BE1030348B1 - NANO-FORMULATION OF SAIKOSAPONIN b1, PREPARATION METHOD AND APPLICATION FOR PREPARING DRUGS FOR PREVENTING AND TREATING HEPATIC FIBROSIS - Google Patents

Abstract

The present invention relates to a nano-formulation of saikosaponin b1, its preparation method and its application for preparing drugs for preventing and treating liver fibrosis, belonging to the field of traditional Chinese medicine. Encapsulating said nano-formulation of saikosaponin b1 according to the present invention with a copolymer of polylactic acid and glycolic acid approved by the FDA, dispersing in a buffer of 2-(N-morpholino) ethanesulfonic acid and reducing the potassium permanganate in manganese dioxide to obtain the nano-formulation of saikosaponin b1, the nano-formulation having a spherical core-shell structure of size of approximately 150 nm.

Description

NANO-FORMULATION OF SAIKOSAPONIN bl, METHOD

PREPARATION AND APPLICATION TO PREPARE

MEDICINES TO PREVENT AND TREAT THE

HEPATIC FIBROSIS

TECHNICAL AREA

The present invention relates to a nano-formulation of saikosaponin b1, its preparation method and its application for preparing drugs for preventing and treating hepatic fibrosis, belonging to the field of traditional Chinese medicine.

TECHNICAL BACKGROUND

[0002] With the increase in the rate of obesity and the frequency of appearance of metabolic syndromes throughout the world, especially in developed countries, hepatic fibrosis and liver cirrhosis have become a serious problem for public health. .

Liver fibrosis is a scarring response to chronic liver injury, characterized by excessive production and deposition of extracellular matrices, which will lead to loss of liver functions and destruction of liver structure, affect oxygen supply and nutrients to the liver, and generated a very anoxic microenvironment in the liver region. During the process of hepatic fibrosis, the anoxic microenvironment of the liver can induce the generation of reactive oxygen and further promote the activation of hepatic stellate cells, thereby accelerating the process of hepatic fibrosis. Therefore, it is of great interest to relieve hepatic tissue anoxia to reverse the process of hepatic fibrosis, thus worthy of further exploration.

[0003] Traditional Chinese medicine has been used for thousands of years in the treatment of liver disease. Certain prescriptions of traditional Chinese medicine BE2029/5705 containing Bupleurum, such as Xiao Chaihu decoction and powder of

Chaihu Shugan have become traditional methods for treating liver disease in Asian countries. Saikosaponin bl is one of the biological active ingredients isolated from Bupleurum, which has good inhibition effect on activated hepatic stellate cells and can be well used in the treatment of liver fibrosis.

However, in the anoxic microenvironment of the liver, the therapeutic effect of

Ssbl (saikosaponin bl) is relatively simple and traditional routes of administration (oral route, injection route, etc.) have disadvantages such as low drug availability and high drug dose. Therefore, there is a need to develop a new composite pharmaceutical formulation, which plays an important role in promoting the therapeutic effect on hepatic fibrosis by improving anoxia in the liver region, reducing the stimulation of

ROS and releasing Ssb1 selectively in the liver region.

DISCLOSURE OF THE INVENTION

[0004] In order to relieve hepatic fibrosis and to reinforce the therapeutic effect on hepatic fibrosis, the present invention proposes a nano-formulation of saikosaponin b1 which has a considerably enhanced therapeutic effect on hepatic fibrosis.

[0005] The present invention also provides a process for preparing the nanoformulation of saikosaponin bl.

[0006] To achieve the above goal, the present invention proposes the following technical solution:

[0007] A nano-formulation of saikosaponin bl is prepared by the following process:

[0008] S1. Dissolve saikosaponin b1 (Ssb1) and a copolymer of polylactic acid and glycolic acid (PLGA) in a solvent, add it dropwise to an aqueous solution of polyvinyl alcohol (PVA) to form microspheres.

