Fermented astragalus mongholicus-loaded nano-particles as well as preparation method and application thereof

Abstract

The invention provides nano-particles loaded with fermented astragalus membranaceus as well as a preparation method and application of the nano-particles, and belongs to the field of traditional Chinese medicines. The invention provides a method for preparing nano-particles loaded with fermented astragalus membranaceus, which comprises the following steps: carrying out fermentation culture on astragalus membranaceus by using a mixed strain of bacillus amyloliquefaciens, lactobacillus rhamnosus and bifidobacterium BB-12, and then wrapping by using thiolated chitosan to obtain the nano-particles loaded with the fermented astragalus membranaceus. The loaded fermented astragalus mongholicus nanoparticles prepared by the method improve the utilization rate of astragalus mongholicus, enhance the drug targeting property, play a good role in protecting active substances of astragalus mongholicus, prolong the half-life period of the drug and prolong the action time of the drug. The nano-particles loaded with the fermented astragalus membranaceus can also be used for preparing a medicine for treating the swine colitis.

Description

Technical Field

[0001] The present invention belongs to the field of traditional Chinese medicine, and particularly relates to a nano traditional Chinese medicine for treating swine colitis. Background Art

[0002] Astragalus membranaceus is a famous local medicinal material in Shanxi Province. The output of Astragalus membranaceus in Shanxi Province accounts for 50% of the national demand, and it is also a traditional Chinese medicine with a relatively large dosage in breeding. Its active ingredients mainly include flavonoids, saponins and polysaccharides. Flavonoid components have significant antioxidant, anti-aging and other effects; saponin components have anti-inflammatory, immunomodulatory, antioxidant and other effects. Astragalus polysaccharides (APS) is one of the main active ingredients of Astragalus membranaceus. In addition to having the characteristics of low toxicity, low drug resistance, low residue and no pollution, it also has a variety of biological activities such as anti-inflammatory, immunomodulatory and antioxidant. At present, the traditional processes such as decocting, boiling, simmering, refining, steaming and soaking are generally used for fermenting Astragalus membranaceus, but the traditional processes will destroy the active substances in Astragalus membranaceus and reduce the medicinal efficacy.

[0003] Modern research shows that the onset time, action intensity and duration of drugs in the body are not only related to the chemical structure of the drugs themselves, but also closely related to the physical state of the drugs. Changing the unit size of drugs is an effective way to change their physical state. After the nano-scale transformation of traditional Chinese medicine, the physicochemical properties and physiological activities of drugs may change significantly, and even change the properties of traditional Chinese medicine to produce new effects. Nanotechnology provides a new idea and approach for the research and development of traditional Chinese medicine. Utilize the disciplinary advantages of materials science, engineering and life science and combine them with each other to conduct research on nano traditional Chinese medicine. Xu Huibi et al. put forward the scientific concept of "nano traditional Chinese medicine" based on this and applied for the first patent on nano traditional Chinese medicine technology. Nano traditional Chinese medicine refers to traditional Chinese medicine manufactured by using nanotechnology with a particle size less than 100 nm. However, there is currently little research on nano traditional Chinese medicine containing the active substances of Astragalus membranaceus.

[0004] Swine colitis is a common disease in medium and small-scale pig farms. Its clinical characteristics are intractable diarrhea, which leads to a decline in the production performance of fattening pigs, and the growth rate and weight in the same pen are not uniform enough. Swine infectious colitis is generally a mixed infection, usually caused by two or more pathogenic microorganisms. The pathogenic bacteria causing colitis include Brachyspira pilosicoli, Lawsonia intracellularis, Salmonella, Yersinia and so on. At present, the conventional treatment method is to mix antibiotics such as neomycin, Liliqing, tetracycline family and furazolidone in the feed. Antibiotics are likely to kill the probiotics in the intestine, resulting in repeated attacks of colitis, and at the same time, it is also easy to cause antibiotic drug residues. Therefore, there is an urgent need for a new veterinary drug with significant effects and no antibiotic residues. Summary of the Invention

[0005] The present invention provides a fermented astragalus nanoparticle loaded, which can effectively treat swine colitis, enrich the types of nano traditional Chinese medicine, and solve the problem of antibiotic residue in the current treatment of swine colitis with antibiotics.

