Preparation method of silybin nanostructure lipid carrier

Abstract

The invention discloses a preparation method of a silybin nanostructure lipid carrier, which comprises the following steps: step 1, heating ethanol and silybin in a water bath, and uniformly stirring to obtain a silybin solution; dispersing a co-emulsifier in deionized water, and heating and stirring in a water bath to obtain a water phase; step 2, adding solid lipid, liquid lipid, a main emulsifier and a co-emulsifier into the silibinin solution, heating for melting, and uniformly stirring to obtain an oil phase; under the condition of constant-temperature stirring, transferring the oil phase into the water phase, mixing the solution, and shearing to obtain a coarse dispersion system; and 3, heating the coarse dispersion system to concentrate the coarse dispersion system into an emulsion. The silibinin nanostructure lipid carrier prepared by the invention can improve the solubility of silibinin, and is good in characterization performance; the preparation cost is controllable, and macro preparation can be realized; the silybin nano-structure lipid carrier can be used for effectively preventing acute liver injury caused by CCl4 of animals.

Description

Technical Field

[0001] This invention pertains to veterinary drug preparation methods, specifically a method for preparing a silymarin nanostructured lipid carrier. Background Technology

[0002] Silybin, also known as silymarin, is extracted from the medicinal plant milk thistle. Silybin can reduce oxidative stress in hepatocytes, enhance their antioxidant activity, protect mitochondrial membrane integrity, and regulate inflammatory pathways, thereby effectively reducing hepatocyte damage. Clinically, silybin is commonly used to treat liver diseases such as acute viral hepatitis, drug-induced hepatitis, alcoholic hepatitis, and chronic hepatitis with cirrhosis. However, silybin is highly soluble in organic solvents such as ethyl acetate, ethanol, methanol, and acetone, but insoluble in water, resulting in poor oral bioavailability and limiting its application in veterinary clinical practice. Therefore, there is an urgent need to develop novel formulations to improve its water solubility and enhance its efficacy.

[0003] Nanostructured lipid carriers (NLCs) are novel lipid nanocarriers that offer advantages over traditional nanocarriers, including high drug loading capacity and encapsulation efficiency, good stability, and safe, non-toxic excipients. The hydrophobic cavity and hydrophilic outer end structure of NLCs can improve the solubility of poorly soluble drugs. Orally administered NLCs can bypass first-pass metabolism through lymphatic absorption, thereby improving drug bioavailability. Furthermore, NLCs can mask bitterness, reducing the stimulation of taste in animals and improving palatability and efficacy. Therefore, NLCs have the potential to serve as carriers for silymarin in the treatment of liver injury in animals.

[0004] Chinese patent application number 2009100177369 discloses a silymarin nanolipid carrier and its preparation method, but it has disadvantages such as long process time and dependence on low temperature conditions, which increases the difficulty and cost of production. Summary of the Invention

[0005] Purpose of the invention: In order to overcome the shortcomings of the existing technology, the purpose of this invention is to provide a method for preparing silymarin nanostructured lipid carriers with high solubility and low cost.

[0006] Technical solution: The preparation method of the silymarin nanostructured lipid carrier of the present invention includes the following steps:

[0007] Step 1: Take ethanol and silymarin, heat in a water bath, stir until homogeneous, and obtain a silymarin solution; take a co-emulsifier, disperse in deionized water, heat in a water bath and stir to obtain an aqueous phase;

[0008] Step 2: Add solid lipids, liquid lipids, primary emulsifier, and co-emulsifier to the silymarin solution, heat to melt, and stir until homogeneous to obtain the oil phase; under constant temperature stirring conditions, transfer the oil phase into the aqueous phase, mix the solution and shear to obtain a coarse dispersion system;

[0009] Step 3: Heat the coarse dispersion system to concentrate it into an emulsion, and obtain silymarin nanostructured lipid carrier.

[0010] Furthermore, in step one, the mass ratio of ethanol to silymarin is 100:1~5.

[0011] Furthermore, in step one, the heating and stirring temperature is 50~55℃, and the water bath stirring time is 10~15min.

