Fermented astragalus membranaceus-loaded nanoparticles, and preparation method and application thereof

Abstract

This invention proposes a method for preparing fermented Astragalus membranaceus nanoparticles and their applications, belonging to the field of traditional Chinese medicine. The invention provides a method for preparing fermented Astragalus membranaceus nanoparticles, where Astragalus membranaceus is fermented using a mixed strain of Bacillus amyloliquefaciens, Lactobacillus rhamnosus, and Bifidobacterium BB-12, followed by encapsulation with thiolated chitosan to obtain fermented Astragalus membranaceus nanoparticles. The fermented Astragalus membranaceus nanoparticles prepared by this method improve the utilization rate of Astragalus membranaceus, enhance drug targeting, and effectively protect the active substances in Astragalus membranaceus, increasing the drug's half-life and prolonging its duration of action. The fermented Astragalus membranaceus nanoparticles provided by this invention can also be used to prepare drugs for treating porcine colitis.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine, and in particular relates to nano-sized traditional Chinese medicine for the treatment of porcine colitis. Background Technology

[0002] Astragalus is a traditional Chinese medicine native to Shanxi Province, which accounts for 50% of the national demand. It is also a widely used medicinal herb in animal husbandry. Its active ingredients mainly include flavonoids, saponins, and polysaccharides. Flavonoids have significant antioxidant and anti-aging effects; saponins have anti-inflammatory, immunomodulatory, and antioxidant effects. Astragalus polysaccharides (APS) are one of the main active ingredients of astragalus. Besides being low in toxicity, unlikely to induce drug resistance, low in residues, and pollution-free, they also possess various biological activities such as anti-inflammatory, immunomodulatory, and antioxidant effects. Currently, the fermentation of astragalus generally uses traditional processes such as decoction, boiling, simmering, refining, steaming, and soaking. However, these traditional processes can destroy the active substances in astragalus, resulting in reduced efficacy.

[0003] Modern research shows that the onset time, intensity, and duration of action of drugs in vivo are closely related not only to the chemical structure of the drug itself but also to its physical state. Changing the unit size of a drug is an effective way to alter its physical state. Nanoparticle-based traditional Chinese medicine (TCM) may significantly change its physicochemical properties and physiological activities, even altering its medicinal properties and producing new efficacy. Nanotechnology provides entirely new ideas and approaches for the research and development of TCM. Research on nano-TCM is being conducted by combining the strengths of materials science, engineering, and life sciences. Xu Huibi et al. proposed the scientific concept of "nano-TCM" and applied for the first patent for nano-TCM technology. Nano-TCM refers to TCM with a particle size of less than 100 nm manufactured using nanotechnology. However, current research on nano-TCM containing Astragalus membranaceus active substances is limited.

[0004] Swine transmissible colitis is a common disease in small and medium-sized pig farms. Its clinical characteristics include persistent diarrhea, leading to decreased production performance in finishing pigs and uneven growth rates and weight distribution within the same pen. Swine transmissible colitis is generally a mixed infection, usually caused by two or more pathogenic microorganisms. Pathogens causing colitis include *Treponema polychaete*, *Lawsonia intracellularis*, *Salmonella*, and *Yersinia*. Current conventional treatment involves mixing antibiotics such as neomycin, sulfadiazine, tetracyclines, and sulfadiazine into the feed. However, antibiotics easily kill beneficial bacteria in the intestines, causing recurrent colitis and often resulting in antibiotic residues. Therefore, there is an urgent need for a new type of veterinary drug that is highly effective and leaves no antibiotic residues. Summary of the Invention

[0005] This invention provides a method for treating porcine colitis by loading fermented Astragalus nanoparticles, which can effectively treat porcine colitis, enriching the types of nano-Chinese medicines and solving the problem of antibiotic residues in the current treatment of porcine colitis with antibiotics.

[0006] A type of fermented Astragalus nanoparticles are obtained by fermenting Astragalus with a mixed strain of Bacillus amyloliquefaciens, Lactobacillus rhamnosus and Bifidobacterium BB-12 and then encapsulating it with thiolated chitosan.