[0006] A fermented astragalus nanoparticle loaded is obtained by fermenting and culturing astragalus with a mixed strain of Bacillus amyloliquefaciens, Lactobacillus rhamnosus and Bifidobacterium BB-12 and then wrapping it with thiolated chitosan.

[0007] A preparation method of the fermented astragalus nanoparticle loaded comprises the following method steps:

[0008] S1: Pulverize astragalus slices and sterilize them under ultraviolet light.

[0009] S2: Dissolve the astragalus powder in a fermentation medium, add a mixed strain of Bacillus amyloliquefaciens, Lactobacillus rhamnosus and Bifidobacterium BB-12, and culture at 37 °C and 180 rpm for 3 d to obtain a primary fermented astragalus sample.

[0010] S3: Wrap the primary fermented astragalus sample with TCS NPs. Mix the primary fermented astragalus sample, CS and PETMP and add them to 100 mL of 1% glacial acetic acid, and continuously stir and react overnight at 30 °C to synthesize the fermented astragalus nanoparticle loaded.

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

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

[0013] Further, the addition amount of the mixed strain of Bacillus amyloliquefaciens, Lactobacillus rhamnosus and Bifidobacterium BB-12 in S2 is 1%.

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

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

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

[0017] Further, the addition amount of the primary fermented astragalus sample in S3 is 1-3 mg.

[0018] Application of the fermented astragalus nanoparticle loaded in the preparation of a drug for treating swine colitis.

[0019] Beneficial effects

[0020] The present invention improves the utilization rate of astragalus by providing a preparation method of fermented astragalus-loaded nanoparticles;

[0021] The present invention provides a fermented astragalus-loaded nanoparticle, which improves the bioavailability of traditional Chinese medicine, enhances the targeting property, protects the active substances of astragalus, increases the half-life of the drug, and prolongs the action time of the drug;

[0022] A fermented astragalus-loaded nanoparticle provided by the present invention can effectively treat colitis in pigs. Description of the Drawings

[0023] Figure 1 It is a linear regression equation diagram of the reference solution;

[0024] Figure 2 It is a result diagram of the polysaccharide concentration change curve;

[0025] Figure 3 It is an infrared spectrum diagram of CS and TCS NPs-astragalus, where A is the infrared spectrum diagram of CS and B is the infrared spectrum diagram of TCS NPs-astragalus;

[0026] Figure 4 It is a 1H NMR spectrum diagram of CS and TCS NPs-astragalus, where A is the 1H NMR spectrum diagram of CS and B is the 1H NMR spectrum diagram of TCS NPs-astragalus;

[0027] Figure 5 It is a result diagram of the morphology of TCS NPs-astragalus detected by TEM;

[0028] Figure 6 It is a result diagram of the release curve of TCS NPs-astragalus in simulated gastric juice and simulated intestinal fluid;

[0029] Figure 7 It is an in vitro intestinal imaging diagram of mice, where Y represents TCS NPs-Cy5.5 and N represents free Cy5.5. Detailed Embodiments

[0030] Example 1. Preparation of fermented astragalus-loaded nanoparticles.

[0031] I. Fermentation process flow of astragalus polysaccharide.

[0032] 1. Preparation of bacterial liquid.

[0033] Prepare an agar slant medium. Under a laminar flow hood, inoculate Bacillus amyloliquefaciens, Lactobacillus rhamnosus, and Bifidobacterium BB-12 (purchased from Shandong Yihao Biotechnology Co., Ltd.) onto the slant medium (the medium formula includes by mass percentage: beef extract 0.5%, peptone 1%, NaCl 0.5%, agar 2%, pH 7.2, sterilized at 121 °C for 20 min). Incubate at 37 °C until the colonies cover the inside of the culture tube, and the activated strains are obtained. Take an appropriate amount of the strains from the slant medium and make a bacterial suspension with sterile water. Measure the concentration of the bacterial suspension using a spectrophotometer. The results are shown in Table 1. After detection by the spectrophotometer, the concentrations of all three bacterial suspensions are close to 1×10 8 cfu / mL, meeting the inoculation concentration. Take 1 mL of each of the three bacterial suspensions and inoculate them into 100 mL of liquid medium, and culture at 37 °C and 180 r / min for 24 h to obtain the bacterial suspension for inoculation and fermentation.

