Preparation method and application of superfine crushed dendrobium nobile

Abstract

The invention provides a preparation method and application of superfine crushed dendrobium nobile, and belongs to the technical field of traditional Chinese medicine processing and application, the preparation method comprises the following steps: 1) airing fresh dendrobium nobile stems, cutting into segments, and drying to obtain dry stem segments; 2) pre-crushing the dried stem segments, performing first screening, and collecting undersize components to obtain fine powder; and (3) carrying out superfine grinding on the fine powder, carrying out secondary sieving, and collecting undersize components as superfine powder. According to the preparation method disclosed by the invention, the structure of the dendrobium nobile is greatly destroyed, the particle size of the powder is reduced, the cell wall breaking rate is increased, and the leaching and release of active ingredients such as polysaccharides, dendrobine, total polyphenols and total flavonoids in the dendrobium nobile are promoted; compared with conventional fine powder, the dendrobium nobile submicron powder prepared by the preparation method disclosed by the invention has the advantage that the liver protection activity of dendrobium nobile can be remarkably enhanced.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine processing and application technology, and in particular relates to a method for preparing ultrafine pulverized Dendrobium nobile and its application. Background Technology

[0002] Dendrobium nobile ( Dendrobium nobile Lindl. Dendrobium nobile is a traditional and precious Chinese medicine with effects such as nourishing yin and clearing heat, benefiting the stomach and promoting the production of body fluids. Modern research shows that it has various pharmacological activities, including antioxidant, immunomodulatory, and hepatoprotective effects. Currently, Dendrobium nobile is often used in medicine or health products after conventional pulverization, but the dissolution rate of its effective components (such as polysaccharides, dendrobine, polyphenols, and flavonoids) is limited, and its bioavailability is not high, which to some extent restricts the full realization of its efficacy. Ultrafine pulverization is an advanced material processing technology that can significantly increase the specific surface area and cell wall breakage rate of medicinal materials, theoretically improving its physicochemical properties and enhancing its biological activity. However, the systematic effects and dose-response relationships between ultrafine pulverization and the physicochemical properties, active ingredient leaching patterns, in vitro antioxidant activity, and protective effects in specific in vivo disease models (such as LPS / D-GalN-induced acute liver injury) of Dendrobium nobile have not yet been clearly studied or have been developed into any products. Summary of the Invention

[0003] In view of this, the object of the present invention is to provide a method for preparing ultrafine Dendrobium nobile powder and its application, wherein the preparation method can significantly enhance the hepatoprotective activity of Dendrobium nobile. A second object of the present invention is to provide the application of ultrafine Dendrobium nobile powder in the preparation of products for the prevention and / or treatment of liver injury (especially LPS / D-GalN-induced acute liver injury).

[0004] This invention provides a method for preparing ultrafine pulverized Dendrobium nobile, comprising the following steps: 1) Cut fresh Dendrobium nobile stems into sections and dry them to obtain dried stem sections; 2) The dried stem segments are pre-crushed and sieved for the first time. The undersize components are collected to obtain fine powder. 3) The fine powder is subjected to ultra-fine grinding, then sieved a second time, and the undersize component is collected as ultra-fine powder.

[0005] Preferably, the drying in step 1) is blower drying or freeze drying.

[0006] Preferably, the temperature of the blower drying is 58~62℃, and the time of the blower drying is 1.5~2.5h.

[0007] Preferably, the pre-crushing in step 2) is intermittent crushing, with a single crushing time of 10~30s, an intermittent cooling time of 30~60s, and a crushing cycle of 5~8 times.

[0008] Preferably, the first sieving in step 2) is sieving through an 80-100 mesh sieve.

[0009] Preferably, in step 3), the second sieving is through a 400-500 mesh sieve.

[0010] The present invention provides Dendrobium nobile ultrafine powder prepared by the preparation method described above, wherein the particle size distribution of the Dendrobium nobile ultrafine powder is D90≤25μm.

[0011] This invention provides the application of Dendrobium nobile ultrafine powder prepared by the above-described preparation method in the preparation of drugs for the prevention and / or treatment of liver injury.

[0012] Preferably, the liver injury is acute liver injury.

[0013] This invention provides the application of Dendrobium nobile ultrafine powder prepared by the above-described preparation method in the preparation of functional foods or health products.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The preparation method of ultrafine pulverized Dendrobium nobile of the present invention includes two pulverization steps: pre-pulverization and ultrafine pulverization. Through the above two pulverization steps, the structure of Dendrobium nobile is greatly destroyed, the particle size of the powder is reduced, the cell wall breakage rate is increased, the specific surface area of ​​the powder and the contact surface with the solvent are increased, and the leaching and release of chemical components such as polysaccharides, dendrobine, total polyphenols and total flavonoids in Dendrobium nobile are promoted. The cell wall breakage rate, hydration (including water holding capacity, water solubility and swelling capacity) and filling capacity of the powder are significantly improved, and the leaching rate of active ingredients and the in vitro antioxidant activity are significantly higher than those of conventionally pulverized powders.