[0007] The preparation method of fermented Astragalus membranaceus nanoparticles is as follows:

[0008] S1: Grind the Astragalus membranaceus slices into powder and sterilize them under ultraviolet light;

[0009] S2: Dissolve Astragalus powder in fermentation medium and add a mixed strain of Bacillus amyloliquefaciens, Lactobacillus rhamnosus and Bifidobacterium BB-12. Incubate at 37℃ and 180rpm for 3 days to obtain primary fermented Astragalus sample.

[0010] S3: The primary sample of fermented Astragalus membranaceus was encapsulated with TCS NPs. The primary sample of fermented Astragalus membranaceus, CS and PETMP were mixed and added to 100 mL of 1% glacial acetic acid. The mixture was stirred overnight at 30 °C to synthesize fermented Astragalus membranaceus nanoparticles.

[0011] Furthermore, the mass-to-volume ratio of the Astragalus powder dissolved in the fermentation medium in S2 is 1:4.

[0012] Furthermore, in the mixed strain of Bacillus amyloliquefaciens, Lactobacillus rhamnosus, and Bifidobacterium BB-12 described in S2, the mixing ratio of the three strains is 1:1:1.

[0013] Furthermore, the mixed strain of Bacillus amyloliquefaciens, Lactobacillus rhamnosus, and Bifidobacterium BB-12 described in S2 was added at 1%.

[0014] Furthermore, the mass fraction of CS in S3 is 1.5%.

[0015] Furthermore, the mass fraction of PETMP in S3 is 0.5%.

[0016] Furthermore, the mass ratio of CS to PETMP in S3 is 12:1.

[0017] Furthermore, the amount of fermented Astragalus membranaceus primary sample added in S3 is 1-3 mg.

[0018] Application of fermented Astragalus nanoparticles loaded with fermentation in the preparation of drugs for treating porcine colitis.

[0019] Beneficial effects

[0020] This invention improves the utilization rate of Astragalus by providing a method for preparing fermented Astragalus nanoparticles.

[0021] This invention provides a method for improving the bioavailability and targeting of traditional Chinese medicine by loading fermented Astragalus nanoparticles, thereby protecting the active substances in Astragalus, increasing the half-life of the drug, and prolonging the duration of action of the drug.

[0022] The present invention provides a method for treating swine colitis by loading fermented astragalus nanoparticles. Attached Figure Description

[0023] Figure 1 The graph shows the linear regression equation for the reference solution.

[0024] Figure 2 This is a graph showing the results of the polysaccharide concentration change curve;

[0025] Figure 3 The infrared spectra of CS and TCS NPs-Astragalus are shown, where A is the infrared spectrum of CS and B is the infrared spectrum of TCS NPs-Astragalus.

[0026] Figure 4 The images show the 1H NMR spectra of CS and TCS NPs-Astragalus membranaceus, where A is the 1H NMR spectrum of CS and B is the 1H NMR spectrum of TCS NPs-Astragalus membranaceus.

[0027] Figure 5 The image shows the morphological results of TCS NPs-Astragalus membranaceus detected by TEM.

[0028] Figure 6 The figure shows the release curves of TCS NPs-Astragalus in simulated gastric and intestinal fluids.

[0029] Figure 7 This is a mouse ex vivo intestinal imaging image, where Y represents TCS NPs-Cy5.5 and N represents free Cy5.5. Detailed Implementation

[0030] Example 1. Preparation of fermented Astragalus nanoparticles.

[0031] I. Fermentation process of Astragalus polysaccharides.

[0032] 1. Preparation of bacterial culture.