[0012] Furthermore, in step one, the co-emulsifiers are Span 20 (S20), Span 80 (S80), and Span 85 (S85).

[0013] Furthermore, in step two, the mass ratio of solid lipids, liquid lipids, primary emulsifier, and co-emulsifier added to the silymarin solution is 1~9:1~9:3~4.5:0.5~2.

[0014] Further, in step two, the solid lipid is any one of lauric acid, stearic acid, palmitic acid, glyceryl monostearate, glyceryl distearate, and glyceryl behenate. Preferably, the solid lipid is lauric acid.

[0015] Further, in step two, the liquid lipid is any one of ethyl acetate, oleic acid, epoxidized soybean oil, isopropyl myristate, and castor oil. Preferably, the liquid lipid is ethyl acetate.

[0016] Further, in step two, the primary emulsifier is Tween 80 (T80) or Tween 85 (T85). Preferably, the primary emulsifier is Tween 85 (T85).

[0017] Further, in step two, the mass ratio of the primary emulsifier to the co-emulsifier is 6-9:1-4, and the mass ratio of solid lipids to liquid lipids is 1-9:1-9. Preferably, the mass ratio of solid lipids to liquid lipids is 3:7.

[0018] Furthermore, in step three, the heating temperature is 75~80℃.

[0019] Preparation principle: Solid lipids, liquid lipids, and lipid-soluble drugs are combined under high temperature conditions to form a homogeneous oil phase mixture. Simultaneously, an emulsifier is dissolved in water to obtain an aqueous phase. The oil phase mixture is then dispersed in the aqueous phase containing the emulsifier, and the emulsion is ultrasonically amplified using an ultrasonic probe to form nanoscale particles.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant features:

[0021] 1. The prepared silybin nanostructured lipid carrier can improve the solubility of silybin and exhibits good characterization performance;

[0022] 2. The preparation cost is controllable, and large-scale preparation can be achieved;

[0023] 3. Silymarin nanostructured lipid carriers can effectively prevent acute liver injury caused by CCl4 in animals. Attached Figure Description

[0024] Figure 1 This is a transmission electron microscope image of the silymarin nanostructured lipid carrier of the present invention;

[0025] Figure 2 This is a hydration particle size distribution diagram of the silymarin nanostructured lipid carrier of the present invention;

[0026] Figure 3 This is a zeta potential distribution diagram of the silymarin nanostructure lipid carrier of the present invention;

[0027] Figure 4 Figure A shows the effect of the silymarin nanostructure lipid carrier of this invention on the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum. Figure B shows the effect on the ALT level and the AST level.

[0028] Figure 5 The study investigated the effects of silybin nanostructured lipid carriers on mouse liver tissue. Specifically, A represents the effect of silybin nanostructured lipid carriers on the superoxide dismutase (SOD) content in mouse liver tissue, B represents the effect of silybin nanostructured lipid carriers on the hydroxyproline (Hyp) content in mouse liver tissue, and C represents the effect of silybin nanostructured lipid carriers on the glutathione peroxidase (GSH-PX) content in mouse liver tissue.

[0029] Figure 6 This study investigated the effects of silymarin nanostructured lipid carriers on pathological changes in mouse liver. Detailed Implementation

[0030] In the following embodiments, silybin: Highly lipophilic, poorly water-soluble, chemically unstable and easily affected by light, temperature, and pH, leading to reduced utilization. Ethanol: A polar organic solvent, a colorless and transparent liquid at room temperature, volatile and flammable, and infinitely miscible with most organic solvents. Lauric acid: A typical medium-chain saturated fatty acid, a white solid at room temperature, possessing both lipophilicity and surface activity, and chemically stable. Ethyl acetate: A moderately polar organic solvent, a colorless and transparent liquid at room temperature, volatile and miscible with most organic solvents. Tween 85: A nonionic surfactant, a yellow oily liquid at room temperature, with good lipophilicity and emulsifying ability, chemically stable, and biocompatible. Span 80: A nonionic lipophilic surfactant, a yellow oily liquid at room temperature, with strong emulsifying ability and high stability.