[0015] The in vitro antioxidant activity (DPPH and ABTS free radical scavenging capacity) of the ultrafine pulverized Dendrobium nobile described in this invention is significantly enhanced compared to that of 80-mesh fine powder. When applied to mice with liver injury, it significantly reduced the levels of ALT and AST in mouse serum (P<0.05), indicating that reduced particle size can enhance the protective effect of Dendrobium nobile against acute liver injury.

[0016] HE staining of the liver showed that the morphology of the liver tissue was significantly improved after administration of Dendrobium nobile with different particle sizes. Specifically, hepatocyte damage, inflammatory cell infiltration, and vascular and sinusoidal congestion and edema were significantly reduced. Moreover, the livers treated with ultrafine Dendrobium nobile were closer to the normal state, indicating that the effect of Dendrobium nobile on improving pathological liver damage is enhanced as the particle size decreases. Attached Figure Description

[0017] Figure 1Photographs of Dendrobium nobile powders with different particle sizes are shown. OF indicates air-dried fine powder; OEF indicates air-dried ultrafine powder; OSF indicates air-dried ultrafine powder; FF indicates freeze-dried fine powder; FEF indicates freeze-dried ultrafine powder; FSF indicates freeze-dried ultrafine powder. The above markings are used in subsequent figures. Figure 2 Scanning electron micrographs of Dendrobium nobile powder with different particle sizes; Figure 3 Angle of repose (A) and angle of slip (B) for Dendrobium nobile powders with different particle sizes; Figure 4 Loose density (A) and tap density (B) of Dendrobium nobile powder with different particle sizes; Figure 5 The swelling capacity (A), water holding capacity (B), and water solubility (C) of Dendrobium nobile powder with different particle sizes; Figure 6 Fourier transform infrared spectra of Dendrobium nobile powder with different particle sizes; Figure 7 The leaching rates of dendrobine from Dendrobium nobile powder with different particle sizes are shown in Figure 1, where A is the standard curve and B is the leaching rate. Figure 8 The extraction rates of polysaccharides from Dendrobium nobile powders of different particle sizes are shown in Figure A, where A is the standard curve and B is the extraction rate. Figure 9 The total polyphenol leaching rate of Dendrobium nobile powder with different particle sizes is represented by A, where A is the standard curve and B is the leaching rate. Figure 10 The total flavonoid leaching rate of Dendrobium nobile powder with different particle sizes is represented by A, where A is the standard curve and B is the leaching rate. Figure 11 The DPPH free radical scavenging activity of Dendrobium nobile powders with different particle sizes was evaluated, where A represents OF, OEF, OSF, and Vc; and B represents FF, FEF, FSF, and Vc. Figure 12 The ABTS free radical scavenging activity of Dendrobium nobile powders with different particle sizes was evaluated, where A represents OF, OEF, OSF, and Vc; and B represents FF, FEF, FSF, and Vc. Figure 13 Serum ALT (A) and AST (B) levels in mice from different treatment groups; Figure 14 The serum GSH content (A) and SOD level (B) of mice in different treatment groups were measured. Figure 15 The images show the morphological observations of the livers of mice in different treatment groups. A represents the epimorphological observation of the mouse liver, B represents the mouse liver index, and C represents the HE staining of the mouse liver. The scale bar in C is 100 μm. Figure 16The levels of oxidative stress in the liver tissue of mice in different treatment groups are represented by A, B, C, and D, respectively. Figure 17 The expression of genes related to inflammation and oxidative stress in mice under different treatment groups is shown, where A~H represent IL-6, IL-1β, NF-κB, TNF-α, TLR4, Nrf2, HO-1, and IFN-γ mRNA, respectively. Detailed Implementation

[0018] This invention provides a method for preparing ultrafine pulverized Dendrobium nobile, comprising the following steps: 1) cutting fresh Dendrobium nobile stems into segments and drying them to obtain dried stem segments; 2) pre-pulverizing the dried stem segments, passing them through a first sieve, collecting the sieve-undersized components to obtain fine powder; 3) subjecting the fine powder to ultrafine pulverization, passing it through a second sieve, and collecting the sieve-undersized components to obtain ultrafine powder.

[0019] In this invention, fresh Dendrobium nobile stems are cut into sections and dried to obtain dried stem sections. Preferably, the fresh Dendrobium nobile is washed and then air-dried. There are no particular limitations on the air-drying process; drying until the surface is free of moisture is sufficient. Preferably, the Dendrobium nobile stems are cut into sections of 0.5-1.5 cm, more preferably 1 cm. The drying process is preferably either forced-air drying or freeze-drying. The forced-air drying temperature is preferably 58-62℃, more preferably 60℃; the forced-air drying time is preferably 1.5-2.5 hours, more preferably 2 hours. The moisture content is measured using a moisture meter, with a moisture content of less than 12% as the drying endpoint.