[0033] Agar slant culture medium was prepared. *Bacillus amyloliquefaciens*, *Lactobacillus rhamnosus*, and *Bifidobacterium BB-12* (purchased from Shandong Yihao Biotechnology Co., Ltd.) were inoculated onto slant culture medium (the medium formula, by weight percentage, includes: 0.5% beef extract, 1% peptone, 0.5% NaCl, 2% agar, pH 7.2, sterilized at 121℃ for 20 min) on a clean bench. The medium was incubated at 37℃ until the inoculum tubes were confluent with colonies, thus obtaining activated bacterial strains. An appropriate amount of the bacterial strain was taken from the slant culture medium and a bacterial suspension was prepared with sterile water. The concentration of the bacterial suspension was determined using a spectrophotometer. The results are shown in Table 1. Spectrophotometer analysis showed that the concentrations of the three bacterial suspensions were all close to 1×10⁻⁶. 8 The cfu / mL concentration was suitable for inoculation. 1 mL of each of the three bacterial suspensions was inoculated into 100 mL of liquid culture medium and incubated at 37℃ and 180 rpm for 24 h to obtain the bacterial solution used for inoculation and fermentation.

[0034] Table 1

[0035] Strains (diluent) <![CDATA[Linear range / (×10 8 cfu / mL)]]> Linear equations <![CDATA[Coefficient of correlation r 2 > Bacillus amyloliquefaciens 1.027-1.113 y = 6.578x + 0.15828 0.99899 Lactobacillus rhamnosus 0.978-1.243 y = 7.331x - 0.17617 0.99962 Bifidobacterium BB-12 1.122-1.535 y = 5.1657x - 0.12582 0.99899 physiological saline 0.002-0.013 y = 5.2836x - 0.01481 0.99954

[0036] 2. Preparation of Astragalus fermentation broth.

[0037] Weigh out an appropriate amount of Astragalus membranaceus slices, grind them into powder, sterilize them under ultraviolet light, and set aside for later use.

[0038] The fermentation medium was formulated as follows (by weight percentage): 0.5% beef extract, 1% peptone, 0.5% NaCl, and 2% agar, pH 7.0, and sterilized at 121℃ for 20 min. Astragalus was added at a rate of 0.25 g per milliliter of medium, i.e., 50.0 g of astragalus per 200 mL of medium. The initial pH of all media was 7.0, and the medium was dispensed into 250 mL Erlenmeyer flasks. To verify the effects of *Bacillus amyloliquefaciens*, *Bifidobacterium BB-12*, and *Lactobacillus rhamnosus* on the polysaccharide content of astragalus in the fermentation broth, seven combinations of different strains were established (mixed strains were mixed at a volume ratio of 1:1 or 1:1:1), as shown in Table 2. The fermentation temperature was 37℃.

[0039] Table 2

[0040] Group strains A Bacillus amyloliquefaciens + Lactobacillus rhamnosus + Bifidobacterium BB-12 B Bacillus amyloliquefaciens C Lactobacillus rhamnosus D Bifidobacterium BB-12 E Lactobacillus rhamnosus + Bifidobacterium BB-12 F Bacillus amyloliquefaciens + Lactobacillus rhamnosus G Bacillus amyloliquefaciens + Bifidobacterium BB-12

[0041] The prepared culture medium was divided into AG groups, with each group repeated three times. The bacterial strains were inoculated into the culture medium at the same time points as shown in Table 1, at an inoculum rate of 1.0%. The culture time after inoculation was 7 days. The content of Astragalus polysaccharides in the fermentation broth was measured daily, and the daily changes in Astragalus polysaccharide content for each group were recorded. The results are shown in Table 3.

[0042] Table 3

[0043]

[0044]

[0045] Weigh 100 mg of D-anhydrous glucose (dried to constant weight at 105℃), add water to a final volume of 100 mL, and shake well to obtain a glucose solution. Pipette 0.2, 0.4, 0.6, 1.0, and 1.4 mL of the glucose solution into 25 mL volumetric flasks, add distilled water to a final volume of 25 mL, and shake well to prepare a series of reference solutions. Pipette 2 mL of each series of reference solutions into test tubes, using distilled water as a blank control, add 1.0 mL of 5% phenol solution, shake well, quickly add 5.0 mL of concentrated sulfuric acid, shake for 2 min, incubate in a boiling water bath for 15 min, then in a cold water bath for 30 min, and measure the absorbance at 490 nm. Perform a linear regression on glucose concentration (C) and absorbance (A), obtaining the regression equation y = 40.0300x + 0.55420(r) 2 =0.99821), such as Figure 1 The results showed that glucose exhibited good linearity in the range of 2.0 μg / mL to 14.0 μg / mL, with an average recovery rate of 97.78% and an RSD of 2.54% (n=6).