[0031] Example 1

[0032] A method for preparing a silymarin nanostructured lipid carrier includes the following steps:

[0033] (1) Add 0.02 g silybin to 0.4 g ethanol, heat and stir at 50 °C for 15 min to obtain a clear silybin solution.

[0034] (2) Add 0.10 g stearic acid, 0.90 g oleic acid and 0.45 g Tween 80 to the silymarin solution in sequence, and continue to heat and stir at 51 °C for 10 min until a homogeneous and transparent oil phase mixture is formed.

[0035] (3) Take 0.05 g of Span 20 and add it to 10 mL of deionized water. Heat and stir at 51 °C to completely dissolve Span 80 in water and obtain a clear aqueous phase.

[0036] (4) At the same temperature, the oil phase was dispersed in the aqueous phase, and the mixed solution was sheared at 15,000 rpm for 5 min using a high shear machine to obtain a coarse dispersion system.

[0037] (5) The coarse dispersion system was heated and concentrated at 75 °C to a system volume of 5 mL to obtain silymarin nanostructure lipid carrier.

[0038] Example 2

[0039] A method for preparing a silymarin nanostructured lipid carrier includes the following steps:

[0040] (1) Add 0.02 g of silybin to 1 g of ethanol, heat and stir at 55 °C for 12 min to obtain a clear silybin solution.

[0041] (2) Add 0.90 g palmitic acid, 0.10 g epoxidized soybean oil and 0.30 g Tween 85 to the silymarin solution in sequence, and continue to heat and stir at 51 °C for 10 min until a homogeneous and transparent oil phase mixture is formed.

[0042] (3) Take 0.20 g of Span 85 and add it to 10 mL of deionized water. Heat and stir at 51 °C to completely dissolve Span 80 in water and obtain a clear aqueous phase.

[0043] (4) At the same temperature, the oil phase was dispersed in the aqueous phase, and the mixed solution was sheared at 15,000 rpm for 5 min using a high shear machine to obtain a coarse dispersion system.

[0044] (5) The coarse dispersion system was heated and concentrated at 80 °C to a system volume of 5 mL to obtain silymarin nanostructure lipid carrier.

[0045] Example 3

[0046] This embodiment aims to explore the optimal solid lipid.

[0047] Accurately weigh 1 g of solid lipids (lauric acid, stearic acid, palmitic acid, glyceryl monostearate, glyceryl distearate, and glyceryl behenate) and place them in vials. Place each vial in a constant-temperature water bath 5 °C above its melting point. Heat the vial to melt the solid lipids into a liquid state. After complete melting, add silybin in 1 mg increments while maintaining constant temperature and stirring. Observe the solubility of silybin in the solid lipids. If silybin is completely dissolved, continue adding until the solid lipids are saturated with silybin. Observe and record the results, and select the solid lipid with the highest drug solubility. The solubility of different solid lipids for silybin is shown in Table 1. The results show that lauric acid has the highest solubility for silybin; therefore, the solid lipid was identified as lauric acid.

[0048] Table 1. Effects of different solid lipids on the solubility of silymarin

[0049]

[0050] Example 4

[0051] This embodiment aims to explore the optimal liquid lipids.

[0052] Accurately weigh 1 g of liquid lipids (ethyl acetate, oleic acid, epoxidized soybean oil, isopropyl myristate, and castor oil) and add them to a vial. Place the vial in a 25 ℃ constant temperature water bath and stir. Add silybin to the liquid lipids at 1 mg increments. Observe the solubility of silybin in the liquid lipids. If silybin is completely dissolved, continue adding until the liquid lipids are saturated with silybin. Observe and record the results, and select the liquid lipid with the highest silybin solubility. The solubility of silybin in different liquid lipids is shown in Table 2. By observing the fluidity, appearance, and solubility of the solution, ethyl acetate was found to be the most effective; therefore, the liquid lipid was identified as ethyl acetate.

[0053] Table 2. Effects of different liquid lipids on the solubility of silymarin

[0054]

[0055] Example 5

[0056] This embodiment aims to explore the optimal ratio of solid lipids to liquid lipids.