[0009] S2. disperse the microspheres in a buffer of 2-(N-morpholino) ethanesulfonic acid (MES) at 0.1-1.0 mol/L, add to this solution dropwise gradually a solution of potassium permanganate at 1-10 mol/L, shake well and centrifuge to obtain the nano-formulation of saikosaponin b1. BE2029/5705

[0010] Encapsulate said nano-formulation of saikosaponin bl according to the present invention with a copolymer of polylactic acid and glycolic acid approved by the

FDA, disperse in 2-(N-morpholino)ethanesulfonic acid buffer and reduce potassium permanganate to manganese dioxide to obtain the saikosaponin b1 nano-formulation, the nano-formulation having a spherical core-shell structure of size approximately 150 nm. The nano-formulation of saikosaponin b1 exhibits significantly enhanced therapeutic effect on liver fibrosis.

[0011] The pharmacological effects of the drug according to the present invention consist in the following: the syndrome of liver stagnation and splenic insufficiency is one of the main syndromes of hepatic fibrosis, Bupleurum was recorded for the first time under the Qing dynasty, with bitter and acrid taste, and slightly cold nature, which returns to the liver and gallbladder meridian, and exhibits effects of relieving external syndromes and reducing fever, clearing liver energy, and to raise the clear IQ, with obvious pharmacological activity. It was found that Bupleurum decoction alone had a good anti-liver fibrosis effect. The results of modern pharmacological studies have shown that the most active ingredient in Bupleurum is saikosaponin. The anti-liver fibrosis effect of Ssbl is an effective practice based on traditional Chinese medicine theories, which can effectively induce apoptosis of activated hepatic stellate cells. However, the clinical therapeutic effect of Ssb1 alone on liver fibrosis is not ideal. In the anoxic microenvironment of the liver, the therapeutic effect of Ssb1 is relatively simple and traditional drug delivery routes ( oral route, injection route, etc.) have the disadvantages such as low drug availability and high drug dose.

The pharmacological basis of the nano-formulation of saikosaponin b1 according to the present invention is that the appearance of fibrosis promotes anoxia of the tissue and aggravates the disease process. Reversing the anoxic state in the liver region can significantly relieve the stimulation on hepatic stellate cells, which is very necessary for the treatment of liver fibrosis. Aiming at the characteristic of anoxia in the hepatic microenvironment, the invention consists of decomposing BE2029/5705 hydrogen peroxide (H202) to generate oxygen under MnO catalysis: reducing the pressure of oxidative stress while relieving anoxia of the tissue, thus reducing the stimulation on hepatic stellate cells at the source, and synergistically reinforcing the therapeutic effect on hepatic fibrosis. The nano-formulation of saikosaponin b1 provided by the present invention can selectively release Ssb1 and MnO: in the liver region, thereby effectively improving drug availability. Moreover,

MnOz catalyzes the decomposition of H,O, in the liver region to generate oxygen, thereby reducing the pressure of oxidative stress while relieving the state of anoxia of the tissue and enhancing the therapeutic effect of Ssb1 on hepatic fibrosis.

Preferably, the mass percentage of PVA in the aqueous PVA solution is 6 to 15 mg/ml.

Preferably, the weight ratio of Ssb1 to PLGA is (2-8): (5-20).

Preferably, the nano-formulation of saikosaponin bl has a spherical core-shell structure of size 150 nm+5 nm.

[0016] A process for preparing the nano-formulation of saikosaponin blm comprises the following steps:

[0017] S1. dissolve satkosaponin b1 (Ssb1) and a copolymer of polylactic acid and glycolic acid (PLGA) in a solvent, add it dropwise to an aqueous solution of polyvinyl alcohol (PVA), shake for 2-3 hours, centrifuge at a speed of 5,000 to 10,000 rpm once the acetone has evaporated, and wash with water to obtain PVA-stabilized PLGA/Ssb1 microspheres;

[0018] S2. disperse the PLGA/Ssb1 microspheres prepared in a MES buffer at 0.1-1.0 mol/L, add to this solution dropwise gradually a solution of potassium permanganate at 1-10 mol/L, so as to generate the manganese dioxide (MnO2) gradually onto the surface of the microspheres by the reduction of the hydroxyl group on the PLGA segment, shake well, then centrifuge, and wash with water to obtain the nano-formulation of saikosaponin b1.