[0020] This invention, after obtaining dried stem segments, pre-pulverizes and first sieves the dried stem segments, collecting the undersized components to obtain fine powder. In this invention, the pre-pulverization is preferably performed using a high-speed universal pulverizer, and the pre-pulverization is preferably intermittent; the single pulverization time is 10-30 seconds, the intermittent cooling time is 30-60 seconds, and the number of pulverization cycles is 5-8 times. The high temperature caused by the pulverizer may damage the plant active ingredients of *Dendrobium nobile*. This invention employs an intermittent pulverization method to reduce the potential loss of plant active substances. The core of the intermittent pulverization method lies in "short time, multiple times, and sufficient cooling." By strictly controlling the single pulverization time and ensuring sufficient intermittent cooling time, the temperature rise during the pulverization process can be effectively suppressed, thereby preserving the phytochemical components in *Dendrobium nobile*. The first sieve in this invention is preferably through an 80-100 mesh sieve.

[0021] In this invention, after obtaining fine powder, the fine powder is subjected to ultrafine grinding and a second sieve, and the undersize component is collected as ultrafine powder. In this invention, the ultrafine grinding is preferably carried out using a WED 100 low-temperature vibration pulverizer with a refrigeration unit, with a particle size of less than 30μm and a grinding temperature of -3℃ to 5℃; the second sieve is preferably passed through a 400-500 mesh sieve, more preferably through a 500 mesh sieve.

[0022] The present invention also provides Dendrobium nobile ultrafine powder prepared by the preparation method described above, wherein the particle size distribution of the Dendrobium nobile ultrafine powder is D90≤25μm.

[0023] The present invention also provides the application of the Dendrobium nobile ultrafine powder prepared by the above preparation method in the preparation of drugs for the prevention and / or treatment of liver injury, wherein the liver injury is preferably acute liver injury, acute liver injury induced by LPS (lipopolysaccharide) and D-GalN (D-galactosamine).

[0024] In this invention, the Dendrobium nobile ultrafine powder exerts its hepatoprotective effect through the following mechanisms: (1) reducing serum transaminase (ALT, AST) levels; (2) increasing the activity of antioxidant enzymes (SOD, CAT) and glutathione (GSH) content in the liver and serum; (3) reducing the content of lipid peroxides (MDA) in the liver; (4) inhibiting the mRNA expression of liver inflammatory factors (IL-6, IL-1β, TNF-α, NF-κB, TLR4, INF-γ); (5) regulating the mRNA expression of oxidative stress-related factors (Nrf2, HO-1); and (6) regulating the MAPK signaling pathway and NOD-like receptor signaling pathway.

[0025] This invention also provides the application of the Dendrobium nobile ultrafine powder prepared by the above preparation method in the preparation of functional foods or health products.

[0026] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1

[0028] The dried Dendrobium nobile raw material was subjected to ultrafine pulverization.

[0029] Freshly collected Dendrobium nobile stems were washed, dried, and cut into sections. They were then dried in a forced-air drying oven at 60°C and freeze-dried in a vacuum freeze dryer. Afterward, they were pulverized using different methods. In the first method, a high-speed universal pulverizer was used to pulverize the Dendrobium nobile, and the powders were then passed through an 80-mesh sieve. The prepared samples were named oven-dried fine powder (OF) and freeze-dried fine powder (FF), respectively. It is important to note that the high temperature generated by the pulverizer may damage the phytochemicals of Dendrobium nobile; therefore, an intermittent pulverization method was used in this process to minimize potential loss of phytochemicals.

[0030] The specific steps are as follows: The dried Dendrobium nobile stem segments were placed in a refrigerator at 4°C for 5 hours beforehand to cool.

[0031] Pre-cool the pulverizer chamber as much as possible.

[0032] Single feed amount: 1 / 3 of the maximum capacity of the crusher.

[0033] Set the pulverization / intermittent time parameters: intermittent cooling time 40s, pulverization cycle number 8 times; the first 4 times, the single pulverization time is 20s; in the last 4 times, as the powder becomes finer, the frictional heat generation will be more intense, and the single time of the subsequent cycle is shortened to 15s.

[0034] Forced cooling operation

[0035] During each intermittent cooling period, the pulverizer lid must be opened to allow the heat accumulated inside the chamber to dissipate quickly.

[0036] In the second method, the *Dendrobium nobile* samples were first pre-pulverized according to the above method, then transferred to a 1.2 L stainless steel container and subjected to ultrafine pulverization using a WED 100 low-temperature vibration pulverizer with a refrigeration unit. The samples were then passed through 300-mesh (extremely fine) and 500-mesh (ultrafine) pharmacopoeia sieves, respectively. The powders obtained from drying the samples were named Oven-dried extremely fine powder (OEF) and Oven-dried super-fine powder (OSF), respectively. The powders obtained from freeze-drying the samples were named Freeze-dried extremely fine powder (FEF) and Freeze-dried super-fine powder (FSF), respectively. The particle size D90 of the ultrafine powder was ≤ 25 μm.