[0057] Weigh out 1 g of a mixture of solid and liquid lipids in ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1 into a vial. Heat and stir in a 51 °C water bath. Once the mixture is completely melted, add silybin at 1 mg / time. Observe the solubility of silybin in the mixture. If silybin is completely dissolved, continue adding until the mixture is saturated with silybin. Observe and record the results, and select the solid-liquid lipid ratio with the highest drug solubility. Due to the properties of silybin, the solubility difference between lauric acid and ethyl acetate in the 1:9–9:1 range is not significant. Therefore, add the same mass of silybin to the mixture of lipids in the 1:9–9:1 range, heat and stir at 51 °C, and observe its flowability. By observing the flowability of the system, the mass ratio of solid lauric acid to liquid ethyl acetate was determined to be 3:7.

[0058] Example 6

[0059] This embodiment aims to screen for the optimal emulsifier.

[0060] Each emulsifier has a fixed HLB value, and each dispersion system has its optimal HLB value. However, the HLB value of a single emulsifier is often insufficient to achieve perfect emulsification. Therefore, one emulsifier is usually used as the main emulsifier, while another is used as a secondary emulsifier to adjust the HLB value, thereby achieving a better emulsification effect. Since this invention aims to prepare an oil-in-water (O / W) emulsion, the main emulsifier was selected from O / W emulsifiers. In this experiment, Tween 20 (T20), Tween 80 (T80), Tween 85 (T85), and Poloxamer 407 (P407) were selected as O / W emulsifiers to prepare nanostructured lipid carriers, and the main emulsifier was selected based on the measured hydration particle size, zeta potential, and polydispersity index.

[0061] (1) Optimization of primary emulsifier

[0062] First, an O / W type emulsifier was selected to emulsify the lipids. Under the condition of a milk fat ratio of 50%, the characterization of the prepared formulation is shown in Table 3. Both T80 and T85 achieved good emulsification effects, exhibiting small hydrated particle size and polydispersity index. Since the formulation emulsified with T80 showed stratification after standing for 48 h, T85 was selected as the main emulsifier.

[0063] Table 3. Effects of different primary emulsifiers on the characterization of silymarin nanostructured lipid carriers

[0064]

[0065] (2) Optimization of co-emulsifiers

[0066] T85 is an O / W type emulsifier, and it exhibits good emulsifying effects when compounded with water-in-oil (W / O) Span emulsifiers. Therefore, Span emulsifiers (S20, S80, and S85) were compounded with T85 respectively, and their hydrated particle size, zeta potential, and polydispersity index were examined. The results are shown in Table 4. S80 showed the best co-emulsifying effect, exhibiting a smaller hydrated particle size and polydispersity index, as well as a higher zeta potential. Therefore, S80 was selected as the co-emulsifier.

[0067] Table 4. Effects of different co-emulsifiers on the characterization of silymarin nanostructured lipid carriers

[0068]

[0069] Example 7

[0070] This embodiment aims to investigate the optimal mass ratio of emulsifier to lipid (milk fat ratio).

[0071] Under the conditions of a solid-liquid lipid ratio of 3:7, a total mixed lipid mass of 1 g, and 10 mL of single-distilled water, the characterization results of the prepared silymarin nanostructured lipid carriers at milk fat ratios of 20%, 30%, 40%, 50%, and 60% are shown in Table 5. With increasing milk fat ratio, the hydrated particle size of the formulation first decreases and then increases. The hydrated particle size is smallest when the milk fat ratio is 50%, therefore, the milk fat ratio was determined to be 50%.

[0072] Table 5. Effect of milk fat ratio on the characterization of silymarin nanostructured lipid carriers

[0073]

[0074] Example 8

[0075] This embodiment aims to explore the optimal ratio of primary emulsifier to co-emulsifier.

[0076] The selected primary emulsifier T85 and co-emulsifier S80 were mixed in ratios of 6:4, 7:3, 8:2, and 9:1, added to a mixture of molten lipids and drugs, heated and stirred, and the hydrated particle size and zeta potential of the prepared silymarin nanostructured lipid carrier were measured. The results are shown in Table 6. When the ratio of primary emulsifier to emulsifier was 7:3, the hydrated particle size and polydispersity index were the smallest, and the zeta potential was the highest.