[0034] Table 1

[0035] Strain (diluent) <![CDATA[Linear range / (×10 8 cfu / mL)]]> Linear equation <![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 Normal saline 0.002-0.013 y = 5.2836x - 0.01481 0.99954

[0036] 2. Preparation of the Astragalus membranaceus fermentation broth.

[0037] Weigh an appropriate amount of Astragalus membranaceus cut pieces, powder them, and sterilize them under ultraviolet light for later use.

[0038] The formula of the fermentation medium is by mass percentage: beef extract 0.5%, peptone 1%, NaCl 0.5%, agar 2%, pH 7.0, sterilized at 121 °C for 20 min. The addition amount of Astragalus membranaceus is 0.25 g of Astragalus membranaceus per milliliter of medium, that is, 50.0 g of Astragalus membranaceus is added to every 200 mL of medium. The initial pH value of the medium is 7.0, and it is filled in 250 mL Erlenmeyer flasks. To verify the effects of Bacillus amyloliquefaciens, Bifidobacterium BB-12, and Lactobacillus rhamnosus on the content of Astragalus polysaccharide in the fermentation broth, set 7 groups of different combinations of strains (the mixed strains are all mixed according to a volume ratio of 1:1 or 1:1:1), and the grouping is shown in Table 2. The fermentation temperature is 37 °C.

[0039] Table 2

[0040] Group Strain 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] Divide the prepared medium into groups A - G, with 3 replicates for each group. Inoculate the strains into the medium at an inoculation amount of 1.0% at the same time points as shown in Table 1. After inoculation, the culture time is 7 days. Measure the content of Astragalus polysaccharide in the fermentation broth every day, record the changes in the content of Astragalus polysaccharide in each group every day, and 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 °C), add water to make the volume up to 100 mL, shake well to obtain a glucose solution. Respectively 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 make the volume up to 25 mL, shake well to prepare a series of reference substance solutions. Respectively pipette 2 mL of each series of reference substance solutions into test tubes, use distilled water as the 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, heat in a boiling water bath for 15 min, cool in a cold water bath for 30 min, and measure the absorbance at 490 nm. Perform linear regression on the glucose concentration (C) and the absorbance (A), and obtain the regression equation as y = 40.0300x + 0.55420 (r 2 = 0.99821), as Figure 1 shown. The results show that glucose has a good linear relationship in the range of 2.0 μg / mL - 14.0 μg / mL, the average recovery rate of sample addition is 97.78%, and the RSD is 2.54% (n = 6).

[0046] According to the OD values of the polysaccharides in the 3 fermentation broths in the table, substitute them into the regression equation to obtain the change in the polysaccharide concentration in the daily fermentation broth. The curve of the change in the polysaccharide concentration is shown in Figure 2 . Draw a curve for the change in the polysaccharide concentration of each group. In the product of the combined fermentation of Bacillus amyloliquefaciens, Lactobacillus rhamnosus, and Bifidobacterium BB - 12 on the third day, the concentration of the polysaccharide substance reaches the peak.

[0047] II. Preparation of the sample solution.

[0048] Take 50 g of astragalus powder and dissolve it in 200 mL of fermentation medium. Prepare a suspension of the mixed strains of Bacillus amyloliquefaciens, Lactobacillus rhamnosus, and Bifidobacterium BB - 12 with sterile water to a concentration of 1×10 8 cfu / mL. Inoculate the suspension according to 1% of the volume of the medium, and culture it in a constant - temperature shaker at 37 °C and 180 rpm for 3 d. Centrifuge the fermentation broth and take the supernatant to obtain the primary sample of fermented astragalus.

[0049] III. Preparation of the sample of the fermented astragalus extract.