Preferably, the mass percentage of PVA in the aqueous PVA solution is 6 to 15 mg/mL. Preferably, the mass percentage of PVA in the aqueous PVA solution is 10 mg/mL. BE2029/5705

Preferably, the weight ratio of Ssbl to PLGA is (2-8): (5-20). Most preferentially, the weight ratio of Ssb1 to PLGA is 1:10.

[0021] A traditional Chinese pharmaceutical composition comprises the nano-formulation of saikosaponin b1 according to 1. The nano-formulation of saikosaponin b1 according to the present invention may be compatible with other pharmaceutical ingredients or excipients commonly used in medicines to prepare medicines to prevent and treat liver fibrosis.

[0022] An application of the nano-formulation of saikosaponin b1 to prepare drugs to prevent and treat hepatic fibrosis is proposed.

[0023] Tests in animals have shown that, compared to the administration of saikosaponin b1l monomer, the nano-formulation of saikosaponin b1 of the present invention considerably reduces inflammatory cell infiltration and collagen deposition in the tissue. , which shows its enhanced therapeutic effect on liver fibrosis. Therefore, the nano-formulation of saikosaponin bl provided by the present invention has reasonable compatibility, and has safety in use shown by pharmacological validations, it can effectively relieve the symptoms of liver fibrosis, reverse the process of fibrosis hepatic without adverse effects reported.

DESCRIPTIONS OF FIGURES

Figure 1 shows a morphological characterization of the nano-formulation of saikosaponin b1 of the present invention, where scale bar = 100 nm;

[0025] Figure 2 shows simulated in vitro release curves of the saikosaponin b1 nanoformulation of the present invention;

[0026] Figure 3 shows a curve of the effect of the nano-formulation of saikosaponin b1 of the present invention to generate oxygen by catalyzing HO; ;

[0027] Figure 4 shows the effect of saikosaponin bl of the present invention on the expression of the Collagen I, α-SMA and Caspase 3 proteins in HSC-T6 cells, where BE2029/5705

Scale bar = 100 nm;

[0028] Figure 5 shows the effect of the nano-formulation of saikosaponin bl of the present invention to relieve the anoxia of the tissue of mice affected by hepatic fibrosis induced by carbon tetrachloride;

[0029] Figure 6 shows the therapeutic effect of the nano-formulation of saikosaponin b1 of the present invention on hepatic fibrosis induced by carbon tetrachloride, where Scale bar = 100 nm; And

[0030] Figure 7 shows an evaluation of the acute toxicity of the nano-formulation of saikosaponin b1 of the present invention on normal mice, where scale bar = 100 nm;

[0031] In the figures, A - White control group; B - Carbon tetrachloride model group; C - Saikosaponin bl monomer group; D - MnO group; ; E - group of PLGA/Ssb1 microspheres; F - Saikosaponin bl nano-formulation group.

DETAILED STATEMENT OF MODE OF ACHIEVEMENT

[0032] The technical solution of the present invention will be described in more detail below via the specific embodiment examples. It will be understood that the implementation of the present invention is not limited to the exemplary embodiments below, and all variations and/or changes in any form made to the present invention must be included in the protection framework of the present invention.

[0033] In the present invention, unless otherwise indicated, all parts and percentages are units of weight, and the materials and raw materials used can be purchased on the market or commonly used in the art. The methods in the exemplary embodiments below, unless otherwise indicated, are methods conventional in the art.

[0034] In order to better illustrate the essence of the present invention, the effects of the present invention will be further illustrated below by the results of tests of the pharmacological BE2029/5705 effects of the pharmaceutical combination of the present invention.

It is important that the present invention provides a nano-formulation of saikosaponin bl, which is prepared by the following process:

[0036] S1. Dissolve saikosaponin b1 (Ssb1) and a copolymer of polylactic acid and glycolic acid (PLGA) in a solvent, add it dropwise to an aqueous solution of polyvinyl alcohol (PVA) to form microspheres.

[0037] S2. disperse the microspheres in a buffer of 2-(N-morpholino) ethanesulfonic acid (MES) at 0.1-1.0 mol/L, add to this solution dropwise gradually a solution of potassium permanganate at 1-10 mol /L, shake well and centrifuge to obtain the nano-formulation of saikosaponin b1.

