Application of radix bupleuri polysaccharide in preparation of medicine for resisting liver transplantation rejection reaction

Abstract

The invention discloses application of bupleurum polysaccharide in preparation of a medicine for resisting liver transplantation rejection, the weight-average molecular weight of the bupleurum polysaccharide is 10-600kDa, and the bupleurum polysaccharide comprises monosaccharides such as mannose, galacturonic acid, glucose, galactose and arabinose. The polysaccharide disclosed by the invention has the effect of resisting liver transplantation rejection, has the potential of inducing immune tolerance of an organism, and is expected to realize final evacuation of an immunosuppressor, so that adverse reaction caused by long-term administration is avoided. The polysaccharide mainly acts on liver macrophages, has tissue specificity, and is non-toxic and good in safety, so that the polysaccharide is expected to be developed into a treatment or adjuvant treatment medicine for resisting liver transplantation immunological rejection with good specificity and low adverse reaction.

Description

Technical Field

[0001] The invention belongs to the field of natural medicines, and in particular relates to the application of bupleurum polysaccharide in the preparation of drugs for resisting liver transplant rejection. Background Art

[0002] Liver transplantation is the most effective means of treating chronic end-stage liver disease, acute liver failure, liver tumors and other serious liver diseases, and the key factor affecting the survival of transplant patients is transplant rejection. According to research, except for identical twins of the donor and recipient, most transplanted livers will experience transplant rejection within 1-2 weeks. If not treated in time, it will lead to transplant liver failure, severe ischemia-reperfusion injury, secondary infection and multiple organ failure, and then death.

[0003] Currently, commonly used drugs include calcineurin inhibitors cyclosporine A (CsA) and tacrolimus (Tac, FK506); mammalian target of rapamycin (mTOR) inhibitors such as sirolimus and everolimus; antimetabolite immunosuppressive drugs such as mycophenolic acid (MPA) and azathioprine (Aza); glucocorticoid drugs such as hydrocortisone sodium succinate, methylprednisolone sodium succinate, etc.; and antibodies such as basiliximab and daclizumab.

[0004] However, current anti-liver rejection drugs mainly inhibit the activation of non-tissue-specific T cells and have no tropism for T cell typing, thus lacking specificity. In addition, due to the systemic distribution of these drugs, while inhibiting rejection in the liver, they also affect the overall immune balance of the body, leading to a significant increase in the incidence of infectious diseases. Long-term use can also cause significant side effects such as liver and kidney damage, neurotoxicity, bone marrow suppression, tumors, etc. Therefore, seeking new strategies with good specificity and low adverse reactions is currently a hot topic in the field of liver transplantation research. Summary of the invention

[0005] The purpose of the present invention is to provide the application of bupleurum polysaccharide in preparing the medicine for resisting liver transplant rejection reaction.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] Application of Bupleurum polysaccharide in the preparation of drugs against liver transplant rejection;

[0008] The weight average molecular weight of the Bupleurum polysaccharide is 10 to 600 kDa;

[0009] The bupleurum polysaccharide comprises monosaccharides such as mannose, galacturonic acid, glucose, galactose and arabinose;

[0010] The bupleurum polysaccharide comprises monosaccharides such as mannose, galacturonic acid, glucose, galactose and arabinose, and the molar ratio thereof is (1-10):(15-25):(30-40):(5-14):(35-45);

[0011] The bupleurum polysaccharide is extracted from bupleurum by alkaline water extraction and alcohol precipitation method;

[0012] The Bupleurum root is distinguished by its processing method, and vinegar Bupleurum root is preferred, as its polysaccharide has a slightly better effect in preventing liver transplant rejection.

[0013] The alkaline water extracted with alkaline water is preferably one or more of ammonia water, sodium hydroxide aqueous solution, and sodium bicarbonate aqueous solution;

[0014] The bupleurum polysaccharide is prepared by the following method:

[0015] Take vinegar bupleurum, defat it, then extract it with alkaline water, concentrate the extract, precipitate it with ethanol solution, re-dissolve the precipitate with water, remove protein by Sevage method, freeze-dry it to obtain total polysaccharide of vinegar bupleurum, and purify the total polysaccharide to obtain the bupleurum polysaccharide of the present invention;

[0016] The degreasing is carried out using 75% ethanol solution;

[0017] The ethanol solution is preferably an 80% ethanol solution;

[0018] The purification is carried out by using a membrane separation system equipped with hollow fiber columns UFP-100-C-4MA and UFP-10-C-4MA.

[0019] The bupleurum polysaccharide can inhibit acute rejection of liver transplantation and reduce the acute rejection score; it can improve liver damage caused by rejection of liver transplantation, inhibit immune hyperactivity factors, and promote the release of immunosuppressive Treg cytokines, thus having the potential to induce immune tolerance in the body;

[0020] The bupleurum polysaccharide is non-toxic to normal liver and kidney cells and shows good safety.