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

[0051] Mix 2 mL of chitosan (CS) solution (1.5 g / 100 mL) and 0.5 mL of pentaerythritol tetrakis(3-mercaptopropionate) (PETMP) solution (0.5 g / 100 mL) in a mass ratio of 12:1, then add 100 mL of 1% glacial acetic acid, and continuously stir and react overnight at 30 °C to synthesize nanoparticles (blank TCS NPs). At the end of the reaction, a milky white gel or solution is formed to obtain the product of the Schiff base reaction. Then, mix 2 L of deionized water with the product of the Schiff base reaction (volume ratio 20:1) to form an emulsion suspension of nanoparticles (sol). Subsequently, dialysis purification is carried out through a cellulose-based membrane filter (MW cut-off value of 10 kDa, Amicon Ultra-15) at 1200 rpm until the pH value of the solution is neutralized to obtain TCS NPs. Finally, store the purified nanoparticles in the form of an aqueous dispersion in a sealed container at 4 °C.

[0052] 2. Synthesize fermented Astragalus membranaceus-loaded nanoparticles (hereinafter referred to as TCS NPs-Astragalus membranaceus).

[0053] In the first step, concentrate the primary sample of fermented Astragalus membranaceus. Lyophilize and concentrate the primary sample of fermented Astragalus membranaceus to make a high-concentration stock solution (30 mg / mL) for later use.

[0054] In the second step, wrap the primary sample of fermented Astragalus membranaceus with TCS NPs. Mix 0.07 mL of the primary sample of fermented Astragalus membranaceus (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) and add them to 100 mL of 1% glacial acetic acid, and continuously stir and react overnight at 30 °C to synthesize TCS NPs-Astragalus membranaceus.

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

[0056] The preparation of TCS NPs-Cy5.5 is to replace the primary sample of fermented Astragalus membranaceus in the above step with 1.5 mg of Cy5.5, and the remaining synthesis steps are the same as above.

[0057] 4. Detect the drug loading and encapsulation efficiency by HPLC.

[0058] Lyophilize the primary sample of fermented Astragalus membranaceus, prepare molecular standard solutions with different concentrations (1.25, 2.5, 5, 50, 100, and 200 μg / mL), perform on-machine testing according to the high-performance liquid chromatography conditions, and measure the ultraviolet absorption and record the peak area at 254 nm. Take the peak area as the ordinate and the concentration of the primary sample of fermented Astragalus membranaceus (C, μg / mL) as the abscissa, perform linear regression, and obtain the linear regression equation of the primary sample of fermented Astragalus membranaceus as P = 49579C + 15754 (R 2= 1). When preparing TCS NPs - Astragalus membranaceus, three groups of experiments were set up. Respectively, 1, 2, or 3 mg of the primary fermented Astragalus membranaceus sample was encapsulated with TCS NPs. Then, the supernatant of the product was collected, and 10 μL was detected by high - performance liquid chromatography. Calculated according to the formula:

[0059] Encapsulation efficiency: EE%=(weight of the primary fermented Astragalus membranaceus sample input - weight of the free primary fermented Astragalus membranaceus sample) / weight of the primary fermented Astragalus membranaceus sample input×100%;

[0060] Drug - loading capacity: DL%=(weight of the primary fermented Astragalus membranaceus sample input - weight of the free primary fermented Astragalus membranaceus sample) / TCS NPs - Astragalus membranaceus×100%;

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

[0062] Table 4

[0063]

[0064] II. Infrared spectrum.

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

[0066] The infrared spectra of chitosan (CS) and TCS NPs - Astragalus membranaceus are as Figure 3 ... Characteristic peaks of the Astragalus membranaceus extract appear in TCS. The results show that the Astragalus membranaceus extract is successfully encapsulated in the carboxymethyl chitosan nanoparticles.

[0067] III. 1H NMR characterization.

[0068] The 1H NMR spectra of CS and TCS NPs - Astragalus membranaceus with D2O as the solvent are as 1 ... Figure 4 In the spectrum, response signal peaks of various sugar protons can be seen from δ 3.5 to δ 1.5. Among them, the signal at δ 4.8 is the strongest, and a group of peaks from δ 4.5 to δ 5.2 is very representative. Among them, δ 5.2 and δ 5.08 are the anomeric proton signals on the α - configuration pyran ring, δ 4.6 and δ 4.4 are the anomeric proton signals on the β - configuration pyran ring; δ 5.4 is the anomeric proton signal on the α - configuration furan ring, indicating that Astragalus membranaceus polysaccharide is composed of multiple monosaccharides.

[0069] IV. Determination of sulfhydryl content.