[0046] Substituting the OD values ​​of polysaccharides in the fermentation broth from Table 3 into the regression equation, the daily changes in polysaccharide concentration in the fermentation broth were obtained. The polysaccharide concentration change curve is shown in the figure. Figure 2 Curves were plotted to show the changes in polysaccharide concentration for each group. The polysaccharide concentration reached its peak in the product on the third day of co-fermentation using a mixed strain of Bacillus amyloliquefaciens, Lactobacillus rhamnosus, and Bifidobacterium BB-12.

[0047] II. Preparation of sample solution.

[0048] Dissolve 50g of Astragalus membranaceus powder in 200mL of fermentation medium. Prepare a 1×10⁻⁶ strain of a mixed bacterial strain of Bacillus amyloliquefaciens, Lactobacillus rhamnosus, and Bifidobacterium BB-12 using sterile water. 8 A suspension of CFU / mL was prepared. The suspension was inoculated at 1% of the culture medium volume and cultured on a shaker at 37°C and 180 rpm for 3 days. The fermentation broth was centrifuged, and the supernatant was collected to obtain the primary sample of fermented Astragalus membranaceus.

[0049] III. Preparation of Fermented Astragalus Extract Samples

[0050] 1. Synthesis of thiolized chitosan (TCS NPs)

[0051] 2 mL of chitosan (CS) solution (1.5 g / 100 mL) and 0.5 mL of pentaerythritol tetrakis(3-mercaptopropionic acid) (PETMP) solution (0.5 g / 100 mL) were mixed at a mass ratio of 12:1, and then 100 mL of 1% glacial acetic acid was added. The mixture was stirred overnight at 30 °C to synthesize nanoparticles (blank TCS NPs). At the end of the reaction, a milky white gel or solution was formed, yielding the product of the Schiff base reaction. Then, 2 L of deionized water was mixed with the product of the Schiff base reaction (volume ratio of 20:1) to form an emulsion suspension of nanoparticles (sol). Subsequently, the solution was purified by dialysis at 1200 rpm through a cellulose-based membrane filter (MW cutoff of 10 kDa, Amicon Ultra-15) until the pH of the solution was neutralized, yielding TCS NPs. Finally, the purified nanoparticles were stored in an aqueous dispersion in a sealed container at 4 °C.

[0052] 2. Synthesis of fermented Astragalus nanoparticles supported on TCS NPs (hereinafter referred to as TCS NPs-Astragalus).

[0053] The first step is to concentrate the primary fermented Astragalus sample. The primary fermented Astragalus sample is freeze-dried and concentrated to make a high-concentration stock solution (30mg / mL) for later use.

[0054] In the second step, the primary fermented Astragalus sample was encapsulated with TCS NPs. 0.07 mL of the primary fermented Astragalus sample (high-concentration stock solution, 30 mg / mL), 2 mL of CS (1.5 g / 100 mL), and 0.5 mL of PETMP (0.5 g / 100 mL) were mixed and added to 100 mL of 1% glacial acetic acid. The mixture was stirred continuously at 30 °C overnight to synthesize TCS NPs-Astragalus.

[0055] 3. Synthesize TCS NPs-Cy5.5.

[0056] The preparation of TCS NPs-Cy5.5 involves replacing the primary fermented Astragalus sample in the above steps with 1.5 mg of Cy5.5, while the remaining synthesis steps are the same as above.

[0057] 4. HPLC was used to determine the drug loading and encapsulation efficiency.