[0038] Example of embodiment 1

[0039] A process for preparing the nano-formulation of saikosaponin blm comprises the following steps:

[0040] S1. dissolve 20 mg of polylactic acid-glycolic acid copolymer (PLGA) and 2 mg of Ssb1 in 500 uL of acetone solution, add it dropwise to an aqueous solution of polyvinyl alcohol (PVA) at 10 mg/mL with stirring, shake for 3 hours, obtain saikosaponin b1 microspheres after the acetone has evaporated, centrifuge at a speed of 5000 rpm for 10 mins, and wash with secondary water for three times to obtain PLGA/Ssb1 microspheres stabilized with PVA;

[0041] S2. disperse the PLGA/Ssb1 microspheres prepared at S1 in a buffer of 2-(N-morpholino) ethanesulfonic acid (MES) 0.1 mol/L (pH=5.9), disperse with ultrasound then shake, add drop by drop gradually to this solution 200 uL of 5 mmol/L potassium permanganate solution, so as to generate manganese dioxide (MnO) gradually on the surface of the microspheres by the reduction of the hydroxyl group on the PLGA segment, stir at room temperature for 24 hours , allow to react with stirring until the solution becomes brownish yellow, centrifuge at a speed of 8000 rpm, wash with secondary water for three times, and disperse with ultrasound to obtain the nano-formulation of saikosaponin b1.

[0042] Application examples

[0043] Test methods: BE2029/5705

[0044] 1. Characterization of the nano-formulation of saikosaponin b1

[0045] Take the PLGA/Ssb1 microspheres and the nano-formulations of saikosaponin b1 synthesized in embodiment 1 and characterize the parameters such as the morphology, the drug release speed and the efficiency of oxygen generation under catalysis.

[0046] 1.1 Morphological characterization

[0047] First of all, disperse the nanoparticles in water, drip on copper mesh and a conductive glass slide, dry with nitrogen, then characterize the microscopy using transmission electron microscopy. (TEM) and field emission scanning electron microscopy (SEM). The results of the characterization are presented in Figure 1.

[0048] As shown in Figure 1, the size of the PLGA/Ssb1 microspheres designed according to the present invention was approximately 150 nm, and in regular spherical shape, and the nano-formulation of saikosaponin bl formed a thin obvious layer of MnO2 on the surface of the PLGA/Ssbl microspheres. Under the SEM microscope, the soft microspheres

PLGA/Ssb1 exhibited agglomeration-fusion, while the nano-formulation of saikosaponin bl covered with the thin layer of MnO2 on the surface was relatively rigid and maintained the spherical shape.

[0049] 1.2 Drug release speed

Load respectively the PLGA/Ssb1 microspheres and the nano-formulation of saikosaponin b1 into dialysis bags having a molecular retention threshold of 2000, respectively, and dialyze with ten volumes of PBS buffer (pH 7.4), take at the defined times then complete with the corresponding volume buffer to continue dialysis. After sampling at the set times, determine the release amount of saikosaponin bl using an ultraviolet spectrophotometer to simulate the drug release effect in liver tissue. The results are presented in Figure 2.

[0051] Figure 2 shows drug release curves from the microspheres

PLGA/Ssbl and the nano-formulation of saikosaponin bl, in which the nano-

saikosaponin bl formulation designed according to the present invention releases Ssbl BE20225705 slowly and uniformly for about 10 hours, thereby prolonging the effective action duration of the drug, which is beneficial for enhancing the therapeutic effect on liver fibrosis.

[0052] 1.3 Efficiency of oxygen generation under catalysis

[0053] The effectiveness of the nano-formulation of saikosaponin b1 in generating oxygen by catalyzing the decomposition of H, O, was measured using a dissolved oxygen meter. Disperse the saikosaponin b1 nano-formulation in an aqueous solution and introduce argon continuously into the aqueous solution for 30 minutes to remove dissolved oxygen until the dissolved oxygen drops below 4 mg/L. Then add different quantities of 3% H,O solution, i.e. 0 mM, 11 mM, 33 mM and 44 mM, monitor the generation of oxygen in real time using a dissolved oxygen meter, read and record every 10 seconds, 50 records in total.