[0077] Table 6. Effect of the ratio of primary emulsifier to co-emulsifier on the characterization of silymarin nanostructured lipid carriers.

[0078]

[0079] Example 9

[0080] A method for preparing a silymarin nanostructured lipid carrier includes the following steps:

[0081] (1) Add 0.02 g silybin to 2.00 g ethanol, heat and stir at 51 °C for 10 min to obtain a clear silybin solution.

[0082] (2) Add 0.30 g lauric acid, 0.70 g ethyl acetate and 0.35 g Tween 85 to the silymarin solution in sequence, and continue to heat and stir at 51 °C for 10 min until a homogeneous and transparent oil phase mixture is formed.

[0083] (3) Take 0.15 g of Span 80 and add it to 10 mL of deionized water. Heat and stir at 51 °C to completely dissolve Span 80 in water to obtain a clear aqueous phase.

[0084] (4) At the same temperature, the oil phase was dispersed in the aqueous phase, and the mixed solution was sheared at 15,000 rpm for 5 min using a high shear machine to obtain a coarse dispersion system.

[0085] (5) The coarse dispersion system was heated and concentrated at 78 °C to a system volume of 5 mL to obtain silymarin nanostructure lipid carrier.

[0086] The morphology of the silybin nanostructured lipid carrier was observed using transmission electron microscopy. 10 μL of the silybin nanostructured lipid carrier prepared in Example 1 was dropped onto a copper grid and allowed to adsorb for 2 min. Excess sample was absorbed with qualitative filter paper, and the sample was air-dried at room temperature for 12 h. The morphology and dispersion of the silybin nanostructured lipid carrier were then observed using transmission electron microscopy. Figure 1 It is known that the silymarin nanostructure lipid carrier is spherical with a particle size of about 50 nm.

[0087] Determination of hydration particle size, zeta potential, and polydispersity index of silybin nanostructured lipid carriers: 1 mL of the nanostructured lipid carrier was added to a sample dish and equilibrated at 25 ℃ for 2 min. The hydration particle size and polydispersity index were measured using a laser particle size potentiometer. 0.85 mL of the silybin nanostructured lipid carrier was placed in a capillary sample dish, and the zeta potential was measured. Figures 2-3 It can be seen that the hydrated particle size of the silybin nanostructured lipid carrier is 108.5±0.6 nm, the polydispersity number is 0.201±0.036, and the zeta potential is -12.7±2.7 mV. Moreover, the silybin nanostructured lipid carrier has good hydrated particle size and zeta potential distribution.

[0088] The content of silybin in the silybin nanostructured lipid carrier was determined to be 13.105±0.005 mg / mL by ultraviolet spectrophotometry, and the encapsulation efficiency was 95.507±0.001%.

[0089] Comparative Example 1

[0090] The method for preparing blank nanostructured lipid carriers is as follows:

[0091] 2.00 g of ethanol was heated and stirred at 51 °C. Then, 0.30 g of lauric acid, 0.70 g of ethyl acetate, and 0.35 g of Tween 85 were added sequentially, and the mixture was heated and stirred at 51 °C for 10 min until a homogeneous, transparent oil phase mixture was formed. 0.15 g of Span 80 was added to 10 mL of deionized water, and the mixture was heated and stirred at 51 °C until Span 80 was completely dissolved in the water, yielding a clear aqueous phase. At the same temperature, the oil phase was dispersed in the aqueous phase, and the mixed solution was sheared at 15000 rpm for 5 min using a high-shear apparatus to obtain a coarse dispersion. The coarse dispersion was concentrated at 78 °C to a volume of 5 mL, yielding a blank nanostructured lipid carrier.

[0092] Application Example 1

[0093] The therapeutic effects of the silymarin nanostructured lipid carriers (Sily-NLCs) prepared in Example 9 were investigated using a CCl4-induced acute liver injury model in mice. The specific experimental steps are as follows:

[0094] (1) Experimental mice and their rearing environment

[0095] Six- to seven-week-old male mice (specific pathogen-free grade, weighing 19-25 g) were purchased from Yangzhou Comparative Medicine Center and housed at the Experimental Animal Center of Nanjing Agricultural University. The mice had free access to food and water.