[0021] The anti-liver transplant rejection drug comprises a pharmaceutically acceptable carrier;

[0022] The carrier includes excipients, preservatives, glidants, disintegrants, etc.;

[0023] The excipients include starch, lactose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, etc.;

[0024] The preservatives include sodium benzoate, ethylparaben, etc.

[0025] The glidant includes magnesium stearate, etc.

[0026] The disintegrant includes sodium carboxymethyl cellulose and the like.

[0027] The anti-liver transplant rejection drug can be prepared into liquid preparations such as oral liquid, and can also be prepared into solid preparations such as tablets, capsules, granules, pills, etc. The administration route is oral. The dosage is 0.1 to 5 g / day, which can be determined by the physician according to the specific conditions of the patient.

[0028] Compared with the prior art, the present invention has the following advantages and effects:

[0029] The polysaccharide of the present invention has the effect of resisting liver transplant rejection reaction, and has the potential to induce immune tolerance of the body, and is expected to achieve the final withdrawal of immunosuppressants, thereby avoiding the adverse reactions caused by long-term administration. The polysaccharide of the present invention mainly acts on liver macrophages with tissue specificity, and is non-toxic and safe. Therefore, the polysaccharide of the present invention is expected to be developed into a therapeutic or auxiliary therapeutic drug for resisting liver transplant immune rejection reaction with good specificity and low adverse reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the molecular weight spectrum of the polysaccharide of the present invention.

[0031] Figure 2 The ultraviolet and infrared spectra of the polysaccharide of the present invention are shown in FIG.

[0032] Figure 3 HPLC spectrum of monosaccharide composition of mixed standard (A) and polysaccharide (B) of the present invention.

[0033] Figure 4 The figure shows the effects of the polysaccharide of the present invention on liver appearance (A), liver pathology (B) and RAI score (C) of rats with liver transplant rejection (NC: blank control group; M: model group; CsA: cyclosporine A group; H: high-dose group of the polysaccharide of the present invention; L: low-dose group of the polysaccharide of the present invention).

[0034] Figure 5 The effects of the polysaccharide of the present invention on liver function indexes and immune factors in rats with liver transplant rejection; (A) ALT; (B) AST; (C) TBIT; (D) TBA; (E) TNF-α; (F) IL-10; (NC: blank control group; M: model group; CsA: cyclosporine A group; H: high-dose group of the polysaccharide of the present invention; L: low-dose group of the polysaccharide of the present invention).

[0035] Figure 6 The invention discloses the influence of the polysaccharide on the expression level of TNF-α and IL-10 factors in Kupffer cells.

[0036] Figure 7 The present invention shows the influence of the polysaccharide and CsA on the activity of normal liver and kidney cells. DETAILED DESCRIPTION

[0037] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0038] In the present invention, the molecular weight, ultraviolet spectrum, infrared spectrum and monosaccharide composition of the polysaccharide are determined by the method reported in the literature (Ni Wei, et al. Acs Omega, 2023, 8(32): 29253-29261).

[0039] Example 1

[0040] The preparation method of bupleurum polysaccharide of the present invention comprises the following steps:

[0041] The extraction was carried out using the method reported in the literature (Ni Wei, et al, Acs Omega, 2023, 8(32): 29253-29261).

[0042] Specifically, 3 kg of vinegar bupleurum root medicinal material is taken, and 75% ethanol is used to defat 3 times and then the vinegar bupleurum root powder is dried, and then one or more mixed extractions are used for ammonia water / sodium hydroxide / sodium bicarbonate, and the extract is concentrated to contain 0.5-1.5 g / mL of crude drug, 80% ethanol precipitation is performed, and the precipitation is re-dissolved in water, and the protein is removed by Sevage method, and after freeze-drying, the total polysaccharide of vinegar bupleurum root is obtained, and the total polysaccharide is dissolved in pure water to form a solution with a concentration of 2.0 mg / mL, and the polysaccharide is intercepted by a membrane separation system equipped with hollow fiber columns UFP-100-C-4MA and UFP-10-C-4MA. The procedure is as follows: install the hollow fiber column, circulate and flush with pure water for 5 minutes, recirculate and flush for 5 minutes until the permeate is neutral, empty the liquid, circulate the permeate at a pump speed of 5%, set the pump speed to 5.0%, and the pump pressure to 0.2 bar, and collect the permeate and the intercepted liquid. After the sample is injected, the column is flushed with pure water for 10 minutes, and then flushed with 0.5M NaOH for 20 minutes and then flushed with pure water for 60 minutes. After the liquid is drained, it is flushed with pure water for 10 minutes and then flushed with pure water until it is neutral. The separation is first performed using a UFP-100-C-4MA hollow fiber column, and the permeate is further separated using a UFP-10-C-4MA hollow fiber column. The retentate is collected and freeze-dried to obtain the polysaccharide of the present invention.