[0054] Figure 3 shows an effect of the nano-formulation of saikosaponin bl of the present invention to generate oxygen by catalyzing the decomposition of hydrogen peroxide. For different concentrations of hydrogen peroxide solutions, the nano-formulations of saikosaponin bl could catalyze the generation of oxygen quickly and efficiently, with high concentrations of hydrogen peroxide corresponding to high speed of oxygen generation, which shows that the nano-formulation of saikosaponin b1 of the present invention could quickly eliminate the hydrogen peroxide accumulated in the liver and reduce the oxidative damage of liver cells, in addition, the oxygen generated under catalysis could relieve insufficient oxygen supply in the liver fibrosis region and adjust the environment of the liver fibrosis region and make it more conducive to maintaining the liver cells in the normal state.

[0055] 2. Cellular tests

[0056] 2.1 Cellular modeling

The model of cellular hepatic fibrosis was constructed by stimulating the activation of HSC-T6 hepatic stellate cells of rats by the recombinant protein induced by transforming growth factor-B (TGF-B). Inoculate HSC-T6 cells onto a 6-well plate at 1 x 10°, leave adherent to the wall for BE2029/5705 overnight, replace with serum-free medium and culture for 12 hours, leave two wells blank, add TGF- B at 10 ng/mL in the other wells to activate for 24 hours, scrape the cells, extract the protein then boil it to denature, detect the expressions of a-smooth muscle actin (a-SMA) and type I collagen (Collagen 1) by Western blot (WB) test to verify the success or failure of modeling hepatic fibrosis activated by HSC-T6 cells.

[0058] 2.2 Anti-hepatic fibrosis effect

[0059] Once the success of the cell modeling has been verified, prepare a cell suspension from the HSC-T6 cells in the logarithmic phase of growth, then inoculate the HSC-T6 cells onto a 6-well plate at 1 x 10°, leave adherent to the wall overnight, replace with serum-free medium, culture for 12 hours, leave one well blank, and add TGF-B at 10 ng/mL to the other wells to activate.

Observe the anti-liver fibrosis efficacy of saikosaponin bl monomer drug in activated HSC-T6 cell wells. Co-culture for 24 hours, scrape the cells, extract the protein, detect the expressions of a-SMA and

Collagen 1, and repeat for three times. HSC-T6 cells secrete a-SMA protein after being stimulated and activated by TGF-B to indicate the activation state of HSC-T6 cells. In addition, HSC-T6 differentiates into fibroblasts, secretes collagen and other substances to form collagen fibers, thereby leading to fibrosis in the liver. Figure 4 shows the effect of saikosaponin bl of the present invention on the expression of Collagen 1, α-SMA and Caspase 3 proteins in HSC-T6 cells, where

Scale bar = 100 nm. Figure 4 shows that saikosaponin b1 monomer could effectively reduce the expression of u-SMA and collagen 1, which shows that saikosaponin b1 exhibited a good inhibition effect on cell activation.

HSC-T6 and therefore presented an anti-liver fibrosis effect.

[0060] After that, determine the protein expression change of apoptosis enzyme 3 (Caspase 3) of HSC-T6 cells by WB test, and repeat for three times. There

Caspase 3 (molecular weight 32KD in normal state) is a key executive protein in the cell apoptosis process, which is activated at the early apoptosis phase and breaks down into Cleaved-Caspase 3 subunits having a molecular weight of 12KD, BE2029/5705 which ultimately mediates cellular apoptosis. Saikosaponin b1 induced the expression of cleaved-caspase 3 subunit in HSC-T6 at a high concentration (15 uM), showing that saikosaponin b1 could induce apoptosis in activated HSC-T6 cells, inhibiting thus liver fibrosis.

[0061] 3. Tests in animals

[0062] 3.1 Modeling of animal tests

[0063] Select 60 Balb/c male mice weighing approximately 20 g aged 5 to 6 weeks, divide them randomly into a white control group; a model group of carbon tetrachloride; a group of saikosaponin bl monomer; a manganese dioxide group; a group of PLGA/Ssb1 microspheres; a saikosaponin bl nano-formulation group, 10 mice for each group. Inject olive oil subcutaneously into mice in the white control group, and inject a mixture at 3mL/kg composed of 40% carbon tetrachloride and olive oil into mice from other groups. to induce liver fibrosis in mice, twice a week for 5 weeks, kill the mice and detect the state of liver fibrosis.