[0096] (2) Establishment of a mouse model of acute liver injury

[0097] Mice were acclimatized for 5 days in an animal room at 25±2 ℃ and then randomly divided into 7 groups (two control groups and five experimental groups), with 7 mice in each group. The control groups were divided into a Control group and a Sily-NLCs control group. Mice in the Control group were administered physiological saline (20 mL / kg) by gavage for 7 consecutive days, while mice in the Sily-NLCs control group were administered Sily-NLCs solution (60 mg / kg) by gavage for 7 consecutive days. The experimental groups were divided into a CCl4 group, a high-dose Sily-NLCs group, a medium-dose Sily-NLCs group, a low-dose Sily-NLCs group, and a silybin (Silybin) raw material group. The CCl4 group was administered physiological saline (20 mL / kg) by gavage for 7 consecutive days. The high, medium, and low-dose Sily-NLCs groups were administered Sily-NLCs solution at 60 mg / kg, 40 mg / kg, and 20 mg / kg, respectively, by gavage for 7 consecutive days. The Silybin raw material group was administered Silybin (60 mg / kg) by gavage for 7 consecutive days. One hour after the last gavage, all experimental group mice were injected intraperitoneally with 1% CCl4 peanut oil solution, while control group mice were injected intraperitoneally with an equal volume of peanut oil.

[0098] (3) The protective effect of silymarin nanostructured lipid carrier on acute liver injury in mice

[0099] Twenty-four hours after mouse modeling, blood was collected by enucleation. All mice were then immediately euthanized by cervical dislocation, and liver tissue was harvested, weighed, washed with pre-cooled physiological saline, and a portion of the tissue was fixed in 4% paraformaldehyde, while another portion was preserved in liquid nitrogen.

[0100] I. Detection of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST)

[0101] Eye blood was left at room temperature for 0.5 h, then incubated overnight at 4 °C. Serum was collected after centrifugation at 3000 rpm for 5 min at 4 °C. The ALT and AST levels in the serum were read using an automated biochemistry analyzer according to the kit instructions. Results are as follows: Figure 4 As shown, compared with the Control group, the ALT and AST levels in the CCl4 model group were significantly increased, indicating that the liver injury model was successfully established. Sily-NLCs can alleviate CCl4-induced liver injury, as evidenced by a dose-dependent decrease in ALT and AST levels. Silybin raw material can also reduce ALT and AST levels, but its efficacy is not as good as that of the high-dose Sily-NLCs group with the same dosage. In addition, there was no significant difference between the Sily-NLCs control group and the Control group, indicating that Sily-NLCs have no effect on the AST and ALT levels in mice, suggesting that they do not cause liver function damage.

[0102] II. Detection of superoxide dismutase (SOD), hydroxyproline (Hpy), and glutathione peroxidase (GSH-PX) levels in liver tissue

[0103] 0.1 g of liver was weighed into 1 mL of tissue extract, and then homogenized at 60 Hz for 20 s using a multi-sample tissue homogenizer. After homogenization, the homogenate was centrifuged at 10000 rpm for 10 min at 4 °C. The supernatant was collected, and the contents of SOD, Hpy, and GSH-PX in the liver were determined according to the kit instructions. Results are as follows: Figure 5 As shown in A-5B, compared with the control group, the levels of SOD and GSH-PX in the CCl4 model group were significantly reduced, proving the successful modeling. Sily-NLCs can increase the levels of SOD and GSH-PX in a dose-dependent manner, thus alleviating liver damage. Silybin API can also increase the levels of SOD and GSH-PX, but the effect is not as good as that of the high-dose group of Sily-NLCs with the same amount of drug.

[0104] In addition, statistical analysis was performed on the Hyp content in mouse liver. For example... Figure 5As shown in Figure C, compared with the Control group, the Hyp content in the CCl4 model group was significantly increased, proving the successful modeling. Sily-NLCs can reduce the Hyp content in liver-injured mice in a dose-dependent manner. Silybin raw material can also reduce the Hyp content in liver-injured mice, but its anti-fibrotic effect is not as good as that of the high-dose group of Sily-NLCs with the same amount of drug.

