant was collected. The supernatant was then centrifuged at 1000 r / min for 20 min. The above combined centrifugation steps were repeated three times, and the supernatant was collected and purified by centrifugation at 12000 r / min for 20 min. S4: The precipitate after purification and centrifugation in S3 was dissolved, vortex-mixed, and centrifuged at 1000 r / min for 20 min. The supernatant was collected and centrifuged at 3000 r / min for 20 min. The above combined centrifugation steps were repeated three times. The supernatant was collected and centrifuged at 12000 r / min for 20 min to obtain ginseng centrifuge solution. The supernatant was collected. S5: The collected supernatant is filtered and purified, the filtrate is freeze-formed, and then freeze-dried to obtain ginseng self-assembled nanobodies.

2. The method for preparing ginseng self-assembled nanobodies according to claim 1, wherein: The sieving in S1 is performed using a 20-40 mesh sieve; the mass ratio of the ginseng powder to distilled water is 1:6-12; the soaking time is 1-3 h; the decoction temperature is 80-120° C.; and the decoction time is 2-3 h.

3. The method for preparing ginseng self-assembled nanobodies according to claim 1, wherein: The mass ratio of the ginseng medicinal material to distilled water in S2 is 1:4-8; the decoction temperature is 80-120° C.; and the decoction time is 1.5-3 h.

4. The method for preparing ginseng self-assembled nanobodies according to claim 1, wherein: The filtration in S5 uses filter membranes with pore sizes of 0.4 μm and 0.22 μm respectively; the freezing molding temperature is -20°C, and the freezing molding time is 6 to 18 hours.

5. The method for preparing ginseng self-assembled nanobodies according to claim 1, wherein: The freeze-drying pressure in S5 is 0.5 MPa, the freeze-drying temperature is -80°C, and the freeze-drying time is 80 to 120 h.

6. A ginseng self-assembling nanobody, characterized in that: The ginseng self-assembling nanobody is prepared by the preparation method according to any one of claims 1 to 5, wherein the ginseng self-assembling nanobody uses ginseng glycoprotein as a carrier and synergistically completes self-assembly with an active ginsenoside composition.

7. The ginseng self-assembled nanobody according to claim 6, characterized in that Based on dry weight, the mass fraction of the ginseng glycoprotein is 72-92%, and the ginseng glycoprotein includes polysaccharides and proteins, and the mass ratio of the polysaccharides to proteins is 14-22:1; the mass fraction of the active ginsenoside composition is 2-7%; the types of the ginsenoside composition include ginsenoside Re, ginsenoside Rg1, ginsenoside Rf, ginsenoside Rb1, ginsenoside mRb1, ginsenoside Rc, ginsenoside Rg2, ginsenoside Ro, ginsenoside Rb2, ginsenoside Rh1, ginsenoside Rd, ginsenoside Rg6, ginsenoside Rg4, ginsenoside Rk3, ginsenoside Rh4, ginsenoside Rg3, 20-glucose-ginsenoside Rf and Panax notoginseng saponin R1. Based on dry weight, the mass fraction of each type of ginsenoside in the ginsenoside composition is: ginsenoside Re 0~9 parts; ginsenoside Rg10~19 parts; ginsenoside Rf 0~5 parts; ginsenoside Rb1 6~20 parts; Ginsenoside mRb1 1-8 parts; Ginsenoside Rc 5-11 parts; Ginsenoside Rg2 2-5 parts; 3-10 parts of ginsenoside Ro; 2-6 parts of ginsenoside Rb2; 1-5 parts of ginsenoside Rh1; 4-7 parts of ginsenoside Rd; 0-1 part of ginsenoside Rg6; 0-2 parts of ginsenoside Rg4; 0-2 parts of ginsenoside Rk3; 0-3 parts of ginsenoside Rh4; 0-4 parts of ginsenoside Rg3; 0-2 parts of 20-glucose-ginsenoside Rf; 0-3 parts of notoginsenoside R1.

8. The ginseng self-assembled nanobody according to claim 7, characterized in that The mass ratio of ginsenoside Re, ginsenoside Rg1, ginsenoside Rf, ginsenoside Rb1, ginsenoside mRb1, ginsenoside Rc, ginsenoside Rg2, ginsenoside Ro, ginsenoside Rb2, ginsenoside Rh1, ginsenoside Rd, ginsenoside Rg6, ginsenoside Rg4, ginsenoside Rk3, ginsenoside Rh4, and ginsenoside Rg3 is 5:10:4:5:1:4:3:3:3:2:4:1:2:2:3:

3.

9. Use of the ginseng self-assembling nanobody according to any one of claims 6 to 8 in the preparation of a drug for treating atopic dermatitis.

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