[0037] Six different powder samples, namely OF, OEF, OSF, FF, FEF and FSF, were prepared according to the above method, and the performance of the above samples was tested.

[0038] 1.1 Scanning electron microscopy observation

[0039] Scanning electron microscopy results of Dendrobium nobile powders with different particle sizes are as follows: Figure 2 As shown, the dried and freeze-dried fine powder exhibits irregular shapes, rough surfaces, and large flakes. After ultrafine pulverization, under the action of mechanical force, the particle size of Dendrobium nobile powder decreases, long fragments are transformed into short fragments, and the flake structure of the ultrafine powder is reduced. After further pulverization, the ultrafine powder has a smooth surface, a round or irregular shape, and most of the particle cells are broken, which helps to improve powder properties, such as solubility and dispersion. The above results indicate that ultrafine pulverization greatly disrupts the structure of Dendrobium nobile, reduces the particle size, increases the cell wall breakage rate, and thus promotes the release of chemical components of Dendrobium nobile.

[0040] 1.2 Liquidity

[0041] The flowability of Dendrobium nobile powder with different particle sizes was evaluated by measuring the angle of repose and the angle of slip. Generally, the smaller the angle of repose and the angle of slip, the better the flowability of the powder. Results are as follows... Figure 3 As shown, OF represents dried fine powder; OEF represents dried ultrafine powder; OSF represents dried ultrafine powder; FF represents freeze-dried fine powder; FEF represents freeze-dried ultrafine powder; FSF represents freeze-dried ultrafine powder; 'a' indicates comparison with a fine powder sample. P <0.05; b indicates comparison with the ultrafine powder sample, P <0.05; ± SD, n=3.

[0042] Depend on Figure 3 As shown in A, the angle of repose of the dried sample first increases and then decreases, while the angle of repose of the freeze-dried sample decreases with decreasing particle size. However, this change is not significant, indicating that decreasing the particle size of Dendrobium nobile powder does not significantly affect its angle of repose. Regarding the angle of slip of the powder, it tends to increase with decreasing particle size. Figure 3 (B) indicates that the flowability of Dendrobium nobile powder decreases as the particle size decreases. This may be because the Dendrobium nobile powder particles become smaller after ultrafine grinding, the specific surface area increases, the internal cohesive groups are easily exposed, and the attraction between particles increases.

[0043] 1.3 Filling properties

[0044] Loose density and tapped density are important parameters for evaluating the filling properties of powders. In the production of tablets or capsules, high loose density and tapped density are generally required to facilitate product formation. The loose density and tapped density of *Dendrobium nobile* powders with different particle sizes were analyzed, and the results are as follows: Figure 4 As shown, Figure 4 In the middle, 'a' indicates a comparison with the fine powder sample. P <0.05; b indicates comparison with the ultrafine powder sample, P <0.05; ± SD, n=3. It can be seen that ultrafine grinding significantly increases the loose density and tap density of Dendrobium nobile powder, which may be due to the reduction in particle size and the reduction in the pore space between particles.

[0045] 1.4 Hydration

[0046] Hydration is a crucial factor affecting the production of powdered pharmaceuticals and food products. Among these, swelling power, water retention capacity, and water solubility reflect the influence of particle size on the hydration properties of powders. Results are as follows... Figure 5 As shown, 'a' represents a comparison with the fine powder sample. P <0.05; b indicates comparison with the ultrafine powder sample, P <0.05; ± SD, n=3. It can be seen that as the particle size decreases, the swelling force and water holding capacity of dried and freeze-dried Dendrobium nobile powder decrease significantly, while the water solubility increases significantly, indicating that the reduction of particle size has a significant effect on improving the hydration of Dendrobium nobile powder.

[0047] 1.5 Fourier Transform Infrared Spectroscopy Analysis

[0048] To characterize whether the chemical composition of Dendrobium nobile changes after ultrafine pulverization, Fourier transform infrared spectroscopy analysis was performed on Dendrobium nobile powders of different particle sizes. The results are as follows: Figure 6 As shown, the overall spectral profiles of Dendrobium nobile powders with different particle sizes are similar, with no new spectral peaks generated and no peaks disappearing. This indicates that ultrafine grinding has little effect on the characteristic absorption of the infrared spectrum of Dendrobium nobile powder, and that ultrafine grinding has not changed the molecular structure of Dendrobium nobile, nor has it generated new chemical groups. Further, some characteristic peaks were assigned, and Table 1 lists the detailed peak positions and assignments of Dendrobium nobile samples with different particle sizes. It can be seen that the spectra are very complex, containing many bands assigned to the main components of Dendrobium nobile, with the peak value at 3418 cm⁻¹. -1 The small peaks in and around the vicinity are related to the stretching vibrations of OH and NH, with wavenumbers around 2923 cm⁻¹. -1 and 2853 cm -1 Both are related to the tensile vibrations of CH and CH2; wavenumbers are around 1739 cm⁻¹. -1 Related to C=O stretching vibrations; wavenumber at 1604 cm⁻¹-1 The vicinity is associated with COO stretching vibrations; wavenumber at 1375 cm⁻¹. -1 The area is associated with symmetrical bending of CH and CH2; wavenumber at 1245 cm⁻¹ -1 and 1054 cm -1 The surrounding area corresponds to CO stretching vibrations; wavenumber is 607 cm⁻¹. -1 The nearby area is attributed to the stretching vibration of OH.