[0070] Determination of the thiol content in TCS NPs-Astragalus membranaceus by the Ellman method: First, dissolve 50 mg of DTNB in 100 mL of PBS buffer (pH 8.0) to prepare the Ellman reagent. Then, mix 1 mL of the TCS NPs-Astragalus membranaceus solution (25 mg / mL) with 6.5 mL of the Ellman reagent. After reacting in the dark at room temperature for 2 hours, measure the sample with a UV spectrophotometer at a wavelength of 410 nm, and calculate the thiol content according to the standard curve, which ranges from 0.2 to 2.4 μg / mL.

[0071] V. Morphology detection: The morphology of TCS NPs-Astragalus membranaceus was detected by TEM.

[0072] Use a pipette to aspirate 20 μL of TCS NPs-Astragalus membranaceus and drop it on a carbon film copper grid for 3 - 5 min, then use a filter paper to absorb the excess liquid and keep it dry at room temperature. Observe under a transmission electron microscope, collect images for analysis, such as Figure 5 (Scale bar: 200 nm). The results show that the TCS NPs-Astragalus membranaceus microspheres all have a good spherical morphology, and there are many protrusions and wrinkles on the surface.

[0073] VI. Particle size and zeta potential detection.

[0074] Use a Malvern nanoparticle size and zeta potential analyzer Zetasizer to detect the diffusion of Brownian motion particles. The instrument model is Zetasizer Nano ZS90, equipped with a He-Ne light source, the light source wavelength is 633 nm, and it is detected at a scattering angle of 90°. The temperature is set at 25 °C. Take 100 μL of the nanoparticle solution in a cuvette and place it in the detection chamber. Repeat the detection 3 times, with 20 cycles each time, and pause for 10 s between cycles. Analyze with Malvern Dispersion Technology Software 4.2 and take the average value. The results are shown in Table 5 - 6. The particle sizes of the TCS NPs-Astragalus membranaceus (Table 5) and TCS NPs-Cy5.5 (Table 6) microspheres are similar, both around 0.9 μm. The particle size distribution of the microspheres is narrow and uniform.

[0075] Table 5

[0076] Hydrodynamic size (nm) PDI Zeta (mV) The 1st time 91.06 0.206 11.8 The 2nd time 91.99 0.187 11.6 The 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) The 1st time 91.46 0.203 13.8 The 2nd time 90.83 0.189 12.1 The 3rd time 90.27 0.207 12.1 Average value 90.85 0.199 12.7

[0079] VII. In vitro release behavior.

[0080] The release behavior of nanoparticles was studied in artificial simulated gastric juice and artificial simulated intestinal juice, respectively. Accurately weigh 10 mg of TCS NPs-Astragalus membranaceus, first add artificial simulated gastric juice (pH 1.2) without pepsin, incubate at 37 °C in a digital constant temperature shaker for 5 h, then add artificial simulated intestinal juice (pH 6.8), and incubate at 37 °C in a digital constant temperature shaker for 24 h. Collect the supernatant at different time points and perform quantitative analysis on the machine.

[0081] The release curves of TCS NPs-Astragalus membranaceus in simulated gastric juice and simulated intestinal juice are shown in Figure 6 . After staying in artificial gastric juice for 5 h, the release rate is about 20%, indicating that TCS NPs-Astragalus membranaceus protects the fermented Astragalus membranaceus extract (primary fermented Astragalus membranaceus sample) well. The release rate of the fermented Astragalus membranaceus extract (primary fermented Astragalus membranaceus sample) in artificial intestinal juice can reach about 63% within 8 h, indicating that TCS NPs-Astragalus membranaceus rapidly releases the fermented Astragalus membranaceus extract in simulated intestinal juice, and its release rate in a neutral environment is comparable to the experimental data of the same type.

[0082] VIII. In vivo targeting behavior.

[0083] For in vitro intestinal imaging of mice, two groups of materials (TCS NPs-Cy5.5, free Cy5.5) were administered by gavage. After 4, 12, and 24 h of administration, the mice were sacrificed and dissected for in vitro intestinal imaging detection, as shown in Figure 7 . After comparison, free TCS NPs-Cy5.5 was basically evenly distributed in the mice after 24 h.