[0043] The molecular weights of the obtained polysaccharides are shown in Figure 1 The polysaccharide of the present invention mainly contains 4 molecular weight peaks, and the weight average molecular weights are 524.99 kDa, 54.93 kDa, 14.46 kDa and 1.48 kDa respectively. The ultraviolet and infrared spectra of the obtained polysaccharide show Figure 2The obtained polysaccharide is composed of mannose, galacturonic acid, glucose, galactose and arabinose, with a molar ratio of 2.27:18.90:34.67:7.31:36.84 ( Figure 3 ).

[0044] Example 2

[0045] Polysaccharide of the present invention inhibits acute rejection reaction of liver transplantation

[0046] The following experiments were carried out using the polysaccharide obtained in Example 1:

[0047] The internationally used Lewis-BN rat model was used to establish a rat liver transplantation acute rejection model, and the BN rats that had successfully undergone orthotopic liver transplantation were randomly divided into a model group, a cyclosporine A group, a high-dose polysaccharide group of the present invention, and a low-dose polysaccharide group of the present invention. The blank control group consisted of BN rats that underwent laparotomy but did not undergo liver transplantation, and the number of rats in each group was 6.

[0048] After the liver transplantation was successful, the drug was administered the next day. The dosage of the cyclosporine A group was 6.923 mg / kg / d, the high dosage of the polysaccharide of the present invention was 60.000 mg / kg / d, the low dosage of the polysaccharide of the present invention was 30.000 mg / kg / d, and the blank control group was given normal saline. The drug was administered continuously for 10 days. The liver was taken on the 10th day after the drug administration, and the appearance of the rats was observed. The liver was fixed in 10% formaldehyde solution, embedded, sliced, and the pathological tissue sections were stained with HE. The liver tissue lesions were observed under an optical microscope, and then the scores were scored according to the grading standard of acute rejection reaction in the Banff scheme [Demetris AJ, et al. Banff scheme forgrading liver allograft rejection: An international consensus document [J]. Hepatology, 1997, 25 (3): 658-663.].

[0049] The total score of the Rejection Activity Index (RAI) is 9 points:

[0050] RAI < 3 points indicates inconclusive acute rejection; 3-5 points indicates mild rejection; 5-7 points indicates moderate rejection; and 7-9 points indicates severe rejection.

[0051] The results showed that the polysaccharide of the present invention can improve the disorder and collapse of liver lobule structure, hepatocyte deformation and necrosis, hepatic sinusoid dilatation and portal edema caused by liver transplant rejection, and can also improve inflammatory cell infiltration, thereby significantly reducing RAI score ( Figure 4 ).

[0052] Example 3

[0053] The polysaccharide of the present invention can improve liver function indicators, inhibit immune activation factors, and promote the release of immunosuppressive factors

[0054] The following experiments were carried out using the polysaccharide obtained in Example 1:

[0055] The modeling and administration method of Example 2 were followed, and the administration was continued for 10 days. Blood was collected from the abdominal aorta on the 10th day after administration to detect changes in liver function indicators (ALT, AST, TBIL and TBA) and immune cytokines (TNF-α and IL-10) in the serum of transplanted rats.

[0056] The results showed that the polysaccharide of the present invention can improve the liver function indexes of transplanted rats, especially reduce the bile acid content, inhibit the content of immune activation factor TNF-α, and increase the content of immunosuppressive factor IL-10 ( Figure 5 ), while CsA only significantly inhibited the content of TNF-α and had no significant effect on the content of IL-10. IL-10 is a factor secreted by Treg cells, and Treg cells are an important factor in inducing immune tolerance. Therefore, the polysaccharide of the present invention may have the effect of inducing immune tolerance, while the positive drug CsA has no such effect.

[0057] Example 4

[0058] The polysaccharide of the present invention inhibits the level of M1 type factors of liver macrophages (Kupffer cells) and increases the level of M2 type factors.

[0059] Kupffer cells were isolated by collagenase in situ liver perfusion method according to Dai et al. [Dai LL, et al. Synthesis of endotoxin receptor CD14 protein in Kupffer cells and its role in alcohol-induced liver disease [J]. World J Gastroenterol, 2003, 9 (3): 622-6.]. Kupffer cells in the logarithmic growth phase were taken and 1×10 6 The cells were inoculated into 48-well plates and divided into a blank control group, an LPS group (1ug / mL), and a polysaccharide of the present invention + LPS (1μg / mL) combined treatment group. The drug group was first pre-administered with different concentrations of the polysaccharide of the present invention (0.1-100μg / mL) for 2h, and then LPS (1μg / mL) was added for co-culture for 24h, the cell supernatant was collected, and the content of TNF-α and IL-10 was detected using an Elisa kit.