[0049] However, the precise peak intensities of Dendrobium nobile powder vary depending on its particle size. The intensities are 1604, 1507, 1425, 1245, 1160, 1054, and 607 cm⁻¹. -1 The wavenumber in the vicinity decreases with decreasing grain size. In contrast, the wavenumbers at 3418, 2923, 2853, 1739, 1458, and 1375 cm⁻¹ decrease. -1 The wavenumbers in the vicinity increase with decreasing particle size. The detailed displacements may be related to the disruption of intramolecular hydrogen bonds in cellulose and hemicellulose, as well as the formation of soluble sugars and new amorphous cellulose due to the mechanical forces generated by ultrafine grinding. Furthermore, the exposure and transformation of hydrophilic groups can effectively improve the hydration capacity of the powder, which may be the material basis for the high antioxidant activity of ultrafine Dendrobium nobile powder.

[0050] Table 1. Main absorption peaks of infrared spectra of Dendrobium nobile powders with different particle sizes

[0051] Ultrafine grinding significantly reduces the particle size of Dendrobium nobile powder, increasing its specific surface area and cell wall breakage rate. Reduced interparticle porosity leads to increased bulk density and tapped density, thus improving filling capacity. Furthermore, the significant increase in specific surface area and cell wall breakage rate improves the diffusion of water-soluble molecules within the particles, thereby promoting the release of a large amount of soluble substances from Dendrobium nobile and enhancing its hydration properties. However, due to the increased specific surface area of ​​Dendrobium nobile powder, its internal cohesive groups are easily exposed, increasing interparticle attraction and reducing its flowability. Fourier transform infrared spectroscopy analysis shows that the molecular structure of Dendrobium nobile powder does not change with decreasing particle size.

[0052] 2.1 Dendrobine leaching rate

[0053] Dendrobine is the hallmark bioactive component of Dendrobium nobile and is considered one of the evaluation standards for its quality, possessing various medicinal effects. For example... Figure 7 As shown, Figure 7 In the middle, 'a' indicates a comparison with the fine powder sample. P <0.05; b indicates comparison with the ultrafine powder sample, P <0.05; ± SD, n=3. It can be seen that the leaching rate of dendrobine varies with different particle sizes of Dendrobium nobile powder. With decreasing particle size, the leaching rate of dendrobine in dried Dendrobium nobile powder shows no significant difference, while the leaching rate of dendrobine in freeze-dried Dendrobium nobile powder shows a significant increasing trend.

[0054] 2.2 Polysaccharide extraction rate

[0055] Besides dendrobine, polysaccharides are also a major medicinal component of Dendrobium nobile. Research results show that the polysaccharide extraction rate of both dried and freeze-dried Dendrobium nobile powders increases with decreasing particle size, exhibiting a significant negative correlation with powder particle size. Figure 8 ). Figure 8 In the text, 'a' indicates a comparison with the fine powder sample. P <0.05; b indicates comparison with the ultrafine powder sample, P <0.05; ±SD, n=3.

[0056] 2.3 Total polyphenol leaching rate

[0057] Polyphenols are natural antioxidants, mainly derived from plants. Their abundance in nature is second only to sugars, making them an extremely important class of compounds. Studies have found that, compared to fine powder, the total polyphenol leaching rate of ultrafine powder is higher in both dried and freeze-dried samples of Dendrobium nobile, while the increase is more significant in ultrafine powder. P <0.05), indicating that ultrafine grinding can significantly improve the total polyphenol extraction rate of Dendrobium nobile. Detailed results are as follows: Figure 9 As shown, 'a' represents a comparison with the fine powder sample. P <0.05; b indicates comparison with the ultrafine powder sample, P <0.05; ± SD, n=3.

[0058] 2.4 Total polyphenol leaching rate

[0059] Flavonoids are another type of natural antioxidant found in plants, possessing various effects such as anti-tumor, analgesic, anti-inflammatory, antioxidant, and anti-aging properties, playing an important role in human health. For example... Figure 10 As shown, Figure 10 In the middle, 'a' indicates a comparison with the fine powder sample. P <0.05; b indicates comparison with the ultrafine powder sample, P <0.05; ± SD, n=3.

[0060] It is evident that, regardless of whether the Dendrobium nobile powder samples were dried or freeze-dried, the total flavonoid extraction rate showed an increasing trend as the particle size decreased. Compared with the ultrafine powder, the increase was more significant in the freeze-dried ultrafine powder. P <0.05).