[0084] IX. Treatment of porcine colitis with TCS NPs-Astragalus membranaceus.

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

[0086] The levels of SOD, CAT, and GST in porcine colon were determined by ELISA. According to the kit instructions, appropriate reagents were added to the blank wells, standard wells, and sample wells. After incubation, washing, and color development, the absorbance was measured at a wavelength of 450 nm using an enzyme-labeled instrument. The standard curve was established based on the standard wells and the concentration was calculated.

[0087] The kits were from Jiangsu Enzyme Immunoassay Industry Co., Ltd.: Porcine Total Superoxide Dismutase (T-SOD) ELISA Kit, Porcine Cathepsin (cath) ELISA Research Kit, 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 membranaceus fermented product nanoparticles on inflammation and pro-inflammatory cytokines in porcine ulcerative colitis

[0092] The levels of TNF-γ, IL-1β, IL-6, MPO, and NO in porcine colon were determined by ELISA. According to the kit instructions, appropriate reagents were added to the blank wells, standard wells, and sample wells, incubated, washed, and developed. Then, the absorbance was measured by an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm. The standard curve was established based on the standard wells, and the concentration was calculated.

[0093] The kits were from Jiangsu Enzyme Immunoassay Industry 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, 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] X. Conclusion

[0098] Chitosan was chemically modified by thiolation to obtain thiolated chitosan. Fourier transform infrared spectroscopy (FTIR) analysis indicated successful modification.

[0099] Both scanning electron microscopy (SEM) and particle size analysis showed that the thiolated chitosan microspheres loaded with fermented Astragalus membranaceus extract had a good spherical morphology, and the average particle size was smaller than that of the unloaded thiolated chitosan.

[0100] The drug loading rate and encapsulation efficiency of the thiolated 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 artificial simulated gastric juice and artificial simulated intestinal juice for 5 and 20 h, respectively, the release rates of the carboxymethyl chitosan microspheres loaded with fermented Astragalus membranaceus extract were approximately 20% and 63%. During the treatment of porcine colitis with fermented Astragalus membranaceus extract nanoparticles, all indicators showed that the fermented Astragalus membranaceus extract nanoparticles played a positive role in the treatment of porcine colitis.

Claims

1. A loaded fermented astragalus nanoparticle, characterized in that: The loaded fermented astragalus nanoparticles are obtained by fermenting astragalus with mixed strains of Bacillus amyloliquefaciens, Lactobacillus rhamnosus and Bifidobacterium BB-12 and then encapsulating with thiolated chitosan.

2. The method for preparing the loaded fermented astragalus nanoparticles according to claim 1, characterized in that: The method steps are as follows: S1: Powder the Astragalus slices and sterilize them under ultraviolet light; S2: dissolving the astragalus powder in the fermentation medium, adding a mixed strain of Bacillus amyloliquefaciens, Lactobacillus rhamnosus and Bifidobacterium BB-12, and culturing at 37°C and 180 rpm for 3 days to obtain a primary sample of fermented astragalus; S3: The fermented Astragalus primary sample was encapsulated by TCS NPs. The fermented Astragalus primary sample, CS and PETMP were mixed and added to 100 mL of 1% glacial acetic acid. The mixture was stirred and reacted overnight at 30°C to synthesize the loaded fermented Astragalus nanoparticles.

3. The method according to claim 2, characterized in that: The mass volume ratio of the astragalus powder in S2 dissolved in the fermentation medium is 1:

4.

4. The method according to claim 2, characterized in that: 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.

5. The method according to claim 2, characterized in that: The addition amount of the mixed strain of Bacillus amyloliquefaciens, Lactobacillus rhamnosus and Bifidobacterium BB-12 described in S2 is 1%.

6. The method according to claim 2, characterized in that: The mass fraction of CS in S3 is 1.5%.

7. The method according to claim 2, characterized in that: The mass fraction of the PETMP in S3 is 0.5%.

8. The method according to claim 2, characterized in that: The mass ratio of CS to PETMP described in S3 is 12:

1.

9. The method according to claim 2, characterized in that: The added amount of the fermented Astragalus primary sample in S3 is 1-3 mg.

10. Use of the loaded fermented astragalus nanoparticles according to claim 1 in the preparation of a drug for treating porcine colitis.

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