[0058] Primary samples of fermented Astragalus membranaceus were lyophilized, and molecular standard solutions of different concentrations (1.25, 2.5, 5, 50, 100, and 200 μg / mL) were prepared. The samples were then analyzed using high-performance liquid chromatography (HPLC) under specific conditions, and the peak area was recorded at 254 nm. A linear regression was performed with peak area (C, μg / mL) as the ordinate and the concentration of the primary fermented Astragalus membranaceus sample (R0) as the abscissa. The linear regression equation for the primary fermented Astragalus membranaceus sample was P = 49579C + 15754(R0). 2=1). Three experimental groups were set up during the preparation of TCS NPs-Astragalus membranaceus: 1, 2, or 3 mg of fermented Astragalus membranaceus primary sample was encapsulated with TCS NPs, and the supernatant of the product was collected. 10 μL of the supernatant was then analyzed by high-performance liquid chromatography (HPLC). The result was calculated according to the formula:

[0059] Encapsulation rate: EE% = (Weight of primary fermented Astragalus sample added - Weight of free primary fermented Astragalus sample) / Weight of primary fermented Astragalus sample added × 100%;

[0060] Drug loading: DL% = (Weight of primary fermented Astragalus sample added - Weight of free primary fermented Astragalus sample) / TCS NPs - Astragalus × 100%;

[0061] The specific results are shown in Table 4.

[0062] Table 4

[0063]

[0064] II. Infrared Spectroscopy.

[0065] The CS and TCS NPs-Astragalus raw materials were dried, compressed into tablets using KBr, and the corresponding infrared peaks were detected.

[0066] Infrared spectra of chitosan (CS), TCS NPs-Astragalus membranaceus are shown below. Figure 3 Characteristic peaks of fermented astragalus extract were observed in the TCS. The results indicate that the fermented astragalus extract was successfully encapsulated within carboxymethyl chitosan nanoparticles.

[0067] III. Characterization by proton nuclear magnetic resonance (NMR) spectroscopy.

[0068] CS and TCS NPs-Astragalus membranaceus using D2O as solvent 1 H NMR spectrum, such as Figure 4 The spectrum shows response signal peaks for various sugar protons in the δ3.5–δ1.5 range, with the strongest signal at δ4.8. A set of peaks in the δ4.5–5.2 range is particularly representative. Specifically, δ5.2 and δ5.08 represent anomeric proton signals from the α-configuration pyran ring, δ4.6 and δ4.4 represent anomeric proton signals from the β-configuration pyran ring, and δ5.4 represents an anomeric proton signal from the α-configuration furan ring. This indicates that Astragalus polysaccharides are composed of multiple monosaccharides.

[0069] IV. Determination of thiol content.

[0070] The thiol content in TCS NPs-Astragalus was determined using the Ellman method: First, 50 mg of DTNB was dissolved in 100 mL of PBS buffer (pH 8.0) to prepare the Ellman reagent. Then, 1 mL of TCS NPs-Astragalus solution (25 mg / mL) was mixed with 6.5 mL of the Ellman reagent. After reacting for 2 hours at room temperature in the dark, the sample was measured at 410 nm using a UV spectrophotometer, and the thiol content was calculated based on the standard curve, ranging from 0.2 to 2.4 μg / mL.

[0071] V. Morphological examination: TEM examination of the morphology of TCS NPs-Astragalus membranaceus.

[0072] Use a pipette to apply 20 μL of TCS NPs-Astragalus membranaceus onto a carbon membrane copper grid. Let it stand for 3-5 minutes, then blot away excess liquid with filter paper and allow it to dry at room temperature. Observe under a transmission electron microscope, acquire images, and analyze them. Figure 5 (Scale bar: 200 nm). The results showed that the TCS NPs-Astragalus microspheres all had good spherical morphology and many protrusions and wrinkles on the surface.

[0073] VI. Particle size potential detection.

[0074] The diffusion of Brownian motion particles was detected using a Malvern Nano potentiometer (Zetasizer). The instrument, a Zetasizer Nano ZS90, was equipped with a He-Ne light source at a wavelength of 633 nm and a scattering angle of 90°. The temperature was set to 25°C. 100 μL of nanoparticle solution was placed in a cuvette and placed in the detection chamber. The detection was repeated three times, with 20 cycles per cycle and a 10-second pause between cycles. Analysis was performed using Malvern Dispersion Technology Software 4.2, and the average values ​​were taken. The results are shown in Tables 5-6. The particle sizes of the TCS NPs-Astragalus (Table 5) and TCS NPs-Cy5.5 (Table 6) microspheres were similar, both around 0.9 μm. The particle size distribution of the microspheres was narrow and uniform.