[0064] 3.2 Administration protocol

The mouse model suffering from hepatic fibrosis was successfully constructed at the end of five weeks of carbon tetrachloride injection. Except for blank control mice, other mice with liver fibrosis were injected caudally intravenously with 100 μL of PBS buffer, saikosaponin bl monomer dispersed in CMC sodium, microspheres solution of manganese dioxide, a solution of PLGA/Ssb1 microspheres and a solution of saikosaponin bl nano-formulation. Administer every week, and at the same time, inject a mixture at 3mL/kg composed of 40% carbon tetrachloride and olive oil abdominally for 3 weeks to maintain the state of liver fibrosis.

[0066] 3.3 Collect for animal tests

[0067] 24 hours after the last injection, lightly anesthetize the mice with chloral hydrate (at a concentration of 4% prepared with physiological serum, 1 ml per 100 g of mice), then take a sample of blood from the heart , kill by BE2029/5705 decapitation, leave the sample at room temperature for 3 hours, centrifuge at 1500 rpm, collect the supernatant plasma and store at 4 °C. Dissect the liver of mice, remove a piece of liver tissue of the same size, fix with 4% paraformaldehyde for one week, and embedding with paraffin. At the same time, collect a 1.0 cm x 1.0 cm x 1.0 cm piece of liver tissue from the same site, homogenize with saline at low temperature, and store the remaining liver tissue at -80°C.

[0068] 3.4 Detection of relevant indicators

[0069] Take a small piece of frozen liver tissue by cutting at the corresponding site, coat it with ICG, cut into slices having a thickness of 4 μm using a cryo-slicer, carry out tissue immunofluorescence (IF) analysis. to detect the expression of anoxia-inducible factor lo (HIF-1a) in the tissue.

HIF-1" is overexpressed by cells in the anoxic state and induces cells to perform oxygen-independent glycolytic metabolism, allowing them to survive the anoxic state. However, in the process of anoxic metabolism, due to insufficient oxygen supply, a large amount of the high-reactivity oxygen will be generated in the mitochondria, stimulating the activation of HSC-T6 cells. HIF-10, as an indicator protein of cell anoxic state, can well indicate cell growth state.

[0070] In Figure 5, the liver tissue of mice model of hepatic fibrosis presents the highest level of HIF-lo fluorescence, in mice injected with manganese dioxide microspheres and the nano-formulations of saikosaponin b1, l The expression of HIF-1a in their liver tissue showed a significant decrease, showing that the manganese dioxide microspheres and saikosaponin bl nano-formulations of the present invention could effectively catalyze hydrogen peroxide to generate the oxygen in the liver, therefore effectively relieve insufficient oxygen supply to liver fibrosis tissue, reduce oxidative stress of liver tissue, and exhibit a good protective effect on the liver.

[0071] Take the liver tissue coated with paraffin, cut into slices having a thickness of 4 μm and carry out an analysis by staining with hematoxylin-eosin

(H&E), in which the nucleus was stained blue and the cytoplasm was stained red. BE2029/5705

Compared with the normal group, a large amount of inflammatory cells were infiltrated into the liver tissue in the model group, which presented as accumulation of numerous nuclei (Figure 6). In treated mice, compared to the administration of saikosaponin b1 monomer, the saikosaponin b1 nano-formulation of the present invention significantly reduced inflammatory cell infiltration. Additionally, prepare the tissue in paraffin sections and perform Masson staining analysis, with collagen in the liver fibrosis region stained blue, and as shown in Figure 6, compared to administration of saikosaponin b1 monomer , the nano-formulation of saikosaponin b1 of the present invention effectively reduces tissue collagen deposition, indicating its enhanced therapeutic effect on liver fibrosis.