[0061] In summary, ultrafine pulverization breaks down the large structure of Dendrobium nobile into small particles, increasing the specific surface area of ​​the powder and the contact area with the solvent, thereby promoting the leaching rate of polysaccharides, dendrobine, total polyphenols, and total flavonoids in Dendrobium nobile.

[0062] Example 2

[0063] In vitro antioxidant activity of ultrafine powder (DPPH and ABTS free radical scavenging ability)

[0064] (1) Determination of DPPH scavenging rate

[0065] The 2,2-diphenyl-1-pyridylhydrazide (DPPH) free radical scavenging activity of Dendrobium nobile powder with different particle sizes was determined using the method reported by Shen et al. Specifically, 1 mL of sample solution at different concentrations (1, 5, 10, 15, 20, 25 mg / mL) was placed in a test tube, and 3.5 mL of 1.9 × 10⁻⁶ ppm solution was added. -4 Mix the mol / L DPPH solution immediately, let it stand in the dark for 30 min, and measure the absorbance at a wavelength of 517 nm. The positive control group was prepared by replacing the sample solution with ascorbic acid. Results are expressed as the percentage of DPPH free radicals scavenged and calculated using the following formula: DPPH removal rate (%) = (A0 - (A1 - A2)) / A0 × 100% In the formula: A0, A1 and A2 are the absorbance of the reference standard (DPPH solution without sample), the sample and the sample without DPPH solution, respectively.

[0066] (2) Determination of ABTS clearance rate

[0067] The method for testing the ABTS free radical cation scavenging activity of Dendrobium nobile powder with different particle sizes was modified according to Zhu et al. An ABTS free radical solution was prepared by mixing 7 mmol / L ABTS solution with 2.45 mmol / L potassium persulfate solution at a 1:1 ratio. + Let it stand at room temperature, protected from light, for 12-16 hours. Before use, mix ABTS with PBS (pH=7.4). + The solution was diluted to an absorbance of 0.68–0.72 at 734 nm. 0.5 mL of different concentrations (1, 5, 10, 15, 20, 25 mg / mL) of *Dendrobium nobile* extract samples were added to 2.5 mL of ABTS free radical solution, mixed thoroughly, and allowed to stand at room temperature for 6 min. The absorbance at 734 nm was immediately recorded. Using ascorbic acid as a positive control, the ABTS free radical cation scavenging activity was calculated according to the following equation: ABTS clearance rate (%) = (A0 - (A1 - A2)) / A0 × 100% In the formula: A0, A1 and A2 are the absorbance of the reference standard (ABTS solution without sample), the sample and the sample without ABTS solution, respectively.

[0068] DPPH free radical scavenging activity

[0069] DPPH is a stable free radical widely used for the quantitative determination of antioxidant capacity in biological samples and food. The DPPH free radical scavenging activity of Dendrobium nobile powders with different particle sizes was compared, and the results are as follows: Figure 11 As shown, Figure 11 In the middle: 'a' indicates a comparison with the fine powder sample. P <0.05; b indicates comparison with the ultrafine powder sample, P <0.05; ± SD, n=3.

[0070] It can be seen that the scavenging activity of Dendrobium nobile powder with different particle sizes against DPPH free radicals increases with decreasing particle size.

[0071] ABTS free radical scavenging activity

[0072] ABTS is a water-soluble free radical that is widely used to evaluate the antioxidant activity of foods, fruits and vegetables, beverages, etc. Figure 12 The ABTS free radical scavenging activity of Dendrobium nobile powders with different particle sizes was demonstrated. Figure 12 In the text, 'a' indicates a comparison with the fine powder sample. P <0.05; b indicates comparison with the ultrafine powder sample, P <0.05; ± SD, n=3. The results showed that the ABTS scavenging activity of Dendrobium nobile powder extract exhibited a similar trend to that of DPPH free radical scavenging activity, that is, the ABTS scavenging activity increased with the decrease of Dendrobium nobile particle size.

[0073] It is evident that Dendrobium nobile possesses certain antioxidant activity, and the in vitro antioxidant activity of Dendrobium nobile powder is enhanced after ultrafine grinding. This provides important guidance for the application of ultrafine grinding technology to Dendrobium nobile and also offers a scientific basis for the further rational development and application of Dendrobium nobile as an antioxidant.