[0075] Table 5

[0076] Hydrodynamic size (nm) PDI Zeta(mV) 1st time 91.06 0.206 11.8 2nd time 91.99 0.187 11.6 3rd time 91.08 0.201 12.3 average value 91.38 0.198 11.9

[0077] Table 6

[0078] Hydrodynamic size (nm) PDI Zeta(mV) 1st time 91.46 0.203 13.8 2nd time 90.83 0.189 12.1 3rd time 90.27 0.207 12.1 average value 90.85 0.199 12.7

[0079] VII. External release behavior.

[0080] The release behavior of nanoparticles was investigated in simulated gastric and intestinal fluids. 10 mg of TCS NPs-Astragalus was accurately weighed and first added to simulated gastric fluid (pH 1.2) without pepsin. The mixture was incubated at 37°C for 5 h in a digitally controlled temperature shaker. Then, simulated intestinal fluid (pH 6.8) was added, and the mixture was incubated at 37°C for 24 h in the same shaker. The supernatant was collected at different time points and subjected to quantitative analysis.

[0081] The release curves of TCS NPs-Astragalus in simulated gastric and intestinal fluids are shown below. Figure 6 After 5 hours in simulated gastric fluid, the release rate was approximately 20%, indicating that TCS NPs-Astragalus effectively protected the fermented Astragalus extract (primary sample). In simulated intestinal fluid, the release rate of the fermented Astragalus extract (primary sample) reached approximately 63% within 8 hours, demonstrating that TCS NPs-Astragalus rapidly releases the fermented Astragalus extract in simulated intestinal fluid, and its release rate in a neutral environment is comparable to data from similar experiments.

[0082] 8. In vivo targeted behavior.

[0083] Mouse ex vivo intestinal imaging: Two groups of materials (TCS NPs-Cy5.5 and free Cy5.5) were administered via gavage. The mice were sacrificed at 4, 12, and 24 hours post-administration, and the ex vivo intestines were then imaged. Figure 7 After comparison, free TCS NPs-Cy5.5 were found to be basically evenly distributed in the mice after 24 hours.

[0084] 9. TCS NPs-Astragalus treatment for swine colitis.

[0085] 1. Effects of TCS NPs-Astragalus on antioxidant enzyme activity in porcine ulcerative colitis.

[0086] The levels of SOD, CAT, and GST in porcine colon were determined by ELISA. Following the kit instructions, appropriate amounts of reagent were added to blank wells, standard wells, and sample wells. After incubation, washing, and color development, the levels were measured using a microplate reader at a wavelength of 450 nm. A standard curve was established based on the standard wells, and the concentrations were calculated.

[0087] The kits used were from Jiangsu Enzyme Immunoassay Co., Ltd.: Porcine Total Superoxide Dismutase (T-SOD) ELISA Kit, Porcine Cathepsin (cath) ELISA Research Kit, and Porcine Glutathione S-transferase (GST) ELISA Research Kit. The results are shown in Table 7. TCS NPs-Astragalus membranaceus has a good therapeutic effect on porcine colitis.

[0088] Table 7

[0089] Grouping <![CDATA[SOD(U / mg -1 )]]> CAT (U / mg-1) <![CDATA[GST(U / g -1 )]]> Blank control group 14.39±1.20 134.23±16.57 0.91±0.09 colitis model group <![CDATA[6.94±0.68 * ]]> <![CDATA[94.73±7.07 * ]]> <![CDATA[0.37±0.05 * ]]> TCS NPs-Astragalus treatment group <![CDATA[13.01±1.51 # ]]> <![CDATA[125.07±9.70 # ]]> <![CDATA[0.73±0.03 # ]]>

[0090] *P < 0.05; #P < 0.05

[0091] 2. Effects of Astragalus Fermentation Nanoparticles on Inflammation and Pro-inflammatory Cytokines in Porcine Ulcerative Colitis

[0092] The levels of TNF-γ, L-1β, L-6, MPO, and NO in porcine colon were determined by ELISA. Following the kit instructions, appropriate amounts of reagents were added to blank wells, standard wells, and sample wells. After incubation, washing, and color development, the levels were measured using a microplate reader at a wavelength of 450 nm. A standard curve was established based on the standard wells, and the concentrations were calculated.