[0072] 3.5 In vivo safety assessment

[0073] The mice were fasted for 12 hours before the tests and randomly distributed into 5 groups according to their body weight: a blank control group, a saikosaponin bl monomer group, a manganese dioxide group, a group of PLGA/Ssb1 microspheres and a group of saikosaponin b1 nano-formulation, 6 mice for each group. Inject the drug intravenously caudally at a dose of 20 mL/kg in each group, and inject an equal volume of PBS intravenously caudally in the PBS control group, once a day for 3 consecutive days, and detect the safety biology of the drug on mice. After injection, collect from animals as described in section "3.3", particularly the following organs: heart, liver, spleen, lungs and kidneys, fix with 4% paraformaldehyde for one week, and coat with paraffin, cut into slices and carry out analysis by H&E staining. Figure 7 shows the results of H&E staining on the main organs of different groups, in histomorphological analysis, there were no obvious changes in the mice in the drug administration group, which showed that the drugs and the Nano-formulations of the present invention did not exhibit obvious toxicity and exhibited good biological safety.

[0074] In conclusion, the nano-formulation of saikosaponin bl of the present BE2029/5705 invention can effectively inhibit and reduce the level of liver fibrosis, has good biological safety and a good liver protection effect.

[0075] Finally, it should be noted that the protection framework of the present invention is not limited to the examples of embodiments below, the examples of embodiments below are only to explain and illustrate the present invention, at least instead of limiting the framework of protection of the present invention, all modifications, equivalent replacements and improvements obtained by those skilled in the art without creative work and respecting the spirits and principles of the present invention must be included within the framework of protection of the present invention.

Claims (10)

CLAIMS BE2029/5705 1. Nano-formulation of saikosaponin b1, characterized in that the nano-formulation of saikosaponin bl is prepared by the following process: S1. Dissolve saikosaponin b1 (Ssb1) and a copolymer of polylactic acid and glycolic acid (PLGA) in a solvent, add it dropwise to an aqueous solution of polyvinyl alcohol (PVA) to form microspheres. S2. disperse the microspheres in a buffer of 2-(N-morpholino) ethanesulfonic acid (MES) at 0.1-1.0 mol/L, add to this solution dropwise gradually a solution of potassium permanganate at 1-10 mol /L, shake well and centrifuge to obtain the nano-formulation of saikosaponin b1. 2. Nano-formulation of saikosaponin b1 according to claim 1, characterized in that the mass percentage of PVA in the aqueous solution of PVA is 6 to 15 mg/mL. 3. Nano-formulation of saikosaponin b1 according to claim 1, characterized in that the weight ratio of Ssbl to PLGA is (2-8): (5-20). 4. Nano-formulation of saikosaponin b1 according to claim 1, characterized in that the nano-formulation of saikosaponin b1 has a spherical core-shell structure of size 150 nm + 5 nm. 5. Process for preparing the nano-formulation of saikosaponin bl according to claim 1, characterized in that it comprises the following steps: S1. dissolve saikosaponin b1 (Ssb1) and a copolymer of polylactic acid and glycolic acid (PLGA) in a solvent, add it dropwise to an aqueous solution of polyvinyl alcohol (PVA), stir for 2-3 hours , centrifuge at a speed of 5,000 to 10,000 rpm after the acetone has evaporated, and wash with water to obtain PVA-stabilized PLGA/Ssb1 microspheres; S2. disperse the PLGA/Ssb1 microspheres prepared in a MES buffer at 0.1-1.0 mol/L, add to this solution drop by drop gradually a solution of potassium permanganate at 1-10 mol/L, so as to generate the dioxide of manganese (MnO2) gradually on the surface of the microspheres by the reduction of the hydroxyl group on the PLGA segment, shake well, then centrifuge, and wash with water to obtain the nano-formulation of saikosaponin b1. BE2029/5705 6. Process for preparing the nano-formulation of saikosaponin bl according to claim 5, characterized in that the percentage by mass of PVA in the aqueous solution of PVA is 6 to 15 mg/mL. 7. Process for preparing the nano-formulation of saikosaponin bl according to claim 5, characterized in that the weight ratio of Ssb1 to PLGA is (2-8): (5- 20). 8. Process for preparing the nano-formulation of saikosaponin bl according to claim 5, characterized in that the weight ratio of Ssb1 to PLGA is 1:10. 9. Traditional Chinese pharmaceutical composition comprising the nano-formulation of saikosaponin b1 according to claim 1. 10. Application of the saikosaponin b1 nano-formulation according to claim 1 to prepare drugs for preventing and treating liver fibrosis is proposed.