[0074] Example 3

[0075] Male BALB / c mice were randomly divided into 6 groups according to body weight: normal group, model group (LPS / D-GalN), silymarin positive control group (100 mg / kg), FF group (9.0 g / kg), FEF group (9.0 g / kg), and FSF group (9.0 g / kg). The blank control group consisted of 10 mice, and the other groups consisted of 13 mice each. The model group and the drug-treated groups were intraperitoneally injected with LPS (10 μg / kg) and D-GalN (700 mg / kg), respectively. The blank control group was intraperitoneally injected with physiological saline. The drug-treated groups were immediately administered the corresponding drug via gavage after intraperitoneal injection of LPS / D-GalN (dosage determined based on previous preliminary experiments). The blank control group and the model group were administered distilled water via gavage. Tissue samples were collected 6 hours later. The success of the model was determined by observing the morphology of the mouse liver tissue and measuring the level of serum transaminases. Serum AST and ALT enzyme activities were measured according to the instructions of the kits from Nanjing Jiancheng Bioengineering Institute. GSH and SOD enzyme activities were measured using ELISA kits from Shanghai Renjie Biotechnology Co., Ltd. Mouse liver lobule tissue was fixed in 10% paraformaldehyde solution, then embedded in paraffin, cut into 4 mm thick slices, and finally stained with hematoxylin and eosin (HE stain). Histopathological changes in the liver tissue were observed under an optical microscope. Liver homogenate was centrifuged at 2500 r / min, and the supernatant was further analyzed using kits from Nanjing Jiancheng Bioengineering Institute to determine SOD and CAT enzyme activities, as well as MDA and GSH contents.

[0076] Serum ALT, AST, SOD enzyme activity levels and GSH content

[0077] Serum transaminase levels are a sensitive indicator for assessing the extent of hepatocyte damage. For example... Figure 13 As shown, the study found that the activities of ALT and AST enzymes were significantly increased in the model group, and this situation was significantly improved after administration of Dendrobium nobile. Compared with the model group, the activities of ALT and AST enzymes in the FF, FEF, and FSF groups were significantly decreased. P <0.05 indicates that Dendrobium nobile has the potential to treat acute liver injury. Furthermore, compared with FF, both FEF and FSF reduced the levels of ALT and AST in mouse serum, with FSF showing a more significant decreasing trend. P The value <0.05 indicates that the reduction in particle size can enhance the protective effect of Dendrobium nobile against acute liver injury.

[0078] Figure 13 In the text, 'a' indicates a comparison with the blank group. P <0.05; b indicates comparison with the model group, P <0.05; c indicates comparison with the freeze-dried fine powder group, P <0.05; ±SD.

[0079] SOD and GSH, as the first line of defense against oxidation, play a crucial role in scavenging oxygen free radicals and maintaining the balance between oxidation and antioxidation. Figure 14 As shown, Figure 14 In the text, 'a' indicates a comparison with the blank group. P <0.05; b indicates comparison with the model group, P <0.05; ± SD.

[0080] After administration of LPS / D-GalN, the levels of SOD and GSH in mouse serum were significantly reduced, while administration of the positive control drug silymarin and Dendrobium nobile extracts of different particle sizes improved both levels to varying degrees. Compared with the FF group, the GSH level in the FEF group did not change significantly, while the SOD level increased slightly, whereas the GSH and SOD levels in the FSF group were significantly increased. P The value <0.05 indicates that Dendrobium nobile can inhibit LPS / D-GalN-induced oxidative stress damage, and that ultrafine grinding can significantly enhance the protective effect of Dendrobium nobile.

[0081] Liver morphological observation

[0082] To observe the protective effect of Dendrobium nobile with different particle sizes against LPS / D-GalN-induced liver injury, the liver was subjected to epimorphological observation and HE staining. The results are as follows: Figure 15 As shown, Figure 15 In the text, 'a' indicates a comparison with the blank group. P <0.05; b indicates comparison with the model group, P <0.05; c indicates comparison with the freeze-dried fine powder group, P <0.05; ± SD. The livers of mice in the normal control group were morphologically normal, with a reddish color and soft texture, while the livers of mice in the model group showed obvious pathological changes, including a brittle texture, a purplish-black color, and significant swelling and congestion. Administration of silymarin and extracts of Dendrobium nobile powder with different particle sizes alleviated the swelling and congestion of the mouse liver tissue, and as the particle size of Dendrobium nobile powder decreased, the morphology of the mouse liver tissue gradually returned to normal. Figure 15 (A) After administration of LPS / D-GalN, the liver index in mice was significantly increased ( P <0.05), while silymarin and Dendrobium nobile of different particle sizes had a retrograde effect, and the liver index of the FSF group was significantly reduced compared with the FF group. P <0.05).

[0083] Furthermore, HE staining of the liver revealed that the hepatocytes in the normal group mice were intact and regularly arranged, without inflammatory cell infiltration or necrosis. In contrast, the livers of the model group mice showed significant inflammatory cell infiltration, focal hepatocyte necrosis, liver tissue structure destruction, loss of cell boundaries, and extensive nuclear pyknosis. Administration of silymarin and Dendrobium nobile with different particle sizes significantly improved liver tissue morphology, specifically by a marked reduction in hepatocyte damage, inflammatory cell infiltration, and vascular and sinusoidal congestion and edema. Compared with the FF group, the livers of the FEF and FSF groups were closer to normal, indicating that the ameliorative effect of Dendrobium nobile on pathological liver damage increased with decreasing particle size.