[0060] The results showed that compared with the model group, the polysaccharide of the present invention significantly reduced the level of M1 cytokines (TNF-α) in Kupffer cells and significantly increased the level of M2 cytokines (IL-10) ( Figure 6 ).

[0061] Example 5

[0062] Comparison of the safety of the polysaccharide of the present invention and CsA, a commonly used drug for liver transplant rejection in clinical practice

[0063] Human normal liver cells LO2 and human embryonic kidney cells HEK293 in logarithmic growth phase were used for the following experiments:

[0064] The cells were inoculated in a 96-well plate at a density of 5000 cells / well and cultured in a 37°C, 5% CO2 incubator for 24 hours. The polysaccharide of the present invention was diluted to a series of concentrations of 0, 1.953125, 3.90625, 7.8125, 15.625, 31.25, 62.5, 125, 250, 500 μg / mL with a culture medium containing 10% FBS, and CsA was diluted to a series of concentrations of 0, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100, 200 μg / mL. The supernatant in the well was discarded, and the cells were grouped according to the above-mentioned polysaccharide and CsA series concentrations, 100 μL / well, and incubated for 24 hours. 20 μL MTT solution (5 mg / mL) was added to each well and incubated at 37°C in the dark for 4 hours. The supernatant in the wells was discarded, and 150 μL of DMSO was added to each well. The wells were shaken for 10 min, and the absorbance of the dissolved crystals was measured at a wavelength of 490 nm using an ELISA reader.

[0065] The proliferation rate of each group was calculated according to the formula:

[0066] Proliferation rate (%) = OD value of the drug administration group / OD value of the control group × 100.

[0067] The results showed that the polysaccharide of the present invention had no effect on the viability of normal liver and kidney cells at 0-500 μg / mL, while CsA at 6.25 μg / mL significantly inhibited the viability of normal liver cells and at 3.125 μg / mL significantly inhibited the viability of normal kidney cells ( Figure 7 ). The results show that CsA has significant toxicity to normal liver and kidney cells, which is consistent with clinical reports. Clinical reports show that liver and kidney toxicity are the main toxic side effects of CsA, which hinders its clinical application; while the polysaccharide of the present invention is basically non-toxic to normal liver and kidney cells, showing better safety.

[0068] Example 6

[0069] A tablet for inhibiting liver transplant rejection reaction is prepared from 200 g of the polysaccharide of Example 1, 300 g of starch, 5 g of magnesium stearate and 10 g of sodium carboxymethyl cellulose by a method known in the art.

[0070] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Application of Bupleurum polysaccharide in the preparation of drugs to prevent liver transplant rejection.

2. The use according to claim 1, characterized in that: The weight average molecular weight of the bupleurum polysaccharide is 10 to 600 kDa.

3. The use according to claim 1, characterized in that: The bupleurum polysaccharide comprises mannose, galacturonic acid, glucose, galactose and arabinose.

4. The use according to claim 1, characterized in that: The bupleurum polysaccharide comprises mannose, galacturonic acid, glucose, galactose and arabinose, and the molar ratio thereof is (1-10):(15-25):(30-40):(5-14):(35-45).

5. The use according to claim 1, characterized in that: The radix bupleuri is vinegar radix bupleuri.

6. The use according to claim 1, characterized in that: The bupleurum polysaccharide is extracted from bupleurum by adopting an alkaline water extraction and alcohol precipitation method.

7. The use according to claim 6, characterized in that: The alkaline water used in the alkaline water extraction is one or more of ammonia water, sodium hydroxide aqueous solution and sodium bicarbonate aqueous solution.

8. The use according to claim 7, characterized in that: The bupleurum polysaccharide is prepared by the following method: Take vinegar bupleurum, defat it, then extract it with alkaline water, concentrate the extract, precipitate it with ethanol solution, re-dissolve the precipitate with water, remove protein with Sevage method, freeze-dry it, and obtain total polysaccharide of vinegar bupleurum. The total polysaccharide is purified to obtain the bupleurum polysaccharide.

9. The use according to claim 8, characterized in that: The purification is carried out by using a membrane separation system equipped with hollow fiber columns UFP-100-C-4MA and UFP-10-C-4MA.

10. The use according to claim 1, characterized in that: The anti-liver transplant rejection drug comprises a pharmaceutically acceptable carrier.

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