[0093] The kits were from Jiangsu Enzyme Immunoassay Co., Ltd.: Porcine Tumor Necrosis Factor γ (TNF-γ) ELISA Research Kit, Porcine Interleukin 1β (IL-1β) ELISA Research Kit, Porcine Interleukin-6 (IL-6) ELISA Research Kit, Porcine Myeloperoxidase (MPO) ELISA Kit, and Serum Nitric Oxide (NO) ELISA Detection Kit. The results are shown in Table 8.

[0094] Table 8

[0095]

[0096] *P < 0.05; #P < 0.05

[0097] 10. Conclusion.

[0098] Thiolization chemical modification of chitosan yielded thiolated chitosan. Infrared spectroscopy analysis confirmed successful modification.

[0099] SEM and particle size analysis both showed that the thiolated chitosan microspheres loaded with fermented Astragalus membranaceus extract had good spherical morphology and the average particle size was smaller than that of unloaded thiolated chitosan.

[0100] The drug loading rate and encapsulation efficiency of thiolized chitosan microspheres at an initial dosage of 2 mg of fermented Astragalus membranaceus extract were 5.10±0.1% and 79.20±0.2%, respectively.

[0101] After soaking in simulated gastric and intestinal fluids for 5 and 20 hours, respectively, the release rates of the carboxymethyl chitosan microspheres loaded with fermented astragalus extract were approximately 20% and 63%, respectively. Various indicators during the treatment of porcine colitis using fermented astragalus extract nanoparticles showed that these nanoparticles played a positive role in the treatment of porcine colitis.

Claims

1. A nanoparticle loaded with fermented Astragalus membranaceus, characterized in that: The loaded fermented Astragalus nanoparticles were obtained by fermenting Astragalus with a mixed strain of Bacillus amyloliquefaciens, Lactobacillus rhamnosus and Bifidobacterium BB-12 and then encapsulating it with thiolated chitosan. The loaded fermented Astragalus nanoparticles are used to prepare a drug for treating porcine colitis. The preparation method of the loaded fermented Astragalus nanoparticles is as follows: S1: Grind the Astragalus membranaceus slices into powder and sterilize them under ultraviolet light; S2: Dissolve Astragalus powder in fermentation medium and add a mixed strain of Bacillus amyloliquefaciens, Lactobacillus rhamnosus and Bifidobacterium BB-12. Incubate at 37℃ and 180rpm for 3 days to obtain primary fermented Astragalus sample. S3: The primary sample of fermented Astragalus membranaceus was encapsulated with TCS NPS. The primary sample of fermented Astragalus membranaceus, CS and PETMP were mixed and added to 100 mL of 1% glacial acetic acid. The mixture was stirred overnight at 30 °C to synthesize nanoparticles loaded with fermented Astragalus membranaceus. The mixing ratio of the three strains in the mixed strain of Bacillus amyloliquefaciens, Lactobacillus rhamnosus, and Bifidobacterium BB-12 described in S2 is 1:1:1; The mass ratio of CS to PETMP in S3 is 12:

1.

2. The loaded fermented astragalus nanoparticles according to claim 1, characterized in that: The mass-to-volume ratio of Astragalus powder dissolved in the fermentation medium in S2 is 1:

4.

3. The loaded fermented astragalus nanoparticles according to claim 1, characterized in that: The mixed strain of Bacillus amyloliquefaciens, Lactobacillus rhamnosus, and Bifidobacterium BB-12 described in S2 was added at 1%.

4. The loaded fermented Astragalus nanoparticles according to claim 1, characterized in that: The mass fraction of CS mentioned in S3 is 1.5%.

5. The loaded fermented Astragalus nanoparticles according to claim 1, characterized in that: The mass fraction of PETMP mentioned in S3 is 0.5%.

6. The loaded fermented astragalus nanoparticles according to claim 1, characterized in that: The amount of fermented Astragalus membranaceus primary sample added in S3 is 1-3 mg.

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