[0084] Liver activity levels of CAT and SOD enzymes, as well as GSH and MDA content

[0085] In addition to the antioxidant enzymes GSH and SOD mentioned above, MDA and CAT are also indicators for assessing the degree of cellular damage from free radical attack and tissue lipid peroxidation. Therefore, the analysis of these four substances in the liver yielded the following results: Figure 16 As shown, Figure 16 In the text, 'a' indicates a comparison with the blank group. P <0.05; b indicates comparison with the model group, P <0.05; c indicates comparison with the freeze-dried fine powder group, P <0.05; d indicates comparison with the freeze-dried ultrafine powder group. P <0.05; ± SD. Treatment with Dendrobium nobile extracts of different particle sizes significantly reversed the LPS / D-GalN-induced decreases in SOD and CAT enzyme activity, GSH content, and MDA content in liver tissue. Specifically, compared to the FF group, the GSH, SOD, and CAT levels gradually increased, while MDA gradually decreased in the FEF and FSF groups. This indicates that Dendrobium nobile can enhance the antioxidant capacity of hepatocytes, reduce oxidative damage, and exert a protective effect against LPS / D-GalN-induced acute liver injury; the smaller the particle size, the stronger the hepatoprotective ability.

[0086] Real-time quantitative PCR was used to verify the expression of genes related to inflammation and oxidative stress.

[0087] The expression of IL-6, IL-1β, NF-κB, TNF-α, TLR4, Nrf2, HO-1, and IFN-γ mRNA in the inflammatory and oxidative stress pathways was detected by quantitative real-time PCR. Figure 17 As shown, Figure 17 CON represents the blank control group; MOD represents the model group; FF represents the freeze-dried fine powder group; FEF represents the freeze-dried ultrafine powder group; FSF represents the freeze-dried ultrafine powder group; 'a' indicates comparison with the blank group. P <0.05; b indicates comparison with the model group,P <0.05; c indicates comparison with the freeze-dried fine powder group, P <0.05; d indicates comparison with the freeze-dried ultrafine powder group. P <0.05; ± SD.

[0088] The results showed that after LPS / D-GalN stimulation, the expression levels of IL-6, IL-1β, NF-κB, TNF-α, TLR4, Nrf2, IFN-γ, and HO-1 mRNA in mouse liver were significantly upregulated, while the expression of these genes could be downregulated by Dendrobium nobile powder of different particle sizes. Among them, the expression levels of IL-1β, TNF-α, TLR4, IFN-γ, and HO-1 mRNA decreased significantly with decreasing particle size.

[0089] As can be seen from the above embodiments, the preparation method of ultrafine pulverized Dendrobium nobile provided by the present invention greatly disrupts the structure of Dendrobium nobile, reduces the particle size of the powder, increases the cell wall breakage rate, and promotes the leaching and release of active ingredients such as polysaccharides, dendrobine, total polyphenols, and total flavonoids in Dendrobium nobile; it also significantly improves the cell wall breakage rate, hydration (including water holding capacity, water solubility, and swelling capacity), and filling properties of the powder. When applied to mice with liver injury, its effect on improving pathological liver damage is stronger than that of conventional fine powder.

[0090] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing ultrafine pulverized Dendrobium nobile, characterized in that, Includes the following steps: 1) Cut fresh Dendrobium nobile stems into sections and dry them to obtain dried stem sections; 2) The dried stem segments are pre-crushed and sieved for the first time. The undersize components are collected to obtain fine powder. 3) The fine powder is subjected to ultra-fine grinding, then sieved a second time, and the undersize component is collected as ultra-fine powder.

2. The preparation method according to claim 1, characterized in that, Step 1) The drying process is either blower drying or freeze drying.

3. The preparation method according to claim 2, characterized in that, The temperature of the blower drying is 58~62℃, and the time of the blower drying is 1.5~2.5h.

4. The preparation method according to claim 1, characterized in that, Step 2) The pre-crushing is intermittent crushing, with a single crushing time of 10~30s, an intermittent cooling time of 30~60s, and a crushing cycle of 5~8 times.

5. The preparation method according to claim 1, characterized in that, Step 2) The first sieving is through an 80-100 mesh sieve.

6. The preparation method according to claim 1, characterized in that, Step 3) The second sieving is through a 400-500 mesh sieve.

7. The Dendrobium nobile ultrafine powder prepared by the preparation method according to any one of claims 1 to 6, characterized in that, The particle size distribution of the Dendrobium nobile ultrafine powder is D90≤25μm.

8. The use of Dendrobium nobile ultrafine powder prepared by the preparation method according to any one of claims 1 to 6 in the preparation of drugs for the prevention and / or treatment of liver injury.

9. The application according to claim 8, characterized in that, The liver injury described is acute liver injury.

10. The application of Dendrobium nobile ultrafine powder prepared by the preparation method according to any one of claims 1 to 6 in the preparation of functional foods or health products.

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