[0105] Finally, among the above indicators, there were no significant differences between the Sily-NLCs control group and the Control group, further demonstrating that Sily-NLCs do not cause significant damage to the liver of mice.

[0106] III. Histopathological Observation of Liver

[0107] Mouse livers were fixed in tissue fixative for 24 hours, then dehydrated and cleared, embedded in paraffin, prepared into slides, stained with hematoxylin and eosin (HE), and observed under a microscope. Figure 6 In the Control group and the Sily-NLCs control group, the liver condition was good, with no inflammatory cell infiltration or hepatocyte necrosis observed histologically, indicating that Sily-NLCs did not cause liver damage. In contrast, the livers of mice in the CCl4 group were yellowish-brown, with a tense liver capsule and blunt, swollen edges. Histopathological examination revealed large areas of necrosis, enlarged and swollen hepatocytes, inflammatory cell infiltration, indistinct cell boundaries, and shrunken nuclei. The liver lesions in mice in the Sily-NLCs group and the Silybin raw material group showed varying degrees of relief. Histopathological examination indicated that the high-dose Sily-NLCs group exhibited the mildest liver lesions, with a significant reduction in the area of ​​liver necrosis.

[0108] The data above show that silymarin nanostructured lipid carrier (60 mg / kg) can prevent and protect against acute liver injury in mice. Example 9 is the optimal example.

Claims

1. A method for preparing a silymarin nanostructured lipid carrier, characterized in that, Includes the following steps: Step 1: Take ethanol and silymarin, heat in a water bath, stir until homogeneous, and obtain a silymarin solution; take a co-emulsifier, disperse in deionized water, heat in a water bath and stir to obtain an aqueous phase; Step 2: Add solid lipids, liquid lipids, main emulsifier, and co-emulsifier to the silymarin solution, heat to melt, and stir evenly to obtain the oil phase; Under constant temperature stirring conditions, the oil phase is transferred into the aqueous phase, and the mixed solution is subjected to shearing to obtain a coarse dispersion system. Step 3: Heat the coarse dispersion system to concentrate it into an emulsion, and obtain silymarin nanostructured lipid carrier.

2. The method for preparing a silymarin nanostructured lipid carrier according to claim 1, characterized in that: In step one, the mass ratio of ethanol to silymarin is 100:1~5.

3. The method for preparing a silymarin nanostructured lipid carrier according to claim 1, characterized in that: In step one, the heating and stirring temperature is 50~55℃, and the water bath stirring time is 10~15min.

4. The method for preparing a silymarin nanostructured lipid carrier according to claim 1, characterized in that: In step one, the co-emulsifiers are Span 20, Span 80, and Span 85.

5. The method for preparing a silymarin nanostructured lipid carrier according to claim 1, characterized in that: In step two, the mass ratio of solid lipids, liquid lipids, primary emulsifier, and co-emulsifier added to the silymarin solution is 1~9:1~9:3~4.5:0.5~2.

6. The method for preparing a silymarin nanostructured lipid carrier according to claim 1, characterized in that: In step two, the solid lipid is any one of lauric acid, stearic acid, palmitic acid, glyceryl monostearate, glyceryl distearate, and glyceryl behenate.

7. The method for preparing a silymarin nanostructured lipid carrier according to claim 1, characterized in that: In step two, the liquid lipid is any one of ethyl acetate, oleic acid, epoxidized soybean oil, isopropyl myristate, and castor oil.

8. The method for preparing a silymarin nanostructured lipid carrier according to claim 1, characterized in that: In step two, the primary emulsifier is Tween 80 or Tween 85.

9. The method for preparing a silymarin nanostructured lipid carrier according to claim 1, characterized in that: In step two, the mass ratio of the primary emulsifier to the co-emulsifier is 6~9:1~4, and the mass ratio of solid lipids to liquid lipids is 1~9:1~9.

10. The method for preparing a silymarin nanostructured lipid carrier according to claim 1, characterized in that: In step three, the heating temperature is 75~80℃.

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